Menstrual Effluent Provides a Novel Diagnostic Window on the Pathogenesis of Endometriosis

Abstract

Endometriosis is a chronic inflammatory disorder characterized by the presence of endometrial-like tissue growing outside of the uterus. Although the cause is unknown, retrograde menstruation leads to deposition of endometrial cells into the peritoneal cavity. Lack of disease recognition and long diagnostic delays (6-10 years) lead to substantial personal, social and financial burdens, as well as delayed treatment. A non-invasive diagnostic for endometriosis is a major unmet clinical need. Here, we assessed whether differences in menstrual effluent-derived stromal fibroblast cells (ME-SFCs) from women with and without endometriosis provide the basis for a non-invasive diagnostic for endometriosis. In addition, we investigated whether treatment of control ME-SFCs with inflammatory cytokines (TNF and IL-1β) could induce an endometriosis-like phenotype. ME-SFCs from laparoscopically diagnosed endometriosis patients exhibit reduced decidualization capacity, measured by IGFBP1 production after exposure to cAMP. A receiver operating characteristic (ROC) curve developed using decidualization data from controls and endometriosis subjects yielded an area under the curve of 0.92. In addition, a significant reduction in ALDH1A1 gene expression and increased podoplanin surface expression were also observed in endometriosis ME-SFCs when compared to control ME-SFCs. These endometriosis-like phenotypes can be reproduced in control ME-SFCs by exposure to inflammatory cytokines and are associated with increased cell migration. These results are consistent with the hypothesis that chronic intrauterine inflammation influences the development of endometriosis lesions following retrograde menstruation. In conclusion, the analysis of ME-SFCs can provide an accurate, rapid, and non-invasive diagnostic for endometriosis and insight into disease pathogenesis.

Single cell analysis of menstrual endometrial tissues defines phenotypes associated with endometriosis

“…IGFBP1, LEFTY2, LUM, DCN , etc). This is consistent with previous studies showing impaired decidualization of cultured endometrial stromal cells obtained from endometrial and ectopic endometriosis biopsies [18, 21], as well as from menstrual effluent [11, 15]. Secondly, there is a striking reduction in the proportion of uNK cells in the ME-derived endometrial tissue of patients with endometriosis compared with controls.…”

“…A deficiency in the decidualization capacity of stromal cells cultured from biopsies of the eutopic endometrium has been reported previously [18], and is also found in ME-derived stromal cells collected at the time of menstruation [11, 15]. Our scRNA-Seq data clearly shows the reduction of the IGFBP1+ -expressing decidualized stromal cell subclusters in endometriosis cases vs. controls (Figure 5C).…”

“…On the other hand, if disease causation is due to retrograde menstruation of abnormal endometrial tissues, it provides an opportunity to explore new therapies using cells derived from menstrual endometrial tissues and cells. For example, we have previously shown that stromal cells grown from ME have persistent differences in decidualization capacity, even after weeks in culture [15]; similarly, treatment of ME-derived stromal cells with TNF and/or IL-1β significantly compromises their subsequent decidualization capacity for weeks [15]. These phenotypic changes can be induced in stromal cells in normal subjects by exposure to inflammatory cytokines.…”

“…Control subjects who self-reported no gynecologic history suggestive of a diagnosis of endometriosis were enrolled as ‘controls’ (N=9). Endometriosis, symptomatic, and control subjects collected their ME using an ‘at home’ ME collection kit for 4-8 hours on the day of their heaviest menstrual flow (typically day 1 or 2 of the cycle) with a menstrual cup (provided by DIVA International), except for one subject who collected ME using a novel menstrual collection sponge (as previously described [15]). After collection, ME was shipped priority overnight at 4 ° C to the laboratory for processing.…”

“…Our previous flow cytometry studies showed that uterine natural killer (uNK) cells were relatively depleted in ME from endometriosis cases vs. controls [11]. In addition, we demonstrated a defect in decidualization capacity of endometrial stromal cells grown from the ME of patients with endometriosis when compared to ME-stromal cells grown from healthy controls [11, 15]. While these earlier results potentially provided a basis for a screening test for endometriosis, these analyses relied on laborious and expensive cell culture and in vitro assays, making them impractical for clinical application.…”