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+This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).Tremor is a prominent phenotype of the twitcher mouse, an authentic genetic model of Globoid-Cell Leukodystrophy (GLD, Krabbe’s disease). In the current study, the tremor was quantified using a force-plate actometer designed to accommodate low-weight mice. The actometer records the force oscillations caused by a mouse’s movements, and the rhythmic structure of the force variations can be revealed. Results showed that twitcher mice had significantly increased power across a broad band of higher frequencies compared to wildtype mice. Bone marrow transplantation (BMT), the only available therapy for GLD, worsened the tremor in the twitcher mice and induced a measureable alteration of movement phenotype in the wildtype mice. These data highlight the damaging effects of conditioning radiation and BMT in the neonatal period. The behavioral methodology used herein provides a quantitative approach for assessing the efficacy of potential therapeutic interventions for Krabbe’s disease. Globoid-cell leukodystrophy (GLD; Krabbe’s disease) is a rapidly progressive demyelinating disease caused by the deficiency of the lysosomal enzyme Galactosylceramidase (GALC) [1]. The disease is characterized by prominent CNS symptoms including delayed development, spasticity, and seizures. In humans, the age of onset of symptoms is usually around 6 months, with inevitable progression and death by around 2-3 years of age. Currently, the only available treatment in humans is bone marrow transplantation (BMT). Although BMT can be relatively effective if initiated in pre-symptomatic patients, it is not a cure and treated children continue to develop clinical signs. One important question is: are the continually progressing clinical signs due to GALC deficiency or are they treatment-related?The murine model used to study GLD is the twitcher mouse which is deficient in the same enzyme as that of humans [2,3]. The eponymous and characteristic feature of the murine model is severe twitching or tremor. Other phenotypic features include decreased body weight, hindlimb ataxia, decreased mobility and a shortened lifespan. Similar to the human disease, the neuropathology consists of demyelination, axonal damage [4,5] with prominent inflammation, and the accumulation of lipid-laden macrophages (globoid-cells) throughout the CNS and the PNS. Although tremor is a prominent phenotype in the twitcher mouse, it has not been thoroughly characterized. Also, little is known about how the tremor is altered after various treatments, especially BMT following harsh conditioning regimens. In the current study, a detailed phenotypic characterization of the tremor in the twitcher mouse was performed using a force-plate actometer [6] that allowed recording of the movement-related force generated by small (7–13 g) mice. The power spectra and the locomotor activity of the twitcher mice were compared to the wildtype mice to reveal several important differences. The study also evaluated the effect of BMT on tremor and locomotor activity in both the twitcher and wild type mice. The results demonstrate that BMT and the harsh conditioning regimens associated with this procedure can superimpose additional clinical defects on this already devastating disease. Galc +/–and wildtype mice on the C57BL/6J background were obtained from The Jackson Laboratory (Bar Harbor, ME) and maintained under the supervision of M.S.S. at Washington University School of Medicine. The galc-/- mice were obtained by galc+/– X galc+/– matings. The galc genotype was determined by twitcher-specific PCR [7]. All animals were allowed ad libitum access to food and water, except during brief (12-min or less) behavioral recording sessions. All animal experiments were approved by Institutional Animal Care and Use Committee at Washington University School of Medicine. Neonatal pups were genotyped on day 2 or 3 and BMT was performed on post natal day 3 or 4. The mice received 400 rads of total body irradiation from a 137Cs source. The animals received an intravenous injection of 106 nucleated bone marrow cells from a sex-matched galc+/+, GFP (+) donor [8] via the superficial temporal vein [9].Harmaline (1-methoxy-3, 4-dihydro-β-carboline, H1392, Sigma, St. Louis, MO) at a dose of 15 mg/kg was injected intraperitoneally 12 minutes before the start of tremor monitoring on post natal day 36. The nomenclature for the various treatment groups and the number of animals used in the study is as follows: (a) Wt- untreated wildtype (n = 28), (b) Twi- untreated mutant (twitcher; n = 34), (c) BmtWt- wildtype mice treated with BMT (n = 12), (d) BmtTwi- twitcher mice treated with BMT (n = 7), (e) WtH- wildtype mice treated with harmaline (n = 11), (f) TwiH- twitcher mice treated with harmaline (n = 8), (g) BmtWtH- BmtWt mice treated with harmaline (n = 11), and (h) BmtTwiH- BmtTwi mice treated with harmaline (n = 7). Since the current study was an extension of the previous study (Reddy et al., 2011), and there was an overlap in the animals that were used in the abovementioned study with the current study. The engraftment levels from the various groups are described in results. The design of the force plate actometer and the principles used in the design were described previously [6]. For the current study a force-plate actometer was custom made to accommodate the relatively low body weight and impaired force production capabilities of the untreated twitcher mice. The mean weight of the twitcher mice at 36 days was 10.4 ± 1.6 grams compared to 16.9 ± 1.8 grams for wildtype mice at this age. The custom-made actometer used a carbon fiber/nomex composite material for the load plate, which weighed 57 g, was 3.2 mm thick and measured 24 cm × 24 cm. The sensing area was 20 cm × 20 cm, and the cage that confined the mouse to the load plate was constructed of 6.4 mm-thick clear polycarbonate with inside dimensions of 20 cm long by 20 cm wide by 15 cm high. A removable clear polycarbonate top was perforated with ventilation holes. The load plate was supported by four Model 31a miniature strain gauge load cells purchased from Honeywell/Sensotec (Columbus, Ohio). The load cells were calibrated to yield a force resolution of 0.2 gram-force. The animals were acclimated for at least 30 minutes in the same room prior to tremor monitoring. Data recording was conducted between 2 pm and 6 pm. For mice given harmaline, the drug or saline was injected 12 minutes prior to recording. Data were collected for 6 min, but only the first minute, when movement was maximal, was used for the tremor analyses. The recordings from the transducers were collected at 100 samples/s. The 12-bit integer raw data files were acquired with a LabMaster interface (Scientific Solutions, Mentor, Ohio) that was controlled by a DOS-based Free Pascal program (http://www.freepascal.org). The data from the raw integer files were converted to text files and formatted by Free Pascal programs for further processing by commercially available software (see Statistics section below). Custom written Free Pascal programs were used to calculate distance traveled and the number of low mobility bouts (see below). The following data were extracted from the raw data files [6]: (a) Fz- the net force exerted by the animal at a particular 0.01-s “instant” was calculated as the sum forces on each of the four transducers that supported the load plate. The digitized Fz data obtained at 100 samples/s for the first minute of the recording session were formatted into 12 consecutive 5.00-s time series. Importantly, for the tremor analyses the Fz time series data were expressed as a percent of each mouse’s body weight. This normalization made it possible to compare the power spectra across genetic and treatment conditions without potential confounding by the body weight differences. Each time series (Fz(t)) was Fourier transformed using the fft function in MATLAB (The Mathworks, Inc., Natick, MA). A 500-point Hanning time-domain data window was used. The resulting 12 power spectra were averaged together to yield a single power spectrum for each mouse. The individual frequencies obtained after Fourier transformation were plotted as a continuous function (power spectrum; see Figure 1C) after filtering to retain frequencies between 2.5 and 30.0 Hz. (b) The bandwidth was defined as the difference between the upper and lower limit of the frequencies where the power was half that of the maximum. (c) The center frequency was calculated as the frequency co-ordinate of the vertical line bisecting the bandwidth. (d) The frequency at peak power was taken as the frequency at which the power was at its maximum. (e) Power between 13 and 20 Hz was obtained by integrating the area under the power spectrum curve between 13 and 20 Hz. The aforementioned power spectrum variables (a–e) were computed for each individual mouse, and these variables were then subjected to standard statistical treatments (see below). Although the tremor analyses were performed for the first minute of force-plate recordings, the variables for tracking and quantifying the mouse’s horizontal movements in the actometer were based on the entire 6-min session. (f) The X-Y position of the mouse on the force plate and (g) the distance traveled by the animal was calculated using the principle of moments and by calculating distance (in mm) between centers of force locations at successive time points 0.5 s apart, respectively [6]. The X-Y locations of the animal at various time points were plotted as a function of time to obtain the (h) trajectory of animal movement. (i) X-Y coordinates of the center of force as a function of time were additionally used to identify a low mobility bout, which was defined in terms of a virtual circle with a radius of 15.0 mm that was centered on the mouse as it moved across the load plate. When 5.00 s elapsed without movement beyond the perimeter of the circle, a low mobility bout was tallied, and the 5.00-s time interval was reset to 0 in order to “look for” the next bout. This measure gives an indication of a mouse’s proclivity to “stay in one place” regardless of the location of that place on the load plate.Characterization of tremor in the Twi mice. Representative Fz time series recording from a Wt mouse (A) and a Twi mouse (B) over 12 seconds (each series is 6 s). The X-axis represents time and the Y-axis represents force the vertical force variation (Fz) recorded by the force plate actometer. A Fourier transformation performed on the Fz time series data yields a power spectrum that shows how much power (variance) the Fz variation contains at each frequency of oscillation. The power spectrum can then be further analyzed to yield peak power, frequency at peak power, center frequency, and bandwidth. These data are represented diagrammatically on a hypothetical power spectrum plot (C). The averaged power spectrum (D) of the Twi mice (solid black line) was shifted towards higher frequencies compared to the Wt mice (dashed gray line). The center frequency (E) was significantly increased in the Twi (open circles) compared to the Wt mice (filled squares). There was a significant increase in the band width (F) in the Twi compared to the Wt group. The peak power (G), frequency at peak power (H) and the power between 13 and 20 Hz (I) were significantly increased in the Twi group compared to the Wt group. The horizontal bars represent the mean and the error bars represent the SEM (***p < 0.001, **p < 0.01).Systat (Systat Software Inc., Chicago, IL, USA) and Graphpad prism (Graphpad Software, Inc., La Jolla, CA) were used for generating graphs and performing statistical analyses. ANOVA or Kruskal-Wallis tests were used to compare different groups, and post-hoc multiple comparisons were done using Bonferroni tests.The qualitative differences in the Fz time series between the Wt and Twi mice are shown in Figure 1A and B. A Fourier transform was applied to the Fz time series to obtain a power spectrum (Figure 1C). The power spectra of different animals were further analyzed to obtain the peak power, frequency of peak power, center frequency, and bandwidth (Figure 1C). The averaged power spectra (Figure 1D) showed that the Twi mice had substantially higher power at higher frequencies compared to the Wt mice. The center frequency and the bandwidth in the Twi group were significantly higher than these variables for the Wt group (Figure 1E and F). The peak power (Figure 1G), frequency of peak power (Figure 1H), and the integrated power between 13 and 20 Hz (Figure 1I) were significantly increased in the Twi group compared to the Wt group (p < 0.001 for all three comparisons; t-test). The data for the Wt group are indicative of normal movements without any visible tremor, whereas the Twi mice exhibited the obvious tremor for which they are named. This analysis shows that the tremor of the twitcher mice manifests itself as relatively broad band, high-frequency force oscillations while on the force-plate.Characterization of locomotor activity in the twitcher mouse. The movement trajectory of representative Wt and Twi mice are shown in (A) and (B), respectively. Each panel represents the movement of the mouse for duration of 1 minute. The box in which each movement trajectory is plotted represents the inside wall of the 20 cm × 20 cm cage that confined the mouse to the load plate. Each point in the panel represents the XY location of the mouse at a certain point of time. The total distance traveled during 6 minutes is shown in (C). The total distance traveled by the Wt mice (filled squares) is significantly higher than that of the Twi mice (open circles). The horizontal bars represent the mean and the error bars represent the SEM (***p < 0.001).The Twi group had decreased locomotion compared to that of the Wt group. Representative trajectories of these two groups are shown in Figure 2A and B. The total distance traveled was significantly lower in the Twi group compared to the Wt group (p < 0.001, t-test; Figure 2C).One way to validate the tremor recorded by this newly developed actometer is by comparing it to a relatively well accepted pharmacological tremor model (harmaline treatment) [10]. Therefore, the tremor in the Twi mouse was compared to the tremor in the wildtype mice injected with harmaline (WtH). Harmaline induces a characteristic narrow-band, near 12-Hz tremor. Comparison of the Twi and the WtH animals revealed a robust between-group difference in the averaged power spectra (Figure 3A). As quantified in terms of the mean of the center frequencies, the Twi mice (15.8 Hz) had significantly higher values than the WtH group (12.2 Hz) (p < 0.001; Bonferroni test, Figure 3B). The WtH group had a near-12.2 Hz narrow peak, and the bandwidth was significantly less than that of the Twi group (p < 0.001; Bonferroni test, Figure 3C). There was no statistically significant difference between the distance traveled and the number of low mobility bouts between the WtH and the Twigroups (data not shown), probably because tremor-inducing doses of harmaline in intact animals suppresses locomotion [10]. Interestingly, when twitcher mice were injected with harmaline (TwiH), there was a blunted response with no statistically significant change in center frequency or bandwidth compared to the Twi group (Figure 3B and C). In the WtH group, the peak power was significantly higher (p < 0.001; Bonferroni test; Figure 3D) while frequency of peak power was significantly lower (p < 0.05; Bonferroni test; Figure 3E) compared to the Twi group. There was no significant difference between the TwiH and WtH groups with respect to the distance traveled or the number of low mobility bouts (data not shown). In order to directly assess the effect of myeloreductive conditioning and BMT on the power spectra, tremor quantification was performed on both wildtype and twitcher mice that received BMT (BmtWt versus BmtTwi). The engraftment in various groups is essentially as reported in the study by Reddy et al., 2011. BmtWt group had a mean +/– standard error engraftment of 20.52 +/– 3.077, BmtTwi group was at 12.1 +/– 1.9 and the AAVBMT group was 16.09 +/– 4.0. The engraftment level in the untransplanted animals was less than 1%. The BmtWt group had greater power in both the frequencies around 10–12 Hz and in the higher frequencies in the 13–20 Hz range compared to the Wt group (Figure 4A). The BmtTwi group had greater power in the near 10 Hz lower frequency range compared to the Twi group. The center frequency of the BmtWt group was significantly increased compared to the Wt group (p < 0.01; Bonferroni test; Figure 4B). The bandwidth variable (data not shown) did not indicate any significant between-group effects. The peak power (Figure 4C) and the power between 13 and 20 Hz (Figure 4E) was significantly increased in the BmtWt group compared to the Wt group but not in the BmtTwi group compared to the Twi group. The frequency of peak power was not significantly different in the BmtWt group compared to the Wt group (Figure 4D). There was no significant difference in the frequency of peak power between the BmtTwi group and the Twi group (Figure 4D). The total distance traveled was significantly decreased (decrement in normal function) in the BmtWt group compared to the Wt group (Figure 4F). Comparison of the tremor of the Twi mice with that of the tremor induced by harmaline. The average power spectrum (A) of the Twi mice (solid black line) reflected a broadband tremor and that of the WtH mice (solid gray line) showed a characteristic narrow band 12 Hz tremor. The response to harmaline was blunted in the TwiH mice (dashed black line). The center frequency (B) and bandwidth (C) were significantly increased in the Twi group (open circles) compared to the WtH group (filled diamonds), which exhibited the expected characteristic narrow band tremor typically induced by harmaline in mice. The peak power (D) was significantly increased in the WtH group compared to the Twi group. The frequency at peak power was significantly decreased in the WtH group compared to the Twi group. There was no significant difference in the center frequency, bandwidth, peak power and the frequency of peak power between the Twi and TwiH groups (plus symbols). Horizontal bars represent the mean and the error bars represent the SEM (***p < 0.001, **p < 0.01 and *p < 0.05).Effect of BMT on power spectra. The averaged power spectrum (A) of the BmtWt group (dashed gray line) is shifted upward and rightward compared to the Wt group (solid gray line). Similarly, the averaged power spectrum in the BmtTwi group (dashed black line) is shifted upward across a broad frequency band compared to the Twi group (solid black line). The center frequency (B) was significantly increased in the BmtWt group (open triangles) compared to the Wt group (filled squares). There was no significant difference between the untreated mut (open circles) and BmtTwi groups (cross marks) in the center frequency. The peak power (C), frequency of peak power (D) and the power between 13 and 20 Hz (E) was significantly increased in the BmtWt group compared to the Wt group. Compared to the Twi group, the BmtTwi group showed no significant difference in the center frequency (B), peak power (C), frequency of peak power (D) and power between 13 and 20 Hz (E). The distance traveled by the BmtWt group was significantly decreased compared to the Wt group (F). The horizontal bars represent the mean and the error bars represent SEM (***p < 0.001, **p < 0.01 and *p < 0.05). The BMT-treated animals that had additional higher frequencies in the averaged power spectra were compared to mice that received harmaline after BMT (BmtWtH and BmtTwiH). Interestingly, the mice in the BmtWtH group were more resistant to harmaline-induced tremors than the WtH group (Figure 5A). The response to harmaline in the BmtTwiH group was similar to the TwiH group (Figure 5A). The center frequency was higher and the bandwidth was broader in the BmtWtH group compared to the WtH group (Figure 5B and C). The peak power was significantly lower in the BmtWtH group compared to the WtH group (Figure 5D). There was no significant difference in the center frequency, bandwidth and peak power between the TwiH and BmtTwiH groups (Figure 5B, C and D). There was no significant difference in the distance traveled or in the number of low mobility bouts in the groups compared in Figure 5 (data not shown). Harmaline tremor response in the BMT-treated animals. Averaged power spectra of various groups treated with harmaline (A). The averaged power spectrum of WtH group (solid gray line) appears different compared to the averaged power spectrum of the other groups in (A). The averaged power spectra of the BmtWtH (solid black line), TwiH (dashed black line), and BmtTwiH (solid gray line) appear very similar to each other. The center frequency (B) and the bandwidth (C) were significantly increased in the BmtWtH group (open triangles) compared to the WtH group (filled squares). The peak power (D) was significantly decreased in the BmtWtH group compared to the WtH group. There was no significant difference in center frequency, bandwidth or peak power between the TwiH (open circles) and BmtTwiH groups (cross marks). Horizontal bars represent the mean and the error bars represent the SEM (***p < 0.001, **p < 0.01 and *p < 0.05).The brains of the various treatment groups were evaluated for the presence of globoid cells (multinucleated macrophages with storage material) and myelination using Periodic acid-Schiff (PAS) and Luxol fast blue (LFB) staining, respectively (Figure 6). When stained for PAS, the cerebellar white matter from a Wt mouse shows no globoid cells (Figure 6A), in contrast the brain from a Twi mouse contains a large number of multinucleate cells with pink staining cytoplasm (Figure 6B, arrowheads). There is no apparent decrease in the number of globoid cells in the BmtTwi mouse compared to the Twi mouse (Figure 6C). When the brains are stained for LFB, the Wt group shows uniform myelination in the cerebellar white matter (asterisk in Figure 6D). In contrast, the architecture of the myelin in the Twi group appears less well organized, probably secondary to inflammatory edema (Figure 6E). In the BmtTwi group, the myelin remains disorganized, similar to that observed in the Twi group (Figure 6F).Histology- PAS and LFB staining. There are no identifiable PAS-positive cells in the cerebellum of the Wt animal (A). In the Twi (B), there are numerous pink multinucleated globoid cells (arrowheads). There is no obvious decrease in the number of PAS-positive cells in the BmtTwi group (C). Comparison of LFB staining in the cerebellar white matter (asterisk in D–F) and cerebellar folia shows a disruption of myelin architecture (blue staining) in the Twi (E) mouse compared to the Wt (D) animal. In the BmtTwi group (F), the myelin architecture remains disrupted, similar to the Twi group.In the current study, a detailed quantitative characterization of the tremor phenotype (movement-related force oscillations) in the twitcher mice was performed using a force-plate actometer. The tremor in the twitcher mouse was seen as increased power at relatively higher frequencies with a broader bandwidth than the movement-related force oscillations exhibited by the wildtype mice moving normally on the force plate. An incidental finding in these studies was that the stereotypical tremor produced by harmaline was significantly altered in the twitcher mice. Harmaline is believed to disrupt the olivocereballar circuits [11]. The altered response of twitcher mice to harmaline is consistent with the deficiency of GALC leading to inflammation and demyelination in the white matter tracts of the cerebellum [12,13] including the olivocerebellar nucleus. The validity of tremor monitoring in evaluating the effects of various therapies was also determined. Hematopoietic stem cell transplantation (either using umbilical cord blood or bone marrow) is the only available therapy for Krabbe’s disease in humans [14,15]. Bone marrow transplantation also prolongs the lifespan in the twitcher mouse [12,16,17]. However, the effect of radiation conditioning (in preparation for BMT) and transplantation of donor bone marrow cells on the prominent tremor phenotype exhibited by the twitcher mouse had not heretofore been determined. Twitcher mice receiving BMT had power spectra that had greater power in higher frequencies, implying a worsening of tremor. This is contradictory to what is observed in terms of other measures like lifespan seen in previous studies [12,13]. Interestingly, wildtype animals receiving only BMT also had altered power spectra and were resistant to the effects of harmaline compared to the wildtype animals that did not receive BMT. Bone marrow transplantation using myeloreductive conditioning (400 rads of total body irradiation) is known to cause cerebellar dysplasia in neonatal mice [18]. The blunted response of the harmaline-treated animals receiving BMT is also consistent with the conditioning regimen causing cerebellar damage, including the olivocerebellar nucleus. Conditioning and BMT could also be adversely affecting some as yet unknown regions in the brain or periphery to give the same abnormal response. The current study thus highlights a possible harmful effect of conditioning in treating the disease. These effects could be explained by the presence of rapidly proliferating cells in the cerebellum during the time of conditioning and BMT [19]. Although, the current study uses myeloreductive conditioning at post natal day 3 or 4, similar and perhaps more severe function-compromising effects could be expected in studies that use fully myeloablative regimens [16,17]. Although conditioning regimens used in newborn children [15] typically do not involve ionizing radiation, the myeloablative drugs are nonetheless highly toxic and may superimpose additional abnormal phenotypes on the already complex Krabbe’s disease presentation. This could further complicate the interpretation of the therapeutic benefits of BMT for GLD. This study establishes the use of a custom-built, ultra-sensitive force-plate actometer in evaluating the effects of various therapies for the twitcher mice. It also emphasizes the need to evaluate the impact of various therapeutic approaches on a wide variety of functions before drawing conclusions on their safety and efficacy. We thank Kevin O’Dell for excellent technical assistance with the animal husbandry. We also thank Haihui “Sophie” Wang for the help with histology. This work was funded by NIH R01 HD055461 and NIH R01 NS043205 (MSS), Hunter’s Hope foundation and National Tay-Sach’s and Allied Diseases Association (ASR), NIH Neuroscience Blueprint Interdisciplinary Center Core Grant HD06217110 to Washington University (DFW), and Center Core Grant HD02528 to the University of Kansas (SCF). 

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+This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).A 59 year-old woman with liver cirrhosis due to hepatitis C, complicated by refractory hepatic hydrothorax was treated with a TIPS (transjugular intrahepatic portosystemic shunt) procedure. The procedure was complicated by substantial gastrointestinal hemorrhage. EGD (esophagogastroduodenoscopy) was performed and revealed hemobilia. A hepatic angiogram was then performed revealing a fistulous tract between a branch of the hepatic artery and biliary tree. Bleeding was successfully stopped by embolization of the bleeding branch of the right hepatic artery. Hemobilia is a rare cause of upper gastrointestinal bleeding with an increasing incidence due to the widespread use of invasive hepatobiliary procedures. Hemobilia is an especially uncommon complication of TIPS procedures. We recommend that in cases of hemobilia after TIPS placement, a physician should immediately evaluate the bleeding to exclude an arterio-biliary fistula.The term hemobilia was first coined by Sandblom [1], when he described bleeding into the biliary tree following trauma. Hemobilia has now become widely recognized due to the improvements in diagnostic modalities and an increased index of clinical suspicion for the disorder. Hemobilia occurs when a fistula forms between a vessel of the splanchnic circulation (hepatic artery or portal vein) and the intrahepatic or extra-hepatic biliary system. Common causes include iatrogenic manipulation of the hepatobiliary system and trauma [2]. Management of hemobilia is aimed to stop bleeding, maintain continuous flow through the biliary system and treat the underlying etiology. Iatrogenic hemobilia after TIPS (transjugular intrahepatic portosystemic shunt) is extremely uncommon but several cases have been reported. [3,4,5]. We report iatrogenic hemobilia as a complication of TIPS procedure [6], which was successfully managed by transarterial embolization.The patient is a 59 year-old female with history of liver cirrhosis due to hepatitis C, which was complicated by refractory ascites, hepatic hydrothorax, and hypertension, who visited to the hospital for shortness of breath and abdominal distension. She was found to have a right-sided pleural effusion. Pleural fluid analysis showed a serum ascites-albumin gradient (SAAG) >1.1, consistent with transudative effusion, most likely hepatic hydrothorax. A TIPS procedure was recommended for the treatment of the patient’s refractory hepatic hydrothorax. Pre-procedure: Total bilirubin 0.6 mg/dL, albumin 3.2 g/dL, ALP (alkaline phosphatase) 70 IU/L, ALT (alanine transaminase) 27 IU/L, AST (aspartate transaminase) 41 IU/L, INR (International Normalized Ratio) 1.5, hemoglobin 16.2 g/dL and serum creatinine 0.4 mg/dL. The TIPS procedure was successfully performed. After the procedure, the patient had multiple episodes of hemetemesis and her hemoglobin dropped to 8.9 g/dL. Packed red blood cell transfusion was administered and an EGD showed fresh, large blood clots emerging from the ampullary orifice consistent with hemobilia (Figure 1).Ampulla of Vater showing hemobilia.She was immediately prepped for a hepatic angiogram with possible embolization. When the selected right hepatic artery that opacified the biliary tree was identified, a slurry of gelfoam and contrast was injected until there was a cessation of blood flow and resolution of the opacification of the biliary tree. See Figure 2 and Figure 3.Sub-selective right hepatic arteriogram shows normal arborization of the selective artery injected. Shunt is in place.Delayed image of the arteriogram demonstrating the opacification of biliary tree, which indicates a fistula between hepatic artery and biliary tree.Transjugular intrahepatic portosystemic shunt (TIPS) has been utilized in the treatment of portal hypertensive complications for more than 20 years. Indications for TIPS determined by controlled trials include management of variceal bleeding, refractory cirrhotic ascites, hepatorenal syndrome, gastric antral vascular ectasia, Budd Chiari syndrome, and refractory hepatic hydrothorax [7].Table 1 shows the reported complications of tips [8].Reported complications of TIPS (transjugular intrahepatic portosystemic shunt)[8].Iatrogenic hemobilia [9] may occur as a result of percutaneous liver procedures, liver or biliary operations, or therapeutic anticoagulation. Given the close proximity of bile duct radicals to the branches of the hepatic artery and portal vein, the substantial incidence of concurrent injury to these structures and fistula formation is not unexpected [10,11]. A 3.8% incidence of hepatic vascular abnormalities was found following percutaneous transhepatic cholangiography [12], a 5.4% incidence of hepatic vascular abnormalities following percutaneous liver biopsy, and a 26.2% incidence following the placement of indwelling transhepatic drainage catheters [13]. The frequency of clinical hemobilia ranges from less than 1% for liver biopsy [14] to 4% for transhepatic cholangiography, 3% to 14% for percutaneous transhepatic catheter drainage and <5% post-TIPS procedure. Our patient had end stage liver disease (ESLD). The TIPS procedure was done as a palliative therapy. In our case, a covered stent was not used. Furthermore, a survival benefit has not been demonstrated in covered stent. Therefore, an uncovered stent was used in our patient as she was not a transplant candidate and stent longevity was not of concern given the palliative nature of the procedure. The clinical presentation of hemobilia includes the Quinke triad: biliary colic, jaundice, and gastrointestinal bleeding, which may range from occult to massive bleeding. The initial diagnosis can be made with endoscopy, bleeding scan, or angiogram. Angiography with possible embolization is the treatment of choice for most cases of hemobilia because it can be both diagnostic and therapeutic [15,16]. Although only 12% of cases are initially diagnosed with endoscopy, it may confirm the diagnosis in an additional 30% of patients and help exclude other causes of upper gastrointestinal tract bleeding [17]. Biliary endoscopic procedures in current practice are helpful in the management of hemobilia [18]. Other diagnostic methods include computed tomography and ultrasonography [19]. If all of these measures fail to provide a diagnosis, or if the patient presents under emergency circumstances such as hemodynamic instability, the surgeon may be forced to do an exploratory laparotomy without a precise preoperative diagnosis.The goals of therapy in cases of hemobilia are to stop the bleeding and to restore bile flow past any clot formation. Modalities used to stop bleeding include angiography with embolization, surgical intervention, and endoscopic electrocoagulation or photocoagulation. In the past, successful embolization of intrahepatic bleeding sites was affected by the technical inability to have selective arterial access. This lead to complications caused by nonselective embolization. However, today, angiography [15] is clearly the most efficacious method for controlling intrahepatic bleeding sources, with success rates above 95%. A complication of embolization includes hepatobiliary necrosis (6%), abscess formation (9%), bleeding (6%), and gallbladder fibrosis (2%).It is interesting to note that hemobilia from a portal venous source, though exceedingly rare, is more likely to require surgical treatment [20]. Finally, surgical therapy should be considered as the treatment of choice when the cause of hemobilia constitutes an independent indication for such treatment, such as cases associated with cholelithiasis, cholecystitis, or resectable neoplasm.The least commonly used option for managing hemobilia is that of expectant observation. Spontaneous cessation of bleeding occurs most often in patients who undergo percutaneous cholangiography or liver biopsy; therefore, this group merits observation as the primary management. Some authors have proposed the prophylactic administration of clot promoters such as absorbable gelatin sponges (Gelfoam, Upjohn, Kalamazoo, MI, USA) into percutaneous puncture tracts during withdrawal of the instruments or drains from the liver to reduces bleeding complication [21]. Endoscopic techniques for controlling hemorrhage and managing clots include nasobiliary drainage, sphincterotomy, and laser photocoagulation using small endoscopes placed through a catheter tract via access to the biliary tree [22]. These methods have only been reported anecdotally and will probably continue to have a role in selected cases. The complications of hemobilia are uncommon and include pancreatitis, cholecystitis, and cholangitis.Physicians should be aware of hemobilia as one of the possible complications of TIPS and should be experienced in its management. Liver parenchymal puncture during a TIPS procedure may damage vascular structures such as the hepatic artery, portal vein, as well as bile duct. Therefore, we recommend that in cases of gastrointestinal hemorrhage after TIPS placement, a suspicion of hemobilia should be high on the differential diagnosis. After the diagnosis of hemobilia is confirmed by an upper endoscopy, hepatic angiogram should be done to visualize arterio-venous-biliary fistula. Today, transarterial embolization is the gold standard in the management of hemobilia. The authors declare no conflict of interest. 

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+This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).“Any fool can know. The point is to understand.”“Why yet another journal?” Because this new journal will be different in that we are not just about new information but about understanding new information. We are online, peer reviewed, with a quick turnaround time from submission to publication and without any limit regarding length! Therefore, I am honored to introduce the Journal of Clinical Medicine (JCM), which has been created to serve as a hub for disseminating new findings and discoveries in clinical medicine to clinicians and medical researchers worldwide. JCM is an international, peer-reviewed, open-access journal published online quarterly that will provide a platform for advances in health care/clinical practices, the study of direct observation of patients and general medical research. This multi-disciplinary journal is aimed at a wide audience of medical researchers and healthcare professionals. Each edition of the journal will present original research and ideas as well as reviews, communications, short notes, and book reviews involving all fields of clinical medicine. Because of our on-line format, there is no limit to publication length. We encourage our authors to publish their findings and results in as much detail as needed so that our readership will truly understand how they performed their research or worked up their patient and why. Additionally we will cover a wide spectrum of subject areas including (1) clinical trials in which we will include the design of the trial; details regarding data collection; discussion of the legal, ethical and regulator issues; and quality assurance issues; (2) medical science research; (3) epidemiolgic studies and reviews; (4) bioethic reviews and discussions; (4) in-depth reviews on preventive medicine, translational medicine, occupational health and palliative medicine; and (5) information regarding medical care and research in developing countries. Each article will undergo a rigorous peer-review process that will incorporate strict ethical policies and standards to ensure that the journal is scholarly. To ensure this process we have gathered a prestigious and international group for our editorial board including: Dr. Emmanuel Andrès (Department of Internal Medicine, Diabetes and Metabolic Diseases, University Hospital of Strasbourg, France), Dr. Michael Berk (School of Medicine, Deakin University, Strategic Research Centre for Psychiatry and Epidemiology, Australia), Dr. David L. Brown, (Cardiovascular diseases, Stony Brook University Medical Center, New York, NY, USA), Dr. Edzard Ernst (Complementary Medicine, Peninsula Medical School, Devon, UK), Dr. Bronwen A. Evans (Department of Child Health, School of Medicine, Cardiff University, Cardiff, UK), Dr. Nuri B. Farber (Department of Psychiatry, Washington University, St. Louis, MO, USA), Dr. Kathleen M. Gillespie (Diabetes and Metabolism, Southmead Hospital, Bristol, UK), Dr. Michael R. Hamblin (Department of Dermatology, Harvard Medical School, Wellman Center for Photomedicine, Massachusetts General Hospital, Boston, MA, USA), Dr. David T. Harris (Department of Immunobiology, Arizona Health Sciences Centre, University of Arizona, Tucson, AZ, USA), Dr. Anthony J. Lembo (Gatrointestinal diseases, Department of Medicine, Beth Israel Deaconess Medical Center, Boston, MA, USA), Dr. Laxmaiah Manchikanti (Pain Management Center, Anesthesiology and Perioperative Medicine, University of Louisville, Paducah, KY, USA), Dr. Pier Mannuccio Mannucci (Hematology, IRCCS Cà Granda Maggiore Policlinico Hospital Foundation, Milano, Italy), Dr. Hans-Jürgen Möller (Department of Psychiatry, University of Munich, Munich, Germany), Dr. Guido Nikkhah (Department of Stereotactic and Functional Neurosurgery, University Medical Center Freiburg Neurocenter, Freiburg, Germany), Dr. Julia Polak (Imperial College Tissue Engineering Centre, Imperial College London, London, UK), Dr. Parminder Raina (Geriatrics, McMaster University, Hamilton, Ontario, Canada), Dr. Russel J. Reiter (Department of Cellular & Structural Biology, The University of Texas Health Science Center, San Antonio, TX, USA), Dr. Mark S. Sands (Washington University School of Medicine, Departments of Medicine and Genetics, St. Louis, MO, USA), Dr. Evan Y. Snyder (Sanford-Burnham Medical Research Institute, Department of Pediatrics, University of California San Diego, La Jolla, CA, USA), Dr. David F. Wozniak (Department of Psychiatry, Washington University School of Medicine, St. Louis, MO, USA) and Dr. Xiao Yan Zhong (Women’s Health, Department of Biomedicine, University Hospital Basel, Basel, Switzerland).Welcome! We invite you to submit your articles and learn along with us as we undertake this new journal. 

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+This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).Background: Adhesion formation is a widely acknowledged risk following abdominal or pelvic surgery. Adhesions in the abdomen or pelvis can cause or contribute to partial or total small bowel obstruction (SBO). These adhesions deter or prevent the passage of nutrients through the digestive tract, and may bind the bowel to the peritoneum, or other organs. Small bowel obstructions can quickly become life-threatening, requiring immediate surgery to resect the bowel, or lyse any adhesions the surgeon can safely access. Bowel repair is an invasive surgery, with risks including bowel rupture, infection, and peritonitis. An additional risk includes the formation of new adhesions during the healing process, creating the potential for subsequent adhesiolysis or SBO surgeries. Objective: Report the use of manual soft tissue physical therapy for the reversal of adhesion-related partial SBOs, and create an initial inquiry into the possibility of nonsurgical lysis of adhesions. Case Reports: Two patients presenting with SBO symptoms due to abdominal adhesions secondary to abdominal and pelvic surgery were treated with manual soft tissue physical therapy focused on decreasing adhesions. Conclusions: Successful treatment with resolution of symptom presentation of partial SBO and sustained results were observed in both patients treated.Peritoneal adhesions are a major contributory factor in the development of small bowel obstruction (SBO) worldwide and are a common post-abdominopelvic surgical occurrence. The literature indicates that 50% to 100% of patients develop adhesions after any abdominal/pelvic surgery [1,2,3,4,5]. A large analysis of the incidence of adhesions which was performed in the 1990’s found that 35% of all open abdominal or pelvic surgery patients were readmitted to the hospital more than twice to treat post-surgical adhesions within 10 years following their initial surgery, with 22% of readmissions requiring surgery occurring within the first year. The report noted “readmissions continued steadily throughout the 10-year period” of the study [6].The severity of post-surgical adhesions are challenging to control, as they are influenced by factors attributed to both the patient and surgical procedure. Despite numerous clinical trials to assess the validity of various strategies, agents and surgical meshes to prevent adhesion formation, the debate continues with no protocol yet shown that prevents the generation of adhesions as the body heals from surgical intervention [2].Adhesions commonly form secondary to the normal wound healing process. They form as a result of fibrin formation at the site of the surgical wound which provides the matrix for fibroblasts to migrate and generate a collagen extracellular matrix (ECM) [2]. It is this ECM and its associated cells that ultimately become the adhesions. Although it is the body’s response to surgery and the healing process in the presence of increased levels of inflammatory cytokines that promotes the formation of adhesions, it is clear that adhesions are not merely nonfunctional scar tissue. It has been found that mature adhesions are complex tissues with ingrowths of capillaries, adipose tissue, smooth muscle and nerve fibers that are typically found in complex regenerating structures [7]. Further, adhesion formation is not always limited to the geography of the locally traumatized tissues. Some patients form additional adhesions at locations proximal or distal to the surgical site. It should be noted that surgery is not the only cause of adhesion formation and that all healing, including surgery, physical trauma, infection and/or inflammation has the ability to form adhesions in the body [8].The standard current treatment for SBO due to adhesions that cannot be resolved by gastric decompression or bowel rest is surgery to lyse the adhesions (adhesiolysis), thereby resolving the obstruction. In some cases, SBO surgery also involves cutting and removing adhered, damaged or necrosed sections of the bowel, followed by resection of the remaining bowel ends. Utilizing surgery to lyse the adhesions caused by an initial surgery and/or to resect the bowel often causes the formation of new adhesions as a part of the wound healing process. This can create and perpetuate an ongoing cycle of healing and adhesion formation, obstruction surgery to remove the offending adhesions or resect the bowel, followed by new healing and new adhesion formation, and so on. In cases of SBO, this cycle can mean repeat hospitalizations to treat a life-threatening condition. The risk of infection due to spillage of bowel contents into the interstitial spaces of the abdomen is not an insignificant complication following bowel resection. Thus, any method that can be used to slow, prevent, or non-surgically release post-surgical adhesions that compromise the bowel is of significant value to physicians and their patients.Adhesions have also been identified as a leading cause of secondary female infertility [9]. The Clear Passage Physical Therapy group has previously reported the successful use of this manual soft tissue physical therapy in the treatment of fallopian tube occlusion and treatment of dyspareunia and dysmenorrhea secondary to adhesions [10,11]. Following publication of those studies, the same manual soft tissue physical therapy was applied to patients with documented abdominal adhesions following recurrent abdominal surgeries, and to patients presenting with current or recurring partial and total SBOs as a non-surgical treatment option.The two patients presented in this retrospective study were required to complete a standard Patient Intake Questionnaire detailing their pain, medical and surgical history. Body Mass Index was calculated for each patient using the online BMI calculator from the NIH National Heart Lung and Blood Institute. [12] Those patients who indicated a history of small bowel obstruction (SBO) on the patient intake form were asked if they were willing to allow us to use their data in a small, investigative, retrospective study. Each patient then signed an Informed Consent document. Frequency, timing and severity of SBO episodes and all abdominal or pelvic surgeries were noted in their charts. Each patient was required to submit all relevant medical records and operative reports from which the medical history presented in this case study is summarized. Both patients presented in this case report had sought medical treatment and diagnostics for adhesions and bowel obstructions from multiple physicians and radiologists prior to manual physical therapy treatment.Previous medical histories for each patient were obtained in accordance with HIPPA regulations. Detailed clinical records were kept of each patient throughout the course of therapy including symptomatic complaints, areas treated, techniques used, and treatment date/duration in accordance with the American Physical Therapy Association guidelines.Upon initial evaluation and discharge assessment, patients were asked to rate their pain on a scale from 0 to 10, with 0 being no pain and 10 being debilitating pain. The patients were also asked about quality of life measures including impact of pain on daily activities and dietary restrictions due to their condition.Findings from the initial evaluation, which included patient history, previous pathology reports and physician diagnoses, and visual, palpatory, postural and movement exams were correlated to determine dysfunctional areas needing treatment. In these cases, restrictions, scars or adhesions were palpated by the therapists, major organs were identified, and decreased mobility of specific organs was determined. The evaluation included layer palpation of the myofascial and visceral structures; starting with the most superficial structures and progressing to the deepest. The development of tactile skills includes the ability to detect tissue and visceral texture abnormalities and restricted mobility [13]. The therapists assessed tissue temperature, moisture, shear, extensibility and texture, throughout the abdominopelvic viscera, with a special focus on areas deep to surgical scars. Testing visceral mobility consisted of the therapist palpating the tissues, then making precise manual movements to assess the ability of the organs to move directly over surrounding organs and tissues. The patients were treated via a site-specific intensive manual physical therapy that occurred for 4 h a day for 5 consecutive days. In the case of these two patients, approximately 70% of the therapists’ time was utilized directly on the adhered areas of the abdomen and pelvis. The manual physical therapy protocols utilized by the therapists have been previously described in other studies where the primary focus of therapy was the manual decrease of adhesions, and the outcomes were a return of normal mobility and motility to previously adhered organs. [10,11,14] Thus, the therapy described in these studies and on those patients may be applied to adhered areas of any of the abdominal or pelvic organs, including the reproductive, digestive, urinary and other organs within and external to the peritoneum.Briefly, the protocol consists of over 200 individual manual techniques focused on creating what we theorize are micro-failure of the adhesive crosslinks, the building blocks of adhesions, by application of various site-specific pressures across adhered areas of the abdominal viscera. Because no means of visual confirmation exists (excluding surgery, which was not performed), macro-failure or deformation of adhesions could only be assumed as visceral mobility improved, and as patients reported increased function and decreased pain. In earlier published studies, the manual physical therapy techniques used for treating abdominal adhesions in patients with histories of adhesion related SBO were used to clear adhesion related blocked fallopian tubes. Independent blinded radiologic tests documented that the patient’s fallopian tubes were no longer blocked by adhesions post treatment.The amount and duration of force that is applied to cause these microfailures can be significant, but varies within the tolerance of each patient and according to the site of the body that is being treated. For example, the same technique applied to the abdomen will use much greater force than when applied to the ovary or fallopian tubes. The manual physical therapy approach is one of whole body treatment, treating not only the abdomen in these patients, but also other areas of the body in which decreased function or mobility was identified during the evaluation. In general, patients received about 14 h of this manual therapy directly to perceived adhered abdominal structures for every 20 h of therapy; the remaining six hours were spent with history review, evaluation, treating other symptomatic areas including nearby myofascial structures (low back, lower extremities, shoulders and neck, etc.), and paperwork. Via palpation and an understanding of the anatomy, therapists focused on treating the adhesions, rather than treating the underlying viscera. Except at their ligamentous attachments and their normal anatomical attachment to other organs, abdominal and pelvic organs should be able to glide freely over each other. When therapists were unable to palpate freely gliding organs, the restrictions were noted as decreased mobility. This frequently occurred at the sites of prior surgeries.While the force used by the therapists was sometimes significant, it is interesting to note that except for some temporary soreness, there have never been any significant adverse events in the hundreds of patients treated with abdominal or pelvic adhesions, using this therapy. This observation seems to refute the commonly held belief among some manual therapists that abdominal structures should only be treated lightly, and infrequently. Notwithstanding, a BMI of above 36 and surgery within 12 weeks preceding therapy are considered contraindications by the clinic for this treatment. Both patients presented in this case report had low BMIs allowing for easy palpation of the internal organs and adhesions, scars and restrictions. In clinical trials and published studies, these techniques increased mobility and decreased pain in patients with significant surgical histories indicating adhesion formation [10,14,15,16].Patients are typically followed for at least a year following treatment via voluntary survey.Patient 1 was a 69 year old Caucasian male with a BMI of 17.7 and a history of GERD, asthma, hypertension, emphysema, recurrent SBO and bilateral inguinal hernias. His surgical history included SBO surgery at birth, childhood appendectomy, 3 laparoscopically repaired hernias from 2008–2010 with a surgical mesh inserted, Nissen fundoplication and partial thyroidectomy in 2010, laparotomy for SBO secondary to extensive adhesions in July 2010 and January 2011. Immediately after surgery in 2011, Patient 1 underwent a small bowel radiographic series that showed an incomplete SBO at the junction of the proximal mid ileum, and dilation throughout the small bowel, likely due to adhesions per the radiologist (Figure 1A). He was still experiencing symptoms of SBO (bloating, nausea, inability to eat normally) three months following the 2011 laparotomy, and reported a significant impact on his quality of life, focused on the changes to his diet that occurred after the SBO. He also reported abdominal tightness and significant intermittent pain (10/10 on a pain scale).Small bowel radiographs of Patient 1 documenting SBO resolution over time. Arrows note areas of obstruction. (A) Before therapy in 2011: incomplete SBO due to adhesions visualized by X-ray showing dilation of the proximal mid ileum. (B) Twelve months after therapy in 2012: mild stricture at the terminal ileum with no other small bowel abnormalities. (C) After 40 h of therapy: normal small bowel series X-ray in 2012.Patient 1 received no further medical interventions after his 2011 radiography prior to receiving this manual physical therapy treatment. Upon initial physical therapy evaluation, he presented with pain; visible scarring superficial to tissues that felt like deep palpable adhesions (consistent with his physician’s 2011 diagnosis of abdominal scarring), decreased visceral mobility (decreased ability for organs to glide normally), and tightness throughout the abdomen; myofascial, osseous and visceral hypomobility; and decreased quality of life due to SBO. He also presented with decreased strength and range of motion in both hips and trunk, and in his cervical, thoracic, and lumbar spine at the initiation of his physical therapy regimen. In 2011, Patient 1 underwent 20 h of intensive manual physical therapy over the course of five days with a focus on improving range of motion, and decreasing scar tissue, adhesions, and soft tissue restrictions. Primary goals of therapy included decreased SBO symptoms and a decrease of future bowel obstructions. At discharge from physical therapy, Patient 1 presented with improved range of motion in trunk, cervical, thoracic, and lumbar spine, and hips by at least 5 degrees for each range of motion measurement (data not shown). Abdominal mobility and tightness had also improved. Upon return to his home state, he underwent follow-up diagnostic evaluations by a blinded, independent radiologist. Findings showed an ileum stricture but no other obstructions in the bowels (Figure 1B). Furthermore, this patient reported no further obstructions or SBO symptoms over the 12 months following therapy.In 2012, following a diagnostic test showing a decreased diameter of the ileum (Figure 1B), Patient 1 was treated again for 20 h over the course of 5 days. Once again, he demonstrated improvement in all areas tested after twenty additional hours of the manual physical therapy including a minimum of 5 degrees increase in all ranges of motion that were measured to be decreased at the re-evaluation (data not shown). Ten days after the conclusion of therapy, the patient underwent a small bowel series, again conducted by a blinded independent radiologist. Results showed a normal bowel, with no kinking, constricting lesions or abnormal masses noted (Figure 1C). Patient 1 reported no further SBO symptoms for one year following the second course of treatment, for a total time of two years with no further medical interventions for SBO or SBO symptoms.Patient 2 was a 49 year old Caucasian female with a BMI of 21.8 and a significant history of recurrent abdominal adhesions as a result of multiple surgeries and injuries including benign lumpectomy of the breast, dilation and curettage, motor vehicle accident with injury to the soft tissue of the chest wall resulting in migraine headaches and fibromyalgia, and injuries to bilateral wrists. She also had a past medical history of systemic lupus, chronic fatigue, fractured right upper extremity and several fingers, left knee surgery, mild arthritis, brown recluse spider bites and multiple miscarriages. Patient 2 underwent the first of a series of surgeries beginning with a breast lumpectomy. Within weeks of this surgery, she was hospitalized as she was unable to keep any food or fluids down and all bowel movements ceased, accompanied by severe bloating and abdominal pain. After six days in the hospital, an emergency exploratory laparotomy revealed adhesion-related bowel obstructions. She had cysts removed from her ovaries and colon, as well as an appendectomy in June 2006. Two weeks later (July 2006) she underwent her second abdominopelvic surgery, the first to treat adhesion-related small bowel obstruction, in which the surgeon resected a fibrous band from her transverse colon to her posterior peritoneum, which had caused an internal herniation. In August 2006, the patient underwent a third abdominopelvic surgery, an exploratory laparotomy for a spigelian hernia, ovarian mass resection, oophorectomy, and adhesiolysis. Despite undergoing two adhesiolysis procedures, the patient continued to experience symptoms of bowel obstruction including abdominal distention, nausea, vomiting, pain and constipation immediately following the surgeries. In January 2007, she was again diagnosed with abdominal adhesions, a diagnosis confirmed during a laparotomy performed in July 2007, which was her fourth abdominopelvic surgery. That surgeon noted and lysed dense intra-abdominal adhesions and scar tissue that were binding the loops of the small intestine; the physician also performed an umbilical hernia repair with mesh. In October 2007 she underwent a fifth abdominopelvic surgery: bilateral inguinal hernia repair.In July of 2008, she underwent a sixth abdominopelvic surgery, another exploratory laparotomy, in which the surgeons noted extensive abdominal adhesions, a nearly complete SBO due to adhesions causing eight to ten acute angle kinks of the bowel and adhesions around the stomach causing it to become elongated, J-shaped and adhered to the anterior abdominal wall. Shortly thereafter, she underwent a complete hysterectomy and oophorectomy due to uterine fibroids and ovarian disease, her seventh abdominopelvic surgery. Thus, she underwent seven abdominopelvic surgeries, most related to adhesions and/or SBO, within a period of 30 months. In November 2008 the patient was diagnosed with recurrent idiopathic abdominal adhesions and informed of the need for an eighth abdominopelvic surgery, a laparotomy for adhesiolysis to treat the recurring SBO symptoms. Feeling that she needed to take another path, she elected to try a manual physical therapy in November 2008 rather than undergo further surgical intervention for her current partial SBO.During the physical therapy initial evaluation, Patient 2 reported constant dull aching abdominopelvic pain averaging 7/10 on the pain scale; intermittent dull aching pain in her torso at 10/10 on the pain scale; and pain in one lower leg that was intermittent dull aching, averaging 5/10 on the pain scale in an area that was bitten by a brown recluse spider. She reported pain with bowel movements, urination and intercourse. She had lost 18 pounds and was on a totally liquid diet at the time of the initial evaluation. She reported pain and a severe decrease in quality of life, beginning with her first surgery (June 2006) that forced her to leave the workforce. She presented with severe abdominal pain; decreased range of motion at the cervical, thoracic, lumbar and sacral regions. Physical therapy palpation and tests revealed severe visible and palpable scars and adhesions, severe restrictions in visceral abdominal mobility; decreased myofascial and osseous mobility and range of motion; and significant postural asymmetries. Current medication at the initiation of treatment was Darvocet-N 100 every 6 h for pain. Like Patient 1, Patient 2 underwent 20 h of intensive manual physical therapy over the course of 5 days with the goals of improving range of motion, flexibility, strength and posture, as well as decreasing adhesions in the bowel and abdominal wall in an attempt to decrease or negate the need for her pending surgery and to prevent a total small bowel obstruction. Again, the therapists used similar techniques as noted in their earlier published studies in which they focused on adhesions affecting endometrial and tubal structures. That is, they treated the adhesions that existed on and within the interstitial spaces, as well as on and within abdominal and pelvic organs, as the primary focus of their therapy.At discharge from physical therapy, Patient 2 reported that pain had decreased by 90% or more, and she was able to return to a normal diet. The therapist noted improvement in range of motion to within functional limits and improved posture, increased visceral mobility, increased myofascial and osseous mobility, and significantly decreased pain with urination, sexual intercourse and bowel movements. Several weeks following discharge from therapy, Patient 2 reported that she had re-gained the 18 pounds she had lost due to her compromised bowel, and she had returned to a normal quality of life. She was able to cancel the scheduled surgery for adhesiolysis and SBO. At her one-year follow-up from therapy, Patient 2 reported experiencing no additional SBOs or SBO symptoms, and that no further surgical interventions were needed or performed.In cases where non-surgical management is unsuccessful, the current standard medical treatment for SBO caused by adhesions is surgery. Unfortunately, surgery is implicated as the most common cause of abdominal adhesions and adhesion-related SBO [5]. Previous studies have shown the decrease in pain associated with abdominal adhesions after surgical adhesiolysis lasted up to one year; the subsequent return of the pain has been hypothesized as due to new, or expanded adhesion formation [17,18,19]. In the US in 2010, 70,194 patients underwent small bowel resection surgery with an average hospitalization of 14.2 days, an average cost of $114,175, and a patient death rate of 6.75%; 89,222 patients underwent surgery for adhesiolysis, an average hospitalization of 8.4 days, an average cost of $65,955, and a patient death rate of 2.3% [20]. Those numbers are staggering when it is considered that, based on prior studies, 35% of those patients that undergo pelvic or abdominal surgery surgery will likely be readmitted for surgical intervention for adhesiolysis over the next 10 years [6].Here we reported the use of a manual physical therapy for the treatment of SBO symptoms in two patients who presented with numerous, frequently recurring bowel obstructions. Furthermore, both patients had entered a cycle of surgery to remove adhesions, followed by the formation of post-surgical adhesions, followed by another adhesiolysis surgery, and so on. In each case, post-surgical adhesion formation occurred more rapidly and more severely with each successive surgery. Patient 2 experienced only 12 weeks between the most recent surgery for SBO and her next partial SBO, when she received her physician’s recommendation for another (eighth) abdominal surgery. Her surgeon had scheduled the next surgery for adhesiolysis when she opted to try this manual physical therapy treatment instead. Both patients reported lasting pain relief and avoidance of further SBOs after therapy that exceeded their prior post-surgical experiences. We hypothesize the positive responses from the therapy were due to the reduction of adhesions and the avoidance of new post-surgical adhesions that were causing SBOs and pain in both patients. The use of this non-surgical procedure appears to be of direct patient benefit, and the cost is significantly lower than surgery (SBO surgery costs $114,175; adhesiolysis surgery costs $65,955; this therapy costs $5,200). Unlike abdominal surgery, the therapy has no associated risks from general anesthesia. Data to date (from approximately 50 patients seeking therapy for SBO) shows that it appears to have little or no risk of major adhesion formation, infection or peritonitis. No hospital stay is required with the therapy, because there is virtually no recovery period. The use of manual physical therapy is not a new concept in the treatment of patients for a wide variety of medical conditions, including the management of adhesions [21,22,23,24,25,26]. Orthopedic physicians refer to manual physical therapy for treatment of adhesive capsulitis [27]. Oncologists call for manual therapy to treat post-mastectomy scars [28]. Thus, this new utilization for this manual physical therapy regimen has some precedent in clinical care. A recently published study suggested that massage therapy reduces inflammation after muscle damage by decreasing the levels of inflammatory cytokines produced [29]. This correlates with our observations that symptoms hypothesized to be attributed to the presence of adhesions do not reoccur after attempted lysis via manual physical therapy. Another study in animals investigated whether a manual massage technique of a surgical area was capable of preventing the formation of adhesions. That study demonstrated fewer adhesions in treated animals as compared to untreated controls [30]. In another study, the manual technique used in these two cases was documented in blinded radiographic reports to open fallopian tubes that were occluded by pelvic adhesions. The long history of clinical physician referrals from other branches of medicine for manual therapy to treat adhesive conditions, along with the studies noted above, support the observation that manual lysis of adhesions may be a viable alternative to SBO surgical treatment.Without visual confirmation, we can only hypothesize that the manual soft tissue physical therapy protocol effectively lyses the adhesions by causing microfailure of the attachments of the individual crosslinks, allowing return of normal anatomical organization, and formation of more normal tissue structures [31,32,33,34]. If true, these micro-failures of crosslinks and macro-failures of adhesions appear to have similar immediate end results for the patient as surgical adhesiolysis. However, manual manipulation of the soft tissue does not appear to activate the level of inflammation observed with surgical healing. Thus, the adhesions appear to be lysed without the significant induction of new adhesion formation that often follows abdominal and pelvic surgery. This hypothesis from our human based case report is supported by historical data in rat models for post-surgical healing in which manual physical therapy performed to soft tissues of the surgical incision site, with animals treated exhibited increased healing as compared to no treatment [35,36]. This therapy also avoids the risks of anesthesia, and the high cost and moderate death rate of surgery. It also appears to minimize the risk of surgical infection, and hospitalization following surgery. Because manual physical therapy has virtually no recovery time, patients are able to recover and participate in daily activities immediately. In follow-up assessments, this return of mobility appears to further deter the reformation of adhesions, and enhance return of more normal tissue organization. This is the same idea that surgeons promote when surgical patients are encouraged to walk and exercise shortly after surgery. Early mobility promotes increased blood flow and an earlier return of normal range of motion. We note that patients who underwent the manual therapy demonstrated increased range of motion in both cases; an early return to normal activities of daily living simply enhanced the benefits.A formal, well-structured clinical trial is currently underway for the investigation of a nonsurgical approach for the treatment and prevention of SBOs to provide statistical significance to the observations from our human efficacy data presented in this case report.Belinda F. Wurn, PT and Lawrence J. Wurn, LMT, are the sole shareholders of Clear Passage Therapies, Inc. Wurn Technique is a Therapeutic Method for Treating Infertility in Humans and Animals, patent pending with the United States Patent and Trademark Office, Application No. 09/887,884 filed June 22, 2001 by Lawrence J. Wurn and Belinda F. Wurn. All other authors declare no conflicts of interest.

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+These authors contributed equally to this work.This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).Human Papilloma Virus (HPV) related verrucae pedis persist, seemingly evading host immune surveillance, yet sometimes disappear with inflammation. The absence, or reduction, of a cellular immune response may explain why verrucae treatments are not uniformly successful and treatment can be difficult even in immune-competent individuals. Little investigation has been undertaken into the potential benefit and efficacy of needling verrucae, a treatment modality causing HPV infected keratinocyte destruction in addition to inducing an assumed enhanced immune response. A review of clinical practice is presented, reporting the treatment method and results of data collected from a retrospective review of 45 patients. Thirty-one (69%) cases demonstrated complete resolution of verrucae following needling treatment. Three patients demonstrated reduction in size and pain whilst 11 showed no improvement. No adverse events were noted. Needling may have a place in the management of verrucae pedis in an adult population but a large scale study utilising objective measures and a control intervention would provide more detailed efficacy data along with a greater understanding of the effects of this treatment on long term immunity. Verrucae (plantar warts) are a notorious source of frustration for both practitioners and patients alike, as no single treatment is completely effective in all patients. Despite a plethora of medical literature on this subject, high quality evidence for the efficacy of almost all treatments is non-existent [1,2]. A review by Lipke [3] also stated that although evidenced based reviews with guidelines have been published, they do not cover treatments that have yet to be subjected to blinded randomized, controlled clinical trials. Moreover, Lipke asserted that lack of robust evidence of a therapy, which has not been subjected to such rigorous scientific testing, does not mean that it is not worth knowing about nor worthy of use in practice, particularly when a specific treatment has been utilised and reported, albeit anecdotally, with a reasonably high clinical success rate. This article is a patient-centred, review of clinical practice of one such verrucae treatment, which was first described by Falknor [4] in 1969, the method of which he termed “needling”. Verrucae are benign tumours, caused by infection of epidermal keratinocytes by the double stranded DNA Human Papilloma Virus (HPV). There are currently more than 100 known types of HPV and these determine the anatomical distribution and morphology of the lesion [5]. The most common warts on the hands and feet are the subtypes 1, 2 and 4 [6]. Infection of the keratinocyte at the basal layer of the epidermis is established through abrasions of the skin surface. Here, the virus remains latent in the cell from 1 to 8 months [7]. As the epidermal cells differentiate and migrate to the surface, the virus is triggered to undergo replication and maturation until it is shed in the exfoliation of the epidermis. The process of virus replication produces proliferation of prickle cells which alters the character of the epidermis, resulting in the visible warty appearance of the verrucae.In most viral infections, the viral proteins within a cell cause damage to the host cell and stimulate production of cytotoxic T cells, which then seek out and destroy the targeted infected cells. However, unlike many viruses, HPV prevents cell lysis as infection spreads through the shedding of infected epithelial cells from the surface of the skin. In other words, there is no (or indeed limited) release of viral proteins to the circulating dentritic cells, and therefore, no (or inadequate) antigen presentation to the immune system. Furthermore, HPV proteins also encode specific functions to inhibit immune responses by inducing specific anti-inflammatory mechanisms by activating T suppressor cells. Frazer [8] explains that “such inhibition would be expected to reduce specific antiviral defence mechanisms and also effective presentation of antigen to the host immune system”.The absence, or reduction, of a cellular response may explain why verrucae treatments are not uniformly successful and treatment can be difficult even in immune-competent individuals. Most treatments work by destroying affected tissues, by either a cytotoxic or physically ablative mode of action. However, tissue damage alone may not be enough to produce the relevant cytokines to destroy latent virus in adjacent cells [9], thus recurrence and further treatment is often required after apparent resolution [10]. Verrucae persist, evading host immune surveillance, but sometimes disappear with inflammation [11]. Sterling et al. [6] stated that despite the lack of antigens, HPV does sometimes induce an immune response and spontaneous regression is often seen, although warts are less likely to resolve in adults and in immuno-suppressed patients. Therefore, research into efficacy of verrucae treatment for children must take into account the incidence of a higher rate of spontaneous regression [12]. Frazer’s work concluded that induction of cell-mediated immunity to early proteins of HPV may prove useful as a therapeutic approach to HPV infection [8]. This is in agreement with Tyring [13] who also stated; “The ideal way to combat HPV infection would be to improve the immune response to the virus so it is specific and directed against early viral proteins.” One way of achieving this would be by better presentation of viral antigens to the immune system. Recent research on successful treatments has been aiming toward creating an enhanced systemic immune response to eradicate the virus [7]. This enhancement is required as although HPV does induce a localised immune response, it is not effective enough to trigger a systemic response because any expression of viral proteins are limited to superficial epithelial cells, thus there is a reduced presentation of these to the immune system. The work of Parton and Sommerville [14] asserted that resolution of a single plantar verruca in children aged 4–14 years could be successfully achieved by lightly debriding the lesion to produce capillary bleeding and then abraded with fine glass paper. Chapman [15] expanded upon this work in 1998 and hypothesised that it should be possible to demonstrate a “whole body response” in patients with several verrucae in that the treatment of just one verruca will lead to the resolution of the untreated verrucae. Twenty-one patients participated in Chapman’s clinical trial with a wider age group range (aged 6–36 years) than that of Parton and Sommerville, who claimed an optimistic 94% of cases cleared within two weeks after one treatment. Chapman reported 43% success rate in participants aged 6–13 years. His conclusion concurred with Stirling’s observation; that treatment is more likely to be successful in patients under 14 years old. The ideal verrucae treatment should result in resolution of all or a great percentage of warts, be painless, need only one or a part of a lesion treated, create no scarring and offer HPV immunity for a lifetime.Falknor [4] first explained a direct needling procedure as a form of physical trauma without the use of chemicals (notwithstanding the use of local anaesthesia). The method he outlined comprised of anaesthetising the verruca, then thrusting in a needle “in dart fashion, so as to penetrate the full depth of the verruca and exiting through the base of the capsule into the fat”. He claimed only two recurrences of 126 lesions treated with this technique. Subsequent published research using this method has been sparse. Skilton and Mehar [16] published a case series of 14 patients with painful verrucae who were treated with the needling technique. They claimed resolution in 50% (7 out of the 14) had complete resolution at 8-week review. This paper retrospectively analyses 46 cases of verruca treated using Falknor’s needling method in a single private podiatry practice. The authors present a retrospective review of 46 cases (34 female, 12 male) treated within a private practice in Hampshire, UK using Falknor’s method. Ethical approval was sought through the National Research Ethics Service for this study. Each patient presented with clinically diagnosed verruca pedis. Treatment was indicated where the patient reported associated pain with the verrucae and subsequent interference with daily activities. However, a minority also requested clinical intervention as they perceived their quality of life was compromised due to cosmetic embarrassment. All treatment options, including no treatment, and the risks and benefits were explicitly explained to each patient. Those opting for needling treatment were enrolled into the study. Informed, signed consent was given by all patients prior to the needling procedure.Data from each patient was collected at baseline (prior to the procedure), one week post-operatively and at their final review to ascertain the following:
+        
+          
+            Previous treatment methods (at baseline only).
+          
+          
+            Location and duration of the lesion(s) (at baseline only).
+          
+          
+            Any discomfort throughout and at one and eight weeks post-operative (recorded as none, mild, moderate, severe).
+          
+          
+            Any post-operative infection or scarring following the procedure (at one and eight weeks).
+          
+          
+            Resolution or reduction in size of lesion (at eight weeks).
+          
+        Previous treatment methods (at baseline only).Location and duration of the lesion(s) (at baseline only).Any discomfort throughout and at one and eight weeks post-operative (recorded as none, mild, moderate, severe).Any post-operative infection or scarring following the procedure (at one and eight weeks).Resolution or reduction in size of lesion (at eight weeks).Local anaesthetic (Mepivacaine® 3%) was administered by tibial nerve block, digital block or local infiltration according to the location of the lesion chosen for needling. Once the area of skin was anaesthetised, any overlying callus was debrided. If the patient presented with mosaic or multiple plantar warts the largest and thickest lesion was selected for treatment. All treatments were undertaken by a single operator (BL).The area surrounding the lesion was first cleansed with povidone-iodine before an empty 27 gauge needle was utilised to puncture through the lesion to the subcutaneous tissue (Figure 1). Each puncture produced pin point bleeding and this was continued until there was no more resistance, or reactive pressure, from the epidermis and the entire lesion was perforated enough to produce a beefy red wound (Figure 2). The total number of punctures varied according to the size of the lesion. Demonstrating the needling technique.Wart. Immediately post-operatively after undergoing needling.Pressure was then applied to the wound with sterile gauze and then dressed with a non-adherent sterile dressing (Melolin®) and fixing tape (Mefix®). A semi compressed felt aperture pad was also applied on weight bearing sites to deflect pressure and reduce post-operative bruising. Each patient was issued with post-operative care sheets and advised to lightly shower and wash the area after keeping the dressing dry for 24 h. Each patient was advised to avoid taking NSAIDs or other anti-inflammatory medication for 48 h to increase the likelihood of a successful controlled inflammatory response. Wound inspection and debridement of any uncomfortable eschar was performed one week later. The final inspection for verrucae resolution was carried out 8 weeks later. Complete resolution was deemed accomplished on return of normal dermatoglyphics to the treated lesion, i.e., uninterrupted skin striae and no pain on lateral compression of the area. Figure 3a–c demonstrate a lesion before, immediately after and six weeks following treatment.(a) Lateral plantar lesion before treatment; (b) Same lateral plantar lesion during treatment; (c) Same lateral plantar lesion resolved, one month after treatment.A total of 46 patients (13 male, 33 female) underwent a standardised needling procedure. The mean age of the cohort was 41.8 years ± SD 12.65 (range 17–66 years). The average patient reported duration of the warts was 5.7 years ± SD 4.15 (range 1–20 years). The locations of all the treated lesions are given in Table 1. A total of 45 patients were available for review at eight weeks post-operatively with one patient lost to follow up.Location of primary (treated) lesions.Of 45 patients, 69% (21 female and 10 male) demonstrated a complete resolution of verrucae (10 patients with single lesions, 8 with mosaic and 13 with multiple types). There was no significant difference in the cure rates between males and females (p = 0.463). Table 2 profiles previous treatments of the resolved and unresolved lesions. Of the 45 patients, 7 (2 single lesions, 2 mosaic lesions and 3 multiple lesions) opted for a second needling treatment as the initial treatment did not fully rid all lesions.Previous treatment profile of resolved (R) and unresolved (U) lesions (n = 45).Lesions from 14 patients (31%) failed to resolve. There was no difference in the mean duration of resolved versus unresolved lesions (mean duration unresolved 7.67 years ± SD 13.26 versus mean resolved 6.68 years ± SD 4.41 (p = 0.7571). Three of the patients with unresolved lesions (7%) reported significant reduction of verrucae size and subsequent pain, signifying a clinical improvement. All treated patients reported their pain level after needling as either “none” (n = 29 [64%]) or “mild” (n = 16 [36%]) describing mild symptoms such as “bruising” or “slight discomfort”. No post-operative infection or scarring was evident on examination or reported by patients post-operatively.This paper represents the largest case series published to date using Falknor’s method since his original paper was published in 1969. The selection of patients from an adult population with a broad age range (17–66 years) has reduced the possibility of spontaneous regression often observed in child population based studies of this kind. The resolution rate following a maximum of two treatments (69%) indicates a good response when compared to other modalities. Using a similar approach of exposing wart virus to the immune system, Nischal et al. [17] auto-implanted debrided wart tissue from sufferers’ feet and implanted into the sub-cutis of the patients forearm or thigh. Analysis of the 27 subjects demonstrated a similar 74% clearance of lesions within three months.The observed positive clinical outcomes, in this current review, suggest that the hypothesis; provocation of a cell-mediated response as the cause of verrucae regression, is a viable premise. Needling just one lesion often produced a “cascade” effect, whereby the remaining untreated lesions also resolved in a number of patients. Thus, it can be suggested that introducing already HPV infected keratinocytes into the subcutaneous layer appears to facilitate a desired immune response in some patients.Currently the practice of needling verrucae is not extensively practiced. This may be due to many factors, including lack of published research, preference to utilise traditional and established treatments, (such as salicylic acid and cryotherapy) or simply because many practitioners require update in anaesthesic skills for ankle block infiltration. Although the short review period of eight weeks post-operatively, potentially does not rule out completely the possibility of reinfection or recurrence, longer term follow up is required. The authors acknowledge that this is small case series and cannot imply effectiveness and any placebo action inherent cannot be fully determined. However, a larger scale investigation, with objective measures using a control intervention, over a longer period of time would provide a more detailed picture of efficacy and long term immunity of the needling technique.Currently, there is no consistently effective treatment for plantar warts. This case series has reviewed the potential for the use of a direct needling technique in the management of plantar warts in an adult population. Following treatment of 45 patients, complete resolution of warts after eight weeks was observed in 31 (69%) cases. This represents a relatively small case series, within a restricted clinical setting. However, a larger scale investigation, with objective measures using a control intervention would provide a more detailed picture of efficacy and long term immunity of this promising technique.The authors would like to acknowledge the assistance of Kevin Kirby DPM for describing the needling technique.The authors declare no conflict of interest.

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+This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).This cross-sectional study explores factors affecting the decision of basketball players to wear ankle support devices (ASDs). A questionnaire regarding attitudes towards ASD usage was developed based on the Health Belief Model (HBM). The questionnaire assessed HBM perceptions (susceptibility, severity, benefits, and barriers) and modifying factors (demographic, personal history of ankle injury, influence of coach to preventive action) that may affect an athlete’s decision to wear ASDs. One hundred forty basketball players competing at the recreational, high school, or university levels completed the questionnaire, with the questionnaires being completed at the basketball gymnasium or at home. It was found that athletes whose coaches enforced ASD use were significantly more likely to wear them (OR: 35.71; 95% CI: 10.01, 127.36), as were athletes who perceived ankle injuries to be severe (OR: 2.77; 95% CI: 1.04, 7.37). Previous injury did not significantly increase the odds of using an ASD. The combined influence of coach enforcement and previous injury had the greatest effect on increasing ASD use. The largest barrier to ASD use was a lack of aesthetic appeal. Strategies aimed at increasing players’ willingness to wear ankle protection should be emphasized among coaches and parents as this may increase use of ASDs.Basketball is one of the most popular sports in the world with over 450 million documented participants playing organized basketball in 213 countries [1]. It is gaining global popularity and has replaced soccer as the most popular sport in Asia and Australia [1].As the number of basketball players has increased so has the burden of injury, particularly amongst young men and women. From 2000 to 2001, basketball was the most frequent cause of sports-related emergency department visits for youth and adolescents in the United States with 395,251 reported cases [2]. Studies have identified ankle injuries as the most prevalent basketball-related injury [3,4,5] due to swift changes in direction while running, the frequent jumping and landing while shooting and rebounding, and the contact with other players [6,7]. Recovery from ankle injuries may result in missed time and residual symptoms, such as pain, instability, and weakness [8]. Other effects include medical expenses, decreased strength, delays in muscle reaction time, disability, and impaired athletic performance [3,8,9]. Ankle support devices (ASDs) include bracing or taping and are used to prevent initial ankle injury or repeat injury [3]. They have been shown to both reduce the incidence and severity of ankle injury, particularly for sprains [10,11,12,13,14]. A recent critical review of ankle sprain prevention suggests that ASDs along with neuromuscular training will achieve the best preventive outcomes [15]. Studies have demonstrated that ankle braces are more supportive, user friendly, and effective than tape [14,16,17]. Use of ankle braces results in a five-fold reduction in the incidence of ankle sprains among soccer players with previous injury [12]. Among college-aged intramural basketball players, taping reduces the incidence of ankle sprains to 14.7 sprains per 1000 player games compared with 32.8 sprains per 1000 player games found in the un-taped athletes [18]. However, common barriers to the use of protective equipment in relevant situations include feelings of discomfort and perceiving the ASD as unnecessary; in contrast, parental influence and younger age increase use [19,20]. It is unknown what specifically influences basketball players to use ASDs. A more behavioural approach towards injury prevention has previously been suggested [21]. The Health Belief Model (HBM) is a theoretical framework used to understand and predict health behaviors [22]. According to this model, the likelihood of an individual engaging in preventive health behavior can be predicted by four individual perceptions: perceived susceptibility to harm, perceived severity of injury, perceived benefits of the action, and perceived barriers to preventive action. A review of 46 studies utilizing the HBM identified perceived barriers to preventive action as the most consistent predictor of protective behavior [22]. The HBM has previously been used to study how psychological factors may affect protective gear use [23]. In this study, we aimed to understand the factors that affect a young basketball player’s decision to wear an ASD by way of an HBM guided questionnaire. We initially interviewed 6 athletes, 2 coaches, 1 sport medicine physician, 1 physiotherapist, and 2 athletic trainers regarding ASDs and barriers to their use. To guide the open-ended discussion process, we asked: “What do you think influences an athlete’s decision to wear ankle protection?” If required, the question was rephrased: “Why do some basketball players choose to not wear ankle protection?” The responses were recorded and analyzed for recurring themes, which were then used to construct the questionnaire. The psychosocial variables identified as potentially influencing basketball players’ decisions to wear ankle protection included: cost of the ASD, impact of appearance with respect to social identity and image, degree of perceived physical comfort/discomfort, perceived negative effects on athletic performance, and coaches’ enforcement (i.e., coaches may force their players to wear an ASD). As per the HBM, perceptions and modifying factors were also examined. Questions to represent each perception in the HBM were developed along with questions to identify modifying factors. Perceptions and their corresponding questions are presented in Table 1. The modifying factors that were identified with questions were demographics, personal history of self-reported ankle injury, and the influence of the coach.Health Belief Model (HBM) perceptions examined in questionnaire using corresponding questions.The initial questionnaire was pilot tested with 6 players from recreational, high school, and university levels. A 5-point Likert-scale was used to quantify each of the four perceptions that influence ASD usage according to the HBM. The final questionnaire used in the study examined the level of perceptions of susceptibility (2 questions), severity (2 questions), and benefit (3 questions), demographic factors (2 questions), and personal history of ankle injury and ASD use (4 questions). Perceived barriers to use were also assessed: appearance (3 questions), performance (1 question), influence of professional athletes (2 questions), comfort (3 questions), and cost (3 questions).The self-report questionnaire was administered by a research assistant to 94 male and 46 female basketball players in one recreational league (ages 11–20), two high school teams (ages 13–21), and three university-varsity teams (ages 18–26). All participants resided in the Toronto area. A research assistant attended the high school and university practices and administered the survey on site. Recreational players were given the survey to complete at home and returned by mail to a research assistant. The questionnaire required approximately 5–10 min to complete. A Likert score of 4 (somewhat agree) and 5 (strongly agree) were defined as important and a Likert score of 1, 2, or 3 was deemed not important. For perceptions that had more than one question (e.g., severity of ankle injuries), a player had to respond with a 4 or 5 for all relevant questions in order for the perception to be classified as important. A chi-square omnibus test was used to determine if there was a significant difference in the proportion of athletes identifying a perception as important between the three levels of players. If a significant difference was observed (p < 0.05), additional chi-square tests were performed to determine where the difference occurred. To examine the various barriers, the analysis was repeated among the players who indicated that they did not currently use ASDs.Forwards selection logistic regression was used to determine which variables (perceptions, coach enforcement, previous ankle injury) best predicted current ASD use. The variable that resulted in the smallest p-value was included in the model first and each additional variable was added one at time. The variable that was the most statistically significant (indicated by the p-value) was retained in the model and this process was repeated until there were 10 ASD users per included variable [24]. STATA 12 software was used to conduct all analyses. Ethical approval was granted by the St Michael’s Hospital Review Board.In total, 140 athletes completed the survey, resulting in a response rate of 76.1%. Demographic data for the participants is summarized in Table 2. Overall, 32.9% of players wore an ASD and 81.4% self-reported a previous ankle injury. Compared with recreational and high school players, a significantly greater proportion of university players wore ASDs (p < 0.01) and had coaches who enforced ASD use (p < 0.01). There was no significant difference in previous ankle injury among the three levels of play. Demographic characteristics of recreational, high school, and university basketball players.ASD: ankle support device; * Significantly greater among university players versus recreational or high school players (p < 0.01).The perceptions relevant to the HBM are presented in Table 3, and are stratified by competition level. Overall, the perceived susceptibility to ankle injury was the most frequently reported perception, with 34.3% of all athletes perceiving basketball as an activity with a heightened risk of ankle injury. Ankle injuries were perceived to be severe injuries by 44.3% of athletes, and 48.1% perceived ASDs as beneficial. In comparing the different competition levels, university athletes were significantly most likely to perceive ankle injuries to be severe (p < 0.01) and ASDs to be beneficial (p < 0.01). There was no significant difference in perceptions between recreational players and high school players. High school athletes were the least likely to perceive that they would benefit from ASDs at 18.8%. Perceptions regarding ankle support devices (ASD) usage among recreational, high school, and university basketball players.ASD: ankle support device; Based on defining “strongly agree” or “somewhat agree” as “important” to the individual; * Significantly greater among university players versus recreational or high school players (p < 0.01).Athletes’ perceived barriers to ASD use are presented in Table 4, stratified by competition level. Overall, 53% of athletes reported at least one barrier to ASD use and the proportion of players reporting a barrier increased by level of competition—high school and university athletes reported a significantly higher proportion of at least one barrier to ASD use as compared to recreational players (p < 0.01). Among all level of players, aesthetic appearances was the most frequently reported barrier to ASD use (29.3%). Cost (12.1%) and comfort (15.0%) were the most infrequently reported barriers. When limited to the 94 athletes reported that they did not use ASDs, the perceived barriers in decreasing order were: aesthetic appearances (27.7%), appearance of weakness (20.2%), performance (17.0%), comfort (11.7%), and cost (6.4%).Reported barriers to ASD usage among recreational, high school, and university basketball players.ASD: ankle support device; Based on defining “strongly agree” or “somewhat agree” as “important” barrier to player; * Significantly higher among high school and university players than recreational players (p < 0.01); ** Significantly lower among recreational players versus high school or university players (p < 0.01).Athletes whose coaches enforced ankle protection were more than five times as likely to use ASDs (87.5% vs. 16.7%, p < 0.001). All university players who reported that their coaches enforced ASD usage also reported wearing ASDs. In addition, basketball players with a previous ankle injury were more than three times likely to use ASDs than those with no history of ankle injury (37.7% vs. 11.5%, p = 0.01). An additive effect between coach enforcement and previous injury was also observed. Athletes who had a previous ankle injury and were made to use ASDs by their coaches (89.7%) were significantly more likely (p < 0.001) to wear ASDs compared with previously injured athletes with no coach enforcement (20.0%) and athletes with neither coach enforcement nor a history of ankle injury (4.4%). In total, 48.6% of athletes believed that NBA and national team players wore ASDs and this did not differ by level of competition (p = 0.116). Several factors predicted ASD usage (Table 5). Athletes had significantly higher odds of wearing ASDs when their coach enforced usage (OR: 35.71; 95% CI: 10.01, 127.36). Athletes who identified cost as a barrier to ASD usage had significantly higher odds of ASD use (OR: 4.66; 95% CI: 1.13, 19.05). There was a significantly increased odds of ASD use among players who perceived ankle injuries to be severe compared with those who did not (OR: 2.77; 95% CI: 1.04. 7.37). The odds of ASD use were higher among players who had sustained a previous injury, although it was not statistically significant (OR: 3.93; 95% CI: 0.81, 19.03).The association between factors that predict ASD usage and actual ASD usage (odds ratios and 95% CI).ASD: ankle support device.The odds of wearing an ASD while playing basketball was significantly higher among those who believed ankle injuries were of a severe nature and ASDs were effective. Additionally, coach enforcement was a significant predictor. The most common reason for not wearing an ASD was because they were aesthetically unappealing. Meeuwisse et al. [25] found that on average all ankle injuries resulted in a loss of 5.5 basketball sessions (games or practices) per injury [25]. Tears to the lateral collateral ligament may require the individual to wear a hinged knee brace or to even undergo surgical treatment [26]. However, 41% of respondents in our study did not consider ankle injuries to be severe. Athletes who perceived ankle injuries as severe were at a greater odds of wearing ankle protection than those who did not. Therefore, player, coach and parent education regarding the severity of ankle injuries may be important in increasing ASD use.Previously established programs such as the Heads Up Hockey Program (an American ice hockey safety initiative) and Smart Hockey (a Canadian ice hockey injury prevention initiative) emphasize the importance of the coach as a role model who demonstrates respect for teammates, opponents, and the game [27,28]. Hockey coaches at the Atom level reported that most effective way to change player behavior was to change their coaching behavior [27,28]. The coach can be the single most important figure in terms of safety promotion—especially when they can mold a young player’s outlook on sport [29]. A role for coaches in endorsing ASD usage has been recommended [14]. Our study showed that players who had coaches who enforced ASD had greater usage, indicating that coaches may serve as a primary force in affecting ASD use and modifying sporting culture. Few recreational and high school players reported that their coaches enforced ASD usage and this is an opportunity for injury prevention programs to inform coaches about the effectiveness of ASDs. New Zealand Netball and Football (soccer) community-level coaches received an injury prevention curriculum as part of their education and 89% of netball and 96% of football coaches reported that the curriculum changed their coaching practices [30]. A coach taught injury prevention program resulted in a significant reduction in youth basketball acute injuries [31]. Therefore, coaches can play a valuable role in injury prevention. Having sustained an ankle injury is a risk factor for subsequent injury [18]. It would be expected that players who had a previous ankle injury would perceive these injuries to be severe compared with players with no previous ankle injury; however, there was no significant difference in perceptions of severity by history of previous injury. The majority of athletes with a prior history of ankle injury felt that ASDs prevented subsequent damage. It has been found that ASDs may be more effective in preventing re-injury than initial injury [12,15]. A significantly higher proportion of university players believed ASDs were beneficial. The HBM predicts that players who believe that ankle protection is effective would be more likely to use ASDs [22]. ASDs can decrease the predisposition of ankle inversion by stiffening the ankle joint to reduce range of motion and preventing the athlete from landing on an inverted foot [17,32]. ASDs may also increase an athlete’s proprioception, thus allowing a greater degree of muscular control [33]. Furthermore, ASDs can also serve as a psychological reminder to the athlete to moderate lower-limb behavior [32]. Our results showed that those who perceived ASDs as beneficial were 1.8 times more likely to wear ankle protection.The literature suggests that perception of the severity of a potential injury and the benefits of preventive action are insufficient to predict the actual use of preventive measures because a person at risk must also have a willingness to engage in pro-health behavior [22]. For example, Dutch pediatricians were aware of bicycle helmet effectiveness and the majority reported professional or personal experience with bicycle injuries, yet 94% did not wear a helmet [34]. We found that only 17% of recreational and high school athletes wore an ASD. This study highlights the critical role of the coach in encouraging ASD use. Organized basketball leagues and camps could provide incentives for ASD use and disincentives for coaches and teams not utilizing ASDs. Second, education is needed to understand the significance and severity of ankle injuries in players and protective effect of ASDs. High school players may underestimate the severity of ankle injuries and rarely use ASDs, and educational interventions should be directed towards both players and their coaches. Since changes in knowledge and attitudes generally precede changes in behaviour [22], these goals could be accomplished by mandatory educational or training sessions given to all players and coaches in organized basketball. Finally, opportunities exist for manufacturers and other stakeholders to address barriers to use such as comfort and cost, as well as placing more emphasis on aesthetic considerations.There were some limitations to this study. Although we had representation from 140 players from a limited number and variety of teams, a larger sample size would allow for a more in-depth exploration of beliefs, behaviours, and associated factors around the use of ASDs. In addition, while the high school and university-level players included male and female players, the recreational players were part of a male-only division. However, a player’s sex was not found to be a significant predictor of ASD use, and therefore it may not be a significant limitation. We did not stratify respondents in to whether they owned ASDs or not. Researchers found that factors responsible for not wearing a bicycle helmets were different for helmet owners (loss of the helmet or helmet not needed) than non-helmet owners (comfort and appearance) [35]. Although we asked about cost as a barrier, we did not determine a player’s or patent’s ability to afford or access ASDs. Interestingly, players who identified cost as a barrier had a significantly higher odds of ASD use. We also did not ask if mandatory ASD use would influence their willingness to play organized basketball.Future studies should include a larger sample size that includes a variety of geographical locations, and cultural and economic conditions. As well, the influential roles of parents, coaches, trainers and other role models such as professional athletes should be examined, as they could play important roles in shifting attitudes towards a willingness to use ASDs. Factors that would influence non-ASD users to use ASDs should be determined. Finally, more economic evaluation of the value of ankle protectors is required. The authors gratefully acknowledge Allison Eisner for her work in revising and editing the manuscript.Funding: This study was partially funded the St Michael’s Hospital Foundation and the Canadian Institutes of Health Research Strategic Team Grant in Applied Injury Research # TIR-103946.The authors declare no conflict of interest. 

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+These authors contributed equally to this work.This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).The objective of this study was to evaluate the correlation between multiple cardiovascular risk factors (MCRFs) and circulating mononuclear cells (CMCs) in asymptomatic coronary artery disease patients. Design and Methods: 126 subjects (54 male), aged 48 to 62 years, with asymptomatic coronary artery disease (CAD) documented previously with angiography, and 25 healthy volunteers were enrolled in the study. The flow cytometric technique was used for predictably distinguishing cell subsets that depend on the expression of CD14, CD34, Tie-2, CD45, and CD309 (VEGFR2). Results: The analysis of the outcome obtained shows a trend of an increase in circulating CD45−CD34+ CMCs and a reduction in CMC population defined as CD14+CD309+ and CD14+CD309+Tie2+ in known asymptomatic CAD patients in comparison with healthy volunteers. Substantial correlations between CD45−CD34+ and conventional cardiovascular risk factors (hs-CRP, T2DM, serum uric acid and hypertension) were found in the patient cohort. The concentrations of CD14+CD309+ and CD14+CD309+Tie2+ CMCs had effect on such factors as T2DM (RR = 1.21; 95% CI = 1.10–1.40; p = 0.008), hs-CRP > 2.54 mg/L (RR = 1.29; 95% CI = 1.12–1.58; p = 0.006), Agatston score index (RR = 1.20; 95% CI = 1.15–1.27; p = 0.034), and occurrence of three and more cardiovascular risk factors (RR = 1.31; 95% CI = 1.12–1.49; p = 0.008). Conclusion: It is postulated that the reduction in circulating CD14+CD309+ and CD14+CD309+Tei2+ CMCs is related to a number of cardiovascular risk factors in asymptomatic patients with known CAD. Multiple cardiovascular risk factors (MCRFs) in cardiovascular diseases, such as chronically increased blood pressure, hyperlipidemia, hyperuricemia, hyperglycemia, and obesity, have a negative impact on the heart exposed to ischemia [1,2]. It has been postulated that hematopoietic-derived progenitor cells have an effect on angiogenesis and tissue repair following several injuries, such as ischemia, reperfusion, inflammation, allograft vasculopathy, atherosclerosis, stroke, etc. [3,4,5,6]. Many studies have demonstrated the presence of circulating endothelial progenitor cells (EPCs) in the peripheral circulation [5,7]. Circulating EPCs have been defined previously using two distinct methods to characterize them, i.e., flow cytometry and cell culture; recently, however, the validity of both definitions has been questioned. Flow cytometric definition is based on the expression of CD34+ on the circulating cells. Usually, the recent studies using flow cytometry have investigated cells that express CD34+ and CD309+(VEGFR-2+). An alternative definition of EPCs describes colonies of spindle-shaped cells appearing after culture of blood-derived mononuclear cells. Сirculating EPCs, however, might not only have bone-marrow origin; they are also transformed from peripheral mononuclear cells that have the capacity to come to the sites of tissue injury and may be differentiated into mature endothelial cells [8,9]. It has been determined that EPCs have been largely characterized by the expression of the primitive hematopoietic progenitor markers CD34+ and VEGF receptor (VEGFR)-2 [10,11]. Basically, CD34+CD45− cells have nonhematopoietic origin; and recently they have been identified as putative EPCs by virtue of their ability to form “late outgrowth” colonies phenotypically and functionally indistinguishable from mature endothelial cell colonies in culture [12,13]. Previous studies have shown an increase in the circulating concentration of population CD34+CD45− cells in patients with established coronary artery disease (CAD) and peripheral artery disease [10,14,15]. Theoretically, CD34+ cells which do not express CD45 by flow cytometry may include cells capable of developing bona fide endothelial cells. Nevertheless, traditional EPC populations, such as CD34+VEGFR-2+ and CD34+VEGFR-2+CD133+, are not related to severity of CAD or clinical outcome in the patients with acute coronary syndrome and unstable angina. On the other hand, the concentrations of proangiogenic monocites may reflect the extent of vascular injury and atheroma burden in this patient population [3,9]. The objective of this study was to evaluate the correlation between MCRF and various types of circulating mononuclear cells (CMCs) that express CD14+VEGFR-2+ and CD14+VEGFR-2+Tie2+ in patients with asymptomatic coronary artery disease.The study population was structured retrospectively after determining the coronary artery disease (CAD) by contrast-enhanced spiral computed tomography angiography in 126 asymptomatic subjects. Twenty five healthy volunteers were enrolled in the study aimed at the verification of the reference average of biological markers. All the subjects gave their written informed consent to participate in the study prior to enrollment. The following are the exclusion criteria: Symptomatic chronic heart failure; left ventricular ejection fraction (LVEF) ≤40%; uncontrolled diabetes mellitus; severe kidney and liver diseases that may affect clinical outcomes; malignancy; unstable angina; Q-wave and non-Q-wave MI within 30 days before the study entry; creatinin plasma level above 440 μmol/L; eGFR index <35 mL/min/m2; brain injury within three months before the enrollment; body mass index above 30 kg/m2 and less than 15 kg/m2; pulmonary edema; tachyarrhythmia; valvular heart disease; thyrotoxicosis; ischemic stroke; intracranial hemorrhage; acute infections; surgery; trauma; all ischemic events within the three previous months; inflammations within the previous month; neoplasm; pregnancy; implanted pacemaker, any disorder that may discontinue the patient’s participation in the study according to investigators; and finally the patient’s refusal to participate in the study or to give his consent for it. The coronary vessel-wall, plaque geometry, and compositional parameters were measured on contrast-enhanced spiral computed tomography (CT) angiography [16]. Contrast-enhanced spiral CT was performed on a Somatom Volum Zoom scanner (Siemens, Erlangen, Germany) with two rows of detectors (32 × 2 CT system) at the time of end-expiratory breath-hold. After noncontrast localization image acquisition, injection of Omnipak nonionic contrast (Amersham Health, Carrigtohill, Ireland) was used to determine the optimal coronary arterial image. Images were reconstructed in 0.6-mm axial slices. The coronary artery calcification was quantified by calculating the Agatston score index and measuring the calcification mass [17]. Calcified atherosclerotic plaque (CAP), high-density noncalcified plaque (HD-NCP), and low-density noncalcified plaque (LD-NCP) were determined. Calcified atherosclerotic plaques were characterized by an attenuation value that was 150 HU (Hounsfield units) or greater for CAP, 30 to 149 HU for HD-NCP U, and 100 to +30 HU for LD-NCP [18,19]. According to recommendation of the American Society of Echocardiography, standard transthoracic echocardiography in B-mode was performed on an ACUSON scanner (Siemens, Erlangen, Germany) using a transducer with a frequency of 2.5–5 MHz. End-diastolic and end-systolic LV volumes were obtained using a two-dimensional reference sector according to Simpson’s method, and the LV ejection fraction (LVEF) was calculated according to conventional methods [20].Estimated glomerular filtration ratio (eGFR) was calculated using the MDRD formula [21].All the samples were placed into EDTA tubes with serum gel for further flow cytometry analysis and mononuclear cell preparation. The whole blood was analyzed: The complete blood count with white blood cell differential count was carried out using the analyzer. After taking for blood chemistry test, all the blood samples were placed in the cooling vacutainer and centrifugated immediately (at a temperature of 4 °C at 6000 rpm for 15 min). After centrifugation, the serum was coded and stored in the refrigerator at a temperature of −70 °C until used.High-sensitive C-RP (hs-C-RP) level was measured by a nephelometric technique and obtained with a “AU640 Analyzer” (Olympus Diagnostic Systems Group, Shizuoka-ken, Japan).Serum uric acid level (SUA) was determined by enzymatic methods using a Beckman Synchron LX20 chemistry analyzer. The analytical average range for SUA was 0.5–12 mg/dL.Concentrations of total cholesterol (TC) and high density lipoprotein (HDL) cholesterol were determined with the Dimension Clinical Chemistry System (Dade Behring Inc., Newark, NJ, USA). Low density lipoprotein (LDL) cholesterol was calculated using the Friedewald formula [22].The flow cytometric technique (FCT) was used for predictably distinguishing circulating cell subsets, which depend on expression of CD45, CD34, CD14, Tie-2, and VEGFR2, using High-Definition Fluorescence Activated Cell Sorter (HD-FACS) methodology [23]. Accordingly, the cells in question were phenotyped on the basis of their forward scatter characteristic (FSC) and side scatter characteristic (SSC) profiles. The cells were directly stained and analyzed for phenotypic expression of surface proteins using anti-human monoclonal antibodies, including anti-CD45 FITS (BD Biosciences, San Jose, CA, USA), anti-CD34 FITS (BD Biosciences), anti-VEGFR-2 known as anti-CD309 (BD Biosciences), anti-Tie2 (BD Biosciences) and anti-CD14 (BD Biosciences). The fluorescence minus one technique was used to provide negative controls and establish positive stain boundaries. After lysis of erythrocytes with Utilize wash solution, the samples were centrifuged at 200× g for 15 min; then they were washed twice with PBS and fixed immediately. Double- or triple-positive events were determined using Boolean principles (“and”, “not”, “or”, etc.). Circulating EPCs were defined as CD34/VEGFR2 positive cells with lack of CD45 expression. From each tube 500,000 events were analyzed. For CD14+ populations, coexpression with Tie-2− and/or VEGFR-2− was determined using quadrant analysis. Mononuclear cells were cultured for functional analysis (CFUs) after FCT. Standardized cell counts were presented as a percentage of the total of the white blood cells count, identified as the total number of all CD45+ cells.A 10-year cardiovascular risk for study patients was calculated using the Framingham General Cardiovascular Risk Score (2008) by on-line calculator.All the statistical analyses were performed in SPSS for Windows v. 20.0 (SPSS Inc., Chicago, IL, USA, 2011). Continuous variables are presented as mean ± SD, mean and 95% CI or median and interquartile range. Categorical variables are expressed as frequencies and percentage. An independent group t-test was used to compare all the interval parameters matching the criteria of normality and homogeneity of variance. For interval parameters that fail to match these criteria, the non-paramentric Mann-Whitney test was used to compare variables. Categorical variables and frequencies were compared using Chi2 test and Fisher exact test of independence. SUA frequencies were normally distributed (using the Kolmogorov-Smirnov test), and data were not positively skewed. Frequencies of CMCs and hs-CRP concentrations were not distributed. The data, however, were not transformed. The potential factors that may be associated with CMCs were identified first with the univariate analysis (ANOVA), and then the independent predictors of a decrease in CMCs were searched with the multivariate one-step backward logistic regression analysis, initially including variables for which a p value < 0.1 was achieved from the univariate analysis. The odds ratio (RR) and confidence intervals (95% CI) were calculated for factors independently associated with a decrease in circulating CMCs. A calculated difference of p < 0.05 was considered significant. General characteristics of study patients are presented in Table 1. Two groups of persons surveyed (healthy volunteers and patients with known CAD) were compared in terms of demographics, smoking, body mass index, CAD family history, mean systolic BP, heart rate, creatinin, fasting glucose and eGFR. Patients with known CAD, however, had hyperlipidaemia (44.4%), arterial hypertension (66.7%), T2DM (36.5%). An increase in HbA1c, serum uric acid, hs-CRP, LDL cholesterol, and triglycerides, as well as a decrease in HDL cholesterol were found in patients with known CAD when compared with healthy volunteers. Mediana of a 10-year Framingham General Cardiovascular Risk in patients with known CAD and in healthy volunteers was 23% and 2% respectively.Baseline angiographic and treatment characteristics of patients with known CAD are presented in Table 2. Calcified atherosclerotic plaques were determined in 96% of patients; HD-NCP and LD-NCP were found in 31% and 25% respectively. The median of the Agatston score index was 586 (95% CI = 401–838). Coronary arteries with plaques were determined in 36.5%; 33.3%; and 20.2% for one vessel, two vessels, three and more vessels respectively. General characteristics of study patients.Note: CAD—coronary artery disease, CI—confidence interval, T2DM—type 2 diabetes mellitus, eGFR—estimated glomerulal filtration ratio, TC—total cholesterol, HbA1c—glycated hemoglobin, LDL—low-density cholesterol, HDL—high-density cholesterol, SUA—serum uric acid, BP—blood pressure, hs-CRP—high sensitive C-reactive protein, LV EF—left ventricular ejection fraction.All the asymptomatic patients were informed about coronary angiography, and they were treated according to current clinical guidelines with diet, lifestyle modification, and drug therapy that included ACE inhibitors/ARBs, aspirin or other antiagregants, statins and metformin if needed.Analysis of the outcomes obtained showed no significant increase in circulating CD34+ subset cells (CD45+CD34+ and CD45−CD34+) in known asymptomatic CAD patients when compared with healthy volunteers (Table 3). The authors suppose that there are decreased CD14+ subsets of CMCs in CAD subjects. Indeed, there is a significant difference between the medians of CD14+CD309+ and CD14+CD309+Tie2+ in both healthy volunteers and patients with known asymptomatic CAD respectively.There were no significant correlations between CD45+CD34+ and demographics, cardiovascular risk factors, Agatston score index, and plaque characteristics in a univariate regression model. The relationship between CD45−CD34+ cells subset frequency and high sensitive C-reactive protein (R = 0.864; p = 0.001), T2DM (R = 0.614; p = 0.001), Agatston score index (R = 0.467; p = 0.001), SUA (R = 0.380; p = 0.002), and arterial hypertension (R = 0.240; p = 0.026) was determined by positive linear regression. A negative correlation was supposed between CD45−CD34+ cell subset frequency and LD-CAP (R = −0.508; p = 0.001), LVEF (R = −0.414; p = 0.001) and smoking (R = −0.222; p = 0.040). There is a significant positive correlation between CD14+CD309+ cell subset frequency and high sensitive C-reactive protein (R = 0.892; p = 0.001), Agatston score index (R = 0.520; p = 0.001), T2DM (R = 0.520; p = 0.001), SUA (R = 0.348; p = 0.002), LDL-cholesterol (R = 0.322; p = 0.001), arterial hypertension (R = 0.280; p = 0.006) and TC (R = 0.260; p = 0.041). A negative correlation between CD14+CD309+ cell subset frequency and LD-CAP (R = −0.591; p = 0.001), LVEF (R = −0.424; p = 0.001), smoking (R = −0.259; p = 0.042), and CD14+CD309+Tie2+ cell subset frequency was found, as well as correlation with high sensitive C-reactive protein (R = 0.92; p = 0.001), Agatston score index (R = 0.538; p = 0.001), T2DM (R = 0.597; p = 0.001), SUA (R = 0.382; p = 0.002), LDL-cholesterol (R = 0.354; p = 0.001), TC (R = 0.258; p = 0.043), CAP (R = −0.598; p = 0.001), LD-CAP (R = −0.594; p = 0.001), LVEF (R = −0.374; p = 0.001), smoking (R = −0.285; p = 0.042) and BMI (R = −0.272; p = 0.046).Baseline Angiographic and Treatment Characteristics of patients with known CAD.Note: CI—confidence interval, ACEI—angiotensin-converting enzyme inhibitor, ARBs—angiotensin-2 receptor blockers, HD-NCP—high-density noncalcified atherosclerotic plaque, LD-NCP—low-density noncalcified atherosclerotic plaque, CAP—calcified atherosclerotic plaques.The potential factors, which may be associated with EPCs determined as CD45−CD34+, CD14+CD309+, and CD14+CD309+Tie2+, were identified as a multivariate one-step backward logistic regression analysis. There was a significant effect of cardiovascular risk factors (T2DM, SUA, TC, hs-CRP, LDL-cholesterol) and Agatston score index on the combined dependent variable (CD45−CD34+, CD14+CD309+, and CD14+CD309+Tie2+ cell subsets) (F = 46.16, p < 0.001; Wilks’ Lambda = 0.05; partial η2 = 0.72). Analysis of each individual dependent variable showed that there was an effect of hs-CRP level (F = 0.39, p = 0.002, partial η2 = 0.52), T2DM (F = 0.38, p = 0.016, partial η2 = 0.33), LDL-cholesterol (F = 0.38, p = 0.018, partial η2 = 0.30) on CD14+CD309+ cells. A significant effect of T2DM (F = 0.41, p = 0.001, partial η2 = 0.60), SUA (F = 0.32, p = 0.024, partial η2 = 0.32), hs-CRP (F = 0.36, p = 0.008, partial η2 = 0.70), LDL-cholesterol (F = 0.36, p = 0.004, partial η2 = 0.32), and Agatston score index (F = 0.34, p = 0.004, partial η2 = 0.31) on CMCs determined as CD14+CD309+Tie2+ was found. There was a relation between cardiovascular risk factors and CMCs determined as CD45−CD34+, while an effect of Agatston score index to CD45−CD34+ cell subset (F = 0.34, p = 0.028, partial η2 = 0.36) was supposed. Frequencies and absolute values of circulating mononuclear cells in the study patient population.Note: The values corespond to medians and a interquartile range (IQR) of 25%–75%. Statistical comparisons are made using Mann-Whitney test with significance levels of 0.05 and 0.01 (for 2-tailed). Note: Values are medians and 25%–75% interquartile range (IQR). Statistical comparisons are made.The odds ratio (OR) and confidence intervals (95% CI) were calculated for factors independently associated with CMC lowering (Table 4).Independent predictors of circulating circulating mononuclear cells (CMCs). The results of multivariate one-step backward logistic regression analysis.Note: OR—odds ratio, CI—confidence interval, MCRFs—multiple cardiovascular risk factors.Since no significant correlations between CD45−CD34+ and cardiovascular risk factors were found in patients with asymptomatic known CAD, multivariate statistics with OR calculations for CMCs determined as CD14+CD309+ and CD14+CD309+Tie2+ was performed. Analysis of the data obtained revealed that subjects with T2DM and calcification of coronary arteries determined with Agatston score index had a 1.16-fold and 1.14-fold increase in OR of CD14+CD309+ CMC lowering in circulation respectively. When three and more cardiovascular risk factors occurred, circulating CD14+CD309+ CMCs lowered more significantly (OR = 1.27; 95% CI = 1.10–1.42; p = 0.009). Contrary to expectations, no significant independent correlation between CMC concentration, and the number of HD-NCP and LD-NCP in the coronary arteries was found. There were a number of cardiovascular risk factors that had an effect on CD14+CD309+Tie2+ CMC lowering. The most important factors were T2DM (OR = 1.20; 95% CI = 1.06–1.34; p = 0.005), hs-CRP >2.54 mg/L (OR = 1.22; 95% CI = 1.06–1.44; p = 0.006), Agatston score index (OR = 1.16; 95% CI = 1.10–1.22; p = 0.044) and the number of MCRF >3 (OR = 1.27; 95% CI = 1.10–1.42; p = 0.009). Finally, the conclusion was made that it is CD14+CD309+Tie2+ CMCs lowering that demonstrated a more pronounced fit with MCRF in cohort of the asymptomatic patients with known CAD.Recent studies show that bone marrow-derived endothelial progenitor cells (EPCs) play an important role in the maintenance of endothelial integrity and atherosclerosis [3,14]. A correlation between MCRF and concentration of CD34+CD45− cells and CD14+CD309+/CD14+CD309+Tie2+ cells was found in asymptomatic coronary artery disease patients. Recently a clinical study showed that there is inverse correlation between the number of circulating CMCs and cardiovascular risk factors [24]. nevertheless, as the number of the existing cardiovascular risk factors varies among patients, simple CMC counts do not describe adequately a vascular disease risk in any clinical condition and, therefore, the CVD risk remains [24,25]. The authors agree with Padfield G. J. et al. [15] who showed that CD34+CD45− cell number was increased in patients with CAD compared with those with normal coronary arteries and correlated well with atheroma. The authors believed that CD14+VEGFR-2+Tie-2+ cells and endothelial cell-colony forming units were increased in patients with acute coronary syndrome; that cell concentrations reflected myocardial necrosis, and did not predict the extent of CAD. Patients with myocardial infarction or acute coronary syndrome, however, were not included in this study. The authors also determined that there was a reduction in CD14+VEGFR-2+Tie-2+ concentration, as well as a trend of an increase in CD34+CD45− cells in the cohort of asymptomatic patients with coronary calcification and known CAD at the early stage. The authors agreed that no exact fit exists between the concentration of the EPCs traditionally determinined and severity of coronary atherosclerosis. The data obtained reflect that the concentration of monocytes with proangiogenic capacity, which were determined as CD14+CD309+/CD14+CD309+Tie2+ cell subsets, is associated more with the number of cardiovascular risk factors than with the calcification of the coronary arteries and plaques types. MCRFs, however, were powerful predictors of circulating CD14+CD309+/CD14+CD309+Tie2+ cells irrespective of the severity of coronary calcification. Provided by Chen, J. Z. et al. [7] and Loomans, C. J. et al. [2], the results clarified a pivotal role of CMC lowering in pathogenesis of vascular complication in patients with diabetes and hyperlipidaemia. Werner, N. and Nickenig, G. [26] demonstrated a correlation between lower CD34+/CD309+ cells in later stages of CAD. Boilson, B. A., Kiernan, T. J., Harbuzariu, A., Nelson, R. E., Lerman, A. and Simari, R. D. [27], presented data which supported this correlation available at a much earlier stage of CAD. Obtained in this study, the data show that MCRFs had the most important prediction value to lower circulating CD14+CD309+/CD14+CD309+Tie2+ cells when compared with isolated cardiovascular risk factors, even those such as T2DM and hs-CRP elevation in asymptomatic patients with known CAD. These findings may support a hypothesis of the impact of MCRFs on disorders of cellular protective mechanisms in subjects at an early stage of CAD.The authors postulate that a reduction in circulating CD14+CD309+ and CD14+CD309+Tei2+ EPCs is related to a number of cardiovascular risk factors in asymptomatic patients with known CAD.This study has some restrictions. The authors believe that a greater cohort is desirable to improve the power of the study. There is a variation in the definition of EPCs, the number of existing cardiovascular risk factors in various patients, and in the interaction between EPCs and other hematopoietic progenitor, inflammatory cells or platelets. The authors suppose that these restrictions might have no significant impact on the study data interpretation.The study was approved by the local ethics committee of State Medical University, Zaporozhye, Ukraine. The study was carried out in conformity with the Declaration of HelsinkiThis research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. The study results were presented as a poster presentation on the 20th International Postgraduate Course of Cardiovascular Disease, Davos, Switzerland, 10–15 February 2013.The authors declare no conflict of interest.

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+This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).FAPA syndrome (periodic fever, aphthous stomatititis, pharyngitis and adenitis) is a relatively new entity described in pediatric patients. In adults, reports of FAPA are limited to rare case reports. The differential diagnosis of FAPA in adults includes Behcet’s syndrome, familial Mediterranean fever (FMF), Hyper IgD syndrome and juvenile rheumatoid arthritis (JRA), i.e., adult Still’s disease. With FAPA syndrome, between episodes patients are completely asymptomatic and serologic inflammatory markers such as erythrocyte sedimentation rate (ESR), C-reactive protein (CRP) and white blood cell (WBC) count are normal. The etiology of FAFA is unknown, but lack of secondary cases or clustering in close contacts, lack of seasonality, and the lack of progression for years argue against an infectious etiology. We describe an extremely rare case of an adult with a recurrent FUO with profuse night sweats and prominent chills due to FAPA syndrome.Fever of unknown origin (FUO) was first defined by Petersdorf, but Knockaert later defined recurrent FUOs as recurrent FUOs with at least 2 weeks between febrile episodes. There are relatively few disorders, usually rheumatic/inflammatory, that cause periodic or recurrent FUOs, e.g., familial Mediterranean fever (FMF), Behcet’s syndrome. Rarely, other conditions, e.g., hypertriglyceridemia may cause recurrent FUOs [1,2] (Table 1).Differential diagnosis of recurrent fevers of unknown origin (FUOs). Adapted from [1,2].Recurrent FUOs are the most difficult of diagnostic challenges. Even after a detail focused diagnostic approach, many recurrent FUOs remain undiagnosed [1]. The value of repeated evaluations cannot be overestimated; findings not initially noticed may later become apparent, e.g., splenomegaly [2].Marshall’s syndrome or FAPA (periodic fever, aphthous stomatititis, pharyngitis and adenitis) was firstdescribed in pediatrics [3]. In adults, reports of FAPA are rare [4,5]. The differential diagnosis of FAPA includes Behcet’s syndrome, Familial Mediterranean Fever (FMF), hyper-IgD syndrome [2,3]. Between episodes, FAPA patients are asymptomatic and laboratory tests are unremarkable. Lack of clustering, seasonality, and progression argue against an infectious etiology [4,5]. We report a case of recurrent FUO in an adult with profuse night sweats and prominent chills due to (FAPA).A 24-year-old male with recurrent FUOs was evaluated for recurrent fevers, chills and night sweats every two months for 3 years. When symptomatic, he has fevers to 103 F, sore throat, cervical lymphadenopathy and malaise for 3 days, followed by 3 days of profuse night sweats and prominent chills. Between episodes, heis completely asymptomatic. He is of Mediterranean descent. Repeated treatments, with antibiotics, colchicine, and non-steroidal anti-inflammatory drugs (NSAIDs) had no effect.Examination was unremarkable, except for a single right posterior cervical node. His WBC count was 3.4 K/mm3, platelet count was 170 K/mm3, and ESR was 7 mm/h. Serum transaminases were normal, as was serum ferritin. HIV testing was negative. Coxsackie A/B titers, Toxoplasma, Bartonella, Epstein-Barr Virus (EBV) and cytomegalovirus (CMV) titers were negative. Serum protein electrophoresis (SPEP) was unremarkable. Anti-nuclear antibody and double stranded DNA titers were negative. Extensive work ups were non-diagnostic by oncology/hematology, infectious disease and rheumatology consultants but none inquired about aphthous ulcers. Since the patient had fever, cervical adenopathy and pharyngitis, suggesting FAPA, we asked him about aphthous ulcers during his febrile episodes. He reported aphthous ulcers occurred only during some attacks, but ulcers were not present with each attack.Recurrent FUOs remain a most difficult diagnostic challenge. The differential diagnosis in this case of recurrent FUO included cyclic neutropenia, hyper-IgD syndrome, Behcet’s syndrome, and FMF. Although of Mediterranean descent, he never had peritonitis, epididymitis, arthritis, and did not respond to colchicine suggesting FMF. Behcet’s was excluded with an unelevated ESR, no genital ulcers, and did not demonstrate pathergy with venipuncture. Causes of recurrent FUOs with cervical adenopathy, e.g., EBV, CMV, systemic lupus erythematosis (SLE), and toxoplasmosis were ruled out.This case of recurrent FUO is particularly interesting because of the rarity in adults of FAPA. Because of his profuse night sweats, we were concerned about TB and lymphoma, but normal ESR, hemogram, SPEP and peripheral blood flow cytometry argued against lymphoma, and his T-spot was negative. FAPA syndrome can be difficult to diagnose because some clinical features may not present during attacks, e.g., aphthous ulcers as occurred with this case. His recurrent FUO was due to FAPA syndrome. He has been managed conservatively for a year, this is his fourth year of recurrent FUOs. FAPA syndrome responds to prednisone or cimetidine.Clinicians should be aware that FAPA syndrome should be added to the list of rare causes of recurrent FUOs [1,2]. To the best of our knowledge this is the first reported case of recurrent FUO due to FAPA syndrome in an adult.The authors declare no conflict of interest.

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+This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).For spinal fusions and the treatment of non-union fractures, biological substrates, scaffolds, or carriers often are applied as a graft to support regeneration of bone. The selection of an appropriate material critically influences cellular function and, ultimately, patient outcomes. Human bone marrow mesenchymal stem cells (BMSCs) are regarded as a critical component of bone healing. However, the interactions of BMSCs and commercial bone matrices are poorly reported. BMSCs were cultured with several commercially available bone substrates (allograft, demineralized bone matrix (DBM), collagen, and various forms of calcium phosphates) for 48 h to understand their response to graft materials during surgical preparation and the first days following implantation (cell retention, gene expression, pH). At 30 and 60 min, bone chips and inorganic substrates supported significantly more cell retention than other materials, while collagen-containing materials became soluble and lost their structure. At 48 h, cells bound to β-tricalcium phosphate-hydroxyapatite (βTCP-HA) and porous hydroxyapatite (HA) granules exhibited osteogenic gene expression statistically similar to bone chips. Through 24 h, the DBM strip and βTCP-collagen became mildly acidic (pH 7.1–7.3), while the DBM poloxamer-putties demonstrated acidity (pH < 5) and the bioglass-containing carrier became basic (pH > 10). The dissolution of DBM and collagen led to a loss of cells, while excessive pH changes potentially diminish cell viability and metabolism. Extracts from DBM-poloxamers induced osteogenic gene expression at 48 h. This study highlights the role that biochemical and structural properties of biomaterials play in cellular function, potentially enhancing or diminishing the efficacy of the overall therapy.The practice of medicine in orthopedic surgery has included bone regeneration since the earliest bone grafts and bone marrow transfers nearly a century ago [1,2,3,4,5,6]. More recently, the disciplines of tissue engineering and regenerative medicine have permitted systematic evaluation of combinations of scaffolds or graft materials with stem and progenitor cells (either implanted with the graft or [7,8] recruited naturally from nearby tissues) [9,10,11,12], and growth factors for the purpose of directed tissue formation. Accordingly, the literature now contains a vast number of examples of successful cell-based therapies in bone tissue, albeit most in an experimental setting. The overall consensus of these studies is that regardless of the cell source employed, it is the cells that are directly responsible for tissue formation, not the scaffold, drug, or surgical instrumentation. In many instances, scientists and clinicians have attempted to supplement or replace cells by administration of potent growth factors, including bone morphogenetic proteins (e.g., BMP-2, BMP-7) [11,12]. Of the numerous cell sources that have been tested for bone tissue engineering, mesenchymal stem or stromal cells (MSCs) have received the most attention. MSCs are multi-potent progenitors of connective tissue such as bone, cartilage, muscle and adipose [13,14,15,16,17,18,19,20,21,22,23,24,25,26,27], and can be harvested from bone marrow, periosteum, synovium, muscle, fat, and other adult tissues [28,29,30,31,32,33]. Of the countless studies performed exploring the potential for MSCs for the repair bone tissue, some have demonstrated that they have remarkable efficacy in the absence of a scaffold [34,35,36,37,38,39] but the general consensus is that a scaffold may be required for confinement of MSCs at the site of injury during initial stages of healing [1,7,10,26,37,38,40,41,42]. Natural graft materials include autograft (tissue harvested from the patient) [6,43,44], allograft (tissue transplanted from another donor, usually a cadaver) [3,4,6], or processed tissue/proteins from cadaveric humans or animals [3]. Autograft is considered the “gold-standard” in fracture repair and spinal fusion, usually requiring the harvest of a portion of the iliac crest [43,44]. This strategy has greatly decreased in popularity and use, as it leads to donor-site morbidity (necrosis at the harvest site) and enduring pain to the patient [45,46]. Allograft products present some risk of disease transmission, inflammation and immune response to the foreign tissue [2,46]. Due to these shortcomings, synthetic biomaterials have been developed comprised of ceramics, proteins, peptides, carbohydrates, and polymers [1,3,7,10,47]. Synthetic materials can be designed to achieve the desired biological and mechanical properties, degradation or bioresorption rates, and tissue integration based on specific applications [3,10]. For orthopedic purposes, synthetic materials consist of calcium phosphates (including hydroxyapatite (HA) and β-tricalcium phosphate (βTCP)), collagen, protein, or hydrophilic polymer-based foams and gels, and hydrophobic polymers (e.g., poly(l-lactic-co-glycolic acid), polyurethanes, poly(propylene fumarate)). Calcium phosphate-based materials are advantageous as they offer natural osteoinductive cues through their ionic crystalline structure while providing a framework to build new tissue upon that is slowly resorbed and remodeled. Gan has reported successful spinal fusions in a human clinical study using autologous BMSCs augmented with βTCP granules [48].The use of graft materials in spine and orthopedic markets has primarily focused on bone-derived or bone-like substrates including allograft bone chips and powderized bone, demineralized bone matrix (DBM, acidified allograft bone), calcium phosphate particles and cements, and collagen sponges [47,49]. Commercially marketed graft materials have come to be known as carriers for their ability to “carry” cells and proteins with them. More accurately, they are also referred to as scaffolds and substrates, as they provide a foundation for cells to lay down new extracellular matrix (ECM) and build mature tissue. Graft materials vary greatly in their form, origin, and biochemical properties. Substrates used in fusions, non-union skeletal defects, and as bone void fillers are often selected by surgeons based on appearance or handling properties rather than their effect on co-implanted or endogenous stem and progenitor cells. In fact, the nature of the interactions between primary human osteoprogenitor cells and most bone tissue substitutes are poorly understood. Another critical, but poorly understood characteristic of common bone substitutes is their degradation rate and consequent release of factors that affect the local microenvironment.To better understand the effects of graft materials on osteoprogenitor cell function, primary human bone marrow MSCs (BMSCs) were seeded onto several commercially available substrates and incubated in a surgically-relevant duration for up to 48 h. Viable BMSC adhesion was measured over at 30 min, 1 h and 48 h by quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR). The expression of bone-related genes was also quantified by RT-PCR at the conclusion of 48 h culture. During this period, the physical state of the material and pH also was also monitored. In another series of experiments, we evaluated the effect of agents released from three injectable poloxamer-based demineralized bone matrix preparations on proliferation and osteogenic gene expression. From these studies, we found that bone substitutes vary widely in their ability to bind BMSCs and activate common osteogenic markers. While some matrices facilitate BMSC binding and enhance osteogenic markers, others fared poorly in physiologically-buffered serum-containing media, losing their three dimensional form within a few minutes. In some cases, the materials also caused substantial changes in the pH of media that affected the viability of cells. Of the injectable materials tested, BMSCs cultured with soluble extracts of all 3 substances reduced cell expansion but two of them enhanced expression of osteogenic markers. From the in vitro data presented here, we predict that while some bone substitutes have some positive attributes with respect to cell binding and osteogenic stimulation, many have potentially undesirable effects that should be seriously considered before utilizing clinically.Human BMSCs preparations were generated from a healthy human donor as previously described [19]. Briefly 2 mL of iliac crest bone marrow was processed by discontinuous density-gradient centrifugation and 20 million cells of the mononuclear fraction were cultured in complete culture media (CCM) consisting of alpha-minimal essential media (Invitrogen, Carlsbad, CA, USA) containing standard concentrations of penicillin and streptomycin (Invitrogen) and 20% (v/v) fetal bovine serum (FBS, Atlanta Biologicals, Norcross, GA, USA). Cells were incubated at 37 °C with 5% (v/v) CO2 with media changes every three days until a density of about 2000 plastic adherent cells per cm2 was attained. Cells were re-plated at 100 per cm2 and allowed to grow for 7–9 doublings before re-passage. The identity of the cell preparations was confirmed by differentiation to mineralizing osteoblasts, adipocytes and chondrocytes and surface phenotype as described previously [19,36,37]. Cryopreserved vials of 1 million BMSCs were stored in liquid nitrogen at passage 2 for this study.Substrates investigated in this study include allograft cancellous bone chips (Allograft Cancellous Bone, IsoTis OrthoBiologics, Irvine, CA, USA), powdered cancellous bone (Cortical-Cancellous Bone Powder, Bone Bank Allografts, San Antonio, TX, USA), DBM strip/sponge (Accell TBM, Integra, Plainsboro, NJ, USA), DBM putty (OrthoBlast II, Citagenix, Laval, QC, Canada), DBM poloxamer-putty (Accell Connexus and OsteoSurge, Integra, Plainsboro, NJ, USA), collagen sponge (DuraGen, Integra, Plainsboro, NJ, USA), βTCP (SBM Bio-1, SBM, Winchester, MA, USA), βTCP-collagen morsels (Mozaik, Integra, Plainsboro, NJ, USA), HA-collagen (NanOss, Pioneer Surgical, Marquette, MI, USA), HA-collagen-bioactive glass (Vitoss BA, Orthovita, Malvern, PA, USA), 60:40 βTCP-HA granules (CymbiCyte, Celling Biosciences, Austin, TX, USA) and porous HA granules (Solum IV, under development by Celling Biosciences, Austin, TX, USA). The porous HA granules have a surface area of 7 m2 per gram of material according to the manufacturer’s specifications.BMSCs (500,000 cells in 1 mL CCM) were mixed with 0.5 mL of each substrate at a ratio of 1:2 (cell suspension to substrate, v/v). The mixture was incubated 30 and 60 min at 37 °C with inversion every 15 min or 48 h at 37 °C with 5% (v/v) CO2. After incubation, excess media was carefully removed and replaced by 2 mL of cation-free phosphate buffered saline (PBS). Samples were centrifuged at 400× g for 5 min, the supernatant was removed, and the process was repeated 2 more times to remove loosely-attached cells. To prevent substrate constituents from interfering with the RNA extraction and final purity, cells were dissociated from matrices using a trypsin/ethylene-diamine-tetraacetic acid dissociation cocktail (0.25% Trypsin-EDTA, Gibco, Grand Island, NY, USA) for 7 min at 37 °C. Cells were recovered by centrifugation followed by extraction of total RNA (High-Pure mRNA extraction kit, Roche Diagnostics, Indianapolis, IN, USA). One tenth of the RNA was then used to generate a mixture of oligo-dT and random-hexamer primed cDNA using a kit (Super-Script III Kit, Invitrogen). The original number of recovered and viable cells was determined by measuring the copies of glyceraldehyde phosphate dehydrogenase (GAPDH) cDNA using qRT-PCR. For this purpose, one fifth of the cDNA was amplified with a previously described primers and conditions [50] (Table 1) with fast SYBR-Green master mix (Applied Biosystems, Foster City, CA, USA) on a Bio-Rad C1000 thermocycler fitted with a CFX96 Real Time System (Bio-Rad, Hercules, CA, USA). Cycle thresholds (Ct) were compared with known standards run in parallel and converted to the original number of cells per sample. The experiment was performed in quadruplicate (n = 4) for each substrate and data were expressed as means with standard error of the mean (SEM).RT-PCR primer sequences for glyceraldehyde phosphate dehydrogenase (GAPDH), Runx2, alkaline phosphatase (ALP), and collagen I.BMSCs and mineralized substrates (cancellous bone chips, βTCP-HA, and porous HA) were incubated as described above in complete medium with 20% FBS for 48 h in a 37 °C incubator with 5% CO2. Copy DNA was prepared as described, and the relative expression levels of collagen I [51], alkaline phosphatase (ALP) [51] and runt-related transcription factor 2 (Runx2) [53] was measured using previously described primers and conditions (Table 1). The genes were chosen to reflect early and intermediate markers of osteogenic differentiation that would be expected to be up-regulated during adherence to substrates over a 48 h period. One microgram of cDNA was added to each reaction, and expression levels relative to monolayer BMSCs were calculated using the 2-delta-delta Ct method, normalizing to GAPDH expression [54].Soluble substrates (2 mL, DBM putty and DBM poloxamer-putty) were dissolved in 10 mL complete medium by incubation for 15 h at 4 °C with frequent inversion. Due to substantial acidity, the extracts were diluted a further 1:10 with medium until the pH reached 7.4. For proliferation studies, cultures of BMSCs (n = 3 per measurement) were established in 6-well 9.5 cm2 tissue culture plates (Invitrogen) by plating at 150 cells per cm2 in DBM conditioned media. Media was changed every 2 days and as a negative control, monolayer BMSCs were cultured in complete medium without DBM conditioning. At day 1, 2, 4 and 8 the cells were recovered by trypsinization and counted by Cy-Quant (Invitrogen, Grand Island, NY, USA) fluorescence incorporation assay as previously described [55]. For expression studies, cultures were established in 55 cm2 plates in the same manner but allowed to proceed for eight full days with media changes every two days. After 4 and 8 days, cells were recovered by trypsinization, total RNA was extracted and expression levels of Runx2, ALP, and type I collagen was measured. The experiment was performed in triplicate (n = 3) for each substrate. Data were calculated using the 2-delta deltaCt method, normalizing to GAPDH expression and expressed as fold-changes relative to control cultures not conditioned with DBM extract.Each of the substrates was incubated at room temperature for 24 h to observe physical changes and dissolution and measure the pH after hydration with a buffered solution. In separate 15 mL vials, each material (2 cc) was submerged in 3 mL human plasma or PBS. Plasma was prepared by centrifuging whole blood (South Texas Bood and Tissue Center, San Antonio, TX, USA) for 10 min at 1000× g. Plasma and PBS-only tubes were prepared and measured at each time point as a control. The tubes were shaken gently every 15 min. The pH of each solution was measured using an Orion-4 pH probe (Thermo Scientific, Waltham, MA, USA) incrementally for 24 h. The experiment was run in triplicate for each substrate.Data were analyzed by one sided analysis of variance (ANOVA) with Tukey or Dunnett’s post-testing using commercially available software (GraphPad Prism version 5.00 for Windows, GraphPad Software, San Diego, CA, USA).The kinetics of BMSC binding and retention is shown in Figure 1. After 30 min of culture, DBM and collagen-based materials had significantly dissolved into the media leaving about 30% of the original solid volume to collect for measurement and consequently, cell recovery was low. The structural degradation of substrates before and after hydration is demonstrated in Figure 2. Synthetic materials made of βTCP, βTCP-HA, and porous HA retained their structural integrity over the entire culture period, and rapidly bound more BMSCs over a 30 min period when compared to cancellous bone chips. After one hour, cell counts on βTCP-HA and porous HA granules were comparable to bone chips and significantly greater than the other substrates examined by the prescribed methods. After 48 h of culture, the bone chips, porous HA and to a lesser extent, βTCP-HA all had significant numbers of cells associated with them, suggesting that these materials could sustain stable constructs. In contrast, stable cultures of BMSCs on βTCP could not be established in our culture system, and the cell retention was diminished to below detectable levels after 24 h.Bone marrow stromal cell retention on insolubilized substrates after 30 min, 60 min, and 48 h of incubation at 37 °C. Error bars represent standard error of the mean (n = 4 per substrate). Asterisk denotes significant difference (* p < 0.05) against the appropriate bone chip measurements by ANOVA and Dunnett post test.Cancellous bone chips, demineralized bone matrix (DBM) putty, DBM-poloxamer putty, collagen, βTCP-collagen-bioactive glass, βTCP-HA granules, and porous HA granules (left to right) prior to hydration with phosphate buffered saline (top row), 5 min after hydration (middle row), and 60 min after hydration (bottom row).To evaluate the short-term differentiation potential of BMSCs bound to substrate materials, the transcription of the early osteogenic transcription factor Runx2, intermediate markers ALP and collagen I were measured by quantitative RT-PCR (Figure 3). Experiments were performed on those materials that could sustain stable cultures (bone chips, βTCP-HA and porous HA) and results were compared to expression levels on tissue culture plastic. All of the osteogenic materials induced modest upregulation of transcription of ALP (1.5–2 fold) and collagen I (1.5–5 fold) when compared to tissue culture plastic but expression of Runx2 was substantially up-regulated by culture (8–10 fold). Relative expression of Runx2, alkaline phosphatase (ALP), and type I collagen as a fold increase over tissue culture plastic by cells incubated with cancellous bone chips, βTCP-HA, and porous HA granules after 48 h of incubation at 37 °C. Error bars represent standard error of the mean (n = 4 per substrate). Asterisks denote significant difference (* p < 0.05, ** p < 0.01) by ANOVA and Tukey post test.Three injectable putty preparations examined in this study for support of BMSC proliferation and differentiation; two DBM poloxamer-putty preparations (Accell Connexus and OsteoSurge, Integra, Plainsboro, NJ, USA) and one DBM putty (OrthoBlast II, Citagenix, Laval, QC, Canada) were studied. Because the materials partially dissolved in media, it was not possible to perform a conventional cell binding assay. We therefore prepared filtered extracts of the samples in growth media by incubation at 4 °C for 15 h. The DBM extract media was incubated with 2D monolayer culture BMSCs. Initial observations indicated that the 2 poloxamer-based putties caused substantial acidity of the extracts (all cells died within 12 h), but this could be buffered by 1:10 dilution in fresh medium. Upon monolayer culture in the presence of the extracts it was apparent that all three samples caused a minor reduction in cell recovery after 4 and 8 days (Figure 4), but the absence of morphological signs of cell death suggested that the effects were due to cell-cycle inhibition rather than necrosis or apoptosis. After 4 and 8 days of culture, the relative transcription levels of Runx2, ALP and collagen I were measured by real time RT-PCR. At day 4, transcriptional levels of all three genes did not significantly differ from the control media. However, after eight days of culture, extracts from all putties caused a modest increase in Runx2 transcription (3–4 fold) when compared with untreated controls (Figure 5). Interestingly, only the DBM-poloxamer extracts caused an appreciable increase in ALP and collagen I at 8 days, suggesting that osteogenesis had progressed further in these cultures. Taken together, these data suggest that although modestly detrimental to BMSC expansion, extracts from the DBM and DBM-poloxamer materials had a positive effect on osteogenesis in vitro if the acidic properties can be maintained within the physiological pH range.Cell growth curves for BMSC cultured in standard medium or 1:10 diluted extracts of DBM or DBM-poloxamer putties through 8 days (n = 4 per substrate). Asterisk denotes significant difference (* p < 0.05) against the no treatment control by ANOVA and Dunnett post test.Relative expression of Runx2, alkaline phosphatase (ALP), and type I collagen as a fold increase over tissue culture plastic by cells cultured in 2D with 1:10 diluted extracts from DBM and DBM-poloxamer putties after four days (A) and eight days (B) of incubation at 37 °C. Error bars represent standard error of the mean (n = 4 per substrate). Asterisks denote significant difference (* p < 0.05, ** p < 0.01) by ANOVA and Tukey post test.The plasma and PBS solution pH of each substrate through 48 h is demonstrated in Table 2. Most substrates remained within the normal physiologically neutral range (pH 7.3–7.5) for the duration of the investigation and with little variation after the initial 5 min in solution. The most acidic products were the DBM strip (pH < 7.2) and the DBM poloxamer-putty (pH < 4.9). The Βtcp-collagen-bioactive glass material elicited a basic response (pH > 10). Average pH values of substrates buffered in human plasma over 48 h (pH values in phosphate buffered saline in parentheses).In an in vitro setting, cell retention was measured on 9 different commercially available carrier substrates at 30 and 60 min, and 48 h (Figure 1). A cell suspension was added to the substrate at a 1:2 volumetric ratio. The first observation was the dissolution of DBM and collagen-containing materials. DBM carriers are synthesized by the acidification of allograft bone with hydrochloric and/or citric acid to remove mineral, preserving a matrix of organic proteins, consisting primarily of primarily collagen fibrils [56,57,58]. Most collagen and collagen-composites are synthesized by the lyophilization (freeze-drying) or precipitation of a solubilized collagen derived from acidified bovine or porcine tissues [59,60]. For DBM and collagen substrates, the introduction of water, saline, blood, or other fluids hydrates the material, making it softer by dissolving a portion of the protein matrix. The hydration of dry collagen matrix can lead to physical constriction of the material, decreasing the average pore size and thereby limiting its accessibility to cells and blood vessels. As the proteins dissolve, the loss of available binding surface area may render the material ineffective as a scaffold to bridge a bony defect. The dissolution of DBM and collagen-containing carriers made evaluation of cell retention beyond early time points impossible. Two of the collagen-containing materials demonstrated cell retention at 30 min comparable to bone chips and the calcium phosphate materials. However, as the materials resorbed into the media, there was little or no physical material to collect for cell quantification. This study aimed to identify the number of cells resident on an implant material through 48 h to provide researchers and physicians insight into the fate of BMSCs. If the cell-bound portions of the materials are resorbed between 60 min and 48 h in vitro, there is likely some corollary to in vivo material resorption and potential cell loss (migration away from the implant site or death). The provision of a stable ECM is essential for sufficient retention of cells at the implant site [37]. The βTCP/HA and HA substrates maintained their mass and geometry in solution through the duration of the study and achieved increasing cell counts with time. The successful binding between cells and ceramic materials can be attributed initially to electrostatic interactions of membrane proteins with calcium and phosphate ions on the granules’ surface. The increased cell counts from 30 to 60 min are related to gradual binding of cells, however, the drastic gain in cell number at 48 h is undoubtedly due to cell proliferation on the substrates as an extracellular matrix is established on the surface of the material. Porous HA granules possess more than 7 m2 of surface area per gram, at least six-fold increase over typical calcium phosphate particulates. The increased surface area permitted greater cell proliferation and spreading, which demonstrated the highest cell counts at the later time point. The second observation upon addition of cell suspension to the substrates was an immediate change in media color for several samples. The culture media used in these experiments contained phenol red pH indicator dye, which is responsive to pH changes associated with depleted nutrients and accumulated waste products. The DBM strip and DBM poloxamer-putty caused the media to change from red to yellow over the first 30 min of incubation, indicating a drop in pH and the acidity of the solution. In contrast, the bioactive glass material rapidly turned the media from red to purple (less than one minute), meaning the solution had become highly basic. This phenomenon was most likely to account for the exceptionally low cell counts for these materials, given that the pH was outside the physiological range for BMSC viability. To test this hypothesis, the experiment was repeated with human plasma and a buffered saline solution (PBS) rather than cell medium, and the pH was closely monitored with an electronic pH probe (Table 2). DBM poloxamer-putty decreased the solution pH below 5, while the DBM strip and βTCP-collagen substrates produced a less acidic effect. Although mild levels of transient acidity is tolerable to cells and is common with inflammation and infection, decreased pH can alter the chemical characteristics of many proteins and drugs, reducing their efficacy. Prolonged exposure to acidic conditions can result in apoptosis and tissue necrosis as well as inhibition of hydroxyapatite deposition during bone remodeling [61]. Conversely, the bioactive glass carrier achieved a solution alkalinity (pH 10) that is known to adversely affect cell viability in vitro, and thus, was likely the cause of very poor cell recovery. These results were surprising given that numerous favorable reports of bioglass and its application in bone engineering [62,63]. In partial explanation of this disparity, simulation studies have suggested that the hydroxyl moieties released through hydrolysis of the glass constituents do exist, but they are rapidly re-sequestered to the surface of the glass [64]. Although this could account for the reduced effects of alkalinity in some situations, the solution alkalinity detected in this study was approximately 300 times higher than physiological levels. Although surgery sites may be slightly acidic, the basicity of this bioglass is orders of magnitude higher. The bioglass components of this substrate were more easily and quickly dissolved than sintered or conjugated analogous materials. While the buffering capacity of blood and the body may negate long term deleterious effects of materials with non-ideal pH, short term incubation of these materials outside the body during graft preparation may impact cells’ viability and their therapeutic effects.It is known that the biological, chemical, and physical properties of the graft materials directly influence the physiology of osteoblast progenitors. For example, nano-scale topological features such as porosity have influenced differentiation mechanisms of attached MSCs [65,66,67] and molecules on the surface may directly modulate receptors or achieve this indirectly through bound plasma proteins, growth factors, or other molecules. In the present study, we observed that BMSC adherence to bone chips, βTCP-HA, and porous HA increased Runx2 expression by 8.5, 10.5, and 8.7-fold when compared to tissue culture plastic monolayer controls. Runx2 is an early-stage transcription factor that activates osteoblastic differentiation [68] and is highly indicative of commitment to an osteoblastic lineage. This level of Runx2 up-regulation was unexpectedly high for a 48 h time point without dexamethasone or bone morphogenic administration supporting the critical role of the solid substratum in addition to biochemical stimulation. On the other hand, the expression of ALP was only moderately increased for these substrates (2.3, 1.7, and 1.8-fold, respectively) but this protein is usually upregulated by BMSCs after more than 48 h or osteogenic stimulation under standard conditions [55]. Bone chips triggered a greater up-regulation of type I collagen than either synthetic substrate (4.6-fold over culture plastic compared to 3.8-fold for βTCP-HA and 1.5-fold for porous HA). We hypothesize that this effect is partially due to existing collagen and ECM proteins within the bone chips and absent from the ceramic particles that may promote further ECM remodeling and synthesis. It was hypothesized that the DBM putties would enhance BMSC proliferation and differentiation given the reported effects of bone extracts on osteoprogenitor function [58,69,70,71,72,73]. However, their rapid resorption and the acidity associated with their fabrication proved incapable of maintaining cell growth in vitro. After dilution of DBM and DBM-poloxamer extracts at 1:10 in standard media, cells were able to proliferate on culture plastic at retarded rates. These reduced growth rates may be attributed to cell cycle changes associated with differentiation as well as the toxicity of the extracted acidic byproducts. After 48 h in culture, the diluted DBM and DBM-poloxamer extracts induced expression of osteogenic genes, validating their osteogenic properties once the acidic effects are mitigated. Several commercially available orthopedic substrates were examined for their interactions with BMSCs within 48 h to mimic cell response after surgical preparation of graft materials and implantation in orthopedic or spinal fusion procedures. DBM and collagen-based substrates quickly resorbed upon hydration, rendering them incapable of cell retention or serving as a lasting scaffold for tissue regeneration in vivo. The DBM and DBM-poloxamer materials also exhibited a release of acidic factors after hydration, preventing cell proliferation without dilution of the DBM extracts. After adjustment of media pH, however, the DBM-poloxamer substrates did elicit an osteogenic effect on plastic-bound cells in terms of Runx2, ALP, and collagen I expression. The DBM putty only increased Runx2 expression compared to standard controls. Cancellous bone chips, βTCP-HA, and porous HA granules supported cell retention and 3D proliferation through the 48 h of observation and induced significantly greater expression of Runx2, ALP, and collagen I compared to controls.The application of mesenchymal stem and progenitor cells is a vital component for regenerative medicine therapies. However, the choice of substrate accompanying the cells can facilitate their success, inhibit their functionality, or cause their premature death depending on the material’s structure, composition, and byproducts. The primary purpose of biomaterial scaffolds is to provide a substrate for cell adhesion and proliferation that guides the growth of new tissue. In non-union fracture and spinal fusion procedures, substrates that quickly dissolve cannot perform either function. A lack of consideration or understanding of cell-substrate interactions may negate the regenerative potential of cell therapies and lead to undesired clinical outcomes. The authors thank Jessica Terrazas and Melissa Samano for assistance with biomaterial analysis and pH experiments. Matthew B. Murphy, Richard K. Suzuki, Theodore T. Sand are employed by a private company (Celling Biosciences) that is licensed to market Graftys and is developing Solum IV. The other authors indicate no potential conflicts of interest.

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+This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).Despite advances in adjuvant therapy for breast cancer, bone remains the most common site of recurrence. The goal of therapy for these patients is palliative and focused on maximizing the duration and quality of their life, while concurrently minimizing any disease or treatment-related complications. Bone metastases predispose patients to reduced survival, pain, impaired quality of life and the development of skeletal-related events. With an increased understanding of the pathophysiology of bone metastasis, effective treatments for their management have evolved and are now in widespread clinical use. This article will discuss the pathogenesis of bone metastases and review the key clinical evidence for the efficacy and safety of currently available systemic bone-targeted therapies in breast cancer patients with an emphasis on bisphosphonates and the receptor activator of nuclear factor kappa B ligand (RANKL) inhibitors. We will also discuss novel strategies and therapies currently in development.Bone remains the most common site of breast cancer recurrence [1,2,3]. The development of bone metastases is associated with reduced survival, impaired quality of life and pain. In addition, bone metastases can undermine the structural integrity of the skeleton and predispose patients to skeletal complications, collectively known as skeletal-related events (SREs). SREs typically include: Radiotherapy or surgery to the bone, pathologic fractures, spinal cord compression and hypercalcemia of malignancy [2,4,5,6]. SREs are associated with significant morbidity and mortality, and their avoidance is an essential component of patient management [7,8,9,10,11].Over the past two decades, bone-targeted agents, such as bisphosphonates and the receptor activator of nuclear kappa ligand (RANKL) inhibitor, denosumab, have emerged as effective options for the treatment of women with breast cancer that has metastasized to the bone [12,13,14]. Their use in combination with anti-cancer therapies (i.e., chemotherapy and hormonal therapy) is supported by clinical trial evidence showing efficacy for reducing the number of SREs and delaying the onset of SREs [15,16]. As a result, these agents are now considered the standard of care for the treatment of breast cancer patients with bone metastases [17].The following review will summarize the major clinical trial evidence for both the bisphosphonates and denosumab for the treatment of bone metastases from breast cancer. We will briefly discuss their proposed mechanisms of action as they relate to the pathophysiology of bone metastases. We will not discuss adjuvant bone-targeted therapies, as this is beyond the scope of this manuscript [18,19]. Finally, we will review some novel treatment strategies and newer therapies currently under investigation that may be entering clinical practice in the future.Normal bone is continuously renewed by remodeling, a tightly controlled process that involves a balance between osteoclast-mediated bone resorption and osteoblast-mediated bone formation [5]. Disturbance in this balance will either lead to net bone loss or formation. Breast cancer cells that metastasize to the bone marrow do not directly cause bone destruction on their own [17,20]. Instead, they alter the bone microenvironment, leading to an uncoupling of the balance between bone resorption and formation [5,17,20,21]. This process is complex, but in essence, the presence of breast cancer cells in the bone ultimately leads to excessive bone resorption via upregulation of osteoclasts leading to characteristic lytic metastases and tumor propagation [5,17,20,21,22]. The disruption of normal bone remodeling by metastatic breast cancer cells ultimately sets up a “vicious cycle” that propagates the process of bone destruction [5,10].Bisphosphonates are stable drug analogues of naturally occurring inorganic pyrophosphate that can: (i) Inhibit processes essential for osteoclast survival and (ii) Promote osteoclast apoptosis [23]. As a result of their anti-osteoclastic effects, bisphosphonates have the ability to modulate bone turn over and diminish tumour-mediated bone resorption and destruction [23]. There are two main classes of bisphosphonates; the amino- and the non-amino bisphosphonates [23,24]. Given their greater potency, the amino-bisphosphonates are generally used clinically [23,24]. There are currently four bisphosphonates available for use to treat women with breast cancer bone metastases; clodronate, pamidronate, ibandronate and zoledronic acid. All except clodronate are amino-containing bisphosphonates [23].Denosumab is a fully human monoclonal antibody that binds to RANKL and prevents it from interacting and activating its receptor, RANK [25,26]. RANK is found on the surface of osteoclasts and osteoclast precursors, and pre-clinical models identified the RANKL-RANK interaction as a key mediator of osteoclastic activation [25,26]. By binding to RANKL, denosumab decreases cancer-induced bone destruction by suppressing osteoclast-mediated bone resorption.Clodronate was the first bisphosphonate widely studied in women with breast cancer metastatic to bone [27,28]. Subsequent larger placebo-controlled trials confirmed that daily oral clodronate was effective for reducing the incidence of SREs [29,30,31] (Table 1).Placebo controlled trials of bisphosphonates available for clinical use.RCT: Randomized controlled trial; No.: Number; vs.: versus; i.v.: Intravenous; HR: Hazard ratio.The first large placebo-controlled randomized trial with pamidronate in women with breast cancer metastases to bone was performed over 20 years ago [31]. In this trial, pamidronate was administered orally on a daily basis instead of the usual intravenous dose on a 3–4 week basis. Despite having to reduce the dose in half from the initially planned 600 mg/day dose (due to significant nausea and vomiting), this study demonstrated that oral pamidronate significantly reduced hypercalcemia, bone pain, pathological fractures and the need for radiotherapy compared to the placebo group [29,31]. Moreover, this study highlighted that oral pamidronate can cause significant gastrointestinal toxicity and also led to further studies using the intravenous formulation.Subsequent trials of pamidronate in patients with breast cancer assessed intravenous administration at three or four week intervals against placebo. The detailed findings from these trials are summarized in Table 1. The cumulative results from these trials showed that pamidronate significantly reduced the incidence of SREs and time to onset of SREs. This was independent of the underlying concurrent systemic chemotherapy or endocrine therapy. None of these trials showed either progression-free or overall survival advantage [32,33,34,35,36].Contemporary practice is to use pamidronate at 90 mg intravenous every 3–4 weeks based on hypercalcemia studies [32,41,42]. The effect of pamidronate given intravenously at a lower dose vs. control on the incidence of SREs in women with metastatic breast cancer has also been investigated in two randomized trials (Table 1) [32,34]. The total dose given per unit time was similar in both trials, as one trial used a dose of 45 mg every three weeks and the other, a dose 60 mg every four weeks. Both trials demonstrated that pamidronate was significantly better than placebo at reducing the number of SREs and delaying the time to an SRE [32,34]. The findings from these studies are important clinically, as they provide the treating physician alternative dose options.Ibandronate is a potent third-generation amino-bisphosphonate with oral and intravenous formulations. It is widely used in Europe and has demonstrated clinical efficacy on reducing and delaying SREs [37,38,39]. The recommended dosing is 50 mg orally per day or 6 mg intravenous every 3–4 weeks [37,38,39]. The results of the main ibandronate placebo-controlled clinical trials are summarized in Table 1.Zoledronic acid is a potent third-generation amino-bisphosphonate that can be administered intravenously over 15 min. Only one large study has investigated zoledronic acid compared to placebo [40]. This was a registration trial in Japan, where placebo was deemed an appropriate comparator arm as, at the time, no bisphosphonate had been approved for the treatment of metastatic bone disease. This study showed that zoledronic acid reduced SRE rates and delayed the time to the first SRE (Table 1) [40].The question of whether one bisphosphonate is more effective than others in controlling SREs in patients with breast cancer has been investigated for zoledronic acid relative to pamidronate [43] and ibandronate [44]. No head to head trial between clodronate and the other bisphosphonates in the metastatic setting have been completed. The main findings from these trials are summarized below and in Table 2.Comparative trials of bone-modifying therapies available for clinical use.RCT: Randomized controlled trial; No.: Number; vs.: versus; i.v.: intravenous; HR: Hazard ratio; NI: Non-inferiority; S: Superiority.Zoledronic acid was compared to pamidronate in 1130 patients with breast cancer and at least one metastatic bone lesion [43]. In this double blind, double dummy trial, patients were randomized to receive either pamidronate 90 mg intravenous or zoledronic acid 4 mg or 8 mg intravenous every 3–4 weeks for one year. Due to excess renal toxicity, the 8 mg dose of zoledronic acid was stopped, and patients in that arm received further treatment with 4 mg instead. The primary efficacy endpoint was the proportion of patients who experienced at least one SRE during the 13 months on the study. Overall, there was no difference between the zoledronic acid and pamidronate groups with respect to the primary endpoint. In the subgroup of patients with only lytic metastases, there was a statistically significant clinical benefit for zoledronic acid over pamidronate (prolonged time to first SRE and reduction in the annual incidence of skeletal events) (Table 2).An extension of this trial to 25 months of follow-up included 412 patients with breast cancer [45]. There was no difference detected in the proportion of patients that experienced at least one SRE (46% in the zoledronic acid group and 49% in the pamidronate group). The skeletal morbidity rate (the ratio of the number of skeletal complications to the time on the trial) was reduced by 40% in the zoledronic acid group; however, this was not statistically significant (0.9 vs. 1.49 events per year). Multiple event analysis showed that zoledronic acid significantly reduced the risk of developing any skeletal complications by an additional 20% compared with pamidronate. The time to first SRE, skeletal morbidity rate and risk of skeletal complications were better controlled in the zoledronic acid group in patients receiving endocrine therapy, but not chemotherapy [45].The results of a large trial comparing oral ibandronate with zoledronic acid were recently reported in abstract form and are summarized in Table 2 [44]. The findings did not demonstrate that oral ibandronate was statistically inferior to zoledronic acid.The pooled analysis of data from eight studies that included 2189 breast cancer patients with bone metastases showed that bisphosphonates reduced the risk of developing an SRE by 17% compared to placebo (risk ratio: 0.83; 95% CI: 0.75–0.93; p = 0.001) [15]. There was no difference between oral or intravenous bisphosphonates (risk ratio: 0.84 vs. 0.83). The pooled data did not show a survival benefit in favor of bisphosphonates relative to placebo-control (risk ratio: 1.01; CI: 0.92–1.11) [15].Two phase II trials of patients with bone metastases demonstrated that denosumab at doses ranging from 30 to 180 mg administered every four or 12 weeks was similar to intravenous bisphosphonates in suppressing bone turnover markers [47,48]. Despite the absence of evidence of differences among the majority of denosumab doses or between the four- and 12-week weekly administration on biomarker response, it was the 120 mg dose of denosumab every four weeks that has been evaluated in subsequent clinical trials.Denosumab (120 mg subcutaneously every four weeks) was compared head to head with zoledronic acid (4 mg intravenous every four weeks) in a pivotal large, double-blind, double-dummy phase III clinical trial involving 2046 women with metastatic breast cancer with bone metastases [46]. The efficacy endpoints were time to first SRE and time to first and subsequent SREs (multiple event analysis). This study showed that denosumab significantly delayed the time to first on-study SRE by 18%, reduced the risk of developing multiple SREs by 23% and delayed the median time to first onset SRE (26.4 months for zoledronic acid vs. not reached for denosumab). Denosumab was also shown to be superior to zoledronic acid in reducing markers of bone resorption after 13 weeks of follow-up. There was no progression-free or overall survival difference between the two arms [46] (Table 2).Bisphosphonates are generally well-tolerated; however, there are some common and potentially serious toxicities associated with their use. The most clinically significant toxicities are: Nephrotoxicity, hypocalcemia, acute phase reactions (fever, pain, fatigue) and osteonecrosis of the jaw (ONJ) [49,50,51,52,53]. These are all more commonly associated with intravenous bisphosphonates. Oral bisphosphonates may also cause significant gastrointestinal upset (nausea and vomiting, epigastric pain, esophagitis) [49,54].Nephrotoxicity typically manifests as a rise in creatinine and occurs in 2%–8% of patients on intravenous bisphosphonates. Patients with underlying renal dysfunction are at greater risk [49]. The American Society of Clinical Oncology clinical practice guideline recommends careful clinical monitoring of renal function in all patients who are on active bisphosphonate treatment and, if renal impairment occurs, recommends discontinuation of the bisphosphonate until the serum creatinine has returned to within 10% of baseline [49]. Intravenous bisphosphonates are not recommended in patients with creatinine clearances less than 30 mL/min [49,50].Bisphosphonates cause a relatively rapid and prolonged drop in serum calcium and are the cornerstone of the modern day treatment of hypercalcemia; however, hypocalcemia may occur in up to 5% of patients on bisphosphonates [49]. Most of the trials that investigated their use in management of bone metastases supplemented the study participants with calcium and vitamin D (Table 1 and Table 2). It is generally recommended that patients on bisphosphonates be on concurrent calcium and vitamin D supplementation and have their serum calcium checked periodically [55,56].Fever, myalgias, bone pain and fatigue are quite common, occurring in up to one third of patients treated with intravenous bisphosphonates, and are most likely to occur following the first infusion [49,51]. They typically subside within 72 h and are believed to be a result of an acute flare of proinflammatory cytokines [51]. Given the likelihood of occurrence, patients about to start therapy with a bisphosphonate should be warned about the potential for developing such symptoms and reassured that they are transient and can be managed with supportive measures as required.Bisphosphonate-induced ONJ is perhaps the most dreaded potential complication of bisphosphonate therapy given its potential morbidity. It is defined as an area of exposed bone in the maxillofacial or mandibular area that has persisted for more than eight weeks, in a patient treated with bisphosphonates and no previous radiation therapy to the jaw [52,53,55]. Risk factors have been identified that may pre-dispose patients to ONJ, and these include: The use of high potency amino-bisphosphonates, prolonged bisphosphonate treatment, concurrent chemotherapy, underlying dental disease and a history of recent invasive dental procedures [53,57]. The incidence of ONJ in patients with breast cancer and bone metastases on a bisphosphonate ranges from 1.2%–2.4%, but can be as high as 10% in those who have multiple risk factors [49,53]. Patients should be screened for risk factors predisposing to ONJ, and any invasive dental procedures should be completed prior to initiation of therapy [53,58]. Patients should also be advised to let their dental care professional know that they are on bisphosphonate therapy. A careful examination of the oral cavity should be routinely performed, and prompt referral to a maxillofacial surgeon should be made for further management if ONJ is suspected.There is also increasing concern around the effects of long-term bone-targeted therapies on bone architecture with an increased risk of fragility fractures [59].One of the proposed benefits of denosumab over bisphosphonates was the reduction in the requirement for renal monitoring [46,47,48]. That being said, however, widespread use of denosumab has only just begun, and its potential “real-world toxicities” (outside of the context of a clinical trial) are still evolving. At this point in time, the major concerning toxicities appear to be symptomatic hypocalcemia, renal toxicity and ONJ [14,16,46,49,60]. In studies, renal toxicity was reported as 4.9% in the denosumab group (as compared to 8.5% in the zoledronic acid group) [46]. Moreover, ad hoc analyses of a phase III trial that investigated denosumab in patients with bone metastases from prostate cancer, solid tumors and multiple myeloma, reported similar renal adverse events in both the denosumab and zoledronic acid groups (9.2% vs. 11.8%) [16]. Given these data, periodic monitoring of renal function and serum calcium should be considered in patients being treated with denosumab.As with the potential for renal toxicity, the incidence of ONJ may also be higher for denosumab than previously thought. The pooled data from the three pivotal phase III trials of denosumab vs. zoledronic acid in metastatic bone disease showed that the incidence of ONJ was similar in both groups, at 1%–2% [60]. However, in the open label extension study of denosumab in patients with bone metastases from breast cancer, the incidence of ONJ of the jaw at five years was as high as 3.5%–4.7% [61].Research focused on the identification of new therapeutic targets and on how to optimize or “personalize” the use of currently available bone-modifying therapies is ongoing [62]. The following section will address some novel agents and therapeutic strategies that are currently under investigation and, also, highlight some of the key questions looking to be answered with regards to optimizing or “personalizing” the use of currently available agents.Although use of denosumab or bisphosphonates has had a significant impact on the quality of life for patients with bone metastases, no prospective and appropriately powered study to date has demonstrated any improvement in either progression-free or overall survival. As such, new potential therapeutic options for bone metastatic patients are actively being investigated. The process of breast cancer mediated bone destruction is complex and involves many mediators, which ultimately lead to deregulation of the process of bone remodeling [63]. The factors mediating this process and how they interact with one another are slowly being elucidated and serve as potential new targets for novel therapies. Clinical trials evaluating novel drugs that affect both breast cancer and bone are ongoing and include many early phase trials [17,64]. The following section will briefly discuss some of these potential new therapies under clinical investigation.Src family kinases are known to play important roles in tumour cell proliferation and invasion, and as such, pharmacologic inhibitors to src family kinases have been developed. More recently, an important role for src has also been demonstrated in osteoclasts, whereby inhibition of src family members with selective tyrosine kinase inhibitors (TKIs) was shown to block osteoclast differentiation from precursor cells [65,66] and inhibit osteolytic tumor growth in preclinical models of bone metastasis [67]. Based on this rationale, phase II studies evaluating the effects of inhibition of src family members have been initiated in advanced breast cancer patients. Dasatinib monotherapy has shown some efficacy in advanced breast cancer patients [68,69,70]. A number of other clinical trials are ongoing with SRC inhibitors to evaluate bone turnover markers as a specified endpoint in addition to tumour responses in breast cancer, including randomized phase II studies with exemestane [71] or letrozole administered with or without dasatinib [72], a randomized phase II study of fulvestrant with or without dasatinib [73], a phase I/II study of dasatinib in combination with zoledronic acid [74] and a phase II study of dasatinib administered either once or twice daily in patients with breast cancer and bone metastases [75]. Saracatinib’s effects on bone markers have been evaluated in a randomized phase II trial versus zoledronic acid in patients with prostate or breast cancer [76] and a phase II study of patients with metastatic hormone receptor-negative or locally advanced unresectable breast cancer [77]. Results of these studies will be eagerly anticipated.Cathepsin K is a serine protease, which is highly expressed by activated osteoclasts and is necessary for the degradation of bone matrix proteins [78]. Inhibition of cathepsin K has been shown to inhibit bone resorption in preclinical animal models [79]. Given that cathepsin K is frequently upregulated in breast cancer and is associated with more invasive disease and increased risk of bone metastasis [80,81], it has become a clinical therapeutic target of interest. Use of the cathepsin K inhibitor, odanacatib, was recently evaluated in women with breast cancer and metastatic bone disease. Patients were randomized 2:1 (double-blind) to oral odanacatib 5 mg daily for four weeks or intravenous zoledronic acid 4 mg given once at study initiation [82]. Evaluation of circulating levels of bone turnover markers (urinary N-telopeptide) demonstrated that odanacatib was equally effective in suppressing bone turnover markers as zoledronic acid in this short-term study and was well-tolerated. Thus additional analyses testing its efficacy alone or in combination with standardly used bone-targeting agents is warranted.The transforming growth factor-beta (TGF-β) family of proteins are known to play a significant role in tumour progression, including the ability to control tumor cell invasion, epithelial to mesenchymal transition and response to therapy [83]. It is also a prime driver of the vicious cycle in bone metastases. A number of inhibitors of TGF-β signalling pathways have been developed and are at various stages of preclinical testing for efficacy in bone metastasis control. Both blockade of TGF-β ligands [84] and inhibition of TGF-β receptor signalling [85,86,87] have demonstrated efficacy in inhibiting bone metastases in models of breast cancer.CXCR4 is a transmembrane G protein-coupled receptor, which has gained recent interest in the cancer metastasis world. Studies have shown that tumour cells that express CXCR4 preferentially metastasize to the bone, where its ligand, CXCL12, is abundantly expressed [88]. The subsequent CXCL12/CXCR4 signaling also enhances cell survival via Akt activation, thereby providing an advantage to newly arrived tumor cells in the bone microenvironment [89]. Disruption of the CXCR4 signaling pathway using neutralizing antibodies or synthetic peptide antagonists have been shown to reduce the formation of lung and bone metastases caused by CXCR4-expressing breast cancer cells in preclinical models [90]. Clinically, a phase I/II clinical trial has determined the tolerability and safety profile of repeated administration of a CXCR4 peptide antagonist in a small cohort of patients (n = 25) with advanced metastatic disease. Some patients had stable disease and tolerability was good [91]. However, the efficacy of CXCR4 blockade in bone metastatic breast cancer patients will await determination in future clinical studies.Many questions regarding the optimization of bone-targeted therapy still remain, especially for the use of bisphosphonates in an era of personalized medicine, where the, “one size fits all approach” of 3–4 weekly systemic therapy from diagnosis of bone metastases until death is no longer ideal [92]. Key questions for both physicians [93] and patients [94] that are currently under investigation include questions on optimal timing and dosing of bone-modifying therapy and what to do with this therapy upon documented disease progression.Therapy de-escalation in appropriate patients is an attractive option, as it has the potential to improve patient quality of life, reduce drug toxicity and to be more fiscally responsible to individual healthcare systems. This issue was investigated in a phase 3, open label, randomised, non-inferiority trial looking at the efficacy and safety of 12-weekly versus 4-weekly zoledronic acid for prolonged treatment of patients with bone metastases from breast cancer (the ZOOM trial) [95]. This trial demonstrated that the skeletal morbidity rate (SMR) was numerically very similar (but statistically non-inferior) in the group of patients who had their zoledronic acid treatment de-escalated to every 12 weeks, as opposed to maintaining it at every four weeks after at least one year of prior treatment, i.e., a less-intensive treatment regimen was equivalent to the standard regimen in women with breast cancer and bone metastases for the primary study endpoint.A recent systematic review, including the ZOOM data, confirmed that due to the heterogeneity of the current published trials, there is currently insufficient evidence to make wide-spread practice changes to less frequent dosing of bone-targeted agents [96]. Other trials addressing this topic, including TRIUMPH (a multicentre study assessing 12-weekly intravenous bisphosphonate therapy in women with low risk bone metastases from breast cancer using bone resorption markers) [97] and OPTIMIZE-2 (a study of zoledronic acid administered monthly versus every 3 months in multiple myeloma and breast cancer patients who were treated with zoledronic acid the prior year) [98], address de-escalation in patients already established on bisphosphonate therapy, while trials, like the Cancer and Leukemia Group B (CALGB) 70604 trial [99], address the de-escalation question in bisphosphonate naive patients.A common clinical question is whether or not to switch bone-targeted agents in patients with either disease progression or occurrence of an SRE, while on a bone-targeted therapy. Data from phase III trials addressing this question is currently lacking. To date, the only data that can answer the question of “switching” are small phase II trials, and definitive conclusions cannot be drawn from them [47,100,101,102]. Switching to a more potent bisphosphonate or from a bisphosphonate to denosumab after progression stands as an attractive alternative; however, such a strategy cannot be definitively recommended or refuted until a large adequately powered clinical trial with appropriate endpoints is completed to test this hypothesis. Our group is currently conducting a randomized, double-blind, placebo-controlled, phase III trial evaluating the palliative benefit of either continuing pamidronate or switching to second-line zoledronic acid in breast cancer patients with high risk bone metastases (the Odyssey Study) [103]. The primary objective of this study is to compare the proportion of high-risk metastatic breast cancer patients with bone metastases that will achieve a decrease in serum C-telopeptide (a surrogate for decreased risk of SREs) in the zoledronic and pamidronate treatment arms. Secondary objectives include determining the proportion of high-risk metastatic breast cancer patients that will achieve a significant improvement in palliative response and assessment of the overall pain control between the experimental group and the control group over the 12-week study period.Bone metastases in breast cancer are common and are associated with increased morbidity and poor prognosis. Directed therapy at minimizing such complications plays a pivotal role in the management of these patients. Over recent decades, increased understanding of the pathological processes involved in bone metastases behavior has led to effective and safe bone-targeted therapies becoming available for use in the palliative management of patients with breast cancer and bone metastases. Based on clinical trial data, at this time, both bisphosphonates and denosumab are commonly used. They have been shown to reduce the number, and delay the onset, of SREs. Both options are relatively safe, as well, and the choice of which one to pursue should be individualized on a patient to patient basis based on factors, such as; personal choice, availability of agent, cost, route of administration, compliance, co-morbidity and clinician preference. It should be noted, however, that neither the bisphosphonates nor denosumab have been shown to affect either progression-free or overall survival.Patients with bone only metastases from breast cancer may survive for many years, so long-term toxicities from bone-targeted therapies are important; especially since these are given in the palliative setting. The potential for toxicity should always be considered against the relative benefit of therapy and all efforts made to identify risk factors that predispose to toxicity and managing them appropriately prior to initiation of bone-modifying therapy.Ongoing studies on the treatment of bone metastases are focused on two areas: Firstly, studies designed to develop a better understanding of the underlying pathophysiology of bone metastasis so that new agents may be developed; and secondly, those utilizing our currently available therapies in a more patient-centred manner. As clinicians in an era where personalized medicine is becoming more widespread, it is time to move away from the thinking of a “one size fits all” approach and start individualizing our treatments to maximize benefit in our patients while simultaneously minimizing unnecessary toxicity. These findings should also hopefully translate into improved care for patients with other malignancies that commonly spread to bone [104]. Studies evaluating novel agents and treatment strategies are ongoing, and the results of these studies are awaited with great anticipation. Mark Clemons has received honoraria for talks (Amgen, Mississauga, ON, Canada; Novartis, Montreal, QC, Canada; AstraZeneca, Mississauga, ON, Canada) and advisory board membership (Amgen, Novartis). Brian Hutton has received honoraria for talks (Amgen, Mississauga, ON, Canada). Demetrios Simos, Christina L. Addison, Iryna Kuchuk and Sasha Mazzarello declare no conflicts of interest.

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+These authors contributed equally to this work.This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).Breast cancer cells preferentially metastasise to the skeleton, owing, in part, to the fertile environment provided by bone. Increased bone turnover releases growth factors that promote tumour cell growth. In turn, tumour cells release factors that stimulate further bone turnover, resulting in a vicious cycle of metastasis growth and bone destruction. The RANK-RANK ligand (RANKL) pathway plays a key role in this cycle, and inhibition of RANKL using the fully-human monoclonal antibody denosumab, has demonstrated efficacy in delaying skeletal complications associated with bone metastases in three phase 3 trials. Preclinical studies suggest that the RANKL pathway also plays a role in breast cancer tumourigenesis and migration to bone. In a subgroup analysis of the negative Adjuvant Zoledronic Acid to Reduce Recurrence (AZURE) trial, the bisphosphonate zoledronic acid showed potential for improving survival in patients who were postmenopausal; however, a prospective study in this patient population is required to validate this observation. Ongoing trials are examining whether adjuvant blockade of the RANKL pathway using denosumab can prevent disease recurrence in patients with high-risk breast cancer. These are building on analogous studies that have shown that denosumab improves bone metastasis-free survival in prostate cancer and suggested that it confers an overall survival benefit in non-small-cell lung cancer.Bone metastases are common in many types of solid tumours and occur in over 70% of individuals with advanced breast cancer [1]. They are associated with debilitating skeletal complications, commonly referred to as skeletal-related events (SREs) and comprising pathologic fracture, radiation to bone, surgery to bone or spinal cord compression [2]. As well as being associated with increased mortality [3,4], SREs can be associated with severe pain, impaired mobility and reduced quality of life [1,5]. Improvements in the management of breast cancer have led to increased survival times for patients with metastatic bone disease, but this means that the life-time risk of developing SREs has also increased.The high frequency of bone metastasis in breast cancer can be attributed in part to patterns of venous circulation, with blood draining from the breast to the spinal veins [2]. The distribution of bone metastases reflects this network of blood flow. Thus, most common metastatic site is the axial skeleton, in particular the spine, sternum and ribs [2,6,7]. Although patterns of venous circulation explain the distribution of metastases to a degree, autopsy studies indicate that, if metastatic sites were solely determined by blood flow, there should be fewer bone metastases than are observed in breast cancer [8].Tracking the migration and growth of tumour cells in a mouse model illustrated that the rate-limiting step in metastasis was not migration and extravasation into tissue, but was the ability of tumour cells to grow in the surrounding tissue [9]. This ability is determined by both the properties of the tumour cells themselves, and of the tissue they have migrated to. In 1889, Stephen Paget proposed the theory that bone provides an environment that is particularly conducive to breast cancer cell growth [10]. Extensive research has since demonstrated that it is the continuous turnover of bone that results in a fertile “soil” in which metastatic cells can “seed”. During bone resorption, the bone matrix releases a variety of growth factors that promote cell proliferation and survival [11,12]. The cells involved in resorption (osteoclasts) also produce angiogenic factors and matrix metalloproteinases that facilitate growth of new vasculature [13], an absolute requirement for tumour progression [8].The favourable environment provided by bone is enriched further through interactions between tumour cells and some of the cells within the bone (osteoclasts and osteoblasts) [12]. These interactions stimulate bone resorption, thereby increasing the availability of tumour-promoting factors, which in turn results in tumour cell proliferation. If left unchecked, these reciprocal interactions result in a self-perpetuating cycle of bone destruction and tumour growth. The RANK ligand (RANKL)/RANK pathway is a key driver of this “vicious cycle” (Figure 1).The “vicious cycle” of bone destruction in metastatic bone disease.RANKL is a member of the tumour necrosis factor family of cytokines. When it binds to its receptor, RANK, expressed on osteoclast precursors, it promotes their differentiation, function and survival [14]. When breast cancer cells grow they produce several factors, such as parathyroid hormone-related peptide [15], that stimulate production of RANKL by stromal and osteoblast-lineage cells [16]. This in turn promotes osteoclastogenesis, increasing bone resorption and resulting in the release of more growth factors and angiogenic mediators that further stimulate tumour cell growth and proliferation [11,12,14]. Animal studies using mouse models of breast cancer metastasis to bone have demonstrated that administration of recombinant osteoprotegerin (OPG), the RANKL decoy receptor, decreases the number of tumour-associated osteoclasts and reduces levels of bone resorption, thus confirming a role for RANKL in tumour-induced, osteoclast-mediated bone destruction [17,18].Denosumab, a fully-human monoclonal antibody that binds RANKL and neutralises its function, has demonstrated efficacy in reducing SREs in three phase 3 trials, including in patients with bone metastases associated with breast cancer, prostate cancer and other solid tumours or multiple myeloma [19,20,21,22], illustrating the part that the RANKL pathway plays in metastatic bone disease (Table 1). In these trials, denosumab was compared with the bisphosphonate zoledronic acid, the previous standard of care for patients with bone metastases. Denosumab significantly delayed the time to both first and subsequent SREs in patients with breast and prostate cancer compared with zoledronic acid, and was non-inferior in delaying SREs in patients with other solid tumours or multiple myeloma. Denosumab also significantly decreased levels of bone turnover in all three studies, as determined by suppression of two bone turnover markers: urine N-telopeptide corrected for creatinine (uNTx), and bone-specific alkaline phosphatase (BSAP) [19,20,21]. Pre-specified analysis of the results from the breast cancer trial found that denosumab significantly delayed progression to moderate or severe pain in patients with no or mild pain at baseline [23]. Individuals in the denosumab arm of the study were also more likely to have an improvement in their quality-of-life scores and less likely to have a decline in quality of life than those in the zoledronic acid arm [24].Results from a phase 3, randomised, double-blind study comparing denosumab with zoledronic acid for the prevention of skeletal-related events in patients with bone metastases associated with breast cancer [19].p Values for superiority were adjusted for multiplicity; CI, confidence interval; HR, hazard ratio; i.v., intravenous; NR, not reached; Q4W, every 4 weeks; RR, rate ratio; s.c., subcutaneous; SRE, skeletal-related event.Pre-clinical evidence suggests that the RANKL pathway not only functions in the establishment and growth of bone metastases, it also plays a role earlier in the breast cancer disease continuum [13]. RANKL and RANK are expressed in a number of cell types, including mammary gland epithelial cells [25]. While hormone-driven proliferation of mammary gland epithelial and stem cells can be partially explained by the autocrine effect that results from progesterone binding its receptor, the majority of proliferating cells are progesterone receptor-negative. This paracrine effect appears to be mediated by the RANKL pathway [26,27]. Moreover, murine studies have revealed a role for RANK and RANKL in hormone-driven mammary gland development during pregnancy (Figure 2) [28].Notably, both RANK and RANKL are also expressed in tumour and stromal cells from human breast cancer [29,30]. Two key studies in mouse models have demonstrated a potential role for the RANKL pathway in mediating progesterone-driven breast cancer. One study used a transgenic model in which RANK was deleted from mammary gland epithelial cells [31]. The other study engineered overexpression of RANK in a mouse model, and used pharmacological inhibition (the RANKL antagonist, RANK-Fc) to block the pathway [29]. Hormonal stimulation (using a synthetic progesterone derivative, medroxyprogesterone acetate (MPA)) markedly increased levels of RANKL in both the transgenic mice overexpressing RANK and the wild-type mice, and triggered epithelial cell proliferation [29,31]. Mice over-expressing RANK had a much higher incidence of mammary tumours following co-administration of MPA and a carcinogen (7,12-dimethylbenzanthracene (DMBA)) than wild-type mice. Blocking the pathway using RANK-Fc dramatically decreased the incidence of tumour formation in both types of mice (Figure 3) [29]. Furthermore, comparing mammary cell proliferation following RANK-Fc inhibition with proliferation following inhibition of the progesterone receptor found that the RANKL pathway was responsible for the majority of the proliferatory effect [29]. Therefore, similar to its role in mammary gland development (Figure 2), the RANKL pathway appears to be a key mediator of progesterone-driven cell proliferation in tumourigenesis.RANKL in mammary gland epithelial cell proliferation. Following the binding of progesterone to its receptor, RANKL is produced and acts in a paracrine fashion to stimulate mammary gland epithelial cell expansion. PR, progesterone receptor. Reprinted from [32].In contrast to the effects seen with overexpression of RANK, mice with mammary gland epithelial cell RANK gene deletion had decreased cell proliferation upon progesterone stimulation compared with wild-type mice. They also exhibited a marked delay in tumour formation and increased overall survival when stimulated with MPA and DMBA (Figure 4) [31]. The protective effect of RANK deletion occurred only if it was deleted from mammary gland epithelia: Deleting RANK from other cell types did not reduce mammary tumour formation. This pattern suggests an additional, cell-specific role of the RANKL pathway that is restricted to mammary gland epithelial cells. Furthermore, administration of zoledronic acid, which has been demonstrated to inhibit the functioning of osteoclasts through the mevalonate pathway by blocking post-translational modification of proteins necessary for their survival [33], had no effect on mammary tumour growth. This again suggests that the RANKL pathway involvement in mammary tumourigenesis is independent of its role in bone physiology [29].Blockade of RANK through pharmacological inhibition or genetic inactivation inhibits tumour formation in mice. Tumour growth following the administration of the carcinogen 7,12-dimethylbenzanthracene (DMBA) and the progesterone derivative medroxyprogesterone acetate (MPA), with and without concomitant treatment with the RANK inhibitor RANK-Fc, in (A) transgenic mice overexpressing RANK and (B) wild-type mice [29]. Reprinted from [29].RANK knock-out from mammary gland epithelia inhibits tumour formation. Tumour growth following the administration of the carcinogen DMBA and the progesterone derivative MPA in mice with inactivated mammary gland epithelial cell RANK expression (RANKΔmam) and in wild-type mice (control) [31]. Reprinted from [31].Results from in vitro and in vivo studies suggest that RANKL may also be involved in tumour cell migration to the skeleton. In vitro stimulation of breast cancer cells with RANKL resulted in actin polymerisation and cell migration, both pre-requisites for metastasis [30]. A mouse model of melanoma (which frequently metastasises to bone) demonstrated that treatment with a recombinant form of the RANKL decoy receptor, OPG, reduced tumour burden in bone and prevented development of paralysis [30]. Interestingly, treatment with zoledronic acid did not reduce tumour burden in bone [30], suggesting that RANKL mediates its chemotactic function through a mechanism independent of its effects on osteoclasts.In support of these roles of the RANKL pathway in tumour progression, analysis of RANK and OPG gene expression in patients with breast cancer found that low levels of RANK and high levels of OPG correlated with improved disease-free survival and overall survival, and that RANK expression positively correlated with the risk of developing bone metastases [34]. A study of RANK overexpression in breast cancer and mammary epithelial cells found that RANK induces de-differentiation of cells and epithelial-mesenchymal transition; changes that are associated with tumourigenesis, and invasion and metastasis, respectively [35].The potential anti-tumour effects of bone-targeted agents have been explored in several trials. Five large, open-label clinical trials, Austrian Breast and Colorectal Cancer Study Group (ABCSG-12), AZURE, immediate versus delayed administration of zoledronic acid in patients with early breast cancer receiving adjuvant letrozole (E-ZO-FAST), Zometa-Femara Adjuvant Synergy Trial (Z-FAST) and (ZO-FAST), have investigated the adjuvant use of zoledronic acid in patients with breast cancer, with mixed results (Table 2) [36,37,38,39]. In ABCSG-12, premenopausal patients receiving goserelin with tamoxifen or anastrozole were randomly assigned to either receive zoledronic acid 4 mg every 6 months, or to have no anti-resorptive therapy. Postmenopausal patients enrolled in E-ZO-FAST, Z-FAST and ZO-FAST were randomly assigned to either receive zoledronic acid upfront (4 mg every 6 months), or to have treatment with zoledronic acid delayed until fracture or a decrease in bone mineral density (BMD) [38]. In AZURE patients received standard therapy with or without zoledronic acid. The bisphosphonate was administered at 4 mg every 3–4 weeks for the first six doses, then every 3–6 months thereafter [37].Improvements in disease-free survival were achieved in the ABCSG-12 and ZO-FAST studies, and an improvement in overall survival was observed in the ABCSG-12 study [36,39]. Further analysis demonstrated, however, that in the ABCSG-12 study, this benefit was confined to patients aged over 40 years [36]. Exploratory analyses of the ZO-FAST results revealed a potential survival advantage for patients who were either over 60 years old or who had been postmenopausal for over 5 years (HR = 0.50; p = 0.022) [39]. Despite having the same study design as ZO-FAST, no improvements in disease-free survival were seen in either E-ZO-FAST or Z-FAST [38]. The recently updated Cochrane analysis by Wong et al. [40] further reviewed these data along with the outcomes from similar studies conducted with oral clodronate. Based on the results of their meta-analysis, the authors concluded that overall, bisphosphonates do not prevent overall disease recurrence (RR 0.97; 95% CI 0.81, 1.16; p = 0.75) or improve survival (RR 0.84; 95% CI 0.68, 1.04; p = 0.11) in early breast cancer when compared with no bisphosphonates. When compared with delayed bisphosphonate treatment, early bisphosphonate treatment did not prevent overall recurrence (RR 0.85; 95% CI 0.49, 1.50; p = 0.58) and was even associated with a trend towards reduced survival (RR 1.45; 95% CI 0.44, 4.70; p = 0.54).Results of five large, open-label clinical trials investigating the adjuvant use of zoledronic acid in patients with breast cancer.BMD, bone mineral density; CI, confidence interval; HR, hazard ratio; LS, lumbar spine; NR, not reported; NS, not significant; SOC, standard of care; vs., versus.AZURE included a heterogeneous group of both premenopausal and postmenopausal patients. In this study, which was discontinued by the data management committee on the grounds of futility, no improvements in disease-free survival or overall survival were seen in the overall study population [37]. However, in an unadjusted sub-group analysis of patients who had been postmenopausal for more than 5 years, improvements in both disease-free survival (HR = 0.75; p = 0.02) and overall survival (HR = 0.74; p = 0.04) were observed. No benefit was seen in any of the other patient subsets (premenopausal, perimenopausal or unknown menopausal status), and there was an adverse effect of zoledronic acid treatment on non-bone invasive disease-free survival in these patients (HR = 1.32). With respect to these findings, Wong et al. [40] noted that combining the data for the post-menopausal subgroups from the AZURE study and the artificially-induced menopausal women in the ABCSG-12 study had a significant relative risk reduction in overall recurrence versus control (RR 0.71; 95% CI 0.59, 0.85; no statistical heterogeneity p = 0.46). However, they cautioned against over interpretation given that oestrogen levels were not measured in these studies and thus such sensitivity analyses are only speculative.The apparent differences in treatment effect according to hormone status are not specific to zoledronic acid: A recent study suggested the bisphosphonate clodronate, which acts via a different pathway to zoledronic acid, may also improve disease-free survival in older patients with breast cancer [41]. It should be note, however, that another study of adjuvant clodronate in patients with primary breast cancer found it was associated with significantly worse overall survival, compared with a control group, although this study did include both premenopausal and postmenopausal patients [42]. The reasons for this possible hormone-specific effect on survival are not fully understood. Some hypotheses suggest oestrogen provides a protective effect on bone that reduces the potential for skeletal metastases, thereby reducing the potential benefit bone-targeted agents would provide. Oestrogen is known to prevent bone turnover; premenopausal women may therefore be less likely to benefit from agents that suppress bone resorption than postmenopausal women. Hormones also influence the cytokine milieu in the bone microenvironment, and so the environment in which the metastatic cells “seed” could potentially be quite different depending on the hormone status of patients [43]. Alternatively, oestrogen could be mediating a pro-tumour effect that counteracts the anti-tumour effect of bisphosphonates. For example, oestrogen can polarise the immune system to a tumour-permissive state by supporting M2 (tumour-promoting) macrophages and increasing cell resistance to cytotoxic natural killer cells [44]. Furthermore, some cytokines, such as transforming growth factor-beta (TGF)-β, are regulated by both hormonal pathways and by the mevalonate pathway, suggesting zoledronic acid and oestrogen may act antagonistically [43]. However, as clodronate does not act via the mevalonate pathway [33], this mechanism is unlikely to mediate the differences in treatment effect for this drug. Establishing whether hormone-dependent treatment effects are specific to all drugs that inhibit bone turnover, or whether the effects are bisphosphonate-specific, may help elucidate the specific pathways involved and improve patient selection. In addition to differences in hormone profile between younger and older women with breast cancer, a recent study examining gene expression found young women with breast cancer are more likely to have high RANKL expression than older women. Therefore, in pre-menopausal women with breast cancer, who are unlikely to benefit from adjuvant bisphosphonates, the RANKL pathway may be a potential therapeutic target [45].The preclinical evidence discussed earlier suggests that denosumab may have a role in preventing disease progression in breast cancer. Furthermore, clinical data from several studies support the pre-clinical evidence suggesting a role for RANKL in tumour progression in prostate cancer. In a phase 3 study of patients with high-risk, non-metastatic, castration-resistant prostate cancer, RANKL blockade using denosumab significantly improved bone metastasis-free survival by 4.2 months compared with placebo (HR = 0.85; p = 0.028), illustrating how manipulation of the RANKL pathway may affect disease progression [46]. A separate analysis of patients undergoing radical prostatectomy found that increased serum concentration of RANKL is an independent prognostic risk factor for biochemical disease recurrence [47]. In addition, a sub-group analysis of clinical data from the phase 3 trial that compared denosumab with zoledronic acid in patients with solid tumours or multiple myeloma found that treatment with denosumab was associated with improved overall survival versus treatment with zoledronic acid in patients with non-small cell lung cancer (NSCLC) (HR = 0.78; p = 0.01) [48]. These outcomes may be a RANKL pathway-specific effect: Results from the Zometa European Study (ZEUS), which compared standard therapy with and without zoledronic acid treatment in patients with high risk prostate cancer, suggest that zoledronic acid treatment does not improve either overall survival or the incidence of bone metastasis [49]. Furthermore, zoledronic acid does not appear to prolong overall or disease-free survival in patients with NSCLC [50].Building on the strong preclinical rationale and analogous studies in other tumour types, the ongoing D-CARE (denosumab as adjuvant treatment for women with high risk early breast cancer receiving neoadjuvant or adjuvant therapy) study is investigating whether adjuvant denosumab (120 mg subcutaneously monthly for the first 6 months and every 3 months thereafter) can prevent disease recurrence in patients with high-risk breast cancer [51,52]. This international, randomised, double-blind phase 3 study is evaluating bone metastasis-free survival, disease-free survival and overall survival in approximately 4500 women with stage II or III breast cancer at high risk of recurrence. The trial has recently completed recruitment of patients. The results of the ABCSG-18 trial will also be of interest; this trial is primarily investigating using a lower dose of denosumab (60 mg every 6 months) to reduce the rate of clinical fracture in patients with non-metastatic breast cancer, but will also report bone metastasis-free, disease-free and overall survival as secondary endpoints [53].There is little doubt that both denosumab and bisphosphonates can delay the occurrence of SREs in patients with bone metastases, thereby reducing pain and improving patients’ quality of life. However, the role of bone-targeted agents in the adjuvant setting is unclear. Data suggest that bisphosphonates may be beneficial in certain subsets of patients with breast cancer, but may cause harm in others. Further studies with sufficient power and prospectively defined endpoints are required to confirm the population that should be targeted and to determine the risk/benefit profile for treatment. Evidence from other tumour types indicates a potential role for denosumab in delaying progression to bone metastasis and possibly in improving overall survival. There is also a strong preclinical rationale for blocking the RANKL pathway at an earlier stage of breast cancer treatment to delay disease progression. Ongoing clinical trials will determine whether manipulating the RANKL pathway at an earlier stage in breast cancer will be a valuable therapeutic strategy.The authors thank Kim Allcott from Oxford PharmaGenesis™ Ltd. (Oxford, UK) who provided medical writing support. Funding for this support was provided by Amgen (Europe) GmbH, Zug, Switzerland.Ian Haynes is an employee of Amgen and holds stock.Roger von Moos has participated in advisory boards for Amgen, Bristol-Myers Squibb, Merck Sharp & Dohme, Novartis, Roche and Sanofi Aventis. He has received unrestricted research grants from Amgen and Roche and received speaker honoraria from Amgen, GlaxoSmithKline and Roche.

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+These authors contributed equally to this work.This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).Introduction: Cardiospheres (CS) are self-assembling clusters of cells that can be grown from cardiac tissue. They contain a heterogeneous cell population that includes cardiac progenitor cells (CPCs) and cardiac fibroblasts. CS and CPCs have been shown to improve cardiac function after myocardial infarction (MI) in experimental models and are now being studied in clinical trials. The effects of aging on the proliferative capacity of CS and CPCs, and the paracrine signaling between cell types, remain incompletely understood. Methods and Results: We compared the growth of CS from young and aging murine hearts at baseline and following MI. The number of CS from young and aging hearts was similar at baseline. However, after MI, young hearts had a dramatic increase in the number of CS that grew, but this proliferative response to MI was virtually abolished in the aging heart. Further, the proportion of cells within the CS that were CPCs (defined as Sca-1(stem cell antigen-1)+/CD45−) was significantly lower in aging hearts than young hearts. Thus the number of available CPCs after culture from aging hearts was substantially lower than from young hearts. Cardiac fibroblasts from aging hearts proliferated more slowly in culture than those from young hearts. We then investigated the interaction between aging cardiac fibroblasts and CPCs. We found no significant paracrine effects on proliferation between these cell types, suggesting the impaired proliferation is a cell-autonomous problem. Conclusions: Aging hearts generate fewer CPCs, and aging CPCs have significantly reduced proliferative potential following MI. Aging cardiac fibroblasts also have reduced proliferative capacity, but these appear to be cell-autonomous problems, not caused by paracrine signaling between cell types.Aging is associated with a higher incidence of myocardial infarction (MI) [1], and higher prevalence of heart failure in those that survive MI [2]. The cellular and molecular mechanisms that underlie the exaggerated left ventricular (LV) remodeling in aging patients remain incompletely described [3]. One potential mechanism that may contribute to post-infarction heart failure in the elderly is stem cell failure [4].We have previously grown cardiospheres (CS), self-assembling clusters of cells, from murine hearts [5,6]. CS contain multiple cell types including cardiac progenitor cells (CPCs) and cardiac fibroblasts [7]. It is thought the heterogeneous nature of the cells contained within CS recapitulates the in vivo stem cell niche, conferring a benefit upon CS over other cell types as a source for cell therapy to achieve myocardial regeneration [7]. Following MI, we have shown that the proliferation rate of CS dramatically increases, with the proportion of CPCs within the CS remaining constant, and that CPC derived from CS can reduce scar size and improve function in the infarcted heart [5]. However, what happens to the growth rate of CS in aging hearts, particularly after MI remains unknown. Recently, cells derived from CS have been used in early clinical trials [8]. Because MI in humans is a disease-state that is strongly associated with aging, it is paramount to understand the effects of aging and MI on CS and CPCs.Cardiac fibroblasts constitute a large proportion of the cells within the adult heart [9], are part of cultured cardiospheres [10], and are known to become dysfunctional with aging [11]. They therefore represent a potential cell of interest that may interact with and influence the behavior of CPCs. In this series of in vitro experiments, we demonstrate an age-related impairment of CS growth, a reduction in the number of CPCs, and we investigate whether this is due to interactions between aging cardiac fibroblasts and CPCs.All procedures were approved by the UCSF Institutional Animal Care and Use Committee.Sca-1+/CD45− cardiac progenitor cells were isolated from male C57BL/6 mice as we have described previously [5,6], based on the original methods used by Messina et al. [12]. For this series of experiments, young mice were 3 months of age and aging mice were 18 months old. The whole heart was removed from the mice and cut into 1–2 mm3 pieces. After being washed with Ca++ Mg++ free phosphate-buffered saline (PBS) and digested three times, 5 min each at 37 °C with 0.25% trypsin (Invitrogen, Carlsbad, CA, USA) and 0.1% collagenase D (Roche Diagnostics, Indianapolis, IN, USA), the tissue pieces were cultured as “explants” on fibronectin (Sigma, St. Louis, MO, USA) coated 6-well plates, 2 wells for each heart in Iscove’s modified Dulbecco’s Medium (IMDM) with 10% fetal bovine serum (FBS) and 0.1 mM β-mercaptoethanol at 37 °C with 5% CO2. A layer of fibroblast-like cells grew from explants, over which small, round phase-bright cells (CS-forming cells) appeared 2 to 4 weeks after initiating the culture. Once the fibroblast-like cells grew to 90% confluence determined visually, the cells surrounding the explants were harvested by two washes with PBS, one wash with 0.53 mmol/L EDTA and one wash with 0.05% trypsin (Invitrogen) at room temperature. The harvested cells were filtered by 70 mm cell strainer (BD Biosciences, San Jose, CA, USA), and then cultured at a density of 1 × 105 cells/mL in each well of 24-well plates coated with Poly-d-Lysine (BD Biosciences) in cardiosphere growth medium (CGM), which included 35% IMDM, 65% DMEM-F12, 3.5% FBS, 0.1 mM β-mercaptoethanol, 2% B27 (Invitrogen), 10 ng/mL epidermal growth factor (R & D systems), 20 ng/mL basic fibroblast growth factor (R & D systems), 40 nmol/L thrombin (R & D systems) and 4 nmol/L cardiotrophin (R & D systems). The number of CSs in each well was counted by visual inspection.CSs were dissociated into single cell suspension by Blendzyme 4 (5.6 u/mL) (Roche). The following phycoerythrin (PE) or allophycocyanin (APC) conjugated rat anti-mouse antibodies and conjugated isotype-matched control antibodies were used: Sca-1-PE, c-kit-PE, CD133-PE, CD34-PE, CD45-APC, Flk-1-APC and CD31-APC (eBioscience, San Diego, CA, USA). The cells were incubated with antibodies for 25 min on ice, washed with PBS containing 0.2% BSA, and analyzed by FACSCabilur with CellQuest software (BD Biosciences).MI was induced surgically by a permanent ligation of the left anterior descending (LAD) coronary artery as we have previously described [5,13]. Briefly, with the animal anesthetized and ventilated, permanent ligation of the LAD is made by a 7–0 suture in the anterior myocardium at 50% of the length of the heart from the anterior-inferior edge of the left atrium to the apex. The chest is then closed and the animal allowed to recover. One week after MI, hearts were harvested for CS culture.Cardiac fibroblasts were isolated from young and aging mice. Whole hearts were removed, placed in cold serum-free Dulbecco’s modified Eagle medium (DMEM; UCSF Cell Culture Facility, San Francisco, CA, USA), and perfused with 2 μg/mL collagenase II in Hank’s buffered salt solution. The left ventricle was dissected away, cut into small pieces, and incubated with the collagenase solution at 37 °C for 10 min. The supernatant was removed and the tissue was incubated with fresh collagenase again to improve yield. The cell suspension was passed through a 100 μm cell strainer to remove undigested tissue. The filtrate was centrifuged at low speed for 2 min to remove cardiomyocytes and then at a higher speed for 10 min to pellet smaller cells. The cells were plated with to allow fibroblasts to attach. After 1 h, medium was changed to remove non-adherent cells. The cells were cultured in DMEM supplemented with 10% fetal bovine serum. Medium was changed every two days.To assess the rate of proliferation of cardiac fibroblasts, 5000 fibroblasts were plated in each well of 24-well plates. 10,000 Sca-1+/CD45− CPCs, or control medium, were seeded in inserts within the wells of the plate. Cardiosphere growth medium was used.To assess the rate of proliferation of CPCs and the interaction with cardiac fibroblasts, CPCs were plated in the wells and cardiac fibroblasts in the inserts. Cardiac fibroblasts (15,000 cells/insert) were first plated in excess of CPCs (10,000 cells/well), and in the second experiment the CPCs (50,000 cells/well) were in higher numbers than cardiac fibroblasts (5000 cells/insert).For all co-culture experiments, cell proliferation was measured by incubating with MTS (Promega, Madison, WI, USA) for 3.5 h. Absorbance at 490 nm was recorded using a plate reader. Proliferation is measured in relative light units (RLU) and then normalized to the appropriate control for each experiment (i.e., the first column in the graph). To investigate the effects of aging on the resident cardiac progenitor pool following MI, whole hearts were removed from young and aging mice, and cut into small pieces as “cardiac explants”. A monolayer of fibroblast-like cells migrated out from the cardiac explants over several weeks in culture. From this monolayer, small, round, phase-bright cells (CS-forming cells) were seen to emerge. We observed that aging cardiac explants took longer time to form a confluent monolayer than young explants in culture (p < 0.001) (Figure 1). We also found that explants from infarcted hearts (1-week post-MI) grew faster than those from non-infarcted hearts in both young and aging mice (p < 0.001), however post-MI explants from aging hearts grew slower than those from young post-MI hearts (p < 0.001, Figure 1).We observed that the total number of cardiospheres (CSs) derived from each heart in young and aging mice at baseline were not significantly different (Figure 2). However, the number of CSs derived from injured hearts was much higher than from non-injured hearts in young mice (p < 0.004), but not in aging mice (p = ns). These results suggest that the number of cardiac progenitors resident within the heart is not significantly decreased at baseline with age; however the ability of CPCs to proliferate in response to acute injury is impaired in the aging heart. Explants from aging hearts grow more slowly. Explants from hearts of aging mice grow to confluence in vitro more slowly both at baseline and after MI. The aging hearts retain some ability to increase the explant outgrowth rate.Age limits the proliferative capacity of cardiac progenitors following MI. (A) The number of CSs from young and aging murine hearts at baseline was not significantly different. The number of CSs from injured young hearts (n = 10) was much more than those from non-injured young hearts (n = 5). Aging blunts the proliferative response seen after MI. The number of CS generated from non-injured (n = 16) or injured (n = 13) aging hearts is similar. Cardiospheres are seen as self-assembling clusters of cells; (B) Bar graph showing the percentage of Sca-1+/CD45− cardiac progenitor cells within CSs from young or aging hearts at baseline or 1-week post-MI by FACS (n = 5–8). Data in figure shown as mean ± SEM.We analyzed the cellular components within CS. Using fluorescence-activated cell sorting (FACS) of dissociated CS, we found that CPCs (Sca-1+/CD45− subpopulation) made up a large fraction of CS cells (~25%) in young mice, both at baseline and post-MI (Figure 2). However, the proportion of Sca-1+/CD45− CPCs in CSs from aging mice was significantly lower than those from young at baseline and 1-week post-MI (p < 0.001) (Figure 2). Cells expressing c-kit+, CD133+, CD34+, Flk1+ and CD31+ in CSs were rare (less than 2%) and were not significantly different in CSs from young and aging mice.To assess the reason for slower outgrowth from cardiac explants with age, we cultured aging cardiac fibroblasts and compared their growth rates compared to young cardiac fibroblasts. Aging cardiac fibroblasts proliferated more slowly than the young fibroblasts (p < 0.01; Figure 3). To simulate the growth of cardiospheres, where CPCs grow alongside cardiac fibroblasts, we co-cultured cardiac fibroblasts with CPCs. The rate of proliferation of cardiac fibroblasts was unaffected by the presence or absence of CPCs (Figure 3), indicating no paracrine signaling effect on fibroblast proliferation.Impaired proliferation of aging cardiac fibroblasts is independentof the presence of CPCs.In this experiment we plated fibroblasts in the well and the CPCs in the inserts. (A) There were more aging fibroblasts early, but the growth rate was slower thanthe young fibroblasts. This was unaffected by the presence of CPCs ((B), n = 4 pergroup). The CPCs in the insert proliferated to the same extent in the presence ofyoung and aging fibroblasts ((C), n = 4 per group).CPCs were plated in non-contact co-culture with cardiac fibroblasts to determine the effect of the fibroblasts on CPC proliferation. The number of viable CPCs approximately doubled from day 3 to day 5 in culture. At both time points, no differences in proliferation rate were detected when co-cultured with young or aging cardiac fibroblasts compared to cell-free medium (Figure 4). To confirm our data and exclude an effect of cell dose, we repeated the experiment using different cell density. In the first experiment, cardiac fibroblasts were in excess of CPCs, and in the second experiment the CPCs were in higher numbers than cardiac fibroblasts (Figure 4). In both cases, we found no paracrine effect from the co-cultured fibroblasts on the proliferation rate of CPCs.Our study has several important findings. First, we show an age-related decrease in the growth rate of CS, and a blunting of the proliferative capacity after MI. Second, we found that CPCs made up a much smaller proportion of CS with age. Third, we show that aging is associated with less proliferative capacity in cardiac fibroblasts. Fourth, we demonstrated a lack or paracrine interaction between cardiac fibroblasts and CPCs in the proliferation of either cell type. These results have important implications for clinical trials of autologous cell therapy in aging patients following MI.Cell therapy shows promise for ameliorating heart failure following MI. MI and heart failure become more prevalent with increasing age [14]. Disappointingly, however, the results of cell therapy in experimental MI are less robust in the aged compared to the results achieved in the young. The inferior results with aging have been attributed to the age of the cells delivered [15,16,17,18], but may also relate to the age of the recipient tissue [19]. A more in depth understanding of the mechanism by which cell therapy is less effective in aging remains elusive. One aspect of autologous CS or CPC therapy that may limit its uptake clinically is the ability to generate enough cells for therapy in the aging population. Our data demonstrate that aging significantly reduces the number of CPCs available for cell therapy. This does not appear to be a problem of just reduced CS growth from slower explant outgrowth, or impairment of cardiac fibroblast proliferation. The low percentage of CPCs in CS demonstrates that there is a defect in proliferation of the CPCs themselves. Much of the early experience with human CPCs has been from biopsies taken from transplanted hearts, which tend to be younger and healthier than many post-MI patients. It is enticing to speculate that more biopsy tissue and longer culture times may overcome this issue in aging patients, but this should be tested in future clinical studies in aging patients. It is noteworthy that we used 18-month-old mice as our aging group. We chose this age because we have previously demonstrated a detectable increase in fibrosis and reduction in cardiac function at this age [20]. These are not very elderly senescent mice, but more middle-aged, as C57/Bl6 live to approximately 30 months of age in captivity [21]. One could reasonably expect further reductions in proliferative capacity as the mice age further. Our data support the use of allogeneic CPCs from young donors for cell therapy aimed at myocardial regeneration. Proliferation of Sca-1+/CD45− cardiac progenitor cells at low (A) and high (B) seeding density. 1 × 104 CPCs were seeded into each well and 1.5 × 104 cardiac fibroblasts were seeded into each insert (A). There was no difference in CPC proliferation when cultured with young or aging fibroblasts compared to cell free medium (n = 4 per group). To exclude an effect of cell density, we repeated the experiment with 5 × 104 CPCs seeded into each well and 5 × 103 cardiac fibroblasts seeded into each insert (B). CPCs proliferated at the same rate regardless of the presence of young or aging fibroblasts (n = 4 per group). Fb = fibroblasts; CPC = cardiac progenitor cells.Aging has significant effects on post-infarction LV remodeling. We have extensively reviewed this topic [3]. Several differences of the aging heart are germane to the findings of this study. First, the aging heart has increased fibrosis at baseline [20], yet the cardiac fibroblasts derived from aging hearts have impaired proliferative capacity. Cieslik and colleagues studied fibroblasts from aging hearts [22] and found that, compared to those derived from young hearts, they had impaired response to transforming growth factor beta (TGFb) but had increased collagen production both at baseline and in response to insulin. Their demonstration of reduced responsiveness of aging cardiac fibroblasts to proliferative factors is congruent with our data, and is also in keeping with the literature regarding fibrosis in the aging heart [20]. Furthermore, TGFb is necessary for formation of CS [23]. One could speculate that reduced TGFb signaling could be a common cause for both the dysfunctional CS and the hypoproliferative fibroblasts in the aging heart, but this remains to be tested in future studies.Our results have implications beyond CS. The interactions, or lack thereof, have wider consequences for our understanding of CPCs residing in the stem cell niche in vivo. The cardiac stem cell niche consists of CPCs, cardiomyocytes, fibroblasts, and other supporting cells [24,25]. It is an intricate three-dimensional structure that provides a specific microenvironment for the maintenance of CPCs. When injury occurs, CPCs can be triggered to mobilize and differentiate into other cell types as needed. We show that there is no effect of the presence of fibroblasts on the proliferation of CPCs. This demonstrates that the defect in CPC proliferation with age is a cell-autonomous problem, rather than an environmental effect from the aging cells surrounding the CPCs. The ratio of CPCs to fibroblasts may influence the amount of secreted molecules, which in turn may affect the level of response. The adult murine heart is made up of about 25% fibroblasts, but CPCs are scarce [9]. Furthermore, the ratio is likely to be different within the stem cell niche. To address the issue of relative cell concentration, we performed our experiments with CPCs in excess and again with fibroblasts in excess, and found cell numbers had no effect. The repeated trials using different cell ratios and growth surfaces demonstrate that the observed result was not merely a dose-response or attachment problem. We recognize two potential limitations to this study; first, our in vitro experiments may create a niche that is different from the in vivo structure and composition of the cardiac stem cell niche. However, we believe it is important to start with single cell interactions before attempting to recapitulate the entire niche in vitro. Second, there may be important interactions between CPCs and fibroblasts that require direct cell-cell contact, such as signaling through gap junctions. This topic requires further study in direct cell-cell contact culture conditions.In conclusion, aging hearts generate fewer CPCs, and aging CPCs have significantly reduced proliferative potential following MI. Aging cardiac fibroblasts also have reduced proliferative capacity, but these appear to be cell-autonomous problems, not caused by paracrine signaling between cell types, as cardiac fibroblasts do not affect the proliferation of CPCs, and CPCs cannot rescue the impaired proliferation of aging fibroblasts.This study was funded by grants from the NIH K08HL090915 (to Andrew Boyle) and the Ellison Medical Foundation (to Andrew Boyle).The authors declare no conflict of interest.

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+This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).Haematopoietic stem cell (HSC) transplantation is an established cell-based therapy for a number of haematological diseases. To enhance this therapy, there is considerable interest in expanding HSCs in artificial niches prior to transplantation. This study compared murine HSC expansion supported through co-culture on monolayers of either undifferentiated mesenchymal stromal cells (MSCs) or osteoblasts. Sorted Lineage− Sca-1+ c-kit+ (LSK) haematopoietic stem/progenitor cells (HPC) demonstrated proliferative capacity on both stromal monolayers with the greatest expansion of LSK shown in cultures supported by osteoblast monolayers. After transplantation, both types of bulk-expanded cultures were capable of engrafting and repopulating lethally irradiated primary and secondary murine recipients. LSKs co-cultured on MSCs showed comparable, but not superior, reconstitution ability to that of freshly isolated LSKs. Surprisingly, however, osteoblast co-cultured LSKs showed significantly poorer haematopoietic reconstitution compared to LSKs co-cultured on MSCs, likely due to a delay in short-term reconstitution. We demonstrated that stromal monolayers can be used to maintain, but not expand, functional HSCs without a need for additional haematopoietic growth factors. We also demonstrated that despite apparently superior in vitro performance, co-injection of bulk cultures of osteoblasts and LSKs in vivo was detrimental to recipient survival and should be avoided in translation to clinical practice.Haematopoietic stem cell (HSC) transplantation is a curative treatment for a number of haematological malignancies, bone marrow aplasia, congenital haemoglobinopathies and immunodeficiencies [1,2]. Umbilical cord blood (UCB) transplantation is a promising alternative to bone marrow (BM) reconstitution for those who lack a human leucocyte antigen (HLA)-matched family member or a living unrelated donor [3]. Allogeneic UCB transplantation has been shown to elicit less frequent, and less severe, graft-versus-host disease (GVHD) than allogeneic marrow or mobilised peripheral blood stem cell transplantation [4,5,6,7,8,9,10]. However, the transplantation of UCB-derived HSCs is often associated with delayed engraftment, especially in adult recipients, mainly due to the small number of HSCs that can be recovered from a single cord. As HSC transplant outcomes are dose-dependent [11], it has been hypothesised that clinical outcomes could be significantly enhanced through ex vivo expansion of UCB-derived HSC prior to transplantation. Numerous studies describe on-going efforts to characterise the stromal support cell composition of the HSC BM niche [12,13,14,15,16,17,18,19,20]. There is mounting evidence that cells of the osteoblast lineage, namely osteoprogenitors or mesenchymal stromal cells (MSCs) likely play the most influential supportive roles [21,22,23] together with endothelial cells which have a critical role in HSC maintenance and proliferation in vascular HSC niches [24,25,26]. The first successful efforts to mimic this complex signal milieu, resulting only in transient in vitro HSC maintenance, were reported by Dexter and colleagues [27,28]. In these studies unselected populations of stromal and haematopoietic cells from whole BM were co-cultured. It is now well established that cell-cell contact between HSCs and BM niche stromal cells is essential for HSC regulation [29,30,31,32]. Therefore, like the studies by Dexter et al., co-culture of HSCs in direct contact with a supportive stromal cell population remains a commonly used in vitro model system and expansion platform. Recent Dexter-type co-cultures have utilised osteoblast-lineage cells, as supportive feeder layers for in vitro HSC maintenance and/or expansion [15,19,33,34]. Nakamura and colleagues [34] successfully co-cultured LSK Flt-3+ HSCs with fresh MSCs thought to be pre-osteoblasts on the basis of Sca-1 and Alcam-1 expression. Likewise, Zhu and colleagues [35] co-cultured Lin− Sca-1+ HSCs with osteoblasts differentiated as in this study, and released the HSCs with collagenase-trypsin treatment before transplantation. Similarly, a number of studies have shown the expansion of phenotypic HSCs when MSCs are used as feeder layers [36,37,38,39]. In an effort to elucidate the relative supportive capacity of undifferentiated MSCs vs. differentiated osteoblasts we used a murine system to directly compare the  in vitro expansion potential of a purified population of HSCs on undifferentiated MSCs or on osteoblast feeder layers.C57BL/6 mice (purchased from the Australian Animal Resource Centre) or inbred C57BL/6 transgenic for green fluorescent protein (GFP) under the control of the ubiquitin promoter (C57BL/6-GFP) were used. All animal experiments were approved by the University of Queensland Animal Ethics Committee.LSK and MSC populations were isolated from C57BL/6 or C57BL/6-GFP mice as previously described by our group [40]. All experiments involving MSCs were performed at passage 8–12. LSKs and MSCs were characterized by morphology, cell surface phenotype and functional capacity as previously published by our group [40]. MSCs were induced into the osteogenic lineage in vitro as follows: 2 × 104 MSCs were seeded in 24-well plates, grown to confluence and cultured for 21 days in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with dexamethasone (0.1 μM), β-glycerol phosphate (100 mM), l-ascorbate-2-phosphate (10 mM), calcium chloride (4 mM), 10% fetal calf serum (FCS) and gentamycin (40 μg/mL, Pfizer, New York, NY, USA). These were fixed in 4% paraformaldehyde (PFA) and stained for the presence of calcified osteoid deposits with Alizarin red S solution [40,41]. Undifferentiated MSCs and MSCs induced into osteoblasts were further characterised according to their gene expression of HSC niche markers including angiopoietin 1 and 2, stem cell factor, jagged-1 and stromal cell-derived factor 1 (CXCL12).Cell sorting and immunophenotype analysis was performed by flow cytometry using fluorochrome-labeled rat-anti mouse monoclonal antibodies (all at 1–2.5 μg/mL) as follows: c-kit allophycocyanin (APC; 2B8; BD, Franklin Lakes, NJ, USA), Sca-1 phycoerythrin cyanine-7 (PE Cy7; D7; BD), CD45 APC (30-F11; BD), CD31 PE (MEC13.3; BD), CD44 PE (IM7; BD), CD11b PE (M1/70; BD), F4/80 Pacific Blue (BM8; eBioscience, San Diego, CA, USA), Gr-1 APC Cy7 (RB6-8C5; BD), CD45R/B220 Pacific Blue (RA3-6B2; BD) and CD5 APC (53–7.3; BD). A biotinylated lineage cocktail (containing CD5 (53–7.3; BD), CD45R/B220 (RA3-6B2; BD), Gr-1 (RB6-8C5; BD) and F4/80 (BM8; eBioscience)) was also used for staining of haematopoietic cells with streptavidin Pacific Blue (Invitrogen, Carlsbad, CA, USA) secondary staining. Cell sorting was performed by fluorescence-activated cell sorting (FACS) on a FACS-ARIA (BD) and immunophenotyping was performed on an LSRII (BD) with results analysed in FlowJo Version 7.5 (Tree Star, Ashland, OR, USA).RNA from was extracted with a Qiagen RNeasy Mini Kit (Qiagen, Venlo, Netherlands) using the manufacturer’s protocol. RNA was pre-incubated with DNase I (Invitrogen) and reverse transcription was performed with oligo-dT and Superscript III (Invitrogen) as described in the manufacturer’s instructions. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed on cDNA using ABsolute™ QPCR SYBR® Green (ABgene, Waltham, MA, USA). Product size and primer sequences used are shown in Table 1. Primers used for characterisation of MSC and osteoblasts (Ob).MSCs were seeded into a 24-well plate and allowed to reach confluence. Osteoblasts were induced from MSC as described above and used for experiments after 2–3 weeks of differentiation. 1 × 103 purified LSKs (500 cells/cm2) were seeded on top of stromal cells in static cultures for 7 days in BioWhittaker X-vivo 10 medium (Lonza, Basel, Switzerland) supplemented with 20% FCS and gentamycin (40 μg/mL). After this period the resultant cell populations were removed from culture, counted and analysed by flow cytometry for lineage-specific haematopoietic markers and LSK markers. Female C57BL/6 recipient mice were administered a split dose of total body irradiation (2 × 550 cGy) 3 h apart via a self-contained Gammacell 40 Exactor (GC-40E) irradiator using a 137caesium radiation source. Twenty-four hours after irradiation, recipients were intravenously injected via the retro-orbital sinus with the appropriate cell populations resuspended in saline supplemented with 2% heat-inactivated FCS and DNase I (10 μg/mL, Roche, Basel, Switzerland). All mice were monitored by weight and clinical scoring over the first 28 days of the experiments and drinking water was supplemented with antibiotics and antifungal agents for the first 14 days. For analysis of functionality of the initial purified LSK population, BM cells and LSKs were isolated as above from C57BL/6-GFP mice. The c-kit depleted cells were sourced from the negative fraction of c-kit magnetic separation (cells devoid of HSCs) and the vehicle group received cell excipient only. For competitive repopulation transplants, 200 GFP+ LSKs or the equivalent number of cells produced in culture from 200 LSKs, were used as the donor population together with 4 × 105 whole bone marrow (BM) cells from C57BL/6 as the competitor cells. Where performed, MSCs and osteoblasts were physically removed from harvested co-cultures prior to transplantation using flow sorting for GFP+ haematopoietic cells. For secondary transplants, BM was harvested from primary recipients, pooled and 2 × 106 cells transplanted into lethally irradiated secondary recipients. All treatment groups were monitored for health and well-being by daily scoring. Tail bleeding was performed 4, 6, 8, 10 and 12 weeks post-transplant and flow cytometry analysis of GFP was used to detect the level of donor (GFP+) and competitive (GFP−) total cells, myeloid cells (CDllb+) and B cells (CD45R/B220+) in the recipient blood. T cell engraftment was not examined because of the known ability of recipient T cells to survive high-dose total body irradiation. Donor populations were deemed to be engrafted if greater than 1% GFP+ cells were observed in blood or BM. Blood, BM and spleen counts were performed on a Sysmex KX-21N Cell Counter (Sysmex Corporation, Kobe, Japan).Snap frozen tissue was crushed into a powder using a pestle and mortar on dry ice and digested by overnight incubation with Proteinase K at 56 °C. The genomic DNA was then extracted using a QIAamp DNA Mini Kit (Qiagen) according to the manufacturer’s protocol (RNA was removed by RNase treatment). The genomic DNA was assessed for the presence of genomic GFP using the primers and fluorescent probe as follows: 5′end: CTGCTGCCCGACAACCA. 3′end: TGTGATCGCGCTTCTCGTT fluorescent probe 5′end: CCCAGTCCGCCCTGAGCAAAGAC. The number of GFP+ cells was then calculated using a standard curve of the amount of GFP genomic DNA from known cell numbers.Either a Mann-Whitney test or Kruskal-Wallis test with Dunn’s Multiple Comparison post-test was used to test statistical significance between groups. This was performed using GraphPad Prism version 4.03 for Windows (GraphPad Software, La Jolla, CA, USA). Results were deemed statistically different if p < 0.01. All continuous data were expressed as mean ± 1 standard deviation (SD).LSK and MSC populations were characterised as per Cook et al. (2012) [40]. Briefly, LSKs were shown to be enriched for a true repopulating population of HSC by transplantation of 200 LSKs into lethally myeloblated recipients and achieving 100% survival for >16 weeks (data not shown). MSCs had a classical fibroblast morphology and were Sca-1+, CD44+, CD45−, CD11b− and CD31−. MSC were also shown to be able to differentiate into osteoblasts, adipocytes and chondrocytes [40,41] (data not shown).The ability of MSCs to differentiate into osteoblasts was confirmed by qRT-PCR for lineage-specific markers. It was shown that MSC-derived osteoblasts expressed higher levels of markers for early osteogenic development, namely RUNX2 and osterix, when compared to undifferentiated MSCs (Figure 1A,B). Furthermore, MSC-derived osteoblasts expressed higher levels of osteocalcin (also known as bone gamma-carboxyglutamate protein), a marker of mature osteoblasts (Figure 1C) [12]. Comparison of mesenchymal stromal cell (MSC) and osteoblast monolayers for bone-related and haematopoietic stem cell (HSC) niche markers. Differentiation into osteoblasts was shown to increase the expression of (A) RUNX2, (B) osterix and (C) osteocalcin when compared to undifferentiated MSCs. MSCs or MSC-derived osteoblasts were examined for expression of HSC niche genes including (D) angiopoietin-1, (E) angiopoietin-2, (F) stem cell factor, (G) jagged-1 and (H) stromal cell-derived factor-1 (CXCL12). Data is shown as mean ± SD with each point representing independent MSC and MSC-derived osteoblast cultures from distinct mice.MSCs and osteoblasts were also assessed by qRT-PCR for their expression of HSC niche markers as an indication of their potential to support HSC growth and proliferation in vitro. A panel of niche-specific markers was selected which included angiopoietin-1, angiopoietin-2, stem cell factor, jagged-1 and SDF-1. All assayed genes were detected in both MSC and osteoblast populations; however, significantly higher expression of all genes was observed in osteoblasts (Figure 1D–H). This suggested that both populations had the propensity to support HSC growth in vitro but that osteoblasts might be a superior candidate for this. For co-culture of LSKs on MSCs or osteoblasts monolayers, only serum was used: No haematopoietic growth factors or any other proteins were added. Co-cultures were optimised for haematopoietic cell expansion with X-vivo-10 medium supplemented with 20% FCS shown to be optimal for proliferation. 1000 LSKs were seeded on confluent MSC or osteoblasts monolayers. Haematopoietic cell expansion occurred on both undifferentiated MSCs and on osteoblasts (Figure 2A,B). After 5 days, cobblestone-like areas of adherent haematopoietic cells were observed (Figure 2A). After 7 days, over 95% of haematopoietic cells were found to be adherent to the stromal layer (Figure 2B). A mean 788-fold increase (±291-fold) in total haematopoietic cells on MSC monolayers was observed with an approximate two-fold higher total expansion (1513 ± 449-fold) when LSKs were co-cultured with osteoblasts compared to undifferentiated MSCs (p = 0.0013) (Figure 2C). This equated to a mean doubling time for haematopoietic cells on MSCs and osteoblasts of 17.5 h and 15.9 h respectively. The expansion appeared to be MSC- or osteoblast-specific, since LSKs co-cultured in the presence of adipocyte-differentiated MSCs showed no expansion of LSKs (data not shown), suggesting that marrow adipocytes are negative regulators of HSC expansion and/or survival, and confirming the findings of two previous studies [42,43]. Similar proportions of LSK cells were found to be present whether undifferentiated MSCs or osteoblasts were used (Figure 2D). However, as a result of the higher total cell expansion on osteoblasts, an increased yield of LSK was obtained in osteoblast/LSK co-cultures (p = 0.003) (Figure 2E). When characterising these expanded cells it was found that a majority of the expanded haematopoietic cells were lineage-committed (Figure 2F). The degree and type of haematopoietic lineage commitment was similar regardless of whether the supporting monolayer was of undifferentiated MSCs or osteoblasts, with a majority of the lineage-committed cells being of the myeloid lineage (Figure 2F).Haematopoietic cell expansion on stromal cell monolayers. (A) Co-cultures of HSCs with undifferentiated MSCs or osteoblasts after 0, 5 and 7 days by light microscopy; (B) Haematopoietic cells adhered to the stromal layer after the culture was washed (n = 8); (C) Total cell fold expansion after 7 days from the original 1000 LSKs was higher on osteoblast than on undifferentiated MSC monolayers (p = 0.0013; n = 8); (D) Flow cytometry of cultures revealed no differences in LSK proportion on either monolayer (n = 8); (E) Expansion of LSK HSCs, as determined by the product of total expansion and LSK proportion per culture, was significantly higher on osteoblasts compared to undifferentiated MSCs (p = 0.003; n = 8); (F) Myeloid, B cell, T cell and erythroid lineage commitment was similar when cultured on either stromal monolayer (n = 3). Data is shown as mean ± SD with each point representing independent co-cultures with from individually isolated LSK, MSC and MSC-derived osteoblast cultures from distinct mice.CD marker profile/phenotype is not always indicative of cell function and thus flow cytometry data cannot be used to identify functional HSC numbers (i.e., those with the ability to repopulate the BM). As such, an in vivo competitive repopulation model was used to compare the functional capacity of 200 LSK expanded on either undifferentiated MSCs or osteoblast monolayers to that of 200 freshly isolated LSKs. At 12 weeks after transplantation we found that LSKs co-cultured for 7 days on MSCs or osteoblasts contained functional HSCs after primary transplantation into myeloablated recipient mice (Figure 3B–D). There was no significant difference between the co-cultured cells and the freshly isolated LSK cells in these experiments (Figure 3B–D), indicating that functional HSCs had been maintained in co-cultures with MSC and osteoblasts despite the absence of exogenous cytokines. As an additional test for the functional capacity of putative HSCs, BM cells were taken from animals that had received a primary transplant of co-cultured LSKs and were transplanted into myeloablated syngeneic secondary hosts. At 12 weeks after total body irradiation and cell infusion mean multi-lineage haematopoietic reconstitution was similar to that following primary transplantation (Figure 3F–H). Multi-lineage reconstitution was again evident for total haematopoietic cells, B cells and myeloid cells (Figure 3F–H) at a similar level to the fresh LSK recipient controls. This reinforced the results from the primary transplants indicating that HSC were maintained during culture with MSC and osteoblast monolayers. However, HSC were not expanded, as engraftment was not increased. The fact that reconstitution in myeloid and B lineages was equivalent proves that these cultured LSK cells were capable of long-term multi-lineage reconstitution. If a bias in either myeloid or B lineage had been observed, the conclusion would be that HSC clonal subtypes with different differentiation potentials (such as α-HSC, β-HSC, γ-HSC and δ-HSC as defined by Benz et al., 2012 [44]) were differentially selected on MSC or osteoblasts. This was not the case in the transplants shown. Therefore, co-cultures on MSC or osteoblasts are likely to maintain all HSC regardless of their clonal subtype. Primary and secondary transplantation of expanded LSKs into lethally myeloablated mice. Competitive repopulation transplant assays were performed in which the donor GFP+ co-cultured cells were co-transplanted with GFP− competitor whole BM and compared to freshly isolated LSK transplants. (A) Primary transplantation of co-cultured cells at 4 weeks showed decreased survival in the osteoblast co-culture group (p = 0.0062; n = 12 pooled from 3 independent experiments). At 12 weeks post-transplant the proportion of GFP+ (B) total haematopoietic cells, (C) B cells and (D) myeloid cells was comparable in each group (n = 6 per group). (E) Secondary transplantation of pooled whole BM from primary recipients also showed markedly poorer survival at 4 weeks post-transplant in recipients of marrow in which the primary recipients had received LSKs co-cultured with osteoblasts compared to those receiving fresh LSKs or marrow from primary recipients that had received marrow co-cultured with undifferentiated MSCs. At 12 weeks post-transplant the proportion of GFP+ (F) total haematopoietic cells, (G) B cells and (H) myeloid cells was similar between recipients of fresh LSKs and secondary recipients of marrow in which the primary recipients had received marrow co-cultured with undifferentiated MSCs. Engraftment of marrow in which the primary recipients had received LSKs co-cultured with osteoblasts was not possible to assess since only 1 such mouse survived to this time point (n = 4 per group). (Mean ± SD).Surprisingly, mice receiving LSKs co-cultured on osteoblasts, while showing haematopoietic engraftment at similar levels to that of freshly isolated LSKs, displayed significantly poorer survival than those receiving LSKs co-cultured on MSC (p = 0.0062) (Figure 3A) due to haematopoietic failure between days 6–10 following transplantation. This was despite all mice receiving 4 × 105 healthy competing BM cells, which theoretically contained 4–6 long-term repopulating HSCs (enough to ensure haematopoietic reconstitution and survival). Thus, we concluded that transplantation of LSK-osteoblast co-cultures compromised survival of recipients of the primary transplants and that this in turn compromised survival of recipients of the secondary transplants (Figure 3A,E respectively). This suggested that the self-renewal capacity of normal (GFP−) HSCs in the survivors of the primary transplants was compromised. We hypothesised that co-culture of LSKs on osteoblasts compromised HSC homing and engraftment. One possibility to explain this was that residual osteoblasts, harvested from the co-cultures, were trapped in the lungs and prevented HSCs accompanying them from reaching sites of haematopoiesis including the bone marrow and spleen. An alternative possibility was that the normal homing mechanisms of HSC was in some way impaired by their co-culture and administration with osteoblasts. Using MSCs from mice transgenic for luciferase, we have previously shown that intravenously injected MSCs are detectable in the lungs within minutes of injection [45]. Thus, we hypothesised that large, matrix-secreting osteoblasts bind HSCs tightly prior to injection and that theses complexes becomes lodged in the small capillaries of the lungs, thus preventing optimal homing of both expanded and competitor-repopulating HSCs to sites of haematopoiesis. To explore this hypothesis, GFP+ osteoblasts or GFP+ MSCs were injected intravenously into lethally irradiated C57BL/6 recipients with unlabelled co-cultured haematopoietic cells and the lungs harvested at 1 day, 3 days or 12 days post-injection. GFP+ MSCs and GFP+ osteoblasts were detected by qRT-PCR for genomic GFP in harvested lung tissue at 1 and 3 days after injection, but were not detectable at 12 days after injection (Figure 4A). These findings are very similar to those using luciferase transgenic MSCs [45]. We next investigated whether MSC and osteoblast entrapment in the lungs was preventing HSC migration to the marrow and spleen. This was performed in a similar manner to the primary transplants (Section 3.3) by transplanting freshly isolated GFP+ LSKs or GFP+ LSKs co-cultured with unlabelled MSCs or osteoblasts together with whole BM competing cells. Assessment of leucocyte counts in the blood, BM and spleen was performed at the typical time of death in the osteoblast co-cultured transplant group (day 8–14 post-transplant). This revealed no observable differences between these groups in cellular reconstitution of blood or BM at day 8 post-transplant (Figure 4B,C). However, at day 8 post-transplant significantly fewer leucocytes were found in the spleens of recipients that had received HSCs co-cultured with osteoblasts compared to recipients of fresh LSKs (p < 0.001) and LSK/MSC co-culture recipients (p < 0.01) (Figure 4D). Since this was a competitive transplant experiment, it appeared that osteoblasts from the co-culture prevented both co-cultured HSCs and competitive unmanipulated HSCs from reaching the spleen and proliferating there. Thus, we concluded that the cause of death in the osteoblast/LSK co-cultured recipients was due to an impairment of short-term reconstitution, particularly in the spleen, rendering recipients susceptible to pancytopenia and subsequent infection. We next explored the question of homing to sites of haematopoiesis (marrow and spleen) of these different populations.Early post-transplant migratory studies of MSC, osteoblasts and cultured LSKs. (A) GFP+ MSCs and osteoblasts were detected in the lungs by qPCR for GFP on days 1 and 3 but not on day 12 after primary transplantation (n = 4 per time-point). Co-cultured donor GFP+ leucocytes were detected in the (B) blood, (C) BM and (D) spleen at eight days post-transplant (n = 4 per group). Leucocyte counts in the spleen 8 days after primary transplantation showed a significant decrease in splenic leucocytes in the osteoblast co-culture group (p = 0.0084) compared to recipients of fresh LSK or recipients of marrow co-cultured with undifferentiated MSCs (n = 10 per experimental group pooled from 3 independent experiments; healthy controls n = 5–6). At 16 h postransplant, significantly less donor GFP+ haematopoietic cell homing was detected in recipients of osteoblast co-cultured LSKs in the (E) spleen and the (F) BM compared to recipients of fresh LSKs and MSC co-cultured LSKs (* p < 0.05; data presented from 1 transplant with 4 recipients per group). Data represent mean ± SD.The above experiments did not show whether the impairment of short-term splenic reconstitution was due to compromised splenic proliferation of haematopoiesis or compromised homing to the spleen. However, since this was a competitive transplant model (i.e., recipients were co-transplanted with 4 × 105 unmanipulated BM cells—A dose that normally allows short- and long-term repopulation), we hypothesised that this was due to compromised homing of both transplanted populations. To explore this, we assessed the homing of fresh GFP+ HSCs, LSKs co-cultured with MSCs and LSKs co-cultured with osteoblasts to the spleen at 16 h post-transplant, a time point at which detectable proliferation was unlikely. This revealed that the transplantation of LSK/osteoblasts co-cultured cells led to significantly reduced homing to the spleen (p = 0.0245) when compared to fresh LSK and LSK-MSC co-culture recipients (Figure 4E). Similar findings were made for BM homing (Figure 4F, p = 0.0154). In conjunction with the low splenic leucocyte counts, this implied that the transplanted competitive BM cells with repopulation ability cultured on osteoblasts did not reach the spleen early post-transplant. This led to complete failure of homing and engraftment of HSC in 50% of the animals and death within 10 days of transplant (Figure 3A). The other 50% of recipients survived for the duration of the study and showed a similar BM reconstitution to that of fresh LSK and MSC-cultured LSK (Figure 3B–D). Hence, we propose that the co-transplantation of osteoblasts significantly inhibits and/or prevents migration of cells with repopulation ability to both the spleen and BM. In order to attempt to confirm this, we then removed osteoblasts from the co-culture prior to injection of the co-cultured LSK.Seven day co-cultures of GFP+ LSKs with unlabelled MSCs or with osteoblasts were initiated as before. The total resulting cells were harvested and the GFP+ haematopoietic cells were isolated by FACS sorting. These cells were then transplanted with whole BM competitors as before. Transplants depleted of osteoblasts now showed 100% survival at 30 days postransplant—The same result as with fresh LSKs and LSKs cultured with undifferentiated MSCs (Figure 5A). These findings showed that removal of osteoblasts from cell suspensions prior to infusion allowed short-term reconstitution. However, removal of MSCs or osteoblasts before transplantation paradoxically compromised the engraftment of the co-culture-expanded donor GFP+ haematopoietic cells (Figure 5B–D). Only a single mouse showed donor GFP+ myeloid engraftment in the MSC-cultured LSK transplanted group (Figure 5D). This suggested that HSC were lost during the sorting process. Since the cells were mono-dispersed prior to sorting, this loss was likely due to shear damage in the sorting process. Alternatively, the removal of osteoblasts may have allowed the “released” LSKs to differentiate more and thus lose their self-renewal potential.In this study we show that purified murine LSKs, a population enriched for HSCs, can be co-cultured with both undifferentiated MSCs or osteoblasts differentiated from MSCs, without the addition of exogenous growth factors and that this can maintain haematopoietic cells which can subsequently contribute to long-term in vivo haematopoiesis in myeloablated recipient mice after both primary and secondary transplantation. However, while both cell types were shown to maintain the number of engrafting HSCs, neither could expand them as demonstrated by in vivo competition repopulating assays. Cells of the osteoblast lineage have previously being shown to be key regulators of the HSC niche [18,20] and thus would appear a good candidate to support ex vivo HSC growth for clinical transplantation purposes, particularly in settings in which timely haematopoietic engraftment is problematic such as after cord blood transplantation in adults [3]. Recently, endothelial cells have been shown to be essential to HSC maintenance and proliferation within the BM niche [24,25,26]. However, the co-cultures in this paper did not include endothelial cells and this could provide an explanation as to why HSCs could only be maintained rather than expanded in this system.Primary transplantation of co-culture LSKs after removal of stromal cells. Co-cultures were initiated with GFP+ LSKs and unlabelled MSCs or osteoblasts. After harvest of cultures, flow cytometry sorting was performed and only cells expressing GFP, containing cultured haematopoietic cells but devoid of undifferentiated MSCs or osteoblasts, were selected. These were then transplanted into myeloablated primary recipients. (A) Four week survival was 100% in each group. However, the proportion of GFP+ LSKs contributing to total haematopoietic cells in the (B) blood, (C) B cells and (D) myeloid cells was significantly less compared to that demonstrated by fresh LSKs. (n = 4 per group; data represent mean ± SD). When comparing co-culture of an undifferentiated MSC monolayer to that of a monolayer of osteoblasts derived from undifferentiated MSCs, osteoblast co-cultures led to a significantly greater increase in cells with the LSK phenotype (over 40-fold from their original starting number). Conversely, however, when LSKs were seeded into cultures with adipocytes derived from MSCs, no growth was observed. This is supported by recent data suggesting that adipocytes have a negative influence on HSC growth [42,43], although a third report demonstrated the opposite effect [46]. Haematopoietic cells adhered to both MSCs and osteoblasts in co-culture, consistent with cobblestone area-forming cells (CAFC) and long-term culture-initiating cell (LTC-IC) assays that have been developed as surrogate in vitro assays in attempts to determine true HSC qualities. Moreover, it has also been shown that the more primitive HSC/HPC populations (as identified through LTC-IC assays) have a higher affinity for stromal monolayers than their committed progeny [29,30,31].In this study we have described for the first time the paradoxical effect of higher in vitro numbers of haematopoietic cells co-cultured with osteoblasts but poorer in vivo survival in competitive serial haematopoietic cell transplantation assays with such LSKs compared to those co-cultured on undifferentiated MSCs. This appeared to be due to poorer homing and, presumably, subsequent proliferation, of the osteoblast co-cultured LSKs on reaching sites of haematopoiesis after intravenous injection into the myeloablated hosts.No significant difference in phenotype was observed with either MSC or osteoblast co-cultured HSCs after a seven-day expansion period. Consistent with previous studies, the majority of the resulting cells appeared committed to the myeloid lineage (Gr-1+ or F4/80+). Very low proportions of lymphoid and erythroid cells were found. These data support the potential of such a system for granulocyte expansion as prevention or treatment of neutropenia.We demonstrated the functional capacity of LSKs co-cultured on undifferentiated MSCs or osteoblasts using in an in vivo competitive repopulation transplant model. We showed that overall engraftment capacity in primary recipients of LSK co-cultured on undifferentiated MSCs was equivalent to that of freshly isolated LSKs both for multi-lineage reconstitution and for LSK engraftment. Although there was an increase in LSK cell number by phenotype (i.e., by flow cytometric analysis) within LSK-MSC co-cultures, the numbers of HSC with true engraftment and repopulation potential were maintained rather than expanded. This effect was further verified in serial transplants where donor cells from primary recipients of LSKs co-cultured on undifferentiated MSCs were able to repopulate secondary hosts at a similar efficiency to that of freshly isolated LSKs. In contrast, however, recipients of LSK co-cultured on osteoblasts displayed significantly lower survival rates after primary in vivo transplantation. This result was both surprising and perplexing as the model used to test the functionality of the cultured cells was a competitive transplant model, meaning that hosts received both manipulated HSCs from the expansion cultures and unmanipulated, freshly isolated HSCs from whole BM. Thus, even if functional HSCs were not maintained in LSK/osteoblast co-cultures, hosts receiving these transplants should still be have been repopulated by the whole BM competing cells. Furthermore, when engraftment in the primary recipients did occur, self-renewal capacity remained compromised as demonstrated by the significantly lower survival observed after secondary transplantation. To explain this, we hypothesised that osteoblasts transplanted concurrently with the donor and competitor haematopoietic populations, adversely influenced the fate of their co-transplanted HSCs. Therefore, we investigated whether a factor present in the LSK/osteoblast co-cultures was either directly causing death, or indirectly preventing both co-cultured HSCs and competing whole BM from homing to, and engrafting in, the marrow and spleen. We further hypothesised that osteoblasts, after intravenous infusion, got trapped in the endothelial capillaries of the lungs, causing pulmonary embolism and subsequent pulmonary ossification post-transplant. We have previously shown that undifferentiated MSCs are trapped in the lungs after intravenous infusion [45]. This was also the case in this study (using stromal cells transgenic for GFP rather than MSCs from luciferase-transgenic mice [45]), in which it was found that both undifferentiated MSCs and osteoblasts were trapped in the lungs for up to 3 days post-transplant. However, these cells not detected in the lungs at 12 days post-transplant, a typical time of death in these recipients. It thus seemed unlikely that osteoblasts were causing significant pulmonary pathology after their intravenous injection. We considered whether transient osteoblast entrapment in the lungs was compromising long-term engraftment ability of the co-injected LSKs. However, this appeared unlikely as more undifferentiated MSCs than osteoblasts were found in the lungs without engraftment being compromised. As indicated above, we noted that the time of death was consistent with that caused by haematopoietic failure in lethally myeloablated recipients (~days 8–14 post-irradiation). Leucocyte counts in the blood, femoral BM and spleen were analysed at day 8 post-transplant. Very few leucocytes were found in the blood or BM, as is typical after myeloablation and there was no significant difference between the different co-cultured groups. However, recipients of osteoblast co-cultured LSKs had significantly fewer leucocytes in the spleen and significantly lower donor cell engraftment in the BM and spleen. Thus, the cause of mortality in these animals was likely due to delayed short-term reconstitution in the spleen with subsequent anaemia and/or infection due to pancytopenia. The reason for this haematopoietic failure was next explored: Donor cell homing to the spleen and BM was investigated at 16 h post-transplant. This showed that significantly lower numbers of donor cells from LSK/osteoblast co-cultures homed to the spleen and BM and that virtually all engraftment was from the competitive whole BM population in these organs. In secondary transplants, these recipients still demonstrated 50% mortality, indicating that co-transplantation of osteoblasts significantly inhibited the proliferation of all cells with repopulation ability, from both the donor and competitor populations, in the spleen and BM.Finally, we showed that transplantation of culture suspensions devoid of stromal cells (physically removed prior to transplant) prevented the mortality observed in recipients of osteoblast co-cultured LSKs. However, while the competitor HSCs successfully engrafted in this case, the repopulation ability of the co-cultured LSKs was compromised and almost all engraftment was from the competing whole BM populations. This indicated that removal of stromal cells from culture suspensions also removed the HSCs with long-term repopulating characteristics. Thus, we have identified a confounding feature that may exist in culture systems that contain osteoblasts and this emphasises the importance of pre-clinical HSC transplant models to identify such limitations.Overall, this co-culture assay has enabled comparison of undifferentiated MSCs and osteoblasts to support HSCs in culture. The system will be useful to further identify constituents that may be used to enhance ex vivo expansion of HSC, and to translate it to the human setting using CD34+ cord blood HSCs co-cultured with human MSCs or their differentiated progeny.We would like to thank Robert Wadley for his assistance with flow cytometry and Mater Research Institute, University of Queensland Scientific Services for laboratory maintenance. Matthew M. Cook was supported by an ANZ Trustees (Postgraduate Research Scholarship for Medical Research in Queensland). Jean-Pierre Levesque and Ingrid G. Winkler are supported by a Senior Research Fellowship (#1044091) and a Career Development Fellowship (#1033736) from the National Health and Medical Research Council of Australia. The project was funded by the Mater Research Institute, University of Queensland.The authors declare no conflict of interest. 

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+This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).Tumor metastasis can occur years after an apparent cure due to a phenomenon known as metastatic tumor dormancy; in which tumor masses or individual tumor cells are growth restricted for extended periods of time. This period of dormancy is induced and maintained by several mechanisms, including: (1) Tumor microenvironment factors such as cytokine expression, immunosurveillance and angiogenesis; (2) Metastasis suppressor gene activity; and (3) Cancer therapeutics. Disseminated tumor cells (DTC) are the key cells that result in dormant tumors. However, many challenges exist towards isolating DTCs for mechanistic studies. The main DTC that may represent the dormant cell is the cancer stem cells (CSC) as they have a slow proliferation rate. In addition to limited knowledge regarding induction of tumor dormancy, there are large gaps in knowledge regarding how tumors escape from dormancy. Emerging research into cancer stem cells, immunotherapy, and metastasis suppressor genes, may lead to new approaches for targeted anti-metastatic therapy to prevent dormancy escape. Overall, an enhanced understanding of tumor dormancy is critical for better targeting and treatment of patients to prevent cancer recurrence.The majority of cancer related deaths are due to metastatic outgrowths of the primary tumor mass that develop years to decades after apparent cures. Metastatic spread of tumors is a well-coordinated sequence of events, where cells shed from primary tumors, enter blood circulation, and spread to distant organs [1,2]. This process is, however, highly inefficient, where the majority of cells are predicted to die upon dissemination. Some disseminated tumor cells (DTC) will immediately begin to proliferate and colonize the new environment, but some DTC, while still viable, will enter a growth arrested state [3,4,5]. These growth arrested cells can remain viable and clinically undetectable for extended periods of time and are termed dormant cells. The dormant cells can awaken years later and resume proliferation and colonization even after the presumably successful treatment of the primary tumor [3,4,5]. In addition to a proliferation-arrested state (G0/G1 arrest) clinical dormancy may be due to micro-metastases where active proliferation is counterbalanced by apoptosis [6]. These metastatic growths are usually more malignant than the primary tumor, having acquired the ability to circumvent conventional therapies and growth barriers from non-permissive microenvironments. Tumor cell dormancy is characterized by solitary cells existing in a quiescent like state accompanied by decreased expression of proliferation markers [6]. Tumor cell dormancy is caused by several events including, microenvironment induced stress, transcriptional program from the primary tumor, and even drug therapies for primary tumor treatment. There has been debate as whether or not this state is quiescence or a reversible senescence [6,7]. Quiescence and senescence are mechanisms to induce cell cycle arrest and therefore could lead to tumor dormancy. Quiescence is defined as reversible cell cycle arrest, while senescence is permanent cell cycle arrest [8]; since senescence is permanent proliferation arrest, it is assumed that tumor cells have evolved the ability to bypass senescence mechanism. Perhaps a combination of both pathways leads to tumor cell dormancy [7].The microenvironment can have a very profound effect on the ability of tumor cells to develop into clinically relevant tumors. This was first hypothesized by Stephen Paget who, in his seed and soil theory, theorized that metastatic tumors (seed) will only grow in microenvironments (soil) for which they are suited [9]. The microenvironment is in direct contact with the tumors cells and thus acts as a critical source of vital signals needed for tumor cell survival and proliferation [10]; adapting to the microenvironment is an essential step in successful metastatic tumor growth. While some tumors have a predisposition towards metastasizing to specific organs [2,11,12,13,14], they may not be able to immediately colonize the new region due to inefficient interactions with the microenvironment [6]. DTCs may encounter a new environment in which they are not compatible with and therefore cannot fully engage the extracellular matrix. For example, Barkan et al. showed that cells incapable of making cytoskeletal rearrangements to fully engage the microenvironment will enter into and remain in a dormant state until they can make the needed modifications [15]. Using breast cancer cell lines D2Al and D2.0R, which exhibit similar proliferation rates in vitro, have different characteristics in vivo D2.0R remain as single quiescent cells for extended periods of time, compared to D2A1 cells which remain dormant for a relatively short time and switch to form rapid growing masses, Barkan et al. showed that these cells differentiate in their ability to express fibronectin and therefore induce β-1 integrin signaling and cytoskeletal rearrangements [15]. Under these conditions, the microenvironment is interpreted as hostile, as the cells only have transient adhesion to the microenvironment, leading to the activation of stress response signaling such as, urokinase-type plasminogen activator receptor (uPAR) deactivation [15,16,17]. uPAR is a metastasis-associated receptor that leads to tumor growth through α5β1 integrin interactions [16]. Low uPAR signaling prevent DTCs from interacting with and activating B1 integrin and downstream signaling events, including cytoskeletal dynamics, reducing microenvironmental interactions [6,15,18]. In addition to down regulation of uPAR signaling, microenvironment-induced stress also leads to p38 activation and ERK1/2 deactivation [19,20,21]. p38 activation has been shown to inhibit tumor progression as it implicated in promoting growth arrest, by activating p53 andp16 signaling, and down regulating cyclin D1 [22,23,24,25]. It has also been implicated in reducing the expression and activation of mitogenic signaling of ERK1/2 [19]. The ratio of ERK1/2 and p38, activation has been shown to predict if a tumor cell will proliferate or enter a dormant state upon dissemination, with a high ratio suggesting proliferation and a low ratio suggesting dormancy [20].Microenvironment induced stress may induce the expression or activation of metastasis suppressor genes (MSGs) [26]. MSGs are genes that prevent the formation of metastases, while having little to no effect on primary tumor formation. MSGs act on a wide range of cellular processes to inhibit metastatic growth including activation of signaling pathways which promote dormancy through cell cycle arrest or deactivating signaling pathways which promote cell proliferation (reviewed in [27]). The MSGs mitogen-activated proteins kinase-kinase (MKK) 4 and MKK6 have been shown to activate p38 signaling [28,29]; with MKK4 also activating and the cyclin-dependent kinase inhibitor p21, inducing cell cycle arrest. MSG N-myc downstream-regulated gene 1 (NDRG1), has been implicated in regulating oncogenic signaling pathways of TGF-β, PI3K, and Ras [30].In addition to activating stress responses through inefficient adhesion/interaction, there are reports that suggest that microenvironments, as part of their normal activity, can secrete factors that are anti-proliferative to DTCs. For example bone marrow stromal cells secrete bone morphogenic protein 7 (BMP7), which has been shown to induce dormancy in prostate cancer tumor cells [31]. The secretion of BMP7 leads to the increase of the metastasis suppressor gene NDGR1, which subsequently leads to an increase in p38 activation, cell cycle inhibitor p21 expression and ultimately cell cycle arrest [31]. Another example, also within the bone, occurs with the secretion of growth arrest-specific 6 (GAS6) by osteoblasts and tumor cells, which induces prostate cancer tumor cell dormancy [32]. Shiozawa et al. showed that GAS6 expression within the bone leads to a decrease in prostate cancer cell proliferation and an increase in chemoresistance [32]. Lim et al. showed that breast cancer cells in contact with bone stromal cells enter G0/G1 arrest by receiving proliferation-inhibiting microRNAs from the stromal cells, a phenomenon that is inhibited when gap junction intercellular communication is inhibited [33].In addition to stressed induced MSG expression, some cells disseminate from the primary tumor with a gene expression profile that is prone to tumor dormancy. Recent studies have found gene expression signatures within primary tumors (in addition to ERK1/2 and p38 ratio) that predict if tumors will produce dormant cells with early or late reoccurrence [34,35]. Kim et al. using gene signatures identified in dormancy models of tumor cell quiescence and angiogenic failure, generated a 49-gene expression profile [34]. Using this gene profile, they have developed a scoring system to determine if tumor will produce late or early reoccurring tumors.Tumor dormancy may arise as a response to cancer treatments [36,37,38,39]. The majority of treatments for cancer targets rapidly dividing cells. To circumvent drug induced death, some cancer cells will undergo cell cycle arrest/dormancy mechanisms that inhibit proliferation to survive. For example, ovarian tumor cells treated with farnesyl tranferase inhibitors (FTIs) undergo tumor dormancy by inducing autophagy [37]. Autophagy, the process of cellular organelle degradation to decrease cellular energy consumption and avoid apoptosis, occurs when cells experience prolonged periods of stress such as low nutrition, toxicity or to avoid anoikis [40,41,42]. This suggests that in order to survive a hostile environment and even drug treatment, tumor cells will induce autophagy, which has been reported to be the gateway to cell cycle arrest and tumor dormancy [42,43,44]. Some chemotherapeutic drugs, have been linked to an increase in p53 expression to induce senescence along with apoptosis in tumor cells [45]; however, there are reports that suggests that p53 induction can also lead to the induction of quiescence [46,47]. Tamoxifen exposure has also been shown to activate p38 [48]; which as mentioned above may lead to dormant cells. This suggests that chemotherapy may cause a subset of tumor cells to enter into quiescence and thus dormancy. Treatment induced dormancy may also be linked to cancer stem cells (CSCs), since these cells are slow cycling compared to the bulk of actively dividing cell within the tumor mass.CSCs represent a small population of cells within a tumor that are responsible for tumor maintenance, as they are fully capable of reconstituting a tumor, unlike the non-stem cell population within a tumor mass [49]. Like adult progenitor cells, these cells are predominately quiescent and may contribute to tumor dormancy, since they are largely resistant to majority of chemotherapies, which typically target rapidly dividing cells [37,50,51]. They can also become quiescent through co-opting target organ progenitor cell mechanism for quiescence, as demonstrated by Shiozawa et al. showed that prostate cancer cells are able to compete with hematopoietic stem cells [52]. After treatment, these cells are then free to slowly divide and rebuild the tumor leading to metastatic growth. As mentioned above, tumor dormancy can be a survival mechanism during therapy, with treatments able to specifically induce dormancy in CSCs [37]. Tumor cells may, as a survival mechanism to conventional drug treatments, spontaneously convert to CSCs. For example, it has been reported that non-stem tumor cells (NSTCs) may spontaneously convert to CSCs [53]. Specifically, Chaffer et al, showed mammary NSTCs (CD4410, CD24+) can spontaneously convert into mammary CSCs (CD44hi, CD24−), and give rise to both stem and non-stem cells [53]. This conflicts with the tenets of the CSC theory, that only CSCs can give rise to both CSCs and NSTCs [54,55]. However, there is some evidence that suggests that stemeness in tumorl cells may be transient, with any cell within a tumor population exhibiting stem-like qualities at any given moment [56]; with this conversion to the CSC phenotype is linked to epithelial-mesenchymal transition (EMT) [57]. Mani et al. demonstrated that forced induction of EMT leads to an increase in the expression of CSC markers and CSC properties [57]. As there are several lines of evidence that chemotherapy can lead to the induction of EMT [58,59,60], it is plausible treatment is enriching for or pushing cells into exhibiting stem-like properties, that would be capable of reconstituting a tumor at a later time point. Unlike tumor cell dormancy, tumor mass dormancy arises from DTCs that are able to proliferate at the metastatic site, but do not continue to progress to a clinically apparent metastasis as their growth is limited due to insufficient angiogenesis or active immunosurveillance. These cells exist as micro-clusters of cells actively proliferating, but not able to grow beyond a few mm as the rate of proliferation is equal to the rate of apoptosis. These micro-masses, like solitary cells, may remain indolent for extended periods of time.It has been long known that the immune system can have profound effects on tumor formation and progression. An active role for the immune system in preventing tumorigenesis is seen in transplant recipients, who after immunosuppressive therapy, spontaneously develop tumors at a higher rate than the general population or develop tumors of donor origin; where donors have no history of cancer [61,62,63,64]. This was also demonstrated in experiments that showed tumor formation and progression was higher in immunodeficient mice vs. immunocompetent mice [65,66,67,68,69,70,71]. Performed mainly by cells of the adaptive immunity, the immune system contributes to dormancy of DTCs by eliminating highly immunogenic tumor cells through cytolysis (reviewed in [66,72,73,74]). Early in the immunoediting process, immune cells are highly intolerant of tumor cells effectively managing to suppress tumor cell growth [74]. However, as the process continues tumor cells with low immunogenicity or tumor specific antigen (TSA) expression begins to emerge, creating a “stale mate” between tumor cells and immune cells. As tumors cells proliferate, immune cells are killing high TSA expressing tumor cells at the same rate [73]. This was shown by transplanting “unedited” tumors from immunodeficient mice and placing them in immunocompetent mice [66]. In these experiments, the unedited tumors were quickly cleared in immunocompetent mice, suggesting that tumors developed in immunocompetent mice are less immunogenic than tumors developed in immunocompromised mice. This was also demonstrated by Kobel et al. where immunocompetent mice, treated with the carcinogen 3′-methylcholanthrene (MCA) at low doses, maintained occult tumors cells without tumor outgrowth for extended periods of time [75]. The mice developed overt tumors after treatment with monoclonal antibodies directed against components of the immune system in the same location as the MCA injections, which provides strong evidence of the ability of the immune system to maintain micro-masses in a clinically apparent dormant state. This was further demonstrated in the DA1-3b mouse leukemia model. In mice vaccinated with DA1-3b cells expressing CD40L or IL-12 followed by a live DA1-3b cell challenge, without overt, showed few dormant tumor cells; which were able to induce AML when isolated and injected into naive mice. This further supports the hypothesis that tumor dormancy can result from a population of tumor cells that persists in balance with the immune system [76].Immune cells are also capable of inducing dormancy and preventing the aggressive outgrowth of metastatic tumors through non-cytotoxic methods [75,77,78,79]. Eyles et al. showed that CD8+ T-cells can prevent metastatic outgrowth in non-orthotopic organs, through cytostatic effects on disseminated tumor cells [74,80]. T-cells have been shown to inhibit cell cycle progression of tumor cells through IFN-γ and TNF mediated signaling, independent of cytotoxicity induction [75,77,78,79]. Muller-Herm et al. showed that IFN-γ producing TNFR+ CD4+ T-cells can inhibit tumor cell proliferation and angiogenesis [77]. The immune system also quickly clears pathogenic infections, which limits inflammatory responses prevent that can ultimately induce tumor cell growth and even induce angiogenesis (reviewed in [74]). There is some controversy surrounding the role of the immune system in metastatic tumor dormancy, since, in theory, metastatic tumors cells should have acquired the needed mutations to circumvent the immune targeting [7]. However, there is evidence to suggest that primary tumors induce changes in the microenvironment, which leads to non-global immune cell tolerance in the primary site that is not granted to metastatic cells in a new microenvironment [81,82]. This was demonstrated in several experiments were mice injected with tumors cells eventually have actively growing tumors, but reject secondary challenges with the same tumor cells at different sites [83,84,85]. In addition to tumor cell killing and inhibition of tumor cell proliferation, immune cells, may also play a role in preventing angiogenesis, as natural killer cells can secrete anti-angiogenic factors [86].As tumor cells proliferate and develop into clinically apparent masses, they must recruit and sustain their own blood supply through a process called angiogenesis [87]. Defined as one of the “hallmarks of cancer”, angiogenesis refers to the sprouting of, or recruitment of new blood vessels from existing vasculature in response to lack of oxygen and nutrients [87]. After reaching approximately 1–2 mm, the tumor becomes deficient in oxygen and nutrients as the nutrients in the microenvironment can no longer support the needs of the micro-metastases [88]. While proliferation competent, tumor cells may not be able to induce angiogenesis due to failure to express or induce the expression of factors necessary for angiogenesis to occur. Angiogenesis is controlled through pro- and anti-angiogenic factors such as vascular endothelial growth factor (VEGF) and angiostatin, respectively, within the tumor microenvironment. It is plausible that due to early shedding of tumor cells from the primary tumor, DTCs may not have acquired the ability to induce angiogenesis in its new microenvironment. In addition to expression of pro-angiogenic factors, angiogenesis is dependent on the proliferation and recruitment of endothelial cells to nearby vasculature in the tumor microenvironment. Png et al. showed that tumor cells expressing microRNA cluster 126 (miR-126) inhibit the recruitment of endothelial cells to the tumor site, through blocking GAS6/MER signaling [89]. They also showed that these cells were proliferation competent and angiogenic competent, when co-transplanted with endothelial cells [89]. Straune et al. also showed that tumor cells with low heat shock protein 27 (HSP27) expression remain non-angiogenic and dormant for extended periods of time in part due to inhibition of endothelial cell proliferation [90]. HSP27 expression lead to an increase in microvessels and proliferating cells within the microvessels [90].As mentioned above the microenvironment can have profound effects on the fate of DTCs, stimulating dormancy induction as well as escape from dormancy. Changes in the microenvironment, especially age-related changes, can induce tumor cells to escape from dormancy. As a host ages, more of their cells enter senescence [91,92,93,94,95], which can lead to an increase the activation of dormant cells. Senescent cells, while proliferation-inhibited, may become highly secretory; secreting high levels of cytokines, chemokines, growth factors and proteases in a phenomenon known as senescence-associated secretory phenotype (SASP) [96]. The secretome includes strong pro-proliferation molecules and pro-inflammatory molecules as well as pro-angiogenic molecules creating an environment that stimulates tumor cell proliferation, and promotes angiogenesis (reviewed in [96]). Changes within the microenvironment can also lead to the mobilization and activation of CSCs. As mentioned earlier, CSCs can co-opt target organ progenitor cell mechanisms for quiescence. As a host ages there are also changes to tissue stem cell niches, leading to increased growth and mobilization of stem cells [97]. This can also lead to the activation and mobilization of CSCs and subsequently, tumor reoccurrence.In addition to age-related changes within a host, diet/and medications can tumor growth and dormancy. A poor diet can lead to an increase in adipose tissue and obesity; which has been linked to tumor reoccurrence in breast cancer [98]. Adipocytes have been shown to cause inflammation responses through secretion of MMP11, as well as pro-inflammatory cytokines IL-6 and IL-1β [99]. Adipose tissue is linked to estrogen secretion; which can also lead to the stimulation of tumor cell proliferation [100].Medications, as mentioned above, may have profound effects on dormancy in tumor cells. Drugs that act as soluble epoxide hydrolase inhibitors to increase vascular epoxyeicosatrienoic acids (EETs), are in clinical trials for use in treatment of cardiovascular disease, and may induce tumor dormancy escape through stimulating angiogenesis [101,102]; allowing tumor cells to proliferate beyond micro-metastases.As mentioned earlier, immune cells target tumor cells with high levels of TSA expression [66,72,73,74]. As the immune system eliminates cells with high levels of tumor specific antigen expression, they are selecting for cells with low to no expression of TSA; giving rise to a population of cells that have the ability to grow without interference from immune cells [103]. In addition to tumor evolution through selection for less immunogenic cells, tumor cells may interact with the microenvironment to create an environment that inhibits the recruitment of immune cells, giving tumor cells selective permission for growth in a particular environment [72,83,84,85]. Cells may also attain the necessary mutations, while dormant, colonize the new microenvironment or induce an angiogenic switch [104,105].There are multiple factors which contribute to tumor dormancy (Figure 1). The microenvironment provides signals to tumor cells which can confer growth arrest through the induction of stress signaling such as p38 activation. The microenvironment can also inhibit the activation of angiogenesis, a necessary step for sustained metastatic growth. The immune system can also lead to dormancy by inhibiting the net proliferation of tumor cells, but not necessarily tumor cell shedding. After an apparent cure, some patients will have circulating tumor cells (CTCs) within their blood system without disease presentation, suggesting that they have dormant tumors; however these cells or dormant tumors may or may not become clinically apparent within their lifetime.Schematic of metastatic tumor dormancy. Some tumor cells will leave the primary tumor without the ability to proliferate in the new microenvironment and remain as solitary cells (tumor cell dormancy), due to microenvironmental-induced stress, microenvironment incompatibility or even a gene expression profile that is prone to dormancy. Some tumor cells can leave the primary site with the ability to proliferate in the new site, but cannot grow beyond a few mm (tumor mass dormancy) due to immunosurveillance or angiogenic failure. Despite a myriad of advances in technology for both treatment and detection, tumor dormancy and escape remains poorly understood. The biggest challenge is detecting and CTCs, solitary DTCs, and micro-metastases within hosts for studies, which can be as low as 1 per 105 cells [106]. Current detection, for CTCs and DTCs is based on enrichment through size restriction or surface antigen recognition, both of which have limitations that may skew the actual concentration of CTCs and DTCs (reviewed in [107]), making it difficult to perform an accurate analysis or manipulate cells for detailed studies. Future studies directed on improving detection techniques and factors that prevent escape from tumor dormancy, in addition to direct targeting of dormant tumor cells, offer unique opportunities to achieve significant therapeutic gains.The authors declare no conflict of interest.

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+This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).In breast cancer patients, bone is the most common site of metastases. Medical therapies are the basic therapy to prevent distant metastases and recurrence and to cure them. Radiotherapy has a primary role in pain relief, recalcification and stabilization of the bone, as well as the reduction of the risk of complications (e.g., bone fractures, spinal cord compression). Bisphosphonates, as potent inhibitors of osteoclastic-mediated bone resorption are a well-established, standard-of-care treatment option to reduce the frequency, severity and time of onset of the skeletal related events in breast cancer patients with bone metastases. Moreover bisphosphonates prevent cancer treatment-induced bone loss. Recent data shows the anti-tumor activity of bisphosphonates, in particular, in postmenopausal women and in older premenopausal women with hormone-sensitive disease treated with ovarian suppression. Pain is the most frequent symptom reported in patients with bone metastases, and its prevention and treatment must be considered at any stage of the disease. The prevention and treatment of bone metastases in breast cancer must consider an integrated multidisciplinary approach.Breast cancer is one of the most commonly diagnosed cancers among women in the industrialized world [1]. At diagnosis, approximately 5%–6% of women present with distant spread, and over 70% of these will have bone metastases (BM) during the course of disease [2].Osteolytic, osteoblastic and mixed forms of metastasis are observed. The signs and symptoms of BM largely depend on the location and the mechanical stress on the affected parts of the bone. It manifests itself in pain, movement restrictions and skeletal-related events (SREs), which are usually defined as pathological fractures, spinal cord compression, bone pain requiring palliative radiotherapy (RT) and orthopedic surgery [3]. The first approach in the management of BM is the prevention of SREs, given their negative impact on quality of life and on worsening of the prognosis. Early diagnosis of BM is advisable with the aim of (1) changing the oncological therapies, (2) assessing and treating pain appropriately, (3) preventing SREs and (4) monitoring the patients over time. Although premature death is inevitable, remissions are frequent, and patients usually require supportive and palliative therapy for many months or years.Treatment of BM and SREs often requires a multimodal approach (Table 1), considering medical, surgical, percutaneous procedures and RT, which plays a central role. Additional application of antiresorptive agents (e.g., bisphosphonates or the receptor activator of nuclear factor-kB ligand inhibitors) has proven successful [3,4].Treatment of Metastatic Bone Disease: Multidisciplinary approach.The signs and symptoms of BM largely depend on the location and the mechanical stress on the affected parts of the bone. Pain is often registered as the first symptom of both osteolytic and osteoblastic BM and may be present in the absence of radiological evidence (standard X-ray) (Figure 1). In the presence of tumor infiltration, of compression of a nervous plexus or of pathological fracture with bone impingement, also, sensory and/or motor deficits can be registered. The degree of bone involvement or destruction, the amount of neural compression and the extent of systemic disease are all considered poor prognostic factors in breast cancer patients [3]. Adjuvant therapies for breast cancer increase the overall survival of patients with this disease, thus increasing the period during which bone lesions may arise. Therefore, in patients with breast cancer, the presence of pain and/or the other aforesaid symptoms cannot be under evaluated, because they can be suggestive of BM until proven otherwise. Bone scintigraphy is used for the diagnostic workup, allowing a total skeletal assessment, which is helpful for confirming the doubt of BM and the extent of the metastatic spread. Traditional X-ray imaging is used for the evaluation of suspiciously enhancing lesions. It permits a differentiation between lytic, blastic or mixed lytic-blastic BM. Computed tomography (CT) also shows smaller osteolysis, allows diagnosis of tumor extension into adjacent soft tissues and permits the evaluation of bone stability better than traditional X-rays. When a myelo-radicular compression is suspected or already diagnosed by CT, magnetic resonance imaging (MRI) has an important role in studying the local extension and the disease diffusion along all the spine.Standard X-ray examples of bone changes detectable in women with breast cancer: Osteoporosis of head femoral areas (a), pathological fracture of proximal third of the right humerus due to a large lytic lesion (b) and blastic bone infiltration of right hemipelvis (c).The goals of the medical therapy after surgical intervention are the prevention of distant bone and visceral metastases. The adjuvant systemic treatment for breast cancer has the aim of reducing the risk of recurrence and death by means of poly-chemotherapy [5], endocrine therapy [5] and biological therapy (trastuzumab) [6,7,8,9,10,11,12,13,14], also through the prevention of BM.Endocrine therapy is indicated in hormone receptor-positive tumors. Tamoxifen administered for five years is the treatment of choice for premenopausal or perimenopausal patients with resected hormone responsive breast cancer, regardless of other features of the tumor. Tamoxifen can also be administered in postmenopausal for 2–3 years, followed by the third-generation aromatase inhibitors (AI) for 3–2 years. In addition, postmenopausal tamoxifen is an alternative to the AI: For patients who refuse the AI and for patients for whom the use of AI is contraindicated. In women with tumors that are estrogen receptor positive, tamoxifen significantly reduces the annual risk of recurrence by 39% and death from breast cancer by 31%, compared to the control, independent of the use of chemotherapy, age, menopausal state, nodal status and the state of the progesterone receptor. At a median follow-up of 15 years, tamoxifen for five years results in a reduction of the absolute risk of recurrence and death of 11.8% and 9.2%, respectively [5].Aromatase inhibitors are indicated in the adjuvant endocrine treatment of women with postmenopausal hormone responsive breast cancer. The option of treatment includes monotherapy for five years or the sequence for 3–2 years after tamoxifen for 2–3 years.Two phase III randomized trials with an “upfront” strategy compared AI for five years (anastrozole in the Arimidex, Tamoxifen, Alone or in Combination (ATAC) trial, letrozole in the study, BIG 1-98) to tamoxifen for five years [15,16]. The ATAC trial showed an absolute advantage in disease-free survival (DFS) at a median follow-up of 100 months by 2.4%, but no significant benefit in overall survival (OS). The BIG 1-98 study showed an absolute advantage in DFS at a median follow-up of 76 months of 2.3% and a significant advantage in OS, but only for censored analysis, i.e., excluding the patients (25, 2% of cases) that had the chance of changing the treatment from tamoxifen to letrozole after the first interim analysis, performed at a median follow-up of 25.8 months.The data from the ATAC and BIG 1-98 were collected in a meta-analysis (9856 patients). Overall, it fixed an absolute advantage in DFS at a median follow-up of five and eight of 2.9% and 3.9%, respectively (p < 0.00001), with no advantage either in OS or in breast cancer mortality. The advantage in DFS was more evident in terms of reducing the risk of developing a contralateral breast cancer (HR = 0.59, p = 0.0009) and local recurrence (HR = 0.70, p = 0.003) and less obvious in terms of reducing the risk of distant recurrence (HR = 0.82, p = 0.002) [17].To date, in adjuvant endocrine therapy in premenopausal patients, the role of luteinizing hormone releasing hormone (LHRH)-analogue in addition to tamoxifen or the combination of chemotherapy and tamoxifen should be considered uncertain. The addition of LHRH analogue to tamoxifen versus tamoxifen alone did not significantly reduce the risk of recurrence (HR = 0.85, p = 0.20) and death after recurrence (HR = 0.84, p = 0.33) [18]. Despite the addition of LHRH analogue to tamoxifen, it seems to have a marginal benefit in terms of recurrence and death; the use of such a combination prevents the increase in plasma levels of estradiol, which occurs with tamoxifen alone, reducing the toxicity that may result from ovarian cysts or metrorrhagia [19]. Combination chemotherapy is superior to single-agent chemotherapy. The regimens containing anthracyclines and taxanes are superior in DFS and in OS compared to regimens without taxanes.The major randomized trials that compared regimens without taxanes to regimens with taxanes in the adjuvant treatment of patients with high risk of relapse (axillary nodes positive or negative) were included in the final meta-analysis, of the Early Breast Cancer Trialists’ Collaborative Group (EBCTCG) [20]. Thirty-three studies were taken into account that enrolled a total of 44,000 patients. Overall, the meta-analysis confirmed a reduction in the risk of relapse by 13% and the risk of death (from any cause) by 11% in favor of taxane-containing regimens. Restricting the analysis to studies in which the taxane (paclitaxel or docetaxel) was added concomitantly or in sequence to anthracyclines and compared to treatments that contain anthracyclines, benefits of taxanes of a similar entity were observed compared to that observed in the overall analysis. However, it was found, in contrast to previous meta-analyzes available [21], that there was a significant impact of anthracyclines doses without taxanes in comparison schemes. The benefit of the addition of taxanes to anthracyclines is maximal when the cumulative dose of anthracyclines in the two comparator arms is similar. In this case, the addition of the taxane determines a reduction of the risk of relapse and death by 16% and 14%, respectively, which results in a significant gain in PFS and OS to eight years by 4.6% and 3.2%, respectively. With an increasing dose of anthracyclines without taxanes in the comparator arm, the benefits in terms of both DFS and OS tend to decrease, canceling when the dose of anthracyclines in the comparator arm is double or more than that in the arm with taxanes.The study by US Oncology [22] is the only one in the adjuvant setting that compared a regimen containing anthracycline (AC: doxorubicin 60 mg/m², cyclophosphamide 600 mg/m² every 21 days for four cycles) with a regimen containing taxanes, but without anthracyclines (TC: cyclophosphamide 600 mg/m2, docetaxel 75 mg/m² every 21 days for four cycles), showing a benefit in DFS and, in a five-year follow-up, even in OS [23]. Therefore, the TC scheme can be taken into account in patients with contraindications to the use of anthracyclines and CMF as an alternative to the scheme.Trastuzumab, a monoclonal antibody for the extracellular domain of HER2, should be administered in patients with operated HER2-positive breast cancer. Globally, almost all studies of trastuzumab in the adjuvant setting have shown, excluding the studies with a smaller sample (PACS-04 and FINHER), a significant advantage in DFS and variable from 6% to 12.8% compared to the control, with administration for a year [23]. The advantage in OS was instead obtained only with the administration of trastuzumab in combination with chemotherapy (taxane), but not in sequence to it, with an absolute advantage variable from 3.2% to 5% at a mean follow up of 4.5 years [20].The benefit of trastuzumab was evident at both local-regional and distant sites, at time to distant recurrence [24].Bisphosphonates (BPs) are a well-established, standard-of-care treatment option to reduce the frequency, severity and time of onset of the SREs in breast cancer patients with BM [2,25,26,27,28,29,30]. From many years, BPs, in particular zoledronic acid (ZOL), have been incorporated into clinical practice recommendations for these patients [31], and denosumab [32,33] has been approved in many countries for the delay of onset of SREs due to BM in breast or prostatic cancer patients.The efficacy and safety of a reduced dosing frequency of ZOL in women with bone metastases due to breast cancer, treated previously with monthly zoledronic acid, was assessed in a phase 3 trial in 62 centers in Italy (ZOOM study) [34]. After one year of a monthly administration of zoledronic acid, for 425 patients, the patients were assigned to the 12-week group (209 patients) and 216 to the four-week group. The results showed that it was possible to maintain the therapeutic effects with the two different schedules of administration. However, median N-terminal telopeptide concentration changed from baseline more in the 12-week group than in the four-week group after 12 months (12.2% vs. 0.0%, p = 0.011) [34]. The effects on N-terminal telopeptide should be investigated further before changing practices, because in previous studies, high levels of N-terminal telopeptide (NTX) were associated with a poorer prognosis and a shorter time to first skeletal-related event and NTX levels decreased in response to treatment with zoledronate [35].In patients treated with BPs or denosumab, preventive dental measures, after dental screening examination [36], are advocated to reduce the osteoncrosis of the jaw (ONJ) incidence [37], due to their efficacy in patients with BM, but not in oncological patients with osteoporosis, yet. Recent recommendations for ONJ include a conservative approach with intermittent prophylactic antibiotic therapy and rinses with oral chlorhexidine and debridement [38]; moreover, a careful sequestrum removal of necrotic bone is recommended [39,40].Bone is the most common site for breast cancer metastases, and the bone microenvironment plays a crucial role in harboring disseminated tumor cells (DTCs). Therefore, agents that affect bone metabolism might not only prevent the development of bone lesions, but also provide meaningful reductions in the risk of relapse, both in bone and beyond [41].Bone homeostasis is a function of osteoblastic and osteoblastic activity. Osteoblastic bone resorption is stimulated by receptor activator of nuclear factor kappa-B ligand (RANKL) and osteoprotegerin (OPG) (a RANKL decoy receptor, which regulates the availability of active RANKL). Estrogens and androgens are able to modify OPG secretion and, thus, to act on osteoblastic activity. Sex hormone levels decrease with age and post menopause. Consequently, OPG levels also decrease, and osteoclastic bone resorption increases, whereas bone mineral density (BMD) decreases, favoring osteopenia and osteoporosis. Moreover, secondary osteoporosis may occur as a result of cancer treatments that decrease sex hormone levels, including chemotherapies, AI, androgen depravation therapy and gonadotropin-releasing hormone (GnRH) agonists [42].Bisphosphonates, through blocking malignant osteolysis and, consequently, reducing bone-derived growth factors and cytokines, prevent bone destruction and would be expected to render the “soil” less hospitable for the growth of the cancer “seed” within bone.Thus, adjuvant breast cancer treatments can have longer-term deleterious effects on bone health. Data of the literature shows that BPs have a role in preventing and treating cancer treatment-induced bone loss (CTIBL). Adjuvant bone protection studies of zoledronic acid have clearly shown effective prevention of aromatase inhibitor-induced bone loss (AIBL) and have additionally provided evidence of an anti-tumor effect [43].Various revisions of the literature have been published on adjuvant bone-targeted therapy to prevent metastasis in breast cancer patients [44,45].The first studies in the adjuvant setting were carried out on oral clodronate to test the potential efficacy of bone-targeted agents in preventing metastasis in early stage breast cancer, with conflicting results [46,47,48].Later, the Australian Breast and Colorectal Cancer Study Group (ABCSG)-12 trial enrolled 1803 premenopausal women to evaluate different endocrine strategies with and without six monthly zoledronic acid treatments on bone health and recurrence endpoints. All patients received ovarian suppression therapy with goserelin and were randomized to receive additional treatment with tamoxifen or anastrozole, either with or without ZOL. At a median time follow-up of 48 months, anticancer effects with ZOL were seen both in bone and beyond (less frequency of distant metastases, of locoregional relapses and contralateral breast cancer) [49]. More than three years after the completion of treatment, further analyses at 62 months [50] and at 84 months [51] showed a persisting benefit in disease-free survival (DFS). The recent analysis of ABCSG-12 suggested a statistically significant age effect on the impact of ZOL on both DFS and OS. In younger premenopausal patients, no statistically significant difference in DFS was observed between the ZOL group and the control group (p = 0.821). Differently among patients aged more than 40 years in whom complete ovarian suppression with goserelin was more likely, ZOL resulted in a 42% reduction (p = 0.013) in the risk of DFS events and a 43% reduction in the risk of death compared with the control group (p = 0.057) [51].The trial “Does adjuvant zoledronic acid reduce recurrence in stage II/III breast cancer?” (AZURE) [52] includes 3360 breast cancer patients, who received chemotherapy and endocrine therapy and were randomly assigned to receive ZOL every 3–4 weeks for six doses and every 3–6 months, until five years or the first evidence of distant metastases. In the intention-to-treat population, the addition of ZOL did not significantly increase DFS compared with standard treatment alone at a median follow-up of 50 months. There was a trend toward an improvement in overall survival in patients treated with ZOL (p = 0.07). In postmenopausal women, in the ZOL arm, significant reduction of locoregional recurrence, new second primaries and non-skeletal distant recurrence was observed (p < 0.001).ZO-FAST [53] is a European study designed to investigate the bone-preserving activity of zoledronic acid during adjuvant therapy with AI in postmenopausal women treated with letrozole and ZOL, 4 mg, every six months or delayed treatment with ZOL following a significant decrease in BMD (control group). After three years’ median follow-up, in addition to the BMD benefits with ZOL (primary endpoint), the upfront-ZOL group had a significant 41% reduction in the risk of DFS events vs. the delayed ZOL group (p = 0.0314).The proper and regular self-reporting assessment of pain is the first step for an effective and individualized treatment. Pain is always a subjective sensation. Individualized pain management should take into account the onset, type, site, duration, intensity and temporal patterns of the pain (from this, it is often possible to define the cause of the pain), concurrent medical conditions and, above all, the subjective perception of the intensity of pain that is not proportional to the type or to the extension of the tissue damage, but that depends on the interaction of physical, cultural and emotional factors.The proper and regular self-reporting assessment of pain (intensity and outcomes) with the help of validated assessment tools is the first step for an effective and individualized treatment. The most frequently used standardized scales are visual analogue scales (VAS), verbal rating scale (VRS) and the numerical rating scale (NRS). The assessment of all components of suffering, such as psychosocial distress, should be considered and evaluated [54]. Principles of pain management are described in Table 2. Figure 2 summarizes the treatment of pain in BM [54]. Analgesic therapy should be administered at any time of the clinical history of the patient and, also, before painful diagnostic procedures, such as bone biopsy.An effective pain-relieving therapy must considering the following issues.Treatment of pain due to bone metastases.Uncomplicated bone metastases are lytic, blastic or mixed lesions without the risk of impending fracture or spinal cord compression.Opioids are the mainstay of analgesic therapy and can be associated with non-opioid drugs, such as paracetamol or nonsteroidal anti-inflammatory drugs, and to adjuvant drugs (for neuropathic pain and symptom control). The role and the utility of weak opioids (i.e., codeine, dihydrocodeine, tramadol) is a point of controversy. Morphine has been placed by the WHO [55,56] on its Essential Drug list. In a comparative study with other strong opioids (hydromorphone, oxycodone), there was no evidence to show the superiority or inferiority of morphine as the first choice opioid. Oral methadone is a useful and safe alternative to morphine. Methadone presents the potential to control pain that is difficult to control with other opioids. Although the oral route of opioid administration is considered the one of choice, intravenous, subcutaneous, rectal, transdermal, sublingual, intranasal and spinal routes must be used in particular situations. Transdermal opioids, such as fentanyl and buprenorphine, are best reserved for patients whose opioid requirements are stable. Switching from one opioid to another can improve analgesia and tolerability [54].A distinctive feature of pain related to BM is that it may be absent or mild at rest, but may become severe or intolerable when exacerbated by different movements or positions (incident pain), such as standing, walking, sitting or turning. Moreover, pain induced by movement may be difficult to treat, even with rescue opioid medications, and is a predictor of poor pain control and leads to varying degrees of functional impairments and psychological distress.Treatment of pain reported on movement in patients with BM is an important clinical problem, because it may be difficult to treat, even with rescue opioid medication, and creates functional impairment, as well as psychological distress.Freedom from pain during movement is particularly difficult to achieve in patients with BM. An insufficient amount of opioids taken at regular intervals, as well as movement can cause acute episodes of pain that are predictable and not classifiable, known as unpredictable breakthrough pain (BTP). These BTP episodes are of moderate to severe intensity, with rapid onset (min) and a short duration (maximum 30 min) [57].Breakthrough pain in cancer patients should be treated using a combination of pharmacological and nonpharmacologic interventions, including a careful optimization of background pain therapy to avoid the exacerbation of pain, due to the final dose analgesic effect, and the prescription of supplemental analgesic rescue therapy to prevent or treat the rapid onset of pain exacerbation.Available pharmacological treatment options include oral, subcutaneous, intravenous transmucosal, buccal or nasal opioids; however, few RCTs are available [54].On evaluating the role of BPs in achieving pain relief in patients with bone metastases, a Cochrane review [58] has identified thirty randomized controlled studies (21 blinded, four open and five active control), for a total of 3682 subjects included. For the proportion of patients with pain relief (eight studies) pooled data showed benefits for the treatment group, with an NNT at four weeks of 11 (95% CI 6–36) and at 12 weeks of seven (95% CI 5–12). In terms of adverse drug reactions, the NNT was 16 (95% CI 12–27) for discontinuation of therapy. Nausea and vomiting were reported in 24 studies, with a non-significant trend for a greater risk in the treatment group. The small number of studies in each subgroup with relevant data limited our ability to explore the most effective BPs and their relative effectiveness for different primary neoplasms. The overall conclusion from the Cochrane review was that there is enough evidence to support the effectiveness of BPs in providing some pain relief for bone metastases. There is insufficient evidence to recommend BPs for an immediate effect, as a first line therapy. Bisphosphonates and denosumab should be considered for the prevention of SREs, also, where analgesics and/or RT are adequate for the management of painful BM [54,59].The goals of palliative RT are pain relief, recalcification and stabilization of the bone, as well as reduction of the risk of complications (e.g., bone fractures, spinal cord compression). The exact time of pain relief derived from RT is actually not known; generally, it is observed within a few days after the start of RT. Instead, radiologically detectable recalcification and stabilization are to be expected at the earliest within 6–12 weeks after termination of RT [60,61].In case of singular or oligo-BM, high-dose RT can stop disease progression with the expected complications of the affected skeletal manifestation (e.g., pathological fracture and spinal cord compression) [2].Radiotherapy has specific and critical efficacy in providing pain relief caused by cancer; however, the effectiveness depends largely on the radiosensitivity of the tumor, and in clinical practice, a difference of radiosensitivity has not been clearly integrated in therapeutic strategy (Figure 2). Painful BM are the most common indication for the use of palliative RT, with numerous prospective trials over the past three decades documenting improvements in pain from 60% to 80% after treatment, and in clinical practice, different fractionation regimens are used [62,63]. Recently, the American Society for Radiation Oncology, reviewing randomized published data regarding the use of RT in patients with BM, showed a pain relief equivalency for different regimens, including 30 Gy in 10 fractions, 24 Gy in six fractions, 20 Gy in five fractions and a single 8-Gy fraction [64,65].Fractionated schedules of RT have been associated with an 8% repeat treatment rate to the same anatomic site because of recurrent pain vs. 20% after a single fraction. However, the long-term results concerning pain relief after re-irradiation were thus comparable with a palliative effect of more than 70% [66,67,68].In clinical practice, the single dose approach optimizes patient and caregiver convenience. Therefore, a hypofractionated schedule can be considered the regimen of choice in patients with BM [69]. Furthermore, repeat irradiation to the same site of bone disease might be safe, effective and less necessary in patients with poor prognosis [70].Recently, the breast cancer expert panel of the German Society for Radiation Oncology has developed guidelines for palliative RT. In their conclusions, the authors confirm that the appropriate fractionation should be chosen with respect to the treatment duration, estimated life expectancy and need for hospitalization [71]. Generally, patients in a good general health status would rather select fractionated irradiation. Therefore, the decision should be taken with the agreement of the radiation oncologist and patient. In conclusion, for patients with low performance status and pain, complex irradiation techniques and multi-fraction are, in general, not of increased value, due to prolonged treatment times, as well as the higher needs of precise positioning and immobilization. Simple techniques with a single dose of RT (i.e., 8 Gy) should be used when analgesia is the main goal of treatment.Fractionated RT regimens (30 Gy in 10 or 20 Gy in five fractions, respectively) can be associated with better results concerning bone recalcification [61,72].Generally, recalcification and stabilization of bone lesions are detectable with X-ray from 6 to 12 weeks after RT. This result is ameliorated by concomitant administration of bisphosphonate, while simultaneous application of denosumab are still outstanding [73]. In the case of expanded BM, with high risk of fractures, stabilization with surgery must be evaluated before RT.Bone metastases are defined as complicated in the case of impending fracture or spinal cord compression.A patient with an impending fracture (i.e., a large lytic metastasis, which can be the cause of pathological fracture) must be evaluated for a surgery stabilization procedure before RT. If the surgeon does not give a surgical indication, RT alone represents the treatment of choice to control local disease and pain (Figure 2) [54].Metastatic spinal cord compression (MSCC) can be induced by tumor infiltration of the spinal space or by intraspinal metastases. Spinal cord compression is one of the most dreaded complications of metastatic cancer, occurring in 5%–10% of all cancer patients during the course of their disease and requiring urgent oncologic care. Autopsy studies suggest that approximately one third of patients with solid tumors may have metastases to the spine, but the clinical evidence of MSCC is estimated in 3% to 7% of patients [74,75]. Approximately 50% of MSCC cases in adults arise from breast, lung or prostate cancer, but MSCC has also been described in patients with lymphoma, melanoma, renal cell carcinoma, thyroid carcinoma, sarcoma and myeloma. Pain accompanies spinal cord compression in approximately 95% of adults and 80% of children with MSCC and usually precedes the diagnosis by days to months. Weakness, the second most common symptom at presentation, usually follows the development of local or radicular pain and generally progresses to plegia over a period of hours to days. Other symptoms of MSCC are sensory loss and incontinence, which typically develop after the pain [76].On suspicion of MSCC, magnetic resonance imaging should be performed. Magnetic resonance imaging has a sensitivity of 93%, a specificity of 97% and an overall diagnostic accuracy of 95% [74].Prognosis is, above all, related to early diagnosis and therapy. The speed of neurologic deficit onset can condition the functional outcome, which is significantly better with slower development of motor dysfunction before RT. One study evidenced that ambulatory recovery occurred in 86% and 35% of patients with a history of >14 days compared with one to seven days, respectively [77]. Survival after MSCC is related to primary tumor type, ranging from 17–20 months for breast, prostate and myeloma to only four months for lung. If untreated, the majority of patients with MSCC becomes paraplegic. Early detection and treatment when the patient is still able to walk results in the highest chance of ambulation.In MSCC, the aim of treatment is to improve the patients’ quality of life through control of back pain and preservation or recovery of motor and sphincter functions. Although it could be questionable if local treatment increases patients’ survival, there is a tight relationship between survival time and functional status. In fact, MSCC patients who had no motor dysfunctions live longer than paraparetic and paraplegic ones and, generally, die of systemic tumors, rather than local progression at the spine [78]. Considering that treatment success is related to the severity of the epidural disease and to the patient clinical condition at the time of diagnosis, it is important to perform diagnosis early and to begin treatment before significant myelopathy develops.In clinical practice, MSCC can be treated with surgery followed by RT or RT alone, and the choice of treatment depends on patient selection. Once the diagnosis of MSCC is made, steroids are generally prescribed to control edema and to lessen pain.Surgery plays an important role in selected cases. On the basis of literature evidence, it can be concluded that initial surgical resection followed by RT should be considered for a carefully selected group of patients that has with single-level MSCC and neurological deficits. Other possible indications for surgery include the necessity of stabilization, vertebral body collapse causing bone impingement on the cord or nerve root, compression recurring after RT and an unknown primary requiring histological confirmation for diagnosis [75]. However, when there are diagnostic doubts, tomography-computed guided percutaneous vertebral biopsy can be an alternative to open surgery to avoid surgical side effects and to reduce incisional pain and the recovery period.Although RT is an effective approach for the majority of MSCC patients, the optimal radiation schedule remains unknown. Except for some particular circumstances, the use of conventional fractionated RT (2 Gy per day to a total dose of 30–50 Gy in 3–5 weeks) has been abandoned in favor of RT regimens requiring a smaller number of fractions. Since 2005, there have been published two phase III randomized multicenter Italian trials [79,80]. The first compared a short-course regimen (i.e., 8 Gy repeated after one week to a total dose of 16 Gy) to a split-course regimen (i.e., 5 Gy × 3, four days rest and then 3 Gy × 5) [79]. The second compared the same short-course regimen to 8 Gy in a single fraction [80]. It is worth noting that both of these studies were performed in patients with short life expectancy (≤6 months) and that responders maintained function until death. While both hypofractionated RT regimens adopted has effective results, the authors concluded that the 8-Gy single fraction is the best option, considering that it is well tolerated, effective and convenient in this setting of patients. Published retrospective and prospective non-randomized data support the above randomized data in that no dose fractionation schedule has demonstrated with a higher ambulation rate [81]. However, some experience suggested that in MSCC patients, the duration of local control is superior, and consequently, the rate of in-field recurrences is lower following long-course RT regimens; these data add further weight to the argument for selecting a patient’s treatment based on prognosis [82]. Recently, it published a score predicting post-RT ambulatory status [83]. It was developed based on 2096 retrospectively evaluated MSCC patients and considered six prognostic factors (i.e., tumor type, interval between tumor diagnosis and MSCC, presence of other bone or visceral metastases at the time of RT, pre-treatment ambulatory status and duration of motor deficits). This scoring system has been validated prospectively for the endpoint survival and ambulatory function. In conclusion, evidence suggests that until further randomized data is available, short-course/single fraction regimens (e.g., 5 × 4 Gy or 1 × 8 Gy) can be used for patients with short life expectancy, while fractionated, higher dose schedules (e.g., 10 × 3 Gy or greater) should be considered for patients with better prognosis.Generally, in MSCC patients, RT is administered with concomitant steroids to lessen back pain, prevent progressive neurologic symptoms and reduce radiation-induced spinal edema [84]. Steroids should be given immediately when the clinical-radiologic diagnosis of MSCC is obtained. Dexamethasone is the most frequently used drug, although the use of methylprednisolone is also reported. The dexamethasone dose ranges from moderate (16 mg/die in two four-times daily parenteral or oral divided doses) to high (36–96 mg/die), eventually preceded by a bolus of 10–100 mg intravenously [74]. The steroids are usually tapered over two weeks. No study has been published comparing high dose to moderate dexamethasone dose. There is only one randomized clinical trial comparing high-dose dexamethasone to no drug in 57 patients with MSCC treated with RT. This trial evidenced that high dose dexamethasone significantly improves post treatment ambulation, but is accompanied by a certain probability (11%) of high toxicity [84]. A phase II trial showed the feasibility of treating patients with MSCC, no neurologic deficits, or only radiculopathy, and no massive invasion of the spine at MRI or CT with RT (3 Gy × 10) without steroids [85]. However, in clinical practice, considering that published studies have shown no difference in outcome between high-dose and moderate-dose dexamethasone and the relatively high incidence of side effects from steroids, above all, in patients with diabetes mellitus, hypertension and peptic ulcer, a moderate dexamethasone dose (e.g., 16 mg/die two-times-daily) is suggested for symptomatic MSCC patients.For treatment of MSCC, chemo-hormonal therapy can be used in combination with RT or alone in adults who are not surgical or radiation candidates, but who have chemo-hormonal sensitive tumors, such as lymphoma, small cell lung carcinoma, myeloma, breast, prostate or germ cell tumors. In children, chemotherapy is the primary treatment for chemo-responsive tumors.Radioisotope treatment can be considered an interesting palliative care option for pain control in patients with generalized BM. Moreover, the published data do not suggest that radioisotope therapy alone can substitute RT in these cases. However, radioisotope treatment is limited by the kind of BM, which must be osteoblastic, documented by a technetium-99 bone scan. A sufficient interval should be kept after a previous myelotoxic chemotherapy or half body irradiation (4–6 weeks). In selected cases, retreatment with radioisotope can be proposed, but should only be performed after the regeneration of blood cell count. This also applies to a planned chemotherapy or RT after radioisotope, since myelosuppression can occur with some delay.A small number of trials have shown that radioisotopes can relieve bone pain in patients with breast cancer and lung cancer, while inconsistent results were produced in patients with hormone-refractory prostate cancer [86,87,88].Particularly, the therapeutic efficacy of radioisotopes (i.e., strontium-89-chloride and 186Re-1,1-hydroxyethylidene bisphosphonate) was evaluated in the palliation of painful BM in 50 patients with breast cancer. The global response rate ranged from 84% to 92%, with a median duration of pain relief of four months (range, 2–14); treatment was safe and feasible, with a moderate hematological toxicity [88].Moreover, a recent systematic review evidenced a small benefit of radioisotopes for complete or partial relief in the short and medium term (1–6 months), with no modification of the analgesics used [89]. Therefore, the use of radioisotope treatment can be appropriate in circumstances in which patients have several sites of painful osteoblastic metastases in an anatomic area greater than that which could be safely treated with external RT.Linear accelerator technology has evolved with multileaf collimation, intensity modulated irradiation, systems of image guidance and robotic technology. These new technologies permit one to administer to a target volume a dose escalation of RT by ensuring radioablative dose delivery to tumor, while, at the same time, avoiding an excessive dose to surrounding critical normal tissue organs. Stereotactic body RT has emerged as a new treatment option in the multidisciplinary management of BM, particularly for lesions located within or adjacent (paraspinal) to vertebrae or the spinal cord. Radiation-induced myelopathy has a relevant late toxicity, because it may result in severe neurologic dysfunction. Higher RT doses, larger doses per fraction and previous exposure to radiation could be associated with a higher probability of developing radiation-induced myelopathy [74].Therefore, stereotactic body RT provides an attractive option to deliver high-dose per fraction radiation, typically in a single dose (i.e., 10–16 Gy) or in hypofractionation (i.e., 9 Gy × 3 fractions or 6 Gy × 5 fractions) [90,91,92]. Moreover, stereotactic body RT was used in selected retrospective single-institution reports, and the endpoints of these studies were also heterogeneous. A radiation therapy oncology group is carrying on a phase II/III study (Radiotherapy Oncology Group—RTOG protocol 0631) of image-guided stereotactic RT versus conformal RT for localized spine metastasis. Considering the little available data on stereotactic RT for spine metastasis and awaiting the results of the aforesaid RTOG study, the American Society for Radiotherapy and Oncology-ASTRO guidelines suggest the use of this new technology only in selected cases (e.g., re-irradiation of spinal metastasis) [64].The goals of surgical intervention in patients with BM are to reduce pain and to improve function if no surgical treatment fails. Treatments for painful BM may not only diminish pain, but also may improve quality of life and independence/mobility and reduce skeletal morbidity, potential pathologic fractures, MSCC and other SREs.The surgical management of established BM must be individually tailored to each patient. Multidisciplinary support and communication with regard to a patient’s performance status and response to oncological treatments will assist the orthopedic surgeon in identifying the surgical intervention. Surgical implantation must not only allow immediate stability for mobilization, but also provide a reliable, durable and long-term construct that matches the expected long-term outcome of the patient. The decision to proceed with surgery is based on a variety of factors, including severity of symptoms, location of tumor, expected morbidity if a fracture were to occur, expectations of the patient and viability of alternative or adjuvant treatments. It is worth noting that the patient must be fit for surgery, and the recovery and rehabilitation phase should not exceed the patient’s life expectancy. Moreover, the surgical construct should be sufficiently durable to last throughout the patient’s lifetime. This can be difficult to estimate and is based on multiple factors, such as age, comorbidities and the extent of visceral and skeletal disease. Even if the patient is expected to survive for >3 months, the pain relief from stabilization of a fractured humerus, femur or tibia is substantial. Asymptomatic lesions need only be followed clinically and radiographically. These lesions can be effectively managed with medical treatment, including RT, bisphosphonate therapy and treatment of primary tumor [93].In clinical practice, patients treated with surgery for BM are routinely submitted to postoperative RT. Although there are quite limited published data describing the outcomes among these patients, postoperative RT was generally associated with patients regaining normal use of their extremity (with or without pain) and undergoing fewer reoperations to the same site [94].In patients with metastatic spine tumor, the goals of surgery include pain relief, restoration and preservation of neurologic function and stabilization of the vertebral column. In appropriately selected patients, surgery has provided a significant improvement of quality of life, pain control, functional status and the ability to undergo adjuvant therapy. Minimally-invasive, or minimal-access, spine surgery is also evolving in the management of patients with spinal metastases and epidural disease not causing cord compression. The aim is to minimize the surgical morbidity, while maintaining efficacy by still performing a radical tumor resection, stabilizing the spine with implants and/or injecting cement via endoscopic technology, which often can be performed as outpatient. Kyphoplasty and vertebroplasty have been used in the treatment of vertebral compression fractures for patients with spine metastases. Both techniques involve the injection of chemical cement (polymethyl methacrylate) into the vertebral body under image guidance. Kyphoplasty involves balloon insertion first into the vertebral body, to create a cavity and to augment the vertebral body, and low pressure cement injection. Vertebroplasty involves high pressure injection of cement into the vertebrae without cavity creation. A randomized trial is ongoing to determine the appropriate use of these procedures. These techniques have been reported to provide immediate and sustainable pain relief from malignant spine compression fracture [95,96]. The up-front spinal stabilization and rapid pain relief is potentially advantageous for subsequent RT, given that patients require strict immobilization and co-operation for RT. A preliminary efficacy and safety for the combined treatment of kyphoplasty and stereotactic body RT has been reported [97]. A novel combination approach was recently reported with samarium-153-ethylene diamine tetramethylene phosphonate mixed with bone cement and injected using kyphoplasty into painful vertebral metastases [98]. Although only the preliminary feasibility can be commented on, this represents an innovative approach to combine stabilization with direct deposition of radiation into a diseased, fractured vertebrae.In patients with MSCC, posterior decompressive laminectomy removes the lamina and posterior spinous process with no attempt to remove the anterior vertebral body tumor. This surgical approach has the disadvantage of not removing the tumor effectively, often resulting in inadequate decompression and the potential of worsening spine stability [99,100,101]. Decompressive laminectomy is usually used urgently to restore the neurological status in patients with widespread metastasis and is reserved as a salvage therapy [102]. Direct circumferential decompressive surgery came on board with advances in surgery and stabilization instrumentation. It is aimed at removing the anterior vertebral body tumor and decompressing the spinal cord. It is usually performed from both anterior and posterior approaches with anterior column reconstruction to stabilize the spine. This surgical approach, together with RT, has been shown to improve the ambulatory function in selected group patients with favorable prognosis. However, this is a major operation with the potential for prolonged hospitalization and rehabilitation.Bone metastases are a common complication of breast cancer, and proper treatment is crucial, considering the long-term survival generally associated with patients affected by this condition. The prevention of SREs represents an important goal to avoid the severe symptoms often related to complications.Various medical approaches are available for this setting of patients. Radiotherapy is a useful treatment option that can effectively control pain and prevent neurological dysfunction. Surgery is a valid tool that has to be considered in selected cases.Early diagnosis and appropriate therapy represent the major goals that can be better achieved by a multidisciplinary team.The authors declare no conflict of interest.

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+This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).Cancer metastasis to the bone develops commonly in patients with various malignancies, and is a major cause of morbidity and diminished quality of life in many affected patients. Emerging treatments for metastatic bone disease have arisen from advances in our understanding of the unique cellular and molecular mechanisms that contribute to the bone metastasis. The tendency of cancer cells to metastasize to bone is probably the end result of many factors including vascular pathways, the highly vascular nature of the bone marrow (which increases the probability that cancer cells will be deposited in bone marrow capillaries), and molecular characteristics of the cancer cells that allow them to adapt to the bone marrow microenvironment. The goals of treating osseous metastases are manifold. Proper treatment can lead to significant improvements in pain control and function, and maintain skeletal integrity. The treatment plan requires a multidisciplinary approach. Widespread metastatic disease necessitates systemic therapy, while a localized problem is best managed with surgery, external beam radiotherapy, or both. Patients with bone metastasis can have prolonged survival, and proper management can have a significant impact on their quality of life. We will review the factors in this article that are promising molecular bone-targeted therapies or will be likely targets for future therapeutic intervention to restore bone remodeling and suppress tumor growth.Cancer metastasis to the bone develops commonly in patients with various malignancies, and is a major cause of morbidity and diminished quality of life in many affected patients. The effective treatment of bone metastasis requires a multidisciplinary approach by medical, surgical and radiation oncologists. Emerging treatments for metastatic bone disease have arisen from advances in our understanding of the unique cellular and molecular mechanisms that contribute to the bone metastasis. The tendency of cancer cells to metastasize to bone is probably the end result of many factors including vascular pathways, the highly vascular nature of the bone marrow (which increases the probability that cancer cells will be deposited in bone marrow capillaries), and molecular characteristics of the cancer cells that allow them to adapt to the bone marrow microenvironment [1,2]. In fact, breast cancer cells have been shown to adopt an osteoblast-like phenotype that may help them survive in the bone marrow [3]. Once in the bone marrow cancer stem cells may remain dormant for prolonged periods of time; however, the specific signals that control cancer stem cells dormancy and reactivation are currently not well understood [1].This metastasis can result in substantial morbidity in the form of skeletal related events that are defined as pathological fractures, spinal cord compression, hypercalcemia or pain requiring radiation or surgery of the bone. Once bone metastases are diagnosed, they are usually incurable and the goal of treatment becomes focused on palliation and prevention of skeletal related events. Treatment modalities for bone metastasis include chemotherapy, hormonal therapy, analgesics, radiotherapy, and orthopedic surgery. In addition, bisphosphonates are potent antiresorptives, used in the prevention of skeletal related events, but they are not completely effective in inhibiting the progression of bone metastasis. A new strategy is bone-targeted therapy. In this review, bone-targeted treatment relating to breast cancer metastasis will be discussed.Breast cancer cells in the bone marrow alter the functions of bone-resorbing (osteoclasts) and bone-forming cells (osteoblasts) and thereby disrupting physiological bone remodeling [4,5]. Breast cancer cells may cause stimulation of osteoclast differentiation and maturation along with secreting factors that inhibit osteoblast differentiation and activity. Their interaction with osteoblasts also induces the release cytokines that promote tumor growth [4,5]. This leads to an imbalance between bone resorption and formation, resulting in enhanced skeletal destruction and as a consequence of osteolysis, occurrence of pathological fractures. Several molecules that are produced by breast cancer—e.g., parathyroid hormone-related protein, interleukins (IL-6, IL-8 and IL-11), cytokines (macrophage stimulating factor (M_CSF)) and prostaglandins—stimulate osteoclast active through the activation of the receptor activator of nuclear factor kB ligand (RANKL)/RANK pathway, which is the primary mediator of osteoclast-mediated bone resorption [5,6]. Breast cancer cells also secrete activin A (a member of transforming growth factor (TGF)-β), noggin (a bone morphogenetic protein (BMP) antagonist) and dikkopf-1 (DKK-1; a wingless (Wnt) protein antagonist), all of them inhibiting osteoblast differentiation [5,7]. As bone is resorbed, growth factors (like TGF-β and insulin-like growth factor-I) stored in the bone matrix are then released and stimulate breast cancer cell proliferation, providing a supportive niche for tumor growth [5,6,7]. The goals of treating osseous metastases are manifold. Proper treatment can lead to significant improvements in pain control and function, and maintain skeletal integrity. The treatment plan requires a multidisciplinary approach. Widespread metastatic disease necessitates systemic therapy, while a localized problem is best managed with surgery, external beam radiotherapy, or both. Patients with bone metastasis can have prolonged survival, and proper management can have a significant impact on their quality of life. The realization that normal cells in the bone microenvironment support the development of skeletal lesions has led to the use of bisphosphonates, as inhibitors of osteoclast-mediated bone resorption, in the treatment of patients with bone metastasis [4]. Nitrogen-containing bisphosphonates (N-BP) specifically inhibit osteoclast farnesyl pyrophosphate synthase activity, a key enzyme in the mevalonate pathway, which causes the inhibition of prenyllation of small GTPases and the subsequent inactivation of osteoclasts. In addition to inhibition of osteoclast activation and function, a growing body of preclinical data suggests that N-BPs exert direct and/or indirect antitumor effects. There is abundant evidence of the inherent antitumor activity of N-BPs in vitro, including induction of tumor cell apoptosis, inhibition of tumor cell proliferation, migration, and invasion [8]. In vitro, N-BPs (zoledronate, risedronate, alendronate, ibandronate) also interfere with all major steps of the angiogenic process, such as endothelial cell migration, proliferation and tube formation [8]. Despite these theoretical advantages, the data are inconsistent on what impact bisphosphonates have on breast cancer outcomes. In a meta-analysis of trials that randomized patients to bisphosphonates or not in the adjuvant setting failed to find significant reductions in the overall number of deaths, bone metastases, overall disease recurrences, distant relapse, visceral recurrences, or local relapses [9]. Nevertheless, in subgroup analysis, use of zoledronic acid was associated with a statistically significant lower risk for disease recurrence but not death or bone metastases rates. However, the role of osteoclast inhibitors in preventing breast cancer recurrence and improving outcomes continues to be an active subject of clinical research. We will review the factors in this article that are promising molecular bone-targeted therapies or will be likely targets for future therapeutic intervention to restore bone remodeling and suppress tumor growth.RANK/RANKL (osteoclast formation), integrin, c-Src, and cathepsin K (osteoclast function), all represent suitable targets for inhibition of pathological bone resorption.RANKL is a transmembrane protein expressed on the surface of osteoblasts that can be cleaved as a soluble form by proteases. Both the membrane-bound and soluble forms of RANKL attach to RANK, a receptor on the cell surface of osteolclast precursors, to stimulate osteoclastogenesis [6]. On the other hand, osteoblasts secrete RANKL inhibitor that inhibits RANKL/RANK interaction. The balance between the RANKL and RANKL inhibitor regulates the process of bone resorption and the imbalance of this process has been seen in many cancers, including breast cancer [5].Denosumab is a fully human monoclonal antibody that specifically targets RANKL. This antibody is currently a very promising potential alternative to bisphosphonates. Several studies have compared the efficacy of denosumab and bisphosphonates in patients with breast cancer, with promising results. A randomized phase II, open label trial, examined sequential osteoclast inhibiting therapy on the biochemical marker of bone resorption, urinary N-telopeptide (uNTx) [10]. One hundred and eleven patients with metastatic bone disease from a variety of tumors including breast with uNTx levels above 50 nmol/L were randomized to either continuing same IV bisphosphonates or switching to denosumab therapy. A significantly greater percentage of patients on denosumab vs. IV bisphosphonates reduced the uNTx below 50 nmol/L [10]. Another study examined non-inferiority as the primary endpoint when compared to zoledronic acid, and superiority as a secondary endpoint [11]. Denosumab met both of these endpoints with denosumab showing a greater delay to first on study SREs than zoledronic acid [11]. Denosumab has been well tolerated in these trials. The risk of osteonecrosis of the jaw was not significantly different between denosumab and zolendronic acid, but the renal toxicity was less frequent with denosumab [11]. Although increased risk of infection and secondary neoplasms with densosumab is a potential concern, since RANKL also regulates immune function involving dendritic cells and T and B cells [12]. Long term follow up is needed to clarify this risk. Cathepsin K is a lysomal cysteine protease highly expressed in osteoclasts that plays a major role in bone resorption [5,13]. The function of cathepsin K in osteoclasts was first revealed by the finding of a loss of function mutation in the human cathepsin K gene in patients with pycnodysostosic, a rare genetic disorder characterized by impaired osteoclastic bone resorption [13]. Also it was noticed that pycnodysostosis phenotype can be reproduced in mice if cathepsin K was genetically impaired. Overall, these findings indicate that cathepsin K plays a key role in osteoclast-mediated bone resorption. After this discovery, it was postulated that molecules that inhibit cathepsin K activity could serve as useful therapeutic agents against diseases associated with excessive levels of bone destruction. Moreover, it was noticed that cathepsin K inhibitors not only inhibit bone resorption but also stimulate bone formation [14]. This way it seems cathepsin K inhibitors have an advantage over other antiresorptive agents in the treatment of diseases associated with bone loss. A number of cathepsin K inhibitors have been examined in animal models of estrogen deficiency osteoporosis. Development of two of these compounds relacatib and balicatib, was stopped because of side effects thought to be due to lack of specificity [15]. An increased incidence of skin rashes and morphea-like skin adverse events has been reported in a phase 2b study of balicatib [16,17]. Three cathepsin K inhibitors are currently in development for treatment of osteoporosis. Odanacatib is in phase 3, ONO-5334 is in phase 2, and MIV-711 is in preclinical development. Thus, direct inhibition of cathepsin K, the enzyme primarily responsible for degradation of bone matrix by osteoclasts, shows promise for treatment of postmenopausal osteoporosis and perhaps other bone diseases. Further development in oncology field is still on hold. c-Src, a member of the non-receptor tyrosine kinase family, has role in a number of signaling pathways that regulate malignant cell proliferation, angiogenesis, adhesion, invasion, motility, survival and metastasis [18,19,20]. It is overexpressed in breast cancer tissue as well as in colon, lung and skin [21]. It positively regulates osteoclasts and negatively regulates osteoblasts via complex signaling pathways [22,23]. It has been seen that targeted disruption of c-Src in mice resulted in development of osteoperosis characterized by thick bone trabeculae secondary to osteoclast dysfunction, suggesting a role for c-Src in bone remodeling [24]. c-Src activity is associated with the capability of breast cancer cells to metastasize to bone. A study showed that a Src gene-expression signature was associated with late onset of bone metastasis in breast cancer, independent of estrogen receptor status or breast cancer subtype [25]. Preclinical studies showed that c-Src inhibiors decrease osteoclastic bone resorption [26]. Therefore, c-Src is a promising target for suppressing tumor-induced osteolysis and tumor growth.Currently, three c-Src inhibitors are in the breast cancer trials: dasatinib, bosutinib and saracatinib. Dasatinib is an oral tyrosine kinase inhibitor of Src family kinases and act at Philadelphia chromosome BCR-ABL [27]. Dasatinib is approved for imatinib-intolerant or resistant chronic myeloid leukemia CML and Philadelphia chromosome-positive acute lymphoblastic leukemia. A phase II study in patients with locally advanced or metastatic triple negative breast cancer with dasatinib (n-44) was completed. Although the results were not very impressive, it was suggested that selected patients may benefit from Src inhibitors [28]. Several phase II studies are ongoing using dasatinib as a single agent or combined with zoledronic acid in breast cancer with bone metastasis, and combined with either aromatase inhibitors or chemotherapeutic agents in metastatic breast cancer. Saracatinib, a dual inhibitor of Src/Abl, has been shown to decrease levels of bone resorption markers in a phase I study in patients with solid tumors [29]. Phase II studies are ongoing to evaluate saracatinib effects in patients with breast cancer and bone metastases. One phase II study is comparing the effects of saracatinib and zoledronic acid in patients with breast and prostate cancers with bone metastasis. Bosutinib is also an oral tyrosine kinase inhibitor of both Src family and ABL. Bosutinib inhibits Src-mediated signaling pathways involved in breast cancer cell proliferation, angiogenesis, growth factor expression, motility, and invasion [30]. It is being assessed in phase III trials for CML and Philadelphia positive acute lymphoblastic leukemia. A phase II study of single agent bosutinib in metastatic breast cancer is under way. The combinations with aromatase inhibitors or chemotherapeutic agents with bosutinib are also being evaluated in metastatic breast cancer patients.Integrins are a family of cell surface receptors that primarily mediate interactions of cells with components of the extracellular matrix. Although osteoclasts express various integrins, it is generally well accepted now that avb3 integrin is a central molecule for osteoclast function [31]. There are several ongoing clinical trials evaluating the anticancer effect integrin antagonists in advanced refractory and metastatic cancers [32,33]. ATN-161, IMGN388 and L-000845704 are being evaluated in phase I/II studies and it would be interesting to examine the effects of these agents in clinical oncology [32,33]. C-X-C chemokine receptor type-4 (CXCR-4) is a chemokine receptor for stromal cell derived factor-1 (SDF-1 or chemokine ligand-12 CXCL-12). Several types of cancer over express CXCR-4 including breast cancer tissue but the normal breast tissue has low expression [34]. Also CXCR-4 is much more highly expressed in bone metastasis than in visceral metastasis [35]. The CXCR-4/SDF-1 axis is an attractive therapeutic target because of its role in bone metastasis. CTCE-9908 is a synthetic peptidic antagonist that reduces the formation of experimental lung and bone metastasis caused by CXCR-4 expressing breast cancer cells [36,37]. In a mouse model, treatment with CTCE-9908 did not reduce the frequency of metastasis, but did decrease both the tumor burden of breast cancer in bone, other organs, and also of the primary breast tumor [36]. A phase I/II study using CCE-9908 was conducted in 25 patients with refractory solid cancers. Tolerance was good and response was modest with progression of disease in 17 patients and stable disease in 5 patients [38]. The short half-life of CTCE-9908 may be a barrier to its use in breast cancer. AMD3100 (PLERIXAFOR) is a CXCR-4 inhibitor that promotes hematopoietic stem cells to mobilize from the bone marrow into the blood stream and is approved for use in autologous transplantation [39]. Clinical trials with this agent alone or with combination of bisphosphonates are also underway. TGF-β binds to a heteromeric complex of transmembrane serine/threonine kinases, the type I and type II receptors, activin receptor-like kinase 5 (ALK5) and transforming growth factor-beta receptor type II TβRII, which phosphorylate and activate the TGF-β specific intracellular signaling mediators Smad2 and Smad3. The phosphorylated Smad2/3 complex then binds Smad4 and translocates to the nucleus, where it regulates the transcription of TGF-β target genes [40]. It regulates the expression of many factors (integrin, IL-6, IL-11, matrix metalloproteinase-1 (MMP-1), CXCR-4) that are involved in bone metastasis formation [40]. Thus, the blockade of TGF-β signaling offers a target for therapeutic intervention to decrease bone metastasis. So far there are no TGF-related drugs in clinical trials for breast cancer with bone metastasis, although TGF inhibitors have been investigated in other types of solid cancers. AP12009 (Trabedersen), a TGF-β2-specific antisense oligonucleotide, showed encouraging results in patients with stage III/IV pancreatic cancer, colorectal cancer and malignant melanoma in a phase I/II study [41]. The SAPPHIRE study (Efficacy and Safety of AP12009 in Adult Patients with Recurrent or Refractory Anaplastic Astrocytoma (WHO grade III) as Compared to Standard Treatment with Temozolomide or Carmustine: A Randomized, Actively Controlled, Open-label Clinical Phase III Study) also randomized refractory astrocytoma patients to AP12009 or standard chemotherapy [42].A positive TGF-β gene-expression signature in estrogen receptor negative primary breast cancer was associated with high risk of metastasis to lung but not to the bone [43]. Novel biomarkers may be useful for assessing the clinical response to TGF-β inhibitors. The osteolytic lesions in bones from breast cancer results not only results from osteoclast-mediated bone resorption but inhibition of osteoblast-mediated bone formation has also a crucial role. This realization led to the development of therapeutic strategies aimed at restoring osteoblast function. The Wnt signaling pathway plays a key role in osteoblastogenesis. Wnt proteins bind frizzled receptor family members and in association with low density lipoprotein receptor-related protein (LRP)5/6, trigger downstream signaling via β-catenin, which includes activation of different genes involved in osteoblastogensis [44]. Elevated levels of DKK-1 were first described in the serum and bone marrow of patients with multiple myeloma [45]. The blockade of DKK-1 using neutralizing antibodies resulted in a decrease of both osteolysis and skeletal tumor growth in a severe combined immunodeficiency (SCID)-hu murine model of multiple myeloma [46]. In addition, DKK-1 antibody treatment increased in osteoblast number, serum human osteocalcin level, and trabecular bone, indicating that this antibody had bone anabolic effects [47]. A clinical trial is evaluating BHQ880 and zoledronic acid in relapsed/refractory multiple myeloma. There is pre-clinical evidence that breast cancer derived DKK-1 inhibits osteoblastogenesis [48]. Further studies are required to examine the importance of DKK-1 as a therapeutic target for breast cancer bone metastasis. It is a member of the TGF-β superfamily of growth factors that is widely present in different cells and tissues [7]. In bone metastasis, activin A produced by tumor cells acts as a stimulator of bone of bone degradation, inhibiting osteoblast differentiation and stimulating osteoclast differentiation [50]. The circulating levels of activin A are significantly higher in patients with breast and prostate cancer than patients without bone metastasis [49]. Therefore, this cytokine may be considered as a potential target for a more selective therapeutic approach in the treatment of skeletal metastasis.The endothelins are family of three small peptides: ET-1, ET-2, and ET-3 [50]. They are produced by different cells types, including breast and prostate cancer cells [50]. ET-1 is involved in the formation of osteoblasts, and it decreases osteoclast activity and motility [50]. ET-1 and ET-2 increase breast cancer cell migration and invasion in vitro [51]. Therefore, there is a rationale in studying the effect of endothelin antagonists in breast cancer patients. However, no clinical trials in breast cancer have been conducted so far. Majority of the patients get excellent palliation for localized metastatic bone pain with external beam radiotherapy. Several randomized trials have shown that a single fraction of 8 Gy is adequate for pain relief [52]. Radiopharmaceuticals are now available for the palliation of metastatic bone pain. Strontium has been shown to be as effective as wide field radiotherapy in prostate cancer [53] and because of the preferential uptake of strontium at sites of new bone formation, is probably most effective for sclerotic metastases. Samarium, which is linked to the bisphosphonates diamine tetramethylene phosphonic acid, has been evaluated in prostate and breast cancer. Samarium is also preferentially taken up at sites of bone formation, and emits both α and γ particles. The former allows imaging of the skeleton and the latter provides the therapeutic effects. Samarium is suitable for outpatient use and it has a significant effect on bone pain and analgesic consumption [54]. Further studies are indicated to compare radioisotope treatment with high-dose bisphosphonates and to determine whether the two treatment approaches complement one another. Bone metastasis is currently incurable and can be complicated by skeletal related events (SREs), which result in substantial morbidity and mortality. Bisphosphonates are currently the standard agents used for bone metastasis to reduce the frequency of the SRCs but they may have anti-tumor effects and could be useful for preventing and treating metastasis to bone and visceral sites. A more thorough understanding of the cellular and molecular mechanisms of bone metastases and bone microenvironment will help in developing novel agents. Given the complexity of the mechanisms of bone metastasis, combinations of drugs with different targets are probably needed to accomplish a successful outcome. This review highlights several molecular components acting at early or late stages during the development and progression of breast cancer bone metastases. These components are the new attractive targets for cancer therapeutics. They could be used in combination with bisphosphonates to efficiently block the development of skeletal lesions in women with breast cancer. The authors declare no conflict of interest. 

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+This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).The antitumor effect of bisphosphonates (BPs) is under increasing scrutiny. Preclinical and clinical evidence has shown that BPs might sensitize breast tumors to chemotherapy. Here, we present a review of current preclinical and clinical evidence for antitumor effects of BPs, and evaluate how BPs might play a role in neoadjuvant treatment of women with breast cancer.Neoadjuvant chemotherapy (NCT) is a generally accepted and worldwide standardized treatment for patients with locally advanced or large operable (stage II–III) breast cancer [1]. NCT is as effective as adjuvant chemotherapy following local treatment in terms of (recurrence-free) survival [2]. Besides the opportunity to study changes in tumor biology and response, NCT has the capability of downstaging breast tumors, facilitating in breast conserving surgery. The antitumor effect of adding bisphosphonates (BPs) to (neo)adjuvant chemotherapy for breast cancer is still under debate. There is emerging preclinical evidence for a synergistic effect of the most potent BP, zoledronic acid, in combination with chemotherapy, when administered after chemotherapy [3]. Clinical results suggest that BPs might improve treatment efficacy in patients with breast cancer [4,5]. Clinically, menopausal and/or hormonal status seem to play a role. The neoadjuvant model is ideal for gaining insight into the biological antitumor mechanisms of BPs in combination with NCT and can aid in defining predictors of response for this treatment strategy. Here, we provide a comprehensive review of preclinical and clinical evidence for the antitumor effects of BPs and a rationale for possible efficacy of BPs in the neoadjuvant setting.BPs are pyrophosphates and can be divided into two subgroups based on the structure of the R2 side chain: non-nitrogen containing BPs (e.g., clodronate), and the more potent nitrogen-containing BPs (e.g., zolendronic acid, alendronate, ibandronate, risedronate), which are widely used in the clinical setting [6]. Zoledronic acid is currently the most potent available BP containing two nitrogen atoms [7,8,9]. As all pyrophosphates, BPs easily bind to the bone mineral with the P–C–P chain at locations showing a high level of bone resorption. BPs inhibit the breakdown of hydroxyapatite, thereby suppressing bone resorption and promoting osteoclast apoptosis [10]. Nitrogen-containing BPs bind to and inhibit farnesyl pyrophosphate synthase (FPPS), which is an important regulatory enzyme of the mevalonate pathway (Figure 1), and which is responsible for the production of lipids needed for the posttranslational modification (prenylation) of proteins and activation of intracellular signaling proteins [11,12,13]. These signaling proteins are essential for cell functioning and survival, and osteoclast apoptosis is induced by inhibiting the posttranslational modification of proteins with isoprenyl [13]. Furthermore, nitrogen-containing BPs induce the production of an adenosine triphosphate analogue (Apppi) that can directly induce apoptosis [14]. In addition, bisphosphonates have been found to inhibit both osteoblast and osteocyte apoptosis [15].BPs have a well-established role in the prevention and treatment of osteoporosis and in the treatment of bone metastases, causing a reduction in pain, hypercalcemia of malignancy and skeletal related events (SRE), such as pain, pathological fractures, and spinal cord compression. These SREs are a major cause of morbidity and a reduced quality of life [7]. In addition to their use in treating osteoporosis and bone metastases, bisphosphonates are gaining recognition for the management of breast cancer through various mechanisms, and their use has grown rapidly in recent years. Needless to say, the mechanisms by which BPs prevent and decrease tumor burden in bone is currently still speculative and under investigation.There is ample evidence to suggest that the mechanism of bone metastases is multifaceted, comprising both bone resorption and bone formation aided by osteoblast and osteoclast activity [16]. In breast cancer, bone metastases are generally characterized by a predominantly osteoclastic activity, with osteolysis the result of osteoclast stimulation. In response, there is some degree of bone formation or bone repair, caused by osteoblasts [16].Mevalonate pathway and inhibition of farnesyl diphosphate synthase by bisphosphonates.Cancer cells produce a range of growth factors and cytokines that increase osteoclast activity [17]. Tumor production of factors including parathyroid hormone (PTH), PTH-related peptide (PTHrP) and interleukins (IL)-1, IL-6 and IL-11 stimulate the production of the cytokine, receptor activator of nuclear factor-KB ligand (RANKL), by osteoblasts and stromal cells. Following stimulation by PTHrP, RANKL induces osteoclast activity. PTHrP also causes a decrease in the production of osteoprotegrin (OPG), a receptor that prevents RANKL from binding to its receptor (RANK) on osteoclast progenitor cells, thereby blocking bone resorption [16].During bone resorption, other potentially tumor-stimulating growth factors such as transforming growth factorbeta (TGF-beta) and insulin-like growth-factor-1 are released by osteoblasts, facilitating tumor cell growth and proliferation, and attracting other tumor cells [17]. BPs may reduce tumor burden and growth by inhibiting this bone turnover. BPs do this, both directly, through the apoptosis of osteoclasts and tumor cells, and indirectly, through alterations in the bone microenvironment (Figure 2). Direct effects include the metabolism of non-nitrogen-containing BPs to an adenosine triphosphate analog that is toxic for macrophages and osteoclasts [14]. Nitrogen-containing BPs also work through several indirect mechanisms. For example, BPs may render the bone microenvironment less favorable for tumor cell growth. Namely, in case of skeletal metastases, a balanced coupling of osteoblastic bone formation and osteoclastic bone resorption is lost [18]. BPs can interrupt this vicious cycle of osteolytic bone loss.In addition, BPs inhibit angiogenesis, as demonstrated in one study, where zoledronic acid was found to reduce circulating levels of vascular endothelial growth factor (VEGF) after the first infusion in patients with metastatic bone disease [19,20]. Lastly, and most importantly in the neoadjuvant setting, BPs may reduce tumor burden by indirectly modulating the immune system. For example, BPs enhance cellular antitumor toxicity by attracting and triggering expression of γ/δ T-cells, which could be an important factor in antigen specificity and the ability to recognize and kill tumor cells [21,22,23]. Furthermore, bisphosphonates are suggested to differentiate monocytes into tumoricidal M1 macrophages [24].Schematic diagram of the interaction between the bone microenvironment and tumor cells.Tumor cells produce cytokines and growth factors which stimulate osteoclastogenesis, inducing an increase in bone resorption and the release of growth factors from the bone matrix. These factors stimulate proliferation, migration and angiogenesis of tumor cells. Bisphosphonates can break this vicious circle by inhibiting osteoclastogenesis and bone resorption. PTHrp = parathormone-related protein; PGE = prostaglandin E; RANKL = receptor activator of nuclear factor κβ ligand; OPG = osteoprotegerin; PDGF = platelet derived growth factor; IGF = insulin-like growth factor; IL = interleukin.Needless to say, there is still a need for more translational research giving insight into the alleged anti-tumor effect of bisphosphonates, and further investigations on the role of BPs are most certainly warranted. The neoadjuvant setting provides a suitable platform for this kind of research.Of particular interest is the potential for BPs to enhance the anti-tumor activity of cytotoxic agents in the context of (neo)adjuvant chemotherapy. In vitro data have shown that clinically relevant concentrations of doxorubicin followed by zoledronic acid consistently induced sequence-dependent synergistic apoptosis of cancer cells across several malignant cell lines [25]. However, the drugs alone, in the reverse sequence, and even given synchronously, had little or no effect on apoptosis [21]. In a mouse model, sequence-dependent synergy between doxorubicin and zoledronic acid was observed with complete inhibition of tumor growth associated with enhanced apoptosis and reduced proliferation and angiogenesis. These effects were statistically more pronounced when the zoledronic acid was administered 24 h after chemotherapy, suggesting that an initial priming of tumor cells by doxorubicin renders them more sensitive to subsequent exposure to zoledronic acid [26]. Possible molecular pathways by which sequential treatment with zoledronic acid and doxorubicin induce tumor cell apoptosis and inhibit proliferation were also shown in an in vivo model of breast tumor growth in the bone [27]. Interestingly, zoledronic acid specifically inhibited the development of bone metastases in an ovariectomy-induced/postmenopausal mouse model [28].Following previous discordant data with the less potent bisphosphonate, clodronate, in the adjuvant setting [29,30,31,32], the Austrian Breast Cancer Study Group-12 trial was the first adjuvant clinical trial to notice an improvement in disease-free survival (DFS), a reduction in distant (non-bone) metastases, locoregional and contralateral relapses, as well as a trend to reduced risk of death, with zoledronic acid (4 mg intraveneously every 6 months for 3 years) added to endocrine treatment with ovarian suppression in premenopausal breast cancer patients [33,34]. The protective effect of zoledronic acid persisted even after a median follow-up of 76 months, with zoledronic-acid-treated patients having a significant reduction in the risk of DFS events (27%) and a significant reduction in the risk of death (41%) when compared with controls [35]. Of note, all patients received goserelin, and were therefore postmenopausal, from an endocrionlogical viewpoint. This has probably contributed largely to the significant benefit of zoledronic acid in these patients. Three other similarly designed trials investigated the effect of delayed vs. upfront zoledronic acid on bone mineral density in postmenopausal breast cancer patients, with disease recurrence as a secondary endpoint (Z-FAST, E-ZO-FAST and ZO-FAST) [36,37,38]. Fewer DFS events with upfront zoledronic acid were only observed in the ZO-FAST study (37% RR, p = 0.05). Based on exploratory analyses, initiating zoledronic acid may have significant DFS benefits. In the AZURE trial, patients were randomized to standard therapy (any (neo)adjuvant chemotherapy and/or endocrine therapy), with or without zoledronic acid during 3 years. At a median follow-up of 59 months, no significant differences in DFS were found in the complete study population [39]. However, when concentrating on the subset of postmenopausal women, a statistically significant difference in DFS was found between the treatment groups (HR 0.74, p = 0.04). These results in postmenopausal women are consistent with the findings in premenopausal women in ABSCG-12 trial, suggesting that efficacy of zoledronic acid treatment is dependent on menopausal status and/or hormonal levels. Recently, this was confirmed in a meta-analysis of phase III studies by Yan et al. in which treatment with zoledronic acid did not improve DFS in breast cancer patients [5]. However, in the postmenopausal group, a significant benefit in terms of DFS (RR 0.75) distant (RR 0.74) and locoregional recurrence (RR 0.51), was found. Different results were found in a meta-analysis by Valachis et al. in which phase II studies were also included [4]. In this study, in which no specific analyses for postmenopausal women were done, zoledronic acid use resulted in a significantly better OS (HR 0.81) in patients with early-stage breast cancer, strengthening the argument for an antitumor effect of zoledronic acid.In a retrospective subset evaluation of patients in the AZURE trial, adding the BP, zoledronic acid, to neoadjuvant chemotherapy resulted in better tumor shrinkage and a doubling of the pathological complete response rate [40]. Patients who were treated with neoadjuvant chemotherapy received zoledronic acid 6 times every 3 or 4 weeks, depending on their chemotherapeutic schedule. Zoledronic acid also seemed to sensitize the tumor to the effects of neoadjuvant chemotherapy, as the pathological complete response rate was nearly doubled. The preliminary results of the AZURE trial have motivated investigators to investigate the possible benefit of zoledronic acid in the neoadjuvant setting. For example, our study group aimed to determine the pathological response of neoadjuvant chemotherapy, with and without zoledronic acid, in the NEOZOTAC trial. Here, HER2-receptor negative patients with stage II or III breast cancer are treated with 6 three weekly cycles TAC (docetaxel, doxorubicin, cyclophosphamide with pegfilgrastim), with or without zoledronic acid 4 mg intravenously administered within 24 h of the start of each cycle. The toxicity data of this study, showing that there is no significant difference in toxicity between the treatment arms, has recently been presented [41]. Biomarker data from biopsies and surgical specimens, as well as blood sera are currently being collected for translational research. Response results from several other neoadjuvant chemotherapy trials are expected soon (Table 1). In a study by Chavez-Macgregor et al. in which patients who were treated with neoadjuvant chemotherapy were retrospectively identified for pCR rate evaluation, 39 patients received bisphosphonates [42]. The pCR rate was higher in the bisphosphonate group than in the non-bisphosponate group, although not statistically significant (25.4% vs. 16%, p = 0.11). Furthermore, the JONIE-1 group recently presented data of their phase III trial comparing neoadjuvant chemotherapy with and without zoledronic acid [43,44]. Interestingly these results not only suggested that postmenopausal women benefit more from zoledronic acid therapy (18.4% vs. 5.1%, p = 0.07), but also that triple-negative bisphosphonate-treated patients respond better than their chemotherapy-only counterpart (35.3% vs. 11.8%, p = 0.06). Aft et al. reported a study in which 120 patients were allocated to a neoadjuvant/adjuvant chemotherapy schedule (with four cycles of neoadjuvant epirubicin plus docetaxel and two cycles of adjuvant epirubicin and docetaxel) with no zoledronic acid or zoledronic acid (4 mg i.v.) every 3 weeks, for 1 year [45]. The primary endpoint was the number of patients with detectable disseminated tumor cells (DTCs) at 3 months. Less DTCs were detected in the zoledronic acid group, suggesting that neoadjuvant treatment with zoledronic acid might affect long-term outcome by preventing metastasis. However no significant difference in pathologic complete response was found (22% in the zoledronic acid arm vs. 16% in the control arm, p = 0.63), although more pathologic complete response was observed in estrogen receptor (ER)-negative/HER2-negative patients (29% in the zoledronic acid arm vs. 11% in the control arm). Interestingly, at 5-year follow up, significantly less death and recurrence events occurred among patients with estrogen receptor-negative tumours, which was not observed in the total study group. Neoadjuvant treatment might therefore indeed have a beneficial effect on long-term outcome [45].As previously mentioned, neoadjuvant studies are valuable for translational research. An example of this in the context of zoledronic acid treatment is the ANZAC study [46]. In this study 40 patients were randomized to neoadjuvant chemotherapy with or without a single infusion of zoledronic acid after the first cycle. This way, short therm biologic effect induced by zoledronic acid could be investigated. The authors found that a greater reduction in serum vascular endothelial growth factor (VEGF) occurred in the zoledronic acid group at day 5 than in the control group, although this effect could not be observed after day 21. Furthermore, the authors investigated serum reproductive hormones within the TGF-beta family (e.g., activin, TGF-beta-1, inhibin and follistatin) and observed that follistatin levels dropped more from baseline in postmenopausal zoledronic acid treated patients, which is interesting considering the still puzzling benefit of zoledronic acid in postmenopausal women. Summary of neoadjuvant studies with chemotherapy in combination with zoledronic acid *.Change in disseminated tumor cells in the bone marrowChange in serum markers of apoptosisChange in tumor markers of apoptosis and proliferationAssessment tumor responseChange in circulating gamma-delta-T-cell activationToxicityDifference in gene expression according to treatment responseImpact of zoledronic acid on relapseEffect of treatment on quality of life inClinical responseTolerabilityDisease-free and overall survivalHeterogeneity of ER/PR and HER2 measurement in core biopsy and surgical specimenLong-term outcome (disease free survival and overall survival)Clinical responseDisease free survivalReduction in Ki67 between preoperative biopsy and operative specimenChanges in serum angiogenesis markersChanges in bone biochemical markersDetection of and changes in circulating tumor cells in peripheral bloodPrediction of pathological response by MRI calculated from the sequence of apparent diffusion coefficient* Data obtained from [47].In summary, there are several features of bisphosphonates which can contribute to an anti-tumor effect and can inhibit tumor growth. This given in combination with still sparse preclinical and clinical evidence for a benefit of neoadjuvant treatment, helps warranting clinical and translational research into this field. In the next few years response results and long-term outcome results of several neoadjuvant chemotherapy trials are expected. Translational research is represented in most of these trials. Hopefully, clinical and translational results will provide more answers to the question whether zoledronic acid in combination with chemotherapy can enhance tumor response.The authors declare no conflict of interest.

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+This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).Multipotent mesenchymal stromal cells or mesenchymal stem cells (MSCs) are adult stem cells exhibiting functional properties that have opened the way for cell-based clinical therapies. MSCs have been reported to exhibit immunosuppressive as well as healing properties, improving angiogenesis and preventing apoptosis or fibrosis through the secretion of paracrine mediators. This review summarizes recent progress on the clinical application of stem cells therapy in some inflammatory and degenerative rheumatic diseases. To date, most of the available data have been obtained in preclinical models and clinical efficacy needs to be evaluated through controlled randomized double-blind trials.Mesenchymal stem cells, or stromal cells (MSCs) are progenitor cells mainly isolated from adult bone marrow (BM), and adipose tissue. MSCs are also present in most adult tissues, including muscles, synovial tissue, placental tissue, and teeth [1]. The last two localizations allow easier collection and therefore an extension of their use. They have several functions: Synthesis of extracellular matrix, immune tolerance, development, inflammation and fibrosis. Each of their properties seems to have promising future therapeutic applications in various diseases (see Figure 1).Schematic representation of mesenchymal stem cell (MSC)-based therapy via intra-articular injection in the treatment of rheumatic diseases. Through the release of trophic factors and cell contact, MSCs may act to reduce cartilage degeneration, osteophyte formation and synovial inflammation. MSCs may also have systemic properties that may be of interest in extra-articular manifestations of rheumatic diseases.MSCs are defined by their functional abilities of differentiation and differ from hematopoietic stem cells by the expression of mesenchymal markers (CD105, CD70, CD90), while lacking expression of CD34, CD45, CD14 monocyte or markers of T or B cells, or the major histocompatibility class II (MHC II) [2,3]. MSCs have a phenotypic heterogeneity with some multipotent properties and are the progenitors of multiple lineages including bone, cartilage, muscle or fat. Understanding the mechanisms of action of MSCs in various diseases can be hampered by their phenotypic heterogeneity since each phenotype may be associated with a different biological response. MSCs are currently being studied for tissue engineering applications, including bone and cartilage repair because of their potential to differentiate into different lineages such as chondrocytes, osteoblasts or adipocytes [3]. Recently, these cells have also been shown to have immunosuppressive and healing capacities, to improve angiogenesis and prevent fibrosis.MSCs have immunomodulatory and immunosuppressive properties and are involved in both the innate and the adaptive immunity [1,4,5]. This immunosuppressive effect is mainly due to the secretion of soluble factors by MSCs and by direct contact with immune cells. MSCs acquire their immunosuppressive properties after exposure to an inflammatory environment. Some cytokines such as tumor necrosis factor alpha (TNF-α), interleukin 1 beta (IL-1-β) or interferon gamma (IFN-γ) are able to activate MSCs [6].Whatever the T cell status (naive or activated), MSCs have a suppressive role in CD4+ and CD8+ T cells by blocking their cell cycle by inhibiting the expression of cyclin D2. Dendritic cells (DCs) are the key antigen presenting cells. MSCs modulate the immune response because they are able to avoid the transformation of CD34+ DC into mature DC (via the co-secretion of IL-4 and granulocyte-macrophage colony-stimulating factor (GM-CSF)) [7]. MSCs also promote a failure of antigen presentation by DCs [7]. MSCs in contact with DC acquire a tolerogenic profile producing a large amount of IL-10 and decreasing the secretion of IL-12 [8]. Finally, MSCs induce the expression of a regulator phenotype (Treg) CD4+CD25+Foxp3+ in different murine models (asthma, diabetes, autoimmune encephalitis, etc.). Recently, our team showed that the beneficial effect observed following MSCs injection in an autoimmune mouse model was associated with the suppression of Th17 cells and the increase in CD4+CD25+Foxp3+ T lymphocytes percentage [9].These suppressive properties of MSCs not only depend on cell contact but also on secretion of regulatory molecules [5]. Adhesion molecules are involved in MSC-mediated immunosuppression. In a MSC and T cell coculture system, activated T cells secrete several inflammatory cytokines, including IFN-γ, TNF-α, and IL-1. The combinations of IFN-γ with TNF-α or IL-1 can upregulate the expression of adhesion molecules: Intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1). Importantly, blocking of the function of ICAM-1 and VCAM-1 significantly reversed the immunosuppressive effect of MSCs in vitro and in vivo [10]. Programmed death-1 (PD-1) is a molecule expressed on various cell types that plays an important role in the negative regulation of immune responses and the maintenance of peripheral tolerance [11]. Our team demonstrated that MSC suppressive effect on mature Th17 cell function and proliferation is contact-dependent and mediated by PD-1 pathway up-regulation on primed MSCs [12]. Thus, these molecules are important for MSC-mediated immunosuppression through cell-cell interactions.Some mediators, indoleamine 2,3-dioxygenase (IDO), nitric oxide synthase (iNOS) as well as the secretion of human leukocyte antigen (HLA-G), transforming growth factor (TGFβ), interleukin-6 (IL-6), protein that stimulates the TNFα gene (TSG6) or prostaglandin E2 (PGE2) have been proposed to play a significant role in the suppressive properties of MSCs. Contradictory results have been reported on the implication of IDO in MSC-mediated immunosuppression. While a key role of IDO is suggested by a majority of studies [13,14], human MSCs deficient in the expression of both IFN-γ receptor 1 and IDO activity still exert important immunomodulatory activity [15]. Secretion of nitric oxide (NO), produced by iNOS in MSC has been shown to inhibit T cell proliferation involving a Stat5-dependent pathway [16]. NO is considered as an important cytotoxic effector molecule, the activity of which is dependent on the presence of IFN-γ [17]. Furthermore, MSCs from iNOS −/− mice have a reduced ability to suppress T cell proliferation, both in vitro and in vivo, in a model of graft-versus-host disease and delayed-type hypersensitivity [17].MSCs seem to be immune privileged cells due to the low expression of MHC and co-stimulation mechanisms of T cell (CD80/CD86, CD40) [4]. Soluble HLA-G also seems to participate in this tolerance [18]. These features help prevent a rapid rejection and immune sensitization. However, transplant rejection of MSCs has been described in the literature in the long term [19]. NK cells may be involved in increasing rejection of allogeneic donor BM cells. MSC had only a partial inhibitory effect on proliferating NK cells. More importantly, activated NK cells could lyse efficiently MSCs [20]. However, MSC may suppress NK activation, and this enhanced by IFN-γ prestimulation. This occurs through multiple mechanisms, including PGE2 secretion [21]. At low concentration of IFNγ or activation by Toll-like receptors (TLR), increased expression of MHC II and stimulation of CD4+ proliferation do not affect enough immunogenicity of MSCs [22]. These findings should be taken into consideration when using MSCs in clinical trials, including whether autologous or allogeneic MSCs should be employed.In tissue repair, MSCs do not seem to have a direct effect, but they stimulate the regenerative properties of resident cells. They have a paracrine effect by reducing the release of pro-inflammatory cytokines and by stimulating the secretion of anti-inflammatory cytokines. MSCs exert their regulatory role by forming a perivascular niche in close contact with endothelial cells and osteoblasts in bone marrow and in close relationship with the immune and hematopoietic stem cells [5,23]. MSCs operate in collaboration with endothelial factors (epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), insulin-like growth factor 1 (IGF-1), stromal cell-derived factor 1 (SDF-1), transforming growth factor (TGF), Angiopoietin 1) to improve their “homing” on damaged sites. The various studies on intravenous injection of MSCs show a redistribution of cells mainly in lungs and a modest uptake by the liver and kidney. However, the biodistribution of MSCs may be modified in some pathological conditions (inflammation, senescence, etc.). Kraitchman et al. found evidence of trafficking of MSCs to the heart after induction of ischemic lesion suggesting the responsibility of chemotactic factors [24].MSC-based cell therapies represent innovative strategies for the treatment of rheumatic diseases for which currently available treatments are limited and rarely restore the full functions of the tissue. In Figure 2, we summarized some therapeutic applications of MSCs in rheumatic diseases.Current therapeutic applications of mesenchymal stem cells (MSCs) in rheumatic diseases.Osteoarthritis (OA) is a multifactorial disease, quite debilitating, which can affect both weight-bearing joints such as knees and non-bearing joints such as hands. Despite a very high prevalence (12% in the age group >60 years), no effective disease modifying OA drug is available and patients must often have a total joint replacement [25]. OA leads to progressive degeneration of the cartilage, sclerosis of the subchondral bone with formation of osteophytes and subchondral cysts. Severe OA causes chronic pain, stiffness, deformation and effusion, leading to reduced function and quality of life.The rationale for using local injection of MSCs for inducing regeneration of OA cartilage is based on a first in vivo study in a caprine model [26]. Whatever the source of MSCs (BM, adipose tissue or synovium), factors secreted by MSCs increased cartilage matrix production by chondrocytes. Adipose-derived stem cells (ASCs) share many properties with MSCs but may easily be collected through liposuction and be used in human trials. Local injection of BM-MSCs or ASCs in the joint is likely to exert several roles: Inhibition of osteophyte formation, decrease in synovial inflammation, reduction in cartilage degeneration with less fibrosis and apoptosis of chondrocytes or stimulation of chondrocyte proliferation and extracellular matrix synthesis. However, neither the exact mechanism of action when ASCs or BM-MSCs are not in direct contact with chondrocytes, nor the identification of possible mediators, have been investigated.Although OA is not considered an inflammatory disease, pro-inflammatory mediators, such as cytokines, metalloproteinases (MMP), reactive oxygen species (ROS), are secreted by OA chondrocytes or synoviocytes and participate in joint tissue alterations. Several pro-inflammatory cytokines are significantly down-regulated in chondrocytes when cultured with ASCs suggesting that ASCs may also be protective through the down-regulation of inflammatory mediators [27]. Interestingly, paracrine factors of BM-MSCs share the same anti-inflammatory effects on OA cartilage and synovial explants in vitro [28]. Moreover, a significant decrease in TGF-β1 secretion by chondrocytes and induction of Hepatocyte Growth Factor (HGF) secretion by ASCs was observed in OA explants [29].Recently, new studies have provided interesting data on animal models. In the first work, a single ASC injection in the knee joint of mice showed significant decrease in synovitis score and cartilage damage probably by suppressing synovial macrophage activation [30]. The local benefits have been replicated in a rabbit model of OA induced by meniscectomy and anterior cruciate ligament transection [31]. The cells were found locally up to 6 months after intra-articular injection [32]. Biodistribution and toxicology studies have confirmed safety of ASCs injections [32].In humans, no controlled studies have been published yet. In order to prevent OA, MSCs have been administered locally in 55 patients undergoing meniscectomy, and absence of local side effects was reported (Chondrogen® Osiris Therapeutics Inc. trial, Columbia, MD, USA). Recently, in Iran, four patients with moderate-to-severe knee OA were selected for a Phase I study [33]. Autologous BM-MSCs were injected in the knee joint. They observed improvement in walking time and reduction in walking pain in three patients. Most importantly, no side effects were reported after 1-year follow-up [33]. Moreover, another Iranian phase I clinical trial recently reported that intra-articular injection of autologous BM-MSCs in six patients with knee OA was safe and improved pain and functional status of the knee. As important, magnetic resonance imaging (MRI) displayed increased cartilage thickness and decreased subchondral oedemas in three out of six patients [34]. All these data support the trophic action of MSCs for protecting cartilage from degradation and stimulating regeneration. A clinical phase I trial has been initiated at the sites of Montpellier (France) and Würzburg (Germany). The principle is based on liposuction, expansion of ASCs and their local autologous injections in knee osteoarthritis. This trial (NCT01585857) has obtained the authorization and agreement of the National Security Agency (ANSM). The study is in progress and preliminary results show excellent tolerance to the local injection. However, due to the low number of patients and the absence of a control group it is too early to draw any conclusion of clinical benefit.Currently, no human clinical trial has been conducted in rheumatoid arthritis (RA). In the collagen-induced arthritis (CIA) murine model, which is representative of RA in humans, contrasted results have been reported. Thus, it has been reported that a single injection of primary murine MSCs prevents the onset of arthritis, which was associated with a decrease in serum pro-inflammatory cytokines and increased Treg [35,36]. The therapeutic benefit of xenogeneic human MSCs, from either adipose tissue or umbilical cord, has also been described [37,38]. However, other studies failed to demonstrate any improvement with MSC treatment. Systemic infusion of the allogeneic C3H10T1/2 cell line increased local inflammation and the clinical signs of arthritis [39]. The immunosuppressive effect of primary murine BM-MSCs seems to depend on genetic backgrounds. In contrast to allogeneic MSCs, syngeneic or partially mismatched MSCs may delay the time of onset of clinical disease and alter disease progression in CIA [40]. Alternatively, the immunomodulatory role of BM-MSCs was reported to be dependent on the window of injection, with therapeutic benefit only when two cell injections on day 18 and 24 were done [36].Systemic Lupus Erythematosus (SLE) is an autoimmune disease with various clinical features that may involve life-threatening, such as renal or neurological manifestations. SLE is characterized by B-cell hyperactivity, autoantibody production as antinuclear, anti-DNA and anti-Sm. Several studies are underway. The first Chinese pilot study was published in 2009 on 4 patients treated with doses of 1 to 10 million cells per kg [41]. Two other cases of refractory SLE treated with autologous MSCs were published [42]. These patients underwent injection of autologous bone marrow MSCs without therapeutic benefit. One patient developed renal failure due to SLE. Tolerance injection was acceptable and the authors observed a slight increase in the circulation of regulatory T cells.More recently, two phase I/II studies were published in lupus nephritis, one of 15 patients with allogeneic MSCs from BM [43] and the other of 16 patients with MSCs derived from umbilical cord [44]. Most patients improved clinically and serologically, but the follow-up was short and pre-treatment may have affected the results.Recently, MSCs have been evaluated in 35 SLE patients with refractory cytopenia, of which 20 had leukopenia and 24 had thrombocytopenia [45]. The authors found a significant improvement in haematological parameters for most patients. Clinical remission was accompanied by an increase in Treg and a decrease in Th17. Two patients died during the study.In all published series, the feasibility and safety are acceptable, although the efficacy remains an unresolved issue. Large prospective randomized studies are needed, particularly in refractory lupus nephritis.The immunosuppressive properties of MSCs have also been recently evaluated by Chinese authors in Sjögren’s syndrome (SS). In a beautiful work, Xu J. et al. have confirmed in a non-obese diabetic (NOD) mice model the benefit of allogeneic MSCs from bone marrow on the modulation of the immune response by promoting the production of Treg lymphocytes and switch to a Th2 profile [46]. They have highlighted a reduction in the production of Th17 after injection of MSCs. They confirmed these results by injection of allogeneic MSCs, derived from umbilical cord, in a heterogeneous panel of 24 patients affected by SS. Eleven patients had refractory dryness syndrome and 13 had systemic involvement of the disease (thrombocytopenia, anemia, hepatitis, tubulopathy, interstitial pneumonia, enteritis and neurological complications). Although patient characteristics, associated treatment during follow-up and the lack of control arm can be criticized, it is the first study suggesting clinical efficacy (visual analog scale (VAS) patient activity score EULAR Sjögren’ syndrome disease activity index (ESSDAI), salivary flow) of MSCs in the Sjögren’s syndrome. In addition, it is very interesting to note biological effectiveness with a sharp and rapid decrease in the production of anti-SSA antibodies.Systemic sclerosis (SSc) is a rare disease where therapeutic benefit can be expected because of the pro-angiogenic and anti-fibrotic effects mediated by MSCs. MSCs isolated from the bone marrow of patients with SSc have differentiation properties similar to MSCs from healthy donor and maintain their immunosuppressive properties on T cells [47]. In these patients, the ability of MSCs to differentiate into endothelial progenitor cells seems reduced as migration functions and pro-angiogenic potential of these progenitors [48]. These data support on the use of autologous or allogeneic MSCs in this disease.Currently, we have few observations of the use of MSCs in patients with SSc. One patient with severe SSc, refractory to conventional treatments, was treated by intravenous injection of allogeneic MSCs [49]. Three months after injection, a significant decrease in the number of ulcers was observed. At 6 months, blood flow to the hands and fingers and transcutaneous partial pressure of oxygen were significantly improved. Rodnan skin score was reduced from 25 to 11. On the other hand, the benefit of the administration of autologous MSC was recently described in one patient by obtaining a regression of the distal necrosis surfaces after the first injection of MSCs [50].Recently, Keyszer et al. reported observations of five patients with refractory SSc who received the injection of MSCs [51]. The treatment appears to be relatively well tolerated. Although their results were quite heterogeneous, it made them consider phase I–II clinical trials in this disease. In France, a National Hospital Clinical Research Program coordinated by D. Farge (Saint-Louis, MO, USA, Paris, France) was held in 2011 and is expected to begin shortly.MSC-based cell therapies represent innovative strategies for the treatment of rheumatic diseases for which currently available treatments are limited and rarely restore the full functions of the tissue. Several studies are ongoing. We have listed in Table 1 all the studies currently under recruitment and referenced in ClinicalTrials.gov registry. We look forward to the results because we need to evaluate clinical efficacy of stem cells through controlled randomized double-blind trials.Summary of studies currently under recruitment in ClinicalTrials.gov using MSC as a therapeutic option in rheumatic diseases. OA: osteoarthritis; RA: rheumatoid arthritis; SLE: systemic lupus erythematosus; AS: ankylosing spondylitis; ASC: adipose stem cell; BM: bone marrow; MSC: mesenchymal stem cell; MPC: mesenchymal precursor cell; UC: umbilical cord; IA: intra-articular; IV: intra-venous.New therapeutic applications of MSCs aim at interfering with immune responses of patients in various inflammatory autoimmune disorders or inhibiting progress of the clinical symptoms in degenerative diseases. Besides current research on mechanisms regulating the therapeutic efficacy of MSCs, more knowledge on migration, biodistribution, survival and safety of MSCs need to be obtained for generalized therapeutic use in rheumatic diseases.We thank Valerie Macioce for her support in linguistic adjustments. Figure 1 and Figure 2 were designed by using pictures of Servier Medical Art service.The authors declare no conflict of interest.

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+This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).A 31-year-old woman presented with a 7-week history of irregular vaginal bleeding without abdominal pain. She had been using the intrauterine contraceptive device (IUD) for the last 3 years. A pregnancy test was positive and subsequent serum beta human chorionic gonadotropin (β-HCG) was 4992 mIU/mL. A transvaginal ultrasound scan demonstrated an empty uterus with an associated adnexal mass but no free fluid. A right primary ovarian ectopic pregnancy was diagnosed a laparoscopy. This was managed laparoscopically using monopolar diathermy hook with conservation of the ovary and minimal blood loss. Ovarian pregnancy is rare, especially in women without the classical risk factors for tubal pregnancy, and efforts should be made to exclude ectopic pregnancy in the absence of abdominal pain in a woman of reproductive age presenting with prolonged and irregular vaginal bleeding. Methods to conserve the ovary are also encouraged in cases of ovarian pregnancy.Ovarian pregnancy is rare and remains a diagnostic and management challenge, especially in settings where health resources are stretched [1]. A potential major surgical and clinical problem is intra-peritoneal bleeding and difficulty in achieving haemostasis to conserve the ovary. This is in addition to the difficulty in accurately arriving at a diagnosis pre-operatively. The aim of this article is to describe a case of an unruptured primary ovarian pregnancy managed laparoscopically using diathermy hook with conservation of the ovary. In addition, it would add to the literature of methods to conserve the ovary in cases of ovarian pregnancy.A 31-year-old African woman presented with a seven-week history of irregular vaginal bleeding without abdominal pain. She was married and gave vaginal birth twice. The patient had been using a copper intrauterine contraceptive device (IUD) for contraception and her medical history was unremarkable. On examination, the patient was haemodynamically stable. The abdomen was soft and non-tender and pelvic examination was unremarkable. A urine pregnancy test was positive with an initial β-HCG of 4992 mIU/mL. A subsequent trans-vaginal ultrasound scan demonstrated normal size uterus with the IUD in the correct position, an endometrium measuring 2.8 mm with no intra-uterine gestation sac. The left ovary appeared normal and a 33.4 mm × 30 mm × 28 mm mass adjacent to the right ovary was noted with no free fluid in the pelvis (Figure 1). The (copper T 380) IUD had been removed and a repeat β-HCG in 48 h was 4232 mIU/mL. Ultrasound image demonstrating a mass (ma) in the right adnexa (A); (B) shows a thin endometrium with a normally sited intrauterine contraceptive device (IUD) (iu) in the uterus (ut).Considering a slow fall of HCG, a diagnostic laparoscopy was planned. Laparoscopy was performed through a 10 mm trans-umbilical port and two further secondary (right-5 mm, left-10 mm adjustable) ports inserted on each iliac fossa, lateral to epigastric vessels. The uterus and both the Fallopian tubes appeared normal without dilatation or signs of intra-tubal hemorrhage and no bleeding from the fimbriae. A haemorrhagic mass (20 mm × 30 mm) of dark red color with intact surface attached to the right ovary was noted (Figure 2). The left ovary clinically appeared normal. The pouch of Douglas contained 30 to 40 mL of serous peritoneal fluid and no evidence of endometriosis or pelvic adhesions was noted. A clinical diagnosis of right ovarian pregnancy was made. Laparoscopic view of an unruptured right ovarian ectopic pregnancy. (Ut = uterus, POD = pouch of Douglas, Ect = ectopic pregnancy, Ov = right ovary, Tu = Fallopian tube).The ovary was stabilized by holding the ovarian ligament with atraumatic grasping forceps. The suspected ovarian pregnancy was dissected off intact from the right ovarian surface, by monopolar diathermy hook with conservation of the ovary. Normal saline irrigation was carried out and ovarian haemostasis checked. There was hardly any intra-operative bleeding noted from the ovary. The specimen retrieved through 10 mm port by means of an endopouch® (Ethicon Endo-Surgery, Norderstedt, Germany). On postoperative day one β-HCG dropped down to 1547 mIU/mL. Histology demonstrated the presence of chorionic villi and ovarian stromal tissue including part of the corpus luteum confirming ovarian ectopic pregnancy. She was followed up for 3 weeks until serum HCG was below 5 mIU/mL.Primary ovarian pregnancy is when the primary nidation occurs in the ovary as opposed to secondary ovarian pregnancy where the initial implantation is in the Fallopian tube and the ovarian attachment is secondary to tubal abortion. Ovarian pregnancies can be associated with IUD in situ (4%–20%), but there is however no direct evidence linking IUD with ovarian pregnancy [2]. Cases of ovarian pregnancies have also been described following ovarian hyperstimulation with gonadotropins or clomiphene citrate treatment and after assisted conception procedures [3,4,5]. The clinical manifestations may be similar to those expected in tubal pregnancy, i.e., abdominal pain and vaginal bleeding, but in this case, the patient conceived with an IUD in situ and presented only with prolonged painless vaginal bleeding. The rarity of this condition, the variability in clinical presentation and the lack of a discriminatory test make the condition more difficult to diagnose pre-operatively [1,3,6]. Moreover, although β-HCG remains a useful adjunct for diagnosis of ectopic pregnancy, there are reports of ruptured ovarian pregnancies even with decreasing β-HCG levels [7]. This clearly adds to the diagnostic dilemma.Most of the clinical diagnosis of an ovarian pregnancy is made during surgery when a haemorrhagic mass is noticed attached to one of the ovaries in the presence of normal looking fallopian tubes. It could be difficult to distinguish an ovarian pregnancy from a haemorrhagic corpus luteum or ovarian cyst [1,3,8]. Since the first publication of laparoscopic surgery for early ovarian pregnancy in 1988 [9], laparoscopic therapy is now the method of choice after diagnosis [10,11]. Laparoscopic surgery includes partial ovariectomy (wedge resection), ovarian cystectomy or blunt dissection of the trophoblastic tissue. Ovariectomy or adnexectomy is considered for advanced or ruptured cases [1,3,6,9]. The main problem during surgery is to procure ovarian haemostasis; which is usually achieved by applying haemostatic suture laparoscopically or using diathermy forceps (bi-polar or mono-polar) [1,3,6]. In general, diathermy or electrosurgery is the use of high frequency electric current to produce heat. These frequencies are usually greater than 100 kHz in order to avoid spasm or paralysis of muscle. With bipolar diathermy, current passes between the tips of the instruments, e.g., forceps unlike monopolar where electricity exits the patient via the indifferent electrode placed on the patient. The main clinical use of diathermy is to cut or destroy tissue to produce coagulation but the actual or specific effects, i.e., coagulation or cutting effect will depend on the current intensity and wave form used. In the case series by Seinera et al. [10], removal of the products using biopsy forceps was the mainstay of treatment at laparoscopy in the majority of cases with haemostasis achieved using thermal coagulation. There were no cases reported of persistent trophoblast [10]. The good outcome from this case series is reassuring as it offers women the opportunity to preserve their fertility. Similarly, in Odejnimi et al.’s [11] case series, 11 out of 12 cases of ovarian pregnancy were successfully managed laparoscopically with conservation of the ovary while one had oophorectomy [11]. In this case, we used diathermy hook (mono-polar) to resect the ovarian pregnancy intact providing diathermy and surgical resection with minimal bleeding and conservation of ovary. Most case reports do not specifically describe the type of surgical instrument used and therefore hard to assess the types of instruments used for conservative management and how they differed if any from ours. However, this is not unexpected because the case reports or series generally focuses on the general management of ovarian pregnancy and their associated outcomes rather than the specific surgical tools or forceps used for their treatment. Whether the type of instrument used is clinically relevant or provides better surgical haemostasis is open to further debate. Certainly, the publication of more case series may provide more insight into ovarian pregnancy management and the associated surgical techniques employed.Primary ovarian pregnancy is very rare, difficult to diagnose and associated with intraoperative complications including bleeding, risks of losing the ovary. In this case, we used diathermy hook (mono-polar) to dissect the ovarian pregnancy intact with conservation of ovary; a technique that is easy to learn by the trainee as well. This case adds to the literature of reports describing methods to conserve the ovary in cases of ovarian pregnancy. More importantly, it also highlights the importance of having an index of suspicion for ectopic pregnancy in women of reproductive age presenting with vaginal bleeding even in the absence of abdominal pain. This is more so the case when other risk factors for ectopic pregnancy, e.g., IUD, are present.The authors declare no conflict of interest. Data in this manuscript was presented as a poster at the annual meeting of the Association of Early Pregnancy Units (AEPU), London, UK in November 2012.

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+These authors contributed equally to this work.This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).With a constellation of stem cell sources available, researchers hope to utilize their potential for cellular repair as a therapeutic target for disease. However, many lab-to-clinic translational considerations must be given in determining their efficacy, variables such as the host response, effects on native tissue, and potential for generating tumors. This review will discuss the current knowledge of stem cell research in neurological disease, mainly stroke, with a focus on the benefits, limitations, and clinical potential.With the increasing diversity of stem cell sources emerging for donor cells in transplantation therapy, many laboratory-to-clinic translational factors must first be considered, dynamics such as the source of the cells, ease of extraction, immunogenicity, capacity for proliferation, and cell yield. These concerns may serve as potential limitations respective to the donor cell origin being considered, proving a particular source to be a more suitable therapy for a specific disease.Harvesting of stem cells may be divided into two domains, allogenic vs. autologous sources (though xenogeneic cells have been previously tested). Autologous stem cells are acquired from the host in which the cells are intended for use, while allogenic cells are procured from an unrelated donor prior to transplantation. As one may expect, the use of allogenic stem cells may predispose an individual to various immunologic complications upon treatment, giving rise to the significant limitation of graft rejection with this method of treatment. Yet, autologous treatments may be limited by their ability for propagation and cell yield. The immunological barriers, such as graft vs. host or required immunosuppression of the host, are of constant consideration in the therapeutic benefits and limitations of stem cell transplantation. Prior research of immunocompromised stroke animals demonstrated inhibition neurogenesis in the cortex endogenously via a CD4+ T cell, but not CD25+ T cell mechanism, supporting the influence of immunodeficiency in reducing stem cell apoptosis [1]. In another study, upon exposure to cyclosporine A, an immunosuppressant, there was enhanced recovery of cortical injury following stroke secondary to endogenous stem cell activity and migration [2]. These papers support the hypothesis that inflammation following a cerebral event may not only damage tissue, but also further disrupt the endogenous neurogenic pathways of repair involving the migration of stem cells from the lateral ventricle regions of the brain, a topic further discussed in the neural stem cell section. This contrasts the proposed mechanism of action for neurotrophic modulators that are believed to furnish a microenvironment conducive to repair, but do not yield new neurons [2].Because of the potential immunological host response to the transplanted cells, much research has been conducted investigating specific cell line’s prospective immunogenicity. Surmounting evidence suggests that the more naive the cell lineage, the less likely the incidence of immunological reaction following transplantation. For instance, umbilical cord blood, due to its immunological immaturity, is less likely to invoke an immunological response and therefore less likely to require immunosuppression. More so, human leukocyte antigen (HLA) matching may be less stringent in umbilical cord blood transplants compared to bone marrow derived transplants and even whole tissue grafts, leading to higher cell viability [3]. Contrasting immunogenicity, some cell lineages may be more immunosuppressive than bone marrow derived stem cells. HLA-G, a contributing factor of immunosuppression [4], is of higher expression in chorionic plate-derived mesenchymal stem cells compared to bone marrow-derived stem cells and adipocyte tissue-derived mesenchymal stem cells [5], suggesting its usefulness as a prognostic indicator of transplant viability in the presence of host immunity [6]. Additionally, placenta-derived mesenchymal stem cells demonstrate less immunomodulation than bone marrow-derived mesenchymal stem cells, suggesting regenerative transplantation potential to be less efficacious [7]. Stroke is a major unmet clinical need with only one current Food and Drug Administration (FDA)-approved drug, the tissue plasminogen activator, efficacy limited to 4.5 h after stroke onset. Accordingly, the challenges of proliferation capacity and cell yield are evident with regards to the optimum delivery time of stem cell therapy. With the current clinical trials of cell therapy for acute stroke mostly targeting a window of 48 h, the potential limitation in generating a sufficient number of autologous cells from freshly harvested tissue for therapy in such a short period is apparent [8]. In view of this limitation, allogeneic transplantation is indicated when contemplating with acute stroke therapy. Alternatively, the extended time required for cell amplification with autologous stem cell transplantation renders it more appropriate for chronic stroke therapy. Nonetheless, regardless of autologous or allogeneic stem cell sources, cell harvesting imparts additional technical challenges. For example, acquiring neural stem cells may require invasive procedures that may be disadvantageous despite the therapeutic potential of the cells.The following sections aim to outline the different tissue sources available for harvesting stem cells, along with their respective benefits, limitations, and prospective use in clinical application as they pertain mainly to neurological diseases, most notably stroke therapy. A review of current stem cells being investigated in neurorestoration has recently been published [9]. We only briefly discuss embryonic and extraembryonic stem cells and focus this paper on our long-standing research interest in adult stem cells.Embryonic stem cells are pluripotent cells derived from the inner cell mass of the blastocyst that arguably serve as the foundation by which the properties of “stemness” are measured in other cell lines. With the potential to differentiate into all three germ layers, transplantation of embryonic stem cells (ESCs) into animal stroke models has demonstrated repair in both vascular [10] and neuronal damage [11], improved functional recovery of deficits [12,13,14,15,16] and provision of neurotrophic, angiogenic, and anti-apoptotic effects [13,14,15,16,17]. These benefits may extend well into potential translational therapy following a cerebral event, giving the cell line a plentiful array of therapeutic actions in terms of modulating a number of diseases including stroke. The distribution of these cells has been demonstrated with imaging techniques in both the brain and the periphery following transplantation in animal stroke models [18,19]. Although ESCs possess the potential for vast differentiation, there are two predominant concerns limiting their use. The ethics of harvesting embryonic stem cells is widely debated. However, recent advancements, discussed later in this review, may further develop and refine methods for producing these cells through retrograde manipulation of mature cell lines, alleviating some of the tension surrounding their use. Additionally, their naive lineage aligns with the stem cell tenet of the more naive the cell, the greater the potential for tumorigenicity, a topic that will be expanded upon in subsequent sections. Stem cells have the potential to form tumors after transplantation. This tumorigenicity is mostly associated with embryonic stem cells and pluripotent stem (iPS) cells [20]. Shortly after transplantation, a dysregulated differentiation of ESCs was found to cause the formation of teratomas containing all types of somatic tissues of the early embryo [21], due likely to the presence of oncogenes and trisomies which are known to have roles in cancer cell formation [22]. In view of this stem cell tumorigenicity, strategies have been explored including predifferentiation of cells to remove stemness or genetic modification to activate anti-oncogenic genes such as Nurr1 to abrogate the neoplastic state or render the cells post-mitotic [23,24]. While this tumorigenicity has been closely associated with ESCs and iPS cells, recent evidence suggests that safety precautions should also be taken with adult stem cells due to possible ectopic tissue formation seen in grafted mesenchymal stem cells (MSCs) in the Central Nervous System (CNS) [25]. These studies highlight a major hurdle in stem cell therapy, and emphasize the importance of closely monitoring stemness and tumorigenicity as we translate cell therapy to the clinic [22,24,26].With the concerns surrounding the use of embryonic tissue to harvest stem cells, researchers have looked to sources external to the embryo to harvest stem cells. Wharton’s jelly (within the umbilical cord), amnion, placenta, and umbilical cord are all rich stem cell sources [27]. As seen with the neural stem cells and mesenchymal stromal cells, extraembryonic stem cells also relate to different germinal layers. The amniotic epithelium is of ectoderm origin while the amnion-derived mesenchymal stromal cells are derived from the mesoderm [28]. Yet, the amnion-derived mesenchymal stromal cells exhibit less endothelial propensity conferring cell specificity [29]. Transplantation of placenta-derived mesenchymal stromal cells in animal models of stroke are believed to supply a microenvironment favorable for endogenous neural repair and replace damaged tissue [30,31,32]. Mesenchymal stromal cells derived from umbilical cord lining develop an immunosuppressive effect while demonstrating functional recovery, increased vascular density, increased expression of vascular endothelial growth factor, and basic fibroblast growth factor in rat stroke models [33,34].Adult stem cells often exist in combination with non-stem cells committed to distinct lineage, creating a heterogeneous environment. Because of this, one challenge in the use of adult-derived stem cells is the purification for the isolation for the particular stem cell of interest. In the following sections we will discuss the variety of adult-derived stem cells (Figure 1), their tissue sources, benefits, limitations, and clinical relevance.Adult stem sources include umbilical cord blood, placenta, amniotic fluid, bone marrow, menstrual blood, breast milk, dental pup, and skin fibroblasts. Most of these cells have been shown to exert neuroprotective effects in stroke animal models and a few have reached clinical trials in stroke patients.A divergent population stem and blast cells constitute the bone marrow. Thus, these cells may be utilized as an admixture or purified upon harvesting. Emerging research demonstrates the ability of bone marrow-derived stem cells, upon injury, to mobilize from the bone marrow (BM) into the peripheral blood. This feature is very practical for harvesting cells, which is currently employed for many immunologic, hematologic, and oncologic clinical applications. With relation to stroke, once in systemic circulation they may migrate to regions of the central nervous system in response to neuronal injury [35]. Cellular components of bone marrow include: hematopoietic stem cells (HSCs), mesenchymal stem cells (MSCs), endothelial progenitor cells (EPCs), and very small embryonic-like stem cells (VSELs) [36]. Here we will outline the aforementioned cell lines in greater detail.Hematopoietic stem cells are found primarily in the bone marrow where they give rise to both the myeloid and lymphoid lineages of blood cells. Cytokines produced by the CNS can incite hematopoietic stem cell mobilization into the blood, from the marrow, in response to a cerebrovascular accident (CVA) [37,38,39,40]. This mobilization may also be influenced by neurotransmitters, notably catecholamines, either through a paracrine mechanism signaling directly into the bone marrow or through systemic sympathetic release into circulation [41]. This cytokine-mediated recruitment of HSCs is applied clinically through treatment with granulocyte-colony stimulating factor [39,40].Abundant mobilization of immature hematopoietic CD34+ colony-forming cells and Long-Term Culture-Initiating Cells (LTC-IC) has been observed from clinical data of acute stroke, with the magnitude of this mobilization correlating with recovery of function. Autologous infusions of bone marrow mononuclear cells in human stroke patients during acute, subacute, and chronic phase of stroke have demonstrated no adverse effects of transplantation [42,43,44,45]. Transplantation of HSCs into animal models of stroke has greatly elucidated the therapeutic benefits of this type of stem cell in regenerative medicine. HSCs intravenously administered were able to increase the survival rate of stroke mice, accompanied by decreased neuronal cell death, and facilitated recovery from paralysis and forelimb weakness [46]. In an effort to reveal the mechanism of action of HSC therapeutic benefit in stroke, HSCs were intravenously administered at 24 h after ischemic stroke in mice which showed grafted cell migration into the spleen and later into ischemic brain parenchyma, expressing microglial but no neural marker proteins [47]. Moreover, transplanted stroke animals displayed significantly smaller infarct volumes and less apoptotic neuronal cell death in peri-infarct areas accompanied by a reduction of invading T cells and macrophages and a downregulated proinflammatory cytokine and chemokine receptor gene transcription within the spleen [47]. These findings indicate that transplanted HSCs exert therapeutic effects in stroke possibly acting via regulation of both central and peripheral (i.e., spleen) inflammation. Bone marrow (BM) derived HSCs are also being considered as potential treatment for diseases affecting cardiovascular tissue, bone, and cartilage among other tissues.Mesenchymal stromal cells were first isolated in bone marrow, but have since been found in nearly every tissue of the body. Here we address the therapeutic application of mesenchymal stromal cells, as well as non-bone marrow derived stem cells, for treatment of stroke.Transplantation of mesenchymal stromal cells into stroke models induces functional recovery of neurological deficits following cerebral ischemia [48,49,50]. The limited differentiation capacity of mesenchymal stromal cells suggests that observed transplantation benefits may be afforded through activation of endogenous repair pathways by secretion of neurotrophic factors which include brain-derived neurotrophic factor (BDNF) [51], nerve growth factor (NGF) [51], vascular endothelial growth factor (VEGF) [52], basic fibroblast growth factor (bFGF, FGF-2) [52], hepatocyte growth factor (HGF) [48,53], and insulin growth factor-1 (IGF-1) [54]. MSCs may recruit primary stem cells from the subventricular and subgranular zones of the brain to the site of injury, while also dampening apoptosis in the penumbral zone of the lesion [51,52]. A clinical trial of intravenous infusion of autologous BM-derived mesenchymal stromal cells in ischemic stroke patients shows significant functional improvement in infused patients without adverse effects in comparison with non-infused patients [55]. In a long-term 5 year follow up, patients infused with mesenchymal stromal cells demonstrated increased survival rates and greater functional improvement compared to non-infused patients [56]. However, the current transplantation techniques are plagued by very low graft survival rates, and therefore, mode of delivery remains a significant limitation of mesenchymal stromal cell-based therapies for stroke [57]. Mesenchymal stem cells have recently been the focus of many research endeavors, due in large part to their accessibility when compared to other stem cells. Mesoderm-derived mesenchymal stromal cells may be extracted from almost any mesenchymal tissue of the body including: bone marrow, placenta, teeth and adipose tissue. This abundance of harvest sites makes MSCs a preferred line for autologous transplantation. However, evidence indicates that harvest location may impart a specific role to mesenchymal stromal cells as a function of various methods of extraction, isolation, and proliferation [58,59,60,61,62]. To this extent, one site of tissue derived mesenchymal stromal cells may be better qualified for a specific therapy than cells derived from another tissue site. Despite their limited differentiation capacity and relatively transient life-span after transplantation, evidence shows that mesenchymal stromal cells promote neurogenesis following ischemic injury [52]. As mentioned previously, benefits may stem from secretion of neurotrophic factors such as BDNF and β-NGF, as well as modulation of vasculature from bone marrow, adipose tissue, skeletal muscle, and myocardium [63].Laboratory findings indicate that neurotrophic factors are involved in the neuroprotective action of stem cells as evidenced by their trophic effects, but additionally their anti-inflammatory and anti-apoptotic effects in animal models of stroke and other neurological disorders [64,65,66]. That BDNF and NGF have been shown as consistently secreted by transplanted stem cells suggest that targeting these two trophic factors’ signaling pathway may further improve the outcome of stem cell therapy. Alternatively, the combination of trophic factor treatment with stem cell transplantation may allow a more robust functional improvement in the clinic. The safety profiles of these trophic factors in the clinic in other disease indications [67,68], and the recognition of their clinical limitations, will guide the clinical trials of this combination therapy for stroke. Of note, BDNF Val(66)Met polymorphism has been implicated in worsened functional outcome in patients with subcortical stroke [69]. Similarly, serum from stroke patients revealed NGF upregulation significantly correlates with clinical and neuroradiological parameters of brain injury [70].In addition to the many potential benefits of menchymal strem cell-based therapies mentioned above, there are also significant risk factors that must be addressed. As with many types of stem cells, the risk of mesenchymal stem cells developing into tumors must be considered. One study showed that a sarcoma developed in the lungs of mice following transplantation of mesenchymal stem cells [71]. Not only the cells themselves but also their secretions may affect tumors. Interleukin-6 (IL-6) and vascular endothelial growth factor (VEGF) secreted from MSCs increases the migration of breast cancer cell lines [72]. Breast cancer cells stimulate de novo secretion of the chemokine CCL5 from mesenchymal stem cells, which then acts in a paracrine fashion on the cancer cells to enhance their motility, invasion, and metastasis [73]. Accordingly, certain types of mesenchymal stem cells may demonstrate greater tendency toward tumorigenicity and promotion of metastasis.Endothelial progenitor cells (EPCs) represent a small population of cells present in the blood that give rise to mature endothelium that lines blood vessels. While in circulation, these cells can be recruited to produce new blood vessels, a term called vasculogenesis. The etiology of stroke is multifaceted. One contributing factor includes the compromise of vascular integrity, leaving a region vulnerable to stroke. With the endothelium regulating the permeability of the blood brain barrier (BBB), the role of endothelial progenitor cells in producing the mature lining of blood vessels is integral in maintaining cerebral homeostasis. Preliminary studies demonstrated that transplanted EPCs were integrated into newly vascularized endothelium of the hind limbs in ischemic animal models [74]. Further research specifies that BM-derived endothelial progenitor cells are likely signaled to sites of new vascularization prior to differentiation [75,76].A correlational study in human ischemic stroke patients indicates that the level of circulating EPCs relates to improvement on the National Institute of Health Stroke Scale [77]. Animal models of stroke show that intravenous transplantation of EPCs reduces cerebral infarcts in stroke diabetic mice [78]. Moreover, EPCs can incorporate to the BBB microvasculature and delay the stroke onset in an ischemic hemorrhagic stroke model [79]. In addition, intravenous infusion of autologous EPCs after stroke in rabbits produces functional improvement, decreases number of apoptotic cells, increases microvessel density in the ischemic boundary area, and reduces infarct area [80].The current hypothesis of very small embryonic-like stem cells is that these pluripotent stem cells are deposited early in embryonic development from an epiblast source, where they function as a reserve that can be accessed in response to physiological stress [81,82]. Investigation is underway using VSELs for stroke therapy in the brain, a region rich in VSEL phenotypic cells [83,84]. VSELs are a great candidate in therapy for cerebral vascular incident because of their potential to differentiate into neurons, oligodendrocytes, and microglia to regenerate damaged CNS [35].However, current restrictions present a challenge in moving forward. Very small embryonic-like stem cells are present in limited quantity, producing a low yield from harvesting. Such an obstacle may be overcome with refining methods of proliferation prior to transplant [35]. An additional challenge is the decreasing population of VSELs present in older age, further contributing to the difficulty of sufficient yield upon harvesting [84]. With endogenous stem cells being located in the subgranular zone (SGZ) of the dentate gyrus, the subventricular zone (SVZ), and the subependymal zone (SEZ) of the spinal cord, the therapeutic potential of NSCs for cerebrovascular accidents seems obvious. Chemokine signals such as stromal-derived factor-1 (SDF-1), vascular endothelial growth factor (VEGF), and angiopoietin are released from ischemic tissue, influencing the course of the SVZ NSCs toward a path along blood vessels to reach the infarcted area [85,86,87,88]. Although endogenous stem cells migrate to the lesion following stroke, there appears to be minimal stem cell survival [89,90,91]. This supports the hypothesis that endogenous neural stem cells may not exert their effects solely by replacement of neuronal tissue, but rather by secreting growth factors that influence repair. Immunological responses may also influence the differentiation of endogenous stem cells. In ex vivo studies, microglia from ischemic brains prompted the maturation of NSCs into neurons [92].Although endogenous NSCs are shown to migrate in response to cellular injury, their effects may be augmented by the addition of exogenous neural stem cells. The literature describes transplantation of NSCs inducing further endogenous stem cell production at the site of injury [93,94,95,96]. However, another study suggests that intravenous infusion of neural progenitor cells decreased neurogenesis despite increasing dendritic length and the number of branch points [97]. This may further support the hypothesis of neurotrophic factors secreted from stem cells exerting a primary effect. Neural stem cells are proven in terms of their therapeutic potential; however, they present a few significant limitations. The difficulty of obtaining the cells may be the greatest challenge. Under most circumstances, harvesting neural stem cells would require an invasive procedure for autologous use while allogenic grafts would require a fetal source or manipulation from another cell source. A possibility to circumvent this problem would be harvesting the stem cells for therapy during a surgical procedure already intended for the patient [98], such as during a temporal lobe resection in which subventricular matter, a known source of stem cells, could be harvested. As with many other stem cells, there is constant concern about the potential to illicit aberrant cell growth, producing tumors upon transplantation. Whereas the less differentiated the cell, the less likely it will invoke a host reaction; however, the more naive the stem cell, the greater its propensity for uncontrolled proliferation. Adult stem cells possess a reduced capacity for proliferation and may be less tumorigenic. However, this presents a problem with producing a sufficient number of cells for transplantation. To traverse these limitations, researchers have developed methods such as: long-term culturing, immortalization, insertion of oncogenes, or even derivation of neural stem cells from other tissues or from pluripotent stem cells.With their availability, ease of harvesting, and ability for autologous and allogenic use, the therapeutic potential of umbilical cord blood is expansive. The heterogeneous mixture of cells comprising cord blood includes hematopoietic progenitors, lymphocytes, monocytes, embryonic-like stem cells, and mecenchymal stromal cells. Yet, cord blood is considered immunologically immature and exerts its effects through immune modulation and reducing inflammation [99].Transplantation of umbilical cord blood-derived stem cells in animal models of stroke has produced encouraging results of functional recovery, reducing infarct size, and higher expression of neuroprotective factors, such as BDNF and VEGF [100,101,102,103]. In other studies, human umbilical cord blood has exhibited protective effects in the rat hippocampus in vitro, while promoting dendritic growth. Additional emerging research is investigating the capabilities of human umbilical cord blood hematopoietic stem cells for functional recovery of dopaminergic neuron morphology of the substantia nigra, caudate, and putamen in an 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) Parkinson’s disease mouse model. After intracardioventricular injection, there was an increase in size and density of tyrosine hydroxylase staining cells of the substantia nigra [104].Adipose tissue derived stem cells have demonstrated the ability to differentiate into neural, glial, and vascular endothelial cells, and also show higher proliferative activity with greater production of VEGF and hepatocyte growth factor in comparison with bone marrow derived stromal cells [98]. In combination with the accessibility, these features make adipocyte-derived stem cells a desirable source for neurovascular therapy. Transplantation of adipose-derived stem cells in ischemic stroke models demonstrates reduction in damage [98]. Additional studies exhibited reduced infarct size, improved neurological function, reduced level of cerebral inflammation, and chronic degeneration in an intracerebral hemorrhage model, substantiating their therapeutic value [105,106].Yet, stem cells derived from adipose tissue are also subject to limitations. It was considered that spontaneous mutations occur with extensive passaging that foster tumorigenesis, potentially leading to cancer [107,108]. Follow-up studies suggest adipose-derived stem cells promote pre-existing cancerous cells, but do not initiate tumorigenesis. In a human clinical trial of spinal cord injury patients, none of the eight patients experienced any adverse events within the three-month follow-up [109].Following many of the factors considered in harvesting stem cells, menstrual blood provides a source with many benefits. With the monthly cycling of the endometrium, the ease and availability for harvesting is a large benefit in the research for therapeutic potential. Stem cells collected from menstrual blood demonstrate multipotency and secrete trophic factors such as VEGF, BDNF, and NT-3 in response to oxygen glucose deprivation (OGD) in an in vitro model of stroke. In such studies, the co-culturing of rat primary neurons with menstrual blood, or its conditioned media, improved survival rate [110]. Further, both intracerebral and intravascular transplantation of menstrual blood-derived stem cells in rat stroke models also enhanced survival and behavioral function [110]. Of note, it has been observed that the adherent fraction of menstrual cells do not lose their karyotypic normality or develop tumorigenic potential even after being expanded through 68 doublings [111].Mammary stem cells (MaSCs) present in tissue of the breast, along with differentiated cells, enter the milk through lactating epithelium. Researchers postulate that these cells enter the breast milk through a combination of migration, cell turnover, and mechanical shearing forces [112,113]. The stem cells of breast milk demonstrate pluripotency similar to that of embryonic stem cell morphology and phenotype and thus allow for differentiation into all three germ layers in vitro [113]. Future research may elucidate therapeutic potentials in line with those of ESCs. Additional benefits results from the noninvasive harvesting of the cells, availability, and potential for autologous transplant. In terms of tumorigenicity of breast milk-derived stem cells, a study has reported that even nine weeks after subcutaneous injection of breast milk-derived stem cells in immunodeficient mice, these cells did not produce tumors [113]. Along this line, subpopulations of pluripotent adult cells and other multilineage stem cells have failed to form teratomas. Further work characterizing breast milk-derived stem cells for any oncogenic activity under different pathological conditions is needed to determine their tumorigenicity. Dental tissue-derived stem cells, such as post-natal dental pulp stem cells (DPSCs) [114], stem cells from exfoliated deciduous teeth (SHED) [115], periodontal ligament stem cells (PDLSCs) [116], stem cells from apical papilla (SCAP) [116,117], and dental follicle precursor cells (DFPCs) [118], which exhibit mesenchymal stromal cell-like capabilities, have been identified (for review, see [119]). Furthermore, dental tissue-derived stem cells have demonstrated differentiation into a variety of cell lines including neural tissue, adipocytes, and odontoblasts [120].The use of dental tissue-derived stem cells have been utilized in the study of animal model middle cerebral artery occlusion (MCAO), demonstrating improved motor function following transplantation into the dorsolateral striatum [121]. Once considered unidirectional, stem cells were thought to progress through a linear maturation process leaving them terminally differentiated. However, current evidence suggests otherwise. Through manipulation, differentiated stem cells may be coerced into a prior state of multipotency. Utilizing the method of transfecting specific transcription factors fibroblasts can be manipulated into their embryonic-like stem cell precursors [122]. This technique has also been applied to umbilical cord blood cells, placental mesenchymal stromal cells, neural stem cells, and adipose-derived precursor cells to increase their potency [123,124]. Further studies in animal models of ischemic stroke demonstrate that some of the benefits in transplanting induced pluripotent stem cells (iPSCs) includes improving sensorimotor functions [125,126], reducing infarct size, reducing pro-inflammatory cytokines, and increasing anti-inflammatory cytokines [125].As noted above, there is speculation for concern when transplanting less differentiated cells. Of particular apprehension is their potential for tumorigenesis and immunogenicity. The transfection technique used to induce retrograde manipulation utilizes transcription factors of known oncogenicity. The finding that transplantation of iPSCs into ischemic brain tissue produces a higher incidence of tumors than in healthy brain tissue, further supports this notion [127]. Transplantation is also limited by rejection by the host, even when autologous cells are grafted [128]. With the aforementioned concerns in mind, emergent research is demonstrating the therapeutic feasibility of vector-free and transgene-free induced pluripotent cells while reducing their tumor potential. A current study investigates the use of these human iPS cell-derived neural progenitor cells (hiPS-NPCs) in a mouse ischemic stroke model after discovering they differentiated into functional neurons in vitro. There was no evidence of tumor formation for 12 months following in vivo transplantation [129].Due to distinct therapeutic potential of individual cell lines, the possibility exists to combine their respective benefits in targeting disease. Mounting literature substantiates the potential for synergistic effects on stem cell survival when co-transplanted. One such study established enhanced stem cell survival when delivered with adipose-derived stem cells [130]. Moreover, co-transplantation therapy may also decrease adverse events. The co-transplantation of bone marrow-derived stromal cells with embryonic stem cells reduced the incidence of tumor production and transplanting neural stem cells with epithelial cells enhanced survival while promoting differentiation [131,132].The ability to enhance therapeutic effects is not limited solely to the use of stem cells. Combination therapy employs the addition of a substrate to enhance the efficacy of the stem cell line being transplanted. Examples include: Combining bone marrow-derived stromal cells with trophic factors to enhance survival and potentiation [133] or providing a scaffold for stem cell adherence [134].The recognition of immunosuppressant factors secreted by certain cells (such as bone marrow and Sertoli cells) supports the use of co-transplantation with an immune-protective cell to allow better graft survival of the cells [135]. Sertoli cells are the germ cells of the testis and it has been shown that they are able to secrete trophic factors that are highly immunosuppressive, and which serve as neuroprotective factors in different animal models of neurological disorders [135,136,137]. As discussed previously, with unique sets of growth factors secreted by stem cells, co-transplantation of stem cells should generate a cocktail of growth factors to be secreted and delivered to the injured brain, thereby affording much more improved therapeutic outcomes. Furthermore, following brain injury, different cell types die or succumb to neurodegeneration, thus warranting the need to transplant multiple cell types. In this case co-transplantation of cells that could differentiate into these multiple cell types will be a logical approach towards replacement of the variety of cells damaged after brain injury [138]. A recent study has demonstrated that neural progenitor cell (NPC) survival and therapeutic support can be enhanced when co-grafted with other genetically modified doxycycline NPCs that can provide bFGF when activated [139,140,141].As combinations for therapy continue to surface and demonstrate effectiveness, many variables still persist. Factors including: optimal dose, route of administration, and sex of donor/recipient, all of which are likely to be contingent upon the cell type being investigated. To date, we have investigated many of these parameters with umbilical cord blood for conditions such as Alzheimer’s disease, Amyotrophic Lateral Sclerosis (ALS), and Sanfilippo syndrome [142], however, there is still much to be ascertained in regards to stroke therapy. To this end, the Stem Cell Therapies as an Emerging Paradigm in Stroke (STEPS) was initiated to resolve these issues and standardize procedures [143,144,145,146]. Throughout this review we discussed how each cell line under investigation has its own unique benefits and limitations associated with use. Some of those limitations are immunogenicity, tumorigenicity, ease of harvesting, and the ability to proliferate cells. Researchers are currently addressing these issues through many of the techniques reviewed; yet there are still limited clinical trials. More so, current research is expanding beyond a single cell line transplant. It is likely that future clinical therapy may include the use of co-transplantation and combination therapy mentioned. Further studies also aim to explore the molecular mechanism of response by native tissue in the presence of stem cells. This may progress the exploration of unique trophic factors produced by the stem cells and their utilization in these novel therapies. In moving forward, research must still be conducted in assessing factors for optimal transplantation parameters and the efficacy of treatment, however, across the literature, it is evident that stem cells provide a promising niche of therapeutic potential.Cesario V. Borlongan holds patents in stem cell technologies for the treatment of neurodegenerative disorders. Cesario V. Borlongan is supported by James and Esther King Foundation for Biomedical Research Program 1KG01-33966 and NIH NINDS RO1 1R01NS071956-01.

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+This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).Even if vaccination is often described as one of the great achievements of public health, results of recent studies have shown that parental acceptance of vaccination is eroding. Health providers’ knowledge and attitudes about vaccines are important determinants of their own vaccine uptake, their intention to recommend vaccines to patients and the vaccine uptake of their patients. The purpose of this article is to compare how midwives and physicians address vaccination with parents during pregnancy and in postpartum visits. Thirty semi-structured interviews were conducted with midwives and physicians practicing in the province of Quebec, Canada. Results of our analysis have shown that physicians adopt an “education-information” stance when discussing vaccination with parents in the attempt to “convince” parents to vaccinate. In contrast, midwives adopted a neutral stance and gave information on the pros and cons of vaccination to parents while leaving the final decision up to them. Findings of this study highlight the fact that physicians and midwives have different views regarding their role and responsibilities concerning vaccination. It may be that neither of these approaches is optimal in promoting vaccination uptake.The decline in vaccine-preventable diseases, along with the increasing number of vaccines offered in child schedules, have challenged the acceptability of vaccination for parents [1]. Parents are often uncertain about the risks and benefits of vaccination and many studies have shown that a significant proportion of parents have doubts regarding vaccination [2,3,4,5]. Recent outbreaks of infectious diseases among unvaccinated individuals also suggest that concerns regarding vaccination are widespread in Canada and in other countries [6,7,8]. Many studies have shown the crucial role that health providers play in the success of vaccination programs, as their recommendations are one of the key determinants of parents’ decision to have their child vaccinated [9,10,11,12,13,14]. In Quebec, Canada, vaccines against 13 diseases are offered free of charge to parents through the universal provincial immunization program. Vaccination is not mandatory and parents are free to accept or decline vaccination for their children. Children vaccination schedules involve five vaccination visits when the child is aged 2, 4, 6, 12 and 18 months old. Most vaccines are administered by nurses in public community health services (CLSC) or by physicians and/or nurses in private offices. Previous studies have also shown that the decision-making process about vaccination of one’s child may begin during pregnancy [4,15,16,17]. Pregnant women in Quebec are usually treated by family physicians or obstetricians-gynecologists. Recently, pregnant women have also begun to receive care from midwives. Midwifery was authorized by law in 1999 in Quebec [18]. It includes prenatal care for normal pregnancies (i.e., pregnancies without any special medical conditions), birth delivery and 6-weeks postpartum monitoring of mothers and newborns. Midwifery practice take place in Maisons de naissance which are locations that welcome pregnant women and their families. They are located in houses in the community, distinct from both the home and the hospital center, but part of the public health system. They are designed to allow for a reasonable number of births each year that preserve an intimate, family and human character [19]. In 2009, 1.9% of births in Quebec were assisted by midwives and the objective of the Quebec Ministry of Health is to increase access to these professionals so that up to 10% of Quebec pregnant women have the assistance of a midwife by 2018 [20]. Since 2008, midwives have also been authorized to administer some vaccines (hepatitis B, measles, mumps and rubella (MMR) vaccine) as well as immunoglobulin against hepatitis B [21]. However, results of two Canadian studies have shown that births assisted by midwives were associated with incomplete vaccination status of the child [22,23].The aim of this study is to compare how midwives and physicians address vaccination with parents during pregnancy and in postpartum visits so as to better understand their potential influence on parents’ decisions about vaccination.This study is based on in-depth interviews conducted with physicians who assist births and with midwives. Interviews with midwives were conducted during the summer and fall of 2010 and interviews with physicians, during the fall of 2011. In Quebec, there are approximately 130 midwives registered with the Ordre des sages-femmes du Québec [24]. Purposive sampling was used to recruit midwives from all 11 maisons de naissance of the province. The physician sample was constituted intentionally. A convenience sample of physicians who assist births or follow newborn babies was constituted by mailing invitations to participate to a random sample of physicians listed on the websites of five health agencies of the province. Participation by midwives and physicians was voluntary, and a small monetary compensation was given to participants. The ethics approval for the study was obtained from the principal author’s institution. All participants provided written informed consent.Interviews were conducted using a semi-structured guide (Table 1). The guide was designed to elicit information about: Academic courses and work experience, practices around pregnancy and birth, approaches to health and prevention and, more specifically, approaches to vaccination. Interviews were loosely conducted and, in an iterative process, the interview guide was adjusted throughout data collection. All interviews were conducted by a research professional trained in anthropology (Maryline Vivion). The sample was constituted purposefully; interviews were conducted with participants having different socioprofessionals characteristics (age, number of year of practice, training, etc.) in order to include different perspectives regarding the main themes of the study. Participants were recruited and interviews were conducted until data saturation was reached within each group of professionals, that is, when no new ideas emerged during the interviews for the main themes [25]. All interviews were audiotaped and transcribed verbatim. A content analysis of transcribed interviews was done using N’Vivo 9.0. All transcribed interviews were read by three authors (Eve Dubé, Chantal Sauvageau and Maryline Vivion). Data codification was performed by Maryline Vivion. Data were organized into main coded themes which followed the interview guide, with a particular focus on vaccination-related themes. After coding a few of the verbatim texts, the coding tree was discussed by the authors (Eve Dubé and Maryline Vivion) and adjusted. Ambiguous verbatim texts were discussed between authors (Eve Dubé and Maryline Vivion).Thirty interviews were conducted: 17 with midwives and 13 with physicians. Interviews lasted on average 60 min with midwives and 30 min with physicians. Interviews with physicians were shorter as they generally had fewer comments on vaccination than midwifes. In addition, as all interviews were conducted at the providers’ office, duration of the interviews was similar to the length of consultation with patients of physicians and midwives. Mean number of years of practice was 9 years for midwives and 16 years for physicians. Physicians and midwives were practicing in different regions (urban and rural) of the province of Quebec. All interviews were conducted in French. Quotes provided in the following sections were selected on the basis of their clear representation of the key themes. Quotes were translated into English and submitted to a back-translation to French to ensure that their meaning was maintained.Semi-structured guide for the interviews with physician and midwives.Both midwives and physicians considered that they have an important role in health promotion and disease prevention. However, how they actualize this role differs greatly. Midwifery philosophy promotes the respect of the normal process of pregnancy, empowerment of mothers and families, informed choice and a personalized approach to health. Indeed, midwives emphasized the importance of parents taking decisions by themselves, and saw their role more as one of providing information. Physicians more often considered their role as that of an “advisor” or an “educator”. Thus, in health promotion, they were more prescriptive in telling parents what they ought or ought not to do. All interviewed midwives viewed their particular approach in opposition to biomedicine and most were critical of biomedical obstetric practices. Midwives stressed their openness to alternative medicine practices and their emphasis on the natural continuum of pregnancy and birth. Interviewed midwives also stressed their personalized approach to health promotion in pregnancy. In contrast, physicians referred more systematically to a list of themes to be addressed with patients. Length of consultation varied greatly between midwives and physicians, with approximately 1 h of consultation each month for midwives compared to 15 min each month for physicians. The fact that midwives’ consultations last longer was perceived by midwives as facilitating health promotion counseling with parents. Differences in the perceptions of midwives and physicians as to their role in health promotion are also reflected in their practical approach to prenatal diagnosis tests. Many prenatal tests are offered free of charge to Quebec pregnant women (e.g., echography, screening for diabetes or trisomy 21). Physicians and midwives have different ways of presenting these tests to parents. Physicians tend to prescribe systematically all available “routine” tests to pregnant women. It is important to note, however, that the approach of physicians was more nuanced regarding genetic screening tests like the trisomy 21 test. The pros and cons of genetic tests were always discussed with patients and, generally, patients’ decisions were treated with respect. In contrast, midwives advocate for a “judicious use” of technology and, with an emphasis on parent empowerment, prefer to give information and leave the decisions on whether or not to undergo the test to parents. Some of the interviewed midwives were also very critical of the routine and systematic use of testing by physicians. When asked what factors have contributed to the decrease in infectious diseases, vaccination was mentioned by all interviewed physicians, along with improved hygiene. In comparison, only three midwives spontaneously answered vaccination. Most midwives attributed the decrease in infectious diseases to hygiene, better food and breastfeeding. When asked about the pros and cons of vaccination, physician and midwife responses were different. All physicians were highly supportive of vaccination while midwife responses indicated doubts regarding the usefulness and safety of vaccination. Among the “pros” of vaccination, the decline or eradication of infectious diseases was mentioned by half of physicians (6/13) and by one out of 5 midwives (3/17). Six midwives and four physicians also talked broadly about the disease prevention offered by vaccination. The fact that vaccines are free and the efficacy and safety of vaccines were mentioned by physicians (3/13) and midwives (6/17) as other arguments in favor of vaccination.Spontaneously, four physicians stated that there were no arguments against vaccination. The others talked about the fact that some vaccines are not included in the free universal vaccination program and thus that parents have to pay for them (5/13) or about the increasing number of vaccines in the child schedules (3/13). Three midwives also mentioned this argument. In fact, the recent inclusion of new vaccines (against varicella and rotavirus) in the Quebec national vaccination program for children raised questions among both physicians and midwives. Ten midwives considered that it was not important to vaccinate children against varicella and four, against rotavirus. Some physicians also partially shared this opinion, describing the varicella vaccine (4/13) and rotavirus vaccine (5/13) as “less important vaccines”. However, six physicians clearly stated that all vaccines were important and four were highly supportive of varicella vaccination. One of them showed a picture of a child with complications from varicella as an argument used to convince parents to have their child vaccinated.Midwives also noted adverse events after vaccination (9/17), the fact that long-term efficacy of vaccines was unknown or deficient (11/17), the lack of “unbiased” information regarding vaccination (9/17) and the fact that vaccines were used to prevent mild diseases (7/17). None of the interviewed physicians mentioned any of these arguments. Physicians and midwife opinions regarding the Quebec provincial vaccination program were also very divergent (Table 2). While all physicians approved of the child vaccination schedule, 12 midwives felt that childhood vaccination starts too early. Physicians were also highly supportive of combined vaccines, which reduce the number of injections for children whereas most interviewed midwives considered that combined vaccines restricted the possibility for parents to choose which antigens their child should receive. Midwives’ and physicians’ opinions regarding some aspects of the Quebec national vaccination program.Finally, all interviewed physicians considered their knowledge on vaccination as sufficient while five out of 17 midwives felt they were lacking knowledge about vaccination.Usually, physicians talk about vaccination with parents during the first postnatal visit which takes place between 2 weeks and 1 month after birth. Midwives usually discuss vaccination with parents at the end of their postnatal follow-up, i.e., 6 weeks after birth. All interviewed physicians stated that they systematically discuss vaccination with parents. However, three midwives told us that they did not consider vaccination a part of their practice while the others systematically or usually discussed vaccination with parents. Nine physicians stated that childhood immunizations were administered in their office. Also, seven midwives mentioned that the vaccines that they were authorized to provide were administered in their office. When vaccination was not administered in an office, physicians and midwives referred parents to the CLSC.As for health promotion, the way that vaccination is presented to parents varied between physicians and midwives (Table 3). Most midwives considered their role as one of giving information to parents without positioning themselves either personally or professionally. As a way of encouraging parents’ informed choice regarding vaccination, interviewed midwives said that they always gave “balanced” information on vaccination. To present the “pros” of vaccination, midwives usually gave documents produced by government agencies and used books or texts produced by alternative medicine practitioners to present the “cons”. Ten midwives also stated that they present vaccination to parents as a choice. In contrast, physicians saw their role as one of explaining, educating and encouraging parents to have their children vaccinated. Few physicians said that they systematically gave written information, as most rely on their professional knowledge and experiences. Physicians also often expressed their personal, pro-vaccination opinions to patients. The different approaches of physicians and midwives regarding vaccination counseling are even more evident when they are asked about their reactions to parents who are opposed to the vaccination of their children. When faced with a parent who refuses to vaccinate his or her child, both physicians and midwives asked them about their reasons for refusal, so as to explore whether their choice was a thoughtful one. However, midwives said that, if they judged that the parents had good reasons not to vaccinate, they would respect this choice in order to maintain a trusting relationship with them. In contrast, physicians acknowledged being very uncomfortable with a parent’s decision to refuse vaccination. Most of them clearly positioned themselves in favor of vaccination and tried to convince parents to change their minds. Communication strategies used and reactions of physicians and midwives when confronted with parents opposed to vaccination are shown in Table 4.Approaches of physicians and midwives regarding vaccination counseling. Communication strategies used and reactions of midwives and physicians when faced with parents who refuse to vaccinate or who have significant doubts regarding vaccination.Despite being considered to be one of the most successful public health measures [16,26,27], vaccination is perceived as unsafe and unnecessary by a growing number of parents. Recent outbreaks of vaccine-preventable diseases in several parts of the developed world have shown the devastating consequences of under- or non-vaccination [28]. In Quebec, vaccination is voluntary and there are no vaccine mandates. Around 80% of two-year-old children in Quebec have received all recommended vaccines [29]. This high rate of childhood vaccination coverage indicates that vaccination remains widely accepted among Quebec parents. However, results of recent surveys have indicated that a significant proportion of them have doubts and concerns about vaccination [30,31]. In 2011, the province of Quebec experienced the largest epidemic of measles in the Americas since this disease was declared eliminated in 2002 [32]. When parents have doubts regarding vaccination, health professionals remains the most trusted source of information [30]. The results of this qualitative study indicate that physicians and midwives have different views regarding their role and responsibilities concerning vaccination. As has already been shown by results of large quantitative studies conducted among Canadian clinicians [33,34], physicians interviewed in this study were strong supporters of childhood vaccination programs. They saw their role as one of promoting vaccination by educating and encouraging parents to vaccinate their children. To convince parents to vaccinate, they expressed their personal opinions and used their professional experience of vaccine-preventable diseases. A parent’s decision to question or refuse vaccination was also very challenging for interviewed physicians. Indeed, results of studies conducted in the US have shown that up to one-third of physicians would refuse to keep parents in their practice who are opposed to vaccination [35,36].In contrast, midwives’ opinions regarding childhood vaccination were more mixed. While the majority of interviewed midwives agreed with the benefits of vaccination in preventing infectious diseases, almost all disagreed with some of the components of Quebec’s childhood vaccination program. For instance, despite the evidence showing the high risk of contracting many vaccine-preventable diseases in the first year of life (e.g., pertussis, meningitis caused by Haemophilus influenzae type B, pneumococcal infection), most midwives considered that vaccination begins too early in life.Interviewed midwives saw their role as one of giving information and presenting the pros and cons of vaccination while leaving the final decision to parents. Indeed, all midwives said they respected parents’ decision to vaccinate their child or not if they judged that this decision was deliberate. Some midwives also considered that vaccination was not part of their practice and did not engage in discussion on this topic with parents. It is also important to note that, despite the fact that midwives are authorized to administer some vaccines, most maisons des naissances do not keep vaccines on their premises. In addition, the follow-up by midwives ends at 6 weeks post-partum, just before the first vaccination visit, planned at 8 weeks. Both these facts, along with possibly less positive attitudes regarding vaccination, could explain, at least partially, why some midwives do not feel involved in vaccination despite their strong commitment to health promotion.The different ways physicians and midwives present vaccination to parents could also be seen as rooted in different views about informed consent and informed choice that are built into biomedical and midwifery philosophies. Medical practice is governed by a code of ethics and a medico-legal guide that present key concepts and fundamental legal principles governing medical practice [37]. The Quebec Immunization Protocol [38] stipulates that health care professionals should inform, in clear language, vaccine recipients about the risks and benefits of vaccines to be administered. Physicians also have the duty to inform all patients about vaccines recommended for them, even if the vaccines are not included in the free universal vaccination program [39]. Informed consent, in biomedicine, focuses on three components: legal, ethical, and administrative compliance [40]. Due to the fact that physicians are at risk of prosecution, legal aspects tend to take an important place in the process. Like physicians, midwives also have a code of ethics and a guide of practical norms by which to conduct their practice [41,42]. These are written in accordance with the midwifery philosophy which is based on a recognition of the natural process of pregnancy and birth. Informed choice and empowerment are key principles of this philosophy. Indeed, Quebec’s midwifery philosophy specifies that midwives “view decisions as a results of a process where responsibilities are shared between women, their family (as defined by women) and health professionals” and insists that midwives should “acknowledge that the final decision belongs to the woman” [19]. In contrast to the medical concept of informed consent, which often implies compliance with a higher authority, the approach of midwifery suggests that women have the power or opportunity to choose among meaningful alternatives [43]. Our study highlighted the importance of the midwife philosophy in guiding the entire practice. The informed choice is one of its principles, and midwives approach many health promotion issues, including vaccination, on the basis of this principle. While Quebec nurses and doctors must recommend vaccination to their patients [39,44], midwives consider their role as that of an information provider instead, presenting the pros and cons of vaccination to parents. However, for consent or choice to be informed, it is not just a question of giving relevant information and letting the patient decide what is best for him or her. It is also about the nature of the information given to the patient and about the way it is presented [45]. In this study, physicians actively promoted vaccination while midwives, in the perspective of informed choice gave information to parents about the pros and cons of vaccination. The nature of information given by physicians and midwives was also largely different. Physicians gave standard information about mild and frequent reactions after vaccination, such as fever or pain at the injection site [38]. In contrast, to illustrate the “cons” of vaccination, midwives gave parents books or texts from alternative medicine practitioners, which are very critical of vaccination and often put forward non evidence-based events attributed to vaccines, such as the onset of autoimmune diseases [46,47]. This disparity in the nature of information regarding potential adverse events after vaccination could certainly lead to different decisions among parents [1]. In addition, it may be hard for parents to get a sense of the very polarized information on vaccination [48]. Besides the nature of the information given to parents, the way of presenting this information could also impact parents’ decisions. Some studies have highlighted that parents find it difficult to have an open discussion about vaccination with their physician and report feeling alienated when vaccines are discussed [49]. In contrast, discussion about vaccination with alternative medicine practitioners, such as naturopaths, was perceived to be more in line with what the parents perceived to be an ideal consultation for their children than was the case for consultations with physicians [50]. In our study, physicians used a prescriptive approach by strongly encouraging parents to vaccinate, sometimes without much openness about parental concerns regarding vaccination. Clearly, some of the interviewed physicians were using a “knowledge deficit approach”, assuming that parents who were uncertain or who refused vaccination lacked knowledge and that their role was to educate them, without much consideration for their opinions and values. Instead of simply delivering standard messages on diseases or vaccines, physicians could develop more dialogue-based approaches which work with and build on the concepts that parents already use to think about vaccination [51]. In contrast, midwives adopted a neutral stance by not positioning themselves, either professionally or personally, for or against vaccination. This approach could be viewed as a form of disengagement by health professionals [52]. Others have highlighted that putting too much emphasis on being neutral could impede real communication between health professionals and patients [53]. Informed choice supposes that patients do take decisions by themselves. However, studies have shown that many patients prefer to delegate their decisions or to defer to the opinions of others, including their health providers [40,54,55] as a way of sharing the burden of health responsibilities [56]. In addition, no information is value-neutral. Studies [53,57] have shown that even an approach that aims at giving balanced information is often prescriptive [43]. As Leask and collaborators have pointed out, there are challenges to ensuring valid consent in the field of vaccination [58]. One of these challenges lies in accommodating different lay and public health views about the relative merits of vaccination in a context where risk-benefit ratios of vaccination are less apparent, as a result of the decline in vaccine-preventable diseases [59]. In addition, as for all health interventions, no vaccine is completely safe or effective and health providers have to communicate these uncertainties to patients [51]. Communicating uncertainties is a very challenging task [60] and there are many issues involved in communicating the risks and benefits of vaccination [1]. One of these is the presentation of vaccination from a top-down population-level intervention perspective to parents who are evaluating the appropriateness of vaccination in relation to their child’s particular health [51]. The inclusion of new vaccines in the childhood program to prevent diseases that could be perceived as mild (e.g., varicella or rotavirus gastroenteritis) is also challenging the communication of risks and benefits of vaccination to parents. Like parents [5,61], midwives and physicians were concerned by the increasing number of vaccines in the childhood schedules. The changes in childhood vaccination schedules and rapid developments in the field of vaccines challenged providers who have to handle a lot of vaccine-related information to be able to ensure that their knowledge is up-to-date. Many articles in the literature have stressed the importance of health providers addressing concerns of vaccine-hesitant patients in a well-managed way and authors have given their tips to providers on how to do so [62,63,64,65]. Although the approaches presented in these articles vary, they do share some common characteristics, such as the importance of maintaining a trustworthy patient-provider relationship and the importance of tailoring the communication to specific patients’ concerns and doubts. This study has strengths and limitations. First, as for all studies relying on qualitative interviews, social desirability bias cannot be excluded. However, the fact that interviews were conducted by a research professional from the anthropological field should have reduced this bias. Second, the sample of participants was constituted by on a voluntary basis, which could lead to a selection bias. Indeed, even if saturation of data was attained, results of this study cannot be extrapolated to all midwives and physicians working in Quebec. Qualitative researches imply a limited number of participants. However, our sample was constituted using diversification criteria, as recommended in qualitative research [66,67]. To our knowledge, this is the first study to explore the approach of Quebec midwives to vaccination using the same interview scheme to be able to compare their opinions with those of physicians. Thus, this study is exploratory in nature. Despite this limitation, as their role is crucial in sustaining the success of vaccination programs, results of this study could be useful to develop educational tools to enhance health providers’ communication about vaccination to new parents. Finally, this study reports only on the point of view of health practitioners. Patients’ opinions about the vaccination discussion with their health provider have not been assessed. The perspective of parents should be explored to have a better understanding of the impact that the discussion by providers has on parental vaccine decision-making.The knowledge and attitudes about vaccines among health providers have previously been shown to be an important determinant of their own vaccine uptake, their intention to recommend the vaccine to their patients and the vaccine uptake of their patients. Results of this study indicate that physicians and midwives have different views regarding their role and responsibilities toward vaccination. Midwifes and physicians need to reflect on how they deliver information on vaccination to parents. Is it done in a paternalistic way or does it support informed parental decision-making? Are they really providing neutral, non-biased, information? Do they listen to parents’ questions and concerns about vaccination with empathy and in a nonjudgmental way? Do they discourse present evidence-based scientifically sound information on vaccination? In the context where parental acceptance of vaccines is apparently eroding, the support of health providers is essential to ensure the success of vaccination programs that rely on high level of vaccine uptake.We would like to thank all participants as well as the Canadian Institutes of Health Research (MOP-115012) and Quebec Ministry of Health for the funding of these studies. The authors declare no conflict of interest.

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+These authors contributed equally to this work.This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).An 87-year old Caucasian male with past medical history of rheumatoid arthritis (RA) and chronic kidney disease presents with left hand erythema, pain, tenderness, induration and edema. Clinically, these hand findings began proximal to the metacarpo-phalangeal joints and extended to the distal wrist. He was noted to have ipsilateral axillary lymph node enlargement but denied any constitutional signs or symptoms. Laboratory markers of inflammation were poor prognostic indicators due to relatively active RA, the use of chronic daily glucocorticoids and weekly adalimumab use. Oral antibiotics were administered with limited success leading to a skin biopsy which reported a hematogenously disseminated fungal panniculitis; cultures grew Cryptococcus neoformans, however, serum cryptococcal antigen was negative. With initial fluconazole treatment, skin findings and lymphadenopathy improved gradually over the next six months. However, the patient’s improvement stagnated and his condition reverted back to the state of initial presentation.An 87-year old Caucasian male, resident in the United States for over 50 years, with past medical history of rheumatoid arthritis (RA) and chronic kidney disease presents with left hand erythema, pain, tenderness, induration and edema. Clinically, these hand findings began proximal to the metacarpo-phalangeal joints and extended to the distal wrist. Erythema was 9 cm with central induration and swelling measuring 2 cm. The patient had no limitation of motion due to swelling, pain, neurologic or vascular compromise. He was noted to have ipsilateral axillary lymph node enlargement but denied any constitutional signs or symptoms. Laboratory markers of inflammation were poor prognostic indicators due to relatively active RA, the use of chronic daily glucocorticoids and weekly adalimumab use. The patient had traveled to Portugal two years ago where he had no contact with farm animals. After withholding adalimumab and treating with escalating spectrum of antibiotics over a month with minimal improvement, an MRI was performed showing infiltration of the ulnar aspect subcutaneous fat without evidence of osseous damage. Therefore, oral antibiotics were continued with limited success leading to a skin biopsy which reported a hematogenously disseminated fungal panniculitis. Both the bacterial and the fungal cultures of the skin biopsy grew Cryptococcus neoformans. On fungal culture, sparse growth of yeast-like colonies was obtained on week three of culture. The yeast grew on Sabouraud dextrose agar at 30 °C. Round, narrow-budding yeasts were seen on wet preparation of the colonies. Urease was positive, and a caffeic acid disk test was positive. The Vitek2 YST card (bioMérieux, Durham, NC, USA; code number 21343) identified the yeast as C. neoformans/C. gattii. Differentiation between C. neoformans and C. gattii was not performed on this isolate, as that practice was only performed for immunocompetent patients with unexplained cryptococcosis. Serum cryptococcal antigen was negative. Antifungal drug susceptibility testing was not requested since it is known that Cryptococcus neoformans is almost universally susceptible to fluconazole, especially when the patient has no history of previous fluconazole administration. With initial fluconazole treatment, skin findings and lymphadenopathy improved gradually over the next six months. However, the patient’s improvement stagnated and his condition reverted back to the state of initial presentation. Given this, his hand was explored surgically and a cryptococcoma was excised in its entirety (histopathologically-confirmed diagnosis of Cryptococcus neoformans infection). The patient was treated with fluconazole for a total of six months after the surgical exploration and within a few post-operative months was pain and swelling free and enjoyed his pre-infected functional state.Cryptococcus species are encapsulated yeasts that infect humans and were first described in 1894. Infection with cryptococcal species presents across a wide spectrum usually as meningoencephalitis and pneumonitis, with cutaneous infections appearing more uncommonly. Soft tissue cryptococcal infections include cellulitis, necrotizing fasciitis, and cryptococcomas [1]. In humans, TNF-α is responsible for macrophage and phagosome activation, it differentiates monocytes into macrophages and leads to macrophage recruitment to sites of infection [2]. Given the fact that cryptococcal species are encapsulated yeasts, eradication occurs primarily through macrophage phagocytosis. In this patient, inhibition of TNF-α with adalimumab led to diminished macrophage function and increased susceptibility to opportunistic infection.Although no cases of cryptococcosis have been detected in 10,050 treated patients in the United States post-marketing database for adalimumab, other cases of invasive cryptococcosis in a patient receiving adalimumab have been documented and reinforce the relationship between TNF-α antagonists and the emergence of severe opportunistic infections [3].Cryptococcus gattii primarily occurs in immunocompetent hosts whereas 90% of Cryptococcus neoformans infections occur in immunocompromised hosts [4]. Differentiation of C. neoformans from C. gattii may be performed via either sequencing or through use of canavanine glycine bromothymol blue (CGB) agar. However, at this point in time, it is not routine for laboratories to perform such differentiation. Differentiation can be performed for isolates cultured from either immunocompetent patients or those patients with significant travel history to the Pacific Northwest region of Canada and the United States. Our patient did not have risk factors at the time for C. gattii and so was not tested. Although clinical human cases of C. gattii are still unusual in areas outside of tropical and subtropical climates as well as the Pacific Northwest, scientists are discovering ecologic niches in temperate climates of Europe and elsewhere which may contribute to transmission of this organism [5]Treatment of patients with mild to moderate cryptococcal infections consists of fluconazole for 6–12 months as first line therapy. Other acceptable regimens include itraconazole, voriconazole, posaconazole and amphotericin B. Surgery remains a viable option for those patients who cannot tolerate antimicrobial therapy or those that fail to respond, as was the case in this patient [5].This case demonstrates the awareness required by physicians to identify uncommon pathogens found in skin infections, especially in those who are immunocompromised [5]. While not widely reported, TNF-α inhibition with adalimumab provides a clinical environment for development of cutaneous cryptococcal disease. A high degree of suspicion for these pathogens is necessary when assessing immunosuppressed patients with skin lesions that fail to improve despite conventional therapy [6].The authors declare no conflict of interest. 

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+This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).Many studies have revealed molecular connections between breast and bone. Genes, important in the control of bone remodeling, such as receptor activator of nuclear kappa (RANK), receptor activator of nuclear kappa ligand (RANKL), vitamin D, bone sialoprotein (BSP), osteopontin (OPN), and calcitonin, are expressed in breast cancer and lactating breast. Epithelial-mesenchymal transition (EMT) and mesenchymal-epithelial transition (MET) effectors play critical roles during embryonic development, postnatal growth, and epithelial homeostasis, but also are involved in a number of pathological conditions, including wound repair, fibrosis, inflammation, as well as cancer progression and bone metastasis. Transforming growth factor β (TGFβ), insulin-like growth factor I & II (IGF I & II), platelet-derived growth factor (PDGF), parathyroid hormone-related protein (PTH(rP)), vascular endothelial growth factor (VEGF), epithelial growth factors II/I (ErbB/EGF), interleukin 6 (IL-6), IL-8, IL-11, IL-1, integrin αvβ3, matrix metalloproteinases (MMPs), catepsin K, hypoxia, notch, Wnt, bone morphogenetic proteins (BMP), and hedgehog signaling pathways are important EMT and MET effectors identified in the bone microenviroment facilitating bone metastasis formation. Recently, Runx2, an essential transcription factor in the regulation of mesenchymal cell differentiation into the osteoblast lineage and proper bone development, is also well-recognized for its expression in breast cancer cells promoting osteolytic bone metastasis. Understanding the precise mechanisms of EMT and MET in the pathogenesis of breast cancer bone metastasis can inform the direction of therapeutic intervention and possibly prevention.Over 80% of the malignant tumors are of epithelial origin and a number of these carcinomas are highly osteotropic. More than 90% of cancer-related death is caused not by the primary tumor, but by their metastases at distant sites [1,2].Although breast cancer mortality has been declining over the past decade, primarily due to earlier detection, adjuvant therapies, and the advent of targeted therapies for estrogen receptor-positive and (epithelial growth factor receptor-2) HER2-positive cancers; many patients relapse after an initial response to standard treatment options. Bone is the most common site of breast cancer metastasis (70%) [3,4,5,6,7,8,9]. Bone metastasis is associated with reduced survival, impaired quality of life, and pain due to skeletal-related events (SREs) [10]. Well-known bone-targeted agents, bisphosphonates and the receptor activator of nuclear kappa ligand (RANKL) inhibitor, denosumab, have emerged as effective options for the treatment of breast cancer patients with bone metastases [11,12,13,14,15,16,17,18]. Tumors contain a subpopulation of cells, called cancer stem cells (CSCs), which have the ability to self-renew and regenerate the tumor. The residual tumors after systemic treatments (hormonotherapy, chemotherapy, or targeted therapy) are enriched for CSCs and have a gene signature with hallmarks of epithelial-mesenchymal transition (EMT)-like properties [19,20,21,22]. Epithelial-mesenchymal transition (EMT) endows metastatic properties upon cancer cells to promote invasion, migration, and subsequent dissemination. The disseminated tumor cells recruited into the target organs may also undergo mesenchymal-epithelial transition (MET) that would favor metastasis formation [23,24,25,26].This article will review the clues and clinical implications of EMT and MET for treatment and possibly prevention of bone metastasis of breast cancer. Much of the evidence for a possible role of EMT in progression of breast cancer and bone metastasis has arisen from studies of in vitro culture of epithelial cell lines. In vivo and clinical evidence has started to accumulate recently.Metastasis is associated with the presence of peripheral blood circulating tumor cells (CTCs) and bone marrow disseminated tumor cells (DTCs) in patients with breast cancer [26,27,28]. Early in the metastatic cascade, cancer cells from the primary tumor acquire invasive properties and gain access to the blood or lymphatic vascular systems, which is aided by neo-angiogenesis and remodeling/destruction of the basement membrane. In the bloodstream (and presumably in lymphatic vessels), intravasated CTCs are capable of surviving and eventually reach “hospitable” distant secondary sites, such as bone, lungs, brain, and liver. Extravasation of CTCs at the secondary site requires recognition of, and adhesion to, vascular endothelial cells followed by matrix degradation [26,29,30,31,32,33,34]. Finally, the CTCs must invade the secondary tissue to become DTCs, typically shown in the bone marrow. All of these processes are evidence of a more motile and plastic “mesenchymal like” phenotype that promotes movement from a syncytial mass and invasion through tissue [35,36].An EMT-like process, first described in embryonic development, is one of the main mechanisms involved in breast cancer metastasis and most likely contributes to metastases from all types of carcinomas [37].EMT can be classified into three subtypes. Type 1 EMT occurs during development and includes the mesenchymal transition of primitive epithelial cells during gastrulation, generation of migrating neural crest cells from neuroepithelial cells, and formation of endocardial cushion tissue from cardiac endothelial cells. Type 2 EMT includes the transition of secondary epithelial (and endothelial) cells to tissue fibroblasts, which can be observed during the processes of wound healing, regeneration, and fibrosis in adult tissues. Type 3 EMT also occurs in adult tissues and involves the mesenchymal transition of epithelial carcinoma cells, leading to generation of metastatic tumor cells [38].BCSCs were originally described by Al-Hajj et al., in 2003 [39]. They isolated a tumorigenic subset of cancer cells from human breast tumors based on the expression of the surface markers CD44+, CD24−/low, and ESA+ (CD, cluster of differentiation; ESA, epithelial specific antigen). The CD44 high/CD24 low phenotype in breast cancer cell has been linked to EMT through the mesenchymal attributes of breast cancer stem cells, which also have dramatically enhanced malignant properties [40,41,42,43,44].Stephen Paget, in 1889, proposed the seed and soil hypothesis: Bone provides the fertile soil in which certain cancer cell seeds prefer to grow [45].Bone is a dynamic tissue that is constantly remodeled through the resorption of old bone by osteoclasts and the subsequent formation of new bone by osteoblasts [46,47].MSCs from BM (bone marrow) can become tumor-associated fibroblasts, have immunosuppressive function, and facilitate metastasis by epithelial-to-mesenchymal transition. Moreover, MSCs generate osteoblasts and osteocytes and regulate osteoclastogenesis. Therefore, MSCs can play an important pro-tumorigenic role in the formation of a microenvironment that promotes BM and bone metastases. MSCs are multipotent progenitor cells, which do not only regulate haematopoietic development, but also give rise to a majority of BM stromal cell lineages. These lineages include osteoblasts, adipocytes, chondrocytes, fibroblasts, endothelial cells, and myocytes. MSCs also release soluble factors that regulate the development and function of osteocytic and osteoclastic lineages, such as IL-1b, IL-6, IL-11, Dkk-1 (Dickkopf-1), Wnt proteins (Wnt 2, 4, 5, 11, 16), TGF-β, FGF-2, PDGF, PGE2, RANKL, LIF, OPG, M-CSF, MIP-1a, and HA. As MSCs may have an essential role in invasion and proliferation of cancer cells, there is close interaction and crosstalk among metastatic cancer cells, the BM microenvironment, and bone [48,49,50,51,52,53]. Bone-derived TGF-β is one of the most abundant growth factors in bone matrix and is a major factor regulator of tumor cell behavior in bone [54,55].TGF-β itself is a regulator of both physiological and pathophysiological EMT. TGF-β elicits its cellular responses by binding to TGF-β type I and type II serine/threonine kinase receptors and phosphorylation of receptor regulated (R-) Smad2 and Smad3. Activated R-Smads form heteromeric complexes with common mediator Smad4, which accumulates in the nucleus, where they control gene expression in a cell type-specific manner [56,57,58,59,60,61,62].Normal mammary gland development is under the influence of hormones, such as estrogen, progesterone, and prolactin, during the stages of prepuberty, puberty, pregnancy, lactation, and involution. A number of genetic pathways control this process, including the RANK/RANKL/OPG pathway. Studies have also demonstrated a key role for the RANK/RANKL/OPG pathway during mammary tumor formation and metastasis [63,64,65,66,67,68]. RANKL is a TNF family member that activates NF-κB and plays a fundamental role in antigen-presenting cells and during osteoclastogenesis. RANKL protein is exclusively expressed in PR-positive cells in the mammary epithelium. RANKL is necessary for the extensive proliferation of HR-negative epithelial cells in response to progesterone stimulation [68]. Another paracrine mediator of PR signaling is calcitonin, a 32-amino-acid peptide hormone involved in calcium homeostasis. Calcitonin expression is induced by progesterone in the luminal cells. The cognate calcitonin receptor is expressed in the myoepithelium, suggesting that calcitonin may act as a paracrine factor in a heterotypic interaction; but its biological function remains to be defined [69]. Taken together, ER and PR, they function in the mammary involvement of the stroma in the case of pubertal ER signaling and possibly more crosstalk with myoepithelial cells in PR-driven epithelial proliferation [63,66].RANKL has been proposed to act as a paracrine mediator of stem cell activation because expression of its cognate receptor, RANK, is enriched in the basal compartment. Type I, type II, and type III or atypical cadherins are expressed in the mammary gland. Type I cadherins include epithelial (E), neural (N), placental (P), and retinal (R) cadherins. E-Cadherin is expressed exclusively in all of the mammary epithelial cells, while P cadherin is expressed in mammary epithelial cells of the alveoli and ducts, but also in the myoepithelial cells. N-Cadherin is expressed in mesenchymal cells of the mammary stroma. R-Cadherin, which was first identified in the retina, is expressed in the mammary epithelial cells. E-Cadherin provides a tight connection between epithelial cells and localizes and interacts with components of the adherens junction [70,71].The functional loss or downregulation of E-cadherin (CDH1) from epithelial cells is considered a hallmark of EMT. E-Cadherin downregulation in cancer cells often occurs as a result of promoter methylation. The dissolution of adherens junctions is a critical step of EMT, with loss/decrease or relocalization of CDH1 as the most commonly used determinant of the EMT phenotype [72,73,74,75,76].All metastatic tumors of invasive ductal carcinoma were seen to be re-expressing E-cadherin irrespective of the E-cadherin status of the primary tumors [71]. Studies, thus, provide proof of principle that the metastatic cascade invokes E-cadherin emergence and, thus, supports a MET-like phenomenon.Vimentin is a key regulator of breast cancer cell migration and a marker for mesenchymal subtype, characteristic of cancer cells that have undergone epithelial-mesenchymal transition. Expression of vimentin is related to reduced expression of E-cadherin and upregulation of N-cadherin [77].MicroRNAs can regulate TGF-β-induced apoptotic and growth suppressive functionality. miRNAs together with other non-coding RNAs (long non-coding RNAs, small nucleolar RNAs, and ultraconserved regions) contribute to carcinogenesis. miRNAs can function both as oncogenes and as tumor suppressors, the involvement of different miRNAs is reported in the formation and regulation of human BCSCs. The microRNA (miR)-200 family is master regulator and inhibits initialing steps of EMT and metastasis [78,79,80,81,82,83]. Down-regulation of the epithelial miR-200 family in primary breast tumors, which leads to repression of E-cadherin, already predispose the cancer to successful metastasis, as evidenced in poorer outcomes [79]. The miR-200 family members have been revealed to promote E-cadherin re-expression via the repression of ZEB family genes, causing inhibition of cancer invasion and metastasis [80,81]. Expression of miR-200 is shown to be epigenetically regulated by histone-modifications and DNA promoter methylation. While let-7 family, miR-200 family, miR-30 family, miR-128, miR-34c, and miR-16 are downregulated in BCSCs; miR-181 and miR-495 are upregulated in BCSCs. The most frequently cited EMT-related miRNAs are those belonging to the miR-200 family, which consists of miR-200a/b/c, miR-141, and miR-429. miR-10b is also highly expressed in metastatic breast cancer cells and promotes tumor invasion and migration [84,85,86,87].Locally invading tumor cells undergoing an EMT proliferate less as they migrate more. EMT can arrest cell proliferation through many EMT regulators such as β-catenin, Snail, and ZEBs [88,89,90].Bone marrow-derived human MSCs promote de novo production of lysyl oxidase (LOX) from human breast carcinoma cells, which is sufficient to enhance the metastasis of otherwise weakly metastatic cancer cells to the lungs and bones.LOX is a copper-dependent amine oxidase that catalyzes the cross-linking of collagens and elastins in the ECM. LOX is an essential component of the CD44-Twist signaling axis, in which extracellular hyaluronan causes nuclear translocation of CD44 in the cancer cells, thus, triggering LOX transcription by associating with its promoter. Processed and enzymatically active LOX, in turn, stimulates Twist transcription, which mediates the MSC-triggered epithelial-to-mesenchymal transition (EMT) of carcinoma cells. Surprisingly, although induction of EMT in breast cancer cells has been tightly associated with the generation of cancer stem cells, it is shown that LOX, despite being critical for EMT, does not contribute to the ability of MSCs to promote the formation of cancer stem cells in the carcinoma cell populations [91].Release of secreted proteins (termed the secretome) appears to underline the progression of the metastatic phenotype [92]. For example, secretion of soluble cytokines and chemokines is known to modulate cell-cell communication at primary and secondary tumor sites, however, a novel suite of extracellular vesicles (EVs) (exosomes) capable of horizontal transfer of information (protein, mRNA, miRNA, and lipid) between cells has been identified as important regulators of the tumor microenvironment [36,93]. EVs have been implicated in modifying the tumor microenvironment to induce angiogenesis and metastasis in breast cancer, as well as facilitate the transfer of oncogenic potential through activation of MAPK and Akt signaling pathways. Given that EVs carry disease specific signatures such as miRNAs, their clinical importances are being investigated.Tumor-associated macrophages regulate breast cancer invasiveness through exosome-mediated delivery of oncogenic miRNAs (oncomiRs). Exosomes commonly contain expression of proteins involved in multivesicular body (MVB) biogenesis (TSG101, Alix), heat shock proteins (Hsp70, Hsp90), cytoskeletal components (actins, tubulins, keratins), adhesion molecules (integrins, tetraspananins), and membrane trafficking regulators (Rabs, annexins). Exosomes can modulate the immune response, control stromal remodeling in the metastatic niche, activate signaling pathways in neighboring cells, and transfer genetic and oncogenic information to recipient cells, increase cell motility [93,94,95,96,97].Vimentin and TGF-β regulates MTHFD2 (methylenetetrahydrofolate dehydrogenase 2) expression in metastatic breast cancer cells. It has been shown that MTHFD2 knockdown reduces cancer stem cell properties of bone metastatic breast cancer cells. Mitochondrial enzyme MTHFD2 has a potential role in breast cancer progression and bone metastasis [77,98]. Overexpression of mesenchymal genes, such as SPARC (secreted protein acidic and rich in cysteine), indicates that breast cancer cells may acquire mesenchymal markers by EMT and by fusion with MSCs; in particular, SPARC has recently been associated with the most aggressive and highly metastatic tumors [36,99].The serine/threonine kinase protein kinase D1 (PKD1) in normal ductal epithelial cells of the breast maintains the epithelial phenotype and prevents epithelial-to-mesenchymal transition (EMT). In addition to its inhibitory effects on EMT, PKD1 negatively affects directed cell migration by blocking actin reorganization processes at the leading edge of migrating cells. Furthermore, the expression and activity of PKD1 regulate the invasiveness of breast cancer cell lines by inhibiting the expression of multiple matrix metalloproteinases (MMPs) [100]. Except for CDH1 (E-cadherin), a variety of proteins that are down-regulated in response to an EMT include plakoglobin (JUP), occludin (OCLN), zonula occludens1 (TJP1), α-catenin (CTNNA3), and claudins 3/4/7 (CLDN-3/4/7). On the other end of the spectrum, the promotion of a mesenchymal-like phenotype is indicated by the up-regulation of proteins such as fibronectin (FN1), CDH2, VIM, ACTA2, and nuclear CTNNB1. The zincfinger proteins Snail1 (SNAI1), Snail2 (SNAI2), Zeb1 (ZEB1), and Zeb2 (ZEB2) each directly repress transcription of CDH1 in mammary cells by binding the E-boxes (CANNTG) located in the CDH1 proximal promoter, as do the basic helix-loop-helix factors E12/E47 (TCF3) and TWIST1. A number of other transcription factors cause relocalization of junctional CDH1, including SIX1, goosecoid (GSC), and forkhead box C2 (FOXC2). Interestingly, knockdown of CDH1 alone is sufficient to induce an EMT, highlighting the significance of repressors of CDH1 in the induction of an EMT. Indirect repression of CDH1 is also accomplished by EMT inducers, including SIX1, GSC, and FOXC2. Recently, p53 (TP53), Twist2 (TWIST2), and forkhead box Q1 (FOXQ1) have been added to this list of oncogenic EMT inducers [22,100]. The transcription factor Runx2 is essential for the formation of the skeleton and it also has a role in the regulation of normal mammary gland gene expression such as the transcription of the mammary gland-specific gene, β-casein. This skeletal transcription factor is aberrantly expressed at high levels in breast cancer cells that aggressively metastasize to the bone environment. In cancer cells, Runx2 activates expression of bone matrix and adhesion proteins, matrix metalloproteinases, and angiogenic factors that have been associated with metastasis [101,102,103,104]. In addition, Runx2 mediates the responses of cells to signaling pathways hyperactive in tumors, including BMP/TGFβ and forms co-regulatory complexes with SMADs and other co-activator and co-repressor proteins to regulate gene transcription contributing to tumor growth in bone and the accompanying osteolytic disease [46]. In addition to transcription factors, several signaling pathways are known to induce an EMT, such as the TGF-β/Smad, receptor tyrosine kinases (epithelial growth factor (EGF), hepatocyte growth factor (HGF), insulin-like growth factor (IGF), fibroblast growth factor (FGF), platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF)), bone morphogenetic proteins (BMP), Wnt, notch and hedgehog, TNF-α/NF-κB, hypoxia-induced Jagged2, and HFI-1α/LOX pathways [105,106,107], Figure 1.Signaling pathways and markers of epithelial to mesenchymal transition (EMT).A process opposite to the initial EMT at the primary tumor site, mesenchymal to epithelial transition (MET), is an evolving and relatively under-investigated mechanism that is considered to contribute substantially to the colonisation of DTCs into metastatic tumors at the secondary site. Recent studies suggest that MET can occur in breast cancer models. As E-cadherin (CDH1) downregulation in cancer cells often occurs as a result of promoter methylation, loss of promoter methylation at the secondary site causes the metastatic cancer cells to re-express E-cadherin through MET [108,109,110,111,112,113,114,115,116,117,118]. A potential demethylating factor has been identified as 1α,25-dihydroxyvitamin D3, which has been shown to promote de novo E-cadherin re-expression in breast cancer cell lines [119,120]. Microenvironmental factors like miRNAs can also contribute to MET at the metastatic site. It is shown that miR-200 promotes Sec23A-positive secretory vesicles, the cargo of which may regulate both autocrine and paracrine pathways to promote establishment, survival, and/or growth of the macrometastases [121,122,123,124,125,126,127,128].EMT and MET may determine dormant or active states of the tumor, respectively, and allow for an indeterminate number of cycles of invasion and metastases formation, Figure 2.Mesenchymal to epithelial transition (MET).Evidence suggests that EMT-associated apoptosis reduction and senescence inhibition contribute largely to therapeutic resistance. Cells that have undergone EMT withstand external insults better, leading these cells to display resistance to chemotherapy and endocrine therapy [19,20,21,22,117,129,130,131]. Tumors that have undergone a MET at a secondary site may be more susceptible to apoptotic insults and, hence, may be treated more successfully with chemotherapeutic drugs. Subclinical tumor may be forced to undergo a MET to facilitate therapy.Thus, it is reported that experimental model systems will be needed to settle this key question as it directly impinges on whether inducing or inhibiting MET would be beneficial in the treatment of breast cancer. Further, the question of whether the MET is stable in the metastases or if these cells show ongoing phenotypic plasticity leading to a second EMT is also open to question.Elimination of tumor cells that exhibit a mesenchymal phenotype could potentially be achieved by blocking the signaling pathways that trigger and/or maintain tumor EMT. In particular, blockade of the IL-8–IL-8R axis appears to be an attractive strategy to disrupt the autocrine positive feedback loop between EMT and IL-8, while simultaneously decreasing the paracrine signals that mesenchymal tumor cells could exert on their surrounding environment. Secretion of IL-8 is also a feature of the tumor stroma, and blockade of IL-8 signaling could be fundamental in lessening the tumor promoting signals originated on stromal fibroblasts, monocytes, neutrophils, and endothelial cells in response to stressful environments, including hypoxia, acidosis, or genotoxic damage. Supporting this strategy, several preclinical studies have already demonstrated the ability of neutralizing antibodies to the IL-8Rs, a humanized antibody against IL-8 (ABXIL-8) and the small-molecule inhibitor repertaxin to inhibit angiogenesis, tumor growth, and metastasis in xenograft tumor models [132]. Members of TGF-β superfamily, which include bone morphogenetic proteins (BMP), are involved in the control of many different biological processes, including cell proliferation, differentiation, apoptosis, and regulation of invasiveness. The homodimeric protein BMP7 induces MET in normal and nontransformed cells.BMP7 expression in patients with primary breast tumors exclusively developing bone metastases is significantly lower than in primary breast tumors developing exclusively visceral (lung and/or liver) metastases. These clinical findings suggest that decreased BMP7 expression may confer a bone metastatic potential to human breast cancer cells. Normal ducts of the breast display strong apical BMP7 protein expression. Functional studies reveal that BMP7 overexpression by breast cancer cells inhibits de novo formation of osteolytic bone metastases and, hence, the metastatic capability of breast cancer cells in in vivo bone metastasis model. BMP7 is able to counteract SMAD-dependent TGF-β signaling. Inactive TGF-β is concentrated and stored in high amounts in extracellular bone matrix and can be released and activated by osteoclastic resorption. Activated bone matrix-derived TGF-β may act as a paracrine growth factor for neighboring osteolytic cancer cells that may have colonized the bone marrow. BMP7 regulates epithelial homeostasis in the human mammary gland by preserving the epithelial phenotype. Decreased BMP7 expression during breast cancer progression may, therefore, contribute to the acquisition of a bone metastatic phenotype. Furthermore, exogenous BMP7 can still inhibit breast cancer growth at the primary site and in bone marrow. Therefore, BMP7 may represent a novel therapeutic molecule for repression of local and bone metastatic growth of human breast cancer [133,134].Related with the bone resorption induced by breast tumor cells, there is evidence that the TRAF inhibitor ABD56 is able to inhibit the M-CSF and RANKL induced osteoclastogenesis enhanced by breast tumor cells. ABD56 acts both on osteoclast precursor blocking the membrane localization and ubiquitination of TRAF6 and subsequent phosphorylation of various factors induced by RANKL as IkB, and on breast tumor cells inhibiting the adhesion, spreading, and migration with no impact on the cell viability. Such dual effect of TRAF inhibitor, not observed for RANKL inhibitor and different from the previously described for bisphosphonates, suggests great therapeutic potential for such inhibitors. There is also evidence that the VEGF inhibitor sunitinib can normalize vascularization of highly osteolytic bone metastatic tumor and improve efficacy of the associated cytotoxic therapy. Jagged1, which is expressed by osteoblasts under the control of by parathyroid hormone signaling pathway co-opted in pathological bone metastasis, can regulate the expansion of hematopoietic stem cells in the bone microenvironment through Notch signaling. The clinical importance of Jagged1 is its association with an increased incidence of breast cancer relapse and bone metastasis. It is shown to be an ideal target for monoclonal antibody therapy as it is a cell-surface protein. TGF-β signaling has been targeted by therapeutic agents currently being tested in clinical trials [135,136,137,138,139,140,141,142,143].EMT transcription and signaling pathways are considered as anticancer drug targets. For EMT transcription pathways; AKT (KTX-O401 (perifosine), VQD-OO2 (API-2), GSK690693, mTOR (RAD 001 (everolimus)), XL-765 (Exelixis)), NF-κβ (OT-304, IMD-0354), β-catenin (ERX-3722), PKC (LY317615 (enzastaurin)), and for EMT signaling pathways; EGFR-1 (erlorinib, gefitinib), ErbB2 (trastuzumab), IGF-1R (CP-751, 871; AMG479), VEGF/VEGFR (bevacizumab, cediranib) Src (dasatinib, bosutinib), and NOTCH (anti-notch-1 monoclonal antibody) are investigated [100,143].Search stategy is restricted with the EMT/MET pathways and breast cancer bone metastasis and treatment or prevention. Only published English-language articles of preclinical and clinical studies as well as reviews were considered eligible.There is growing implication of EMT and MET in the progression of breast carcinoma and bone metastases both in studies with experimental models and humans. New surrogate markers are needed to define different stages during the transition from the epithelial to mesenchymal phenotype, and the reverse transition.The interrelationship between CSCs, embryonic signaling pathways, and EMT/MET pathways offers a continuum of potential therapeutic targets for breast carcinoma related bone diseases. Theraupetic delivery of microRNAs, “differentiation-inducing” agents such as HDAC inhibitors, antibody-directected to the cytoskeletal markers or manipulating the skeletal transcription factors, such as Runx2, by depletion either chemically or by RNA interference can be potential strategies to treat and/or prevent breast cancer bone metastases.The author declare no conflict of interest.

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+This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).Bone loss is common in patients with breast cancer. Bone modifying agents (BMAs), such as bisphosphonates and denosumab, have been shown to reverse or stabilize bone loss and may be useful in the primary and metastatic settings. The purpose of this review is to provide clear evidence-based strategies for the management of bone loss and its symptoms in breast cancer. A systematic review of clinical trials and meta-analyses published between 1996 and 2012 was conducted of MEDLINE and EMBASE. Reference lists were hand-searched for additional publications. Recommendations were developed based on the best available evidence. Zoledronate, pamidronate, clodronate, and denosumab are recommended for metastatic breast cancer patients; however, no one agent can be recommended over another. Zoledronate or any oral bisphosphonate and denosumab should be considered in primary breast cancer patients who are postmenopausal on aromatase inhibitor therapy and have a high risk of fracture and/or a low bone mineral density and in premenopausal primary breast cancer patients who become amenorrheic after therapy. No one agent can be recommended over another. BMAs are not currently recommended as adjuvant therapy in primary breast cancer for the purpose of improving survival, although a major Early Breast Cancer Cooperative Trialists’ Group meta-analysis is underway which may impact future practice. Adverse events can be managed with appropriate supportive care.Optimization of bone health is an important aspect of care to consider for patients with a breast cancer diagnosis at any stage. In patients with primary breast cancer, treatment associated bone loss can occur either with adjuvant endocrine therapy (e.g., aromatase inhibitors), or in patients with a reduction of ovarian function due to surgery or chemotherapy. Bone loss in this setting is due to enhanced bone turnover secondary to estrogen decline [1]. Other factors, unrelated to breast cancer, that may also contribute to the risk of bone loss, include age (>65 years), race (Caucasian), low body mass index (<20 kg/m2), family and personal history of fractures, menopausal status, oral corticosteroid use, history of osteoporosis, and smoking [2]. In the primary breast cancer setting, bone loss can present (as is the case with osteopenia or osteoporosis) as bone fractures, bone pain, and/or impaired mobility. The rate of vertebral fractures is five times higher among women with breast cancer, compared to the general population [3]. As such, there is an impact on overall quality of life for these patients.Bone health can also be compromised in the metastatic breast cancer setting as a result of malignant cells stimulating bone resorption via osteoclasts; this breakdown of bone can stimulate further tumour growth, leading to an increased risk of skeletal related events such as pain, vertebral and long bone fracture (with associated morbidity), spinal cord compression, and hypercalcemia [4,5]. Among patients with metastatic breast cancer, the incidence of bone metastases at some point during the course of the disease is approximately 73% [6].To date, efforts to optimize bone health have focused on bisphosphonates and biological agents, such as denosumab. Although the effectiveness of bone modifying agents (BMAs) in improving bone health has been established, uncertainties still remain (e.g., ideal time to initiate therapy, duration of therapy, modification of therapy, etc.). The purpose of this paper is to provide clear evidence-based strategies for the management of bone loss and symptoms of bone loss in patients with a breast cancer diagnosis. Specifically, the following questions are addressed: (1) In patients with metastatic breast cancer, when should bone modifying agents (BMAs) be used? Which BMAs should be considered and for how long? Should the BMA be switched after a skeletal-related event (SRE) or documentation of disease progression in bone? (2) In patients with primary breast cancer, how and when should fracture risk be assessed? Is there a role for BMAs in these populations and, if so, which agents should be considered and for how long? How should treatment with BMAs be monitored for effectiveness? (3) Should BMAs be used as adjuvant therapy to improve breast cancer-related outcomes? (4) What adverse events can occur with BMA use and should be disclosed to patients? What is the frequency of these adverse events with the different agents and schedules of administration? How should these adverse events be managed?The review process for this guideline was developed, based on: (1) The National Institute for Health and Clinical Excellence (NICE) overview of clinical guideline development for stakeholders, the public, and the National Health Service (NHS, England) [7]; (2) Cummings and Rivara’s methodology on reviewing manuscripts for Archives of Pediatrics & Adolescent Medicine (2002) [8]; and (3) The Appraisal of Guidelines for Research and Evaluation (AGREE) collaboration [9]. With that methodological foundation, the guideline recommendations were drafted by a medical oncologist from the Tom Baker Cancer Centre, Calgary, Alberta, with support from a research methodologist and an expert panel of physicians from the Province of Alberta Breast Tumour Team. Members of the Breast Tumour Team include medical oncologists, radiation oncologists, surgeons, pathologists, psychosocial oncologists, nurses, and pharmacists. The evidence base for the guideline was informed by a systematic review of the literature. The literature will be periodically reviewed and the guideline will be updated as new or compelling evidence is identified.A systematic search for relevant literature related to breast cancer and bone health was conducted of MEDLINE (1996 to 2012 July) and EMBASE (1996 to 2012 July). The search included the terms “breast neoplasm” and “zoledronic acid” or “zoledronate” or “clodronate” or “clodronic acid” or “alendronate” or “alendronic acid” or “pamidronate” or “pamidronic acid” or “ibandronate” or “ibandronic acid” or “denosumab”. The search was limited to randomized controlled trials and meta-analyses. Reference lists of key clinical trials were also hand-searched for additional literature. Guidelines on this topic were identified from a search of the National Guidelines Clearinghouse database, as well as individual guideline developers’ websites.Articles were selected for inclusion in the review of the evidence if they reported outcomes related to survival, bone mineral density or skeletal related events, or adverse events. Indirect evidence with other oncologic-patient populations treated with BMAs was also deemed relevant if insufficient primary evidence was available. Articles were excluded from the review of the evidence if they were qualitative or descriptive studies, opinion papers, letters, or editorials. Due to a lack of translation services, non-English language articles were excluded from the review of the evidence.A total of 57 published articles were deemed relevant to inform the role of BMAs in breast cancer. Twenty-nine publications focused on the use of BMAs in the prevention of treatment-related bone loss in patients with primary breast cancer (Table 1); thirteen publications examined the efficacy of BMAs in preventing skeletal related events (SREs) in patients with metastatic breast cancer (Table 2); and fifteen publications looked at the use of BMAs in the adjuvant setting in patients with primary breast cancer (Table 3).The literature search identified seven clinical practice guidelines that provided recommendations on the use of bisphosphonates in the setting of breast cancer; these guidelines were developed by: American Society of Clinical Oncology (ASCO) [10,11], the National Comprehensive Cancer Network (NCCN) [12], Cancer Care Ontario (CCO) [13], the British Columbia Cancer Agency (BCCA) [14], the International Society of Geriatric Oncology (ISGO) [15], the European Expert Panel (EEP) [16], and Cancer Care Australia [17]. Following the initial search, an additional Canadian guideline, BONUS 6, was published by a panel of experts in the field [18] and ASCO released an updated version of their guideline [11]. The World Health Organization’s Fracture Risk Assessment Tool (FRAX) [19] was also identified by the literature search and was included in the review in order to establish patients at risk of fracture.Evidence on the use of bone modifying agents (BMAs) in patients with treatment-related bone loss from breast cancer.Note: ZOL—zoledronate; RIS—risedronate; PAM—pamidronate; CLO—clodronate; IBA—ibandronate; DEN—denosumab; ALE—alendronate; q—every; m—months; w—weeks; d—day; BMD—bone mineral density; SC—subcutaneous; IV—intravenous.Evidence on the use of BMAs in patients with metastasis-related bone loss from breast cancer.Note: ZOL—zoledronate; PAM—pamidronate; CLO—clodronate; IBA—ibandronate; DEN—denosumab; w—weeks; d—day; SC—subcutaneous; IV—intravenous; SRE—skeletal related events/complications; OS—overall survival.Evidence on the use of BMAs as adjuvant therapy in patients with primary breast cancer.Note: ZOL—zoledronate; PAM—pamidronate; CLO—clodronate; IV—intravenous; w—weeks; d—day; m—months; DFS—disease-free survival; OS—overall survival; RFS—recurrence-free survival; MFS—metastasis-free survival; SRE—skeletal related events.Bone loss can occur in breast cancer due to treatment, especially estrogen suppressing therapy and certain chemotherapies, or due to the disease itself [1,4]; however, bone modifying agents such as bisphosphonates and denosumab offer patients with breast cancer a way to treat and potentially prevent bone loss. The following discussion of the literature will highlight trials demonstrating the effectiveness and safety of these bone modifying agents and describe data on less established areas, such as the timing of therapy, the duration of therapy, and the use of therapy as an adjuvant to prolong survival.The effectiveness of bisphosphonates as bone modifying agents has been well established. Trials employing the use of zoledronate, risedronate, alendronate, ibandronate, clodronate, and pamidronate in patients receiving aromatase inhibitors or chemotherapy have demonstrated that these agents increase bone mineral density and decrease bone turnover and the risk of fractures. Zoledronate (4 mg IV, every six months) significantly increased bone mineral density at the hip and spine (+3.9% and +4.0%, respectively, vs. baseline) in early stage premenopausal patients with ovarian suppression (n = 404) at five years follow-up, as compared to placebo which incurred significant losses at these sites (−4.1% and −6.3%, respectively, vs. baseline) [63,64,65,66]. Others have observed similar changes in bone mineral density with the use of zoledronate among premenopausal patients [30,31]. Pamidronate (60 mg IV, every three months), but not risedronate (35 mg per week, orally), was able to achieve a significant 1.9% increase in bone mineral density at the spine, but not at hip, among premenopausal patients undergoing chemotherapy [25,34]. No significant difference in the rate of fractures has been observed among the premenopausal group in any of these studies. There have been mixed results regarding the markers of bone turnover (i.e., N-terminal telopeptide (NTX), C-terminal cross-linked telopeptide of type I collagen (CTX-I), N-terminal propeptide of type I collagen (P1NP), and bone alkaline phosphatase (AP)) [63,64,65,66,77].Among post-menopausal women receiving aromatase inhibitors, a variety of bisphosphonates have been tested and show similar improvements in bone loss. Zoledronate (4 mg IV, every six months) was shown to maintain or significantly increase (+2.66% vs. baseline) bone mineral density at the spine [20,22]. Similarly, in the Study of Anastrozole with the Bisphosphonate Risedronate (SABRE) and Risedronate’s Effect on Bone in Women with Breast Cancer (ReBBeCa) trials, risedronate demonstrated significantly better maintenance or increases in bone mineral density, vs. placebo (2.5%–2.9% higher than placebo for hip and 1.6%–4.0% higher than placebo for spine); significant improvements in NTX, CTX-I, PINP, and bone AP, indicating a reduction in bone turnover, were also observed [21,32,33]. The Anastrozole-Induced Bone Loss (ARIBON) trial likewise demonstrated improvements in bone mineral density at the hip and spine (4.5% higher than placebo for hip and 6.2% higher than placebo for spine) with ibandronate (150 mg orally, every 28 days); in addition, ibandronate improved serum levels of NTX, CTX-I, bone AP, as well as T-score [27]. Oral clodronate (1600 mg per day) also led to a significant improvement in bone mineral density at the hip, spine, and femoral neck among postmenopausal patients [29,55,73]. Finally, alendronate has also demonstrated efficacy among postmenopausal patients in maintaining or increasing bone mineral density at the spine and femoral neck [26]. To date, no trials comparing one bisphosphonate with another have been conducted in the setting of treatment-related bone loss; bisphosphonates are generally thought to be comparable in terms of efficacy and the decision to use one agent over another is often related to route of administration or other factors that could affect compliance.The ratio of receptor osteoprotegerin (OPG) to receptor activator of nuclear factor-kappa B ligand (RANKL) plays a role in osteoclastogenesis: When RANKL levels are high, bone loss will occur. However, by altering the ratio in favor of OPG by inhibiting RANKL, bone loss can be prevented [78]. Therefore, biological agents that inhibit RANKL may prevent bone loss due to treatment or metastases. Denosumab, a RANKL inhibitor, has demonstrated efficacy among postmenopausal patients receiving aromatase inhibitors. As compared to placebo, denosumab (60 mg SC, every six months) significantly increased bone mineral density of the hip (+4.7% vs. placebo), spine (+7.6% vs. placebo), wrist (+6.1% vs. placebo), and femoral neck (+3.6% vs. placebo) [23,24]. In this trial, denosumab also significantly improved serum levels of CTX (−91% vs. +9% for placebo) and PINP (−29% vs. −2% for placebo), but did not significantly improve the rate of fractures.Recommended agents for the prevention and management of bone loss include zoledronate (IV 4 mg over no less than 15 min every 6–12 months) and denosumab (60 mg SC every 6 months). Any oral bisphosphonate is acceptable as well, including clodronate (1600 mg per day), risedronate (35 mg per day), or alendronate (70 mg per day). The route of administration should be left to the discretion of the treating physician, taking into account compliance with treatment, cost of treatment, and patient preference.The timing of therapy (upfront vs. delayed) has been evaluated in several trials, among postmenopausal patients receiving aromatase inhibitors. The Zometa-Femara Adjuvant Synergy Trials (Z-FAST, ZO-FAST, and EZO-FAST) [38,39,40,41,42,43,44,46,47] and a National Cancer Institute (NCI) trial [48] compared zoledronate upfront (i.e., immediately) with delayed (i.e., following a fracture or a decrease in bone mineral density). Immediate zoledronate (4 mg IV, every six months) significantly increased bone mineral density at both the hip (5.4%–6.7% higher than delayed) and the spine (8.6%–9.3% higher than delayed) in the ZO-FAST and Z-FAST trials, as well as in meta-analysis of the combined data (hip: 3.4% higher than delayed; spine: 5.1% higher than delayed) [45]. The EZO-FAST and NCI trials also reported significant improvements in bone mineral density at the hip and spine. Where reported, there were no significant differences in T-scores, fracture rates, or overall survival (OS) for any of these studies.Bone loss can occur in metastatic breast cancer as a result of malignant cells stimulating bone resorption, which in turn can lead to further tumour growth [4]. Bone modifying agents may, therefore, be able to reverse or stabilize bone loss in patients with bone metastases. In a trial comparing clodronate (1600 mg per day, orally) with placebo, clodronate was shown to reduce the incidence of skeletal-related events (SREs) among patients with bone metastases from breast cancer [61]. Zoledronate (4 mg IV, every four weeks) was shown to significantly decrease the fracture rate (25.4% vs. 38.9% for placebo) and lower the incidence of one or more SREs (29.8% vs. 49.6% for placebo) among breast cancer patients with bone metastases [52]. Likewise, pamidronate (90 mg IV every three to four weeks for 24 cycles) significantly decreased the fracture rate (40% vs. 52% for placebo), the level of bone AP (−33% vs. +5% for placebo), and the incidence of one or more SREs (51% vs. 64% for placebo) [56]. Similar significant positive changes in the fracture rate and incidence of SREs were observed for ibandronate (2 or 6 mg every three to four weeks) [55]. Zoledronate, pamidronate, and ibandronate were then compared with denosumab and demonstrated similar efficacy, in terms of OS, at six months, among breast cancer patients with bone metastases (85% for denosumab vs. 81% for bisphosphonates) [49]. Monthly SC denosumab (120 mg) has been shown to be superior to monthly IV zoledronate in delaying time to first SRE and cumulative SREs [50].Bone modifying agents are recommended for patients with breast cancer with evidence of bone metastases. The presence of non-bone metastases is not a clear indication for the use of bone modifying agents; however bone modifying agents can be used when osteopenia (i.e., T-score between −1.0 and −2.5) or osteoporosis (i.e., T-score less than −2.5) is present. The FDA has approved zoledronic acid (5 mg once every 2 years) for the prevention of postmenopausal osteoporosis [79]; however, recommendations cannot be made favoring one agent over another. Acceptable agents and dosing regimens for bone metastases include zoledronate (4 mg IV over no less than 15 min every 4 weeks), pamidronate (90 mg IV over no less than 2 h every 3 to 4 weeks), clodronate (1600 mg per day orally), and denosumab (120 mg SC every four weeks). There are several advantages and limitations to the different agents and routes of administration. The route of administration should be left to the discretion of the treating physician, taking into account compliance with treatment, cost of treatment, and patient preference.The use of bone modifying agents as an adjuvant to standard therapy has also been investigated. The Austrian Breast and Colorectal Cancer Study Group Trial 12 (ABCSG-12) looked at the effect of adding zoledronate (4 mg IV, every six months) to endocrine therapy in early stage premenopausal patients with ovarian suppression (n = 404); after a median follow-up of 47.8 months, the trial demonstrated that zoledronate provided a gain in DFS (90.8% for endocrine therapy alone vs. 94.0% for endocrine therapy with zoledronate), but not a reduction in the risk of death (hazard ratio, 0.60; 95% CI, 0.32 to 1.11; p = 0.11). The addition of zoledronate resulted in a relative reduction of 35% in the risk of recurrence (hazard ratio, 0.65; 95% CI, 0.46–0.92; p = 0.01) [63,64,65,66]. Clodronate (1600 mg per day, orally) taken over a duration of two years significantly improved OS (81.5% vs. 76.1% for placebo) and reduced the incidence of bone metastases (9.6% vs. 13.5% for placebo) among patients with primary operable breast cancer [73,74]. However, pamidronate (150 mg per day, orally) did not improve OS or the rate of bone metastases, vs. placebo [70]. Similarly, among patients with breast cancer (stages II–III) in the Adjuvant Zoledronic Acid to Reduce Recurrence (AZURE) trial, zoledronate (4 mg every 3–4 weeks for six doses, before or after surgery, followed by 4 mg every 3 months for eight doses and then every 6 months for five doses) did not improve DFS (77% vs. 77%) or OS (85.4% vs. 83.1%) as compared to standard therapy; however, a sub-group analysis of postmenopausal patients revealed a significant improvement with treatment [67,68]. The National Surgical Adjuvant Breast and Bowel Project (NSABP) B-34 trial recently published the 7.5 year (median 90.7 months follow up) analysis of survival data among early breast cancer patients treated with oral clodronate (1600 mg per day) for three years or placebo, with similar findings of a sub-group advantage in postmenopausal patients for distant metastases-free interval in the treated patients [62]. Overall, evidence for the use of bisphosphonates as adjuvant therapy for prevention of bone metastases in women with breast cancer is undergoing review and meta-analysis to determine if benefit exists in subgroups such as postmenopausal patients. Ongoing research includes the Denosumab as Adjuvant Treatment for Women with High Risk Early Breast Cancer Receiving Neoadjuvant or Adjuvant Therapy (D-CARE) trial, which randomizes patients with bone metastasis to denosumab or placebo and follows patients for the main outcomes of DFS and bone metastasis-free survival [80]. Until such data are available, the use of bone modifying agents is not recommended outside of a clinical trial for patients with breast cancer as a standard adjuvant therapy to improve recurrence or survival rates.Adverse events associated with bisphosphonate use (oral or intravenous) and denosumab are typically mild and manageable, but include arthralgia, fever, thrombosis, bone pain, fatigue/tiredness, nausea, and gastrointestinal symptoms [20,21,24,25,26,27,29,36,81]. Patients undergoing therapy with bone modifying agents should be monitored throughout therapy for changes in renal function (i.e., creatinine clearance, serum calcitriol, serum calcium) [9,82,83,84,85]. In addition, patients with poor dental hygiene or poor dental health may be at increased risk of osteonecrosis of the jaw, especially those patients receiving potent IV BMAs [86,87]; therefore, patients should undergo preventive dentistry before starting treatment with a bone modifying agent and dentition should be monitored during therapy [9]. Furthermore, dental extractions should be avoided if possible and carried out by an experienced dentist. The Zoledronic Acid for Prolonged Treatment of Patients with Bone Metastases from Breast Cancer (ZOOM) trial recently compared 4-weekly and 12-weekly zoledronic acid (4 mg) in patients with breast cancer with one or more bone metastases (n = 425) and showed that 12-weekly treatment was not inferior to 4-weekly treatment. However, the trial was underpowered to identify clinically significant differences in grade 3–4 toxicity (i.e., bone pain, nausea, and asthenia), renal adverse events, or osteonecrosis of the jaw. N-Terminal telopeptide concentration did change significantly from baseline to 12 months in the 12-weekly group vs. the 4-weekly group (median 12.2% vs. 0.0%, respectively; p = 0.011) [88].Recent observations have raised the possibility of atypical fractures occurring in patients on long-term BMAs [89]; among patients with atypical fracture, the prevalence of bisphosphonate use was 90% [90]. Ongoing surveillance for atypical and minimal trauma fractures is warranted in patients undergoing therapy with BMAs.Baseline bone mineral density (BMD) testing and fracture risk assessment is recommended for patients with primary breast cancer for whom therapy with agents that suppress ovarian function is given, including premenopausal women with premature ovarian failure or ovarian suppression with luteinizing hormone releasing hormone analogue (LHRHA) and postmenopausal women on aromatase inhibitors (AIs). BMD testing in other postmenopausal women with primary breast cancer is recommended according to the Canadian Osteoporosis screening guidelines using a BMD cut off of T-score <−2.5 [91]. BMD is calculated using a dual-energy X-ray absorptiometry (DEXA) scan. Fracture risk should be assessed using the World Health Organization Fracture Risk Assessment Tool [19]. Repeat BMD testing should be performed as follows, in patients for whom pharmacotherapy with bone modifying agents is deemed to be not beneficial: Every five years in low risk patients (10-year risk <10% based on FRAX score) and every one to three years in moderate risk patients (10-year risk 10%–20% based on FRAX score).BMAs should be considered for the following patients with primary breast cancer: Premenopausal OR postmenopausal at high risk (i.e., 10-year fracture risk >20% OR prior fragility fracture of hip or spine OR more than one fragility fracture) and postmenopausal at moderate risk (i.e., 10-year fracture risk 10%–20%) OR a T-score lower than −2.0, AND undergoing aromatase inhibitor therapy for breast cancer. As per the Canadian Osteoporosis guidelines [91], exercise, adequate calcium (1200 mg per day total, diet plus supplements) intake, and vitamin D (1000 IU per day) supplementation are also recommended. For patients with primary breast cancer, no recommendations can be made favoring one bone modifying agent over another. Acceptable agents and dosing regimens for bone loss include: zoledronate (intravenous 4 mg over no less than 15 min, every 6–12 months), any oral bisphosphonate, denosumab (subcutaneous 60 mg, every 6 months). The route of administration should be left to the discretion of the treating physician, taking into account compliance with treatment, cost of treatment, and patient preference. There is no data on the optimal duration of therapy with bone-modifying agents for patients with primary breast cancer with treatment-related bone loss. Most randomized controlled trials have used durations of two to three years and none have compared one time period with another.In patients with primary breast cancer undergoing therapy with a bone modifying agent, BMD can be checked every two years. However, in patients with osteopenia, BMD should be checked annually.In patients with metastatic breast cancer, bone modifying agents (BMAs) are recommended upon confirmation of bone metastases; the presence of non-bone metastases is not an indication for the use of bone modifying agents.For patients with breast cancer with bone metastases, no recommendations can be made favoring one agent over another. Acceptable agents and dosing regimens for bone metastases include: zoledronate (intravenous 4 mg over no less than 15 min, monthly), pamidronate (intravenous 90 mg over no less than 2 h, monthly), clodronate (oral 1600 mg, daily) and denosumab (subcutaneous SC 120 mg, monthly). There are advantages and limitations to the different agents and routes of administration. The agent and route of administration should be left to the discretion of the treating physician, taking into account compliance with treatment, cost of treatment, and patient preference. BMAs should be continued in patients with breast cancer with bone metastases until there is evidence of a substantial decline in performance status. In the case of progressive disease, a change in systemic anti-cancer treatment is warranted with continuation of BMAs.In patients with breast cancer with bone metastases who have experienced a skeletal-related event (SRE) or progression in bone metastases, switching from one bisphosphonate to another is currently not recommended, since no double-blind data are available to support this strategy. However, some oncologists are switching from a bisphosphonate to denosumab at progression, based on proven better efficacy [53].Outside of a clinical trial, bone modifying agents are not recommended at this time for patients with primary breast cancer, as a standard adjuvant therapy to improve recurrence or survival rates. The Early Breast Cancer Cooperative Trialists’ Group meta-analysis of adjuvant bisphosphonate trials is ongoing and may impact future practice.Patients undergoing therapy with BMAs should be aware that the most common adverse events include nausea, fatigue, arthralgia, back pain, pyrexia, bone pain, vomiting, anemia, diarrhea, dyspnea, extremity pain, and constipation. Patients should also be monitored for changes in renal function (i.e., creatinine clearance). In addition, patients with poor dental hygiene or poor dental health may be at increased risk of osteonecrosis of the jaw and should undertake preventive dentistry before starting treatment with a bone modifying agent and avoid dental extraction. Adverse events should be managed with appropriate supportive care. Denosumab does not require specific monitoring of renal function but as with bisphosphonates, patients should be monitored for rare instances of symptomatic hypocalcemia.The authors acknowledge the contributions of the Province of Alberta Breast Tumour Team and the Guideline Utilization Resource Unit in the development of this clinical practice guideline.Alexander H. G. Paterson has received honoraria from Bayer, Amgen, Roche Diagnostics, and Novartis. Melissa A. Shea-Budgell has no conflict of interest to declare.

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+This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).For a long time, the stem cell regenerative paradigm has been based on the assumption that progenitor cells play a critical role in tissue repair by means of their plasticity and differentiation potential. However, recent works suggest that the mechanism underlying the benefits of stem cell transplantation might relate to a paracrine modulatory effect rather than the replacement of affected cells at the site of injury. Therefore, mounting evidence that stem cells may act as a reservoir of trophic signals released to modulate the surrounding tissue has led to a paradigm shift in regenerative medicine. Attention has been shifted from analysis of the stem cell genome to understanding the stem cell “secretome”, which is represented by the growth factors, cytokines and chemokines produced through paracrine secretion. Insights into paracrine-mediated repair support a new approach in regenerative medicine and the isolation and administration of specific stem cell-derived paracrine factors may represent an extremely promising strategy, introducing paracrine-based therapy as a novel and feasible clinical application. In this review, we will discuss the regenerative potential of fetal and adult stem cells, with particular attention to their secretome.Stem cell therapy has been shown to promote a substantial regenerative effect on damaged tissues and organs in several animal models. A large number of studies have provided strong evidence that stem cells (and in particular mesenchymal stem cells, MSC) can mediate tissue repair through the modulation of the local environment, influencing the immune/inflammatory response, sustaining angiogenesis and establishing co-operative effects with the resident cells, overall resulting in a significant cytoprotective and pro-survival beneficial influence [1,2]. The mechanism through which this regenerative result is achieved seems to rely more on the secretion of specific bioactive factors, rather than the direct differentiation of the transplanted stem cells in the host tissue, given the low incidence and poor efficiency of their survival and therapeutically relevant level of engraftment. This hypothesis is defined as paracrine effect and the composite set of the cytokines, chemokines and growth/trophic factors orchestrating many biological activities and secreted from stem cells (or shed from their membranes) is defined as secretome.Direct evidence that stem cell-derived secretome plays a key role in mediating the regenerative effects observed in vivo has been demonstrated by a consistent body of studies on cardiovascular, renal, liver and lung injury, as well as in neurodegenerative disease models [3,4,5,6,7]. As proof of principle, some reports have demonstrated that the administration of stem cell-conditioned medium, which contains all the bioactive factors released by the cells in culture, can exert the same regenerative effect obtained with cell transplantation. Hence, current interest towards investigating the intercellular interactions underlying the paracrine effect is driving attention from the stem cell genome to the stem cell secretome, focusing on the cell-to-cell communication mechanisms.In this scenario, microvesicles have been described as key regulators of the stem cell paracrine activity. The term of extracellular microvesicles (MVs) was first introduced to indicate nano-sized bodies released as shedding vesicles by various cell types into the extracellular environment. They include: (i) Exosomes, which are 30–100 nm diameter vesicles of endocytic origin obtained upon fusion of multivesicular bodies (MVB) with the cell membrane; (ii) Ectosomes (shedding vesicles), which are 100 nm–1 μm diameter vesicles directly shed from the cell membrane; and (iii) Apoptotic blebs, 1–5 μm diameter vesicles secreted by cells undergoing apoptosis [8]. Some confusion still exists in the literature regarding the distinction between exosomes and MVs. The difference between these two terms is based on the vesicle size: Exosomes are within 100 nm while microvesicles range from 100–1000 nm, but because this is still quite a novel research field, these definitions are flexible [9]. Microvesicles were first identified in sheep reticulocytes and described later on as mediators of the communication and activation processess involving B-lymphocytes and T-cell [10,11]. MVs were shown to be secreted by a variety of stem and somatic cells, either constitutively or when stimulated during activation or apoptosis; as well, they can be found in most of the physiological body fluids [10,11,12]. In recent years, exosomes have been specifically characterized with parameters other than their diameter size, such as the presence of a bi-lipid membrane similar to the plasma membrane, a specific flotation density of 1.1–1.18 g/mL on a sucrose gradient and an evolutionarily conserved set of markers including molecules from the tetraspanin family (such as CD81, CD63, CD9) and others like Alix, as well as cell type-specific antigens derived from the parental cell they originate from [13]. More recently, MVs, and in particular exosomes, have been described as playing a pivotal role in inter-cellular communication between stem cells and injured cells via paracrine signalling [12]. Exosomes were demostrated to contain proteins, bioactive factors, mRNAs and microRNAs reflecting the functionality of the cell producing them; they can transfer their content into recipient cells, resulting in the modulation of their protein synthesis and they were shown to act as carriers of the active component of the stem cell-conditioned medium and vehicles of the paracrine factors influencing the responder cells. As a matter of fact, MVs and exosomes, derived from stem cell-conditioned medium, exerted a beneficial influence, which is comparable to the regenerative effects obtained with stem cell transplantation in several preclinical disease models [14]. MVs and exosomes have recently captivated attention from the research community because of their paracrine factors content, thus suggesting them as a new therapeutic delivery tool. In this scenario, the fact that MSC, among many other stem cell types, are known to secrete MVs and exosomes provide a good rationale for testing their therapeutic potential in different animal models; thus, the analysis of the stem cell secretome and their MVs/exosomes’ content is a fast developing field, fuelled by a growing interest towards the clinical potential of this new, promising strategy for regenerative medicine.In this review, we will discuss the latest and more relevant findings on the paracrine potential of the fetal and adult stem cells’ secretome for tissue regeneration and repair.Stem cells are undifferentiated cells characterized by the capability of producing new cells with equal stem functionality (by self-renewal) or with restricted more specialized features via the expression of a specific committed phenotype (by differentiation) [15]. According to their origin and differentiation properties, they can be distinguished into embryonic stem cells, which are pluripotent, giving rise to all derivatives of the three primary germ layers and with unlimited self-renewal potential, or adult stem cells, which are found in several somatic tissues in the adult organism and are multipotent, with more restricted self-renewal potential [15,16]. The possibility of deriving multi- and pluripotent cells from fetal tissues, with properties intermediate between embryonic and adult stem cells, has recently been described by several groups [17]. Fetal tissue can be an attractive source of stem cells for therapy because of its pluripotency, proliferative ability and lack of immunogenicity. It could be obtained from a direct biopsy of the fetus during gestation or from fetal annexes such as umbilical cord blood, term placenta, villi and amniotic fluid; as the first procedure is associated with a defined morbidity/mortality and ethical issues, the other sources are preferred and represent an easily accessible and abundant font of progenitors. Stem cells with a therapeutic potential for regenerative medicine have been identified in term placenta, umbilical cord blood and amniotic membrane and fluid [18,19,20,21]. Particularly, human umbilical cord blood and placenta are well known sources of stem cells with potentials similar to bone marrow-derived mesenchymal stem cells [18,22] and they have been recently described as actively engaged in tissue repair mechanisms via the secretion of therapeutic bioactive factors. Human multipotent placenta mesenchymal cell-conditioned medium was demonstrated to contain paracrine factors from the IL6 superfamily and significant levels of pro-angiogenic bioactive molecules, such as vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), basic-fibroblast growth factor (bFGF), transforming growth factor-β (TGF-β) and insulin-like growth factor-1 (IGF-1). The therapeutic relevance of their secretome has been investigated in several preclinical studies, showing a significant cytoprotective potential in a model of oxidative stress on placental endothelial cells, via activation of anti-apoptotic IL6ST/STAT3 and superoxide dismutase 2 (SOD2) expression in the responder cells; similarly, human MSC demonstrated to produce exosomes under hypoxic conditions, resulting in endothelial cell migration and tube formation while improving the wound healing and vascular regeneration process in a diabetic mouse model [23,24,25]. Human placenta and amniotic membrane-derived mesenchymal stem cells (AMSCs) have been shown to possess peculiar modulatory properties as well, such as the regulation of T-cell proliferation and dose-dependent inhibition of peripheral blood mononuclear cell-mediated immune responses [26,27]. In a preclinical transgenic model of Alzheimer’s disease, these cells also demonstrated long-term beneficial effects following transplantation, suggesting a significant paracrine role in re-instructing the host compromised immune system and secreting amyloid beta-degrading enzymes and high levels of TGF-β and matrix metallopeptidase 9 (MMP9) [28]. Therapeutic transplantation of cord-blood derived progenitors has been broadly described in different ischemic and oxidative stress models with encouraging results. Conditioned medium from human cord blood-derived endothelial stem cells were shown to exert mitogenic and chemotactic effects on keratinocytes and fibroblasts, possibly mediated by the significant level of the platelet-derived growth factors (PDGF)-α, PDGF-β and of the keratinocyte growth factor (KGF) cytokines strongly expressed in it. The paracrine factors released by umbilical cord blood-derived progenitor cells confirmed their beneficial potential also by accelerating the wound healing and the neovascularization process while exerting a renoprotective effect in a diabetic mouse model [29,30]. Another interesting source of human fetal MSC is represented by the perivascular connective tissue of the umbilical cord, the Wharton’s jelly [31]. These mesenchymal progenitors, defined as human umbilical cord perivascular cells (HUCPVCs), were demonstrated to exert significant proliferative effects on primary cultures of neurons and glial cells and a remarkable neuroprotector influence following transplantation into animal models of spinal cord injury and Parkinson’s disease. Their paracrine potential was mainly expressed via the increase of human neutrophil-activating protein-2 (NAP-2), neurotrophin-3 (NT-3), bFGF and glial derived neurotrophic factor (GDNF) at the site of injury [32,33]. Recent studies have further confirmed the peculiar potential of the HUCPVC secretome, showing that these stem cells preferentially express factors related to neuroprotection, neurogenesis and angiogenesis, with a dramatic influence on the metabolic viability of hippocampal neurons via the robust expression of nerve growth factor (NGF) [34,35]. HUCPVC have also shown therapeutic benefits when transplanted into a mouse preclinical model of splinted wound, as they accelerated the wound healing process by supporting collagen deposition and angiogenesis by paracrine mechanisms. Likewise, treatment with their conditioned medium alone provided similar results as stimulating anti-inflammatory M2 macrophages and sustaining angiogenesis through the expression of tissue-repairing cytokines like interleukin (IL)-10, transforming growth factor (TGF)-β1, vascular endothelial growth factor (VEGF)-1 and angiopoietin-1 at the healing wound site [36]. Furthermore, human stem cells derived form the umbilical cord perivascular fraction (PCs), together with mesenchymal stem cells from the cord blood, also demonstrated regenerative properties by mediating short- and long-term therapeutic benefits in a paracrine manner in a bronchopulmonary dysplasia model. The cells conditioned medium was shown to prevent the arrest of lung angiogenesis and to support the alveolar architecture in neonatal rats exposed to hyperoxia, thus preserving lung function at six months [37]. In particular, the remarkable cytoprotective effect of the exosomes isolated from human umbilical cord-MSC has also been recently described, showing that these microvesicles can exert an important therapeutic benefit in ameliorating oxidative stress, improving cell survival and reducing apoptosis and necrosis in the proximal kidney tubules of a rat model of cisplatin-induced nephrotoxicity [38].Along with fetal tissue, amniotic fluid (AF) is another source of stem cells with suitable potential for therapeutic applications [39]. Amniotic fluid is known to contain multiple cell types derived from the developing fetus and it represents an alternative source of immature progenitor cells that can be easily collected during amniocentesis, a well established technique for prenatal diagnosis with low risk both for the foetus and the mother [40], as well as during C-section procedures at term. In the last few years, several groups have presented various studies demonstrating the presence of heterogeneous progenitors in the amniotic fluid, mainly with mesenchymal characteristics [41,42,43,44,45]. In particular, pluripotent stem cells from the amniotic fluid (AFS, Amniotic Fluid Stem cells) can be isolated using discarded back-up amniocentesis samples by selection for the expression of the membrane stem cell factor receptor c-kit [46]. AFS share some embryonic stem cell properties, such as the expression of pluripotency stem markers like SSEA4 and OCT4, high proliferative and self-renewal potential and in vitro differentiation into adipogenic, osteogenic, myogenic, endothelial, neural and hepatic lineages; furthermore, they were recently demonstrated to give rise to induced pluripotent stem cells (iPS) via non-viral methods [46,47]. Recently, a growing body of work has suggested a therapeutic role for the secretory profile of these cells. The first detailed description of the paracrine potential of the amniotic fluid mesenchymal stem cells comes from a study on skin wound repair, describing the cells-conditioned medium as a powerful source of a broad array of cytokines and chemokines, such as IL-8, IL-6, TGF-β, tumor necrosis factor receptor I (TNFRI), VEGF and EGF, with relevant roles in instructing dermal fibroblasts during wound healing, following activation of the TGF-β/SMAD2 pathway [48]. The human c-kit+ AFS cell secretome has been also partially characterized, showing a significant enrichment for pro-angiogenic soluble factors, such as monocyte chemoattractant protein-1 (MCP-1), stromal cell-derived factor-1 (SDF-1) and VEGF in a dose-dependent manner. Indeed, the secretome of these cells provided a remarkable paracrine effect in vivo in a limb ischemia-reperfusion rmouse model, by enhancing vasculogenesis via chemo-attraction of host endothelial precursor cells [49]. These results were further confirmed in a another preclinical ischemic model of fascio-cutaneous flap where the topical administration of human c-kit+ AFS cell-conditioned medium provided significant improvement in the perfusion level of the injured area, together with recruitment of local progenitor cells and subsequent differentiation into endothelial lineage with capillary formation [50]. Moreover, the amniotic fluid stem cell secretome proved to promote tissue repair not only by mediating pro-angiogenic regenerative effects, but also via the local and systemic modulation of inflammation, as shown in a mouse model of acute hepatic failure. Indeed, in this work, the AF mesenchymal stem cell secretory profile showed to be significantly enriched for a variety of interleukins (IL-10, IL-27, IL-17E, IL-12p70, IL-1β and IL-1ra) and liver regenerative mediators (MCP-1, SDF-1, platelet-derived endothelial cell growth factor (PD-ECGF), tissue inhibitors of metalloproteinase, TIMP1 and TIMP2, fibroblast growth factor 7 (FGF7), and EGF) [51]. The cytoprotective paracrine influence of the AFS cells was also validated in other injury preclinical models of lung, renal, intestine and heart diseases [52,53,54,55,56]. Of particular interest, human AFS cells and their conditioned medium were shown to exert a remarkable pro-survival and anti-apoptotic effect on the ischemic cardiac tissue in an experimental model of acute myocardial infarction in rat, resulting in a significant decrease of the infarct size and cardiomyocyte death within two hours of treatment with a putative paracrine effector identified in thymosin beta 4, a cardioprotective factor actively secreted by these cells [55]. Similarly, remarkable results were obtained in a study evaluating the regenerative role of rat AFS cells in rat neonates affected by necrotising enterocolitis. Cell transplantation and the injection of their conditioned medium resulted in modulation of the cyclooxygenase-2 enzymatic activity in the host stromal cells, causing a decrease of bowel inflammation, tissue necrosis and damage with significant improvement of intestinal function [56]. A well-characterized regenerative role for the amniotic stem cell-derived exosomes has also been reported using an in vitro co-culture system with human amniotic mesenchymal stem cells and primary fibroblasts from a patient affected by cystinosis with a lysosomal cystine transporter mutation (CTNS −/−). In this study, the amount of altered cell cystine detected in culture was significantly reduced, suggesting a paracrine effect of the amniotic stem cells on the mutant fibroblasts, which was mediated by the microvesicles derived from the stem cells and transferring wild type cystinosin and CTNS transcript to the affected target cells [57]. In light of these considerations, fetal stem cells possess a promising paracrine capacity which may be the principal mechanism contributing to tissue repair, in addition to their multipotent differentiation potential. Considering their remarkable in vitro self-renewal properties, these stem cells may represent an ideal candidate for paracrine therapy, in order to isolate and scale-up putative drug-like formulation from their secretome for future regenerative medicine applications. Adult stem cells are involved in the maintenance of homeostasis and physiological repair of somatic tissues and organs through a fine balance between self-renewal and differentiation; they reside in specialized niches, a term used to describe the complex interaction of different cells, matrix components and biological factors in distinct tissue locations [58]. After an insult, stem cells can actively exit the niche and become activated in order to mediate tissue regeneration; in particular, activated stem cells have been demonstrated to secrete immunomodulatory and trophic factors, which can support the repair process by endogenous mechanisms [59]. Among the different adult stem cell populations described so far, bone marrow-derived mesenchymal stem cells (BM-MSC) have been broadly described as a clinically effective therapeutic agent for a variety of tissue injuries. MSC are routinely derived from primary cultures of adult tissues, such as bone marrow and adipose tissue, along with other stromal and mesodermal sources [60]. According to the International Society for Cellular Therapy (ISCT), adult MSC can be characterized based on their specific culture conditions and immunophenotypic features, along with their mesodermal differentiation potential. These criteria include the expression of the mesenchymal markers CD105, CD73 and CD90 with lack of mature hematopoietic antigens such as CD45, CD34, CD14, CD11b, CD79a, CD19 and HLA-DR and the in vitro potential to give rise to adipogenic, chondrogenic and osteogenic lineages [61]. Nevertheless, a clear cell surface marker to identify a true MSC has not been identified yet. In addition to their multipotent transdifferentiation potential, adult MSC have also been shown to play a key role as cellular modulators in tissue repair mechanisms. An increasing body of work based on lineage trace studies has showed that transplantation of adult MSC can exert a remarkably beneficial effect in different injury models, despite their poor engraftment and in vivo low level of differentiation in the long term. Such a therapeutic effect seems to be mediated by the MSC paracrine potential to secrete trophic factors, which sustain local neovascularization, inhibit cell death with modulation of the immune response and mobilization and survival of host cells [62,63]. In a rat model of myocardial infarction, transplantation of allogeneic MSC demonstrated beneficial effects through their paracrine activity, improving left ventricular function at four weeks after transplantation [64]. The cardioprotective role of the adult MSC secretome was further confirmed by exposing cardiomyocytes to genetically modified MSC-conditioned medium under hypoxic conditions [65]; in particular, MSC-conditioned medium also promoted in vitro proliferation and migration of endothelial cells in a dose-dependent manner with VEGF and bFGF being the key factors implicated in this process. Likewise, in a murine model of hindlimb ischemia, the factors released into the adult MSC-conditioned medium enhanced collateral flow recovery and remodeling, thus attenuating muscle atrophy [2]. An additional confirmation of the therapeutic role of the MSC secretome has also been reported in a preclinical model of acute renal failure, where cell transplantation improved the general outcome via paracrine effects influencing the modulation of the inflammatory, vascular and apoptotic/necrotic processes related to the ischemic kidney injury. Notably, none of the transplanted MSC differentiated into mature tubular or endothelial phenotypes, while the expression of pro-inflammatory molecules such as IL-1β, TNF-α, IFN-γ and of inducible nitric oxide synthase were significantly reduced in the treated kidneys, with concurrent induction of the anti-inflammatory cytokines IL-10, bFGF, TGF-α, and Bcl-2 [66]. In light of these results, the characterization and therapeutic use of MSC, with respect to their ability to produce and secrete bioactive factors, inspired their description as “site-regulated, multidrug dispensaries, or injury drugstores” [67]. It has also been recently demonstrated that the intrinsic capacity of MSC to activate endogenous regenerative mechanisms and to induce the mobilization of host cells is critically dependent on their commitment level, highlighting the importance of carefully investigating the differences in the soluble morphogens used in the culture conditions. The addition of basic-fibroblast growth factor (bFGF or FGF-2) to primary bone marrow-derived MSC cultures proved to be a key element in order to select in vitro specific MSC subpopulations with potential to induce the host regenerative response in vivo. Implantation of constructs seeded with bFGF-selected MSC led to formation of bone of host origin through the activation of an endochondral ossification process, whereas an intramembranous ossification directly performed by the seeded cells was observed in implanted constructs with unselected MSC [68]. Moreover, bFGF-selected mouse MSC, when ectopically implanted in immunocompetent syngeneic hosts were demonstrated to exert a potent anti-inflammatory effect towards mobilized macrophages through the release of prostaglandin E2 (PGE2); MSC-released PGE2 induced a functional switch of macrophages from a pro-inflammatory to a pro-resolving phenotype and this event triggered a cascade of cellular events resulting in the creation of a bone regenerative niche via the mobilization of host bone marrow-derived endothelial and osteogenic cells [69]. A similar anti-inflammatory effect was also described in a mouse model of sepsis, where systematically injected MSC induced IL-10 production in host macrophages [70]. Moreover, it has been reported that intravenous (iv) or intraperitoneal (ip) injection of MSC resulted in a substantial paracrine effect, reducing inflammation and increasing vascularization in a rat model of sterile injury to the cornea: Transplanted MSC reduced the damage to the cornea, without showing significant engraftment in the host tissue, suggesting that their pro-resolving effect was mainly due to the secretion of TNF-α stimulated gene/protein 6 (TSG6) [71]. It is well known that the extracellular microenvironment contains solutions of proteins, polysaccharides as well as vesicles containing proteins, mRNA, and micro-RNA [72]. Recent reports identified in the release of MVs and exosomes a novel and co-operative paracrine mechanism carried out by adult MSC [12,73]. The first study that investigated the therapeutic effect of adult MSC-derived MVs was performed in a rat model of a glycerol-induced acute kidney injury. In this work, systemic injection of MVs induced epigenetic changes in the resident host cells by horizontal transfer of mRNA, leading to cell cycle restoration and activation of tissue regenerative programs [74]. In a follow-up study, the same group also demonstrated that, immediately after ischemia and reperfusion injury, a single injection of MVs obtained from adult MSC could prevent both acute and chronic kidney disease in a rat model [75]. Moreover, recent studies proposed that the beneficial paracrine effect observed on cardiovascular cells in several preclinical models of cardiac ischemia and/or disease following MSC transplantation, might be mainly mediated by these stem cell-derived MVs. In a preclinical model of myocardial ischemia/reperfusion (I/R) injury, the injection of human MSC-conditioned medium led to a 60% reduction in the myocardial infarct size, showing that only the fraction of the conditioned medium containing products in the range of 100–220 nm size provided cardioprotection [76]. A more recent study also highlighted the cardiac regenerative properties of human MSC-exosomes, showing that, in a mouse model of cardiac I/R injury, the treatment with intact exosomes resulted in the restoration of bioenergetics, reduction of oxidative stress and activation of pro-survival signaling pathways, overall enhancing the cardiac function [77]. The therapeutic effect of MSC-derived MVs was further confirmed in other animal models of acute myocardial injury, where their administration resulted in a cardio-protective effect dependent on the release of cytokines, bioactive molecules and growth factors, mediating regenerative effects similarly to MSC or MSC-conditioned medium transplantation [12]. MVs and exosomes released by MSC are currently being tested in several other preclinical injury models, such as stroke [78], liver fibrosis [79], rheumatic diseases [80], and acute lung injury [81]. The wide range of therapeutic effects mediated by MSC-derived trophic factors and MVs/exosomes suggests that their potential for clinical applications is much broader than those currently identified. Hopefully, scientific advances in the understanding of the paracrine properties of MSC will pave the way to innovative clinical strategies, where stem cell-based paracrine therapy could actively contribute to regenerate/repair the appropriate tissue, with adult MSC already a broadly investigated candidate for clinical trials and regenerative medicine strategies.Various adult and fetal progenitor cells have been demonstrated to exert cytoprotective, pro-survival, anti-inflammatory, anti-apoptotic and angiogenetic effects on injured cells by secreting and delivering regenerative factors within MVs/exosomes. More recently, another mechanism underlying the paracrine hypothesis has been proposed, suggesting that the bioactive factors secreted by the exogenous transplanted stem cells, and contained in their conditioned medium, might act not only on the injured host cells, but also by targeting an endogenous progenitor population residing in the tissue, in order to recruit and activate them to initiate/sustain the repair process, overall improving the functional outcome by autocrine effects. This synergistic result, which is based on the intercellular crosstalk via paracrine signalling between the exogenous stem cells and the host endogenous progenitors, has been described in different cell therapy preclinical studies, in particular in cardiovascular, neural, bone and revascularization models. The restoration of cardiac progenitor cell (CPC) populations and the activation of the molecular pathways within their repair programme have been recently described following the paracrine effect mediated by transplanted adult bone marrow-derived stem cells in the setting of myocardial ischemic injury [82,83,84,85]. This approach resulted in two mechanisms of endogenous cell proliferation, targeting both CPC and the surviving pre-existing cardiomyocytes [86]. Transplantation of bone marrow MSC was also shown to promote tissue repair through secretion of paracrine factors and reactivation of local progenitors in an experimental model of glaucoma, as well as in an ectopic bone regenerative study [69,87,88]. Likewise, fetal stem cells such as AFS cells have been shown to trigger a chemo-attractive response resulting in the recruitment of CD31+/VEGFR2+ and CD31+/CD34+ host endothelial progenitor cells into ischemic subcutaneous tissues in rats and of mesodermal precursors committed to fat, muscle, fibrous tissue and immature bone lineages, following subcutaneous implantation onto hydroxyapatite scaffolds into nude mice [50,89]. Hence, these observations support the developing idea that harnessing the stem cell secretome can lead to a double beneficial influence on the injured tissue (1) by direct modulation of the local environment, thus providing an immediate cytoprotective, angiogenic and anti-inflammatory effect during the acute phase following injury and (2) by boosting the local resident stem/progenitor cells, with the aim of achieving a more pronounced tissue-specific repair programme and instructing the stimulation of resident endogenous progenitors as a critical mechanism for future development of pharmacological paracrine therapy.An additional feature of the paracrine therapy is represented by the characterization of the signalling mechanisms that orchestrate the activity of the stem cell secretome, and in this scenario, MV/exosome secretion represents a crucial aspect. Microvesicles and exosomes currently represent a very intriguing and timely focus in stem cell biology, as they are considered the key component mediating stem cell-derived paracrine effects on target cells via cell-to-cell signalling. Different studies have described them as active components of the pro-angiogenic and regenerative effects exerted by stem cells in animal models of myocardial ischemia/reperfusion injury or corneal assay [12,90,91,92,93,94]. Their role as paracrine effectors is based on evidence that they can transfer—locally or through biologic fluids—not only bioactive molecules, but also genetic information between the secreting and the responding cell; therefore, they are emerging as interesting mediators of cell-to-cell communication, either via proteomic or genomic interactions [95]. Several independent studies have recently reported that MVs and exosomes can be involved in the transfer of genetic material in the form of mRNAs and microRNAs (miRNAs). miRNAs are a novel class of small non-coding post-transcription regulating RNAs, which can be involved in cell proliferation, differentiation, development, and death. A growing body of work supports the idea that stem cell-derived MVs/exosomes can modulate or reprogram the phenotype of a recipient somatic cell via the horizontal transfer of small RNAs, in particular of miRNAs [95,96,97]. Therefore, it might be reasonable to assume that stem cells can exert their regenerative effects on the injured tissue by paracrine mechanisms via delivering certain transcripts which can up- or down-regulate specific pathways in the target cells, such as local cell de-differentiation and cell cycle re-entry, while promoting cell survival, angiogenesis and endogenous tissue repair activation. This hypothesis has been validated in several preclinical studies. In an experimental animal model of renal ischemia/reperfusion injury, adult endothelial progenitor cell-derived MVs were shown to restore the angiogenic program in quiescent cells by the horizontal transfer of mRNA and to prevent acute kidney injury via a mechanism involving the angiogenic miRNAs miR-126 and miR-296; the renoprotective effect achieved with the microvesicle treatment was lost after treatment with RNAse or transfection with specific miR-antagomirs, suggesting a key role of the miRNAs shuttled by the MVs in this regenerative process [98,99]. Similar results were obtained in a murine model of hindlimb ischemia, where treatment with the MVs isolated from adult stem cells and containg miR-126 and miR-296 resulted in significantly enhanced perfusion and reduced damage of the muscle [100]. More recently, adult MSC were demonstrated to be able to communicate with brain parenchymal cells and to influence neurite outgrowth by transfer of miR-133b via exosomes; loss of expression of miR-133b in the MSC and luciferase assay confirmed that the results obtained in the study were attributed to the delivery of functional miRNAs from the MSC-derived exosomes to the neural cells [101]. Adult rat MSC overexpressing GATA-4 also showed to mediate cytoprotective and pro-survival effects on hypoxic cardiomyocytes via a miRNA-associated mechanism, in which the expression of the anti-apoptotic miR-221 transferred by MVs significantly enhanced cardioprotection by reducing the levels of p53 in the treated cells [102]. By acting as a shuttle for the transfer of genetic information, stem cell-derived MVs/exosomes play a critical role in the intercellular communication phase of the paracrine effect. Since miRNAs are post-trascriptional regulators inducing epigenetic changes in the target cells that survive the injury, they can be envisioned as a novel strategy to modulate and activate the tissue endogenous regenerative programme.In the last few years, significant effort has been invested in stem cell-based therapy for tissue regeneration following injury, trauma and chronic disease. However, much scepticism still surrounds cell transplantation- and tissue engineering-based methods, due to their poor clinical feasibility, namely limitations associated with safety, immunogenicity, and, in many cases, limited efficacy of the injected cells. A strong limiting factor to stem cell-base therapy is also represented by the high cost of the procedure to treat each patient that precludes application to a large number of people within public and private health social systems. In this scenario, isolation and administration of specific stem cell-derived regenerative factors for future paracrine pharmacological therapy may represent an extremely promising strategy with the clinical advantage of limiting many of the safety concerns associated with the transplantation of viable replicating cells. Indeed, this approach represents a more tractable model to still obtain stem cell-mediated regenerative effects but through the cell-free delivery of soluble molecules, shifting attention from cell therapy to paracrine pharmacological treatment. Insights into paracrine-mediated repair support a new approach for regenerative medicine, via the identification of suitable cocktails of stem cell-derived trophic factors to trigger endogenous tissue repair mechanisms, thus introducing paracrine therapy as a novel and feasible clinical read-out for patients suffering from injuries and traumas. In addition, patients suffering from diseases such as ischemic injuries and/or traumas need prompt therapeutic intervention. Therefore, it would be ideal to have access to “off-the-shelf” products ready for administration on demand, and by taking advantage of our knowledge of the stem cell secretome, we could envisage the use of stem cells as “drug-stores” to produce specific soluble paracrine factors, which can ultimately be administered orally or systemically. As well, this strategy removes the invasive or additional surgical procedures generally required for cell-therapy or tissue engineering methods, together with the risks associated with them. While paracrine therapy may represent a novel “cell-free” approach for tissue regeneration, it is worth considering that cytokine therapy strategies, based on the delivery of multiple/high doses of a single trophic factor, have not yet met expectations, due to problems related to appropriate delivery, pharmacokinetics and in vivo stability of such molecules. Therefore, it is crucial to optimize the stem cell culture protocols in vitro, in order to produce the right amount, concentration and combination of paracrine factors and to obtain the required therapeutic effect once administrated in vivo [103].In this scenario, MVs and exosomes may represent an ideal vehicle for paracrine therapy, since they can be used as immunologically inert nano-carriers with the intrinsic capacity to carry a significant amount of stem cell-derived bioactive molecules (proteins, lipids, mRNAs and miRNAs), while protecting these from the degradative enzymes/chemicals and bypassing biological barriers for the in vivo targeting of specific tissues. As a matter of fact, exosomes show several features of the ideal drug carrier, such as membrane penetration potential, intrinsic homing ability, long circulating half-life and the possibility of undergoing membrane modifications in order to enhance targeting of specific cell types [92]. Despite their promising and appealing therapeutic potential, the biological characterization of the stem cell-derived MVs and exosomes still represents a novel approach in regenerative medicine and the necessary proteomic and genomic data are either still lacking or requiring more comprehensive analysis. Currently, only one detailed proteomic analysis of human adult MSC-derived MVs has been reported, identifying about 730 MV proteins, including 43 surface receptors and signaling molecules that control self-renewal and differentiation of MSCs. The results of this analysis suggested that MV protein content might relate to cellular processes such as cell proliferation, adhesion, migration, and morphogenesis [104]. mRNA and miRNA microarray characterisation of MSC-derived MVs is another exploratory approach which is fastly developing [9]. One of the first studies using human embryonic stem cell-derived MSC and based on the detailed microarray analysis of their MVs described a shared expression in the MV miRNA content with that of the parent cells [97]. In a similar study, the RNA contained within the MVs secreted by human bone marrow-MSC and liver resident stem cells was profiled for 365 known human miRNAs. The analysis resulted in 41 miRNAs co-expressed in the MVs and in the parental cells, which could be involved in multi-organ development, cell survival, differentiation and, to some extent, immune system modulation [105]. Due to the enormous potential of paracrine factors and MVs released by MSC for future regenerative therapeutic purposes, there is an urgent need for a detailed identification of such elements, and the exponential increase in exosomal studies witnessed in recent years has required the scientific community to have access to reliable protocols for exosomal isolation and purification procedures. In order to provide such information, ExoCarta, a manually curated web-based database, has been recently created as a catalogue of exosomal proteins, RNA and lipids [8]. With this bioinformatic tool, the information on exosomal isolation/purification procedures, samples used and exosomal molecular components, such as proteins, mRNAs and miRNAs reported in previously published studies, can be easily obtained. Similarly to ExoCarta, another online dataset of extracellular vesicles, defined as Vesiclepedia, will be soon available for consultation, and with the assistance of the International Society of Extracellular Vesicles (ISEV), the current confusion on the definition of MVs and exosomes should be resolved by the introduction of a standardised nomenclature, in association with stringent purification protocols for exosome isolation [106].Another crucial aspect to be evaluated, when harnessing the stem cell secretome for therapeutic purposes, is defining suitable strategies to stimulate the cells to release significant amounts of the desired paracrine factors in their conditioned medium or microvesicles, in order to provide tissue regeneration, as represented in Figure 1. Most of these approaches are performed in vitro and are referred to as preconditioning methods [103]. Serum deprivation and starvation have been broadly described in the recent literature as common strategies to induce the secretion of paracrine factors and MVs from cultured stem cells. For example, bone marrow MSC cultured under these stress conditions were shown to undergo epigenetic changes and to significantly upregulate the expression of the pro-angiogenic and anti-apoptotic factors IGF1 and leptin [107]. Moreover, the medium conditioned by MSC cultured under serum-deprived conditions denoted an angiogenic potential higher than that obtained in standard conditions in an ex vivo rat aortic and in a chick chorioallantoic membrane angiogenesis assay [108]. To mimic the in vivo ischemic environment, in which transplanted stem cells are likely to exert their beneficial effect, hypoxia preconditioning has been broadly used by subjecting MSC to physiological conditions of low oxygen tension (<5%) in vitro. This strategy induced adult and fetal MSC to secrete higher levels of pro-angiogenic and pro-survival cytokines, such as VEGF and bFGF, to counteract hypoxic effects, while activating survival pathways with genes like Akt, HO-1 and Hsp70 [25,109,110]. Although preconditioning via hypoxia exposure was shown to significantly increase stem cell secretion of bioactive trophic factors, a general consensus on this method has not yet been obtained, as there is still considerable variation in the hypoxic protocols and results published so far. Further analyses are required to determine whether the hypoxia preconditioning results can be mantained in the long term in order to produce a clinically relevant result [103]. To improve the efficiency of the MSC secretome for cardiac repair, genetic manipulation has also been suggested, using transgenes for conditional overexpression of genes like Akt, IGF-1, VEGF GATA-4 and SDF-1. This approach was validated in an ischemic injury model, resulting in sustained paracrine effect mediated by trophic and angiogenic molecules secreted from MSC, along with mobilization of endogenous c-kit+ and CD31+ progenitors and reduced ventricular remodeling [111,112,113,114,115]. Despite the encouraging results achieved with this startegy, the sustained paracrine effects might be limited to the overexpression of a single specific gene, the expression levels of which might not correlate with the concentration of the desired secreted factors, in addition to the risk of creating secondary, undesired effects; moreover, the potential use of viral vectors can limit the clinical translation of this approach, given the safety concerns associated with this technique. Molecular stimulation of stem cells using a set of cytokines, chemokines and growth factors playing a key role during the inflammatory response or involved in the development of ischemic injury and disease, have also been used to trigger and sustain MSC paracrine secretion in vitro. Preconditioning MSC with pro-inflammatory cytokines such as TGF-α, TNF-α and lipopolysaccharide (LPS) resulted in an increased production of VEGF, IL-6, IL-8 and MCP-1 [116,117,118,119], while stimulation of mouse MSC with addition of bFGF in the culture medium resulted in selection of specific subpopulations with increased paracrine potential in terms of inducing the host regenerative process [68]. Similarly, co-colture with apoptotic cells has been adopted to prime MSC against apoptotic cytokines, in order to stimulate the secretion of pro-survival factors, such as STC-1 [120]. Nevertheless, more comprehensive studies are still needed to fully elucidate this approach, as the synergistic effects created in vitro using different cocktails of signalling cues in order to mimick the injury setting might differ from the actual situation in vivo. A further relevant point to be considered for future stem cell-derived paracrine therapy regards the use of primary stem cells to isolate specific therapeutic factors. As a matter of fact, adult and fetal MSC (although the latter to a lesser extent) are characterised by significant, yet limited, proliferative and self-renewal potential. Thus, a scale-up production of MSC-derived paracrine factors for future applications would involve the use of repeated isolation of cells with the risk of batch-to-batch variation [92]. In order to avoid the finite expansion capacity of MSC and to improve the yield of the secreted regenerative factors, immortalization of primary human stem cells has been recently suggested. Human embryonic stem cell-derived MSCs were transfected with a MYC gene to demonstrate that, once immortalized, they can increase their proliferative rate, while maintaining the production of exosomes therapeutically effective in reducing the infarct size in a mouse model of cardiac I/R injury [121].Lastly, the technology available to analyse the stem cell secretome will be an instrumental role for future paracrine therapy, since the cell culture medium conditioned by adult and fetal MSC is a subject of intensive characterization in the search for specific released factors and MVs/exosomes that might be used for regenerative medicine purposes. In this scenario, the techniques applicable to stem cell secretomics mainly focus both on proteomic analysis and microarrays for small RNAs screening, coupled with bioinformatic computational approaches [122]. Proteomic investigation of MSC secretome is currently based on the analysis of cell-conditioned medium via protein/peptide separation techniques (such as 2-dimensional gel electrophoresis and liquid chromatography), followed by specific protein identification by mass spectrometry and immunological assays like ELISA (Enzyme-Linked ImmunoSorbent Assay) and western blotting. Recent advances in mass spectrometry have led to more specific techniques for relative protein quantification, such as, for example, the SILAC (Stable Isotope Labelling by Amino acids in Cell culture) methodology, which is based on the incorporation of the essential aminoacid arginine and/or lysine, which are labelled with the non-radioactive carbon-13, nitrogen-15 or deuterium isotopes into the newly synthesized proteins during cell culture [123]. This method can provide detailed comparison and quantification of protein levels between samples, thus offering more comprehensive data on the cell secretome profile. As well, antibody-based assays have been significantly improved to detect specific antigens and proteins that are present in the cell-conditioned medium at very low concentrations. Multiplex immunological assay, in the form of microarray or microbeads formulations like the Luminex® methodology, have been recently developed in order to simultaneously measure several hundreds of compounds in multiple samples, with the possibility of detecting cytokine concentration less than 1 picogram/mL [122]. Schematic representation of paracrine therapy using adult or fetal mesenchymal stem cells. After isolation, stem cells are expanded in vitro and stimulated in order to release trophic factors into their conditioned medium via microvesicles/exosomes secretion. Factors directly released in the conditioned medium or contained in the microvesicles may be further isolated and use as therapeutic agent for tissue regeneration and repair. As for the proteomic characterization of the stem cell-derived MVs and exosomes, their profiling is based on the same techniques as for the measurement of soluble secreted peptides, such as one dimensional sodium dodecyl sulphate—polyacrylamide gel electrophoresis (1D SDS PAGE), liquid chromatography and mass spectrometry; at present, only a few studies have been published so far, such as those involving neural stem cells, adipose tissue and human embyonic stem cell-derived MSC [118,124,125]. MSC-derived exosomes have been shown to shuttle not only proteins but also small RNA between cells, resulting in the epigenetic modulation of the recipient cells [96]. The profiling of RNA content within the stem cell-derived exosomes has been mainly described through the use of molecular techniques, like microarray and qRT-PCR analysis [97,126]. Another critical tool for the proteomic and genomic profiling of the stem cell secretome is represented by bioinformatics, which has undergone significant development in recent years. Several web databases and search engines have been recently created in order to interpret the large amount of data generated by the analysis of the MSC secretome. Software such as DAVID (the Database for Annotation, Visualisation and Integrated Discovery), the Ingenuity Pathway Analysis (Ingenuity® Systems), Bioinformatic Harvester and the Panther (Protein Analysis Through Evolutionary Relationships) have been recently developed to provide clusterings of groups, maps of signalling pathways and entries for possible interacting proteins in order to predict the putative roles of the stem cell secreted factors in metabolism, inflammation, immune response, development, tissue repair and regeneration [122]. Although much progress has been made in terms of the technology and the tools currently available for the screening of the MSC secretome, a comprehensive database of their secreted paracrine factors and small RNAs has to be yet generated as reference. Moreover, it has to be considered that each technique presents some limits, either in the instrument sensitivity or in the specificity of the method itself; therefore, a more complete characterization of the MSC secretory profile will need a comprehensive combination of several different approaches at proteomic, genomic and computational levels.Over the past few years, we have witnessed scientists and physicians expressing increasing enthusiasm for the therapeutic potential of stem cells. Traditional cell therapy is based on the belief that, when healthy cells are injected into patients, they can stimulate the body’s own healing process. Recent data available on database about clinical studies of human participants conducted around the world (Clinical Trials.gov, August 2013) showed that more of 4600 stem cell-based therapeutic approaches are globally active, and 300 of those are MSC-related. Based on advanced cell technology, promising curative effects and ethical issues, MSC have become the most common cell source in cell-based treatment. In light of these considerations, many biotechnology companies are supporting stem cell therapy and focusing on producing and commercializing human cell technology to treat degeneration of tissue and organs. Although much effort has been invested into preclinical research, several concerns and risks associated with the direct use of stem cells still need to be addressed. As well, many questions remain unanswered, such as the potential of the transplanted stem cells to generate fully functional new cells in the patient’s damaged organ. In addition, we also need to consider a manufacturing risk: Stem cell therapy requires Good Manufacturing Practice (GMP facility) with a consequent drastic increase in the costs of cellular technology. Thus, despite the extensive research developed in the last 20 years and the encouraging results obtained with cell therapy for the regeneration of tissue such as the epithelium (i.e., skin repair following burns or cornea regeneration), many concerns still exist related to the clinical application of stem cells for the repair of mesodermal tissue and organs with many researchers currently suggesting that the use of MSCs should be reconsidered. The clinical potential offered by MSC for therapeutic applications includes diverse clinical targets, and in most of the current ongoing clinical trials, stem cells seem to mostly contribute in modelling the in vivo regenerative microenvironment by secreting bioactive molecules acting on angiogenesis, inflammatory and local immune response. Furthermore, to establish the basis of a future paracrine pharmacological therapy, it is of crucial relevance to identify the most suitable stem cell font. The ideal stem cell source should be selected upon consideration of their paracrine potential and the feasibility of their isolation, together with their in vitro self-renewal properties. Both adult mesenchymal stem cells and fetal stem cells unambiguously fulfil these criteria, as they can be easily isolated, expanded and cryopreserved while retaining a stable karyotype and low immunogenic profile. While adult stem cells (such as bone marrow mesenchymal stem cells or adipose stem cells) are isolated from biopsy obtained by surgical procedures, fetal stem cells (like amniotic fluid or umbilical cord stem cells) can be harvested from leftover samples from prenatal diagnosis or discarded tissue collected at birth, avoiding all the surgical risks associated with the first source, which might retain some level of morbidity. At the same time, despite the fact that fetal stem cells retain a more immature potential and higher self-renewal properties compared to progenitor cells from adult sources, the characterisation of their secretome is incomplete and further studies need to be performed.Finally, although stem cell-derived paracrine therapy may represent an extremely exciting and novel therapeutic strategy, several aspects are yet to be addressed before clinical use of their secreted factors and MVs is considered. The large-scale production of specific paracrine molecules (proteins, lipids, small RNAs) obtained from cultured stem cells needs to be defined in detail; the regenerative potential of different preparations of stem cell-derived MVs and trophic factors has to be investigated and experiments to assess the long-term safety, bio-distribution and persistency of the therapeutic effects in vivo must be performed and evaluated accurately [127]. Furthermore, our knowledge of the MSC secretory profile is mainly based on in vitro studies, thus it has to be considered that the secretome of cells cultured under specific controlled conditions might be quite different in terms of composition, concentration and timing from that in vivo, where stem cells can modulate their paracrine potential in response to the stimuli released by the injured microenvironment. Hence, it will be particularly important to profile the MSC secretome in vivo and to develop more sensitive techniques in order to characterise the dynamic expression of MSC-secreted factors and their qualitative and quantitative changes in response to the local milieu.In this review, we discussed how the number of studies exploiting fetal and adult MSC therapeutic potential based on the release of regenerative trophic factors, rather than their direct trans-differentiation capacity, is rapidly evolving including several clinical applications, such as graft versus host disease (GVHD), myocardial infarct, stroke, acute kidney, lung failure, wound healing, rheumatoid arthritis, and multiple sclerosis [67]. It is reasonable to conclude that the protective and restorative benefits mediated by adult and fetal stem cells are partially due to paracrine effects obtained by the release of specific cytokine, chemokine andgrowth factors, which are likely exerted via exosome and microvesicles secretion. At the same time, the advantage of using cell-based therapy relies on the possibility of providing the physiological concentrations of trophic factors to injured tissue at the appropriate timing; these synergistic effects may provide a regenerative microenvironment in the damaged tissue as well as recruit resident progenitor cells for tissue repair.The paracrine potential of adult and fetal stem cells might be then considered as a new regenerative medicine approach for future clinical use, in order to cure specific disease or congenital defects.Partially funded by PRIN 2010–2011 Project from the Italian Ministry of University and Research (MIUR), protocol number 20102MFT8x_004. We would like to thank Nicola Smart from University of Oxford for the critical reading of the manuscript and help with editorial comments.The authors declare no conflict of interest. 

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+This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).A common clinical problem concerns the utility of repeat lumbar puncture (LP) in adults with acute bacterial meningitis (ABM), e.g., pneumococcal meningitis [1]. An LP is initially done for diagnostic purposes in patients with suspected ABM, i.e., diagnostic lumbar puncture (DLP). A repeat LP (RLP) may be done 1–3 days after the initial DLP, if the patient shows no improvement. If a patient with ABM is not doing well after three days, adequacy of antimicrobial therapy is the main concern. Other reasons for RLP is to detect possible intracranial complications of ABM unrelated to adequacy of therapy [1,2]. Cerebrospinal fluid (CSF) Gram stain and culture aside, the single most important CSF diagnostic parameter of ABM is the CSF lactic acid level [3,4,5,6], even with a negative CSF Gram stain. While the usual CSF lactic acid level breakpoint is 2.2 mmol/L, for ABM a cutoff of 6 mmol/L is highly sensitive/specific and rapidly/accurately differentiates ABM from partially treated bacterial meningitis (PTBM), as well as aseptic acute meningitis/encephalitis [6]. Typically, initial DLP CSF lactic acid levels in S. pneumoniae ABM are highly elevated (>12 mmol/L). Equally important, should the CSF lactic acid levels obtained on RLP have greatly decreased, this is predictive of cure. In the case of ABM, the most important CSF as predictor of therapeutic efficacy and good prognosis are CSF glucose and CSF lactic acid levels [6]. The CSF white blood cell (WBC) count and differential counts are much less sensitive predictors of therapeutic efficacy, as CSF pleocytosis may persist for weeks despite effective antibiotic therapy.Antibiotic penetration into CSF depends on the antibiotic’s molecular size, lipid solubility and degree of blood brain barrier (BBB) permeability. The best index of BBB penetrability is CSF albumin, i.e., the higher the CSF albumin, the greater BBB penetrability [7]. This is clinically relevant since some pneumococcal strains may be penicillin resistant S. pneumoniae (PRSP), and are often empirically treated with “high dose” vancomycin. In spite of the “high dose” vancomycin, CSF penetration is not assured as only ~15% of vancomycin simultaneous serum levels penetrate the CSF in the presence of inflammation, i.e., ABM and <1% penetrates the BBB in the absence of inflammation, i.e., no ABM. Therefore, increasing the dose of an inflammation dependent antibiotic, e.g., vancomycin, is likely to be ineffective in the case of PRSP ABM. In contrast, “meningeal doses” of third generation cephalosporins or meropenem penetrate the CSF if PRSP is administered in therapeutic concentrations [8].In ABM, RLP with a marked decrease in CSF lactic acid levels, is predictive of cure. Clinically this is important since the CSF Gram stains may remain positive for RLP [1]. A persistently positive Gram stain for RLP may mislead any physician unaware of the problem into changing therapy, but markedly decreased CSF lactic acid levels are predicative of the therapeutic effectiveness of the cure and antibiotic [6,7,9].Unless the patient is clearly not improving or has even deteriorated, RLP is usually not necessary. CSF lactic acid levels are helpful both diagnostically in the DLP and prognostically in the RLP, and helpful in confirming whether antimicrobial therapy is effective and predictive of cure. The treatment for “penicillin resistant” pneumococci (PRSP) remains as penicillin (or a β lactam given in “meningeal doses”). In contrast to vancomycin, in “meningeal doses” of third generation cephalosporins, e.g., ceftriaxone 2 g (IV) q12 h, the penicillin non-susceptibility and resistance breakpoints for PRSP are well below achievable CSF concentrations [10]. CSF levels following 2 g of ceftriaxone are approximately 257 mcg/mL which is well above the minimal inhibitory concentration (MIC) of even highly resistant (PRSP) in CSF [8].

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+This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).Bone is the most common site of metastasis from breast cancer. Bone metastases from breast cancer are associated with skeletal-related events (SREs) including pathological fractures, spinal cord compression, surgery and radiotherapy to bone, as well as bone pain and hypercalcemia, leading to impaired mobility and reduced quality of life. Greater understanding of the pathophysiology of bone metastases has led to the discovery and clinical utility of bone-targeted agents such as bisphosphonates and the receptor activator of nuclear factor kappa-B ligand (RANK-L) antibody, denosumab. Both are now a routine part of the treatment of breast cancer bone metastases to reduce SREs. With regards to prevention, there is no evidence that oral bisphosphonates can prevent bone metastases in advanced breast cancer without skeletal involvement. Several phase III clinical trials have evaluated bisphosphonates as adjuvant therapy in early breast cancer to prevent bone metastases. The current published data do not support the routine use of bisphosphonates in unselected patients with early breast cancer for metastasis prevention. However, significant benefit of adjuvant bisphosphonates has been consistently observed in the postmenopausal or ovarian suppression subgroup across multiple clinical trials, which raises the hypothesis that its greatest anti-tumor effect is in a low estrogen microenvironment. An individual patient data meta-analysis will be required to confirm survival benefit in this setting. This review summarizes the key evidence for current clinical practice and future directions. Breast cancer is the most common malignancy and the leading cause of cancer death among women worldwide [1]. The incidence is highest in North America, Australia, New Zealand, western and northern Europe, where as many as one in eight women develop breast cancer [2,3]. Bone is the most common site of breast cancer metastasis, with over 70% of patients who died from breast cancer found to have bone metastases on postmortem examination [4]. In women with early breast cancer, risk factors for developing bone metastases include the presence of significant nodal disease with greater than four involved axillary lymph nodes at initial diagnosis, primary tumor size greater than 2 cm, estrogen receptor positive progesterone receptor negative tumor and younger age [5,6]. The presence of significant nodal disease has the highest cumulative incidence of bone metastases: 15% at 2 years and 41% at 10 years [5]. The median survival for patients with breast cancer and bone metastases is approximately 2 years [7,8]. Patients with bone-only metastases have a significantly better outcome than those with visceral metastases [8] and highly selected series have reported an average survival of 72 months [9]. However, bone metastases from breast cancer are associated with significant morbidity including immobility and the development of SREs, which are defined as the development of pathological fractures, spinal cord compression, the need for surgery and radiotherapy to bone. When bone pain and hypercalcemia are included as SREs as in older definitions, SREs occur in over 50% of patients with breast cancer bone metastases [10]. Given the frequency of breast cancer bone metastases, its negative impact on quality of life, the relatively longer survival of these patients and the burden of SREs to society, much research has focused on the pathophysiology, treatment and prevention of bone metastases from breast cancer over the last two decades. This paper discusses key research findings and summarizes the data from randomized controlled trials for evidence-based clinical practice and future directions. Normal bone formation is a coordinated dynamic process of active bone production by osteoblasts and bone remodeling and resorption by osteoclasts. This fine balance is mediated by a variety of local and systemic factors such as transforming growth factor-beta (TGF-β), insulin growth factor (IGF), bone morphogenic protein, platelet-derived growth factor (PDGF), prostaglandin and parathyroid hormone, as well as RANK-L, a key factor for osteoclast production [11]. Bone metastases disrupt this complex interplay through an organized and multistep process involving tumor intravasation, cell survival in the circulatory system, extravasation into surrounding tissue, initiation and maintenance of growth, vascularization and angiogenesis [12]. Key gene expression signatures identified in this process include C-X-C chemokine receptor type 4 (CXCR4), fibroblast growth factor 5, connective tissue-derived growth factor, interleukin-11, matrix metalloproteinase (MMP)-1, follistatin, A disintegrin and metalloproteinase with thrombospondin motifs 1 (ADAMTS1) and proteoglycan-1, all of which are overexpressed by at least four-fold when compared with the same cell lines that have not metastasized to bone [13].The ���seed and soil” hypothesis was first proposed in 1889 by Stephen Paget, who suggested that the “seeds (tumor) can only live and grow if they fall on congenial soil (optimal bone microenvironment)” [14]. This concept was further expanded in the “metastatic niche” model by Psaila and Lyden, who explained the relationship between the disseminating seed (tumor) and metastatic soil (bone) through a continual supply of growth factors from the microenvironment, loss of apoptotic signals and the recruitment of endothelial progenitor cells [15].Osteolytic and osteoblastic metastases result in excessive bone resorption and formation respectively, both at the expense of quality bone formation, mineralization and organization. Radiologically, approximately 48% of bone metastases from breast cancer are purely osteolytic, 13% are purely osteoblastic and 38% are mixed osteoblastic and osteolytic [16]. Histologically and biochemically the two processes coexist irrespective of lytic or blastic radiological appearance [17]. Key mediators of the osteolytic tumor pathway include the parathyroid hormone-related peptide (PTHrP), which upregulates RANK-L from osteoblasts and stromal cells, resulting in down regulation of osteoprotegerin, activation of osteoclasts, production of TGF-β and IGF which in turn promotes tumor cell growth and further release of PTHrP, creating a “vicious cycle of bone metastases” [18] (Figure 1). The osteoblastic pathway is less well studied and much of its research has been focused on prostate cancer. Several mediators produced by tumors are thought to play important roles in the pathogenesis, including endothelin-1 (ET-1), bone morphogenic protein (BMP), fibroblast growth factor (FGF) and PDGF, as well as the Wnt protein pathway through negative regulation of Dickkopf-1 (Dkk1) [19].An improved understanding of the pathophysiology of bone metastases from breast cancer has ushered the development and clinical use of bone-targeted agents in this area and the specific pathways elucidated provide potential targets for future bone-targeted therapy. The optimal treatment of bone metastases from breast cancer involves the integration of local, systemic anti-cancer therapy, bone-targeted agents and supportive care through a multidisciplinary team of surgeons, radiation oncologists, medical oncologists, palliative care physicians, radiologists, cancer nurses and coordinators. Treatment is palliative and is aimed at preventing SREs, reducing pain and suffering, preventing disability and improving quality of life.The vicious cycle of bone metastases. In the osteolytic vicious cycle, tumor cells secrete parathyroid hormone-related peptide (PTHrP) and other factors including interleukins, prostaglandin E, tumor necrosis factor and macrophage-stimulating factor. PTHrP induces osteoclastogenesis by upregulation of RANK-L. The activated osteoclasts in turn produce TGF-β and IGF, which promotes cancer cell growth. In the osteoblastic vicious cycle, breast cancer cells produce osteoblast-stimulating factors such as bone morphogenic protein (BMP), fibroblast growth factor (FGF) and platelet-derived growth factor (PDGF). PTHrP is also overexpressed. It activates ET-1, which down regulates Dkk1, a negative regulator of osteoblastogenesis. The activated osteoblasts in turn produce factors including interleukin-6 (IL-6), monocyte chemotactic protein-1 (MCP-1), vascular endothelial growth factor (VEGF), macrophage inflammatory protein-2 (MIP-2); which facilitate breast cancer cell colonization and survival upon arrival in the bone microenvironment. In reality, there is a complex interplay between the two cycles [11,19,20,21]. Reproduced with permission from the Journal of Breast Cancer: Targets and Therapy [18]. Bisphosphonates are potent osteoclast inhibitors and an important class of bone-targeted agents used to reduce the frequency of SREs, improve bone pain and serve as an established treatment for hypercalcemia of malignancy [22,23] (Table 1). Non-nitrogen containing bisphosphonates, such as clodronate and etidronate, are converted intracellularly into methylene-containing analogs of adenosine triphosphate (ATP), which accumulate within macrophages and osteoclasts causing direct apoptosis [18]. Nitrogen-containing bisphosphonates, including pamidronate, ibandronate and zoledronic acid, also inhibit farnesyl diphosphate synthase, a rate-limiting enzyme of the mevalonate pathway, preventing protein prenylation of small guanosine triphosphatase (GTPase) such as Ras, Rho and Rab, which are important signaling proteins that regulate cell survival in osteoclasts [18,24]. In vitro, at higher concentrations, nitrogen-containing bisphosphonates inhibit osteoblasts, epithelial and endothelial cells as well as breast tumor cells, in part explaining their potential anti-tumor properties [24].Bisphosphonates are defined by their P-C-P conformation, which renders them high affinity to the hydroxyapatite in the bone mineral. Bisphosphonates contain two side chains, R1 being the variable structure that determines the potency of the compound (top left of each structure), and R2 being the short addition that increases the bone affinity (bottom left of each structure). Nitrogen-containing R1 improves the potency by at least 100 fold, and OH-containing R2 significantly increases the affinity to bone. Additional abbreviations: MBC, metastatic breast cancer; IV, intravenous; PO, oral. Reproduced with permission from the Journal of Breast Cancer: Targets and Therapy [18].The effects of bisphosphonates on SREs have been extensively studied in metastatic breast cancer over the last two decades using a variety of agents. In a 2012 Cochrane systematic review and meta-analysis, data from 19 randomized controlled trials (RCTs) and 6646 patients were incorporated to evaluate the effects of bisphosphonates or denosumab on SREs from breast cancer bone metastases [25] (Figure 2). For women with advanced breast cancer and clinically evident bone metastases, bisphosphonates significantly reduced the incidence and rate of SREs (excluding hypercalcemia) by 15% as compared to placebo control (risk ratio (RR) 0.85; 95% CI 0.77–0.94; p = 0.001) [25]. Efficacy in reducing SREs was demonstrated for both parenteral (RR 0.83; p = 0.008) and oral (RR 0.84; p = 0.0007) routes of administration compared to control. Individual drug effects on SREs were shown for intravenous (IV) zoledronic acid 4 mg (RR 0.59), IV pamidronate 90 mg (RR 0.77), IV ibandronate 6 mg (RR 0.80), oral ibandronate (RR 0.86) and oral clodronate (RR 0.85) [25]. Few trials have directly compared agents.A large multi-center randomized, double-blind, placebo-controlled trial of patients with bone metastases from breast cancer and multiple myeloma (n = 1130) led by Rosen et al. [26] compared 4 mg or 8 mg IV zoledronic acid to 90 mg IV pamidronate, every 3–4 weeks for up to two years. After a protocol modification due to concerns about renal toxicity with the 8 mg zoledronic acid, 4 mg zoledronic acid was shown to be equivalent in efficacy in terms of SREs and tolerability including incidence of renal impairment, when compared to pamidronate in the overall population [26]. In the lytic metastases from breast cancer subgroup (n = 528), zoledronic acid produced a significant prolongation of time to first skeletal related event (SRE) (310 versus 174 days; p = 0.013), significant reduction in skeletal morbidity rate (1.2 versus 2.4 events; p = 0.008) and a significant reduction in the SRE rate (p = 0.010) when compared to pamidronate [27]. Skeletal morbidity rate was significantly lower when zoledronic acid was combined with radiotherapy (0.47 versus 0.71 events, p = 0.018) or with hormone therapy (0.33 versus 0.58 events, p = 0.015), suggesting synergism between zoledronic acid and other anti-cancer therapies in preventing skeletal complications [26]. In a more recent phase III trial, the zoledronic acid versus oral ibandronate comparative evaluation (ZICE) study (n = 1405), oral ibandronate was shown to be inferior to zoledronic acid in terms of the primary endpoint of SRE rate (0.543 versus 0.444, HR (hazard ratio) 1.22; 95% CI 1.04–1.45; p = 0.017) [28].Forest plot of comparison: Overall risk of SREs (excluding hypercalcemia) from breast cancer bone metastases: bisphosphonate versus control. Reproduced with permission from the ©Cochrane Collaboration [25]. The question of when to start a bisphosphonate, and when to stop have, yet to be answered by RCTs. In the exploratory retrospective analysis of the zoledronic acid versus pamidronate trial led by Rosen et al. [26], patients with one prior SRE were found to be at significantly higher risk (HR 2.08) of developing an on-study SRE than patients with no prior SRE [29]. This suggests starting bisphosphonates early may be warranted rather than waiting for a SRE to occur [18]. The American Society of Clinical Oncology (ASCO) guidelines, the Cancer Australia National Breast and Ovarian Cancer Centre (NBOCC) guidelines and the International Expert Panel guidelines all recommend starting bisphosphonates at the first radiographic sign of bone metastasis [30,31,32] (Table 2). As for the duration of bisphosphonates, there is currently a paucity of data on their use beyond 2 years, which is the treatment duration most commonly set in RCTs. However, this should not be a contraindication to continual therapy in individual patients and is encouraged by consensus guidelines [30,31,32]. The standard dosing of zoledronic acid is 4 mg every 3–4 weeks [30]. A recent randomized controlled trial (RCT) from Italy demonstrated non-inferiority of reduced frequency dosing at every 12 weeks in the second year [33].In the 2012 Cochrane meta-analysis, few serious adverse events were reported and many were disease or chemotherapy related. Fever and asymptomatic hypocalcemia were the most commonly reported side-effects in women receiving IV pamidronate. Gastrointestinal toxicity was the most frequently reported side-effect of oral bisphosphonates, while acute-phase reactions were more common with IV bisphosphonates [25]. When calcium and vitamin D supplementation were not given, hypocalcemia was more common with zoledronic acid (39% versus 7%) compared to placebo [34], however no significant hypocalcemia was seen when calcium and vitamin D supplementation was instituted [27]. Renal toxicity was the main issue with IV zoledronic acid with incidence at 8.5% [35] and was related to dose and infusion time [26]. Osteonecrosis of the jaw (ONJ) was rare at 1.4% after objective assessment [35]. Established guidelines recommend cessation of bisphosphonates prior to invasive dental treatments or avoidance of such procedures during bisphosphonate therapy [36]. Existing guideline recommendations for bisphosphonate use in metastatic breast cancer patients with bone metastases. Additional abbreviations: CT, computed tomography; MR, magnetic resonance; ZOL, zoledronic acid; IBA, ibandronate; PAM, pamidronate; CLO, clodronate; DMB, denosumab. Reproduced with permission from the Journal of Breast Cancer: Targets and Therapy [18].Lytic disease on X-rayAbnormal bone scan with CT/MR showing bone destructionIV PAM 90 mg every 3–4 weeks ORIV ZOL 4 mg every 3–4 weeks ORSC DMB 120 mg every 4 weeksIV preferable (ZOL, IBA, PAM)PO for patients who cannot or need not attend hospital care (CLO, IBA)While bisphosphonates as a group significantly reduced incidence and rates of SREs, they do not affect survival in women with bone metastases from breast cancer (RR 1.01; 95% CI 0.92–1.11) [25].Denosumab, a fully human monoclonal antibody to RANK-L, has been shown in preclinical studies and clinical trials to inhibit osteoclast-mediated bone destruction [37]. Its superior suppression of bone turnover (urinary N-telopeptide <50 nM) compared to zoledronic acid (71% versus 29%) was demonstrated in a randomized Phase II trial involving breast cancer, prostate cancer and multiple myeloma patients [38]. In a landmark Phase III trial led by Stopeck et al. [35], 2046 patients with bone metastases from breast cancer were randomized to subcutaneous (SC) denosumab 120 mg or IV zoledronic acid 4 mg every 4 weeks. Denosumab significantly delayed first on-study SREs compared to zoledronic acid and the study met both its primary endpoint of non-inferiority (HR 0.82, 95% CI 0.71–0.95; p < 0.001) and secondary endpoint of superiority (HR 0.82, p = 0.01). Denosumab extended the median time to development of first on-study SRE compared to zoledronic acid (32.4 versus 26.4 months) [35]. In addition, denosumab prolonged the time to developing moderate or severe pain compared to zoledronic acid (HR 0.78; p = 0.002) [39]. The 2012 Cochrane meta-analysis included 3 RCTs and 2345 patients comparing denosumab and IV bisphosphonates, showed a significant reduction in the risk of developing a SRE by 22% favoring denosumab (RR 0.78; 95% CI 0.72–0.85; p < 0.00001) [25] (Figure 3). Forest plot of comparison: Overall risk of SREs in breast cancer bone metastases: denosumab versus bisphosphonate. Reproduced with permission from ©Cochrane Collaboration [25].The incidence of adverse events were similar between denosumab and zoledronic acid in the study by Stopeck et al. [35]. There was no significant difference in the rate of ONJ (2% versus 1.4%, p = 0.39) and denosumab was associated with significantly less renal toxicity (4.9% versus 8.5%, p = 0.001) and fewer acute-phase reactions (10.4% versus 27.3%) [35]. Toothache and hypocalcemia were more common with denosumab and for the latter, adequate daily calcium and vitamin supplementation was emphasized. The most common adverse reactions in patients receiving denosumab were fatigue, asthenia, hypophosphatemia and nausea [35]. Denosumab’s improved efficacy over zoledronic acid, ease of administration and more favorable renal toxicity profile have resulted in the ASCO guidelines recommending it as a first-line option in the management of bone metastases from breast cancer [30].Key points for clinical practice:
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+              Zoledronic acid is the most potent and effective bisphosphonate in preventing SREs. Standard dose is given IV 4 mg every 3–4 weeks for 2 years and to continue if performance status remains adequate;
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+              Denosumab given 120 mg SC every 4 weeks, has superior efficacy over zoledronic acid in preventing SREs;
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+            
+              Calcium and vitamin D supplementation could prevent treatment related hypocalcemia;
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+              While ONJ is rare at 2% or less, invasive dental procedures should be avoided during bisphosphonate or denosumab therapy;
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+              Bisphosphonates do not improve survival in women with metastatic breast cancer.
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+		  Zoledronic acid is the most potent and effective bisphosphonate in preventing SREs. Standard dose is given IV 4 mg every 3–4 weeks for 2 years and to continue if performance status remains adequate;Denosumab given 120 mg SC every 4 weeks, has superior efficacy over zoledronic acid in preventing SREs;Calcium and vitamin D supplementation could prevent treatment related hypocalcemia;While ONJ is rare at 2% or less, invasive dental procedures should be avoided during bisphosphonate or denosumab therapy;Bisphosphonates do not improve survival in women with metastatic breast cancer.Intractable bone pain occurs in 50%–90% of patients suffering from bone metastases from breast cancer [40]. Bone pain can be poorly localized, of deep aching quality and patients often experience episodes of stabbing discomfort particularly worse at night not necessarily relieved by lying down [40]. The unique pathophysiology of bone pain involves spinal cord astrocytosis, enhanced neuronal activity through c-Fos expression and sensitization of the central dorsal horn of the spinal cord mediated by dynorphin, a pro-hyperalgesic peptide [41]. It is believed that both tumor-induced damage as well as tumor-produced factors such as endothelin-1 have important roles in the pathophysiology of bone pain [21]. A 2002 Cochrane meta-analysis specifically examining bisphosphonate effects on bone pain evaluated the effects of etidronate, pamidronate and clodronate in 30 RCTs encompassing 3682 patients with breast, prostate and lung cancers, multiple myeloma and cancer of unknown primary [42]. There was significant pain relief among patients with metastatic breast cancer who received bisphosphonate therapy (OR (odds ratio) 1.83, 95% CI 1.11–3.04). In the subgroup analysis of the three bisphosphonates, the response was significant for oral clodronate (OR 3.26, 95% CI 1.80–5.09), but not for intravenous pamidronate (OR 2.35, 95% CI 0.77–7.15) and the trend was unfavorable for etidronate (OR 0.28, 95% CI 0.01–7.67) [42]. However, Lipton et al. demonstrated in two RCTs (n = 751) that pamidronate significantly reduced the pain score (−0.07, p = 0.015) and the analgesia score (−0.06, p = 0.001) at 24 months [43]. Zoledronic acid has been shown to provide significant and sustained pain relief, and improve quality of life [44]. In addition, it demonstrated efficacy as a second-line agent after failure on pamidronate or clodronate [45]. Both oral and intravenous ibandronate were shown to reduce bone pain, peaking within 8–12 weeks and providing the longest time of sustained pain relief for at least 96 weeks [46]. Quality of life was significantly improved in patients who received intravenous ibandronate [47]. In the study by Stopeck et al., while denosumab delayed the onset of pain compared to zoledronic acid, the median time to pain improvement was similar between treatment arms (82 versus 85 days: HR 1.02; p = 0.72) [35].External beam radiotherapy is an established treatment for bone pain secondary to bone metastases, with pain relief commonly achieved within 4–6 weeks. Re-treatment is possible if pain recurs [48]. A single fraction (8 Gy/1 fraction) was shown in a systematic review to be equivalent to multiple fractions (20 Gy/5 fractions) in achieving an overall response in pain (58% versus 59%, 95% CI 0.95–1.03), but the retreatment rate was 2.5-fold higher after single fraction treatment (p < 0.00001) [49]. A meta-analysis established 8 Gy as the standard dose in a single fraction after demonstrating its superior pain response rates compared to 4 Gy [50]. Re-treatment with single fraction radiotherapy was recently demonstrated to be non-inferior to multiple fractions [51]. Given its equal efficacy, patient convenience and cost effectiveness, single fraction radiotherapy using a dose of 8 Gy to provide palliation from painful bone metastases is supported by the Guidelines from the American Society of Radiation Oncology [52]. Bone-targeted radiopharmaceuticals, such as strontium-89, samarium-153 and radium-223 have been developed for palliation of refractory bone pain. These are thought to act as a substitute for hydroxyapatite in bone, with greater uptake in osteoblastic metastases where new reactive bone is formed [18]. While the clinical evidence is most established in metastatic prostate cancer, one study involving 100 patients (40 with metastatic breast cancer) randomized to strontium-89 or samarium-153 showed improvement in performance status (Karnofsky score +20) and reduction in pain (visual analog scale −4) with more favorable results for osteoblastic than mixed metastases [53]. Spinal cord compression is a potentially devastating complication which occurs in up to 8% of patients with metastatic breast cancer [54]. A multidisciplinary team approach with experienced surgeons, radiation oncologists, medical oncologists, palliative care physicians, cancer nurses and coordinators is often necessary. Optimal management consists of high dose corticosteroids, magnetic resonance imaging to confirm diagnosis, prompt surgical decompression and radiotherapy [18]. In a landmark RCT led by Patchell et al. [55], surgery followed by radiotherapy demonstrated a significantly better post-treatment ambulatory rate (84% versus 57%, p < 0.001) compared with radiotherapy alone, with significantly higher continence rate (OR 0.47, p = 0.016), superior functional ability (OR 0.24, p = 0.0006) and superior motor strength (OR 0.28, p = 0.001) [55]. Survival time was also significantly longer in the combined modality group (126 days versus 100 days, OR 0.60, p = 0.033) [55]. However, patients with very radiosensitive tumors, multiple areas of spinal cord compression or total paraplegia for longer than 48 h were excluded from the study. Therefore, combined modality treatment with upfront surgery should be offered for fit and functional patients with spinal cord compression, while radiotherapy alone is best reserved for the unfit, already incapacitated patients with poor prognosis [18].Effective systemic anti-cancer therapy is paramount in the management of bone metastases. While chemotherapy is an integral part of systemic treatment, the role of endocrine therapy is particularly important in bone-only or bone-predominant metastases from breast cancer [18]. Among patients with recurrent breast cancer, those with estrogen receptor (ER)-positive tumors are twice as likely to develop bone metastases as those with ER-negative tumors [56]. Microarray studies in breast cancer also showed that bone metastases occur far more frequently in ER-positive tumors (luminal type 68%), compared with human epidermal growth factor receptor 2 (HER2) positive tumors (20%), basal tumors (7%) and normal molecular subtypes (6%) [57]. The genes upregulated for ER-positive bone metastases are entirely different from those for HER2-positive or basal subtype bone metastases, suggesting a distinct molecular pathway for ER-positive tumors to metastasize to bone [18,57]. Current guidelines recommend endocrine therapy in preference to chemotherapy for women with ER-positive advanced breast cancer, except in the presence of rapidly progressive visceral disease [58]. In the Breast Cancer Trials of Oral Everolimus-2 (BOLERO-2) study published in 2012, the inhibition of the mammalian target of rapamycin (mTOR) pathway using everolimus in addition to exemestane has been demonstrated as an effective systemic treatment for patients with ER-positive metastatic breast cancer who were previously endocrine resistant [59]. Further research in the molecular pathways of bone metastases from ER-positive tumors, including the phosphatidylinositol 3-kinase (PI3K) and mTOR pathways, may provide insights to new therapeutic targets to bone metastases.Approximately 55% of breast cancers exhibit TGF-β activity via a 153-gene TGF-β response signature [18]. The overproduction of this multi-function cytokine in the setting of bone metastases from breast cancer induces osteolysis and angiogenesis via mothers against decapentaplegic homolog 3 (Smad 3), which in turn drives epithelial-mesenchymal transition and tumor invasion via multiple cell signaling pathways [60]. TGF-β monoclonal antibodies or tyrosine kinase inhibitors (TKI) strongly inhibit bone metastases from basal like breast cancer in mouse models [61]. These are a promising new class of agents that may help halt the “osteolytic vicious cycle” and are currently entering into early phase clinical trials [18,62]. Src is a non-receptor tyrosine kinase that promotes cellular proliferation, differentiation, motility and survival. High levels of Src are implicated in breast cancer osteoclastic activity and activation of endothelial growth factor receptor, HER2, PI3K/mTOR pathways [18]. Src activation was also demonstrated to be associated with late-onset bone metastases in breast cancer [63]. Dasatinib, a multi-targeted Src TKI, had been shown in vivo to inhibit osteoclast differentiation and rapidly lowers calcium levels [64]. Several phase I and II trials of dasatinib are currently running, evaluating its role as a bone-targeted agent in addition to zoledronic acid in the treatment of bone metastases from breast cancer [62]. Saracatinib is a dual specific Src/abl TKI which is also being studied in several phase II trials in bone metastases from breast cancer, evaluating its role as a bone-targeted agent compared to zoledronic acid, and as systemic anti-cancer therapy in addition to aromatase inhibitors [18,62]. The Wnt pathway plays an important role in osteoblastogenesis. The production of a key protein of the pathway, Dkk1, was first shown to be associated with lytic bone lesions from patients with multiple myeloma [65]. Dkk1 secretion by breast cancer cell lines and high circulating levels were subsequently shown be associated with osteolytic metastases from breast cancer [66]. A clinical trial of a Dkk1 neutralizing antibody BHQ880 is ongoing in patients with lytic lesions from multiple myeloma (NCT00741377) [62]. Further studies are required to evaluate this pathway as a potential therapeutic target in the treatment of bone metastases from breast cancer.Other potential novel agents include Cathepsin K inhibitors and CXCR4 antagonists, both showing good preclinical effects on bone turnovers and entering early phase clinical trials [18,65]. New biomarker assays may help guide clinicians to select high risk patients for bone metastasis complications and allow better use of bone-targeted agents, refining the use of “personalized medicine” [18].Key points for clinical practice:
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+            Bisphosphonates and denosumab improve pain in women with bone metastases from breast cancer;
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+            Bisphosphonates may improve quality of life, as was demonstrated with IV ibandronate;
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+            8 Gy single fraction external beam radiotherapy is an effective means of palliation for bone pain;
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+            Combined surgery and radiotherapy for spinal cord compression is superior to radiotherapy alone in terms of functional outcomes;
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+            Optimal treatment of bone metastases involves integration of bone-targeted agents with local and systemic therapy and supportive care through a multidisciplinary team.
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+		Bisphosphonates and denosumab improve pain in women with bone metastases from breast cancer;Bisphosphonates may improve quality of life, as was demonstrated with IV ibandronate;8 Gy single fraction external beam radiotherapy is an effective means of palliation for bone pain;Combined surgery and radiotherapy for spinal cord compression is superior to radiotherapy alone in terms of functional outcomes;Optimal treatment of bone metastases involves integration of bone-targeted agents with local and systemic therapy and supportive care through a multidisciplinary team.The 2012 Cochrane meta-analysis included three studies which evaluated oral bisphosphonates in women with advanced breast cancer without clinically evident bone metastasis [25]. The three RCTs comprised of 320 evaluable patients who received oral clodronate or oral pamidronate compared to placebo. The pooled meta-analysis showed no significant reduction in the incidence of skeletal metastases (RR 0.99; 95% CI 0.67 to 1.47; p = 0.97) and no significant difference in survival (RR 0.91; 95% CI 0.75 to 1.11; p = 0.36). One study assessed quality of life using a validated questionnaire and found no significant difference between oral pamidronate and placebo control [67]. Given the available evidence, the Cancer Australia NBOCC Guidelines do not support the use of bisphosphonates to prevent bone metastases or SREs in women with metastatic breast cancer without clinically evident bone metastasis [31]. Whether more potent modern agents such as zoledronic acid or denosumab can be effective in this setting has not been formally tested in RCTs.Key points for clinical practice:
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+          Current evidence do not support the use of bisphosphonates to prevent bone metastases in women with advanced breast cancer without bone metastasis.
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+	  Current evidence do not support the use of bisphosphonates to prevent bone metastases in women with advanced breast cancer without bone metastasis.Dormant cancer cells are an important source of local and systemic breast cancer recurrence [68,69]. The bone marrow provides a unique microenvironment and acts as a niche or sanctuary for disseminated tumor cells (DTC) through a complex interplay of bone and tumor-derived growth factors and cytokines [70]. In an individual patient data pooled analysis of 4703 patients, bone marrow micrometastasis at the time of early breast cancer diagnosis was shown to correlate with increased risk of disease recurrence and poor prognosis [71]. Preclinical studies have suggested that bisphosphonates may hinder the development of bone metastases by a direct anti-tumor effect and by modifying the bone microenvironment to become a less accommodating host to cancer cell survival and proliferation [72,73]. There is evidence in vitro that bisphosphonates can inhibit tumor adhesion, invasion, induce tumor apoptosis and exert an anti-angiogenic effect [72]. Bisphosphonates may also have immunomodulatory effects, with continual activation of gamma-delta effect or T cells after a single dose of zoledronic acid in an ex vivo model of disease-free breast cancer patients [74]. Zoledronic acid, the nitrogen-containing bisphosphonate, demonstrated synergy with chemotherapy when it caused a 10-fold increase in tumor apoptosis in-vitro when administered 24 h after doxorubicin [75].Translational studies also demonstrated the effects of zoledronic acid in reducing the prevalence and survival of DTCs in the bone marrow [76]. In a phase II RCT involving 120 women with stage II or III breast cancer, the addition of zoledronic acid to neoadjuvant chemotherapy was associated with a higher rate of elimination of DTCs from the bone marrow (70% versus 53%, p = 0.054) [77]. In another study, zoledronic acid after adjuvant chemotherapy significantly reduced the prevalence of DTCs at 12 and 24 months compared to baseline (p < 0.001) [78].These preclinical and translational evidence collectively provided rationale for conducting clinical trials examining bisphosphonates in the adjuvant setting, to prevent metastases and ultimately improve overall survival. Three RCTs commenced in the 1990s examining the effects of clodronate 1600 mg daily as adjuvant therapy for early breast cancer produced discordant results. In the study by Diel et al., where 302 early breast cancer patients with detectable tumor cells in the bone marrow were randomized to 2 years of oral clodronate or placebo, oral clodronate initially improved bone metastasis-free survival (p = 0.003) [79] which became statistically insignificant after 8.5 years of follow-up (HR 0.90, p = 0.770) [80]. It did however produce a durable improvement in overall survival (OS) (HR 0.50, p = 0.04) [79,80]. The Powles et al. study, randomized 1069 patients to 2 years of oral clodronate or placebo, oral clodronate produced significant improvements in both bone metastasis-free survival (HR 0.692, p = 0.043) and OS (HR 0.743, p = 0.041) [81,82]. However, in the Saarto et al. study, where 299 patients were randomized to 3 years of oral clodronate and placebo, clodronate did not improve metastasis-free survival (HR 1.23, p = 0.35) and OS (HR 1.33, p = 0.13), yet was associated with a significant increase in visceral metastasis (HR 1.61, p = 0.015) [83,84]. This study was criticized for its methodology and imbalance of baseline characteristics between treatment arms, with the clodronate group having more ER-negative patients (35% versus 25%), more post-menopausal women (52% versus 43%) who did not receive chemotherapy in this study [18]. In a meta-analysis published in 2007, adjuvant clodronate did not significantly improve bone metastasis-free survival (HR 0.68, 95% CI 0.38–1.23) or OS (HR 0.75, 95% CI 0.31–1.82) although the trend was favorable [85]. The National Surgical Adjuvant Breast and Bowel Project (NSABP) B-34 was the largest RCT conducted using adjuvant clodronate, where 3323 women were randomized to 3 years of oral clodronate 1600 mg daily or placebo. The results published in 2012 after a median follow-up of 90.7 months showed no difference in disease-free survival (DFS) (HR 0.91, p = 0.27) or OS (0.84, p = 0.13) [86]. However, in a pre-planned subgroup analysis of women aged 50 years or older, the clodronate arm showed significantly reduced bone metastasis-free interval (HR 0.62, p = 0.027), non-bone metastasis-free interval (HR 0.63, p = 0.014) but not OS (HR 0.80, p = 0.094). The study investigators hypothesized that adjuvant clodronate may have anti-cancer benefits for older postmenopausal women with a low estrogen bone microenvironment [86].In the German Adjuvant Intergroup Node-Positive (GAIN) Study, 3023 patients with node-positive breast cancer receiving adjuvant dose-dense chemotherapy were randomly assigned 2:1 to adjuvant oral ibandronate 50 mg daily for two years or observation [87]. There were no significant differences in DFS (HR 0.945, p = 0.589) or OS (HR 1.040, p = 0.803) between the two arms. However, a trend towards improved DFS was observed in postmenopausal women older than 60 years (HR = 0.75, 95% CI 0.49–1.14) or women younger than 40 years (HR = 0.70, 95% CI 0.44–1.13), some of whom were given ovarian suppression therapy with a luteinizing hormone-releasing hormone (LHRH) agonist, rendering a low estrogen bone microenvironment [87]. The Austrian Breast and Colorectal Cancer Study Group trial-12 (ABCSG-12) was the first RCT to report the effects of adjuvant zoledronic acid in early breast cancer. 1803 premenopausal women with ER-positive early breast cancer treated with either tamoxifen or anastrozole in combination with ovarian suppression using the LHRH agonist goserelin were randomized to receive 3 years of intravenous zoledronic acid 4 mg every 6 months or observation [88]. Gnant et al. showed that after 76 months of median follow-up, women who received zoledronic acid had a significantly improved DFS (HR 0.73, p = 0.021) and OS (HR 0.59; p = 0.042) compared to endocrine therapy alone [89,90]. A pre-planned subgroup analysis showed that the survival benefit of zoledronic acid was restricted to women older than 40 years at study entry (n = 1390) and no benefit was seen in women under the age of 40 years (n = 413) [89,90]. Gnant hypothesized that very young women may have incomplete ovarian suppression hence a higher estrogen bone microenvironment, which may explain the ineffectiveness of zoledronic acid in this group [90,91]. Three companion trials, the Zometa-Femara Adjuvant Synergy Trials (Z-FAST, ZO-FAST and E-ZO-FAST) conducted across North America, the UK, Europe and worldwide evaluated the effects of immediate versus delayed zoledronic acid 4 mg every 6 months for 5 years in postmenopausal women with early breast cancer receiving an aromatase inhibitor. The smaller Z-FAST (n = 602) and E-ZO-FAST (n = 527) studies achieved their primary endpoint of increased bone mineral density but did not show a significant difference in DFS with immediate zoledronic acid [92,93]. However, the larger ZO-FAST (n = 1065) study demonstrated a significant reduction of DFS events (HR 0.66, p = 0.0375) with immediate zoledronic acid, and fewer local and distant recurrences compared to delayed zoledronic acid which was initiated after a fracture or reduced bone mineral density [94].The Adjuvant Zoledronic Acid to Reduce Recurrence (AZURE) trial led by Coleman et al. [95] was the largest RCT conducted using adjuvant zoledronic acid. 3360 patients with early breast cancer were randomized to standard adjuvant systemic therapy with or without zoledronic acid, given 4 mg every 3 to 4 weeks for 6 doses then every 3 to 6 months to complete 5 years. The addition of zoledronic acid did not significantly improve DFS (HR 0.98; p = 0.79) or OS (HR 0.85; p = 0.07) in the overall population [95]. However, pre-planned subgroup analysis showed that in women who were at least 5 years postmenopausal (n = 1041), zoledronic acid significantly improved DFS (HR 0.75; p = 0.02) and OS (HR 0.74; p = 0.04) [95]. While the AZURE and NSABP-B34 adjuvant bisphosphonates trials were demonstrably negative in the overall population, benefit in the postmenopausal or ovarian suppression subgroup is consistently observed across multiple trials. This raises the hypothesis that the greatest anti-tumor benefit of adjuvant bisphosphonates is in a low estrogen microenvironment. A low estrogen state may, through unknown mechanisms, allow alterations in bone microenvironment, “the soil”, to become less conducive to tumor “seeding” and metastases [70,90].The 2012 Cochrane meta-analysis including 9 RCTs did not show any significant benefit of adjuvant bisphosphonates in preventing bone metastases (RR 0.94; p = 0.36), or overall disease recurrence (RR 0.97; p = 0.75) or OS (RR 0.84; p = 0.11) in the overall early breast cancer population [25]. Two recent meta-analyses of RCTs of adjuvant zoledronic acid have yielded conflicting results. Yan et al. [96] meta-analyzed 5 studies (n = 7354) and demonstrated no significant improvement in OS, DFS or bone metastasis-free survival with the use of adjuvant zoledronic acid compared to control. However, in the postmenopausal subgroup, the addition of zoledronic acid significantly improved DFS (RR 0.763, p < 0.001), locoregional recurrence (RR 0.508; p = 0.001) and distant recurrence (HR 0.744; p = 0.003), but there was no significant improvement in OS (RR 0.811; p = 0.286) [96]. Valachis et al. [97] meta-analyzed 15 studies (n = 9197) and demonstrated a significant improved OS (HR 0.81; p = 0.007) but no difference in DFS (HR 0.86; p = 0.15) or incidence of bone metastases (HR 0.94; p = 0.74) [97]. The differences in inclusion criteria may have accounted for the discrepancy of results in the two meta-analyses. Of note, the high levels of heterogeneity of studies (I2 = 67.3% for DFS in Yan et al., I2 = 55% for DFS in Valachis et al.) [96,97] is an issue for both meta-analyses, and has made it difficult for a firm conclusion to be drawn. Nevertheless, meta-analyses have concluded that adjuvant zoledronic acid is well tolerated in the adjuvant setting, with the rate of ONJ at 0.52% [97], which is lower than in the metastatic setting [35]. Overall, the meta-analyses conducted based on published data do not provide definitive answers to the question and the available evidence do not support the routine use of adjuvant bisphosphonates in unselected patients with early breast cancer.While the results of other adjuvant bisphosphonate studies such as the Southwest Oncology Group-0307 (SWOG-0307) and the German Simultaenous Study of Gemcitabine-Docetaxel Combination Adjuvant Treatment, as well as Extended Bisphosphonate and Surveillance (SUCCESS) trials are awaited, an individual patient data meta-analysis will be required to definitively answer the question on adjuvant bisphosphonates in early breast cancer. This is necessary in order to minimize publication bias and the effects of heterogeneity of included studies, as were shown to be problematic in the meta-analyses conducted using published data. Particular attention is required in the subgroup of women with a low estrogen bone microenvironment, either through natural menopause or medical ovarian suppression. Denosumab has been shown to improve bone mineral density and is safe and well tolerated when compared to placebo in patients with non-metastatic breast cancer [98]. The effects of adjuvant denosumab on recurrence and survival are currently being investigated, including the large RCT, the Study of Denosumab as Adjuvant Treatment for Women with High Risk Early Breast Cancer Receiving Neoadjuvant or Adjuvant Therapy (D-CARE), with results expected after 2016 [62]. Genomic and proteomic profiling have been shown to have prognostic potential correlating with disease progression as well as predictive potential for specific treatment benefits [99]. In the future, these technologies may help guide clinicians to select patients to treat aggressively with bone-targeted agents and to selectively omit unnecessary treatment. While health care costs in oncology are rising globally, personalized medicine in the molecular age may potentially be cost-effective and cost-saving [100]. Key points for clinical practice:
+          
+            
+              Current evidence do not support the routine use of adjuvant bisphosphonates in unselected women with early breast cancer;
+			
+            
+              The incidence of ONJ in the adjuvant setting is very rare, in the order of 0.52%;
+                     
+            
+              Adjuvant bisphosphonates may provide survival benefit in the subgroup of women with low estrogen bone microenvironment, either through natural menopause or ovarian suppression;
+            
+            
+              Individual patient data meta-analysis will be required to definitively address this question.
+            
+          
+		  Current evidence do not support the routine use of adjuvant bisphosphonates in unselected women with early breast cancer;The incidence of ONJ in the adjuvant setting is very rare, in the order of 0.52%;Adjuvant bisphosphonates may provide survival benefit in the subgroup of women with low estrogen bone microenvironment, either through natural menopause or ovarian suppression;Individual patient data meta-analysis will be required to definitively address this question.Bone metastasis from breast cancer is a common condition and is associated with incurable disease, significant complications, morbidity and reduced quality of life. Treatment of bone metastases is palliative and is aimed at reducing SREs, preserving mobility and improving quality of life. Greater understanding of the pathophysiology of bone metastases has led to the discovery and clinical utility of safe and effective bone-targeted agents such as bisphosphonates and denosumab. The integration of bone-targeted agents with other local, systemic anti-cancer therapy and supportive care is important for the optimal treatment of bone metastases. While potentially more effective bone-targeted agents are being developed, prognostic and predictive biomarkers may help guide future directions on personalized treatment of bone metastases from breast cancer. While there is no evidence for the use of bisphosphonates for the prevention of bone metastases in advanced breast cancer without skeletal involvement, its use in the adjuvant setting has generated worldwide interest in recent years. As preclinical and translational data suggested potential therapeutic effects of bisphosphonates on tumor cells and the bone microenvironment, multiple large scale RCTs involving bisphosphonates as adjuvant therapy in early breast cancer have been completed. While bisphosphonates have failed to conclusively show any survival benefit in unselected early breast cancer patients, consistent benefits in reducing bone recurrence and/or improving survival have been observed in the subgroup of women with a low estrogen bone microenvironment, either through natural menopause or ovarian suppression. As we await the results of a few more ongoing RCTs, an individual patient data meta-analysis will be most useful to answer this important question in early breast cancer management and bone metastasis prevention. The authors declare no conflict of interest. 

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+This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).Prolonged oligohydramnios following extreme preterm prelabour rupture of membranes (EPPROM) is traditionally associated with a high morbidity and mortality to both the mother and the baby. The clinical maternal evaluation and fetal ultrasound assessment may provide important prognostic information for the clinicians and should be taken into account when counselling the patients so as to provide them with enough information to make decision of continuing or interrupting the pregnancy. Current financial constraints on the National Healthcare Service (NHS) resources make it imperative for clinical decision-makers and budgetary planners to make the right decision of continuing or terminating a second trimester pre-viability amniorrhexis for desperate parents. To assess the economic consequences following EPPROM, the risk of infection to both baby and mother, psychological impact on the parents and associated complications and further disability after delivery on this fragile group of patients to the NHS resources. We review the clinical course, outcome, and the challenges to parents and health care professionals on three pregnancies complicated by EPPROM, occurring before 24 weeks’ gestation with a membrane rupture to delivery interval (latent period) of 14 days or more. The anticipated birth of an extremely premature infant poses many challenges for parents and health care professionals. As parents are faced with difficult decisions that can have a long-term impact on the infant, family and country’s resources, it is critical to provide the type of information and support that is needed by them. Taking all these into consideration with the period of ventilation and respiratory assistance in Neonatal Intensive Care Unit (NICU) is essential to provide maximum chances for survival, minimizing the risk for long term sequelae of the neonate and provides the parents enough time to decide on making the right decision with the associated guidance of the healthcare provider.Preterm prelabour rupture of the membranes (PPROM), particularly at very early gestations, presents a management problem for obstetricians and neonatologists alike with inherent risks to both mother and foetus. About six in 1000 pregnancies are complicated by PPROM in the second trimester [1]. The management of this group of patients in obstetrics is one of “watchful waiting”, involving monitoring for signs of developing infection, the administration of antibiotics and antenatal corticosteroids, the use of tocolytics and the delivery of the infant once there is any sign of infection or an acceptable maturity is reached [2,3,4].Overall, there has been traditionally poor outcome in neonatal survival following second trimester PPROM with a number of published series quoting figures of between 20% and 55% [5,6]. Mortality outcomes of >90% have been recorded in infants following rupture of membranes prior to 25 weeks, with a latency period to delivery of over 14 days with persistently confirmed severe oligohydramnios [7]. Parents need to be aware of this when termination is discussed as one of the management options in severe cases of PPROM. More recent studies have showed an improved neonatal outcome following the increased use of antenatal corticosteroids, improvements in antenatal care and monitoring, postnatal surfactant therapy, and general improvement in neonatal intensive care [8,9]. The risks to the infant following EPPROM are associated with the gestation at amniorhexis and gestation at delivery with added risks of infection, limb contractures, cord compression during labour, and pulmonary hypoplasia. These adverse sequelae combined with preterm birth impose a considerable burden on finite National Healthcare Service (NHS) resources. Assessments of the economic consequences following EPPROM, the risk of infection to both baby and mother, psychological impact on the parents, associated complications, and disability of the baby after delivery could provide an invaluable resource for clinical decision-makers and budgetary or service planners on the NHS.We review the clinical course, outcome, and the challenges to parents and health care professionals on three pregnancies complicated by EPPROM, the first published by Engemise et al. [10] occurring before 24 weeks’ gestation with a membrane rupture to delivery interval (latent period) of 14 days or more.A 34-year-old woman with severe Crohn’s disease, grade IV endometriosis, and bilateral tubal obstruction, booked for antenatal care at 13 weeks gestation. This was her first pregnancy following four attempts of in vitro fertilization and embryo transfer (IVF). Routine antenatal blood investigations were unremarkable. Her blood group was A-negative. Pregnancy was uncomplicated until 17 weeks gestation when she presented with spontaneous PPROM. This was confirmed by the presence of a pool of clear liquor in the vagina and a positive nitrazine test. High vaginal swab cultures for bacteria were negative. Ultrasound scans confirmed a singleton pregnancy with oligohydramnios. There was complete anhydramnion at 19 weeks gestation, and this rendered assessment of foetal anatomy difficult.The couple was counselled on the poor outcome and risks of infection to the mother and foetus but expressed the wish to continue with the pregnancy. The risks of significant perinatal mortality and neonatal morbidity associated with chronic anhydramnios and the poor outcome associated with extreme prematurity was fully discussed by the neonatal team. The risks were based on the risk of infection to both mother and the foetus, up to date evidence and the ultrasound findings.She was commenced on erythromycin 250 mg eight hourly, and managed expectantly as an outpatient with twice daily temperature checks at home, as well as serial full blood counts (FBC), serum C-reactive protein (CRP) and weekly low vaginal swabs. Two weekly growth scan showed a normally growing foetus, with visible breathing movements, and chest circumference growing along the 50th centile. Abdominal circumference (AC) measurement in foetuses with oligohydramnios may be technically difficult and less reproducible as the abdominal profile may be significantly deformed due to compression, thus, it is probably more reproducible to use ratios based upon head circumference rather than AC as the fetal head is more rigid, even in such conditions [11]. Liquor volume was never measurable due to continuous amniotic fluid leak and anhydramnios.She remained well until 24 weeks gestation when she was admitted into hospital following a painful antepartum bleed. There was no clinical or laboratory evidence of chorioamnionitis. She was managed conservatively, with bed rest in hospital, and prophylactic antibiotics. Two doses of 12 mg intra-muscular. Betamethasone were given at 24 weeks, 24 h apart, in order to facilitate foetal lung maturity, and minimise neonatal respiratory distress syndrome (NRDS). Anti D immunoglobulin was also administered to prevent rhesus isoimmunization. She remained in hospital and pregnancy continued largely uneventful until 28 weeks gestation when she had a major placenta abruption; associated with foetal heart decelerations on the cardiotocogram. A live male infant weighing 1100 g was delivered by emergency caesarean section, with an Apgar score of 4 at 1 min. He was electively intubated immediately and given a dose of surfactant. A diagnosis of pulmonary hypoplasia was made on the basis of immediate onset of severe respiratory distress syndrome (RDS) requiring high ventilator pressures (MAP = 18) and no improvement in oxygenation or lung compliance after two doses of surfactant. Plain chest X-ray showed small lung fields with elevated diaphragms and a bell shaped thorax; highly suggestive of the diagnosis of pulmonary hypoplasia.Apart from his compressed ears and mildly depressed tip of the nose, (mild degree of Potter’s features), there were no other gross skeletal deformities. The second day of life was complicated by pneumothorax probably secondary to the high-pressure ventilation. This resolved with no sequelae following chest tube drainage. He was extubated after 14 days of ventilation but required nasal continuous positive airway pressure ventilation for another 80 days. He tolerated bilateral inguinal herniotomy at the age of 130 days and was discharged home, self-ventilating in air and in good health; with a follow-up appointment for developmental assessment.A 33-year-old woman para 4 (two term and two preterm at 34 and 28 weeks) all normal deliveries, one miscarriage, and one termination booked for antenatal care at eight weeks and five days gestation. Routine antenatal blood investigations were unremarkable. Her blood group was A Rhesus positive. The patient developed a urinary tract infection at 15 weeks and three days and was treated with antibiotics. At 22 weeks of gestation she presented with a history suggestive of spontaneous PPROM. This was confirmed by the presence of trickling of clear liquor through the cervix on speculum examination. High vaginal swab cultures taking at the time for infection were negative. Ultrasound scans confirmed a singleton pregnancy with complete oligohydramnios which made assessment of foetal anatomy difficult.The couple was counselled on the outcome and risks of infection to the mother and foetus but expressed the wish to continue with the pregnancy. The risks of significant perinatal mortality and neonatal morbidity associated with chronic oligohydramnios and the outcome associated with extreme prematurity was also fully discussed with the couple.She was commenced on erythromycin 250 mg, six hourly, and managed expectantly as inpatient with twice daily temperature checks, twice weekly FBC and serum CRP. Two weekly growth scan showed a normal growing foetus, oligohydramnios with visible breathing movements, and chest and abdominal circumference growing along the 50th centile. Abdominal circumference (AC) measurement in foetuses with oligohydramnios may be technically difficult and less reproducible as the abdominal profile may be significantly deformed due to compression so it is probably more reproducible to use ratios based upon head circumference rather than AC as the fetal head is more rigid, even in such conditions [11].Two doses of 12 mg intra-muscular betamethasone were given 24 h apart at 23 weeks in order to facilitate foetal lung maturity, and minimise NRDS. Liquor volume was never measurable due to continuous amniotic fluid leak and anhydramnios. While in hospital at 25 weeks and six days gestation she developed mild lower abdominal pain with minimal vaginal bleeding with no clinical or laboratory evidence of chorioamnionitis. At 27 weeks and 6 days, the bleeding was severe with moderate to severe intermittent lower abdominal pain. On bimanual vaginal examination the presentation was breech and cervix was 9 cm dilated with adequate contraction.Cardiotocogram was normal and patient was delivered by breech extraction of a male infant weighing 1126 g with Apgar of 4, 7, and 10 at 1, 5, and 10 min respectively. He was electively intubated at 10 min of life and given a dose of surfactant and then transferred to the special baby care unit (SCBU).In SCBU he was treated for presumed sepsis and briefly ventilated for 10 h for RDS and required nasal continuous positive airway pressure (CPAP) for 29 days, and oxygen until day 41. Echocardiography revealed a small patent ductus arteriosus (PDA) and ultrasound of the liver showed an abnormality, which was presumed to be a haemangioma. The patient was treated with diuretics and for gastro-oesophageal reflux disease and was discharged home, self-ventilating on air after 69 days in SCBU weighing 2250 g; with a follow-up appointment for developmental assessment in six weeks.A 42-year-old A rhesus negative woman with minimal endometriosis, multiple uterine surgeries for removal of uterine fibroids, MTHFR—(methylene-tetra-hydro-folate-reductase) homozygous mutation with factor V Leiden deficiency booked for antenatal care at six weeks gestation following three embryo transfers. She was a para (Never carried a pregnancy beyond the viability stage is the 0 and the 3 is the three miscarriages as indicated) 0 + 3, three miscarriages with the fourth attempt of IVF. This was a triplet pregnancy with a monochorionic diamniotic (MCDA) twins’ and a singleton. The patient was placed on daily 60 mg of subcutaneous clexane and 75 mg of oral aspirin until 24 weeks gestation when her aspirin was stopped. Routine antenatal blood investigations were unremarkable.On routine scan at 15 weeks, the MCDA twins’ were found to have developed severe Twin-to-Twin Transfusion (TTTs) with a 17.3% growth discrepancy and a velamentous cord insertion of the recipient twin. The couple was counselled on the condition, as well as the chances of survival of the MCDA twins’ either by expectant management or surgery. The later was agreed and performed at 16 weeks gestation with laser ablation of the communicating vessels.At 18 weeks of gestation the patient presented into gynecology emergency clinic (GEAC) with history suggestive of PPROM, which was later confirmed by the presence of clear liquor in the vagina. High vaginal swab for cultures were negative for infection. Ultrasound scans confirmed a reduced liquor of maximum amniotic fluid index (AFI) of 3 cm in the MCDA donor twin, 11 cm of the recipient twin, and normal liquor volume in the singleton, with the later lying at the basal end of the uterus. Baseline FBC and CRP were taken and their results showed no sign of infection.The couple was counselled on the poor outcome and risks of infection to the mother and foetus but expressed their desire to continue with the pregnancy. The risks of significant perinatal mortality and neonatal morbidity associated with persistent reduced liquor and extreme prematurity were also discussed by the neonatal team.She was commenced on clindamycin 150 mg, six hourly throughout her stay in hospital, as she was allergy to penicillin, erythromycin, and metronidazole, and managed expectantly while in hospital as inpatient with routine daily vital signs, twice weekly FBC and serum CRP, as well as weekly low vaginal swabs. A weekly growth, liquor, and viability scan showed normal growing foetuses and maximum AFI depth of 3 cm in the MCDA twins with normal liquor in the singleton, with visible breathing movements and chest circumference of all three foetuses growing along the 50th centile.At 23 weeks and four days gestation, two doses of 12 mg intra-muscular betamethasone were given, 24 h apart, to facilitate foetal lung maturity and minimise NRDS. She also had anti D immunoglobulin administered to prevent rhesus isoimmunization. At 27 weeks and three days the vagina liquor was blood stained with intermittent abdominal pain and ultrasound scan revealed a minor placenta abruption. She remained in hospital and pregnancy continued until 28 weeks and five days gestation when she had an elective caesarean section for worsening vaginal bleeding and lower abdominal pain with the delivery of three live female infants weighing 1350 g, 1165 g, and 1038 g, respectively, with cord blood and base excess (Table 1). Her estimated blood loss (EBL) during the procedure was 2 L. They were electively intubated immediately and each given a dose of surfactant and transferred to SCBU.Birth weight and venous and arterial cord bloods of the triplets.She was the singleton of the triplet with Apgar score of 9 and 10, in 1 and 5 min, respectively, at delivery. She was immediately electively ventilated due to prematurity with a dose of surfactant given at 9 min of life and transferred to SCBU. In SCBU she was treated for presumed sepsis and developed RDS for possible pulmonary hypoplasia requiring CPAP and oxygen until day 85. She developed chronic lung disease (CLD) in the process and has been treated for gastro-oesophageal reflux disease (GORD). She also developed grade IV intraventricular haemorrhage on day eight and later developed a parencephalic cyst which did not progress to hydrocephalus during her stay in SCBU. Echocardiography revealed a small PDA, which had closed by the 21st day of life. She has been discharged home, self-ventilating on air after 98 days in SCBU, weighing 3.260 g; with a follow-up appointment for developmental assessment in six weeks.She was the MCDA recipient twin with triplet C with Apgar scores of 7 and 9, at 1 and 5 min, after delivery. She was immediately electively ventilated due to prematurity and given a dose of surfactant and then transfer to SCBU. In SCBU she was treated for presumed sepsis and developed RDS. However, she developed pulmonary hypertension and hypotension, which was not responding to maximal con ventilation and inotropes but had a good response with nitric oxide and high frequency ventilation following transfer to the tertiary centre. Following the RDS, she developed CLD and was treated with diuretics and erythromycin from days 47 to 57 and for GORD. She has been discharged home, self-ventilating on air from day 85 after 98 days in SCBU, weighing 3.210 g; with a follow-up appointment for developmental assessment in six weeks.She was the MCDA donor twin with triplet B with Apgar scores of 6 and 8, at 1 and 5 min, after delivery. As noticed above (Table 1) this triplet revealed signs of fetal distressed as evidence by the cord arterial PH. She was immediately electively ventilated due to prematurity and given a dose of surfactant and then transferred to the SCBU. In SCBU she was treated for presumed sepsis and developed RDS and possible pulmonary hypoplasia requiring CPAP and oxygen. She then developed CLD and was treated with diuretics and erythromycin from day 43 to 53 and for GORD. She was discharged home, self-ventilating on air from day 85 after 98 days in SCBU, weighing 3.082 g; with a follow-up appointment for developmental assessment in six weeks.EPPROM before 24 weeks occurs in less than 1% of pregnancies [12]. Most cases deliver spontaneously before one week [13,14] with expectant management not applicable. The optimal management of the remaining pregnancies is still controversial because pulmonary hypoplasia and extreme prematurity limit survival and increase perinatal morbidity. The decision to continue or terminate a pregnancy complicated by EPPROM prior to foetal viability is fraught with difficulties due to the medical, socioeconomics, and emotional problems associated with it. The adverse sequelae resulting from EPPROM impose a considerable burden on finite NHS resources and the country. Over the past few years, advances in neonatal intensive care have led to a dramatic increase in neonatal survival at early gestation; therefore termination of second trimester pre-viability amniorrhexis in a desperate family situation needs some consideration.Assessments of the economic consequences of preterm birth with parents’ involvement could provide an invaluable resource for clinical decision-makers and budgetary or service planners on the NHS. The risk of infection to both baby and mother, psychological impact on the parents’ lives and the associated complications and disability on the baby following delivery at extreme prematurity further weighs on the fragile economy system.The current financial constraints on NHS resources make it imperative to define better health care outcomes achieved and the costs of achieving these outcomes. In the last decade, there has been an increase in aggressive obstetric management and neonatal resuscitation for threatened preterm births [15]. According to American College of Obstetricians and Gynecologists (ACOG) there is sufficient evidence to inform parents that the survival rate for newborns increases from 0% at 21 weeks to 75% at 25 weeks, with or without major disability [16]. Associated costs impose a significant burden on multiple sectors of the US economy and include long-term hospital, outpatient medical, developmental, and educational expenses. A full characterization of all costs is necessary when evaluating the cost-effectiveness of preterm delivery, prevention therapies and when estimating the impact that increasing preterm birth rates and extreme preterm survivorship will have on future expenditures.Neonatal and postneonatal hospital and outpatient costs that are associated with preterm birth and, related, low birth weight, have been well characterized. Estimates of neonatal inpatient costs for children who are born preterm range from approximately $11,000 to $18,000 (2,003 dollars) per birth, compared with $1,300 to $1,900 (2,003 dollars) per term birth [17,18,19]. Rogowski et al., in [20], estimated the cost of rehospitalizations and outpatient care during the first year for preterm infants who are born <1500 g to be approximately $8,000 (1,987 dollars) per child. Lewett et al. [21] estimated that each low birth weight child costs an average of approximately $290 more than a higher birth weight child for inpatient medical care during the preschool years. These are even higher in pregnancies complicated by EPPROM prior to foetal viability, as these patients have extremely long hospital admission as in these cases with continuous use of the health care resources and expertise. Accommodation and other resources are allocated to the infants according to the need.Amongst infants with disability, mean health service costs for the entire follow-up period were estimated at £14,510 for the lowest birth weight group (<1000 g), £12,051 for the intermediate birth weight group (1000–1500 g) and £7,178 for the highest birth weight group (>1500 g). Relatively little is known about the economic impact of preterm birth outside of the health sector [22]. Moreover, there is no empirical evidence that focuses on the economic impact of extreme preterm birth, which is of increasing relevance in the modern perinatal care context.Counselling may produce confusion and ambiguity for parents. While this is a very real and stressful event for them, the physicians talks about hypothetical situations and uncertainty. The fear of what may happen to the baby in terms of morbidity and suffering is often mixed with a fear of losing the baby. As a result, many women will wax and wane on their decision to continue the pregnancy. Given the confusion and stress that parents may be experiencing there are many things that healthcare providers need to consider. First, it is the parents who will have to accept the ramifications of their decisions for the rest of their lives although the life time financial burden of a disable child rests in the nation’s economy. The medical and obstetrics history of the mother, as well as the risk of chorioamnionitis to the mother and the foetus, up to date literature evidence, and the ultrasound findings will help both the healthcare provider and the couple in deciding on either terminating or continuing the pregnancy.Taking these into consideration render the management of pregnancies complicated by EPPROM controversial and either option has risks for the mother and baby. Parents need to be counselled on the benefits and burdens either decision (termination or continuation) will have on their future life. This is so as the most critical interval in fetal lung development, the canalicular phase, which occurs between 16 and 28 weeks’ gestation, is delayed following EPPROM, thus, causing the likes of pulmonary hypoplasia. Pulmonary hypoplasia poses a serious threat to the live of the fetus with the mortality rate estimated to be 70% (55%–100%) [23]. The lethal form of pulmonary hypoplasia is only proven by autopsy as there is poor sensitivity and specificity of imaging techniques to predict this condition is poor [24]. Vergani, in 2012, proposed that the most accurate prediction of lethal pulmonary hypoplasia in utero might be achieved by different combinations of clinical, ultrasound, and MRI parameters as there is no single test to achieve that at the moment [25]. The spirituality and support from both the parents and family may also help them to cope at this difficult time [26]. In a study that explored the effects of high risk pregnancies on families, it was found that family support had a positive impact on the mothers’ ability to manage their current situation [26]. The fragility of the parent’s condition, both physiologically and psychologically at this point need’s consideration by the healthcare provider with the experienced team members giving the leading role. Despite the best care, adverse outcomes occur and should be anticipated by all involved with the possibility that the infant may die in utero or may not be viable at birth.Microbial invasion of the amniotic cavity is present in one third of patients with PPROM and is strongly associated with impending preterm delivery, adverse pregnancy, and neonatal outcome [27,28,29]. The risk of infection to the mother and the unborn baby should be anticipated with the babies more susceptible. This susceptibility is more pronounced in preterm babies who have been potentially exposed to maternal flora following a breach in the amniotic membrane secondary to prolonged PPROM. This has made the prognosis for a normal pregnancy where the membranes rupture at 14 weeks dismal primarily due to the risk of miscarriage secondary to infection. Even with appropriate antibiotic treatment, approximately 50% of pregnancies are delivered each subsequent week following PPROM. Therefore parent should be aware that, when the membranes rupture before 20 weeks of gestation the probability of reaching viability is <5% [30], hence, the need for good decision in this stressful situation.Chorioamnionitis, when present, regardless of its infectious aetiology, challenges the functional integrity of the membranes, making them vulnerable to other environmental insults with pathologic consequences during the pregnancy. Bacteria from the vagina can access foetal membranes by ascending the cervical canal and, then, infect amniotic fluid and foetal blood resulting in inflammation. This inflammation will cause the release of inflammatory markers like cytokines. Normal level of cytokines and their receptors, found in central nervous system cells, are important for brain development and function. They influence inflammatory response as well as neuron and glial cell development. Elevated levels of proinflammatory cytokines in amniotic fluid, cord blood, or neonatal blood indicate the presence of a systemic foetal inflammatory response. A persistent neuroinflammatory response may result when the inflammation signal is transmitted across the blood-brain barrier [31]. This is associated with intraventricular haemorrhage, white matter damage, and cerebral palsy [32,33]. When associated with hypoxic events, cytokines negatively affect functional outcome, such as early psychomotor development [34]. High concentrations of specific cytokines, such as interleukin-1β, interleukin-6, and interleukin-8, have been associated with abnormal neurodevelopment at 6, 12, and 30 months of age [35,36,37]. These cytokines are not routinely determined in patients with EPPROM as persistence high level in the maternal blood and amniotic fluid may indicate a possible abnormal psychomotor development in later life [35,36,37]. White matter injury appears to be the most common brain abnormality in preterm infants with PPROM, and a major predictor of smaller volumes along with gestational age [38].A second reason for dismal prognosis is the risk of neonatal death secondary to pulmonary hypoplasia when pregnancy becomes viable. The chance of pulmonary hypoplasia is lessened if the fluid re-accumulates before 24 weeks of gestation. One study using a multivariate analysis suggested that the likelihood for neonate survival increases by 2.7 (95% CI 1.45 to 4.65) for every 5-mm increase in the depth of amniotic fluid during the follow up from rupture up to the 24th week of gestation [39]. Despite dismal prognosis, however, expectant management for EPPROM at 14 weeks may be appropriate if the parents are well-informed and aware of the risks in both the mother and the unborn baby. In time of decision making the healthcare provider need to consider the emotional attachment to the unborn baby and the psychological impact this will have on the parents’ future lives.Preterm infants are at increased risk of other range of adverse neonatal outcomes including retinopathy of prematurity [40], necrotizing enterocolitis, and neonatal sepsis [41]. In later life, they are at increased risk of motor and sensory impairment [42,43], learning difficulties [44,45,46,47,48], and behavioural problems [49,50,51,52]. These should be borne in mind when counselling the couple as this helps them to make the right decision with no future regret.The maternal morbidity and mortality risk of chorioamnionitis, retained placenta, postpartum hemorrhage, placental abruption, and classical caesarean section also increases in this group of patients. These risks need to be explained to the parents with their complications (such as endoparametrities, septic pelvic thrombophlebitis, and the possibility of hysterectomy in very rare cases). The risk of chorioamnionitis is up to 30% in some studies [53,54], which may reflect to a more widespread use of antibiotics in recent years in this group of patient. The overall frequency of placental abruption of 4.0%–6.8% as reported by several authors [55,56,57,58], occurred in two of these patients. Scar dehiscence in future pregnancies following a classical caesarean need not be ignored.Our small series of three treated successfully cases without recourse to the complication associated with EPPROM may be the subject to publication bias and may not represent widespread medical opinion. This makes the acceptable outcome individually determined and parents should be aware that the mortality for infants with rupture of membranes prior to 25 weeks and a latency period of over 14 days with severe oligohydramnios is in excess of 90% in some studies [7]. The limit; expectancy; risks of infections to the mother, and chances of infant survival is always a consensus for debate in EPPROM. Thus, the clinical maternal evaluation and the fetal ultrasound assessment, as well as other risks, such as chorioamnionitis may provide important prognostic information for the clinicians and should be taken into account when counselling the patients so as to provide them with enough information to make decision of continuing or interrupting the pregnancy.The anticipated birth of an extremely premature infant also poses many challenges for the parents, government and health care professionals. As parents are faced with difficult decisions that can have a long-term impact on the infant and family, it is critical to provide the type of information and support that is needed by them. Taking all these into consideration parents should be given enough time to decide on making the right decision with the associated guidance of the healthcare provider without forgetting all the risks involved to both the mother and the premature infant.The authors declare no conflict of interest.

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+This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).Preservation of adult stem cells pools is critical for maintaining tissue homeostasis into old age. Exhaustion of adult stem cell pools as a result of deranged metabolic signaling, premature senescence as a response to oncogenic insults to the somatic genome, and other causes contribute to tissue degeneration with age. Both progeria, an extreme example of early-onset aging, and heritable longevity have provided avenues to study regulation of the aging program and its impact on adult stem cell compartments. In this review, we discuss recent findings concerning the effects of aging on stem cells, contributions of stem cells to age-related pathologies, examples of signaling pathways at work in these processes, and lessons about cellular aging gleaned from the development and refinement of cellular reprogramming technologies. We highlight emerging therapeutic approaches to manipulation of key signaling pathways corrupting or exhausting adult stem cells, as well as other approaches targeted at maintaining robust stem cell pools to extend not only lifespan but healthspan.Diseases of aging constitute a huge burden for society, both in terms of economic cost and quality life-years of the population. The need for therapies to prevent and/or correct age-related molecular events leading to these diseases is growing. These include metabolic syndrome and diabetes, atherosclerosis, neurodegenerative diseases, osteoporosis, and cancer (Table 1, [1]). Onset of these diseases is highly correlated to advancing age across organ systems. Though molecular mechanisms contributing to cancer formation have been extensively studied, the mechanisms underlying age-related disease on the whole have only begun to be elucidated [2]. Molecular changes associated with age include telomere dysfunction, oxidative stress and deranged mitochondrial metabolism, inflammation, and cellular senescence, as well as altered signaling of sirtuins, insulin/insulin-like growth factor-1 (IGF-1), and the mammalian target of rapamycin (mTOR) pathway [3,4,5].Conserved aging phenotypes. Reprinted by permission from Macmillan Publishers Ltd.: Nature [1], copyright 2008. A three-pronged approach exists to combatting diseases of aging in the clinic, and further research can improve all three areas. The first arm is prevention of age-related disease through better understanding of the molecular causes of systemic aging and age-related disease. The second is pharmacologic intervention to reverse, correct, or prevent age-related disease. The third, in lieu of therapies that prevent and correct age-related molecular changes, is to surgically repair degenerated tissues. This includes engineering cells and tissues ex vivo to replace or augment regeneration of those in diseased and injured solid organs, nervous system components, and musculoskeletal structures [6,7,8,9,10]. The use of adult stem cells for this third approach, as well as the suspected regenerative functions of adult stem cells in vivo, has led researchers to closely consider the effects of systemic aging on the stem cell pools of an individual. Stem or progenitor cells are generally considered highly promising candidate cells for regenerative applications not only because they possess a high proliferative capacity and the potential to differentiate into other cell types [11,12,13,14], but also because they can be sourced autologously, eliminating any concerns about rejection or need for immunosuppressive therapy [8,15,16,17,18,19]. They possess innate immunomodulatory properties, home to sites of injury or inflammation, and direct the cells around them to begin a repair process via the production of bioactive factors and signaling molecules. Stromal stem cells are currently in clinical use as gene delivery agents to enhance tissue regeneration, to destroy cancer cells, and to regenerate cartilage and bone, and hematopoietic stem cells (HSCs) have been in clinical use for many years to reconstitute the immune system in cancer and other illnesses. However, studies of adult stem cells show that they do not fully retain their proliferative and multi-lineage differentiative capabilities in aging humans or after prolonged ex vivo propagation. Genetically or epigenetically modifying adult stem cells either to rejuvenate those of an elderly individual or to confer resistance to cellular aging during ex vivo propagation would yield a favorable cell source for regenerative medicine applications. Understanding the events that contribute to stem cell aging and developing methods to reverse those changes will also facilitate development of therapies to maintain in vivo adult stem cell pools as people age.Adult stem cells are thought to reside in vivo as self-renewing pools and facilitate repair/replacement of damaged tissues over the lifespan of the organism. Stem cell quiescence lies on one end of a spectrum of self-renewal potential spanning from quiescence, to robust proliferation, to senescence, and death. Maintaining stem cell quiescence is essential for preserving the long-term self-renewal potential of the stem cell pool in a number of organ systems, such as the brain, bone marrow, musculoskeletal system, and skin [20,21]. There is an emerging body of evidence that altered and decreased function of adult stem cells in vivo secondary to accumulated metabolic stress plays an important role in the initiation of diseases of aging [22,23]. This is true in multiple organ systems. For example, in bone studies on the osteoblastic versus osteoclastic differentiation of progenitors in aging mouse models have shown that, over time, osteoblastic potential of stromal progenitors decreases, while osteoclastic differentiation of hematopoietic progenitors increases. This suggests an organismal aging program that results in common diseases of aging, including decreased bone quality [24]. Another example is in the immune system, where clonal diseases of myeloid stem cells occur more frequently and become more resistant to therapy with increasing age [25]. The hypothesis is now being investigated that this is caused by age-related genomic instability, causing a defective DNA damage response that results in abnormal differentiation of HSCs (reviewed in [26]). It would appear that the primary hit to adult stem cells during aging is to their proliferative/self-renewal potential more than their ability to undergo terminal differentiation effectively, although this is somewhat lineage-dependent. HSC populations in mice have been shown to actually increase in number and frequency with age, but with reduced ability to divide, delayed cell cycle progression, and age-related genetic changes in cell cycle regulators such as p21 and p18 [27]. In humans lower numbers of neuronal progenitor cells have been found in aged brains compared to young brains, but this population is still responsive and proliferates in response to ischemic injury [28]. Circulating hematopoietic progenitors were shown to increase more dramatically in younger patients after cardiopulmonary bypass graft than in older patients, and advanced age was associated with impaired coronary microvascular response to vascular endothelial growth factor (VEGF) [29]. Conversely, advanced age has been associated with a higher S-phase fraction of circulating HSCs in patients with aplastic anemia, but this predisposed them to dysplasia and conversion to acute myeloid leukemia, indicative of abnormal HSC function [30]. Studies of adult stem cell isolation yield in elderly individuals have shown that equivalent numbers of adipose-derived mesenchymal stem cells (MSCs) can be isolated from older individuals undergoing vascular surgical procedures as from young, healthy individuals [31,32]. The question remains whether those cells can be adequately expanded in tissue culture and whether they are able to mobilize, proliferate, and effect tissue repair in vivo when they are needed. In fact these same studies [31,32] have shown that while advanced age does not impact availability of stem cells, fat from patients with diabetes yielded significantly fewer stem cells than fat from non-diabetic patients. This would suggest that stem cells in the context of aging should probably be considered distinctly from stem cells in the context of diseases of aging. Advanced glycation endproducts, which accumulate in the setting of advanced age or diabetes, have been shown to directly impair HSC function and induce MSC apoptosis [33,34].Other studies have shown that patients of increasing age yield adipose-derived MSCs that can be differentiated. However, frequently these studies do not compare the quality of differentiation to that achieved with cells from younger patients, leaving the question of whether differentiation potential has declined unanswered [35]. In one study looking at the efficacy of MSC transplantation following myocardial infarction, cells from aged donors did not perform as well as cells from younger donors [36]. Similarly, MSCs obtained from young individuals have been induced to undergo neuroectodermal differentiation in vitro, but this effect cannot be replicated in MSCs from elderly individuals [37]. A study demonstrating that lineage fate of MSCs from human donors was unaffected by donor age also found that activation from quiescence, including replicative function and quality of differentiation, was negatively impacted by advanced donor age [38]. A further complication in teasing apart the effects of aging on adult stem cell compartments is the changing interaction between stem cell types and with an aging immune system (reviewed in [39]). For example, the health and age of marrow-derived stromal stem cells has been shown to have an impact on the quality of HSCs, both in vivo and upon co-culture ex vivo [40,41]. Chronic pro-inflammatory cues, such as circulating inflammatory cytokines, which are upregulated in aged individuals [42], may both dysregulate the differentiation of stromal cells, and in turn negatively impact their ability to support hematopoietic progenitors, resulting in further dysregulation of the immune compartment. Mouse models of premature aging have demonstrated induction of NF-κB signaling and secretion of high levels of pro-inflammatory cytokines as a causative factor in the accelerated aging phenotype [43].Network analyses of signaling pathways differentially regulated in aging suggest that, rather than being a tightly regulated, well-defined program, aging may reflect a destabilization of other programs over time. On the other hand, extreme differences in lifespan between evolutionarily closely related species would argue that there is a dominant central aging program that determines organismal lifespan. Research indicating that survival to old age is not correlated with absence of risk alleles for common age-related diseases, such as cancer, coronary artery disease, and type 2 diabetes also supports the idea of a prevailing aging program [44]. Similarly, gene expression profiling has been done in adult stem cells to examine the effects of age in the setting of osteoarthritis and vice versa, revealing that different sets of genes were differentially regulated in association with either aging or osteoarthritis [45,46]. The pathways associated with aging were closely associated with glycan metabolism, in contrast to osteoarthritis, which was heavily associated with aberrations in immune signaling genes and regulators of self-renewal and differentiation, such as Wnt-related transcripts. Another gene expression profiling study looking specifically in human skin showed sex-specific age-related changes, with females displaying increased expression of pro-inflammatory genes that was not observed in males [47]. A recent meta-analysis of genome-wide association studies performed to identify polymorphisms associated with diseases of aging revealed that genes associated with multiple diseases known to occur in elderly individuals are generally associated with pathways regulating either inflammation or cell senescence, with the most highly significant susceptibility locations mapping to regulators of senescence, leading the authors to conclude that germline genetic heterogeneity in regulators of these pathways contributes significantly to onset of age-related disease [3].Some of the most pertinent research to understanding the molecular mechanisms underlying aging, rather than the molecular effects resulting from aging, is in the area of heritable longevity and premature aging in humans. Many genetic variants, the value of which is unknown, have been identified in areas associated with longevity and disease resistance, including dietary restriction, metabolism, autophagy, stem cell activation, tumor suppression, DNA methylation, progeroid diseases, stress response, and neural processes [44,48]. One of these variants, a single nucleotide polymorphism (SNP) in the gene TOMM40, was found not to be directly linked to decreased longevity, but instead reflects a linkage disequilibrium with multiple isoforms of the APOE gene that are deleterious to longevity and have been strongly associated with elevated cholesterol, cardiovascular disease, Alzheimer’s disease, and cognitive decline and dementia, as well as serum IGF-1 levels in women [49]. Several genetic variants in the insulin/IGF-1 pathway have been associated with longevity or increased healthspan and include multiple SNPs from nine different genes along this signaling axis: AKT1, FOXO1A, FOXO3A, GHR, GHRHR, IGF1R, IGFBP3, IGFBP4, and PTEN. Indeed, common SNPs in AKT1 and FOXO3A have consistently been associated with longevity in three independent cohorts [50], as well as a SNP in the CAMKIV gene, which in vitro has been shown to activate AKT, SIRT1, and FOXO3A [44]. A SNP in the MNPP1 gene, which codes for an enzyme similar to phosphatase and tensin homolog (PTEN) that regulates intracellular levels of polyphosphates, critical for determining the rate of cell proliferation, has also been associated with longevity in meta-analyses of large-scale genome-wide association studies.Progeria, or premature aging, reflects an opposite outcome from long lifespan or long healthspan. Individuals with Hutchinson-Gilford progeria syndrome (HGPS), caused by a point mutation in LMNA, the gene for the lamin A nuclear envelope protein, experience premature aging as a result of nuclear defects that lead to impaired cell division and transcriptional deregulation (reviewed in [51]). This point mutation activates a cryptic splice donor site, leading to production of a dominant negative form of the lamin A protein which has been named progerin; this splice variant is also expressed at low levels in normal individuals, accumulates in some cell types with normal aging, and is expressed at higher levels in several human cancer cell lines [52]. Similarly, individuals with Werner’s syndrome, who display adult-onset progeria, have a defect in the WRNp protein, which is critical for DNA replication and repair. In both aging syndromes, telomere shortening and DNA damage synergistically destabilize the genome, leading to accelerated p53-dependent senescence and apoptosis; this phenotype has been rescued in experimental models by over-expression of hTERT or p53 inactivation [53,54,55], and this process has also been documented in normal human fibroblasts [56]. Increased rates of nuclear DNA damage in all cell types, in combination with impaired stem cell regeneration of damaged tissues, are thought to be directly responsible for the accelerated aging phenotype that is observed. As a result this disease has inspired the generation of several aging models, both transgenic animal models and in vitro systems employing induced pluripotent stem (iPS) cells derived from fibroblasts of HGPS patients [57,58,59,60,61,62,63,64]. From these models, it has been learned that high rates of cellular senescence and apoptosis due to increased nuclear DNA damage correlate very well with decreased lifespan, independent of increased rates of cancer, whereas models with comparatively low rates of cellular senescence and apoptosis display increased lifespan [55,65]. Comparison of tissue phenotypes observed in normal aging and HGPS suggests that lamin A may play an important role in sensing and transducing stress response signals critical for adult stem cell and niche maintenance in all individuals [66]. Progerin has been demonstrated to accumulate in MSCs, vascular smooth muscle cells, and fibroblasts, both in in vitro disease models and in human subjects, in association with disease and other signs of aging in the skin, musculoskeletal, and cardiovascular systems [59,67,68]. MSCs have been shown not only to be most susceptible to progerin accumulation and failed cell division, but also more susceptible to oxidative and other kinds of stress in the context of progerin accumulation both in vitro and in vivo. In the absence of normal lamin A or abundance of progerin, mild oxidative stress is sufficient to induce nuclear disorganization and premature senescence, confirming the importance of this protein for maintaining tolerance to reactive oxygen species [69]. These factors combine to effectively wipe out this adult stem cell pool in HPGS patients, leaving them with a critical deficit in tissue regeneration [59,70], and it is likely this same process plays a role in progressively declining MSC function with normal aging. A wholly separate but potentially instructive area of research involves those studies focused on ex vivo aging of adult stem cells. Ex vivo stem cell aging has been shown to be very similar to in vivo stem cell aging in rodent models, but this has not held true on a molecular level in every study done with human cells. For example, telomere shortening, which drives cellular senescence in cultured human cells, is not observed in rodent cells clearly undergoing replicative senescence [71]. Despite this finding, there does appear to be an association between ex vivo cell senescence and organismal lifespan, and studies of telomerase mutations in humans have revealed an association with diseases of aging in which tissue compartments require a high degree of cell self-renewal [72]. Similarly, short telomeres have been linked with some tissue-specific degenerative diseases, and telomere length is evaluated as a clinical parameter in determining therapeutic approaches (reviewed in [73]). Ablation of senescent cells in progeroid mice has been shown to delay or rescue the aging phenotype at the organismal level, implicating senescent cells in the pathogenesis of age-related disease in vivo [74]. Taken together, these findings suggest that the study of ex vivo senescence could yield information pertinent to in vivo aging. Examination of adult stem cell proliferation in ex vivo tissue culture have shown that MSC proliferation declines precipitously after repeated passaging [75]. Studies of differential gene expression between early and late passage MSCs showed progressive down-regulation of genes associated with self-renewal, such as OCT4, SOX2, and TERT and up-regulation of genes associated with osteogenic potential; this was accompanied by an increased propensity for spontaneous osteogenic differentiation and decreased proliferation over time [76]. The authors of this work noted a concomitant increase of epigenetic dysregulation of histone H3 acetylation in association with these differentially regulated genes, and correction of this dysregulation with fibroblast growth factor (FGF) administration during culture, resulting in promotion of proliferation and suppression of spontaneous osteogenesis.Adult stem cells experience many stressful insults in the course of a lifetime of tissue repair. Regulation of energy metabolism is critical to withstanding stress, which comes in the form of nutrient deprivation, oxidative stress, DNA damage, pathogens, and other stressors. Studies on the molecular causes/effects of aging in adult stem cells have shown that in aged subjects these cells display an altered proteome, with proteins involved in cytoskeletal organization and anti-oxidant defense being age-dependent and associated with functional impairment of the cell, including decreased responsiveness to physical environmental cues and decreased resistance to oxidative stress [77]. Mesenchymal stem cells (MSCs) from both bone marrow and adipose tissue have been shown to have reduced capacity for oxidative stress with increasing donor age [78,79,80]. Studies in patients undergoing percutaneous coronary intervention after myocardical infarction have shown that self-renewal capacity and therapeutic efficacy of autologous bone marrow-derived MSCs can be correlated with blood gas levels in the marrow niche. This indicates that the function of these cells is highly dependent on their redox status [81]. Oxidative stress is increasingly being recognized as a fundamental underlying component of the aging process, leading to hyperactivity of pro-growth pathways like insulin/IGF-1 and mTOR, subsequent accumulation of toxic aggregates and cellular debris, and ultimately activation of cell death/survival pathways leading to apoptosis, necrosis, or autophagy (reviewed in [82]). Insulin/IGF-1, mTOR, FoxO, AMP-activated protein kinase (AMPK), and the sirtuin pathways all play a role in stem cell maintenance and differentiation through their sensing and regulation of energy availability in times of stress (reviewed in [83,84,85]), and these same pathways have been associated with advancing age in humans (Figure 1, [86]). Studies of two independent cohorts testing the expression of mTOR-related transcripts in aging found robust associations for genes involved in insulin signaling (PTEN, PI3K, PDK1), ribosomal biogenesis (S6K), lipid metabolism (SREBF1), cellular apoptosis (SGK1), angiogenesis (VEGFB), insulin production and sensitivity (FOXO), cellular stress response (HIF1A) and cytoskeletal remodeling (PKC), all of which were negatively correlated with advancing age, and for genes involved in inhibition of ribosomal components (4EBP1) and inflammatory mediators (STAT3), which were positively correlated with advancing age [87].Environmental cues and evolutionarily conserved pathways that regulate the aging process in diverse eukaryotic phyla. Reprinted by permission from Macmillan Publishers Ltd.: Cell Death and Differentiation [86], copyright 2008.In response to sublethal stress-induced insults, cells must remove or repair damaged components in order to reestablish homeostasis. Autophagy is one of the processes by which cells accomplish stress-induced metabolic adaption, and it has been identified as a critical mechanism for maintenance of stem cell function with aging [88]. Basal levels of autophagy are higher in adult stem cells from many tissue types compared to terminally differentiated cells, and autophagy is down-regulated during differentiation of adult stem cells. mTORC1, AMPK, and the sirtuins have all been shown to differentially regulate autophagy in response to nutrient stress, suggesting one possible connection between starvation and resistance to aging (reviewed in [89]). The sirtuins are a family of nicotinamide adenine dinucleotide (NAD+)-dependent deacetylases which are critical for maintaining cellular homeostasis in the face of age-related metabolic and other stressors, helping to prevent diseases of aging, but they are not thought to be involved in regulation of organismal lifespan (reviewed in [90]). Sirt1 coordinates stress responses and cell metabolism and regulates replicative senescence, is found in much higher levels in stem cells than differentiated cells, and is down-regulated upon differentiation of stem cells [91]. HSCs are dependent upon Sirt1 for maintenance of their undifferentiated state through elimination of reactive oxygen species, FoxO activation, and p53 inhibition [92]. In the case of embryonic development or tissue revascularization following ischemic injury, Sirt1 promotes endothelial progenitor branching and proliferation, although it is not required for endothelial cell differentiation [84]. These effects of Sirt1 are a result of its negative regulation of downstream effectors such as FoxO and Notch proteins. Resveratrol, a known Sirt1 agonist, has been shown to enhance osteogenic differentiation over adipogenic differentiation of MSCs, thereby conferring bone-protective effects and highlighting the importance of Sirt1 and its downstream target FoxO3 in preventing age-related osteoporosis [93]. Sirt1 confers sensitivity to insulin when over-expressed, and has been shown to be significantly down-regulated in cells resistant to insulin [94]. Given the critical function of this enzyme for maintaining robust adult stem cell pools and regulating their differentiation in multiple organ systems, its down-regulation in the context of insulin resistance provides one clue as to why metabolic disease is so damaging for regenerative processes in aging individuals.Regulation of organismal longevity is coordinated through many intersecting signaling pathways that maintain a tight balance between carcinogenesis and apoptosis in individual cells [95,96]. For a stem cell, which over the course of its existence travels on the self-renewal spectrum from unlimited proliferative potential to senescence and ultimately death, many of these pathways are at work in opposition to each other all the time (Figure 2, [97]). A number of the well characterized pathways that control cell proliferation in cancer are now being examined for their role in regulating stem cell renewal and aging. One group took advantage of this similarity in regulatory networks between cancer cells and stem cells to study the effect of anti-aging reagents on induction and maintenance of self-renewal behavior and underlying mechanisms in stable cancer cell lines. They found that BMI1, a well-known proto-oncogene and critical regulator of self-renewal in multiple adult stem cells populations, took over the epigenetic program in cells retrogressing to a more primitive state as a result of the anti-aging treatments [98]. This proto-oncogene has also been identified as a critical promoter of osteogenesis through its coordinated stimulation of SIRT1 expression and inhibition of p16, p19, and p27 in response to pararthyroid hormone related peptide (PTHrP) signaling [99], resulting in enhanced proliferation, decreased apoptosis, and decreased adipogenic differentiation of MSCs [100]. (a) PTEN is active in quiescent long term-HSCs and represses protein kinase B (PKB) signaling towards downstream components (such as mTOR and FoxO). Active FoxO programs cells to remain quiescent by cell-cycle repression and other mechanisms, but also allows survival by switching to a metabolic program of gluconeogenesis and fatty acid metabolism, together with elimination of reactive oxygen species (ROS). Ataxia telangiectasia mutated (ATM) may function in conjunction with FoxO through an as yet undefined mechanism, probably involving regulation of ATM expression by FoxO. Proteins active in LT-HSCs are shown in white elipses; (b) HSCs are driven to proliferate after loss of PTEN or FoxO. PKB and other downstream phosphoinositide 3-kinase (PI(3)K) events are active in this situation (indicated in white elipses). Loss of FoxO results in increased intracellular ROS levels, which in turn activates p38. If PI(3)K signaling is required to drive LT-HSCs into proliferation under normal conditions, what external niche signals would do so are unknown. However, PI(3)K signaling may function in cooperation with other signaling pathways, and this is illustrated by c-Myc—a downstream target of Wnt signaling. Myc represses FoxO, and may also independently regulate control of proliferation; (c) Differentiation of lineage-restricted cells further continues and is guided by extracellular signals such as interleukins (ILs) and various colony stimulating factor (CSFs); (d) After executing their function, all hematopoietic cells die and regeneration begins. Modified with permission from Macmillan Publishers Ltd.: Nature Cell Biology [97], copyright 2007.Ultimately aging at the cellular level is suspected to result from a disruption of the balance between alternative cellular states (reviewed in [101]), with proto-oncogenes that promote stem cell function, such as BMI1 and Wnt/β-catenin, operating in opposition to tumor suppressor genes that induce death or senescence in stem cells, such as INK4A (p16Ink4a) and ARF (p19Arf) [102]. In the hematopoietic system, aging has been closely linked with impaired repair response to DNA damage, leading to increased propensity for dysplastic syndromes and ultimately cancer [26]. The occurrence of cellular senescence, in contrast to quiescence or proliferation, is thought to be a protective response against oncogenic insults. Expression of INK4A has been shown to increase with age, and this progressive increase in tumor suppressor activity independent of levels of proto-oncogene expression may account for reduced stem cell activity with aging [102]. The tumor suppressor AIMP3/p18, endogenous levels of which increase in aged human tissues, drives cells to senescence when overexpressed. In transgenic mice p18 promotes a progeroid phenotype through selective degradation of normal lamin A [103]. A frequently raised concern about the therapeutic use of autologous adult stem cells from aged individuals is precisely that with the life-long accumulation of potentially mutagenic insults to their DNA, if they are still capable of robust self-renewal, they might pose an increased risk for cancer upon mobilization or exogenous activation [104]. That said, several studies have indicated that over the course of human development, as the need for growth decreases and the risk of oncogenesis increases, requirements for critical tumor suppressor mechanisms change, with adult stem cells displaying dependence on self-renewal regulatory signaling pathways that are not necessary in embryonic or even fetal stem cells [105].Just like the constant struggle between carcinogenesis and apoptosis at the cellular level, growth versus somatic preservation is also balanced at the organismal level throughout life. One emerging hypothesis of aging is that it reflects tissue dysfunction due to hypertrophy and hyperplasia, rather than tissue damage [106], plus senescence resulting from prolonged hypertrophic arrest [107]. Insulin/IGF-1 signaling, turned on at the systemic level in response to glucose or growth hormone, is a potent stimulator of cell growth and proliferation via the Akt-TOR pathway, regulating organismal growth in childhood and anabolic metabolism in adulthood. As discussed above, gene variants along this pathway have been associated with longevity (age ≥92 years) in clinical cohorts [50]. Interestingly, reduced IGF-1 levels are present not only in extremely long-lived individuals, but also in progeroid individuals. This illustrates that suppression of this axis is not a causative factor in increasing or decreasing lifespan and healthspan, but rather an adaptive response against accumulating DNA damage at the expense of growth and other metabolically expensive processes [108,109,110]. Adult stem cells are highly responsive to insulin/IGF-1 signaling and programmed to replicate and repair; both metabolically costly activities. As somatic DNA is exposed to a lifetime of potentially mutagenic hits, these metabolically active cells are increasingly caught in the balance between cancer and metabolic disease, the hallmark of which is insulin resistance. Pharmacologic agents targeting the insulin/IGF-1 axis—both neutralizing monoclonal antibodies against IGF-1/IGF-1R and tyrosine kinase inhibitors which target the insulin receptor and IGF-1R—have been developed to treat cancer, but in clinical trials to evaluate these agents, a common side effect was hyperglycemia due to inhibition of insulin signaling [111]. Similarly, metformin, an agent already in use for the treatment of type 2 diabetes, has been shown to reduce the incidence of cancer, in part through AMPK-dependent inhibition of mTOR (thus cell growth) and AMPK-independent cell cycle arrest [112], but also in part through decreased levels of insulin and insulin resistance (reviewed in [113,114,115]). Manipulating this pathway to combat disease in aging humans is fraught with complications, and creative, highly specific approaches are required to avoid trading one disease for another. This is especially important to consider in the case of stem cells. Chemotherapy-resistant cancer stem cells, characterized by a high degree of metabolic flexibility, have been shown to be very sensitive to metformin [116,117]. One of the proposed mechanisms of action of metformin on cancer stem cells is interference with TGFβ-induced epithelial-to-mesenchymal transition [118], and metformin has been shown to prevent transcriptional activation of OCT4 through AMPK activation [119]. These properties of metformin should generate concern about the effects on adult stem cell populations during its use for treatment of diabetes and cancer, but studies have also shown that while metformin-induced AMPK activation interferes with mechanisms critical for cancer stem-cell related tumorigenesis, adult stem cells may be less susceptible to disruption by metformin [120]. Accumulating evidence suggests that pathways governing self-renewal have distinct effects on normal stem cells and cancer stem cells even within the same tissue. In their study demonstrating adult stem cell dependence on PTEN in the hematopoietic compartment, in contrast to cancer stem cells, Yilmaz et al. [121] discussed several mechanisms through which maintenance of normal adult stem cells may be different from that of cancer stem cells. They suggested that persistent activation of PI(3)K in the absence of PTEN inhibition may lead to the accelerated exit of normal HSCs from the progenitor pool [121], and later showed that PTEN deficiency induces senescence and apoptosis in normal HSCs via increased expression of the cell cycle-regulating tumor suppressors p16 and p53 [122], in contrast to other cells from the hematopoietic compartment. Effects of PTEN deficiency on the HSC pool could be rescued by rapamycin, indicating events downstream of mTOR are responsible, for example changes in Akt signaling. It is known that mTOR inhibition activates FoxO signaling, resulting in increased stress resistance and longevity in invertebrate models [123]. It was hypothesized that prolonged rapamycin treatment might actually be inhibiting Akt signaling through mTORC2 rather than activating Akt through mTORC1, leading to loss of FoxO function, an attractive explanation for the accelerated stem cell aging observed with PTEN deficiency [124]. However, thus far this has not been shown to be the case in PTEN-deficient HSCs [105,122]. Cancer stem cells are able to escape this process through secondary mutations that attenuate mTOR-dependent tumor suppressive mechanisms [122]. Similar results have been obtained in HSCs with deletion of the cell cycle regulator p21, showing that control of cell cycle entry under conditions of stress is crucial for maintenance of stem cell quiescence and prevention of premature deletion of an entire adult stem cell pool [125]. Indeed, even in the case of pluripotent cells, metformin appears to have split effects: When administered to mice after iPS cell transplantation, metformin prevented teratoma formation but did not interfere with tissue formation from all three germ layers [126]. The findings are controversial regarding the effect of metformin on the differentiation of adult stem cells. In one study on rat marrow-derived MSCs, metformin enhanced osteogenesis at the expense of adipogenesis, presumably through modulation of peroxisome proliferator-activated receptor (PPAR)γ activity [127], opposite of the effect observed with glitazones, which activate PPARγ and can lead to bone loss [128]. However in another study of human and rabbit MSCs, metformin did not induce osteogenesis, while 5-aminoimidazole-4-carboxyamide ribonucleoside (AICAR), a small molecule activator of AMPK, induced robust osteogenic differentiation even in the absence of induction medium [129]. In studies of primary osteoblasts, activation of AMPK signaling was observed during early differentiation events, but chemical induction of AMPK with metformin blocked terminal differentiation and matrix mineralization [130]. Even in the absence of pharmacologic intervention targeting these pathways, metabolic disease is frequently the trade-off associated with oncogenic resistance. Perturbation of the aforementioned lifespan determinant pathways, such as SIRT1, insulin/IGF-1, FoxO and mTOR, leads to the development of metabolic syndrome features in mice [131]. Metabolic syndrome—characterized by the triumvirate of high cholesterol, high blood pressure, and high fasting blood glucose—and type 2 diabetes in turn lead to accelerated aging. In the case of full-blown type 2 diabetes, this accelerated aging is evidenced at the cellular level by slower DNA unwinding, increased collagen cross-linking, capillary basement membrane thickening, increased oxidative damage, and decreased Na+K+-ATPase activity, and at the organismal level by an increased incidence of cataracts, vascular disease and associated events (myocardial infarction, stroke, and pressure ulcers), cognitive decline, hip fracture, pain, incontinence, infections, and depression [132].A vicious cycle then evolves whereupon metabolic disease can in turn indirectly increase oxidative stress and associated dysregulation of adult stem cell function [133]. An expanded adipose compartment produces higher levels of free radicals, leading to oxidative stress, one of the effects of which is to disrupt adipocytokine production. Adiponectin, an adipocytokine down-regulated in obesity and metabolic syndrome, is an important regulator of glucose and fatty acid metabolism, and in combination with other adipose-derived hormones, such as leptin, prevents insulin resistance (reviewed in [134]). One of the critical functions of adiponectin is to oppose the actions of angiotensin II, local (adipose) over-production of which also contributes to a pro-inflammatory state and increases oxidative stress in vivo (reviewed in [135]). The resultant disruption to homeostasis has many downstream effects that further increase inflammation and co-opt adult stem cells in worsening the situation. First, trafficking of multiple types of adult stem cells is likely altered in response to the inflammatory adipocytokines (IFNα, TNFα, IL-6) up-regulated during this process, leading some to speculate about adult stem cell exhaustion and the resulting impairment of tissue repair as the primary mechanism underlying long-term effects of metabolic disease, and in a less fulminant way the aging process in general [23]. Additionally, fate of adult stem cells is differentially regulated in this environment. For example, enhanced production in metabolic syndrome of 20-hydroxy-5,8,11,14-eicosatetraenoic acid (20-HETE) by the cytochrome P450 system and its cyclooxygenase-2-derived product 20-OH-PGE2 act to bias MSCs toward adipogenic differentiation through up-regulation of PPARγ and β-catenin, resulting in compounded inflammation-driven adipogenesis and impaired peripheral tissue maintenance through loss of otherwise uncommitted progenitors in patients with these disorders [136,137,138]. Activation of PPARγ has been shown to impair IGF-1 signaling in the marrow microenvironment [138], further contributing to skeletal loss, disruption of metabolic homeostasis, and potentially altering organismal lifespan [139]. In this way adult stem cells contribute to the pathogenesis of metabolic disease and also are impaired in their physiologic function by the presence of metabolic disease.Despite the occurrence of insulin resistance and other attempts by cells to thwart oncogenic transformation in an aging metabolic system, metabolic disease is frequently associated with a higher incidence of cancers [140], particularly in sites with a high degree of cell metabolism and/or turnover. In part this reflects not a causal relationship but twin manifestations of a stressed system struggling and failing to restore homeostasis. However, the peripheral insulin resistance of metabolic disease also drives cancer growth through a decrease in hormone binding globulins (thus higher free steroid hormone levels), dysregulation of inflammatory cytokine and steroid and peptide hormone levels, and most importantly compensatory hyperinsulinemia, with many cancer cell types expressing high levels of insulin receptors and IGF receptors [141]. Large chromosomal clonal mosaic events, the incidence of which has been shown to increase with age [142], have been associated both with type 2 diabetes and with an increased risk of blood and solid organ cancers [143,144]. Clonal mosaicism in the blood compartment in particular further contributes to cancer formation, as well as increased susceptibility to disease in general, by leading to a reduced number of immune cell clones in circulation and resultant immunosenescence with age [142].As the MSC pool is skewed away from an osteogenic fate toward an adipogenic one in metabolic disease, there is massive disruption of signaling pathways that have implications for adult stem cells beyond differentiation. One of these pathways is the Wnt pathway, which is not only critical during development for axial patterning, but is also critical in stem cell fate determination. Many developmental events in stem cells are regulated by Wnt signaling, including self-renewal, differentiation, aging [145,146], and senescence [147,148,149]. Extensive crosstalk has been documented between Wnt signaling and FGF, prostaglandin E2 (PGE2), bone morphogenetic protein (BMP), Notch [150,151], TGF-β, and SMAD signaling pathways, with the common downstream target of this crosstalk being β-catenin [152,153]. In canonical Wnt signaling, β-catenin interacts with members of the T cell factor/lymphoid enhancing factor (TCF/LEF) transcription factor family to enhance expression of their target genes [146], which in turn regulate cell proliferation, carcinogenesis, differentiation, embryonic patterning, and stem cell maintenance [154,155,156]. The effects of Wnt signaling, however, have been shown to be highly tissue-specific and Wnt-specific. In the case of hair follicle stem cells in the skin, signaling through Wnt1 activated stem cell hyperproliferation via an mTOR-dependent mechanism, but long-term this activation of mTOR led to stem cell exhaustion and senescence [147]. This finding led the authors to conclude that while Wnt signaling can be a potent stimulus for stem cell proliferation, prolonged mTOR activation may serve as a protective mechanism to prevent tumor formation. The cost of this, of course, is exhaustion and depletion of that stem cell pool, ultimately resulting in impaired regeneration and aging of the tissue. In patients with acute myeloid leukemia, aberrant Wnt/β-catenin signaling, which controls self-renewal in the HSC pool, was higher in patients with unfavorable karyotypes and predicted a shortened survival [157].In HGPS the tissue-specific patterns of accelerated aging point to a defect in MSC function. The defect in this adult stem cell compartment has been shown to be the result of both impaired self-renewal and dysregulated differentiation resulting from aberrant Notch and Wnt signaling [158]. Disruption of the nuclear lamina by progerin was shown to be directly responsible for downstream activation of Notch signaling effectors, spurring uncontrolled sporadic differentiation of MSCs along all three germ layers and enhanced osteogenesis at the expense of adipogenesis when differentiation was directed [159]. Wnt signaling was found to be severely disrupted in the progeroid Zmpste24−/− mouse model (deletion of this enzyme causes restrictive dermopathy in humans), where the absence of normal lamin A resulted in an absence of active nuclear β-catenin in follicular stem cells, leading to down-regulation of cyclin D1 and repression of Akt and mTOR activation [160]. Further work has shown that LEF1 is down-regulated as a result of this impaired nuclear translocation/retention of β-catenin; the absence of this transcription factor-activator complex in adult cells markedly reduced activation of canonical Wnt targets [161]. In this setting of severe Wnt inhibition, stem cells were not reduced in number, but instead entered a senescent state earlier and failed to proliferate, resulting in exhaustion of the functional stem cell compartment. The authors of this study also discovered increased apoptosis of the support cells in the stem cell microenvironment, which they suggested is another negative impact of defects in critical fate-determining signaling pathways that enable communication between tissue-resident stem cells and their niches.Another mouse model of accelerated aging also points to aberrant Wnt signaling as a causative factor in degeneration due to stem cell defects, but from a different perspective. The Klotho mouse, which lacks klotho, a transmembrane and secreted β-glucuronidase involved in regulating insulin sensitivity among other functions, displays an accelerated aging phenotype, including short lifespan, infertility, arteriosclerosis, skin atrophy, osteoporosis, and emphysema [162]. Analysis of tissue-resident stem cells from multiple organs in Klotho mice revealed that they were reduced in number and displayed abundant senescence-associated markers prematurely [163]. Klotho was found to be a secreted Wnt antagonist capable of binding Wnts1, 3, 4, and 5a, and over-activation of Wnt signaling in Klotho mice drove tissue-resident stem cells into an early senescent phenotype, resulting in lack of self-renewal and stem cell compartment exhaustion. Taken together with findings regarding aberrant Wnt signaling in the Zmpste24−/− mouse, these studies reveal the exquisite sensitivity of adult stem cell pools to the fate-determining effects of Wnt signaling, too much or too little of which results in failed maintenance of quiescent progenitors in adulthood. Perturbation of this pathway one way or the other has been demonstrated to result in adult stem cell aging and exhaustion, along with impaired differentiation, in the muscle compartment [164], the hematopoietic compartment [165,166], the vasculature [161], and the skeletal system [161,167] in addition to the skin, the gut, and the kidney. It is suspected that these processes unfold in normal aging as well as accelerated aging phenotypes, especially given the accumulation of low levels of progerin over time in normal individuals and declining serum levels of klotho in human aging [168].Another mechanism leading to altered Wnt signaling in aging is a shift in β-catenin binding to favor FoxO transcription factor signaling over the canonical Wnt pathway, mediated by TCF/LEF transcription factor signaling, in response to increasing oxidative stress. The FoxOs are a family of transcription factors regulating several of the previously discussed intersecting pathways, and have been shown to coordinate cell response in tumor suppression, metabolism, and organismal longevity (reviewed in [2,169,170]). In this capacity FoxO signaling acts to resist cellular stress opposite the growth-promoting signaling of mTOR [171]. FoxO transcription factors are downstream targets of insulin, growth factors, and nutrient and oxidative stress stimuli and in turn regulate several fundamental processes, depending on the cell type, including gluconeogenesis, neuropeptide secretion, cell cycle arrest, atrophy, autophagy, apoptosis, and stress resistance [169,172,173] (Figure 3, [173]). Furthermore, the FoxOs are an interesting family of transcription factors in that several are ubiquitously expressed, but display both highly specialized and universal functions in distinct cell types, as well as distinct and redundant functions which can be attributed to different FoxOs within the same cell type [2].Roles of FoxO transcription factors in cells and in the organism. FoxO transcription factors trigger a variety of cellular processes by upregulating a series of target genes (in italics). The cellular responses elicited by FoxO affect a variety of organismal processes, including tumor suppression, longevity, development and metabolism. Reprinted by permission from Macmillan Publishers Ltd.: Oncogene [173], copyright 2008.Studies on the effect of oxidative stress on adult stem cells suggest that it drives a shift in binding of the cellular β-catenin pool from the TCF/LEF family to the FoxO family [2,145,174]. As a cofactor in FoxO-mediated transcription, β-catenin facilitates defense against oxidative and other cellular stresses [175,176,177]. Diversion of β-catenin binding from TCF/LEF to FoxO, combined with inhibitory feedback to β-catenin/TCF signaling, serves to diminish canonical Wnt signaling [145,146,178,179]. This redirection of β-catenin function secondary to oxidative stress and resulting change in the cell’s transcriptional program have been implicated in several aging-related disease processes and cell senescence [97,146,149]. This transcriptional shift is particularly important in osteoporosis, where canonical Wnt signaling in osteoblasts is critical for skeletal homeostasis. This is one example of how increased activation of FoxO signaling by oxidative stress accumulated with age serves to directly undermine maintenance of an entire organ system, independent of the more broadly discussed “oxidative hypothesis of senescence”, wherein reactive oxygen species drive cells into arrest by activating FoxO signaling.FoxO1 is of particular importance in age-associated skeletal disease. Osteoporosis, for example, is characterized by decreasing bone mass, which is attributed both to declining numbers of osteoblasts and declining function of osteoblasts. FoxO1 is critical for regulating osteoblast proliferation in the face of age-related oxidative stress and declining resistance to the effects of oxidative stress, both through its regulation of protein synthesis via interaction with the osteoblast-specific transcription factor ATF4 and through suppression of stress-induced p53 signaling which would otherwise lead to cell cycle arrest [180].Several FoxOs regulate organismal metabolic function and thus play a role in metabolic diseases of aging and resistance to those diseases. FoxO1, through its regulation of osteocalcin secretion by osteoblasts, impacts pancreatic β-cell proliferation, insulin secretion, and insulin sensitivity [181]. In the case of atherosclerosis and vascular diseases of aging, FoxOs are important downstream effectors of PI(3)K, AMPK, and c-Jun N-terminal kinase (JNK) signaling. In the endothelium FoxO1 serves as a negative regulator of angiogenic behavior; this suppressive function, which is enhanced by activities of FoxO3 and FoxO4, is critical for organized vessel growth during development and repair [84]. FoxO1, FoxO3, and FoxO4 are also required to maintain endothelial quiescence within healthy vessels, and formation and remodeling of the endothelial barrier function is regulated by FoxO1 in a β-catenin-dependent manner.FoxO transcription factors also play a role in attenuating diseases of aging through their regulation of the immune system and its progenitor pools (reviewed in [182]). In general, FoxOs regulate survival, cell cycle progression, and resistance to stress in immune progenitors and differentiated immune cells, as they do in many other cells types, consistent with a decline in overall immune function and proliferative capacity of the HSC pool with aging. FoxO transcription factors also regulate immune activity via specialized functions in different cell types. FoxO1 specifically regulates development and trafficking of B and T lymphocytes through several mechanisms, including survival and homing of pre-B cell and naive T cells in response to growth factor receptor regulation, pre-B cell maturation by induction of Rag genes and B cell receptor recombination, and B cell class-switch recombination and somatic hypermutation in response to germinal center formation. FoxO3 has been extensively studied in the immune system, and regulates not only lymphocyte function, but also innate immunity. Like FoxO1, FoxO3 regulates T and B lymphocyte survival and cell cycle progression, but FoxO3 additionally controls survival and entry of memory T cells into a quiescent state, critical for later response to infection. This particular function of FoxO3 is of great interest in the human immunodeficiency virus (HIV) field, as inhibitors of FoxO3 might be used to prolong survival of memory T cells during chronic HIV infection. FoxO3 also has similar roles in B cells, and is likely important for terminating immune responses to infection, and possibly for controlling lymphocyte responses that would result in autoimmunity. FoxO3 also has critical specific regulatory functions in innate immune cells: Controlling the number of neutrophils, monocyte/macrophages, dendritic cells (DCs), and erythrocyte progenitors (in opposition to erythropoietin signaling); directing neutrophil migration; and regulating inflammatory cytokine secretion by DCs in response to coinhibitory molecules [2,182]. Immune-specific functions of FoxO transcription factors, including the examples outlined here, are not well understood in the context of aging and increased metabolic stress, but may also contribute to the overall decline of cell-specific immunity observed in elderly individuals, including decreased maintenance of the HSC pool, increased susceptibility to infection, reactivation of latent viruses, and decreased immune surveillance with respect to cancer.In addition to their place on a spectrum of proliferative capacity, stem cells also exist on a spectrum of differentiation bounded by terminally differentiated unipotent effector cells at one extreme and pluripotent embryonic stem cells (ESCs) at the other [183]. Pluripotent cells, which can differentiate to any cell in the body, are thought to be extremely rare in adult mammals. Much work has investigated directed epigenetic manipulation of cell fate, inducing a cell to follow a completely different transcriptional program and as a result shift to an entirely different phenotype and spectrum of activity. The recent development of nuclear reprogramming methods used to generate iPS cells has created new opportunities for regenerative medicine using stem cells, but the mechanisms underpinning cell reprogramming remain incompletely understood, and many areas where stem cell manipulation can enhance regenerative medicine have yet to be explored [184,185]. Introduction of a cocktail of pluripotency-maintaining transcription factors, likely in combination with a series of stochastic epigenetic events, can direct terminally differentiated cells to revert to a state similar to that of an ESC [184,186,187,188,189], resulting in iPS cells that are pluripotent and germ-line competent, and exhibit the capacity for chimerism and teratoma formation and a gene expression profile characteristic of ESCs [190]. The exact nature of the pluripotency induction steps that take place during and subsequent to the expression of the exogenously transduced reprogramming transcription factors is an active area of research. Whether the two key “stemness” features of iPS cells, i.e., proliferative capacity and multi-lineage differentiation potency, arise from specific epigenetic events during the reprogramming process and result from the synergistic action of more than one of the reprogramming factors is unknown. Full reprogramming, i.e., the production of iPS cells, requires a minimal period of expression of these reprogramming transcription factors; this period was discovered by Yamanaka’s group to be significantly longer for induction of pluripotency in human cells compared to mouse cells, taking approximately three weeks [189]. Recent studies have also focused on the screening of small molecules capable of reprogramming [191,192], with one group achieving successful reprogramming using a combination of seven compounds [193]. However, iPS cells do not make ideal starting material for regenerative medicine or cell therapy [194]. Like ESCs, it is technically challenging to direct them to undergo exclusive differentiation along a specific cellular lineage, and they exhibit a shift in the self-renewal spectrum that confers a high risk of carcinogenesis, frequently forming tumors in animals [190,195]. It is possible that in the future we may be able to achieve partial reprogramming, resulting in the acquisition of renewed proliferative capacity and an increased differentiation lineage potential, but without other characteristics of ESCs and fully reprogrammed iPS cells, such as the capacity for chimerism and teratoma formation.Partial reprogramming is the process of moving an adult cell on the spectrum of differentiation from limited multipotency toward pluripotency, without returning it to the completely pluripotent state of an ESC. Several groups are engaged in studying how partial reprogramming can most effectively be induced in adult stem cells, how it alters the transcriptional program and phenotype of adult stem cells, and how this approach may be used to preserve the potency, proliferative capacity, and regenerative utility of adult stem cells as they are cultured in vitro (reviewed in [196]). Observations from Yamanaka’s work in mouse cells suggests that selection based on expression of FBX15 yields partially reprogrammed iPS cells [197]. The definition of partial reprogramming described in that work was that partially reprogrammed iPS cells formed teratomas but lacked the ability to generate adult chimeric mice. A lesser degree of partial reprogramming has been described in umbilical cord blood cells cultured in medium supplemented with FGF4, SCF, and FLT-3 ligand [198]. These cells exhibited increased binding of acetylated histones H3 and H4 at the OCT4 promoter and upregulation of OCT4 and Nanog expression, but their reprogramming was considered a partial event because they exhibited DNA hypermethylation in the OCT4 gene region, and continued H3 and H4 acetylation at promoter regions for markers of terminal differentiation. Other studies have achieved partial reprogramming by administration of growth factors or transcription factors to redirect a non-pluripotent progenitor cell to a pluripotent phenotype [198,199,200]. Growth factor-induced partial reprogramming has been used to enhance plasticity in peripheral blood monocytes and subsequently to convert them to immature β endocrine cells [199]. During the observed limited life span of increased plasticity, these cells exhibited up-regulation of pluripotency markers. Recently some groups have undertaken to “directly reprogram” or transdifferentiate cells from one terminally differentiated phenotype to another using both developmental and lineage-specific transcription factors for therapeutic application in specific organ systems [201,202,203,204,205,206,207,208,209]. Partial reprogramming is a potentially promising approach to confer some of the desirable properties of ESCs onto adult stem cells or terminally differentiated effector cells, but it is evident that controlling partial reprogramming and resulting changes in potency requires a more complete understanding of underlying regulatory mechanisms. Discovery of mechanisms by which reprogramming events redefine the transcriptional program in adult cells, particularly signaling related not only to potency, but to telomere maintenance, oxidative stress, and senescence, will aid in generating techniques to increase the longevity of the adult stem cell in culture and preserve those cells in vivo [210,211,212,213,214,215]. Regulation of stem cell pluripotency and differentiation has been studied at the transcriptional and epigenetic level in ESCs, particularly mouse ESCs [216,217,218,219,220,221,222,223]. High-throughput sequencing methodologies are now used to characterize whole networks of regulation in ESCs [224,225] and analyze the roles of overlapping and interactive regulatory networks in determining stem cell fate, including the role of microRNAs [226] and epigenetic marks (reviewed in [227]). Regulatory networks in reprogrammed cells are also now being studied using genome-wide analytical tools, and initial results from studies of iPS cells derived from aged individuals suggests that reprogramming can undo many, though not all, effects of age (reviewed in [228]). SIRT1, critical for maintenance of stemness in multiple types of adult stem cells, is post-transcriptionally up-regulated during the reprogramming process [91]. Reprogramming of aged HSCs to iPS cells with subsequent re-derivation of HSCs showed comparable function to endogenous blastocyst-derived HSCs in marrow reconstitution assays [229]. Perhaps the most critical lesson regarding stem cell aging and loss of self-renewal gleaned from reprogramming research has been that cell aging as we know it is a largely reversible process, characterized not by permanent genetic mutations so much as progressive epigenetic inflexibility. Studies of transcriptional reprogramming efficiency have proven very instructive in the area of methods for enhancing cell stemness and overcoming senescence. Several pathways controlling onset of cellular senescence must be differentially regulated to achieve reprogramming, including telomerase, p53, and mitochondrial/oxidative stress pathways [230]. Telomere length as a measure of cellular aging has revealed interesting differences between reprogrammed pluripotent cells and their embryonic counterparts. Many widely used human iPS cell lines derived from somatic cells display prematurely aged telomeres compared to hESCs with accompanying differential regulation of genes regulating telomere length. iPS cell clones derived from an hESC-derived mortal clone (for isogenic comparison) largely followed the same pattern with the exception of one clone spontaneously displaying levels of telomerase activity comparable to the parent hESC line, with maintenance of longer telomere length in culture [231]. From this finding we have learned that current reprogramming methods do not always result in iPS cells where the aging process has been fully reversed, but that further—likely stochastic—epigenetic events can enable full reversal of cell aging. Discovery of those specific events that result in maintenance of long telomeres is a relatively focused research problem that is likely solvable with the massive generation of transcriptional network data currently underway. Comparative studies of reprogramming in aged cells from multiple organs in mice have thus far demonstrated that age is an impediment to efficient reprogramming. However, many groups have successfully generated bona fide iPS cells from somatic cells of aged human subjects [228], and with ever-improving techniques have even demonstrated comparable reprogramming efficiency in fibroblasts from young versus old patients [232].With respect to oxidative stress and mitochondrial function, the observation has been made that iPS cells rely on a Warburg-type switch to glycolytic metabolism. During reprogramming of fibroblasts to iPS cells, repression of H+-ATPase and up-regulation of the lipogenic enzymes acetyl-CoA carboxylase and fatty acid synthase is observed, as is the case in cells from many types of cancer, and inhibition of these lipogenic enzymes greatly decreases reprogramming efficiency [233]. Studies of mitochondria within human iPS cells have revealed that they revert to an immature state similar to those of an ESC, complete with reduced oxidative damage, contributing significantly to rejuvenation of the cell [228,230]. Pharmacologic induction of autophagy has also been shown to enhance reprogramming efficiency, perhaps through elimination of older, damaged mitochondria [88]. Metformin, an AMPK activator, has been shown to decrease reprogramming efficiency in multiple studies [119], despite the fact that AMPK activation induces endogenous antioxidant expression and reduces intracellular reactive oxygen species [234]. When activated, AMPK, which functions as a master sensor and regulator of intracellular changes in energy status, prevents transcriptional activation of OCT4 (though not other reprogramming transcription factors) and prevents somatic cells from making the energetic switch to glycolysis, thereby effectively blocking reprogramming [119]. This is highly instructive for two reasons. First, the malignant component of teratomas derived from implanted iPS cells are driven by OCT4, and application of metformin to iPS cells (after reprogramming) has been used to suppress or block entirely the formation of iPS-derived teratomas [126], suggesting that this well characterized FDA-approved drug might enable clinical application of iPS cells without risk of carcinoma. Second, studies on the effects of metformin and other AMPK activators such as 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) on the reprogramming process have illuminated a critical path to achieving pluripotency: appropriation of energetic capital. It has long been known that many types of stem cells are able to survive in harsh, energetically unfavorable conditions such as hypoxia because of their ability to rely heavily on glycolysis (provided they are not calorically restricted), but the discovery that a particular metabolic phenotype is required for supporting the energetic requirements of the reprogramming process has resulted in the understanding that being able to readily shift to a glycolytic metabolic phenotype is a defining property of stem cells. The implications of these findings to the study of stem cells in aging are enormous, because manipulating the metabolic phenotype of a cell as a strategy to restore its function is an approachable problem.Ascorbate, a potent antioxidant, has been shown to accelerate the kinetics of reprogramming and to alleviate cell senescence by reducing levels of p53 [235]. Curcumin, another antioxidant, has been shown to have similar effects on reprogramming efficiency [236]. It is possible that ascorbate enhances reprogramming in part through reduction of reactive oxygen species, but more likely by increasing the rate of transcriptome changes through other mechanisms: It is a cofactor for several enzymes, including collagen prolyl hydroxylases, HIF (hypoxia-inducible factor) prolyl hydroxylases, and histone demethylases [237], and may facilitate histone demethylation. Epigenetic modifiers, such as valproic acid, have been shown to enhance reprogramming efficiency, either alone or in combination with antioxidants. This further supports the idea that enabling histone demethylation confers epigenetic flexibility and enhances the ability of the cell to dramatically shift its transcriptional program [235]. Regulation of senescence and metabolic state through the mTOR hub seems to be of particular importance during reprogramming and is a pathway that can be fine-tuned to direct cell fate (reviewed in [238]). mTOR inhibitors, such as rapamycin and resveratrol (which is also a sirtuin activator), are known to slow cellular senescence in response to DNA damage by limiting the accumulation of p16 and p21, thereby enabling entry into a reversible quiescent state rather than an irreversible senescent state. These same compounds have been shown to increase the efficiency of reprogramming, in addition to other sirtuin activators, antioxidants, autophagy inducers, and PI(3)K inhibitors [236]. Interestingly, although it enhances reprogramming of normal somatic cells, resveratrol inhibits the stemness, epithelial-mesenchymal transition, and metabolic reprogramming of cancer stem cells to glycolysis through activation of p53, again highlighting the innate differences between the molecular circuitry of normal stem cells and cancer stem cells, a finding that can potentially be exploited for therapeutic purposes [239].Given the growing evidence that many diseases of aging may reflect adult stem cell exhaustion, it is not surprising there is great interest in restoring adult stem cell function to ameliorate these conditions and regenerate aged tissues [23]. Adoptive transfer of fetal MSCs into adult mice has been shown to extend median lifespan of the animals [240]. Adult stem cell mobilization and transplant are two obvious strategies that have achieved moderate success for certain types of injury and disease in humans, and many types of adult stem cells have been utilized for this purpose [241]. MSC cellular therapy has proven to be safe for a number of vascular disorders, such as coronary artery disease, peripheral vascular disease, erectile dysfunction, and stroke, and is an attractive option for patients who are poor surgical candidates [242,243,244,245,246]. Despite these successes, the problem remains that adult stem cells from elderly donors, the very people who most frequently require enhanced peripheral stem cell function for tissue repair, undergo changes in their functional capacity as a result of aging (reviewed in [104]). This decline in functional capacity, therefore therapeutic utility, has been combatted using some surprisingly simple interventions: Conditioning with hypoxia prior to transplant, for example, has been extensively documented as effective for reducing reactive oxygen species production by adult stem cells and improving their therapeutic efficacy in many in vivo ischemia and other disease models [247,248,249]. This has proven sufficient to counteract the impaired oxidative stress resistance of MSCs from elderly donors [78]. Likewise, the use of naturally occurring antioxidant polyphenols, such as curcumin, has been documented to suppress inducible oxidative stress in human MSCs ex vivo and may prove to be a safe method for reducing oxidative damage to the in vivo MSC pool [250]. Rejuvenation of aged human MSCs has been achieved by seeding cell scaffolds with proangiogenic growth factors, resulting in improved functional capacity of the aged cells after implantation into an infarcted rat heart compared to aged cells seeded on untreated scaffolds [251]. Systemic administration of growth factors has also proven effective for restoring aged MSCs in vivo; in the case of senile osteoporosis, intraperitoneal injections of rhBMP2 were sufficient to reverse the osteoporotic phenotype, and this effect was mediated by an expanded MSC pool displaying increased proliferation and decreased apoptosis [252]. Ex vivo genetic modification has also been used to overexpress rejuvenating factors in aged bone marrow- and adipose-derived MSCs prior to therapeutic delivery. Transplantation of aged MSCs overexpressing telomerase and/or myocardin was more efficacious in stimulating arteriogenesis and blood flow in a limb ischemia model than transplantation of control aged MSCs [253]. A similar study achieved increased angiogenesis and less adverse matrix remodeling in a rat model of myocardial infarction using aged MSCs transfected with TIMP3 or  VEGF [254]. The idea has been raised that it might be possible to exploit reprogramming techniques for renewal of the in vivo stem cell pool to combat diseases of aging [255]. While full reprogramming of stem cells in vivo to restore tissues degenerated as a result of age is not likely to manifest clinically until highly efficient reprogramming can be achieved through delivery mechanisms other than lentiviral vectors, the idea of “direct reprogramming” of cell fate in specific tissues in vivo has been pursued using developmental regulators that redirect a cell’s terminally differentiated state rather than returning the cell to a pluripotent state—what is known in the adult stem cell world as transdifferentiation, as opposed to dedifferentiation. This approach has been employed successfully to convert pancreatic exocrine cells to endocrine cells, rescuing the hyperglycemic phenotype in a mouse model of diabetes [256]. Generation of stem cells resistant to the phenotypic changes that accompany replicative senescence, such as arrested proliferation and decreased differentiation potential, would create a more ideal cell type for use in stem cell-based tissue engineering and cell therapy. Ex vivo reprogramming to achieve a kind of cell “reset” may in the future yield this improved cell source. iPS cells generated from HGPS patient fibroblasts display no evidence of progerin accumulation, nuclear envelope and epigenetic defects, or accelerated aging, suggesting this approach can in fact be used to reset an aged cell [58]. In the case of HGPS-iPS cells, differentiation results in the rapid accumulation of progerin and restoration of the accelerated aging phenotype [257]. However, this would not be an issue with physiologically aged donor cells. iPS cells derived from young and old non-progeroid human fibroblasts displayed no differences in mitotic activity after differentiation back to a fibroblast phenotype, suggesting that reprogramming is a successful approach to reset aged cells to a youthful phenotype in physiologically aged donors. In this study an excisable vector was used, further illustrating what might be a feasible approach to ex vivo rejuvenation of aged cells [232].Stem cell rejuvenation techniques are also needed in situations where it is preferable to use cells from a specific donor who happens to be of advanced age, since HSC donor age is correlated with adverse events after infusion [258]. Meeting this need is critical for transplants with autologous or human leukocyte antigen (HLA)-matched sibling HSCs from elderly donors, which result in better outcomes in leukemia and lymphoma than HSCs from an HLA-matched unrelated younger donor [259]. Several groups have investigated the use of reprogramming transcription factors to restore differentiation potential and proliferative capacity of adult stem cells from aging donors. In one such study, Nanog was over-expressed in adult marrow-derived MSCs, resulting in reversal of lost myogenic differentiation potential and enhancement of proliferation comparable to that observed in neonatal marrow-derived MSCs [260]. It remains to be seen if other approaches to dedifferentiation will restore an unblemished phenotype to cells to the same degree that reprogramming appears to. Calorie restriction as a therapeutic intervention to delay aging and extend lifespan has been extensively studied in animal models but, at levels that would confer significant clinical benefit, is unlikely to gain much traction due to low rates of adherence. Pharmacologic agents to reduce nutrient intake or absorption might be employed to this end (reviewed in [261]). The effects of diet and exercise to reduce body weight and correct metabolic disease on adult stem cell populations are unknown, although reduction in visceral fat has been shown to correct endocrine functions of adipocytes. Enhancement of PPARδ signaling has been suggested as an adjunct therapy to boost catabolism in visceral adipose tissue, perhaps in part through differentiation of adipose-resident MSCs to mitochondria-enriched small adipocytes [261]. To this end PPARδ agonists have been tested in clinical trials, but despite protective effects against obesity and diabetes, development was discontinued due to multi-organ cancer formation in animal models [262,263]. Other studies have investigated the use of pharmacologic agents to mimic the molecular benefits of calorie restriction for extending lifespan and healthspan (Figure 4, [264]). A recent report described extension of lifespan and healthspan in male mice with administration of metformin beginning in middle age [265]; past work has established this same phenomenon in invertebrates [266]. At the cellular level, treated mice displayed increased AMPK activity, decreased oxidative damage, and a transcriptomic shift mimicking the effects of calorie restriction. As a result the mice maintained sensitivity to insulin and low levels of systemic inflammation into old age. It should be noted that the dose of metformin used to achieve these effects resulted in serum drug levels an order of magnitude higher than what is typically achieved in patients when the drug is used as an antidiabetic therapy; a ten-fold higher dose proved toxic rather than beneficial in this study. As is the case for many pathways regulating longevity, cellular aging, and oncogenic resistance, the degree to which AMPK signaling is altered is likely to require a fine balance between too much and too little. This, combined with concerns about the pleiotropic effects of metformin in vivo which manifest differently with short-term versus long-term use, means that significant work is still needed before this potentially attractive therapy for systemic anti-aging can be safely employed.The growth hormone (GH)—insulin-like growth factor I (IGF1) pathway and its signaling cascade, which involves PI(3)K and Akt, can modulate longevity and cancer in model systems. Calorific restriction inhibits GH-IGF1 signaling and can also inhibit mTOR and activate AMP-activated protein kinase (AMPK) and sirtuin 1 (SIRT1). Interactions between components of these pathways, and with SIRT1, remain incompletely understood. The role of SIRT1 in modulating mammalian ageing has not been demonstrated, and it seems to have a dual role in cancer. DNA repair systems and DNA damage checkpoints prevent the DNA damage accumulation that contributes to cancer and ageing, although possibly through different cellular mechanisms. Reprinted by permission from Macmillan Publishers Ltd.: Nature Reviews Cancer [264], copyright 2013.The emerging field of translational epigenetics is aimed at correcting heritable but potentially reversible “epimutations” with chemical modifiers, and is gaining some traction in diseases of aging such as metabolic syndrome (reviewed in [267]) and cancer (reviewed in [268]) because it offers the possibility of targeting some of the same cell processes as transcriptional reprogramming without the associated risks of introducing exogenous genetic material. The treatment of epigenetically disrupted stem cells in cancer in particular may yield tremendous clinical benefit as the number of epigenetic modifiers grows, allowing for more specific targeting of known associated epimutations. In the area of metabolic disease, epigenetic modification with nucleic acids or small molecules may allow for extending the healthspan if not the lifespan of patients. The demethylase UTX-1, the activity of which increases in mid-life, removes gene inactivating marks such as histone H3 trimethylation on lysine 27 (H3K27me3) on members of the insulin/IGF-1 signaling pathway, enhancing their activity and resulting in decreased FoxO activity and age-related cellular decline. Restoration of H3K4me3 on an insulin-like receptor gene in C. elegans has been shown to decrease insulin/IGF-1 signaling, resetting the cell to a more naive epigenetic state and ultimately extending the life of the animal by 30% [139]. Although this effect was achieved through the use of RNA interference, the authors expressed optimism that in the near future small molecules might be employed to target epigenetic marks and/or modifying enzymes in a similarly specific strategy. Similarly, the histone methyltransferase SUV39H1 is protected from proteasomal degradation by enhanced binding to progerin in Zmpste24−/− mice, resulting in increased H3K9me3 levels and compromised genome maintenance, which leads to accelerated senescence [269]. Targeting of SUV39H1 in this study resulted in amelioration of the progeroid phenotype in Zmpste24−/− mice, including reduction of bone loss and extension of lifespan by 60%, suggesting a similar strategy might be useful in the context of normal aging. In an analogous approach, H4K16 hypoacetylation was targeted in the same mouse model to ameliorate the progeroid phenotype, with overexpression of the histone acetyltransferase Mof or addition of the histone deacetylase inhibitor sodium butyrate to drinking water promoting repair of damaged DNA and resulting in reduced evidence of disease [270]. Hypoacetylation of this mark was also found in aged wild-type mice, suggesting that aberrant histone acetylation may play a role in physiologic aging and administration of histone deacetylase (HDAC) inhibitors may have therapeutic value in disease of aging.Genetic modification strategies have specifically targeted known regulators of senescence and lifespan to combat diseases of aging. Preventing senescence, clearing senescent cells, or interfering with the senescence-associated secretory phenotype, in which cells release inflammatory mediators such as cytokines and matrix metalloproteinases, are all approaches that might lessen the contribution of cellular aging to chronic illness [271]. Given the complexity of the signaling crosstalk regulating senescence and associated events, identification of therapeutically targetable elements in this network—the “senectome”—is proceeding at multiple levels, the most recent of which includes senescence-associated micro-RNAs, which could be manipulated or used as clinical biomarkers [272]. In non-healing diabetic skin ulcers, siRNA- or vivo-Morpholino antisense-based gene therapy targeting of CAV1 or PTRF, which are both turned on by oxidative stress in diabetic fibroblasts and induce p53-dependent premature senescence, inhibited senescence and accelerated ulcer repair [273]. Atherosclerosis, especially in the context of type 2 diabetes, is related to endothelial senescence and has been reduced using a variety of interventions targeting nitric oxide levels and bioavailability in the endothelial microenvironment, including eNOS gene therapy [274]. Gene therapy to induce telomerase activity in CD8 T cells, which undergo premature senescence in the context of HIV infection, results in enhanced proliferation and increased antiviral function [275]. While preliminary research in this area has not resulted in karyotypic changes or wildly altered growth kinetics of the CD8 compartment, the authors of this study emphasized the need for pharmacologic approaches that would mimic these effects without the need for TERT gene therapy due to the obvious risks. The use of pharmacologic agents to modulate senescence-associated pathways is a promising avenue to counteract the effects of aging in the clinic. Mice of both genders treated with rapamycin starting in mid to late life display extended lifespan and reduced incidence of cancer [276]. Treatment of cells from HGPS patients with rapamycin, results in enhanced clearance of the mutant protein progerin by autophagy and delayed onset of senescence [277]. Conversely, activation of Akt-mTOR signaling through inhibition of isoprenylcysteine carboxyl methyltransferase, the enzyme which processes prelamin A to lamin A and enables trafficking to the nuclear rim, in Zmpste24−/− mice also delayed onset of senescence and improved disease phenotype. This suggests the Akt-mTOR axis, like the Wnt axis, is finely tuned and must be carefully manipulated to achieve therapeutic benefit [278]. Rapamycin is unlikely to be utilized extensively as an anti-aging therapeutic due to its side effects, which include hyperlipidemia and immunosuppression; however, newer analogs of rapamycin (rapalogs) are in development and may find use as anti-aging compounds, along with other agents that inhibit mTOR (reviewed in [279]). Pharmacologic activation of SIRT1 in a rat model of diabetes restored endothelial differentiation, pro-angiogenic chemokine secretion, and in vivo angiogenic activity of bone marrow-derived early outgrowth cells to that of cells from control animals [280]. Pharmacologic blockade of angiotensin II signaling through its type I receptor, which is used clinically to lower blood pressure and prevent insulin resistance in metabolic syndrome, also inhibits adipogenesis in adipose- and bone marrow-derived MSCs, both preventing further pathologic expansion of adipose tissue and helping to maintain an uncommitted progenitor pool for tissue homeostasis and repair [281]. In opposition to the enhanced production of 20-HETE by the cytochrome P450 system observed in metabolic syndrome, MSCs generate P450-derived epoxyeicosatrienoic acids (EETs) from arachidonic acids, and when administered exogenously these lipid mediators have been shown to decrease adipocyte differentiation of MSCs via an increase in heme oxygenase-1 and decrease in PPARγ, C/EBPα, and Fas and to reprogram adipocyte stem cells to a new phenotype displaying a smaller cell size, increased secretion of adiponectin, and decreased secretion of inflammatory cytokines [282]. EET agonists have also been shown to reverse a metabolic syndrome phenotype in an obese animal model, highlighting the therapeutic potential of targeting production of these molecules in the adult stem cell pool to combat this age-related disease phenotype in humans [283].Given the complex and sometimes unpredictable nature of these emerging pharmaceutical and genetic approaches to age-related disease therapy, sometimes the simplest approaches to maintaining health are best. In the lifelong struggle between growth-promoting signaling pathways and stress resistance pathways, sleep has a critical place in determining the balance [171]. mTOR and FoxO signaling are turned on in distinct temporal windows during early and late sleep, respectively, in response to alterations in somatotrophic signaling, suggesting that a good night’s sleep truly does have restorative powers. Adult stem cells have been demonstrated to undergo significant circadian regulation in multiple studies, with HSCs, marrow- and adipose-derived MSCs, and cancer stem cells all subject to transcriptome modulation by core circadian regulatory proteins (reviewed in [284]). ESCs, in contrast to adult stem cells, are not subject to circadian regulation and have been shown to acquire molecular circadian oscillation upon differentiation; subsequent transcriptional reprogramming with Sox2, Klf4, Oct3/4, and c-Myc genes was shown to suppress circadian cycling, literally resetting the internal clock in the resulting iPS cells [285]. Interestingly, action of core circadian regulatory proteins on physiologic cellular processes is opposed by SIRT1 with aging (reviewed in [286]), and control of central circadian cycling by SIRT1 in the brain decays over time [287]. In general circadian rhythms break down with age, coinciding with the development of metabolic derangements and potentially contributing significantly to the organismal aging process (reviewed in [288]). Research into the impact of macro-environmental factors, such as organismal circadian rhythms, on stem cell niches may open new therapeutic avenues for manipulating stem cell fate in diseases of aging [289]. As therapeutic approaches go, good sleep hygiene is safe and free from side effects, cost-effective, and potentially contributes more than we currently know to the long-term maintenance of adult stem cell compartments.Adult stem cells serve to replenish and direct repair at sites of tissue injury throughout the body, and exhaustion of dysfunction of an adult stem cell population in vivo with age results in degenerative disease. Several finely tuned and contextually regulated pathways coordinate the activities of tissue-resident adult stem cell pools over time in response to a host of cellular stressors in an effort to maintain the balance between growth-promoting function and oncogenic resistance. Manipulation of one or more of these pathways has the potential to prevent or reverse the impact of advancing age on adult stem cell function, but is fraught with the difficulty of tipping the balance toward metabolic derangement, or more likely toward cancer formation. Harvest and manipulation of adult stem cells ex vivo for use in regenerative medicine is a piecemeal approach to addressing systemic age-related chronic illnesses, but for now may prove to be a safer approach. In this regard, it is noteworthy that the clinical safety of HSCs and MSCs has been well documented, not in the least on the basis of decades of successful clinical outcomes of heterologous bone marrow transplantation. Further development of therapeutic approaches to maintain these cells in vivo requires that the mechanistic basis of their age-related degeneration or renewal be understood. This is an area continually being informed by studies of early-onset aging syndromes and of families exhibiting extreme longevity. Transcriptional reprogramming, which effectively wipes away all signs of age from most cell types, is also yielding valuable insights into what makes a cell young or old. Rejuvenating stem cells to stave off aging safely will require highly innovative approaches, but the results of this research will have far-reaching implications for regenerative medicine. This work was supported in part by Commonwealth of Pennsylvania Department of Health. The authors declare no conflict of interest.

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+This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).Bipolar disorder is a severe affective disorder which can present in adolescence, or sometimes earlier, and often requires a pharmacotherapeutic approach. The phenomenology of bipolar disorder in children and adolescents appears to differ from that of adult patients, prompting the need for specific pharmacotherapy guidelines for long-term management in this patient population. Current treatment guidelines were mainly developed based on evidence from studies in adult patients, highlighting the requirement for further research into the pharmacotherapy of children and adolescents with bipolar disorder. This review compares and critically analyzes the available guidelines, discussing the recommended medication classes, their mechanisms of action, side effect profiles and evidence base.Bipolar disorder (BD), previously known as manic depression, is a complex psychiatric illness characterized by alternating episodes of mania and depression. Manic symptoms commonly include racing thoughts, delusions of self-grandeur, partaking in risky behaviors and reduced need for sleep. In contrast, during depressive periods, patients commonly experience reduced mood, changes in appetite, irritability and anhedonia. BD is considered to be one of the most disabling psychiatric disorders, associated with a suicide rate approximately 20–30 times that of the general population [1]. Overall lifetime prevalence is thought to be around 4% [2], with typical onset occurring during late adolescence or early adulthood. Prevalence of BD in children and adolescents alone is thought to be around 2% [3,4]; however there is controversy surrounding the diagnosis of BD in this patient population. Although there is no clear consensus as to how many types of BD exist even in adult patients, current classification systems acknowledge the existence of two BD subtypes [5]. BD type I is characterized by the presence of one or more manic episodes, whilst depressive or hypomanic episodes occur frequently but are not required for diagnosis. In BD type II there are no manic episodes, but one or more hypomanic episodes and one or more major depressive episodes. Hypomanic episodes do not go to the full extremes of mania (i.e., do not usually cause severe social or occupational impairment, and are not associated with psychotic features).The first controversial issue is about diagnostic criteria, as not all professionals agree on the usefulness of the criteria set out in the Diagnostic and Statistical Manual of Mental Disorders (DSM) [5]. Specifically, it has been suggested that symptoms of mania presenting in childhood are not easily distinguishable from those of attention deficit hyperactivity disorder (ADHD) and other neuropsychiatric disorders [6]. With regard to BD subtypes, there is reasonable agreement surrounding the diagnosis of BD type I in children and adolescents, however there is doubt concerning the validity of a broad diagnosis of bipolar spectrum disorder. In the United Kingdom (UK), the National Institute for Health and Care Excellence (NICE) [7] suggested that a broader bipolar diagnosis could be unreliable and therefore not useful. This is somewhat in contrast with the prevalent view in the United States, where other BD diagnoses, such as BD type II and BD-not otherwise specified, are widely accepted and in recent years there has seen a 40-fold increase [8]. The exact reason for this phenomenon is yet unknown; possible explanations include the wider application of the diagnostic criteria to children and adolescents, and previous under-diagnosis of the condition [9]. In the absence of accepted diagnostic criteria specific to children and adolescents, in pediatric practice BD is generally diagnosed based on adult criteria. In DSM-5, bipolar and related disorders are given a chapter on their own (between depressive disorders and schizophrenia spectrum disorders), where bipolar-like phenomena that do not fulfill the diagnostic criteria for BD type I, II or cyclothymic disorder (i.e., short-duration hypomanic episodes and major depressive episodes, hypomanic episodes with insufficient symptoms and major depressive episodes, hypomanic episode without prior major depressive episode, and short-duration cyclothymia) are summarized under the label “other specified bipolar and related disorders”. In this new edition of the DSM, the symptoms which have to be present to fulfill the formal criteria for a hypomanic or manic episode have been specified: While in the past only a distinct period of abnormally and persistently elevated, expansive or irritable mood was necessary, these symptoms now have to be present in association with persistently increased (goal-directed) activity or energy [10].Early-onset BD tends to be clinically more severe than later-onset forms and patients tend to have more frequent episodes [11]. Furthermore, mood swings and episodes of mixed mania and depression appear to be more frequent in younger patients [12]. Importantly, risk of suicide is also higher in early-onset BD [11]. Differences in BD phenomenology between children/adolescents and adult patients should therefore reflect in separate management guidelines for these two patient groups. Management of BC includes integrated behavioral and pharmacological treatment. The most commonly used behavioral therapies include targeted psychotherapy, family therapy, interpersonal and social rhythm therapy and group co-education, which have been shown to be effective, especially as an adjunct to pharmacological therapy [7,8]. This review will focus specifically on currently available guidelines for long-term pharmacological therapy of BD in children and adolescents.There are four main classes of medications indicated for the management of BD in children and adolescents. These include mood stabilizers (primarily lithium), atypical antipsychotics, anticonvulsant drugs and antidepressants. The mechanisms of action along with guidelines for their use in children and adolescents will be reviewed separately for each class of medication. The treatment guidelines which will be discussed here include those published by the National Institute for Health and Care Excellence (NICE) [7], the American Academy for Child and Adolescent Psychiatry (AACAP) [3], and the Child and Adolescent Bipolar Foundation (CABF) [14] (Table 1). A further set of guidelines for the diagnosis and treatment of BD in adults have been compiled by the British Association of Psychopharmacology (BAP) [8]. However, although these guidelines contain information about the diagnosis of BD in children and adolescents, they do not make any specific recommendations regarding treatment of the disorder in this patient population, and therefore will not be discussed in this review.Summary of current clinical guidelines for the pharmacological treatment of bipolar disorder in children and adolescents.CABF, Child and Adolescent Bipolar Foundation; NICE, National Institute for Health and Care Excellence; AACAP, American Academy for Child and Adolescent Psychiatry.Among the most commonly prescribed pharmacological treatments for BD are lithium salts, most commonly lithium carbonate. A number of lithium salts are used medically as mood stabilizing drugs, and are commonly referred to simply as “lithium”. The mechanism of action of lithium is complex, and multi-faceted, as it acts by mimicking the role of various cations, entering cells and interfering with transmitter release, thereby inhibiting of a number of enzymes within signal transduction pathways. This is thought to decrease neuronal over-excitability, thus reducing the symptoms of mania [15]. Lithium has been used in the treatment of BD since the 1960s, and a large number of studies have demonstrated its superiority to placebo in long-term treatment of BD [16,17], in the prevention of relapses [17] and of acute manic episodes [18]. Lithium is currently the only drug used in the treatment of BD licensed in both the UK and USA for patients with a diagnosis of BD above 12 years of age. A limited number of double-blind randomized controlled trials have been carried out to assess the efficacy of lithium as a treatment for BD in children and adolescents. Most of these studies are however limited by small sample sizes and variability in diagnostic criteria. Despite these limitations, lithium remains the most widely investigated treatment for BD in this age group. Clinical research has shown that lithium has a number of significant adverse effects, for which patients should be monitored closely. These include tremor, weight gain and dehydration. It also has a relatively narrow therapeutic index and therefore precise dosing is required, alongside monitoring of blood lithium levels. NICE recommends that lithium should be used as a second-line agent in the long-term treatment of children and adolescents, whereas AACAP and the CABF guidelines suggest lithium as one of the possible first-line agents.Second-generation or atypical antipsychotics, especially olanzapine, quetiapine and risperidone, have recently been advocated for the treatment of BD [19]. Atypical antipsychotics mainly differ from their first-generation counterparts in their improved safety profile. Side effects of atypical antipsychotics include tardive dyskinesia and extrapyramidal symptoms (although to a lower extent than first generation antipsychotics), weight gain and metabolic changes. The exact mechanisms of action of atypical antipsychotics are not fully understood and vary between individual drugs. All antipsychotics have an inhibitory action on the dopaminergic neurotransmitter system; however the receptors involved, and the affinity to which they bind, differs between agents [20]. Few studies have specifically evaluated the efficacy of atypical antipsychotics in the treatment of BD in children and adolescents, and the recommendations published in the guidelines are primarily extrapolated from adult patients’ data. Aripiprazole, olanzapine, risperidone and quetiapine have been shown to be effective in the management of acute mania in adults, whilst olanzapine has also been proven useful in the maintenance therapy for BD [13]. The NICE guidelines suggest that due to their superior safety profile compared to lithium, atypical antipsychotics should be the first-line treatment for the long-term maintenance of BD in children and adolescents. CABF recommendations suggest that atypical antipsychotics may be used as a first-line treatment, as an alternative to lithium or anticonvulsants. No preference is suggested for any specific drug, as the comparative efficacy of the drugs had not been sufficiently investigated. Finally, the AACAP guidelines suggest that atypical antipsychotics, without specifying individual agents, may be used as an adjunct or alternative to pharmacotherapy with lithium and sodium valproate.Anticonvulsants are medications originally developed to prevent and treat epileptic seizures. They are however increasingly being used in conditions other than epilepsy, such as neuropathic pain and BD, due to their mood stabilizing properties [21]. Common side effects of anticonvulsants include drowsiness, weight gain, dizziness and diplopia. Sodium valproate also poses considerable caution due to its teratogenic potential. These medications appear to be particularly effective for the treatment of acute manic episodes and prevention of further episodes. Anticonvulsant drugs most commonly recommended in clinical guidelines concerning the treatment of BD in children and adolescents include sodium valproate, carbamazepine and lamotrigine. Sodium valproate with its GABAergic mechanism of action [22], is the most frequently recommended anticonvulsant in the treatment of BD. In the NICE guidelines, sodium valproate is considered as a second line agent (in patients who do not respond to atypical antipsychotic monotherapy) as an alternative to lithium, in male patients. Lithium alone is recommended as a second-line treatment in female patients due to the teratogenic effects of sodium valproate. The AACAP guidelines provide different recommendations, suggesting that valproate may be used as a first-line agent alongside lithium, with or without antipsychotics. The use of lamotrigine, a sodium channel blocker with known antidepressant action, is also mentioned as an effective maintenance therapy for children and adolescents with BD. The guidelines published by the CABF also recommend sodium valproate as a second or third line treatment, which may be complemented by other anticonvulsants such as carbamazepine (a stabilizer of voltage-gated sodium channels).The majority of the drugs mentioned above are effective in the treatment of mania, however depression is also an issue of primary importance in BD. Antidepressant drugs, most commonly selective serotonin reuptake inhibitors (SSRIs) are also commonly prescribed in patients with BD. These antidepressants work by preventing reuptake of serotonin back into the presynaptic membrane, therefore increasing the amount of serotonin available to be taken up by the postsynaptic membrane [23]. SSRIs are relatively safe medications; however occasional side effects can include headache, nausea, agitation and sexual dysfunction. In the NICE guidelines, it is recommended that in children and adolescents with bipolar depression, initial treatment should consist of their normal mood stabilizer alongside psychotherapy. If after four weeks the response is minimal, SSRI treatment is recommended. Fluoxetine is recommended in the first instance, and if this produces no response, alternatives such as sertraline or citalopram may be tried. The AACAP guidelines recommend SSRIs as a useful adjunct when the patient is taking at least one mood stabilizer. Their published article also mentions the demonstrated efficacy of the combination of olanzapine, an atypical antipsychotic, and fluoxetine in treating bipolar depression in adults with BD, suggesting that this may also be a worthwhile combination in treatment of children and adolescents. The CABF produced no algorithm for pharmacotherapy of bipolar depression, as they did for mania, due to insufficient evidence pertaining to antidepressant treatment in this patient group. They did however, mention the potential benefits of SSRIs or bupropion, an atypical antidepressant thought to stimulate release and prevent reuptake of both dopamine and noradrenaline.Clinical guidelines produced by NICE consist of recommendations on treatment of specific conditions within the NHS in the United Kingdom. They also publish guidance pertaining to public health and interventional procedures, alongside appraisals of medical technology. Their guidance is intended for use not only by health care professionals, but also by local authorities, employers or any other professional body. NICE Clinical Guideline 38 was published in 2006 and covers management strategies for patients with BD, with focus on treatment of BD in adults, children and adolescents in primary and secondary care. These guidelines are based on high quality studies, reflecting the best available evidence, and include recommendations on both pharmacotherapy and psychological therapy, as well as advice on self-help and the support available for family and carers.The “Practice Parameter for the Assessment and Treatment of Children and Adolescents with Bipolar Disorder” was developed by the AACAP, and published in the Journal of the American Academy for Child and Adolescent Psychiatry in 2007. The focus of these guidelines was to encourage best practices in child mental health, and consist of a number of recommendations pertaining to the diagnosis and management of children and adolescents with BD. They are based mainly on studies assessing treatments of BD in adult patients, and suggest that treatment of children and adolescents should consist of a drug approved by the Food and Drug Administration (FDA) for use in adults. They suggest that the chosen drug should be selected based on six factors, including evidence of efficacy, phase of the illness, presence of confounding presentation, drug’s side-effect profile, patient’s medication history and personal/family preference.The CABF, a child psychiatric workgroup, developed a set of guidelines consisting of two treatment algorithms regarding BD in children and adolescents. These algorithms suggest treatment pathways for manic episodes, with or without psychosis, in children and adolescents with BD. They were compiled by CABF members along with 20 clinicians and were published in the Journal of the American Academy for Child and Adolescent Psychiatry in 2005. These treatment guidelines are very concise, specific and easy to follow. Furthermore, the authors have recognized the paucity of large-scale studies involving this patient population, and did not produce an algorithm for depression in BD. However, the algorithms they did produce were based primarily on studies involving children and adolescents, and not solely adult studies.These three sets of guidelines were published by three different organizations, and have a slightly different remit. Both the AACAP and the CABF guidelines were developed specifically for the treatment of children and adolescents with BD, whereas the NICE clinical guidelines are primarily focused on treatment of adults with BD, with a short section pertaining to children and adolescents. The differences and similarities between each set of guidelines deserve a few comments, along with a discussion of the discrepancies. Lithium is the most frequently prescribed medication for adult BD, however there are differences concerning its use in children and adolescents across these guidelines: AACAP and CABF recommend lithium as a first-line agent in the long-term treatment of BD in children and adolescents, whereas NICE recommends an atypical antipsychotic with fewer adverse effects such as weight gain and elevated prolactin. This seems to suggest that the NICE guidelines prioritizes patient safety and compliance (reduced undesired effects), in addition to the evidence base for treatment. Furthermore, the NICE guidelines have stipulated that sodium valproate is not recommended for treatment of female patients due to its teratogenic effects, whereas neither the AACAP nor the CABF made any distinction between management of male and female patients. Apart from a few differences, these guidelines are on the whole consistent, recommending the same drug classes, and where specific drugs are indicated, these are generally the same. As the guidelines were all produced at around similar times, it is likely that they are based on similar, or largely overlapping, research studies. Importantly, all three guidelines recognize the lack of evidence specific to children and adolescents, and base their clinical recommendations on evidenced derived from adult studies. There is a clear need for data from more double-blind, randomized controlled trials in order to update evidence-based recommendations. Overall, these guidelines call for greater attention from both clinicians and researchers in the field towards the early diagnosis of BD in childhood and adolescence and the consideration of early intervention and long term follow up as required, based on the subtle aspects of individual presentation of the condition.At present, very few double-blind randomized controlled trials have been carried out in child and adolescent patients with BD. Available guidelines have been largely developed based on studies carried out on adult samples. It is generally accepted that clinical features of BD in children and adolescents varies significantly from those of adult-onset, confirming the need for more trials specifically in the younger age-groups. Inevitably, this raises ethical issues as children and adolescents with BD can be a vulnerable patient group, both due to their psychiatric condition and their young age, and may lack the capacity to appropriately consent to participation in research. The choice of management strategies is also complicated by the fact that affective disorders, including BD, are a common psychiatric co-morbidity in patients with underlying neurological disorders [24]. Finally, all three sets of guidelines analyzed in this review were published more than five years ago, highlighting the need for updated evidence-based recommendations.The authors declare no conflict of interest.

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+This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).Little is known about women’s comparative attitudes towards prenatal testing for different categories of genetic disorders. We interviewed women who delivered healthy infants within the past year and assessed attitudes towards prenatal screening and diagnostic testing, as well as pregnancy termination, for Down syndrome (DS), fragile X (FraX), cystic fibrosis (CF), spinal muscular atrophy (SMA), phenylketonuria (PKU) and congenital heart defects (CHD). Ninety-five women aged 21 to 48 years participated, of whom 60% were Caucasian, 23% Asian, 10% Latina and 7% African American; 82% were college graduates. Ninety-five to ninety-eight percent indicated that they would have screening for each condition, and the majority would have amniocentesis (64% for PKU to 72% for SMA). Inclinations regarding pregnancy termination varied by condition: Whereas only 10% reported they would probably or definitely terminate a pregnancy for CHD, 41% indicated they would do so for DS and 62% for SMA. Most women in this cohort reported that they would undergo screening for all six conditions presented, the majority without the intent to terminate an affected pregnancy. These women were least inclined to terminate treatable disorders (PKU, CHD) versus those associated with intellectual disability (DS, FraX) and were most likely to terminate for SMA, typically lethal in childhood.With recent advances in molecular genetics, carrier screening and prenatal diagnosis is now available for a broad array of disorders. Screening is currently recommended in pregnancy for a number of genetic (single gene or Mendelian) disorders, chromosomal abnormalities and structural birth defects in the fetus [1,2,3,4]. It is generally agreed that the following criteria should be met for screening programs to be effective: (1) A disorder of sufficient severity to warrant screening; (2) A high frequency of carriers in the screened population; (3) The availability of an inexpensive and dependable test with low false-negative and false-positive results; (4) Access to genetic counseling for couples identified as carriers; (5) The availability of prenatal diagnosis; and (6) The acceptance and voluntary participation by the population targeted for screening [5].In practice, disease incidence and severity and the availability of an effective screening test will often culminate in an expert opinion that population screening for a given disorder should be recommended, with little data collected regarding the attitudes of the target population. Despite increased recognition of the importance of involving patients in medical decisions [6,7,8], consideration of patient attitudes and preferences is not currently employed to determine prenatal genetic screening policies, an area that is particularly value-laden and where such considerations are arguably the most important. Another assumption underlying much of the discussion surrounding prenatal genetic testing is that screening tests should focus on disorders for which women would elect to terminate their pregnancy or which are severe enough that termination of pregnancy is considered a reasonable option by the medical profession. Whether the target population shares this assumption is unknown. While previous studies have retrospectively evaluated differences in the rate of termination of pregnancy for disorders of increased severity or those that include intellectual disability [9,10,11], few to no data exist on how women compare or value testing for different categories of conditions and whether they associate a desire for testing with an inclination toward termination for a given condition.The objective of this project was to better understand women’s attitudes regarding prenatal genetic testing for a broad range of congenital disorders, including those that are potentially treatable, those that include substantial intellectual disability as a primary feature and those that are primarily medical disorders with a shortened life expectancy. In particular, the study sought to determine what women who had recently given birth would be inclined to do in their next pregnancy if they were offered the option of screening, diagnostic testing and termination of pregnancy for affected fetuses with a broad array of congenital disorders with varying characteristics.Between September 2010 and May 2012, we interviewed Kaiser Permanente Northern California (KPNC) members aged 18–48 years old who had given birth within the past year. Kaiser Permanente Northern California (KPNC) is a large, integrated healthcare organization that provides care for more than 3.2 million residents, including approximately 35,000 pregnant women, per year. Except for the lowest and highest income earners, the KPNC membership is representative of the total population in the region. To recruit participants, letters were sent to all women who had given birth to healthy infants and who had had a postpartum visit at Kaiser San Francisco within the past 12 months. Although decision making regarding testing typically takes place during pregnancy, we recruited postpartum women due to concern about the potential for raising pregnant women’s anxieties about birth defects that might affect their infants, including those for which testing is not yet available or is not routinely offered.Standard prenatal counseling at Kaiser involves the offer of screening for single gene disorders as recommended by the American College of Obstetricians and Gynecologists (ACOG), and a discussion and the offer of screening and diagnostic tests for aneuploidy, which are offered regardless of maternal age. Thus, all participants would have been offered screening for neural tube defects, Down syndrome and cystic fibrosis. Screening for fragile X (FraX) and spinal muscular atrophy (SMA) is not routinely offered, nor is specific screening for congenital heart defects (CHD), other than with nuchal translucency and routine second trimester anatomy ultrasound.Our goal was to determine patient attitudes toward testing and termination for six congenital disorders of varying severity. The study included “prototype” conditions that reflected the types of disabilities for which testing is currently available or may become available in the future. The six disorders that were included (Down syndrome (DS), phenylketonuria (PKU), congenital heart disease (CHD), spinal muscular atrophy (SMA), fragile X syndrome (FraX) and cystic fibrosis (CF)) are common and were selected as they represent different characteristics with regards to severity, the presence of cognitive disability, the presence of physical abnormalities, the availability of treatment and life expectancy (Table 1).Trained research assistants interviewed each participant individually. After signing informed consent, each participant completed a self- or interviewer-administered demographic/attitudinal survey instrument, which included questions regarding whether and how well they knew an individual with any of the six disorders under investigation. The conditions were then presented to the participant in random order, using a computerized tool. For each disorder, the name of the condition was provided along with a description of its characteristics (its effects on appearance, cognition and mental health, behavior, physical and medical health and the life expectancy), the prevalence of the condition and whether it was associated with maternal age and race/ethnicity.Following the description of each disorder, the participants were asked whether they thought they would elect to have a blood (screening) test or an amniocentesis (diagnostic test) to detect the disorder and whether they would opt to terminate an affected pregnancy. Response options included “definitely” and “probably” would have, “uncertain” and “probably” and “definitely” would not have screening, diagnostic testing or a termination. For simplicity, participants were not asked to consider decisions regarding amniocentesis based on characteristics of screening tests, but rather to consider whether the disorder was severe enough to warrant diagnostic testing.To assess general prenatal testing and abortion attitudes, participants were presented with a series of statements for which they were asked to indicate their agreement using a 5-point Likert scale ranging from “strongly disagree” to “strongly agree”. The “value of testing information” scale consisted of a single item: “Having prenatal testing is a way to make me less anxious during my pregnancy”. We assessed abortion attitude by asking about agreement with the statement: “Abortion should be generally available to any woman who wants one”.We conducted descriptive analyses to profile the sample, including the examination of means and proportions, measures of variability and confidence intervals around these estimates. Approval was obtained from the Kaiser Permanente Institutional Review Board (IRB) for the study.Disorders and key characteristics presented to participants.* Refers to medical treatment, as opposed to educational interventions; ID = intellectual disability; PKU = phenylketonuria; CHD = congenital heart defect; SMA = spinal muscular atrophy; rx = treatment.We recruited and interviewed 95 women who had delivered healthy infants within the previous year. Participants ranged from 21 to 48 years of age and were racially and ethnically diverse; 60% were Caucasian, 23% were Asian, 10% were Latina and 7% were African American. From a socio-economic perspective, they were less diverse, with most having college degrees, an income >$100,000/year and being employed and married or living with a significant other. For 39%, the recent pregnancy was their only prior pregnancy (Table 2). In all, 27 (28%) reported having had one or more abortions.In terms of their familiarity with the genetic conditions of interest, more than half reported knowing someone with Down syndrome and 12% indicated that they had a relative with the condition. Nearly half (45%) reported knowing someone with a congenital heart defect, 13% knew someone with cystic fibrosis and 27% reported knowing someone with another congenital or genetic disorder. These ranged from minor conditions, such as color blindness, to a range of genetic disorders, such as hemophilia and sickle cell disease. Relatively few subjects knew or were related to anyone with SMA, PKU or fragile X syndrome (Table 3).Sociodemographic characteristics of the population.The number of subjects who are related to or know someone with congenital disorders.SMA = spinal muscular atrophy; PKU = phenylketonuria; CHD = congenital heart defect; * A broad range of other inherited or congenital disorders, such as hemophilia, color blindness and spina bifida.With regard to personal experience with testing, 98% reported having had testing for Down syndrome, 37% had had testing for cystic fibrosis, 21% reported testing for Tay-Sachs disease and 16% had had an Ashkenazi Jewish panel. Fewer than 10% had had testing for fragile X syndrome or spinal muscular atrophy.In exploring attitudes toward prenatal testing and pregnancy termination, 81% of the participants reported somewhat or strongly agreeing with the statement, “Having prenatal testing is a way to make me less anxious during my pregnancy”. In addition, 85% somewhat or strongly agreed with the statement, “Abortion should be generally available to any woman who wants one”.Most participants indicated that they would opt to have a screening test for each of these conditions (95%–98%, depending on the specific test), and the majority also indicated that they would have amniocentesis (ranging from 64% for PKU to 73% for SMA). Inclinations regarding pregnancy termination varied much more substantially by condition: While only 11% of the participants indicated that they would choose to terminate a pregnancy for a congenital heart defect, 41% would be inclined to do so for Down syndrome and 65% for SMA (Figure 1). While the number of subjects that were moderately or very familiar with these conditions was relatively small, knowing someone with CF and SMA was associated with an increased likelihood of having had testing for the disorder (p = 0.002 for CF and p = 0.01 for SMA). This was despite the fact that these were unrelated acquaintances, rather than related individuals, which would indicate an increased risk that our subject would be a carrier of the condition. The association of familiarity with an affected individual and testing for the condition was not found with the other disorders.Proportion of patients indicating they would definitely or probably undergoing screening (blood test), diagnostic testing (amniocentesis) or pregnancy termination for each condition.While over 95% of women in this cohort indicated that they would choose to undergo screening for all of the conditions presented to them, the majority would do so without intent to terminate an affected fetus. Women were less inclined to terminate treatable disorders, such as PKU and congenital heart defects, as compared to those associated with intellectual disability (Down syndrome and fragile X syndrome). Patients were most inclined to terminate for SMA, which is typically lethal in childhood. Only for SMA would more than half of women terminate their pregnancy; for all of the other disorders presented, even those who would choose diagnostic testing did not feel they were likely to terminate based on the results. This represents a substantially different view of the purpose of prenatal genetic testing than what is typically discussed by the medical community; both advocates and critics of prenatal genetic testing commonly describe the goal as identifying affected fetuses with an intent to abort those that are abnormal. In this study, most patients clearly did not view the purpose of such testing as identifying those that should be terminated.Few previous studies have investigated the attitudes of patients regarding screening or testing for different categories of disorders, although some studies have reported results similar to this one with regard to differences in the uptake of screening, diagnostic testing and pregnancy termination. A study in the UK focusing on 109 relatives of individuals affected with CF found that while most (75%) would undergo screening for this disorder, fewer than half would have a pregnancy termination [12]. Another study of the parents of children with CF found that while most would have a prenatal diagnosis, the decision to have an abortion was far more difficult [13]. In a study conducted to gauge consumers’ opinions towards genetic testing for diseases and enhancements, patients were asked to indicate traits and conditions for which they would choose reproductive genetic testing. Most, but not all, respondents were pregnant women intending to have prenatal diagnostic procedures. The majority indicated that they would elect to have prenatal genetic testing for mental retardation (75%), deafness (54%), blindness (56%), heart disease (52%) and cancer (51%); 49.3% would choose testing for a condition that resulted in death by five years of age. Those findings were similar to the results in this report, although these investigators did not distinguish between screening and diagnostic testing and did not assess whether the desire for reproductive testing was associated with the intent to terminate an affected pregnancy [14].It appears that some disparity exists between prospective parents and their care providers. During pregnancy, prospective parents seek reassurance that their fetus is developing normally. Providers, on the other hand, frequently see the purpose of prenatal genetic testing as identifying abnormalities for which the patient would choose to terminate a pregnancy. In a review of reproductive genetic counseling, Pergament et al. noted that “Decisions made after genetic counseling have limited parameters: parents must consider diagnostic testing after positive screening for a genetic disorder and then either continue the pregnancy or electively terminate, if the pregnancy is affected” [15]. Many providers, as part of the conversation about the choice to undergo testing, recommend against it for disorders if the patient indicates that she is unlikely to elect an abortion if the specific abnormality is identified.In a systematic review of reproductive genetic testing, Green et al. note that “Clearer consensus is required about the knowledge that is necessary and sufficient for women to have when they are making decisions about prenatal and newborn genetic testing [16]. Put more bluntly, what is it that people do need to know and whose business is it to decide that? Professionals have been preoccupied with conveying certain kinds of information (e.g., procedural matters, risk estimates) but have virtually ignored others (e.g., what it might be like to bring up a child with the condition in question), and an approach based on parents’ needs rather than staff needs is long overdue” [16]. This study is a step toward collecting data to improve and inform such an updated approach.The study is not without limitations. The sample size was relatively small, and the participants were all recruited from one Kaiser site in Northern California. While the cohort was racially and ethnically diverse, for the most part, they were highly educated. The intent originally was to use generic descriptions of the conditions to avoid prior perceptions of the significance of these disorders. However, in focus groups conducted prior to the study, it was evident that many women quickly recognized the various disorders, so ultimately, the conditions were labeled; the participants may therefore place value on each condition that has been colored by prior experience, associations or preconceptions. In addition, the study was based on hypothetical situations, and decisions regarding pregnancy termination might be quite different when women are presented with an actual diagnosis of an affected pregnancy compared to the hypothetical situation posed in the study.While the field of genetics, and prenatal genetics in particular, has evolved at a remarkable rate, the approach to the assessment of how to best implement new tests and technologies has lagged. The field has continued with a paradigm of offering individual tests for single conditions, with the assumption that the primary outcome to consider is pregnancy termination. It appears from this study that the primary consideration of this cohort of patients is quite different and that there is value to information about fetal disorders that extends beyond a decision regarding pregnancy continuation or termination.This project was supported by a grant from the Kaiser Community Benefits Program.The authors declare no conflict of interest.

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+This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).This study examines the association between state and county unemployment rates and individuals’ body weight status during the latest recession in the U.S. We used the U.S. Behavioral Risk Factor Surveillance System (BRFSS) data in 2007, 2009 and 2011, which were collected from 722,692 American adults aged 18 or older. Overweight and obesity were defined as body mass index (BMI) ≥25, and ≥30, respectively. Multivariate linear and logistic regressions were applied to assess the association between BMI, risks of overweight and obesity, and state and county unemployment rates. State unemployment rates were negatively associated with individual BMI across years, while county unemployment rates were significantly positively associated with BMI and obesity rates in all years (p < 0.05). However, the scale of the positive relationship was reduced in 2009 and 2011. Stratified analyses were conducted among adults with employment and without employment. The unemployed group’s body weight status was not related to state- and county-level economic conditions in most times. In the pooled analyses with all three years’ data, the relationship between unemployment rates and body weight status were consistently reduced after the recession of 2008–2009. Our results indicated that macroeconomic conditions at different levels can have different associations with individuals’ obesity risk across time. The scale, length and depth of the latest economic recession has exceeded all recessions since the Great Depression [1]. The U.S. economy reached its worst level in 26 years, while the national unemployment rate reached 9.3% in 2009, a 60.3% jump from the previous year [2]. This prolonged recession will further increase unemployment and poverty, potentially resulting in significant health consequences affecting millions of Americans [3,4]. The relationship between economic cycles and health outcomes has warranted continuous interest since the Great Depression in the 1930s. Earlier studies have demonstrated the negative impact of economic recessions on general health outcomes. Brenner demonstrated this association with time-series studies, observing increases in psychiatric hospital admission, infant mortality rates and deaths in recessions due to cardiovascular disease, cirrhosis, suicide and homicide [5,6,7]. Catalano and Dooley’s study found that an economic contraction negatively affects illness and injury rates by increasing the incidence of undesirable job and financial events for middle-income respondents only [8]. Likewise, Gerdtham and Johannsson found a highly significant effect of unemployment on mortality [9].However, more recent evidence suggests that sudden or short-run economic upturns, instead of recessions, negatively affect health by creating more job stress, less time for self-care activities such as eating well or exercising, overindulgence in unhealthy food, and work-related accidents [10,11,12,13]. Ruhm [10] reported that state unemployment rates are significantly and negatively related to total mortality and to 8 out of 10 specific causes of fatality. A one percentage point rise in unemployment reduces the total death rate by 0.5% [10]. Ruhm also demonstrated that a rise in unemployment predicts reductions in the prevalence of medical problems, decline in acute morbidities and bed-days, and declines in ischemic heart disease and intervertebral disk disorder [14]. He clarified that while in the short-run, economic expansions are more likely to place stress on individuals, resulting in adverse health effects, permanent gains in economic improvements provide higher-level health through technological innovations, greater access to care and improved purchasing ability for items that provide greater safety, like newer automobiles [15]. Regarding economic recession and obesity, researchers have painted a mixed picture in the literature. Ruhm used the Behavioral Risk Factor Surveillance System (BRFSS) 1987–1995 data and found that during economic downturns people have more time to exercise and prepare healthy meals and are thus more likely to maintain a healthy weight [10]. Subsequently, he used data on adults from the BRFSS 1987–2000 to demonstrate that excess weight and physical inactivity decline when economic conditions improve [16]. Hruschka also used BRFSS data and compared the annual growth rates of BMI during 2004–2007 and 2008–2010 [17]. His results indicated that the annual change in BMI was significantly reduced across income groups after the 2008 recession. However, a Finnish study using individual microdata from 1978–2002 found that improvement in economic conditions produced a decrease in BMI [18]. Charles and DeCicca used data from the National Health Interview Survey (NHIS) during 1997–2001 and found that an increase in unemployment rates was associated with an increase in body weight status among those least likely to be employed (low-income and low-education) and African Americans [19]. The contrary findings in the literature suggest that further research is needed.The most recent recession (2008–2009) was the severest since the Great Recession in the 1930s in terms of its length (18 months) [1]. Therefore, the recession in 2008–2009 provided a unique opportunity to examine the association between economic conditions and obesity. There are concerns that a major economic downturn could impose additional risks of obesity due to people having a lower dietary quality and engaging in less physical activity [20]. However, others have debated the actual health impact of the latest recession [21,22,23,24]. To provide more evidence to clarify the associations between economic conditions and obesity, we examined the associations between obesity and state or county unemployment rates using cross-sectional waves of the Behavioral Risk Factor Surveillance System (BRFSS) in 2007, 2009 and 2011, which encompass the periods before, during, and after the recession. The BRFSS, a cross-sectional telephone survey of non-institutionalized American adults aged 18 years or older, has been an annual survey conducted by the Centers for Disease Control and Prevention (CDC) since 1984 that provides representative data at the national and state levels on health behaviors, preventive health practices and risk factors for the leading causes of death in the U.S. [25]. A multi-stage, disproportionate stratified sample (DSS) design was employed by the BRFSS consistently across years. After telephone numbers were randomly selected, computer-assisted telephone interviews were conducted by trained professionals in each state. After the interviews, the data were compiled at the CDC, which processed and prepared the national data for that year. More details about the BRFSS data collection are available [25]. Three waves of BRFSS data, 2007, 2009 and 2011, were used in this study. The national unemployment rate in 2007 was 4.6%, while the numbers in 2009 and 2011 were 9.3% and 8.9%, respectively, so these three years captured the window period before and after the recession of 2008–2009. We linked publicly available BRFSS data with Local Area Unemployment Statistics (LAUS) for state and county unemployment rates [2]. Outcome variables: Body mass index (BMI) was defined as weight (kg)/height2 (m2); overweight if BMI ≥25; and obesity if BMI ≥30.Key exposure variables: State and county unemployment rates. Covariates: Socio-demographic variables, such as employment status, race/ethnicity, education, age, and income group, were controlled. Employment status in the BRFSS was classified as “employed for wages”, “self-employed”, “out of work for more than 1 year”, “out of work for less than 1 year”, “a homemaker”, a “student” and “retired”. In our analyses, the “employed” groups included adults who were employed for wages or self-employed; the “unemployed” groups included adults who were “out of work for more than 1 year” and “out of work for less than 1 year”. Since the retired group was no longer associated with labor market outcomes, we removed them from the analyses. Since the unemployment rates can affect both employed and unemployed adults’ lifestyles, we stratified the analyses for the employed and the unemployed groups. Race/Ethnicity was categorized as “non-Hispanic whites”, “non-Hispanic blacks”, “Hispanics” and “Other”. Education was classified as “<high school”, “high school”, “some college”, and “≥college graduate”. Income groups were created as “<$15,000”, “$15,000–$25,000”, “$25,000–$35,000”, “$35,000–$50,000” and “≥$50,000”. We also controlled the state per capita Gross Domestic Product (GDP) as the proxy for state-level economic indicators, since the GDP may fluctuate in business cycles. For other unobserved heterogeneity at the state level, we used dummies as a control in the regressions. Smoking behavior, current smoker or not, was also added as a control for health behavior and an indicator for general health. Our analysis was conducted using Stata, Version 11 (Stata Press, College Station, TX, USA) and took into account the complex survey design of the BRFSS. First, we calculated the descriptive statistics of the study samples from the BRFSS in each year. Next, we conducted multivariate linear and logistic regression to examine the associations between BMI, overweight and obesity with state or county unemployment rates across years. Since the BRFSS is a cross-sectional survey, we are unable to conduct a longitudinal study to examine how the unemployment changes were associated with changes in individual body weight status. To address this limitation partially, we pooled the data from 2007, 2009 and 2011 and added the interactive terms of unemployment and survey years to test whether the relationship between the unemployment rates and body weight status changed across the two years. Due to the high correlation between state and county unemployment rates, we conducted the regression separately for state- and county-level unemployment rates. Table 1 presents the summary statistics of the subjects interviewed in the BRFSS 2007 to 2011 and the pooled sample of the three years. Our analysis sample included individuals with a mean age of 41.96 years in the pooled sample. Approximately half of them were men; 66.24% of them were non-Hispanic white, 10.88% were non-Hispanic black, 16.89% were Hispanics. The unemployment rates were 5.22% in 2007, 9.84% in 2009, and 10.28% in the pooled sample. The almost doubled unemployment rates after 2008 reflected the severity of the latest recession. Although the recession officially ended in June 2009 based on the National Bureau of Economic Research, labor market conditions were not immediately improved. The proportions of homemakers were essentially the same, while greater proportions of the surveyed individuals were students (4.63% in 2007, 5.00% in 2009, and 5.83% in 2011). Descriptive Statistics of the Behavioral Risk Factor Surveillance System (BRFSS) 2007, 2009 and 2011.Table 2 presents the results from the linear regression between BMI and the state and county unemployment rates across years. Each cell in Table 2 represents one regression result. In all years for the total group, state unemployment rates had a negative relationship with BMI, although the coefficient was not significant in 2009. However, the signs of these coefficients varied by gender and employment groups. Among men, the state unemployment rate was not a significant factor associated with BMI in all years, although the results were significant among women. Among the employed adults, the state unemployment rates were negatively associated with BMI and the coefficients were significant in 2007 and 2011. For unemployed adults, the signs of the coefficients were mixed for state unemployment rates. The coefficients in 2009 and 2011 were positive, while the coefficient in 2007 was negative. County unemployment had a more consistent association with BMI. In all years, the coefficients were all positive in all gender and employment groups, except the unemployment groups in 2007 (beta = −0.03, p > 0.05). The positive associations indicate that on average, individuals living in counties with higher unemployment rates had a greater BMI and the relationship was statistically significant in all groups except for the unemployed groups. For all the significant groups, the scale of the coefficients was much smaller in 2009 and 2011 than in 2007, e.g., the total group’s beta of county unemployment was reduced to 0.10 in 2009 and 2011, down from 0.19 in 2007. The same patterns were found in men, women and the employed groups, which indicate that the associations between county unemployment rates and individual BMIs were weakened by the economic recession in 2008, although the county unemployment rates remained a significant predictor.Linear relationship between body mass index and state and county unemployment (unemp) rates among BRFSS participants in 2007, 2009 and 2011.*** <0.001; ** <0.01; * <0.05; Models controlled for age, gender, race/ethnicity, income, education, state level GDP per capita, smoking behavior, state dummies and employment status when the analysis was not conducted; Overweight: BMI ≥25; Obesity: BMI ≥30.The associations between unemployment rates and risks of overweight were similar in Table 3 as the results in Table 2. Adults in states with higher unemployment rates were less likely to be overweight, especially for women and employed adults. Among men and unemployed group, there was no significant relationship between state unemployment rates and overweight. The odds ratios (ORs) for county unemployment rates were consistently positive, but they were not significant among men in 2007 and unemployed groups in 2007 and 2009. Table 4 presents the results with obesity as the outcome. The signs of state unemployment rates were more mixed than in Table 2 and Table 3, while the significance levels were reduced compared with the results in Table 2 and Table 3. In general, state unemployment rates were not significantly associated with obesity, except among women. In 2007 and 2009, women in states with higher unemployment rates were significantly less likely to be obese, while this relationship became insignificant in 2011. County unemployment rates remained a significantly positive risk factor for obesity in all groups, except the unemployed adults. However, the scales of the ORs were clearly reduced in 2009 and 2011 compared with those in 2007. Individual risk of overweight among BRFSS participants in 2007, 2009 and 2011 by state and county unemployment rates.*** <0.001; ** <0.01; * <0.05; Models controlled for age, gender, race/ethnicity, income, education, state level GDP per capita, smoking behavior, state dummies and employment status when the analysis was not conducted; Overweight: BMI ≥25; Obesity: BMI ≥30.Individual risk of obesity among BRFSS participants in 2007, 2009 and 2011 by state and county unemployment rates.*** <0.001; ** <0.01; * <0.05; Models controlled for age, gender, race/ethnicity, income, education, state level GDP per capita, smoking behavior, state dummies and employment status when the analysis was not conducted; Obesity: BMI ≥30.To test formally whether the association between unemployment rates and body weight status changed across three waves, we pooled the data of 2007, 2009 and 2011. The interactive terms were added in the analyses, while 2009 was used as the reference year. The results are presented in Table 5. For total groups, the interactive terms of state unemployment rates with year 2007 was negative for BMI, overweight and obesity, while the interactive term was significant for BMI. However, the interactive terms between state unemployment rates and year 2011 were highly significant for all three definitions of body weight status (p < 0.001), which means the relationship between unemployment rates and obesity was significantly reduced after the recession. The interactive terms between county unemployment rates and year 2007 were all significantly positive for all body weight statuses (beta > 0, and OR >1), and those of year 2011 were all significantly negative (beta < 0 and OR < 1). Therefore, the relationship between county unemployment rates and obesity was stronger prior to the recession, while weaker after the recession. Similar patterns were found for men, women and the employed group, with the unemployed group as an exception. Among the unemployed group, none of the interactive terms were significant, which indicates the relationship between unemployment rates and body weight status did not change during the economic recession. Pooled analysis of relationship between body mass index, body weight status and state- and county unemployment rates in the BRFSS adults in 2007, 2009 and 2011 by participant characteristics.*** <0.001; ** <0.01; * <0.05; Models controlled for age, gender, race/ethnicity, income, education, state level GDP per capita, smoking behavior, state dummies and employment status when the analysis was not conducted; Overweight: BMI ≥25; Obesity: BMI ≥30.In summary, comparing the associations of unemployment rates with individual body weight status during, before and after the economic recession of 2008–2009, we found the following patterns: First, the positive relationship between the county unemployment rates and body weight status was reduced after the economic recession; Second, the reduction of the relationship between state unemployment rates and body weight status after the recession was more evident among women and the employed adults; Lastly, little significant relationship was found among the unemployed adults, and this pattern did not change after the recession. Using three waves of the latest nationally representative data, our study suggests that the recent economic recession was associated with American adults’ body weight status. The association was different at the state and the county levels and changed in the periods before, during, and after the latest recession of 2008–2009. We found that the negative association between state unemployment and individual body weight status, especially among women and employed adults. These findings echo previous research about the health benefits of recession if the economic condition is indicated by the state unemployment rate [10,16]. At the county level, higher unemployment rates were still associated with higher BMI and obesity risks. However, the scale of this association was reduced as well in the recession, which is consistent with the latest finding that the annual growth rates of body weight slowed after the recession [17]. The opposite directions of economic conditions and obesity at different levels (state and county) reflect the recent debate on the health outcomes of the current recession [19]. Our results indicate that the state economic condition may not bring a significant shock to an individual’s body weight status, but worse local economic conditions can be a significant risk factor for obesity, especially in a booming time [18]. In boom times or in recession, there was little significant relationship between state or county unemployment and unemployed adults’ body weight status. There has been no clear answer in the literature to explain why unemployed adults’ body weight status is not associated with macroeconomic conditions. One possible explanation is that the detrimental health effect of unemployment alone is dominant, so that macroeconomic conditions may not have an additional impact on unemployed individuals’ health [26]. Another possibility is that unemployed adults may rely on federal assistance programs, such as the Supplemental Nutrition Assistance Program (SNAP), which does not vary significantly across regions. With the massive levels of unemployment in the severe recession in 2008–2009, the unemployed might thus be more homogeneous in dietary intake and physical activity than employed adults, regardless of local- or state-level economic conditions [16,27]. Since regional economic condition is little associated with obesity risks of the unemployed, standard programs across the nation can be implemented to promote a healthy lifestyle among adults without employment. One caveat of our study is that we did not examine change in employment status and its association with obesity risk, although the literature suggests that changes in individual employment status across business cycles may cause changes in lifestyles and health afterwards [10,15,18]. Researchers in the future may use more longitudinal data to understand fully the changes in health status among unemployed adults across business cycles. On the other hand, employed adults’ body weight status was more likely to be associated with state and county unemployment rates. They were more likely to be obese in counties with higher unemployment rates. Literature has documented the causal effect of economic recession and its detrimental effect on mental health, including stress [28]. Biological studies have indicated that chronic stress may induce human beings to increase their food intake as a comforting mechanism [29]. Therefore, employed adults in counties with high unemployment rates may work longer hours and/or experience more stress due to greater job insecurity. However, the latest recession of 2008–2009 was so severe that the economic conditions may have become quite miserable in most counties, which might have leveled the association between county unemployment rates and individuals’ obesity risk. That explains why the scale of the relationship at the county level was reduced in or after the recession compared with that in 2007. A few limitations should be acknowledged: First, the BRFSS collects self-reported body weights and heights based upon telephone surveys, which suffer reporting bias. Literature suggests that women are more likely to underreport their weight, while men are more likely to over-report their height [30]. Therefore, the interpretation of our results should be taken with caution due to the possible self-reporting bias in the original BRFSS data; Second, the BRFSS is a cross-sectional survey, so we were unable to track the same individuals across time and observe their changes in body weight status; Third, our study is one of the first studies that examine the latest economic recession and its health impact with regard to obesity. However, we only focused on the association instead of the causation between unemployment rates and body weight status. A longitudinal study with panel data may fully establish causality between economic recessions and individual health risks. Finally, the mechanism of how macroeconomic conditions affect individuals’ health status is complex and understudied, i.e., how the regional economic shocks get “under the skin” is still an unknown. The seemingly contradictory findings at the state and county levels could be also due to the heterogeneity in other unobserved factors. This line of research is beyond the question solely of economics, and more multidisciplinary research is desirable. In summary, our study provided a preliminary look at the association between state- and county-level unemployment rates and adults’ body weight status during one of the most severe recessions since the Great Depressions in the 1930s. Our results indicate that the recession did not bring significant increases in obesity risk across gender and employment groups, which provides important evidence in the debate on the health impact of the latest recession.This study was supported in part by the National Institutes of Health/the National Institute of Diabetes and Digestive and Kidney Diseases (NIH/NIDDK, R01DK81335-01A1), National Institutes of Health/the Eunice Kennedy Shriver National Institute of Child Health and Human Development (NIH/NICHD, R01HD064685-01A1 and U54 HD070725-01).The authors declare no conflict of interest. 

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+This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).It has now been over 50 years since it was discovered that Down syndrome is caused by an extra chromosome 21, i.e., trisomy 21. In the interim, it has become clear that in the majority of cases, the extra chromosome is inherited from the mother, and there is, in this respect, a strong maternal age effect. Numerous investigations have been devoted to clarifying the underlying mechanism, most recently suggesting that this situation is exceedingly complex, involving both biological and environmental factors. On the other hand, it has also been proposed that germinal trisomy 21 mosaicism, arising during the very early stages of maternal oogenesis with accumulation of trisomy 21 germ cells during subsequent development, may be the main predisposing factor. We present data here on the incidence of trisomy 21 mosaicism in a cohort of normal fetal ovarian samples, indicating that an accumulation of trisomy 21 germ cells does indeed take place during fetal oogenesis, i.e., from the first to the second trimester of pregnancy. We presume that this accumulation of trisomy 21 (T21) cells is caused by their delay in maturation and lagging behind the normal cells. We further presume that this trend continues during the third trimester of pregnancy and postnatally, up until ovulation, thereby explaining the maternal age effect in Down syndrome.It is well known that aneuploidy is common in humans, leading to reproductive failure, intrauterine deaths and live born offspring affected by congenital defects and learning disabilities. We have focused our attention on trisomy 21 Down syndrome, this being the most common aneuploidy condition in the live born human population. It is clear that the majority of trisomy 21 (T21) conceptions are caused by a segregation error in maternal oocytes with an increasing risk dependent on maternal age. However, in spite of numerous investigations to this effect, the exact mechanism underlying this meiotic segregation error has not been clarified. Instead, it has become generally accepted that many different biological and environmental factors may be involved in giving rise to aneuploidy (reviewed in [1,2,3,4,5]). In contrast, we have recently proposed that in any one woman, the most likely precursor is the degree of T21 mosaicism that originally existed in her ovaries during her own fetal development [6]. In other words, we advocate that the crucial predisposing factor may be the proportion of immature oocytes with three rather than two chromosomes 21 that are available in the ovarian cortex to undergo the first meiotic division, taking place just before ovulation. Following on from Zheng and Byers [7], we have further proposed that the so-called maternal age effect is due to an accumulation of T21 oocytes during prenatal and postnatal development [8]. In other words, we advocate that the crucial predisposing factor may be the age-related proportion of oocytes with three rather than two chromosomes 21 that are available in the ovarian cortex to undergo the first meiotic division, taking place just before ovulation. The net effect of these two mechanisms is that even a low percentage of trisomic cells present in gonadal tissue or germinal cells significantly increases the risk of aneuploidy in the offspring [9].In a previous study, using fluorescence in situ hybridization (FISH), we identified the occurrence of T21 germ cells in eight fetal ovaries, obtained following social termination of pregnancy (TOP) in the second trimester [6]. This study has now been extended to include 12 additional fetal ovaries, where TOP was performed during the first trimester of pregnancy. We show that in these cases, where TOP has taken place at an earlier gestational age, the incidence of T21 mosaicism is significantly lower. Thus, in accordance with our hypothesis, there is an apparent accumulation in the degree of T21 mosaicism during normal fetal oogenesis.We have used FISH with two chromosome 21-specific probes, labelled in different colors (red and green) in order to assess the incidence of T21 in fetal ovaries obtained following TOP for a social reason at the clinical gestational age of 9–11 weeks.In this new series, we have recorded the incidence of T21 cell nuclei in 12 fetal ovarian samples, obtained during the first trimester of pregnancy. Examples of cell nuclei with T21 in comparison to the normal diploid are given in Figure 1. We identified a mean number of only 0.066% cell nuclei, showing T21 with a range of 0.00%–0.14% and an standard deviation (SD) of 0.045% in a total cell population of 27,042 (Table 1). This result is highly statistically significantly different (p < 0.0001) from the results (average, 0.54%; range, 0.20%–0.88%; SD, 0.23) obtained in our previous investigation of eight cases, ascertained after TOP in the second trimester (Figure 2 in [6]).Illustrations of cell nuclei containing a different chromosome 21 copy number: Two red-green signals indicative of disomy 21 (left) and three red-green signals indicative of trisomy 21 (middle and right).Chromosome 21 copy number by fluorescence in situ hybridization (FISH) analysis of fetal ovarian cell nuclei. T21, trisomy 21.* The cell nuclei scored as having 2 red/2 green have been recorded as likely to be normal disomic, but may include some nuclei having 3 green or 1 green, considered most likely to be false positive or false negative green.The cell nuclei scored as having a 1 red/1 green signal were recorded as either false negative monosomy 21 (due to the pairing of the two chromosomes 21) or true monosomy 21 [10,11] and those with 1 red/2 green and 3 red/2 green as likely false negative or false positive signals.It should be added that in obtaining the estimate of T21 mosaicism, we have excluded cell nuclei showing deviations from the ones with three red together with three green signals, as these might be artifactual (Table 1). We also used a complementary chromosome 18-specific probe to differentiate between true T21 cell nuclei in relation to the potential occurrence of dual trisomy/triploidy. No such nuclei were identified in this series of fetal ovarian cells.Ideally, any hypothesis on the origin of trisomy 21 Down syndrome should address and seek to explain the following issues.We suggest that the reason for the preponderance of the maternal origin is the substantial sex difference in degree of T21 germinal mosaicism with a much higher incidence in fetal ovaries than testes [12]. To our knowledge, there are no explicit hypotheses in the literature, as regards this question, other than the suggestion that maternal meiotic chromosome mal-segregation is more common than paternal and that the mechanisms of origin may therefore be different.In our view, the changes in maternal recombination patterns, as seen by family linkage analysis, are most readily explained by the expected patterns in a T21 oocyte in relation to that in a normal disomy oocyte [6,8]. These maternal recombination patterns are firmly laid down in her fetal oocytes and cannot be altered by any factors later during development. This also includes the meiotic divisions, taking place just before ovulation and after fertilization. In our view, it is therefore unlikely that any factors, such as cohesion deficiency (further discussed below), may play a causative role in the mal-segregation process.We have suggested that the increased recurrence risk in younger women is likely to be caused by a higher incidence of fetal oogonial/oocyte T21 mosaicism [6]; reviewed in [9]. It has been previously documented that such women may show somatic T21 mosaicism, as well [13]. On the basis of cytogenetic/molecular data and also maternal and grandmaternal ages in Down syndrome families, Kovaleva [14] suggests that the normal grandmothers were older and proposed that they conceived offspring that were trisomic, but these conceptions subsequently became mosaics by so-called post-zygotic rescue. To date, it is not clear, however, to what extent such generalized trisomy 21, involving both germinal and somatic cell populations, may, in fact, explain an increased recurrence risk in younger mothers.In order to explain the so-called maternal age effect, we have proposed that there is an accumulation of T21 cells at prenatal and postnatal oocyte development, leading to a higher proportion at later maternal ages [8]. We have here shown that such an accumulation does indeed occur from the first to the second trimester of pregnancy in a sample of ovaries from fetuses with a normal mitotic karyotype (Figure 2). It remains to be investigated whether or not any additional accumulation may take place during postnatal development. As of yet, we have not been able to obtain the relevant cellular samples for this type of study, i.e., primary oocytes in ovaries from women at different biological ages.Accumulation of T21 oogonia/oocytes during fetal oogenesis. The graph shows the mean number of T21 oogonia/oocytes scored in samples of fetal ovaries during the first trimester (Table 1) in comparison to those during the second trimester (Table 1 in [6]). Note the apparent accumulation in incidence of T21 germ cells during the progression of oogenesis from the first to the second trimester of pregnancy.The occurrence of maternal germinal mosaicism as a predisposing factor has previously been implicated in a number of T21 (and other aneuploidy) cases through studies of oocytes and polar bodies, obtained for research in connection with in vitro fertilization (IVF) treatment [9,15]. It is noteworthy, though, that recent studies on human oocytes, retrieved in relation to IVF treatment, indicate that the types and frequency of maternal meiotic segregation errors differ substantially from those occurring in natural conception [2].Much of the work, aiming to explain the mechanism(s) underlying the origin of aneuploidy with special reference to T21 Down syndrome, has been performed on other mammalian species, in particular using mouse models (reviewed in [16,17]). Special attention has been paid to the possibility that the maternal age effect is caused by an age-related deficiency in the cohesion complex, normally holding chromatids of the meiotic bivalents together until the first meiotic anaphase (see, e.g., [18,19]). No such age-related cohesion deficiency has, however, been noticed in the only study performed so far on human oocytes [20].Human fetal ovarian samples were obtained from the Medical Research Council (MRC)/Wellcome Trust funded Human Developmental Biology Resource (HDBR, London, UK) with appropriate maternal written consent and approval from the local National Health Service (NHS) Health Authority Ethics Committee.The samples were transported from the clinic to the HDBR resource and were staged, dissected and the tissue frozen on dry ice and stored at −80 °C following a minimum time delay, usually within 2 h of the termination of pregnancy. The age of the fetal samples was estimated following the staging guidelines of Hern [21]. A snip of tissue from the sample was taken prior to freezing to perform cytogenetic analysis to determine the mitotic karyotype. All the samples had a normal female karyotype (including Case 11 with the variant 46,XX,inv(9)(p11q12)).The frozen samples were transported to our laboratory on dry ice and stored at −80 °C until analysis. Microscopy slides were prepared according to the technology described by Papadogiannakis et al. [22].Microscopy slides for FISH analysis were fixed in 70% ethanol for 30 min and treated with pepsin (0.1 mg/mL) in 0.01 M HCl for 2 min at 37 °C. After additional washing in phosphate-buffered saline (PBS), paraformaldehyde (1%) fixation and dehydration through a series of alcohol, the slides were left to air-dry at room temperature.Hybridization was performed according to the manufacturer’s instructions with two DNA probes positioned near the end of the long arm of chromosome 21 and labelled in SpectrumOrange and SpectrumGreen, respectively (Catalogue No. 32-190002, Abbot Molecular Inc., Des Plaines, IL, USA, and Cytocell, Catalogue No. LPT21QG/R, Cytocell Technologies Ltd., Cambridge, UK). A chromosome 18 centromeric probe labelled in SpectrumAqua was added to be able to differentiate between trisomy and triploidy (Catalogue No. 32-131018, CEP 18 (D18Z1) SpectrumAqua Probe). The DNA probes were mixed and added to the slides followed by denaturation, hybridization and post-hybridization washing. After dehydration, slides were mounted in glycerol containing 2.3% DABCO (1,4-diazabicyclo-(2,2,2) octane), as antifade and DAPI (4,6-diamino-2-phenyl-indole) 0.5 mg/mL for nuclear counterstaining. The cells selected for scoring were defined by the morphology of the nuclei, those being the only ones having large round or roundish nuclei [23].Microscopy analysis was performed on a Zeiss Axioplan 2 microscope. Large cell nuclei, isolated from each other, were initially screened using the spectrum red light filter (Rhodamine). Images were captured and processed using the computer program, AxioVision. If an abnormal number of signals were detected using the red light filter, the spectrum green light filter (FITC) was switched on. Cell nuclei showing a dual red plus a green signal were scored as normal disomy 21, while those showing three dual signals were scored as T21, provided that the cell did not contain three chromosome 18 signals, which could be due to dual trisomy/triploidy. Any other combination of signals was considered likely to be artifactual and excluded. A minimum of 2000 cells per slide was analyzed.In conclusion, it may seem unlikely that we will get to grips with the true origin of T21 Down syndrome, including, in particular, the maternal age effect, until such time as direct investigation on primary oocytes in the ovarian cortex may be performed. We would suggest that one possible way to obtain further information in this regard would be to record the incidence of T21 mosaicism in primary oocytes of the ovarian cortex, following oophorectomy in women at different biological ages. According to our own hypothesis [8], we would then expect to find an accumulation of T21 oocytes related to the increasing biological age of these women. Finally, it should be added that Rowsey et al. [24] stress that the type of technology that we have used for the counts of chromosome copy number is not applicable to cells at the pachytene stage of meiosis when homologues are synapsed. Further work is required to resolve this question.The project was conceived of by Maj A. Hultén, who also wrote the first versions of the manuscript. All authors have seen and agreed to the final version. The preparation of the microscopy slides, as well as the microscopy analysis was undertaken by Linn Öijerstedt and the statistical analysis of the data performed by Jon Jonasson.The human fetal ovarian material was provided by the Joint MRC (grant G0700089)/Wellcome Trust (grant GR082557) Human Developmental Biology Resource (http://hdbr.org).All authors declare no conflict of interest.

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+This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).As new technologies enable the development of non-invasive prenatal diagnosis (NIPD) for cystic fibrosis (CF), research examining stakeholder views is essential for the preparation of implementation strategies. Here, we compare the views of potential service users with those of health professionals who provide counselling for prenatal tests. A questionnaire incorporating a discrete choice experiment examined preferences for key attributes of NIPD and explored views on NIPD for CF. Adult patients (n = 92) and carriers of CF (n = 50) were recruited from one children’s and one adult NHS specialist CF centre. Health professionals (n = 70) were recruited via an e-mail invitation to relevant professional bodies. The key attribute affecting service user testing preferences was no miscarriage risk, while for health professionals, accuracy and early testing were important. The uptake of NIPD by service users was predicted to be high and includes couples that would currently decline invasive testing. Many service users (47%) and health professionals (55.2%) thought the availability of NIPD for CF would increase the pressure to undergo prenatal testing. Most service users (68.5%) thought NIPD for CF should be offered to all pregnant women, whereas more health professionals (68.2%) thought NIPD should be reserved for known carrier couples. The implications for clinical practice are discussed.Cystic fibrosis (CF) is an autosomal recessive disorder caused by mutations in the CFTR gene. The prevalence is one in 2500 to one in 3500 live births, with a carrier frequency of one in 25 for those with Northern European ancestry [1]. While CF impacts many body systems, the lungs and digestive system are particularly affected. People with CF suffer frequent chest infections, resulting in progressive lung function decline and ultimate respiratory failure. Multi-disciplinary care is vital, focussing on slowing lung function decline, the maintenance of adequate nutrition and psychosocial support, with lung transplantation as a potential ultimate treatment option [2]. Outcomes for people with CF continue to improve, and median survival, which is currently 43 years in the United Kingdom (UK), has significantly improved as a result [3,4].Testing for CF in pregnancy can be used to plan and prepare for the birth of an affected child or for the option of terminating the pregnancy. People who are carriers of CF and those affected with CF value the availability of prenatal diagnosis for couples with a high genetic risk of having a child with CF [5]. However, prenatal testing for CF is currently reliant on invasive diagnostic tests (chorionic villus sampling or amniocentesis), which have a risk of miscarriage of around 0.5% to 1% [6], and is not performed before 11 weeks in pregnancy in the UK. In the near future, prenatal testing for CF could be performed using non-invasive prenatal diagnosis (NIPD) based on the analysis of cell-free DNA (cfDNA) in maternal plasma. NIPD only requires a maternal blood sample and, thus, avoids the risk of miscarriage associated with invasive diagnostic tests and can be performed early in pregnancy (7–9 weeks). NIPD has been shown to be successful for the diagnosis of CF in cases where parents carry different CF mutations, where it has been used to determine the presence or absence of the paternal mutation [7,8,9]. When parents carry the same mutation, NIPD is more difficult, due to the high background of maternally-derived mutant alleles. New technologies, such as digital PCR and next generation sequencing (NGS), are bringing the possibility of using NIPD for CF closer to clinical implementation by allowing the quantification of normal and mutation alleles (for a review, see [10]).Research examining stakeholder views is critical for the development of implementation strategies for NIPD for CF and other single gene disorders. Discrete choice experiments (DCE) are a valuable tool for exploring stakeholder preferences and understanding the drivers of healthcare decisions [11]. A DCE provides an opportunity to explore real-life decision making by presenting a series of hypothetical healthcare options with differing attributes and asking participants to indicate which one they would choose, thus enabling insight into people’s preferences and the importance they place on particular attributes [12]. For example, DCEs have been used to examine preferences for screening and diagnostic tests for Down syndrome [13,14,15,16,17,18]. Our previous research has used qualitative approaches to explore the opinions of service users [19] and health professionals [20] around NIPD for sickle cell disease, thalassaemia and CF. The current study utilises a larger scale quantitative approach that incorporates a DCE to compare the views of patients with CF and carriers of CF with those of health professionals who provide counselling for prenatal testing for CF (genetic counsellors and clinical geneticists). Here, we explore the preferences for the key attributes of diagnostic tests for CF, examine views on NIPD, including expected uptake and how testing should be offered, and assess differences in preferences between stakeholders, to inform strategies for appropriate implementation.Ethics approval was obtained from the National Research Ethics Service Committee (10/H0714/3). The study design and analysis followed DCE guidelines [12,21,22].Data was collected from three groups of participants: (1) adult patients with CF; (2) carriers of CF; and (3) health professionals who offer prenatal testing for CF (genetic counsellors and clinical geneticists). Adult patients and carriers of CF were recruited from one children’s and one adult NHS specialist CF centre: the Cystic Fibrosis Unit at Great Ormond Street Hospital (GOSH) and the West Midlands Adult Cystic Fibrosis Centre at Birmingham Heartlands Hospital. This was a convenient sample drawn from English speaking patients and carriers of CF aged 18 or over who were attending the specialist CF centre for a clinical appointment. Participants were asked to anonymously complete the questionnaire while waiting to see a clinician or to complete it at home and return it via reply-paid post. Details of those given a reply-paid envelope were not recorded, so follow-up was not possible. Health professionals were recruited via an invitation to participate sent by e-mail with a link to the online questionnaire to the member lists of the Association of Genetic Nurse Counsellors (AGNC) (300 recipients) and the Clinical Genetics Society (CGS) (430 recipients).The questionnaire comprised three sections: (1) DCE choice sets; (2) structured questions about prenatal testing and NIPD; and (3) demographic questions. Attributes for the DCE component of the questionnaire were selected following a series of focus groups with carriers of single gene disorders, where one of the topics raised for discussion was the perceived importance of particular attributes of prenatal tests [19]. The three attributes people felt to be most important were safety, accuracy and time in pregnancy when the test result is received. These attributes form the basis of the DCE. The levels selected for safety (a small risk of miscarriage or no risk of miscarriage), accuracy (90%, 95%, 98% or 100%) and time of test results (8 weeks, 10 weeks, 12 weeks or 14 weeks) represent clinically feasible ranges. The DCE design follows the approach of Street and Burgess [23]. Two attributes had four levels and one attribute had two levels (Supplementary Material, Figure S1A). The number of possible combinations of attributes and levels was statistically reduced from 32 (42 × 21) to eight scenarios using an orthogonal fractional main effects design [24], and a shift of one level was to create eight additional scenarios. The two sets of scenarios were then randomly paired to form the eight choice sets. All levels of each attribute occur with equal frequency (level balance), and within each individual choice set, there is no overlap in attribute levels (minimal overlap). One of the choice sets had a clearly superior test as an option, and this was used as an internal consistency check. The questionnaires for adult patients with CF and carriers of CF asked which test they would prefer to have, and the questionnaires for health professionals asked which test they would prefer to offer. Participants were asked to choose between Test A, Test B or neither (Supplementary Material, Figure S1B). Inclusion of the neither option makes the choice more realistic.The structured questions around prenatal tests included ranking five attributes of prenatal tests (early testing, accuracy, financial cost, safety and comprehensive information) in order of importance and views on prenatal testing for CF, including views on the pressure to have prenatal testing for CF and who should be offered NIPD for CF. In addition, the service users were asked whether they have had or would have an invasive test for CF and if they would have NIPD if it became available. Demographic questions for service users included age, gender, ethnicity, education and number of children. Demographic questions for health professionals included job title, years in role, age and gender. For the questionnaire, NIPD was described as follows: “NIPD is a non-invasive test that will allow us to use a normal blood sample from the mother’s arm to determine whether or not the baby has CF. Because it is a blood test, there is no risk of miscarriage. Non-invasive tests to identify CF are being developed and are not currently available”. The questionnaires took approximately 20 min to complete. Questionnaires were piloted with 20 carriers of CF to determine whether they could be readily understood, and participants were asked if there were any other important attributes of prenatal tests that were not covered in the questionnaire. No changes were made following the pilot.The DCE preference data were analysed using a conditional logit regression model [25] that included a constant term to reflect the “neither” option [26]. For data entry, the levels for accuracy and time of results were mean centred, and the risk of miscarriage was effect coded. The sign (+ or −) of the coefficients generated in the regression analysis indicates the direction of the preference for each attribute. We anticipated positive coefficients for accuracy and no miscarriage risk, as we expected participants to prefer tests with greater accuracy and safer tests. We anticipated a negative coefficient for the timing attribute, as we expected participants to prefer an earlier test. The preferences of adult patients with CF and carriers of CF were compared to each other and to those of health professionals. In addition, we determined which participants considered multiple attributes when choosing between tests (“traders”) and those who made choices on the basis of one attribute only (“non-traders”). To explore the trade-offs that participants were willing to make between test attributes, we calculated the marginal rates of substitution (MRS) as the ratio of the coefficients of two attributes. The MRS allows direct assessment of how much of one attribute participants are willing to trade for one unit of another attribute and enables a comparison of different attributes on a common scale [21].Other analyses involved descriptive statistics on single items. On the agreement scale, “Strongly agree” and “Agree”, as well as “Strongly disagree” and “Disagree” were collapsed together to yield a binary index of agreement. The relationship between variables was examined using a chi-square test. For all tests, p-values < 0.05 were considered statistically significant. The software package Stata 10.0 [27] (StataCorp., College Station, Texas, TX, USA) was used to perform all analyses.The response rate for patients recruited through the adult CF centre was 91.59% (98/107) and 83.64% (46/55) through the children’s CF centre. The response rate for health professionals was 7.3% (22/300) through the AGNC mailing list and 11.2% (48/430) through the CGS mailing list. Questionnaires were excluded if the consistency question was not answered as expected (patients n = 1; health professionals n = 0) or if the respondents did not complete the choice set (patients n = 1; health professionals n = 0). Consequently, a total of 142 service user and 70 health professional questionnaires were included in the analysis. Demographic information for health professionals and service users is summarised in the Supplementary Material, Tables S1 and S2, respectively.Both service users and health professionals prefer a test with greater accuracy, early testing and no risk of miscarriage (Table 1). These results meet the a priori expectations and, thus, support the theoretical validity of the models. All coefficients were statistically significant for both groups. The comparison of service user and health professional regression results shows a statistically significant difference between the coefficients for all three attributes (Table 1). The comparison of people affected with CF and people who are carriers of CF indicated that people affected by CF placed a greater emphasis on safety, but there was no significant difference for accuracy or timing (Table 2). For those affected with CF, the comparison based on gender found that males placed a greater emphasis on safety, but there was no significant difference for accuracy and timing (Table 2). There were not sufficient males who were carriers of CF to allow for a comparison of men and women for the carriers of CF. The participants’ willingness to trade between the attributes was considered (Supplementary Material, Table S3). Notably, 48.9% of people affected with CF and 40.0% of carriers of CF chose tests based solely on there being no miscarriage risk.Conditional logit analysis regression results for service users and health professionals.CI, confidence interval; a The number of observations = 3408; pseudo-R2 = 0.4966; b The number of observations = 1695; pseudo-R2 = 0.4746.Conditional logit analysis regression results to compare service user subgroups. CF, cystic fibrosis.CI, confidence interval; * Coefficient significant at <0.05; All other coefficients significant at <0.0001; a The number of observations = 2208; pseudo-R2 = 0.4961; b The number of observations = 1224; pseudo-R2 = 0.5221; c The number of observations = 1032; pseudo-R2 = 0.4234; d The number of observations = 1176; pseudo-R2 = 0.5692.Calculation of the MRS confirmed the service user’s strong preference for a test with no risk of miscarriage. Service users were prepared to wait longer and accept lower accuracy compared to health professionals for a test that had no risk of miscarriage (Table 3).Marginal rates of substitution.Participants were asked to rank five test attributes in order of importance: early testing, accuracy, financial cost, safety and comprehensive information (Supplementary Material, Tables S4 and S5). Rankings supported the regression results, with 62% of service users ranking safety highest, whereas 81% of health professionals ranked accuracy as the most important attribute. Cost was ranked lowest by both groups.Service users were asked about their willingness to have prenatal testing for CF (Table 4). More than half of the participants (56.5%) said they would not have an invasive diagnostic test for CF. Amongst those that said they would have an invasive test, the most common reason given was “to plan and prepare for the possible birth of a baby with CF” (62.3%), with a smaller proportion (32.1%) having testing to “help make a decision about whether or not to continue the pregnancy”. Over half of the participants said they would never have an invasive test because of the risk of miscarriage. When asked whether they would have NIPD for CF if it was available, 94.9% of participants said they would and 90% said that they would be prepared to pay for the test. Around 10% of respondents would not be prepared to pay for NIPD. There was no significant difference between the responses of those affected with CF and those who were carriers of CF.Service user uptake of prenatal testing. NIPD, non-invasive prenatal diagnosis.Service users and health professionals were asked their views on the pressure to have prenatal testing for CF and whether the introduction of NIPD would increase this pressure (Table 5). The majority of both service users (60.6%) and health professionals (74.6%) thought that there was no pressure on couples at risk of having a child with CF to have prenatal testing. Service users who felt there was pressure to have testing reported that the three main sources of this pressure were a partner, family members and health professionals. Approximately half of the service users (47.0%) and half of the health professionals (55.2%) thought that the pressure to have prenatal testing would increase if NIPD for CF became available. There were no significant differences between the responses of health professionals, people affected with CF or people who are carriers of CF.Views on the pressure to have prenatal testing.* Participants could choose up to three responses.Service users and health professionals were asked who they thought should be offered NIPD for CF (Figure 1). The majority of health professionals (68.2%) thought that NIPD should only be offered to pregnant women who are known to be carriers of CF, whereas the majority of participants who are affected with CF (60.1%) or carriers of CF (76.9%) thought NIPD for CF should be offered to all pregnant women.Who should be offered NIPD for CF?In this study, we have examined the preferences of potential service users and health professionals for prenatal tests for CF. A DCE was designed to explore the relative importance placed on key attributes of hypothetical diagnostic tests for CF, taking into account the key clinical features of NIPD relative to invasive tests. While both service users and health professionals preferred safe tests with high accuracy that were conducted early in pregnancy, differences between groups arose in the emphasis placed on each of these attributes. Service users were prepared to wait longer and accept lower accuracy than health professionals if the test had no risk of miscarriage. The importance of safe testing for service users was further highlighted, as approximately half of this group did not trade on the test choices and chose only those tests that had no risk of miscarriage. These findings are analogous to those of a DCE study looking at preferences for attributes of invasive and non-invasive tests for Down’s syndrome, wherein accuracy was the key driver of decisions for health professionals and safety was most important for pregnant women [18]. Health professionals offering NIPD for CF in the future need to be aware that their views about the importance of test attributes may differ from the views of their clients. As safety is such a clear priority when service users are making decisions about whether to have prenatal testing, care must be taken to ensure that the other attributes of NIPD, including the disadvantages, are presented.More than half of the service users indicated that they would not have invasive testing for CF and that the risk of miscarriage was a barrier to testing. In addition, our findings, like those of other studies [5,28,29,30,31,32], indicate that while many couples who would have invasive testing for CF make this choice to guide decisions about termination of pregnancy, a large proportion take up testing so that they can plan and prepare for the birth of a baby affected with CF and would not consider termination. Accordingly, over 90% of participants said they would have NIPD for CF if it was offered. Overall, these results suggest that uptake of NIPD is likely to be high and will include many couples who would currently decline invasive testing due to the risk of miscarriage and a large proportion who would want testing for information only. This supports the findings of qualitative studies, which also suggested that uptake of prenatal testing for single gene disorders would increase if NIPD became available [19,20]. The high predicted uptake for NIPD does, however, need to be interpreted with caution, since predicted uptake may differ from actual uptake, as has been reported in other studies of genetic testing [33,34,35]. As such, research to monitor actual uptake when NIPD for CF is implemented will be important.Other studies have shown that reproductive choices around prenatal diagnosis for CF and termination of pregnancy change over time, and what people said they would do was often different from their final decisions [29]. This notion of reproductive choices being dynamic is particularly important to note if a large proportion of couples who would not previously have had prenatal testing take up NIPD, as many may find themselves making a decision about the termination of pregnancy, which they would not have been confronted with in the past. For these reasons, it will be important to take time in pre-test counselling to talk through the implications of the test and the possibility that people may change their mind about the termination of pregnancy when the test result is given. It is also critical that individualised support is available through post-test counselling.One of the concerns most often raised about NIPD is that people may feel pressured to have the test merely because it is easy to do and has no risk of miscarriage [36,37,38]. Indeed, previous research around NIPD for the single gene disorders, CF, sickle cell disease and thalassaemia, highlighted concerns from both potential service users and health professionals that the perceived ease of a blood test may bring increased pressure to have diagnostic testing during pregnancy [19,20]. In the current study, this concern was again voiced, with the majority of health professionals (55.2%) and 43.5% of service users feeling that the availability of NIPD would increase the pressure to have a prenatal test for CF. In addition, the DCE results indicate that the option of a test with no risk of miscarriage is highly influential. Health professionals offering NIPD for CF need to be conscious of the potential for NIPD to engender feelings of pressure and take care when counselling to ensure that this is not the motivation for prenatal testing. Being aware and willing to adjust approaches to counselling is particularly important, as service users cited health professionals as one of the three main sources of pressure to have prenatal testing. In particular, it is important not to “sell” NIPD by virtue of its safety, so that couples can make prenatal testing choices in line with their personal beliefs and values.There was a significant difference between service user and provider views on who should be offered NIPD for CF, with service users suggesting NIPD should be offered to all pregnant women and health professionals feeling this test should be reserved for known carriers. Further research is needed to understand the nuances of this difference. It is possible that health professionals may be considering wider issues, such as cost to the NHS. Moreover, service users may be responding to the fact that there is no carrier screening programme for CF in the UK and carrier status is often not picked up until after the birth of a baby with CF, as 95% of carriers have no family history of the condition [39]. Prenatal carrier screening for CF is recommended for all pregnant women in some countries, including the U.S. [40] and Australia [41], but is not routinely offered in the UK. The UK’s National Screening Committee is currently reviewing carrier screening for CF in pregnancy [42]. Previous research conducted in the UK has shown that people affected with CF and parents of children with CF support population carrier screening during pregnancy [43]. The cost of NIPD would be a key factor in determining whether the test could be offered to all pregnant women. In addition, it is not yet clear how many CF mutations it will be possible to test for with NIPD and whether this will match current carrier screening panels that test for 23 different CF mutations.A number of issues may limit the generalizability of our findings. Most notably, the response rate for health professional recruitment was low (9.7%). However, this response rate is equivalent to similar studies recruiting health professionals by e-mail invitation to member lists of professional bodies [44]. The low response rate may mean there is a sampling bias towards those with pre-existing interests or particular concerns about NIPD, and as such, the responses may not represent the wider views of genetic specialists. The response rate for potential service user recruitment was high for both people affected with CF and people who are carriers of CF. However, while approximately half of the participants who were affected with CF were men, only a small number of men who are carriers of CF were recruited. This may impact findings, as there is evidence to suggest that men and women may have conflicting views and can take on different roles in reproductive decision making about CF [5,32]. Gender differences were in fact seen in this study, as men affected with CF were found to place a greater emphasis on test safety than women affected with CF. Infertility and the need for assisted reproductive technology for men who are affected with CF are likely to be major factors in their additional emphasis on choosing safe prenatal tests; however, further research to explore the motivations is needed. It would also be valuable to see if this gender-based difference is present in carriers of CF. In addition, as our recruitment focussed on adult and paediatric CF clinics, we are missing the views of parents of children with CF in the case where the child has died. In addition, only a small number of carriers of CF who do not have children were recruited, and the views of this group may differ to those of carriers with children who may have more direct experience of living with CF. As with any stated preference study, the choices made by participants do not necessarily reflect the choices that would be made in real life. The DCE only included three attributes of prenatal tests, when real-life decisions must incorporate many more considerations. In addition, the DCE design does not explore the reasoning behind the choices made and give insight into how the tests were perceived. Finally, as discussed earlier, participants’ stated uptake of NIPD is hypothetical and may not necessarily correlate with actual uptake.Successful implementation of NIPD for CF and other single gene disorders must consider the views and preferences of a range of stakeholders. In this study, we found that potential service users and health professionals place different values on the test attributes of safety, accuracy and timing when making decisions about prenatal testing for CF. Implementation of NIPD for CF into clinical practice must consider these differences to ensure that the needs of all stakeholders are met. It was clear that when making decisions about testing, service users placed great emphasis on test safety. While it is likely that as a safe test, NIPD will be welcomed by couples and uptake will be high, pre-test counselling must include issues beyond safety to address the concerns raised regarding the availability of NIPD increasing the pressure to undergo prenatal testing. Even for service user groups who are clearly very familiar with the condition, pre-test counselling and informed consent processes that allow time for reflection are needed to make certain that attributes of the test beyond safety are discussed and that the implications of the result are considered. The importance of thorough pre- and post-test counselling emphasises the need to maintain specialist care pathways for the delivery of NIPD, so testing is offered by health professionals specifically trained in counselling for prenatal testing. Future research at the time of clinical implementation will be required to guide continued service delivery, as predicted uptake may not reflect what actually happens in practice. In addition, discussion and debate is needed as to how NIPD is offered, as service users are clearly in favour of testing being available to all pregnant women, regardless of whether the carrier status of CF is known.We are grateful to the parents of children with CF, adults with CF and health professionals who participated in the study. We also thank the staff assisting with the research at each hospital site; in particular, Charlie Dawson at Great Ormond Street Hospital and Yvonne Newey at Birmingham Heartlands Hospital. This manuscript presents independent research funded by the National Institute for Health Research (NIHR) under the Programme Grants for Applied Research Programme (RP-PG-0707-10107) (the “RAPID” project). Lyn S. Chitty is partially funded by the Great Ormond Street Hospital Children’s Charity and the NIHR Biomedical Research Centre at Great Ormond Street Hospital. The views expressed are those of the authors and not necessarily those of the NHS, the NIHR or the Department of Health.The authors declare no conflict of interest.

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+This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).Objective: Post-operative laminectomy wounds are frequently accompanied by seromas. Post-operative wound drainage may be colonized or infected. The differentiation of wound colonization from infection is difficult for non-infectious disease physicians. Methods: External chart reviewers classified 31/1531 laminectomies (over three years) as post-operative wound infections. We re-evaluated these cases using infectious disease criteria, i.e., while pathogens may be cultured from both colonized and infected wounds, only wound infections have a purulent discharge with abundant white blood cells (WBCs) on Gram stain. Colonized wounds have positive wound cultures but no/few WBCs on Gram stain. Results: We found only 11/31 actual wound infections, the remainder were not bona fide wound infections, but were colonized seromas. Conclusion: Post-laminectomy colonized seromas that are culture positive for one or more organisms often mimic wound infections. In the era of public reporting of nosocomial infections, it is important that external reviewers differentiate colonization from infection to provide regulatory agencies with accurate data.Laminectomies are among the most complicated orthopedic/neurosurgical procedures. Complicated surgical spine procedures are technically difficult and are of long duration which increases the potential for infectious complications, i.e., deep wound infections [1,2,3,4]. Post-operative laminectomy wound infections may be due to several procedure related factors, e.g., procedure duration/complexity and may be due to host related factors, e.g., age, weight, diabetes and host defense status. With a good aseptic surgical technique, prophylactic antibiotics are given to minimize post operative wound infections [5,6,7,8,9]. We became interested in clinically differentiating colonized from infected seromas, which are a particular problem in post-laminectomy surgery, to avoid needless antibiotic therapy and to accurately classify these wounds.During the past three years (2010–2012) a total of 1531 laminectomies were performed in our 600 bed university-affiliated teaching hospital. Non-infection control (IC) and non-infectious disease (ID) external chart reviewers reported a relatively high incidence of wound infections post-laminectomy. Since the number of cases seemed unusually high, we re-reviewed 31 cases classified as post-laminectomy wound infections to determine the number of actual infections. In our review, the duration of the case, age, weight, comorbidities, history of previous infections, individual surgeons, laminectomy location and pre-operative antibiotic regimens were analyzed. In addition, microbiological data were correlated with the clinical findings and pre-operative antibiotic regimens.Thirty one potential post-laminectomy wound infections identified over a three year period were reviewed; of these only 11 cases met infectious disease criteria for wound infections (Table 1) [10]. Among the 11 post-laminectomy wound infections, we found no procedure or individual surgeon related common denominators. Furthermore, a variety of wound isolates were identified; Klebsiella pneumoniae, methicillin resistant Staphylococcus aureus (MRSA), methicillin sensitive Staphylococcus aureus (MSSA), coagulase negative staphylococci (CoNS), and E. coli. There was no relationship between the spectrum of the prophylactic antibiotics used and organism cultured from colonized or infected wounds (Table 2, Table 3 and Table 4).Winthrop-university hospital, recent experience with potential post-laminectomy wound infections.Potential post-laminectomy wound infections (2010–2012).* Some cases had multiple isolates; MSSA = methicillin sensitive Staphylococcus aureu; MRSA = methicillin resistant Staphylococcus aureus; VRE = vancomycin resistant enterococci; CoNS = coagulase negative staphylocci.Differential diagnostic features of post-operative laminectomy wounds: colonization vs. infection *.* Any post-operative patient may have fever, leukocytosis, wound erythema/induration not related to wound infection. WBCs = white blood cells.Skin/Wound organisms that should be considered as commensals/colonizers †.† Considered a bona fide infection only if a purulent wound exudate has abundant WBCs on Gram stain.Skin colonization is the rule in the microbial milieu of the hospital. Seromas are common post-laminectomy and positive wound or seroma cultures often represent colonization by skin organisms. Non-infection control and non-infectious disease external chart reviewers, who evaluated 31 patients, were often confused by discordant seroma fluid microbiology results, i.e., Gram stain (number of white blood cells (WBCs) and organisms) and wound/fluid culture results.The most common cause of misdiagnosis of post-operative laminectomy wound infections was colonized seromas. External chart reviewers (non-infection control and non-infectious disease personnel) had difficulties in correlating clinical and microbiologic data to diagnose or rule out post-laminectomy wound infections. Post-operatively, the skin surrounding incisions may be erythematous, but unless infected is not warm or tender. Furthermore, unlike non-laminectomy post-operative wound infections, laminectomies were commonly complicated by seromas. Colonized seromas draining through the wound may mimic wound infection, but seroma fluid is clear or serosanguineous, but not purulent.Colonization of wounds and seromas is common but is not preventable by utilizing pre-operative prophylactic antibiotics. Wound Gram stains demonstrating few/no polymorphonuclear cells (PMNs) with positive wound cultures from a body fluid without the clinical criteria of infection are not indicative of wound infection. When the signs of infection are absent, e.g., no surrounding skin erythema, warmth and tenderness, microorganisms cultured from non-purulent seroma fluid indicate colonization but not infection.Fever also caused diagnostic confusion for external reviewers in attributing fever/leukocytosis in post-laminectomy patients with positive wound cultures to wound infection. However, post-laminectomy patients often have low-grade fevers due to a variety of other non-wound related infectious or non-infectious causes, e.g., inflammation, atelectasis, phlebitis. In our experience, external chart reviewers have difficulty in interpreting and correlating wound cultures results with microbiologic results, i.e., wound culture vs. wound Gram stains to differentiate post-laminectomy wound infections from colonized seromas.All authors declare no conflict of interest.

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+This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).Stress urinary incontinence is a significant social, medical, and economic problem. It is caused, at least in part, by degeneration of the sphincter muscle controlling the tightness of the urinary bladder. This muscular degeneration is characterized by a loss of muscle cells and a surplus of a fibrous connective tissue. In Western countries approximately 15% of all females and 10% of males are affected. The incidence is significantly higher among senior citizens, and more than 25% of the elderly suffer from incontinence. When other therapies, such as physical exercise, pharmacological intervention, or electrophysiological stimulation of the sphincter fail to improve the patient’s conditions, a cell-based therapy may improve the function of the sphincter muscle. Here, we briefly summarize current knowledge on stem cells suitable for therapy of urinary incontinence: mesenchymal stromal cells, urine-derived stem cells, and muscle-derived satellite cells. In addition, we report on ways to improve techniques for surgical navigation, injection of cells in the sphincter muscle, sensors for evaluation of post-treatment therapeutic outcome, and perspectives derived from recent pre-clinical studies.Stress urinary incontinence (SUI) is not only a major hindrance for any individual affected, but it is a very large social, medical, and economic burden to society [1]. SUI is associated with multifactorial pathologies [2,3], including structural changes in the muscle’s composition, loss of the muscle cells, a surplus of the collagenous fibrous connective tissue, changes in vasculature or enervation, and mechanical load. Current treatment strategies involve muscular training, electrophysiological stimulation, and pharmacological interventions to improve neural activation of the sphincter in female patients. For women, other therapeutic options include mechanical support of the urethra to increase sphincter function but require surgical application of slings or injection of bulking agents. Another problem of treatment of SUI with slings is that it can lead to complications, especially in the long term, and has raised concerns against this type of therapy [4]. Comparably, injection of bulking agents in the urinary sphincter muscle did not meet clinical expectations, although some benefit was noted [5]. For men, artificial urinary sphincter devices were introduced in the early seventies, more than forty years ago [6]. Although some success in treatment of iatrogenic male incontinence after prostatectomy was noted, the design of this type of product and of other mechanical prostheses are repeatedly changed, improved and re-designed, due to notable complications or insufficient long term treatment [7]. Therefore, one can conclude that although there is some improvement for the patients with current treatment regimens, they do not treat the cause of the disease: malfunction of the sphincter complex.Pre-clinical studies of SUI with stem/stromal cells or progenitor cells have yielded promising results [8,9,10,11,12,13,14,15] suggesting that a cell-based regimen could potentially treat the cause of SUI for some patients. Although experts suggest that cell-based therapies for treatment of SUI are at an experimental stage [16,17,18], recently, several centers reported results on clinical trials [19,20,21,22,23,24,25] using different types of cells including muscle derived stem cells [19,21,25], mesenchymal stromal cells (MSC) [24,26], or myoblasts and fibroblasts [23]. Some of these trials did not meet the quality measures expected from proof-of-principal studies and were retracted [24,27]. However, overall, based on clinical trials [21,22] and positive results from pre-clinical studies involving satellite cells or adipose-derived MSC [11,12,13,14,15], recently a clinical phase II study was initiated [26]. However, it is still unclear what the best approach to cellular therapy for treatment of SUI is. In order to explore the potential of any cell-based therapies for treatment of SUI, it is critical to: (i) Define the optimal type of cell required for regeneration of the sphincter muscle; (ii) Develop gentle but precise surgical techniques to apply the cells without interfering with its already weakened function; and (iii) Improve strategies for exactly determining if the muscular function has improved during follow-up treatment.Here, we discuss three different types of cells: bone marrow-derived mesenchymal stromal cells, urine-derived stem cells, and muscle-derived satellite cells, and their potential to strengthen the urinary sphincter muscle. In addition we briefly report on specific techniques for surgical navigation and stem cell injection methods, as well as on the development of sensors for monitoring the sphincter muscle post-stem cell treatment, and on a pre-clinical model system.Mesenchymal stromal cells (MSC), also called multipotent stromal cells or mesenchymal stem cells, were described for the first time by Friedenstein and colleagues some 40 years ago as fibroblast precursors isolated from mouse bone marrow [28]. Later, MSCs from bone marrow were described as osteogenic precursors [29,30,31,32] and, in 1999, the multi-lineage differentiation capacity of human bone marrow-derived MSC (bmMSC) was reported [33]. MSCs were also detected in adipose tissue [34], term placenta [35], and other sources [36,37]. MSCs modulate immune responses [38,39,40,41], and may serve as a source for growth factors during wound healing [42,43] and tissue regeneration [43,44]. The regenerative potential of MSC was investigated in different tissues, including bone [45], cartilage [46], cardiac tissue [47,48], and MSCs even facilitated outgrowth of neurons [49]. The success reported with MSC applications in the above mentioned studies motivated research to utilize MSC from adipose tissue (called adipose-derived stem cells, ADSC) [14,15] or from bone marrow [50,51,52,53,54] for treatment of urinary incontinence in pre-clinical in vivo studies [14,15,51,52]. Post-treatment assessments in these feasibility studies ranged from one week [52] to 13 weeks [53], included different types of cells (ADSC or bmMSC), and different models of incontinence [9]. Therefore, comparison of results reported in these studies must be interpreted with care. However, overall, the application of mesenchymal stromal cells in animal models suffering from experimentally induced urinary incontinence seemed to be beneficial. However, larger and randomized cohorts, and longer follow-up are required to acquire a clearer picture on the risk versus benefit ratio.In terms of the cell types used, application of ADSC or bmMSC may yield several advantages over therapies with muscular progenitor cells including satellite cells or myoblasts in this clinical context: Autologous ADSC or bmMSC can be obtained without intolerable side effects, in sufficient numbers and with an adequate cell quality from patients suffering from SUI (Table 1). Furthermore, bmMSC or ADSC may be applied as undifferentiated progenitor cells, or after myogenic differentiation, in vitro (Figure 1). However, the efficacy of myogenic differentiation of both, bmMSC and ADSC in either smooth or striated muscle (like) cells under GMP-compliant conditions, is not yet state-of-the-art. Selected features of human stem or progenitor cells suitable for regeneration of the urinary sphincter in the context cell based therapies for stress urinary incontinence (* SMC: smooth muscle cell).Another benefit of MSC is that, depending on the numbers of cells to be injected (also see Section 2.2.), only a short period of time for expansion MSC is required. Routinely, approximately 1 × 107 total mononuclear cells can be obtained from an average bone marrow aspirate (mean 18 ± 3 mL). This will yield more than 107 proliferation- and differentiation-competent MSC in less than two weeks of in vitro culture. For clinical application, these cells have to be expanded under GMP-compliant conditions [61,62]. Comparably, from approximately 6 g of human subcutaneous adipose tissue, approximately 1 × 105–1 × 106 ADSC can be obtained in one week of in vitro cultivation. In some cases, MSC derived from term placenta (pMSC) might be an interesting alternative cell source [35]. Within two to three weeks of expansion, about 1 × 107 pMSC can be generated from 100 g of human term placenta. As childbirth is a significant risk factor for SUI, application of autologous MSC from the endometrial part of the term placenta might eventually become a preventive SUI regimen [63]. Therefore, the regenerative potential of MSC from human term placenta has been investigated in more detail, recently, as well [35,41,64,65].Selected progenitor cells, sources, and myogenic differentiation. Suitable progenitor cells, sometimes also referred to as “stem” cells, can be isolated from different sources, such as bone marrow, adipose tissue, striated muscle, or urine, to generate bone marrow-derived mesenchymal stromal cells (bmMSC), adipose-derived stem cells (ADSC), satellite cells (SC), or urine-derived stem cells (USC). After a primary expansion the quality of bmMSC, ADSC, and USC is explored by adipogenic (“A”), chondrogenic (“C”) and osteogenic (“O”) differentiation of the stem cells, and by detection of the expression of the inclusion/exclusion cell surface antigens [34,37,55]. For SC expression of lineage-specific marker antigens is investigated (not shown). The bmMSC, ADSC, SC, or USC are then either applied as progenitor cells, or incubated in differentiation media to generate smooth muscle cells (SMC), for example, from bmMSC, ADSC or USC, or myoblasts (MB) and multinucleated myofibres from SC.Of late, a seemingly unusual source of progenitor cells was described and its potential for regenerative regimens was explored [57,58,66,67,68,69]. Collecting urine samples over three consecutive days from adult males enabled researchers to investigate several protocols for harvesting urine-derived stem cells (USC), their preservation and storage, in vitro culture, and characterization and differentiation capacity to generate mature cells with urothel-like and a smooth-muscle like phenotypes [57]. Ex vivo, human USC express a panel of cell surface markers such as CD44, CD73, CD90, and CD105, but lacked CD31, CD34, and CD45, suggesting that these cells are of mesenchymal origin and seem closely related to MSC including ADSC (Table 1). CD73 and CD90 were found on preserved USC as well [57]. USC incubated in differentiation media enriched with transforming growth factor-β (TGF-β) plus platelet-derived growth factor-BB (PDGF-BB) induced smooth muscle cell differentiation (Figure 1) as shown by expression of desmin, calponin, smoothelin, myosin, and α-smooth muscle actin [57]. In addition to generation of smooth muscle-like cells in vitro [57], USC were also tested for their in vivo regeneration capacities and the generation of endothelial cells (as investigated by expression of CD31 and von Willebrand factor) and differentiation to striated muscle-like cells (as explored by detection of desmin, MyoD, and Myf-5) [58]. Furthermore, incubation of USC with epidermal growth factor (EGF) induced urothelial cells that expressed the urothelial markers uroplacin, cytokeratins-7, -13, and -20, and the epithelial antigens cingulin and E-cadherin [57]. As the USCs were cloned prior to induction of differentiation, the USCs may be capable of differentiation along two distinct cellular lineages: (i) The mesenchymal lineage of cells, derived from the mesoderm; and (ii) The epithelial cell lineage, derived from the endoderm. However, such an adult progenitor cell plasticity or the trans-differentiation of adult somatic cells across germ line borders are a matter of debate [70], and some people would argue that, at least in bulk USC preparations, stem cells from different germ lines are collected in the starting samples, enabling preferred outgrowth of either the mesenchymal/muscular or the epithelial/urothelial cells. However, by all means, this rather academic discussion distracts from the clinical potential of USC. If applied successfully to ameliorate urinary incontinence in pre-clinical studies, USC may be an interesting alternative to bmMSC, ADSC, or satellite cells (see below) in future urological or gynecological regenerative medicine.The omega-shaped urinary sphincter muscle consists of smooth-muscle and striated muscle fibers [71]. Therefore, regeneration and/or performance of both parts of this muscle may be required to gain efficient and long lasting sphincter function. Differentiation of functional striated muscle cells from ADSC or MSC has been reported [14,50,72,73,74]. However, the efficacy in production of bona fide striated muscle cells from MSC in vitro is rather low. Additionally, the differentiation protocols presented to the scientific public so far either employ recombinant techniques [73], xenobiotic serum or growth factors in the media [72,73], cancerous components [14,50,52], or combinations thereof, to induce myogenic differentiation. A probably safer way to generate striated muscle tissue from progenitor cells may be through the isolation and expansion of satellite cells (SC), as these cells are the natural precursors for generation and regeneration of striated muscle tissue [59,60,75,76]. Satellite cells reside between the basal lamina and sarcolemma in muscle fibers of striated muscle tissue and are activated on demand by growth factors or other stimuli including hypoxia or injury to generate multinucleated myofibers. Therefore, the potential of satellite cells or muscle-derived cells for treatment of SUI has been explored in pre-clinical [11,77,78,79] and even clinical pilot studies [19,21,23,80]. In a study for management of male urinary incontinence, 46% of patients treated with autologous muscle-derived cells did not show improvement, but adverse effects were not at all observed [25]. However, in this study the patients received a mixed cell population, and only approximately 50% of the cells injected expressed typical muscular marker genes [25]. Follow-up in the clinical studies employing “muscular progenitor cells” ranging from one to four years also showed promising results [21,80]. However, failure of therapy in some of these cases may be associated with the individual blend or unclear cellular phenotypes of the cells injected. However, experts agree that striated muscle-derived stem cells or satellite cells do yield clinical benefit for patients suffering from SUI or other forms of incontinence. However, large numbers of satellite cells are required for the injection of cells for the treatment of incontinence. This may result in a severe impact on the healthy muscle at sites where satellite cells are harvested. Therefore some protocols for cell-based treatment of incontinence favor the use of stem or progenitor cells from other sources that yield less side effects, such as bone marrow or adipose tissue. Accordingly, the currently ongoing clinical phase II trial HULPURO employs autologous adipose tissue derived MSC [26], but not satellite cells (Table 1).The typical steps for cell therapy include the production of the cells, cell implantation, and, finally, following and measuring the treatment’s results. A general trend towards minimally invasive surgery is preferred and, therefore, surgeries are often performed by aid of an endoscope and other high-end imaging instruments [19,25]. Therewith, cell implantation in the urethral sphincter can be performed by a needle/syringe, which is manually pushed through an endoscope to the site of cell injection. From a technical point of view, this procedure using a linearly guided needle is fairly straightforward. However, accurate control of the position of cell injection and distribution of the cells within the sphincter muscle appears a demanding challenge. Furthermore, using needles for injection of cells inherits the risk of tissue damage, possibly accompanied by bleeding.Based on existing navigation systems for sphincter cell therapy, current research and development are being dedicated to develop novel navigation and cell injection methods for a more precise and less invasive treatment. The workflow for future treatments may look as follows: First, the exact individual anatomical conditions of the patient to be treated and the exact area for injections of the cells in the sphincter are recorded and marked out by MRI and ultrasound imaging (USI) to generate a virtual three dimensional “map”. During treatment, the exact position of the instruments can be visualized as an overlay of the endoscope image, and the “map” generated by the MRI, fused in real-time with the topical USI data. With a tracking system attached to the endoscope and the injection needle, the delicate sphincter muscle can be located and cells can be injected with high precision.Although optimal positioning of the injection needle is very important, a second aspect of cellular application in the urethral sphincter may be critical for the overall success of such a regimen as well: Optimal spatial distribution of the cells in the tissue itself. Thus far, this has not yet been thoroughly investigated for sphincter regeneration [81]. This is probably due to the fact that many pre-clinical studies employed rodents [11,14,15,51,52,53,54,77,82,83]. In such models, controlled studies comparing, e.g., injection of cells in one site of the sphincter versus several injections at different angles or positions, cell dose escalation studies [81], or combinations of cells and biomaterials cannot be addressed due to the anatomical size limitations in these animals. Therefore, these surgical aspects need to be addressed in larger animal models [84]. In addition, a gentle, yet not penetrative, cell injection technology might be helpful as well. In humans, the sphincter muscles measures only a few millimeters in width and thickness [71]. A needle may easily cut through the tissue and cause the cells to be delivered into the peritoneal space. To overcome this limitation of cell injections by needles, new cell application technologies may facilitate the cell application and, at the same time, improve clinical outcome. For example, one possible method would be to “shoot” cells through an endoscopic air pressure nozzle into the tissue. Variation of pressure and volume of the air impulse, design of duct, droplet size, and cell density in the injection fluid may allow to optimization of depth, density, and distribution of the injected cells.An objective and reproducible measurement of the sphincter muscle strength is crucial for the assessment of the overall effect of cell-based therapy. Further, a spatially resolved reconstruction of the sphincter muscle strength has the potential to allow the location of weak spots and facilitate the targeted application of stem cells. Currently, no methods are available to measure the spatial distribution of the sphincter muscle strength. Studies investigating the leak point pressure, which can be interpreted as sphincter muscles strength, and its connection to incontinence, exist [85]. However, as leak point pressure is only measured via an intravesical catheter, no data exists on the spatial distribution of the sphincter muscle strength. A novel approach, based on a mathematical model of the urethra and microtip catheter, may overcome this problem. Urethral pressure profilometry is a common tool in the diagnosis for urinary incontinence and can deliver data on the state of the sphincter muscle. To this end, a special catheter is inserted into the urethra and, while it is being slowly retracted, the pressure along the urethra is measured. Several types of catheters capable of measuring pressure inside the urethra are known [86]. One type, the microtip catheter, uses electric pressure transducers on the circumference to measure the local pressure exerted by the sphincter muscle through the urethra on the catheter. Usually, up to four pressure sensors are placed on the catheter’s circumference. However, raw data obtained by those catheters is often difficult to interpret diagnostically. Sensor noise may obscure details and possible angular fluctuations in the pressure profile may not be detected due to limited angular resolution of the sensor arrangement and gaps between the sensors. Additionally, different pressure levels were measured in different directions inside the urethra, the cause of which is still unclear [87]. As those differences cannot be explained through direction-dependent fluctuations of the muscle’s strength, one might have to consider them as artifacts caused by bending of the catheter [88]. An alternative to this approach may be to process the measured data through a mathematical model of the urethra to obtain the pressure profile on the outside of the urethra, which is exerted by the sphincter muscle. This approach can also measure different pressure levels in several directions inside the urethra. The first approach to a mathematical model is to describe the urethra as a linear-elastic isotropic hollow cylinder. The system of partial differential equations governing this problem is solved numerically. Therefore the model is discretized through the Finite-Element-Method (FEM). In order to solve this problem, boundary conditions have to be defined. As the catheter is placed inside the urethra, both pressure profile and displacement of the inner boundary are known. However, no information on the pressure profile and displacement on the outside is available. This leads to a so-called inverse problem, which violates at least one condition for a well-posed problem [89]. That means that the solution of this problem is numerically unstable, i.e., tiny variations in the input data cause arbitrarily large variations in the results. Therefore, special algorithms, which are able to recover stability of the solution, have to be developed to solve this problem.Inverse elasticity problems have been studied extensively in literature [90]. They have been used in some medical applications, including for instance elastography of artery walls [91,92,93]. However, to the best of the authors’ knowledge, inverse algorithms have not been used in urodynamics. Therefore, we propose a mathematical model and an inverse algorithm to reconstruct the spatial pressure profile on the outside of the urethra from measured pressure on the inside. Furthermore, due to the limited angular resolution of the sensor arrangement, data is not continuously available on the inside of the urethra. Hence, sensor positions and areal sensitivity characteristics have to be taken explicitly into account.In a second approach, experimental data can be used to test and validate the developed algorithms. Data is obtained for instance on a special test stand, which allows data collection under comparatively well-known and reproducible conditions (Figure 2). Measurements can be conducted with a custom-made microtip catheter with eight pressure sensors on the circumference allowing at the least double the angular resolution compared to standard devices. The test stand mechanically resembles the female urethra. A soft silicone tube emulates the urethra. Strings of different diameters can be wound around the tube in various configurations. They are tightened by electric motors and emulate the sphincter. With this setup, different pressure profiles can be created on the outside of the tube and measured by the catheter.The results of the inverse algorithm are post-processed to three-dimensional figures depicting the deformed shape of the urethra (Figure 3). A color map indicates the pressure level at any given point on the surface. With this method, physicians can easily pinpoint abnormalities and identify differences between before and after the stem cell treatment. CAD-Rendering of the test stand.3D-Representation of the deformed urethra model under pressure.Many of the currently used reconstructive techniques for treatment of SUI at this point mainly cure the symptoms, instead of functionally regenerating the tissue [94]. Application of regenerative or even differentiated cells directly into the urethral sphincter muscle could potentially recover the function of the sphincter muscle [26]. As tissue samples from patients treated for SUI with cells cannot be harvested without a risk to harm the injected sphincter muscle, mechanisms of such regenerative regimen have to be explored in detail in animal models, such as for instance minipigs (Figure 4). In large animal models the cells can be injected under visual control into the urethral wall using standard clinical instruments. In addition, large animal model allow a comparison of different patterns of cell injections: Cell injections in one site versus injections in several sites [25], variation of numbers of cells, and injection volume. The cells can be labeled by paramagnetic particles [95], fluorescent dyes [96], or by recombinant techniques prior to the injection [97]. This enables the investigators to follow the cells after injection in life animals and to localize them by MRI, and visualize the cells in vivo. In addition, cells labeled with fluorescent dyes can be localized easily ex vivo in tissue samples by microscopy. Moreover, due to the anatomical features of this model, urethra pressure profiles (UPP) in treated vs. untreated animals can be monitored again by standard clinical instruments to measure the urodynamic function. Cell application and functional read-out. (A) Transurethral injection of cells during cystoscopy procedure under visual control. Defined volumes of labeled cell suspension can be applied via a syringe (asterisk) and needle; (B) Urethral pressure profiles (UPP) can continuously be measured by a balloon catheter automatically retracted from the bladder to the rhabdosphincter and urethra.The goal of pre-clinical studies for cell based sphincter therapies is to develop safe and precise injection technologies and to investigate the behavior and fate of the cells injected. To come as close as possible to the real clinical situation one can even use human cells in a suitable animal model. This however may need some sort of immunosuppression or an immuno-deficient recipient [98]. Based on the hypothesis that human MSC may facilitate endogenous repair mechanisms of porcine sphincter muscles, regimens for mild yet sufficient immuno-suppression were developed. Thus, precise intramuscular application of undifferentiated human MSC for example into the porcine rhabdosphincter may lead to the induction of myogenic in vivo differentiation of the cells injected, or result in activation of resident repair mechanisms. This problem—MSC as regeneration promoting cells [43,99] or their functional integration by in vivo differentiation [10,54]—represents one of main challenges in realizing this xenogenic transplantation animal model. Moreover, assessing and controlling the fate of the cells after injection is an additional task for quality management and monitoring of the treatment. Achieving these objectives, together with the translation of the entire procedure to clinical routine therapy, will bring this stem cell-based regenerative therapy closer to patients suffering from incontinence compared to any other approach attempted before.Mechanical devices are not yet a satisfactory solution to treat stress urinary incontinence. Due to the demographic changes in Western societies, the incidence and prevalence of SUI are rising. Therefore safe and effective novel treatments are needed. Stem cell based therapies may tackle this problem.We thank Tanja Abruzzese, Sabine Maurer, and Leon Gustafson for excellent technical support of the MSC projects. Our own work, briefly mentioned in this review, was supported in part by grants from the German Research Council (DFG), the Federal Ministry of Education and Research (BMBF), and in part by institutional funding.The authors declare no conflict of interest.

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+This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).The editors of the Journal of Clinical Medicine would like to express their sincere gratitude to the following reviewers for assessing manuscripts in 2013:
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+            Almog, Nava
+            Drago, Denise
+            Hirai, Yuji
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+            Amadori, Dino
+            Economou, Athina
+            Houssiau, Frederic A.
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+            Antebi, Ben
+            Esteban, Andrés
+            Hubalek, Michael
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+            Bernal-Mizrachi, Carlos
+            Evans, Mark
+            Ingvild, Ingvild
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+            Bourin, P.
+            Facchini, Andrea
+            Josson, Sajni
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+            Bouza, Emilio
+            Fong, Daniel Tik-Pui
+            Kasten, Mary J.
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+            Bove, Geoffrey
+            Foroozan, Rod
+            Kim, Han Su
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+          
+            Bower, Chris
+            Frölich, J.
+            Kohn, Michael R.
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+            Brodowicz, Thomas
+            Füllen, Georg
+            Kusuma, Sravanti
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+            Broggini, Massimo
+            Galanter, Cathryn A.
+            Kwon, Tae Gyun
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+            Brufsky, Adam
+            Goetzl, Laura
+            Languino, Lucia
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+            Califaretti, Nadia
+            Goldstein, Benjamin I.
+            Laurie, Karen L.
+          
+          
+            Casimiro, Sandra
+            Gräs, Søren
+            Lee, Moon Soo
+          
+          
+            Cavoretto, Paolo
+            Gunetti, Monica
+            Li, Gang
+          
+          
+            Connelly, Kim
+            Hagmann, Sébastien
+            Lipton, Allan
+          
+          
+            Conover, Joanne
+            Halperin, John
+            Lu, Baisong
+          
+          
+            Cousens, Nicole
+            Harper, Gordon
+            Lungu, Codrin
+          
+          
+            Cunha, Burke A.
+            Hashmi, Farina
+            Luparello, Claudio
+          
+          
+            Dahlström, Lisen Arnheim
+            Haubitz, Sebastian
+            Lutz, Stephen
+          
+          
+            Davies, Robert D.
+            Hematti, Peiman
+            Magann, Everett F.
+          
+          
+            De Geest, Bart
+            Henry, David
+            Mccloskey, Eugene
+          
+          
+            Dennis, Kristopher
+            Hernigou, Philippe
+            Mlineritsch, Brigitte
+          
+          
+            Montgomery, Scott
+            Rincón, Olga Gómez del
+            Takebe, Naoko
+          
+          
+            Morris, Patrick G.
+            Rodriguez, Anne-marie
+            Teich, Steven
+          
+          
+            Mortara, Franco
+            Rosenblatt, Jon E.
+            Trial, Joann
+          
+          
+            Narod, Steven
+            Salgado, A. J.
+            Truong, Daniel
+          
+          
+            Ngim, Chin Fang
+            Schul, Wouter
+            Vagefi, Reza
+          
+          
+            Nilar, Shahul
+            Schuleri, Karl
+            Van Poznak, Catherine
+          
+          
+            OBrien, Louise
+            Selvaraj, Vimal
+            Verhagen, Ealm
+          
+          
+            Odejinmi, F.
+            Sesti, Francesco
+            Wang, Qun
+          
+          
+            Padfield, Gareth J.
+            Shaak, Thomas L.
+            Wang, Yun
+          
+          
+            Park, Won
+            Sonne, Susan C.
+            Watanabe, Shigeru
+          
+          
+            Pergament, Eugene
+            Sprick, Martin R.
+            Wilson, Kumanan
+          
+          
+            Pergolizzi, Stefano
+            Springett, Kate
+            Yasuhara, Takao
+          
+          
+            Polo, Jose
+            Sridharan, Shankar
+            Yennamalli, Ragothaman
+          
+          
+            Pontikoglou, Charalampos
+            Stoddard, Joel
+            Yu, Seongjin
+          
+          
+            Porpora, M. G.
+            Stoeger, Herbert
+            Zhang, Jian
+          
+          
+            Powles, Trevor
+            Subbulakshmi, Subbulakshmi
+            Zhang, Yong
+          
+          
+            Radisky, Derek
+            Sundar, Krishna M.
+            Zhu, Xuegong
+          
+          
+            Reed, Elizabeth
+            Sykes, Jane
+             
+          
+        
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+This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).This report evaluates whether classification tree algorithms (CTA) may improve the identification of individuals at risk for bipolar spectrum disorders (BPSD). Analyses used the Longitudinal Assessment of Manic Symptoms (LAMS) cohort (629 youth, 148 with BPSD and 481 without BPSD). Parent ratings of mania symptoms, stressful life events, parenting stress, and parental history of mania were included as risk factors. Comparable overall accuracy was observed for CTA (75.4%) relative to logistic regression (77.6%). However, CTA showed increased sensitivity (0.28 vs. 0.18) at the expense of slightly decreased specificity and positive predictive power. The advantage of CTA algorithms for clinical decision making is demonstrated by the combinations of predictors most useful for altering the probability of BPSD. The 24% sample probability of BPSD was substantially decreased in youth with low screening and baseline parent ratings of mania, negative parental history of mania, and low levels of stressful life events (2%). High screening plus high baseline parent-rated mania nearly doubled the BPSD probability (46%). Future work will benefit from examining additional, powerful predictors, such as alternative data sources (e.g., clinician ratings, neurocognitive test data); these may increase the clinical utility of CTA models further.Diagnosis of bipolar spectrum disorders (BPSD) in youth remains a difficult but important clinical responsibility. Assessment strategies are needed that are inexpensive, easy to implement, have good predictive power, and simultaneously combine multiple pieces of clinical information to increase or decrease the probability of BPSD. This presents a significant challenge because the optimal statistical methods for combining multi-faceted clinical information, such as logistic regression, are often difficult to implement in typical clinical practice and do not map clearly onto clinical decision making [1].In a recent paper by Fristad and colleagues [2], logistic regression analyses suggested decreasing incremental value in predicting BPSD using information from parent ratings of mania at screening and baseline, family history of parental mania, and parenting stress. This work suggested an initial assessment strategy that may be clinically useful for screening out individuals with low probability of BPSD and identifying individuals with higher probability of BPSD who would benefit from more detailed assessment. However, several possibilities exist for implementing this assessment strategy. The simplest approach would be to examine results for each significant predictor and implicitly weight their importance. Unfortunately, large volumes of literature argue against this approach to clinical decision making [3,4,5]. For example, decisions based on clinical intuition tend to be inaccurate or highly variable and are extremely sensitive to the base rate of the condition in the setting where the decision is being made [6,7].More detailed and defensible methods involve explicit weighting of predictors. One weighting approach would be to use the regression weights derived from logistic regression analyses. Although familiar within the research community, logistic regression analyses are difficult to implement in clinical practice. They require generating a complex combination of scores multiplied by regression coefficients to produce post-test probabilities of having BPSD. Additionally, logistic regression estimates are optimized for the study participants from which they are derived and thus, may not replicate well across clinical settings [8]. Logistic regression assumes a linear relationship between predictors (test scores) and the transformed odds of the criterion (diagnosis). In contrast to logistic regression, newer methods, such as classification tree approaches, can accommodate both linear and non-linear relationships. Finally, application of regression weights requires that complete information about all of the variables in the model is available for the new clinical case. For instance, if the regression model includes family history of mood disorder as a predictor, then weights for other variables are adjusted based on inclusion of family history in the model, and the model cannot be used with cases that are missing family history (or any other predictor variable). Clearly, alternative methods are needed that are empirically defensible, easier to implement, and that better approximate clinical decision-making.The purpose of the present paper is to extend the work of Fristad and colleagues [2] by using classification tree analyses (CTA). CTA is an iterative method that optimizes classification at each step by selecting the strongest classifier variable while simultaneously avoiding over-fitting through the use of bootstrap re-sampling, and providing results in a clinically-useful tree diagram form [9]. CTA-derived algorithms have the potential to produce equally or more accurate overall classification relative to logistic regression, provide a superior balance of sensitivity and positive predictive power (important for identifying low base rate conditions), and provide visual tree diagrams that can be helpful for simplifying the integration of complex clinical information during the decision process (e.g., [10,11]). We anticipate that CTA models will more clearly delineate how parent ratings of mania, parental history of mania, stressful life events, and parenting stress can be integrated to increase or decrease the probability of BPSD—providing concrete recommendations for clinical decision-making.For this study, the same sample, study design, and data collection procedures were used as in the paper by Fristad and colleagues [2]. Institutional Review Boards at each of the four Longitudinal Assessment of Manic Symptoms (LAMS) sites (Case Western Reserve University, Cincinnati Children’s Hospital Medical Center, the Ohio State University, and the University of Pittsburgh Medical Center/Western Psychiatric Institute and Clinic) approved all protocol procedures.The source population consisted of all eligible youth aged 6 through 12 years visiting 9 child outpatient mental health clinics (2 in Cleveland, 1 in Cincinnati, 5 in Columbus and 1 in Pittsburgh) associated with universities in the LAMS study. Exclusion criteria included: (1) a prior visit to the same clinic within the preceding year; (2) not being accompanied by a parent or legal guardian; (3) having a parent who did not understand or speak English; and (4) having a sibling or other child living in their household who had already participated in LAMS screening [12].Caregivers completed the Parent General Behavior Inventory 10-Item Mania Scale (PGBI-10M) [13] to screen for elevated symptoms of mania (ESM). Items comprising the PGBI-10M describe hypomanic, manic, and biphasic symptoms and have been reported to discriminate BPSD in youth from other diagnoses [13]. Items are scored from 0 (“never or hardly ever”) to 3 (“very often or almost constantly”); total scores range from 0 to 30 with higher scores indicating greater symptoms. Each patient whose parent/guardian rated the child ≥12 (ESM+) on the screening PGBI-10M was invited to participate in the longitudinal portion of the LAMS study. A smaller comparison group of patients who scored 11 or lower (ESM−) matched on age, sex, race, ethnicity, and Medicaid status was also selected. Of 1124 children who screened ESM+, 621 or 55% accepted the invitation. Information including age, sex, race, ethnicity, and health insurance status was obtained from parents/guardians. There were no socio-demographic differences between children/families agreeing to enroll in the longitudinal study and those who did not. The mean time interval between screening and baseline assessment was 45.5 (SD = 41.4) days. ESM− children were selected from the available pool to match on age (±2 years), sex, race/ethnicity and insurance status; 86 children without ESM (ESM−) were included in the longitudinal cohort [12]. It was anticipated that some, but not all children who were ESM+ would receive diagnoses of a BPSD upon completion of the baseline assessment described below.Risk factors eligible to be included in predictive models were: parent reports of mania symptoms at screening and baseline using the PGBI-10M (range 0–30) [13], parent history of mania (PHM; any history present vs. absent), Parent Stress Survey (PSS; [14]) scores ranging from 0 (no stress) to 100 (high stress), and Stressful Life Events Schedule (SLES; [15]) scores (range 0–34). The criterion was BPSD diagnosis (BPSD vs. no BPSD), collapsing bipolar I, bipolar II, cyclothymic disorder, and bipolar not otherwise specified (NOS) into BPSD and all other diagnoses or no diagnosis into no BPSD. All diagnoses followed strict DSM-IV criteria [16], with NOS following the operational definitions used in the Course and Outcome of Bipolar Youth study [17].Children and their guardians were administered the Schedule for Affective Disorders and Schizophrenia for School-Age Children-Present and Lifetime Episode (K-SADS-PL) [18] with additional depression and manic symptom items derived from the Washington University Kiddie Schedule for Affective Disorders (WASH-U K-SADS) [19,20]. Items assessing nonverbal communication, the child’s relationship with others, shared enjoyment, and social-emotional reciprocity according to DSM-IV criteria were added to the KSADS-PL to screen for pervasive developmental disorders (PDDs). The resulting instrument, the K-SADS-PL-W, is a semi-structured interview that assesses current and lifetime psychiatric diagnoses and time course of each illness. In this report, only current diagnoses at baseline assessment are presented.Unmodified DSM-IV diagnostic criteria were used. Criteria for BP-NOS were clarified to follow those criteria used in the Course and Outcome of Bipolar Youth study (COBY) [21] and were operationalized as follows: (a) elated mood plus two associated symptoms of mania (e.g., grandiosity, decreased need for sleep, pressured speech, racing thoughts, increased goal-directed activity), or irritable mood plus three associated symptoms of mania; (b) change in the participant’s level of functioning (increase or decrease); (c) symptoms must be present for ≥4 h within a 24-h period; and (d) the participant must have had ≥4 such episodes, or a total of four days of the above-noted symptom intensity in his/her lifetime. To prevent post-training rater drift, all interviewers rated taped administrations of the K-SADS-PL-W taped at each site throughout the course of the study. Kappas were: 0.82 for K-SADS-PL-W psychiatric diagnoses in general and 0.93 for bipolar diagnoses in particular. These are within acceptable levels [22].All diagnoses were reviewed and confirmed by a licensed child psychiatrist or psychologist.The PGBI-10M, described above, was administered at screening and baseline.The Family History Screen (FHS) [23,24] collected information on 15 proxy psychiatric disorders and suicidal behavior in biological parents. In this study, symptoms of DSM-IV-TR defined mania were evaluated. Individuals were considered positive for a parental history of mania (PHM) if one (score = 0.5) or both (score = 1.0) parents endorsed yes for “extreme elated mood” plus 3 supporting symptoms (extreme or non-extreme- more talkative, inflated self-esteem, decreased need for sleep, racing thoughts, distractible, restless, and excessive involvement in pleasurable activities) or yes for “extreme irritability” plus four supporting symptoms. In cases where there were missing data, if enough symptoms were known to meet the above criteria, the parent was scored positively. If the parent could not meet criteria even if missing symptoms were available (e.g., if neither extreme elated mood nor extreme irritable mood was endorsed), the parent was scored negatively. If there was uncertainty regarding whether or not a parent would or would not meet criteria if missing symptoms were known, the parent was scored as unknown and that child was not included in this sample.The Parent Stress Survey (PSS) [14] was used to assess parental stress related to raising a psychiatrically impaired child. The PSS is a 25-item parent self-report instrument; each item has a yes or no response to document occurrence of the event, followed by a Likert-type scale, with response choices ranging from 0 (not at all stressful) to 4 (very stressful), to measure the severity of stress experienced. The total score is the sum of the 25 Likert-type items and ranges from 0 (no stress) to 100 (high stress). The questionnaire has a coefficient alpha of 0.87 [25].The Stressful Life Events Schedule (SLES) [15] collected information on occurrence, date of occurrence, duration, and perceived threat of events experienced by the youth. The SLES also allows for the determination of whether an event was dependent on behaviors of the child or adolescent. The SLES has shown good test-retest reliability (κ = 0.68).CTAs were computed using optimal data analysis methods [9]. CTAs, although less familiar to many clinical researchers than logistic regression, provide several advantages, including: comparable or better accuracy of prediction relative to logistic regression [26,27,28,29]; ability to model both linear and non-linear relationships between the predictor and criterion; inclusion of predictors only in parts of the model where they improve classification [9]; maintenance of experiment-wise Type I error at 0.05 using a generalized multiple comparison correction to minimize over-fitting; use of jackknife leave-one-out bootstrap re-sampling to identify the most stable predictors and attempt to maximize generalizability of the CTA model; and visual output of classification rules—in the form of a decision tree—that can be easily implemented by clinicians [29]. CTA is conceptually related to other types of classification analyses such as Q-ROC, classification and regression tree, bagging, and random forests [1,30].Classification tree building via univariate optimal data analysis methods is an iterative process involving the following steps: (1) Find the optimal cut-point for the strongest predictor by examining all possible predictors. The strongest predictor is identified based upon maximum classification accuracy across jackknife (bootstrap) re-sampling.(2) Find the optimal cut-point to create branches that maximize classification accuracy for that predictor. For example, screening and baseline PGBI-10M scores may be expected to be the most powerful predictors. For each PGBI-10M variable, a cut-point is identified (e.g., screening PGBI-10M <12 vs. ≥12), creating two branches. Each branch includes individuals who are correctly and incorrectly classified as having or not having a BPSD diagnosis. The cut-point maximizes classification accuracy for PGBI-10M scores.(3) Examine each branch separately to find the next largest predictor that significantly improves classification. For example, for individuals with elevated screening PGBI-10M scores (≥12), elevated baseline PGBI-10M scores may provide the largest increase in classification accuracy. This offers an opportunity for improvement over logistic regression and other methods, in that different variables could be selected to optimize performance in different branches. Gender might be important in refining classification of high scorers on the PGBI-10M, but not relevant to the low scorers, for example.(4) Identify the optimal cut-point for this next predictor. Each cut-point represents new branches added to the tree.(5) Repeat this process for individuals with low screening PGBI-10M scores (<12) and each available branch until no additional predictors add significantly to a branch.(6) When the tree model is completed, “prune” branches using a Bonferroni adjustment of Type I error to minimize overfitting the tree model.(7) Compute and visually display the overall predictive performance and classification accuracy of the final tree so clinicians can use this information in decision making. At the end of each branch, the proportions of individuals with and without BPSD are provided. These proportions represent posterior (post-test) probabilities because the relevant risk factors for BPSD have been considered. Posterior probabilities can be compared to the anterior probability (pre-test probability or sample base rate) to evaluate the change in identification of BPSD.In the present study, CTA results were compared to those obtained using hierarchical logistic regression [2]. Sensitivity, Specificity, Positive (PPV) and Negative Predictive Values (NPV) were computed to compare classification accuracy across methods.Table 1 presents the LAMS sample description, separately for children with a baseline BPSD diagnosis and children without a baseline BPSD diagnosis. Children with BPSD were older and more likely to be female than the rest of the sample (albeit still only 42% female). They were less likely to be diagnosed with a disruptive behavior disorder and were slightly, albeit non-significantly, less likely to be diagnosed with an autism spectrum disorder. There were no other differences in demographics or baseline diagnoses. Given that previous analyses of these data [2] found only minor effects of demographic factors, demographic factors were not included in CTA and logistic regression model comparisons.Longitudinal Assessment of Manic Symptoms (LAMS) sample description, separately for children with and without bipolar spectrum disorders (BPSD).Figure 1 presents the CTA decision-tree diagram with all significant predictors. Both screening and baseline PGBI-10M were significant in the first iteration (p < 0.001). Because screening PGBI-10M is collected first, it was entered as the first branch point. Interestingly, PHM entered as a predictor for low screening PGBI-10M scores, but not for high scores. PSS scores did not enter in any branch, suggesting it may account only for variance in the BPSD diagnosis that was already accounted for by PGBI-10M scores and PHM. SLES scores improved prediction only for individuals with the lowest BPSD probability (low screening and baseline PGBI-10M scores, negative PHM).Classification tree analysis decision tree predicting the presence vs. absence of a Bipolar Spectrum Disorder (BPSD).Note: PHM = parent history of mania scored as any history present or absent; PGBI-10M = Parent General Behavior Inventory—10 item mania form. Scores on the PGBI-10M infrequently yielded non-integer values if a parent selected two Likert scale responses (ex. scores of 2 and 3 circled). For this reason, we present cut scores as defined empirically with non-integer values. Assessment and treatment examples in the above figure are NOT evidence-based recommendations but rather represent possibilities that MIGHT map to different levels of the post-test probability continuum if the evidence-base was clear. Other examples of potential clinical actions exist, such as withholding, or using with caution, antidepressants or stimulants for youth with moderate risk of BPSD.Several classification tree branches appear to be helpful in increasing or decreasing the probability of BPSD. For example, the 24% sample probability of BPSD was substantially decreased in youth with low screening and baseline PGBI-10M scores, negative PHM, and low levels of stressful life events (2%); for those with low screening and baseline PGBI-10M scores, negative PHM, and high stressful life events (8%); and for those with high screening but fairly low baseline PGBI-10M scores (6%). These branches represent a substantial proportion of the sample (Total n = 202; No BPSD = 193, BPSD = 9) and provide excellent negative predictive power (0.96). High screening coupled with high baseline PGBI-10M scores nearly doubled the probability of BPSD (from 24% to 46%). Similarly, a low screening score followed by a high baseline score and positive PHM almost doubled the probability (43%). However, in both cases the probability was still below 50%. It is also crucial to note that all of the branches on the tree are clinically useful for detection of BPSD. For example, considering only increased risk branches (rightward branches), after the first step (screening PGBI-10M) only 38% of BPSD cases are identified as increased risk.Table 2 presents a comparison of diagnostic efficiency statistics between logistic regression and classification tree analyses. Logistic regression and CTA produced comparable overall accuracy (77.6% vs. 75.4%, respectively). However, unlike logistic regression, classification tree analyses (CTA) strike a balance between sensitivity and positive predictive value (PPV) while maximizing weighted accuracy and accounting for the base rate of BPSD. Relative to logistic regression, CTA increased sensitivity (+0.10) while decreasing specificity (−0.06). The balance of positive and negative predictive power was better for logistic regression than CTA due to the restrictive fashion in which accuracy was computed for CTA (all high scores on PGBI-10M required for predicting BPSD).Diagnostic efficiency statistics for logistic regression and classification tree analyses (CTA) (N = 621).Note: For CTA, overall diagnostic efficiency statistics were computed with bipolar spectrum disorders (BPSD) as the target when all branches were positive (high PGBI-10M scores, positive PHM); otherwise no BPSD was the target. PPV (2+ signs) was computed as the proportion of BPSD cases when high scores for baseline PGBI-10M and positive PHM were present. NPV (3− or 4− signs) was computed as the proportion of non-BPSD cases when at least 3 negative branches (low PGBI-10M and SLES scores, negative family history).The pragmatic advantage of CTA becomes clear when combining the last two rows of Table 1 with inspection of Figure 1. CTA permits examination of the most useful combinations of predictors and cut scores for decreasing and increasing the probability of BPSD. For example, screening and baseline PGBI-10M scores, negative PHM, and low SLES scores are very helpful for “ruling out” bipolar disorder (only 3 BPSD cases out of 121 total youth) in a high base rate clinical setting such as the present sample (24% BPSD). CTA is also helpful for showing that several conflicting combinations of predictors (e.g., low screening and baseline PGBI-10M scores with a positive PHM) are not helpful for altering the probability of BPSD. The present study demonstrated the utility of CTA in combining the available clinical information to inform a comprehensive assessment. Relative to logistic regression, which requires the cumbersome application of a linear formula, including calculation of the log odds, CTA provides a diagram that can be easily implemented by clinicians. This diagram offers clear information about the positive and negative predictive value of particular combinations of predictors, including both continuous (PGBI-10M, PSS, SLES) and categorical (PHM) predictors. Clinical use of CTA diagrams are also vastly superior to alternative approaches based on mental combinations of predictors or using odds ratios or relative risks in an iterative fashion. This is because CTA explicitly accounts for the shared variance among significant predictors, whereas an iterative approach is prone to over-or under-estimating the probability of BPSD. Furthermore, because CTA classifications are derived using a jack-knife leave-one-out procedure, previous applications have suggested that they may be more likely to generalize to other settings than sample-dependent logistic regression estimates [26,28]. The approach is also more accurate than any single-gate screening using any of the available predictors.The CTA approach navigates a huge number of possible combinations of scores—more than 9,800,000 in this example based on permutations of the five raw score inputs, or 32 combinations if each were dichotomized and treated as high risk versus low risk. CTA combines these predictors and produces estimates that would be impossible to duplicate intuitively, and would be tedious to calculate manually. For example, two low PGBI-10M scores can over-ride even extremely high levels of stress to indicate a low probability of BPSD (<10%), but they are cancelled out by positive family history of mania (23% probability of BPSD, similar to the starting base rate). Conversely, high scores on both the screen and first visit PGBI-10M double the risk of BPSD, and they obviate the need to consider stressful life events with regard to the diagnosis. The potential information conveyed by stressful life events is statistically redundant with the information captured by the elevated PGBI-10M with regard to BPSD diagnoses. This approach also lends to generating even simpler rules of thumb for clinicians. For example, if the second (baseline) PGBI-10M score is >11.5, then the probability of BPSD is at least 26%, a level that would suggest the need for more detailed evaluation.CTA analyses can readily be combined with the decision-threshold model advocated by Evidence Based Medicine [31,32]. In this framework, the clinician estimates the probability of having a BPSD along a continuum from nearly 0% to nearly 100%. There are two major decision-making thresholds along the continuum, marking changes in the next action that the clinician takes with the patient. One is the Wait-Test Threshold. When the probability falls below this threshold, then the diagnosis is considered “ruled out”—sufficiently unlikely to not require further evaluation, let alone active treatment. Probabilities above this threshold indicate more intensive evaluation, until new information either reduces the probability of BPSD to fall below the Wait-Test Threshold, or else increases the probability to the point that it crosses the second threshold, the Test-Treat Threshold. At that point, active treatment of BPSD would likely be indicated. EBM authorities point out that the position of these thresholds can vary depending on the risks and benefits attached to testing and treatment. It also is possible to map these thresholds onto different “doses” of assessment and treatment [33,34,35].Connecting the CTA results with the EBM threshold approach shows that the CTA can quickly sort the clinical information to divide cases into “low risk” or “wait zone” versus middling risk “test more” cases. This is accomplished using a brief, public domain screen, then augmenting with family history or a stress inventory to reduce false negatives. Cases in the low risk zone all met criteria for at least one Axis I disorder. Appropriate treatments for these other conditions could start with increased confidence that they would not exacerbate an unrecognized bipolar disorder. The other cases, falling in the “Test Zone”, would be appropriate for more intensive evaluation before beginning acute treatment. Options here would include referral to a psychiatrist or specialist, conducting a semi-structured diagnostic interview to systematically explore possible mood disorder, and/or initiation of intensive retrospective or prospective mood monitoring. Semi-structured interviews are likely to be particularly helpful given the low reliability of typical clinical interviewing with regard to bipolar disorder [5,36]. Despite clinician reservations, semi-structured approaches are well-tolerated by patients [37]. These cases also might warrant initiation of psychotherapy focused on improving mood regulation [38], enhancing family communication [39], or coaching about mood and behavior management strategies [40]. Each of these are likely to be helpful if there proves to be a BPSD involved, and each is more likely to help than harm if instead there is “only” unipolar depression, attention-deficit/hyperactivity disorder, a disruptive behavior disorder, or some similar clinical issue.As an example of a possible clinical application, a parent may be sent a PGBI-10M to be completed prior to their child’s initial appointment. Subsequently, the parent may be given the PGBI-10M and the SLES to be completed at the appointment. Based on findings from these measures, elevated PGBI-SF scores at screening (>21.5) and baseline (>17.5) virtually doubles the probability of BPSD from the sample base rate of 24% to a posterior probability of 46% (i.e., probability after accounting for the risk factors).In a world of decreasing resources and increasing managed care, this would aid in prioritizing who should receive further evaluation for BPSD. Alternatively, a patient with low scores on the PGBI-10M at screening (≤21.5) and baseline (≤9.5), coupled with a negative family history in the biological parents and a low score on the SLES (≤9.5), decreases probability of BPSD almost 12-fold (0.02 posterior probability). The CTA approach identifies cases for more intensive follow-up, and this may provide sufficient “medical necessity” to facilitate reimbursement by third party payors.Prior to widespread implementation, the CTA results from the present study will require replication and extension to other clinical and community samples. Analyses should also proceed in larger samples where CTA analyses can be re-computed in specific demographic sub-samples. This will be particularly important given that Fristad and colleagues [2] identified small but potentially important differences in the value of predictors across demographics. Fortunately, if replicated, adoption in typical clinical practice is likely to yield a favorable trade-off between a slight increase in evaluation time (consulting the CTA diagram) and a substantial increase in the accuracy of clinical decision making.The primary limitations of the present study were the lack of follow-up information on BPSD and the modest number of predictors evaluated. Additional waves of LAMS data collection will be useful for extending the present analyses to evaluate both prediction of all BPSD cases and prediction of new onset BPSD cases. The number of predictors was limited by the available information at baseline, but was also chosen for comparability to the logistic regression analysis results of Fristad and colleagues [2]. Future work would benefit from examining additional enhancements to the CTA approach, such as generating PGBI-10M symptom measurements via computerized adaptive testing implementing item response theory, rather than static paper-pencil questionnaires. Additional research is also needed examining other behavioral, neurocognitive, or biological potential predictors of BPSD in youth.The present study demonstrates the utility of CTA in conjunction with brief, readily attained assessments in modifying the probability of a BPSD diagnosis. In the present context, CTA had greater utility and is more clinically useful than logistic regression. Future work will benefit from examining additional, powerful predictors, such as clinician ratings, neurocognitive test data, and/or structural and functional neuroimaging patterns. Addition of more powerful predictors is likely to further enhance the accuracy, clinical utility, and caregiver acceptability of the diagnostic process. This work should also consider the developmental course and development of BPSD, a crucial next step for analyses of the LAMS sample.This study was supported by NIMH awards: Case Western Reserve University: R01 MH073967-06A1; Cincinnati Children’s Hospital Medical Center: R01 MH073816-06A1; Ohio State University: R01 MH073801-06A1; and University of Pittsburgh: R01 MH073953-06A1. The findings and conclusions presented in this paper are those of the authors alone, and do not necessarily reflect the opinions of NIMH.Thomas W. Frazier has received federal funding or research support from, acted as a consultant to, received travel support from, and/or received a speaker’s honorarium from the Simons Foundation, Ingalls Foundation, Forest Laboratories, Ecoeos, IntegraGen, Shire Development, Bristol-Myers Squibb, National Institutes of Health, and the Brain and Behavior Research Foundation. L. Eugene Arnold receives or has received research support, acted as a consultant and/or served on a speaker’s bureau for Abbott, Celgene, Lilly, McNeil, Novartis, Neuropharm, Organon, Otsuka, Shire, Sigma Tau, and Targacept. Boris Birmaher receives or has received research support, acted as a consultant and/or served on a speaker’s bureau for Forest Laboratories, Inc. and Schering Plough. Robert L. Findling receives or has received research support, acted as a consultant and/or served on a speaker’s bureau for Abbott, Addrenex, AstraZeneca, Biovail, Bristol-Myers Squibb, Forest, GlaxoSmithKline, Johnson & Johnson, KemPharm Lilly, Lundbeck, Neuropharm, Novartis, Organon, Otsuka, Pfizer, Sanofi-Aventis, Sepracore, Shire, Solvay, Supernus Pharmaceuticals, Validus, and Wyeth. Eric A. Youngstrom has consulted with Lundbeck, Fristad, Horwitz, Kowatch, Axelson, and the other authors have no financial interests to disclose.

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+This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).Frontotemporal neural systems are highly implicated in the emotional dysregulation characteristic of bipolar disorder (BD). Convergent genetic, postmortem, behavioral and neuroimaging evidence suggests abnormalities in the development of frontotemporal white matter (WM) in the pathophysiology of BD. This review discusses evidence for the involvement of abnormal WM development in BD during adolescence, with a focus on frontotemporal WM. Findings from diffusion tensor imaging (DTI) studies in adults and adolescents are reviewed to explore possible progressive WM abnormalities in the disorder. Intra- and interhemispheric frontotemporal abnormalities were reported in adults with BD. Although evidence in children and adolescents with BD to date has been limited, similar intrahemispheric and interhemispheric findings have also been reported. The findings in youths suggest that these abnormalities may represent a trait marker present early in the course of BD. Functional connectivity studies, demonstrating a relationship between WM abnormalities and frontotemporal dysfunction in BD, and DTI studies of vulnerability in first-degree relatives of individuals with BD, are discussed. Together, findings suggest the involvement of abnormal frontotemporal WM development in the pathophysiology of BD and that these abnormalities may be early trait markers of vulnerability; however, more studies are critically needed.Bipolar disorder (BD) can be a severe and disabling mood disorder, associated with detrimental outcomes, such as high rates of hospitalizations, substance abuse and suicide [1,2,3]. It is a recurrent illness that is characterized by manic or hypomanic episodes, which may alternate with depressive episodes and periods of normal mood, or euthymia. Dysregulated affective states are central to the acute episodes: euphoric or highly irritable states during mania or sad states during depression. The affective changes are accompanied by shifts in motivation, impulse regulation, energy, and activity levels, in addition to changes in sleep and appetite, implicating the brain regions that subserve emotional regulation and these associated functions.BD shows a peak in emergence during adolescence and early adulthood [4,5], implicating this epoch as highly important in the development of the disorder. Early onset of BD has been of particular concern because it is associated with a more severe course of the disorder than later presentations, which may have alternate etiologies [6,7]. Therefore, early detection of the illness is of great importance and adolescence has been a major focus within BD-research. Understanding of the development of BD during this epoch could provide insights into the pathophysiology of the disorder. In youth diagnosed with BD, there is a strong overlap in clinical symptoms with other disorders, including attention deficit hyperactivity disorder and major depressive disorder. Misdiagnosis can lead to interventions that have the potential to worsen the outcome of BD [8,9,10]. Therefore, the detection of trait markers early in the disease course might be of importance in adolescents to ensure they receive needed treatments and avoid treatments that could be detrimental.Convergent evidence supports a central role for altered development of frontotemporal neural systems in BD. Early studies of patients with lesions and seizure foci in anterior cortical and mesial temporal regions, and with lesions in white matter (WM) tracts connecting these regions, reported behavioral symptoms similar to those seen in BD [11,12,13,14,15,16,17,18]. Findings from neuroimaging studies support abnormalities in frontotemporal systems, including abundant findings of abnormal structure and functioning in the amygdala and ventral prefrontal cortex (vPFC) [19]. Recently, studies have suggested that a progression in regional brain abnormalities is present in BD. For gray matter (GM) structures, there is initial evidence that subcortical abnormalities, including the amygdala, may be present by adolescence, while PFC abnormalities, including in the vPFC and more rostral PFC regions, may progress during adolescence and into early adulthood [20,21,22,23,24].Structural magnetic resonance imaging (MRI) studies and postmortem studies support the involvement of abnormal WM in adults with BD. There is little data available on whether abnormalities in WM appear early and whether they show a progression during adolescence in BD. WM connections continue to develop into adolescence and well into adulthood [25,26,27,28,29,30], suggesting that WM abnormalities in BD might be influenced by developmental changes. In the last decade, the advent of diffusion tensor imaging (DTI) techniques has provided the opportunity to investigate the structural integrity of WM tracts in vivo more specifically. Studies have emerged in which this method is applied not only to adults with BD, but also to the study of youths with BD.This article will review studies that implicate abnormal WM development in frontotemporal neural systems in BD, with a focus on what can be learned about WM in adolescents with BD. DTI findings in adolescents will be discussed in the context of findings in adults to explore the involvement of WM development in the disorder. Although DTI studies will be the focus of the review, studies using other structural imaging techniques will also be included to allow comparisons of DTI findings within the context of other work in the field. Furthermore, functional connectivity studies will be reviewed to explore the relationship between DTI findings and frontotemporal dysfunction in individuals with BD. Finally, this article will examine genetic studies related to WM and neuroimaging studies of first-degree relatives of BD individuals, considered at-risk for the disorder, as BD is a highly heritable illness [31,32] and these studies may help identify possible markers for early detection or even prevention of BD. Together, the studies reviewed support neurodevelopmental mechanisms underlying frontotemporal WM pathology in BD.Emotional dysregulation is the characteristic feature of BD, suggesting the involvement of abnormalities in frontotemporal brain regions, especially the amygdala and vPFC, which are central to emotional regulation. The amygdala plays an early and important role in emotional processing [33,34,35,36,37], while the vPFC integrates information from the amygdala and other brain regions that provide information about emotional and motivational relevance of stimuli, and synthesizes an adaptive executive feedback to regulate amygdala and other subcortical responses [38,39]. The involvement of frontotemporal abnormalities has been further implicated in BD by behavioral neuroanatomical and neuroimaging studies. Early reports of lesions within the PFC, particularly in the vPFC, provided descriptions of symptoms similar to those seen in BD, including depressive symptoms and manic-like symptoms such as inappropriate euphoria [11,12,13,17,18]. BD-type symptoms were also observed in patients with seizures with foci in the mesial temporal lobe, including in the amygdala [13,15,16]. As the vPFC and the amygdala are highly interconnected brain regions [40], these findings suggest that abnormalities within both the vPFC and amygdala and/or their connections might contribute to BD. Indeed, subsequent findings from structural and functional MRI studies in adults with BD have converged in demonstrating abnormalities in each of these frontotemporal neural system components in individuals with BD [19].Recent evidence suggests a developmental progression in frontotemporal abnormalities in BD. Subcortical brain structures mature earlier than PFC structures and PFC structures continue to show dynamic maturational changes over adolescence. Therefore, it was theorized that, consistent with the pattern of maturation of brain structures, frontotemporal system abnormalities in BD might emerge earlier in subcortical structures and PFC abnormalities might progress during adolescence [21]. Preliminary structural and functional neuroimaging studies support this progression, with amygdala abnormalities demonstrated in adolescents with BD and PFC abnormalities appearing to progress during adolescence and early adulthood [20,21,22,24]. However, these studies are cross-sectional and must be considered as models of longitudinal outcomes with caution. There is a previous longitudinal study that also supports this model; however, the sample size was small and the significance threshold modest, so findings should be considered preliminary and in need of replication in a larger cohort [23].In addition to GM developmental changes during adolescence, evidence supports dynamic maturational changes in WM connections between the amygdala and vPFC [27]. Human postmortem and neuroimaging studies have shown that myelination of these connections continues through adolescence and well into adulthood in the normal developing brain [25,26,28,29,30]. The substantial developmental changes in these connections over adolescence and young adulthood suggest that if WM connections are involved in the developmental pathophysiology of BD, they may particularly show changes in their expression during these epochs in the disorder [22,27,29,41,42].For more than a century, lesions in frontal WM and frontal-subcortical connections have been described in association with both depressive symptoms and manic-like states, including disinhibition and inappropriate excitement and laughter [14]. WM connections within frontotemporal neural systems have since been implicated in the pathophysiology of BD, as the frontotemporal GM structures in which abnormalities have been shown in BD are highly interconnected within these systems. Furthermore, postmortem studies have demonstrated reductions in glia cells, especially oligodendrocytes, and downregulation of genes related to oligodendrocytes and myelination, particularly in frontal brain regions, in individuals with BD [43,44,45,46,47]. These findings suggest abnormalities in myelin synthesis and axonal survival in individuals with BD and implicate the involvement of abnormal WM in the pathology of the disorder.Frontotemporal WM tracts can be divided into intra- and interhemispheric, i.e., connecting brain structures within one hemisphere or providing connection between the two hemispheres, respectively. Important intrahemispheric WM bundles in the frontotemporal neural circuitry include the uncinate fasciculus (UF) and the cingulum bundle (CB); both carry major connections between the amygdala and vPFC and thus are WM structures especially implicated in BD. Frontotemporal interhemispheric WM abnormalities are also highly implicated in the pathophysiology of the disorder, as the anterior corpus callosum (CC) provides major right-left vPFC connections [48]. Furthermore, studies have identified more widely distributed connections from the amygdala and vPFC, such as to more dorsal PFC, hippocampus, striatum, thalamus, cerebellum and hypothalamus, areas associated with motivational behaviors, biological rhythm and neurovegetative processes [40,49,50,51,52]. Thus, this may explain why impaired amygdala-vPFC connectivity could lead not only to emotional dysregulation, but also to a broader range of symptoms seen in BD.Studies of structural abnormalities in WM in adults with BD have shown both decreases in volume of WM within ventral frontal regions [22], as well as decreases in CC area and signal intensity, i.e., mean signal intensity thought to reflect myelination [53,54,55]. These implicate both intra- and interhemispheric frontotemporal WM abnormalities in the pathology of BD in adults. Additionally, decreased signal intensity in the CC in BD children and adolescents has been reported [56], suggesting that altered myelination may occur during neurodevelopment in BD and that these WM abnormalities may be an early feature of the disorder.Other WM abnormalities reported in MRI studies of BD are WM hyperintensities (WMH). These are hyper-intense bright spots that may reflect brain regions of increased water density, possibly because of local altered vascular permeability or other processes [57,58]. However, the etiology of WMH remains unclear. WMH have been associated with aging [58,59], and an increase in their occurrence has also been reported in several studies in adults with BD [60,61,62,63,64,65,66,67,68,69,70]. These findings might suggest that the accumulation of WMH might be associated with aging processes in the disorder [71]. Furthermore, an association between increased WMH and previous suicide attempts has been reported [62], suggesting the involvement of WMH in risk for suicide attempts in BD individuals. Some of these studies reported WMH in the deep frontal WM, further suggesting that tracts connecting fronto-cortical and subcortical regions are affected in the disorder [61,68]. Evidence related to WMH in youths with BD is contradictory. In some studies of children and adolescents with BD, significant increases in WMH number were not detected [72,73]. There have also been reports of increases in WMH in children and adolescents with BD [74,75]. This suggests that WMH may involve pathophysiological processes other than aging or may be associated with particular subtypes of BD, such as early onset. However, the involvement of WMH early in the disorder remains unclear.DTI studies have started to focus on structural integrity differences between WM in adolescents with BD, compared to healthy comparison (HC) adolescents (Table 1). DTI studies provide noninvasive measures of the organization of WM [76]. Fractional anisotropy (FA) is a common DTI measure that provides information on the structural integrity and coherence of fibers within WM regions [77]. Some studies report additional measures to provide more insight on diffusivity in WM, such as apparent diffusion coefficient (ADC) or mean diffusivity (MD), radial diffusivity (RD) and axial diffusivity (AD); however, FA is the most consistently reported measure and therefore the main focus of this review.Frontotemporal WM abnormalities implicated in BD, both intra- and interhemispheric, have been reported in many adult DTI studies. Reduced UF and neighboring orbitofrontal WM integrity [78,79,80,81,82,83,84,85,86,87,88] and reduced anterior CB integrity [86,89,90,91,92] have been among the most consistent WM integrity findings in adults with BD. Studies of adults with BD have also shown intrahemispheric WM abnormalities in connections between dorsal frontal regions, to regions such as the striatum or thalamus via connections within structures, including the anterior limb of the internal capsule (ALIC) [82,87,93,94]. More abundant interhemispheric findings include reduced structural integrity in the anterior CC in adults with BD, and supports abnormal interhemispheric frontotemporal circuitry in the disorder [70,87,88,94,95,96,97,98,99]. In addition to tensor-based diffusion MRI studies, recent research in adults with BD has also utilized alternative methodologies, such as high-angular resolution diffusion imaging (HARDI) and diffusion spectrum imaging (DSI), which are able to further parse heterogeneous diffusion directions and crossing fiber tracts [99,100]. These techniques are promising and may potentially provide additional insight into WM abnormalities in BD.Diffusion tensor imaging (DTI) studies of children and adolescents with bipolar disorder and/or at-risk for bipolar disorder.Studies of children and adolescents with BD have also shown findings of reduced intrahemispheric structural integrity in ventral frontal WM regions, compared to HC children and adolescents [102,104] (Table 1). Decreased FA values have been reported in pericingulate regions, including the CB, in children and adolescents with BD [102,107,109]. These decreases in WM structural integrity include findings in prepubertal children [102,107]. This might be an indication that intrahemispheric frontotemporal WM abnormalities are already present in childhood, representing some of the earliest markers of the disorder.Abnormalities in additional tracts have also been demonstrated in children and adolescents with BD, including tracts to other connection sites of frontotemporal circuitry, such as to more dorsal frontal regions, basal ganglia, thalamus and posterior association cortices [102,105,106]. Reduced structural integrity in superior frontal regions has been reported in BD adolescents [103]. Reductions of FA values have also been reported in frontal projections through the anterior corona radiata in children and adolescents [105,106], and in a group that included both adolescents and young adults [98], with BD. In contrast to findings in adults, abnormalities in the ALIC have not been a consistent finding in children and adolescents with BD. Studies have reported both no differences [105] and reduced structural integrity of the ALIC in children and adolescents with BD compared to HC youths [110]. The latter study included both adults and youths with BD and compared early- and late-onset of BD and reported lower FA values in the early-onset group, suggesting that early- and late-onset BD might represent different subtypes within the bipolar spectrum with different pathophysiologies, potentially accounting for some conflicting results in the literature.Findings of frontotemporal interhemispheric WM abnormalities in adolescents with BD have also started to emerge. Lower FA values of interhemispheric connections in a group with both children and adolescents with BD have been found in the anterior commissure (AC) [108]. Decreases in structural integrity in the anterior CC have been reported in prepubertal children and adolescents [106,108]. The AC and the anterior CC link the right and left temporal lobes and prefrontal lobes respectively, and therefore could play a role in the frontotemporal neural circuitry suggested in BD. Findings suggest that reduced integrity of frontotemporal interhemispheric WM bundles might be present early in the disease course.Functional connectivity neuroimaging studies further implicate the involvement of abnormal frontotemporal WM connections in BD. Functional connectivity MRI measures can be derived from the degree that activity is coordinated in time between different brain regions. Functional neuroimaging studies have provided support for trait abnormalities in functional connectivity between the amygdala and vPFC regions in both adults [81,111,112,113,114,115,116] and children and adolescents [117,118,119] with BD across mood states.Several studies have aimed to examine the relationship between DTI results and dysfunction in BD. An investigation combining DTI and functional connectivity data showed an association between reduced structural integrity in the UF with decreases in the functional connectivity between the vPFC and the amygdala during processing of emotional stimuli by adults with BD [81]. This multimodal neuroimaging study suggests that the WM aberrations may contribute to disruptions in the ability of the frontotemporal brain structures to work together in the regulation of responses to emotional stimuli. Hemispheric lateralization of processing of positive and negative emotions is evident in normal affective processing, and the balance between the hemispheres has been proposed to be important in healthy emotional regulation [120]. A low frequency resting state functional connectivity study showed increased interhemispheric correlations between left and right vPFC in adults with BD, relative to healthy individuals [111]. This finding suggests that interhemispheric abnormalities might contribute to impaired hemispheric integration and the hemispherically-lateralized dysfunction in acute mood states of the disorder [48,111].A functional connectivity study investigating children and adolescents with BD showed less functional connectivity between the amygdala and posterior association cortices during an emotional face identification task [121]. This finding suggests that youth with BD may have impaired communication in neural systems critical to processing faces and emotional stimuli and that WM connections to posterior regions may be early abnormalities in BD. As connections from posterior associations structures to amygdala may carry information used to associate stimuli with emotional information [122], abnormalities in these connections could contribute to abnormalities in the development of emotional associations to environmental stimuli. More multimodal studies are needed to further delineate the relationship between WM abnormalities and dysfunction in BD.Increasing evidence that genes related to WM and frontotemporal connectivity are associated with BD further implicate frontotemporal neural circuitry in the disorder. BD has strong genetic contributions with heritability estimates varying from 40% to 70% [123]. Studies have suggested that genes involved in cytoarchitecture of frontotemporal WM structures might be associated with an increased risk of BD [124]. For example, the neuregulin 1 (NRG1) gene plays a key role in neurodevelopmental processes in WM brain connections, such as in axonal guidance and myelination [125,126,127]. NRG1 is suggested to influence the susceptibility to BD and seems especially associated with psychotic features [128]. Abnormal frontotemporal WM volume, including in the CB and regions in the CC, has been found to be associated with a single nucleotide polymorphism (SNP) in the NRG1 gene (rs35753505) in adults with BD [129]. DTI evidence showed that another NRG1 SNP (rs6994992) is associated with reduced WM density and integrity in the ALIC [130], suggesting that NRG1 may increase susceptibility to psychopathology by altering connections between PFC and other brain regions. Another gene of particular interest in BD is the CACNA1C gene, which has been reported in genome-wide association studies to be related to BD [131,132]. The CACNA1C gene is implicated in the development and plasticity of the frontotemporal neural circuitry [133]. CACNAIC rs1006737 SNP variation has been associated with altered frontotemporal functional connectivity between the amygdala and vPFC [134]. This finding implicates variations in gene expression in the neural circuitry associated with BD, and that altered gene expression associated with BD may lead to abnormalities in WM connections, including in their development and plasticity, as well as in associated frontotemporal functional connectivity.The high heritability rates in BD are supported by several longitudinal studies, showing offspring of individuals with BD to be at greater risk to develop BD [135,136]. First-degree relatives of affected BD individuals are considered to have more than a ten-fold higher risk of developing BD than the general population [137]. Thus, youths at-risk for BD (AR-BD), as they have a first-degree relative with BD but they themselves have not yet developed BD, are of particular interest to investigate. The presence of neurodevelopmental abnormalities in AR-BD youth, prior to the onset of acute episodes of the disorder, was suggested by a prospective study of a large cohort [138]. AR-BD youths might show brain differences that are apparent even before onset of mood episodes, revealing abnormalities associated with vulnerability to the disorder and minimizing confounds, such as the possibility that abnormalities are a result of having experienced an acute episode or medication exposure.Structural MRI studies in AR-BD adults have shown reduced WM volume in the left hemisphere [139] and WMH abnormalities [140,141], suggesting that WM disconnectivity might also be present in AR-BD individuals. However, only limited DTI studies have to date explored structural integrity in AR-BD individuals with BD.In DTI studies, frontotemporal structural integrity abnormalities have been reported in both adults with BD and their AR-BD adult relatives, both showing decreased FA values in the right UF [87]. Reductions of structural integrity in the ALIC and in frontal connections to posterior association cortices have also been reported in adult AR-BD individuals [87,94,142,143]. Decreased structural integrity found in the CC in BD individuals has not been detected in their relatives [87]; however, increased genetic liability for BD was reported to be associated with a trend towards reduced FA in the anterior CC, with intermediate values for AR-BD adults [94]. This suggests the possibility that intrahemispheric frontotemporal connections are more associated with genetic heritability than interhemispheric WM connections. The frontal system WM findings in AR-BD are consistent with reports of cognitive dysfunction in AR-BD adults [144]. Future research to combine tests of cognitive and behavioral functions with neuroimaging may help to identify relationships between cognitive dysfunction and underlying WM abnormalities.To date, only limited numbers of studies have been reported that explore WM in AR-BD youths (Table 1). One study reported altered WM in the left CC during early adolescence in AR-BD youths, compared to youths with no family history of BD, with the AR-BD youths showing decreases in FA with age, while the low-risk youths showed an increase of FA with age [101], suggesting altered WM developmental trajectories in the AR-BD adolescents. Another study reported decreased structural integrity in bundles connecting frontal cortices with posterior association cortices in both prepubertal children with BD and AR-BD prepubertal children [102]. These results raise the intriguing possibility that abnormalities in WM connections may provide an early marker for vulnerability to BD. However, due to the limited DTI research conducted in AR-BD subjects, it remains unclear whether WM abnormalities are involved in the vulnerability for developing BD and whether they could potentially be a biomarker for the disorder.While GM volumetric and functional changes have been a focus of neuroimaging research in BD, recent neuroimaging data have been converging to suggest WM abnormalities may be important in the developmental pathophysiology of BD and have potential as early biomarkers. Intrahemispheric WM abnormalities in UF and CB are especially implicated in the disorder, and additional abnormalities in connections to dorsal frontal regions, striatum and thalamus, as well as to posterior association cortices, have also been shown in adults and adolescents. Interhemispheric anterior CC abnormalities have also been repeatedly shown in adults with the disorder, although less evidence is available in adolescents. WM abnormalities are associated with frontotemporal system dysfunction, as well as associated behaviors. Genetic variations associated with both the development of WM connections and with BD susceptibility have also shown association with frontotemporal structural and functional connection abnormalities, suggesting potential genetic mechanisms that may underlie connection abnormalities. The intrahemispheric abnormalities show promise as possible early markers of vulnerability to BD. There is less data to support interhemispheric abnormalities in vulnerability.More research is critically needed. Longitudinal studies of adolescents with BD could help to identify the processes involved in the neurodevelopment of BD, contributing to understanding of the progression of abnormalities and the factors that contribute to them. Longitudinal studies of AR-BD children are especially needed. These could help to identify neurodevelopmental processes involved in the transition from risk to developing the disorder for children who go onto develop BD, as well as protective processes for those who do not. Increased sample sizes, as well as consideration of various heterogeneous demographic and clinic features, are needed. It will be important to investigate associations between specific regional findings with symptom and behavioral domains to provide a dimensional understanding of neuropathological mechanisms of BD.Measures assessed in imaging studies have been limited, such as a focus primarily on FA in DTI studies. As additional measures may help to clarify the types of pathophysiological processes involved have been informative in studies of adults, and have shown some differences in studies of youths, they will be important to include in future studies. Previous studies have varied in other aspect of the imaging acquisition and analyses methods, including variation in the statistical thresholds used. The field will benefit from increasingly sensitive and specific measures of white matter features, and of studies with larger samples with more stringent statistical thresholds.In summary, research on WM in BD supports an important role for frontotemporal WM. Neurodevelopmental abnormalities that affect trajectories of WM development during adolescence are implicated in the emergence of BD during this epoch. Future studies might reveal important insights into the pathophysiology of BD and identify brain differences and mechanisms to target for early identification, intervention and prevention strategies.This work was supported by research grants from the National Institute of Health R01MH69747 (HPB), R01MH070902 (HPB), RC1MH088366 (HPB), RL1DA024856 (HPB), T32MH014276 (ETCL) and CTSA UL1RR0249139 from the NIH National Center for Research Resources (HPB) and the National Alliance for Research in Schizophrenia and Depression (HPB), American Foundation for Suicide Prevention (HPB), International Bipolar Foundation (HPB), Attias Family Foundation (HPB) and Women’s Health Research at Yale (HPB).The authors declare no conflict of interest.

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+This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).Pediatric bipolar disorder (BD) rates have notably increased over the past three decades. Given the significant morbidity and mortality associated with BD, efforts are needed to identify factors useful in earlier detection to help address this serious public health concern. Sleep is particularly important to consider given the sequelae of disrupted sleep on normative functioning and that sleep is included in diagnostic criteria for both Major Depressive and Manic Episodes. Here, we examine one component of sleep—i.e., circadian phase preference with the behavioral construct of morningness/eveningness (M/E). In comparing 30 BD and 45 typically developing control (TDC) participants, ages 7–17 years, on the Morningness-Eveningness Scale for Children (MESC), no between-group differences emerged. Similar results were found when comparing three groups (BD−ADHD; BD+ADHD; TDC). Consistent with data available on circadian phase preference in adults with BD, however, we found that BD adolescents, ages 13 years and older, endorsed significantly greater eveningness compared to their TDC peers. While the current findings are limited by reliance on subjective report and the high-rate of comorbid ADHD among the BD group, this finding that BD teens demonstrate an exaggerated shift towards eveningness than would be developmentally expected is important. Future studies should compare the circadian rhythms across the lifespan for individuals diagnosed with BD, as well as identify the point at which BD youth part ways with their healthy peers in terms of phase preference. In addition, given our BD sample was overall euthymic, it may be that M/E is more state vs. trait specific in latency age youth. Further work would benefit from assessing circadian functioning using a combination of rating forms and laboratory-based measures. Improved understanding of sleep in BD may identify behavioral targets for inclusion in prevention and intervention protocols.Although the incidence of mania was once considered negligible in childhood and adolescence, rates of pediatric bipolar disorder (BD) have notably increased over the past three decades. For example, the percentage of children and adolescents discharged from psychiatric hospitals in the U.S. with a diagnosis of BD surged from less than 10% in the mid-1990s to more than 20% in the mid-2000s [1]. The growing prevalence of pediatric BD is especially concerning given the significant morbidity and mortality associated with BD, including high rates of suicidality, academic and social impairment, lower perceived quality of life, and high health care expenditures [2,3,4].Despite increasing data supporting pharmacological and psychotherapy treatment options for youth once diagnosed with BD, efforts are needed to identify factors useful in earlier detection to help address this serious public health concern. Towards that end, sleep is of particular interest given the sequelae of disrupted sleep on daily psychosocial functioning (e.g., poor academic performance, decreased emotion/behavior regulation [5,6]) and that sleep alterations are included in the Diagnostic and Statistical Manual, 5th edition [7] criteria for both Major Depressive and Manic Episodes. Data exist not only documenting sleep disturbances (e.g., insomnia, irregular sleep schedules) as common among BD youth, but also indicating its importance as a prodrome [8,9]. In a recent study of 82 youth diagnosed with BD, for example, nearly half of parents retrospectively reported sleep disruption (e.g., insomnia, parasomnias) as the first observed symptom [10]. Furthermore, in comparing 100 youth at high-risk for BD by virtue of having a parent diagnosed with BD Type I to 112 healthy controls with no parental history of BD, Shaw and colleagues [11] showed that decreased need for sleep differentiated those at high- vs. low-risk during a 10-year follow-up period. Studies with adults diagnosed with BD also report that sleep disturbance (i.e., induction of sleep deprivation) may contribute to relapse/the onset of subsequent mood episodes [12]. In fact, interpersonal and social rhythm therapy (IPSRT) [13] was developed based upon the adult literature showing erratic sleep-wake cycles in BD and with the goal of increasing consistency in an individual’s sleep and daily functioning as a means to minimize risk of symptom recurrence.Borbély’s [14] widely recognized two-process model of sleep provides a framework to advance what is known about sleep in childhood. Within this framework, the homeostatic component (Process S) regulates the need for sleep and the circadian component (Process C) manages the timing of sleep onset and offset. Interestingly though, studies-to-date focused on the outcomes of disrupted sleep in children are best tied to the model’s homeostatic component (Process S) as researchers specifically investigate the role of sleep loss, decreased sleep quality, and subsequent daytime sleepiness. Less is known about the model’s circadian component (Process C) in children’s psychosocial outcomes.The endogenous circadian system can be studied with intensive and sometimes invasive lab-based measures (e.g., core body temperature, melatonin and cortisol levels, forced desynchrony) [15,16,17]. However, a more appropriate first step and less intensive proxy is to measure the behavioral construct of morningness/eveningness (M/E), which gauges an individual’s circadian phase preference by acknowledging the timing of optimal daily functioning [18,19]. While M/E is presumed to be normally distributed in the general population, studies suggest that adults with BD endorse more extreme evening phase preference (i.e., time preference for sleep and wake—“lark” vs. “owl”) compared to their control counterparts (see [20] for a more detailed review of adult-specific findings) [21,22]. Further, Ashman et al. reported evidence of such phase delay in a sample of rapid cycling BD adults such that morning activities in BD adults were more delayed compared to controls [23]. Greater eveningness in BD adults is also associated with heightened clinical severity (e.g., lower global assessment of functioning scores and more self-reported symptoms of depression) [21,24]. Moreover, adults diagnosed with BD assigned to IPSRT for stabilization of mood symptoms went significantly longer during the two-year study without experiencing a new mood episode compared to those BD I adults assigned to a clinical management protocol (i.e., provided psychoeducation and non-specific support) [25].Despite existing studies documenting sleep dysfunction in children and adolescents with BD (e.g., decreased sleep duration, initial insomnia; see [8]), little is known about circadian phase preference specific to pediatric BD. To address this gap in knowledge, we evaluated the phase preference of youth with BD vs. typically developing control (TDC) participants without psychopathology. Given the dearth of such comparative studies, our study was intended as a first step towards understanding potential alterations in chronobiology associated with BD in children that might guide subsequent studies with lab-based measures. Based on the adult BD literature [18,19], we hypothesized that BD youth would have greater preferences for eveningness compared to TDC participants.Participants ages 7 to 17 years were enrolled in an institutional review board-approved study performed at Bradley Hospital and Brown University. After the study was explained and before participation, parents and children gave written informed consent and assent. Participants were recruited through advertisements distributed to local physicians’ offices and placed on local/national websites.BD (N = 30) inclusion criteria were: (1) Meeting DSM-IV-TR criteria for BD, including history of at least one episode of hypomania (>4 days) or mania (>7 days) wherein the child exhibited abnormally elevated or expansive mood as well as three or more DSM-IV criterion “B” mania symptoms; and (2) The presence of a primary caretaker to grant consent and participate in the research process. Children with irritability only, without elevated or expansive mood, were excluded from this group as were children with BD “not otherwise specified”. Thus, all BD participants met the criteria of Leibenluft et al. narrow-phenotype BD [26].Additional exclusion criteria were: (1) A pervasive developmental disorder diagnosis; (2) Psychosis interfering with the child’s capacity to comply with study procedures; and IQ < 70 (the latter applied for both BD and TDC groups).TDC (N = 45) inclusion criterion was a negative psychiatric history in the control participant and their first-degree relatives.Following a telephone interview to ascertain relevant symptoms, potential participants were invited for a more comprehensive on-site screening that included the Schedule for Affective Disorders and Schizophrenia for School-Aged Children, Lifetime Version (K-SADS-PL) administered by doctoral-level clinicians (M.D. or Ph.D.) with established inter-rater reliability (kappa ≥ 0.85; DPD, KLK) [27].Current mood symptoms were assessed for the BD participants with the Young Mania Rating Scale (YMRS) and Children’s Depression Rating Scale—Revised (CDRS-R) [28,29].The Wechsler Abbreviated Scale of Intelligence (WASI) was administered to every participant as an estimate of cognitive ability [30]. Youth completed several rating forms including the Conners’ Rating Scales—Revised (CRS-R) [31] and a self-report of pubertal development [32]. Parents also completed ratings of their child’s psychosocial functioning and provided demographic information. Socioeconomic status (SES) was categorized according to the Hollingshead Index [33].The Morningness-Eveningness Scale for Children (MESC) was the primary outcome measure for this study [34]. The MESC is a self-report measure that assesses the construct of morningness/eveningness (M/E) as a proxy for circadian phase preference and was based upon the accepted adult scale of M/E developed by Smith and colleagues [35]. The scale consists of 10 multiple-choice questions (e.g., “Your parents have decided to let you set your own bed time. What time would you pick?”) and asks youth to base their answers on how they have felt during the recent weeks. The MESC has been shown to have both adequate reliability and validity with children and adolescents ages 8 to 16 years [34,36,37]. Scores can range from 10 (extreme evening or E-type) to 43 (extreme morning or M-type). Internal reliability of the MESC (Cronbach’s alpha) for the current sample was 0.79 and scores ranged from 17 to 40. To better describe youth categorized as extreme chronotypes (i.e., M- or E-type), cut-off scores were calculated based upon the 10th and 90th percentiles of the current TDC sample distribution in line with procedures established by previous research [38,39]. The 10th percentile of MESC scores = 22 and 90th percentile of MESC scores = 37.4.We performed an analysis of covariance (ANCOVA) using group (BD, TDC) as the fixed factor and MESC scores as the dependent measure. Age was selected as the covariate based on its negative association with MESC scores in this sample (r = −0.34 to −0.52, p = 0.004 to 0.02), as well as the known role of age on circadian phase preference [40]. Pubertal development, also negatively associated with MESC scores in this sample (r = −0.32 to −0.38, p < 0.05), was not included as a covariate given its significant positive association with age so as to limit issues of multi-collinearity.Given the wide range of ages represented in our sample, we performed exploratory ANOVAs using group as the fixed factor and MESC scores as the dependent measure with samples limited to latency age youth (<13 years old) and then adolescents (ages 13 years and older). Also, given the high rate of co-occurrence of BD and ADHD, we sub-divided the BD group into those with (BD+ADHD) and without (BD−ADHD) ADHD, and performed a post-hoc analysis of circadian phase preference via an ANCOVA with group as the fixed factor, age as the covariate, and MESC scores as the dependent variable.The sample included 75 youth ranging in age from 7 to 17 years: N = 30 in the BD group and N = 45 in the TDC group. There were no between-group differences in age, sex, or race/ethnicity (see Table 1). Regardless of diagnostic group, MESC scores were negatively correlated with pubertal status (r = −0.33, p < 0.01) and age (r = −0.41, p < 0.01), indicating that as youth developed/aged, their preferences shifted towards greater eveningness.All youth in the BD group met criteria for type I BD (although type II was not exclusionary for this study). The BD group was overall euthymic by mood rating during study participation (CDRSmean = 30.0 ± 11.7, YMRSmean = 8.3 ± 7.3). Of the 30 BD youths, 23 (77%) were euthymic (YMRS ≤ 12, CDRS < 40), 2 (7%) were depressed (YMRS < 12, CDRS ≥ 40), 3 (10%) were hypomanic (YMRS = 13–24, CDRS < 40), and 2 (7%) were in a mixed mood state (YMRS > 12, CDRS ≥ 40) during study participation. Mood state for the BD group did not significantly correlate with MESC scores (r = −0.07, p = 0.73). CDRS and YMRS scores were also not independently associated with MESC scores among BD youths (rCDRS = −0.01, p = 0.97 and rYMRS = 0.22, p = 0.26).Demographic information for the Bipolar Disorder (BD) and typically developing control (TDC) groups.ANCOVA assessing between-group differences in circadian phase preference while controlling for age did not show a main effect of diagnostic group on the MESC [F(1,71) = 1.49, p = 0.23, partial eta2 = 0.02]. Based on 10th (MESC scores < 22; E-types) and 90th (MESC scores > 37.4; M-types) percentiles calculated for our TDC sample, the BD (Mmesc = 27.35 ± 4.77) youth were best characterized as intermediate types, such that they did not endorse strong tendencies toward morningness or eveningness. In fact, only 3 BD participants described themselves as E-types, and only 1 BD participant described themselves as M-types.Post-hoc analyses to further evaluate the effect of age showed a significant between-group difference on MESC [F(1,39) = 4.70, p < 0 .04, partial eta2 = 0.11] when the sample was limited to adolescents (13 years or older). Specifically, BD teens (n = 17) reported significantly greater eveningness (Mmesc = 25.00 ± 4.26) compared to TDC teens (n = 24; Mmesc = 28.13 ± 4.74). There was no between-group difference on MESC [F(1,25) = 0.36, p = 0.55, partial eta2 = 0.01] when the sample was limited to latency age (<13 years old) BD (n = 6; Mmesc = 32.17 ± 3.54) or TDC (n = 21; Mmesc = 30.76 ± 5.34) youth.Post-hoc analyses to evaluate the effect of comorbid ADHD among age- and gender-matched groups of BD+ADHD, BD−ADHD, and TDC participants (N = 10 per group) did not show significant between-group differences on the MESC [F(2,26) = 0.58, p = 0.57, partial eta2 = 0.04], nor did MESC scores significantly correlate with CRS-R ADHD Index scores (rBD+ADHD = 0.06, p = 0.88 and rBD−ADHD = −0.12, p = 0.79).Our study is an initial attempt to understand the circadian rhythms of youth with BD. Contrary to our hypothesis that BD youth would be characterized by greater eveningness compared to TDC participants, we did not find significant between-group differences in self-reported MESC scores when comparing the entire sample of BD youths to the entire sample of TDC participants. Restricting our sample to just teenagers, but not latency-age youth, however, showed that BD participants had significantly greater eveningness than TDC participants. Additional post-hoc analyses did not demonstrate a significant effect of ADHD comorbidity when sub-dividing the BD sample into participants with and without comorbid ADHD. Caution is urged in over-interpreting these latter results, given potential type I and II errors, as well as the need to corroborate self-reported information with other biological assessments of circadian rhythms, such as dim light melatonin onset phase.While perhaps not a trait marker specific to all of pediatric BD given the null results when including a wide age range of participants, it is possible that M/E transitions into a more important role as youth with BD age. In line with extensive efforts documenting the general phase delay common to adolescence, we found a negative association between MESC scores and age, as well as pubertal status for the overall sample [18,39,41]. We then found that BD teens, but not latency-age youth, are consistent with circadian phase data available for adults with BD as they reported significantly greater eveningness compared to TDC teens. For example, Ahn et al. and Wood et al. reported lower M/E scores among BD adults compared to TDC adults, reflecting circadian phase delay and greater eveningness preferences [21,22]. The current finding suggests an exaggerated response of a developmentally expected change—much like the assumption that psychopathology represents an exaggeration of normal traits [42]. Future studies are needed to compare circadian functioning across the lifespan for individuals diagnosed with BD to better understand the timing of this exaggerated transition towards greater eveningness, as well as the possible implications of intervening on an adolescent’s phase delay on their adult course of BD.In addition to identifying the timing at which the shift towards greater eveningness for BD youth exceeds that of their TDC peers, further exploration of M/E as a trait vs. state phenomenon is likely important for future studies. Wood and colleagues found that M/E scores were negatively associated with depression ratings and positively associated with global assessment of functioning scores for their BD group, suggesting that greater eveningness is linked to current depression severity and impairment in adults. Similarly, Ashman and colleagues found that, while morning activities were delayed in BD adults compared to controls, the activities were notably more delayed during periods of depression compared to (hypo)mania [23]. It is possible that our finding that current depression symptoms of BD youth did not correlate with MESC scores is related to our inclusion of those who were generally euthymic during study participation. Perhaps then greater circadian preference towards extreme E-type reported during euthymic states is a marker for the severity of sleep disruption possible during mood episodes. Additional studies using longitudinal designs and including BD youths across mood states (mania, depression, and euthymia), similar to the work of Leibenluft’s group who followed a sample of BD adults over 18-months to naturalistically assess the interaction of mood and sleep variables (including duration), are warranted to determine the timing or onset of sleep problems in children and adolescents with BD, given that such sleep disturbances are among the most common prodrome of mania among BD adults [8,12,43].Our study has several limitations. First, our study relied on subjective report forms of circadian phase preference and psychiatric symptoms. Given that this is the first step in understanding sleep and circadian rhythms in BD youths, future work should pair questionnaires with laboratory-based or more objective methods of assessment. Actigraphy specifically has proven a useful tool in capturing sleep and circadian rhythm data with BD adults within their naturalistic settings. For example, Jones and colleagues found BD adults to have greater variability in their circadian patterns compared to control participants [44]. Gathering such data from BD teens might afford better understanding of how their general circadian phase preference interacts with actual sleep behavior (e.g., sleep onset consistency on school vs. weekend days) to impact mood and psychosocial functioning [45]. Future studies should also include evaluation of genetic moderators, given the link between psychiatric symptoms, circadian phase preference (M- vs. E-type), and genes previously implicated in circadian clock function. For example, the CLOCK gene may be of particular interest, as past findings have shown that BD adults with the C allele of CLOCK3111 T/C demonstrate greater eveningness, delayed sleep onset, and decreased total sleep [46].The high rate of co-morbid ADHD in our BD group is another limitation. However, our sample’s level of ADHD comorbidity aligns with prior studies, and thus findings may generalize better than if we had excluded BD youths with comorbid ADHD [9,47]. To address this potential limitation, we conducted post-hoc analyses of BD youths with and without comorbid ADHD. These analyses failed to reveal M/E differences among youth with BD without comorbid ADHD, BD with comorbid ADHD, and TDC, possibly due to type II error and reflective of the small group sizes (N = 10 in each). Future studies are needed to explore the circadian timing system among larger diagnostic groups. Moreover, available data on circadian functioning indicate delayed phase preference in ADHD children and adults who report difficulties with sleep onset and offset, with greater eveningness positively associated with symptom severity, particularly increased inattention [48]. This association was not found in the current sample, as MESC scores did not significantly correlate with CRS-S scores. Future studies might clarify this association further by including teacher report of behavior and direct observation, as well as by evaluating the independent and joint impact of endorsed M/E and actual sleep behavior. The latter is highlighted because data suggest the most common subjectively-reported sleep issues in ADHD are irregular sleep patterns, delayed sleep onset, and longer nighttime duration of sleep [49,50,51].In summary, contrary to our hypothesis, we did not find between-group differences in circadian phase preference among BD and TDC youths when including a wide pediatric age range. However, BD adolescents endorsed significantly greater eveningness compared to their TDC peers—a finding consistent with BD adult-based circadian phase data. Future work is needed to identify the point at which BD youth part ways with their non-psychiatric peers and begin to demonstrate this exaggerated shift towards eveningness, as well as the possible ways in which an individual’s course of BD is altered if their phase delay is addressed sooner rather than later. Also, to determine if M/E is a state vs. trait phenomenon, future work is needed to determine the role of mood state in circadian phase preference by studying BD youths while currently euthymic, manic, and depressed and to longitudinally assess the role of sleep in the prediction of mood episodes and their severity.This project was supported by Award Number K22MH074945 from the National Institutes of Mental Health and also departmental funds from EP Bradley Hospital/Brown University (East Providence, RI, USA). We are also grateful for the time and effort of all participants and their families.The authors declare no conflict of interest.

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+These authors contributed equally to this paper.This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).Background: An intervention trial found a trend for shorter length of stay (LOS) in patients with community-acquired pneumonia (CAP) when the CURB65 score was combined with the prognostic biomarker proadrenomedullin (ProADM) (CURB65-A). However, the efficacy and safety of CURB65-A in real life situations remains unclear. Methods: From September, 2011, until April, 2012, we performed a post-study prospective observational quality control survey at the cantonal Hospital of Aarau, Switzerland of consecutive adults with CAP. The primary endpoint was length of stay (LOS) during the index hospitalization and within 30 days. We compared the results with two well-defined historic cohorts of CAP patients hospitalized in the same hospital with the use of multivariate regression, namely 83 patients in the observation study without ProADM (OPTIMA I) and the 169 patients in the intervention study (OPTIMA II RCT). Results: A total of 89 patients with confirmed CAP were included. As compared to patients with CURB65 only observed in the OPTIMA I study, adjusted regression analysis showed a significant shorter initial LOS (7.5 vs. 10.4 days; −2.32; 95% CI, −4.51 to −0.13; p = 0.04) when CURB65-A was used in clinical routine. No significant differences were found for LOS within 30 days. There were no significant differences in safety outcomes in regard to mortality and ICU admission between the cohorts. Conclusion: This post-study survey provides evidence that the use of ProADM in combination with CURB65 (CURB65-A) in “real life” situations reduces initial LOS compared to the CURB65 score alone without apparent negative effects on patient safety.Community-acquired pneumonia (CAP) is one of the most common infectious diseases associated with high morbidity, mortality and financial burden [1]. Different clinical risk stratification scores for management of CAP, prediction of mortality, and the need for hospitalization were developed in the last years and are recommended by guidelines [2]. Among limitations of clinical risk scores are their static behavior and poor memorability. In contrast biomarkers are objective, dynamic, and easily measurable. ProADM improved the prognostic accuracy of the pneumonia severity index (PSI) [3] and seemed to be a useful risk stratification tool [4]. In addition, the performance of the biomarkers ProADM and procalcitonin were comparable with the established clinical scores PSI and CURB65 [5,6,7]. We previously combined ProADM cut-offs with CURB65 classes to the CURB65-A score [8] and developed an algorithm to reduce and shorten hospitalizations in patients with low medical risk (OPTIMA I observation study) [9,10].Hospitalization rates and length of stay (LOS) are affected by medical, biopsychosocial, and functional factors, as well as by the preferences of patients and their relatives [10,11,12,13]. Therefore, our algorithm included the post-acute care discharge score (PACD on admission and day three) [14] and the self-care index (SPI = “Selbstpflegeindex”, outpatients and during the ward stay) [15].Nurse-led units (NLU) are already implemented in the United Kingdom and Scandinavia as institutional settings for patients with low medical but predominantly nursing care needs [16]. In nurse-led care nurses are responsible for the coordination and steering of patient care [9,10,17].From 2010 to 2011, we conducted a single-center proof-of-concept randomized controlled trial (OPTIMA II RCT). 313 patients with LRTI were enrolled. This trial showed a trend for reduction of LOS during the initial encounter (0.5 days) and for overall hospitalizations (0.7 days) within 90 days in the ProADM-enhanced intervention group compared to the control group [17].However, results from an RCT may not unconditionally be generalized because of exclusion criteria or non-enrollment and are frequently not adequately implemented in daily practice. Therefore, we performed a post-study surveillance to investigate the real-life effectiveness of our ProADM-enhanced algorithm for site of care decision in patients with CAP after completion of the OPTIMA II RCT. We also compared LOS with historic patient cohorts from the previous OPTIMA I and OPTIMA II RCT studies.Herein, we performed observational post-study quality surveillance at the Medical University Department of the public, cantonal Hospital of Aarau, a tertiary care 600-bed hospital in Northern Switzerland. Consecutive adult patients with CAP presenting to the ED were enrolled from September 2011 to April 2012. There were no exclusion criteria. Patients were registered on a password-secured website by the treating physician. All patients were triaged according to the algorithm consisting of medical (CURB65-A including ProADM on admission as described previously [8]), biopsychosocial (PACD), and functional (SPI) criteria (Figure 1).Predefined medical, biopsychosocial, and organizational criteria and patient’s preference could be used to optionally overrule triage decisions and transfer patients to higher risk classes. Patient’s preference had priority for the final triage decision [17]. Treating physicians and nurses were reminded of correct application of the triage algorithm including ProADM values, stability, and overruling criteria. Nursing staff received ongoing training on correct use of biopsychosocial and functional criteria.Medical stability was evaluated twice daily throughout hospitalization. The discharge management was also evaluated daily based on PACD, SPI, and the clinical judgment about need of social worker involvement. On day 3, additional ProADM values were used to reassign each patient to the appropriate risk class and PACD for the biopsychosocial risk according to the triage-algorithm. Patients were considered appropriate for discharge if stability criteria were fulfilled for 24 h during hospitalization (Figure 1).PACD: Post-acute care discharge score; SPI: “Selbstpflegeindex” self-care deficit score; ProADM: Proadrenomedullin; ICU: Intensive care unit.Admission to ICU, based on respiratory (respiratory rate ≥ 30/min and/or SO2 < 90% with 6 L O2/min) or hemodynamic instability (systolic blood pressure for ≥1 h <90 mmHg despite adequate volume resuscitation or vasopressor requirement);Life-threatening co-morbidity, i.e., imminent death; complications (abscess, empyema); for COPD GOLD III & IV; O2-saturation < 90% despite 30 min intensive treatment;Acute illness requiring hospitalization independent from CAP;Comorbidity, i.e., immunodeficiency (neutrophils < 500/μL; if HIV+: CD4 < 350/μL, leukemia, lymphoma, myeloma, cytotoxic medications, hemodialysis), pneumonia within last 6 weeks, hospitalization independent of indication within the last week, other significant lung disease (cancer, fibrosis, bronchiectasis, tuberculosis, pulmonary embolism, cavitary lung disease);Confusion, delirium, or intravenous drug use.Algorithm of risk assessment for triage decisions on admission and during hospitalization.Criteria requiring intensive nursing care, i.e., dementia, recurrent falls, pressure ulcer, and inability to reliably take medications;SPI score < 32 points in patients with a low PACD score (<8);Deficit of mobility or self-care requiring treatment.Waiting for placement in a non-acute medical care facility (holiday bed, rehabilitation, nursing home, home health care);Waiting for laboratory results, imaging studies or consultant examinations.Patient’s or relative’s concerns about safety at home;Lack of supporting social network;Financial reasons.Site of care was determined by biopsychosocial and organizational factors in patients who were otherwise appropriate for discharge.All patients underwent a standardized quality control phone interview on days 30 and 180. Informed consent was waived by the local ethics committee (EKAG 2010/045, EKAG 2009/074, Kantonale Ethikkommission Aargau).ProADM was measured on admission, and on day 3, in the laboratory from EDTA plasma with a commercially available immunoassay (MR-ProADM, Thermofisher Scientific-BRAHMS AG, Hennigsdorf, Germany) with a functional assay sensitivity of 0.12 μg/L [4]. Results were routinely available around the clock within 1.5 h, upon ordering.CAP was defined as a new infiltrate on chest radiograph and at least one respiratory symptom and an auscultatory sign or a sign of systemic infection in patients with symptoms of a lower respiratory tract infection [18].Patients were considered medically stable as described in Figure 1 [17].The primary endpoint was LOS of index hospitalization and within 30 days. The secondary endpoints were intensive care unit (ICU) admission and all-cause mortality.Discrete variables were expressed as counts (percentage) and continuous variables as medians or means and standard deviations or interquartile range, unless stated otherwise. We used a linear regression model adjusted for sex, age, initial levels of procalcitonin and albumin, as well as the CURB65 score, to compare the LOS of index hospitalization and within 30 days of this cohort with the previously published cohorts from a prospective observational quality control study (OPTIMA I) and a randomized controlled trial (OPTIMA II RCT).For the adverse outcomes death and intensive care unit (ICU) admission, logistic regression analyses were performed to assess the safety of the use of the algorithm.Statistical analyses were performed using STATA version 12.1 (Stata Corp, College Station, TX, USA). All the testing was 2-tailed, and p < 0.05 was considered statistically significant.A total of 115 patients with CAP were enrolled in this study, 25 had a final diagnosis other than CAP, and one patient was included twice. Therefore, we analyzed a total of 89 patients with CAP.We compared our results with the 83 patients of the OPTIMA I observation study and the 169 patients of the OPTIMA II intervention study with CAP as final diagnosis who completed 30 days follow-up; four patients had been lost to follow-up between day 30 and day 90 and had therefore not been included in the previously published article [17]. Baseline characteristics in these groups are shown in Table 1.Baseline characteristics.Thirteen of 89 patients (14.6%) of the post-study surveillance OPTIMA III had a low medical risk according to the CURB65-A score (CURB65-A class I) and qualified for outpatient treatment at home with or without home health care, treatment in a non-medical care center (health resort, rehabilitation, nursing home) or in the NLU. Forty-eight of 89 patients (53.9%) were assigned to the intermediate risk group (CURB65-A class II) qualifying for a short-term hospitalization. Twenty-eight of 89 patients (31.5%) were assigned to the high-risk group (CURB65-A class III) who were supposed to be hospitalized.Of the 13 patients in the low medical risk group, only three were treated as outpatients due to the presence of biopsychosocial and organizational criteria or patient’s preference. One patient in the intermediate medical risk group was treated as outpatient as well.We compared the length of stay within 30 days after enrollment in the patients of OPTIMA III observation study with two well-defined historic cohorts of CAP patients hospitalized in the same hospital from OPTIMA II RCT and OPTIMA I studies. Adjusted regression analysis for age, sex, initial levels of procalcitonin and albumin, as well as the CURB65 score, showed a significantly shorter LOS during index hospitalization in patients of OPTIMA III study compared to OPTIMA I (7.5 vs. 10.4 days; adjusted regression coefficient, −2.32; 95% CI, −4.51 to −0.13; p = 0.04). Regarding the initial LOS no significant differences were found for comparisons with OPTIMA II RCT intervention group (7.5 vs. 8.4 days; adjusted regression coefficient, 0.07; 95% CI, −2.16 to 2.3; p = 0.95) and control group (7.5 vs. 8.7 days; adjusted regression coefficient, −0.94; 95% CI, −3.06 to 1.17; p = 0.38). With the use of the CURB65-A score in our triage algorithm there was a non-significant trend for shorter LOS within 30 days compared to the OPTIMA I observation without ProADM (Table 2, Figure 2).Efficacy and safety outcome in the observation with ProADM (OPTIMA III) compared to former studies. All analyses are adjusted for age, sex, CURB65, albumin, and procalcitonin.Length of stay during index hospitalization and within 30 days after admission.With the use of the ProADM-enhanced algorithm, there was no significant increase of mortality within 30 days, as shown in the logistic regression model in Table 2.The rate of patients admitted to the ICU was the same in the three studies, as shown in Table 2.The aim of our study was to describe the effects of biomarker-enhanced triage decisions over time on clinical outcome and length of stay during a randomized controlled trial and after the effort of its implementation into clinical routine. As previously described, we developed the algorithm with the observational cohort of OPTIMA I study [9] and introduced the algorithm in our center in the study setting of the OPTIMA II study [17]. In the historic comparison, we analyzed only patients with CAP to avoid the heterogeneity of patients with non-pneumonic lower respiratory tract infections (LRTI). There was likely a learning process, and over time, confidence in applying the algorithm in clinical care was established, after the treating physicians and nurses had gained personal experience and were provided with the results of the randomized controlled trial. This might not only relate to the use of ProADM alone but also to greater attention to the discharge process itself with confidence from early and safe discharges. Aujesky et al. showed that the adherence to the recommendations according to the PSI risk score for site of treatment decisions was low [19]. Similarly, Karmakar et al. [20] showed only 5% application of the CURB65 score in a New Zealand hospital. Our group showed already in the international multicenter ProREAL post-study surveillance that after testing of an algorithm in the ProHOSP RCT, the clinical routine could be changed in the participating centers [21]. Hansson et al. [22] showed in case of acute appendicitis that the results of a clinical trial can lead to a change in clinical practice. After initially overruling an antibiotic-only-algorithm by performing primary appendectomy in almost 50% of the time during a randomized trial [22], surgeons in the same hospital network changed their practice to primarily only use antibiotics in 79% of patients after this was shown to be successful in the previous randomized trials [23].However, in contrast to the reduction in overall LOS, there remained reluctance by the treating physicians to discharge patients from the emergency room and to treat patients entirely as outpatients.Our main finding in the historic comparison was a significantly shorter LOS in the observational cohort with ProADM guidance (OPTIMA III) compared to the observational cohort without ProADM guidance (OPTIMA I). The comparison between the current observational study, using ProADM guidance, with the OPTIMA II RCT study results is more complicated as several aspects need to be considered. Overall, there was no difference compared to either the OPTIMA II intervention group or the OPTIMA II control group. As previously stated in the discussion part of the OPTIMA II article [17], the OPTIMA II RCT used an interdisciplinary risk assessment bundle and compared it with a highly competitive, guideline-conforming and strictly reinforced control group, which by itself optimized LOS. In contrast, the current OPTIMA III study was an observational cohort without exclusion criteria and without reinforced algorithm adherence and even though we controlled for age, sex, initial levels of procalcitonin and albumin, as well as the CURB65 score, we cannot exclude unmeasured differences between the two populations or differences due to other unmeasured confounders. On the other hand, there was likely a learning effect with greater experience and confidence from the results of the previously RCT. Therefore, and in view of the lack of a significant difference even between the intervention group and the control group within the OPTIMA II RCT, it is not surprising to find no difference between LOS in OPTIMA III and the OPTIMA II control group.Moreover the non-significant trend for increased mortality is unlikely related to the implementation of our algorithm but rather a selection bias of differential patient populations included in a clinical trial and in a post-study surveillance. In OPTIMA III, we included patients with severe comorbidities and immunodeficiency including terminally ill patients who were formerly excluded in the RCT. With adjustment for known confounders for mortality, there was no significant increase of mortality over time.There were no major structural, organizational or strategic alterations in our hospital, but from 2009 to 2012 there was a trend for shorter LOS 30 days after enrollment and even significantly shorter LOS of initial hospitalization with the use of the CURB65-A score. Of note, diagnosis related groups (DRGs) have been introduced in Switzerland in January, 2012, but a similar financing system had already been in place for years in our canton throughout the observed time periods.We show the development, implementation and use of the algorithm with proadrenomedullin enhanced CURB65 score over four years in our publications. The description of the derivation of the biomarker enhanced score, clinical trial, and post-study surveillance could be used as a model for new clinical scores and algorithms in LRTI and other diagnosis [24,25]. The studies (OPTIMA I–III) had well characterized cohorts, similar methodology and overall 341 patients with radiologically confirmed CAP.The study was performed in a single centre, and the results were compared to previous studies from the same centre. Therefore, our results have a lack of generalizability. Another limitation is the small sample size. We focused on patients with radiologically confirmed pneumonia in order to avoid heterogeneity. Prior to widespread implementation, our algorithm has to be tested in different settings, ideally in multicenter studies with sufficient sample sizes.In our study, we show that the implementation of a biomarker-enhanced triage algorithm in clinical routine was feasible. Effective and early triage for site of care and a timely discharge after hospital admission are important to avoid nosocomial complications and reduce healthcare costs in the time of DRGs. Our studies contribute to the development of biomarker-guided and safe triage algorithms with objective criteria. These promising results are the basis for further research to improve triage decisions [26].We are grateful to all physicians, nurses, social workers and patients who participated in this study. Especially, we thank Petra Tobias and the staff of the emergency room, Susanne Schirlo and the staff of the medical clinic, Renate Hunziker, Martha Kaeslin and the central laboratory of the Cantonal Hospital of Aarau for their very helpful assistance and technical support. We thank Susanne Hochuli, head of the Department for Gesundheit und Soziales, Kanton Aargau, for financial support.Daniel Widmer and Daniel Drozdov wrote and contributed equally to this manuscript. All authors amended and commented on the manuscript and approved the final version.Swiss National Science Foundation: 32003B_135222/1; Department of Health, Kanton Aargau; Forschungsrat and Medical University Department Kantonsspital Aarau.This is an investigator-initiated quality control survey. To exclude any conflict of interest, no commercial sponsor had any involvement in design and conduct of the quality control survey, i.e., collection, management, analysis, and interpretation of the data; preparation, decision to submit, review, or approval of the manuscript.For other studies unrelated to this quality control survey Werner Albrich, Philipp Schuetz and Beat Mueller received support from BRAHMS Thermofisher and from bioMérieux to attend meetings and fulfilled speaking engagements and served as consultants for BRAHMS Thermofisher. Beat Müller received research support from BRAHMS Thermofisher. All other authors have not disclosed any conflicts of interests.

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+This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).Preimplantation genetic diagnosis was developed nearly a quarter-century ago as an alternative form of prenatal diagnosis that is carried out on embryos. Initially offered for diagnosis in couples at-risk for single gene genetic disorders, such as cystic fibrosis, spinal muscular atrophy and Huntington disease, preimplantation genetic diagnosis (PGD) has most frequently been employed in assisted reproduction for detection of chromosome aneuploidy from advancing maternal age or structural chromosome rearrangements. Major improvements have been seen in PGD analysis with movement away from older, less effective technologies, such as fluorescence in situ hybridization (FISH), to newer molecular tools, such as DNA microarrays and next generation sequencing. Improved results have also started to be seen with decreasing use of Day 3 blastomere biopsy in favor of polar body or Day 5 trophectoderm biopsy. Discussions regarding the scientific, ethical, legal and social issues surrounding the use of sequence data from embryo biopsy have begun and must continue to avoid concern regarding eugenic or inappropriate use of this technology.Preimplantation genetic diagnosis (PGD) is a form of prenatal diagnosis that is performed on early embryos created by in vitro fertilization (IVF). In comparison to other established methods of prenatal diagnosis, such as chorionic villus sampling and amniocentesis, PGD is not performed on an ongoing intrauterine pregnancy in the late first or early second trimester, but on embryos developing in the IVF laboratory prior to transfer to the uterus. Despite some misconception to the contrary, PGD is not a therapeutic procedure for embryos; there are no changes to the DNA or any other genetic-related structures. It is solely a diagnostic procedure that can identify whether a specific embryo carries a single gene disorder for which the couple is at-risk or a chromosome abnormality that could lead to either failed implantation, subsequent miscarriage or the birth of a child with physical and/or developmental disability. This information is used by the couple and their physicians to make decisions on which embryo(s) should be transferred to the uterus and will with high likelihood result in a normal pregnancy. Since multiple embryos are created in IVF, PGD has a distinct numerical advantage over testing of a single ongoing pregnancy. The greater the number of embryos created, the greater the chance that genetically normal embryos can be identified. The level of selection is that of choosing which embryos can be transferred in a fresh IVF cycle or cryopreserved for future use, versus those predicted to be affected with an abnormality. This is in contrast to chorionic villus sampling or amniocentesis, where a decision has to be made whether to terminate an ongoing affected pregnancy. For many couples, PGD is the more, or only, acceptable choice.Early research in PGD dates back to Edwards and Gardner [1], who, in 1968, biopsied a rabbit blastocyst and performed Barr body analysis to determine gender. PGD became feasible in humans with the first successful IVF by Edwards and Steptoe in 1978 [2]. Other research on PGD was performed over the next 10 years on mouse blastomeres, including the demonstration by Monk and Handyside in 1988 of the feasibility of using PGD to detect a single gene disorder [3]. The first successful application of PGD in humans was by Handyside and colleagues in 1990 [4], who performed sexing of embryos by polymerase chain reaction (PCR) for the presence of the Y chromosome sequence to avoid males affected with X-linked adrenoleukodystrophy and X-linked mental retardation. Shortly thereafter, successful PGD was reported for cystic fibrosis [5], α-1 antitrypsin deficiency [6] and many other single gene disorders.The other area where PGD has played a major role in assisted reproduction is for the detection of chromosome abnormalities in embryos due to inherited structural rearrangements, such as translocations, inversions or duplications/deletions, as well as age-related aneuploidy, with the goal of improving the outcome of IVF. This was made possible by the use of fluorescence in situ hybridization (FISH) in biopsied blastomeres by Griffin [7] and Griffo [8], who initially used probes for the X and Y chromosome to determine gender, and later by Munne [9], who developed protocols for multicolor-FISH to screen up to five chromosomes simultaneously. FISH has, for the most part, been replaced by molecular technologies for aneuploidy testing, including array comparative genomic hybridization (aCGH), single nucleotide polymorphism (SNP) arrays, quantitative PCR (qPCR) and, most recently, next generation sequencing (NGS)-based protocols. The potential applications of PGD have expanded dramatically with the easy availability of sequence-based information on pathologic sequence changes and copy number variations, along with the remarkable ability of current sequencing platforms to rapidly generate large amounts of sequence data.In this review, I will trace the development of PGD from a boutique technique performed in only a few centers worldwide, to an established prenatal diagnostic procedure practiced, at least in some form, by the majority of IVF centers. The changes that have occurred in the indications for PGD and the type/timing of embryo biopsy will be discussed along with the introduction of advanced DNA analytic technology, including comparative genomic hybridization (CGH) and next generation sequencing (NGS). Finally, we can only start to predict what the future holds for PGD; however, it is clear that discussion is needed to more carefully focus on the ultimate goal(s) of PGD, or the technology will be viewed skeptically as a precursor to eugenics and genetic engineering of children to produce “designer babies”.The actual number of PGD cycles that have been performed to date can only be estimated. In the U.S., until recently, there was no requirement (nor mechanism) to report the use of PGD in the clinical data submitted to the Society for Assisted Reproductive Technology (SART), which publishes a database of IVF cycles and outcomes for U.S. clinics. Two international working groups for PGD have been formed with the goal of collecting and mining data on PGD cycles performed and to act as a forum for participating PGD centers to exchange information, develop quality control measures and best practice standards, as well as to provide educational opportunities and hands-on workshops. The ESHRE PGD Consortium was established in 1997 and has collected data each year thereafter. At the 2013 ESHRE conference, the most recent data from 115 registered centers was reported [10]. The dataset contained information on 51,589 PGD cycles with the breakdown of indications shown in Table 1.Data from the ESHRE Preimplantation Genetic Diagnosis (PGD) Consortium, including their 14th data collection (Traeger-Synodinos et al. 2013 [10]) and the recorded indications for PGD in 51,589 cycles.In addition to the ESHRE PGD Consortium, Verlinsky and Kuliev in Chicago had organized an International Working Group on PGD, which held meetings dedicated to education and advancement of the field of PGD. In 2003, this group became the PGD International Society (PGDIS) and held its first meeting in 2005. On their website (www.pgdis.org), the organization currently estimates that approximately 100,000 PGD cycles have been performed worldwide over the past 23 years. Nearly 80% of these cycles have been performed for aneuploidy screening, 12% for single gene disorders, 6% for chromosome rearrangements and 2% for sibling human leukocyte antigen (HLA) matching. Both this and the ESHRE surveys (which likely contain overlapping data) confirm that aneuploidy testing is the major indication for PGD. This is, however, performed more frequently in the United States as opposed to Europe, where this indication had decreased for the previous seven years, due to concerns regarding the accuracy of FISH-based chromosomal analysis and the lack of data showing an increase in the delivery rate in IVF cycles where FISH-PGD was used as compared to matched IVF cycles where PGD was not performed.A survey from the Johns Hopkins Public Policy Center developed by Baruch et al. [11] was mailed to 415 ART clinics in the Unites States in 2008 to determine how many clinics utilized PGD for various indications. Of the 137 responders who offered PGD services, the indications are shown in Table 2.Results of a survey of 137 in vitro fertilization (IVF) centers regarding the indications for which PGD was offered in their clinic. PGD for aneuploidy testing, genetic disorders and structural chromosome rearrangements was performed by the majority of the respondents [11].* HLA = human leukocyte antigen.Of the 186 clinics that responded to the survey, 74% indicated that they have provided PGD services. All clinics performing >500 cycles per year reported that they have offered PGD. The single most common indication was using PGD for aneuploidy testing to attempt to improve the outcome of IVF or for couples with recurrent pregnancy loss or failed IVF. Eighty-two percent of clinics indicated that they perform PGD for couples at-risk for autosomal single gene disorders, such as cystic fibrosis, Tay Sachs disease, sickle cell disease and Myotonic dystrophy. Single gene disorders comprised 12% of all PGD cycles in the U.S. Twenty-eight percent of clinics offered IVF/PGD to avoid adult onset disorders, such as Huntington disease, hereditary breast and ovarian cancer and Alzheimer disease. Using PGD to screen embryos for diseases that will not develop until adulthood or for mutations that confer a heightened risk (as opposed to a certainty) for developing a particular disease raises issues of how to weigh the possible benefits of PGD to the future child and adult against the known and unknown risks of PGD and IVF. Having a genetic mutation associated with a particular disease, such as hereditary breast cancer, does not mean there is a certainty that the disease would develop, and children with adult onset disorders could expect to remain healthy for decades before symptoms, if any, would begin.Some couples have used PGD to attempt to have a baby who is an immunological match for an existing seriously ill child, where the baby’s cord blood is then used for stem cell transplantation. This use of PGD is known as human leukocyte antigen (HLA) typing. This type of PGD was first reported by Verlinsky [12,13], and overall, 23% of IVF clinics have performed PGD for HLA typing in conjunction with genetic analysis to ensure the baby will also be free of the genetic disease affecting the older sibling. Some families have sought HLA typing to have a baby who is an HLA match for an older child when the disease is not inherited and for which the future baby is not at risk. Six percent of IVF clinics have provided PGD in such cases. Conceiving a child for the purpose of curing an older sibling is controversial; when one child has been selected to serve as an immunological match for another, the term “savior baby” is frequently used. It has been argued that in cases where the disease affecting the older sibling has no hereditary basis, any potential risks from IVF or embryo biopsy would be imposed upon the younger child without any benefit to that child [14].PGD can be used to select the sex of an embryo, either to avoid a genetic disease in males caused by a mutation on the X chromosome (X-linked disease) or simply to satisfy the gender preference of the future parents. When PGD for sex selection is done in the absence of medical indications, it is often referred to as “non-medical sex selection” or “social sexing”. According to the survey, 42% of IVF clinics have provided PGD for non-medical sex selection. Of all PGD cycles reported in 2005, non-medical sex selection was performed in 9% of cases [15]. Nearly one-half of the clinics reported that they will provide gender selection under any circumstances, while others will only provide “family balancing” for a second or subsequent child of the under-represented sex already present in the family. The use of PGD for sex selection has triggered ethical concerns that sex selection amounts to sex discrimination. It is also assumed that most couples would desire a male child. While this may be true for cultural reasons in some ethnic groups, most studies in the U.S. have shown that there is a nearly equal desire for females as for males among couples wishing to select the gender of their child through PGD [16] or in cases of fetal reduction [17]. Non-medical sex selection is prohibited in Europe, China and Australia, although since sex chromosome information is reported in aneuploidy testing, non-reported cases are likely to occur.An uncommon use of PGD involves the selection of an embryo for the presence of a particular disease or disability, such as deafness or dwarfism, in order that a child would share that characteristic with his or her parents. Only four IVF clinics in the Baruch et al. 2008 survey [11] indicated that they have provided this service. Of all the controversial uses of PGD, this one occurs least often, but nevertheless attracts significant public attention. Most IVF clinicians have indicated that they will not provide PGD to families who seek to select for a disability, since in their mind, the goal of PGD is to help produce healthy children.Utilization of PGD services is regulated or prohibited in many countries based on national and/or local laws [18]. The majority of countries require that PGD be limited to conditions that produce significant, incurable medical illness and in which there is a significant risk that the fetus will be affected with the condition through typical Mendelian inheritance. In France, India, the Netherlands and the U.K., access to PGD is regulated by local law and allowed only with a specific license. Austria, Switzerland and Western Australia prohibit PGD. In Germany, only as many embryos may be generated in vitro, as will be transferred in the same IVF cycle (up to a maximum of three), and since 2011, analysis of the first polar body can be used to select oocytes for fertilization. PGD for serious genetic disorders has also recently been allowed. Switzerland has among the most restrictive legislation in the world on medically-assisted reproduction, and the development of more than three embryos outside the mother’s body is prohibited. Italy’s IVF regulations passed in 2004 require that all fertilized embryos be transferred, and therefore, PGD was limited only to analysis of the first polar body prior to fertilization. In 2009, PGD for single gene disorders and translocations was allowed after an Italian Supreme Court decision that the restrictions were unconstitutional [19]. For a further discussion of European regulation of PGD, see Corveleyn, et al. [20,21].Decisions regarding the optimal time to perform PGD biopsy and testing involve careful consideration of multiple factors, including: (1) When in embryologic development is the abnormality first identifiable? For example, mitotic nondisjunction will be missed in polar body analysis; (2) Are genetic changes in the specimen indicative of abnormalities in the embryo as a whole? Chromosome mosaicism in the blastomere and the capacity of the embryo for self-correction are key questions in this situation; (3) Does the timing of the biopsy relative to transfer allow sufficient time for the analysis? (4) Does the biopsy itself compromise the ability of the embryo to develop normally [22]?PGD may be performed at three different embryonic developmental stages. The first involves biopsy of the polar bodies just prior to conception (first polar body) and after fertilization (second polar body). Day 3 cleavage cell biopsy involves blastomere removal at the 5–8 cell embryonic stage. Finally, trophectoderm or blastocyst biopsy is performed on Day 5–6 embryos that consist of approximately 120 cells. Five to eight cells are removed from the trophectoderm as it “hatches” through the zona pellucida.The first and second polar bodies are by-products of the meiosis of the egg, and their removal is believed to be less harmful to the embryo than either blastomere or trophectoderm biopsy, which, by their nature, reduce the embryonic cell number [23]. Polar body biopsy is performed early in the IVF process, which can allow extra time for PGD testing to be performed prior to the fresh transfer. The basis of polar body screening is that any abnormality identified is associated with a corresponding error in the oocytes. For example, if the polar body shows two copies of chromosome 21, the corresponding oocytes will have no copies, and an embryo will be monosomic for chromosome 21. For women who carry recessive single gene mutations or are affected with a dominant genetic disorder, if the mutation is present in the polar body DNA, it is assumed that the egg carries the normal allele and, thus, will lead to a normal embryo [24].The first polar body is present before fertilization, and therefore, with its biopsy, one can obtain pre-conceptual information on the egg, which can then guide clinicians as to which oocytes should undergo fertilization [6]. This is particularly useful where limited numbers of embryos can be created due to local regulations. The limitations of this approach are that no information on the paternal DNA contribution can be deduced, nor will mitotic nondisjunction be detected. Especially for embryonic chromosome anomalies, the first and second polar body need to be evaluated, which doubles the cost of the analysis. Even in this case, the interpretations of the results from polar body analysis are not entirely straightforward. This is because not all errors in the oocyte occur from nondisjunction in the first phase of meiosis [25]. Premature separation of sister chromatids in Meiosis I has been shown to be a significant cause of aneuploidy in oocytes and complicates the interpretation of polar biopsy results [22,26,27]. The polar body biopsy process requires entering the perivitelline space through an opening in the zona pellucida produced by either laser or mechanical dissection. This latter method was perfected by Yuri Verlinsky of the Chicago Reproductive Genetics Institute, who used two perpendicular cuts in the zona to create a flap to introduce a biopsy pipette. The two polar bodies can be obtained at the same time or sequentially. The optimal time for biopsy seems to be 6–9 h post fertilization [28]. After that time, the first polar body has a tendency to degenerate, compromising FISH or DNA-based testing.Due to the limitations on PGD in Europe described above and because of the frequent finding of chromosomal mosaicism in cleavage stage embryos, an ESHRE-sponsored clinical trial in Europe was undertaken to access whether polar body diagnosis by array CGH provided a reliable, accurate and timely methodology to determine the maternal component of the chromosomal status of the embryo [29]. This showed that polar body-based PGD provided acceptable accuracy, but still greater than 10% of normal embryos could be given an incorrect diagnosis of aneuploidy [30].Cleavage stage biopsy is the most widely used biopsy technique for PGD and involves the removal of a single embryonic cell on Day 3 of development (6–8 cell stage) after opening of the zona pellucida with either laser, mechanical dissection or exposure to acidic Tyrode’s solution. The blastomere is removed after introduction of the biopsy pipette by aspiration or by extrusion of the cell with pressure on the outside of the zona [31]. The embryos just prior to biopsy are transferred to calcium/magnesium-free media to aid in blastomere removal [32]. Some centers removed two blastomeres, until this was shown to produce a greater decline in live birth than a single-cell biopsy [33,34]. Cleavage-stage biopsy, unlike PB biopsy, is able to detect maternally and paternally-derived chromosome defects, as well as some mitotic defects. Biopsy on Day 3 embryos allows 2–3 days for PGD analysis to be completed if a fresh embryo transfer is desired.The presence of embryo mosaicism is a major limiting factor in the interpretation of PGD results in cleavage-stage embryos. Mosaicism affects 15%–80% of embryos at Day 3, and significant mosaicism remains present at the Day 5 blastocyst stage [35,36,37]. This raises the question as to whether a biopsied blastomere is an accurate representation of the embryo as a whole. In addition, it appears that the ability of the early embryo to undergo self-correction by either selective apoptosis or allocation of abnormal cells to the trophectoderm is limited [38].The consideration of cleavage stage blastomere biopsy must take into account the impact of the biopsy on embryo progression, implantation and development. Most investigators were initially encouraged by the early results of Hardy [39], which suggested that blastomere biopsy did not inhibit the ability of embryos to progress to the blastocyst stage. This data, however, was based on good quality embryos and likely does not accurately represent the spectrum of embryo quality seen in a typical IVF cycle. Cohen et al. [34] pointed out that based on a blastomere cryopreservation survival model developed by Edgar [40], the biopsy of a single cell at the eight-cell stage would lead to a decrease in implantation of 12.5%, and a two-cell blastomere biopsy would produce a 25% decrease in implantation. This coupled with the significant incidence of mosaicism in cleavage stage embryos, as well as the high incidence of allele dropout (ADO, see below) led to the interest in obtaining a multicellular trophectoderm biopsy of the Day 5 blastocyst as an alternative to single-cell blastomere biopsy for use in PGD.The human blastocyst contains approximately 130 cells distributed between the inner cell mass, which will develop into the fetus proper, and the surrounding trophectoderm cells, which will become the placenta and fetal membranes. Clinical use of blastocyst biopsy was initially reported by McArthur and the Sydney IVF group [41,42] and entails the biopsy of 5–10 trophectoderm cells on Day 5 or 6 after laser assisted hatching on Day 3, which creates a 25–30 μm opening in the zona pellucida, allowing herniation of the trophectoderm cells through the zona. The cells are stretched out in a biopsy pipette and removed with a laser. Since trophectoderm cells are removed and cells from the inner cell mass are avoided, it is believed that blastocyst biopsy causes less harm to the embryo than Day 3 (blastomere) biopsy. Trophectoderm biopsy has the advantage of collecting multiple cells, which can mitigate the effects of mosaicism and ADO when genetic analysis is performed by PCR. In addition, since only about 50% of fertilized embryos will progress to blastocysts, fewer embryos are biopsied compared to Day 3 blastomere or polar body biopsy. This can also be a disadvantage, as laboratories performing blastocyst biopsy must be capable of embryo culture to Day 5 or 6. Trophectoderm biopsy will also result in fewer embryos being available for transfer, since compacting embryos and morulas, which can occasionally produce pregnancies with Day 5 transfers, are generally excluded from analysis.Scott et al. [43] have provided evidence for the superiority of blastocyst biopsy in a randomized clinical trial using a paired design where each patient acted as their own control group. In this study, couples undergoing IVF had two embryos selected from the same treatment cycle for transfer on either Day 3 or Day 5. Prior to the embryo transfer, one of the two chosen embryos randomly underwent biopsy, either on Day 3 (blastomere) or Day 5 (trophectoderm). The biopsied material did not undergo aneuploidy testing, but instead, DNA fingerprinting was performed, so that it could be determined which embryo implanted in singleton conceptions. The DNA pattern of the delivered child was determined from prenatal testing or fetal DNA obtained from maternal serum and was correlated with either the implanted or non-implanted embryo. Evaluation of biopsy performed at the Day 3 cleavage stage (blastomere) showed that it resulted in a relative decrease in embryo implantation of 39%. For biopsies performed at the Day 5 (blastocyst) stage, there was no difference in implantation and delivery rates between biopsied and non-biopsied embryos. These results provide strong evidence that cleavage-stage biopsy is more detrimental to the embryo than biopsy at the blastocyst stage, and this leads to poorer clinical outcomes. It is likely that more PGD centers will adopt trophectoderm biopsy in the future. The most recent ESHRE dataset [10] indicated that only 2.3% of biopsies were performed on Day 5 or 6, as compared to 79.8% at the Day 3 cleavage-cell stage.PCR has been the preferred method of diagnosis in PGD for single gene disorders. Nearly all genetically inherited single gene disorders where sequence information on the mutation is known can be detected by PGD. Diagnostic protocols for over 200 different single gene disorders have been reported, with the most common being for cystic fibrosis, spinal muscular atrophy, hemoglobin disorders, such as β-thalassemia, Huntington disease, fragile X syndrome and myotonic dystrophy. As opposed to the analysis of DNA from blood or amniocentesis/CVS samples, PCR in PGD usually begins from very limited amounts of DNA found in blastomere or trophectoderm biopsies. Single cells contain approximately six picograms of DNA, and for many PCR tests, 250 nanograms or more of DNA is required as the starting material. This implies that roughly a 40,000-fold amplification in DNA concentration is needed. One problem that became apparent was that the large number of PCR cycles needed to generate a product led to an increasing rate of the introduction of errors in the DNA. This was overcome by the development of “nested” PCR by Holding and Monk [44]. This technique uses a first round of amplification with an outer set of primers, followed by a second round using a set of inner primers, which when using the first round product as a template, amplifies a smaller PCR product inside the outer primers, which is robustly produced with high accuracy.The low initial starting concentration of DNA in embryo samples predisposes PCR to several other complicating factors, such as contamination with external DNA and allele dropout (ADO). Contaminating DNA can originate from the technician performing the PCR, from carryover of other PCR products or controls from earlier amplifications or from cumulus cells or sperm adherent to the zona. Careful cleaning of the embryo prior to fertilization and the use of intra-cytoplasmic sperm injection (ISCI) of a single sperm for oocyte fertilization can avoid this latter type of contamination. Operators typically dress in surgical gowns and perform the PCRs in laminar flow hoods situated in “clean rooms” to limit external DNA contamination.Allele drop out (ADO) refers to preferential amplification or failure of the amplification of one of the alleles present in a heterozygous sample. This could lead to misdiagnosis of a heterozygous embryo as a homozygous affected sample, in which case, a normal embryo would be excluded from the cohort of embryos available for transfer. In the case of a dominant genetic disease, ADO can cause an abnormal embryo to be misdiagnosed and transferred in error as “normal”. ADO can occur if the annealing of one of the two PCR primers is less efficient. Alternatively, inefficient cell lysis or degenerated DNA can theoretically lead to ADO. The frequency of ADO is variable and, in PCR of single blastomeres, can be seen in 5% to over 20% of amplifications [45,46].The early evaluation of PCR products included the analysis of products of radioactive-labelled primers or nucleotides, as well as ethidium bromide staining of DNA in agarose or polyacrylamide gels after restriction enzyme digestion. These methods have been replaced by fluorescent labeled probes or nucleotides, yielding products detected and sized by capillary electrophoresis. The most frequently utilized method for mutation detection is DNA primer-extension minisequencing [46] in which a probe is developed that extends up one base prior to the first nucleotide of the gene alteration. Single base-pair changes or small deletions/duplications can then be detected by the extension of the primer with fluorescent-labeled dideoxynucleotides, which will identify the next base in the target sequence and then stop, due to the chain-termination effect of dideoxynucleotides. Commercially available kits, such as the ABI SNapShot™ protocol (Life Technologies, Carlsbad, CA, USA), can be used for this purpose. Deletions and duplications can also be detected by that sizing of a PCR product, including the region of interest. The PCR reactions include primers for multiple target sites, not only for the area containing the mutation under consideration, but for closely linked polymorphic short tandem repeat (STR) markers, which can be used to detect DNA contamination and ADO [46]. This multiplex fluorescent PCR protocol led to a significant decrease in the number of analyses compromised by contamination or ADO.A pretest work-up typically involves obtaining DNA samples from the at-risk couple, as well as an affected child or grandparents to allow phasing of the STR haplotype with the mutation being tested. This allows the determination of the two maternal and two paternal haplotypes, as shown in Figure 1. This analysis aids in the interpretation of the results of PGD testing, although recombination of STR markers with the genetic mutation can be seen.Pre-PGD workup for a family with a previous child with spinal muscular atrophy. Panel (a) shows how the study of both parents and grandparents allows the phasing of the SMN mutation relative to polymorphic short tandem repeat (STR) markers; panel (b) shows the maternal and paternal haplotypes M1, M2, P1 and P2 and the distance of the STR markers from the SMN gene; panel (c) shows the four predicted fetal haplotypes. These reflect a Hardy–Weinberg equilibrium of one homozygous non-carrier, two heterozygous carriers and one that is homozygous and affected. Short tandem repeat markers linked with the SMN mutation are shown in red. DEL indicates the presense of the exon 7 (840 C>T) mutation.Newer PCR protocols have been developed using whole genome amplification (WGA) as a universal first step after biopsy of a single blastomere or 5–9 cells from a trophectoderm biopsy. This allows the analysis of a number of different sequences, such as compound heterozygous mutations of the CFTR gene, as well as allowing parallel testing of single gene disorders and linked STR markers or aneuploidy testing by aCGH. There are a number of methods for WGA that have been developed. Early protocols included primer extension PCR [47], degenerative oligonucleotide primed (DOP) PCR [48] or blunt end ligation and PCR amplification (GenomePlex™, Rubicon Genomics, Ann Arbor, MI, USA) [49]. All of these methods were dependent on PCR and carried the potential for uneven representation of the genome by preferential amplification or ADO. Multiple displacement amplification (MDA) is a WGA technology based on isothermal “rolling amplification” after the binding of random hexamers to the target sequence [50,51]. Despite the isothermal amplification in MDA protocols, ADO rates have been found to be at least 4% with SNPs [51], while others found a much higher ADO rate closer to 25% for linked STR markers used for PGD haplotyping [52,53]. The significant issues with ADO in single gene disorder PGD can be alleviated by the inclusion of multiple cells in the biopsy sample, which is why many centers are now moving toward trophectoderm biopsy, where 5–10 cells are typically obtained. In our lab, using trophectoderm biopsy and GenomePlex™ amplification, we have encountered an ADO rate of approximately 5% in single gene disorder PGD. Zong et al. [54] have reported on a new WGA method of multiple annealing and looping-based amplification (MALBAC), which is reported to significantly reduce amplification bias and provide 85%–95% genome coverage with 25X sequencing depth compared to 72% coverage at the same sequencing depth for the MDA protocol.The GenomePlex™ system presents another issue, in that the average size of the amplified product is 500–1000 bp. In using this DNA for single gene disorder testing, we have found it important to limit the amplicon size, such that PCR products are smaller than the average WGA DNA product length and, therefore, contained in a single fragment. The use of larger amplicons leads to the failure of the PCR to include the desired sequence. Tran et al. [55] showed that this approach is successful in developing PGD tests and analyzing results for different classes of mutations, including single base pair changes, deletions/duplications and triplet repeat expansion mutations along with STR repeats, all with a low ADO rate.It is likely that all WGA protocols result in some biased amplification, leading to decreased genomic coverage. Newer products are now being developed to address this bias, as well as producing highly accurate and representative amplification. As interest in more extensive sequencing in PGD develops, these issues will become more critical; however, at the moment, more limited evaluation, such as single gene sequencing and shotgun sequencing-chromosome counting protocols, seems to produce acceptable results with some current WGA methods (see below).The most common use of PGD involves the analysis of embryos for chromosome aneuploidy arising from either meiotic mal-segregation in oocytes or (less commonly) sperm or mitotic abnormalities of embryos after syngamy, which results in mosaicism; defined as the presence of two or more cytogenetically defined cell lines in the same embryo. Chromosome aneuploidy is strongly correlated with maternal age, however; mitotic abnormalities seem to be age-independent [56]. Embryos that are aneuploid often result in developmental arrest, failed implantation or subsequent miscarriage [57]. Chromosome analysis of miscarriage products by aCGH shows that approximately 70% of first trimester miscarriage is due to aneuploidy [58,59]. Since chromosome anomalies are the major cause of unsuccessful IVF or pregnancy loss, it was proposed that identification and preferential transfer of euploid embryos in IVF would lead to higher implantation and birth rates with a lower miscarriage rate [9,60]. This concept was felt to make perfect sense, and it was initially expected that aneuploidy testing would result in a major improvement in IVF outcome, especially in couples with recurrent pregnancy loss or recurrent IVF failure. Since standard metaphase karyotyping cannot be rapidly performed on polar bodies, blastomeres or trophectoderm cells, a different technology of interphase FISH was introduced to evaluate the chromosomal content of embryonic cells. Clinically, FISH was first performed for sexing, using X and Y probes to avoid the transfer of embryos with X-linked genetic disease. Shortly thereafter, more extensive chromosome testing was developed using protocols that score 5–12 chromosomes in multiple rounds of hybridization using centromeric or locus-specific FISH probes [7,8,9]. Testing was limited by the fact that there were only five visible spectrum fluorochromes available to label FISH probes, so testing needed to be performed in multiple sequential rounds of hybridization, stripping and re-hybridization; each round performed with decreasing efficiency.This form of PGD, which did not involve genetic disease testing and was designed to improve the outcome of IVF, was termed preimplantation genetic screening (PGS). The term was first used in Europe, apparently to distinguish chromosome testing from single gene disorder analysis, which was considered a less controversial use of PGD. The name PGS is a misnomer, as aneuploidy testing by PGD is clearly not a screening test in that clinical action (i.e., the non-transfer of embryos diagnosed as abnormal) is taken based on the results of the testing. This is in contrast with the classical definition of screening tests, which alters one’s odds of having a specific condition without providing a definitive, actionable diagnosis. An example of a screening test is first trimester combined screening (ultrasound and blood analyte measurement), which does not provide definitive chromosomal analysis, but adjusts a patient’s prior risk based on maternal age alone.Most chromosome studies performed prior to 2007 on cleavage stage embryo biopsy samples utilized FISH for analysis and showed significant levels of mosaicism throughout preimplantation development [36,61]. A systematic review of chromosomal mosaicism in preimplantation embryos, where every cell of an un-transferred embryo was analyzed [35], showed that only 22% of embryos were uniformly diploid, 73% were mosaic and 5% had multiple non-mosaic abnormalities. Of those that were mosaic, nearly half had diploid/aneuploid mosaicism. Comparative genomic hybridization (CGH) is an alternative method of chromosome analysis originally developed to karyotype solid tumors. DNA from a test sample and control sample are labeled with either green or red fluorochromes. The DNAs are both hybridized in equal concentration to either metaphase chromosomes (mCGH) or to chromosome specific DNA probes fixed to a microscope slide (aCGH) [62]. The fluorescent intensity of the two fluorochromes is measured; if equal, a yellow color is obtained. A red or green color is seen if either the test or control DNA is present in excess. A commercial aCGH protocol (24Sure™ Bluegnome, Cambridge, UK) has been developed specifically for PGD, which contains about 3600 very robust BAC clones (average size of 150 kb) spaced at approximately 1 MB intervals with 25% genomic coverage and a resolution of 4–10 MB. The protocol can be completed in as little as 12 h, allowing a fresh Day 6 transfer after Day 5 trophectoderm biopsy. The results are presented as log2 ratios, as shown in Figure 2.Early studies using mCGH of all blastomeres of non-transferred embryos [48,63] revealed that only 25% of the Day 3 embryos were totally euploid and that as high as two thirds demonstrated mosaicism. In one-half of these embryos, there were no normal cells (aneuploid/aneuploid mosaic), and 25% were diploid/aneuploid mosaics. These results led many to question whether the analysis of a single Day 3 blastomere was a good indicator of the developmental potential of the embryo.Array comparative genomic hybridization (aCGH) tracing after trophectoderm biopsy: (a) normal male embryo (female embryo control in blue); (b) female embryo with monosomy for chromosome 20 (male control in red); (c) an excellent quality blastocyst showing chaotic chromosome abnormalities. Nearly every chromosome is aneuploid.Early data using aneuploidy testing with FISH in women of advanced reproductive age seemed to indicate a trend toward improvement in implantation and pregnancy rates [64,65,66,67,68]. These studies were mostly small, case-control studies in patients with advanced reproductive age or recurrent pregnancy loss. Despite somewhat encouraging results, statistical power could not be generated, and therefore, it was recommended that randomized clinical trials (RCT) be undertaken to prove that aneuploidy testing via PGD led to an increase in the birth of healthy children. Staessen et al. [31] in 2004 reported the first RCT comparing the results of blastocyst transfer with or without PGD using a FISH panel of seven chromosomes after the Day 3 cleavage stage biopsy of two cells. The results showed improved implantation and clinical pregnancy in the PGD group, but the difference was felt to not be of statistical significance. This group later performed a second RCT employing only single cell embryo biopsy, which produced improved results compared to the biopsy of two cells, but did not demonstrate any statistically significant benefit for the implantation or ongoing pregnancy in the PGD group, as compared to controls without PGD [33]. Overall, 10 RCTs have been performed to evaluate the efficacy of aneuploidy testing (for a summary table, see Fragouli and Wells [69]). The 2007 multicenter double-blind RCT performed by Mastenbroek et al. [70] reported that Day 3 blastomere PGD actually led to a decrease in the ongoing pregnancy rate, with the PGD group having a 25% ongoing pregnancy rate, as compared to 37% for the control group. This study garnered much media attention and caused much alarm among patients contemplating chromosome-based PGD. It was pointed out that the study was flawed on multiple levels, from patient selection, lack of embryo biopsy experience, selection of incorrect chromosomes for study and a high incidence of no results [71,72]. Some investigators pointed out technical issues with the RCTs already performed and felt that strict adherence to their protocol was necessary to show improved pregnancy rates with FISH [73].At this point in time, there was a re-evaluation of the technology of the cleavage stage PGD for chromosomal aneuploidy testing. Relevant factors included: (1) The reduction in the implantation of embryos after blastomere biopsy; (2) The experience of the embryology laboratory with blastomere biopsy; (3) Proper cell fixation and cell lysis technique; (4) Hybridization issues, such as failure, split or overlapping signals, or nuclear damage with micronuclei formation; (5) The selection of optimal probes for FISH analysis (the use of seven probes for chromosome X, Y, 13, 16, 18, 21 and 22 was shown to account for 72% of chromosome abnormalities seen in spontaneous miscarriage [74]); and (6) The presence of chromosomal mosaicism, which leads to decreased accuracy and poorer pregnancy outcome [75,76].The clinical utility of FISH-based PGD for chromosome abnormalities was seriously questioned, and it was recognized that in order to keep recommending PGD for patients with advanced reproductive age, recurrent miscarriage or multiple failed IVF cycles, a new RCT was needed using a different source of embryonic tissue for analysis [77,78]. In Europe, the analysis of polar bodies was selected, due to the legal restrictions on IVF and PGD present in many countries. Other groups turned their attention to the blastocyst and trophectoderm biopsy. At the same time, researchers in the field again considered CGH for chromosome analysis. As noted above, in 2000, mCGH was used by Wells and Delhanty [48] and Voullaire and colleagues [63] to analyze DNA obtained from multiple blastomeres of untransferred embryos after degenerate oligonucleotide primed (DOP) PCR. These studies indicated that 51% of cleavage stage embryos were aneuploid in every cell, and 24% contained a mixture of normal and abnormal cells, with only 25% being uniformly normal. Although superior in detecting chromosome anomalies compared to FISH with limited panels of chromosomes, mCGH is very labor and time intensive, thus requiring all embryos to be cryopreserved with a later frozen embryo transfer using embryos determined to be normal. Originally, this protocol was complicated by the fact that cryopreservation of biopsied embryos was very poor with low survival [79]. This improved dramatically with the introduction of embryo vitrification, with survival rates now over 90% and implantation similar to that of fresh embryo transfer [80].Renewed interest in CGH resulted in this technology being validated for use in polar bodies [81,82,83], blastomeres and blastocysts [84,85,86]. Analytic improvement was accomplished with the evolution of mCGH to aCGH, where hybridization was performed on chips containing thousands of DNA segments fixed on a glass slide. Gutierrez-Mateo et al. [87] studied single blastomeres by aCGH after WGA in non-transferred embryos and compared the results to those obtained by FISH. They showed that aCGH detected 42% more chromosome defects, as compared to a panel of 12 FISH probes. Fragouli and colleagues [86] investigated the chromosomal status of blastocysts with mCGH. Some of these embryos had an abnormal Day 3 FISH result, which was confirmed by CGH, while the majority were donated, untested embryos, which were felt to represent what might be obtained in clinical practice. The aneuploidy rate of the blastocysts was 38.8%, which was lower than the 51% seen in Day 3 embryos [48,63]. Aneuploidy of all chromosomes was seen in blastocysts, including those of the larger chromosomes, as well as chaotic abnormalities (see Figure 2c). These findings dispelled previous beliefs that culturing to the blastocyst stage would effectively select against embryos with chromosome anomalies. The authors also showed the concordance of chromosome errors in the ICM and TE, indicating that preferential allocation of abnormal cells away from the ICM to the TE is not seen in humans.In addition to mCGH and aCGH, single nucleotide polymorphism (SNP) arrays were validated to perform 24 chromosome analysis. SNPs are sequence variants that exist in the population, where at a particular locus, one of two (or more) nucleotides is commonly found. Biallelic SNPs, where one of two bases is seen, have proven to be useful in molecular cytogenetic analysis. The relative intensity of the two alleles, typically referred to as A and B, can be measured at heterozygous loci to detect duplications or deletions of chromosomal regions. SNP arrays also can reveal the parental origin of chromosomal errors by genotyping the parents. Rabinowitz and colleagues [56] have developed a bioinformatics algorithm using parental genotypes and reported on the chromosome number analysis of blastomeres from cleavage stage embryos. This showed increasing aneuploidy with maternal age originating from meiotic nondisjunction of the oocyte. SNP arrays also have the advantage of being able to detect polyploidy and uniparental disomy (UPD), which cannot be seen with CGH. Treff and colleagues [49] validated a high-density array with 262,000 SNPs using 99 WGA amplified single cells from chromosomally-defined lymphoblastoid lines. Three hundred thirty five WGA blastomeres were also studied with only 37.3% being euploid. This group also compared the performance of SNP arrays with that of interphase FISH by examining 160 blastomeres from arrested embryos and concluded that the SNP array was more accurate and FISH overestimated the aneuploidy rate [88]. Finally, CGH and SNP arrays demonstrated the significant mosaicism of cleavage stage embryos, which could lead to misdiagnosis and the non-transfer of potentially viable embryos.Biopsy of embryos at the blastocyst stage was considered after the development of sequential media that allowed in vitro culture to Day 5 or 6 after fertilization. These embryos were felt to have higher developmental potential, and the testing of trophectoderm cells could lead to improved clinical outcome with less damage to the embryo. Fragouli and colleagues [85] studied 64 non-transferred blastocysts by mCGH, aCGH and a nine-probe FISH panel and showed that 42% of blastocysts were uniformly euploid, 30% uniformly aneuploidy, 17% were diploid-aneuploid mosaics and 15.4% composed of different aneuploid cells. FISH results were concordant with CGH in 10 of 12 embryos; however, in the remaining two, only the sex chromosomes were consistent. Northrup and colleagues [38] used SNP arrays to study 50 blastocysts and demonstrated that 62% of the blastocysts were uniformly euploid, 7.8% uniformly aneuploid and the remaining 30% mosaic, containing either diploid/aneuploid or aneuploid/aneuploid mosaicism.Studies evaluating the ability of aCGH to detect mosaicism in blastocysts have been reported by Mamas et al. [89] and Novik et al. [90]. These studies created models of mosaic blastocysts by either mixing cells or DNA from euploid and aneuploid cell lines or embryos confirmed to be uniformly monosomic by FISH. The results showed that aCGH using the 24Sure protocol was able to clearly detect mosaicism when the proportion of abnormal cells was approximately 25%–30%.As trophectoderm biopsy appeared to produce a more accurate assessment of the chromosomal status of the embryo, clinical studies were undertaken to evaluate the outcome of array-based PGD in infertile couples and those with recurrent pregnancy loss or repeated IVF failure. Schoolcraft and colleagues [91] studied 45 couples with infertility that underwent CGH-based PGD with trophectoderm biopsy and compared the results to 113 couples who had blastocyst transfer without PGD. Nearly 70% of the PGD couples achieved a clinical pregnancy as compared to 45% in couples where PGD was not performed. A clinical study in infertile couples using trophectoderm biopsy, SNP microarray analysis, embryo vitrification and subsequent frozen embryo transfer (FET) was reported by Schoolcraft and colleagues [92]. In the first 100 FETs, the biochemical pregnancy rate was 87% with a 73% clinical pregnancy rate (positive fetal heart rate) and a 2.3% miscarriage rate.To avoid the need for embryo vitrification and later FET, Treff and colleagues [93] developed a new method for 24 chromosome analysis of blastocysts using real-time quantitative PCR (qPCR). This method used multiplex PCR in a 384-well plate format to amplify two areas on each arm of all chromosomes. The analysis can be completed in 4 h, allowing for fresh embryo transfer. Each of the methods for 24 chromosome analysis described above has their own specific advantages and drawbacks. An excellent summary comparing the different technologies has been published by Handyside [94].The results of early and ongoing clinical trials of blastocyst biopsy and 24 chromosome analysis on improving implantation and ongoing pregnancy have been encouraging [95]. Harton et al. [96] have collected data from multiple IVF centers on patients undergoing IVF with PGD by aCGH after cleavage stage or trophectoderm biopsy. The pregnancy rates were higher for trophectoderm biopsy, but for both, the transfer of euploid embryos blunted the typical decrease in implantation and ongoing pregnancy associated with advancing maternal age. In other words, if euploid embryos were identified, their implantation potential was relatively independent of maternal age. This is what would be expected if aneuploidy was the major cause of implantation failure and miscarriage. Of course, as maternal age increased, so did the number of women who did not produce any euploid embryos. Scott et al. [97] have reported on an RCT involving 155 couples who were randomized to receive a Day 5 blastocyst transfer without PGD (control group) or a trophectoderm biopsy, comprehensive chromosomal screening by four-hour qPCR and Day 6 embryo transfer (study group). The rate of implantation and delivery was 66.4% with PGD and 47.9% in the control group. Delivery per cycle was 84.7% for the PGD group versus 67.5% in the control group.These results also highlight one of the most important benefits of the increased implantation and lower miscarriage rate after 24 chromosome analysis; namely, the ability to optimize elective single embryos transfer (eSET). The goal is to reduce the incidence of twin gestation, which is associated with a 5–10-fold increase in the risk for fetal and maternal complications, including gestational diabetes, preeclampsia, premature delivery and low birth weight [98]. Since embryos that have a normal chromosome analysis have the highest developmental potential, it makes sense that the transfer of euploid blastocysts will optimize eSET. An RCT for eSET in good prognosis women <35 years of age was carried out by Yang et al. [99], who compared the transfer of a single embryo at the blastocyst stage on Day 6 that was either tested by aCGH or assessed by morphologic criteria only. In the aCGH group, the ongoing (>20 gestational weeks) pregnancy rate was 69.1% compared to 41.7% in the morphology only group. Another RCT was carried out by Forman et al. [100] in 205 women <43 years of age, but with normal ovarian reserve testing. The study group had chromosome testing by qPCR with subsequent eSET, while the control group transferred their two morphologically best embryos. The ongoing pregnancy rate per patient was similar (60.7% in the eSET group versus 65.1% after the two-embryo non-aCGH tested transfer); however, the multiple pregnancy rate was significantly higher in the two embryo transfer group (53.4%) versus the PGD eSET group (0%). The conclusion was that 24 chromosome screening after trophectoderm biopsy and eSET produced as high a pregnancy rate as the transfer of two untested embryos without encountering the increased risk for twin gestations.Structural chromosome rearrangements include reciprocal and Robertsonian translocations and inversions. These can be seen in approximately 1/500 live-born infants and 1/250 prenatal samples [101]. Individuals who carry balanced chromosome translocations (or inversions) generally have no clinical findings related to the translocation, but will produce high rates of abnormal gametes after meiotic segregation. The involved chromosomes will orient at the metaphase plate in a quadrivalent pattern and will segregate to the two daughter cells in one of 30 or so segregation patterns. These can contain areas of chromosome deletion or duplication, which can lead to pregnancy loss, failed implantation, apparent infertility or the birth of a child with physical and/or developmental disability.Scriven and colleagues [102] published a method for the evaluation of segregation in translocations involving FISH using commercially available specific centromeric and sub-telomeric probes. Although this technique could identify embryos that were balanced for the involved chromosomes, it was subject to the same technical limitations described above for aneuploidy testing utilizing FISH. In addition, no information was provided for chromosomes not involved in the translocation, which could produce aneuploidy and decreased reproductive potential. Fiorentino et al. [103] developed a PCR-based method to evaluate translocations using STR markers that flanked the translocation breakpoints along with others to evaluate the copy number of other chromosomes. This was applied in 27 PGD cycles where 18 couples achieved a clinical pregnancy. Shortly thereafter, mCGH or aCGH was applied after WGA, and this analysis allowed not only aneuploidy evaluation of the translocated chromosomes, but also detected age-related aneuploidy in other chromosomes not involved in the translocation. In the report of Alfarawati et al. [104], where 16 couples with translocations underwent 20 cycles of IVF/PGD, 22% of embryos were chromosomally normal and 28.9% were balanced for the translocation, but had aneuploidy of other chromosomes. Fiorentino et al. [105] reported on 28 PGD cycles for translocation in 24 couples with aCGH and found that 16% of embryos were normal for all chromosomes, while 27.3% were normal for the translocation, but showed aneuploidy in other chromosomes. Treff et al. [106] validated an SNP array for the detection of translocations and evaluated 19 IVF/PGD cycles from 15 patients carrying a translocation. Of 122 normally developing blastocysts, 50.8% were normal or balanced for the translocation and 32% were euploid in the remaining chromosomes. Overall, including arrested embryos, 15.2% of all embryos produced were chromosomally normal. In 12 cases where the embryo transfer of normal embryos was carried out, a clinical pregnancy rate of 75% was seen. The implication of these results was that by using FISH for the PGD of translocations, approximately half of the embryos that were determined to be “normal” actually contained aneuploidy for other chromosomes not involved in the translocation and, therefore, not detected by the FISH analysis used in their case. Less than one quarter of the embryos produced in these cycles therefore were truly euploid and capable of producing a healthy pregnancy. The reproductive outcome in couples carrying a translocation is likely dependent on the shape of the quadrivalent and subsequent modes of segregation of the specific translocation and the risk of producing a viable abnormal gestation. Scriven et al. [107] argue that for fertile couples carrying translocations with a low risk of producing viable abnormal gestations, natural conception may be capable of producing a normal child in a shorter time and with lower cost than the use of IVF/PGD.Rapid advances in DNA sequencing technology have made it possible to generate very large amounts of sequence data with the use of high-throughput NGS and bioinformatics tools. The recent approval by the United States FDA of the Illumina MiSeq DNA sequencing platform is an indication that NGS will become integrated into multiple fields of medicine, including PGD. Typically, NGS protocols involve the fragmentation of the DNA to be sequenced into 100–200 base pair segments. Linker sequences, including molecular “barcodes” and oligonucleotides to attach the fragment to the flow cell, are ligated onto the fragmented DNA. On the flow cell surface, hundreds of thousands of the fragments are sequenced in parallel reactions involving the successive addition of fluorescent nucleotides and ultra-high resolution imaging of the successively added base. This approach is called “sequencing by synthesis”. The results are compared to a reference genome using a bioinformatics algorithm, and the protocol is repeated until a sufficient read depth is obtained by the sequencing of other fragments from the same genomic region. The use of barcodes allows multiple samples from different analyses to be sequenced simultaneously in the same flow cell, allowing efficiency of cost.For PGD of single gene disorders using NGS, sequencing of the region containing the mutation until sufficient read depth is accomplished to be confident that the base calling is straightforward. Methods are now being developed to enrich WGA samples for specific genomic regions, where single gene mutations are present to increase the read depth of these sequences [108]. Treff and colleagues [109] evaluated NGS-based PGD for single gene disorders in six couples at-risk of transmitting either autosomal recessive disease (Walker Warburg syndrome, cystic fibrosis, familial dysautonomia), dominant disease (neurofibromatosis 1) or X-linked hypophosphatemic rickets to their children. NGS sequencing was carried out on excess embryonic DNA from trophectoderm biopsy using semiconductor sequencing technology with the Ion Torrent Personal Genome Machine (Life Technologies, Carlsbad, CA, USA). Aneuploidy testing was performed by qPCR. The genetic disease results were compared with Taqman allelic discrimination assays or PGD results from an outside reference laboratory. In all cases, the NGS results were perfectly consistent with those of the other two methodologies.To determine chromosome copy number by NGS, the shotgun sequencing-chromosome mapping protocol originally developed by Fan et al. [110] for non-invasive diagnosis of cell-free fetal DNA in maternal circulation can be used. In this protocol, WGA embryo biopsy material is randomly fragmented, and the sequencing of 33–36 base pairs is carried out to allow the mapping of the fragment to the chromosome of origin. The number of fragments that map to a particular chromosome should be proportional to the copy number of that chromosome, with trisomic or monosomic chromosomes having more or less fragments, respectively. Since the DNA involved will contain only fetal sequence, as opposed to the situation in cell-free fetal DNA testing, where only 10% of the DNA is fetal in origin, relatively low average read depth and genomic coverage is adequate to accurately assess chromosome copy number. Yin and colleagues [111] studied trophectoderm biopsy samples from 38 donated blastocysts from 16 IVF cycles by both NGS and SNP microarray, with qPCR being used to define any inconsistencies between the two protocols. High throughput sequencing was performed using an Illumina HiSeq 2000 sequencer. An average of 9.3 million reads per embryo was obtained with an average 0.07× sequencing depth and 5.5% coverage of the whole genome. Results showed that 26 of the embryos (68.4%) were determined to be completely euploid, and 12 contained chromosome errors (31.6%). The euploid embryos were correctly identified by NGS and SNP array, and consistent abnormalities were identified in six uniformly aneuploid embryos. Segmental aneuploidy was detected by NGS and qPCR, but missed by the SNP array in two embryos, possibly due to bias from WGA. This group (Li et al. [112]) then used the NGS protocol clinically in 41 couples, analyzing trophectoderm biopsied WGA DNA, generating 8.2 million reads per embryo, with 5.5% genomic coverage. Uniformly euploid embryos were identified in 47.3% of biopsied blastocysts, and an ongoing pregnancy rate of 58.5% was recorded in 24 women who received a transfer.Combining both aneuploidy testing and genetic disease diagnosis, Wells and colleagues [113] have reported the births of children to couples at-risk for cystic fibrosis or mitochondrial DNA defects after PGD. NGS using the Ion Torrent platform was performed on trophectoderm biopsy samples after MDA WGA to diagnose chromosome aneuploidy, as well as direct sequencing of the mutations present in the family. It was noted that the high throughput of the NGS system allowed simultaneous genetic analysis of up to 100 embryos, which could significantly reduce the cost of PGD to two-thirds of the current cost using aCGH. In addition, the pre-workup for single gene disorder PGD would no longer be needed, adding additional savings.The above studies have demonstrated that NGS-based PGD can be accomplished accurately and with very high throughput. This will likely herald the move of PGD technology from PCR and aCGH to NGS analysis as the cost of sequencing continues to decline. A very significant question to be answered at this time is what will PGD look like in five to 10 years? As molecular technologies continue to evolve and allow us to accumulate huge amounts of sequence data, we need to decide what will be the role of PGD in the future. Some have suggested that PGD should be a part of all IVF cycles, especially where eSET is recommended. It is not clear, however, that universal PGD makes sense in terms of the extra cost for patients and the small number of qualified laboratories currently able to perform the procedure.It has also been suggested that additional genetic screening be added to current PGD aneuploidy testing. In this regard, we will probably follow the experience of classical prenatal diagnosis, which is now, and will be in the future, most frequently performed by cell-free testing of fetal DNA in the maternal plasma. It has been discussed that several genomics providers will shortly introduce testing for chromosome microdeletion syndromes (DiGeorge/velocardiofacial syndrome, Williams syndrome, etc.) to their current aneuploidy screening protocol. It would be possible to do multiplex enrichment for these genomic abnormalities, which could be detected by NGS sequencing, likely in a protocol with embryo vitrification, data analysis and subsequent frozen embryo transfer.The use of PGD for severe genetic disorders has always been non-controversial, and chromosome aneuploidy testing is only currently beginning to be more widely accepted as data accumulates from RCTs showing improved pregnancy and delivery rates. Other uses of PGD have been viewed more skeptically, such as HLA matching, adult onset genetic disorders, conditions with variable penetrance and non-medical sex selection. There is concern that PGD could be used for other non-medical conditions, such as height, hair color, memory or athletic ability, producing what has been called “designer babies”. As our understanding of genomic information improves, new prenatal tests will be developed, which are likely to be DNA sequence-based, and many could be adapted for prenatal or preimplantation diagnosis. It is not clear, however, whether PGD is the appropriate clinical setting to introduce new genetic testing strategies that have not yet been validated in the prenatal diagnostic clinic.While the use of PGD to prevent the transmission of known serious genetic disease in a family is considered appropriate, the situation becomes less clear as more extensive genetic screening of embryos by technologies, such as whole exome or whole genome screening, become possible in the context of PGD. Questions arise regarding the moral obligation of the parents to perform extensive genetic testing on their embryos, simply because it is possible, as well as the obligation of health professionals to provide such testing.Preliminary discussions regarding the future use of PGD and an ethical framework for its application have been carried out with panels of experts in the field [114,115,116]. Although there are few definitive conclusions from these meetings, the participants were unified in their belief that unless focused goals for the use of PGD are established, the procedure will encounter significant resistance for what will be perceived by some as eugenic pandering to parents’ fantasies about the “perfect” child [115]. In addition, extensive genomic sequencing will undoubtedly reveal unintended findings that may be clinically significant, or alternatively, variants of unknown clinical significance may be identified. This underscores the need for carefully obtaining the consent of couples for PGD along with both pre- and post-test genetic counseling to make sure the results are correctly interpreted and clearly explained.PGD was initially performed nearly 25 years ago as an alternative for the prenatal diagnosis of single gene disorders in ongoing intrauterine gestations with the potential interruption of affected pregnancies. In the second phase of PGD development, cycles were performed for the detection of aneuploidy to improve the outcome of IVF in patients with translocations, advanced reproductive age, recurrent IVF failure or recurrent pregnancy loss. While there has been the general acceptance of PGD for genetic disorders, there has been skepticism of the benefit of PGD for chromosome aneuploidy, specifically the lack in multiple RCTs of a statistically significant improvement in the live birth rate with PGD, as compared to IVF without testing. With the analytic technology evolving from FISH-based testing to aCGH and SNP microarrays and, most recently, NGS, the quality of the results has improved. This has also been achieved with a change in the tissue biopsied from Day 3 blastomeres to Day 5 trophectoderm or polar bodies, which causes less harm to the embryo and, in the case of trophectoderm biopsy, provides a multi-cellular sample, which can mitigate the mosaicism common in cleavage stage embryos, as well as lower the risk of ADO. Initial reports have shown improved implantation and live birth rates with lower miscarriage rates in IVF when chromosomal PGD is performed. The identification and transfer of euploid embryos appears to blunt the effect of advancing maternal age and is the ideal method to select embryos for use in eSET to reduce the incidence of multiple gestations with IVF.With the increasing use of NGS in PGD, both single gene disorder and chromosomal testing can be performed simultaneously on the same sequencing platform without the need for the pre-test workup of single gene disorders. In the near future, NGS is likely to be used to identify embryos with microdeletion syndromes or common pathologic copy number variations. New developments in sequencing technology and bioinformatics will likely allow even more sequence information to be rapidly generated from embryos. The objectives of such testing and the role that PGD should play in IVF will need to be further defined and validated, or the technology could be used for the identification of non-medical traits, such as stature, memory, hair and eye color or athletic ability. Without focused goals, PGD could be considered a mechanism to attempt the selection of the “perfect” child and will invoke the specter of “designer babies”.The author declares no conflicts of interest.

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+This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).The diagnosis of pediatric bipolar disorder (PBD) has increased dramatically in community-treated youth in the past 20 years. No previous study has assessed the trend in PBD subtype diagnoses or the impact of clinician-reported behavioral comorbidities (BC) on psychotropic medication prescribing patterns. This study aims: (1) to characterize national trends in PBD visits in relation to PBD subtypes; and (2) to assess differences in socio-demographic PBD subtype diagnostic patterns and psychotropic medications prescribed in PBD visits with and without behavioral comorbidities (w/w/o BC). PBD visits for 1999–2010 from the National Ambulatory Medical Care Survey (NAMCS) data were assessed using population-weighted chi-square and logistic regression analyses. While PBD visit rates were stable across 12 years, the proportional shift of subtype diagnosis from Bipolar I (89.0%) in 1999–2002 to Bipolar Not Otherwise Specified (NOS) (74.1%) in 2007–2010 was notable. Compared with PBD without behavioral comorbidities (w/o BC), PBD visits w/BC had greater proportions of the bipolar-NOS subtype, more males, 2–14-year-olds, and more publicly-insured visits. The prescription of antipsychotics (60% vs. 61%) was common in PBD visits regardless of the presence of behavioral comorbidities. Stimulants were the predominant class prescribed for PBD visits with BC (67.8% vs. 9.4%). Antidepressants were significantly greater in PBD visits without BC (41.6% vs. 21.0%). Overall one-third of PBD youth visits were prescribed antipsychotics concomitant with other psychotropic classes. Behavioral conditions accompanying PBD visits were prominent, suggesting the need for monitoring and evaluating the outcomes of complex medication regimens in community populations.Pediatric bipolar disorder (PBD) is a serious mental disorder that can lead to disruption in the lives of children and adolescents [1]. Bipolar disorder, once considered rare in adolescents and younger children, has been increasingly diagnosed in community populations over the last decade [2,3]. Blader and Carlson [2] reported population-adjusted rates of hospital discharges of children with a primary diagnosis of PBD that increased linearly over eight years. The U.S. rate between 1996 and 2004 grew 5.6-fold for children and four-fold for adolescents [2]. The study by Moreno et al. showed that the annual estimate of PBD office visits increased 40-fold from 1994–1995 to 2002–2003 [3]. This rapid increase of PBD diagnosis has raised concerns of the over-diagnosis of this disorder among children and adolescents [3,4]. The overlapping of symptoms, e.g., distractibility, pressured speech, and irritability, have made clinicians and researchers aware of the difficulty of diagnosing comorbid behavior conditions, like attention-deficit hyperactivity disorder (ADHD) in children with PBD [3,4,5,6]. Recently, Dusetzina et al. analyzed private insurance claims in 2007 for youth <18 years of age. Among 16,641 youth with clinician-reported PBD, approximately 30% had a comorbid diagnosis of ADHD [7]. The distribution of the PBD subtype was PBD-I/PBD-II/PBD-Not Otherwise Specified (NOS) 38.3%/11.2%/50.5%. Along with the PBD diagnostic growth, there has been a corresponding increase in medication treatment patterns, consistent with the expanded use of antipsychotics [8,9] and concomitant psychotropic class use regardless of diagnosis [10].The high prevalence of PBD-NOS subtype raises the question about its diagnostic reliability. NOS diagnoses do not meet the defined criteria in the Diagnostic and Statistical Manual of Mental Disorders (DSM-IV) [11]. These diagnoses are primarily based on sub-threshold symptom levels. Additionally, increased comorbid conditions due to overlapping of symptoms can indicate greater severity and justify additional medications, which can lead to increased risks associated with drug combinations [12].Despite the high visibility of this topic, no information exists on the temporal trends in PBD subtypes, the impact of the presence of behavioral comorbidity in youth diagnosed with PBD and the change in psychotropic medications prescribed during these visits over 12 years. Therefore, this study aims: (a) to assess the time trends in PBD and its subtypes across 12 years from a national sample of physician office visit data; and (b) to assess the impact of the presence of behavioral comorbidities on PBD subtypes, patient visit characteristics and psychotropic medication prescription patterns. Data were drawn from annual U.S. National Ambulatory Medical Care Surveys (NAMCS) for 1999–2010. The NAMCS is a federally sponsored survey conducted by the National Center for Health Statistics (NCHS). NAMCS data are based on a multistage probability sampling design collected from non-federally employed physicians engaged in direct patient care during a randomly assigned one-week reporting period [13]. During this period, data for a systematic random sample of visits are recorded by the physician or office staff on an encounter form provided for that purpose. Data are obtained on selected patients’ demographic characteristics; physician-reported diagnoses, prescribed medications and services provided. Following NCHS recommendations, NAMCS medical visit data from contiguous years are combined to produce stable estimates. In order to estimate the temporal trends of youth visits with a diagnosis of PBD, the data were grouped into three four-year periods as follows: 1999–2002, 2003–2006 and 2007–2010. Each visit is assigned a value, the sum of which projects to an estimate of the total medical visits nationally. This value is referred to as weighted value estimation (WVE). The weighting procedures produce essentially unbiased national estimates and are derived from the following components: (1) an inflated estimate based on national census; (2) adjustment for non-response; (3) a ratio adjustment to fixed totals; and (4) weight smoothing [14]. We included a weighted column percentage (WC%), 95% confidence interval (CI) of WVE along with the number (N) of unweighted visits. Estimates based on fewer than 30 visits are unreliable. There were 424 PBD visits identified from total visits (N = 47,386) for 2–19 years olds. Across the study years, survey response rates varied between 58.3% and 70.4%, with a median response rate of 62.5%. The ratio of physicians to reported visits was 1:22 for the study years. For the present study, age was categorized as 2–9, 10–14 and 15–19 years. Race/ethnicity was categorized as white and non-white (African American, Hispanic, Native American, Pacific Islander and Asian or more than one race). Data regarding sources of payment for the visit were collapsed into 2 mutually exclusive categories: private insurance, including self-payment, and public insurance (Medicare, Medicaid, other government insurance, no charge and unknown payment source). Physician specialties were grouped as psychiatry and non-psychiatry (general practice, family practice, pediatrics, neurology and other specialties). Four regions were defined by U.S. census categories as Northeast, South, Midwest and West-Pacific.Office visit psychiatric diagnoses were recorded by treating physicians according to the International Classification of Diseases, Ninth Revision, Clinical Modification (ICD-9-CM) codes. The visits were classified as bipolar mania (296.0, 296.1 and 296.4), bipolar depression (296.5), bipolar mixed (296.6) and bipolar episode unspecified (296.7). Subtype groupings included the following: Bipolar I (296.0, 296.1, 296.4, 296.5, 296.6, 296.7), Bipolar II (296.89) and Bipolar-NOS (296.80).Psychiatric comorbidities were identified for PBD visits that had additional clinician-reported codes of mental disorders. Comorbid psychiatric diagnoses were categorized as: 314–314.99 for attention deficit hyperactivity disorder (ADHD); 312.00–312.49 and 312.80–312.99 for conduct disorder (CD); 313.81 for oppositional defiant disorder (ODD); 296.2–296.3, 300.4, 311 for depression; 300–300.3, 300.5–300.9 and 309.81 for anxiety disorders. All other ICD-9-CM codes between 290 and 319, excluding the above-mentioned categories, were labeled as “other psychiatric disorder”. Up to three diagnoses could be recorded for each pediatric bipolar disorder visit. Patient visits were categorized as PBD with behavior disorder (with co-morbid diagnosis of ADHD, CD or ODD) and without behavior disorder (PBD with other psychiatric comorbid conditions) for the analysis of PBD w/w/o BC.Psychotropic medications prescribed for the treatment of PBD include seven classes: lithium, antipsychotics (atypical and conventional antipsychotics (ATP)), anticonvulsant-mood stabilizers (ATC-MS), antidepressants (ATD), anti-anxiety/hypnotics, alpha-agonists and stimulants. Additionally, they were categorized as monotherapy or ATP-concomitant regimens. ATC-MS included carbamazepine, oxcarbazepine, divalproex, valproic acid, lamotrigine and topiramate. PBD visits with prescribed psychotropic medication classes were population weighted and reported as column percentages with a 95% confidence interval (CI). Furthermore, the most frequently prescribed ATP regimens concomitant with other psychotropic classes were reported as column percentages. Population-weighted trends in PBD visits were assessed for 1999 to 2002, 2003 to 2006 and 2007 to 2010 using the chi-square statistic. Likewise, in the same time-periods, proportional differences in PBD subtypes were assessed. Subsequently, to study the impact of behavioral comorbidities on PBD visits, we combined the most recent years (2003–2010) to improve statistical reliability. In this analysis, population weighted differences in socio-demographic, clinical, administrative and prescribed psychotropic medication class characteristics of PBD visits with and without behavioral comorbidity were assessed with chi-square statistics. Population-weighted multivariable logistic regression modeling was employed to report the adjusted odds ratio (AOR) with 95% CI for PBD with behavioral comorbidity vs. PBD w/o behavioral comorbidity, adjusting for age group, gender, race/ethnicity, payment type, region and PBD subtype. SAS version 9.2 (SAS Institute, Inc., North Carolina, Cary) was used for all analyses in this study. The percentage of PBD visits as a proportion of total pediatric visits remained stable among youths in recent study years: 0.4% (95% CI, 0.2–0.6) in 1999–2002 and 0.4% (95% CI, 0.3–0.5) in 2007–2010 (data not shown). By contrast, there was a proportional shift of subtype diagnosis from PBD-I (89.0%) in 1999–2002 to PBD-NOS (74.1%) in 2007–2010 (χ2 = 60.3, df = 4, p < 0.0001) (Figure 1). Specifically, PBD-I decreased from 89.0% to 18.3%, whereas PBD-NOS significantly increased from 2.6% to 74.1%. In the most recent period, 2007–2010, the PBD-I/PBD-II/PBD-NOS distribution was 18.3%/7.6%/74.1%, showing the great preponderance of NOS in recent years. Proportional distribution of pediatric bipolar disorder (PBD) visits according to subtype diagnoses in three time periods a.a Data are from the National Ambulatory Medical Care Survey; NOS, not otherwise specified; * Represents unreliable estimates, due to a small sample size.Table 1 shows the demographic, clinical and administrative characteristics of PBD youth visits with and without a behavior disorder for the eight-year period, 2003–2010. Compared to PBD visits without behavioral comorbidity, PBD visits with behavioral comorbidities represented greater proportions of the Bipolar-NOS subtype (60.8% vs. 45.3%), as well as more males (69.7% vs. 46.5%), 2–14-year-olds (65.2% vs. 31.5%) and publicly-insured youth (55.0% vs. 40.0%). Regardless of comorbid behavioral status, more than 75% of PBD visits were made to psychiatrists. The prescription of antipsychotics was common (60.0% vs. 61.0%) in PBD visits regardless of the presence of comorbid behavior disorders. While stimulants were the predominant prescribed class for PBD visits with comorbid behavioral disorder (67.8% vs. 9.4%), antidepressants were significantly greater in PBD visits without comorbid behavioral disorders (20.9% vs. 41.6%). Table 2 illustrates that antipsychotic monotherapy was more common among PBD without behavioral comorbidity. More specifically, ATP with concomitant stimulants was the leading regimen for PBD with behavioral comorbidities compared with PBD without behavioral comorbidities (p < 0.0001). Overall one-third or more of PBD visits had antipsychotic (ATP) regimens with one or more concomitant psychotropic classes (data not shown). The use of ATP with anticonvulsant-mood stabilizers or antidepressants did not differ according to behavioral comorbid status.Demographics, clinical characteristics and prescribed psychotropic medication classes in office-based PBD visits with and without behavioral comorbidities for 2003–2010. a N = 318.a Data are from the National Ambulatory Medical Care Survey; PBD, pediatric bipolar disorder; † Represents unreliable estimates, due to a small sample size; N, number; WC%, weighted column percentage; NS, not significant.Antipsychotic (ATP) drug regimens prescribed for PBD with and without behavioral comorbidities during 2003–2010; N = 318.PBD, pediatric bipolar disorder; N, number; WC%, weighted column percentage; ATP, antipsychotics; ATC-MS, anticonvulsant-mood stabilizers; ATD, antidepressants; † Represents unreliable estimates due to small sample sizes; NS, not significant.Table 3 displays the results of the logistic regression analysis showing that the adjusted odds of having a behavioral comorbidity was significantly (AOR = 2.3 (95% CI, 1.2, 4.0)) greater in Bipolar-NOS than in the reference group, Bipolar I and II. The adjusted odds of having PBD with behavioral comorbidities was 5.3 (95% CI, 2.7, 10.6) times greater in 2–9-year-olds compared with older youth; and 2.3 (95% CI, 1.3, 4.0) times greater among males. In a separate model (data not shown), PBD visits with behavior disorders had 21 times (AOR = 21.0 (95% CI, 10.1, 43.3)) greater adjusted odds of having prescribed stimulants.Adjusted odds ratios (AOR) of behavioral comorbidity vs. no behavioral comorbidity in PBD visits.AOR, adjusted odds ratio (adjusted for age group, gender, race/ethnicity, payment type, region and PBD subtypes); CI, confidence interval; NOS, not otherwise specified.There are three major findings from this study of pediatric bipolar disorder (PBD) in community treated populations across the United States. The first documents the dramatic change in the medical visit PBD diagnosis by subtype from 1999–2002 to 2007–2010. It is not unusual or unexpected that the largest PBD subtype would be “Not Otherwise Specified” (NOS), since this is the case in the epidemiologic survey research of adolescents [15] and clinical pediatric psychiatry assessments [16,17]. However, this was not the case in earlier national medical visit data from 1999–2002, when 89% of such visits were associated with a diagnosis of PBD-I, manic or mixed manic type. By contrast, over the next eight years, the diagnosis of PBD-I fell to 18.3%, while PBD-NOS rose dramatically from 2.6% to 74.0% of the total (Figure 1). PBD-NOS is diagnosed when the number of reported bipolar features are subthreshold (below the cut-off number required for a diagnosis) or when the duration of the bipolar episode is below the length of time criterion defined by the DSM. Stringaris and colleagues [18] using data from a national survey of youth in the UK evaluated those with manic-like episodes that met impairment criteria, but whose episode length was below the diagnostic threshold. The resultant findings were that only a few youth met the criteria for BP-I/BP-II and that the number who met diagnostic criteria for BP-NOS was 10 times that number. The authors concluded that youth diagnosed with BP-NOS are in all likelihood a distinct entity separate from youth meeting the full criteria for bipolar disorder.The relative increase in PBD-NOS likely reflects shifting attitudes about the appropriateness of PBD-I or PBD-II for chronic disruptive behavior problems characterized by hyperactivity and irritability [19,20]. PBD-NOS may become the default diagnosis when ADHD severity, particularly aggression, is a major problem [21]. Among newly diagnosed PBD youth, one-third had a prior behavior diagnosis [22], further supporting the close relationship of PBD and behavior disorders. Finally, experts have debated whether the labeling as PBD symptoms could more properly be identified as severe disruptive behavior disorder [23]; the DSM-5 reflects this perspective in its promulgation of disruptive mood dysregulation disorder, which was initially referred to as temper dysregulation disorder [24].The second major finding is that behavior disorders (i.e., ADHD, ODD and CD) are frequently comorbid with PBD and that those with, compared with those without this comorbidity are significantly more often male, preadolescent and are diagnosed with the PBD-NOS subtype (Table 2). PBD with comorbid ADHD has been analyzed by Kent and Craddock in terms of the overlapping of some ADHD and manic-like symptoms in the DSM-5 and ICD-10 [5] and by Pataki and Carlson in DSM-5 [25]. These analyses are consistent with earlier cohort studies in Italy [26], the UK [18] and in U.S. adolescent PBD hospital admissions [27]. The third finding of note relates to the complexity of the prescribed medication regimens, which has been recognized as challenging [28]. PBD in community treatment is reported to involve 3.4 concomitant medications on average [29]. In the current study, antipsychotic medications had a similar prescribing rate for PBD youth with and without behavioral comorbidities (60% vs. 61%), but their combination with other classes, e.g., stimulants and anticonvulsant-mood stabilizers, raises questions. Concomitant use of antipsychotic and anticonvulsants is common [29,30], despite the mixed evidence of anticonvulsant efficacy, safety and tolerability in either short-term or long-term pediatric use [31], although the point has been disputed [32]. Perhaps single drug clinical trial comparisons to placebo are sufficient to justify monotherapy, but are they adequate to support combinations? Research on the use of anticonvulsants in PBD in prepubertal children has not been encouraging, due to the failure to enroll or sustain participation [33]. In addition, West et al. found a differential benefit for risperidone compared with divalproex in 8–18-year-olds diagnosed with PBD with comorbid behavioral symptoms, particularly aggression [34]. The potential pharmacologic interaction of dopamine agonists (stimulants) and dopamine blockers (antipsychotics) warrants greater understanding in terms of short-term and long-term effectiveness, safety and tolerability [35]. Close monitoring of complex multidrug regimens is recommended by the American Academy of Child and Adolescent Psychiatry (AACAP) practice parameter for pediatric bipolar disorder [36], particularly where atypical antipsychotics are combined with other potent classes [37].This study has several limitations. First, NAMCS survey analyses are based on outpatient physician visits rather than on patients as the unit of analysis. While a 1:1 relationship of persons to visits is not possible, trend analyses across time are accurate. Second, diagnoses in the NAMCS are based on the judgment of treating clinicians rather than on a research-level assessment. Third, because the surveys are cross-sectional and cover a limited timeframe, no information is available concerning the duration of medication use. Fourth, different phases of bipolar disorder (euthymic, manic, hypomanic, depressed) were not available as variables. Finally, a major issue of this NAMCS study relates to the modest statistical power of the individual NAMCS survey years to assess PBD, a relatively rare pediatric condition. To overcome this problem, the data were grouped as four-year periods for trends and as the most recent eight years from 2003 to 2010 for PBD w/w/o BC. Due to the low visit number, we could not distinguish between combinations of psychotropic medications prescribed for PBD with and without behavioral comorbidities. Potential interactions among the study variables are not explored due to the limited sample size. The value of NAMCS is its sophisticated sampling design, reporting of diagnostic and treatment information by health professionals and national scope across many years. Predictions of fewer psychiatric drugs in the pipeline and the waning interest of the pharmaceutical industry in psychiatric drug development [38] may energize clinical research efforts to create the infrastructure and methodology for robust outcomes research. Despite the calls for outcomes research, physicians are trained largely in an individual person model. Nevertheless, that model can be supplemented with training in a population-based model. In effect, this approach would extend beyond clinical trials into post-marketing surveillance, i.e., outcomes research. Along with emerging electronic medical record capacity, brief computerized records of parent-recorded perspectives could accompany physician assessment of the outcome using epidemiologic methods to monitor individuals, as well as prospective cohorts. Federal research calls for proposals from the relatively new Patient-Centered Outcomes Research Center (PCORI) feature patient (or family) perspectives in treatment research and aim to reduce the gaps in existing knowledge of PBD [39]. Child psychiatry training programs can engage their trainees to use brief, patient-oriented monitoring systems to include the parent assessment of symptoms, functioning and adverse events in models that are practical and can be incorporated in community practice settings, despite their time constraints [40].Pediatric bipolar disorder treatment in community-treated populations would benefit from clinical monitoring and evaluation studies of outcomes to better understand the shift to a preponderance of the PBD-NOS subtype and the prominent occurrence of PBD with behavioral comorbidity. The accompanying complexity in drug combinations, e.g., antipsychotics and stimulants, that is shown here demands the evaluation of benefits, safety and tolerability.The authors declare no conflict of interest.

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+This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).The Chinese inland, where low child obesity and overweight rates were reported in earlier studies, has recently experienced rapid economy changes. This may impact children’s health. In the present study, we investigated the obesity rate, metabolic health status, and their impacts on carotid intima-media thickness (IMT) and non-alcoholic fatty liver disease (NAFLD) among children from Yueyang, an inland city of China. We found that the obesity rate was about 5% for both 7- and 11-year olds. Overweightness rates were 9.5% and 11.5% for the 7- and 11-year olds, respectively. Clinical and laboratory examinations revealed significant differences among different weight groups in the 11-year old volunteers, which were absent in the 7-year olds. Further statistical analysis showed that: age, BMI, blood pressure, triglyceride level, and metabolic abnormality were positively correlated to carotid IMT; triglyceride level, obesity, male, and the number of metabolic abnormalities were independent risk factors for NAFLD in these children. Our study suggests that: childhood overweightness and obesity are now epidemic in Yueyang, which have contributed to increased carotid IMT and may also increased NAFLD incidents; and serum triglyceride level is a critical factor in the development of childhood NAFLD. Thus, childhood metabolic health warrants further vigorous research in the inland of China.Economic development impacts every aspect of human life including public health. Since implementing the “Reform and Open-up” policy in China, in 1979, the country has changed tremendously, particularly on the economic front. The Chinese economic boom first occurred in the coast cities and then in big cities during the 1990s, which led to improvements of living standards among the people. Such improvements often accompany changes of life-style and dietary habit (particularly increased meat consumption), which, in turn, unleashes a great impact on public health. Indeed, studies reported a rising prevalence of obesity and being overweight among young children and adolescents from 1985 to 2000 in Beijing and the coast areas, which was absent in the less developed inland cities [1,2]. However, since joining the WTO (World Trade Organization) in 2001, the economic boom has spread even deeper into the vast Chinese inland, where a majority of Chinese resides but no current data are available regarding their children’s health status.Particularly, there is scarce information regarding child metabolic health in China [3]. We are interested in children’s metabolic health conditions in the Chinese inland, which would provide forward predictions on certain potential future health problems. In addition these problems could be addressed in our current preventive care. Metabolic syndrome is one of the most common metabolic disorders, including obesity, hypertension, dyslipidemia, insulin resistance (IR), and type 2 diabetes. Metabolic syndrome associates with adverse cardiovascular outcomes, which can be traced back to early vascular changes in young children and adolescents with the condition [4]. It is well known that dyslipidemia causes artery impairments and even atherosclerosis [5,6]. Lipid deposition in artery intima may start during early childhood, which accumulate and eventually lead to serious pathological damages during adulthood [7,8]. Increased carotid intima-media thickness (IMT) is positively correlated to coronary artery disease in adults [9,10]. Researchers in the pediatrics field also measure carotid IMT to monitor children’s health [6,8]. Non-alcoholic fatty liver disease (NAFLD) is also a common complication of metabolic syndrome [11,12], with an average global prevalence about 20% [13]. However, NAFLD prevalence in children has been rarely studied.In the present study, we set out to determine the prevalence of childhood overweightness, obesity, and metabolic syndrome, and their relationships to carotid IMT and NAFLD, in Yueyang city of Hunan Province, a mid-size city in China’s inland. This study may provide a window to understand childhood obesity and metabolic problems in this vast area.From September to November of 2006, 2124 7-year old and 1884 11-year old students from 5 randomly selected elementary schools in Yueyang city were screened for overweight and obesity using the standard BMI (body mass index) protocol (We chose these two age groups to control study cost while still maintain a significant age difference). Height (Ht), weight (Wt), waist circumference (WC), hip circumference (HC) were measured for all subjects. BMI (kg/m2) was calculated by Wt/Ht2. Three weight groups were then classified by comparing each individual BMI value to the average BMI value of the same sex and age population obtained in the current study: overweight group, BMI ≥ 85% of the average; obesity group, BMI ≥ 95% of the average; the rest belong to the normal group. Then, 206 pupil volunteers (randomly selected from all those participated in the initial screening) from different weight groups were further examined for blood pressure (BP), fasting plasma glucose (FPG), lipid-profile including high density cholesterol (HDL-C), low density cholesterol (LDL-C), cholesterol (CHOL), triglyceride (TG), plus biochemical analysis on transaminase activities, such as alanine aminotransferase (ALT) and aspartate aminotransferase (AST). According to their laboratory test results, these volunteers were further classified into 4 metabolic sub-groups, based on the guidelines (see below in diagnosis of metabolic syndrome) from International Diabetes Foundation on diagnosis of metabolic syndrome (MS) in children and adolescents [14]: MS (≥3 abnormalities), M2 (2 abnormalities), M1 (1 abnormality), M0 (no abnormality). Meanwhile, a SIEMENS G60S ultrasound machine was used to measure carotid IMTs and obtain liver imagines from the volunteers. The studying protocol was pre-approved by the Research Ethics Committee of the First People’s Hospital in Yueyang City. Parent consents from all participating children were obtained prior to their participation in this study.We used the following guidelines to diagnose metabolic syndrome [14]: For children from 10 to <16 years old, a positive diagnosis was made if an individual was presented with abdominal obesity plus any two or more of abnormalities: (1) abdominal obesity: WC ≥ 90% of the average; (2) blood pressure: Systolic ≥ 130 mmHg or diastolic ≥ 85 mmHg; (3) HDL-C < 1.03 mmol/L (40 mg/dL); (4) TG: ≥ 1.7 mmol/L (150 mg/dL); (5) glucose ≥ 5.6 mmol/L (100 mg/dL), or known as T2DM (type 2 diabetes mellitus), or FPG (fasting plasma glucose) ≥ 5.6 mmol/L, and positive on a further oral glucose tolerance test (OGTT).For children <10 years old, metabolic syndrome could not be diagnosed. However, further measurements were made if there was a family history of metabolic syndrome, T2DM, dyslipidaemia, cardiovascular disease, hypertension and/or obesity, and the numbers of metabolic abnormalities were recorded for these children.We followed the guidelines to diagnose NAFLD (except liver biopsy was not performed due to the associated high cost) published by Angulo [13]: (1) No drink or average daily consumption less than 40 g ethanol (less than 20 g in females); (2) Excluded the exogenous factors, such as drugs, poison, infection, such as hepatitis B or C; (3) Liver ultrasound image appeared of fatty suffusion; (4) Patients with existing risk factors for metabolic disorders had slightly elevated levels of fasting ALT (>28 U/L for boys and 24 U/L for girls); (5) Patients had risk factors, such as rapid weight gain, central obesity, impaired fasting plasma glucose, dyslipidemia, and hypertension.An individual would be diagnosed as NAFLD: If he or she satisfied the first, second, and any one of the third and fourth; or the fifth together with the third and/or fourth being improved after changing lifestyle. (Determination of liver fatty suffusion: Sonography was conducted by a qualified radiologist using a SIEMENS G60S ultrasound machine. Increased reflectivity of the hepatic parenchyma on conventional plane images was classified as positive for liver fatty suffusion).The SPSS 11.5 package (SPSS Inc., Chicago, IL, USA) was used for statistical analysis. Student’s t-test was used to compare two groups after homoscedasticity test; analysis of variance (ANOVA) and analysis of covariance (COANOVA) were used to compare multiple groups. For comparison between proportions, the χ2 test was used. Pearson analysis and logistical regression were used to evaluate the correlation and relationship between different factors. p < 0.05 was deemed significant.For initial screening, we studied a total of 4008 students—Among them 2180 males (54.4%) and 1828 female (45.6%) with an average BMI of 16.82 ± 2.69 (kg/m2). Table 1 shows the prevalent rates of being overweight and obesity among different age and sex groups were calculated. Comparing the prevalence of overweight/obesity between different sex groups, we obtained a χ2 value of 93.00 (p < 0.001), indicating the prevalence of overweight/obesity in males was higher than in females.After initial screening, 206 volunteers from different weight groups were randomly selected for further detailed clinical tests. Table 2 summarizes the detailed results of the clinical tests on 97 7-year old and 109 11-year old volunteer pupils—Among them 114 males and 92 females. In the 7-year old children, BMI, WC, and HC were significantly different among different weight groups, while SBP (systolic blood pressure), DBP (diastolic), PP (pulse pressure), TG, HDL-C, and FPG showed no significant difference. In the 11-year olds, BMI, WC, HC, SBP, DBP, PP, and TG were all significantly different among different weight groups. However, no significant difference was found in HDL-C or FPG among different weight groups in the 11-year olds.Prevalence rates of being overweight and obesity in 7- and 11-year old Chinese children of Yueyang city.# Compared with 7-year old female, the prevalence of normal, being overweight, and obese p < 0.001; * Compared with 11-year old female, the prevalence of normal, being overweight, and obese p < 0.001.Clinical features in different weight groups of 7- and 11-year old volunteers.N: normal; O: overweight; F: obese. All numbers are expressed as mean ± SD. ☆: compared to normal group, p < 0.05; *: compared to overweight group, p < 0.05; ★: compared to obese group, p < 0.05; BMI: body mass index; BP: blood pressure; DBP: diastolic; BP: FINS, fasting insulin; FPG: fasting plasma glucose; HC: hip circumference; Ht: Height; HDL-C: high density cholesterol; HOMA-IR: insulin resistance index; PP: pulse pressure; SBP: systolic blood pressure; TG: triglyceride; Wt, weight; WC: waist circumference.There was a general trend that the male pupils had thicker carotid IMT than the females in the study. However, this sex difference was only statistically significant in the 11-year old children (boys 0.466 ± 0.066, girls 0.46 ± 0.061; p < 0.05). Even though there was no MS sub-group in 7-year old children, variance analysis on IMT in other different metabolic sub-groups revealed no significant difference (p = 0.084) in children of this age group. However, Student’s t test did show that the IMT in sub-group M2 was slightly and significantly (p = 0.028) thicker than in sub-group M0 (the normal) in the 7-year olds. Variance analysis on IMT among different metabolic sub-groups from the 11-year old children revealed a significant difference (p = 0.034). Among the 11-year olds, the IMT in MS sub-group was thicker than that in the sub-group M0 or M1 (p = 0.004 or 0.030, respectively). These data indicate that the carotid IMT thickens rapidly as metabolic conditions become worse in children.Table 3 shows the relationship between IMT and various clinical features by Pearson analysis. Age, BMI, WC, HC, SBP, DBP, PP, and TG were all positively correlated to IMT (p < 0.05); metabolic abnormality was also positively correlated to IMT (p = 0.004). However, HDL-C and FPG had no significant correlation with IMT (p = 0.264 and 0.643, respectively).Relationship between clinical features and carotid intima-media thickness (IMT) in Children from Yueyang.After collecting detailed clinic information from 206 volunteers, Pearson analysis was used to determine the relationship between each clinical feature and carotid IMT (intima-media thickness). BMI: body mass index; BP, blood pressure; DBP: diastolic BP; FPG, fasting plasma glucose; HC: hip circumference; Ht: Height; HDL-C: high density cholesterol; MS: metabolic syndrome; SBP: systolic blood pressure; TG: triglyceride; WC: waist circumference.We found the overall prevalence of NAFLD was about 7.7% in the 7-year old children studied and detected no significant difference among different BMI or metabolic groups (p = 0.638). However, we did detect significant differences in NAFLD prevalence among different groups in the 11-year old children (p = 0.006). When grouped by BMI, the prevalence of NAFLD in the obese group of 11-year olds was 31.11%, which was much higher than the normal group (p < 0.001). When compared by their metabolic status for the 11-year old children, we found that the prevalence of NAFLD in MS subgroup was significant higher than M0 subgroup (50.0% vs. 8.0%, p = 0.001). Using NAFLD prevalence as a dependent variable and other clinical features as independent variables in a logistic-regression analysis, we found that TG levels, obesity, male, and the number of metabolic abnormalities were positively related to NAFLD (Table 4). As shown in Table 4, the chance to have NAFLD increases as the TG level arises: when TG level arises 1 mmol/L, the chance to have NAFLD increases 13 times. Meanwhile, obese children have 5.4 fold more chances to get NAFLD than normal children; males are 4.8 times more likely than females to have NAFLD. Finally, the number of metabolic abnormalities increases by one, the chance of having NAFLD increases by 2.4 fold (Table 4).Logistic-regression analysis of different parameters on non-alcoholic fatty liver disease (NAFLD) incidents.Logistic-regression was used to analyze the impact of each clinical variable on NAFLD incidents among the volunteers. Several significant variables are identified and listed here: X2, sex; X3, obesity; X4, the number of metabolic abnormality; X12, triglyceride level. Abbreviations: OR, odds ratio; CI, confidence interval; b, co-efficient estimates; NAFLD, non-alcoholic fatty liver disease.This report represents the first attempt to study obesity and metabolic abnormalities in children living in a Chinese inland city since the country joined the WTO in 2001. The prevalence of childhood overweight/obesity in Yueyang city is currently high, and is getting closer to that of Beijing in 2000 reported by Ji et al. [1,2]. This finding indicates that recent economic advancement in the Chinese inland may have already impacted the health of children living in this vast area as evident by their increased obesity and overweight rates. Our findings showed trends similar to some previous studies in different locations: the prevalence of overweight/obese in boys is higher than in girls [15]; and carotid IMTs in obese children were thicker than in normal children [16]. In addition, for the first time, we have identified several important risk factors that contribute to the development of NAFLD in children. Among them, serum TG level is of particularly importance.Carotid IMT is a faithful indicator to evaluate cardiovascular health status [4,10]. Currently using high frequency ultrasound to measure carotid IMT is the best approach to detect early atherosclerosis, as ultrasound is more accurate in measuring artery thickness than Doppler ultrasound or angiography [10]. Previous studies reported that patients with metabolic syndrome showed significant difference in arterial compliance and artery IMT [17,18]. Zhu et al. [16] found that IMTs in obese children were thicker than in normal children. Our study further showed that the IMT was positively correlated to age, BMI, WC, HC, SBP, DBP, PP, and TG. We also found that there was no correlation between HDL-C and IMT in children, which, however, are negatively correlated in adults [19]. This may be due to the young age of the subjects studied and/or the low numbers, particularly the limited cases of metabolic syndrome in children. No correlation was detected between FPG and IMT in the present study, which is in agreement with a previous study reported from Germany [20]. The aggregation of metabolic abnormality was also found to be positively correlated to IMT in our study, which implicates multiple risk factors for vascular problem. As the number of risk factors increases, blood vessel impairments would gradually become more and more evident, particularly as patients grow older (see more discussion below). After adjusting diet and exercise, children’s vascular function can be greatly improved. Thus, metabolic abnormalities in children are usually reversible, and should be detected and treated rigorously as early as possible (mostly by changing lifestyle).The other focus in our study was NAFLD—A problem also closely related to metabolic syndrome. The pathophysiological development of NAFLD includes initial lipid accumulation and mitochondrion impairment in liver cells, which may lead to cell death and result in non-alcoholic steatohepatitis and fibrosis (two hallmarks of NAFLD) [21]. In our study, the prevalence of NAFLD in obese children was significant higher, which is in agreement with other reports [22,23]. In addition, we found that as the number of metabolic abnormalities increase, the prevalence of NAFLD is ascending. This phenomenon was also observed by others [24]. More importantly, we are the first to show that TG and male are new independent risk factors for NAFLD in Chinese children. Particularly, increased serum TG level dramatically promoted NAFLD incidents (Table 4). Therefore, it seems we should pay more attention to children who have high serum TG levels, especially to male Chinese children. These results are consistent with the well-known Chinese culture bias on overfeeding male children.Age is also an important factor in metabolic abnormality. We have detected different metabolic abnormality profiles in two different age groups of children (Table 2) and found that metabolic abnormality is a risk factor for both carotid IMT thickening and NAFLD incidents. Though in this snap-shot study we have not investigated how long obesity will last, obesity is a long standing feature unless the individual changes his or her life style. Thus, dyslipidemia would become more pronounced as the children grow older. Various lipids especially cholesterol, would be deposited in artery intima, and induce their thickening, which would lead to confined vessels and increased resistance at acroteric-vessles. These would enhance the pathophysiological development of numerous cardiovascular disorders such as atherosclerosis and hypertension [7]. In addition, excessive visceral fat mass was reported to have close relationship with metabolic abnormality and insulin resistance [25,26]. Thus, glucose metabolic abnormality could also be developed when obese children grow older. As mentioned above, metabolic abnormities are usually reversible in children via changing lifestyles. It is also worthy to note that physical activities may modify the evolution of NAFLD in adults [27]. Finally, in the literature, there are controversial reports regarding the NAFLD-IMT association in adults [28,29]. A recent report indicated that, NAFLD severity did not correlated with increased IMT in adult patients with normal or elevated gamma-glutamyltransferase activity [30]. While we have not studied this relationship in our study, it would be interesting to explore the difference between children and adults in that regard. These data suggest a complex relationship between NAFLD and IMT involving numerous factors.Childhood overweight and obesity in Yueyang city are epidemic now, accompanying with various metabolic abnormalities that contribute to increased carotid IMT and NAFLD incidents among the children. While detecting similar risk factors for carotid IMT reported by others, we, for the first time, have identified several important risk factors for NAFLD in children, of which increased serum TG level is of critical importance. Thus, we cannot ignore the negative side effects brought out with the development of a good economy. It is pressing now for the public in the vast Chinese inland area to act together in preventing childhood overweightness and obesity, which will greatly benefit combat against cardiovascular diseases, diabetes, and other metabolic disorders that are rising in the Chinese society.alanine aminotransferaseanalysis of varianceaspartate aminotransferasebody mass indexblood pressurecholesterolanalysis of covariancediastolic BPfasting plasma glucosehip circumferenceHeighthigh density cholesterolinsulin resistanceintima-media thicknesslow density cholesterolmetabolic syndromenon-alcoholic fatty liver diseaseoral glucose tolerance testpulse pressuresystolic blood pressuretype 2 diabetes mellitustriglycerideweightwaist circumferenceWe thank Qiong Sun, Xin-Min Yan and Yu-Ping Li for their excellent help in both initial screening and clinical examinations, and Xiang-Lin Liu in statistical analysis.The authors declare no conflict of interest

Med-MDPI/jcm_1/jcm-03-02-00334.txt

@@ -0,0 +1,38 @@
+This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).Pediatric bipolar disorder is a diagnosis that arose in the mid 1990s in the USA and has mostly remained confined to that nation. In this article a young American man (under a pseudonym) describes his experience of having the diagnosis throughout his adolescent years. His story was conveyed via correspondence and a meeting with the author, an Australian child psychiatrist. The young American’s story reveals several issues that afflict contemporary psychiatry, particularly in the USA, where social and economic factors have contributed to the rise of a dominant biomedical paradigm—or “biologism”. This focus on the “bio” to the relative exclusion of the “psychosocial” in both diagnosis and treatment can have serious consequences as this young man’s story attests. The author explores aspects of his tale to analyze how the pediatric bipolar disorder “epidemic” arose and became emblematic of a dominant biologism. This narrative points to the need, depending on the service and country, to return to or retain/improve a balanced biopsychosocial perspective in child and adolescent mental health. Child psychiatry needs to advocate for health systems that support deeper listening to our patients. Then we can explore with them the full range of contextual factors that contribute to symptoms of individual and family distress.This article arises from a dialogue between myself, an Australian child and adolescent psychiatrist, and a young American man about his experience of psychiatric treatment over the course of his adolescent years. But in a way it starts much earlier with an article I read during psychiatry training in 1990. The article in the Australian and New Zealand Journal of Psychiatry by Australian professor Derek Silove left a distinct impression on me [1]. Silove reported:
+	  
+      “A recent study visit to North America impressed on me the seriousness with which Australian psychiatry should consider the recent ideological shift in the USA to an extreme biological model of mental disorders. … In its most doctrinaire form, this monotheistic biologism rejects (or worse still, pays condescending lip service to) the roles of social, cultural and psychological factors in the genesis and treatment of psychiatric disorders and relegates mentalistic notions to the epiphenomenal waste heap.”
+	  
+	  “A recent study visit to North America impressed on me the seriousness with which Australian psychiatry should consider the recent ideological shift in the USA to an extreme biological model of mental disorders. … In its most doctrinaire form, this monotheistic biologism rejects (or worse still, pays condescending lip service to) the roles of social, cultural and psychological factors in the genesis and treatment of psychiatric disorders and relegates mentalistic notions to the epiphenomenal waste heap.”Sixteen years later I was reminded of Silove’s article when child psychiatric colleagues in Australia became aware of a controversial new diagnosis, “Pediatric Bipolar Disorder” (PBD), emanating from the USA. The death of a 4-year old girl in Boston on 3 psychotropics for PBD further highlighted the controversy. Colleagues and I critiqued a guest editorial favourable to PBD [2] that was published in the Australian and New Zealand Journal of Psychiatry [3], published an article on PBD “a controversy from America” [4] and conducted a survey of Australian and New Zealand child & adolescent psychiatrists on this issue [5].My interest in PBD has led me to meet many excellent U.S. child psychiatry colleagues. Most hold views similar to the systemic biopsychosocial perspective I acquired in my training and practice of the profession here in Australia. They too express deep concern about the PBD “epidemic” in their nation. This article is not aimed at disparaging U.S. psychiatry. Nor is it to discount the true cases of bipolar disorder in young people that we see. However, I, like my American friends and other international colleagues, am motivated by a desire to see our field retain a balanced perspective. The PBD diagnostic epidemic is emblematic of the pressures and problems besetting the field. The DSM-5 [6] introduced a new diagnosis, Disruptive Mood Dysregulation Disorder (DMDD) specifically to curb the overdiagnosis of bipolar disorder in children and adolescents in the USA.Because the USA leads many global trends, the PBD epidemic offers valuable lessons to global psychiatry and mental health care. Diagnostic upcoding factors—financial, social and bureaucratic pressures that foster increased use of particular diagnoses—are an international phenomenon. PBD and DMDD have their corollary in Australia, where an epidemic of Autistic Spectrum Disorder (ASD) relates to diagnostic upcoding factors embedded in educational and welfare benefits for children and families and health insurance rebates to health providers who diagnose ASD [7]. PBD did not receive sustained academic support in Australia or New Zealand and thus overdiagnosis of ASD seems to have played a similar role, though with much less accompanying medication.In 2008, I posted some thoughts on a mental health website forum discussion of PBD. A 20 years old young American man wrote eloquently on the forum of his personal experience. We corresponded by email and in a 2013 study trip of my own to the USA I met with him and heard his story face to face.The young man, whom I shall call “Adam” (not his real name), is now in his mid 20th. His verbal recollections were virtually word for word what he’d reported in the emails 5 years earlier. He is doing well in his university studies, is widely read and very knowledgeable about psychiatry and health related politics. He has had no psychiatric diagnosis, nor any psychotropic medication, since leaving home in 2008. However he still struggles with the iatrogenic trauma of the diagnosis in his life. He recalled “about 30 hospital admissions” during the period of the PBD diagnosis. He was continuing to benefit from psychotherapy and apart from a sense of profound regret for a lost adolescence, he’d had no symptoms that would meet criteria for an “Axis I” psychiatric disorder in the past 5 years.Adam’s narrative is his subjective experience, and thus reliant on memory. However, he did show me several discharge summaries of his hospital admissions that corroborated his story. The documents included concern that Adam was suffering a degenerative neurological disorder at a time he was on multiple psychotropic medications but apparently without consideration of the cognitive impairing effects of the pharmacotherapy.I shall now let “Adam” speak for himself, having only edited his emailed story for de-identification and to reduce repetition. The discussion will focus on the issues this articulate young man’s account raises.I don’t mind sharing most anything about how my extensive psychiatric contact has affected me. I’m almost 21 now. I was 12 when first diagnosed. I had suffered depression and anxiety including severe OCD, which has since disappeared. It should also be mentioned I come from a screwed-up family and was physically abused by a sibling. Parents divorced young. My mother had a lot of issues, etc. So it goes without saying there was a lot the psychiatrist should have asked if he was ever so inclined. But unfortunately, he holds a faculty appointment at (edited—A PBD oriented child and adolescent psychiatry clinic).Within about three months, I was on 8 different medications at one time. Very scientific treatment—all the best—several anticonvulsants, several antipsychotics, a couple of antidepressants and lithium too.Things got so bad, that I ended up being referred to the neurology department, for different opinions about strange symptoms I began having on this cocktail. Which resulted in their giving me a working diagnosis of some kind of mitochondrial myopathy. “Bipolar plus mitochondrial disease” as it went. Which I have been told only recently could have been precipitated by the huge amounts of divalproex I was taking. The symptoms quickly disappeared when I coincidentally stopped the drug for unrelated reasons. Oh well, but it is a clear illustration of what one of the “best” academic medical centers in the world has to offer a struggling young boy.Despite the sedation I survived high school and graduated near the top of my class.I guess the biggest deficit this has left me with is sort of skewing the trajectory of my life. My mother fed into my “being sick” and gained a lot of collateral from it. But worse still, it caused complete neglect of any other possible causes of my problems. My parents in many ways tended to over-interpret every solitary behavior as part of the “disease”. Everything in my life was screened through the filter of this immaterial “disease”. I had enough stacked against me when I was so overwhelmed that they brought me to the psychiatrist in the first place. The neglect of my underlying depression and its being made worse by all the sedating drugs just caused me to just sort of collapse in on myself. And despite being well-liked, I had a difficult time establishing friendships in high school and elsewhere. I had to quit my swim team (when I was 12), something I was amazingly successful at and would have gone far with.Meanwhile, none of this had the potential to correct itself because of my parents’ own problems. So I have suffered for a long time and have been ostracized from my family.I just think my case is so typical, because of the path things took, and the fact that I was diagnosed and treated by someone who is rubbing elbows everyday with world leading “experts” on this thing. Clearly, when a disturbed child walks into your office, divalproex, risperidone, and some basic parental psychoeducation, is not going to mean recovery for that child. But yet that’s what their guidelines for “treatment” essentially are.And to think, there’s a trauma clinic right down the street—where I’ve gotten some treatment—and a stone’s throw away, they’re condemning kids to a diminished life. I’m personally of the belief that the children they’re treating are NOT exceptional in any way, and have problems that could easily be ascribed to factors these people have no interest in considering in a serious manner. If everyone at their clinic presented with classical mania, (edit—the researchers) wouldn’t be famous for anything. So they definitely do not have a clinic full of those kids.I never really believed the label myself like on an intellectual level, because like most young people, I always felt there was a reason for my behavior. I started to put some odd pieces of the puzzle together, like: I have this “disease” and it only manifests itself at home in the presence of 2–3 people that happen to be a part of my life. Then I began to wonder why I had never had another “manic” episode after a few years and realized that adults with the disorder don’t always go years on end without a relapse of that kind of “episode”.I did however sort of believe it, only because if you tell a kid something long enough, they’ll start to believe it. And of course, if I question my craziness, that’s part of the “illness”. So I got put in a double bind that really did make me feel like I was trapped or going crazy. Many of the arguments with my mother that would land me in hospital began several hours before as an argument solely about wanting to stop my medicines. There is always context.But the worst part of this, which I have only been recently able to shake within the last year (2008/9), is the defectiveness I felt. Just kind of in some core way. Like I’m totally different. When I was younger, that feeling was a lot stronger and more prominent. Now I feel like a fool for even having given thought after eight years to the question of whether I might go to sleep one night and wake up manic. I decided with my (new) psychiatrist’s support a year ago to stop my medicines. I’m not doing especially well now, but I have at least been able to shake the feelings the diagnosis itself carved into me. The same can’t be said for its physical and social effects though.I am also gay. And this focus on an immaterial disease brought the issue into my own mind prematurely because of all this psychiatric treatment and it ensured that my family and doctors would completely neglect it (the focus was the “disease”). It made something that isn’t normally a cakewalk something extraordinarily difficult and complicated.As far as ownership of my behaviors and emotions go, I never believed the diagnosis on an intellectual level and I always knew there were reasons for my behavior, I just couldn’t really recognize them or name them. So I think a question like that would, sadly, be better asked of my parents. How did it affect their perception of everything? It didn’t make me feel not responsible for my actions and on some level I was at least partly sure I wasn’t some defective, degenerating, out-of-control machine.The mitochondrial disorder thing was a disaster. The testing and consultation dragged out for months. At one point my mom told me they didn’t know if my brain would keep “degenerating”. In effect, “you’re gonna die”. And my psychiatrist was really out to lunch on that one, again. So that experience just profoundly deepened my ignored depression.I always had terrible sedation from the anticonvulsants and atypical antipsychotics. The sedation from divalproex was unmanageable and had a deadening effect. When I was initially on 7 or 8 drugs, I had terrible tremors, severe memory problems and my head was about as functional as a block of lead.One very embarrassing problem, which I imagine divalproex is involved with and which my psychiatrist certainly never imagined asking about, was my pubic hair began to fall out. Yep. The amount and frequency that came out was not normal. It was not good.I also had severe weight gain. From my first contact with these psychotropics, after only 4 months I gained over 50 lbs. I would subsequently lose it when I would stop the medication myself and then gain it back when I was forced back on the medications. This cycle repeated itself 5 times over 8 years. Obviously I couldn’t go back to swimming. Having almost qualified for national swimming championships a year before my diagnosis, I didn’t recognize myself as the cow I was forced to become. This was very troubling. I lost control of my body. After one cycle I gained about 85 lbs in 6 months.I had sexual dysfunction that would only abate when I stopped the drugs. Every SSRI drug I happened to be put on completely obliterated my sex drive. They were the worst.I also wonder having never had my prolactin levels tested and having been on risperidone and divalproex for about 7 growing years whether I should get my bone mineral density tested.I am in psychotherapy, and with a good psychotherapist (finally!). It’s helping a lot.Sorry for being so long-winded, but that’s the basic extent of things. And I don’t mind you sharing any of it. I read your papers and letters published in the journals, and I have to tell you it gave me a lot of hope and sort of made me feel like the world is a little less crazy.In the face-to-face meeting in 2013, Adam said that his siblings, now all adults, had worked through their issues (partly with therapy) and were reconciled on very good terms. They now had shared insight into the intergenerational patterns of disrupted attachment involving their grandparents and parents. The precipitant to their mother’s investment in Adam’s PBD diagnosis appeared to be a bereavement crisis following the deaths of the maternal grandparents. Adam said he and his siblings were concerned about their mother, who, after Adam left the home, developed a preoccupation with a range of medical complaints and sought out different medical specialists despite normal tests for her alleged medical disorders.Adam also recalled that early in his treatment he received an SSRI that caused him to have akathisia and agitation with insomnia causing intense frustration—but no core symptoms of mania such as euphoria, flight of ideas or grandiosity. This was diagnosed as “mania”. Afterwards, he never had the reaction to the same extent with further SSRIs. From my inquiries in our 2013 discussion he described how he had never had any core manic symptoms at any point.If some readers remain skeptical of Adam’s story and his current wellbeing then a mental state examination is worth adding. Across a dinner table over a couple of hours, both I and my psychiatrist colleague (Dr. Anja Kriegeskotten) found ourselves communicating with a very genuine, perfectly sane and intelligent young man with absolutely normal emotional reactivity and good sense of humor. He showed deep insight into the social dynamics of his family and the health system that had engulfed his adolescence. A warm and candid rapport was easily established.Adam said: “there was a lot the psychiatrist should have asked about”. Psychiatric symptoms do not occur in a vacuum. In Adam’s words—“there is always context”.The political history of psychiatry that led to DSM-III in 1980 explains why psychiatric nosology became decontextualized. Broadly speaking psychiatric nosology has been a struggle between two different perspectives, embodied in (1) Emil Kraepelin’s more “medical model” of categorization by symptoms and course of illness, and (2) the “psychobiological” model of Adolph Meyer who advocated that psychiatric interviews should start with a developmental history and the context of the patient’s life. DSM-III adopted a nomothetic, “neo-Kraepelinian” model of diagnosis, based on symptom criteria checklists. This arose out of the need for reliability in diagnosis following an era dominated by psychoanalysis and subjectively inferred psychodynamic conflicts. There was also great geographical variation in the diagnosis of schizophrenia between the USA and Europe that called for more strictly defined diagnostic methodology. But lost was the “Meyerian” ideographic model for diagnosis (partly embodied in DSM-I and DSM-II) that viewed psychiatric syndromes as arising out of individual lives with multiple interactive biopsychosocial causations [8].Greater reliability of syndrome description does not necessarily mean greater validity of diagnosis. Similar symptomatic presentations can have differing causation in different individuals. The introductions in the DSM-III and DSM-IV manuals specifically warn against reification of diagnoses, and that the DSM must “not be used in a cookbook fashion” [9]. Adam is not alone to suffer from misdiagnosis or diagnosis without consideration of context. The recent publication of DSM-5 occurred amidst controversy. Thousands of mental health clinicians and over 50 mental health organizations signed an online open letter protesting the decontextualized nature of the DSM, the open letter stated:
+		
+        “… (taxonomic) systems such as this (DSM-5) are based on identifying problems as located within individuals. This misses the relational context of problems and the undeniable social causation of many such problems.”
+		[10]
+		
+		“… (taxonomic) systems such as this (DSM-5) are based on identifying problems as located within individuals. This misses the relational context of problems and the undeniable social causation of many such problems.”Robert Spitzer, head of the APA’s DSM-III committee, that emphasized the nomothetic over the ideographic in psychiatric nosology, recently revised his viewpoint in a foreword to the book The Loss of Sadness: How Psychiatry Transformed Normal Sorrow into Major Depressive Disorder [11]:
+		
+        “(this book) has forced me to rethink my own position. … The very success of the DSM and its descriptive criteria … has allowed psychiatry to ignore basic conceptual issues … especially the question of how to distinguish disorder from normal suffering. … DSM diagnostic criteria … ignored any reference to the context in which they developed.”
+		
+		“(this book) has forced me to rethink my own position. … The very success of the DSM and its descriptive criteria … has allowed psychiatry to ignore basic conceptual issues … especially the question of how to distinguish disorder from normal suffering. … DSM diagnostic criteria … ignored any reference to the context in which they developed.”Adam and his family had a lot of relational suffering. It may have been beyond the norm for healthier families. But the suffering was embedded in intergenerational family dynamics. Now in their mid to late twenties Adam and his siblings have insight into these dynamics. That insight has been liberating for them.The head of the DSM-IV committee, Allen Frances, has criticized aspects of DSM-5. He also criticized PBD [12]. Although Frances noted that strict adherence to DSM-IV criteria would’ve ruled out PBD, the nomothetic and by default biomedical model of DSM-IV allowed PBD to flourish within the Bipolar Disorder—Not Otherwise Specified (BD-NOS) category. A recent article [13] goes further to criticize the nomothetic medical model:
+        “A classic criticism against medicalization applies: the “medical gaze” locates the problem and the place of treatment within the individual child, and neglects possible social dimensions of the problem.”
+		
+		“A classic criticism against medicalization applies: the “medical gaze” locates the problem and the place of treatment within the individual child, and neglects possible social dimensions of the problem.”Several factors appear to have fueled the PBD epidemic: The pharmaceutical industry’s influence on research, medical education and consumer groups; a desire for a blame-free biological explanation to distressing family problems; a human individual and societal need to repress trauma; and diagnostic upcoding in the U.S. health system that rations treatment according to DSM diagnoses [14]. To this could be added academic hubris: Adam noted that by defining a “new” disorder, the academic child psychiatric center that he attended gained a degree of fame.The PBD academic literature is grossly lacking in research into contextual factors. A systematic review [15] of over a thousand PBD articles for terms such as attachment theory, maltreatment and child neglect found these terms to be almost completely absent. PTSD, trauma and child abuse terms were infrequently referred to and generally only in passing. Rates of physical abuse and sexual abuse in cohorts of research subjects from the two academic child psychiatric centers that pioneered PBD were far below rates in community surveys and emotional abuse appears to have not been considered at all. The methodology in PBD research leans heavily on structured parent interviews. As in research, so in clinical practice. As Adam informed me, the sessions with his psychiatrist involved his mother and the psychiatrist discussing his symptoms and little space for he to ever talk about the physical and emotional abuse by his brother, or the background to the conflict with his mother.DSM-5 has introduced DMDD with the primary rationale to curb the diagnosis of PBD. However DMDD still embodies the same decontextualized model. A similar systematic literature review of 76 articles found minimal mention of attachment, maltreatment and parenting and family dynamic factors [16]. It seems possible that without recognition of context, a child could go through a similar experience to Adam with a DMDD label. In contrast, another diagnosis submitted for inclusion in DSM-5, Developmental Trauma Disorder (DTD) [17], embedded contextual factors in its criteria. The DSM-5 committee rejected DTD mainly on the basis that symptoms overlapped with other disorders, even though the same critique has been leveled at DMDD [18]. It appears that many researchers prefer to count symptoms rather than explore where they come from.Adam described a staggering amount of psychotropic polypharmacy with a litany of side-effects. The treatment Adam received could trigger Medical Board investigation in Australia, yet Adam informed me his legal inquiries indicated his treatment would be deemed “standard practice” where he lived. Nonetheless there is increasing criticism of these medication practices with reports of iatrogenic morbidity and mortality in the U.S. media [19] and academic literature [20]. A health system that forces many child psychiatrists into brief “med checks” is seen as a serious problem. An op-ed in the Los Angeles Times by A/Prof Laurel Williams expounds on these problems [21].Adam had an akathisia/agitation reaction to an SSRI at age 12. These are now well described in the literature [22,23]. However in the 1990s there was dispute about such reactions, and pharmaceutical manufacturers tended to deny the existence of SSRI induced agitation. I recall seeing several adolescents develop the reaction when I worked on a mood disorders unit for young people in the mid-1990s. At the time I prescribed SSRIs liberally. We now know that at least some published SSRI drug trials suppressed data about these reactions [24]. Patients like Adam suffered if their treating psychiatrists were kept in the dark about side-effects by the academic literature. For example, I recall prescribing quetiapine to help patients on antipsychotics lose weight—on the basis of fraudulent studies sponsored by AstraZeneca (London, UK), the manufacturer of quetiapine (Seroquel) [25].Adam eloquently describes the impact of the diagnosis upon his sense of identity and familial relationships. The central task of adolescence is individuation [26]. Identity development can be severely damaged by a misdiagnosis of PBD, where one’s every thought and feeling can be doubted as whether it is a part of self or, as Adam says, some “immaterial disease”. As Adam also indicates, the impact of sedating medications on subjective experience adds to the impairment. Despite his success at university and the psychotherapy that has helped him work through his family conflicts, he still feels a disturbing lack of connection with his sedated adolescent years.This damage to identity formation in children with PBD diagnoses has been noted [27,28]. Even where biomedical explanations may be warranted, there is evidence that a biomedical explanation is likely to foster greater rather than less stigma and induce “prognostic pessimism” [29]. Adam is at the crest of a tsunami of thousands who’ve grown up with the PBD diagnosis. Many of these young adults do not have the resources Adam has marshaled. It is an area that demands further research. With PBD and other diagnoses psychiatrists are often faced with having to “undiagnose” patients, and given the entanglement of label with identity the task of “undiagnosing” requires tact and much support [30].It is traditional wisdom in child psychiatry that parents often project unresolved issues onto their offspring. The children may identify and act out accordingly. Some extreme versions of this can lead to “Munchausen’s syndrome by proxy”, where a parent, through having an ill child, vicariously gains desired attention from respected medical experts for unmet and disavowed dependency needs. It appears that once Adam left the home his mother produced spurious medical symptoms and diagnoses for herself, in other words her own likely case of Munchausen’s disorder.An early critique of PBD [31] noted that not only could parents have a psychological investment in the PBD diagnosis, but so too could a range of others including the pharmaceutical industry, academic child psychiatry, schools and consumer advocacy groups. The authors speculated whether PBD may be a “variant on Munchausen’s syndrome”.This is not to say that there need be any negligence or mal-intent at all. Factors operate at systemic and subconscious levels. Adam’s mother, his doctors and others no doubt acted with Adam’s best interests in mind. A dominant paradigm is hard to see when you’re living and working within it.Silove [1] (1990) in his prophetic article on psychiatric trends in North America, referenced both the eminent U.S. child psychiatrist Eisenberg [32] and a president of the Canadian Psychiatric Association, Lipowski [33], both of whom used the terms “brainless psychiatry” and “mindless psychiatry”. The mid 20th century hegemony of Freudian psychoanalysis tended at its extreme to be a “brainless” model that Eisenberg and Lipowski were highly critical of. But the thrust of their late 1980s warnings concerned the rise of “mindless” psychiatry, or, as Silove called it, “biologism”.What is it but “biologism” that influenced Adam’s psychiatrist and other doctors to misconstrue parent-child conflict as mania, prescribe him so much medication and misdiagnose polypharmacy side-effects as a neurological disorder involving months of high-tech investigations?In addition to being a method of inquiry, science is a social process and there is a vast research literature concerning the sociology of science. Scientific disciplines do not build on knowledge in a purely linear fashion, but at times undergo dramatic upheavals according to paradigm shifts [34]. The dominant paradigm governs what is acceptable to study, research, publish and practice. Softer sciences like psychiatry can be more susceptible to extreme paradigm shifts. The history of psychiatry reflects this. The issue is not simply an academic one (pun intended). What is emphasized in teaching and research plays out in practice—with real life consequences, as Adam well describes.Silove [1] described a narrowing of psychiatric training by 1990 in the USA:
+		
+        “In the area of teaching, North American clinicians schooled in more comprehensive clinical traditions of yesteryear, express fears that training programmes in psychiatry offer little more than instruction in matching formula-based “diagnoses” to specified pharmacological treatments.”
+		
+		“In the area of teaching, North American clinicians schooled in more comprehensive clinical traditions of yesteryear, express fears that training programmes in psychiatry offer little more than instruction in matching formula-based “diagnoses” to specified pharmacological treatments.”Silove was hopeful Australasian psychiatry’s grounding in the “eclectic” biopsychosocial model could buffer it from biologism. In the years since Silove’s warning, Australian and New Zealand psychiatrists in training have still had to pass written case histories, including long-term psychotherapy cases. The oral viva exam still incorporates “long cases” with real life patients. The presentation of a diagnostic case formulation in these exams—a narrative of the patient’s psychopathology within the developmental biopsychosocial context—is still more valued by the RANZCP examiners, as I know from my time as a case histories examiner, than symptom criteria-based diagnoses such as in DSM-5 or ICD-10.Nonetheless biologism in psychiatry is a global issue. Boyce [35], in a presidential address to the RANZCP annual congress titled “Restoring Wisdom to the Practice of Psychiatry”, noted in Australia and New Zealand there had also been a:
+		
+        “… dumbing down” of psychiatry (due to) “increased service demand, the deification of DSM, the influence of the pharmaceutical industry, a misunderstanding of evidence-based medicine, managerialism and the influence of consumerism.”
+		
+		“… dumbing down” of psychiatry (due to) “increased service demand, the deification of DSM, the influence of the pharmaceutical industry, a misunderstanding of evidence-based medicine, managerialism and the influence of consumerism.”However unlike Australasia where the focus is still generally on clinical need, the U.S. health insurance industry rations treatment according to DSM diagnoses and U.S. academic psychiatry and education has been more dependent on pharmaceutical funding than in Australasia.On my recent study trip to the USA I was privileged to visit some centers of excellent holistic psychiatric training, but these may not reflect the norm. At the 2013 APA annual meeting in San Francisco, a psychiatric resident told me how his group had been practicing for their board exams. Their experienced tutor asked for the “diagnostic formulation” for the patient who was interviewed, but none of the residents had heard of a “formulation” in their entire psychiatric training. I was also informed that the U.S. National Board of Medical Specialties (NBMS) exams were going to be devoid of real life patients, using written clinical vignettes in future.Of U.S. psychiatry training, Tasman [36] wrote:
+		
+        “Many fear that we are in danger of training a generation of psychiatrists and physicians who lack basic psychotherapeutic skills or a framework for understanding mental functioning from a psychodynamic perspective.”
+		
+		“Many fear that we are in danger of training a generation of psychiatrists and physicians who lack basic psychotherapeutic skills or a framework for understanding mental functioning from a psychodynamic perspective.”The loss of the biopsychosocial diagnostic formulation compounds the demise of psychodynamic theory in psychiatric training. In practice this means that the patient’s inner life is devalued or ignored, surface symptoms are taken at face value and underlying causation and meanings may remain unexplored. This could explain why a highly qualified psychiatrist with strong academic credentials and with the best intentions, could, as Adam describes, fail to explore his inner thoughts and feelings and the family context.Psychiatry needs a paradigm shift to one that is neither “brainless” nor mindless”. Bracken et al. [37] described the dominant paradigm in psychiatry as a “technological paradigm” that has relegated relationships, meanings and values to secondary concerns and focused on symptomatology and interventions “independent of context”. They argued psychiatry must break free from the constraints of this technological paradigm:
+	  
+      “Psychiatry is not neurology, it is not a medicine of the brain. Although mental health problems undoubtedly have a biological dimension, in their very nature they reach beyond the brain to involve social, cultural and psychological dimensions. These cannot always be grasped through the epistemology of biomedicine.”
+	  
+	  “Psychiatry is not neurology, it is not a medicine of the brain. Although mental health problems undoubtedly have a biological dimension, in their very nature they reach beyond the brain to involve social, cultural and psychological dimensions. These cannot always be grasped through the epistemology of biomedicine.”It should be obvious actually.Stepping out into the San Franciscan sunshine at the 2013 APA conference, I was greeted by several hundred protestors chanting in loud unison: “APA, APA, how many kids did you drug today?” The protestors were from the Scientology backed Citizens Commission for Human Rights (CCHR). Whilst I did not entertain joining them—I am a psychotropic prescriber after all—I couldn’t help but ponder the question that echoed around the surrounding skyscrapers.I heard that Prof. Joel Paris, editor-in-chief of the Canadian Journal of Psychiatry stated in a presentation at the 2012 APA annual meeting:
+	  
+      “When psychiatrists 50 years from now look back on our current era in psychiatry, they will understand that the diagnosis of pediatric bipolar disorder is the greatest scandal to ever befall psychiatry.” Prof. Paris confirmed: “This is exactly what I said.”
+      —Personal Communication [38]
+	  
+	  “When psychiatrists 50 years from now look back on our current era in psychiatry, they will understand that the diagnosis of pediatric bipolar disorder is the greatest scandal to ever befall psychiatry.” Prof. Paris confirmed: “This is exactly what I said.”What Adam went through was scandalous, even if well-meaning. But his story demands action now and shouldn’t have to wait for the verdict of history. He is at the crest of a tsunami of young people who have been affected by the PBD diagnosis. Others are starting to voice their stories as in documentaries like “Letters from Generation Rx” [39]. Their stories need to be heard. Psychiatry needs to be grounded in listening to our patients. By listening to their full stories and by understanding the full context of whatever problems are brought forth, we may offer more tailored beneficial assistance across the biopsychosocial spectrum, and, at the very least, do no harm.I am very grateful to “Adam” who has shown courage in sharing his story and allowing me to view documentary evidence of his treatment. I am also grateful to Anja Kriegeskotten for comments regarding the manuscript.The author declares no conflict of interest.

Med-MDPI/jcm_1/jcm-03-02-00348.txt

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+This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).Translocation carriers have an increased risk of miscarriage or the birth of a child with congenital anomalies. Preimplantation genetic diagnosis (PGD) is performed in translocation carriers to select for balanced embryos and, thus, increase the chance of an ongoing pregnancy. However, a common experience is that reciprocal translocation carriers produce a high percentage of unbalanced embryos, which cannot be transferred. Therefore, the pregnancy rates in PGD in this patient group are low. In a cohort of 85 reciprocal translocation carriers undergoing PGD we have searched for cytogenetic characteristics of the translocations that can predict the percentage of balanced embryos. Using shape algorithms, the most likely segregation mode per translocation was determined. Shape algorithm, breakpoint location, and relative chromosome segment sizes proved not to be independent predictors of the percentage of balanced embryos. The ratio of the relative sizes of the translocated segments of both translocation chromosomes can give some insight into the chance of transferable embryos: Very asymmetrical translocations have a higher risk of unbalanced products (p = 0.048). Counseling of the couples on the pros and cons of all their reproductive options remains very important.Since 1997 preimplantation genetic diagnosis (PGD) has been performed worldwide to allow translocation carriers to conceive balanced offspring and decrease the risk of miscarriages. However, it has become clear that reciprocal translocation carriers produce many unbalanced embryos [1], and PGD offers acceptable pregnancy rates only if the woman responds to ovarian hyperstimulation by producing many oocytes. Thus far, no prediction model for the success of a PGD treatment in translocation carriers has been postulated.Translocations are present in 0.2% of the general (neonatal) population [2,3]. Carriers of Robertsonian and reciprocal translocations have an increased risk of a miscarriage or the birth of a child with congenital anomalies caused by an unbalanced karyotype. Furthermore, translocation carriers can suffer from infertility, which is especially true in male carriers of Robertsonian translocations [4].To significantly reduce the chance of conceiving unbalanced offspring PGD can be performed. In this procedure, an in vitro fertilization (IVF) treatment is executed and the embryos are tested for their genetic make-up. Only normal or balanced embryos are transferred into the uterus. Some studies have shown that PGD is effective in reducing the number of miscarriages and children born with an unbalanced translocation in comparison to spontaneous conception [1,5,6,7]. However, other studies indicate that the live birth rate after PGD is not significantly different from spontaneous conception [8]. This might be explained by the fact that in translocation carriers, especially reciprocal translocation carriers, 67% to over 80% of embryos are found to be chromosomally abnormal [1,9,10,11]. In translocation carriers the pregnancy rate after oocyte collection is significantly lower (17%–24%) than after embryo transfer (26%–33%), reflecting the high proportion of abnormal embryos that are unsuitable for transfer [1,10].The unbalanced products are the result of the segregation of the chromosomes involved in the translocation during the first meiotic cell division of gametogenesis. Reciprocal translocation chromosomes pair their homologous segments, forming a quadrivalent figure, which can segregate in five different ways: alternate (producing balanced or normal gametes), adjacent-1 and adjacent-2, 3:1, and 4:0, all of which will produce unbalanced gametes [12]. Of the 32 possible segregation products, only one results in a completely normal genotype, and one in a balanced genotype of the gamete. Depending on the chromosomes involved, their size and the location of the breakpoints, translocations are prone to segregate in one or more of the five modes mentioned. The chromosomes involved in a Robertsonian translocation pair their homologous segments forming a trivalent figure, which usually undergoes 2:1 segregation, while 3:0 segregation is extremely rare. “Alternate” 2:1 segregation results in normal or balanced gametes, while adjacent segregation produces four types of unbalanced gametes. If the chromosomes involved are of approximately the same size (symmetrical Robertsonian translocation), the translocation is more prone to segregate in an alternate way than if the chromosomes are of different size (asymmetrical Robertsonian translocation) [13].Most reciprocal translocations are private, i.e., unique for the family. Thus, in contrast to Robertsonian translocations and a few recurring reciprocal translocations, for most reciprocal translocations no empirical data on the risks for unbalanced offspring exist. For each individual couple, a clinical geneticist estimates the risk of viable unbalanced offspring based on the translocation at hand with its unique breakpoints and expected segregation modes, and the family history. These estimates are based on spontaneous conceptions, without the use of assisted reproductive technology (ART) [14,15,16]. From this experience, for example, it is evident that the risk of unbalanced offspring due to 3:1 segregation is higher if the translocation carrier is female [17,18,19]. This was recently also shown for couples who underwent PGD. In this study, 3:1 segregation was three times more likely in female translocation carriers [1]. However, from studies on segregation modes in sperm of male translocation carriers it appeared that the empirical risk estimates based on live born and stillborn babies cannot be used to predict the unbalanced outcome in gametes and early pregnancy products [20]. Since the advent of PGD, studies on segregation modes in preimplantation embryos have been published [9,20,21,22]. They report differences in segregation modes according to the gender of the reciprocal translocation carrier, although these differences did not affect the proportion of balanced embryos in PGD and the take-home baby rate [10].We hypothesized that differentiation of reciprocal translocations based on particular characteristics may be used in predicting the chance of transferable embryos in PGD cycles, and subsequent live birth rates.We performed a retrospective cohort study in all couples in the Netherlands undergoing PGD for translocations from October, 1997, until December, 2010. Only couples that had at least four embryos biopsied were included in the study. Couples in which both partners were carriers of a translocation were excluded, as well as PGD cycles for complex translocations (i.e., more than two chromosomes involved or more than two breakpoints) or translocations involving a sex chromosome. The biopsy results of 85 couples with a reciprocal translocation were compared with the results in 35 couples with a Robertsonian translocation. Before PGD treatment started, all couples gave informed consent for use of their clinical data for research purposes.Structural chromosomal abnormalities were detected by routine karyotyping of lymphocytes.All reciprocal autosomal 2-breakpoint translocations were categorized using different systems as explained in Table 1 by two authors (Elsbeth Dul, Conny van Ravenswaaij-Arts) individually.Possible categories of reciprocal translocations.# Derivative chromosome; a Based on Jalbert et al. [15]; b Based on Anton et al. [14]; c Sizes were determined in megabases (Mb) based on UCSC Genome Bioinformatics [23].The shape algorithms used were based on Jalbert et al. [15], viz. the ratio of the sum of the centric segments to the sum of the translocated segments and the ratio of the shortest centric segment to the shortest translocated segment, and on Anton et al., regarding the symmetry of the quadrivalent [14]. Quadrivalents of translocations in our database that appeared comparable to the quadrivalents shown in the publication of Anton et al. [14] were categorized likewise. In general, a translocation is considered to potentially have a 3:1 segregation mode if the longest axis of separation, as seen in the quadrivalent, is resulting in a 3:1 segregation [15]. This is usually the case when the quadrivalent is highly asymmetric. Breakpoint locations and relative chromosome segment sizes, including and excluding heterochromatic and variable regions, were measured using the International Standing Committee on Human Cytogenetic Nomenclature (ISCN) ideograms [24]. In order to calculate the ratio of the relative sizes of both translocated segments and the total lengths of the translocated segments, the lengths of the translocated segments and the total chromosomal lengths were determined in megabases (Mb), based on UCSC Genome Bioinformatics [23].Controlled ovarian hyperstimulation was performed using a long desensitization protocol with GnRH agonists, recombinant FSH and hCG. Oocytes were retrieved transvaginally 36 h after hCG administration. Fertilization was performed by IVF or ICSI. After three days of culture, the embryos were biopsied and one or two blastomeres aspirated. Genetic analysis was performed according to the PGD guidelines [25] and probes were chosen based on the specific translocation. Embryos were scored, based on their FISH signals, as balanced, unbalanced, or inconclusive [12,26].A linear regression analysis was performed, looking for independent predictors of the percentage of balanced embryos per couple. The predictors included in the model were the gender of the translocation carrier and the classifications summarized in Table 1. Statistical significance was defined as p < 0.05.PGD was performed in 85 couples with 83 different reciprocal translocations. Two times, two couples carried the same translocation. In one of these sets, both carriers were female, in the other set the gender of the carrier differed. No couples with translocations between homolog chromosomes were referred for PGD during the study period. There were 41 female carriers and 44 male carriers. As a control group we analyzed 35 couples with Robertsonian translocations: 27 symmetrical, all (13;14), and eight asymmetrical (D;G). Most of the Robertsonian translocation carriers were male (25/35).Table 2 summarizes the results of the biopsied embryos.Results of the preimplantation genetic diagnosis (PGD) analysis of 120 couples.a Embryos that were biopsied, but not included because of: haploidy, triploidy, tetraploidy; no nucleus biopsied or lysis of blastomere. This never resulted in less than four embryos biopsied per couple.Table 3 shows the results of the univariate regression analysis of the reciprocal translocations (for the complete data of the univariate regression analysis see Supplementary Table S1).Univariate linear regression analysis for 85 reciprocal translocation carriers, regarding the percentage of balanced embryos in PGD analysis.a p-Value of category compared to the reference category; ref = reference category.The type of translocation was an independent predictor for the percentage of balanced embryos per couple. Both reciprocal and asymmetrical Robertsonian translocations produced significantly less balanced embryos than symmetrical Robertsonian translocations. There was no significant difference in the percentage of balanced embryos between male and female reciprocal translocation carriers. None of the studied classification systems showed a significant relation with the percentage of balanced embryos per couple at PGD analysis, except for the ratio of the relative sizes of the translocated segments (p-value = 0.048). This shows that with a higher ratio, the chance of balanced embryos declines. This indicates that if the relative translocation segment sizes are very different from each other, the percentage of unbalanced embryos increases.In this study, we focused on the part of the PGD trajectory from oocyte retrieval to embryo transfer, as that is the success-limiting part in PGD for reciprocal translocations. The chance of an ongoing pregnancy after embryo transfer is comparable to PGD for other genetic indications [10]. Unfortunately, we had no information on the reason why karyotyping was performed in the couples. It is likely that the most frequent indication was recurrent miscarriage, though a previous child with an unbalanced translocation, a familial translocation, or infertility might have been indications. Infertility might influence the frequency of balanced gametes, as might be concluded from the offspring and PGD results in male carriers of a Robertsonian translocation [27,28,29]. An increased frequency of infertility is seen in male carriers of Robertsonian translocation (13;14) and they have more balanced offspring, both in contrast to female carriers of this Robertsonian translocation. However, such a relation has not been described for reciprocal translocations. X-Autosome translocations can be associated with infertility, but we excluded translocations with sex chromosomes from this study.The size of the translocated segments, calculated as the sum of the lengths from the telomeres to the breakpoints, did not influence the percentage of balanced embryos. One might hypothesize that this variable is a predictor of viability of unbalanced offspring, but that was not the subject of our study. On the other hand, a very large total length of translocated segments might result in embryos not reaching the biopsy stage of PGD, thus resulting in more balanced embryos and a positive correlation between total length and percentage of balanced embryos. Such an association was not found.Research into segregation modes of translocation chromosomes in cleavage stage embryos is scarce, and studies consist of small numbers of embryos. Thus far, none of the studies have made clear whether the quadrivalent figure or the sizes of the translocated segments can predict the chance of balanced embryos during a PGD procedure. It is quite possible that other research into this subject has been undertaken, but not published, due to the unpredictability of translocations.With this study, we have tried to find cytogenetic predictors for the percentage of balanced embryos in reciprocal translocation carriers. We hypothesized that more unbalanced embryos are produced in translocations that are more asymmetrical. A theoretical explanation for this might be that the alternate segregation, resulting in normal and balanced gametes, is more likely to occur in symmetric quadrivalents. Therefore, we expected the categories that reflect asymmetry in the variables “pachytene-diagram” and “quadrivalent” to produce more unbalanced embryos. However, in these variables, we did not find a significant difference between asymmetrical and symmetrical quadrivalents. This may be caused by the small numbers in the different categories of these variables. The calculation of the ratio between the exchanged segments of the translocation is more objective and reproducible. Furthermore, it is a linear parameter, which means that the cohort can be analyzed as a whole. In the present study, this calculated ratio of the relative sizes of the translocated segments is the only predictor of success in PGD in reciprocal translocation carriers. This ratio suggests that with a more asymmetrical quadrivalent, the chance of balanced embryos declines. By performing a multivariate regression analysis, the stepwise removal of non-significant factors has been evaluated. No other independent predictors of balanced embryos were found. Whether the ratio of translocated segments is indeed a good predictor of balanced embryos has to be studied in larger cohorts, preferably by using international databases, such as the European Society of Human Reproduction and Embryology (ESHRE) PGD Consortium data.Another reason for not finding a correlation between the percentage of balanced embryos and the two ways of predicting the segregation mode of the translocation can be that the prediction models of Jalbert [15] and Anton [14] are based on live born and stillborn babies. Their categorizing of a translocation into a certain segregation mode, thus, does not exclude that other segregation modes contribute to the chromosomal make-up of the gametes of the carrier.As the percentage of balanced embryos in our study was low (11.4%), we could only perform a univariate analysis. Therefore we could not formulate a prediction model for the chance of balanced embryos per individual translocation. The percentage of balanced embryos found in our study is in accordance with the data collection from the ESHRE PGD Consortium [10]. Larger groups of embryos are necessary to allow for multivariate analysis into the relation between the type of reciprocal translocation and the percentage of balanced embryos.We have shown that the number of transferable embryos in PGD treatment in reciprocal translocation carriers is difficult to predict from the characteristics of the individual translocations. The ratio of the relative sizes of the translocated segments of both chromosomes involved in the translocation can give some insight into the chance of transferable embryos. The implications are that for translocation carriers the pregnancy rates per PGD cycle are low, irrespective of the translocation characteristics, due to a high number of untransferable embryos. Further studies are needed to establish whether the ratio of translocated segments can be used in the PGD counseling of couples. A sufficient response to oocyte stimulation remains an important requisite to increase the chance of transferable embryos. The counseling of couples with translocations remains very important, so they can decide which reproductive option (PGD, gamete donation, or natural conception) suits them best.This study was supported by research grants from Merck Sharpe and Dohme BV, Ferring Pharmaceuticals, and Merck Serono, The Netherlands.The authors declare no conflict of interest.

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+This article is an open-access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).Hepatic encephalopathy (HE) is a frequent complication in cirrhotic patients undergoing transjugular intrahepatic portosystemic shunt (TIPS). Hyponatremia (HN) is a known contributing risk factor for the development of HE. Predictive factors, especially the effect of HN, for the development of overt HE within one week of TIPS placement were assessed. A single-center, retrospective chart review of 71 patients with cirrhosis who underwent TIPS creation from 2006–2011 for non-variceal bleeding indications was conducted. Baseline clinical and laboratory characteristics were collected. Factors associated with overt HE within one week were identified, and a multivariate model was constructed. Seventy one patients who underwent 81 TIPS procedures were evaluated. Fifteen patients developed overt HE within one week. Factors predictive of overt HE within one week included pre-TIPS Na, total bilirubin and Model for End-stage Liver Disease (MELD)-Na. The odds ratio for developing HE with pre-TIPS Na <135 mEq/L was 8.6. Among patients with pre-TIPS Na <125 mEq/L, 125–129.9 mEq/L, 130–134.9 mEq/L and ≥135 mEq/L, the incidence of HE within one week was 37.5%, 25%, 25% and 3.4%, respectively. Lower pre-TIPS Na, higher total bilirubin and higher MELD-Na values were associated with the development of overt HE post-TIPS within one week. TIPS in hyponatremic patients should be undertaken with caution.Hepatic encephalopathy (HE) is a frequent complication of liver disease affecting 50%–70% of patients with cirrhosis [1]. Defined by the West Haven criteria, HE represents a range of neuropsychiatric abnormalities, from mildly abnormal psychometric testing, to severe neurologic dysfunction, in which asterixis, impaired cognition, lethargy and coma are present [2]. These latter grades have been termed overt HE.Multiple mechanisms have been proposed for the development of HE [3]. The most important contributor is the presence of an elevated serum ammonia level due to impaired hepatic function and portosystemic shunting. Hyperammonemia leads to HE by altering the osmotic gradient within astrocytes, changing intracellular glutamine-glutamate metabolism, decreasing cerebral blood flow and cerebral oxygen metabolism [3,4,5]. Elevated serum ammonia leads to the conversion and accumulation of glutamine from glutamate. Glutamine, an active organic osmolyte, leads to astrocytic edema and impairs neurotransmission [6,7].Dilutional hyponatremia (HN), due to the non-osmotic release of arginine vasopressin (AVP) into the bloodstream, occurs in 30% of cirrhotics and is associated with severe portal hypertension. In non-cirrhotic patients, HN is associated with a spectrum of neurologic findings that closely mirrors HE. Clinically, HN exacerbates HE in cirrhosis, as it worsens ammonia-induced brain edema by depleting organic osmolytes and impairing astrocyte osmotic compensatory ability [8]. HN is directly correlated with the incidence of HE and predictive of its subsequent development [8,9].The transjugular intrahepatic portosystemic shunt (TIPS) involves a procedure in which an expandable metal stent is placed within the liver to provide a communicating pathway from the portal to the hepatic vein used to treat complications of portal hypertension, such as variceal bleeding and refractory ascites [10,11,12]. A major complication of TIPS is the development of encephalopathy [12,13,14]. Risk factors for the development of HE after TIPS include older age, a history of HE, a low portal pressure gradient after TIPS, higher Child-Pugh class, high creatinine, low albumin levels and low serum sodium (Na) [13,14,15]. Given the shared pathophysiologic process by which HN and hyperammonemic states lead to neurologic dysfunction via astrocytic edema, we speculated that the presence of HN at the time of TIPS would increase the incidence of overt encephalopathy immediately after TIPS. In this study, we assess risk factors, specifically the effect of HN, for the development of overt HE within one week in patients undergoing TIPS.The Vascular and Interventional Radiology database of patients undergoing the placement of a TIPS between January 2006 and December 2011, at the New York University Langone Medical Center was retrospectively reviewed for patients undergoing the procedure electively for non-variceal bleeding indications. Patients on renal-replacement therapy, requiring mechanical ventilation, or post-liver transplant were excluded, as these factors are known to potentiate the development of HE. We deliberately excluded variceal bleeding in this study to limit factors outside the TIPS creation influencing the development of early overt hepatic encephalopathy. Variceal bleeding in an emergent setting may confound the symptoms of overt encephalopathy. Indications for TIPS in our cohort in large part include refractory ascites and partial portal vein thrombosis, for the maintenance of the listing status for transplantation. In the latter case, patients may have had undergone TIPS in the setting of mild HE after closely weighing risks and benefits. All patients underwent TIPS following consent by the same team of interventional radiologists. The study protocol was approved by the New York University School of Medicine Institutional Review Board (IRB).Demographic, clinical and biochemical data were collected from medical records using data collection forms, including the history of overt HE, HE therapy, ascites, indications for TIPS creation, Child-Pugh (CP) score, the Model for End-stage Liver Disease (MELD) score and MELD-Na (Table 1). The presence of cirrhosis was based on clinical data (i.e., a history of liver disease, the results of imaging, laboratory studies and liver biopsy). A history of overt HE required the documentation of an altered level of consciousness, confusion, disorientation or coma. The Na level and the average of serum Na concentration for the 3 days prior to TIPS insertion (pre-TIPS Na) were recorded. Daily serum sodium for 3 days prior to TIPS was not always available, as some patients in this cohort had ambulatory pre-procedural testing. In those cases, the average of Na levels drawn within 3 days of the procedure were used. A normal Na level was defined as ≥135 mEq/L. Patients with an Na level 130 to <135 mEq/L were considered as having mild HN, 125 to <130 mEq/L as moderate HN and <125 mEq/L as severe HN.Patient records were assessed for the development of overt HE as defined by the documentation of an altered level of consciousness, somnolence, confusion, disorientation or coma. Early overt HE (EOE) was defined as its development within the first week after TIPS. Patients did not undergo psychometric testing. Prophylactic use of Lactulose and/or Rifaximin following TIPS was recorded. The length of stay after TIPS and in-hospital mortality were also assessed.Demographics and clinical characteristics of patients undergoing transjugular intrahepatic portosystemic shunt (TIPS) insertion for non-variceal bleeding indications. Patients managed with medical therapy were categorized as West-Haven Grade 2. EOE, early overt HE; HE, hepatic encephalopathy; MELD, Model for End-stage Liver Disease; HCV, hepatitis C virus; HBV, hepatitis B virus; AST, aspartate aminotransferase; ALT, alanine aminotransferase; INR, international normalized ratio.* Note that several patients had multiple factors contributing to liver cirrhosis and indications for TIPS; ** Pre-TIPS Na indicates the average of serum Na concentration for the three days prior to TIPS insertion. Serum [Na+] denotes the sodium level on the day TIPS was performed.Descriptive statistics were used to summarize patient characteristics. The univariate logistic regression model was used to assess the factors associated with the development of EOE. Pearson correlation analysis was conducted on identified risk factors. All potential risk factors were eligible to be included in the multivariate logistic regression model, where variables were selected by minimizing Akaike’s information criterion. Odds ratios (OR) were calculated to determine the relative risk of developing EOE for each of the predictive variables. The area under the receiver operating characteristic curve (AUC) was calculated as an indication of the discriminative power of the logistic predictive models. Statistical significance of tests was claimed when p  < 0.05. Statistical analyses were conducted using SAS (Version 9.2, Cary, NC: SAS Institute Inc., 2009).Seventy-one subjects undergoing 81 successful TIPS creations met the inclusion criteria. Patient characteristics are presented in Table 1. Indications for TIPS included refractory ascites (N = 69), hydrothorax (N = 14), portal vein thrombosis (N = 7) and TIPS stenosis (N = 3). Twenty-five patients (35%) had no evidence or active treatment for HE. Forty-six (65%) were receiving Lactulose and/or Rifaximin prior to TIPS insertion, among which, 23 had a documented clinical history of overt HE, while others were started on therapy prophylactically by their physician, with clinically evident signs of mild HE prior to TIPS (Table 1). No patients had overt HE at the time of TIPS insertion. Among the 25 patients who were previously not on treatment for HE, 21 (84%) received Lactulose and Rifaximin immediately following TIPS. Normal Na levels at the time of TIPS were observed in 41% of patients (N = 33), and HN was present in 59% (N = 48) (mild, 34%; moderate, 16%; severe, 9%). Pre-TIPS Na levels were normal in 36% (N = 29) (mild, 34%; moderate, 20%; severe, 10%). The mean Child-Pugh (CP) score was 11 ± 2 (range: 7–14) (CP Class A, zero; CP Class B, 27; CP Class C, 54). The mean MELD score was 17 ± 4 (range: 6–29), and the mean MELD-Na was 21 ± 5 (range: 6–33).EOE occurred in 15 of 81 total TIPS creations (18.5%). The relationship between the various clinical variables and EOE are presented in Table 2. Significant factors included pre-TIPS Na, MELD-Na scores and total bilirubin (p  < 0.05). Fourteen of the 15 patients (93.3%) who developed EOE had a pre-TIPS Na <135 mEq/L. Among patients with pre-TIPS Na ≥135 mEq/L, 130 to <135 mEq/L, 125 to <130 mEq/L and Na <125 mEq/L and the incidence of EOE was 3.4%, 25.0%, 25.0% and 37.5%, respectively (Figure 1). The incidence of EOE also increased with increasing MELD-Na (<15, 0%; 15–19, 12%; 20–24, 16%; ≥25, 39%) (Figure 2). Age, albumin, CP scores, history of overt HE and creatinine levels did not significantly differ between patients with and without EOE (p  > 0.05).Factors associated with early overt encephalopathy and length of stay as determined by Wald (χ2) univariate regression analysis. Pre-TIPS Na indicates the average of serum Na concentration for the three days prior to TIPS insertion; SBP, spontaneous bacterial peritonitis; PVT, portal vein thrombosis; HCC, hepatocellular carcinoma; HE, hepatic encephalopathy. * p  < 0.01; LOS, length of stay.Percentage of patients developing early overt encephalopathy (EOE) by pre-TIPS Na (mEq/L).Percentage of patients developing early overt encephalopathy (EOE) by MELD-Na group.Paired correlation analysis conducted on the three predictor variables revealed that they were strongly correlated with each other (p < 0.001 in all cases). To reduce the co-linearity between the predictors, continuous variables were converted into categorical variables, and combinations of categorical and continuous variables were utilized to build a multivariate model. A model selection procedure was used to identify total bilirubin (continuous variable) and a pre-TIPS Na with a cutoff value of 135 mEq/L (categorical value) as the best-fit model with an AUC = 0.712 (Table 3). EOE was therefore best predicted by the total bilirubin concentration and a mean serum Na concentration for the three days prior to TIPS insertion (pre-TIPS Na) of <135 mEq/L.(A) In the best fit multivariate model, pre-TIPS Na was divided into ≥135 and a <135 (mEq/dL) categorical variable, and total bilirubin was included as a continuous variable; (B) Odds-ratio estimates.(A) (B) The average length of stay (LOS) of patients with and without EOE was 15.4 ± 19.8 days and 4.4 ± 5.1 days, respectively (p  < 0.001). Correlations of the various clinical parameters with post-TIPS LOS are presented in Table 3. Factors closely associated with LOS on univariate analysis included low pre-TIPS Na and high total bilirubin, MELD, MELD-Na and CP scores (p < 0.05).Four (4.9%) of the 81 patients undergoing TIPS were associated with hospital mortality, including three patients (75%) with EOE. Prognostic variables predicting death in the univariate analysis were high total bilirubin, MELD, MELD-Na and CP scores.A transjugular intrahepatic portosystemic shunt (TIPS) is an effective therapy for complications of portal hypertension and is frequently employed as a temporizing measure for patients awaiting liver transplantation. The most common complication of TIPS creation is the development of HE, occurring in 30%–60% of patients within one year [13]. The development of overt HE following TIPS creation is associated with significant morbidity and is a predictor of mortality [14,16,17,18]. As a result, the identification of pre-procedural risk factors that are potentially modifiable is of paramount importance.In this retrospective study, we identify the factors predictive of EOE within one week of TIPS. The short post-procedural timeframe distinguishes acute risk factors for post-TIPS encephalopathy from variables associated with the chronic progression of cirrhosis. Among the 81 cases undergoing TIPS for non-variceal bleeding indications, fifteen developed EOE. The most significant predictors included pre-TIPS Na and total bilirubin levels. Of those who developed EOE, 93.3% had a pre-TIPS Na <135 mEq/L. In addition, there was an inverse relationship between the severity of HN and the risk of EOE. EOE was observed in 38%, 25%, 25% and 3% of patients with pre-TIPS Na levels of <125, 125–129.9, 130–134.9 and ≥135 mEq/L, respectively. Furthermore, low pre-TIPS Na and high total bilirubin levels were important predictors of post-procedural LOS, independent of MELD score. Although the small number of deaths precluded a meaningful analysis for the factors predictive of hospital mortality, three of the four patients who died had HN.Hyperammonemia and resultant astrocytic swelling are central events in the proposed models for HE pathogenesis. Because of the presence of glutamine synthetase within astrocytes, elevated ammonia levels lead to increased intracellular concentrations of glutamine, resulting in increased intracellular osmolality [19]. The increased osmolality leads to the passage of extracellular fluid into the intracellular compartment, intracellular depletion of compensatory organic osmolytes, such as myo-inositol, and astrocytic swelling and dysfunction [20,21]. The low-grade cerebral edema impairs neurotransmission and clinically manifests as HE [3]. The model of astrocyte edema leading to HE is supported by magnetic resonance studies in cirrhotic patients that demonstrate increased glutamine/glutamate signal and myo-inositol depletion, consistent with partially compensated glial edema [8,22,23,24]. Moreover, cirrhotics with lower brain myo-inositol have a significantly higher probability of developing overt HE compared with those with higher myo-inositol levels at three months [8].Hyponatremia (HN) is the most commonly encountered electrolyte abnormality in hospitalized patients and associated with a range of neurologic manifestations, due to increased brain edema associated with myo-inositol depletion. Symptoms closely parallel those of HE and range from mild disturbances in gait and attention to depressed sensorium and seizures in severe cases [25,26,27]. Animal studies have demonstrated that severe neurological symptoms develop in hyponatremic individuals when additional stressors, such as hypoxia, are concurrently present [28].Dilutional HN commonly develops in patients with advanced cirrhosis and portal hypertension as a result of the non-osmotic release of vasopressin, due to decreased effective blood volume from splanchnic vasodilation [29,30,31]. It is especially common in patients with refractory ascites in whom TIPS may be indicated. In these patients, diuretic therapy exacerbates the process by inducing intravascular volume depletion and non-osmotic release of AVP [32]. In addition, diuretics impair the reclamation of sodium and chloride, leaving increased free water reabsorption, due to the increased release of AVP being unopposed [32].HN is closely associated with the development of HE in cirrhosis, and a higher incidence of HE is observed among patients with HN [9]. In a prospective study of 997 patients, HN (serum sodium <135 mEq/L) was associated with both refractory ascites and a greater frequency of HE. Although patients with serum sodium <130 mEq/L had the greatest frequency of these complications, the frequency was also increased in patients with a mild reduction in serum sodium levels (131–135 mEq/L) [9]. Finally, a strong inverse relationship between serum sodium and the incidence of HE was observed [9]. In a prospective study of 61 patients with cirrhosis followed over a one-year period, HN (serum sodium <130 mEq/L) was the strongest independent predictor for the development of overt HE [8].HN exacerbates the effects of increased ammonia levels and increases the risk of HE in cirrhotic patients by further depleting the cerebral concentration of organic osmolytes, especially myo-inositol, and the osmoregulatory capacity of cerebral astrocytes [8,20,33,34]. Patients with lower brain myo-inositol levels have a higher probability of developing overt HE compared with those with higher myo-inositol (83% vs. 31% at three months) [6]. Among outpatient cirrhotic patients, HN has been shown to have a detrimental impact on psychomotor abilities in individuals with HE [35]. It has been proposed that HN increases the severity of astrocytic edema by imposing an additional osmoregulatory stressor in the setting of pre-existing astrocytic edema resultant from hyperammonemia [8]. Quantitative electroencephalography has been used as a tool to measure neurotransmission impairments in cirrhotics and is predictive of the occurrence of overt HE. A recent study demonstrated that high ammonia and low sodium levels were the strongest predictors of EEG alterations in cirrhotic patients [36]. While HN may not be sufficient to trigger HE alone, it is a “second hit”, which further precipitates the progression of HE [8,33].The compensated osmotic balance resultant from chronic hepatic dysfunction may be offset following TIPS creation in which a large ammonia load is acutely delivered to cerebral astrocytes. The resultant edema may rarely be accompanied by a rise in intracranial pressure, resulting in seizures and death [37]. More commonly, worsening or new-onset overt HE develops after TIPS creation, occurring in 30%–60% over one year [13,38,39,40]. Most cases of post-TIPS HE occur during the early post-TIPS period, especially within one to three months of TIPS [40,41]. In a prospective study of 87 patients undergoing TIPS, HE occurred with an incidence of 50.5% at one year, with 17% experiencing HE during the same hospitalization of TIPS placement and 76% of episodes occurring within three months [42]. Among 55 patients followed prospectively for HE after TIPS, the cumulative rate of HE in the first three months after TIPS increased from 23.6% (prior to TIPS creation) to 50.9%. De novo HE developed in 30.9% of patients, and the proportion of overt HE increased from 10% to 22.5% by one month following TIPS [38]. Finally, in a study of 77 patients undergoing TIPS, the overall incidence of clinically significant new or worsened encephalopathy after TIPS was 23%, with a mean time to the onset of encephalopathy of 26 days. Among those who developed HE, 90% did so within 45 days of TIPS [38].Numerous studies have attempted to identify risk factors for the development of post-TIPS HE over time periods ranging from one month to one year [13,40,41,42,43]. A recent review found that increased age, a history of HE and a higher Child-Pugh class/score were the most robust predictors for post-TIPS HE [13]. Other studies have demonstrated high creatinine levels, low albumin levels and low serum Na to be independent predictive factors for the occurrence of HE after TIPS [13,14]. Among 87 patients followed for a mean of 30.9 months, risk factors that have been identified for post-TIPS HE included older age and ascites as an indication [39]. The long follow-up period in these studies, however, and the natural history of end-stage liver disease make it difficult to distinguish whether the onset of post-TIPS HE is associated with the rapid progression of liver disease or with the identified pre-procedural risk factors.Animal models of cirrhosis demonstrate that chronic hyponatremia worsens brain edema following portacaval anastomosis [7]. In an analysis of 70 patients with cirrhosis and refractory ascites comparing TIPS with large-volume paracentesis, hyponatremia, serum bilirubin and serum creatinine were independently associated with the development of HE [43]. Riggio et al. also reported HN as a risk factor for post-TIPS HE, but the relationship of the timing of the HE episodes with HN was not provided [13]. Our study is the first to evaluate risk factors for overt HE during the early post-procedure period. In this study, we speculated that HN would play an important role in the development of EOE, because the procedure corrects the ineffective intravascular volume depletion that is the cause for HN. Of note, Riggio et al. did not find a relationship between HN and refractory HE. Because HN was most likely corrected after the procedure, one would not expect it to be related to a long-term condition, such as refractory HE. In contrast to prior studies, older age, a history of HE, a high Child-Pugh score, low albumin or high creatinine were not associated with post-TIPS HE. Possible explanations for this discrepancy include: (i) the small number of HE events in our cohort; (ii) these risk factors predisposing to the onset of HE outside the one-week follow-up; and (iii) these risk factors being harbingers of progressing liver disease and not the development of acute, post-procedure HE.Our findings are subject to several limitations. Our study was a retrospective chart review with a relatively small number of subjects. Moreover, the development of HE was based on the clinical documentation of HE in the medical record and not prospective psychometric testing. Despite these limitations, we demonstrate that pre-TIPS Na is a predictor of EOE following TIPS creation and, of increasing importance in an era of increasing medical expenditures, of increased LOS. In addition to HE, HN has been found to be an independent predictor of mortality in patients undergoing TIPS. A study of 68 patients undergoing TIPS for variceal hemorrhage revealed that sodium <135 mEq/L was a significant predictor of both 30-day (30% vs. 5%) and one-year (59% vs. 14%) mortality [14].TIPS insertion in patients with HN should be undertaken with caution. Future studies are required to determine whether the correction of pre-TIPS HN will decrease the risk of EOE.Jonathan Merola: Study concept and design; acquisition of data; analysis/data interpretation; drafting of the manuscript; critical revision of the manuscript for important intellectual content.Noami Chaudhary: Study concept and design; acquisition of data; analysis/data interpretation; critical revision of the manuscript for important intellectual content.Meng Qian: Statistical analysis.Alexander Jow: Study concept and design; acquisition of data.Katherine Barboza: Statistical analysis.Hearns Charles: Critical revision of the manuscript for important intellectual content.Lewis Teperman: Study concept and design.Samuel Sigal: Study concept and design; acquisition of data; analysis/data interpretation; drafting of the manuscript; critical revision of the manuscript for important intellectual content; study supervision.The authors declare no conflict of interest.

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+This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).Reprogramming somatic cells to induced pluripotent stem cells (iPSCs) or direct reprogramming to desired cell types are powerful and new in vitro methods for the study of human disease, cell replacement therapy, and drug development. Both methods to reprogram cells are unconstrained by the ethical and social questions raised by embryonic stem cells. iPSC technology promises to enable personalized autologous cell therapy and has the potential to revolutionize cell replacement therapy and regenerative medicine. Potential applications of iPSC technology are rapidly increasing in ambition from discrete cell replacement applications to the iPSC assisted bioengineering of body organs for personalized autologous body organ transplant. Recent work has demonstrated that the generation of organs from iPSCs is a future possibility. The development of embryonic-like organ structures bioengineered from iPSCs has been achieved, such as an early brain structure (cerebral organoids), bone, optic vesicle-like structures (eye), cardiac muscle tissue (heart), primitive pancreas islet cells, a tooth-like structure (teeth), and functional liver buds (liver). Thus, iPSC technology offers, in the future, the powerful and unique possibility to make body organs for transplantation removing the need for organ donation and immune suppressing drugs. Whilst it is clear that iPSCs are rapidly becoming the lead cell type for research into cell replacement therapy and body organ transplantation strategies in humans, it is not known whether (1) such transplants will stimulate host immune responses; and (2) whether this technology will be capable of the bioengineering of a complete and fully functional human organ. This review will not focus on reprogramming to iPSCs, of which a plethora of reviews can be found, but instead focus on the latest developments in direct reprogramming of cells, the bioengineering of body organs from iPSCs, and an analysis of the immune response induced by iPSC-derived cells and tissues.Transdifferentiation, direct reprogramming or direct lineage reprogramming are the three different terms used for describing when the overexpression of certain factors makes a fully differentiated cell change its transcriptional and protein profile to directly convert it from one cell type into another without intermediate progenitor stages [1]. The historical origin of cell transdifferentiation has been elegantly reviewed before, clearly setting the stage for the future development and advancement of the field [2]. Many cell types have been made using direct reprogramming methodology as described below.The first report of direct reprogramming or transdifferentiation was that describing the conversion of fibroblasts, chondrocytes, and retinal epithelium into skeletal muscle by the transfection of the transcription factor MyoD [3]. The transdifferentiation into cardiomyocytes has not proved so simple as a master regulator gene for cardiomyocytes, similar to MyoD for skeletal muscle determination, has not been found. The group of Srivastava designed a screening strategy in which a group of 14 candidate genes were tested in different combinations for the cardiomyocyte induction capacity [4]. After fine-tuning to find the best combination with the minimal number of genes, three factors, Gata4, Mef2c, and Tbx5, were identified that together induce direct reprogramming of source cells into cardiomyocytes. The direct reprogramming was successful from both dermal and cardiac fibroblasts and the resulting cardiomyocytes had features similar to neonatal cardiomyocytes including contractility. This strategy has several advantages compared to deriving cardiomyocytes from iPSCs, including a greater efficiency and speed in cardiomyocyte generation.The demonstration of in vitro direct reprogramming to neurons was first reported in 2002 when the conversion of astrocytes into neurons, by over-expression of Pax6 was described [5]. Because astrocytes share a common cell lineage to neurons, they require minimal manipulation to directly reprogram them to neurons and may not be the most feasible source of starting cells for transdifferentiation. More accessible cell types for direct reprogramming to neurons include; (i) fibroblasts by direct reprogrammed using Brn2, Ascl1, and Myt1l (BAM) [6]; (ii) hepatocytes using also BAM [7]; (iii) pericytes using Sox2 and Mash1 [8]; and cord blood using Sox2 and c-Myc [9].Furthermore, fibroblasts can be directly lineage-reprogrammed into spinal motor neurons using Ascl1, Brn2, Myt1l, Lhx3, Hb9, Isl1, and Ngn2 [10]. Of particular interest is the ability to convert one neuronal subtype into another, namely embryonic and early postnatal callosal projection neurons into corticofugal projection neurons by overexpression of Fezf2 [11], indicating that there is a period after post-mitotic development when neurons can change their subtype. The demonstration that this is a stable conversion, at least during this post-mitotic period, remains to be done.Current methodologies employed in direct reprogramming carry a number of concerns when considered for clinical application. Formerly, all transdifferentiation strategies had been achieved using doxycycline-inducible lentiviral vectors. Fears of genotoxic integration and tumorigenicity associated with this method have been voiced. Testing of new non-viral methods for converting fibroblasts into neurons, by using plasmids as a gene carriers coding for BAM [12], by microRNA mediated conversion [13] and by using chemical compounds alone [14], have all been tried. These methods begin to pave the way for future clinical application and further work in this direction is warranted.To date, it has been shown in vitro that neurons made from either mouse or human cells by direct reprogramming methods are electrophysiologically active, form synapses in vitro and express markers of post-mitotic neurons. There exists evidence of induced-dopaminergic neurons partially integrating with local neuronal circuitry after ectopic transplantation in mouse striatum, suggesting that direct reprogramming methods can make functional neurons that integrate successfully [15].The challenge remains to demonstrate direct reprogramming in vivo to generate human neurons, as been demonstrated in mice, and, thus, circumvent the need for cell transplants [11,16,17]. Moreover, future work to achieve the production of different classes of neurons, that are specifically lost in distinct neurodegenerative disorders, is warranted.A leading laboratory in the field of direct reprogramming of cells, has transdifferentiated a B lymphocyte cell line into macrophage-like cells at 100% efficiency, within two to three days, using an estradiol-inducible form of C/EBPalpha [2,18]. They demonstrated that the reprogrammed cells are larger, contain altered organelle and cytoskeletal structures, are phagocytic, and exhibit an inflammatory response. They conclude that the robustness and speed of their system make it a versatile tool to study biochemical and biological aspects of lineage reprogramming [18]. Interestingly, the same group has taken this finding further and demonstrated transdifferentiation of leukemia cell lines into macrophages, thus, impairing their tumorigenicity [19]. This work leads to the exciting idea of using cellular transdifferentiation as a method to treat cancer [19].Hepatocytes and pancreatic islet β-cells are two endoderm-derived cell types that are the subject of much attention due to their indispensable physiological functions and their association with various diseases. The adult β cells have very limited regenerative ability, which is insufficient to compensate for cell loss and is thus the cause of many diseases, including diabetes. For this reason, there has been a large effort to find new sources of β cells and other pancreatic cells. Until recently, efficient treatments for restoring the cellular functions of these cell types were unknown. Clinical studies have now demonstrated that cell transplantation-based therapy can support and restore functions of failed liver and pancreatic islets. Recent advances in hepatocyte and β cell transdifferentiation have provided valuable insights into how to regenerate and restore the normal function of the liver and the pancreas [20].It has been reported that mouse embryonic and adult fibroblasts can be directly reprogrammed into functional hepatocyte-like cells using a combination of endodermal and hepatic transcription factors [21,22]. In both reports, hepatocyte-like cells exhibited typical hepatocyte morphology, gene expression and protein secretions. These cells may resemble an immature stage of hepatocyte differentiation. However, similar human hepatocyte-like cells have yet to be generated. Mesenchymal stem cells (MSC) are an alternative source of hepatic cells. The hepatocyte-like cells transdifferentiated from MSC can engraft into the parenchyma of the liver and at least partially restore liver functions in injury models in vivo [23]. Melton and his colleagues reported that re-activation of three transcription factors (Ngn3, Pdx1, and Mafa) in vivo was able to reprogram pancreatic exocrine cells into endocrine β cells in adult mice. They utilized a cell tracking system and demonstrated that overexpression of the three key pancreatic factors converted acinar cells to insulin-producing β cells. Moreover, these β cells rescued mice from streptozotocin-induced diabetes, demonstrating a clear functional capacity of the cells [24].Another source of new pancreatic cells is the inter-endocrine transition in pancreatic islets. Recent work has demonstrated that new β cells arose from healthy α cells in islets through transdifferentiation, which is induced by fatal β-cells ablation, as in diabetes [25]. Their results argue that a deep lesion (total or near-total β-cell ablation) causes the release of some form of signal that allows prolonged and substantial β-cell regeneration. Such inter-endocrine spontaneous adult cell conversion could be harnessed towards methods of producing β-cells for diabetes therapies, either in differentiation settings in vitro or induced regeneration [25].As liver and pancreas arise from the same bi-potential precursors in the anterior endoderm, it is reasonable to speculate that two closely related tissues may be inter-convertible. For example, the glucocorticoid dexamethasone can efficiently induce rat pancreatic exocrine cells into hepatocytes [26], and there have been reports of hepatocyte transdifferentiation of hepatocytes into pancreatic β cells involving several key pancreatic factors [27,28]. These encouraging recent advance in liver and pancreatic cell transdifferentiation should promote continued development of cell replacement techniques for the future treatment of many liver and pancreas related diseases.It has recently been described the conversion of human foreskin fibroblast into retinal pigment epithelium (RPE)-like cells by defined factors [29]. They developed an RPE-specific Best1::GFP reporter, which could faithfully represent human RPE lineage commitment during human embryonic stem cell differentiation. Using this reporter system, they showed that a defined set of transcription factors can reprogram human fibroblasts into Best1:GFP+ve colonies. They demonstrated that these Best1:GFP+ve cells formed pigmented monolayer epithelium exhibiting molecular features of RPE lineage.In the initial experiment, six RPE developmental transcription factors; Rax, Crx, Pax6, Mitf, Otx2, and Nrl were used [29]. Despite transfection of these factors through retroviruses, pGZ-BEST1-GFP through lentivirus and culturing on Matrigel-coated plates and in hESC culture medium to prime RPE lineage conversion, there was no morphological change and no Best1:GFP+ve cells. In the next experiment, epigenetic “plasticity” was increased with the addition of cMyc and Klf4 factors. The efficiency of RPE induction was altered by removal of certain combination of the six transcription factors. It was found that cMyc, Mitf, Otx2, Rax, and Crx were critical for reprogramming of human fibroblasts into Best1::GFP+ cells, while Klf4, Nrl, and Pax6, individually, were not as important and could potentially be omitted or replaced by other factors without major impact of induced RPE phenotype at day 35 post-transduction [29]. In addition, they observed further maturation of the induced RPE-like cells when they were maintained in medium containing retinoic acid and sonic hedgehog after day 21 [29]. This offers hope that there may be a safer combination of small molecules and transcription factors that can be used to induce RPE lineage from somatic cells for therapeutic application. However, the compulsory requirement of the cMyc oncogene and Klf4 to the transcription factor cocktail limits the clinical utility of this approach.In vivo direct reprogramming strategies involve cell transdifferentiation conducted not in vitro but in an adult organ of a living organism. One of the first examples of in vivo direct reprogramming (transdifferentiation) was in mice by targeting differentiated pancreatic exocrine cells and making cells that closely-resembled β cells by the expression of Ngn3, Pdx1, and Mafa [24]. The first insulin positive cells appeared as early as day three after gene induction, however, the induced β-cells did not organize into islet structures but remained as single cells or small clusters. More recently, hESCs were differentiated in vitro into pancreatic progenitors that were engrafted into mice, they matured in vivo into insulin producing pancreatic endocrine cells and achieved normoglycemia 30 weeks after transplantation [30]. The field of in vivo direct reprogramming offers advantages over in vitro transdifferentiation prior to delivery of cells, however, this technology still requires a lot of work to make functionally safe cells. As an alternative approach to in vivo direct reprogramming, the bioengineering of organs from iPSCs may become an easier and quicker approach towards future tissue regeneration strategies.There have been many attempts to bioengineer different organs from iPSCs such as brain, liver, heart, and other tissues listed below. In general, the successful creation of tissues and organs from iPSCs requires:
+	  
+	  
+      An appropriate mixture of cells to recapitulate cell-cell interaction during organ development. This can be achieved either by differentiating the cells to early progenitors or by mixing them with other cell types or tissues.
+	  
+	  
+      Providing a 3D scaffold or giving the right conditions for assembly in 3D.
+	  
+	  
+      Providing the right extracellular matrix that resembles that of embryonic organogenesis.
+	  
+	  
+	  An appropriate mixture of cells to recapitulate cell-cell interaction during organ development. This can be achieved either by differentiating the cells to early progenitors or by mixing them with other cell types or tissues.Providing a 3D scaffold or giving the right conditions for assembly in 3D.Providing the right extracellular matrix that resembles that of embryonic organogenesis.The ability to generate functional cardiac tissue by in vitro tissue engineering with donor cardiomyocytes has been known for more than ten years [31,32,33,34] but more recent developments include ex vivo [35] and in vivo [36] methods, which also provide the vascular and fibrous elements of cardiac tissue. Cell sheet techniques allowed for iPSC-derived cardiomyocytes to be delivered to the porcine heart demonstrating acute benefits [37] and contractile and vascularized human cardiac organoids have also been created from iPSCs [38,39], which provide longer-term survival and contractility. However the ability to create whole functional hearts by means of tissue bioengineering has proven elusive. The closest result to this complex whole organ bioengineering task has been engineered heart tissue obtained by using human iPSC-derived from multipotential cardiovascular progenitors (MCP) implanted into a decellularized donor mouse heart [40]. In contrast to previous work that used donor cardiomyocytes, MCP—the earliest cardiac progenitors in heart development—gave rise to cardiomyocytes, smooth muscle cells and endothelial cells. The decellularized heart provided a 3D architecture and the complex natural extracellular matrix, which promoted cardiomyocyte proliferation, differentiation and myofilament formation. This use of native cardiac scaffold also avoided the biocompatibility problems of some artificial scaffold materials. The resulting heart tissues presented the requisite features of rhythmic mechanical force generation, electrophysiological characteristics and response to drugs that make it a valuable model for the study of heart development and drug screening. Nevertheless, several improvements still have to be made to enable its use in regenerative medicine: heart fibroblasts are missing, since they do not derive from MCP and a functional cardiac conduction system has not been demonstrated. As a result, the mechanical force generated is insufficient and these model organs lack a coordinated electrical propagation necessary for synchronization of the tissues.Although very much in the early stages of development as a complete tissue, functional islets of langerhans have also been created in vitro from iPSCs [41]. A protocol has been developed to differentiate iPSCs into glucose-responsive functional islets, with a 3D structure similar to adult pancreatic islets and that secreted insulin and improved blood glucose levels in hyperglycemic mice. The protocol involved a two-step cell culture method; first the differentiation of iPSCs to immature pancreatic cells and then second, culture in specific conditions that allow islet formation. The islets exhibited distinct 3D structural features similar to adult pancreatic islets and secreted insulin in response to glucose concentrations. Mice transplanted with the iPSC-derived islets normalized their blood glucose levels after nine days [41].Cerebral organoids have been made by culturing in a 3D system the neuroectoderm derived from human iPSC [42]. First, iPSCs were differentiated into embryoid bodies and from those neuroectoderm was derived. Neuroectodermal tissue was cultured in a 3D scaffold system of matrigel droplets and then transferred to a spinning bioreactor. The resulting three-dimensional tissue presented heterogeneous regions similar to human brain that were discrete but interdependent. These cerebral organoids, which the authors do not intend to use for regenerative purposes, recapitulated features of human cortical development. As mice and human brains have highly complex and integrated structures and developments, these organoids could be good candidates for the study of human brain development and modeling of brain disorders such as microencephaly.A recent and exciting development in the field of bioengineering organs has been the creation of a liver bud which when transplanted into mice was able to rescue drug-induced liver failure [43]. In this case the researcher co-cultured iPSCs differentiated to hepatic endoderm with human mesenchymal stem cells and human umbilical endothelial cells. Co-cultured cells, when plated in a bi-dimensional matrigel layer self-organized into a three-dimensional system that the authors termed iPSC-derived liver buds. These liver buds were able to produce liver specific proteins such as albumin and were able to metabolize drugs ketoprofen and debrisoquine. Notably, the presence of human umbilical endothelial cells in the starting cell mixture provided the iPSC-derived liver buds with vessels that, when implanted in the mice, connected with the host vessels within 48 h. This vascular system, together with the 3D structure seems to be the key for successful engraftment and maturation.iPSCs have been used to generate optic vesicle-like structures that generated retinal cell types suitable for in vitro studies and disease modeling [44]. The authors differentiated iPSCs into 3D optical vesicles with the capacity of self-assemble into rudimentary neuroretinal structures and which expressed markers of intercellular communication. The use of retinal pigmented epithelial (RPE) cells to treat eye disease is currently being evaluated in clinical trials, leading the way for future research to develop the bio-engineered eye structures.The generation of complex tooth-like structures has been recently created from human iPSCs [45]. The authors developed a bioengineering protocol that combines iPSC-derived dental epithelial sheets with embryonic dental mesenchyme. The epithelial sheets gave raise to ameoblasts that produced the enamel component of the tooth and the embryonic dental mesenchyme produced the dentin-pulp complex and peridontium, as well as provided odontogenic signals to the epithelial sheets. The formed regenerative teeth were very similar in structure and mineral content to normal teeth and presented comparable hardness. As the sources of endogenous dental epithelial cells are scarce, iPSCs can provide a promising source of dental epithelial seed cells for use in tooth tissue engineering.The bone tissue-engineering field has also made use of iPSC technology to create functional bone substitutes [46]. To make bone, iPSCs were differentiated into mesenchymal progenitors that were subsequently grown in an osteoconductive scaffold-perfusion bioreactor. The dense bone-like structure that formed inside the scaffold presented a mature bone molecular pattern. When implanted into mice, the engineered bone-tissue was stable over the 12 weeks of the study, without differentiation into other lineages, displayed vascular ingrowth and connective tissue development, and presented signs of initiation of scaffold resorption.3D gut organoids have also been created from iPSC [47]. A protocol has been developed in which iPSCs were differentiated into intestinal tissue following a sequential protocol mimicking embryonic development. Spontaneous three-dimensional spheroids formed that were transferred to three-dimensional culture systems known to promote intestinal formation. The resulting intestinal tissue presented a cellular composition similar to that of foetal intestine and also presented absorptive and secretory functions. However these gut organoids have not been tested for engraftment and function in vivo.As iPSC technology is advancing to become a future tool for clinical therapy, concerns about the susceptibility to immune rejection of iPSC grafts are increasing. As data accumulates, it has become apparent that iPSCs may not be as immune-privileged as initially thought [48]. Despite the sourcing of cells for iPSC treatment from the recipients, therefore overcoming HLA incompatibility, there remains the possibility that the reprogramming process itself might render the grafts or cells immunogenic [48]. Moreover, potential sources of innate proinflammatory “danger” signals that can lead to immune activation may be provided through the use of retroviral or episomal methods to make iPSC. Consistent with this, it has recently been demonstrated in mice that autologous transplantation of iPSCs, generated by episomal or retroviral vectors, elicited anti-graft T cell responses potent enough to prevent the formation of teratomas [49]. Interestingly, this was not observed with embryonic stem cells (ESC), where autologous transplantation in a syngeneic recipient did not elicit an immune response, suggesting the method to reprogram cells to the pluripotent state itself influences the ontogeny of immune responses within the host. Furthermore, the authors also found evidence of abnormal gene expression in some cells differentiated from iPSCs that were able to induce T-cell-dependent immune response in syngeneic recipients [49].Recently, mouse iPSCs generated by lentivirus vectors or episomal vectors have been differentiated into representative cell types of the three germs layers and examined their immunogenicity in vitro and in vivo by transplantation into syngeneic recipients [50]. They found that differentiated cells derived from syngeneic iPSCs were not rejected after transplantation [50]. Moreover, model transplantation experiments were performed using various iPSC-derived differentiated tissues and immune-mediated rejections have not been observed. In further studies no differences in immunogenicity or transplantation success were found between differentiated skin and bone marrow tissue derived from integration-free mouse iPSCs (generated by episomal vectors) compared to that of ESC-derived tissue. This study did not observe any differences between the two groups in regards to the rate of transplantation success [51].A very recent advance in this field has been a description of the induction of dopaminergic (DA) neurons from nonhuman primate iPSC by directed differentiation in vitro, and the comparison of the autologous and allogeneic transplantation into the brains of nonhuman primates [52]. Autologous transplantation of the iPSC-derived cells generated a minimal immune response compared with allografts in nonhuman primate brains in the absence of immunosupression. This data also entertains the idea that immunosupression is not necessary for autologous transplantation of iPSC-derived neural cells into brain. Moreover, they compared the immunogenicity of iPSCs generated with retroviral vectors versus episomal vectors. They detected that autologous grafts derived from iPSCs generated by retroviral vectors were infiltrated by a large number of IBA1+ and CD45+ microglia in comparison with those generated by episomal vectors, probably due to the residual expression of transgenes. This work suggests that residual transgenes can be immunogenic, for this reason it is crucial to use integration-free iPSCs [52].Despite these recent advances, research to date has primarily focused on T cell mediated responses directed against stem grafts. However, many questions remain poorly understood, for example the role of NK cell mediated rejection in iPSC based transplantation technology. This seems to be especially of interest as the HLA class I and II expression on ESCs has been shown to vary depending on their maturation status [53]. Although a low expression of HLA has been thought to partially explain the reduced immunogenicity of ESCs, this reduced expression of HLA may in turn render these cells more vulnerable to NK cell mediated killing.In summary, there is now encouraging evidence that grafts from terminally differentiated cells derived from syngeneic iPSCs can circumvent acute rejection in animal models. However, these studies also highlight the “devil in the detail” and show that even subtle differences in the generation of iPSCs may render them more or less immunogenic. Consequently, the future of the study of immunity against iPSCs is complicated by the simple truth that iPSCs are dynamic antigenic targets and that the immune response elicited by those cells will likely differ based on their generation, differentiation, age, and survival in vivo.Both methods of direct reprogramming to desired cells types or full reprogramming to iPSCs and then differentiating to cells for replacement therapy have pros and cons for future clinical application. The length of time required for generation and differentiation of iPSCs into the desired cell type is the main handicap of iPSCs in comparison to directly reprogrammed cells. In addition differentiation of iPSCs to some cell types have not been achieved, for example, blood and functional sperm, with cells remaining in undifferentiated embryological cell states. Moreover, direct reprogramming avoids going through the big hurdle of the transient tumorigenic state of iPSCs. For this reason, direct reprogrammed cells could be a better choice in the clinical application for human cell replacement therapy for neurodegenerative disorders like Parkinson’s disease, Huntington’s disease, and amyotrophic lateral sclerosis.However, directly reprogrammed cells are also susceptible to chromosomal aberrations [54,55] and all methods for direct reprogramming of cells to date have used viral transduction methods to insert genes into the genome of cells. Another con for direct reprogramming is that some cells have been shown to maintain an epigenetic memory of their cell of origin, like neurons, questioning their stability over time [56]. Moreover, with direct reprogramming the intermediate cell steps that form during the process are difficult to identify because the molecular markers they express remain unknown [1]. Therefore, iPSCs may be the cell choice for future regenerative medicine applications, given that they are well defined, characterized, and that their proliferative capacity is an advantage over post mitotically inactive direct reprogrammed cells.Perhaps one of the major advantages of iPSCs will be for engineering of human organs for transplantation. The co-culturing of more than one cell type has proven successful for making a functional liver bud and although iPSCs were used in this study not all the cells used in the mix were derived from iPSCs [43]. Combining two or more cell types all derived from iPSCs to make complex organs like the liver, brain or heart may be needed in the near future. If this is possible, the powerful ability to efficiently produce pluripotent cells to seed the growth of autologous body organs for transplant medicine may be achievable in the near future.Michael J. Edel is supported in part by the Program Ramon y Cajal (RYC-2010-06512) and project grant BFU2011-26596. Centre of Cell Therapy and Regenerative Medicine (CCTRM), University of Western Australia (UWA), Perth, WA, Australia. National Health and Medical Research Centre project grant 1024817 to Rodney J. Dilley. National Health and Medical Research Centre Early Career Fellowship to Fred Kuanfu Chen (APP1054712). Support of the McCusker Charitable Foundation to Michaela Lucas.Ana Belen Alvarez Palomo: Direct reprogramming and iPSC reprogramming; Michaela Lucas and Andrew Lucas: Immune response of iPSC; Rodney J. Dilley: Bioengineering of body organs; Samuel McLenachan and Fred Kuanfu Chen: RPE and direct reprogramming and iPSC reprogramming; Jordi Requena and Marti Farrera Sal: Direct reprogramming and iPSC reprogramming; Inaki Alvarez Dolores Jaraquemada: Immune response of iPSC; Michael J. Edel: Direct reprogramming, iPSC reprogramming and coordination of paper.The authors declare no conflict of interest.

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+This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).Prenatal screening is often misconstrued by patients as screening for trisomy 21 alone; however, other chromosomal anomalies are often detected. This study aimed to systematically review the literature and use diagnostic meta-analysis to derive pooled detection and false positive rates for aneuploidies other than trisomy 21 with different prenatal screening tests. Non-invasive prenatal testing had the highest detection (DR) and lowest false positive (FPR) rates for trisomy 13 (DR: 90.3%; FPR: 0.2%), trisomy 18 (DR: 98.1%; FPR: 0.2%), and 45,X (DR: 92.2%; FPR: 0.1%); however, most estimates came from high-risk samples. The first trimester combined test also had high DRs for all conditions studied (trisomy 13 DR: 83.1%; FPR: 4.4%; trisomy 18 DR: 91.9%; FPR: 3.5%; 45,X DR: 70.1%; FPR: 5.4%; triploidy DR: 100%; FPR: 6.3%). Second trimester triple screening had the lowest DRs and highest FPRs for all conditions (trisomy 13 DR: 43.9%; FPR: 8.1%; trisomy 18 DR: 70.5%; FPR: 3.3%; 45,X DR: 77.2%; FPR: 9.3%). Prenatal screening tests differ in their ability to accurately detect chromosomal anomalies. Patients should be counseled about the ability of prenatal screening to detect anomalies other than trisomy 21 prior to undergoing screening.Prenatal screening for fetal aneuploidy aims to identify women at increased risk of carrying a fetus with a chromosomal anomaly and limit the offer of invasive diagnostic tests (with their associated risk of miscarriage) to those women at high-risk. National practice guidelines currently recommend that all pregnant women be offered prenatal screening for aneuploidy [1,2]. Methods for prenatal screening have evolved rapidly in recent decades from screening based on maternal age alone, to serum screening, to a combination of maternal serum and ultrasound based measures, and most recently to isolated cell free fetal DNA (cffDNA) from maternal plasma [3,4,5].Prenatal screening for fetal aneuploidy is most frequently discussed in terms of prenatal screening for trisomy 21 (Down syndrome) as this is the most common form of fetal aneuploidy and this condition has clinical implications for the health of the fetus [3,4]. However, counseling issues arise when anomalies are detected that were not discussed during the informed consent process, given that parents would be unprepared for such a result. Parents consistently report that they do not have enough information about prenatal screening, and may be surprised when they undergo a screening test for trisomy 21 only to find out that their fetus has another (and potentially more severe) anomaly [6,7,8,9]. A good understanding of prenatal screening, prior to accessing this test, is important, because if the results are positive, parents must make important and complex decisions about invasive testing which carries a small, but substantial risk of pregnancy loss, and potentially pregnancy termination [9,10]. Counseling about conditions other than trisomy 21 that may be detected through prenatal screening is complicated for a variety of reasons; the clinical implications of balanced chromosome rearrangements are not always known, the risk of specific anomalies is extremely low, and the reported detection rates and false positive rates for different anomalies vary widely in the literature [10]. This study aimed to systematically review the literature and use diagnostic meta-analysis to derive pooled detection and false positive rates for fetal aneuploidies other than trisomy 21 using standard prenatal screening tests. This information may be helpful to care providers as they discuss the relative strengths and limitations of different forms of prenatal screening with their patients.Relevant English-language literature was identified through a systemic search of Medline (1946–2013) and Embase (1974–2013) in November 2013. Reference lists of included articles were examined to identify additional relevant articles that may have been missed in the electronic search. The search terms included generic terms (prenatal diagnosis, antenatal diagnosis, prenatal screening, antenatal screening) as well as specific terms related to the screening test used (first trimester screening, aneuploidy screening, integrated screening, sequential screening, non-invasive prenatal testing, serum screening, combined screening, genetic screening, quad screen) or the chromosomal anomaly (Klinefelter syndrome, tetraploidy, translocation, triploidy, trisomy 13, trisomy 18, Turner syndrome). Truncation symbols were used to include all possible variations of the search term (i.e., screen, screening, screened).Studies were eligible for inclusion if they provided the data necessary to calculate the number of true positives, false positives, false negatives and true negatives for a chromosomal anomaly other than trisomy 21 using a currently available prenatal screening test (see Table 1). Studies that exclusively used maternal serum alpha fetoprotein (MS-AFP) or a combination of MS-AFP and human chorionic gonadotrophin (hCG) (double test) were deemed outdated and were not included in the review. Two reviewers (Amy Metcalfe, Catriona Hippman) independently reviewed all titles and abstracts for potential inclusion. Full-text review was undertaken for any article deemed potentially eligible by either reviewer. Data extraction and verification was performed by the same reviewers. Specific study elements extracted included: screening test used, detection rate, number of women screened, number of screen positive women, number of aneuploid fetuses, study location, and time period of data collection.Prenatal screening tests eligible for inclusion in systematic review.AFP = alpha-fetoprotein; cffDNA = cell free fetal DNA; hCG = human chorionic gonadatrophin; NT = nuchal translucency; PAPP-A = pregnancy associated plasma protein A; uE3 = unconjugated estriol.Guidelines for the Meta-analysis of Observational Studies in Epidemiology (MOOSE) [11] and Studies of Diagnostic Accuracy (STARD) [12] were followed.The accuracy of prenatal screening tests is typically evaluated using both the detection rate (sensitivity) and the false positive rate (1-specificity). As these measures are correlated, a bivariate random effects model was used to derive pooled estimates and generate summary receiver operating characteristic (SROC) curves [13]. Studies were weighted based on their total sample size, as large sample sizes allow for more precise estimates of sensitivity and specificity [14]. For a detailed description of the technical specifications of the model, readers are referred to [14]. A minimum of four studies were required to derive pooled detection and false positive rates. All analyses were conducting using Stata 12 SE (StataCorp LP, College Station, TX, USA).Sixty-five articles met all inclusion criteria and were included in the systematic review (Figure 1). Trisomy 18 was the most common aneuploidy eligible for inclusion in the meta-analysis for which literature was available, while the first trimester combined test was the most common prenatal screening test eligible for inclusion in the meta-analysis for which literature was available (Figure 1). Detailed information on all included studies can be found in the Appendix A.Flow diagram of study selection.Overall, 31 studies reported data on trisomy 13, including 16 studies that utilized the first trimester combined test [15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30], 8 utilizing the second trimester triple screen [31,32,33,34,35,36,37,38], and 9 using non-invasive prenatal testing (NIPT) [39,40,41,42,43,44,45] (Table 2 and Appendix B). Two studies utilizing NIPT [39,45] included data on two patient groups (a training set and a validation set); hence were included as two distinct studies in the diagnostic meta-analysis.Substantial variation was noted in the definition of a “screen positive” test for trisomy 13 using the first trimester combined test—definitions for specific risk levels for trisomy 13 included risks ≥1/200 [19], risks of trisomy 13 or 18 ≥1/150 [15,21] or 1/200 [25], or risks of trisomy 13, 18 or 21 ≥1/250 [24] or 1/300 [23,27]. No studies using the second trimester triple screen included a specific risk algorithm for trisomy 13; however, variation in the definition of “screen positive” for trisomy 21 or trisomy 21/trisomy 18 was observed. Studies differed in that some reported cut-off values based on the risk of trisomy 13 at the time of the test (either the first trimester or the second trimester) or at term; however, this only partially explains the differences observed in cut-off values.NIPT exhibited the strongest overall test performance with a pooled detection rate of 90.3% and a false positive rate <1% (Table 2). The first trimester combined test had a significantly higher detection rate (83.1% vs. 43.9%) and a lower false positive rate (4.4% vs. 8.1%) than the second trimester triple screen, although statistical significance was not achieved for the false positive rate (p > 0.05) (Table 2).Pooled results for trisomy 13.Trisomy 18 was the most frequently reported condition in eligible studies. Data was obtained from 61 studies, including 30 that utilized the first trimester combined test [15,16,17,18,19,20,21,23,24,25,26,27,28,29,30,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60], 17 that used the second trimester triple screen [31,32,33,34,35,36,37,38,61,62,63,64,65,66,67,68,69], 12 that contained data on NIPT [40,41,42,43,44,45,70,71,72,73,74], 3 studies that used the second trimester quadruple screen [47,75,76], and 2 studies that reported data on patients undergoing integrated or sequential screening [52,77] (Table 3 and Appendix B). One of the studies reporting on NIPT included data from two separate patient groups [72], and is included as two separate studies in the diagnostic meta-analysis.Similar to screening for trisomy 13, substantial heterogeneity was observed in the definition for a “screen positive” test for trisomy 18. Positive risk scores for the first trimester combined screen ranged from risks ≥1/100 to ≥1/300. Approximately half of the studies using the second trimester triple screen that reported their definition for “screen positive” results used a patient-specific risk (risks ≥1/100 or ≥1/200), while the remaining studies used a fixed cut-off level based on levels of serum analytes (typically AFP ≤ 0.75 MoM, hCG ≤ 0.55 MoM, and uE3 ≤ 0.60 MoM). Again, studies differed in that some reported cut-off values based on the risk of trisomy 18 at the time of the test (either the first trimester or the second trimester) or at term.Pooled results for trisomy 18.NIPT exhibited the best overall test performance with a pooled detection rate of 98.1% and false positive rate of <1% (Table 3). The first trimester combined test outperformed the second trimester triple screen, as it had a significantly higher detection rate (91.9% vs. 70.5%), with no difference in the false positive rate (p > 0.05) (Table 3). There were an insufficient number of studies to derive pooled estimates on the detection rate and false positive rate for trisomy 18 using the second trimester quadruple test, but the observed detection rate ranged from 44.4% to 100% with false positive rates ranging from 0.5% to 9.6% [47,75,76]. Pooled results from the triple screen population may be generalizable to women screened with the quadruple test, as Inhibin A is not part of the trisomy 18 screening protocol.There were an insufficient number of studies using integrated/sequential screening to determine pooled detection and false positive rates; however, the observed detection rate was 100% in both studies using integrated/sequential screening, while the observed false positive rates ranged from 3.7% to 7.3% [52,77].Twenty studies included data on Turner syndrome, 6 that utilized the first trimester combined screen [19,23,26,30,49,51], 9 that used the second trimester triple screen [31,32,33,34,35,36,66,78,79], 4 that involved NIPT [40,42,43,45], and a single study that used the second trimester quadruple screen [76] (Table 4 and Appendix B).Pooled results for 45,X.With the exception of NIPT, none of the other screening tests involved a specific risk cut-off to identify patients at increased risk of carrying a fetus with Turner syndrome. Turner syndrome is considered an incidental finding amongst patients who screen positive for trisomy 13, 18 or 21 with the first trimester combined test and the second trimester triple and quadruple screens. This has implications for the interpretation of test results in a clinical setting.NIPT had the best overall test performance with a detection rate of 92.2% and a false positive rate of <0.1% (Table 4). A significant difference was not observed between the first trimester combined test and the second trimester triple screen for detection rate, although the first trimester combined test had a significantly lower false positive rate (p < 0.05) (Table 4). The single study that used the second trimester quadruple screen had an observed detection rate of 66.7% and a false positive rate of 9.6% [76].Finally, 11 studies provided data on triploidy: 7 of which used the first trimester combined test [19,20,23,24,27,30,51], 3 utilized the second trimester triple screen [32,33,35] and 1 used the second trimester quadruple screen [76]. Triploidy was an incidental finding amongst women who were screen positive for trisomy 13, 18 or 21. This has implications for the interpretation of test results in a clinical setting and how patients should be counseled following a positive screening test.Seven studies, representing 93,796 women and 15 affected fetuses, contained data on the first trimester combined screen and could be pooled using diagnostic meta-analysis [19,20,23,24,27,30,51]. These studies had a pooled detection rate of 100.0% (99.9–100.0) and a pooled false positive rate of 6.3% (4.9–8.0) (Appendix B). Comparable results were also observed for the other screening tests, even though sufficient data were not available to derive pooled estimates. Observed detection rates ranged from 98.1% to 100% with observed false positive rates of 2.6%–10.6% for the second trimester triple screen [32,33,35]; while the single study that used the quadruple screen reported a detection rate of 100% and a false positive rate of 9.6%.The results of this systematic review and diagnostic meta-analysis confirm the general consensus that for all conditions, NIPT is a superior test in terms of detection rate and false positive rate than other screening tests for aneuploidies, with the caveat that most studies have been performed in high risk populations. However, NIPT is subject to a higher rate of test failures than other prenatal screening tests and currently only provides results for a limited number of aneuploidies. The dramatically higher median prevalence of aneuploidies used in studies of NIPT to date has important implications on test performance in a low-risk setting. While this is unlikely to impact the overall detection rate and false positive rate, positive predictive values (odds of being affected given a positive result) are particularly sensitive to the prevalence of the condition being studied [80,81]. Additionally, pooled results for NIPT did not display 100% detection rates for any condition, indicating the importance of confirmatory invasive testing. Furthermore, due to the high cost of NIPT, many centers continue to utilize other forms of prenatal screening. Nonetheless, having data on pooled detection rates and false positives rates may be helpful when counseling patients.Multiple studies have indicated that up to half of the chromosomal anomalies identified through invasive testing for abnormal prenatal screening results or increased maternal age are not autosomal aneuploidies [15,82,83]. Several authors have expressed concern that moving away from serum and ultrasound based screening to prenatal screening based exclusively on NIPT might miss the detection of rare chromosomal anomalies [5,73,84]. Furthermore, extreme levels of serum analytes are associated with adverse obstetrical outcomes and may be useful to help triage patients into higher levels of prenatal care [85,86,87,88]. Contingent screening with NIPT might provide a compromise in terms of maintaining the benefits of existing prenatal screening programs, while reducing the number of women who proceed onto invasive testing, but the current costs of NIPT make this prohibitive to implement in many population-based screening programs. While the costs of NIPT will likely decrease over time, the current cost of NIPT is $795 (Canadian dollars) in contrast to $303 for the first trimester combined test and $15 for the second trimester triple screen [89,90].Current clinical practice guidelines recommend that prenatal screening should be offered through an informed consent process; in particular, the Society of Obstetricians and Gynecologists of Canada has issued counseling recommendations specifically detailing that all women who are offered prenatal screening should be told that all women have some risk of having a fetus affected by trisomy 21, 18, or 13 [91]. We suggest additional pre-screening counseling recommendations that women should be informed that, while prenatal screening tests have been developed to specifically target the detection of trisomy 21, 18, and 13, other chromosomal anomalies and obstetrical risks may be detected by the screen as well. In particular, it would be worth discussing sex chromosome aneuploidy and triploidy in light of available data, but some mention of the breadth of unexpected results would enhance the informed consent process.This study has limitations. The search specifically excluded terms related to trisomy 21—this was done on purpose to restrict the number of abstracts identified and because the goal of this review was to look at chromosomal anomalies other than trisomy 21. This may have resulted in some relevant articles not being identified in the initial search; however, the reference lists of accepted articles were hand searched to identify other relevant articles. The quality of the pooled results is a direct reflection of the data included in the original articles; the authors of many studies made the assumption that false negative cases would be brought to their attention through cytogenetic databases or birth certificates and did not actively follow-up all women screened. Additionally, the raw data on true positives, false positives, false negatives and true negatives sometimes had to be back-calculated based on reported detection and false positive rates; this may have resulted in some minor inaccuracies. The model used to generate pooled detection and false positive rates does not specifically account for differences in cut-off values or the specific screening algorithm used to define a positive or negative screening test [14]. However, this is reflected in the different estimates of sensitivity and specificity obtained from individual studies and the heterogeneity of these estimates is directly modeled and used to derive the pooled estimates [14]. Multiple between-study differences were observed in terms of the maternal age distribution, inclusion of pregnancies that ultimately resulted in spontaneous abortion and of multiple gestation pregnancies, test uniformity (specifically related to the use of free β hCG vs. total hCG), and test quality standards (specifically related to the use of nuchal translucency). While a random effects model was used to derive pooled estimates, this model addresses statistical heterogeneity, not clinical heterogeneity in the underlying populations. Finally, the search was limited to English language articles which may limit the generalizability of the results. However, the included studies came from North America, Europe, Australia and Asia, indicating wide geographic coverage.In conclusion, while prenatal screening tests are often described to patients in terms of trisomy 21, they do (to varying degrees) identify other chromosomal anomalies. Providing this information to patients prior to screening can help them make an informed choice about accessing prenatal screening and, in some contexts, which screening test is preferable to them.Amy Metcalfe holds a fellowship award from the Canadian Institutes of Health Research. Catriona Hippman is supported by the Women’s Health Research Institute.The authors declare no conflict of interest.Included studies.The Summary Receiver Operator Characteristic (SROC) curves below plot the sensitivity (detection rate) of a given test against 1-specificity (false positive rate) of a given test for detecting the condition of interest [13]. Circles represent the point estimate from included studies; the size of the circle is related to the sample size of the study [13]. Diamonds represent the pooled detection rate and false positive rate [13]. The dashed lines represent the 95% confidence region surrounding the pooled effect estimate, while the dotted lines represent the 95% prediction region that would encompass the true detection rate and false positive rates in future studies [13]. The HSROC curve represents the summary receiver operator characteristic curve obtained from the model [13].Summary Receiver Operator Characteristic (SROC) Curves for the detection of trisomy 13 using (A) first trimester combined test; (B) second trimester triple screen; and (C) non-invasive prenatal testing.Summary Receiver Operator Characteristic (SROC) Curves for the detection of trisomy 18 using (A) first trimester combined test; (B) second trimester triple screen; and (C) non-invasive prenatal testing.Summary Receiver Operator Characteristic (SROC) Curves for the detection of 45,X using (A) first trimester combined test; (B) second trimester triple screen; and (C) non-invasive prenatal testing.Summary Receiver Operator Characteristic (SROC) Curves for the detection of triploidy using the first trimester combined test.