909: Methylation profile of circulating cfDNA at term demonstrates relatively low placental contribution to hypomethylated cfDNA

The fetal fraction of circulating cell-free DNA (cfDNA) is placental in origin, and is a critical variable in noninvasive prenatal testing (NIPT). Although fetal cfDNA is known to be hypomethylated relative to maternal cfDNA, the methylation profile of circulating cfDNA in maternal plasma has not been well studied. We sought to determine whether hypomethylated cfDNA in maternal plasma: (i) could serve as a proxy for fetal fraction and (ii) was reduced in maternal obesity, as the fetal fraction of circulating cfDNA is known to be decreased in the setting of maternal obesity. In this prospective pilot study, maternal plasma was collected at admission and 24-48h postpartum (PP) from women admitted for delivery at term. Subjects were enrolled into 4 groups: lean (pre-pregnancy BMI <25 kg/m2) no labor (Lean-NL) (n=15); lean labor (Lean-L) (n=18); obese (pre-pregnancy BMI ≥30 kg/m2) no labor (OB- NL) (n=16); and obese labor (OB-L) (n=11). Total cfDNA was isolated and genome-wide methylation levels determined using 0.2x coverage whole-genome bisulfite sequencing (WGBS) in all 60 antepartum (AP) samples and in matching PP samples in ∼5 patients per study group. Calibration was performed using serial dilutions of fully methylated and unmethylated DNA. Hypomethylated cfDNA fraction was significantly lower in OB vs lean pregnant women (27.0 ±0.5% vs 28.5±0.3%, p=0.008). Hypomethylated fraction was significantly higher in AP vs PP samples overall, and when stratified by BMI (Fig 1A/B), demonstrating that the placenta contributes to hypomethylated cfDNA in maternal circulation. However, matched analyses of hypomethylation before and after delivery demonstrated a relatively small contribution of placental cfDNA to the overall hypomethylated fraction, ∼15%. The lower hypomethylated cfDNA fraction in OB patients is consistent with our prior observation of reduced placental cfDNA release in a mouse model of diet-induced obesity. Contrary to expectations, the placenta accounted for a relatively low proportion of all hypomethylated cfDNA in maternal plasma at term, suggesting that hypomethylation is not a good proxy for the fetal fraction. The finding of 20-25% hypomethylated cfDNA in non-pregnant healthy women is consistent with the oncology literature. Understanding the impact of pregnancy and maternal obesity on the methylation profile of cfDNA may have broader implications for NIPT, as well as for oncology researchers.