When the post-surgical outcome was analysed, the MIDA Score retained its independent prognostic significance from age and In conclusion, DMR management requires extensive prognostic assessment.

12,13 The MIDA Score, developed using the largest co- horts of DMR medically and surgically treated ever analysed, can for the first time position a given patient within a continuous spec- trum of short- and long-term mortality risk, overall, under medical management and after surgery, providing incremental and inde- pendent prognostic information.

Our findings-generated by analysis of data collected at multiple centres from different contin- ents-may contribute to improve DMR management, by not only facilitating guidelines application but also providing a useful tool to test new therapeutic options for patients with DMR in homoge- neous categories of risk.


In degenerative mitral regurgitation (DMR), lack of mortality scores predicting death favours misperception of individual patients’ risk and inappropriate decision-making.

The Mitral Regurgitation International Database (MIDA) registries include 3666 patients with pure, isolated, DMR consecutively diagnosed by echocardiography at tertiary centres.

The MIDA Score was derived from the MIDA-Flail-Registry by weighting all guideline-provided prognostic markers, and externally validated in the MIDA-BNP-Registry.

The MIDA Score ranged from 0 to 12 depending on accumulating risk factors.

In predicting total mortality post-diagnosis, the MIDA Score showed excellent concordance both in Derivation Cohort (c = 0.78) and Validation Cohort (c = 0.81).

In the whole MIDA population, 1-year mortality with Scores 0, 7–8, and 11–12 was 0.4, 17, and 48% under medical management and 1, 7, and 14% after surgery, respectively (P < 0.001).

Five-year survival with Scores 0, 7–8, and 11–12 was 98 ± 1, 57 ± 4, and 21 ± 10% under medical management and 99 ± 1, 82 ± 2, and 57 ± 9% after surgery (P < 0.001).

In models including all guideline-provided prognostic markers and the EuroScoreII, the MIDA Score provided incremental prognostic information (P < 0.002).

The MIDA Score may represent an innovative tool for DMR management, being able to position a given patient within a continuous spectrum of short- and long-term mortality risk, either under medical or surgical management.


Mitral regurgitation is a public health problem, as the most frequent valvular disease leading to frequent referrals, hospitalizations, and cardiac surgery, and its burden is expected to increase with ageing of the population.

Degenerative mitral regurgitation (DMR) with mitral valve prolapse is the most frequent organic mitral regurgitation and the most repairable, but it is often undertreated in routine clinical practice.

This prevalent under treatment in both Europe and North America has multiple causes, but is favoured by sub-optimal risk assessment.

In DMR, individual risk factors have been identified and endorsed by clinical guidelines.

However, while guidelines strongly recommend a combination of those risk factors in an integrated process, there has so far been no attempt at deriving and validating a risk score satisfying such unmet need.

The lack of an integrated risk score usable at diagnosis contrasts with the availability of surgical risk scores, which are nevertheless instrumental mainly in surgical decisions.

Current unavailability of a mortality score in DMR should not be surprising, since very large cohorts of patients with uniform diagnosis and comprehensive clinical data are required, warranting multi-centre collaboration to obtain separate cohorts for the derivation and validation process (the two mandatory steps to obtain a score and to make it applicable to daily practice).

The international MIDA registries provide the unique opportunity to analyse a comprehensive set of risk markers (prospectively and homogeneously collected) in an integrated manner, allowing derivation and subsequent external validation of a MIDA Score specifically applicable to DMR.

Accordingly, we aimed at developing a risk score easily derivable from routinely collected clinical/echocardiographic parameters endorsed by guidelines and at validating this score externally.

We also wanted to verify the hypothesis that a user-friendly MIDA mortality score is independently and incrementally predictive of short- and long-term survival after diagnosis of DMR.

In summary, we aimed at providing for the first time a tool able to position a given patient within a continuous spectrum of mortality risk whether assigned to surgical treatment or to continuing medical therapy.


Mitral regurgitation (MR) is the most frequent valvular disease in developed countries [1], [2].

The degenerative aetiology predominates in primary MR, therefore its management represents an important public health issue [3].

It has been observed both in Europe and in the USA that there is an underuse of surgery in patients with MR when indicated.

This occurs with the frequent finding that risk evaluation is not adequate, leading to undertreatment of the sickest patients while conversely lower risk patients are often overtreated [4]-[7].

The search for an improvement of risk stratification in patients with degenerative MR was the aim of the study published by Grigioni et al. in this issue of the journal [8].

The goal was to define a score to predict early and late mortality.

The authors should be congratulated for putting together what is the largest study ever published on degenerative MR since it included >3000 patients.

The patient population comes from two registries: the MIDA Flail registry (2472 patients) was used as a derivation cohort for the score, and the MIDA BNP registry (1194 patients) served as the validation cohort.


Among the potential limitations of the present study we should take into account the fact that the inclusion period covers more than two decades, which may lead to significant changes in practices as regards both diagnosis and management.

Although no specific information was given about co-morbidities, the patient population seems to be at relatively low risk since EuroSCORE II ranged from 1 to a maximum of 2.48, mean age was 66 years, a large proportion of patients were asymptomatic, and mean left ventricular ejection fraction was 64%.

Thus it is likely that the high-risk patients currently treated using percutaneous device are under-represented here.

We also lack precision as regards the methods used for grading the severity of MR; however, it was assessed using an integrative approach in experienced teams.

In addition, most patients had flail leaflets which usually results in severe MR.


The MIDA score was derived from the derivation cohort by weighing clinical and echocardiographic parameters which are in the guidelines: age >_65 years, left atrium diameter >_55 mm, left ventricular ejection fraction >_60%, left ventricular end-diastolic diameter >_40 mm, heart failure symptoms, atrial fibrillation, and right ventricular systolic pressure >_50 mmHg.

All criteria corresponding to recommendations for surgery in guidelines were strongly associated with outcome, and this is a further proof of the validity of guidelines and of the relevance of the proposed thresholds [9], [10].

The positive aspects of this score are that it includes both clinical and echo variables taking into account all criteria together with a weight which is proportional to their prognostic impact.

The MIDA score is simple, thus it may be expected to be user-friendly which may facilitate its use in practice.

The score showed a good discrimination and also a good calibration, with a C index of 0.78 and 0.81, respectively.


Finally it appears that the MIDA score adds incremental value in comparison with the guideline-provided prognostic markers and the EuroSCORE II for predicting 1- and 5-year mortality.

The fact that the MIDA score has a good predictive performance of 1-year mortality in medically treated patients is an important contribution in decision-making since it allows for a better assessment of the respective outcomes of operated and medically treated patients.

The predictive value of the MIDA score in operated patients further highlights the prognostic impact of the consequences of MR on the left ventricle, left atrium, and pulmonary pressures.

This is a further incentive to consider early surgery in severe MR without waiting for overt symptoms or left ventricular dysfunction.


A number of methodological limitations do, however, exist, among which one of the most important is the fact that the populations in the derivation and validation cohorts were significantly different as regards important characteristics such as gender (males: 72% in the derivation cohort vs. 65% in the validation cohort), symptoms (asymptomatic patients: 67% vs. 43%), and number of patients with flail leaflets (96% vs. 77%).

It is understandable that performing such a large study was at the price of some heterogeneity and it would be theoretically desirable to concentrate on a more homogenous population with flail leaflets only, severe MR assessed using precise quantification methods, and including only asymptomatic patients where decision-making is more difficult, while surgery is theoretically indicated when MR is severe and symptoms are present.

Finally, percutaneous treatment should be part of the treatment performed since it has a growing role in current practice when affordable [11].


Beyond the issues intrinsic to the value of this score, it is a bit of an oversimplification to think that a score can summarize all the factors in the decision, even more so when addressing a population of elderly patients with a large number of co-morbidities, which are not taken into account in the MIDA score.

A simple score could be used more often but it will not provide the solution for the management of a specific patient [12], [13].

In such cases, the only way to proceed is the judgement of a Heart Team integrating a number of clinical, anatomic, and procedural variables.

Such a process was recently put forward and is now recommended in the new ESC EACTS guidelines on the management of valvular disease [14].


The suggestion that undertreatment is largely due to the absence of such a score also somewhat oversimplifies the problem because the reasons are probably multiple.

In the MIDA registry, patients who were denied surgery were at higher operative risk, as shown by differences in risk scores.

This finding was the same in the Euro Heart Survey, in which the EuroSCORE was higher in patients referred to surgery than in those medically treated, although all of them had a theoretical indication for surgery [4], [5].

We share the opinion of the authors that this finding suggests that the operative risk is overstressed in decision-making.

High-risk patients are also likely to have a particularly poor outcome under medical therapy, but this does not seem to be fully taken into account by the practitioners in charge of the therapeutic decision.


A process needs to be followed to overcome undertreatment of MR.

The first step would be to identify gaps in knowledge.

This can be done by awareness studies performed among primary care physicians and cardiologists [15].

A similar effort was made in Europe and was presented during the last annual congress.

In parallel, another way to identify the gaps in knowledge will come from registries such as the Euro Heart Survey and its second phase which was just finalized and included >7000 patients throughout Europe, the results of which are to be expected next year.

This identification of the gaps will facilitate the development of new educational programmes specifically targeting the gaps derived from awareness studies and registries.

The efficacy of educational programmes will then be evaluated based on changes in knowledge and, more importantly, practices and outcomes to be evaluated by repeated registries.

Thus this study contributes to an improvement in our knowledge in the management of degenerative MR, but there is still a lot of work to be done.


Male lemurs have well-developed scent glands on the wrists (an- tebrachial scent glands) and shoulders (brachial scent glands).

The antebrachial gland is mainly composed of apocrine glands and secretes small quantities of a highly volatile clear fluid, whereas the brachial gland is composed of sebaceous glands that produce a brown greasy paste [5, 6].

Using these secretions, lemurs perform scent marking to advertise their social rank [7, 8] or reproductive state, as well as to demarcate territories or mediate intergroup spacing [9].

During the breeding season, male lemurs rub their wrists to mark their territory.

They rub their antebrachial and brachial scent glands on their own tails and then wave their tails to spread the glandular volatiles that threaten other males [10, 11].

With these behaviors, the male lemurs display their dominance status to potential rivals within the small multimale-multifemale groups.

Taken together, vola- tiles in glandular secretions seem to play a role in male-male communications among lemurs [12, 13].

However, male-female communications through the glandular secretions of lemurs had not yet been well examined.


Meanwhile, several studies have attempted to analyze the chemical components of lemur secretions at a molecular level.

Using gas chromatography-mass spectrometry (GC-MS) anal- ysis, long-chain acids and esters were found in the anogenital se- cretions of lemurs; the chemical profiles were distinguishable be- tween the sexes [14, 15].

The male brachial secretions have individual differences in their relative concentrations of compo- nents and, thus, are likely used for individual recognition [9, 16].

Scordato et al. [ 9] analyzed four glandular secretions (brachial, Current Biology 30, 2131-2138, June 8, 2020 ª 2020 Elsevier Inc. 2131 ll A B C D E F G H


To determine whether the degree of females' interest in male scent marks changes seasonally, the duration that females snif- fed male scent marks during the breeding season was compared with the duration of sniffing during the nonbreeding season at the Japan Monkey Centre (JMC).

We defined the breeding season af- ter the date on which estrous individuals were confirmed within the group.

In previous studies, November to February is the breeding season and March to October is the nonbreeding sea- son in the Northern Hemisphere [20, 21] and, in our cases, the breeding season began from late October to November.

Multi- male-multifemale (8 females and 4 males) groups are naturally reared in an outdoor enclosure at the center (Figure 1A), and focal male scent marks were observed.

Females sniffed male scent marks during the breeding season significantly longer than during the nonbreeding season (p = 0.038; Figures 1B and S1A).

This result indicates that female lemurs are more attracted to male scent gland secretion during the breeding season.


Next, secretions were collected, using a micropipette tip, from the antebrachial scent gland on the wrist of three male lemurs (Bon, Doitsu, and Afu) at the Research Institute of Evolutionary Biology at the Tokyo University of Agriculture (RIEB) and one male (Dan) at Yumemigasaki Zoological Park (YZP) (Figures 1C and 1D).

Se- cretions were collected twice per month from January 2013 to December 2014 and once per month from June 2018 to February 2019 (Figure 1E).

The volume of secretions tended to be larger dur- ing the breeding season (data not shown).

Each collected sample was used for GC-MS analysis and/or behavioral assay, as described in Figure 1E.

The smell of secretions collected during the breeding season was described as more fruity, floral, and sweet than those during the nonbreeding season, which were evaluated as being more bitter, leathery, animalic, and green by human senses.

Therefore, female lemurs may use this odor differ- ence as a signal to judge the timing of mating.


To examine the attractiveness of male secretions to females, the sniffing behavior of two female lemurs toward each of the male antebrachial samples (i.e., eight and six samples derived from the breeding and nonbreeding seasons, respectively, from Bon and Doitsu) was observed during the breeding season at the RIEB (Figures 1E-1G and S1B).

Before the behavioral assay, each female was isolated for approximately 1 h so she could habituate to the testing environment.

Then, the female was pre- sented with a cotton pad containing 1 mL of male secretions for 2 min; the duration of cotton pad sniffing was recorded (Figures 1F and 1G).

The total average time that the females sniffed the cotton pads that had secretions from the breeding season was significantly longer than that for the cotton pads with secretions from the nonbreeding season, or for the control cotton pads.

The sniffing frequency and the number of times the cotton pads were sniffed were variable among the samples, whereas the duration of each sniffing event was always longer for samples from the breeding season, which resulted in a longer total sniffing dura- tion.

After that, we presented cotton pads with secretions to two other females reared at the JMC, and the result was the same (Figure 1H).

These results suggest that female lemurs sniff male secretions derived from the breeding season longer than secretions from the nonbreeding season.


Next, an attempt was made to determine the chemical profiles of compounds in male antebrachial secretions that females were attracted to during the breeding season.

To identify the key com- pound(s), the profiles of volatile chemicals emitted from the male secretions obtained during the breeding and nonbreeding sea- sons were compared using GC-MS.

The GC-MS analyses demonstrated that among the many peaks that were present, 50 appeared in more than two analyzed Bon (age 9) samples; these 50 peaks were analyzed further (Table S1).


Next, the relative median peak area difference between breeding- and nonbreeding-season samples was determined.

In samples from Bon (age 9), the amounts of #21 (dodecanal), #26 (12-methyltridecanal), and #28 (tetradecanal) were higher during the breeding season than the nonbreeding season, whereas the amount of #24 (acetamide) was higher during the nonbreeding sea- son (Figures 2 and S2).

In samples from Doitsu (age 17), the amount of #26 was also higher during the breeding season, and #21 and #28 tended to be higher (Figure 2C; Table S2).

Samples from Afu (age 9) and Dan (age 23) also showed the same tendencies for #21, #26, and #28 (Figure 2C; Tables S3 and S4).

Moreover, principal- component analysis results for seven breeding-season and 10 nonbreeding-season samples from Bon showed that compounds #21, #24, #26, and #28 had a strong effect on the differences seen between the breeding and nonbreeding seasons (Figure S3).


The total ion chromatograms of Bon's samples are shown in Figure 2A.

Figure 2B shows the overlaid chromatograms of Bon's samples in which the chemical identities of #21, #24, #26, and #28 were confirmed using the retention times and mass spectra of standard samples.

The scents of #21, #26, and #28 were green, floral/fruity, and floral/green, respectively, according to the GC-MS olfactometry analysis and were Medians (bands inside boxes), quartiles (boxes), and 1.5 times the interquartile ranges (whiskers) and outliers (circles and scores beside arrows) are shown in the boxplots.

identical to those of standard samples (Figure 2B).

The scents of these compounds were also consistent with the breeding-sea- son samples evaluated by human senses.

Based on these re- sults, three C12 and C14 aldehydes are strong candidate com- pounds for female lemur attraction.


It was hypothesized that one cause of increased dodecanal, 12-methyltridecanal, and tetradecanal secretion was the higher testosterone levels reported in male lemurs during the breeding season.

To test this hypothesis, the serum testosterone level was manipulated by injecting testosterone intramuscularly into the 9-year-old male (Bon) from July to August, which corresponded to the nonbreeding season.

The testosterone level was higher during the breeding season in both the 9- and 17-year-old males (Bon and Doistu), as previously reported, although the level in the 17-year-old male was much lower than in the 9-year-old male, which was likely due to aging (Figure 3A).

The testosterone injection into the 9-year-old male during the nonbreeding season resulted in a higher serum testosterone level, and that level was similar to that in the 17-year-old male (Figure 3A).


GC-MS was used to quantify the amounts of dodecanal, A B C Report acetamide, 12-methyltridecanal, and tetradecanal in secre- tions at the indicated time after testosterone injection (Fig- ure 3A).

12-methyltridecanal and acetamide demonstrated a significant increase and decrease, respectively, in testos- terone-boosted samples (Figure 3B).

Tetradecanal had a nonsignificant tendency to increase.

No change for dodecanal was observed (Figure 3B).

These results are consistent with the observation that the amounts of 12-methyltridecanal in the 17-year-old male lemur were lower than those in the 9-year-old male (Figure 2C), which is likely due to a lower testosterone level (Figure 3A).


To determine the female sniffing behavior associated with the above candidate compounds, two female lemurs (the same indi- viduals as in Figures 1F and 1G) were presented with cotton pads that contained different amounts of dodecanal, 12-methyltride- canal, or tetradecanal for 2 min and the sniffing duration was measured at the RIEB.

No increase in sniffing duration was observed for dodecanal, whereas significant increases were observed for 12-methyltridecanal and tetradecanal at amounts greater than 490 and 300 ng, respectively (Figure 4A).

A measurement of 490 ng of 12-methyltridecanal is equivalent to 20-100 mL of breeding-season secretion.

These results suggest A B that 12-methyltridecanal and tetradeca- nal are key compounds in the male ante- brachial glandular secretions that elicit attractive behaviors among female lemurs during the breeding season.


Finally, the effect of the above candidate aldehyde com- pounds was examined in relatively more natural and social conditions in an indoor enclosure at the JMC (Figure 4B).

To determine whether females were interested in these alde- hydes, they were presented with cotton pads that contained different amounts of dodecanal, 12-methyltridecanal, or tetra- decanal for 5 min (Figure 4B).

In the breeding season, no in- crease in the total number of sniffing events per trial, or the to- tal sniffing duration per trial, was observed for each compound, whereas conspicuous increases were observed for the three-compound mixture (Figure 4B, upper panel).

Moreover, the duration of females' responses to any single compound was not different from their response to the control solution, whereas females sniffed significantly longer at mix- tures with concentrations of 3-10 ppm for 10 mL solutions, which is equivalent to a 3 to 30 mL breeding-season secretion.

On the other hand, females in the nonbreeding season were not interested in these compounds (Figure 4B, lower panel).

These results support the hypothesis that the identified C12 and C14 aldehydes are key compounds in male antebrachial ll Current Biology 30, 2131-2138, June 8, 2020 2135 Report glandular secretions that elicit attraction behaviors of female lemurs during the breeding season.


Despite the results of many studies indicating intraspecies chemical communications among primates, no research where the phero- mone-like molecules were identified had been published.

This was partially due to the complexity of volatile compounds contained in body odors or secretions and the difficulty of performing reliable behavioral experiments in primates.

This study used lemurs, with unique exocrine scent glands and a specific marking behavior, to successfully identify volatile long-chain aldehydes that increased A B during the breeding season in a testos- terone-dependent manner and appeared to be major male-specific odorants involved in female attraction during the lemur-breeding season.

It is conceivable that the identified C12 and C14 aldehydes are putative sex pheromones that aid male-female interactions among lemurs.


There have been several reports of GC- MS analyses of secreting glands in lemurs.

Hayes et al. [ 15] reported that anogenital gland secre- tions contained long-chain alcohols and esters that showed sex differences.

Scordato et al. [ 9] reported that brachial secre- tions were thick and viscous, containing squalene and choles- terol derivatives, whereas labial and scrotal secretions contained organic acids and esters.

They also determined that antebrachial secretions were highly volatile and contained fewer compounds than other glands [9].

Boulet et al. [ 14] reported 338 and 203 semiochemicals in labial and scrotal secretions, respectively.

None of these studies, however, reported the specific com- pounds that are involved in sexual communication.


Three C12 and C14 aldehydes identified in this study have not previously been detected in the secretions of lemurs or in any secreting ll 2136 Current Biology 30, 2131-2138, June 8, 2020 Report gland of other primate species (Mandrillus sphinx [22]; Callithrix jacchus [23]).

There are, however, some reports of these com- pounds being detected in animals other than primates.

Dodeca- nal, tetradecanal, and 12-methyltridecanal have been reported to exist in lamb wool [24] and were suggested to be involved in the recognition of newborn sheep by their mothers.

Tetradecanal is known as a sex pheromone in mantis and Helicoverpa assulta [25, 26].

Thus, although the identified aldehydes are novel find- ings in primate species, long-chain fatty aldehydes appear to be used as communication tools widely throughout the animal kingdom.


It has been reported that the volatile composition of male brachial gland secretions correlates with testosterone level [9, 20].

Consistently, testosterone administration in the 9-year-old male during the nonbreeding season resulted in an increase in the amount of the C12 and C14 aldehydes that were secreted.

We also observed that the volume of antebrachial gland secre- tions increased in both the breeding season and the testos- terone-effective period.

In contrast, the seasonal change in the amount of C12 and C14 aldehyde secretion from the 17-year- old male was small.


The reproductive age of ring-tailed lemurs starts at 2-4 years [27, 28].

The mean (±SD) lifespan of females was 4.9 ± 4.9 years and the longest was 20 years.

Male lemurs rarely live beyond 10 years in the wild, although the maximum lifespan in captivity has been recorded as 37 years among females and 35 years among males [29].

Therefore, the 9- and 17-year-old male subjects of this study could be stratified into prime adult and old age, respectively, and the testosterone- dependent change is a signal from young reproductive males toward females.


On the female side, young females are inter- ested in males' secretions during the breeding season, whereas old females that have passed their reproductive age equally snif- fed secretions during the breeding and nonbreeding seasons (data not shown).

Therefore, both young reproductive males and females appear to benefit from their mating choice, and sub- sequent reproductive behaviors demonstrated during the breeding season by males are associated with the production of testosterone-dependent C12 and C14 aldehydes, which females sense.


Can we say that the identified C12 and C14 aldehydes in lemurs are pheromones?

The classical definition of a pheromone is a sub- stance that is secreted by an individual and received by a second individual of the same species (conspecific) to induce a specific re- action, such as a stereotyped behavior or endocrinological change.

The identified C12 and C14 aldehydes seem to support this definition.

Future studies on behaviors or endocrine changes might help us to argue more explicitly about whether the identified C12 and C14 aldehydes in lemurs are sex pheromones.

Nonethe- less, the identification of key volatiles used in sexual communica- tions among lemurs introduces a way to better understand chem- ical communications among primates.


Detailed methods are provided in the online version of this paper and include the following:


Pheromones mediate a wide range of functions across the animal kingdom [1], and such chemosensory communication is especially widespread among mammals [2].


In a recent paper in Current Biology, Shirasu, Ito et al. [ 3] describe the results of a series of chemical and behavioral studies that identified three aldehyde odors released from the wrist gland of ring-tailed lemurs (Lemur catta) that could represent the first identified sex pheromones in male primates.


Observations of a captive group and controlled presentations of isolated male scent samples showed captive female lemurs sniffing antebrachial scent marks longer on average during the breeding season.


Comparison of the chemical profiles of antebrachial secretions between breeding- and non-breeding-season samples revealed three aldehydes putatively responsible for the female response, the concentration of one of these subsequently shown to increase following testosterone injection of one male.


Average sniffing duration of two females increased slightly with increasing concentrations of two of the three aldehydes in one experiment, and so did the response of seven other females to swabs with mixtures of the three compounds, compared to individually presented aldehydes.


From these results, the authors conclude that “it is conceivable that the identified C12 and C14 aldehydes are putative sex pheromones that aid male–female interactions among lemurs.”


Here, I argue that, in fact, more data are needed to determine whether antebrachial marking and these substances are actually involved in mediating the attractiveness of males to females during the breeding season.


My specific concerns pertain to several aspects of the methods that produce ambiguous results and conclusions that are too strong, especially when considering the broader context of lemur biology.


First, there are issues with reported methodology.

Subjects used to explore the response to male scents, for example, were not equally familiar with each other because the scent of one male was presented to females kept at another institution.

Thus, variation in female sniffing behavior may have (also) been due to variable familiarity with the scent donors [4].

It also remains unknown whether the samples from the (non-)breeding season and the unspecified control were presented simultaneously or sequentially, and if the latter, in which order.


Moreover, most data were derived from a single male, where repeated sampling revealed a difference in aldehyde concentrations between seasons.

These patterns were not replicated in three other males, however.

The same criticism applies to a testosterone challenge, which included only one male but no control.

Because samples from a single male were also used to assess the behavioral response of only two females in most behavioral assays, the general study design also raises substantial concerns about pseudoreplication.


Thus, the empirical basis for this study’s claims may be considered interesting, but preliminary.


These experiments raise more challenging questions still about methodology.

Importantly, it remains unknown whether longer duration or higher frequency of sniffing represent greater interest, motivation, unfamiliarity or animals’ challenges in sampling.

Are five sniffing events of two seconds conjointly equivalent to one investigation over ten seconds?

The authors chose duration as their measure because it was less variable than frequency; but there is no a priori reason for favoring one measure over the other.

Explicit statements of underlying assumptions and cautious interpretation are needed.


Second, claims that “this could be the first sex pheromone in primates” ignore results of earlier work and reflect a lack of familiarity with certain aspects of the mating system of ring-tailed lemurs.

It is possible that the authors wanted to emphasize the fact that their study might be the first to chemically identify a primate sex pheromone because experimental evidence for the olfactory communication of sexual status of female primates has been first presented more than 50 years ago [5].

While Darwin [6] considered “structures that produce sounds and odors with the sole purpose of attracting females” to represent examples of sexually selected traits, it has proven difficult to unambiguously demonstrate the function of putative mammalian male sex pheromones.

If the aldehydes identified by Shirasu, Ito et al. [ 3] do act as male sex pheromones, their putative function remains obscure.

Ring-tailed lemurs live in bisexual groups year-round; there is no need for males to attract females to them, like calling frogs, for example, and females are presumably familiar with all permanently co-resident males and their odors.

Further, these substances did not elicit immediate mating, and sniffing is not a response specific to these signals; hence, they had no releaser effect [1].

A potential priming effect, as has been demonstrated for several substances in male urine that accelerate female puberty and synchronize estrus in other mammals [7], for example, is unlikely, because female lemur reproductive physiology is under photoperiodic control.

Thus, the reported seasonal changes in the bouquets of male antebrachial secretions are most likely not used by the females “as a signal to judge the timing of mating” [3].


This leaves the possibility that the aldehydes advertise male identity and quality and subsequently bias female mate choice.

Females may evaluate males over months prior to mating [8], priming their later mate choice.

However, while there is a link between male odors from scrotal glands and genetic quality [9], female ring-tailed lemurs mate promiscuously with all males of their group during their one-day annual estrus after carefully choosing their first mates [8].


Also, as all males produce a mixture of similar compounds, males could only differ in amounts or ratios of chemicals produced; but the female response did not increase linearly across a >30-fold range of concentrations and was not consistent across experiments; the experiment with two females indicated that they sniffed more in response to higher concentrations of two individual components, whereas the group of seven females appeared to sniff more in response to lesser concentrations of the mixture (but not to any concentration of the individual components).


Finally, tail-waving is exhibited only by males, it occurs far more frequently among male dyads (‘stink fights’), and the top-ranking male exhibits this behavior most often.

Further, males’ antebrachial secretions rubbed on substrates are much more often inspected and overmarked by other males than by females [10].


The authors’ failure to test the response of males biased their study design from the start toward testing only one potential function of this signal.

Without similar study of male responses, it remains unknown whether the compounds identified by Shirasu, Ito et al. [ 3] have a specific function in male–female communication.


Following from all the foregoing, I find the conclusions presented in their paper suggestive at best.

This study should be replicated with larger samples that include male responses to these scents and controls for both familiarity and social relations, as well as for the multi-modal nature of this fascinating display.


We sincerely appreciate the constructive comments made by Peter Kappeler [1] regarding our paper, “Key male glandular odorants attracting female ring-tailed lemurs” [2].

We largely agree with the points raised in these comments, and believe these should be considered as critical discussion that would enable a more reasonable assessment of our findings.


With regard to the first point, as we have clearly indicated in our paper, at present, we cannot justifiably claim that the odors identified in lemurs are sex pheromones, given that we have yet to establish why the lemurs are attracted to these odors.

Although we have verified that females show greater sniffing behavior only during the breeding season, it is still unclear whether this behavior is associated with heightened sexual activity and results in a greater number of mating opportunities.

In this regard, it will be necessary for us to determine more precisely how the behavior of females changes as a consequence of sniffing.

It is also conceivable that these odors are utilized for male–male communication, given their possible testosterone dependency.

There are admittedly certain limitations to primate studies particularly with respect to increasing the number of animals and developing suitable methodologies to examine behavioral traits under natural conditions, and we readily acknowledge that our presumptions regarding the identity of the odors were somewhat premature.

Nevertheless, we are confident that further extensive studies will enable us to confirm whether the identified odors are pheromones.


With respect to the second point raised, we agree that chemical communication in non-human primates is well established.

In this regard, from the perspective of chemical structures, a mixture of short-chain fatty acids emitted from female vaginal secretions was reported to serve as a female pheromone in rhesus monkeys [3].

However, on the basis of studies of anosmic rhesus monkeys, other researchers subsequently refuted their results [4].

To the best of our knowledge, there have been no further reports regarding the key substances involved in chemical communication among primates.


Numerous behavioral studies on primates, including those investigating territorial and sexual behavior among conspecifics, have, nevertheless, provided compelling evidence to indicate that chemical communication is involved.

Thus, on the basis of our detailed chemical analyses and structural identification of key substances, we propose that these substances may be involved in chemical communication among non-human primates.


As Kappeler [1] correctly states, our findings regarding the nature of the odor we identified, and its putative role in lemur behavior, are essentially preliminary and that there are many questions left unanswered that will need to be addressed in the future.


We would, however, like to clarify some of the methodological points raised by Kappeler [1].


Firstly, regarding the issue of the variation in female sniffing behavior, the two male samples were obtained from the same institute, whereas the samples were sniffed by females in two different institutes and all the females showed longer sniffing duration.


Secondly, with respect to the sample presentation method, the samples were presented sequentially in a randomized order.


Thirdly, in our analysis of the amounts of aldehydes produced, we detected significant differences in 12-methyltridecanal in different seasons, and found that amounts increased in response to hormone treatment.


In contrast, the other two aldehydes merely showed tendencies in each of the animals analyzed.


We re-analyzed the data using the combined data obtained from two animals (Bon and Doitsu).


To minimize the effects of individuality, we standardized the peak area values (z values) for each male sample, and compared breeding and non-breeding samples by combining the data from two animals, and accordingly detected significant differences for each of the three compounds, namely, dodecanal (q = 0.032), 12-methyltridecanal (q = 0.032), and tetradecanal (q = 0.032).


Analyses were performed using the Brunner–Munzel test, and false discovery rates (q values) were calculated from the p values using the Benjamini–Hochberg procedure.


Finally, with regards to the control for testosterone, we agree that its use would be desirable.


Unfortunately, however, it is difficult to obtain permission for the use of this compound in our centers.


The main focus of this study, which we hope is appreciated as solid, is the chemistry part, which took several years, shedding a light on olfactory communication at the molecular level in a primate.


About 3%- 4% of fetuses have breech presentation at term.

1 In 2000, the Term Breech Trial (TBT) was published in The Lancet.

2 This study included 121 centers in 26 countries, and showed that planned ce- sarean sections were associated with less neonatal mortality and morbidity than planned vaginal births for breech- presenting fe- tuses at term.

The maternal outcome was equal for the two delivery methods.

This study resulted in a huge decrease in breech deliver- ies worldwide.


Six years later, a prospective observational study comparing planned vaginal and cesarean breech deliveries in France and Belgium found a low rate of neonatal mortality and morbid- ity in both groups.

This study concluded that it was safe to deliver selected breech- presenting fetuses vaginally, in modern, well- equipped obstetrical units at term.


After the TBT was published, there were discussions in Norway about whether the study could be applied to Norwegian obstetrical practice.

An expert group from the Norwegian center of Medical Method Evaluation reviewed the literature on delivery methods for breech- presenting fetuses.

The expert group concluded that both the selection process for breech deliveries and the experience of obstetricians in Scandinavia dif- fered from those described in the TBT.

The expert group concluded that vaginal delivery of breech- presenting fetuses could be advised for Norwegian women, provided that there was a rigorous selection process, careful birth monitoring and the presence of qualified clini- cians.

5 Authors of a Registry- based Cohort Study in Norway came to the same conclusion.


The frequency of vaginal breech deliveries in Norway decreased after the TBT, but has stabilized between 30% and 34% after 2010.

7 Induction of labor (IOL) in vertex position increased from 20% to 24% in Norway during our study period.

7 Induction is associated with increased rates of cesarean section in international retrospec- tive register studies, but not in randomized controlled trials.

8,9 It is unclear whether the high cesarean rates are due to induction itself or to confounding by indication.

10 IOL has become an accepted pro- cedure in breech presentations in Norway.

There is limited evidence, solely from case series, on the safety of breech induction.

11,12 This paper seeks to add to that body of evidence.

We aimed to compare the outcomes of spontaneous and induced breech deliveries and to describe variations in induction rates over time.


This was a retrospective cohort study, evaluating the management of labor and birth outcomes in breech presentations at Trondheim University Hospital (St. Olavs Hospital), Norway, from 1 January 2012 to 31 December 2019.

The obstetric ward had responsibil- ity for patients in the Central Norway Regional Health Authority, with approximately 3700 births each year.

Standardized, quality- controlled procedures were used to collect and record information relating to births and deliveries in an electronic birth journal.


Women with a single fetus in breech presentation were first recommended an external cephalic version, and if external cephalic version failed, a vaginal birth was recommended for women with un- complicated pregnancies.

The selection criteria for vaginal breech deliveries at the hospital all through the study period included ges- tational age of more than 34 weeks, estimated birthweight between 2000 and 4500 g, and complete or frank breech presentation.

Fetal growth restriction was not a criterion for exclusion.

Cesarean sec- tion was not recommended at the hospital before pregnancy week 25.

Between pregnancy weeks 25 and 34 a vaginal birth was allowed in women with their cervix fully dilated and the presenting part of the fetus at a low station at admission.


Pelvimetry was performed using computer tomography in women who had not given birth vaginally previously and with a planned vag- inal breech delivery after 37 weeks of gestation.

The local guidelines for pelvimetry criteria follow the Norwegian guidelines for breech deliveries.

The following measurements are suggested when pelvim- etry is used to determine if vaginal breech delivery is an option (pelv- imetry is not mandatory): the pelvic inlet sagittal diameter should be larger than 11 cm and the pelvic outlet, measured as the sum of the interspinous distance, the intertuberous diameter, and the sagittal pelvic outlet diameter, should be larger than 31.5 cm.


The planned delivery route was thoroughly discussed with the woman at the outpatient clinic during the third trimester.

In the case of an unknown breech presentation at admittance, the mode of de- livery was determined by the doctor on call after discussion with the woman, following the same criteria.

Pelvimetry was not performed in women arriving late in labor.


Induction of labor was an approved option in the hospital guide- lines, and the indications for induction were similar for cephalic and breech presentations.

The induction procedure started with a bal- loon catheter, misoprostol or dinoprostone administered vaginally in women with an unripe cervix.

In women with a ripe cervix the recommendation for cephalic induction was amniotomy, followed by oxytocin infusion.

However, amniotomy was usually avoided as a start procedure in breech inductions because of the risk of umbilical cord prolapse.


Intrapartum cesarean section rates were similar in induced and spontaneous breech deliveries.

There was no signifi- cant difference in metabolic acidosis nor in pH <7.0 in the umbilical artery when comparing the two groups.

The main outcome was intrapartum cesarean section (ICS), and secondary outcomes were postpartum hemorrhage, sphincter rup- tures, Apgar scores, pH in the umbilical artery, and metabolic aci- dosis.

Metabolic acidosis was defined as pH <7.0 combined with a base excess lower than −12.

All data were obtained from the hospital birth journal, Natus (CSAM).


We performed an a priori power analysis based on an estimate of 30% ICS in spontaneous breech deliveries and 45% ICS in induced breech deliveries and an induction rate of 20% in planned vaginal births.

Using a chi- squared test, α = 0.05 and power = 0.80, 515 women were needed in the study (412 spontaneous labors and 103 induced labors).

Categorical variables were compared using a chi- squared test or Fisher's exact test and continuous variables were compared with a Mann- Whitney U test.

Variations over time were analyzed with linear- by- linear association.

The association between ICS and IOL was analyzed using logistic regression and adjusted for potential confounders such as parity, maternal age, body mass index, ges- tational age, and ethnicity.

Duration of labor in spontaneous and induced labors was analyzed with survival methods and compared with a log- rank test.

Cesarean sections and labors with duration >15 hours were censored.


Data were analyzed with the statistical software package SPSS statistics version 26.0. ( IBM SPSS) and P values <.05 were consid- ered statistically significant.


The study was approved by the Regional Committees for Medical and Health Research Ethics on 12 September 2019 (2019/7190) and approved by the health authority at St. Olavs Hospital on 12 December 2019.


In all, 30 769 women delivered at Trondheim University Hospital during the study period.

The number of annual deliveries varied from 3556 to 4021.

The frequency of preterm deliveries (before pregnancy week 37) was 4.7% in women with a single fetus in ce- phalic presentation.

The mean birthweight was 3535 g and 1.6% of neonates had an Apgar score of <7 at 5 minutes, 3.2% had pH <7.10, 1.3% had pH <7.05, and 0.5% had pH <7.0 in the umbilical artery.


Of the women, 1061 had a singleton fetus in breech presen- tation (3.5% of the total births).

We excluded seven women with intrauterine fetal death before the start of the labor, leaving 1054 women in the study population.

In all, 210/1054 (20%) women de- livered before 37 weeks of gestation.

Figure 1 presents a flow chart of the study population.


A vaginal birth was planned in 606 (57.5%) women and cesarean section was planned in 448 (42.5%) women; a further 350 of the 448 neonates were delivered by prelabor cesar- ean section.

Four women with a planned cesarean section delivered vaginally.

Because the labor started before the scheduled date, 94 neonates were delivered with ICS.

In all, 22 women delivered vag- inally before pregnancy week 34, of whom 14 were in week 23 or 24.


Characteristics of the study population are presented in Table 1.

Variations in the frequency of overall cesarean sections, planned vaginal births, successful vaginal births, and frequency of induced labors are shown in Figure 2 for nulliparous women and in Figure 3 for parous women.


We did not observe any significant change in induction rates during the study period (P = .10 and P = .55) in nulliparous women and parous women, respectively.

Table 2 compares the outcome of planned cesarean section and planned vaginal delivery.


Induction of labor was performed in 127/606 (21.0%) women with planned vaginal birth.

The indications for induction were pro- longed prelabor rupture of membranes (21/127), postterm pregnan- cies (17), oligohydramnios (13), hypertensive disorders (11), diabetes (4), growth restriction (6), large fetuses (4), maternal request (3), or a combination of indications (48).

The induction started with the use of a balloon catheter in 70 cases, vaginal misoprostol in 46 cases, di- noprostone in seven cases, oxytocin in three cases, and amniotomy in one case.


We found no significant difference in the frequency of ICS when comparing induced and spontaneous deliveries: 48.0% vs 45.7%, re- spectively (P = .64).

Table 3 compares the outcomes of labor and neonates in spontaneous and induced labors.

Logistic regression analyses are presented in Table 4.

Unadjusted and adjusted odd ratios for an ICS in women with induced labors were 1.08 (95% CI 0.73- 1.60) and 1.65 (95% CI 0.92- 2.94), respectively.

The median birthweight was significantly higher in induced labors than in labors with spontaneous onset, 3452 g vs 3120 g, respectively (P < .01).

The median length of the pregnancy was significantly longer in in- duced labors than in labors with spontaneous onset with gestational age of 283 days vs 274 days, respectively (P < .01).


Three fetuses with planned vaginal birth died during labor, two after spontaneous onset of labor and one after induced labor.

All three were extremely preterm, two in pregnancy week 23 and one in week 25.

The fetus in pregnancy week 25 had spontaneous onset of labor and was delivered by emergency ICS.

Birthweight was 575 g, but the baby was stillborn.


Arterial pH was measured in 489/606 (81%) of the planned vagi- nal deliveries.

The median pH in the umbilical artery was significantly lower in neonates with induced labors compared with neonates with spontaneous labors (7.22 vs 7.25; P = .02), the frequency of pH <7.05 was 7.0% vs 2.9% (P = .05) in the two groups.

The frequency of pH <7.0 was not significantly different between neonates with induced and spontaneous labors; 2.6% vs 0.8% (P = .14), respectively.


Three neonates with planned vaginal birth had metabolic acidosis.

In the first case, labor started spontaneously in pregnancy week 41 and the woman delivered vaginally.

This child was treated with hypothermia, had normal magnetic resonance imaging of the brain after 5 days and was without any sequelae at the age of 3 years.

The second neo- nate was delivered in week 32, after spontaneous onset of labor, followed by ICS because of preterm labor and bleeding.

In the third case, labor was induced in pregnancy week 38.

The neonate was delivered with ICS because of fetal distress.

We have not received


In nulliparous women with planned vaginal birth, the estimated median duration of the active phase of labor was 547 minutes (95% CI 466- 628 minutes) in induced labors vs 478 minutes (95% CI 434- 522 minutes) in spontaneous labors (log- rank test; P = .44).

In parous women with planned vaginal birth, the estimated median duration was 214 minutes (95% CI 49- 379 minutes) in induced labors and 264 minutes (95% CI 205- 323 minutes) in spontaneous labors (log- rank test; P = .89).


When comparing induced and spontaneous breech deliveries, we found no difference in the frequency of ICS.

There was no signifi- cant difference in the frequency of pH <7.0 and the frequency of metabolic acidosis was <1% in both groups.

Intrapartum deaths oc- curred only in extremely preterm cases.


The TBT led to a change in the practice of delivering term breech fetuses towards more planned cesarean sections in many coun- tries.

3 A study from Ireland found a significant increase in planned cesarean section in nulliparous and parous women when compar- ing the time periods before and after the TBT.

13 In Norway, 57% of breech- presenting fetuses were delivered by cesarean section from 1991 to 2000.

The cesarean section rate increased to 67.6% during 2001- 2010 and was stable at 67.8% during 2011- 2018.


A few publications have studied the safety of induction of breech deliveries.

A study from France and Belgium compared induction of breech labor with planned cesarean section.

They found no signifi- cant differences in maternal or fetal outcome, and supported IOL as an alternative for breech- presenting fetuses.

12 We found no difference in the ICS rate when comparing sponta- neous and induced labors.

This is different from what was found in a systematic review and meta- analysis of seven studies from 2018, 11 which found that the cesarean section rate increased when labor was induced.

The neonatal outcomes were similar in both groups.


We observed a significantly higher median birthweight in in- duced labors than in spontaneous labors, probably because of higher gestational age.

The Apgar score was significantly higher in neonates with planned cesarean section compared with neonates with planned vaginal birth.

This is in accordance with international studies as well as other Norwegian studies.

14- 17 The median pH in the umbilical artery was significantly lower in induced labors com- pared with labors with spontaneous onset.

The meta- analysis from 2018 did not find any statistically significant differences in pH in the umbilical artery when comparing induced and spontaneous la- bors.

11 A Dutch population- based study found increased perinatal death when comparing induced breech deliveries with spontaneous breech deliveries.

17 We observed three stillbirths in the group with planned vaginal birth.

Two of these births were spontaneous, one was induced.

All three fetuses were extremely premature.

We be- lieve that neither the selection of a planned vaginal birth nor the choice of induction had any consequence for their outcome.


National guidelines have different recommendations for IOL in breech deliveries.

The Royal College of Obstetricians and Gynaecologists guidelines state that women should be informed that IOL is normally not recommended.

18 The Canadian guidelines of 2009 did not recommend IOL because of a paucity of evidence.


The 2019 Canadian guidelines list the limited evidence and recom- mend IOL as an acceptable option.

19 In France, breech presentation at term is not a contraindication to IOL.

20 In view of the worldwide increase in cesarean section rates for breech deliveries, it is important to consider the possible con- sequences that the first cesarean section may have for subse- quent labors.

According to a meta- analysis from 2018, subsequent labors following a cesarean section will show an increased risk of hysterectomy, abnormal placentation, uterine rupture, stillbirth, and premature birth.

21 Another result of the increasing trend towards planned cesarean sections in breech deliveries is the potential lack of training in vagi- nal delivery for obstetrical staff.

Around 20% of breech presentations were undiagnosed at admission, 22 some leading to difficult abdominal deliveries.

23 The American College of Obstetricians and Gynecologists notes the decreasing number of practitioners with the skills and ex- perience to perform vaginal breech delivery.

24 Obstetrical skills to manage vaginal breech delivery should be as widespread as breech presentation.


Suboptimal antenatal care and/or management of in- hospital deliveries is more common in deliveries with intrapartum or neonatal death than in deliveries where the offspring survives.

Multiple methods have been tried in the search for improved birth outcomes for breech- presenting fetuses.

A retrospective co- hort study published in 2017 compared vaginal delivery in the dor- sal position with vaginal delivery in an upright position.

The paper concluded that upright vaginal breech delivery was associated with reductions in maternal and neonatal injuries, the cesarean rate, du- ration of the second stage of labor, and the number of maneuvers required. 27 This method seems promising, and has the potential to rekindle vaginal breech deliveries.

Especially in low- resource coun- tries, it is important to avoid unnecessary operative interventions.


The main strength of our study is a large study population in a hospital where all clinicians on call have maintained skills in vaginal breech deliveries.

To the best of our knowledge, this is also the first Norwegian study to focus on induction of breech presentations.


The most important limitation is that we do not have long- term follow- up results for the cohort.

Other limitations are a retrospec- tive design and missing umbilical cord samplings in around 20% of the fetuses studied.

Pelvimetry was routinely performed after preg- nancy week 37 in nulliparous women with a planned vaginal birth, but unfortunately we lack information about the frequency of pel- vimetry in women with unknown breech presentation at admission.


Another limitation is that data on transfer of neonates to the neona- tal intensive care unit are lacking.


When comparing the outcome of spontaneous and induced breech deliveries, we found no significant difference in the frequency of ICS.

Further, we did not find any significant differences in pH <7.0 or in metabolic acidosis between the two groups.

Lastly, we did not observe any significant change in induction rates over time in either nulliparous or parous women.


Guidance of women with breech presentation is a subject of continuous discussion.

Welle-Strand et al [1] concluded that there was no significant difference for cesarean section rates or umbilical artery pH lower than 7.0 when comparing induction of breech with spontaneous delivery in their Norwegian cohort.

However, the difference in pH lower than 7.0, which was 2.6% and 0.8% respectively, combined with a significant difference in pH measured as a continuous variable clearly shows that the induced cohort had a clinically significant lower pH. It is expected that induction could lead to stronger contractions and therefore to lower pH or even to increased risk of head entrapment.

When there is biological plausibility and other outcomes support the result, it has to be taken into account when interpreting the results.


Regarding cesarean section, the adjusted analyses showed a 65% increased risk in the women who underwent induction even though confidence intervals covered no effect.

As increased risk of cesarean section has also been found in other studies [2], it seems too simplified to conclude that “no significant effect was found.

” A recent review found increased risk of admission to neonatal intensive care units [2] in a cohort of only 700 induced women.

Hence, there is considerable uncertainty of how induction affects outcomes.


Of real concern is that the rate of Apgar score below 7 after 5 minutes was 11% in the planned vaginal group compared with only 3% in the planned cesarean section group in the current study.

The rate of pH below 7.05 was 7% in women with induced breech delivery.

This seems extremely high as we would expect that the women offered induction were carefully screened for pregnancy complications associated with intrauterine distress.

As stated by the authors, randomized controlled trials [3] have shown increased risk associated with planned vaginal breech delivery compared with cesarean section.

The study was heavily criticized and followed by observational studies arguing that the risks could be eliminated through selection.

However, a meta-analysis of observational studies has confirmed that vaginal delivery is riskier in a “real-life” set-up [4].


A three times increase in the rate of Apgar lower than 7 after 5 minutes was also found in a Belgian/French cohort study that supposedly showed the safety of breech deliveries [5].

The real question is how many mothers would choose a vaginal delivery if they knew.


We would like to thank Schroll and Krebs for their comments on our manuscript [1].

Delivery mode in breech-presenting fetuses will continue to be discussed.

An expert group from the Norwegian Center of Medical Method Evaluation reviewed the literature in 2003 and concluded that vaginal delivery could be advised provided there is a qualified selection, careful birth monitoring and the presence of qualified personnel [2].


We presented results from a large Norwegian hospital in 2012-2019 and we do agree that 11% of newborns with 5-minute Apgar score <7 in women with planned vaginal birth is a high frequency.

The duration of the expulsive phase is longer in vaginal breech deliveries than in cephalic presenting fetuses.

It is recommended that the birth attendant should maintain a hands-off position until the fetal umbilicus is visible and then deliver the baby with active maneuvers [3].


Planned vaginal breech birth implies an increased risk of short-term fetal morbidity, low 5-minute Apgar score, and a low umbilical arterial pH compared with planned cesarean section [4].

We observed 5-minute pH <7.05 in 3.9% of planned vaginal births but metabolic acidosis in only three newborns (0.6%) [5].

This means that the majority with low pH had a respiratory acidosis with a low risk of long-term adverse outcome.


The association between induced cephalic presentations and cesarean sections is discussed.

In register-based studies, the frequency of cesarean sections is higher in induced labors, but this is not confirmed in randomized studies, probably due to confounding by indications [6].

We did a power analysis before investigating our population and supposed intrapartum cesarean section rates would be 30% in spontaneous labors vs 45% in induced labors.

The actual figures were 46% and 48%, respectively, and the difference was not significant after adjusting for confounders.

The most important newborn outcome is the frequency of metabolic acidosis, which occurred in only one of 127 newborns among induced labors and in two of 479 in spontaneous labors [5].


Our objective was not to discuss whether vaginal breech deliveries still should be recommended, but to compare spontaneous and induced breech deliveries.

We think that induction of labor is an acceptable procedure in labor wards practicing vaginal breech deliveries.

This is in accordance with recommendations in the recently published NFOG textbook for medical students, which states that spontaneous onset of labor is optimal, but induction of labor for selected patients may be feasible [3].


A literature search was carried out using Medline (1946-January 2014), Embase (1974-January 2014) and the Cochrane Library.

The research question was, 'What is the evidence for the use of specific therapeu- tic interventions in the management of perioperative pain in procedures that have a high risk of chronic pain and whether these interventions reduce chronic post-surgical pain?' We used a combination of key- words and subject headings.

The subjects of interest included amputation, mastectomy, thoracotomy, peri- operative, pain, neuropathic, chronic pain, pre- emptive, epidural, regional and the relevant drug names and classes.

All subject headings were exam- ined for relevant terms that are related to the types of operation performed and the types of intervention.

Articles were limited to randomized controlled trials (RCTs) in adult human participants.

Additional studies were identified by manually tracking references from published papers.

On some occasions, where a manu- script was unavailable, the authors were contacted directly.


Studies were also excluded in which subjects were recruited after the perioperative period and those with follow-up periods of less than 2 months were excluded.

Studies with high risk of bias as assessed by the Cochrane Risk of Bias Tool (Higgins and Green, 2011) were excluded.

Additional studies were identi- fied by manually tracking references from published papers.

On some occasions, where a manuscript was unavailable, the authors were contacted directly.


All of the trials were reviewed independently by each of the authors and consensus was reached as to the inclusion and interpretation of each individual trial.

This review focused only on surgical procedures associated with a high incidence of chronic pain: amputation, mastectomy and thoracotomy.

Studies investigating acute pain and chronic pain were ana- lysed, whereas studies in which subjects were recruited after the perioperative week and those with follow-up periods of less than 2 months were excluded.

Once all of the trials were collated and ana- lysed, an assessment was made of the potential to perform a meta-analysis of the quantitative efficacy of specific interventions.

• Appropriate dose regimes of gabapentinoids, antidepressants, local anaesthetics and regional anaesthesia may potentially reduce pain.


• Ketamine and intercostal cryoanalgesia were not effective at reducing chronic pain.


• Total intravenous anaesthesia may be beneficial.


The combined literature search identified 598 records and a further 6 additional records were identified through the manual tracking of references.

After the duplicates were removed, 331 records were screened, and of these, 252 excluded as being clearly irrelevant.

A total of 79 records were therefore assessed for eligi- bility and of these 47 were excluded (the most common reasons were as follows: not RCT, inappro- priate study design, high risk of bias, inadequate size, not related to pain and/or chronic pain not assessed).

Thirty-two trials met the eligibility criteria and were included in the qualitative analysis.


Of the 32 trials, 5 (16%) studied amputation, 17 (53%) studied thoracotomy and 10 (31%) studied mastectomy.

Details of all studies are summarized in Tables 1 and 2.

The mean sample size was 89 (±12 SD) subjects (range 36-343).

The mean duration of patient follow-up in the trials was 7 (±1 SD) months following the surgical procedure (range 2-15).

Four of the trials had more than two study arms and (reflecting the complexity of clinical practice) 13 trials incorporated multiple interventions within the same study.

The outcome measures varied between studies; the visual analogue scale (VAS; 0-100 mm) was used in 22 trials; a version of the numerical rating scale (NRS; 0-10) was used in 20 trials; and the verbal rating score (0-3 = nil/mild/moderate/severe) was used in four trials.

Pain at rest and movement was assessed in nine trials and quantitative sensory testing was used in eight trials.

A multidimensional score such as the McGill Pain Questionnaire was used in nine trials.


Five of the trials studied the impact of the gabapenti- noids and the dose regimen and duration of therapy was different in every trial (Fassoulaki et al., 2002, 2005; Nikolajsen et al., 2006; Amr and Yousef, 2010; Kinney et al., 2012).

One trial incorporated the anti- depressant venlafaxine (Amr and Yousef, 2010).


Six trials studied the impact of ketamine; two consisted of intravenous ketamine without epidural analgesia (Hayes et al., 2004; Dualé et al., 2009), two consisted of intravenous ketamine in addition to epidural anal- gesia (Joseph et al., 2012; Mendola et al., 2012) and two examined the effect of epidural ketamine (Wilson et al., 2008; Ryu et al., 2011).


Six trials studied the impact of cryoanalgesia in thoracotomy.

Of these, two trials compared cryoanalgesia against control treat- ment with intravenous opioids (Gwak et al., 2004; Sepsas et al., 2013), one trial compared cryoanalgesia to epidural analgesia (Ju et al., 2008), two trials com- pared the effect of cryoanalgesia versus placebo in thoracotomy patients who all had epidural analgesia (Yang et al., 2004; Mustola et al., 2011) and one trial compared cryoanalgesia to non-divided intercostal muscle flap (Lu et al., 2013).


Six trials examined the impact of local anaesthetic; of these, three incorpo- rated infiltration with differing doses of ropivacaine in mastectomy (Albi-Feldzer et al., 2013; Baudry et al., 2008; Vigneau et al., 2011), one trial studied the impact of lidocaine infiltration in thoracotomy patients that already had a thoracic epidural in situ (Cerfolio et al., 2003), one trial examined the effect of intravenous lidocaine (Grigoras et al., 2012) and the remaining one assessed the impact of topical EMLA (eutectic mixture of local anaesthetic) cream in mas- tectomy patients (Fassoulaki et al., 2000).


The impact of regional analgesia was studied in eight trials (two amputation, two mastectomy and four thoracotomy).

Three trials examined whether commencing an epi- dural prior to thoracotomy was better than commenc- ing it at the end of the procedure (Obata et al., 1999; Ochroch et al., 2002; Senturk et al., 2002).

One trial compared pre-surgical epidural and low-dose intraop- erative remifentanil infusion versus post-surgical epi- dural and high-dose intraoperative remifentanil infusion (Salengros et al., 2010).

One trial compared epidural analgesia to epidural saline plus standard management of thoracotomy pain (Nikolajsen et al., 1997).

Two trials examined the impact of paraverte- bral block for mastectomy patients (Kairaluoma et al., 2006; Ibarra et al., 2011).

One trial had five study arms that studied epidural therapy, fentanyl patient- controlled analgesia (PCA) and standard therapy before, during and after amputation (Karanikolas et al., 2011).

A single trial examined the impact of total intravenous anaesthesia (TIVA) versus inhala- tional anaesthesia in thoracotomy patients with tho- racic epidurals in situ (Song et al., 2012).


In patients after mastectomy, gabapentinoids reduced acute pain in three trials (Fassoulaki et al., 2002, 2005; Amr and Yousef, 2010).

Gabapentinoids also reduced the overall incidence of chronic pain in one (Fassoulaki et al., 2002) and the incidence of chronic burning pain in another (Fassoulaki et al., 2005).

There was no difference in NSAID use between groups during the first week.

Gabapentin did not reduce acute pain in two trials where epidural analgesia was used for amputation or thoracotomy, respectively (Nikolajsen et al., 2006; Kinney et al., 2012).

In the former, therapeutic doses of gabapentin were used, but the patients had chronic pain preoperatively secondary to vascular disease.

In the latter, only a single dose of gabapentin was given perioperatively.

The antidepressant venlafaxine (37.5 mg) was associated with significantly lower post-mastectomy pain after 7 days, and after 6 months, the incidence of post-mastectomy burning and stabbing pain was significantly lower (Amr and Yousef, 2010).


Seven trials investigated the acute and chronic impact of regional anaesthesia including whether it could have a beneficial pre-emptive effect.

One study found that although epidural analgesia was effective in the acute perioperative setting, it did not reduce chronic phantom limb pain in patients who had chronic pain preoperatively secondary to vascular disease (Nikolajsen et al., 1997).

In contrast, two trials found that epidural analgesia reduced acute and chronic pain versus control therapy after thoracotomy and ampu- tation, respectively (Senturk et al., 2002; Karanikolas et al., 2011).

Four trials examined whether the timing of epidural analgesia could impact on acute and chronic pain (Obata et al., 1999; Ochroch et al., 2002; Senturk et al., 2002; Karanikolas et al., 2011).

One of these trials found that it made no difference to acute or chronic pain whether the epidural was commenced immediately before or after the thoracotomy proce- dure (Ochroch et al., 2002).

In contrast, the remaining three trials found that the optimization of analgesia preoperatively had a beneficial impact on both acute and chronic pain (Obata et al., 1999; Senturk et al., 2002; Karanikolas et al., 2011).


Two trials of mastec- tomy patients investigated preoperative paravertebral block.

The first trial found that paravertebral block significantly reduced acute pain and chronic pain 1 year later compared with sham block (Kairaluoma et al., 2006).

The second trial found that paravertebral block had no significant impact on acute pain, but reduced the prevalence of chronic pain, compared with control (Ibarra et al., 2011).

One trial compared preoperative epidural commencement with low-dose intravenous remifentanil infusion versus post- operative epidural commencement with a high-dose intravenous remifentanil infusion for thoracotomy patients (Salengros et al., 2010).

They found no differ- ence in acute pain, but high-dose remifentanil with a post-operative epidural was associated with a higher incidence of chronic pain.

In addition, they found that the surface area of chest wall allodynia was signifi- cantly larger for the high-dose remifentanil group during the first post-operative month.


A single large trial of thoracotomy patients receiving thoracic epidurals compared TIVA with propofol and remifentanil against inhalational anaesthesia with sevoflurane (Song et al., 2012).

There was no differ- ence in acute pain between the two groups, but the incidence of chronic pain was significantly reduced at 3- and 6-month assessments in the TIVA group.


Gabapentinoids, antidepressants, local anaesthetics and regional anaesthesia have the potential to reduce the severity of both acute and chronic pain for patients undergoing amputation, thoracotomy or mastectomy.

Ketamine and cryoanalgesia do not appear to be promising agents for reducing chronic pain.

TIVA with propofol may have the potential to reduce the inci- dence of chronic pain, whereas the effect of opioids such as remifentanil is unclear.


This systematic review was limited to amputation, mastectomy and thoracotomy because these opera- tions are considered to be associated with a combina- tion of nociceptive and neuropathic symptoms and carry a high risk of chronic pain.

If it is accepted that the severity of acute pain is correlated with the risk of developing chronic pain, then it follows that the peri- operative period is a logical target for interventions aimed at reducing or preventing the phenomenon.

The positive and the negative results of this review are both important to guide future clinical practice.

Spe- cifically, it is useful to note the ineffective or harmful therapies so that they may be avoided and further trials of them may also be avoided.


The trials included in this systematic review were of varying quality, but addressed the majority of conven- tional methodological standards.

However, the hetero- geneity of the trial methodology and outcome measures ruled out the performance of a meaningful meta-analysis.


Despite the lack of consistency and comparability between trials, several consistent themes emerge in the results.

Acute pain in the peri- operative period after amputation, mastectomy and thoracotomy appears to be a mix of nociceptive and neuropathic pain.

The acute neuropathic component is potentially responsive to agents that work on neu- ropathic symptoms, but not nociceptive symptoms.

The acute neuropathic component may be relatively resistant to conventional analgesics, but is potentially published by John Wiley & Sons Ltd on behalf of treatable, and if the correct intervention is utilized in the correct manner (dose/duration/timing), it has the potential to reduce the development of chronic pain.

A number of trials included in this review studied a single, brief intervention, which was typically not enough to achieve long-term benefits, potentially due to the fact that the process of neuroplasticity that can continue for days or weeks post-operatively.

The days following an operation appear to be as important as the day of the operation itself, i.e., the potential ben- efits of a pain-free initial 12 h could be made obsolete if the pain is severe thereafter.

Furthermore, neuro- pathic symptoms may take several days to manifest post-operatively, thus emphasizing the importance of recognition and referral to a vigilant and cognizant acute pain service.


There were mixed results for the gabapentinoids; these agents reduced acute post-operative pain unless an effective epidural was present, in which case no sig- nificant difference was observed.

Within the trials, gabapentinoids were often given once only or at sub- therapeutic doses.

However, if an appropriate thera- peutic dose was administered for a longer period of time, the efficacy appeared greater.

This is in keeping with other studies of acute post-mastectomy pain reported in the literature (Dirks et al., 2002; Grover et al., 2009; Kim et al., 2011).


Of note, vascular patients with chronic pain preoperatively continued to have chronic pain post-operatively despite gabapen- tin.

Separately, the antidepressant venlafaxine was associated with enhanced analgesia 1 week after mas- tectomy and after 6 months despite being adminis- tered at the potentially sub-therapeutic dose of 37.5 mg.


Interpretation of the trials examining the potential benefits of regional analgesia as a single group was challenging due to the inherent heterogeneity of trial methodology.


Effective regional techniques in the hands of skilled clinicians can ablate the peak noxious barrage (Bonhomme et al., 2006; Wennervirta et al., 2008) of pain in the perioperative period and poten- tially minimize pathological neural plasticity impli- cated in the genesis of chronic pain (Woolf and Salter, 2000; Scholz and Woolf, 2007; Harvey and Dickenson, 2008; Ivani et al., 2008).


Unsurprisingly, the majority of the trials included in this review found regional analgesia to be beneficial for reducing perioperative pain and subsequent chronic symptoms.


The single trial that found epidural analgesia to be no better than standard analgesia comprised vascular patients with pre-existing chronic pain prior to amputation.


It is perhaps unsurprising that a few days of epidural anal- gesia did not impact on the chronic symptomatology of these vasculopaths.


Several trials examined the timing of regional intervention.


In particular, the complex trial by Karanikolas et al. ( 2011) indicated the benefits of early epidural insertion and continued infusion for the entire perioperative period.


However, the same trial also found that if patients were managed aggressively and effectively with systemic opioids, an acceptable outcome could also be achieved.


Early commencement of regional analgesia may offer some benefits for post-operative pain and may have other advantages such as facilitation of balanced anaesthe- sia, minimization of sympathetic stress on the cardio- vascular system and potential reduction of blood loss.


They are also particularly useful in patients who are opioid tolerant.


Optimal duration of regional analgesia is yet to be determined and potential benefits may be lost if epidural infusions are discontinued too early for logistical reasons.


Infiltration of local anaesthetic could provide a brief period of analgesia, but did not impact on the risk of developing chronic pain.

In contrast, intraoperative intravenous lidocaine and perioperative EMLA cream reduced the incidence of chronic pain following mas- tectomy, without making a significant impact on acute pain.


Remifentanil infusions have become a popular com- ponent of balanced anaesthesia for a multitude of sur- gical procedures.

However, the theoretical concept of opioid-induced hyperalgesia appeared to be confirmed in a relevant clinical setting in the trial by Salengros et al. ( 2010).

Indeed, high-dose intraoperative remifentanil was also associated with an increased risk of chronic post-surgical pain.


In contrast, TIVA with propofol and remifentanil was associated with a reduced incidence of chronic post-thoracotomy pain than inhalational anaesthesia with sevoflurane (Song et al., 2012).

Given the phenomenon of opioid- induced hyperalgesia (Ballantyne and Mao, 2003; Angst and Clark, 2006; Raghavan et al., 2011), the potential benefit of TIVA is more likely to be propofol- mediated, potentially via glutamate, GABAA and glycine receptors (Song et al., 2012).


While ketamine is an effective co-analgesic agent for acute pain and has the advantage that it may be used by patients in whom the oral route is unavailable, it did not reduce the risk of chronic post-surgical pain and its use in general is limited by dysphoric side effects.

Ketamine also offered no additional benefit in the presence of effective epidural analgesia.

Of even greater concern is intercostal nerve cryoanalgesia, which could arguably be renamed as 'cryoneuralgia' and perhaps be considered as an adjunctive therapy only in the setting of palliative care.


Current mainstream perioperative management for amputation, thoracotomy and mastectomy is highly variable, but the choices that anaesthetists make may have long-term implications for patients under their care.

Standard practices that rely primarily on opioids and NSAIDs for perioperative analgesia may be inad- equate for patients at risk of neuropathic pain.

Sepa- rately, techniques such as cryoanalgesia should be avoided.

Gabapentinoids and antidepressants (which are not considered to be effective for nociceptive symptoms), as well as local anaesthetics and regional anaesthesia, may potentially reduce the severity of both acute and chronic pain for patients undergoing operations associated with a high risk of chronic post- surgical pain.

However, some pharmacological agents may take a number of hours to be of benefit and therefore therapeutic dosages should be used for several weeks in many instances.


There have been promising results for the use of gabapentinoids in other conditions (Brogly et al., 2008; Buvanendran et al., 2010; Gray et al., 2011) but anti-neuropathic pain drugs have not been proven to be beneficial for surgical interventions that are pre- dominantly associated with nociceptive pain such as minor gynaecological procedures.


Cases of neuro- pathic pain may be discovered using simple screening tools routinely, e.g., DN4 or LANSS (Bennett, 2001; Bouhassira et al., 2005), and by identifying those who are experiencing a disproportionate level of pain.


Appropriate interventions at this stage may provide superior analgesia and facilitate optimum recovery.


Not all cases of chronic post-surgical neuropathic pain may be prevented, but the risk may be reduced.


This may decrease the burden on society and the amount of financial resources spent on the management of chronic pain syndromes.


Future trials in this field should focus on conditions with a high risk of neuropathic pain, e.g., amputation, thoracotomy and mastectomy. They should use appro- priately high dosages of medications, and therapy could commence prior to surgery and continue for several weeks.

Patient follow-up should be for a minimum of 6 months and a much more comprehen- sive and multidimensional assessment made of the character and severity of pain at rest and movement with an emphasis on level of function and basic neu- rophysiological testing (Melzack, 1987; Wilder-Smith et al., 2003; Kehlet and Rathmell, 2010; Wildgaard et al., 2011).


All authors had an active role in all aspects of the literature review which included a detailed discussion of the results and the manuscript.


Dear Editor,


Humble, Dalton and Li’s extensive systematic review regarding chronic post-operative pain [1] included more randomized controlled trials (RCTs) than a similar Cochrane review by Andreae and Andreae [2].

Twelve to four thoracotomy RCTs, seven to five breast surgery RCTs and five to three amputation RCTs identified and reviewed by Humble et al. [ 1] compared to Andreae and Andreae [2] respectively.

Ironically, it is Humble’s search method that fell short of the recommendation from the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) [6]:


Present full electronic search strategy for at least one database, including any limits used, such that it could be repeated.


A literature search in MEDLINE, EMBASE and the Cochrane Library, using a combination of keywords and subject headings [1], does not contain sufficient information for the interested reader to validate its findings.

This flawed methodology also prevents the original authors from periodically updating their review, the second and often underappreciated component of the Cochrane Challenge [4].


Repeatable electronic search strategies allow for transparency as to which RCTs were identified and excluded.

Andreae and Andreae used the Cochrane Collaboration’s recommended optimally sensitive search strategy (OSSS): a 29-line algorithm, consisted of three strategies, of increasing sensitivity but decreasing specificity.

As a result, by repeating their search, it can be determined that of the 12 RCTs that were included in Humble’s review, they were identified by the OSSS but were not utilized.

Conversely, there were four RCTs in Andreae’s review that were not in Humble’s review.

It is uncertain if these RCTs were identified and excluded by Humble or if it was a failure of the search method to identify them.


Ideally, a unified search method that is simple and easy to use, with high sensitivity and specificity should be agreed upon.

My co-workers and I [3] have independently assessed the OSSS and found it to be highly sensitive (99.6%) with moderate specificity (78%), compared to manual hand searches in analgesia-related research conducted by Oxford University [5].

The acceptance of OSSS by reviewers has been less than enthusiastic.

So much so that the Cochrane Collaboration revised the OSSS to the highly sensitive search strategy (HSSS), essentially the first of the three search strategies of the OSSS, with minor adjustments.

The HSSS is inherently problematic, preventing it to be the unified search method: it is only applicable in MEDLINE/PubMed.

The HSSS relies upon the publication type ‘randomized controlled trial.pt’, a subject heading that is manually identified by trained librarians of the US Library of Medicine.


An enormous undertaking that is labour-intensive, time-consuming and expensive.

This publication type does not exist in EMBASE, arguably the most comprehensive bibliographic database.

Despite zero results from three of the eight lines of algorithm, all of Humble’s 24 RCTs were contained within the 2041 resultant articles identified, line 14 of the EMBASE search.


I have previously proposed an alternate search strategy that can be applied in MEDLINE and EMBASE and validated in thoracotomy-related RCTs: (double blind$ or random$).af. [ 3, 7] This search strategy relies upon the authors of RCTs to declare it so, in the title, abstract or any other ‘fields’.

It was validated to be highly sensitive (96%) and specific (98%) compared to hand searching [3].

In the context of Humble and Andreae’s systematic reviews, this search strategy identified all of the RCTs in EMBASE alone.

The latter introduce an interesting dilemma on two fronts.

Firstly, are the enormous efforts by the USNLM in manually tagging ‘randomized controlled trial.pt’ justified? Secondly, with further confirmation, it may be that searching EMBASE will be sufficient and the ever-expanding number of medical bibliographic databases (e.g. Cochrane Library, CENTRAL, CINAHL) or for that matter MEDLINE and PubMed may be redundant.


T. K. F. Chow
Adjunct Senior Lecture, Department of Anaesthesia and Perioperative Medicine, Monash University, Melbourne, Australia


We thank Dr Chow for his interest in our work.

Dr Chow is indeed correct that our review included more randomized controlled trials than another review that he mentions (Andreae, 2013) and with which we are of course familiar.

However, we disagree with a number of his opinions.

Specifically, our search methodology was carried out as per PRISMA recommendations as documented in some detail in our submission to the EJP and this was subject to thorough peer review.


Chow is correct in the sense that it is difficult to perform literature searches in a way that includes every pertinent article, but this is a problem for literature reviews in general and not of specific relevance to our review alone.

Of note, we realized this limitation and sought to enhance capture of missed trials by incorporating methods such as manual tracking of references lists of known papers, as mentioned in our results section [5].

We took this pragmatic approach with the view that it was better to include these papers than to exclude them for the sake of what could be considered illusory methodological purity, given the inherent pitfalls of searching large databases, as Chow himself discusses in his letter [2][4].


We draw to his attention the fact that our systematic review contains a total of 32 RCTs, not 24 as indicated by Dr Chow’s letter.

However, Dr Chow poses a good question concerning the four RCTs from Andreae’s review that were excluded from our paper.

We can confirm that we were aware of and did indeed review all the RCTs within Andreae’s review.

We excluded specific publications for good reason.

Within the reference frame of our review, there are in fact only three RCTs in Andreae’s review that we could have included.


These are: Katsuly-Liapis, Pinzur and Reuben.

The first of these was only an abstract and had highly questionable methodology, as detailed by Andreae [1].

The second one had significantly suboptimal methodology and only included a total of 21 patients.

The third paper was by an author that is well known to have received a prison sentence for falsification of data and therefore it was thought best to exclude that one too.


Finally, we reiterate our appreciation of his interest in our work.

We recognize the numerous search strategies and their individual merits.

We also point out another tool by Chow that has been overlooked, the ‘DBRCT.af’ [3] and would encourage other researchers to consider doing the same in the interest of collaboration so that we can improve care for our patients.

This was indeed the motivation behind our own systematic review, which has generated much interest already.

We hope that it will actually influence anaesthetic practice internationally and continue to stimulate ongoing research in this interesting field.


The most widely employed methods for dietary inference include mesowear, the visual scoring of macroscopic tooth wear (Fortelius & Solounias, 2000; Fraser et al., 2014; Kaiser & Fortelius, 2003; Kaiser & Solounias, 2003), and microwear, the quantification of microscopic marks on the chewing surfaces of the teeth at either low or high magnification (Grine & Kay, 1988; Merceron, Blondel, Bonis, Koufos, & Viriot, 2005; Semprebon et al., 2004; Solounias & Semprebon, 2002; Ungar et al., 2003).


Both mesowear and micro- wear have proven to be extremely useful tools for inferring the di- etary preferences of extant and extinct mammals (and nonmammals) from numerous families (DeSantis & Haupt, 2014; DeSantis et al., 2015; Donohue et al., 2013; Fortelius & Solounias, 2000; Fraser & Theodor, 2010, 2011, 2013; Fraser et al., 2009, 2014; Grine & Kay, 1988; Haupt et al., 2013; Hedberg & DeSantis, 2017; Semprebon et al., 2004; Solounias et al., 1995).


The mesowear method, as proposed by Fortelius and Solounias (2000), approximates the diets of herbivorous "hoofed mammals" (including artiodactyls, perissodactyls, with newer iterations also including proboscideans) by quantifying the relative amounts of attritional and abrasive wear apparent on the cusps of the cheek teeth (premolars and molars).

Mammalian mastication is driven by asymmetric contraction of the masseter, pterygoideus, and tempo- ralis muscles on the working and balancing sides, which produces a transverse movement of the mandible (Janis, 1979; Rensberger, 1973; Smith, 1993).

During the chewing stroke, the cheek teeth ex- perience both attrition and abrasion (Fortelius & Solounias, 2000).

Attrition occurs during the interaction of opposing wear facets and results in cusp sharpening as the facets slide past one another.

Abrasion occurs during the interaction of the teeth with ingested items including food particles, resulting in the dulling of the cusps (Fortelius & Solounias, 2000).


The original Fortelius and Solounias (2000) mesowear method involves visual categorization of tooth cusp relief (the height dif- ference between the intercusp valleys and the tip of the cusp) and shape (sharp, round, blunt) (Fortelius & Solounias, 2000).

Individuals that experience high attrition (browsers) tend to have high relief and sharp cusps while individuals that experience high abrasion (graz- ers) have low relief and blunt cusps.

Intermediate or mixed feeders (10%-90% grass consumption) tend to have low- or high- relief cusps with rounded tips, depending on whether their diet is dominated by grasses or browse (Fortelius & Solounias, 2000).

Mesowear yields fairly high rates of correct classification (~60%-80%) of ruminant artiodactyls among the dietary categories of browser, grazer, and mixed feeders (Fraser & Theodor, 2011; Fraser et al., 2014).


Microwear is the quantification of microscopic wear features on the teeth that result from masticating different foods with different physical properties (Solounias & Semprebon, 2002).

Traditionally, microwear analysis involves the quantification of the numbers of pits and scratches on the tooth surface within predefined counting areas or grids as identified by one or more human observers (Grine & Kay, 1988; Solounias & Semprebon, 2002).

Pits are thought to be formed as a result of enamel on enamel contact during which small fragments of enamel are chipped away, while scratches are thought to be formed by abrasive items such as phytoliths or exogenous grit being scraped across the enamel surface (Hoffman et al., 2015; Solounias & Semprebon, 2002).

Traditional microwear analysis may be carried out at either high (Grine & Kay, 1988) or low magnifica- tion (Semprebon et al., 2004; Solounias & Semprebon, 2002).

The low- magnification method has been widely employed due to low cost and the accessibility of light microscopes, whereas the high- magnification method requires a scanning electron microscope (SEM).

Low- magnification microwear methods yield intermediate re- sults of successful dietary classification for ruminants (~64%) but, in combination with other dietary metrics such as mesowear, yield very high rates of correct classification (~90%) (Fraser & Theodor, 2011).


Studies of tooth microwear have also grown to include three- dimensional textural analysis of tooth surfaces at high magnification (Ungar, Simon, & Cooper, 1991).

A popular implementation of this approach is known as dental microwear textural analysis (DMTA), for which the tooth surface is scanned across the z- axis using a white- light confocal profiler generating a point cloud matrix (Scott et al., 2006).

Point cloud matrices are then analyzed using scale- sensitive fractal analysis, which operates on the idea that apparent length, area, and volume can change at varying observational scales, and that these changes correlate to the material properties of consumed food (Ungar et al., 2003).

For example, a surface that seems smooth 5358 | FRASER Et Al. at the coarse scale of a human observer, such as a rug, might seem rough and varied at the scale of an ant.


Implementation of DMTA has been limited by the low number of available microscopes and the high cost of the analytical software required to compute the neces- sary textural values (SurFract, Norwich, VT, USA).

However, DMTA is shown to be an effective method for differentiating a wide array of mammalian taxa based on dietary preference including artiodac- tyls (Scott, 2012; Ungar, Merceron, et al., 2007; Ungar, Scott, et al., 2007; Ungar et al., 2012), shrews (Withnell & Ungar, 2014), primates (Scott, Teaford, & Ungar, 2012), carnivorans (Desantis, Schubert, Scott, & Ungar, 2012; Schubert, Ungar, & DeSantis, 2010; Ungar, Scott, Schubert, & Stynder, 2010), marsupials (Prideaux et al., 2009), and xenarthrans (Haupt et al., 2013).


Mesowear and microwear data were collected from the literature (Appendix S1).

Mesowear data are heavily biased toward "hoofed mammals," given that they are devised for dietary inference in se- lenodont and lophodont perissodactyls and artiodactyls.

Although newer mesowear methods have been developed for proboscideans (Saarinen et al., 2015), the phylogenetic breadth of these datasets is too small for the analyses used herein.

Furthermore, given that Proboscidean mesowear is scored on a different scale, the data cannot easily be integrated into other existing mesowear datasets.

Similarly, we have not included the mesowear III method (Solounias, Tariq, Hou, Danowitz, & Harrison, 2014) because the training data- set is too small for a statistically robust analysis of phylogenetic sig- nal.

Unlike mesowear, the microwear data (low magnification and textural) encompass mammals from most dietary guilds and most major clades (Appendix S1).


The Fortelius and Solounias (2000) mesowear method involves visually categorizing the cheek teeth based on two variables: cusp relief (the height difference between the intercusp valleys and the tip of the cusp) and cusp shape (Fortelius & Solounias, 2000).

Cusp relief categories are high and low [cutoffs are discussed in Fortelius and Solounias (2000)].

The cusp- shape categories are sharp, rounded, and blunt.

Sharp cusps are defined as those with little or no round- ing between the mesial and distal wear facets.

Rounded cusps are defined as those with a smooth cusp tip intervening between the mesial and distal wear facets.

Blunted cusps are defined as those in which the wear facets had been worn away (Fortelius & Solounias, 2000).