The role of small extracelllular vesicles in endometriosis

Endometriosis, defined as endometrial-like tissue outside the uterus, causes pain and/ or subfertility in 10% of reproductive-age women (Zondervan, Becker and Missmer, 2020). To date, the cause is unknown, resulting in inadequate diagnostic methods and treatment options (E. Liu et al., 2015; Gupta et al., 2016; Nisenblat, Bossuyt, et al., 2016; Nisenblat, Prentice, et al., 2016). I propose an aetiology similar to cancer’s ‘seed and soil’ hypothesis (Hoshino et al., 2015), requiring interaction between eutopic endometrium, the peritoneal fluid (PF) and the prospective lesion site, mediated by small extracellular vesicles (sEVs). SEVs are cell-derived vesicles 30-150 nm in size that contain genetic material (RNA, DNA) and protein (Colombo, Raposo and Théry, 2014) and influence gene expression and behaviour in target cells (Zomer et al., 2015). Their cargo and surface proteins mirror the expression within their cell of origin. This DPhil thesis attempts to demonstrate the presence of PF-derived sEVs in endometriosis and control samples, to characterise their protein content and to understand the function of PF-derived sEVs in the pathophysiology of endometriosis. This involved an initial feasibility study (Nazri et al., 2020), an optimisation of sEV isolation methods, and a larger study which employed the optimised sEV isolation methods to investigate the differences between PF-derived sEVs in women with and without endometriosis. Methods: Samples and data were collected according to WERF EPHect protocols (Becker et al., 2014; Fassbender et al., 2014; Rahmioglu et al., 2014; Vitonis et al., 2014) from women who consented to be part of ENDOX, a study into the biology of Endometriosis at Oxford study (Rahmioglu et al., 2017). In the pilot (feasibility) study, PF-derived sEVs were isolated from 28 women and were pooled into six groups according to disease stage and cycle phase (control, rASRM stages I-II and III-IV endometriosis in proliferative and secretory phases). Following the optimisation of protocols for the isolation and characterisation of PF-derived sEVs, no pooling of PF samples occurred in the larger, main study involving 63 women. With the addition of the menstrual phase, PF-derived sEVs from these 63 women were analysed in nine groups according to disease stage and cycle phase (control, rASRM stages I-II and III-IV endometriosis, each in proliferative, secretory, and menstrual phases). PF-derived sEVs were purified using size exclusion chromatography (SEC) and analysed by nanoparticle tracking analysis (NTA), immunoblotting and mass spectrometry. TEM imaging was done for samples in the pilot study. Results: SEVs are present in PF of women with and without endometriosis in both pilot and main studies. PF-derived SEVs are positive for marker proteins programmed cell death 6-interacting protein (ALIX), CD9 and syntenin. Differences in concentration of PF-derived sEVs and protein content were observed in different disease stages and cycle phases in both studies, though the results found in the pilot study were not replicated in the main study. Notably, this could be due to the pooling of samples and the smaller sample size in the pilot study. Therefore, higher weightage is ascribed to the results of the main study when in doubt, as PF samples were not pooled, and an additional cycle phase was added to the analysis. In the main study, higher sEV concentrations were found in endometriosis versus control. PF-derived sEV concentrations were highest in stage III-IV endometriosis, followed by stage I-II endometriosis and control, irrespective of the cycle phase (P = 0.0210). The mode size of PF particles in women with endometriosis was 130 ± 8.7 nm, whereas in women without endometriosis, it was 134 ± 2.12 nm. A gradual decrease of sEV concentration in stage III-IV endometriosis was observed as these samples experienced the transition from proliferative to secretory cycle phases. The reverse was seen with stage I-II endometriosis as these samples transitioned from proliferative to secretory cycle phases. Proteomic analysis showed distinct distribution patterns of proteins within endometriosis PF-derived sEVs compared to controls. CD44, complement C1q, and leucine-rich repeat neuronal protein 4 (LRRN4) are sEV proteins with the top three highest VIP scores (variable importance projection, as per PLS-DA plot) uniquely found within the endometriosis population. Unfortunately, functional assays for understanding the role of these PF-derived sEVs in endometriosis need further optimisation. I hypothesise that endometriosis-specific PF-derived sEVs taken up by mesothelial cells in the peritoneal cavity induce epithelial-mesenchymal transition (EMT), thus allowing endometrial cells in PF to adhere and invade the PMC and establish an endometrial lesion. This follows several studies investigating the role of CD44, complement C1q and LRRN4 in endometriosis and cancer metastases. The presence of endometriosis-specific PF-derived sEV CD44, complement C1q, and LRRN4 needs to be validated via ELISA or immunoblotting. Conclusion: PF-derived sEVs differ between endometriosis and control patients. Concentrations vary regardless of cycle phase and disease stage, and this difference appears to be reflected in the proteomics analysis. Correlating PF-derived sEVs in endometriosis versus control groups will aid our understanding of endometriosis and biomarker identification to diagnose endometriosis.