Molecular identification of bacterial species associated with infertility in women in Al-Najaf Province via 16S rRNA sequencing

Abstract Background Infertility affects up to 25% of couples in developing countries, and bacterial vaginitis is increasingly recognized as a catalyst for reproductive failure. However, molecular profiling of the vaginal microbiota in Iraqi women remains limited. Objective To molecularly identify and compare vaginal bacterial species in infertile women with bacterial vaginitis and fertile controls in Al-Najaf Province, Iraq, using 16S rRNA gene sequencing. Methods In this case–control study (January–November 2023), women were enrolled. Two hundred and ten infertile women with clinically diagnosed bacterial vaginitis and 100 age-matched fertile controls were enrolled (UNAJ/REC/2023/012). Mid-vaginal swabs underwent DNA extraction (FavorPrep™). The 16S rRNA gene was PCR-amplified (primers 27F/1492R) and Sanger-sequenced. Sequences were quality-trimmed (Q > 20), assembled, and taxonomically assigned via BLASTn and SILVA. Relative abundances and alpha‑diversity (Shannon, Simpson) were computed in QIIME 2; group comparisons used chi‑square and Mann–Whitney U tests in SPSS v25 (α = 0.05). Results Of 310 swabs, 210 (67.7%) yielded high-quality amplicons. Infertile cases showed significantly lower Lactobacillus abundance (median 22.4% vs. 68.7% in controls; p < 0.001) and higher prevalence of Escherichia coli (45.2%), Klebsiella oxytoca (18.6%), Alcaligenes faecalis (12.3%), and exclusive detection of clostridial species. Alpha‑diversity was greater in cases (Shannon: 2.15 vs. 1.02; p < 0.001). Conclusion Infertile women with bacterial vaginitis exhibit a disrupted vaginal microbiota—marked by depletion of Lactobacilli and enrichment of opportunistic pathogens. These findings support integration of molecular diagnostics and microbiome‑targeted therapies in infertility management in resource-limited settings. Graphical Abstract