Methods: DNA-M was measured in blood from Isle of Wight birth cohort participants during the second half of pregnancy (weeks 22-38), using Illumina Infinium HumanMethylation450 beadchip. First, DNA-M for two consecutive pregnancies were compared in eight mothers using linear mixed models to detect changes (p≤0.05) in cytosine-phosphate-guanine (CpG) sites in Th1 (155 CpGs, 19 genes), Th2 (77 CpGs, 12 genes), Th17 (106 CpGs, 15 genes) and Treg (10 CpGs, 2 genes). Then, in a cross-sectional analysis (n=106) using linear models, we investigated whether the order of pregnancy influences the methylation of 24 CpGs. To rule out false positive results, we adjusted for multiple testing by controlling false discovery rate (FDR).
Results: Among 348 CpGs in T cell pathways, 76 CpGs were differentially methylated and 24 CpGs survived in FDR for two consecutive pregnancies. In cross-sectional analyses, four CpGs were differentially methylated and three CpGs survived in FDR out of 24 CpGs. Three CpGs survived FDR in both analyses, one each was from the IL21R and IL17D genes in the Th17 pathway and one from GATA3 in the Th2 pathway.
Conclusions: Our findings suggest that maternal methylation of specific CpGs varies with birth-order. This study allows to better understand epigenetic changes during pregnancy, which may contribute to prevent allergic diseases in offspring.