Ali, H.; Sujan, M. S. I.; Nahar, K.; Ahmed, M. F.; Azmuda, N.; Akter, S.; Adnan, N.

The cervicovaginal microbiome is pivotal to reproductive health, yet its dynamics in HPV-negative women with gynaecological disorders remain underexplored. We investigated microbial diversity and taxonomic shifts in HPV-negative women from Bangladesh using 16S rRNA gene sequencing and shotgun metagenomics. Of 224 women screened, 136 were HPV-negative; 29 underwent 16S profiling, and three infertility-associated cases were further analyzed by shotgun metagenomics. Healthy controls exhibited low alpha diversity and a Lactobacillus-dominated profile (98.2%), reflecting ecological stability. In contrast, pathological cases displayed significantly elevated richness and evenness, reduced Lactobacillus (28.0%), and enrichment of anaerobic and opportunistic taxa, including Bifidobacterium (23.4%), Achromobacter (12.9%) and Sneathia (7.5%). Distinct microbial signatures emerged across clinical subgroups: pelvic inflammatory disease was enriched in Bifidobacterium, intra-menstrual bleeding retained moderate Lactobacillus, while infertility exhibited prominent dominance of Achromobacter (45.5%). Shotgun metagenomics confirmed Achromobacter spp. (A. ruhlandii, A. dolens, A. xylosoxidans) as the predominant taxa (84.9%) in infertility cases, accompanied by depletion of protective Lactobacillus. Functional inference revealed conserved metabolic backbones but disease-specific enrichment of stress-response and biosynthetic pathways, particularly in infertility and PID. Co-occurrence network analysis identified condition-specific microbial consortia, with Achromobacter forming infertility-associated clusters. This study represents the first integrated application of amplicon and shotgun metagenomic approaches to profile the cervicovaginal microbiota in HPV-negative women. It identifies Achromobacter as a potential microbial biomarker of infertility and highlights the urgent need for microbiome-informed diagnostics and targeted interventions to restore cervicovaginal homeostasis.