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BackgroundAssisted reproductive technologies are associated with altered methylation in term placenta. However, it is unclear whether methylation patterns are the result of fertility treatments or intrauterine environment. Thus, we set out to determine whether there are differences in the first-trimester placenta that may be altered by the underlying fertility treatments. Genome-wide DNA methylation analyses from chorionic villus sampling (CVS) from matched singleton pregnancies conceived using in vitro fertilization (IVF), non-IVF fertility treatment (NIFT), or those conceived spontaneously were performed using Illumina Infinium HumanMethylation450 BeadChip from 15 matched CVS samples. Nanofluidic quantitative polymerase chain reaction (qPCR) of differently methylated genes was performed in a confirmatory cohort of 23 IVF conceptions and 24 NIFT conceptions.ResultsGlobal methylation was similar among the IVF, NIFT, and spontaneous conceptions. However, differential methylation from IVF and NIFT pregnancies was present at 34 CpG sites, which was significantly different. Of those, 14 corresponded to known genes, with methylation changes detected at multiple loci in 3 genes, anaphase-promoting complex subunit 2 ( ANAPC2), C-X-C motif chemokine ligand 14 ( CXCL14), and regulating synaptic membrane exocytosis 1 ( RIMS1). Nanofluidic qPCR of differentially methylated genes identified pre T-cell antigen receptor alpha ( PTCRA) to be significantly downregulated in IVF versus NIFT conceptions.ConclusionAlthough global methylation patterns are similar, there are differences in methylation of specific genes in IVF compared to NIFT conceptions, leading to altered gene expression. PTCRA was differentially methylated and downregulated in IVF conceptions, warranting further investigation. It remains to be determined whether these changes affect placentation and whether it is due to the more profound underlying infertility requiring IVF, yet these data provide unique insight into the first-trimester placental epigenome.

INTRODUCTION: Gut microbiome changes are linked to obesity, but findings are based on stool data. In this article, we analyzed the duodenal microbiome and serum biomarkers in subjects with normal weight, overweight, and obesity. METHODS: Duodenal aspirates and serum samples were obtained from subjects undergoing standard-of-care esophagogastroduodenoscopy without colon preparation. Aspirate DNAs were analyzed by 16S rRNA and shotgun sequencing. Predicted microbial metabolic functions and serum levels of metabolic and inflammatory biomarkers were also assessed. RESULTS: Subjects with normal weight (N = 105), overweight (N = 67), and obesity (N = 42) were identified. Overweight-specific duodenal microbial features include lower relative abundance (RA) of Bifidobacterium species and Escherichia coli strain K-12 and higher Lactobacillus intestinalis, L. johnsonii, and Prevotella loescheii RA. Obesity-specific features include higher Lactobacillus gasseri RA and lower L. reuteri (subspecies rodentium), Alloprevotella rava, and Leptotrichia spp RA. Escalation features (progressive changes from normal weight through obesity) include decreasing Bacteroides pyogenes, Staphylococcus hominis, and unknown Faecalibacterium species RA, increasing RA of unknown Lactobacillus and Mycobacterium species, and decreasing microbial potential for biogenic amines metabolism. De-escalation features (direction of change altered in normal to overweight and overweight to obesity) include Lactobacillus acidophilus, L. hominis, L. iners, and Bifidobacterium dentium. An unknown Lactobacillus species is associated with type IIa dyslipidemia and overweight, whereas Alloprevotella rava is associated with type IIb and IV dyslipidemias. DISCUSSION: Direct analysis of the duodenal microbiome has identified key genera associated with overweight and obesity, including some previously identified in stool, e.g., Bifidobacterium and Lactobacillus. Specific species and strains exhibit differing associations with overweight and obesity, including escalation and de-escalation features that may represent targets for future study and therapeutics. [ABSTRACT FROM AUTHOR]

BackgroundMultiple testing to understand global changes in gene expression based on genetic and epigenetic modifications is evolving. Chorionic villi, obtained for prenatal testing, is limited, but can be used to understand ongoing human pregnancies. However, optimal storage, processing and utilization of CVS for multiple platform testing have not been established.ResultsLeftover CVS samples were flash-frozen or preserved in RNAlater. Modifications to standard isolation kits were performed to isolate quality DNA and RNA from samples as small as 2-5 mg. RNAlater samples had significantly higher RNA yields and quality and were successfully used in microarray and RNA-sequencing (RNA-seq). RNA-seq libraries generated using 200 versus 800-ng RNA showed similar biological coefficients of variation. RNAlater samples had lower DNA yields and quality, which improved by heating the elution buffer to 70 °C. Purification of DNA was not necessary for bisulfite-conversion and genome-wide methylation profiling. CVS cells were propagated and continue to express genes found in freshly isolated chorionic villi.ConclusionsCVS samples preserved in RNAlater are superior. Our optimized techniques provide specimens for genetic, epigenetic and gene expression studies from a single small sample which can be used to develop diagnostics and treatments using a systems biology approach in the prenatal period. © 2016 John Wiley & Sons, Ltd.

Background/aimsCampylobacter jejuni infection is a leading cause of acute gastroenteritis, which is a trigger for post-infectious irritable bowel syndrome (PI-IBS). Cytolethal distending toxin (CDT) is expressed by enteric pathogens that cause PI-IBS. We used a rat model of PI-IBS to investigate the role of CDT in long-term altered stool form and bowel phenotypes.MethodsAdult Sprague-Dawley rats were gavaged with wildtype C. jejuni (C+), a C. jejunicdtB knockout (CDT-) or saline vehicle (controls). Four months after gavage, stool from 3 consecutive days was assessed for stool form and percent wet weight. Rectal tissue was analyzed for intraepithelial lymphocytes, and small intestinal tissue was stained with anti-c-kit for deep muscular plexus interstitial cells of Cajal (DMP-ICC).ResultsAll 3 groups showed similar colonization and clearance parameters. Average 3-day stool dry weights were similar in all 3 groups, but day-to-day variability in stool form and stool dry weight were significantly different in the C+ group vs both controls (P < 0.01) and the CDT- roup (P < 0.01), but were not different in the CDT- vs controls. Similarly, rectal lymphocytes were significantly higher after C. jejuni (C+) infection vs both controls (P < 0.01) and CDT-exposed rats (P < 0.05). The counts in the latter 2 groups were not significantly different. Finally, c-kit staining revealed that DMP-ICC were reduced only in rats exposed to wildtype C. jejuni.ConclusionsIn this rat model of PI-IBS, CDT appears to play a role in the development of chronic altered bowel patterns, mild chronic rectal inflammation and reduction in DMP-ICC.

Despite accelerated research in small intestinal bacterial overgrowth (SIBO), questions remain regarding optimal diagnostic approaches and definitions. Here, we aim to define SIBO using small bowel culture and sequencing, identifying specific contributory microbes, in the context of gastrointestinal symptoms. Subjects undergoing esophagogastroduodenoscopy (without colonoscopy) were recruited and completed symptom severity questionnaires. Duodenal aspirates were plated on MacConkey and blood agar. Aspirate DNA was analyzed by 16S ribosomal RNA and shotgun sequencing. Microbial network connectivity for different SIBO thresholds and predicted microbial metabolic functions were also assessed. A total of 385 subjects with <103 colony forming units (CFU)/mL on MacConkey agar and 98 subjects with ≥103 CFU/mL, including ≥103 to <105 CFU/mL (N = 66) and ≥105 CFU/mL (N = 32), were identified. Duodenal microbial α-diversity progressively decreased, and relative abundance of Escherichia/Shigella and Klebsiella increased, in subjects with ≥103 to <105 CFU/mL and ≥105 CFU/mL. Microbial network connectivity also progressively decreased in these subjects, driven by the increased relative abundance of Escherichia (P <.0001) and Klebsiella (P =.0018). Microbial metabolic pathways for carbohydrate fermentation, hydrogen production, and hydrogen sulfide production were enhanced in subjects with ≥103 CFU/mL and correlated with symptoms. Shotgun sequencing (N = 38) identified 2 main Escherichia coli strains and 2 Klebsiella species representing 40.24% of all duodenal bacteria in subjects with ≥103 CFU/mL. Our findings confirm ≥103 CFU/mL is the optimal SIBO threshold, associated with gastrointestinal symptoms, significantly decreased microbial diversity, and network disruption. Microbial hydrogen- and hydrogen sulfide–related pathways were enhanced in SIBO subjects, supporting past studies. Remarkably few specific E coli and Klebsiella strains/species appear to dominate the microbiome in SIBO, and correlate with abdominal pain, diarrhea, and bloating severities. [Display omitted] [ABSTRACT FROM AUTHOR]

Diabetes represents one of the most significant, and rapidly escalating, global healthcare crises we face today. Diabetes already affects one-tenth of the world's adults—more than 537 million people, numbers that have tripled since 2000 and are estimated to reach 643 million by 2030. Type 2 diabetes (T2D), the most prevalent form, is a complex disease with numerous contributing factors, including genetics, epigenetics, diet, lifestyle, medication use, and socioeconomic factors. In addition, the gut microbiome has emerged as a significant potential contributing factor in T2D development and progression. Gut microbes and their metabolites strongly influence host metabolism and immune function, and are now known to contribute to vitamin biosynthesis, gut hormone production, satiety, maintenance of gut barrier integrity, and protection against pathogens, as well as digestion and nutrient absorption. In turn, gut microbes are influenced by diet and lifestyle factors such as alcohol and medication use, including antibiotic use and the consumption of probiotics and prebiotics. Here we review current evidence regarding changes in microbial populations in T2D and the mechanisms by which gut microbes influence glucose metabolism and insulin resistance, including inflammation, gut permeability, and bile acid production. We also explore the interrelationships between gut microbes and different T2D medications and other interventions, including prebiotics, probiotics, and bariatric surgery. Lastly, we explore the particular role of the small bowel in digestion and metabolism and the importance of studying small bowel microbes directly in our search to find metabolically relevant biomarkers and therapeutic targets for T2D. [ABSTRACT FROM AUTHOR]

Forkhead L2 (FOXL2) is expressed in the ovary and acts as a transcriptional repressor of the steroidogenic acute regulatory (STAR) gene, a marker of granulosa cell differentiation. Human FOXL2 mutations that produce truncated proteins lacking the COOH terminus result in blepharophimosis/ptosis/epicanthus inversus (BPES) syndrome type I, which is associated with premature ovarian failure (POF). In this study, we investigated whether FOXL2's activity as a transcriptional repressor is regulated by phosphorylation. We found that FOXL2 is phosphorylated at a serine residue and, using yeast two-hybrid screening, identified LATS1 as a potential FOXL2-interacting protein. LATS1 is a serine/threonine kinase whose deletion in mice results in an ovarian phenotype similar to POF. Using coimmunoprecipitation and kinase assays, we confirmed that LATS 1 binds to FOXL2 and demonstrated that LATS1 phosphorylates FOXL2 at a serine residue. Moreover, we found that FOXL2 and LATS1 are coexpressed in developing mouse gonads and in granulosa cells of small and medium follicles in the mouse ovary. Last, we demonstrated that coexpression with LATS1 enhances FOXL2's activity as a repressor of the StAR promoter, and this results from the kinase activity of LATS1. These results provide novel evidence that FOXL2 is phosphorylated by LATS1 and that this phosphorylation enhances the transcriptional repression of the StAR gene, a marker of granulosa cell differentiation. These data support our hypothesis that phosphorylation of FOXL2 may be a control mechanism regulating the rate of granulosa cell differentiation and hence, follicle maturation, and its dysregulation may contribute to accelerated follicular development and POF in BPES type I. large tumor suppressor gene 1 phosphorylation; transcriptional regulation; forkhead L2; granulosa cell; premature ovarian failure doi: 10.1152/ajpendo.00534.2009.