Genome-wide methylation profiling of maternal cell-free DNA using methylated DNA sequencing (MeD-seq) indicates a placental and immune-cell signature

Background: Placental-originated cell-free DNA (cfDNA) provides unique opportunities to study (epi)genetic placental programming remotely, but studies investigating the cfDNA methylome are scarce and usually technologically challenging. Methylated DNA sequencing (MeD-seq) is well compatible with low cfDNA concentrations and has a high genome-wide coverage. We therefore aim to investigate the feasibility of genome-wide methylation profiling of first trimester maternal cfDNA using MeD-seq, by identifying placental-specific methylation marks in cfDNA. Methods: We collected cfDNA from nonpregnant controls (female n = 6, male n = 12) and pregnant women (n = 10), first trimester placentas (n = 10), and paired preconceptional and first trimester buffy coats (total n = 20). Differentially methylated regions (DMRs) were identified between pregnant and nonpregnant women. We investigated placental-specific markers in maternal cfDNA, including RASSF1 promoter and Y-chromosomal methylation, and studied overlap with placental and buffy coat DNA methylation. Results: We identified 436 DMRs between cfDNA from pregnant and nonpregnant women, which were validated using male cfDNA. RASSF1 promoter methylation was higher in maternal cfDNA (fold change 2.87, unpaired t-test p < .0001). Differential methylation of Y-chromosomal sequences could determine fetal sex. DMRs in maternal cfDNA showed large overlap with DNA methylation of these regions in placentas and buffy coats. Sixteen DMRs in maternal cfDNA were specifically found only in placentas. These novel potential placental-specific DMRs were more prominent than RASSF1. Conclusions: MeD-seq can detect (novel) genome-wide placental DNA methylation marks and determine fetal sex in maternal cfDNA. Our results indicate a placental and immune-cell contribution to the pregnancy-specific cfDNA methylation signature. This study supports future research into maternal cfDNA methylation.