Shannon, M. J.; Beristain, A. G.

Background: The human placenta is an essential organ for fetal development and pregnancy success. Across gestation, blastocyst-derived trophoblasts facilitate the major functions of the placenta. Specifically, invasive trophoblast subtypes, called extravillous trophoblasts (EVT), play central roles in coordinating nutrient accessibility and maternal immunomodulation to semi-allogeneic fetal and placental tissues. Like the fetus, these trophoblasts can be chromosomally male (XY) or female (XX). While male and female trophoblasts are associated with distinct placental gene signatures, specific differences between male and female EVT have not been defined across the first trimester. Methods: To understand how male and female EVT differ, we subjected male and female first trimester EVT cell preparations to bulk RNA sequencing. Concurrently, publicly available single-cell RNA sequencing datasets of first trimester placental and decidual tissues were utilized to resolve EVT differentiation and EVT subtype-specific sex differences. Candidate genes were then selected and immuno-localized to specific regions and cell populations in male and female placentas. Results: We found that before week 10 of gestation, both male and female EVT lineage cells increase expression of transcripts associated with cell proliferation. Sex-related gene differences within this early developmental time-point are restricted to genes residing on sex chromosomes. Following week 10 of gestation, there is a broad up-regulation of genes linked to immunoregulation in male and female EVT. However, within this later developmental period, autosomal gene differences appear in relation to biological sex. We go on to show that these sex-dependent autosomal gene differences influence EVT-maternal cell signalling within the uterus whereby pregnancies exposed to a male placenta demonstrate more complex MIF and CD99 as well as angiogenesis-associated VEGF cell-cell signals between male EVT and the female maternal immune and non-immune cells found throughout the uterus. Conclusions: These findings resolve early first trimester EVT lineage trophoblast sex differences and highlight a developmental timepoint that is critical to male and female autosomal gene expression.