Project description:Background: While the luminal microbiome composition in the human cervicovaginal tract has been defined, the presence and impact of tissue-adherent ectocervical microbiota remain incompletely understood. Studies of luminal and tissue-associated bacteria in the gastrointestinal tract suggest that they may have distinct roles in health and disease. Here, we performed a multi-omics characterization of paired luminal and tissue samples collected from a clinically well-characterized cohort of Kenyan women. Results: We identified a tissue-adherent bacterial microbiome, with a higher alpha diversity than the luminal microbiome, in which dominant genera overall included Gardnerella and Lactobacillus, followed by Prevotella, Atopobium, and Sneathia. About half of the L. iners dominated luminal samples had a corresponding Gardnerella dominated tissue microbiome. Broadly, the tissue-adherent microbiome was associated with fewer differentially expressed host genes than the luminal microbiome. Gene set enrichment analysis revealed that L. crispatus-dominated tissue-adherent communities were associated with protein translation and antimicrobial activity, whereas a highly diverse microbiome was associated with epithelial remodeling and pro-inflammatory pathways. Communities dominated by L. iners and Gardnerella were associated with low host transcriptional activity. Tissue-adherent microbiomes dominated by Lactobacillus and Gardnerella correlated with host protein profiles associated with epithelial barrier stability, and with a more pro-inflammatory profile for the Gardnerella-dominated microbiome group. Tissue samples with a highly diverse composition had a protein profile representing cell proliferation and pro-inflammatory activity. Conclusion: We identified ectocervical tissue-adherent bacterial communities in all study participants. These communities were distinct from cervicovaginal luminal microbiota in a significant proportion of individuals. This difference could possibly explain that L. iners dominant luminal communities have a high probability of transitioning to high diverse bacterial communities including high abundance of Gardnerella. By performing integrative multi-omics analyses we further revealed that bacterial communities at both sites correlated with distinct host gene expression and protein levels. The tissue-adherent bacterial community is similar to vaginal biofilms that significantly impact women’s reproductive and sexual health.

Project description:Background - Prepregnancy overweight and obesity promote deleterious health impacts on both mothers during pregnancy and the offspring. Significant changes in the maternal peripheral blood mononuclear cells (PBMCs) gene expression due to obesity are well-known. However, during pregnancy the impact of overweight on immune cell gene expression and its association with maternal and infant outcomes is not well explored. Methods – Blood samples were collected from healthy normal weight (NW, BMI 18.5-24.9) or overweight (OW, BMI 25-29.9) 2nd parity pregnant women at 12, 24 and 36 weeks of pregnancy. PBMCs were isolated from the blood and subjected to mRNA sequencing. Maternal and infant microbiota were analyzed by 16S rRNA gene sequencing. Integrative multi-omics data analysis was performed to evaluate the association of gene expression with maternal diet, gut microbiota, milk composition, and infant gut microbiota. Results - Gene expression analysis revealed that 453 genes were differentially expressed in the OW women compared to NW women at 12 weeks of pregnancy, out of which 354 were upregulated and 99 were downregulated. Several up-regulated genes in the OW group were enriched in inflammatory, chemokine-mediated signaling and regulation of interleukin-8 production-related pathways. At 36 weeks of pregnancy healthy eating index score was positively associated with several genes that include, DTD1, ELOC, GALNT8, ITGA6-AS1, KRT17P2, NPW, POT1-AS1 and RPL26. In addition, at 36 weeks of pregnancy, genes involved in adipocyte functions, such as NG2 and SMTNL1, were negatively correlated to human milk 2’FL and total fucosylated oligosaccharides content collected at 1 month postnatally. Furthermore, infant Akkermansia was positively associated with maternal PBMC anti-inflammatory genes that include CPS1 and RAB7B, at 12 and 36 weeks of pregnancy. Conclusions – These findings suggest that prepregnancy overweight impacts the immune cell gene expression profile, particularly at 12 weeks of pregnancy. Further, deciphering the complex association of PBMC’s gene expression levels with maternal gut microbiome and milk composition and infant gut microbiome may aid in developing strategies to mitigate obesity-mediated effects.

Project description:The vagina is an interactive interface with the environment, and as such is covered by a protective epithelial surface. This surface is colonized by bacteria and other microorganisms which, through a variety of mechanisms serve to further protect the host from invasion by pathogens. Alterations in the normal vaginal microflora, particularly those associated with bacterial vaginosis, are thought to contribute to risk of spontaneous pregnancy loss in the second trimester and spontaneous preterm birth. Additionally, alterations in the vaginal microbiome may increase the likelihood of transmission of certain agents including human immunodeficiency virus type 1 (HIV-1). There are physiologic alterations in host condition (e.g., menopause and pregnancy), which are beginning to be investigated as potential selective conditions for change in the "normal" flora, and their impact on disease susceptibility and transmission remains to be more definitively elucidated. The effects of chronically abnormal physiologic states (e.g., diabetes mellitus) on normal vaginal flora have not been well described or studied. Finally, an almost unexplored area of inquiry is the genetic contribution, including race/ethnicity, to the establishment and maintenance of a "normal" vaginal flora, under normal and physiologically altered circumstances. Our research will shed light on how the vaginal microbiome contributes to adverse obstetrical outcomes and sexually transmitted infections in diverse populations. This project addresses the following questions: First: Do the genes of the host contribute to the composition of the vaginal microbiome? We hypothesize that a woman's genetic composition significantly affects the ability of certain commensal, parasitic and pathogenic microbes to colonize and/or infect the genital tract. Thus, we will compare and quantify the microbial populations inhabiting the vaginas of monozygotic and dizygotic twins from the Mid Atlantic Twin Registry, and, in the process, address the question of whether there is a relationship between the microbiomes of the vagina, mouth and GI tract. Second: What changes in the vaginal microbiome are associated with common physiological perturbations or non-infectious pathological states of the host?We hypothesize that "altered" physiologic (pregnancy, menopause) and pathologic (chronic disease, hysterectomy) conditions, or environmental "exposures" (exogenous hormones, antibiotics, chronic immunosuppressant, smoking; douching) can predictably alter the vaginal microenvironment. These alterations will lead to changes in microbial populations within the vagina. Changes in the microbial populations may have impacts, positive or more likely negative, on the spontaneous and future well-being of the affected individual. We are characterizing the effects of these "altered" physiologic and pathologic conditions, and environmental exposures, on the composition of the vaginal microbiome. Third: What changes in the vaginal microbiome are associated with relevant infectious diseases and conditions? We are testing the hypothesis that infectious diseases predictably alter the vaginal microbiome, and that these changes have an impact on the disease susceptibility, process, and outcome. A predilection for bacterial vaginosis, vaginitis, HIV infection, or other sexually transmitted diseases, is likely associated with a women's vaginal microbial composition. Thus, we will characterize samples from women with a variety of these infectious conditions to determine the contribution of their micriobiomes to the disease process and susceptibility. We are addressing these questions using a combination of high throughput 'nextgen' sequencing technologies, including the Roche 454 FLX and the upgraded Illumina Genome Analyzer II instruments currently installed in the Nucleic Acids Research Facilities at VCU. Thus, segments of the 16S rRNA genes will be amplified from the complex samples taken from various target sites in and around the vagina of each study participant. These segments will be sequenced and subjected to taxonomic classification protocols to identify and quantify the bacterial taxa present in each sample. Additionally, total DNA isolated from these samples will be subjected to shotgun sequence analysis to empirically reconstruct the metabolic potential of these microbiomes. Finally, specific bacterial clones will be completely sequenced and analyzed to associate the unavoidable strain and isolate diversity with the clinical phenotypes presented.

Project description:Objective To characterise the vaginal microbiota, mucosal metabolome and host immune response in early pregnancy and investigate its relationship with adverse pregnancy outcomes, including ectopic pregnancy, within the context of pregnancies of unknown location (PUL). DesignProspective cohort study Setting Queen Charlotte’s and Chelsea Hospital, Imperial College Healthcare NHS Trust, London, UK. Population Ninety-one pregnancies of which 22 patients had a favourable outcome of a viable intrauterine pregnancy (VIUP). The remainder had an adverse outcome including 15 with a non-viable intrauterine pregnancy (NVIUP, i.e. miscarriage), 26 a failed PUL (FPUL), 20 an ectopic pregnancy (EP) and 8 a persistent PUL (PPUL). Methods Two matching pairs of vaginal swab samples were collected from women as early as four weeks gestation in pregnancies that resulted in an ectopic pregnancy, miscarriage or viable intrauterine pregnancy pregnancies matched for age, gestation and body mass index. Sequencing of the V1-V2 region of the 16S rRNA gene amplicon was used to characterize and compare vaginal bacterial compositions. The second of the pair of vaginal swabs was used sequentially first for DESI-MS direct-on swab untargeted metabolite profiling followed by extraction of the protein content for quantitative analysis of chemokine and cytokine levels using a 15-plex Luminex immune-profiling assay. Results Adverse final pregnancy outcomes were associated with reduced Lactobacillus spp. abundance (100% vs. 75.4%, p-value=9.83 x 10-3) and higher Shannon α-diversity (p-value=1.10 x 10-3) when compared to viable pregnancies. This association was independent of vaginal bleeding and observed prior to the diagnosis of the final pregnancy outcome (i.e. before the pregnancy was visible on ultrasound). Ectopic pregnancy had an even stronger association with Lactobacillus spp. depletion when compared to viable pregnancies (30% vs 0%, p-value =5.52 x 10-3). Although a strong immune mediator signature, which included MMP-1, IL-6, CCL-2/MCP-1, TNF-α, amongst others was observed in adverse pregnancy outcomes, vaginal bleeding was identified as a major confounder and adjustment of models for bleeding removed the association with outcome. Vaginal bleeding was also found to impact metabolic profiles, increasing the abundances of multiple lipid species. The prediction of outcome based on metabolic profiles was not possible but metabolic profiles were predictive with high accuracy of vaginal microbial composition at the genera level (L. dominant vs. L. deplete), and metabolic correlates of host immune activation were identified, mainly composed of lipids. Conclusions Early pregnancy vaginal microbiome communities dominated by L. crispatus or L. gasseri were observed in women with a PUL who go onto have a viable intrauterine pregnancy. Conversely, a vaginal microbiota deplete in Lactobacillus spp or dominated by L. iners is associated with a diagnosis of ectopic pregnancy in a PUL population. These findings suggest that vaginal microbiota composition is a risk factor for ectopic pregnancy. Vaginal bleeding is an inevitable cofounding factor that must be taken into consideration when performing multi-omic analysis of vaginal mucosal samples in similar clinical populations. Immune and metabolic profiles were particularly impacted by bleeding and bleeding could greatly impact the diagnostic usefulness of immune marker profiling. Further studies are required to clarify the role of microbes and infection in implantation and ectopic pregnancy, as well as determine the mechanistic pathways by which sub-optimal vaginal microbial composition increases risk. Through the integration of metataxonomics, metabolomic and immune profiling data obtained from corresponding samples, our findings demonstrate the robust predictive capacity of specific metabolome signatures. These signatures enable the simultaneous prediction of both the composition of the vaginal microbiome and the inflammatory status of the host, even in the presence of bleeding. The data derived from direct on-swab metabolic profiling using DESI-MS holds promise for swiftly stratifying the risk of early pregnancy loss by rapidly assessing the dynamics between the vaginal microbiota and host. Further validation, however, is essential for future studies to confirm this potential. Linked cross omic data sets:Meta-taxonomics data associated with this study are available in the European Nucleotide Archive (ENA): accession number PRJEB72306.

Project description:To comprehensively describe both host gene expression and microbiome composition in a single sample, parallel experimental and computational workflows -mRNA sequencing and either 16S or metagenomics- have been traditionally applied. Here, following in vitro validation of the reliability of Poly(A)-enriched mRNA sequencing in reconstructing the microbiota composition on a defined microbial mock community, we process a cohort of 30 vulvar mRNA-seq samples to fully analyze not only the transcriptome of the vulvar cells, but also the composition of microbial communities and their parallel changes. This three-level analysis on the very same specimens further enables a gene-level exploration of the reciprocal molecular crosstalk between host and microbes. The vulvar milieu represents an area of emerging research for its role in health and disease. Being at the anatomical intersection between the vaginal milieu and the perineal area, the vulvar microbiome displays an intermediate signature, with contamination from both ecosystems. Using this unified framework, we reveal marked heterogeneity and high inter-individual variability in the vulvar microbiota of healthy individuals, identifying community state types that mirror those described in the vaginal ecosystem. Importantly, we show that distinct microbial configurations are associated with specific host transcriptional programs: Lactobacillus crispatus dominated communities correlate with epithelial differentiation and barrier integrity, whereas communities enriched in taxa associated with dysbiosis exhibit transcriptional signatures linked to inflammation. Beyond providing new biological insights into an understudied anatomical niche, our study introduces a broadly applicable strategy with substantial impact for the field. With tens of thousands of human RNA-seq datasets already available in public repositories, our approach enables retrospective extraction of microbiome information and host-microbe interaction signals from existing transcriptomic data, without the need for additional sequencing or specialized microbiome protocols. This unlocks a powerful and cost-effective opportunity to revisit archived RNA-seq studies across tissues, diseases, and low-biomass environments, revealing previously inaccessible layers of host-microbiome crosstalk and maximizing the scientific value of data that already exist.

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BRAID Cervical microbiome

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