RNA sequencing reveals the developmental onset of autosomal gene expression
differences in male and female extravillous trophoblasts
Authors: Shannon Matthew J. , Beristain Alexander G. 
Published: January 2025

ABSTRACT

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.

HIGHLIGHTS

First trimester male and female placenta cells were compared
using bulk and single-cell transcriptomics

Gestational age and placental sex are the leading drivers of
variation in gene expression

Sex-related differences in autosomal genes generally arise on
week 10 of gestation

Sex-related differences in trophoblast and uterine cell
crosstalk are driven by autosomal gene differences arising after
week 10 of gestation

PLAIN ENGLISH SUMMARY
The human placenta is a temporary organ that forms during pregnancy.
Importantly, the placenta acts as a surrogate for not yet functioning fetal
organ systems (i.e., the heart, lungs, and kidneys) while they mature within
the fetus. Because the placenta develops from cells of the early embryo, it
can be biologically male or female. Male and female placentas are
genetically different from each other, where these differences may
differentially influence placental functions and pregnancy health through
unknown mechanisms. Therefore, this study aimed to understand how male and
female placenta cells differ at the level of gene expression. We find that
on week 10 of gestation, but not before, female placenta cells express a
different repertoire of genes compared to male placenta cells. We show that
these differences potentially affect how cells of the placenta and uterus
interact with each other. Taken together, these results identify a
developmental timepoint in pregnancy where the biological sex of the
placenta may instruct subtle differences in how male or female placenta
cells communicate with the maternal compartment in pregnancy.

Summary Statement
Bulk and single-cell RNA sequencing provides comprehensive comparison
between uncultured male and female HLA-G-purified trophoblasts derived from
first-trimester human placentas.