Quantification of Protein Biomarkers in SonoPODography Fluid Using Single Molecule Arrays for Endometriosis

BACKGROUNDPatients with endometriosis experience chronic, inflammatory symptoms including severe pelvic pain and infertility, caused by endometrial-like tissue growth outside the uterus. Endometriosis remains poorly understood, largely attributed to disease heterogeneity and dependence on qualitative technologies for research. There is an unmet clinical need for quantitative, biomarker-based methods for improved endometriosis diagnosis, prognosis, and treatment. We describe the biomarker analysis of a novel biofluid for endometriosis research, sonoPODography (SPG) fluid, which is a fluid collected using culdocentesis from the rectouterine pouch upon saline infusion through the uterus and fallopian tubes. METHODSSingle molecule array (Simoa) assays were used to quantify TNF, IL-1{beta}, VEGF, and CA125 in SPG fluid of 33 endometriosis patients. Simoa assays were validated using dilution-linearity and spike-and-recovery experiments in pooled and individual SPG fluid samples from endometriosis patients. The agreement of CA125 measurements by Simoa and enzyme-linked immunosorbent assay (ELISA) was assessed using Spearman correlation and Bland-Altman analyses. RESULTSSimoa assays were validated in SPG fluid and dilution factors 2x (TNF), 4x (IL-1{beta}), 8x (VEGF), 600x (CA125) were selected for biomarker quantification. Median biomarker concentrations in SPG fluid from endometriosis patients were 0.276 pg/mL (TNF), 0.21 pg/mL (IL-1{beta}), 30.55 pg/mL (VEGF), and 1,810 pg/mL (CA125). Simoa and ELISA CA125 measurements were highly correlated ({rho} = 0.80, p < 0.0001). CONCLUSIONWe present the feasibility and potential of SPG fluid as a novel source of biomarkers for endometriosis, which can enable quantitative clinical technologies for endometriosis diagnosis, prognosis, and treatment.