Monosomy X in isogenic human iPSC-derived trophoblast model impacts expression modules preserved in human placenta

Abstract

Mammalian sex chromosomes encode homologous X/Y gene pairs that were retained on the Y chromosome in males and escape X chromosome inactivation (XCI) in females. Inferred to reflect X/Y pair dosage sensitivity, monosomy X is a leading cause of miscarriage in humans with near full penetrance. This phenotype is shared with many other mammals but not the mouse, which offers sophisticated genetic tools to generate sex chromosomal aneuploidy but also tolerates its developmental impact. To address this critical gap, we generated X-monosomic human induced pluripotent stem cells (hiPSCs) alongside otherwise isogenic euploid controls from male and female mosaic samples. Phased genomic variants in these hiPSC panels enable systematic investigation of X/Y dosage-sensitive features using in vitro models of human development. Here, we demonstrate the utility of these validated hiPSC lines to test how X/Y-linked gene dosage impacts a widely used model for human syncytiotrophoblast development. While these isogenic panels trigger a GATA2/3- and TFAP2A/C-driven trophoblast gene circuit irrespective of karyotype, differential expression implicates monosomy X in altered levels of placental genes and in secretion of placental growth factor (PlGF) and human chorionic gonadotropin (hCG). Remarkably, weighted gene coexpression network modules that significantly reflect these changes are also preserved in first-trimester chorionic villi and term placenta. Our results suggest monosomy X may skew trophoblast cell type composition and function, and that the combined haploinsufficiency of the pseudoautosomal region likely plays a key role in these changes.

Keywords: Turner syndrome; X chromosome inactivation; monosomy X; placenta; trophoblast.

Fig. 1.

Cytogenomic validation of monosomy X and isogenic euploid hiPSC panels. (A) Schematic of reprogramming and hiPSC characterization. (B) International System for Human Cytogenomic Nomenclature (ISCN) call from the CytoSNP-850k (Illumina) platform of hiPSC clones used in this study. (C) XIST expression by RT-qPCR as a percentage of GAPDH (Mann–Whitney U test P values).

Fig. 2.

Moderately impaired hCG and PlGF secretion and TB markers in monosomy X. (A) Secreted hCG ELISA in mIU/mL normalized to total of RNA (per microgram) harvested from the same well. (B) Secreted PlGF ELISA in picograms per milliliter (pg/mL) per microgram of RNA, as in A. Mann–Whitney U test P values in A and B compare 45,X samples to otherwise isogenic euploid controls (denoted by brackets). (C) TBL RNA-seq vst counts of genes relevant to BAP-induced cell fates by line for female and male1 samples. (D) Expression of genes in C by RT-qPCR as a percentage of GAPDH (Mann–Whitney U test P values) for male2 samples. Boxes denote interquartile range (in C and D).

Fig. 3.

X-chromosomal gene dosage in female- and male-derived TBL cells. (A) PCA of 45,X and isogenic euploid control TBL RNA-seq data. Respective symbols and colors indicate karyotype and cell line. (B) Median-vst normalized expression values for PAR, X-pair, and X-specific escapee genes, which are grouped by the independent escapee calls in all 46,XX lines, 2/3 or a single female euploid line (all, 2/3, 1/3). Escapee annotation panel indicates reported XCI states across multiple studies (“reported,” from ref. 59), as in SI Appendix, Fig. S7, and from refs. – for “VarOther”). Heatmap column annotation denotes hiPSC lines (XX6 values bordered in black; names of escapees silenced in XX6 bolded). Left-most barplot panels report LAF and three Centered barplot panels report log2-fold change in expression (Log2FC) in 45,X samples relative to isogenic euploid controls (XX19/23 or XX6, or male1XY). (C) Left: Autosomal-median normalized vst ratio of X-linked genes subject to XCI (“silenced”), relative to autosomal, X-specific escapees, PAR, and X-linked pair genes. Significant differences to female 45,X denoted by Mann–Whitney U test P value (≤0.1). Right: X:autosome ratios normalized by gene length (fpkm) compares each class of genes within each condition (see text), with median X:A ratio denoted next to each boxplot.

Fig. 4.

Transcriptome-wide impact of monosomy X by differential expression. (A) Median-vst normalized expression (“vst diff.”) of all differentially expressed genes (DEGs, DESeq2 p.adj ≤ 0.05, abs(log2FC) ≥ 0.3) in 45,X lines relative to their isogenic euploid controls (“fXO,” “m1XO”), and female-derived 45,X relative to male1 46,XY (“fXO-m1XY”). Number of overlapping and concordant DEGs identified in all three (Top) or any two comparisons listed as a fraction alongside sign-test P value, Left of DESeq2 Wald barplot panels. DEGs concordant across comparisons are annotated in dark gray, discordant in light gray. (B) GSEA against the Wikipathway collection for each comparison, as well as gene list reranked by the average Wald statistic from all three equally weighted sets (“aveXO”), and control comparison of male- and female-derived 45,X samples (“XOXO”). Bubble position, color, and size denote the signed log10-scaled GSEA p.adjust, normalized enrichment score, and number of core genes, respectively, and are plotted opposite of abbreviated Wikipathway titles. (C) Significant GO terms (beige) relevant to cilia, lysosomes, or autophagy and genes colored by m1XO Wald statistic (red for higher, blue for lower expression). D as in C for fXO Wald score-based GO terms.

Fig. 5.

Correlation of gene modules sensitive to monosomy X and their preservation in primary placenta. (A) Significant (P ≤ 0.05) Spearman ρ coefficients relating escapee gene classes (All, PAR, X-pair, X-specific) to secreted PlGF and hCG levels, and to cycling (86), and cell type markers from early human embryonic studies (50, 51, 53, 87), suffixed by first author’s last initial (W/X/Z/C). Cell fates abbreviated for cyto-TB (CTB), early amnion (E-AM), epiblast (EPI), extravillous TB (EVT), mixed (MIX), primordial endoderm (PE), and syncytio-TB (STB). (B) Signed network from WGCNA plotted over module assignments, DESeq2 Wald stats (fXO, m1XO, fXO-m1XY, and aveXO; red >0, blue <0), and Pearson correlation with PlGF and hCG levels, and each median-normalized marker set (from A). (C) Z summary statistic for preservation of modules (from B) in another BAP dataset (90), (sex-stratified) first trimester chorionic villi sampling (66), and two studies of term placenta (91, 92), the latter further stratified by sex, birthweight, or randomized (“rand”) to control datasets of similar size. (D) Correlation coefficient (kME) of each escapee with its assigned module eigengene, and enrichment analysis (−log10-scaled Fisher P value, above heatmap) for module assignment of escapee classes (same genes and reported XCI annotation as in Fig. 3B).