New procedure for the endometriosis diagnosis

Abstract

Previous studies revealed that when endometrial cells from GFP (Green Fluorescent Protein) mice were introduced into peritoneal cavity of RFP (Red Fluorescent Protein) mice to induce a murine model of endometriosis, GFP+ cells epithelial cells were aberrantly found in the stromal compartment of the eutopic endometrium of RFP mice two, three and four months after surgery. Arrays and immunofluorescence (IF) of these aberrant cells showed that GFP+ cells co-expressed cytokeratin (CK, epithelial marker) and Leucine-rich Repeat Containing G protein-Coupled Receptor 5 (LGR5, stem cell marker of the intestine). The aim of this study was to prove wether this process occurs in the human endometrium of patients affected with endometriosis and to characterize this type of cells in order to is to develop a non-invasive diagnostic test for this disease. Experimental case-control study. Uterine Aspirates from women with endometriosis (n=26) and healthy donors (n=11) were collected from women undergoing surgery for endometriosis or ovum pick-up for donation. Endometriotic patients were dividen in ovarian endometriosis (n=9), pelvic endometriosis (n=3), Deep Infiltrating Endometriosis (n=9) and Adenomyosis (n=4). Samples were aspirates was washed with 1x PBS and placed into 4% formaldehyde for fixation and paraffin embedding. Paraffin-embedded fixed samples were sectioned for hematoxylin and eosin (H&E) staining to determine the stage of the menstrual cycle and for immunofluorescence experiments. Dating of phases of the menstrual cycle was determined by two pathologists according to the criteria of Noyes.Subsequently Immunofluorescence (IF) staining was performed to co-localize LGR5 with E-Cadherine and LGR5 with Citokeratine in the epithelial and stromal compartment. Additionally LGR5+ cells were isolated through cell sorting and mRNA was sequenced. IF analysis on paraffin embedded tissue of mice confirmed that GFP+ cells co-localize with LGR5 and E-cadherin (ECAD, epithelial marker). LGR5+ cells in epithelium and stroma of human eutopic endometrium from patients and donors by FACS and IF. LGR5+ cells co-localize aberrantly with ECAD and CK in the stroma from 17 and 22 of 26 endometriotic patients respectively, whereas there is no colocalization in none of 11 donors. We also performed a pilot study using RNA-sequencing of the LGR5+/- cells from uterine biopsies from four patients of each type of endometriosis and healthy donors in order to identify a genetic signature of the disease and its subgroups. An aberrant expression of epithelial cells was found in the eutopic endometrium of women affected with endometriosis compared to healthy women. These aberrant cells were isolated and their experession was compared to healthy endometrium and each different subtype of endometriosis revealing that each type have a characteristic genetic signature that may led us to potentially establish an new and less invasive diagnostic tool for the diagnosis of endomteriosis.