The most prevalent supplements were sports drinks (42.2%), followed by energy bars (35.3%) and caffeine-containing products (31.6%). However, a greater consumption of group A supplements  and group B supplements  was observed in male compared to female handball players. Supplements categorized as medical supplements were more commonly consumed in professional vs. amateur players . Additionally, a higher consumption of group B supplements was observed in professional compared to amateur players . Handball players revealed a moderate use of supplements while sex and competitive level slighted changed the pattern of supplements use. A high portion of handball players use supplements as fuel during exercise and reported the use of caffeine-containing supplements to enhance performance.Sports supplements are commonly used by elite athletes with the main goal of enhancing sport performance. Supplements use might be substantially different depending on the sport discipline, sex, and competitive level. To date, data about prevalence and the most-commonly used supplements in handball are scarce. Thus, the aim of this investigation was to determine the patterns of supplements use by handball players of both sexes and with different competitive levels: One hundred and eighty-seven handball players (112 men and 75 women) of different competitive levels  completed a validated self-administered questionnaire about supplements use. Supplements were classified according to the categorization of the Australian Institute of Sport (AIS). Overall, 59.9% of the handball players ( Handball is an intermittent team sport that combines short bouts of high intensity exercise interspersed with periods of moderate-to-low exercise intensity . AlthougSports supplements can be defined as a food, food component, or nutrient that is purposefully ingested, in addition to the habitually consumed diet, with the aim of achieving a specific physical performance or health benefit . SupplemThe use of supplements in endurance sports has been extensively studied , while kTo our knowledge, only one previous study has analyzed the use of supplements in handball players . In thisA total of 187 players (112 men and 75 women) participated in this investigation. The sample was divided into two groups: one group composed of 106 professional players ) and the second group, composed of 81 amateur players (Spanish Second and Third Divisions). Participants were recruited with the help of the Spanish Handball Federation which distributed an email with specific instructions and the online version of the questionnaire to all the men’s and women’s First Division Teams of ASOBAL, and to other non-professional handball teams. Potential participants also distributed the email with the questionnaire among teammates and thus, it was unfeasible to record the exact number of athletes solicited for this investigation. From the total of questionnaires received (189), 1.06% were removed because they were incomplete, or had been incorrectly filled out, for a total of 187 valid questionnaires for analysis. This research study was approved by the Ethics Committee of Alfonso X El Sabio University and complied with the Declaration of Helsinki.All handball players voluntarily participated in this investigation and they were only required to complete a self-report questionnaire about their use of supplements. The questionnaire used in this investigation has been previously validated to assess the prevalence and patterns of supplements use by athletes  and it hp value. Chi-square tests (χ2) were applied to identify differences in categorical variables . Once participants had filled out the questionnaire, their responses regarding the type of supplements used were categorized according to the ABCD system of scientific evidence established by the Australian Institute of Sport (AIS) [p < 0.05. The statistical analysis was performed using the Statistical Package for Social Sciences .Data are presented as means and standard deviation (SD) for the participant characteristics, whereas frequencies and percentages have been used for the remaining variables. The Kolmogorov-Smirnov test was used to assess the normal distribution of the data, while homoscedasticity was obtained with Levene’s test. Data were organized by sex (men vs. women) and competitive level . Comparisons for the continuous variables such as number of supplements used, were analyzed using two-way ANOVA (level × sex). The Bonferroni post-hoc test was used to detect pairwise differences in the case of a significant rt (AIS) . Additiop = 0.762), nor between amateur vs. professional handball players . In addition, no significant differences were found in the number of supplements used in men compared to women . However, professional players reported the use of a higher number of supplements than amateur players . There were no significant differences in the frequency distribution in men vs. women (p = 0.217) nor in professional vs. amateur (p = 0.098) according to the number of supplements used (nts used .p = 0.146). In the group handball players that reported the use of at least one supplement (n = 112), the likelihood of this supplement being used to enhance sports performance was 4.25-fold higher (p < 0.001) than being used for improving health, and 4.96-fold higher (p < 0.001) than for improving physical appearance. The frequency of professional players that reported the use of supplements to enhance performance (51.9%) was higher than the frequency in amateurs that used supplements with this aim . According to the purchase sites, statistical differences were reported by sex (p = 0.021) and by competitive level , improving health (12.7%), and improving physical appearance (10.9%) with no sex differences in the frequency of the reasons to use supplements (p = 0.109). The reliance on the strength and conditioning coach was higher in professional than in amateur players while a higher reliance on themselves and on internet was found in amateur players . Regarding the category sport performance supplements, a greater consumption of group A  and group B  supplements was reported in men than in women handball players. In the category of group B supplements, professional handball players also consumed more supplements than amateur players . Finally, low and equal consumption of group C supplements was reported in all categories of handball players.p < 0.050). Regarding sex, a higher consumption was observed in male handball players in terms of creatine and L-carnitine than in female handball players .The aim of this study was to determine the prevalence of supplements use in professional and amateur handball players to characterize the use of supplements in handball according to sex and competitive level. Previously, Sekulic et al.  reportedThe Australian Institute of Sport has proposed an ABCD categorization system to catalogue each supplement according to the existing level of scientific evidence regarding its effectiveness to fulfil a performance or health benefit . By usinOverall, the most frequent reason to use dietary supplements in this sample of competitive handball players was the intention of obtaining a performance benefit, which was several-fold higher to the use of supplements to enhance health or for aesthetic reasons. Sousa et al. , in profIn relation to the site of purchase, subtle differences were observed depending on the competitive level of the players. The specialized nutrition stores were the preferred place of purchase for professionals and these also were the most common sites of purchase in amateur handball players . HoweverIn handball players, the most-consumed supplements were sports drinks followed by energy bars and caffeine-containing products. The use of these supplements was high irrespective of players’ sex and competitive level . Sports The current investigation has several limitations that should be discussed to correctly apply the outcomes in competitive handball. First, we used a validated and reliable questionnaire to assess the use of dietary supplements in elite athletes . HoweverOverall, handball players reported a moderate use of supplements and there were no significant differences in the prevalence of use between men and women nor between professional vs. amateur handball players. As a whole group, handball players mainly consumed sports drinks, energy bars, and caffeine with the aim of increasing performance. However, sex and competitive level affected some patterns of use and the most-consumed supplements in handball. A higher proportion of male handball players used creatine while female handball players relied more on non-specialized shops to purchase supplements. Professional handball players used a higher number of supplements, primarily consulted their strength and conditioning coach as a source of information when planning to use sport supplementation, and nutrition stores were the most common place of purchase. On the contrary, a high proportion of amateur handball players relied on themselves to plan supplements use. The relatively low use of supplements with a low scientific level of efficacy suggests that this sample of handball players was well informed about the most effective supplements in their sports.


Anaerobutyricum soehngenii on glucose metabolism in 24 subjects with metabolic syndrome. We found that treatment with A. soehngenii was safe and observed a significant correlation between the measured fecal abundance of administered A. soehngenii and improvement in peripheral insulin sensitivity after 4 weeks of treatment. This was accompanied by an altered microbiota composition and a change in bile acid metabolism. Finally, we show that metabolic response upon administration of A. soehngenii (defined as improved insulin sensitivity 4 weeks after A. soehngenii intake) is dependent on microbiota composition at baseline. These data in humans are promising, but additional studies are needed to reproduce our findings and to investigate long-term effects, as well as other modes of delivery.Dysbiosis of the intestinal microbiota has been implicated in insulin resistance, although evidence regarding causality in humans is scarce. We performed a phase I/II dose-finding and safety study on the effect of oral intake of the anaerobic butyrogenic strain  However, the role of the intestinal microbiota and the mechanism mediating its impact on metabolic function in humans is still poorly understood. The technique of fecal microbiota transplantation (FMT) has provided insights as to the effects of gut microbiota on human metabolism4. In a pilot study, we showed that lean donor gut microbiota infusion was associated with an increase of relative abundance of Anaerobutyricum spp. in the  intestine, which was directly correlated with improvement in peripheral insulin sensitivity (Rd)5.The development of culture-independent approaches using high-throughput sequencingAnaerobutyricum soehngenii  is an anaerobic Gram-positive, catalase-negative bacterium belonging to the clostridial cluster XIVa of the phylum Firmicutes6. A. soehngenii has bile acid sodium symporter and choloylglycine hydrolase genes and is therefore capable of affecting host bile acid metabolism9. It is a butyrate-producing species, but in contrast to other well known human isolates such as Roseburia and Faecalibacterium spp. that produce butyrate from sugars, A. soehngenii has the capacity to produce butyrate from d- and l-lactate and acetate in an acid environment8, making it more likely to survive the passage through the gastrointestinal tract and the related exposure to low pH values. It is known that treatment-naive insulin resistant subjects have increased small intestinal levels of lactate-producing bacteria10, as well as increased intestinal lactate levels11. Moreover, human subjects with insulin resistance are characterized by increased production of lactate, which correlates with glucose metabolism12. A. soehngenii can convert a potentially damaging acid  into butyrate, a short-chain fatty acid (SCFA), which with known beneficial effects on glucose metabolism13, thus underscoring the potential therapeutic validity and mode of action of this strain. Previously, the safety and efficacy of oral A. soehngenii supplementation was shown in an animal model of insulin resistance by performing 4-week daily oral administrations in a dose-finding study in db/db mice14. Moreover, we observed a dose-dependent effect of A. soehngenii on improved insulin sensitivity in correspondence with fecal A. soehngenii levels. In these treated db/db mice we also observed beneficial effects on the expression of liver genes involved in lipolysis and steatosis, as well as changes in bile acids14.A. soehngenii strain orally ingested once daily for 4 weeks in treatment-naive males with metabolic syndrome. Our primary objective was to assess safety and to study the potential clinical impact on insulin sensitivity, as well as on lipolysis upon 4 weeks daily oral treatment with A. soehngenii. Changes in bile acid metabolism, MRI-measured liver fat content, and bowel habits were also studied. Finally, changes in intestinal microbiota composition, Anaerobutyricum spp. growth rates, and persistence of administered A. soehngenii up to 2 weeks after cessation of treatment were measured.While knowledge regarding the relationship between bacteria and metabolism in rodent models is rapidly increasing, confirmed causality of gut microbiota strains involved in human metabolism is still limited. Thus, in order to investigate the validity of murine data for human insulin sensitivity, we performed a single-blinded phase I/II dose-finding trial to determine the safety, efficacy, and optimal dosage of a live 7 cells/day, 109 cells/day, or 1011 cells/day of A. soehngenii each day for 4 weeks. During the trial, three subjects were excluded (one subject from each group) due to technical difficulties resulting in incomplete measurements and thus 24 were left for analyses. No significant differences were found in baseline characteristics between groups  and the relative abundance of A. soehngenii in the fecal microbiome  were significantly different among dose groups and were highest in the subjects who received the highest dose. Fecal A. soehngenii levels as determined by quantitative PCR (qPCR) were significantly increased after 4 weeks compared to baseline in all dosage groups (p = 0.012), with the highest increase in the high-dose group . The estimated ratio of secreted/ingested A. soehngenii cells was found to be significantly higher in the low-dose group compared to the medium group (Wilcoxon p = 0.015) and the high-dose groups   or Rd in either of the dose groups . There was a trend for a positive correlation between relative abundance of administered A. soehngenii and delta Rd, as well as the relative change in Rd   during the first step of the clamp and whole-body glucose rate of disposal (Rd) during the second step , from a median of 178 umol/g to 161 umol/g   among the different study groups, nor any significant between-group differences 4 weeks after treatment . Microbial composition at baseline and after 4 weeks of A. soehngenii administration is shown in Supplementary Fig. Shotgun metagenomic sequencing was performed on DNA from fecal samples taken at baseline and after 4 weeks of A. soehngenii present in the vials (measured in the high-dose samples) had a strong replication signal of 1.8, showing that ~80% of the cells in this sample were undergoing DNA replication, in line with the most probable number (MPN) culture-based assays showing high viability. Replication signal could only be determined jointly for all Anaerobutyricum spp.  and was variable but consistently lower than in the administered drink. This indicates that Anaerobutyricum spp. in the fecal samples did not have a replication activity as high as that of the administered A. soehngenii. Post-treatment specimens did not exhibit a significantly different replication signal compared to pre-treatment samples (p = 0.74). However, the high-dose post-treatment samples containing over 25% A. soehngenii . There was a borderline significant difference in replication signal between the different dose groups after treatment , with the high-dose group showing a significantly higher Anaerobutyricum spp. replication signal compared to the low-dose group  were labeled as showing “No change”. By this classification, five of the subjects showed an increase in Rd upon A. soehgenii administration , eight subjects showed a decrease, and 11 subjects showed no change. Microbial composition at baseline and after 4 weeks of A. soehngenii administration stratified by these Rd response groups is shown in Supplementary Fig. A. soehngenii, when taking baseline fecal microbiota composition into account. The abundance of three intestinal microbial species in baseline fecal samples significantly correlated with clinical response : Eubacterium rectale and Lachnospiraceae spp. showed a direct correlation , whereas Prevotella copri showed an inverse correlation . Interestingly, subjects showing a significant increase in Rd had around 3 times lower median baseline abundances of P. copri and Ruminococcaceae spp. (Supplementary Table p = 1.00) or after 4 weeks of A. soehngeni administration (Wilcoxon p = 0.75). There were no significant differences between responders and non-responders in total, conjugated, or unconjugated bile acids at either baseline or at week 4 .We next performed an exploratory post hoc analysis to study which microbiota composition characteristics would differentiate metabolic responders from non-responders upon A. soehngenii in metabolic syndrome subjects and found that daily ingestion of increasing A. soehngenii doses for 1 month was associated with increased fecal levels of A. soehngenii with greatest efficacy in the subjects who received the highest dose. The increase was transient . Combined with the good safety profile, our data imply that the highest dose of the A. soehngenii is well tolerated and may be an additional treatment for insulin resistance.In this phase I/II single (only participant) blinded pilot trial, we tested safety and efficacy of 5. We show that this approach is feasible, safe, and may induce a beneficial cardiovascular profile based on the introduced metabolically active bacterial strain. Interestingly, a recent paper showed that the increase in the levels of fecal propionate was causally linked to insulin resistance15, in line with our finding of decreased propionate upon A. soehngenii administration. However, both fecal and plasma SCFAs are notoriously difficult to measure due to volatility and assay detection limits16. Thus, the reduction in blood pressure in the highest dose group might be driven by SCFA-producing fecal bacterial strains17, despite not finding a significant effect on fecal SCFA levels. Finally, although the treatment efficacy of single-strain A. soehngenii was smaller than our findings on improved Rd upon lean donor FMT10, a recent FMT study from another group underscored these findings and showed that an enrichment of Anaerobutyricum spp. was associated with altered bile acids and clinical efficacy upon donor FMT in patients with ulcerative colitis18. In line, our results are similar to other human single-strain intervention studies demonstrating (in a subset of patients) an effect on insulin sensitivity after 12 weeks of supplementation with a high dose of Lactobacillus reuteri DSM 1793819. Upon the highest dose of A. soehngenii, half of metabolic syndrome subjects showed a significant improvement in glucose metabolism, paralleled by a concomitant increase in fecal levels of A. soehngenii after 4 weeks. This was corroborated by recent studies21 showing that engraftment of administered bacterial strains is only seen in a subset of treated subjects and depends on baseline fecal microbiota composition allowing engraftment and driving cohabitation between the endogenous microbiota and the exogenous bacterial strains21. We thus calculated that if baseline microbiota composition indeed drives the efficacy of engraftment of the A. soehngenii, we would need to treat about 20 metabolic syndrome subjects with this specific baseline microbiota composition with a dose of 1011 cells/day to be able to detect a significant increase in Rd. In contrast to our murine study14 and although fecal A. soehngenii increased significantly after 4 weeks of treatment, we observed no changes in fecal butyrate. While we cannot rule out that the effect of A. soehngenii administration is due to butyrate production16 from lactate and acetate in the small intestine7, we also observed that, upon A. soehngenii administration, plasma bile acid concentrations in the medium dose group changed with a predominant increase in plasma secondary bile acids, known to associate with improved glucose metabolism in insulin resistant subjects22. It has been previously observed in a human intervention trial using B. infantis that high concentrations (1010 CFU) of bacterial strains can induce a crowding effect resulting in less efficient dispersion of the bacteria in the intestine and thus in different clinical effects23. The fact that the medium group showed the most pronounced changes in bile acid composition may signify that it is the dose that best drives the endogenous-exogenous bacterial strain intestinal milieu for generation of secondary bile acids. Moreover, the reduction in fecal propionate levels in the same medium dose group, but not in other dose groups, aligns with this finding, although it may have been caused by the small number of subjects per treatment group. It has been increasingly recognized that intestinal microbiota play an important role in bile acid metabolism by synthesizing secondary bile acids from primary bile acids via deconjugation and dihydroxylation24. Next to their role in intestinal fat absorption, bile acids are crucial regulators of glucose and energy homeostasis24, and recent studies have shown that disturbances in bile acid metabolism may contribute to the pathogenesis of type 2 diabetes26. In line, our data in insulin resistant mice demonstrated that oral A. soehngenii treatment significantly changed levels of plasma bile acid14 and a previous probiotic human trial likewise showed altered plasma bile acids upon use of L. reuteri DSM 1793819.This study takes the reductionist approach of reintroducing a bacterial therapeutic strain in metabolic syndrome subjects based on previous intervention trialsA. soehngenii cells present in the fecal sample after 4 weeks of A. soehngenii administration was orders of magnitude higher than the daily intake of A. soehngenii cells per day  compared to that of Anaerobutyricum spp. in feces likely reflects lower growth rates of Anaerobutyricum spp. (including A. soehngenii) in the limited substrate and high competitive environment of the gut. A. soehngenii was grown in large scale production in a sucrose-based medium, found in a previous study to be protective during frozen storage27.Interestingly, the estimated number of A. soehngenii was recently published and underlined significant differences compared to the (endogenous) A. hallii6. Altogether with the different SCFA production pattern and bacterial wall fatty acid membrane composition of A. soehngenii, and in line with our clinical findings, these data strongly suggest that A. soehngenii has specific properties. Nevertheless, the dose-dependent increase in fecal A. soehngenii levels upon treatment was not associated with major changes in gut microbiota diversity, consistent with the observations from our mice study14. However, we observed an inverse correlation between baseline abundance of P. copri and the change in Rd . Also, a comparison of responders and non-responders at baseline found that responders had around 65% less P. copri when compared to non-responders  will have greater therapeutic efficacy.Our study has several limitations, including the nature of its study design, single-blinded for the participant only. Although not as powerful as a randomized clinical trial (RCT) in determining treatment effect, a single-blinded, dose-escalation study was chosen instead due to ethical considerations, as an important aim of this study was to determine whether a high daily dose of 1014. When all treatment groups were pooled, we observed a positive correlation between fecal A. soehngenii abundance and Rd. These results suggest that modulating the microbiota in humans may improve glucose metabolism and could therefore constitute a therapeutic modality in the treatment of type 2 diabetes. More research is needed on long-term effects and modes of delivery, which were beyond the scope of the current study. Nevertheless, we show here that using the current administration, high-dosed A. soehngenii is partially able to survive gastrointestinal tract passage and is accompanied by a beneficial safety profile. This provides a rationale for future A. soehngenii high-dose intervention trials in treatment-naive human subjects with metabolic syndrome.In conclusion, in this proof-of-concept pilot study, humans with metabolic syndrome were treated with a bacterial strain selected based on microbiota findings from our previous studiesn = 27) aged 21–69 years with metabolic syndrome31 not on any medication with a body mass index (BMI) between 25 and 43 kg/m2 and waist circumference > 102 cm, as well as either increased fasting plasma glucose (FPG) ≥ 5.6 mmol/l or increased fasting triglycerides ≥ 1.7 mmol/l, were recruited via local advertisements. Also, HOMA was calculated as an extra screening marker of insulin resistance (HOMA > 2.5). Exclusion criteria were the use of any medication, such as the use of proton pomp inhibitors (PPIs), statins, antihypertensives, oral anticoagulants, and antibiotics in the last 3 months, substance abuse , and history of cholecystectomy or any chronic disorder with the exception of common obesity-related conditions. Only males were included as changes in female hormone concentrations in  women have a disturbing effect on the insulin sensitivity32. Study participants were requested not to alter their physical exercise patterns after inclusion. All participants provided written informed consent and all study procedures were approved by the IRB (ethics committee) of the Amsterdam University Medical Center and conducted in accordance with the Declaration of Helsinki. The study was prospectively registered at the Dutch Trial registry .Caucasian male subjects  and a 1 ml per liter of a vitamin solution . A. soehngenii cells were harvested by microfiltration, washed with phosphate-buffered saline (PBS), and finally stored in PBS containing 10% glycerol at concentrations of either 106 cells/ml (low dose), 108 cells/ml (middle dose), or 1010 cells/ml (high dose) in 10 ml tubes at −80° C. A. soehngenii was handled under strict anaerobic conditions, which were maintained during all stages of the production of the concentrated cells: during growth, microfiltration, glycerol mixing, and filling of the tubes with a nitrogen atmosphere. The viability of A. soehngenii in randomly selected high-dose tubes  was tested every 6 months using MPN analysis in YCFA medium. MPN analyses were performed in duplicate in anoxic YCFA medium containing sucrose incubated at 37° C for 5 days. For the lowest dilutions three tubes were used, ranging up to 10−11. Growth was scored by visual and microscopic inspection. High-dose tubes stayed constant at 1010 cells/ml, within the errors of the MPN method. The drink containing A. soehngenii was shotgun sequenced using the same protocol as the study subjects’ fecal samples.The cells were obtained by culturing A. soehngenii strain L2-7 (NCBI taxonomy id 105843)6 at a concentration of either 106 cells/ml (low dose), 108 cells/ml (middle dose), or 1010 cells/ml (high dose). Thus, each subject received 107 cells/day (low dose), 109 cells/day (middle dose), or 1011 cells/day (high dose) once-daily for the duration of the 4 weeks administration period. Study subjects received the tubes containing the live A. soehngenii cells in frozen form and stored these in their home freezer at −20° C. Prior to use, a single 10 ml tube was thawed, the contents were mixed with 100 ml of milk and consumed fully. These concentrations are comparable to other human probiotics trials19. At every study contact, subjects visited the clinical research unit after an overnight fast, underwent routine physical examination, and completed a questionnaire regarding bowel habits (Irritable Bowel Syndrome (IBS) ROME III criteria and IBS Quality of Life (IBS-QOL) questionnaires)33. After an overnight fast, blood samples were drawn for plasma biochemistry and hematology, markers of inflammation, lipid and glucose metabolism, liver enzymes, and kidney function. Also, at baseline and after 4 weeks, resting energy expenditure (REE) as well as glucose and lipolysis fluxes were determined during a 2-step hyperinsulinemic euglycemic stable isotope-based clamp, and non-invasive magnetic resonance spectroscopy (MRS) of the liver was performed to measure IHTG content, as described in the The study was set up as a phase I/II single center single (only participant) blinded dose-escalation trial in order to study treatment efficacy of each dose for future clinical trials. Subjects were assigned to 1 of 3 treatment arms Fig.  where th5, as well as on the variance of the clinical measurements (stable isotope hyperinsulinemic clamp), we estimated that at least eight subjects per group were needed. A sample size of nine subjects per dose group was chosen to account for potential drop-outs. Within-group changes were tested with paired Wilcoxon-signed rank tests, while Mann–Whitney U-tests were used to compare independent groups. Kruskal–Wallis tests were used for between-group comparisons of baseline characteristics as well for between-group comparisons of relative changes. Spearman’s rho was used for correlation analysis. The significance level  used was 0.05. Power calculations and statistics are described in further detail in Based on the effect size seen upon lean donor FMT in a previous study34. Plasma bile acids were measured in fasting plasma at baseline, 4 weeks, and 6 weeks after treatment. SCFA and bile acid measurements are described in detail in Fecal SCFAs were measured in fresh (directly frozen at −20° C) morning stool samples at baseline and 4 weeks. SCFAs were separated using liquid–liquid extraction and measured using high-performance liquid chromatography-ultraviolet (HPLC-UV)A. soehngenii strain L2-7  that was selected based on (i) its presence in the genome of A. soehngenii L2-7 and absence in A. hallii and other Anaerobutyricum (meta)genomes, (ii) the usefulness of primers that were obtained by Primer-Blast, including a start with 1–2 GC pairs, melting temperature between 50–60° C, not >5° C difference between the melting temperatures of the forward/reverse primers, and absence of primer complementary regions, and (iii) successful amplification in samples with A. soehngenii L2-7 (with appropriate melting temperature of the qPCR amplicons) and the absence of amplification in negative controls. The minimal detection level was set to 3 × 10−5 ng DNA (corresponding to around 7000 gene copies), and the standard curves had a dynamic range of up to 1 ng DNA. Standards of A. soehngenii DNA ranging from 2 × 10−5 ng of DNA (corresponding to around 5000 gene copies) to 8 ng of DNA (2 × 109 gene copies) were used. The baseline samples taken prior to the A. soehngenii intervention were all below the detection level. The gene copies were determined based on the standard curve of A. soehngenii L2-7 DNA.The qPCR target DNA region was a unique sequence present in A. soehngenii strain and endogenous Anaerobutyricum spp. were identified. Anaerobutyricum spp. growth dynamics in the administered drink, as well as in subject feces were calculated using the peak-to-trough ratio method by inferring replication activity35. Metagenomic data was also used to estimate the ratio of secreted/ingested A. soehngenii cells. All DNA extraction, library preparation, and metagenomic methods are described in detail in DNA was extracted from fecal samples taken at baseline, 4 weeks, and 6 weeks after treatment. Subsequent shotgun metagenomic sequencing was performed by Clinical Microbiomics  on an Illumina HiSeq 2500 using paired-end 150 bp reads. Single-nucleotide variants (SNVs) distinguishing between the administered Further information on research design is available in the Supplementary InformationReporting SummarySupplementary Data 1


Given their importance in biology, an analytical approach to follow and understand this complex signaling hub is desirable. Here, capillary electrophoresis (CE) coupled to electrospray ionization mass spectrometry (ESI-MS) is implemented to analyze complex mixtures of InsPs and PP-InsPs with high sensitivity. Stable isotope labeled (SIL) internal standards allow for matrix-independent quantitative assignment. The method is validated in wild-type and knockout mammalian cell lines and in model organisms. SIL-CE-ESI-MS enables the accurate monitoring of InsPs and PP-InsPs arising from compartmentalized cellular synthesis pathways, by feeding cells with either [13C6]-myo-inositol or [13C6]-D-glucose. In doing so, we provide evidence for the existence of unknown inositol synthesis pathways in mammals, highlighting the potential of this method to dissect inositol phosphate metabolism and signalling.The analysis of  Myo-Inositol phosphates (InsPs) and pyrophosphates (PP-InsPs) are important second messengers but their analysis remains challenging. Here, the authors develop a capillary electrophoresis-mass spectrometry method for the identification and quantitation of InsP and PP-InsP isomers in cells and tissues. Myo-Inositol (inositol hereafter) phosphates (InsPs) are second messengers involved in signaling processes in eukaryotes1. In principle, 63 phosphorylated InsPs can be generated by sequential phosphorylation of the OH groups of inositol, resulting in significant analytical ramifications. Moreover, the fully phosphorylated inositol hexakisphosphate InsP6, the usually most abundant species, can be further phosphorylated to diphospho-inositol polyphosphates (PP-InsPs), called inositol pyrophosphates  or two ((PP)2-InsP4) phosphoric anhydride (P-anhydride) bonds in addition to the phosphate esters 2-InsP4. PP-InsPs are considered metabolic messengers, whose functions have recently become the focus of intense research5. Across species, they are signals in diverse processes including the regulation of energy metabolism and phosphate homeostasis8. Other organisms, e.g. the social amoeba Dictyostelium discoideum, produce distinct PP-InsP isomers , and manual scintillation counting of individual fractions11. While this approach is sensitive, it requires a dedicated radioactive suite, is expensive, and labor and time consuming. Moreover, it is blind to inositol generated endogenously from d-glucose-6-phosphate by inositol-3-phosphate synthase 1 (ISYNA1). Therefore, post-column derivatization UV-detection approaches have been developed to avoid radiolabeling13.The metabolic complexity of InsP turnover and their low abundance, in combination with the absence of a chromophore, and high charge density, have hampered research into these signaling molecules. The most widely applied quantitation technology relies on metabolic labeling of cells using tritiated [6 and PP-InsPs14. Different InsP pools generated from exogenously acquired or endogenously synthesized inositol can potentially be monitored using chromatography coupled to mass spectrometry (LC-MS) after feeding cells with “heavier” species such as [13C6]-inositol. However, standard SAX-HPLC using water–salt-based gradients is incompatible with MS detection and MS-compatible volatile buffers do not currently enable isomer assignment15. HPLC-MS/MS and hydrophilic interaction liquid chromatography coupled to MS (HILIC-MS/MS) unfortunately result in a suboptimal separation of the analytes, obliterating InsP isomer identity16.Recently, it was shown that inositol tetrakisphosphate 1-kinase 1 (ITPK1), which is present in Asgard archaea, social amoeba, plants, and animals, sequentially phosphorylates Ins(3)P ultimately leading to InsP17 was instrumental in the discovery and establishment of InsP3 as the Ca2+ release factor18. The separation of higher InsPs by gel electrophoresis (PAGE) is another possibility; it, however, does not have the resolving power to distinguish PP-InsP regioisomers and does not detect lower InsPs due to staining inefficiency19.The development of resolving methods using an electric field to separate the differentially charged InsPs has been pursued. High-voltage paper chromatographyA capillary electrophoresis (CE) MS method is described herein, that complements and significantly improves analytical approaches in the field. It does not require derivatization, benefits from the separation efficiency of charged analytes by CE, and enables accurate isomer identification and quantitation using stable-isotope-labeled (SIL) reference compounds, even in complex matrices. This setup also enables stable isotope pulse labeling experiments to analyze the amount of endogenously synthesized inositol over time.3, but the method was not developed further20. We now introduce a capillary electrophoresis coupled to electrospray ionization mass spectrometry (CE-ESI-MS) method, using a bare fused silica capillary and a simple background electrolyte (BGE), for parallel analyses of PP-InsPs and InsPs.CE is known as an effective separation tool for phosphate-containing molecules. An early attempt to implement CE was made in the study of InsP3, InsP4, InsP4, InsP4, InsP5, InsP5, InsP5, InsP5, InsP6, 5-PP-InsP5, 1-PP-InsP5, and 1,5-(PP)2-InsP4, was resolved with a BGE (35 mM ammonium acetate adjusted to pH 9.7 with NH4OH) by applying a 30 kV voltage over a regular bare fused silica capillary with a length of 100 cm. Detection of analytes was achieved with an ESI-TOF-MS instrument in the negative ionization mode. A stable CE separation current (23 µA) and ESI spray current (2.1 µA) were maintained with a sheath flow CE-ESI-MS interface. The limits of quantitation (LOQs) for different InsPs were 150–500 nM .A set of PP-InsP and InsP standards Fig. , represe5)P4, Ins,3,4,5,6PUCL and HCT116NIH) that have been shown to possess different PP-InsP levels21. The CE-ESI-MS method was fully compatible with current state-of-the-art InsP extraction by perchloric acid followed by enrichment with TiO2 2-InsP4, [13C6]5-PP-InsP5, [13C6]1-PP-InsP5, [13C6]InsP6, and [13C6]InsP5 (ref. 22). Application of SIL standards is crucial, as the assignment of InsPs, particularly regioisomers, now becomes unambiguous. Spiking with precise amounts of SIL standards into a biological extract also enables a reliable quantitative assessment, since they compensate for matrix effects and analyte loss. InsP5, InsP6, and 5-PP-InsP5 were assigned by their isotopic pattern, accurate mass, and identical migration time with spiked SIL standards. Excellent resolution and column efficiency were obtained: 1.5 × 104, 4.6 × 104, 3.0 × 104 theoretical plates per meter for 5-PP-InsP5, InsP6, and InsP5, isolated from HCT116NIH extract, respectively 2-InsP4  and before measurement (post-spiking) P5 and InsP5 or InsP5 were readily distinguished from InsP5 P4, InsP4, and InsP4 P4 Fig. .NIH extract were evaluated, with mean relative standard deviations (RSDs) of 3.0% (Day 1), 3.1% (Day 2), and 6.3% (Day 3) for three technical replicates, and a mean RSD of 3.6% for technical replicates from five individual days  and rapid analysis time (30 min) per run, measurement of technical replicates is feasible. The intra- and interday repeatabilities of the method analyzing HCT116ays Fig. . A compa6, synthesize 5-PP-InsP5. The PPIP5Ks are bifunctional (kinase/phosphatase) enzymes that, by acting on position 1, mainly convert 5-PP-InsP5 into 1,5-(PP)2-InsP4. CE-ESI-MS analysis of HCT116UCLIP6K1,2/−− confirms prior observations that PP-InsPs are absent 27. CE-ESI-MS analysis of NaF-treated HCT116UCL  with concomitant reduction in InsP6. We observed a reduction in InsP5 levels and appearance of 5-PP-InsP4 P5: consistent with this, CE-ESI-MS analysis of NaF-treated HCT116UCLIP6K1,2/−− also failed to detect any 5-PP-InsP4. However, confirming a previous observation26, 1-PP-InsP5 became detectable in HCT116UCLIP6K1,2/−− after NaF treatment P4 levels in NaF-treated HCT116NIH cells . However, InsP5 levels were surprisingly variable, potentially reflecting different functional roles PP5, InsP65, InsP6, and 5-PP-InsP5 were the main InsP species. Comparably low InsP levels were detected in muscle. Analysis of InsP6 and 5-PP-InsP5 extracted from tissues performed by PAGE lack the sensitivity, resolution, and dynamic range, evidenced by CE-ESI-MS. The analysis of InsPs from animal organs or tissues cannot be performed by SAX-HPLC, prohibited by cost, feasibility, and ethical considerations, since it requires feeding a mouse with [3H]-inositol.We quantified the amount of InsPs and PP-InsPs in mouse organs, including liver, brain, muscle, kidney, and spleen  that suppresses the InsP signals 2-InsP4 were detectable. In agreement with the literature32, 5-PP-InsP5 and 1/3,5-(PP)2-InsP4 were around 3% and 1% of the InsP6 level, respectively  and in plants defective in inositol pentakisphosphate 2-kinase (AtIPK1) or the ATP-binding cassette (ABC) transporter 5 (AtMRP5)33 that transports InsP6 into the vacuole 2-InsP4 were readily detected in the shoot extracts of A. thaliana Col-0 seedlings. Surprisingly, comparable levels of 5-PP-InsP5 and 1/3-PP-InsP5 were observed. This represents a significant deviation from the general notion, derived from mammalian cell analysis, that 1-PP-InsP5 represents the minor isomeric species. The atipk1 mutant displayed decreased levels of PP-InsPs and InsP6, and a robust increase in InsP5 s. In atipk1 plants, 5-PP-InsP4 was also detected by CE-ESI-MS, which was previously unsuccessfully tracked by [3H]-inositol labeling and SAX-HPLC analysis34. Analysis of atmrp5 shoots revealed the expected elevated levels of 5-PP-InsP5 and 1/3,5-(PP)2-InsP4, but not of 1/3-PP-InsP5. This analysis provides insights into single isomer PP-InsP alterations in plants, underlining the value of the SIL-CE-ESI-MS method for dissecting PP-InsP isomers and relative abundances.We analyzed the InsPs present in shoots of D. discoideum possesses large amounts of PP-InsPs. However, this model organism contains different PP-InsPs isomers from those in mammals, yeast, and plants, such as 6-PP-InsP5 and 5,6-(PP)2-InsP4 (refs. 35). This complexity is a challenge for ideal CE separation conditions. Employing a different BGE with decreased ionic strength and pH (30 mM ammonium acetate adjusted to pH 9.0 with NH4OH) led to enhanced resolution of InsP6 and more anionic species. Under these conditions, all possible PP-InsP5 isomers, including non-natural ones obtained by chemical synthesis38, were separated and could thus be assigned in complex matrices 2-InsP4 isomers, 5,4/6-(PP)2-InsP4, and 1,5-(PP)2-InsP4. Peak assignment and quantitation were conducted in a single run (30 min), and accurate mass information was provided simultaneously , in inositol-free DMEM in the presence of 1, 10, and 100 µM [13C6]-inositol. We observed a dose-dependent incorporation of [13C6]-inositol into the [13C6]InsP5 and [13C6]InsP6 pools  in inositol-free DMEM for 1, 3, and 5 days P, detected as [13C6]InsP5 and [13C6]InsP6. Note that often 1 µM or less of inositol is used in [3H]-inositol labeling for SAX-HPLC experiments42. Our analysis, therefore, reveals that up to 60% of InsPs are not detected by traditional methods if inositol concentration is kept low to improve [3H]-inositol labeling efficiency. In the presence of a tenfold higher exogenous inositol concentration (10 μM), endogenously generated inositol contributed 15% to the InsP5 and InsP6 pools. Strikingly, we detected [13C6]InsP5 and [13C6]InsP6 in both ISYNA1−/−KO1 and ISYNA1−/−KO2, although to a lesser extent than in wild-type cells  called ISYNA1 in mammals converts glucose-6-phosphate to Ins(3)Pnes Fig. , and thenes Fig. . Titratines Fig. . To stud-MS Fig. . In wildlls Fig. . This un3H]-labeled inositol is fed to cells. This approach, however, misses inositol endogenously synthesized from glucose, is restricted to specialized laboratories, is expensive, and time consuming. While other approaches, such as [13C] labeling for NMR, PAGE, and HILIC-MS, have recently been developed to provide alternatives, a transformative approach is still missing.The analysis of InsP and PP-InsP turnover in cells and tissues is a daunting challenge. Radiolabeling followed by SAX-HPLC has so far been the method of choice, as it provides the sensitivity needed for meaningful analyses of the less abundant species, with the advantage of selectively visualizing only InsPs and PP-InsPs when  NMR) were purchased from Sichem. InsP4 and InsP4 were obtained from Cayman. 1-PP-InsP5, 5-PP-InsP5, 6-PP-InsP5, and 2-PP-InsP5 were synthesized in-house (Jessen)38. SIL internal standards (IS) [13C6]1,5-(PP)2-InsP4, [13C6]5-PP-InsP5, [13C6]1-PP-InsP5, [13C6] InsP6, and [13C6]InsP5 with purities higher than 96% were synthesized in-house (Fiedler)29. Concentrations of stock solutions of InsP and PP-InsP standards for quantitation were determined by [1H] NMR and/or [31P] NMR as described below. Fused silica capillaries were obtained from CS-Chromatographie.InsPAll experiments were performed on a bare-fused silica capillary with a length of 100 cm  on an Agilent 7100 capillary electrophoresis system coupled to a Q-TOF  equipped with a commercial CE-MS adapter and sprayer kit from Agilent. Data were collected with Agilent OpenLAB CDS Chemstation 2.3.53 and Agilent MassHunter Workstation Acquisition for Q-TOF B.04.00.S. cerevisiae and A. thaliana extracts. BGE B (30 mM ammonium acetate titrated by ammonia solution to pH 9.0) was used for D. discoideum. Samples were injected by applying 50 mbar pressure for 10 s, corresponding to 0.5% of the total capillary volume (10 nL). In the study of the endogenous inositol synthesis, samples were injected with 100 mbar pressure for 10 s (20 nL). After sample injection, a BGE post-plug was introduced by applying 50 mbar for 2 s. For each analysis, a constant CE current of either 23 µA (BGE A) or 19 µA (BGE B) was established by applying 30 kV over the capillary, which was kept at a constant temperature of 25 °C.Prior to use, the capillary was flushed for 10 min with 1 N NaOH, followed by water for 10 min, and BGE for 15 min. BGE A (35 mM ammonium acetate titrated by ammonia solution to pH 9.7) was employed for the analysis of all mammalian cell and tissue extracts, as well as −, 112.9855), and . Exact mass data were acquired at a rate of 1.5 or 1 spectra/s over a 60−1000 m/z range. Extracted ion electropherograms (EIEs) were created using a 10-p.p.m. mass tolerance window for theoretical masses corresponding to the targeted inositol pyrophosphates and inositol phosphates.The sheath liquid was composed of a water–isopropanol (1:1) mixture spiked with mass references. It was introduced at a constant flow rate of 1.5 µL/min. ESI-TOF-MS was conducted in the negative ionization mode; the capillary voltage was set to −3000 V and stable ESI spray current at 2.1 µA. For TOF-MS, the fragmentor, skimmer, and Oct RFV voltage was set to 140, 60, and 750 V, respectively. The temperature and flow rate of drying gas was 250 °C and 8 L/min, respectively. Nebulizer gas pressure was 8 psi. Automatic recalibration of each acquired spectrum was performed using reference masses of reference standards P5 in biological samples was achieved by accurate mass, isotopic pattern, and identical migration time. InsP4, InsP4, and 4/6-PP-InsP5 in biological samples were assigned by accurate mass and identical migration time with spiked standards. 5-PP-InsP4 and 4/6,5-(PP)2-InsP4 in biological samples were assigned by accurate mass and based on previous research26.Peak assignment of 1/3,5-(PP)12C / IS peak area (Area)13C is <5 to ensure a linear relationship. IS stock solutions of 125 µM [13C6]5-PP-InsP5, 500 µM [13C6]InsP6, and 500 µM [13C6]InsP5 were used for the spiking experiments; 0.4 µL of IS stock solution was added into 10 µL samples before measurement. 5 µM [13C6]5-PP-InsP5, 20 µM [13C6]InsP6 and 20 µM [13C6]InsP5 were the final concentrations inside samples. The calibration curve for each analyte was constructed at eight levels by regression of nominal analyte concentration against a ratio of analyte peak area (Area)12C / IS peak area (Area)13C P5). For quantitation, two technical replicates were conducted for each sample.Quantitation of PP-InsP and InsP in mammalian cells was performed with known amounts of individual isotopic standards spiked into the samples. The amount of isotopic IS that had to be added was delineated from the concentration of the respective analyte in the sample. Ratio of analyte peak area (Area)S. cerevisiae, A. thaliana, and D. discoideum extracts was performed by comparing analyte peak areas with the respective peak areas of SIL IS with known concentrations. Concentrations of SIL IS solutions were determined by quantitative 31P and 1H NMR against a certified reference standard .Quantitation of InsP and PP-InsP in tissue extracts, NIH and HCT116UCL were originally acquired from American Type Culture Collection  and defined by tandem DNA repeat sequences21. From these lines, HCT116NIHPPIP5K/−− cells43 and HCT116UCLIP6K1,2/−− cells44 were derived. HeLa , HT29 , HEK293T , PC3 , and MCF7  cells were kind gifts from departmental colleagues.HCT116NIH and HCT116NIHPPIP5K/−− cells43 were grown in DMEM (Gibco) supplemented with 4.5 g/L glucose and 10% heat inactivated FBS (Gibco). HCT116UCL (obtained from European Collection of Authenticated Cell Cultures [ECACC]) and HCT116UCLIP6K1,2/−− cells44 were cultured in DMEM supplemented with 10% FBS (Sigma) and 4.5 g/L glucose. All cells were grown in a humidified atmosphere with 5% CO2. InsP levels were modulated by incubating the cells for 60 min with 10 mM NaF or for 30 min with 2.5 µM quercetin28 prior to harvesting.HCT11613C6]-inositol labeling experiments, inositol-free DMEM  with 10% dialyzed FBS (Sigma) was used. Normal inositol (Sigma) or [13C6]-inositol29 were supplemented as appropriate. Cells were acclimatized to 10 μM inositol in inositol-free DMEM for 1 week before starting labeling experiments. For [13C6]-glucose (Sigma) labeling experiments, DMEM lacking both inositol and glucose (Thermo Fisher) was used, using 10% dialyzed FBS. Cells were washed twice in the relevant starvation medium before incubation.For inositol limitation or -inositol radiolabeling was carried out as previously described25. Briefly, cells were seeded into 6-well plates and grown in the presence of [3H]-inositol for 5 days to ~80% confluence. Treatment with NaF (10 mM) was for 1 h. Cells were then washed with ice-cold PBS and extracted with perchloric acid, and after neutralization processed for SAX-HPLC analysis.Analysis of InsP pathways after  in 1% acetic acid) was used to confirm equal loading. Membranes were blocked for 1 h in 5% non-fat milk in TBS-T , then blotted for the following primary antibodies at 1:100–1:1000 overnight in 3% milk: ISYNA1 (sc 271830), actin . Secondary antibodies acquired from Sigma, anti-goat IgG−peroxidase conjugated (A5420) and anti-mouse IgG–peroxidase conjugated (A9044) were diluted in 3% milk. The signal was detected using Luminata Crescendo Western Substrate (Merck Millipore) and Amersham Hyperfilm (VWR) and a film developer.Cells were lysed in TX buffer  supplemented with protease and phosphatase inhibitor cocktails (Sigma). Lysates were cleared by centrifugation at 18,000 × Further information on research design is available in the Supplementary InformationPeer Review FileReporting Summary


The present study examines the relationship between drinking behaviour and drinking refusal self-efficacy among university students, with a focus on the influential roles of sports type and gender. A total of 354 students from Seoul National University of Science and Technology volunteered and participated in this study. The Korean Alcohol Use Disorders Identification Test (AUDIT-K), the Korean Drinking Refusal Self-Efficacy (DRSE) Questionnaire-Revised, and a questionnaire measuring participation in sports activities were administered to measure students’ alcohol drinking behaviours, refusal self-efficacy and sport-related activities, respectively. The data were analysed using descriptive statistics, two-way ANOVAs, correlation analysis, and regression analysis. The subdomains of DRSE had significant influences on binge drinking. Specifically, low DRSE in situations involving social pressure played a key role in predicting increased binge drinking. There was also a significant relationship between sports type and drinking behaviour, but only for male students. Those who participated in individual sports binge drank less than those who participated in team sports. Finally, gender differences were also revealed in drinking behaviour. Male students were more likely to binge drink compared to their female counterparts. This study offers a foundation for practical interventions to help control problematic drinking behaviour among university students. Alcohol is the most readily available and broadly used intoxicant, and excessive drinking is a major cause of health problems around the world . Binge dIn addition, it has been shown that university students’ drinking behaviour varies with gender ,10. WagoThe alcohol expectancy theory suggests that drinking behaviour is caused by individual beliefs and expectations regarding the effects of alcohol . FurtherConsiderable research has been conducted on the relationship between the above-mentioned sub-factors of DRSE and drinking behaviour ,22. For Another interesting component of drinking behaviour is the fact that drinking is associated with one’s level of physical activity and sports participation ,26. DingLimited research has been conducted on the relationships between DRSE, sports type, and drinking behaviour among university students. Furthermore, the majority of studies have been conducted in Western countries. Research on this issue is lacking across Eastern countries such as Korea, where drinking behaviour and related DRSE have become a concern among young adults. Previous Korean studies have mainly dealt with prevalence, frequency, and amount of drinking reported by students as opposed to assessments of related factors such as perceived confidence in refusing drinks and sports participation. Therefore, the present study investigates the relationship between drinking behaviour and DRSE among Korean university students, with a particular focus on the influential role of sport type and gender.Mage = 20.90 years, SD = 1.36) from Seoul Science and Technology (Seoultech) voluntarily participated in this study. The participants included 231 students who were involved with sports with the common sports activities being basketball, baseball, soccer, swimming, and yoga. However, the remaining did not participate in any physical activity. Seoultech is a public university located in Nowon-gu, Seoul, South Korea. It is the fifth-largest campus in the universities of Seoul and was ranked second among the public universities in 2018. Seoultech houses six colleges, 23 departments and seven graduate schools, with the enrolment rate of 11,500 students annually. Sample size calculation was conducted using G*Power 3.1.7 . The expected correlation between binge drinking and DRSEQ’s subscales was 0.20 [A total of 354 university students  and conducted in accordance with the Declaration of Helsinki using a cross-sectional study design. Participants were recruited via (a) a press release issued through the university, (b) recruitment flyers posted on university websites, and (c) an announcement made through classes with information about the study in January 2020. Recruitment occurred over one and a half months, and those who were interested in the study during this time period were added to a wait-list database. Inclusion criteria included fluency in the Korean language and being 18 years of age or older. Informed consent forms were given out before the study began. A total of 400 students gave their consent to participate. Of those, 354 students (88.50%) completed paper-and-pencil surveys. The remaining 40 (11.50%) were disqualified because they failed to complete the consent and survey forms. The estimated time to complete the survey was 10–20 min.Korean Alcohol Use Disorders Identification Test (AUDIT-K). In order to assess students’ binge drinking, two items were taken from the AUDIT-K developed by Kim and colleagues . The firDrinking Refusal Self-Efficacy Questionnaire-Revised (DRSEQ-R). DRSEQ-R was originally developed by Oei and colleagues (2005) and was translated into Korean . It was Participation in Sports Activities. In order to assess students’ participation in sports activities, the following question was used: “Have you participated in a regular sports activity, at least 3 times per week, in the last 2 months? If so, please write down the name of the activity.” The answers were then classified into non-participation, team sports, and individual sports.All analyses were conducted using SPSS Version 25.0 , and statistical significance was set at α = 0.05. Descriptive statistics were used to compare participants’ basic information, and an exploratory factor analysis was conducted on the DRSEQ-R. In order to assess the relationship between binge drinking and DRSE, Pearson correlations were analysed. A multiple linear regression analysis was employed to investigate the influence of DRSE subdomains (factors) on binge drinking. A two-way ANOVA (gender (2) × sport type (3)) was conducted to assess any effects of gender and sport type on DRSE and binge drinking. Before running the ANOVA and multiple linear regression analyses, normal distribution and homogeneity of variance assumptions were tested.F = 3.31, p < 0.05; with a main effect of sport type, F = 3.59, p < 0.05, but no main effect of gender, F = 3.31, p > 0.05 (see t(88) = 3.87, p < 0.001, and male students participating in team sports tended to binge-drink more frequently than those who participated in individual sports, t(58.90) = 3.54, p = 0.001. In contrast, female students’ binge drinking behavior did not differ as a function of sports type.An interaction between gender and sport type was observed, 0.05 see . In ordeF = 3.06, p < 0.05. Moreover, a main effect of sport type was observed, F = 6.98, p < 0.01 (see t(96.09) = −4.54, p < 0.001. Conversely, for female students, sports-type did not influence DRSE scores during situations involving emotional relief. There were no significant effects of sports type or gender on opportunistic drinking, F = 2.20, p > 0.05 and social pressure, F = 0.01, p > 0.05, in DRSE.There was a significant interaction between gender and sports type on the emotional relief of the DRSE subdomains, 0.01 see . When poR2 = 0.23). Meanwhile, only social pressure had a significant impact on alcohol drinking among their female counterparts .Since problematic alcohol drinking habits acquired during university years can lead to unhealthy habits later in adulthood, assessing drinking behaviour among young adults is an important step for preventing harmful health-related behaviour in the future ,7. ThereOur retrospective study revealed that DRSE subdomains had negative influences on binge drinking. In particular, low DRSE in social pressure situations played a key role in predicting increased binge drinking. This result did not differ between male and female students. For male students with low DRSE, when there is an opportunity to drink, the social pressure factor will influence the students to binge drinking. These findings are similar to previous research showing that lower DRSE is associated with higher alcohol consumption ,22. SpecIn a study comparing cognitive patterns related to DRSE among Asian and Caucasian university students , social Results from the current study indicated a significant relationship between sports type and drinking behaviour, but only for male students. Specifically, male students participating in individual sports binge drank less than those participating in team sports. Kim and Lee  reportedThe current findings also revealed that male students were more likely to binge drink compared to their female counterparts. The significant gender differences in binge drinking could be associated with differentiated drinking norms . AlthougAs with binge drinking, DRSE in situations involving emotional relief was also higher among students participating in sports than those who did not. In particular, this difference was significant for individuals engaging in team sports and was more prominent among male students. Engaging in sports is associated with diminished anxiety, depression, and stress and an increased sense of one’s own self-concept, self-esteem, and self-efficacy ,42,43. SThe present findings add to the literature emphasising the importance of DRSE in understanding university students’ drinking behaviour. However, several limitations should be addressed in future research. A cross-sectional research design was employed in the present study; thus, caution must be taken with inferring causal relationships. Longitudinal studies are necessary to determine the causative role of self-efficacy on drinking. In addition, while the measures used underwent a rigorous and systematic translation and validation process, all measures were self-reported, which could lead to biases in interpretation or social desirability. Finally, as the primary focus of this study was to explore drinking behaviour among Korean university students and identify the possible relationship between drinking behaviour and DRSE, and interactions between DRSE and other demographic factors were not examined.Results show that DRSE subdomains have negative influences on binge drinking; low DRSE in social pressure situations played a key role in predicting increased binge drinking. The ideas and issues identified are partially consistent with results from previous Western studies in the same field. Male students were more likely to binge drink compared to their female counterparts. Therefore, the present study offers a starting point for practical interventions to help control problematic drinking behaviour among university students.


Quality of life after ileal pouch-anal anastomosis (IPAA) surgery is generally good. However, patients can be troubled by pouch-related symptoms and pouch disorders that can be inflammatory, mechanical/surgical, and functional. Management of patients with IPAA begins with measures to maintain a healthy pouch such as optimizing pouch function, providing tailored advice on a healthy diet and lifestyle, screening for and addressing metabolic complications of IPAA, pouch surveillance, and risk stratification for risk of pouchitis and pouch failure. Pouchitis is the most common inflammatory disorder. Primary pouchitis is a spectrum currently classified into three progressive phases—an antibiotic-responsive, an antibiotic-dependent, and an antibiotic-refractory phase. It is predominately microbially mediated in acute antibiotic-responsive pouchitis and predominately immune mediated in chronic antibiotic-refractory pouchitis (CARP). Secondary prophylaxis is recommended for recurrent antibiotic-responsive and for antibiotic-dependent pouchitis. Secondary causes of antibiotic-refractory pouchitis should be ruled out before a diagnosis of CARP is made. CARP is best classified as primary sclerosing cholangitis associated, immunoglobulin G4-associated, and autoimmune. Primary sclerosing cholangitis-associated CARP can be treated with budesonide or oral vancomycin. Early recognition of immunoglobulin G4-associated pouchitis minimizes ineffective antibiotic use. Autoimmune CARP can be managed in a manner similar to UC. The current place of immunosuppressives in the treatment algorithm depends on availability and early access to biological agents. Vedolizumab and ustekinumab are the preferred first- and second-line biologics for autoimmune CARP owing to their efficacy, better side effect profile, and low immunogenicity and need for concomitant immunomodulatory therapy. Antitumor necrosis factor should be reserved for autoimmune CARP failing the above and for CD of the pouch. There are no guidelines for the surveillance of pouches for dysplasia. Incidence varies based on a patient's risk. Since incidence is low, a risk-stratified approach is recommended. Restorative proctocolectomy with ileal pouch-anal anastomosis (IPAA) is the preferred surgical treatment for most patients with ulcerative colitis (UC) and familial adenomatous polyposis (FAP). Quality of life (QOL) after colectomy and IPAA is generally good , 2. HoweAn ileoanal pouch is created from 2(J), 3(S), or 4(W) limbs of the small intestine. Of these three pouch designs, the J pouch is the most popular owing to the ease of its creation and reliability of its function. S pouch has the advantage of an additional 2–3 cm of small bowel that can be connected to the anorectal transition zone, reducing anastomotic tension, and improving blood supply in those with a short mesentery. However, suboptimal evacuation and more challenging construction have led to it largely being replaced by the J pouch. The W pouch has largely been abandoned.Quality of life following IPAA surgery is generally good and, in some studies, reported to approach that of the general population at 12 months . Indeed,Antidiarrheal medications such as loperamide, diphenoxylate/atropine, and codeine can be used to help reduce pouch frequency. Evidence supporting their efficacy is sparse. Loperamide is most widely used and, at a dose of 8 mg/day, has been shown to reduce pouch frequency and total stool weight .Supplemental fibers like psyllium husk are frequently prescribed by colorectal surgeons to reduce frequency and improve stool consistency. Psyllium husk is a water-soluble fiber that is minimally fermentable. The reduced frequency and increased stool consistency can be explained by its effects on slowing upper gastrointestinal transit and increased stool bulk through water trapping. However, tolerability and efficacy of supplemental and dietary fibers are not universal among patients with some paradoxically developing loose stools and bloating. This could be related to the amount of fiber and associated small intestinal bacterial overgrowth (SIBO) . We recoThere is currently no standardized dietary advice for IPAA patients. Observational studies suggest that most IPAA patients have at least one intolerable dietary substance negatively impacting pouch function , 6. HoweIn addition to meal contents, meal volume, frequency, and timing influence pouch frequency. One study demonstrated a positive correlation between meal volume, meal frequency, and late night meals and pouch frequency, recommending no more than three meals a day with the last at least 2 h before bedtime .Patients with healthy and inflamed IPAAs have a higher risk of iron deficiency anemia (IDA). Other causes of anemia include B12 deficiency, which has been reported in up to 25% of pouch patients . PatientThe risk of developing pouchitis, the most common disorder of IPAA, varies among patients. Numerous risk factors have been identified. Assessing for the presence or absence of these risk factors can help guide the need for primary and secondary prophylaxis for pouchitis and manage patient expectations. Some risk factors such as the NOD2/CARD15 mutation  and certPrimary Sclerosing Cholangitis (PSC): A positive association between pouchitis and PSC has been reported in numerous studies. The cumulative incidence of acute pouchitis at 10 years has been reported to be 70–80% , 16. MosP < 0.01) : EIMs are a risk factor for acute and chronic pouchitis , 20. In  < 0.01) . De novoouchitis . EIMs arP < 0.01 in one study ” . Immunogne study .Extensive colitis and backwash ileitis: The association of extent of colitis and back wash ileitis and acute and chronic pouchitis is unclear. Some studies have found extensive colitis to be a risk for acute and chronic pouchitis , 24. OthCorticosteroid exposure before proctocolectomy: Steroid dependence and high monthly steroid dose (defined as ≥ 500 mg/month before colectomy) have been associated with acute and chronic pouchitis, respectively, possibly reflecting more aggressive underlying autoimmune disease , 30.9/L, it was found on multivariate analysis to be an independent risk factor for chronic pouchitis   .P < 0.005)  compared with those who did not develop pouchitis  . In a Ja< 0.005) . In the < 0.005) .Sex: Male sex is associated with acute and chronic pouchitis . A shortP < 0.001)  .Postoperative non-steroidal anti-inflammatory drug use: Defined as more than 1 week of regular NSAIDs postoperatively, NSAID use has been associated with chronic pouchitis .Smoking status: The association of smoking and acute and chronic pouchitis is interesting. Smoking is known to have a protective effect in UC and a detrimental effect on the natural course of Crohn's disease (CD). The protective effect in UC is unclear, but smoking or nicotine reduces gut mucosal permeability and hence the antigen load triggering a mucosal immune response . ChronicChronic antibiotic-refractory pouchitis, which is immune mediated, has several shared etiopathological risk factors. Patients with these risk factors have a primed immune system with a lower threshold for initiating and maintaining an abnormal mucosal immune response. Therefore, patients harboring one or more of these risk factors should be counseled about primary prophylaxis of pouchitis, as is discussed below. Similarly, those harboring one or more risk factors who have acute antibiotic-responsive pouchitis (<4 episodes of acute pouchitis a year) can also be counseled about early commencement of secondary prophylaxis.Lactobacillus spp., Bifidobacterium spp., Streptococcus salivarius spp., and Thermophilus spp.) at a dose of 3 g/day was found in one randomized placebo-controlled trial of 40 patients to be associated with a lower pouchitis rate at 12 months (10%) compared with placebo (40%), P = 0.04  over a period of 24 months   .There are no data on the efficacy of mesalazine in primary prophylaxis. The efficacy of sulfasalazine as a primary prophylactic agent was assessed in a retrospective case series where only 15% of the 20 patients on sulfasalazine  developed pouchitis compared with 65% of the 31 controls at a median follow-up of 68 months (10–104) .In conclusion, in patients with one or more risk factors for pouchitis, we recommend primary prophylaxis using probiotics. Since VSL#3 has the strongest available data, we recommend VSL#3 at a dose of 3 g daily. Other probiotics can be tried if VSL#3 is unavailable or costly. Alternatively, sulfasalazine can be used as the 5ASA of choice. We do not recommend using oral antibiotics as primary prophylaxis. This should be combined with dietary advice aimed at achieving a diet balanced in fermentable fibers to ensure a favorable microbial community.The indications for and measures used in secondary pouchitis prophylaxis are discussed below.Pouch disorders can be classified as inflammatory, surgical/mechanical, and functional. Inflammatory disorders include pouchitis, cuffitis, and CD of the pouch. Surgical and mechanical disorders can be broadly divided into obstructive complications and leakage and fistula-related complications. Functional disorders include irritable pouch syndrome (IPS) and pelvic dyssynergia.While laboratory tests are needed to investigate most pouch-related disorders, the most appropriate diagnostic test depends on the presenting signs and symptoms in an individual patient, as outlined in Diarrhea, cramps, urgency, and incontinence symptoms: pouchitis, cuffitis, CD, and IPS—best investigated with pouchoscopy and biopsy.Dyschezia, incomplete evacuation, bloating, obstructive symptoms: stricture, floppy pouch complex, and pelvic dyssynergia—best investigated with anopouch manometry and barium defecography.Clostridioides difficile, abscess, sinus fistula. CARP, cuffitis, and CD of the pouch rarely present with these symptoms—best investigated with fecal microscopy, culture, and sensitivity/C. difficile toxin and MRI of the pelvis.Fever, night sweats, coccygeal pain, leukocytosis: pathogens cytomegalovirus (CMV)/Pouchoscopy: Can be performed with a gastroscope or a colonoscope although we prefer the former. The three areas to examine include the prepouch ileum, the pouch body, and the cuff. A normal J pouch has an owl-eye appearance. Retroflexion is useful to assess the rectal cuff and essential if fistula is suspected. Biopsies should be taken from the three examined areas, biopsying away from suture lines.Imaging: The utility of cross-sectional imaging such as MRI or CT scan of the pelvis is mainly to investigate early and late mechanical or surgical complications as well as suspected perianal or peripouch complications of CD. Barium defecography is useful when investigating obstructive pouch-related disorders.Laboratory investigations:Bloods: Useful laboratory tests include full blood evaluation, urea and electrolytes, liver function tests, C-reactive protein. Patients with anemia should be further evaluated for underlying causes, especially iron deficiency anemia and B12 deficiency.Stool.Clostridioides difficile toxin is particularly important in patients exhibiting fever or who are refractory to antibiotics.n = 10), 290 ± 131 μg/g among patients who have had at least one episode of pouchitis (n = 15), and 832 ± 422 μg/g among patients who have recurrent episodes of pouchitis (≥ 4 episodes/year)  who presented for routine pouchoscopy surveillance, fecal calprotectin was statistically significantly higher in patients with active pouchitis compared to those with inactive pouchitis. Receiver operating characteristic analysis demonstrated that a fecal calprotectin threshold of 92.5 μg/g was 80% sensitive and a 76.5 specific for the diagnosis of pouchitis [Pouchitis Disease Activity Index (PDAI) ≥ 7] . In anotes/year) . We can Functional investigations: Anopouch manometry, balloon expulsion test, barium, or MR defecography are all investigations used to investigate patients with chronic dyschezia and are detailed below.Inflammation of the pouch is the most common pouch-related disorder with around 50–60% of UC patients and 20% of FAP patients suffering at least one episode at 10 years, a fifth of whom go on to develop chronic pouchitis . A usefuThis is defined as idiopathic inflammation of the pouch. Although etiopathogenesis is not completely understood, it is believed to be an abnormal immune response to some aspect of the pouch microbiome. It appears that early on, inflammation is largely microbially mediated as evident by the efficacy of antibiotics. Over time, inflammation can become predominately immune mediated, necessitating the addition of immunosuppressants. The pathogenesis of pouchitis and its subtypes are outlined in Around 25% of chronic pouchitis are secondary to underlying conditions that need to be investigated and ruled out before a diagnosis of CARP is made. These include the following:Ischemia: Ischemia is one of the most common causes of secondary chronic pouchitis. It is characterized by asymmetric inflammation of the pouch involving the distal half, the afferent limb, or staple line . Risk fade novo. The risk of de novo CD of the pouch in patients diagnosed with UC preoperatively is ~6%  of patients initially diagnosed as having UC were diagnosed with CD upon reviewing their colectomy specimen before creating an IPAA . A two- y is ~6%  and in ty is ~6% . Known rsiae-IgA , 56, 57.a) Inflammatory CD of the pouch results in chronic pouch inflammation that may be associated with prepouch ileitis (PI) and deep ulcers in the pouch that is refractory to combination antibiotics for 4 weeks.b) Fibrostenotic CD results in ulcerated strictures anywhere in the jejunum, ileum, pouch inlet, or mid-pouch, associated with inflammation and/or ulcers of the afferent limb in the absence of NSAID use.c) Fistulae attributed to CD are non-anastomotic, developing at least 6 months after ileostomy closure in the absence of postoperative complications such as pelvic sepsis, leaks, or sinuses.The diagnosis of CD currently rests on a combination of clinical, endoscopic, histological, and radiological features. Fibrostenotic and fistulizing CD presenting in the fashion described above can usually be diagnosed endoscopically and radiologically. Crohn's disease presenting with chronic pouchitis can be harder to diagnose and distinguish from primary CARP, especially given that granulomas are only seen in 12–13% of cases, and transmural inflammation on radiological assessment is seen in both CD and CARP  of the pC. difficile infection. The presence of fever should raise the suspicion of CMV and C. difficile infections.Infections: CMV and C. difficile infection (CDI) is a common cause of secondary pouchitis reported in as many as 18% of patients (a) patients . Oral vapatients , 62.b) CMV infection: CMV infection is rarely associated with pouchitis. The main risk factor is immunosuppression. On pouchoscopy, there is pouchitis and often ulcerating PI . DiagnosNonsteroidal anti-inflammatory drugs (NSAIDs): Regular use of NSAIDs postoperatively, defined as daily use of more than 1-week post-IPAA, has been found to be associated with acute and chronic pouchitis . Furtherde novo in patients with IPAA  or ciprofloxacin  . CiproflProbiotics have been tried in acute pouchitis. High-dose VSL#3 at a dose of 3 g twice daily was found to be effective in a 4-week open-label trial , but a rDietary intervention is another potential alternative. In patients with IPAA, there is emerging evidence implicating the relative and absolute concentration of the microbial metabolites hydrogen sulfide (H2S) and butyrate, a short-chain fatty acid (SCFA), in the pathogenesis of pouchitis. Studies have shown an association between H2S production and the number and severity of pouchitis episodes . ReducedPatients failing to respond to 2 weeks of one of the antibiotics can be treated with the other agent for 2–4 weeks. Patients failing metronidazole should be treated with ciprofloxacin. Patients failing ciprofloxacin can be treated with metronidazole, although we prefer using tinidazole, as it appears to be better tolerated and more efficacious against potentially resistant microbes . PatientThe etiopathogenesis of idiopathic pouchitis is better thought of as a spectrum, whereby it is predominately microbially mediated in antibiotic-responsive pouchitis and predominately immune mediated in CARP. Antibiotic-dependent pouchitis is somewhere in between see , with trAny of the antibiotics used for the treatment of antibiotic-responsive pouchitis can be used at the lowest needed dose to maintain remission in antibiotic-dependent pouchitis. However, prolonged use of metronidazole and ciprofloxacin is associated with potential adverse effects such as peripheral neuropathy and tendinopathy, respectively. In a study that followed 39 patients with antibiotic-dependent pouchitis on metronidazole or ciprofloxacin for 1 year, adverse effects were reported in 11 (28%) patients, and antibiotic resistance was found in at least one stool sample of 28 78%) patients . Rifaxim% patientAlternative approaches that bypass the need for antibiotics in antibiotic-dependent pouchitis through attaining and maintaining a healthy microbiota spectrum or microbiota function include (i) the use of probiotics, (ii) the use of the potentially healthy products of microbiota fermentation such as butyrate, or (iii) the use compounds that bind or inactivate the potentially harmful products of microbiota metabolism such as bismuth that binds H2S.Bacteroides, coliforms, Clostridioides, enterococci, or total aerobes and anaerobes in responders despite the increased fecal concentration of all eight strains of ingested bacteria, suggesting that protection was not mediated by “suppression of endogenous luminal bacteria”  and prebiotic (fructooligosaccharides) capsules resulted in complete resolution of symptoms and reversal of endoscopic and histological features in 10 patients with chronic antibiotic-dependent and antibiotic-refractory pouchitis  four times daily (qid) resulted in 100-fold reductions in H2S release  or tinidazole (250 mg/day) for those who cannot obtain rifaximin or when it has failed. Owing to the side effects from long-term use, we would not recommend using ciprofloxacin or tinidazole continuously for more than 1 year. Oral bismuth subsalicylate at a dose of 250 mg (three times daily or qid) is used as third line following oral antibiotics. Until we have more robust data on the dose, efficacy, and tolerability of fibers and SCFA, we recommend combining all these measures with dietary advice on a diet rich in fermentable fibers, individually adjusting the quantity and type of fibers according to the patient's tolerability. Finally, since antibiotic-dependent pouchitis is both microbially and immune mediated, it is reasonable to add measures addressing the immune response to any of the above in an attempt to help patients remain in remission while discontinuing medications with potential side effects such as ciprofloxacin.Patients partially responding to measures targeting the pouch microbiome or those on long-term antibiotics wanting to reduce or discontinue them can be treated with measures aimed at suppressing the mucosal immune response.Topical and oral mesalazines have been tried in patients with CARP showing a 50% remission rate . Sulfasan = 6) and chronic pouchitis (n = 8) associated with PSC. Patients were treated with 9 mg/day of budesonide for 1–3 months and maintained on 3–6 mg/day for 9 months. At 1 year, 75% maintained remission including all of those with acute pouchitis and six of eight of those with chronic pouchitis. An 8-week course of oral budesonide controlled ileal release (9 mg/day) was also successful in inducing remission in 75% of patients with autoimmune CARP. The use of budesonide can therefore be tried, although more data on long-term efficacy and safety are needed before this is a standard recommendation.Budesonide enemas at a dose of 2 mg/100 ml a day for 6 weeks were found to be non-inferior and more tolerable than metronidazole for the management of acute pouchitis . Oral buIn CARP, pouch microbiota may still play a role in driving inflammation, as evident by some response to antibiotics, but the disease is predominately immune mediated and is sometimes referred to as immune-mediated pouchitis. Therefore, it is best managed with medications that address the mucosal immune response. The classification of CARP into PSC-associated CARP, IgG4-associated CARP, and autoimmune CARP helps guide management.a) PSC-Associated CARPBudesonide: As detailed above, budesonide has been shown to be effective in inducing and maintaining remission in PSC-associated CARP , but theVancomycin: Oral vancomycin  is successfully used to achieve and maintain remission in PSC-associated pouchitis/enteritis at the Cleveland Clinic Center for Ileal Pouch Disorders . We haveb) IgG4-Associated PouchitisIgG4-associated pouchitis was first described by Shen et al. . This isc) Autoimmune CARPThe management of autoimmune CARP shares a great deal of similarity to that of UC.® suppositories , Rowasa® enemas , and oral Pentasa®  have been tried in patients with CARP, demonstrating remission rates of 50% , 6-mercaptopurine (50–100 mg/day), and oral or subcutaneous methotrexate (7.5–25 mg/week) have been used as second-line therapy for autoimmune CARP, particularly in those with extra intestinal manifestations. There is, however, a paucity of data on the use of immunomodulator monotherapy for pouchitis . In contBiological agents: To this date, no randomized controlled studies assessing the effectiveness of biological therapy for CARP exist. Most available data come from small observational studies.a) Vedolizumab: In the largest observational study, 20 patients with chronic, antibiotic-dependent, or refractory pouchitis were treated with vedolizumab using the standard IBD dose in 10 centers in Germany. At 14 weeks, the overall reported response rate  was 64% with a drop of median PDAI from 10 to 3 and discontinuation of antibiotics in 17 out of 19 patients. In addition, no serious side effects or intolerances were reported . Other cP = 0.002). The endoscopic response was a decrease in ulcerated surface from >10 to <10% (b) Ustekinumab: In the largest observational study, 24 patients with CARP (including 2 with PSC-associated CARP) were treated with ustekinumab using standard CD dosing. There was a 50% clinical and endoscopic response. The clinical response demonstrated was an improvement in median pouch frequency from 8 to 6 ( to <10% .n = 12) and adalimumab (n = 3) were assessed. At week 14, clinically relevant remission, defined as a mPDAI <5 and a reduction of mPDAI ≥ 2 points from baseline, was achieved in 43.5% of the infliximab group and 38.5% of the adalimumab group. In the long term, 40.7% discontinued anti-TNF therapy due to intolerance or drug reaction (c) Anti-TNF: In a systematic review, the short- and long-term efficacy of anti-TNF therapy  in CARP were analyzed. Short-term efficacy was defined as clinical remission at week 8. Long-term efficacy was defined as clinical remission at the end of year. Short-term efficacy was 10%, and long-term efficacy was 37%. There was significant heterogeneity among the studies. For example, one study assessing the short- and long-term efficacy of infliximab on 24 CARP patients showed an 88% clinical response rate  at week 10 with 56% maintaining this response at a 20-month median follow-up. In a more recent study, not included in the meta-analysis, the efficacy and tolerability of infliximab  is defined as acute or chronic inflammation of the prepouch ileum extending in a contiguous fashion from the pouch inlet beyond 2 cm and up to as much as 30, and in one study 50 cm , 115. ItA basic understanding of surgical and mechanical complications is useful when managing symptomatic pouch patients. This helps facilitate the most appropriate diagnostic test and the best effective treatment, be it medical or surgical. It is useful to broadly divide these disorders into obstructive and leakage-related septic complications. The complications, their risk factors, best diagnostic investigation, and recommended treatment are outlined in de novo after IPAA in some of patients. Poorly absorbed carbohydrates and fibers can be fermented by bacteria releasing gas and increasing stool bulk, exacerbating bloating, and pouch frequency. Indeed, most patients do report improved pouch symptoms of frequency urgency and bloating with a diet low in carbohydrates and fibers and high in meat. Interestingly, supplemental fibers like psyllium husk, frequently prescribed by colorectal surgeons, can reduce frequency and improve stool consistency in pouch patients when used in small amounts. These poorly fermentable fibers can slow the gastrointestinal transit and increase stool bulk through water-trapping effects. Therefore, use of poorly fermentable fibers can be tried particularly if bloating is not a predominate symptom. Some foods such as bananas, potatoes, pasta, and bread have been reported to decrease stool consistency or “thicken stools” and therefore may be tried to see if this helps reduce frequency  or non-relaxing pelvic floor dysfunction is an underdiagnosed pouch disorder , 17. It There is, at present, no standard criteria for the diagnosis of non-relaxing pelvic floor dysfunction in IPAA patients. Although not validated for IPAA, the same tests used for the diagnosis of DD in patients with an intact colon have been used in IPAA patients using the same normal reference ranges based on healthy controls. These tests include anorectal manometry (ARM) or anopouch manometry, the balloon expulsion test, and barium or magnetic resonance defecography. Abnormal ARM, defined as paradoxical contractions, and failed balloon expulsion were found in one study in 50–60% of patients with functional pouch disorders presenting with dyschezia . In anotThe exact incidence of pouch dysplasia and pouch cancer is not clear. In the two largest cohort studies, at 20 years, the incidence of pouch dysplasia was 2.2% in the Cleveland Clinic cohort and 6.9% in the Dutch cohort , 128. ThThe single most important risk factor for pouch dysplasia and cancer is colitis-associated neoplasia before colectomy. In The Cleveland Clinic cohort, colitis-associated neoplasia was associated with pouch dysplasia and pouch cancer with hazard ratios of 3.62  and 13.43 , respectively. In the Dutch study cohort, colitis-associated neoplasia was similarly associated with dysplasia and cancer of the pouch with hazard ratios of 3.76  and 24.69 , respectively , 128.Other risk factors have included concurrent PSC, chronic inflammation of the cuff or the pouch, and mucosal villous atrophy . InteresPouchoscopy with biopsy is the test of choice for pouch neoplasia surveillance. Neoplastic lesions may appear as depressed, slightly raised lesions or, if advanced, appear mass-like. However, they can also be flat and invisible on endoscopy. In a retrospective study of 11 patients with pouch cancer, 3 (27.3%) had no endoscopically visible lesions at the time of cancer diagnosis . The useThere are no unifying consensus recommendations for pouch neoplasia surveillance. Pouch cancer carries a high mortality . PouchosIncludes patients with previous colitis-associated neoplasia before or at colectomy, history of indefinite dysplasia of pouch or focal low-grade dysplasia of the pouch. Pouchoscopy is recommended every year.Includes patients with chronic pouchitis, cuffitis, severe mucosal atrophy, previous biopsies showing hyperplastic or serrated changes in the cuff or pouch, concurrent PSC, and family history of colorectal cancer. Pouchoscopy is recommended every 1–2 years.None of the above. Can undergo pouchoscopy every 3 years commencing 10 years after IPAA surgery.The management of pouch adenocarcinoma is surgical and includes abdominoperineal resection with permanent ileostomy. The need for neo or adjuvant chemotherapy remains unclear due to the rarity of the disease. Because pouch high-grade dysplasia is considered a marker for concurrent or subsequent pouch carcinoma, once confirmed, the recommended treatment is pouch excision . EndoscoC. difficile are associated with fever and night sweats. Other secondary causes include celiac disease, NSAID, and CD of the pouch. Crohn's disease of the pouch can be inflammatory, fibrostenosing, and fistulizing. CARP is best classified as PSC associated, IgG4 associated, and autoimmune. The former two are often associated with PI. PSC-associated CARP and PI can be treated with budesonide or oral vancomycin. Early recognition of IgG4-associated pouchitis minimizes antibiotic use. Budesonide seems to improve inflammation and should be used as first line. Step-up therapy includes immunosuppressive and biologics including anti-TNFs, vedolizumab, and ustekinumab. Autoimmune CARP can be managed in a manner similar to UC. First line includes topical and oral 5ASAs, followed by oral or topical budesonide. There are limited data on the efficacy of immunosuppressives. The current place of immunosuppressives in the treatment algorithm depends on availability and early access to biological agents. Vedolizumab and ustekinumab are the preferred first- and second-line biologics for autoimmune CARP owing to their efficacy, better side effect profile, and low immunogenicity, and need for concomitant immunomodulatory therapy. Anti-TNF should be reserved for autoimmune CARP failing the above and for CD of the pouch. There are no guidelines for the surveillance of pouches for dysplasia. Incidence varies based on a patient's risk. Pouch cancer carries a high mortality. Pouchoscopy surveillance can diagnose dysplasia allowing early intervention. Since incidence is low, however, a risk-stratified approach is recommended.Quality of life after IPAA surgery is generally good. However, patients can be troubled by pouch-related symptoms and pouch disorders that can be inflammatory, mechanical/surgical, and functional. Maintaining a healthy pouch includes optimizing pouch function, providing advice on a healthy diet and lifestyle, screening for and addressing metabolic complications of IPAA, pouch surveillance, and risk stratification for risk of pouchitis and pouch failure. Patients harboring one or more risk factors for pouchitis can be offered primary prophylaxis. Pouchitis is the most common inflammatory disorder. Primary pouchitis is best classified according to antibiotic response into antibiotic responsive, antibiotic dependent, and antibiotic refractory. This is a spectrum of the same disease. It is predominately microbially mediated early on in acute antibiotic-responsive pouchitis and ends up becoming predominately immune mediated in CARP. Secondary prophylaxis is recommended for recurrent antibiotic-responsive and for antibiotic-dependent pouchitis. Probiotics are first-line secondary as prophylactic agents, followed by the antibiotic rifaximin and then bismuth. Prebiotics such as fibers are best combined with any of the above and delivered in the form of a healthy diet that can be individualized based on patients' tolerance of fermentable fibers. Secondary causes of antibiotic-refractory pouchitis should be ruled out before a diagnosis of CARP is made. Ischemic pouchitis is one of the most common causes. Infections such as CMV and All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.