PAX1/JAM3 methylation in cervical exfoliated cells: a robust diagnostic biomarker for cervical high-grade lesions associated with vaginal dysbiosis

Methods Participants were recruited from the Gynecologic Colposcopy Outpatient Clinic of Beijing Anzhen Hospital, Capital Medical University, from February 2024 to December 2024. The clinical performance of the CISCER assay and the vaginal microecology composition of the colposcopy-referred subjects were analyzed

Results The methylation levels of PAX1 and JAM3 were elevated in participants with cervical intraepithelial neoplasia 2 (CIN2). When ��CtPAX1?��?6.6 or ��CtJAM3?��?10.0, the methylation levels for PAX1 or JAM3 were high (with low ��Ct) in �R?CIN2 compared to ��?CIN1 (��CtPAX1: 4.25 vs. 9.35; ��CtJAM3: 9.34 vs. 13.2) groups, reflecting higher methylation in high-grade lesions. For the detection of CIN2?+?and CIN3?+?lesions, the CISCER assay showed high discriminative accuracy, with AUC values of 0.826 and 0.852, respectively. Notably, the CISCER assay demonstrated superior specificity compared to hrHPV testing (89.1% vs. 7.3% for CIN2-), addressing a major limitation of current screening. Elevated PAX1/JAM3 methylation levels are associated with vaginal dysbiosis��characterized by Lactobacillus depletion and Gardnerella enrichment��and are potential strong predictors of CIN2+

Conclusions The PAX1/JAM3 methylation (CISCER) assay for cervical exfoliated cells has robust diagnostic association with high-grade cervical lesions (CIN2+) and correlates with vaginal dysbiosis biomarkers, including Gardnerella enrichment and Lactobacillus depletion, supporting its potential as a molecular triage tool for stratifying hrHPV-positive women.