Single Cell Transcriptomic Modelling of the Fallopian Tube Epithelium Identifies Cellular Specialisation, Novel Differentiation Trajectories, and Gene Network Associations with Ectopic Pregnancy

Abstract

STUDY QUESTIONCan network modelling of single cell transcriptomic data identify cellular developmental trajectories of fallopian tube (FT) epithelium and reveal functional and pathological divergence from the endometrium? SUMMARY ANSWERA bidirectional secretory and ciliated differentiation trajectory was apparent from a novel OVGP1+ progenitor population of FT epithelial cells. A causal network model of whole transcriptome action in the FT and endometrium revealed specific functional divergence between secretory cells of these tissues. The network model reflected the latest ectopic pregnancy genome wide association study (GWAS), invoking MUC1 and other candidate genes in mature secretory cells for ectopic and eutopic implantation. WHAT IS KNOWN ALREADYThe fallopian tube forms the in vivo peri-conceptual environment, which has a significant impact on programming offspring health. The fallopian tube epithelium establishes this environment, however the epithelial cell types are poorly characterised in health and disease. STUDY DESIGN, SIZE, DURATIONPublicly available benign FT single cell RNA sequencing (scRNA-seq) samples from thirteen women across three studies were combined. Endometrial scRNA-seq samples from thirteen women from one study were used to demonstrate transcriptomic differences between the epithelia of the two tissues. Network models of transcriptomic action were constructed with hypergraphs. PARTICIPANTS/MATERIALS, SETTING, METHODSA meta-analysis of FT scRNA-seq samples was performed to identify epithelial populations. Differential gene expression assessed differences between fallopian tube and endometrial epithelial scRNA-seq data. Functional differences between secretory cells in the tissues were characterised using hypergraph models. To identify associations with ectopic pregnancy, expression quantitative trait loci (eQTLs) from a recent GWAS were mapped onto the network models. MAIN RESULTS AND THE ROLE OF CHANCEEpithelial cells (n=14,360) were clustered into 8 secretory and ciliated epithelial populations in the meta-analysis of 3 scRNA-seq datasets. A novel OVGP1+ epithelial progenitor cell was also identified, and its bi-directional differentiation to mature secretory or mature ciliated populations was mapped by RNA velocity analysis. This progenitor exhibited a high velocity magnitude (12.47) and low confidence (0.69), a combination strongly indicative of multipotent progenitor status. Comparing FT epithelial cells with endometrial epithelial cells revealed 5.3-fold fewer shared genes between FT and endometrial glandular secretory cells than between FT and endometrial ciliated cells, suggesting functional divergence of secretory cells along the reproductive tract. Hypergraphs were used to identify highly coordinated regions of the transcriptome robustly associated with functional gene networks. In the FT secretory cells, these networks were enriched for lipid (FDR<0.002) and immune (FDR<0.00007) related pathways. We mapped eQTLs from a GWAS meta-analysis of 7070 women with ectopic pregnancy over a range of significance (P = 1.68 x 10-21- 5.8 x 10-4) to the hypergraphs of FT and endometrium. Of the 22 genes present in the hypergraphs, 13 of these clustered as highly coordinated genes. This demonstrated the functional importance of MUC1 in the FT and endometrium, (GWAS Study P = 5.32x10-9) and identified additional genes (SLC7A2, CLDN1, GLS, PEX6, PLXNA4, NR2F1, CLGN, PGGHG, ANKRD36) implicated in ectopic pregnancy and eutopic pregnancy. LIMITATIONS, REASONS FOR CAUTIONThe sample size of reproductive age women was limited in previous studies, and though causal network modelling was used and previous mechanistic data supports candidate gene involvement, no in vitro or in vivo validation of candidate was performed. WIDER IMPLICATIONS OF THE FINDINGSThese findings consolidate the existing single cell transcriptomic datasets of the FT to provide a comprehensive understanding of epithelial populations and define functionally distinct secretory cells that contribute to the peri-conceptual environment of the FT. We further implicate the role of MUC1 and secretory cells in ectopic pregnancy and suggest future targets for investigating embryo implantation in the FT and endometrium.