Integrative analysis of vaginal and cervical microbiota reveals microbial signatures and network aberrations associated with unexplained recurrent spontaneous abortion

Alterations in the female reproductive tract microbiota have been associated with unexplained recurrent spontaneous abortion (URSA), yet the microbial features characterizing non-pregnant women with URSA remain poorly defined. In this study, we employed 16S rRNA gene sequencing to systematically profile vaginal and cervical bacterial communities in non-pregnant women with URSA and controls. The URSA group exhibited reduced Shannon diversity, particularly in vaginal samples, reflecting diminished community evenness rather than a loss of richness, while beta diversity analyses identified disease status as the primary determinant of microbial composition. Taxonomic analysis revealed a lower relative abundance of Actinobacteriota and higher relative abundances of Proteobacteria and selected anaerobic taxa in URSA. LEfSe analysis further suggested differences in the species-level composition of Lactobacillus between the Control and URSA groups. Additional enrichment of Gram-negative bacteria such as Escherichia–Shigella and Comamonadaceae, and anaerobic Clostridia was observed in URSA. The URSA and Control networks differed in node and edge counts, connectivity patterns, and modular organization. Chronic endometritis (CE) and age as significant drivers of microbial community structure, with genera such as Kingella , Pelomonas , and Propionimicrobium showing strong associations with CE and SAB. Collectively, these findings suggest that URSA is associated with differences in reproductive tract bacterial composition and co-occurrence patterns in the non-pregnant state. These insights provide a preliminary foundation for developing microbiome-based preconception screening strategies and targeted therapeutic interventions to improve reproductive outcomes.