Low methylation levels among HPV-vaccinated women with cervical HSIL 151 7 methylation testing. IDENTIFICATION OF CERVICAL LESIONS AND SELECTION OF HPV16/18 VACCINATED CONTROLS By the end of 2021, a total of 46 cases with cervical lesions had been identified through extended follow-up, including, 26 cases of LSIL and 20 cases of HSIL, with 32 histopathologically confirmed from cone biopsies after age 25. For this nested case-control study, eight 25-year-old controls with no cervical lesion record were matched for each of the 46 cervical LSIL/HSIL cases. Controls were matched based on place of residence and birth cohort from the HPV16/18 vaccinated females participating in the HPV-vaccinated women’s screening trial (NCT02149030). The larger control group selection provides the opportunity to stratify possible vaccine and non-vaccine hrHPV types separately within the cases and controls, and to facilitate more robust comparisons, thus helping to mitigate potential biases or confounding factors. The control women had previously undergone pelvic examination with cervical sampling at age 18 during the communityrandomised trial (NCT00534639) 22. HPV DNA ANALYSES Cervical samples collected during the third follow-up visit at age 25 were subjected to HPV DNA analysis using the modified general primer polymerase chain reaction (PCR), followed by Luminex, detecting 12 carcinogenic HPV types (HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58 and 59); 13 probably carcinogenic HPV types (HPV26, 30, 34, 53, 66, 67, 68, 69, 70, 73, 82, 85 and 97) and 18 non-carcinogenic HPV types (HPV6, 11, 40, 42, 43, 54, 61, 62, 71, 72, 74, 81, 83, 86, 87, 89, 90 and 91). DNA METHYLATION ANALYSES The same cervical samples that were HPV genotyped were analysed for DNA methylation. Extracted DNA underwent bisulphite conversion to convert unmethylated cytosines to uracils using the EZ DNA methylation kit (Zymo Research, Irvine, USA). DNA methylation of CpG islands from EPB41L3 and viral late genes (L1 and L2) of HPV16, HPV18, HPV31, and HPV33 were then amplified and pyrosequenced on a PyroMark Q96ID (Qiagen, Hilden, Germany) as previously described 23. Methylation levels of FAM19A4 and miR1242 were determined using the QIAsure Methylation Test (QIAGEN, Hilden, Germany) via quantitative methylation-specific PCR (qMSP) according to the instructions of the manufacturer.
RkJQdWJsaXNoZXIy MjY0ODMw