Preconception prenatal vitamin use was a significant modifier of IRS2 methylation and associated DMRs overlapped ASD DMRs in placenta. For (A) and (B), the x-axis represents maternal prenatal vitamins use during the first month of pregnancy, while the y-axis shows the percent methylation. (A) CYP2E1 DMR methylation was not significantly altered by P1 prenatal vitamin use. (B) Higher percent methylation at IRS2 DMR was significantly associated with not taking prenatal vitamins at P1 (two-tailed t-test, P = 0.039), which is in the same direction as ASD risk. (C) DMRs identified based on P1 prenatal vitamins use were associated with 587 genes, which showed a significant overlap with ASD DMR associated genes (Fisher’s exact test, P < 2.528e-16). (D) GO and pathway analysis were performed on the overlapped gene list (60 genes) (Supplementary Material, Table 11) between placenta ASD DMR and P1 prenatal vitamin DMR-associated genes for enrichment by Fisher’s exact test with -log (P-value) represented on the x-axis. Genes in each GO term are shown within each bar.