Sexually Dimorphic Crosstalk at the Maternal-Fetal Interface

Abstract

Context: Crosstalk through receptor ligand interactions at the maternal-fetal interface is impacted by fetal sex. This affects placentation in the first trimester and differences in outcomes. Sexually dimorphic signaling at early stages of placentation are not defined.

Objective: Investigate the impact of fetal sex on maternal-fetal crosstalk.

Design: Receptors/ligands at the maternal-fetal surface were identified from sexually dimorphic genes between fetal sexes in the first trimester placenta and defined in each cell type using single-cell RNA-Sequencing (scRNA-Seq).

Setting: Academic institution.

Samples: Late first trimester (~10-13 weeks) placenta (fetal) and decidua (maternal) from uncomplicated ongoing pregnancies.

Main outcome measures: Transcriptomic profiling at tissue and single-cell level; immunohistochemistry of select proteins.

Results: We identified 91 sexually dimorphic receptor-ligand pairs across the maternal-fetal interface. We examined fetal sex differences in 5 major cell types (trophoblasts, stromal cells, Hofbauer cells, antigen-presenting cells, and endothelial cells). Ligands from the CC family chemokine ligand (CCL) family were most highly representative in females, with their receptors present on the maternal surface. Sexually dimorphic trophoblast transcripts, Mucin-15 (MUC15) and notum, palmitoleoyl-protein carboxylesterase (NOTUM) were also most highly expressed in syncytiotrophoblasts and extra-villous trophoblasts respectively. Gene Ontology (GO) analysis using sexually dimorphic genes in individual cell types identified cytokine mediated signaling pathways to be most representative in female trophoblasts. Upstream analysis demonstrated TGFB1 and estradiol to affect all cell types, but dihydrotestosterone, produced by the male fetus, was an upstream regulator most significant for the trophoblast population.

Conclusions: Maternal-fetal crosstalk exhibits sexual dimorphism during placentation early in gestation.

Keywords: first trimester placenta; human pregnancy; placenta cell types; receptor-ligand; sex differences; single-cell RNA sequencing.

Figure 1.

Sexually dimorphic receptor-ligand interactions at the maternal-fetal interface. (A, B) Circos plots showing interactions between sexually dimorphic placenta genes and the maternal decidua. Arrowheads point to receptors. Overlap with upstream regulators is highlighted with bold text and color links. Other genes are not bold, with gray links. Grids represent fold-change sex differences (thick grid, blue-black-pink) and decidua FPKM (thin grid, white-purple, starting at FPKM = 1). (A) Female-upregulated placenta crosstalk to decidua. (B) Male-upregulated placenta crosstalk to decidua.

Figure 2.

Single-cell transcriptomic profiles and sexual dimorphism of the first trimester placenta. (A-i) Placental cell clusters visualized by t-Distributed Stochastic Neighbor Embedding (t-SNE) using scran and scater packages. Colors indicate cell types. (A-ii) The tSNE of male and female placenta cells with males colored in blue and females in pink. (B) The top sexually dimorphic genes (FDR <0.01, ILog₂FCI >1) in each cluster are presented as a heatmap. Black bars on the left of the heatmap indicate genes on sex chromosomes. (C) Sexually dimorphic ligands in the first trimester placental cells interact with decidua-expressed receptors.

Figure 3.

Sexual dimorphism in the first trimester trophoblasts. (A) tSNE of trophoblast subclusters using scran and scater packages. Colors indicate subclusters within the trophoblast population. (B) Pseudotime reconstruction of the developmental trajectory in the trophoblast population shows trophoblast subclusters (T1~T7), indicated by colors. The timepoints 1 and 2 represent hierarchical branching events. Pseudotemporal trajectory indicates developmental timeline. The distribution patterns of cells expressing EVT marker gene HLA-G and STB marker gene ERVFRD-1 in the pseudotemporal trajectory. (C) Representative images of MUC15 and NOTUM immunostaining in 3 female placentas and 4 male placentas, bar = 100 um. (D) The top 10 enriched GO terms are presented as −log₁₀(P value) for either sex in trophoblasts. (Bonferroni-corrected for P < 0.05).