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

ABSTRACT The vaginal microbiome plays a central role in reproductive health. Vaginal microbiome dysbiosis is associated with 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 vaginal samples from a cervical dysplasia cohort, we identified thirteen Lactobacillus crispatus accessory genes significantly associated with 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 and shotgun metagenomic sequencing for uncovering intraspecies microbial variation relevant to reproductive health. IMPORTANCE The vaginal microbiome plays a critical role in reproductive health, and different bacteria from the same species can carry different genes that influence how the strains interact with the host and other microbes. These strain-level differences are often overlooked when microbiomes are analyzed only at the species level. Existing genomic reference databases are heavily biased toward gut and environmental bacteria, leaving the genetic diversity of vaginal microbes understudied. We built a specialized reference database from over 18,000 vaginal bacterial genomes that better reflects this diversity. We then applied this resource to quantify gene-level variation in vaginal samples from a cervical dysplasia cohort. Focusing on Lactobacillus crispatus, a prevalent and often beneficial vaginal species, we identified thirteen genes that were more common in women with cervical dysplasia than in controls. This work demonstrates that body site-specific genomic resources are essential for uncovering strain-level bacterial differences relevant to reproductive health. Competing Interest Statement The authors have declared no competing interest. Footnotes Figures and figure legends improved, and minor language changes and clarification throughout the manuscript. Additional supplemental figures and tables were added. Exploration of the influence of metagenome-assembled genomes (MAGs) vs. isolate genomes on pangenome size was added to the results section.