Efficacy of 5-aminolevulinic acid photodynamic therapy for the treatment of female lower reproductive tract intraepithelial lesions and its predictive biomarker: DNA methylation

Objective This prospective study aimed to evaluate the efficacy of 5-aminolevulinic acid-mediated photodynamic therapy (ALA-PDT) for cervical/vaginal intraepithelial neoplasia (CIN/VaIN) and explore the predictive value of pretreatment PAX1/JAM3 methylation status in exfoliated cells for treatment outcomes. Methods A total of 516 patients with histologically confirmed high-grade squamous intraepithelial lesion (HSIL: CIN2/3, VaIN2/3) who received ALA-PDT between September 2024 and February 2026 were enrolled. Pretreatment exfoliated cells were collected for HR-HPV detection, ThinPrep cytology test (TCT), and PAX1/JAM3 methylation analysis via multiplex real-time quantitative PCR. All patients received two courses of ALA-PDT consisting of six treatment sessions in total at 7–14-day intervals. Each session was performed with 20% ALA topical application for 3 h followed by irradiation at 80 mW/cm 2 , 635 nm, for 25–30 min. Efficacy was evaluated by pathological regression, HR-HPV clearance at 6/12 months, and factors influencing outcomes were analyzed using univariate/multivariate regression and ROC curves. Results The overall disease remission rates were 92.21% (CIN2), 88.68% (CIN3), 78.57% (VaIN2), and 66.67% (VaIN3). The HR-HPV clearance rates at 6 months were 60.79% for CIN2, 61.90% for CIN3, 52.38% for VaIN2, and 50.00% for VaIN3; the corresponding 12-month clearance rates were 71.72, 74.36, 58.54, and 50.00%, respectively. Univariate analysis identified prior cervical surgery, HPV16/18 positivity, TCT ≥ LSIL, and multiple lesions as adverse factors for CIN2. TCT ≥ LSIL, and multiple lesions were associated with poorer outcomes in VaIN2/3. PAX1/JAM3 hypomethylation was significantly associated with higher remission rates ( P < 0.001). Pretreatment PAX1/JAM3 hypermethylation showed predictive value for incomplete response to ALA-PDT. Combined methylation detection showed optimal predictive performance (AUC: 0.861 for CIN2, 0.734 for CIN3, 0.777 for VaIN2, 0.750 for VaIN3). Conclusion ALA-PDT is effective for HSIL with favorable HPV clearance. Pretreatment PAX1/JAM3 methylation status serves as a reliable non-invasive biomarker for predicting PDT efficacy, supporting individualized clinical decision-making.