Cell type proportions rather than DNA methylation in the cord blood show significant associations with severe preeclampsia.

Preeclampsia (PE) is a severe pregnancy complication that threatens maternal and neonatal health. Previous epigenome-wide association studies (EWAS) on PE have produced inconsistent results, possibly due to inadequate adjustment for confounders. Here, we analyzed DNA methylation changes in cord blood from newborns affected by PE, using a multi-ethnic cohort from Hawaii. We comprehensively adjusted for clinical variables (maternal age, BMI, parity) and estimated cell proportions. Additionally, we re-analyzed two public datasets with similar adjustments and conducted a meta-analysis combining all three datasets to increase statistical power. To further address confounding by gestational age, we also included idiopathic preterm samples as controls. After adjusting for cell type proportions and clinical characteristics, all previously reported significant CpG methylation changes associated with severe PE disappeared across our data, the two public datasets, and the meta-analysis. This result remained even after including idiopathic preterm samples. Instead, severe PE was associated with shifts in CD8T and natural killer (NK) cell proportions. We validated this lack of CpG changes using multiple published cord blood methylation datasets. Moreover, we observed that gestational progression itself is accompanied by significant changes in granulocyte, nRBC, CD8T, and B cell proportions. In summary, our study demonstrates that many previously reported DNA methylation changes in severe PE are artifacts caused by confounding factors such as cell type heterogeneity and gestational age. Severe PE is associated with changes in cell proportions rather than direct methylation alterations. These findings emphasize the importance of rigorous confounder adjustment in EWAS.