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

This study investigates the role of reproductive tract microbiota in the pathophysiology of ovarian endometrioma (chocolate cysts, CC) and adenomyosis (AM). We characterized and compared microbial communities across four distinct anatomical sites--cervical canal, posterior fornix, ascites fluid, and endometrium --among patients with CC, AM, and healthy controls (NC) using 16S rRNA gene sequencing. Our analysis aimed to identify specific microbial signatures and diversity shifts associated with these prevalent gynecological disorders. Furthermore, we explored the functional consequences of these microbial alterations by co-culturing representative bacterial species (Lactobacillus helveticus from NC, Enterococcus faecalis from AM, and Enterobacteriaceae from CC) with human endometrial stromal cells (T-HESC) in vitro. We assessed the impact on cell viability using CCK-8 assays and profiled transcriptomic changes via RNA-sequencing (RNA-seq), followed by GO/KEGG pathway analysis and qRT-PCR/ELISA validation. The findings reveal significant, site-specific microbial dysbiosis in women with CC and AM, and demonstrate that disease-associated bacteria can directly impair endometrial stromal cell viability and induce unique gene expression patterns related to inflammation, proliferation, and metabolism. This research provides functional evidence supporting the contribution of the reproductive tract microbiota to the complex pathophysiology of endometriosis and adenomyosis.