Placental Transcriptome Profiling in Subtypes of Diabetic Pregnancies Is Strongly Confounded by Fetal Sex

Abstract

The placenta is a temporary organ with a unique structure and function to ensure healthy fetal development. Placental dysfunction is involved in pre-eclampsia (PE), fetal growth restriction, preterm birth, and gestational diabetes mellitus (GDM). A diabetic state affects maternal and fetal health and may lead to functional alterations of placental metabolism, inflammation, hypoxia, and weight, amplifying the fetal stress. The placental molecular adaptations to the diabetic environment and the adaptive spatio-temporal consequences to elevated glucose or insulin are largely unknown (2). We aimed to identify gene expression signatures related to the diabetic placental pathology of placentas from women with diabetes mellitus. Human placenta samples (n = 77) consisting of healthy controls, women with either gestational diabetes mellitus (GDM), type 1 or type 2 diabetes, and women with GDM, type 1 or type 2 diabetes and superimposed PE were collected. Interestingly, gene expression differences quantified by total RNA sequencing were mainly driven by fetal sex rather than clinical diagnosis. Association of the principal components with a full set of clinical patient data identified fetal sex as the single main explanatory variable. Accordingly, placentas complicated by type 1 and type 2 diabetes showed only few differentially expressed genes, while possible effects of GDM and diabetic pregnancy complicated by PE were not identifiable in this cohort. We conclude that fetal sex has a prominent effect on the placental transcriptome, dominating and confounding gene expression signatures resulting from diabetes mellitus in settings of well-controlled diabetic disease. Our results support the notion of placenta as a sexual dimorphic organ.

Keywords: RNA sequencing; diabetes mellitus; human; placenta; pregnancy.

Figure 1

The effect of diabetes on the placental transcriptome in comparison to healthy controls. MA-plot showing the log2-fold change over mean expression for all genes for the diabetes group (DIAB) without PE (n = 32) versus CTL (n = 29). DIAB = patients with DM1, DM2 and GDM; CTL = healthy controls.

Figure 2

Diabetes subgroups show an altered placental transcriptome in comparison to healthy controls. (a–f) MA-plots showing log2-fold change as a function of mean expression for the diabetes subgroups DM1 (n = 17), DM2 (n = 3), GDM (n = 12) versus CTL (n = 29). DEGs (adj. p-value < 0.05) are marked in red. (g) Gene-set enrichment analysis with tmod for up- and down-regulated genes in these contrasts. The adjusted p-value is color-coded and the AUC statistic is displayed as the dot size. GDM = gestational diabetes mellitus; DM1 = type I diabetes mellitus; DM2 = type II diabetes mellitus; CTL = healthy controls.

Figure 3

The effect of diabetes superimposed with preeclampsia on the placental transcriptome in comparison to healthy controls. (a) MA-plot shows log2-fold change over mean expression for all diabetic placenta samples superimposed with PE (n = 16) versus CTL (n = 29). DEGs are highlighted in red. (b) The gene-set enrichment analysis displays significantly altered gene sets in this comparison. PE = includes patients with DM1 + PE, DM2 + PE and GDM + PE; CTL = healthy controls.

Figure 4

Placental samples group mainly due to fetal sex. (a) Principal component analysis (PCA) displays the highest amount of explained variance (10.39%) between samples in PC1 and the second highest (7.76%) in PC2. Placental samples cluster according to the fetal sex. (b) PC4 and PC3 do not cluster subjects into groups in the PCA. Black border: CTL (n = 29, circle); GDM (n = 12, square); DM1 (n = 17, triangle pointed top); DM2 (n = 3, triangle pointed bottom). Green border: GDM + PE (n = 4, square); DM1 + PE (n = 8, triangle pointed top); DM2 + PE (n = 4, triangle pointed bottom). Fetal sex is indicated in blue = male and red = female. (c) Percentage of explained variance by each PC from PC1 to PC10. Diabetes types are indicated with symbols. CTL = healthy control; GDM = gestational diabetes mellitus; DM1 = Diabetes mellitus type 1; DM2 = diabetes mellitus type 2.

Figure 5

Association of principal components to Y-chromosome linked genes and fetal sex. (a) Gene-set enrichment analysis shows how principal components (PC) are associated with particular gene sets. PC1 and PC2 are strongly influenced by Y-chromosome genes. The effect size (AUC) is shown as dot size and the color indicates significance (adjusted p-value). (b) The heat map shows the contribution of clinical (meta data) and quality control parameters to the PCs. The impact (“importance”) is color-coded with low contribution in grey and high contribution in red. Fetal sex clearly contributes to PC1. The figure is based on all CTL (n = 29) and diabetic placenta samples without PE (n = 32).

Figure 6

Fetal sex effect on gene expression in placenta. The MA-plot displays the effect of fetal sex on gene expression in placentas with male (n = 36) or female fetuses (n = 41). Differential genes (adj. p-value < 0.05) are marked in red. Positive log2-fold change indicates male-specific expression; negative log2-fold changes indicate female-specific expression.