Dysfunctional intercellular communication and metabolic signaling pathways in thin endometrium

Abstract

Background: The endometrial thickness is a key factor for successful implantation. Thin endometrium is associated with lower implantation rate and pregnancy rate. Lacking of a better understanding for the cellular and molecular mechanisms of thin endometrium, managing patients with thin endometrium still represents a major challenge for clinicians. Methods: In this study, we combined four single-cell RNA sequencing (scRNA-seq) and one bulk sequencing (bulk-seq) data for thin endometrium to perform an integrated analysis for endometrial cells in proliferating phase. Cell proportion and differentially expressed genes (DEGs) were analyzed to determine the disease-specific cell type and signaling pathways. The cell-cell communication among cell types were inferred by "CellChat" to illustrate the differential intercellular communication under normal and thin endometrium conditions. GSEA and GSVA were applied to identify dysfunctional signals and metabolic pathways before and after thin endometrium. Results: Integration of scRNA-seq identified eight cell types. The proportion of stromal cells showed a significant difference between normal and thin endometrial tissue. The DEGs in diverse cell types revealed enriched pathways in a cell-specific manner. Aberrant cell-cell signaling transduction was found in almost all cell types, especially in immune cells and epithelial cells. Furthermore, dysfunctional metabolic signaling pathways were induced in a cell-type dependent way. The down-regulation of carbohydrate metabolism and nucleotide metabolism was observed and the energy metabolism switch was indicated. Conclusion: Conclusively, we discover dysfunctional signals and metabolic pathways in thin endometrium, providing insight into mechanisms and therapeutic strategies for the atrophic endometrium.

Keywords: cell diversity; cell-cell communication; metabolic signaling; single-cell sequencing; thin endometrium.

FIGURE 1

Integration of scRNA-seq projects for human normal endometrial tissue and thin endometrial tissue. (A) Workflow of scRNA-seq and bulk-seq data processing and analyzing for thin endometrial tissue. UMAP visualization showing the integrated effects of 66,711 cells in projects (B), samples (C), and conditions. (D). (E) UMAP of cells demonstrating 8 major cell types. Str, stromal cell; Lymph, lymphoid cells; Epi, epithelial cells; pStr, proliferating stromal cells; Mono/macro, mono/macrophages; Peri, pericytes; Endo, endothelial cells; and Cili_Epi, ciliated epithelial cells. (F) Violin plot showing the expression level of the marker genes in each cell type.

FIGURE 2

Differential cell population in thin endometrial tissue. (A) The cell type composition for each sample. (B) The sample profiles in individual projects. (C) The proportions of cell types in normal and thin endometrial tissues. *p < 0.05. The functional annotation for the upregulated (D) and downregulated (E) DEGs in stromal cells.

FIGURE 3

The analysis for DEGs in clusters and under thin endometrium condition. (A) Dot plot showing the up- and downregulated genes across all eight clusters in thin endometrial tissue. The DEGs were colored by clusters and labeled by the top5 up- and downregulated DEGs in each cluster. (B) KEGG enrichment of upregulated and downregulated DEGs across clusters. Violin plot showing the expression level of genes in enriched KEGG pathways: cell cycle (C), MAPK signaling pathway (D), and cGMP- PKG signaling pathway (E).

FIGURE 4

Bulk-seq revealed the enriched pathways in thin endometrium. GSEA analysis showing the pathways enriched in the top (A) and bottom (B,C) of the ranked list, with the corresponding up- or downregulated genes. Left, combo chart showing the running enrichment score and ranked list metric for the ordered fold change gene list with 5 pathway genes labeled; right, the expression level of the representative genes in each cluster.

FIGURE 5

Dysfunctional cell-cell communication in thin endometrium. Heatmap depicting differential incoming (A) and outgoing (B) signaling patterns in each cluster. Representative networks for enhanced (C,D) and reduced (E,F) signaling pathways. Left, circle plot showing the signals in cell-cell interactions between normal and thin endometrium; right, the expression level of ligand-receptor genes in individual signals. (G) Bar graph demonstrating the relative information flow of each signaling pathway between normal and thin endometrial tissues.

FIGURE 6

The metabolic alteration in individual cell types under thin endometrium condition. (A) Heatmap showing the differential metabolic pathways in each cell type. (B) Bar plot showing the dramatically changed metabolic pathways with the metabolic gene sets from the KEGG database in epithelial cells, ciliated epithelial cells, and endothelial cells. Violin plot showing the representative metabolic pathways in energy metabolism (C), carbohydrate metabolism (D), nucleotide metabolism (E), and amino acid metabolism (F).