O-028 Small extracellular vesicles (sEV) in peritoneal fluid and peripheral blood as biomarkers of endometriosis

Abstract

Abstract Study question Can we identify lesion-derived endometriosis-specific sEV cargo in peritoneal fluid of women with endometriosis, and detect the same endometriosis-specific cargo in sEV from peripheral blood? Summary answer Endometriosis-specific sEV are present in PF. Identification in peripheral blood is challenging due to low abundance in comparison to blood-borne sEV. What is known already Endometriosis is characterised by intraperitoneal lesions of endometrial tissue. Although ultrasound detection of severe endometriosis is possible, the final diagnosis currently relies on invasive laparoscopy as the gold standard, which leads to a delay of 8-10 years after women first present to their GPs with symptoms. We have previously shown that the peritoneal fluid (PF) of women with endometriosis contains endometriosis-specific sEV. If these can be traced in blood samples, endometriosis-specific sEV could serve as biomarkers of endometriosis, particularly for stages I and II, which are difficult to detect by ultrasound. Study design, size, duration This is an observational pilot study of 29 patients in total. From March 2021 until August 2021, we collected PF samples from 11 controls and 16 endometriosis cases and blood samples from 12 controls and 11 endometriosis cases. Of these, 19 were paired samples (blood and PF from the same participant). Participants/materials, setting, methods Women >18 years of age undergoing laparoscopic surgery for endometriosis or unrelated conditions were invited to participate (HREC 08078B/HREC 10148B). Exclusion criteria were pregnancy, malignancy, and menopause. Purified, validated sEV were analysed by label based quantitative proteomics Tandem Mass Tag (TMT). Data analysis was performed using Proteome Discoverer v2.4 (Thermo Fisher), statistical analysis using the R (LIMMA), with a protein false discovery rate of 1% and a quantitative threshold of an adjusted p-value <0.05. Main results and the role of chance We ascertained the identity of sEV by TEM and NTA (mode size 121.8 ± 18.0 nm (blood, n = 5) and 155.9 ± 37.2 (PF, n = 6)), and signals for syntenin and ALIX in immunoblots as well as expression of CD9, CD63 and CD81 in capture bead-flow cytometry. Proteomics analysis identified 9145 proteins groups across all sample groups, of these proteins 5,429 are consistently quantifiable, with 602 significantly different between comparisons (adjusted P-value <0.05). PCA showed separation of samples by type rather than batch. Fractionated analysis by SPS-MS3 methodology identified 7064 protein groups, with 3408 proteins consistently quantified across sample groups. Of these, 602 were found to differ significantly across all comparisons. In PF, 533 proteins changed significantly in abundance (245 up and 288 down), while in blood, four proteins changed in abundance. Limitations, reasons for caution The main limitation of the study is in its small sample size, which meant that we could not stratify data according to endometriosis stage. Due to limited amount of protein within sEV samples, not all assays could be done on all samples. Wider implications of the findings Our findings suggest that our methodology makes it feasible to define a protein signature of endometriosis based on sEV cargoes drawn from blood samples. This would represent a non-invasive biomarker of endometriosis and could help diagnose the condition, or rule out endometriosis as the origin of symptoms in the future. Trial registration number not applicable