Reproductive tract microbiota dysbiosis in ovarian endometrioma and adenomyosis: multi-site 16S rRNA profiling and functional impact of key bacterial species on human endometrial stromal cells

To characterize the reproductive tract microbiota in patients with ovarian chocolate cysts (CC) and adenomyosis (AM) compared to controls (NC), and investigate the in vitro effects of representative differential bacteria on human endometrial stromal cells (T-HESC). Microbiota profiles from the cervical canal, posterior fornix, ascites, and endometrium of 20 CC, 20 AM, and 20 NC patients were analyzed using 16S rRNA sequencing. Bioinformatics analyses included diversity assessments and differential abundance testing using a robust consensus approach with LEfSe, ALDEx2, and ANCOM-BC to mitigate compositional bias. Representative bacteria from differential genera (Lactobacillus sp. (NC), Enterococcus sp. (AM), and a strain from the Enterobacteriaceae family (CC)) were co-cultured with T-HESCs. Cell viability (CCK-8) and transcriptomic changes (RNA-seq, GO/KEGG analysis) were assessed. Key DEGs were validated via qRT-PCR and ELISA. Significant alterations in microbial composition and diversity were observed in CC and AM groups across different sites compared to NC. Our robust differential abundance analysis identified site-specific biomarkers, including Lactobacillaceae enrichment in the NC endometrium and the genus Enterococcus in the AM posterior fornix. RNA-seq revealed distinct transcriptomic reprogramming: CC co-culture altered genes like ADH1B, MYOM1, PTGFR, ACLY, enriching pathways like mismatch repair; AM co-culture affected CEBPB, SERPINE1, S100A1, CKS1B, enriching MAPK signaling and cancer pathways. Validation confirmed these changes at the mRNA and protein levels. Endometriosis (CC and AM) is associated with distinct reproductive tract microbiota alterations. Specific bacteria linked to AM and CC impair endometrial stromal cell viability and induce unique transcriptomic changes in vitro, suggesting microbial dysbiosis contributes to endometriosis pathophysiology by affecting endometrial cell function.