Biology subjects

Ruane, P. T.

Publications and source records attributed to Ruane, P. T..

5 recordsLinked to original sources

bioRxiv2026

Fallopian Tube Organoid-Integrated Microfluidic Platform for Real-Time Imaging and Outflow Analysis

The fallopian tube is a dynamic, hormonally regulated epithelial conduit whose luminal microenvironment governs oocyte transport, fertilisation, early embryo development, and the initiation of gynaecological diseases including ectopic pregnancy, hydrosalpinx, and serous tubal intraepithelial carcinoma (STIC), yet existing models such as 2D monolayers, intact organoids, and static Transwell systems fail to recapitulate a polarised epithelium lining a perfused lumen under physiological fluid shear while preserving native organoid secretory function. We present a robust and accessible workflow integrating mechanically opened fallopian tube organoids into a custom-designed polydimethylsiloxane (PDMS) microfluidic device, in which organoid fragments form epithelial sheets and re-establish luminal-like architecture under controlled perfusion. The focus of this study is organoid integration and the characterisation of the resulting organoid-derived epithelium, rather than establishing chip cultures from individual cell lines. As this epithelium is built from expandable organoid stock rather than primary tissue, which typically survives only a few days in culture, the approach removes a major bottleneck to establishing organ-on-chip models, and we maintain viable cultures for up to 10 days. Epithelial sheets reproducibly covered more than 90% of the channel area by day 6 (n = 5 devices) and formed continuous ZO-1-positive tight junctions. The platform supports longitudinal brightfield and fluorescence microscopy at subcellular resolution, continuous collection of conditioned flow-through for ELISA, and quantitative analysis of cell coverage and health using CellProfiler software. We characterise the organoid-derived epithelium by benchmarking against tissue culture (TC) formats, comparing nuclear morphology on-chip with conventional well culture, and showing that the damage-associated cytokine interleukin-1 alpha (IL1 alpha) was undetectable in chip outflow, as in intact organoid secretions, indicating that cell health on-chip is comparable to that of intact organoids. Computational flow modelling characterises perfusion through the device and predicts how factors secreted by healthy epithelium and by tumour cells are distributed along the channel. The feasibility of tumour epithelium co culture was explored using OVCAR3 ovarian cancer spheroids as a proof of concept model of early tumour interaction with the tubal epithelium. Together, these results establish the technical feasibility of the platform and support its further development as an accessible system for studying reproductive health conditions affecting the fallopian tube, including tubal damage from infection, endometriosis, STIC lesions, and occlusion.

bioengineering↗

bioRxiv2024

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

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.

developmental biology↗

bioRxiv2024

The embryo-derived protein PDI is highly conserved among placental mammals and alters the function of the endometrium in species with different implantation strategies.

Pregnancy establishment in mammals requires a complex sequence of events, including bi-lateral embryo-maternal communication, leading up to implantation. This is the time when most pregnancy loss occurs in mammals (including humans and food production species) and dysregulation in embryo-maternal communication contributes to pregnancy loss. Embryo-derived factors modify the function of the endometrium for pregnancy success. We hypothesise that these previously unexplored conceptus-derived proteins may be involved in altering the function of the endometrium to facilitate early pregnancy events in mammals with different early pregnancy phenotypes. Here, we show that protein disulphide-isomerase (PDI) is a highly conserved protein among mammals, and provide evidence for a species-specific roles for PDI in endometrial function in mammals with different implantation strategies. We show how PDI alters the endometrial transcriptome in human and bovine in vitro in a species-specific manner, and using a microfluidic approach we demonstrate that it alters the secretome capability of the endometrium. We also provide evidence from in vitro assays using human-derived cells that MNS1, a transcript commonly downregulated in response to PDI in human and bovine endometrial epithelial cells, may be involved in the attachment (but not invasion) phase of implantation. We propose that the trophoblast-derived protein PDI, is involved in supporting the modulation of the uterine luminal fluid secreted by the endometrium to support conceptus nourishment, and also in the process of embryo attachment to the uterine lumen for pregnancy success in mammals. SIGNIFICANCE STATEMENTWe provide evidence that a highly conserved protein (PDI) alters the endometrial transcriptome in a species- and cell-specific manner. Exposure of endometrial epithelia to PDI altered genes belonging to immune modulatory, pro-inflammatory, and adhesion-pathways. One transcript, MNS1, was commonly downregulated in endometrial epithelia from species with superficial (bovine) and invasive (human) implantation morphologies. Knockdown of MNS1 expression in humans epithelia altered the ability of human trophoblast BeWo spheroids to attach suggesting a mechanism by which PDI affects implantation in human and bovine. In addition, using a microfluidics approach we have shown that PDI alters the secretome in a species-specific manner demonstrating PDI alters a key function of the endometrium in mammals.

developmental biology↗

bioRxiv2021

Macrophages in the uterus are functionally specialised and continually replenished from the circulation

Macrophages are innate immune cells that fight infection but also regulate tissue regeneration and remodelling. In the uterus, although tissue remodelling is essential for establishment and maintenance of pregnancy, the specialisation of macrophages is not well characterised compared to other mucosal tissues. Here we show that uterine macrophages are functionally specialised, expressing multiple markers of alternative activation associated with tissue remodelling and repair, and responding more highly to the type 2 cytokine IL-4 than other mucosal tissue macrophages. Uterine macrophages were continuously replenished from circulating bone marrow-derived CCR2+ monocytes that fluctuated dramatically in number throughout the reproductive cycle, and had properties distinct from the macrophages that they became, including differential responses to microbial stimulation. Importantly, many of these properties of uterine monocytes and macrophages were conserved between mice and humans. These findings further our understanding of immune regulation of uterine tissue integrity and have important implications for differences in immune responses to infections at different phases of the reproductive cycle. SUMMARYUterine macrophages are specialised, alternatively activated cells that are replenished from circulating bone marrow-derived monocytes. Monocyte and macrophage properties fluctuate markedly throughout the reproductive cycle, with many features conserved between mice and humans, and exhibiting differential responses to microbial stimulation.

immunology↗

bioRxiv2020

Trophectoderm differentiation to invasive syncytiotrophoblast is induced by endometrial epithelial cells during human embryo implantation

At implantation, trophoblast derived from the trophectoderm of the blastocyst-stage embryo invades the endometrium to establish pregnancy. To understand how embryos breach the endometrial epithelium, we modelled human implantation using blastocysts or trophoblast stem cell spheroids cultured with endometrial epithelial cells (EEC). Blastocyst invasion of the EEC layer was initiated by multinuclear syncytiotrophoblast. Spheroids also invaded the epithelium with syncytiotrophoblast, and EEC induced upregulation of syncytiotrophoblast markers. Modelling implantation in silico using blastocyst and EEC transcriptomes revealed gene networks that exhibited greater connectivity and organisation in trophectoderm of the polar region of the embryonic axis. However, gene ontologies and machine learning suggested that EEC drives syncytiotrophoblast differentiation in polar and mural trophectoderm. This is the first evidence for endometrial epithelium-induced trophectoderm differentiation to invasive syncytiotrophoblast as the cellular mechanism of embryonic breaching of the endometrium in humans, with implications for reproductive medicine and our understanding of human embryonic development.

developmental biology↗