To examine the association of weight loss magnitude with changes in cardiometabolic risk markers in overweight and obese women from low socioeconomic areas engaged in a lifestyle intervention. 2) who completed a 12-month lifestyle intervention in low socioeconomic communities in Klang Valley, Malaysia. Analysis of covariance (ANCOVA) was used to compare changes of cardiometabolic risk factors across weight change categories  within intervention and control group.  Analyses were performed on 243 women  across the weight change categories were observed within the intervention group at six months postintervention. Participants who lost 5 to 20% of weight had the greatest improvements in those risk markers  compared to those who did not. Those who lost >2% to <5% weight reduced more waist circumference (−4.24 cm CI: −5.44 to −3.04) and fasting insulin (−0.36 μU/mL CI: −1.95 to 1.24) than those who maintained or gained weight. No significant association was detected in changes of risk markers across the weight change categories within the control group except for waist circumference and adiponectin.  A graded association for changes in waist circumference, fasting insulin, and total cholesterol ( Weight loss of >2 to <5% obtained through lifestyle intervention may represent a reasonable initial weight loss target for women in the low socioeconomic community as it led to improvements in selected risk markers, particularly of diabetes risk. Obesity has become a significant public health threat around the globe. Despite reducing the quality of life , obesityBody weight loss of at least 3% from the baseline level is considered as clinically relevant as such amount associated with improvements in multiple cardiometabolic risk markers , 7. As rHowever, the data regarding weight loss intervention and its effect on cardiometabolic risk markers among overweight and obese women in the low socioeconomic community are scant. Tackling the obesity problem among low-income women presents unique challenges that warrant attention if the weight loss interventions are to be effective. The most common perceived barriers to healthy eating and physical activity encountered by these women were related to cost , motivatHence, the purpose of the present study was to (1) determine the overall prevalence of participants achieving weight loss of 5 to 10%, weight loss of >2 to <5%, weight maintained (±2%) and weight gained (>2%) among overweight and obese women following a 6-month lifestyle intervention and a 6-month maintenance period; (2) examine the correlations between per cent weight loss and change in cardiometabolic risk markers after 12 months; and (3) compare the mean changes of those markers in women who achieved 5 to 20% weight loss to those who did not in both study groups.N = 243). The study was divided into two phases: weight loss intervention phase (first 6 months) and maintenance phase (6–12 months). During the weight loss intervention phase, participants attended monthly 1-hour individual diet counselling and moderate physical activities (in a group format), separately. The participants were advised on a reduced-calorie diet  through education on food labelling, portion control, and food substitution (low fat and low sugar choices). They were also encouraged to do moderate physical activities, 7 days/week. Self-monitoring tools such as pedometer, 3-day food diary, and 3-day physical activity diary with MET calendar were given to the participants to monitor their diet and physical activities. During the maintenance phase, these activities were not supervised by the study researchers, and the frequency of contact was reduced to 2 sessions (at months 9 and 12). The control group received general women's health seminars during the follow-up sessions. The participants were recruited based on the following criteria: (i) body mass index (BMI): 25 to 39.99 kg/m2, (ii) age: 18 to 59 years, (iii) not working/housewives, and (iv) able to speak Malay/English. Those who were morbidly obese (≥40 kg/m2), pregnant, currently on weight management regime, or had physical disability and comorbidities  were excluded from the study. Anthropometric measurements and cardiometabolic risk markers were assessed at baseline, 6, and 12 months and subsequently evaluated by study phases  and weight maintenance phase (6 to 12 months)). All study participants provided written informed consent before the study conduct. The research protocol was approved by the Malaysian Research Ethics Committee (MREC), trial no. NMRR-13-726-16391.This study utilised data from a lifestyle intervention among overweight and obese women in low socioeconomic areas in Klang Valley, namely, the My Body is Fit and Fabulous at home (MyBFF@home) conducted from the year 2013 to 2015. Information on study design and recruitment details were published elsewhere . BrieflyWeight was measured in light clothing and without shoes using a digital scale in kilograms (kg) . Waist circumference (WC) was measured in centimetres (cm) at the midpoint between the lower rib and the iliac crest in triplicate. Measurement details were previously reported [α). Venous blood samples were collected after 10 hours of fasting. FPG, TC, HDL-C, LDL-C, and triglycerides were analysed on clinical chemistry analyser  using appropriate reagents, calibrators, and controls . Plasma insulin was measured using TOSOH AIA-360 system analyser . Insulin resistance was assessed using the homeostasis model assessment of insulin resistance (HOMA-IR) which is calculated as fasting glucose (mmol/L) × insulin (μU/mL)/22.5 [α were measured by enzyme-linked immunosorbent assay (ELISA)  according to the manufacturer's instruction. hs-CRP levels were determined by a high sensitivity ELISA .Cardiometabolic risk markers assessed in this study including fasting plasma glucose (FPG), fasting insulin, HOMA-IR as an indicator for insulin resistance, lipid profiles, and adipokines such as adiponectin, high sensitivity C-Reactive Protein (hs-CRP), and tumour necrosis factor-alpha (TNF-mL)/22.5 . The serDescriptive data were tabulated as means ± standard deviation (SD) or 95% confidence intervals (95% CI) as appropriate. The per cent weight change was calculated as weight at time 2–weight at time (1)/weight at time 1 × 100%. The participants in both study groups were stratified into four weight change categories: (1) >2% gain, (2) ±2% maintain, (3) >2 to <5% loss, and (4) 5 to 20% loss of initial body weight . Due to The baseline characteristics across the weight change category were compared using analysis of variance (ANOVA), and statistically different variables were used as covariates in the later analysis. The distribution of participants in the intervention and control group by weight change category were compared using the chi-square test. The correlations between the magnitude of weight loss and cardiometabolic risk markers were assessed using Spearman's (nonadjusted) and partial correlation  by study groups.p < 0.05.The mean changes in the outcome variables were stratified by weight change category, and the differences were evaluated using analysis of covariance (ANCOVA) within intervention and control groups. The covariates within the statistical model included age, baseline weight, and baseline measurements of variable outcomes. The ANCOVA analyses were verified for the equal group variance, independence of covariate and treatment effects, and homogeneity of regression slopes. The results were presented in estimated marginal means with 95% CI. All statistical analyses were conducted using Statistical Package for Social Science (SPSS) for Windows© version 22.0 . The analyses were tested at two-sided, and the significance level was set at The distribution of participants in each weight loss category was summarised in However, the proportion of those losing >2 to <5% and 5 to 20% weight dropped by 1.5% and 7.1% in the intervention and control groups, respectively, during the weight maintenance phase. While the proportion of weight gained >2% increased approximately threefold in both study groups, only a slight decrease was observed in the proportion of participants who maintained their weight during this phase. However, the chi-square test revealed no statistically significant differences across weight loss categories between study groups in both study phases.r = 0.366), fasting insulin (r = 0.228), HOMA-IR (r = 0.270), and FPG (r = 0.228) after adjusting for covariates, whereas weak correlations were found for changes in other lipid measures and inflammatory markers. Significant correlations were also observed in the control group between change in weight and changes in WC, fasting insulin, and HOMA-IR (0.218 ≤ |r| ≤ 0.278). However, the magnitude of these correlations was smaller compared to the intervention group. The changes in other risk markers showed a weak correlation to change in weight.Correlations of the magnitude of changes between weight in percentage and cardiometabolic risk markers after 12 months were presented in p=0.002, for all variables) across the weight change categories were observed within the intervention group where participants who lost 5 to 20% weight showed the most favourable changes compared to other weight change categories. In direct comparison, improvements in WC and fasting insulin of participants who lost >2% to <5% were greater than those who maintained ±2% weight and gained >2% weight, albeit insignificant difference between the categories. In contrast, the magnitude of improvements in other markers did not significantly differ across the weight change categories.p < 0.001). No significant association was detected in changes of other markers across the weight change categories.Similarly, the control group exhibited a graded association for changes in WC where more reduction in WC occurred with higher weight loss. Those who lost 5 to 20% of initial weight increased adiponectin levels substantially than those who gained or maintained weight (p < 0.01 for both groups). Thereby participants who lost between >2 and <5% and 5 to 20% weight had more WC reduction than those who maintained and gained weight, whereas no significant differences were found in mean changes of other risk markers when compared across the weight change categories during this phase. While in the control group, there was a significant difference between mean changes in HDL-C (p=0.018), the improvement was observed in those who gained weight compared to the other weight change categories.The mean changes in cardiometabolic risk markers across categories of weight change during the weight loss maintenance phase were presented in The present study showed that the majority of participants either lost >2 to <5% of weight or maintained their weight  following a 6-month lifestyle intervention, and the proportion was higher than that in the control group. Although such an amount of weight loss was trivial, it was observed that the lifestyle intervention assessed in this study may have prevented further weight gain. Prevention of weight gain is equally essential in a weight loss programme which not only may attenuate the obesity rate but also reduce the risk of chronic diseases including T2DM, CVD, cancer, and nontraumatic death . HoweverOf interest, participants in the control group showed improvement in weight whereby the proportions of those losing 5 to 20% weight were relatively higher in both study phases compared to the participants in the intervention group. Unanticipated improvement in the control group was common in a controlled intervention study as individuals who are willing to participate often have a strong desire and motivation to change their habits . As a maNonetheless, the proportion of individuals achieving at least 5% weight loss in the present study was lower compared to other intervention studies among overweight and obese women in the low-income community ranging from 19% to almost 50% –26. ThosThe magnitude of weight change after one-year was found significantly correlated to WC, fasting insulin, HOMA-IR, FPG, and triglycerides in the intervention group. The findings suggest that changes in those markers can be attributed to weight loss induced through lifestyle intervention. Significant correlations were also observed in the control group between change in weight and improvements in WC, fasting insulin, and HOMA-IR. The correlations within the control group were expected due to similar average weight loss to the intervention group  after one year as reported in the main study , whereasFurthermore, a significant graded association was found between weight loss and changes in WC, fasting insulin, and TC following six months of intervention but the association disappeared during the weight maintenance phase. More reduction was seen in WC as more weight loss occurred and this clear association was found in both study groups. Interestingly, WC reduced in each weight loss category during both study phases. Also, those who lost >2 to <5% of weight reduced WC by more than 3 cm which is likely to alleviate the aggregation of risk factors that contribute to metabolic syndrome . The fin2 showed significant beneficial changes in lipid profiles following modest weight loss at 2 to 3 years follow-up. However, the review concluded that the association diminished over the long term suggesting other lifestyle factors such as dietary fat intake, per se caloric restriction, and physical activity that also need to be sustained.For lipid profiles, only TC showed a significant association with weight loss during the weight loss intervention phase within the intervention group. TC was greatly reduced (−0.59 mmol/L 95% CI: −0.99 to −0.19) in individuals who lost between 5 to 20% compared to other weight change categories. Conversely, Swift et al.  reportedThere are several limitations to the present study worth mentioning. The present study utilised data from a quasiexperimental study which does not involve randomisation. Although the study design offers the feasibility to conduct in the community, it may have introduced a bias in selection and limits external validity. Nevertheless, no compelling differences were observed in the baseline characteristics and most of the variables outcomes between the study groups . The basProjek Perumahan Rakyat/Awam (PPR/PPA), a low socioeconomic community in Klang Valley which were predominantly Malay and were healthy upon participation in the intervention. Therefore, the findings were unable to be generalised to other populations of low-income women residing in different geographic locations. This study was also not powered to detect significant differences between groups in cardiometabolic risk markers as it was in accordance with the main study . Hence, the insignificant relationships between weight changes and improvements in some of the cardiometabolic risk markers should be interpreted with caution.Also, although the study locations of the intervention and control groups were separated geographically , there may be a chance of control contamination as the radius of the study locations were just below 30 kilometres. To limit the contamination between the groups, participants were followed up at their respective residences on different days. The present study included women living in Though it was clearly shown that the degree of improvement in selected risk markers was related to the magnitude of weight loss, other factors such as changes in diet and physical activity may also result in positive impacts on those risk markers. However, it is impossible to discriminate the effects of weight loss per se with other impacts from the intervention components in the programme promoted. The present study focused on weight change per se to address the benefits of clinically relevant weight loss on cardiometabolic risk markers. However, other indicators of adiposity including body fat mass and visceral fat mass may have relationships with the magnitude of weight loss and more relevant to changes in cardiometabolic risk markers.The strengths of the present study include the first weight loss intervention study focusing on low socioeconomic women, a group with increasing rates of obesity as well as having a challenging environment for a healthy lifestyle. Furthermore, the findings owing to limited obesity research among adults in Malaysia whereby most studies were conducted as cross-sectional, qualitative, and systematic reviews and only a small number of intervention studies were currently published . PerhapsThe findings of the present study suggest that weight loss of >2% to <5% obtained through a short-term lifestyle intervention could improve risk markers including WC and glucose homeostasis in overweight and obese women of low socioeconomic level. The findings also reaffirm the benefits of a more achievable weight loss target to encourage women in this group to change their lifestyle to a healthier one.


The fermentation quality and chemical composition were analyzed after 7, 14, 30, and 60 d, respectively. The 60-d silages were subjected to an aerobic stability test and fermented with buffered rumen fluid to measure the digestibility.The mulberry leaves were ensiled either untreated (control) or treated with 1×10Inoculating lactic acid bacteria (LAB) resulted in more rapid increase in lactic acid concentrations and decline in pH of mulberry leaves silage as compared control. Higher acetic acid and lower ethanol and ammonia nitrogen concentrations (p<0.05) were observed in the LAB-inoculated silages as opposed to control during ensiling. The LAB-inoculated silages contained lower water-soluble carbohydrates compared with control during the first 14 d of ensiling, and lower neutral detergent fibre (p<0.05) concentrations as compared with non-LAB inoculated silages. Adding molasses alone increased (p<0.05) the digestibility of dry matter (DM). The aerobic stability of mulberry leaves silage was increased by LAB inoculation, whereas decreased by adding glucose or molasses.The LAB inoculation improved fermentation quality and aerobic stability of mulberry leaves silage, while adding glucose or molasses failed to affect the fermentation and impaired the aerobic stability. Inoculating LAB alone is recommended for mulberry leaves especially when ensiled at a relatively high DM. The rapid development of China’s economy has triggered increasing demand for animal products from consumers. One challenge for further improvement of animal products has been the lack of adequate and high-quality green fodder. Exploring novel feed resources capable of replacing regular poor-quality fodder is important to alleviate this constraint.Morus alba L.) originated from China and has been used widely in agriculture. They can grow well under various climate conditions ranging from temperate to tropic [Mulberry . Five plots of mulberry trees were randomly selected. On November, 2, 2016, the entire plant was harvested when reached approximately 120 cm, with a stubble of 20 cm above soil level. Leaves and stems were separated in a separator machine and the leaves were subsequently transported to laboratory. After wilting at room temperature (25°C) for 6 h, the mulberry leaves were chopped into a length of 2 to 3 cm with a forage chopper .Mulberry trees were cultivated in the experimental field of Nanjing Agricultural University , which contained 10 plots (200 mLactobacillus plantarum). The application rate was 1.0×106 colony forming units (cfu)/g of FM, according to the manufacturer’s specification. For the preparation of each silo, approximately 620 g of mulberry leaves were packed into a laboratory silo  and manually compacted to a uniform density at 278 kg DM/m3. After being sealed with plastic caps and adhesive tapes, the silos were stored at ambient temperature (18°C to 22°C). Five replicates per treatment were randomly selected and opened after 7, 14, 30, and 60 d, respectively. The remaining silos were used for aerobic stability test after 60 d of ensiling.Chopped mulberry leaves were divided into six groups. The six groups were randomly assigned to the following treatments: i) control (without additive); ii) L, LAB inoculant; iii) G, glucose; iv) M, molasses; v) L+G, a combination of L and G; or vi) L+M, a combination of L and M. The application rates of glucose and molasses were 1% and 3%, based on fresh matter (FM), respectively. The LAB inoculant used was supplied by a commercial company ; the dried samples were analyzed for crude protein , neutral detergent fibre  and acid detergent fibre  with the method of Association of Official Analytical Chemists . The wat3-N) was measured using the method of Sun et al [2SO4, 0.5 mL/min).The second subsample (35 g) was extracted in 70 mL of deionized water at 4°C for 24 h to obtain the cold extract, which was used for analyzing silage fermentation parameters according to the procedure of Chen et al . The pH un et al  and the The microbial populations were determined in the fresh mulberry leaves after ten-fold serially diluted to optimal ranges with sterilized saline solution (0.85% NaCl). The LAB was counted on de Man, Rogosa and Sharpe agar medium after incubation in an anaerobic incubator at 37°C for 3 d. Yeasts and aerobic bacteria were enumerated on potato dextrose agar and Agar medium, respectively, after incubation at 37°C for 2 d. All microbiological data were log10 transformed.2 environment and then mixed with the 1:2 (v/v) buffer solution prepared as the description of Menke [2. After 72 h of incubation, samples were taken out from serum bottles and gently rinsed with cold tap water. Bags were weighed to determine in vitro DM digestibility (DM-D) after dried in oven at 65°C for 48 h. The in vitro NDF digestibility (NDF-D) were determined as losses in the NDF during the in vitro digestion process.Ground 60-d silage samples were put into filter bags . Bags were heat-sealed and transported to 130-mL serum bottles. Rumen fluid was collected from four Boer male goats fed twice daily with a diet that contained 30% corn silage, 20% alfalfa silage, 40% corn grain and 10% soybean meal plus supplemental vitamins and minerals. The rumen fluid was strained through four layers of cheesecloth at 39°C under COof Menke . Sixty mMulberry leaves silage from each treatment was loosely filled into an open plastic bucket. A thermocouple wire was placed at the center of the silage biomass to record the temperature every 30 min for 7 d using a data logger . Ambient temperature was recorded from a thermocouple wire in an empty bucket. To avoid contamination and allow air to penetrate the silage mass, the plastic bucket was covered with a double layer of cheesecloth. Aerobic stability was calculated as the number of hours before the temperature of the silage mass rose 2°C above ambient temperature.ij = μ+Si+Aj+Si×Aj+ɛij with μ the overall mean; Si the effect of treatment; Aj the effect of ensilage time; Si×Aj the effect of the interaction between treatment and ensilage time; ɛij the residual error. Tukey’s multiple comparison was used for the means separation. Significant differences were declared when p<0.05.Statistical analysis was performed using the MIXED procedure of Statistical Analysis System . Silage chemical compositions were subjected to a two-way analysis of variance for a 5×4 (treatment×ensilage time) factorial arrangement using the following model: YThe chemical and microbial compositions of fresh mulberry leaves are shown in 3-N accumulated gradually in all the silages with increasing time of ensiling. LAB-inoculated silages  contained numerically or significantly lower ethanol concentrations as opposed to control, G and M silage during ensiling. Inoculating LAB distinctly depressed the production of NH3-N, as indicated by the lower (p<0.05) NH3-N concentrations in the L, L+G, and L+M silages as opposed to the control, G and M silages during ensiling.The dynamics of fermentation parameters during ensiling of mulberry leaves are shown in Dynamics of DM and WSC concentrations of mulberry leaves during ensiling are given in in vitro digestibility of mulberry leaves ensiled for 60 d are displayed in The effects of additives on the CP, NDF, ADF, and 10 cfu/g FM and the content of WSC is 7.0% DM. However, other important factors, such as DM content and the epiphytic LAB compositions, could also affect the resultant fermentation [10 cfu/g FM) and adequate WSC concentration (11.8% DM).The epiphytic LAB numbers and WSC content have become significant factors in predicting the adequacy of silage fermentation and determining whether or not to apply additives to ensilage materials . Chen etLactobacillus plantarum triggered vigorous LA fermentation. Based on the research of Meeske et al [Lactobacillus plantarum were detectable even when the water activity of the media was 0.949. These observations suggested that, compared with epiphytic LAB, Lactobacillus plantarum has a higher ability to thrive in high DM silage. In the experiment, despite mulberry leaves being wilted for only 6 h, a relatively higher DM (38.5% vs 21.5% DM) content was obtained as compared with that in study of Zhang et al [Lactobacillus plantarum to mulberry leaves provided sufficient number of LAB with high osmotolerant ability; thus, it could be expected to improve the fermentation quality.After 60 d of ensiling high pH 5.96) and low LA concentration (2.61% DM) indicated poor fermentation quality of the control silage. Despite more fermentable substrate provided by molasses and glucose addition, these treatments failed to induce intense LA fermentation suggesting that fermentable sugar was not the limiting factor for improved fermentation quality of mulberry leaves silage. In contrast, inoculation with ke et al , when th6 and lowke et al  found thLactobacillus plantarum, used in the experiment, belongs to lactobacilli and is capable of quickly producing large amounts of LA by fermenting a wide variety of substrates. In fact, the Lactobacillus plantarum is a facultative homofermentation species. They can adapt to diverse conditions by altering metabolisms. Under normal conditions of high sugar content and limited access to oxygen, pyruvate is reduced to LA, a metabolic pathway known as homolactic fermentation. However, when available sugars become limited, Lactobacillus plantarum can alter its metabolism to pentose phosphate pathway, which is characterized by initial dehydrogenation, followed by decarboxylation leading to significant amounts of other end products, including CO2, ethanol, or AA [Lactobacillus plantarum, explaining the highest AA concentration of L silage after 7 d of ensiling.The l, or AA . In the Ethanol can be produced from glucose by yeasts and enterobacteria. Lower ethanol content in LAB-inoculated silages as opposed to non-inoculated LAB silages might be ascribed to the restriction of the ethanol-producing microorganisms by rapid pH decline. These results were similarly obtained by Cao et al , who rep3-N is indicative of proteolytic activity and amino acid deamination and decarboxylation, which typically reduce the nutritive value of silages. The satisfactory ranges of NH3-N (<10% TN) within all the silages indicated no extensive proteolysis occurred. This low extent of protein degradation was possibly associated with the depression of protease activity resulting from the high DM. It was observed that NH3-N concentrations in LAB-inoculated silages were reduced in contrast to non-LAB inoculated silages and this might be attributed to rapid decline of pH that suppressed deamination and decarboxylation of amino acids. Similarly, Guo et al [3-N concentrations during the early stage of ensiling compared with control. In the present experiment, the BA and PA concentrations in all the silages were below the detection limit, indicating that clostridia and other undesirable organisms did not develope in large numbers.The NHuo et al  reportedLactobacillus plantarum for LA production. These results were in accordance with Filya et al [Due to the molasses addition, M silage exhibited highest DM during ensiling. During the first 7 d of ensiling WSC concentration in LAB-inoculated silages were apparently lower compared with non-LAB inoculated silages. The decrease in WSC concentrations coincided with the increase in LA concentrations, suggesting that WSC was largely consumed by ya et al , who fouya et al .After 60 d of ensiling, lower NDF contents in the LAB-inoculated silages were observed in comparison to those in non-LAB inoculated silages. This was likely owing to acid hydrolysis of more digestible cell wall fractions during ensiling. It has been found that arabinose, a key component involved in crossing linking between lignin and arabinoxylans, is sensitive to acid hydrolysis . Solubilin vitro incubation [in vitro digestibility of NDF was unaffected by the treatments. Weinberg et al [Digestibility parameters of forages are considered to be essential values in estimation of their nutritive value to ruminants. The digestibility of silage is influenced by the amount of fermentable sugar and CP available for rumen microbial degradation during cubation . Sahoo acubation  previousrg et al  evaluateAerobic stability is of great importance because it is not only a potential cause of nutrient and DM losses, it also leads to health risks to animals and humans due to mycotoxins produced by undesirable microorganisms. In the experiment, high DM restricted the extent of fermentation, resulting in high concentration of residual WSC in the resultant silage, which was a potential source of readily available substrate for the growth of aerobic microflora when the silages are exposed to air. This may explain the poor aerobic stability of the control silage. Likewise, M treatment increased the WSC concentration retained in the resultant silage, which was probably responsible for the decreased aerobic stability compared with control. In contrast, LAB inoculation reduced residual WSC concentrations, contributing to the increased aerobic stability. Furthermore, it is well known that AA is one of the most effective substances for inhibition of spoilage microorganisms . IncreasThe LAB inoculation improved fermentation quality and aerobic stability of mulberry leaves silage, while adding glucose or molasses failed to affect the fermentation and impaired the aerobic stability. Based on the results obtained in the experiment, inoculating LAB alone is advisable especially when mulberry leaves are ensiled at a relatively high DM.


The relationship between maximal muscle strength (MMS) and bone mineral density (BMD) in males remains unclear. Therefore, the aim of this cross-sectional study was to investigate the association of MMS, using 3 fundamental compound exercises, and body composition with BMD in Chinese male adults. One hundred forty-seven Chinese male adults aged 20 to 47 years were recruited. Total and regional BMD and body composition were measured using dual-energy X-ray absorptiometry. Measurements of MMS included bench press, deadlift, and squat 1-repetition maximum (1RM). Bench press, deadlift, squat 1RM, fat mass (FM), and lean mass (LM) had a significant positive association with BMD. Intriguingly, squat 1RM was found to have a stronger association than bench press or deadlift 1RM, whereas bench press 1RM was found as the strongest determinant of BMD at the forearm sites. Furthermore, LM was found to be stronger related with BMD than FM. Our findings identify LM, FM and MMS are positively associated with BMD and squat may serve as a simple, most efficient strategy to optimize peak total body BMD, while bench press fit best for forearm BMD. Our results validate the benefits of MMS training in males and underscores site-specific effects on BMD levels. These findings emphasize the need for prospective studies to investigate the maximum therapeutic potential and sex specific modifiers of MMS training. Although osteoporosis disproportionately affects women, 1 out of 8 men over the age of 50 experience a fragility fracture, with the most common sites as forearm, spine, and hip.,4 In contrast to the rapid bone loss experienced by postmenopausal women, men will undergo slower bone loss starting at the sixth decade of age at an average rate of 0.5% to 1.0% of total bone mass per year.–7 Recently, the rising incidence of osteoporotic fractures in men has kindled interest in preventative measures for the male population.Osteoporosis is a disease characterized by reduced bone mass and microarchitectural defects whose cumulative effects result in brittle bones with an increased risk of fragility fractures. Both plyometric high-impact exercises and traditional strength training have been reported to increase bone mineral density (BMD) at the hip and lumbar spine in young adults.–12 Resistance training has been suggested as an effective way for preventing bone loss.–15 Maximal muscle strength (MMS), measured as 1-repetition maximum (1RM), has been reported to correlate with BMD in women.–18 Maximal strength training has also been reported as a simple and effective training method for improvement of BMD in young adult women and postmenopausal women with osteoporosis or osteopenia.,20 Furthermore, hand grip strength and leg power has been associated with increasing BMD at the lumbar spine and total hip in men, respectively. With accumulating evidence suggesting the association between muscle strength and BMD, which intervention is most effective remains unclear.Current guidelines recommend strength training and weight-bearing exercises for the prevention of bone loss and maintaining bone mass in patients with osteoporosis.In this present study, we aimed to investigate the association between MMS and body composition and BMD in Chinese male adults. We aimed to find out association between MMS and site specific BMD.22.1From January 2013 to May 2015, a total of 147 Chinese male adults were recruited in Zhejiang Provincial People's Hospital. All participants were from 20 to 47 years old. Those with known metabolic bone diseases or those under any medications likely to influence BMD were excluded from the study. Three candidates were excluded because of history of injury with their upper or lower legs. All of the participants have at least 1-year experience with strength training and are familiar with exercises such as the bench press, deadlift, and squat. All of the participants were informed about the details of procedure and written informed consent in accordance with the Declaration of Helsinki was obtained from all of the participants for the subsequent submission for publication. Our study was approved by the Ethics Committee of the Fuyang First People's Hospital and Zhejiang Provincial People's Hospital.2.2,23 Briefly, height was measured without shoes to the nearest 0.1 cm, weight with only light clothing to the nearest 0.1 kg (Detecto). All values were recorded as the mean of the 3 measures. BMI was calculated as body weight (in kilograms) divided by height (in meters) squared. Dual-energy X-ray absorptiometry  was used to measure lean mass (LM), fat mass (FM) and total and regional BMD through whole-body scans. Regional BMD refers to the mean BMD in the regions of forearm, spine and hip. DXA was calibrated daily using a standard phantom provided by the manufacturer.All participants completed a questionnaire on demographic study. Participants who smoked at least 1 cigarette per day or drank alcohol once a week for at least 6 months were defined as smokers or drinkers. None of the participants were heavy smokers or drinkers. Physical measurements were obtained based on standardized protocol as previous described. loading for the tests were increased or decreased by 2.5 to 5 kg based on subject's self-reported 1 RM performance during the previous 6 months. The rest periods between the trials were around 3 to 5 minutes to avoid possible fatigue. When a participant successfully performed a 1RM, weight was increased by 2.5 kg and the exercise was performed again until the participant was unable to perform the exercise. The traditional bench press was performed in the standard supine position on the flat bench . Participants lowered the bar until touching the sternum and pressed the weight away from the torso until the elbows were fully extended. No pause between the descent and ascent of the barbell was required, and bouncing the weight off the chest disqualified that attempt. An acceptable deadlift was determined by the participants lifting the loaded barbell from the floor until their knees were fully extended. An acceptable squat was judged by lifting the weight through a range of motion in which the greater trochanter of the femur was lowered to the same level as the knee  and coming back to a standing position in which, knees were fully extended. Two experienced spotters stood beside the barbell for safety during the whole exercise and verbally guided the participants to maintain proper form during the maximal lift. The heaviest weight for which a participant could successful perform a 1RM was recorded as the MMS for that exercise. We measured the 3 MMS in order of bench press, deadlift, and squat. The rest period between 2 different MMS measurements was 30 minutes to allow for adequate recovery. All the measurements of one participant were recorded in a single day and the results were collected by the same investigator.MSS for bench presses, deadlifts and squats were determined for each participant by measuring the 1 RM for each exercise. Following multiple warm-up trials described previously,2.3Z test. For linear multivariate regression, we had 7 predictors, in which one was of interest and would be tested, assuming a partial correlation of 0.4. One hundred thirty-eight samples are required for a simple correlation and 83 samples are required for linear multivariate regression, respectively. SPSS  was used for analysis. All statistical tests were 2-tailed, and P < .05 were considered significant.Summary statistics of participants were presented as mean ± standard deviation or numbers  Table  as appro33.1The basic characteristics of 147 participants are shown in Table 3.2r = 0.43, P < .001 versus r = 0.21, P < .01 and r = 0.25, P < .01 for hip BMD, while r = 0.51, P < .001 versus r = 0.27, P < .01 and r = 0.33, P < .01 for total BMD). The bench press has a stronger association with forearm BMD than the deadlift or squat . Regarding body composition, LM has a stronger association with spine, hip and total BMD than FM . Age is associated with spine and hip BMD  while not with forearm and total BMD. BMI was associated with spine, hip and total BMD  while not with forearm BMD, as shown in Table Correlations between MMS, body composition and BMD are shown in Table 3.3In order to mitigate the effects of potential confounders such as age, height, weight, BMI, alcohol consumption and cigarette smoking, multiple linear regression was performed including these as explanatory variables along with MMS, and body composition. Table 4In this study, we demonstrated that both MMS and body composition are significantly positively associated with regional and total BMD. Squat 1RM had a stronger association than bench press or deadlift with femoral neck BMD, whereas bench press 1RM had the strongest association with BMD at the forearm sites. Furthermore, LM body composition was more strongly associated with BMD than FM body composition.–18,25–27 Maximal strength training is therefore recommended as a simple and effective training method for improvement of BMD.,20 However, a previous study demonstrates that squats and deadlifts are effective in increasing BMD in young healthy men, while similar benefits were not observed in women who followed the same protocol. This suggests that either the mechanism of the bone anabolic effects of MMS strength training are different among men and women or that the thresholds for reacting to MMS stimulus are different among the sexes. Additionally, MMS are correlated with regional BMD and not total BMD, implying local rather than systemic effects. Furthermore, while most studies were based on Caucasian populations and focused on women , these results can not necessarily be extrapolated to Asian populations or men. Intriguingly, a study based on Chinese adolescent girls indicated that LM was a stronger independent determinant than FM of bone mineral content, whereas handgrip muscle strength was found to be a significant determinant of bone mineral content at the forearm. These observations are partially consistent with our findings in this study. We investigated the 1RM of the 3 most popular exercises used in strength training. MMS of bench press predominantly affects upper body strength, whereas deadlifts and squats targets core musculature and lower body strength. Our findings indicated that MMS of bench press, deadlift and squat were all positively associated with regional and total BMD. 1RM of squat was stronger associated with BMD than bench press or deadlift, whereas 1RM of bench press as the strongest determinant of BMD at the forearm sites. We also found both LM and FM were positively associated with regional and total BMD, whereas LM has stronger association with BMD than FM.Accumulating evidences demonstrates that muscle strength is positively related with BMD.The major strength of our study is that all the measurements were obtained by DXA by 1 technician using 1 densitometer and all MMS were performed in the same gym by 1 fitness trainer. Additionally, no participant had taken a drug known to interfere with muscle strength, bone homeostasis, fat or lean tissue metabolism. Finally, all the participants were Chinese adult male population living in the same region. A limitation of this cross-sectional study is that causal inferences between MMS and BMD are not possible. Additionally, this study design cannot interrogate what magnitude of changes in MMS are necessary for therapeutic benefit. Furthermore, we did not analyze the lifestyle of participants, such as diet style, physical exercise, calcium supplementation, or vitamin D levels, which may also affect BMD. Additionally, our participants are all Chinese male adult and our results may not be generalized to other ethnicities. Finally, bone turnover markers were not measured in this study. Thus, while the association with increased BMD is purportedly due to the anabolic effects of strength training, suppression of bone catabolism cannot be strictly excluded.To the best of our knowledge, this is the first investigation addressing the association between MMS and body composition with BMD in Chinese male adults. We conclude that MMS was positively associated with BMD. Our findings lend further support for high-load resistance training for increasing peak bone mass prior to age-related bone loss. Our results indicate that squats may serve as a simple exercise to incorporate in a strength training regimen to optimize peak total body BMD, while focusing on the bench press should be considered to mitigate forearm BMD. Further research should focus on longitudinal studies to determine if MMS training prior to the onset of bone mass decline can increase peak bone mass to levels that delay the onset of osteoporosis. Furthermore, additional research is needed to determine if beginning MMS straining after the onset of bone mass decline can rescue or slow the progression towards fragile bones. Finally, given that BMD response to MMS may be sex dependent, mechanistic studies focusing on the link between MMS and BMD in men and women are needed to fully elucidate causal relationships.I thank Dr. Michael-John G. Beltejar from University of Rochester Medical Center for his assistance and thoughtful recommendations. I thank Dr. Jiayin Zheng from Fred Hutchinson Cancer Research Center for his help with statistical analysis.Conceptualization: Fei Chen, Qi Su, Yulan Tu, Jun Zhang, Guokang Xu.Data curation: Fei Chen, Qi Su, Yulan Tu, Tingxiao Zhao, Yazeng Huang.Funding acquisition: Guokang Xu.Investigation: Qi Su, Yulan Tu, Xinji Chen, Yazeng Huang, Guokang Xu.Methodology: Fei Chen, Qi Su, Yulan Tu, Jun Zhang, Xinji Chen, Yazeng Huang, Guokang Xu.Writing – original draft: Fei Chen, Qi Su, Yulan Tu, Jun Zhang, Guokang Xu.Writing – review & editing: Xinji Chen, Tingxiao Zhao, Yazeng Huang, Guokang Xu.


Obesity susceptibility genes are highly expressed in the brain suggesting that they might exert their influence on body weight through eating-related behaviors.To examine whether the genetic susceptibility to obesity is mediated by eating behavior patterns.BMI) using almost 1 million single nucleotide polymorphisms. We used principal component analyses to identify eating behavior patterns, twin modeling to decompose correlations into genetic and environmental components, and structural equation modeling to test mediation models between the PRSBMI, eating behavior patterns, and obesity measures.Participants were 3977 twins , aged 31–37 y, from wave 5 of the FinnTwin16 study. They self-reported their height and weight, eating behaviors (15 items), diet quality, and self-measured their waist circumference (WC). For 1055 twins with genome-wide data, we constructed a polygenic risk score for BMI (PRSh2 = 36–48%) eating behavior patterns labeled “snacking,” “infrequent and unhealthy eating,” “avoidant eating,” and “emotional and external eating.” The highest phenotypic correlation with obesity measures was found for the snacking behavior pattern , largely due to genetic factors in common (bivariate h2 > 70%). The snacking behavior pattern partially mediated the association between the PRSBMI and obesity measures .We identified 4 moderately heritable (Eating behavior patterns share a common genetic liability with obesity measures and are moderately heritable. Genetic susceptibility to obesity can be partly mediated by an eating pattern characterized by frequent snacking. Obesity prevention efforts might therefore benefit from focusing on eating behavior change, particularly in genetically susceptible individuals. SubsRecent advances in genetic studies have increased our knowledge of common susceptibility genetic loci associated with obesity. The meta-analysis of genome-wide association studies (GWASs) of BMI from Locke et al.  identifiA few earlier studies have focused on the associations between eating behaviors and genetic susceptibility to obesity. Two studies performed in adults have reported that uncontrolled eating and emotional eating mediate the association between a polygenic risk score (PRS) comprising 90 BMI-associated loci and obesity measures , 22. A mn = ∼5600 twin individuals) . We excluded participants with unknown zygosity, eating styles, diet quality, and BMI. In mediation analyses we excluded twin individuals with missing genome-wide data. In quantitative twin genetic analyses, we excluded those without a co-twin and without information on zygosity, eating styles, and diet quality. The final data  cohort including Finnish twins born in 1975–1979 and identified from the population register  . The FT1nal data . The datn = 42) of FinnTwin16 participants. Subscales of those instruments were significantly correlated with our eating style items except for those describing health-conscious/avoidant eating and 1 item regarding snacking. However, in the study from Keski-Rahkonen et al.  was calculated. Waist circumference (WC) was self-measured midway between the lowest rib and the iliac crest with a tape. The participants received a tape and instructions by mail on how to self-measure WC. The validity of self-reported height, weight, and WC was previously demonstrated in a subsample of FT16 participants . Genotyping quality control batches were applied for SNPs [minor allele frequency (MAF)]. First, we removed every genetic variant with a call rate <0.975 (batches 1 and 3) or <0.95 (batch 2). Second, we removed every sample with a call rate <0.98 (batch 1) or <0.95 (batches 2 and 3). Finally, variants were filtered by their MAF <0.01 with Hardy–Weinberg equilibrium P value <1e-06. Samples from all batches with heterozygosity test method-of-moments F coefficient estimate values <-0.03 or >0.05 (batches 1 and 2) or ±4 SD from the mean (batch 3) were removed, along with the samples that failed the sex check or were among the multidimensional scaling principal component analysis outliers. Genotyped autosomal variants after quality control were 475,526 (batch 1), 239,894 (batch 2), and 388,673 (batch 3), with the following number of samples remaining for imputation: 2617 (batch 1), 5328 (batch 2), and 8218 (batch 3).Genotyping was performed in the United States, United Kingdom, and Finland  (BMI) and 1 for waist-to-hip ratio (WHR) adjusted for BMI (PRSWHR). The total numbers of genetic variants used for the PRS calculations were 996,919 for BMI and 1,148,565 for WHR adjusted for BMI, with the reweighted effect sizes available from 692,578 and 484,563 samples, respectively  between each genetic variant . We did  27,284) . GWAS su 27,284)  with an 2 test for categorical variables.General characteristics of the study sample are presented as means and 95% CIs for continuous variables and as numbers and percentages for categorical variables. Differences between men and women were determined using the adjusted Wald test for continuous variables and Pearson χEating behavior patterns were derived by principal component analyses (PCAs) using 15 eating styles and 1 indicator of diet quality (DQS). Based on eigenvalues >1.0 and scree plot analyses, we retained 4 components. We calculated factor loadings after a varimax rotation to simplify and facilitate their interpretability. Factor loadings (≥0.30) were considered to contribute to the eating behavior pattern and were used to label the 4 components.We used quantitative genetic twin modeling, based on linear structural equations, to estimate the heritability of eating behavior patterns and obesity measures . ClassicWe started the analyses by computing univariate models . Becausera) and shared environmental (rc) correlations  and obesity measures through eating behavior patterns , direct effects (c), and indirect or mediation effects (ab) were calculated to show how much of the association between the PRS and the obesity measure was mediated by the different eating behavior patterns. Bias corrected estimates and 95% CIs were calculated using the bootstrapping approach with 1000 draws. All of the models were estimated for every eating behavior pattern and for both obesity measures using both PRSs, and the results are presented as total, direct, and indirect effects. We added interaction terms to test whether the mediation models were similar in men and women. Because some of the interaction terms were significant (P < 0.05), we present the mediation models separately by sex in Supplemental Table 3.Structural equation modeling (SEM) analyses were conducted to identify potential mediation models between the PRSs . There were no differences between men and women with respect to standardized PRSs. Results of the PCAs of eating behavior patterns, which included 15 questions on eating styles and the DQS, are shown in Supplemental Figure 1).ICCs were higher in MZ twins than DZ twins, indicating the presence of genetic influences . Heritabrp = 0.35 and 0.32, respectively) and the emotional and external eating behavior pattern . Thus, we decomposed these 4 correlations into genetic and environmental components  , and a higher loading on the snacking behavior pattern was associated with a higher BMI and WC . These 2 pathways together represent the indirect effect, which was β = 0.06 for BMI and β = 0.05 for WC (A more detailed description of the pathways of the mediation model for the snacking behavior pattern is shown in 5 for WC .BMI and obesity measures (data not shown).When the analysis was limited to the mediation model through the DQS, there was no mediation in the association between the PRSBMI and BMI. The infrequent and unhealthy eating behavior pattern significantly mediated the association between the PRSBMI and BMI and WC in men but not in women, with higher mediation percentages (6.1% for BMI and 10.0% for WC) in men compared with the percentages in the overall sample (3.4% for BMI and 4.2% for WC). The mediation through the emotional and external eating behavior pattern was statistically significant in women for BMI. Despite these differences, when analyzing the models through the snacking, the infrequent and unhealthy, and the emotional and external eating behavior patterns, the 95% CIs were overlapping in both men and women.When analyzing men and women separately , we obseBMI was not associated with WC adjusted for BMI (data not shown). The associations between the PRSWHR and WC adjusted for BMI are shown in Supplemental Table 4. Despite an overall association between the 4 eating behavior patterns and WC adjusted for BMI, eating behavior patterns did not mediate any of these pathways.The PRSBMI and obesity measures was partly mediated by the snacking behavior pattern in men and women and to a lesser extent by the infrequent and unhealthy eating behavior pattern in men. The association between the PRSBMI and BMI was also partly mediated by the emotional and external eating pattern. In the twin modeling, the associations between the snacking and the emotional and external eating behavior patterns and BMI and WC were largely explained by genetic factors, supporting the inference that the snacking and the emotional and external eating behavior patterns and obesity share a common underlying genetic architecture. Another important finding was that eating behavior patterns were moderately heritable, with the remaining variance accounted for by nonshared environmental factors.In this cross-sectional study of healthy young adults, we showed that the association between the most recent PRSBMI based on <100 SNPs, that have previously reported mediation by undesirable eating behavior traits . Another study showed heritability estimates of ∼43% for restrained eating (Eating behavior patterns were moderately heritable, suggesting that they are influenced by both genetic and environmental factors. In general, our heritability estimates of eating behavior patterns appeared to be somewhat lower than those measured with the Three-Factor Eating Questionnaire , 51. In d eating , somewhad eating .Previous research suggests that genetic factors from heritability estimates provide more evidence for the genetic contribution to obesity susceptibility than genetic variants from GWASs, due to the large difference in the percentage of variance explained between them . Twin stOur study has several potential limitations. The main limitation is its cross-sectional design; we therefore cannot confirm the causal directions of the relations between eating behavior patterns and obesity measures. However, a previous longitudinal study tested the reciprocity between restrained eating and BMI and WC to determine their potential relations, and they indicated that restrained eating was a marker for previous weight gain instead of a trigger of future weight gain . Future Our study has some important strengths. To our knowledge, this is the first study in adults that tested whether the genetic susceptibility to obesity is mediated by eating behaviors by using a PRS that included almost 1 million SNPs representing the whole genome. We included a combination of twin modeling with GWASs analyses based on very different underlying assumptions. The robustness of our results based on these different methods provides further evidence on the common genetic background of eating behaviors and obesity measures. Finally, the inclusion of diet quality in addition to eating behavior patterns allowed us to assess an additional aspect of diet.In conclusion, our findings support that eating behavior patterns are moderately heritable. Snacking, and to a lesser extent infrequent and unhealthy and emotional and external eating behavior patterns, share a common genetic liability with obesity measures. Obesity prevention efforts might benefit from focusing on eating behavior change, particularly in genetically susceptible individuals. Future longitudinal and intervention studies will be beneficial to understand how eating behavior patterns and genetic susceptibility to obesity impact life-long weight outcomes.nqaa181_Supplemental_FileClick here for additional data file.


The liver biopsies during bariatric surgery showed that 53.52% did not present NAFLD, 30.16% had NASH, 15.98% isolated steatosis and 0.34% liver cirrhosis. The median BMI of the longitudinal cohort decreased from 37.9 ± 2.21 kg/m2 at the time of bariatric surgery (T1) to 25.69 ± 3.79 kg/m2 after 21 ± 22 months after the procedure (T2). The prevalence of NAFLD in T1 was 50%, and 16.67% in T2. The histological area of collagen fiber was lower in T2 compared to T1 (p = 0.0152) in the majority of patients, which was also illustrated by immunohistochemistry for Kupffer cell and myofibroblast formation markers. These findings confirmed the NAFLD regression after bariatric surgery and, for the first time, showed the amelioration of these features using more accurate histopathological techniques.Non-alcoholic fatty liver disease (NAFLD) is a chronic disease with several degrees of histological features which may progress to cirrhosis. Obesity is an important risk factor and although NAFLD has no specific pharmacological treatment, bariatric surgery has been associated with NAFLD regression in severely obese patients. However, few longitudinal histological studies support this finding. Therefore, firstly, a retrospective study was performed including clinical and histological data of 895 obese patients who underwent bariatric surgery. In addition, histological analyses of 30 patient’s liver biopsies were evaluated at two timepoints (T1 and T2). The retrospective analysis of the total number of patients revealed that the average body mass index (BMI) was 35.91 ± 2.81 kg/m There are four clinical-pathological features which are usually followed by NAFLD course: non-alcoholic steatosis (NAFL), non-alcoholic steatohepatitis (NASH), NASH-related cirrhosis and hepatocellular carcinoma (HCC). Among them, obesity has been linked not only to initial stages of the disease, but also to its progression, leading to an increased morbidity and mortality. Moreover, NAFLD is strongly associated with insulin resistance, type 2 diabetes (T2D) and the incident cardiovascular disease (CVD)6. The worldwide prevalence of NAFLD and NASH in the general population has been estimated to span from 6.3–33% and 3–5%, respectively. This estimate is increasing with the rise in the incidence of obesity and T2D, so that the prevalence of the NAFLD may be over 85% among the morbid obese and 75.6% in patients with T2D regardless of obesity7. In the United States, the prevalence of obesity was 39.8% in 2016 and affected about 93.3 million of adults, while it has also been observed that the NAFLD/NASH is becoming the leading indication for liver transplantation8. So far, there have been no statistical data in the literature on the Brazilian obese population in relation to the prevalence and progression of the NAFLD.Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease whose prevalence has been associated to the global obesity epidemic6. Bariatric surgery, by leading to significant weight loss and metabolic changes related to the release of incretins and adipokines, as well as decreasing chronic inflammation, can lead to a reduction of hepatic fat deposits. It is currently considered the gold standard treatment option for refractory morbid obesity, as it leads to a significant improvement and/or resolution of several obesity-related comorbidities12.Furthermore, no pharmacological agents have been approved for long-term treatment of NAFLD. The adoption of healthy lifestyles, such as dietary modifications, regular physical activity, and gradual weight loss, is considered the main clinical recommendation and an initial step for the management of NAFLDTherefore, this study aimed to characterize the NAFLD of obese Brazilian patients and to analyze the histologic evolution throughout the spectrum of NAFLD, while assessing the effects of bariatric surgery on the attenuation of this liver disease.This study included the clinical and demographic data of 895 Brazilian morbidly obese patients, of a total initial number of 954 patients who underwent RYGB bariatric surgery with liver biopsy during the procedure Table . Twenty-2. The overall mortality rate was 0.33% (3 patients). Regarding the laboratory tests, plasma albumin (ALB), aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (AF), gamma glutamyl transferase (GGT), glucose (GLU), total bilirubin (TB), total protein (PROT), triglyceride (TGC) and total cholesterol (COL) concentrations were analyzed (see Table The median age of the 895 patients was 38.1 ± 10.2 years-old and 739 (82.5%) were women. All patients included in the study had their BMI equal to or greater than 30 at the time of surgery, and the average BMI was 35.91 ± 2.81 kg/m13, as shown in Fig. The anatomopathological findings for the histological diagnosis and fibrosis staging of NAFLD of liver biopsies were described as recommended by the American Association for the Study of Liver Diseases (AASLD) and the European Association for the Study of the Liver (EASL), which was based on an validated histological score: the FLIP (fatty liver inhibition of progression) Steatosis, Activity, and Fibrosis Score2. The median age at the time of the second surgery (T2) was 43 ± 9 years-old and the median BMI was 25.69 ± 3.79 kg/m2. Ten (33%) were ex-smokers. The median time between the liver biopsy collections during the surgeries (T1 and T2) was 21 ± 22 months, ranging from 3 to 82 months. There were no significant statistical differences regarding age, gender and smoking habit in the evaluated groups. However, there was a statistical difference between these groups regarding BMI (p < 0.001). There is no mortality after bariatric surgery in this subgroup of patients. However, there were mild to moderate complications in their follow-up such as incisional hernia (14 patients); gastric ring migration (6 patients); intestinal obstruction ; anemia (3 patients), anastomotic ulcers (1 patient); depressive disorder (2 patients) and cholelithiasis (2 patients). One of these patients presented both incisional hernia and cholelithiasis.In a second step of this study, we compared clinical and histopathological findings of obese patients at two timepoints, at the time of bariatric surgery (T1) and after T2) the procedure (n = 30). As shown in Table  the procAll groups were evaluated for serum levels of liver function markers, which are expressed as median values and variations showed in Table Liver biopsy evaluation was also performed, as shown in Fig. 13. These findings were classified as: no fibrosis (0), zone 3 mild perisinusoidal (1 A), zone 3 moderate perisinusoidal (1B), portal or periportal fibrosis (1C), Zone 3 plus portal/periportal (2), bridging (3) and cirrhosis (4). At the time of bariatric surgery (T1), 5 patients (16.6%) did not present fibrosis, 8 patients (26.66%) had fibrosis at stage 1 A, 2 patients (6.66%) at stage 1B, 5 patients (16.66%) at stage 1C, 9 patients (30%) at stage 2 and 1 patient (3.33%) at stage 3. At timepoint T2, 7 patients (23.33%) did not present fibrosis, 10 patients (33.33%) had fibrosis at stage 1 A, 3 patients (10%) at stage 1B, 1 patient (3.33%) at stage 1C, 7 patients (23.33%) at stage 2 and 2 patients (6.66%) at stage 3.The stage of fibrosis was also determined by the anatomopathological findings based on the Fibrosis Score, recommended by the American Association for the Study of Liver Diseases (AASLD) and the European Association for the Study of the Liver (EASL)14. We can readily observe this feature by hematoxylin and eosin (H&E) (10×) in the Fig. 14, is shown by H&E (40×). These histological alterations were evidenced together in the majority of the morbidly obese patients with NAFLD who underwent bariatric surgery (T1), and in the minority of the patients in T2, as previously shown in the Fig. The hepatocytes with accumulated triglyceride vacuoles are the histological feature that ties together all of the various types of NAFLDMoreover, representative photomicrographs of Masson’s Trichrome staining in T1 and T2 are shown in Fig. To better evaluate the hepatic lesions along the follow-up after the bariatric surgery in the paired cohort, we applied immunohistochemistry for alpha-smooth muscle actin (α-SMA) and for a specific cell-surface marker for macrophages (EMR1) highly and constitutively expressed on Kupffer’s cells in the liver, besides H&E and Masson’s Trichrome staining. The representative images of immunohistochemistry for Kupffer’s cell (EMR1) and myofibroblast formation (α-SMA) markers also illustrate NAFLD regression Fig. .Figure 615.The prevalence of overweight and obesity has steadily increased in the recent decades, regardless of gender, age or level of country development. For this reason, obesity has been considered a major public health problem worldwide. There are more than 2 billion overweight people and 64% of them are in developing countries17. In the last decades, the relevance of the intestinal microbiota has been suggested as another possible factor involved in the increase of obesity. However, its impact on the treatment of obesity has been negligible18. Special attention should be given to children, due to the considerable reduction in life expectancy and quality related to obesity19. While overweight and obesity showed an increase in prevalence to 27.5% among adults, the infant population’s prevalence reached the high rate of 47.1% between 1980 and 201315.A successful public health model has not been yet identified to reverse this epidemic, mainly because it is a multifactorial disease, which depends on changes in lifestyle, such as less caloric intake and greater energy expenditure15. These data have justified the increased investment in public health for the prevention and treatment of obesity and its comorbidities. Obesity is related to several diseases, such as cardiovascular, orthopedic, diabetes and several types of cancer20, being associated with 14–20% of all cancer deaths21. Obesity is the main risk factor for NAFLD, while other factors such as ethnicity , gender , type 2 diabetes (22.51%), hyperlipidemia (69.16%), hypertension (39.34%), and metabolic syndrome (42.54%) has less impact24. NAFLD is considered the hepatic phenotype of metabolic syndrome, which is directly related with insulin resistance and disturbances in glucose and lipid metabolism. The high worldwide incidence of NAFLD and its correlation with obesity and its comorbidities emphasize the importance to evaluate the impact of the hepatic features in this affection. To our knowledge, there is no other study that has identified and correlated such detailed aspects of the NAFLD in a large Brazilian cohort. All individuals who underwent bariatric surgery in this study participated in a preoperative weight loss program which lasts 4 to 12 weeks, includes weekly follow-ups, and is carried out by a multidisciplinary team. Individuals underwent surgery once a minimum of 10% preoperative weight loss was achieved, or with the minimal BMI of 35 kg/m2 for subjects with obesity-related morbidities or 40 kg/m2 for those free of comorbidities. This may justify the low mortality rate (0.33%) after bariatric surgery in this study, since the weight loss may assist to attenuate the associated comorbidities.Brazil is the fifth nation in absolute number of obese individuals, behind the USA, China, India and Russia26. Nonetheless, it is far from being free of risk, although the incidence of major complications reduced over time as well, mostly after the development of minimally invasive approaches. Early morbidity such as anastomotic leaks and thromboembolic events may occur in up to 2–4% of all operated individuals and, as such, are the most dreaded perioperative complications28. Late morbidity is more related to nutritional issues, such as protein, iron, and vitamin deficiencies, which are also associated with a poor compliance to follow-up, as well as surgical complications, such as intestinal obstruction caused by adhesions or internal hernias, abdominal wall hernias and gallstones. Significant hepatic impairment is unusual after the mainstream procedures, but there are a number of cases after more malabsorptive operations, mainly the biliopancreatric diversions31.Bariatric surgery has largely evolved over time and nowadays its perioperative mortality is arguably as low as 0.2%, which is similar to what is observed after routinely performed operations such as gallbladder removal and hysterectomy, and leads to an overall 40% reduction of all-cause mortality35. The prevalence of liver diseases secondary to alcohol consumption remained stable, and the better diagnosis and subsequent development of vaccine and treatment for viral hepatitis decreased the liver injury due to this affection worldwide in the recent decades. On the other hand, liver disease associated with obesity and the consequent evolution to steatosis, steatohepatitis, cirrhosis and hepatocellular carcinoma has become one of the main causes of liver failure and transplantation in recent years, with the aggravating possibility of recurrence in the transplanted organ37. In our cross-sectional retrospective study, the NAFLD incidence evaluated by histological examination was 46.48% among the morbidly obese patients who underwent bariatric surgery, and 50% in the first timepoint of the paired longitudinal study. This incidence in bariatric patients was verified in other studies with smaller sample sizes43. While most of the studies evaluated NAFLD after bariatric surgery by ultrasound scan or another image or laboratory methods, our group performed liver biopsy at two distinct timepoints. Some serum levels of liver function markers were decreased in T2 compared to T1, that corroborate with the improvement of NAFLD verified in histological analysis in T2. Thus, there was a decrease in ALT (p = 0.0117), GGT (p = 0.0072), TGC (p = 0.0216) and COL (p = 0.0031) serum levels after bariatric surgery. The decreased serum albumin (p = 0.0014) and protein levels (p = 0.0109) may be explained by the decrease of protein intake and absorption after bariatric surgery.Described only in the 1980s, NALFD is currently considered the main liver disease worldwide, with prevalence in the general population of up to 25%, and analyzing only obese patients, the prevalence exceeds 50%44. This condition is aggravated because although the evolution of these changes towards liver cirrhosis and hepatocellular carcinoma is defined24, unfortunately it is not yet possible to predict which patients will remain stable and which will progress to cirrhosis and cancer. Since access to diagnostic tests is scarce in our country for a large part of the population, several patients receive their diagnosis only after the secondary symptoms of liver failure and cirrhosis appear.Since steatosis (NAFL) and steatohepatitis (NASH) are asymptomatic in the vast majority of patients, we have a population of a few billion inhabitants with such diseases without a proper diagnosis45. Only 18% of the 895 patients included in the study were considered diabetic at the time of surgery, thus the histopathological changes found and corresponding liver damage may be attributed to the morbid obesity.Although bariatric surgery provides NAFLD amelioration, the literature is controversial about what surgical technique is more effective or may be equally efficacious in ameliorating NAFLD. All the patients included in this study underwent the same surgical procedure (RYGB bariatric surgery), and the weight loss outcomes were comparable to the literature46. As a screening method, ultrasound scan is a non-invasive, easily accessible and low-cost method to diagnose patients with hepatic steatosis, associated with the investigation of other causes of liver disease such as viral hepatitis, alcohol consumption and hepatotoxic drugs. However, liver biopsy, despite being invasive and not without complications, has been considered the gold standard for diagnosis of the disease and is the only method able to differentiate non-alcoholic steatohepatitis from steatosis, to evaluate inflammation and to stage fibrosis46.Given the importance of assessing liver function and morphology in susceptible patients, several methods have been developed, such as laboratory tests and indexes based on these tests, in addition to imaging tests such as ultrasound scan, nuclear magnetic resonance imaging and elastography47. In this study, we identified the earliest stages of hepatic fibrosis in morbid obese patients during and after bariatric surgery, showing positive staining to α-SMA in both timepoints, but less evident in T2 when compared to T1. Likewise, the staining for Kupffer cells marker (EMR1) showed positivity in both groups, but less in T2 than in T1. Kupffer cells are central players in the progression of steatosis to non-alcoholic steatohepatitis (NASH) and fibrosis48. Therefore, the immunohistochemistry analysis illustrated the results of the histopathological changes classification of the NAFLD based on H&E and Masson’s Trichome staining during and after the bariatric surgery. These findings better characterized the NAFLD histological regression that occurred in the majority of the cases of our longitudinal paired study.In addition to the evaluation of NAFLD’s prevalence among morbidly obese patients who were submitted to the RYGB bariatric surgery in a large cohort, the present study identified the histological grading and evolution of the hepatic lesions in a subset of patients. There was a significant reduction of collagen fiber deposition in the liver biopsies after bariatric surgery, besides decreased hepatic steatosis and hepatocyte ballooning, showing NAFLD regression after the procedure. Moreover, immunohistochemistry for α-SMA and EMR1 corroborated these findings. α-SMA is expressed by hepatic stellate cells, reflects their activation to myofibroblast-like cell and has been directly related to experimental liver fibrogenesis and to human fibrosis in chronic liver disease49. This general limitation of the bariatric practice was also present within the current study, since the individuals which underwent a second biopsy were not regularly accompanied and thus there are no available data in many time points except for both perioperative situations. However, this cohort has a decent size, and a longitudinal dataset as the one presented is of value for the scientific community, as it shows results of liver histology after bariatric surgery. The preoperative weight loss may also have influenced the histological findings and the relatively low prevalence of NAFLD in the entire study population, as well as explain the low BMI at surgery in comparison with other studies.Most of this study’s limitations are related to its retrospective character. Some variables for the applied histological score were not evaluated by the pathologists and, therefore, were not in the medical records, having been considered not informed, as pointed out in the Supplementary Information Files. Unfortunately, the late follow-up of bariatric surgery is reportedly erratic, since most patients do not appropriately comply with the appointed consultations52. The benefit of a routine second liver biopsy at a determined timepoint of the follow-up after bariatric surgery should be confirmed in future large longitudinal prospective studies; however, given the extremely low risk of liver biopsy complications in individuals who would otherwise have to undergo another surgery after their primary bariatric procedure , this seems to be justifiable within this context.The blood liver function tests may to estimate, preoperatively, patients who are prone to unfavorable evolution after bariatric surgery. However, the liver biopsy collected during the bariatric surgery with specific histological analysis may better identify the different types of lesions and map the patients with high risk for hepatic complications in the follow-up after this procedure. Moreover, this histologically focused study may bring new insights on the effects of weight loss in the NAFLD regression. There are a few cases of bariatric patients in the literature who develop hepatic injury after the procedure, and this observation needs further investigation based on the molecular and metabolic pathways involved in this process. Based on the currently available evidence, the occurrence of severe liver disease is unusual and even anecdotal after the mainstream bariatric procedures, but not so rare after predominantly malabsportive operations. The mechanisms of this worsening are yet to be determined, but seem to be related to early rapid weight loss, a degree of protein malnutrition, the lack of hepatotrophic factors, and the effect of high levels of mobilized circulating free fatty acids after surgery, as well as changes in gut microbiota and intestinal bacterial overgrowthWe retrospectively analyzed the clinical and histopathological data from 954 Brazilian patients who underwent Roux-en-Y gastric bypass (RYGB) bariatric surgery in the Clinical Hospital of the University of Campinas between the years 2008 and 2019, by the same surgical team. Bariatric surgery was indicated according to the National Institutes of Health consensus statement. The patients included in this observational study had a BMI equal to or greater than 30 at the time of surgery. The wedge liver biopsy was performed at the time of bariatric surgery.All bariatric procedures followed the same technique and were performed by an open approach. The main features of the Roux-en-Y gastric bypass (RYGB) were a 30 mL gastric pouch, a 100-cm biliopancreatic loop, a 150-cm alimentary limb and a common channel comprised of the remainder of the small intestine.For the longitudinal observational prospective study, 30 patients who underwent bariatric surgery (T1) and a second abdominal surgical procedure after bariatric surgery (T2), such as cholecystectomy (7%), incisional hernia correction (50%), cholecystectomy + incisional hernia correction (3%), exploratory laparotomy (37%) and removal of gastric band (3%), were submitted to another liver biopsy during this procedure.13 for both cohorts of patients. The specifications for the adopted criteria are detailed in the Supplementary Information File. Immunohistochemical staining of the liver biopsies from patients of the longitudinal cohort were performed to better characterize the NAFLD. These data correlate with liver disease stage and overtime evolution.Clinical and demographic data, laboratory records and liver biopsy histopathological findings were collected from both cohorts. Histologic parameters to classify the NAFLD were evaluated to analyze the effect of bariatric surgery and weight loss on the evolution of this liver disease. NAFLD was estimated by hematoxylin and eosin (H&E) and Masson’s Trichrome staining, using the histopathological score recommended by the American Association for the Study of Liver Diseases (AASLD) and the European Association for the Study of the Liver (EASL)The exclusion criteria of the study were as follows: aged below 18 or over 80 years old; history of alcohol abuse; use of hepatotoxic drugs; history of chronic viral hepatitis and primary liver disease. In addition to these criteria, patients with BMI equal to or greater than 30 were excluded from the control group. All procedures performed in studies involving human participants were in accordance with the Declaration of Helsinki and its later amendments. The Ethics Committee from the University of Campinas approved the study (reference number:58184516.2.0000.5404). The study was carried out at the University of Campinas, Surgery Department and IBD Research Laboratory of the School of Medical Sciences.Paraffin-embedded liver blocks of 9 patients who underwent bariatric surgery (T1) and a second abdominal surgery in their follow-up (T2) were used for staining assays. For histological analysis, 4 µm-thick sections were stained with hematoxylin and eosin (H&E) and Masson’s Trichrome dye. Photomicrographs were taken using Zeiss Axioplan 2 microscope with digital camera (Olympus DP – 72) and control software (Cellsens). Fields of higher magnification  were scanned and three random fields of higher magnification were selected for quantitative analysis, which was performed through the percentage of staining pixel volume in a panchromatic objective field of higher magnification 4× by ImageJ2 software.Paraffin-embedded liver blocks of 12 patients who underwent bariatric surgery (T1) and a second abdominal surgery in the follow-up (T2) were used for immunostaining. For this, 4 µm-thick sections were pre-treated for de-paraffinization, rehydration, and epitope retrieval using citrate buffer. A warming step of 20 min at 95 °C was performed. Sections were blocked for 45 min with 1% bovine serum albumin (BSA) or with animal-free blocking solution (Vector Laboratories). Samples were incubated overnight at 4 °C using the following commercially available antibodies: anti-human EGF-like molecule containing mucin-like hormone receptor 1  and anti-human Smooth Muscle Actin  followed by incubation with biotinylated anti-mouse  or anti-rat . Signal detection was determined using the immunoperoxidase detection system (Vector Laboratories), followed by incubation with 3,3’-diaminobenzidine (DAB) solution (Dako). Slides were mounted with Mounting Medium (Dako). Photomicrographs were taken using Zeiss Axioplan 2 microscope with digital camera (Olympus DP – 72) and control software (Cellsens). Fields of higher magnification  were scanned.The results were reported as median with interquartile ranges. To test for distributional adequacy, the Kolmogorov-Smirnov test was used to investigate if the data followed a normal distribution or a Gaussian distribution (p > 0.1). Data were analyzed using the non-parametric Mann-Whitney test. The level of significance was set at p < 0.05.Ethical approval by the ethic board of the University of Campinas (UNICAMP) and consent of patients are included in the original publications.This study was approved by the Ethics Committee of the University of Campinas (UNICAMP), all patients signed the informed consent form, and were performed in accordance with the Declaration of Helsinki.Supplementary information.Supplementary information.Supplementary information.


While carbon dioxide degassing is typically associated with extensional tectonics, volcanoes, and geothermal areas, CH4 seepage mostly occurs in petroleum-bearing sedimentary basins, but the role of tectonics in degassing is known only for some case studies at local scale. Here, we perform a global scale geospatial analysis to assess how the presence of hydrocarbon fields, basin geodynamics and the type of faults control CH4 seepage. Combining georeferenced data of global inventories of onshore seeps, faults, sedimentary basins, petroleum fields and heat flow, we find that hydrocarbon seeps prevail in petroleum fields within convergent basins with heat flow ≤ 98 mW m−2, and along any type of brittle tectonic structure, mostly in reverse fault settings. Areas potentially hosting additional seeps and microseepage are identified through a global seepage favourability model.Earth’s hydrocarbon degassing through gas-oil seeps, mud volcanoes and diffuse microseepage is a major natural source of methane (CH 4 seepage preferably develops in convergent basins, while gas seeps can occur along any brittle tectonic structure.CH4 seepage mostly occurs in petroleum-bearing sedimentary basins, but the role of tectonics in degassing is mostly only known at a local scale. Here, the authors conduct a global scale analysis of seeps, faults, sedimentary basins, petroleum fields and heat flow, and find that geological CH CO2 degassing from volcanic and geothermal areas may have played a climatic role over geological-time scales8 but it appears to represent a minimal component in the present-day global CO2 atmospheric budget; global geo-CO2 emission (likely < 1000 Mt yr−1)4 is three orders of magnitude lower than the total CO2 emissions from natural plus anthropogenic sources9. Quite another matter is the relative importance of geological CH4 (hereafter geo-CH4) emissions: bottom-up and top-down estimates suggest that geo-CH4 emission globally amount to ~45 Mt yr−110, about 8% of total  CH4 sources (~560 Mt yr−1)11. Lower geo-CH4 emissions derived from preindustrial-era ice core radiocarbon 14CH4 analyses12 opened a debate, suggesting the need of further checks of global geo-CH4 estimates. In this respect, an important step is improving the knowledge of global seepage distribution and extension, and its controlling geological factors. While geo-CO2 degassing is mainly controlled by extensional tectonics, along normal faults and rift systems that mostly drive CO2 release from crustal geothermal reservoirs, magmatic chambers and the mantle8, geo-CH4 degassing (seepage) takes place primarily in sedimentary, petroleum (oil and gas)-rich basins, where CH4 can have a microbial or thermogenic origin15. However, the role of the basin geodynamics (convergent or divergent) and the type of faulting  on gas seepage was examined only sporadically, without statistical quantification on a global scale16. CH4 migration and seepage are primarily driven by advection (Darcy’s flow), controlled by gas pressure and rock permeability related to fracture networks and faults15. Whether a specific type of tectonics and fault  is needed for CH4 degassing is unknown. Addressing this issue is critical to assess the pathways of CH4 release and to identify potential, not yet inventoried, geo-CH4 emission regions, including those hosting the invisible, diffuse exhalations (microseepage)18. Knowing the area and spatial distribution of methane seepage is fundamental for both atmospheric methane budget studies and petroleum exploration. In the first case, it allows to refine global and regional estimates of geo-CH4 emission to the atmosphere: the area where the emission takes place represents in fact the activity to be associated to the average gas flux, over that area (emission factors), in bottom-up gas emission estimates11. This is particularly critical for microseepage, which represents a major class of geo-CH4 source, estimated in the order of 15–33 Mt yr−1 10. In the second case, maps of spatial distribution of seepage in sedimentary basins may drive the exploration for the identification of subsurface petroleum reservoirs14. Knowing the type of tectonics favouring hydrocarbon seepage is also important in the study of potential methane source regions on other planets as Mars, where recent atmospheric CH4 detections have raised the question on what are the tectonic features that may have released the gas19.Among the natural sources of greenhouse gases that may contribute to climate changes, geological emissions of carbon dioxide , heat flow and fault type. The work does not include offshore seeps because available inventories, referring to relatively wide areas, do not report geographic coordinates of individual seeps6. The role of tectonics is investigated by fault density maps and by examining the spatial association between seeps and type of faulting .20  occur in convergent basins Table , althoug21. Divergent basins host 758 seeps , of which 70% is oil, 26% gas and 4% mud volcanoes  occurs in areas with heat flow ≤98 mWm−2  occur within the area covered by petroleum fields Fig. ; Table 1% occur w24. Therefore, we have evaluated the spatial occurrence of faults within convergent and divergent basins and within the petroleum fields. We built a fault density map (1° × 1°) as a proxy of brittle tectonics occurrence using onshore, active and non-active faults from a worldwide fault database integrating published global and regional datasets  is mapped by using a kernel density algorithm26 . This is likely due to the presence, in convergent basins, of longer faults, mainly thrusts associated with orogenic belts. These fault systems concentrate along the edges of thrust-and-fold belts and may be related to ruptures from shallow decollement layers up to the surface with multiple short-cuts, back-thrusts, and ancillary high-angle fracturing27. Petroleum fields show a mean fault density value (202.7 Nf kmf km−2) comparable to that of convergent basins. Interestingly, seeps occur for any value of fault density. This means that both isolated faults and regions with fault clusters can host seeps  of the fault.The distance between each seep and the nearest fault is calculated by proximity analysis 24. In our database, for example, we recognise 134 oil seeps located at distances >100 km from the nearest fault (most of them in the cratonic Siberian oil provinces). The association between gas seeps and normal faults  and, in particular, the Analytical Hierarchy Process (AHP)  Mt yr−1)10. The main uncertainty in the global CH4 source strength is related to the limited knowledge of the global microseepage area. Our model suggests a high probability (≥50%) microseepage area of 8.1 Mkm2, a value consistent with that previously predicted by process-based modelling (8.6 Mkm2)6.The model has been validated by verifying that the number of known (inventoried) seeps increases with seepage favourability. The test shows that about 70% of the seeps fall in the probability exceeding 50% Fig. . The mod16. We also checked specific examples where the type of fault in correspondence with the seepage is known. For example, gas seepage along normal faults occurs in the Katakolo Bay in Greece36 and in the Tiber Delta in Italy37. The Giswil seep (Switzerland) is an example of gas exhalation along a strike-slip fault38. Mud volcanoes along thrust faults were studied in particular in Northern Italy and Azerbaijan21.As mentioned above, the results of our global geospatial analysis are consistent with previous qualitative and local scale observations41. Other factors, including lithology, rheology, fault activity and self-sealing processes  control the gas permeability of a fault43.We have shown that the tectonics style does not significantly affect the methane release. In fact, while compressional and extensional stresses may play a role in controlling water and oil migration , they do not significantly affect the gas-bearing property of faults, as a fault that is impermeable to water can be permeable to gas, as well known in studies on gas flow in fractured media45. This is consistent with our analysis that showed no relationship between fault density and seepage.The role of fault activity could not be examined in our geospatial analyses, because available datasets include only active faults (GEM dataset) or do not distinguish active from inactive faults. However, gas migration studies demonstrated that non-active, ancient faults in stable and poorly fractured terrains  can also be gas-bearing4 can potentially be released by any type of fault is a key conclusion in fixing the criteria to search areas of CH4 release on Mars, where reverse and normal faults are recognised46. Recent CH4 spike detections in the Martian atmosphere have raised the question about the tectonic structures potentially favouring the release of methane19. By analogy with what is observed on Earth, any type of fault on Mars, regardless the fault density of the area, may potentially be methane-bearing. Therefore, even isolated faults, either the extensional faults of the Tharsis region and along the Martian dichotomy, or the compressional faults in the lowlands  and in the highlands  can potentially host gas seepage. Like on Earth, potential mud volcanism on Mars should preferentially occur within reverse fault areas. Morphological structures resembling mud volcanoes were actually identified in regions with compressional tectonic settings49. Whether methane source rocks, which are essential for Earth’s seepage, do exist on Mars as yet remains an unknown factor49.The fact that CHReported input data refer to a series of geological and geographic databases available in shapefile , csv, ASCII, and/or grid formats, managed and elaborated in ArcGIS Pro (Copyright © 1999-2018 Esri Inc.). Geographic coordinates of all datasets are expressed in decimal longitude and latitude in the GCS WGS 1984 world projection system. All raster maps are reported in 1° × 1° square grid format. The source of the several datasets  are described in Data Availability.All datasets were analysed by using geospatial and geostatistical analyses in GIS environment. The output raster values of 1° × 1° grid maps were extracted at each seep location using the Spatial Analyst Tool .f·kmf km−2)  to define the distance of each seep from the nearest fault in order to identify the faulting style  of the seep near-field.Exploratory Spatial Data Analysis allows by charts, graphs, and tables to explore and interpret the correlation among different datasets. In this work, bar charts were used to highlight the spatial relationships between seeps and basins. Histograms and normal probability plots were used to highlight the statistical distribution of the seep distances from the nearest fault system bearing the information of the faulting style   MultiCriteria Decision Analysis (SMCDA) to identify the most favourable areas for seepage based on the results of the geospatial analysis, highlighting the relationships among seeps, basins, petroleum fields, heat flow, fault density and fault type  have geographic coordinates provided by GPS measurement with uncertainty in the orders of a few metres. For about 14% of seeps (386 seeps) the exact position was not available and geographic coordinates were assigned58. New Zealand fault database was intended to be represented at 1:250,000 by the designers (GNS Science). Metadata do not report uncertainty and data accuracy59. Other used fault databases do not report any indication of uncertainty and accuracy and thus may not have been subject to any verification or other quality control process. Gaps in the global fault distribution may occur due to the fact that some individual datasets mostly report undefined faults (not included in our integrated dataset), as it happens for Africa, Australia and North America. The final, integrated fault database was checked in terms of geometric duplicates by using the Data Reviewer tool available in ArcGIS. The results of this analysis do not return geometric duplicates.The uncertainty in fault location in the individual datasets used for our global inventory is generally not quantified. The GEM database reports an accuracy corresponding to the denominator of map scale. Since faults were typically compiled and digitised from different maps ranging from 1:100,000 to 1: 1,000,000 scale, the accuracy of the fault location should be in the order of 1–10 km. Australia dataset reports a location accuracy between 50 and 1000 m, depending on the quality and scale of the original source data60, released by the International Heat Flow Commission61, indicated that the location of heat flow measurements is of uncertain origin and that the references, as cited in Global Heat Flow Database, were incomplete. GIS capture and quality control were carried out by the Cartographic Services (part of the Geography Department) at Oklahoma State University.The first version of the Heat Flow dataset compiled by[https://www.prio.org/Data/Geographical-and-Resource-Datasets/Petroleum-Dataset/Petroleum-Dataset-v-12]62 did not quantify the uncertainty of the petroleum field locations. In the original dataset, several of the sources for field locations were not provided in GIS readable format , and thus they were digitised from original pdf maps. Details are reported at [https://www.prio.org/Global/upload/CSCW/Data/Geographical/codebook.pdf].The dataset repository of petroleum fields Supplementary InformationPeer Review File