Sex-influenced DNA methylation differs by placental cell type

Background Sex differences in the function and morphology of the human placenta can lead to sex differences in pregnancy outcomes. X chromosome inactivation (XCI) is the primary mechanism for dosage compensation between the sexes, and is strongly associated with X-chromosome promoter DNA methylation (DNAme) in somatic cells. However, in the placenta, low X-chromosome promoter DNAme has been reported. The placenta is a complex organ consisting of cells of different developmental origins, but the sex differences in DNAme by specific cell types have not been investigated. Methods We examined sex-influenced DNAme from 18-19 samples each of endothelial, stromal, cytotrophoblast and Hofbauer cells, sorted from term placentas, as well as matched whole chorionic villi. We also compared these profiles with data from 65 endothelial cell samples from placental chorionic plate arteries and veins (XX=16, XY=13) and umbilical cord veins (XX=22, XY=14). All data were derived from Illumina Infinium HumanMethylation450 or EPIC DNAme arrays. Sex-stratified analyses of the X/Y and autosomal DNAme were undertaken to identify DNAme differences associated with sex chromosome complement. Results The DNAme distribution on the X-chromosome differed by cell type in a manner that reflected differing developmental origins. Three distinct patterns were observed in XX placental cells reflecting origins from extraembryonic mesoderm (endothelial/stromal), trophectoderm (cytotrophoblast) and epiblast (Hofbauer cells), the latter of which shared a similar distribution with blood and umbilical endothelial cells. Interestingly, the typical XCI-associated DNAme at promoter CpG islands (CGI) on the X-chromosome of XX cells was absent for endothelial/stromal cells and present only at low levels in trophoblasts, suggesting that de novo establishment of promoter-CGI DNAme on the X-chromosome may differ by the developmental origins of each cell type. Y-chromosome DNAme also varied by cell type. Conclusion The lack of promoter DNAme in extraembryonic mesoderm-derived cells (endothelial/stromal) suggests a distinct developmentalorigin of these populations relative to the other placental and umbilical cell types. Autosomal DNAme also showed cell-type differences consistent with a common developmental origin of endothelial/stromal cells distinct from other placental cell types. This work suggests the effects of sex chromosome complement on pregnancy outcomes may differ by placental cell type.