Cellular heterogeneity and dynamics of the human uterus in healthy premenopausal women

Abstract

The human uterus is a complex and dynamic organ whose lining grows, remodels, and regenerates every menstrual cycle or upon tissue damage. Here, we applied single-cell RNA sequencing to profile more the 50,000 uterine cells from both the endometrium and myometrium of five healthy premenopausal individuals, and jointly analyzed the data with a previously published dataset from 15 subjects. The resulting normal uterus cell atlas contains more than 167K cells, representing the lymphatic endothelium, blood endothelium, stromal, ciliated epithelium, unciliated epithelium, and immune cell populations. Focused analyses within each major cell type and comparisons with subtype labels from prior studies allowed us to document supporting evidence, resolve naming conflicts, and propose a consensus annotation system of 39 subtypes. We release their gene expression centroids, differentially expressed genes, and messenger Ribonucleic Acid (mRNA) patterns of literature-based markers as a shared community resource. We identify multiple potential progenitor cells: compartment-wide progenitors for each major cell type and potential cross-lineage multipotent stromal progenitors that may replenish the epithelial, stromal, and endothelial compartments. Furthermore, many cell types and subtypes exhibit shifts in cell number and transcriptomes across different phases of the menstrual cycle. Finally, comparisons between premenopausal, postpartum, and postmenopausal samples revealed substantial alterations in tissue composition, particularly in the proportions of stromal, endothelial, and immune cells. The cell taxonomy and molecular markers we report here are expected to inform studies of both basic biology of uterine function and its disorders.

Keywords: endometrium; menstrual cycle; myometrium; uterine homeostasis; uterine stem cells.

Fig. 1.

Cell types and markers identified from scRNA-seq analysis of five donors. (A) Study overview, including sample and data collection, integration with prior data from 15 donors to generate a consensus atlas, and comparison with a postpartum and a postmenopausal sample. (B) Identification of five major cell types from global clustering, visualized in Uniform Manifold Approximation and Projection (UMAP). The epithelial cells are split into ciliated (4_1) and unciliated epithelial cells (4). (C) Top 150 differentially expressed genes for each major cell type, shown as a gene-centroid heatmap for per-gene standardized values (data in Dataset S3A). (D) Dot plot of literature-based marker genes used to annotate the major cell types. (E) Identification of cell subtypes by focused reclustering of each major cell type, shown in UMAP, from Left to Right, for stromal, unciliated epithelial, blood endothelial, and immune cells.

Fig. 2.

Construction of a 20 sample consensus cell atlas. (A) Identification of five major cell types, shown in UMAP. Epithelial cells are split into the ciliated (Epi-1) and unciliated cells. (B) Identification of cell subtypes by focused reclustering of the four major cell type, shown in separate UMAP plots. The panel for stromal cells used a randomly down-sampled set of stromal cells for better clarity, while the corresponding UMAP for all stromal cells is shown in SI Appendix, Fig. S7B. (C) Subtype-specific expression of literature-based marker genes used to annotate the cell subtypes. Shown are per-gene standardized values from the 20-sample integrated analysis (data in Dataset S7). (D) Comparison of the relative abundance of cell subtypes across samples of different cycle phases. Subtype fractions are calculated for the four major cell types separately (Top to Bottom) and averaged over the samples of the same tissue source (endometrium or myometrium) and the same cycle phase (Dataset S4). Values were colored with shades of gray.

Fig. 3.

Potential differentiation trajectories inferred from subtype correlations and pseudotime analysis. (A) Global comparison of 39 subtypes to highlight dual-character subtypes that may “bridge” across major cell types. Shown in the heatmap of 39-by-39 pairwise rank correlation values among the 39 subtype centroids. (B) Joint UMAP projection of cells in all five major cell types, with pseudotime from Monocle3 shown in a color gradient (blue-early; yellow-late), and inferred trajectories shown as green lines. (C) Pseudotime-based inference of differentiation trajectories for stromal, epithelial (including the ciliated cells), and blood endothelial cells. Note: the Pseudotime analysis for stromal cells shown here is based on subsampled cells, with UMAP the same as in Fig. 2B. Parallel results for all stromal cells are in SI Appendix, Fig. S7A. (D) SCENIC-based TF activities differ across stromal, epithelial, blood endothelial, and immune cell subtypes. Shown are TF-subtype centroid heatmaps for per-TF standardized values (data in Dataset S12). Color range is (−3.2, 3.2) for stromal cells. and (−3, 3) for the other three.

Fig. 4.

Cross-phase DE analysis for individual cell subtypes and for select signaling pathways. (A) Phase- and subtype-resolved centroid heatmaps for stromal (Left) and epithelial (Right) cells. P: proliferative; ES: early secretory: MLS: mid-late secretory. Shown are per-gene standardized values for 1,308 and 2,190 genes that are significantly phase-regulated (Materials and Methods). Centroids are ordered first by phase then by subtype. Genes are ordered loosely by k-means clustering. (B) Cross-phase and -subtype patterns of relevant genes in four of the known signaling pathways: Hedgehog, WNT, IGF, and RA. Shown are dot plots ordered, from Left to Right, first by subtype then by phase. Complementary plots for endothelial and immune cells are in SI Appendix, Fig. S10 A–D.

Fig. 5.

Altered tissue composition in a postpartum and a postmenopausal uterus sample. (A) Relative fractions of the major cell types across four types of uterus samples: endometrium and myometrium samples from the 20 healthy donors, a postpartum sample, and a postmenopausal sample. (B) Relative fractions of subtypes, for immune, stromal, and blood endothelial subtypes. Data for individual samples from the premenopausal donors are in Dataset S4B.