DNA methylation profiling of placental villi from karyotypically normal recurrent miscarriage

Miscarriage occurs in 15-20% of clinical pregnancies. While chromosomal errors are observed in over 50%, causes of karyotypically normal losses are poorly understood. DNA methylation undergoes reprogramming during development and must be appropriately set to maintain a healthy pregnancy. We hypothesize that aberrant DNA methylation may cause karyotypically normal miscarriage, particularly among women experiencing recurrent miscarriage (RM). DNA methylation in first trimester chorionic villi was assessed in chromosomally normal miscarriages from women with RM (N=33) or isolated miscarriage (M, N=21), and elective terminations (TA, N=16). Differentially methylated candidate loci were identified using the Illumina Infinium HumanMethylation27 BeadChip array by comparing 10 RM to 10 TA samples. Follow up showed a significant increase in methylation in RM and M compared to TA placentae at the CYP1A2 (p=0.002) and AXL (p=0.02) promoters and decrease at the DEFB1 (p=0.008) promoter. Gene function analysis showed an enrichment of imprinted genes (p=9.53E-10) and genes previously associated with RM (p=9.51E-06). DNA methylation was evaluated at 7 imprinted loci using bisulfite pyrosequencing. An increase of outliers at these loci was observed in RM (3.9%) compared to M (0%) and TA (0.9%) (p=0.02), with increased average methylation at the H19/IGF2 ICR1 in M samples (p<0.0001). Altered DNA methylation in the placenta at specific loci, as well as global dysregulation in specific cases, may contribute to or be a consequence of placental insufficiency in karyotypically normal miscarriage. First-trimester placental villi samples from karyotypically normal miscarriages from recurrent miscarriage patients (N, N=10) and chromosomally normal elective terminations (PZET, N=10).