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Competing interests | The authors declare no competing interests. | PMC10656569 | ||
References | PMC10656569 | |||
Objectives
| Behcet’s disease | ATHEROSCLEROSIS | To examine the serum irisin level in a group of Behcet’s disease patients, its association with illness parameters, and its utility in diagnosing subclinical atherosclerosis. | PMC9823020 |
Methods | This randomized case–control study included 50 patients and 50 age- and sex-matched controls. Carotid Doppler ultrasound for the measurement of the carotid artery intima-media thickness (CIMT) and ankle-brachial pressure index (ABPI) were performed. A clinical evaluation, lipogram, and serum irisin were also performed. | PMC9823020 | ||
Results | ATHEROSCLEROSIS | Between the patients and the control group, there was a significant difference in CIMT, S. irisin level, and ankle-brachial pressure index; however, gender and BMI did not significantly affect CIMT, ABPI, or S. irisin level. CIMT demonstrated a substantial negative correlation with both S. irisin and ABPI (With a sensitivity of up to 94.30% and a specificity of 93.30%, the ROC analysis revealed that a decrease in S. irisin level in Behcet’s patients was indicative of subclinical atherosclerosis. The drop in the ABPI level demonstrated a sensitivity of up to 94.30% and a specificity of 100%. | PMC9823020 | |
Conclusion | atherosclerosis, Behcet’s illness | ATHEROSCLEROSIS | Subclinical atherosclerosis is prevalent among Egyptian Behcet’s patients, and S. irisin can be employed as a biomarker for diagnosing subclinical atherosclerosis in Behcet’s illness.
• • • | PMC9823020 |
Keywords | Open access funding provided by The Science, Technology & Innovation Funding Authority (STDF) in cooperation with The Egyptian Knowledge Bank (EKB). | PMC9823020 | ||
Introduction | thrombosis, vascular damage, Vasculitis, inflammation, atherosclerotic, CVD, Behcet’s disease, hyperglycemia, metabolic disease, Behcet’s illness, endothelial cell dysfunction, atherosclerosis, platelet aggregation | CARDIOVASCULAR DISEASE, DYSFUNCTION, INSULIN RESISTANCE, THROMBOSIS, HYPERCOAGULABILITY, PLATELET DYSFUNCTION, ENDOTHELIAL DYSFUNCTION, INFLAMMATION, INFLAMMATORY DISORDER, VASCULAR PROBLEM, METABOLIC DISEASE, CHRONIC SPONTANEOUS URTICARIA, HYPERCHOLESTEROLEMIA, CAROTID ATHEROSCLEROSIS, CVD, VASCULITIS, ENDOTHELIAL DYSFUNCTION, HYPERGLYCEMIA, INFLAMMATIONS, ATHEROSCLEROSIS, CHRONIC INFLAMMATORY DISORDER | Behcet’s disease (BD) is a chronic, relapsing, inflammatory disorder characterized by systemic involvement, vascular damage, and endothelial cell dysfunction [Cardiovascular involvement ranges from 7 to 46% [Chronic inflammatory disorders are characterized by systemic inflammation that leads to cardiovascular disease (CVD) by well-established mechanisms: accelerated atherosclerosis, insulin resistance (IR), platelet dysfunction, hypercoagulability, hypercholesterolemia, and hyperglycemia [Endothelial dysfunction is a well-recognized indicator of subclinical vascular atherosclerosis; it is determined by the intima-media thickness of the common carotid artery (CIMT).Endothelial dysfunction is the first manifestation of BD’s vascular problems. It greatly contributes to the onset and progression of vascular damage in many body locations, resulting in metabolic disease consequences [Vasculitis and inflammations of the artery wall can increase platelet aggregation and inhibit fibrinolysis, resulting in thrombosis [Subclinical atherosclerosis is an early sign of atherosclerotic load, and its prompt diagnosis can postpone or prevent the development of overt cardiovascular disease [Irisin is a myokine secreted by the liver, kidney, heart, skeletal muscles, and skin in response to exercise [In 2015, Lee HJ et al. established the association between circulating irisin levels, endothelial dysfunctions, and subclinical atherosclerosis in non-diabetic adult patients by finding a substantial correlation between carotid atherosclerosis and serum irisin levels in dialysis patients [Recently Altay et al. investigated the relationship between inflammation and irisin in serum samples from chronic spontaneous urticaria (CSU) patients, they observed lower irisin levels in the CSU group, and they raised a suggestion that a decrease in irisin levels may be decisive for CSU [The purpose of this study is to determine the link between serum irisin level and subclinical atherosclerosis in patients with Behcet’s illness and to compare serum irisin to other modalities for diagnosing subclinical atherosclerosis and endothelial dysfunctions. | PMC9823020 |
Patients and procedures
| Behcet’s, fasting blood glucose, Behcet Syndrome | STERILE, DISEASE, BEHCET SYNDROME | It is a case–control study that included 50 Behcet’s patients diagnosed according to 2006 classification criteria (ICBD, 2006) [The Ethics Committee of our university’s medical school approved the study under the number IBR ♯ S20-154, and all participants provided written informed permission.All subjects underwent a comprehensive clinical examination, and disease activity was assessed using the Behcet Syndrome Activity Score (BSAS) [Each participant’s 10 ml of venous blood was drawn under aseptic conditions using sterile disposable gloves for laboratory examinations.A: Routine investigations, liver function tests (LFT), kidney function tests (KFT), fasting blood glucose, lipid profile including cholesterol, triglyceride, high density lipoprotein (HDL), and low-density lipoprotein (LDL) utilizing the Cobas c311 Chemistry Analyser System (Roche Diagnostic GmbH, Indianopolis, IN, USA).The B complete blood count was performed by CELL-DYN Abbott (Abbott Laboratories, Diagnostic Division IL, USA) and the ESR was performed using a westergren tube.C: The serum irisin concentration was determined with an irisin ELISA kit and an ELISA Thermo Fisher, Scientific Multiscan EX Microplate Reader, OY, FI-O1621, Vantaa, Finland. | PMC9823020 |
Evaluation procedure
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prepare all reagents, working standards, blanks, and samples according to the instructions in the preceding sections.refer to the assay layout sheet to calculate the number of wells to place any unused wells and desiccant back into the pouch, reseal the pouch, and store the wells at 4C.add 50 mL of the standard to the testing standard well, followed by 10 mL of the testing sample (sample final dilution is fivefold). Pipette the sample to the wells, avoiding the well walls as much as possible, and mix gently.cover with the included adhesive strip and incubate for 30 min at 37 °C.configure liquid, 30 times dilute wash solution with distilled water to configure liquid.washing, remove the adhesive strip, discard the liquid, and then pipette washing buffer into each well. Allow to stand for 30 s and then drain.add enzymes, pipette 50 l of HRP-conjugate reagent into each well, excluding the well designated as a blank.incubate, operation with fourwashing, using number 6add 50 ml of chromogen solution A and 50 ml of chromogen solution B to each well, avoiding light for 15 min at 37 °C.stop the reaction by pipetting 50 ml of stop solution into each well (the blue change to yellow).Calculate the value of the blank well as zero. Read absorbance at 450 nm 15 min after pipetting stop solution. | PMC9823020 | ||
Diagnostic imaging techniques for atherosclerosis | arterial disease, PAD | PAD, ARTERIAL DISEASE, PLAQUE | A: Carotid Doppler ultrasound, for the measurement of carotid artery intima-media thickness (CIMT): CIMT is defined as a low-level echo gray band that does not project into the arterial lumen. CIMT was measured during the diastolic phase as the distance between the leading edge of the first and second echogenic lines of the far walls of the distal segment of the common carotid artery, the carotid bifurcation, and the internal using superficial multifrequency linear array transducer [5–8 MHz] with a sonographic apparatus [Aplio 500 Canon medical system, Japan]. CIMT tests were done on artery segments devoid of plaque. To eliminate examiner bias, all examinations and measures have been conducted by the same examiner. Age-dependent normal range is used as the reference for CIMT. The identification of a carotid atherosclerotic plaque is defined as a focal IMT of 50% or > 0.5 mm relative to the adjacent artery wall, or an absolute IMT of > 1.5 mm [B: Evaluation of ankle/brachial BP index utilizing blood pressure cuff Doppler pulse volume recording (PVR) ultrasound. Grayscale duplex Doppler is used to map arterial disease and to solve problems.The index is a ratio of the pressure in the highest ankle artery / the highest brachial artery.1.0–1.4: normal0.91–0.99 borderline ≤ 0.9: abnormal (i.e., PAD)0.4 to 0.9: moderate to mild PAD < 0.4: indicative of severe PAD | PMC9823020 |
Analytical statistics | Quantitative data were expressed as the mean, standard deviation, median, and range. The student | PMC9823020 | ||
The analysis of regression | atherosclerosis, Behcet’s disease, Behcet’s illness | REGRESSION, ATHEROSCLEROSIS |
Univariate linear regression examination of factors influencing CIMT in Behect’s patients revealed a significant positive relationship with age (Multivariate linear regression analysis of factors affecting CIMT in patients with Behcet’s disease (include significant variable in univariate) revealed that age of patient (The most important predictors of subclinical atherosclerosis in patients with Behcet’s illness are male gender, platelet count, S. irisin, and ankle brachial pressure index, as demonstrated in Table The ROC analysis (Fig. Multivariate logistic regression analysis of factor predicting subclinical atherosclerosis in patient with Behcet’s disease include significant variable in univariate analysisMultivariate logistic regression test. Statistical significant difference (ROC analysis of S. irisin and ankle brachial pressure index in predicting subclinical atherosclerosis. Roc curve analysis test. Statistically significant difference ( | PMC9823020 |
Discussion | Obesity, CVDs, stroke, atherosclerosis, hyperlipidaemia, atherosclerotic, CVD, cardiovascular lesions, Behcet’s disease, Behcet’s illness, cardiac infarction, T2DM | CARDIAC INFARCTION, HYPERGLYCEMIA, OBESITY, CARDIOVASCULAR DISEASES, CVD, STROKE, DISEASE, HYPERLIPIDAEMIA, CHRONIC INFLAMMATORY DISEASE, REMISSION, INSULIN RESISTANCE, VOGT, REGRESSION, ATHEROSCLEROSIS, TYPE 2 DIABETES, ENDOTHELIAL DYSFUNCTION | Increased risk for cardiovascular lesions (CVD) in patients with pre-existing chronic inflammatory diseases at younger ages observed in some studies raised the assumption about endothelial dysfunction as a common initial lesion in the development of atherosclerosis [There are few studies examining S. irisin as a biomarker for diagnosing subclinical atherosclerosis in Behcet’s illness. In our study, we intended to investigate this topic.We did not find a significant relationship between serum irisin level and disease activity score, nor did we find a relationship between CIMT and disease activity score. The BSAS score of the included patients had a mean value of 5.6, and a range of 3 to 9 indicates that they are in remission and have been on treatment for more than six months. These results concur with Icli et al.’s findings [Subclinical atherosclerosis can begin early in life and be unnoticed until a cardiac infarction or stroke [Hyperglycemia and hyperlipidaemia are known to accelerate the atherosclerotic process by direct endothelial dysfunction augmenting cytokine release with increasing lipid peroxidation and causing oxidative damage [There were no documented cases of Q-T dispersion; therefore, the atherosclerotic changes in our patients are asymptomatic and a sufficient narrowing of coronaries may be required for symptoms to appear; our results agreed with [Obesity is a condition characterized by excessive fat buildup and is frequently accompanied with insulin resistance (IR), type 2 diabetes (T2DM), and cardiovascular diseases (CVDs) [The non-significant difference for ankle brachial pressure index between those of BMI 25 and those of BMI > 25 was cleared by the controlled blood glucose level, controlled blood lipid, and blood pressure in our patients.In the same instance, serum irisin levels did not differ significantly between those with a BMI 25 and those with a BMI > 25 (The regression analysis of factors affecting CIMT in a patient with Behcet’s disease revealed that the most influential factors on the disease were the patient’s age and ESR level. The patient’s age is one of the risk factors for atherosclerosis and can be determined by CIMT level [For characteristics that indicate subclinical atherosclerosis, male gender and decreasing S levels are predictive. Irisin, and decreasing ankle brachial pressure index agreed with Vogt et al. [Icli et al. [We also found a very strong correlation between CIMT, an indicator of subclinical atherosclerosis, and decreased irisin levels in BD patients.With a sensitivity of 94.30% and specificity of 93.33%, the declining level of S. irisin in patients can be utilized as a predictor of subclinical atherosclerosis. This finding agreed with Ankle Brachial Index Collaboration, 2008 [ | PMC9823020 |
Author contribution | A. I., S. A. | Conceptualization, methodology, supervision, validation, writing evaluation, and editing by Mohamed A. I. Data curation, formal analysis, research methodology, resources, and original draft writing by Ola M. Laboratory analysis, methodology, review, and editing by Ahmed S. The radiological investigation, methodology, visualization, review, and editing by Hisham A. A. Project management, supervision, validation, and original draft writing by Esam A. A. Review of Methodology by Ahmed R. R. Methodology, project administration, visualization, writing an original draft, review, editing, and submission of the manuscript are the responsibilities of Hanan S. A. | PMC9823020 | |
Funding | Open access funding provided by The Science, Technology & Innovation Funding Authority (STDF) in cooperation with The Egyptian Knowledge Bank (EKB). | PMC9823020 | ||
Data availability | Not available. | PMC9823020 | ||
Declarations | PMC9823020 | |||
Ethical approval | The study was authorized by the Ethics Committee of the faculty of medicine at Sohag university in Egypt, with no IBR ♯ S20-154, and all participants provided written informed consent. | PMC9823020 | ||
Disclosures | None. | PMC9823020 | ||
References | PMC9823020 | |||
1. Introduction | HF, muscle atrophy, human diseases, fatigue, illness, Malnutrition, cancer, inflammation, sarcopenia, Sarcopenia, malnutrition, reduced skeletal muscle mass, heart failure, coronary heart disease, impaired muscle function | MUSCLE ATROPHY, MALNUTRITION, CANCER, INFLAMMATION, SARCOPENIA, FRAGILITY, ADVERSE EFFECTS, SARCOPENIA, MALNUTRITION, HEART FAILURE, CORONARY HEART DISEASE | Background: Malnutrition and sarcopenia frequently affect patients with heart failure (HF), in which clinical outcomes and survival is decreased. Thus, appropriate nutritional screening and early nutrition support are highly recommended. Currently, nutritional support is not a standard of care in patients with HF, and the use of commercially available oral supplements (OSs) could provide an additional benefit to medical treatment in these patients. Aim: To compare the effect of the Mediterranean diet in combination with hypercaloric, hyperproteic OS in patients with HF. Patients and methods: An open label, controlled clinical study in which patients were randomly assigned to receive a Mediterranean diet (control group) vs. hypercaloric, hyperproteic OS (intervention group) for twenty-four weeks. Thirty-eight patients were included; epidemiological, clinical, anthropometric, ultrasound (muscle echography of the rectus femoris muscle of the quadriceps and abdominal adipose tissue), and biochemical evaluations were performed. All patients received additional supplementation with vitamin D. Results: Baseline malnutrition according to the GLIM criteria was observed in 30% of patients, while 65.8% presented with sarcopenia. Body cell mass, lean mass, and body mass increased in the intervention group (absolute increase of 0.5, Nutritional support is essential in several human diseases [Sarcopenia is characterized by decreased muscle strength and fatigue due to reduced skeletal muscle mass, which is accompanied by muscle atrophy and decreased quality of muscle tissue. Specifically, muscle fibers are replaced by fibrotic tissue, which results in increased fragility and impaired muscle function [Currently, there are no specific guidelines or recommendations concerning nutritional support in HF [In the 1960s, the Seven Countries Study revealed that coronary heart disease and cancer rates were not elevated in a cohort of four Mediterranean areas: Crete and Corfu in Greece, Dalmatia in Croatia, and Montegiorgio in Italy. Specifically, at that moment, the Greek diet had the highest content of olive oil and was high in fruit, the Dalmatian diet was highest in fish, and the Italian diet was high in vegetables. These results represent the base of the Mediterranean diet, which recommends the consumption of plant food (fruits, vegetables, whole-grain cereals, nuts, and legumes); olive oil as the principal source of fat; moderate amounts of fish and poultry; and relatively low consumption of red meat [The European and American Societies of Enteral and Parenteral Nutrition state that oral nutritional supplements provide additional nutrients, including protein and energy, for people who are not meeting their nutrition needs through food alone, due to decreased appetite or food intake, increased nutrition needs, or poor absorption of nutrients due to illness. Thus, when oral feeding is insufficient, oral nutritional supplementation is indicated; specifically, hypercaloric (1.5–2 kcal/mL) and, if necessary, hyperproteic oral supplements (OS) are generally recommended, since they can improve inflammation status, QoL, and survival in HF patients [Interestingly, a previous multicenter clinical trial has suggested that diet optimization, specific nutritional recommendations, and nutritional supplement prescriptions (when necessary) in patients with HF may have a prognostic benefit during and after hospital admission [Interestingly, it has been described that in patients with HF, left ventricular ejection fraction (LVEF) has a positive predictive value for adverse effects [ | PMC10781070 |
2. Materials and Methods | PMC10781070 | |||
2.1. Patients | end-stage kidney and/or liver disease | MAY | This study was approved by the Ethics Committee of the Reina Sofia University Hospital (Cordoba, Spain; reference number 5164 approved on 21 October 2021 and updated on 30 May 2023). It was conducted in accordance with the Declaration of Helsinki and according to national and international guidelines. This is a prospective open label study, wherein a written informed consent was signed by every individual before inclusion in the study. All patients received information before the inclusion and were included only if they agreed to participate. The inclusion criteria were patients of both sexes, age > 18 y-old < 85 y-old, LVEF < 50%, and a hospital admission due to HF in the previous 6 months. Exclusion criteria were end-stage kidney and/or liver disease. The sample size was calculated based on the usual number of admissions due to HF for 6 months in this hospital (specifically during the winter and spring seasons). Thirty-eight consecutive patients were included. A schematic overview of the study is depicted in | PMC10781070 |
2.2. Study Design | This was a randomized, open, controlled, clinical trial. Patients were randomly assigned by the clinical investigator to receive either a Mediterranean diet alone or a Mediterranean diet plus two hypercaloric, hyperproteic OSs per day with a 1:1 allocation for twenty-four weeks. Specifically, the OS was composed with a mixture of slow-release carbohydrates and fiber; the specific composition of the OS is presented in Nutritional evaluation was performed by the same endocrinologist in all cases. Physical examination included body composition analysis (bioelectrical bioimpedance, and abdominal, arm and calf circumferences), functional tests (up-and-go test and handgrip strength) and nutritional ultrasound of abdominal adipose tissue and rectus femoris (RF) muscle of the quadriceps. Specifically, vectorial bioelectrical bioimpedance (BIA) was performed using a NUTRILAB-Akern impedanciometer (Akern, Pis, Italy); for handgrip strength, a Jamar | PMC10781070 | ||
2.3. Outcomes | high-density lipoprotein | The primary outcome was to evaluate the change in lean mass of patients after both nutritional interventions. Secondary outcomes included changes in other parameters of body composition (fat mass, water, bone, phase angle, body cell mass (BCME), extracellular mass (ECME)); anthropometric parameters (calf, arm, and abdominal circumference); LVEF assessed by transthoracic ultrasound, biochemical nutritional parameters (hemoglobin, lymphocytes, total cholesterol, total high-density lipoprotein (HDL) cholesterol, low-density lipoprotein (LDL) cholesterol, triglycerides, transferrin, ferritin, albumin, prealbumin | PMC10781070 | |
2.4. Statistical Analysis | PAD | Between-group comparisons were analyzed using the Mann–Whitney U test (nonparametric data). Paired analysis was performed by Wilcoxon test (nonparametric data). The chi-squared test was used to compare categorical data. Statistical analyses were performed using SPSS statistical software version 20, and Graph Pad Prism version 6. Data are expressed as medians with interquartile ranges and percentages. Absolute differences in some parameters were calculated using mean values. For specific group analysis, the absolute number is also expressed in brackets. | PMC10781070 | |
3. Results | PMC10781070 | |||
3.1. Baseline Characteristics of the Groups | gastrointestinal symptoms, abdominal pain, sarcopenia, Sarcopenia, malnutrition, weight loss | SARCOPENIA, SARCOPENIA, MALNUTRITION | Thirty-eight patients were included. Most of them were male (71.1%) with a median age of 67.5 y-old. Both groups were comparable in most clinical variables (Significantly, there were no significant differences between baseline LVEF and NT-proBNP levels between both groups. Despite this, control patients tended to complain more frequently about abdominal pain (21.1% vs. 0%, Baseline malnutrition according to the GLIM criteria was observed only in 24% of patients, while 65.8% presented with sarcopenia. Specifically, most patients presented handgrip strength < p5 (28.9%), followed by p25 (23.1%), p50 (21.1%) and p10 (13.2%). Handgrip strength > p50 was observed only in 13.2% of the evaluated patients. There were no statistically significant differences in the distribution of sarcopenia and malnutrition in both groups, but sarcopenia tended to be higher in the control group (78.9% vs. 52.6%; Sarcopenia was associated with increased age and increased C-RP serum levels, but not with NT-proBNP levels or LVEF (At baseline, NT-proBNP levels negatively correlated with some BIA parameters including body weight, BMI, BCME, ECME, and fat-, lean- and bone mass; anthropometric parameters including abdominal and arm circumference; both functional parameters, increased time in the up-and-go test and decreased handgrip strength, and muscle ultrasound parameters including RF area, circumference, X-axis; and adipose tissue. NT-proBNP also negatively correlated with hemoglobin, albumin, prealbumin, and triglycerides; in contrast, it positively correlated with C-RP serum levels. LVEF negatively correlated only with serum hemoglobin (Adherence to OS was assessed every 21 days. At the end of the study, 71.1% of patients took two supplements per day, 9.8% one per day, 1.9% one and a half per day, and 0.5% half an OS per day.After twenty-four weeks of intervention, there were no significant differences between the prevalence of new hospital admissions, weight loss, or gastrointestinal symptoms; the number of resting hours still tended to be higher in the control group (median 8 vs. 4.5 h/day, At the end of the study, NT-proBNP levels negatively correlated with lymphocytes, serum prealbumin, and triglycerides, while LVEF negatively correlated with RF X-axis in controls ( | PMC10781070 |
3.2. Primary and Secondary Outcomes | Body weight significantly increased in the whole cohort at the end of the study (79 (69–85) vs. 81 (68–88) kg, No statistically significant differences were observed In abdominal, arm, or calf perimeters after 24 weeks of intervention (According to the RF ultrasound, adipose tissue, muscle area, and muscle circumference tended to decrease in the intervention group; specifically, adipose tissue decreased 0.1 cm (Regarding functionality tests, no significant change in handgrip strength was observed, but mobility significantly improved in all patients by 12.6 s according to the up-and-go test (Regarding the biochemical data, hemoglobin increased by 0.4 mg/dL in all patients (Self-reported QoL significantly increased in all patients, from 72.5 (50–85) at baseline to 80 (80–90) (When heart functionality was evaluated, LVEF increased in the whole cohort (38.7% ± 16.6 vs. 42.2% ± 8.9, Age- and sex-adjusted analysis revealed that nutritional support, baseline LVEF, NT-proBNP, body composition parameters, and functionality tests were not associated with mortality or new hospital admissions in this cohort (some representative parameters are depicted in | PMC10781070 | ||
4. Discussion | obesity, muscle mass, ankle swelling, fatigue, cardiac cachexia, elevated intra-cardiac pressures, cachexia, inflammation, sarcopenia, reduced cardiac output, cardiac abnormality, HF [, breathlessness, head and neck cancer, malnutrition, Heart Failure | OBESITY, ANKLE SWELLING, MALNOURISHED, PERIPHERAL EDEMA, DISEASE, CLINICAL SYNDROME, SARCOPENIA, INFLAMMATION, MALNOURISHED, HEAD AND NECK CANCER, MALNUTRITION, HEART FAILURE | The European Society of Cardiology defines HF as “a clinical syndrome characterized by typical symptoms (e.g., breathlessness, ankle swelling and fatigue) that may be accompanied by signs (e.g., elevated jugular venous pressure, pulmonary crackles and peripheral edema) caused by a structural and/or functional cardiac abnormality, resulting in a reduced cardiac output and/or elevated intra-cardiac pressures at rest or during stress” [These patients frequently present malnutrition, within a range of 10–50%, depending on the stage of the disease and according to the parameters used to define malnutrition. In patients with advanced HF, it can progress to cardiac cachexia. Both malnutrition and cachexia have been associated with worse prognosis, period of hospital stay, readmission rate, in-hospital mortality, and overall mortality [Additionally, some patients do not lose weight, despite presenting with overweight or obesity accompanied with sarcopenia. Thus, muscle mass is decreased in quality, quantity, and functionality independently of body weight [A prevalence of sarcopenia of 34% has been previously reported in HF, and is higher in males (37% vs. 33% in females) [According to the Nutritional Intervention Program in Malnourished Patients Admitted for Heart Failure (PICNIC study), individualized nutritional intervention during and after HF admission may have a prognostic benefit. This multicenter clinical trial included 120 malnourished hospitalized patients with acute HF. Patients were randomized to conventional treatment or nutritional intervention (diet optimization, specific recommendations, and nutritional supplement prescriptions if necessary). At the end of the study, all-cause mortality or readmission for worsening of HF was significantly lower in the intervention group (27.1%) compared with the control group (60.7%) [In this context, most experts and cardiology societies recommend a Mediterranean-style diet in these patients, probably due to its anti-inflammatory properties [Generally, it is recommended that hyperproteic OSs could be administered in patients with HF and insufficient oral intake [It is known that patients with HF have elevated circulating levels of pro-inflammatory cytokines; specifically, the innate and adaptive immune systems are activated in HF. Thus, treatment strategies targeting inflammation are a matter of interest in these patients [In clinical nutrition, some nutritional formulas are recommended in specific situations; for example, immunonutrition is currently a standard of care in fast-track abdominal surgery programs, or after surgery in patients with head and neck cancer [This study has some limitations: first the number of participants in each group, which can limit the findings of this study; furthermore, some baseline variables tended to be different in both groups despite there being no statistically significant differences. Ideally, a study with matched controls should be performed, considering that it would not reflect the regular clinical practice. Additionally, we did not supervise the diet daily intake of the patients. Finally, the nutritional intervention was not evaluated alone, which is a frequent limitation in studies regarding nutrition; it was not possible to evaluate the nutritional intervention in an isolated manner, since rehabilitation is currently considered a standard of care in patients with HF [ | PMC10781070 |
5. Conclusions | lean mass gain, sarcopenia, malnutrition | SARCOPENIA, MALNUTRITION | Taken together, our results reveal that a nutritional intervention with the Mediterranean diet in patients with HF results in improvement of functionality, QoL, and cardiac function. Furthermore, the use of a hypercaloric, hyperproteic OS (with slow-release carbohydrates, omega-3, and omega-6) is associated with lean mass gain, cell mass gain, improvement in biochemical nutritional parameters, and a more significant improvement in functionality, QoL, and LVEF, and a decrease in NT-proBNP serum levels, after 24 weeks of intervention. Regular recommendations of the Mediterranean diet should be provided to all patients after HF admission. Furthermore, malnutrition and sarcopenia screening should be regularly implemented before discharge, in order to recommend the use of OSs in patients with (or at risk of) malnutrition or sarcopenia. Additional head-to-head studies, with a larger sample size and comparing different OS formulas, should be performed. | PMC10781070 |
Supplementary Materials | The following supporting information can be downloaded at: Click here for additional data file. | PMC10781070 | ||
Author Contributions | Conceptualization; A.D.H.-M., C.M.J. and M.J.M.P.; Funding acquisition; A.D.H.-M.; Practical performance: M.C.C., J.L.A., A.D.H.-M., G.M.G., C.M.J. and M.Á.G.M.; Formal and data analysis: A.D.H.-M.; Preparation manuscript: A.D.H.-M., M.J.M.P. and C.M.J.; Critical review of manuscript: A.D.H.-M., M.J.M.P. and A.C.C.; Final review of the manuscript: all authors. All authors have read and agreed to the published version of the manuscript. | PMC10781070 | ||
Institutional Review Board Statement | MAY | This study was approved by the Ethics Committee of the Reina Sofia University Hospital (Cordoba, Spain; reference number 5164 approved on 21 October 2021 and updated on 30 May 2023). ClinicalTrials.gov number: NCT05848960. | PMC10781070 | |
Informed Consent Statement | Informed consent was obtained from all subjects involved in the study. | PMC10781070 | ||
Data Availability Statement | Data are contained within the article. | PMC10781070 | ||
Conflicts of Interest | The authors declare no conflicts of interest. | PMC10781070 | ||
References | sarcopenia | REGRESSION, SARCOPENIA | Schematic overview of the study and treatment arms.Study design.Clinical associations between sarcopenia with age, serum C-RP levels, serum NT-proBNP, and LVEF determined by transthoracic ultrasound. Legend: C-RP: c-reactive protein; ns: non-significative; *: Significant clinical correlations between serum NT-proBNP levels and LVEF and nutritional parameters at baseline (Change in LVEF and serum NT-pro BNP serum levels after 24 weeks of nutritional intervention with Mediterranean diet (control group) and Mediterranean diet plus two hypercaloric, hyperproteic oral supplements (Oss). Legend: *: Baseline clinical characteristics of the patients. Comparison between groups based on the nutritional intervention.Legend: Categorical data are presented as percentages and the absolute number in brackets.Clinical characteristics of the patients twenty-four weeks after nutritional support.Legend: Categorical data are presented in percentages and the absolute number in brackets.Morphofunctional assessment of the nutritional status at baseline and six months after nutritional treatment.Legend: RF: rectus femoris. p1 refers to the comparison between all patients at baseline and after twenty-four weeks; p2 refers to the comparison between patients of the control group (Mediterranean diet) at baseline and after twenty-four weeks; p3 refers to the comparison between patients of the intervention group (Mediterranean diet plus oral nutritional supplementation) at baseline and after twenty-four weeks.Biochemical analysis at baseline and six months after nutritional support.p1 refers to the comparison between all patients at baseline and after twenty-four weeks; p2 refers to the comparison between patients of the control group (Mediterranean diet) at baseline and after twenty-four weeks; p3 refers to the comparison between patients of the intervention group (Mediterranean diet plus oral nutritional supplementation) at baseline and after twenty-four weeks.Multivariate logistic regression for mortality and new hospital admissions in patients with HF that received nutritional support after adjusting by age and sex. | PMC10781070 |
Background | COMPLICATION | Out-of-plane (OOP) approach is frequently used for ultrasound-guided insertion of central venous catheter (CVC) owing to its simplicity but does not avoid mechanical complication. In-plane (IP) approach might improve safety of insertion; however, it is less easy to master. We assessed, a homemade needle guide device aimed to improve CVC insertion using IP approach. | PMC10543855 | |
Method | We evaluated in a randomized simulation trial, the impact of a homemade needle guide on internal jugular, subclavian and femoral vein puncture, using three approaches: out-of-plane free hand (OOP-FH), in-plane free hand (IP-FH), and in-plane needle guided (IP-NG). Success at first pass, the number of needle redirections and arterial punctures was recorded. Time elapsed (i) from skin contact to first skin puncture, (ii) from skin puncture to successful venous puncture and (iii) from skin contact to venous return were measured. | PMC10543855 | ||
Results | Thirty operators performed 270 punctures. IP-NG approach resulted in high success rate at first pass (jugular: 80%, subclavian: 95% and femoral: 100%) which was higher than success rate observed with OOP-FH and IP-FH regardless of the site ( | PMC10543855 | ||
Conclusion | In this simulation study, IP approach using a homemade needle guide for ultrasound-guided central vein puncture improved success rate at first pass, reduced the number of punctures/redirections and shortened the procedure duration compared to OOP and IP free-hand approaches. | PMC10543855 | ||
Supplementary Information | The online version contains supplementary material available at 10.1186/s13054-023-04661-w. | PMC10543855 | ||
Keywords | PMC10543855 | |||
Introduction | perforation | Current guidelines recommend ultrasound guidance for central venous catheter (CVC) insertion [For optimal safety, ultrasound-guided CVC insertion requires to visualize the vessel and the needle. Two approaches are available to visualize the needle: the out-of-plane (OOP) approach and the in-plane (IP) approach [Conversely, with IP approach, the needle is within the plane of the ultrasound beam and is tracked from skin's perforation to vessel penetration. As a result, IP approach might enhance safety by minimizing the risk of injury to adjacent structures. However, IP approach requires accurate needle alignment with the ultrasound beam making this approach highly demanding. Needle guiding devices or methods to facilitate IP approach are available but tend to be expensive (several hundred €), sometimes specific to particular probes, and remain poorly investigated so far [ | PMC10543855 | |
Methods | angulation | We designed an open-source 3D printed needle guide to facilitate IP-CVC insertion. The 7.5-MHz linear probe HFL38 available on the M-Turbo® device (Sonosite, Bothewell, MA) was scanned with an EinScan Pro 2X device. Scanning process required two hours per model. We obtained the 3D mold of the probe. The needle guide made of poly lactic acid was wrapped around the digitalized probe using Autodesk Fusion 360. A dedicated needle-guiding railway was incorporated into the mold along the narrow side of the probe. The railway must allow adjustments in needle angulation, while also maintaining tightly the needle in the ultrasound beam. Finally, the needle guide was 3D printed on a Prusa I3mkiiiS printer (Prusa Research, Praha, CZ). Six needle guide iterations were tested before obtaining the perfect fit for the probe (Additional file We conducted a prospective, randomized study among residents and board-certified physicians from either ICU or emergency department to assess the needle guide in the setting of central venous puncture performed on inanimate manikin. All the participants had received a training in ultrasound-guided CVC insertion, but their proficiency was inhomogeneous. The participants were classified according on whether they had performed more or less than twenty CVCs insertion on patients. All participants were given a one-hour US-CVC lecture about US-guided IP and OOP approaches on simulator.Two simulators were used i) (the Blue Phantom II, CAE Healthcare St. Louis, MO) which allows internal jugular and subclavian puncture and ii) (the Gen II Femoral Vascular Access Ultrasound Training Model, CAE Healthcare St. Louis, MO) which permits femoral vein puncture. Blue fluid return confirmed venous puncture, whereas return of red fluid ruled in arterial puncture. Before the study, the participants were given a 10-min session to the needle guide and the simulators. Each operator performed needle puncture of the jugular, subclavian and femoral veins on the simulators, with three different techniques, assigned in random order: out-of-plane free hand (OP-FH), in-plane free hand (IP-FH) and in-plane with needle guide (IP-NG) (Additional file We recorded: success rate at first pass, number of needle redirections (and skin breaches) and duration of different parts of the puncture procedure (i) from skin contact to first skin puncture, (ii) from first skin puncture to successful venous puncture and (iii) from skin contact to successful venous return (entire puncture procedure). Uncomplicated puncture was defined as puncture (with venous return) performed in less than 120 s without arterial puncture.Data are expressed as median [1st and 3rd interquartile]. Success rates are compared using Chi-2 or Fisher exact test as required. The number of needle re-directions and the procedure time are compared using bilateral Wilcoxon matched pairs test. Significance is set for p less than 0.05. | PMC10543855 | |
Discussion | injury of posterior venous wall | STERILE | Whereas guidelines actually recommend ultrasound use for CVC insertion, some uncertainty remains for the best approach to use between OOP and IP [Out-of-plane approach is most often used for jugular and femoral CVC insertion owing to its convenience. However, it is associated with risk of injury of posterior venous wall [We demonstrate here that, compared to IP-FH and OOP-FH, IP-NG approach significantly increases success rate at first pass in all sites. This is in keeping with previous studies reporting similar success rate with guiding system for jugular (82% and 81%) [Accidental arterial puncture with the NG-IP approach was observed only once at the jugular and subclavian site and never at the femoral site. Using a different needle guiding system, Augoustides et al. reported a rate of accidental arterial puncture at jugular site of 10%, similar to the rate observed with the landmark approach [The study reported here presents anyway several limits, the most important being that we should confirm our data in the clinical setting. Moreover the device was not used under a sterile sheath. This has not been investigated in the present study. The prototype had first to be assessed in a simulation model before being tested in the real life.We chose to include operators with limited proficiency considering that whether guide improves performance this would be true especially among operators with limited experience. Compared to IP-FH and OOP-FH, IP-NG translated in shorter total procedure duration only for subclavian puncture whatever proficiency. It should also be outlined that all IP-NG subclavian punctures (considered as the more complex puncture) performed by operators with the smallest experience were completed in less than 48 s. This confirms that needle-guiding interest is relevant especially for subclavian puncture [This study presents the first ultrasound needle-guiding device for IP (CVC) insertion, constructed using an open-source and homemade method. The cost of production and ease of construction are noteworthy. This approach could allow every operator to obtain a guiding device that is adapted to his ultrasound probes.The online version contains supplementary material
| PMC10543855 |
Author contributions | Antoine Villa and Eric Maury made substantial contributions in conception, study design and data gathering. Vladimir Hermand performed the process of the 3D printing. Antoine Villa wrote the manuscript. All authors corrected and approved the final version of the manuscript. | PMC10543855 | ||
Funding | No funding for the present study. | PMC10543855 | ||
Availability of data and materials | Datasets were collected on an Xcel file are stored by AV and EM and can be accessed upon request. | PMC10543855 | ||
Declarations | PMC10543855 | |||
Ethical approval and consent to participate | This simulation study was conducted on inanimate manikin and did not require ethical approval. | PMC10543855 | ||
Competing interests | The authors certify that there is no conflict of interest with any financial organization regarding the material discussed in the manuscript. | PMC10543855 | ||
References | PMC10543855 | |||
Background | T2DM | TYPE 2 DIABETES MELLITUS | Dipeptidyl peptidase-4 inhibitors (DPP-4i) have become firmly established in treatment algorithms and national guidelines for improving glycemic control in type 2 diabetes mellitus (T2DM).To report the findings from a multicenter, randomized, double-blind, placebo-controlled phase 3 clinical trial, which was designed to assess the efficacy and safety of a novel DPP-4 inhibitor fotagliptin in treatment-naive patients with T2DM. | PMC10563289 |
Methods | T2DM | Patients with T2DM were randomized to receive fotagliptin ( | PMC10563289 | |
Results | hypoglycemia | HYPOGLYCEMIA, ADVERSE EVENTS | After 24 weeks, mean decreases in HbA1c from baseline were -0.70% for fotagliptin, -0.72% for alogliptin and -0.26% for placebo. Estimated mean treatment differences in HbA1c were -0.44% (95% confidence interval [CI]: -0.62% to -0.27%) for fotagliptin versus placebo, and -0.46% (95% CI: -0.67% to -0.26%) for alogliptin versus placebo, and 0.02% (95%CI: -0.16% to 0.19%; upper limit of 95%CI < margin of 0.4%) for fotagliptin versus alogliptin. So fotagliptin was non-inferior to alogliptin. Compared with subjects with placebo (15.5%), significantly more patients with fotagliptin (37.0%) and alogliptin (35.5%) achieved HbA1c < 7.0% after 24 weeks of treatment. During the whole 52 weeks of treatment, the overall incidence of hypoglycemia was low for both of the fotagliptin and alogliptin groups (1.0% each). No drug-related serious adverse events were observed in any treatment group. | PMC10563289 |
Conclusions | T2DM | In summary, the study demonstrated improvement in glycemic control and a favorable safety profile for fotagliptin in treatment-naive patients with T2DM. | PMC10563289 | |
Trial registration | ClinicalTrail.gov NCT05782192. | PMC10563289 | ||
Supplementary Information | The online version contains supplementary material available at 10.1186/s12916-023-03089-x. | PMC10563289 | ||
Keywords | PMC10563289 | |||
Background | T2DM | TYPE 2 DIABETES MELLITUS | The prevalence of type 2 diabetes mellitus (T2DM) has increased markedly with an estimated number of 347 million individuals worldwide in 2008, and forecasted to increase to 7079 individuals per 100, 000 by 2030 [Dipeptidyl peptidase-4 inhibitors (DPP-4i) could improve glycemic control by preventing the rapid degradation of incretin hormones and inhibiting glucagon secretion [This is the first phase 3 randomized, double-blind, placebo-controlled clinical study to evaluate the efficacy and long-term safety of fotagliptin in treatment-naive T2DM patients uncontrolled with diet and exercise intervention, comprising 24 weeks of double-blind treatment period followed by an open-label treatment period, making up a total of 52 weeks. | PMC10563289 |
Methods | PMC10563289 | |||
Study design and randomization | T2DM | TYPE 2 DIABETES MELLITUS | Patients with type 2 diabetes mellitus (T2DM) were randomized to receive fotagliptin, alogliptin or placebo. Subjects who met the inclusion criteria would enter the 4 weeks of placebo run-in period. At the end of the run-in period, a baseline enrollment evaluation was performed. Upon evaluation, all eligible subjects would enter the 24 weeks of double-blind treatment period and were randomized into the fotagliptin group (12 mg once daily) or alogliptin group (25 mg once daily) or placebo group at a 2:1:1 ratio. Randomization and drug dispensation were performed with an interactive web response system (IWRS;eBalance version 5.3.7). A stratified randomization method with the permuted block randomization algorithm was used. The blocks were dynamically allocated to each site and stratum from the randomization list. A unique ID number was provided by the vendor and marked on the medication box. Using central randomization, randomization codes were assigned to eligible participants by the IWRS system based on stratification factors (baseline HbA1c level < 8.5% or ≥ 8.5%) and the block size. After 24 weeks of double-blind treatment, subjects would enter the extended open-label treatment period. Subjects in the placebo group were to be switched to fotagliptin (12 mg once daily) treatment, while patients in the fotagliptin and alogliptin groups continued the same treatment until the end of the whole 52 weeks.The trial was conducted in accordance with the | PMC10563289 |
Population | impaired renal or hepatic function, diabetes, T2DM, unawareness | RECURRENT SEVERE HYPOGLYCAEMIA, COMPLICATIONS, DIABETES | Subjects who were 18–75 years old and with previously untreated T2DM (no oral or injected anti-diabetes treatment before 8 weeks of randomisation) were eligible for screening. After a 4-week diet and exercise run-in period, eligible study participants with poor glycemic control (hemoglobin A1c [HbA1c] values of 7.5% to 10.5% inclusive and fasting blood glucose [FBG] ≤ 13.9 mmol/L) were included in the trail. Main exclusion criteria were: treatment with any antihyperglycaemic medication within the run-in period of the trial; hypoglycaemic unawareness or recurrent severe hypoglycaemia; anaphylactic reaction or contraindication to any IMP or placebo; impaired renal or hepatic function; acute or severe chronic complications of diabetes. The full inclusion and exclusion criteria are avaliable in Supplementary table | PMC10563289 |
Outcomes and assessments | hypoglycemia, T2DM | HYPOGLYCEMIA, EVENTS, SECONDARY, ADVERSE EVENTS | The primary efficacy endpoint was to evaluate the HbA1c from baseline to Week 24 in T2DM patients treated with fotagliptin 12 mg/day, in parallel control with alogliptin and placebo. The secondary endpoints were change in HbA1c from baseline to Week 52; change in FBG from baseline to Week 52; occurrence of hypoglycemia from baseline to Week 52; change in bodyweight from baseline to Week 52. All adverse events were followed up by investigators and have been sufficiently characterised. The following adverse events were to be reported: serious adverse events occured in more than 2% of the patients in either treatment group; any adverse events occurred in more than 5% patients in either treatment group; any other safety events of special interest. | PMC10563289 |
Statistical analysis | hypoglycemic | ADVERSE EVENTS | The primary objective was to determine the superiority of fotagliptin over placebo in the change of HbA1c from baseline to Week 24, and the two-sided test of superiority/non-inferiority design was adopted with α = 0.05 and β = 0.20. According to the phase 3 clinical trial results of other drugs in the same class, it is estimated that the primary efficacy endpoint of HbA1c difference between fotagliptin and placebo is 0.45% [The primary analysis was performed in the full analysis set (FAS), which received at least one dose of the study drug and had at least one posttreatment measurement of the endpoint during the double-blind treatment period. Missing data were imputed using the last observation carried forward (LOCF). HbA1c values at follow-up visits of subjects received rescue were treated as missing values. As a dependent variable, the change of HbA1c from baseline to Week 24 was analyzed using the analysis of covariance (ANCOVA) model. With the treatment group as the fixed effect and the baseline HbA1c as a linear covariate, we assessed the least squares mean and standard error of the primary efficacy endpoint of each treatment group, as well as the differences of the primary efficacy endpoint and its standard error or 95% confidence interval between groups.Descriptive statistics (frequency and percentage) were used to summarize the patient demographics, incidence of adverse events and hypoglycemic episodes by treatment group. Continuous variables, such as physical examination and clinical laboratory evaluations, were summarized by means ± standard deviation (SD) for normally distributed data or medians (interquartile ranges) for non-normally distributed data. All statistical tests were two-sided, with | PMC10563289 |
Results | T2DM, SD | REGRESSION, EVENTS, SECONDARY, ADVERSE EVENTS | In all, a total of 836 patients with T2DM from 56 sites were screened for eligibility. After the run-in period, 458 patients were randomized in the double-blind period as follows: 230 patients in the fotagliptin group, 113 patients in the alogliptin group, and 115 patients in the placebo group. In the fotagliptin group, alogliptin group and placebo group, 19 (8.2%), 14 (12.3%), and 9 (7.8%) patients prematurely discontinued the study and the most common reason for study discontinuation was voluntary withdrawal (9, 10, and 9 patients, respectively). There were 91% (416/458) patients compliant with the study drug dosing throughout the double-blind period while 94% (389/416) of these patients completed the whole 52-week treatment period. Details of the patients’ disposition are showed in Fig. Study patient dispositionDemographic and clinical characteristics of the patients at baseline1 Data were means ± SD or medians (interquartile ranges) for skewed variables or numbers (proportions) for categorical variables2 P for trend was calculated for the linear regression analysis tests across the groups. 3 The primary efficacy endpoint was change from baseline in HbA1c at Week 24. As shown in Fig. Differences in primary clinical end points (Full-analysis-set). Compared with subjects with placebo, significantly more patients with fotagliptin and alogliptin achieved the HbA1c targets (< 7.0% and ≤ 6.5%) after 24 weeks of treatment: 20.7%, 20.0% and 4.4% patients achieved HbA1c ≤ 6.5% in the fotagliptin group, alogliptin group and placebo group, respectively; 37.0%, 35.5% and 15.5% patients achieved HbA1c < 7.0% in the fotagliptin group, alogliptin group and placebo group, respectively (Fig. Differences in secondary endpoints. (Full-analysis-set). In contrast to placebo, fotagliptin and alogliptin resulted in significantly greater decrease in mean FBG after 24 weeks of treatment (Fig. Changes in additional efficacy measures from baseline to Week 24 and Week 52 are provided in Table Change in additional secondary measures of efficacy from baseline to week 24 and week 52Table Summary of clinical adverse events in the treated set of safety population1 The safety population was defined as patients who took at least one dose of therapeutic drug or placebo2 Data are number (% of total participants in treatment group)3 *4 This table lists serious adverse events that were present in more than 2% of the patients in either trial group, adverse events that were considered to be related to therapeutic drug or placebo in more than 5% of the patients in either trial group, and any other safety events of special interest | PMC10563289 |
Discussion | T2DM, diabetes | EVENTS, DIABETES | The multicenter clinical study showed that in T2DM patients inadequately controlled with diet and exercise intervention, fotagliptin 12 mg once daily for 24 weeks provided superior glycemic control compared with placebo, as assessed by reductions in HbA1c and FBG. No clinically significant difference in the improvements in clinical response of glycemic control was observed between the fotagliptin group and the alogliptin group.HbA1c represents the most powerful predictor of diabetes related outcomes and mortality [Fotagliptin is a novel highly selective DPP-4 inhibitor under clinical development for the increased levels of intact bioactive GIP and GLP-1. Based on previous studies, fotagliptin could increase active GLP-1 concentrations and have no obvious influence on DPP-8 and DPP-9, thus making it safer to treat T2DM [However, limitations existed in the present trial. First of all, there is a potential limitation in interpreting effect of fotagliptin compared with placebo on risk of major cardiovascular (CV) events, as the study duration may be too short to modulate CV related clinical outcomes. Four previous CV outcome trials of DPP-4 inhibitors have demonstrated a non-inferior risk of a composite CV outcome when compared with placebo [ | PMC10563289 |
Conclusion | hypoglycemia, weight gain, T2DM | HYPOGLYCEMIA | Overall, the trial demonstrated improvement in glycemic control for fotagliptin monotherapy with 12 mg once daily in previously untreated T2DM patients uncontrolled with lifestyle intervention. Furthermore, fotagliptin treatment was not associated with greater risk of hypoglycemia episodes and weight gain, as compared with placebo and alogliptin. Thus, fotagliptin is a potential new approach for the treatment of T2DM patients who are inadequately controlled with diet and exercise intervention. | PMC10563289 |
Acknowledgements | We are indebted to the participants in the present study for their outstanding support and to our colleagues for their valuable assistance. Medical writing and editorial support were provided by Xiangnan Zhou (Clinical Operation Director at Shenzhen Salubris Pharmaceuticals), Chongyang Deng (Medical Manager at Shenzhen Salubris Pharmaceuticals) and Yue Yang (Statistician at Shenzhen Salubris Pharmaceuticals). | PMC10563289 | ||
Authors’ contributions | Protocol design: LY, MX, KS and WX. Project Administration: WX and JS. Methodology and Resources: all authors. Data analysis: KS and LY. Validation: all authors. Manuscript writing: KS. Review and editing: all authors. The authors read and approved the final manuscript. | PMC10563289 | ||
Funding | This study was funded by Shenzhen Salubris Pharmaceuticals Co.,Ltd. The study was also funded in part, by grants from the Shenzhen Science and Technology Program for Undertake the National Science and Technology Major Project (CJGJZD20210408091600001). | PMC10563289 | ||
Availability of data and materials | The data that support the findings of this study are available on request from the corresponding authors after the completion of the study. The data are not publicly available due to privacy or ethical restrictions. | PMC10563289 | ||
Declarations | PMC10563289 | |||
Ethics approval and consent to participate | The study was approved by the ethics committee at each study site and conducted according to the Declaration of Helsinki, Guidelines for Good Clinical Practice, and local laws and regulations. All patients provided written informed consents. | PMC10563289 | ||
Consent for publication | All authors believe that the manuscript represents valid work and have reviewed and approved the final version. The work has not been published previously, and is not under consideration for publication elsewhere, in part or in whole. | PMC10563289 | ||
Competing interests | Wenjie Xu and Jingchao Sun were reported being employed at Shenzhen Salubris Pharmaceuticals. The remaining authors declare no competing interests. | PMC10563289 | ||
References | PMC10563289 | |||
Objective | A wide array of methods exist for processing and analysing DNA methylation data. We aimed to perform a systematic comparison of the behaviour of these methods, using cord blood DNAm from the LIMIT RCT, in relation to detecting hypothesised effects of interest (intervention and pre-pregnancy maternal BMI) as well as effects known to be spurious, and known to be present. | PMC9901304 | ||
Methods | DNAm data, from 645 cord blood samples analysed using Illumina 450K BeadChip arrays, were normalised using three different methods (with probe filtering undertaken pre- or post- normalisation). Batch effects were handled with a supervised algorithm, an unsupervised algorithm, or adjustment in the analysis model. Analysis was undertaken with and without adjustment for estimated cell type proportions. The effects estimated included intervention and BMI (effects of interest in the original study), infant sex and randomly assigned groups. Data processing and analysis methods were compared in relation to number and identity of differentially methylated probes, rankings of probes by p value and log-fold-change, and distributions of | PMC9901304 | ||
Results | There were differences corresponding to each of the processing and analysis choices. Importantly, some combinations of data processing choices resulted in a substantial number of spurious ‘significant’ findings. We recommend greater emphasis on replication and greater use of sensitivity analyses. | PMC9901304 | ||
Introduction and Background | overweight or obese | DISEASE, EARLY PREGNANCY | With the advent of high-throughput assays, epigenome-wide DNA methylation studies have become more popular, and researchers are now investigating the effects on DNA methylation (DNAm) of a wide range of environmental exposures and physiological conditions, with particular interest in the contribution of epigenetic mechanisms such as DNAm to the early life origins of health and disease. The ability to perform EWAS is particularly useful in relation to conditions where associated differences in DNAm are likely to be fairly modest (We recently investigated the effect of an antenatal diet and lifestyle intervention, and of maternal early pregnancy BMI, on neonatal cord blood DNA methylation in infants of mothers who were overweight or obese in early pregnancy (While previous studies comparing different methods have also produced different findings, these have tended to consider only one element of the processing and analysis pipeline (such as normalisation, or batch correction) in isolation. In addition, they have tended to concentrate on the tendency for some methods to produce results which are likely to be spurious (false positives), while often being unable to definitively confirm this due to the lack of known truth regarding the presence and magnitude of differential methylation effects.We therefore set out to investigate the impact of different data-processing choices in a more systematic way, looking at the effect of combinations of data processing choices on findings specifically regarding statistically significant differentially methylated probes (DMPs), and of the behaviour of these combinations in cases where effects are known to be either absent or present, as well as their behaviour in relation to our effects of interest (maternal BMI and lifestyle intervention). We were able to create a scenario in which effects were known to be absent by randomly assigning samples to groupings. We could not similarly ensure a scenario where effects were known to be present (as the truth regarding the existence, location and magnitude of any effects in our samples is not known); however we investigated effects of infant sex as a rough proxy, since infant sex is known to have substantial effects on DNAm which can be detected by the 450K array ( | PMC9901304 |
Data and Methods | PMC9901304 | |||
The LIMIT randomised controlled trial | LGA | EARLY PREGNANCY | The LIMIT study was a randomised, controlled trial of an antenatal diet and lifestyle intervention for women with early pregnancy BMI ≥25.0 kg/mThe primary outcome of the LIMIT study was birth of a large for gestational age (LGA) infant. There were no significant differences observed between the groups in relation to this outcome; however, a significantly lower incidence of high birthweight (>4 kg) was observed in the Lifestyle Advice group, with a Relative Risk of 0.82 (95% CI: 0.68, 0.99, As previously outlined in the companion paper (For the additional analyses investigating known spurious effects, artificial (fake) groups were created by assigning samples based on random draws from binomial distributions. The first grouping (‘Tortoiseshell’ All data processing and analyses were undertaken using R version 4.0 ( | PMC9901304 |
Probe and sample filtering | The Probes were filtered using multiple criteria. Firstly, probes were excluded if they had a detection Probe filtering was performed either after normalisation (post-filtered) or prior to normalisation (pre-filtered). The one exception for pre-filtering was when normalising using the BMIQ method, where probes on the X and Y chromosome were retained as this was required in order for the function to run. | PMC9901304 | ||
Normalisation | Normalisation involves making changes to the raw data in order to remove artifactual variation. In the case of Illumina 450K BeadChip arrays, this requires correcting for the presence of two different probe types. Infinium I probes use the same colour signal for methylated and unmethylated CpG and are often used for regions of high CpG density, while Infinium II probes use different colours to differentiate between methylated and unmethylated states (Numerous methods exist for normalising Illumina BeadChip array data, but there is little consensus or guidance on which should be employed in a given context. The main advice is that between-array methods, which normalise across samples, are preferable when global differences between samples are expected, while within-array methods, which normalise probes within each sample, are better suited to effects in which the majority of genes will not be differentially expressed. (Both Subset Quantile Normalisation and Subset-Within-Array-Normalisation were performed using functions in the | PMC9901304 | ||
Batch effects | Batch effects arise when samples are processed in separate groups, creating unwanted variation due, for example, to different reagents, different plates or different scanner settings (There are 12 Illumina 450K arrays (samples) per chip (this is reduced to eight arrays per chip for the more recent 850K array); thus most studies involving large numbers of samples must be run on multiple chips. This introduces extra variability to the data, and may also confound the actual effects of interest, if samples from different groups are not evenly distributed between the batches. These effects must be accounted for in order to obtain valid estimates of the effects of interest.Unlike probe filtering and normalisation, batch effects can be handled at the analysis stage, by adjusting for batch in the analysis model. However, it is also common to address batch effects at the data-processing stage, using a batch-correction algorithm, with the resulting data considered to be free of batch effects (For each of the normalised datasets ( | PMC9901304 | ||
Flowchart of data processing and analysis. | Combinations of data-processing and analysis choices, consisting of six normalised datasets (SQN, BMIQ or SWAN, with probe filering before or afterwards), use or non-use of ComBat processing (supervised or unsupervised), and analysis with either an unadjusted model, a model adjusted for batch, or a model adjusted for batch and cell type proportion. | PMC9901304 | ||
Statistical analysis | Differential methylation was investigated probe-wise using linear models with empirical Bayes variance correction as implemented in the For each contrast in each model, the number and identity (where applicable) of any differentially methylated probes (DMPs) were obtained. For detection of DMPs, The findings from different data-processing choices were then compared along five dimensions:Number and identity of differentially methylated probes (DMPs); for infant sex, the direction of differential methylation (‘down’, corresponding to negative t-statistics or lower methylation in females, Consistency of rankings by Consistency of rankings by logFC for ‘top 10’ probes, as well as the consistency of the logFC estimates. This gives an indication of whether the estimates of effect are similar between methods.Overall distribution of Overall distribution of logFC estimates. Under the null hypothesis of no effect, logFC estimates should be roughly normally distributed around 0. If effects are present, there will be more estimates far away from 0 (with the direction depending on whether the effect is one of hypomethylation or hypermethylation, and the distance depending on the strength of the effect). | PMC9901304 | ||
Number of DMPs for infant sex (female), by probe filtering method, batch correction method, normalisation method and cell type method. |
No adjustment beyond the correction for batch as implemented in the ComBat algorithm. | PMC9901304 | ||
DMPs for effect of maternal BMI in the standard care group. | PMC9901304 | |||
DMPs for fake groups: ‘short-haired’ in ‘Tabby’. | PMC9901304 | |||
Probes ranked in top 10 by | For each probe the rank is given by pre- | PMC9901304 | ||
Top 10 probes by LogFC: infant sex. | Largest LogFC for Infant Sex (female), by normalisation and batch correction method. | PMC9901304 | ||
Top 10 probes by LogFC: BMI in standard care. | Largest LogFC for effect of BMI in standard care group, by normalisation and batch correction method. | PMC9901304 | ||
Top 10 probes by LogFC: ‘short-haired’ in ‘Tabby’. | Largest LogFC for effect of ‘Short-Haired’ in ‘Tabby’ group, by normalisation and batch correction method. | PMC9901304 | ||
Distribution of unadjusted P values by normalisation and batch correction method, for batch and cell adjusted models. | Only models from data where probe filtering was performed post-normalisation are included. The model is one adjusting for batch (either explicitly in the model or | PMC9901304 | ||
Distribution of log-fold-change estimates by normalisation and batch correction method, for batch and cell adjusted models. | Only models from data where probe filtering was performed post-normalisation are included. The model is one adjusting for batch (either explicitly in the model or Below we discuss the effect of each dimension (probe filtering, normalisation, batch effects, cell type correction) on results. | PMC9901304 |
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