Gut microbiome in endometriosis: a cohort study on 1000 individuals.

BACKGROUND: Endometriosis, defined as the presence of endometrial-like tissue outside of the uterus, is one of the most prevalent gynecological disorders. Although different theories have been proposed, its pathogenesis is not clear. Novel studies indicate that the gut microbiome may be involved in the etiology of endometriosis; nevertheless, the connection between microbes, their dysbiosis, and the development of endometriosis is understudied. This case-control study analyzed the gut microbiome in women with and without endometriosis to identify microbial targets involved in the disease. METHODS: A subsample of 1000 women from the Estonian Microbiome cohort, including 136 women with endometriosis and 864 control women, was analyzed. Microbial composition was determined by shotgun metagenomics and microbial functional pathways were annotated using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. Partitioning Around Medoids (PAM) algorithm was performed to cluster the microbial profile of the Estonian population. The alpha- and beta-diversity and differential abundance analyses were performed to assess the gut microbiome (species and KEGG orthologies (KO)) in both groups. Metagenomic reads were mapped to estrobolome-related enzymes' sequences to study potential microbiome-estrogen metabolism axis alterations in endometriosis. RESULTS: Diversity analyses did not detect significant differences between women with and without endometriosis (alpha-diversity: all p-values > 0.05; beta-diversity: PERMANOVA, both R 2 < 0.0007, p-values > 0.05). No differential species or pathways were detected after multiple testing adjustment (all FDR p-values > 0.05). Sensitivity analysis excluding women at menopause (> 50 years) confirmed our results. Estrobolome-associated enzymes' sequence reads were not significantly different between groups (all FDR p-values > 0.05). CONCLUSIONS: Our findings do not provide enough evidence to support the existence of a gut microbiome-dependent mechanism directly implicated in the pathogenesis of endometriosis. To the best of our knowledge, this is the largest metagenome study on endometriosis conducted to date.

Association of Accelerometer-Determined Physical Activity and Sedentary Behavior With the Gut Microbiome in Middle-Aged Women: A Compositional Data Approach.

The beneficial effects of physical activity (PA) on gut microbiome have been reported, nevertheless the findings are inconsistent, with the main limitation of subjective methods for assessing PA. It is well accepted that using an objective assessment of PA reduces the measurement error and also allows objective assessment of sedentary behavior (SB). We aimed to study the associations between accelerometer-assessed behaviors (i.e., SB, light-intensity physical activity [LPA] and moderate-to-vigorous physical activity [MVPA]) with the gut microbiome using compositional data analysis, a novel approach that enables to study these behaviors accounting for their inter-dependency. This cross-sectional study included 289 women from the Northern Finland Birth Cohort 1966. Physical activity was measured during 14 days by wrist-worn accelerometers. Analyses based on the combined effect of MVPA and SB, and compositional data analyses in association with the gut microbiome data were performed. The microbial alpha- and beta-diversity were not significantly different between the MVPA-SB groups, and no differentially abundant microorganisms were detected. Compositional data analysis did not show any significant associations between any movement behavior (relative to the others) on microbial alpha-diversity. Butyrate-producing bacteria such as Agathobacter and Lachnospiraceae CAG56 were significantly more abundant when reallocating time from LPA or SB to MVPA (γ = 0.609 and 0.113, both p-values = 0.007). While PA and SB were not associated with microbial diversity, we found associations of these behaviors with specific gut bacteria, suggesting that PA of at least moderate intensity (i.e., MVPA) could increase the abundance of short-chain fatty acid-producing microbes.

Cell-Free Microbial DNA Analysis: Effects of Blood Plasma and Serum Quantity, Biobanking Protocols, and Isolation Kits.

Recent studies highlight the presence of bacterial sequences in the human blood, suggesting potential clinical significance for circulating microbial signatures. These sequences could presumably serve in the diagnosis, prediction, or monitoring of various health conditions. Ensuring the similarity of samples before bacterial analysis is crucial, especially when combining samples from different biobanks prepared under varying conditions (such as different DNA extraction kits, centrifugation conditions, blood collection tubes, etc.). In this study, we aimed to analyze the impact of different sample collection and nucleic acid extraction criteria (blood collection tube, centrifugation, input volume, and DNA extraction kit) on circulating bacterial composition. Blood samples from four healthy individuals were collected into three different sample collection tubes: K2EDTA plasma tube, sodium citrate plasma tube, and gel tube for blood serum. Tubes were centrifugated at standard and double centrifugation conditions. DNA extraction was performed using 100, 200, and 500 µL plasma/serum input volumes. DNA extraction was performed using three different isolation kits: Norgen plasma/serum cell-free circulating DNA purification micro kit, Applied Biosystems MagMAX cell-free DNA isolation kit, and Qiagen QIAamp MinElute cell-free circulating DNA mini kit. All samples were subjected to 16S rRNA V1-V2 library preparation and sequencing. In total, 216 DNA and 18 water control samples were included in the study. According to PERMANOVA, PCoA, Mann-Whitney, and FDR tests the effect of the DNA extraction kit on the microbiota composition was the greatest, whereas the type of blood collection tube, centrifugation type, and sample input volume for the extraction had minor effects. Samples extracted with the Norgen DNA extraction kit were enriched with Gram-negative bacteria, whereas samples extracted with the Qiagen and MagMAX kits were enriched with Gram-positive bacteria. Bacterial profiles of samples prepared with the Qiagen and MagMAX DNA extraction kits were more similar, whereas samples prepared with the Norgen DNA extraction kit were significantly different from other groups.

A history of repeated antibiotic usage leads to microbiota-dependent mucus defects.

Recent evidence indicates that repeated antibiotic usage lowers microbial diversity and ultimately changes the gut microbiota community. However, the physiological effects of repeated - but not recent - antibiotic usage on microbiota-mediated mucosal barrier function are largely unknown. By selecting human individuals from the deeply phenotyped Estonian Microbiome Cohort (EstMB), we here utilized human-to-mouse fecal microbiota transplantation to explore long-term impacts of repeated antibiotic use on intestinal mucus function. While a healthy mucus layer protects the intestinal epithelium against infection and inflammation, using ex vivo mucus function analyses of viable colonic tissue explants, we show that microbiota from humans with a history of repeated antibiotic use causes reduced mucus growth rate and increased mucus penetrability compared to healthy controls in the transplanted mice. Moreover, shotgun metagenomic sequencing identified a significantly altered microbiota composition in the antibiotic-shaped microbial community, with known mucus-utilizing bacteria, including Akkermansia muciniphila and Bacteroides fragilis, dominating in the gut. The altered microbiota composition was further characterized by a distinct metabolite profile, which may be caused by differential mucus degradation capacity. Consequently, our proof-of-concept study suggests that long-term antibiotic use in humans can result in an altered microbial community that has reduced capacity to maintain proper mucus function in the gut.