The endometriosis type characterised by intraperitoneal superficial lesions is virtually impossible to detect through ultrasound, and the need for invasive laparoscopy delays diagnosis by 8-10 years. Having shown that peritoneal fluid (PF) of women with endometriosis contains endometriosis-specific sEV, we now isolated protein from sEV in PF and blood, and tested candidate biomarkers for endometriosis.
Women aged 18-50 years undergoing laparoscopic surgery for endometriosis or unrelated conditions were invited to participate in our study. After informed consent, sEV from matched PF and blood samples were isolated by differential ultracentrifugation, validated by Western Blotting, nanosight tracking analysis (NTA) and transmission electron microscopy (TEM), and analysed through label-based, quantitative proteomics. Data analysis was performed using Proteome Discoverer v2.4 in combination with statistical analysis by R (limma), considering a protein false discovery rate of 1% and a quantitative threshold of adjusted p-value <0.05.
We included 25 paired samples (n=11 controls, n=14 endometriosis) in the proteomics cohort. sEV identified by TEM showed a mode size of 121.8±18.0 nm (blood, n=5) and 155.9±37.2 (PF, n=6), and contained syntenin, ALIX, CD9, CD63 and CD81. Proteomics identified 7064 protein groups, with 3408 proteins consistently quantified across sample groups. Of these, 602 were found to differ significantly across all comparisons (Padj<0.05). In PF, 533 proteins changed significantly in abundance, while in blood, four proteins changed in abundance, with proteinase 3 (PRTN3), the receptor of IL-32, significantly downregulated in blood-derived sEV from endometriosis patients. We are now validating the marker in a new cohort (n=21 controls, n=11 endometriosis to date).