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

This prospective single-center study aimed to investigate the clinical correlation between PAX1/JAM3 methylation (CISCER) assay and clinically evaluated vaginal microecological features in women referred for colposcopy, and to assess the potential complementary diagnostic value for detecting high-grade cervical lesions. 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. The methylation levels of PAX1 and JAM3 were elevated in participants with cervical intraepithelial neoplasia 2 (CIN2). When ΔCtPAX1 ≤ 6.6 or ΔCtJAM3 ≤ 10.0, the methylation levels for PAX1 or JAM3 were high (with low ΔCt) in ≧ CIN2 compared to ≤ CIN1 (ΔCtPAX1: 4.25 vs. 9.35; ΔCtJAM3: 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+. 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.