A molecular atlas of the human postmenopausal fallopian tube and ovary from single-cell RNA and ATAC sequencing

Abstract

As part of the Human Cell Atlas Initiative, our goal is to generate single-cell transcriptomics (single-cell RNA sequencing [scRNA-seq], 86,708 cells) and regulatory (single-cell assay on transposase accessible chromatin sequencing [scATAC-seq], 59,830 cells) profiles of the normal postmenopausal ovary and fallopian tube (FT). The FT contains 11 major cell types, and the ovary contains 6. The dominating cell type in the FT and ovary is the stromal cell, which expresses aging-associated genes. FT epithelial cells express multiple ovarian cancer risk-associated genes (CCDC170, RND3, TACC2, STK33, and ADGB) and show active communication between fimbrial epithelial cells and ovarian stromal cells. Integrated single-cell transcriptomics and chromatin accessibility data show that the regulatory landscape of the fimbriae is different from other anatomic regions. Cell types with similar gene expression in the FT display transcriptional profiles. These findings allow us to disentangle the cellular makeup of the postmenopausal FT and ovary, advancing our knowledge of gynecologic diseases in menopause.

Keywords: CP: Cell biology; CP: Molecular biology; carcinoma; endometriosis; fallopian tubes; female; ovarian epithelial; ovarian neoplasms; ovary; post-menopause; reproductive health; scATA-seq; scRNA-seq; single-cell analysis.

Figure 1.. Single-cell RNA sequencing (scRNA-seq) reveals cell types of the normal human postmenopausal FTs and ovaries

(A) Intraoperative image of the right ovary, right side of the uterine fundus, I (the FT segment closest to the uterus), A, and the distal end of the FT (F). The fimbrial end extends over the ovary, allowing direct contact between the epithelium of the F (arrow) and the ovarian surface. (B) Cell types found in the normal postmenopausal FT. A UMAP plot shows the 22 cell clusters identified in the FT using scRNA-seq. Data include F (n = 6), A (n = 6), and I (n = 6) for seven donors. Cell types are abbreviated as follows: ST1–ST5, 5 clusters of stromal cells; T/NK1–T/NK3, 3 clusters of T and natural killer cells; SE, secretory epithelial; LE, lymphatic endothelial; SM, smooth muscle; MP, macrophage; P/V1–P/V3, 3 clusters of pericytes and vascular SM cells; CE, ciliated epithelial; EN1–EN4, 4 clusters of endothelial cells; B/P, B and plasma cells; MA, mast. (C) Dot plot showing common gene expression markers found in IM and non-IM cell subtypes in the FT as identified by HIPPO analysis. (D) Representative cross-section of a postmenopausal ovary (scale bar, 1,000 μm). In the medullary stroma, multiple arteries (arrowheads) and unresorbed corpora albicantia (asterisks) can be seen. Corpora albicantia show signs of multiple lifetime ovulation events that present as lobulated, eosinophilic structures composed of dense collagen fibers with occasional admixed fibroblasts. Inset: atrophic cortex at high magnification (scale bar, 100 μm). In the cellular stroma, no follicles are present, and only a few ovarian surface epithelial cells remain after surgical manipulation (black arrow). (E) Right panel: cell types found in the normal postmenopausal ovary (n = 6). A UMAP plot shows the 17 cell clusters identified in the ovary using scRNA-seq. Cell types are abbreviated as follows: ST1–ST10, 10 clusters of ST cells; IM1/2, 2 clusters of immune cells; PE1/2, perivascular EN cells. Left panel: graphic depiction of cell subclusters and number of cells identified in each subcluster in the ovary using scRNA-seq. (F and G) Relative abundance of the 17 cell clusters identified in the postmenopausal ovary (F) and of the 22 cell clusters found in the postmenopausal FT (G) using scRNA-seq. The graphs show the individual percentage of each cell type by the individual donor. See also Figure S1 and Tables S1, S2, S3, S4, and S5.

Figure 2.. Gene expression in the I, A, and F regions of the postmenopausal FT

(A) Left panel: graphic depiction of cell subclusters and number of cells identified in each subcluster by anatomic region (I, A, and F) using scRNA-seq. Right panel: gross anatomic image of a normal FT, indicating the anatomic regions sampled and their corresponding cross-section H&E staining (1:40). Dashed lines around the FT lumen indicate the epithelium. (B) UMAP of the 22 cellular clusters identified by scRNA-seq, divided by anatomic regions in the FT. (C) Dot plot showing normalized expression levels of marker genes in common cell types identified in the I (n = 6), A (n = 6), and F (n = 6). SE and CE cells are framed. Also shown is a violin plot for Reticulon 1 (RTN1). See also Figure S2.

Figure 3.. scRNA expression of genes identified in genome-wide association studies (GWASs) in different regions of the postmenopausal FT and ovary

(A and B) Dot plot showing cell type-specific expression of disease-specific genes as curated by GWASs in the FT (A) by anatomic region (I, A, F) and ovary (B). (C) H&E and RNA fluorescence in situ hybridization (FISH) of decorin (DCN) and CCDC80 in the FT F. Every dot corresponds to one RNA transcript. Nuclei are stained with DAPI (cyan). Transcripts for DCN are shown in yellow and CCDC80 in magenta. Dashed white lines separate the epithelium from stroma in the FT (scale bars, 20 μm). See also Figure S3 and Tables S7 and S8.

Figure 4.. Ligand-receptor interactions and senescence-related gene expression in the FT and ovary

(A and B) Ligand-receptor interactions between different cell populations in the postmenopausal FT by anatomic region (A) and ovary (B) as detected by CellPhoneDB. y axis, ligands (red) and receptors (black); x axis, cell types with ligand-receptor interactions separated by underline. Black boxes indicate examples of significant changes between anatomic regions. (C) Ovary-F interactions. A heatmap shows the number of interactions detected by CellPhoneDB among different cell types in the ovary and with SE and CE cells in the F of the FT. (D) Ligand-receptor interactions between F and ovary, as detected by CellPhoneDB. (E) Aging and senescence-related gene expression in the ovary. O, ovary. See also Figure S4 and Tables S7 and S9.

Figure 5.. Single-cell assay of transposase-accessible chromatin sequencing (scATAC-seq). Annotation of cell-types by label transfer from scRNA-seq in the postmenopausal FT and O.

(A and B) UMAP plot profiling of 41,515 cells (n = 4: I, A, F) identifying 18 cell clusters in the FT (A) and18,315 cells (n = 3) identifying 13 cell clusters in the O (B) using scATAC-seq. The labels before “–” are transferred from scRNA-seq data, and scATAC-seq-specific cluster labels are added as subgroup numbers after “–” (for example, there are now SE cell clusters −1 and −2). (C and D) Relative abundance of the 18 cell clusters in the postmenopausal FT (C) and 13 cell clusters found in the O (D) using scATAC-seq after integrated analysis (label transfer from scRNA-seq). The graph shows the individual percentage of each cell type by the donor. (E and F) Heatmaps based on scATAC-seq data. Shown is TF activity by cell type in the FT including all 869 motifs available in the cisBP database (E) and specific TFs in the O (F). See also Figure S5 and Tables S5, S6, S10, and S11.

Figure 6.. Clustering, cell type annotation, and transcription factor (TF) analysis of scATAC-seq in the different anatomic regions of the postmenopausal FT

(A) UMAP plot showing the 18 cell clusters identified in the I (n = 4), A (n = 3), and F (n = 4) by scATAC-seq after label transfer from scRNA-seq. (B) Heatmap showing TF activity in the I, A, and F of the FT by cell type. Enrichment results were obtained from the cisBP database and contain 869 TF motifs. The order of TFs is the same across anatomic sites. (C) Heatmap showing selected TFs in the I, A, and F. (D) Dot plot showing chromatin accessibility of TFs of GWAS genes in Figure 3 by anatomic site and cell type. See also Figure S6 and Table S7.