Identification of novel differentiation trajectories and gene network associations with ectopic pregnancy in fallopian tube epithelium

Abstract

Study question: Can 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 answer: A bidirectional differentiation trajectory originating from a novel OVGP1+ progenitor population of FT epithelial cells was uncovered, and causal network modelling of whole-transcriptome activity in the FT and endometrium revealed functional divergence between their secretory epithelial cells, with implications for ectopic pregnancy candidate genes.

What is known already: The FT forms the in vivo peri-conceptual environment, which has a significant impact on programming offspring health. The FT epithelium establishes this environment; however, the epithelial cell types are poorly characterized in health and disease.

Study design, size, duration: Publicly available, benign FT single-cell RNA sequencing (scRNA-seq) samples from 13 women across three previous studies were combined. Endometrial scRNA-seq samples from 13 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, methods: A meta-analysis of FT scRNA-seq samples was performed to identify epithelial populations. Differential gene expression assessed differences between FT and endometrial epithelial scRNA-seq data. Functional differences between secretory cells in the tissues were characterized 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 chance: Epithelial cells (n = 14 360) were clustered into eight secretory and ciliated epithelial populations in the meta-analysis of three scRNA-seq datasets. A novel OVGP1+ epithelial progenitor cell was also identified, and its bidirectional 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-related (false discovery rate (FDR) < 0.002) and immune-related (FDR < 0.00007) pathways. We mapped eQTLs from a GWAS meta-analysis of 7070 women with ectopic pregnancy over a range of significance (P = 1.68 × 10-21-5.8 × 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 = 5.32 × 10-9) and identified additional genes (SLC7A2, CLDN1, GLS, PEX6, PLXNA4, NR2F1, CLGN, PGGHG, and ANKRD36) implicated in ectopic pregnancy and eutopic pregnancy.

Limitations, reasons for caution: The 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 findings: These 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. This study further implicates the role of MUC1 and secretory cells in ectopic pregnancy and suggests future targets for investigating embryo implantation in the FT and endometrium.

Study funding/competing interest(s): No funding was received for this study. The authors do not disclose any competing interests.

Trial registration number: N/A.

Keywords: endometrium; fallopian tube; hypergraph; implantation; network modelling; single-cell transcriptomics; tubal ectopic pregnancy.