Expanding vaginal microbiome pangenomes via a custom MIDAS database reveals Lactobacillus crispatus accessory genes associated with cervical dysplasia

The vaginal microbiome plays a central role in reproductive health. Vaginal microbiome dysbiosis increases risk of many adverse reproductive health outcomes, but most studies have focused on associations at the species level. The potential contribution of intraspecies microbial variation, especially gene content differences across bacterial strains, remains underexplored in reproductive health contexts. The Metagenomic Intra-Species Diversity Analysis (MIDAS) framework enables such analyses but depends on comprehensive reference databases. We constructed a MIDAS-compatible pangenome database from over 18,000 genomes in the Vaginal Microbiome Genome Collection (VMGC). Compared to the Genome Taxonomy Database (GTDB)-derived reference, the VMGC database expanded pangenomes of prevalent vaginal species, better capturing vaginal-specific intraspecies diversity. Applying this database to a cervical dysplasia cohort, we identified thirteen Lactobacillus crispatus accessory genes significantly associated with increased risk of cervical dysplasia, including a HicAB toxin-antitoxin system, three transcriptional regulators, and three phage-derived genes. These findings highlight the utility of body site-specific reference resources for uncovering intraspecies microbial variation relevant to reproductive health.