Deciphering the endometrial niche of human thin endometrium at single-cell resolution

Abstract

Thin endometrium has been widely recognized as a critical cause of infertility, recurrent pregnancy loss, and placental abnormalities; however, access to effective treatment is a formidable challenge due to the rudimentary understanding of the pathogenesis of thin endometrium. Here, we profiled the transcriptomes of human endometrial cells at single-cell resolution to characterize cell types, their communications, and the underlying mechanism of endometrial growth in normal and thin endometrium during the proliferative phase. Stromal cells were the most abundant cell type in the endometrium, with a subpopulation of proliferating stromal cells whose cell cycle signaling pathways were compromised in thin endometrium. Both single-cell RNA sequencing and experimental verification revealed cellular senescence in the stroma and epithelium accompanied by collagen overdeposition around blood vessels. Moreover, decreased numbers of macrophages and natural killer cells further exacerbated endometrial thinness. In addition, our results uncovered aberrant SEMA3, EGF, PTN, and TWEAK signaling pathways as causes for the insufficient proliferation of the endometrium. Together, these data provide insight into therapeutic strategies for endometrial regeneration and growth to treat thin endometrium.

Keywords: cell proliferation; cellular senescence; single-cell sequencing; thin endometrium.

Fig. 1.

Characterization of the different types of cells in normal endometrial samples. (A) Summary of the sample origins and analysis workflow. (B) UMAP of cells with the associated cell types in samples of normal endometrium (n = 3). Macro, macrophage; Lymph, lymphatic endothelial cell; Endo, endothelial cell; Peri, perivascular cell; Str, stromal cell. (C) Expression of classical marker genes of each cell type in the endometrial samples (n = 3). (D–F) Representative immunofluorescence images of markers for stromal cells (WT1, HAND2) and immune cells (CD45) (D), epithelial cells (E-CAD) and cell mitosis (pH3) (E), T cells (CD8), NK cells (CD56 and GNLY), and macrophages (CD163 and CD14) (F) in normal endometrium (n = 5). (G) The distribution of COL1A1 in normal endometrium by UMAP. (H) The expression pattern of highly expressed genes in pStr analyzed using the TCSeq package in R. The expression of these genes was normalized to the Z-score, and the color indicated the membership values representing the degree of genes belonging to this cluster. (I) Functional enrichment of highly expressed genes in pStr compared to Str and Peri based on H. (J) Expression of CCNB1 and PCNA in distinct types of cells shown by violinplot. (K) Immunohistochemistry of CCNB1 and immunofluorescence markers of stromal cells (CD10) and pH3 in normal endometrium (n = 5). (L) The distribution of LIF in normal endometrium by UMAP. (M) The expression pattern of highly expressed genes in GE compared to LE and Cili_Epi analyzed using the TCSeq package in R. Expression of these genes was normalized to Z-score, and the color indicated the membership values representing the degree of genes belonging to this cluster. (N) The functional enrichment of specific highly expressed genes in GE compared to LE and Cili_Epi based on M. (Scale bars, 100 μm.)

Fig. 2.

Aberrant changes in cell proportions and gene expression patterns of stroma in thin endometrium. (A) The proportion of each cell type in normal and thin endometrium. (B) The fractions of each type of cell in stromal niches (Str, pStr, and Peri) in normal and thin endometrium. (C) Functional enrichment analysis of DEGs among Str, pStr, and Peri. (D and E) The distribution of POLR2I (D) and DIO2 (E) in normal and thin endometrium by UMAP. (F and G) Expression of POLR2I (F) and DIO2 (G) in Str, pStr, and Peri between normal and thin endometrium shown by violinplot. Data are presented as the mean ± SEM, **P < 0.01, ***P < 0.001. (H) mRNA expression levels of POLR2I, NM4, and MKI67 between normal and thin endometrium examined by RT-qPCR (n = 10 per group). Data are presented as the mean ± SEM, *P < 0.05, ***P < 0.001. (I) Cell cycle analysis of Str, pStr, and Peri in normal and thin endometrium. (J) Expression of PCNA in G1, S, G2/M phase in pStr between normal and thin endometrium. (K) Colony numbers counted by clone formation assay (n = 6 per group). Data are presented as the mean ± SEM, ***P < 0.001.

Fig. 3.

Abnormal changes in cell proportions and gene expression patterns of epithelia and immune cells in thin endometrium. (A) The fractions of each cell type in epithelial niches (GE, LE, and Cili_Epi) in normal and thin endometrium. (B) KEGG enrichment of up-regulated and down-regulated DEGs in GE, LE, and Cili_Epi. (C) Expression of LDHA, LDHB, and CCNB1 in GE, LE, and Cili_Epi of normal and thin endometrium shown by violinplot. Data are presented as the mean ± SEM, *P < 0.05, **P < 0.01, ***P < 0.001. (D) Expression of PTGS2 in GE, LE, and Cili_Epi of normal and thin endometrium shown by violinplot. (E) Cell cycle analysis of GE, LE, and Cili_Epi in normal and thin endometrium. (F) Expression of PCNA in G1, S, G2/M phase in GE, LE, and Cili_Epi between normal and thin endometrium. (G) Costaining of progesterone receptor with CD4, CD14, or EOMES in normal and thin endometrium by immunofluorescence (n = 5 per group). (Scale bars, 100 μm.) Data are presented as the mean ± SEM, *P < 0.05, **P < 0.01. (H and I) Flow cytometry analysis of the proportion of T cells (CD3+ cells) and NK cells (CD56+ cells) between normal and thin endometrium (n = 5 per group). Data are presented as the mean ± SEM, **P < 0.01.

Fig. 4.

Cell-to-cell connections in normal and thin endometrium. (A and B) Abundance of connections between different cell types in normal (A) and thin (B) samples shown by CellphoneDB. (C) Detailed connections between the indicated cells in normal endometrium. (D) Comparisons of dot plots for the indicated ligand–receptor interactions between normal and thin endometrium.

Fig. 5.

Abnormal incoming signaling leads to insufficient thickness in thin endometrium. (A) Heatmap of incoming signal patterns of all cell types in normal endometrium shown by CellChat. (B) Circle plot showing the inferred EGF signaling networks among different cell types in normal endometrium. (C) Expression of ligand–receptor pairs of EGF signaling among each cell type in normal endometrium. (D) Western blot analysis of the phosphorylation of ERK, STAT3, mTOR, and AKT (p-ERK, p-STAT3, p-S6, and p-AKT) in normal and thin endometrium (n = 7 per group). (E) Immunohistochemical staining for p-ERK, p-STAT3, p-S6, and pAKT in both normal and thin endometrium (n = 5 per group). (Scale bars, 100 μm.) (F) CCK-8 analysis of hESCs after treatment with signaling inhibitors of AKT (3CAI or LY294002), ERK (PD98059 or U0126), mTOR (PF-4708671 or rapamycin), and STAT3 (cryptotanshinone or S3I) for 48 h. Data are presented as the mean ± SEM, **P < 0.01, ***P < 0.001.

Fig. 6.

The role of IHH in the growth of the endometrium. (A) Heatmap of the outgoing signaling patterns of all cell types in normal endometrium as shown using CellChat. (B) Circle plot showing the inferred HH signaling networks among different cell types in normal endometrium. (C) Expression of the ligand–receptor pair of HH signaling among different cell types in normal endometrium. (D) The effects of the signaling inhibitor cyctopamine on HH signaling were confirmed by Western blot. (E and F) Cell cycle and CCK-8 analysis of hESCs after treatment with cyclopamine (E) or recombinant IHH (F) for 48 h. Data are presented as the mean ± SEM, *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 7.

Disrupted outgoing signaling contributes to insufficient thickness in thin endometrium. (A) Heatmap of the outgoing signaling patterns of all cell types in thin endometrium as shown using CellChat. (B) Abnormal SEMA3 signaling networks among each cell type in thin endometrium. (C) Violinplot showing expression of the ligand–receptor pair of SEMA3 signaling among different cell types in normal and thin endometrium. (D) Heatmap of incoming signal patterns of all cell types in normal endometrium as shown using CellChat. (E) Circle plot showing the inferred PTN signaling pathway network in normal endometrium. (F) Immunohistochemical staining for SEMA3B and PTN in normal and thin endometrium during the proliferative phase (n = 5 per group). (Scale bars, 100 μm.) (G) Cell cycle and CCK-8 analysis of hESCs after treatment with SEMA3B protein for 48 h. Data are presented as the mean ± SEM, *P < 0.05, ***P < 0.001. (H) A schematic illustration showing cell type–specific genes involved in endometrial homeostasis in normal and thin endometrium.