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 (TNF and IL-1β) 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.

Keywords: ALDH1A1; endometriosis; endometrium; inflammatory cytokines; non-invasive diagnostic; podoplanin; stromal fibroblast cells.

Figure 1

ME-SFCs from endometriosis patients and symptomatic, undiagnosed patients exhibit defective decidualization. (A) ME-SFCs cultured from healthy controls (CTRL, n = 23), surgically diagnosed endometriosis patients (ENDO, n = 24) and women with symptoms suggestive of endometriosis, but not diagnosed (SYMPTO, n = 9) were treated with vehicle and cAMP (0.5 mM) in decidualization media. After 24 h, decidualization was determined by measuring IGFBP1 concentrations in the culture supernatants by ELISA. Decidualization capacity for healthy controls (white box) and endometriosis cases (gray box) are shown as the ratio of cAMP-IGFBP1:vehicle-IGFBP1 using Tukey box and whisker plots (box = interquartile range [25th and 75th percentile]; horizontal line = median; upper and lower whiskers indicate range without outliers; outliers = •). Significance was determined using the Kruskal-Wallis test followed by Dunn's Multiple Comparisons post hoc comparison. **P < 0.01; ***P < 0.001. (B) Receiving operator characteristic curve or ROC curve shows the visual representation of the decidualization (IGFBP1) data for the healthy controls and the surgically diagnosed endometriosis patients in (A). The area under the curve (AUC) = 0.92.

Figure 2

ALDH1A1 gene expression is reduced and PDPN surface expression is increased by ME-SFCs from endometriosis patients. (A) ALDH1A1 mRNA expression. Confluent monolayers of ME-SFCs from healthy controls (CTRL, n = 34) and surgically diagnosed endometriosis patients (ENDO, n = 30) were incubated decidualization media for 24 h and then analyzed for ALDH1A1 mRNA expression by RT-qPCR. Relative differences in gene expression for each subject were normalized to expression levels of a housekeeping gene, HPRT1. Data are shown for controls (white box) and endometriosis cases (gray box) using Tukey box and whisker plots (box = interquartile range; horizontal line = median; upper and lower whiskers indicate range without outliers; outliers = •). (B) Podoplanin (PDPN) surface expression. Confluent monolayers of ME-SFCs from healthy controls (CTRL, n = 7) and surgically diagnosed endometriosis patients (ENDO, n = 7) were analyzed in duplicate for PDPN surface expression by flow cytometry. MFI data (corrected for isotype control) are shown for controls (white boxes) and endometriosis subjects (gray box) using Tukey box and whisker plots (box = interquartile range; horizontal line = median; upper and lower whiskers indicate range without outliers; outliers = •). Significance was determined using unpaired students t-test with Welch's correction for unequal variances. *P < 0.05; **P < 0.01.

Figure 3

Treatment of control ME-SFCs with TNF induces an endometriosis-like phenotype. (A) Inhibition of IGFBP1 by TNF. Healthy control ME-SFCs (n = 5) were treated with vehicle (Veh) and TNF (10 ng/ml) in separate flasks on days 1 and 3. On day 7 ME-SFCs were lifted, washed and plated in decidualization media. Decidualization capacity was analyzed after treating the vehicle- and TNF-treated cells with vehicle and cAMP (0.5 mM), as described in Figure 1A. After 24 h culture supernatants were analyzed for IGFBP1 concentrations by ELISA. Decidualization capacity (the ratio of cAMP-IGFBP1:vehicle-IGFBP1) for each subject under both conditions was determined as in Figure 1A. (B) Inhibition of ALDH1A1 mRNA expression by TNF. Healthy control ME-SFCs (n = 19) were treated with vehicle (Veh) and TNF (10 ng/ml), in separate flasks, on days 1 and 3. On day 7 cells were lifted, washed and plated in decidualization media for 24 h and then analyzed for ALDH1A1 mRNA expression by real time qPCR as in Figure 2A. Relative differences in ALDH1A1 gene expression for each pair of cells (for each subject under both conditions, vehicle-treated and TNF-treated) were normalized to expression levels of a housekeeping gene, HPRT1. (C) Enhanced PDPN surface expression by TNF. Healthy control ME-SFCs (n = 9) were treated with vehicle (Veh) and TNF (10 ng/ml) for 48 h in separate flasks, and then analyzed for PDPN surface expression by flow cytometry. Data are shown as MFI (corrected for isotype control) for each subject under both conditions (vehicle-treated and TNF-treated). For (A–C), paired data points (after vehicle treatment and TNF treatment) are shown for the cells from each subject. (A–C) Significance was determined using paired students t-test. *P < 0.05; **P < 0.01; ***P < 0.001. (D) Representative histograms of PDPN surface expression after exposure of control ME-SFCs to vehicle and TNF. Healthy control ME-SFCs were treated with vehicle and TNF (1 ng/ml or 10 ng/ml) in separate flasks for 48 h, and then analyzed for PDPN surface expression by flow cytometry. The histogram plot for isotype control, PDPN-vehicle, PDPN-TNF (1 ng/ml), and PDPN-TNF (10 ng/ml) with each mean MFI is shown.

Figure 4

ME-SFCs from endometriosis patients exhibit enhanced cell migration compared to control ME-SFCs; treatment of control ME-SFCs with TNF promotes cell migration. (A) Increased cell migration by ME-SFCs from endometriosis patients. Healthy control ME-SFCs (CTRL, n = 15) and endometriosis ME-SFCs (ENDO, n = 16) were analyzed for cell migration using the scratch assay method as described in the methods section. Monolayers were photographed at time 0 and after 20 h (see Supplemental Figure 2). Images were imported into Image J and cell migration was determined as % wound or scratch closure. Data are shown for ME-SFCs from the controls (white box) and endometriosis subjects (gray box) using Tukey box and whisker plots (box = interquartile range; horizontal line = median; upper and lower whiskers indicate range without outliers; outliers = •). Significance was determined using unpaired students t-tests with Welch's correction. (B) Increased migration of control ME-SFCs after exposure to TNF. Healthy control ME-SFCs (n = 6) were treated with vehicle and TNF (10 ng/ml), in separate flasks, on days 1 and 3. On day 7 cells were analyzed for cell migration using the scratch assay method as described in A. Data are shown as paired data points (after vehicle treatment and TNF treatment) for each healthy control. Significance was determined using a paired students t-test (B). **P < 0.01; ***P < 0.001.

Figure 5

Endometriosis ME-SFCs are more adherent to fibronectin than control ME-SFCs. Increased adherence of ME-SFCs to fibronectin in endometriosis patients. ME-SFCs from healthy controls (CTRL, n = 12) and ME-SFCs from endometriosis patients (ENDO, n = 9) were analyzed for adherence to fibronectin-coated plates after a 20 min incubation. Non-adherent cells were removed by washing and the % of adherent cells (vs. total cells added) was determined by counting using CyQUANT compared to a standard curve of known cell numbers. Data are shown as the mean % adhesion for the control subjects (white box) and endometriosis subjects (gray box) using Tukey box and whisker plots (box = interquartile range; horizontal line = median; upper and lower whiskers indicate range without outliers). No outliers were observed. Significance was determined using an unpaired t-test with Welch's correction **P < 0.01.

Figure 6

Treatment of control ME-SFCs with cytokines induces persistent changes in decidualization capacity reflecting an endometriosis-like phenotype. Healthy control ME-SFCs (n = 9) were treated with (A) vehicle (Veh) and TNF (1 ng/ml) or (B) vehicle (Veh) and IL-1β (1 ng/ml) (n = 8) on days 1 and 3; on day 7 cells were then cultured and passaged in normal growth media. At 1 week (Wk 1), 2 weeks (Wk 2), and 3 weeks (Wk 3) post treatment, each subject's ME-SFCs were assessed for decidualization capacity following treatment with vehicle or cAMP (0.5 mM), as described in Figure 1A. Each subject's data plotted individually are shown in the Supplemental Figure 4. Culture supernatants were analyzed for IGFBP1 (pg/ml) levels by ELISA after 24 hrs. Data are shown as IGFBP1 values for each group's ME-SFCs [vehicle-treated (white box) vs. TNF-treated (gray box), or vehicle-treated (white box) vs. IL-1β-treated (gray box) at weeks 1, 2, or 3 using Tukey box and whisker plots (box = interquartile range; horizontal line = median; upper and lower whiskers indicate range without outliers; outliers = •)]. A persistent reduction in IGFBP1 production over 3 weeks is observed in responses to TNF (***P < 0.001) and IL-1β (**P < 0.01) compared with vehicle as shown in Supplemental Figure 3.