72 Chapter 3 analysis. Strategy (I) was labelled positive if cytology was ≥ASC-US (i.e., ≥BMD). Strategy (II) was labelled positive if HPV16/18 was present. Strategy (III) was labelled positive if predicted probability was above the predefined threshold of the ASCL1/LHX8 marker panel. Strategy (IV) was labelled positive if HPV16/18 was present or the cytology result was positive. Strategy (V) was labelled positive if HPV16/18 was present or the methylation result was positive. Primary clinical end-point was CIN3+. Additional analysis with CIN2 or worse (CIN2+) as endpoint was performed. Controls were defined as ≤CIN1, including women with no histology, normal histology and CIN1. All statistical analyses were performed using SPSS software for Windows (version 26.0; IBM SPSS Inc., Chicago, Il, USA), STATA (version 14.1; StataCorp, College Station, Texas, USA) and R open source software version 3.6.1 17. ST6GALNAC5 ASCL1 LHX8 (B) (A) (C) 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 NS *** *** *** Methylation level (sqrt transformed) no CIN n = 433 CIN1 n = 119 CIN2 n = 72 CIN3 n = 88 CxCa n = 3 NS *** *** *** NS *** *** *** SST GHSR ZIC1 (E) (D) (F) 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 NS *** *** *** Methylation level (sqrt transformed) NS *** *** *** NS *** *** *** no CIN n = 433 CIN1 n = 119 CIN2 n = 72 CIN3 n = 88 CxCa n = 3 no CIN n = 433 CIN1 n = 119 CIN2 n = 72 CIN3 n = 88 CxCa n = 3 no CIN n = 433 CIN1 n = 119 CIN2 n = 72 CIN3 n = 88 CxCa n = 3 no CIN n = 433 CIN1 n = 119 CIN2 n = 72 CIN3 n = 88 CxCa n = 3 no CIN n = 433 CIN1 n = 119 CIN2 n = 72 CIN3 n = 88 CxCa n = 3 Figure 3.2 Methylation levels increase with severity of underlying cervical disease. DNA methylation levels of ASCL1 (A), LHX8 (B), ST6GALNAC5 (C), GHSR (D), ZIC1 (E) and SST (F) represented by the square-root transformed Cq-ratios (y-axis) in the different histology categories (x-axis). Differences between histological categories upon Kruskal-Wallis omnibus test, followed by post hoc testing using the Wilcoxon rank-sum test: *, p value < 0.05; **, p value < 0.01; ***, p value < 0.001; NS, not significant. o outlier sample. Abbreviations: CIN, cervical intraepithelial neoplasia; CxCa, cervical carcinoma; n, number of; no CIN, women with no histology or normal histology; sqrt, square-root

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