Spatial transcriptomic profiles of mouse uterine microenvironments at pregnancy day 7.5†

Abstract

Uterine dysfunctions lead to fertility disorders and pregnancy complications. Normal uterine functions at pregnancy depend on crosstalk among multiple cell types in uterine microenvironments. Here, we performed the spatial transcriptomics and single-cell RNA-seq assays to determine local gene expression profiles at the embryo implantation site of the mouse uterus on pregnancy day 7.5 (D7.5). The spatial transcriptomic annotation identified 11 domains of distinct gene signatures, including a mesometrial myometrium, an anti-mesometrial myometrium, a mesometrial decidua enriched with natural killer cells, a vascular sinus zone for maternal vessel remodeling, a fetal-maternal interface, a primary decidual zone, a transition decidual zone, a secondary decidual zone, undifferentiated stroma, uterine glands, and the embryo. The scRNA-Seq identified 12 types of cells in the D7.5 uterus including three types of stromal fibroblasts with differentiated and undifferentiated markers, one cluster of epithelium including luminal and glandular epithelium, mesothelium, endothelia, pericytes, myelomonocytic cell, natural killer cells, and lymphocyte B. These single-cell RNA signatures were then utilized to deconvolute the cell-type compositions of each individual uterine microenvironment. Functional annotation assays on spatial transcriptomic data revealed uterine microenvironments with distinguished metabolic preferences, immune responses, and various cellular behaviors that are regulated by region-specific endocrine and paracrine signals. Global interactome among regions is also projected based on the spatial transcriptomic data. This study provides high-resolution transcriptome profiles with locality information at the embryo implantation site to facilitate further investigations on molecular mechanisms for normal pregnancy progression.

Keywords: decidua; microenvironment; postimplantation; scRNA-Seq; spatial transcriptomics; uterus.

Graphical Abstract

Figure 1

Visium spatial transcriptomics of D7.5 uterus from four different mice. Histology (Hematoxylin and Eosin staining) of four uterine sections (A). The spatial transcriptomic results of four uterine sections (B). The dots of the same uterine compartment were labeled with one color. The heatmap of DEGs in different microenvironments (C). The colors from the blue to yellow correspond to the gene expression levels from low to high.

Figure 2

UMAP analysis of scRNA-Seq of D7.5 mouse uterus from two Prl8a2^icreSun1GFP^LsL/+ mice and one Sun1GFP^LsL/+ mouse together. The dots with the same color are from the same cellular clusters. Mo: Myeloid cells; Str1, Stroma 1; Str2, Stroma 2; Str3: Stroma 3; Endo, Endothelium; Epi, Epithelium; Peri, Pericyte; Den: Dendritic cells; Meso, Mesothelium; LyB, Lymphocyte B.

Figure 3

scRNA-Seq cell clustered mapped to Visium spatial transcriptomics. The redder the color is, the more likely the cell type is located at this position. Str1, Stroma 1 (A); Str2, Stroma 2 (B); Str3, Stroma 3 (C); Endo, Endothelium (D); Peri: Pericyte (E); Epi: Epithelium (F); Meso: Mesothelium (G); RBC (H); Mo: Myeloid cells (I); NK cells (J); Den: Dendritic cells (K); LyB: Lymphocyte B.

Figure 4

Functional annotation of uterine microenvironment. IPA analysis results of all uterine microenvironment (A). The color is associated with the activations score. The redder the color is, the more activated it is. The greener the color is, the more suppressed it is. The size of each dot is correlated with the P-values, in which the bigger the size, the lower the P-value. IPA upstream and pathway analysis results of DEGs from PDZ compared to udStr, TDZ compared to udStr, and SDZ compared to udStr (B). IPA upstream and pathway analysis results of DEGs from PDZ compared to SDZ (C). The y-axis indicates the activation z score. IPA analysis results of DEGs from PDZ compared to SDZ. The x-axis indicates the activation Z score. Red color is activation, and green is inhibition. .

Figure 5

Endocrine/paracrine regulations in uterine microenvironment. Heatmap of receptor expressions in uterus (A). Ligand and/or receptor activity predicted by IPA upstream analysis (B). The color is associated with the activation score. The redder the color is, the more activated it is. The greener the color is, the more suppressed it is. The size of each dot is correlated with the P-values, in which the bigger the size, the lower the P-value.

Figure 6

Communications of the uterine microenvironment. The global communication of the uterine microenvironments (A). The arrow points from the ligand producer to the receptor producer. The color of the arrow is the same as the ligand producer. The thickness of the line is positively correlated with the communication score. The ligand–receptor pairs in embryonic (B), mesometrial (C), and anti-mesometrial (D) regions. The strength of the ligand–receptor signals is associated with the color of the circle. The color of the circle from blue to red corresponds to the communication from weak to strong. Immune: Immune cells.