Fig. 4

Sex-specific effect of ART/hypofertility on DNA methylation at imprinting control regions (ICRs). DNA methylation within the maternally methylated KCNQ1OT1 ICR (97 CpGs) (A) and paternally methylated H19/IGF2 ICR (106 CpGs) (B) was compared between ART and control groups after sex stratification. To avoid potential skewing caused by missing data, methylation at each CpG was averaged across samples for each CpG. Box plot bodies extend from the 25th to 75th percentiles, with the whiskers extending to the minimum and maximum data values; + represents the mean, and the line within the box represents the median of all CpGs. Two-way ANOVA with Bonferroni correction for multiple comparisons was used to compare ART and control groups for males and females; **p < 0.01, ****p < 0.0001. UCSC Genome Browser view of the ICR of KCNQ1OT1 (C) and H19/IGF2 (D) with DNA methylation differences between ART and control groups shown for each captured CpG. Custom tracks indicate CpG sites (green), CpGs analysed by the Illumina HumanMethylation450 array (red), CpGs analysed by the Illumina HumanMethylationEPIC array (black), the sites captured in our study by the MCC-seq capture (gold), and Sperm Dynamic CpG sites (light green). A data point represents the methylation difference between ART and control groups at a CpG, which was calculated by averaging all replicates for each group and using the mean values to compute the difference (ART-Control). Means ± SEMs values are shown, and each graph contains a smoothing spline curve (using 8 knots) to demonstrate the overall methylation difference trend across the loci