Endometrial stromal cell inflammatory phenotype during severe ovarian endometriosis as a cause of endometriosis-associated infertility

Abstract

Research question

Do endometrial stromal cells from primary infertile patients with severe ovarian endometriosis display differential secretory profiles of inflammation-associated cytokines during the implantation window that may cause infertility?

Design

Forty-eight cytokines were measured in conditioned medium of isolated endometrial stromal cells obtained from primary infertile patients without endometriosis (control group, n = 12) or with stage IV ovarian endometriosis (ovarian endometriosis group, n = 14) using multiplex assays. Key cytokines showing differential secretory profiles were validated using Western immunoblotting. Cellular phenotypic validation was carried out in vitro by comparing proliferation and migration capacity between control (n = 6) and ovarian endometriosis (n = 7) groups.

Results

CCL3, CCL4, CCL5, CXCL10, FGF2, IFNG, IL1RN, IL5, TNFA, and VEGF could be detected only in the conditioned media of stromal cells obtained from the ovarian endometriosis group. Among other cytokines detected in the conditioned media of both groups, CCL2 (P = 0.0018), CSF3 (P = 0.0017), IL1B (P = 0.0066), IL4 (P = 0.036), IL6 (P = 0.0039) and IL13 (P = 0.036) were found to be higher, whereas the concentration of IL18 was lower (P = 0.023) in the ovarian endometriosis group. Concentrations of CCL2, IL1B, IL4 and IL13 in conditioned medium reflected significant diagnostic performance for predicting ovarian endometriosis. Cellular phenotypic validation in vitro revealed an enhanced proliferative phenotype (P = 0.046) with no change in cell migratory capacity of endometrial stromal cells from the ovarian endometriosis group.

Conclusions

Endometrial stromal cells derived from severe ovarian endometriosis samples displayed a hyperinflammatory and hyperproliferative bias in the endometrial stroma during the ‘window of implantation’ putatively causing loss of fecundability.

Introduction

Endometriosis is a common inflammatory disease characterized by the presence of endometrial tissue outside the uterine cavity, the ovary being a common site. Endometriosis has been estimated to affect up to 10% of women of reproductive age (Cramer and Missmer, 2002). An irrefutable clinical association exists between endometriosis and infertility. The prevalence of endometriosis may reach up to 50% in women with infertility, and more than one-third of women with endometriosis have infertility, whereas up to 80% of unexplained infertility may be associated with endometriosis (Evans and Decherney, 2017; Zondervan et al., 2018). The fecundity rate in normal fertile couples of reproductive age is around 20–25%, whereas the fecundity rate in women with untreated endometriosis is reportedly somewhere between 2% and 10% (Hughes et al., 1993; Macer and Taylor, 2012; Practice Committee of the American Society for Reproductive Medicine, 2012). Women with mild endometriosis generally show a significantly lower probability (36%) of pregnancy over 3 years than women with unexplained infertility (55%) (Akande et al., 2004; Macer and Taylor, 2012).

Reported studies using assisted reproductive technology (ART) yielded rather inconclusive results. In a meta-analysis of pregnancy rates in women with and without endometriosis who were undergoing an IVF and embryo transfer programme, Hamdan et al. (2015) reported similar reproductive outcomes between the groups, whereas Vassilopoulou et al. (2018) reported a diverse pattern of success in endometriosis patients. In the presence of endometriosis, the clinical pregnancy rate after IVF and embryo transfer was reportedly lower than in controls. The prognosis was better for mild endometriosis compared with more advanced stages, and the success of surgical procedure plus ART or spontaneous pregnancy was higher than in ART without surgical intervention (Rossi and Prefumo, 2016; Alborzi et al., 2019). It has been suggested that chronic inflammation in the peritoneal environment during endometriosis may result in poor ovarian function, as well as low oocyte and embryo quality (Garrido et al., 2002; Giacomini et al., 2017; Sanchez et al., 2017; Freis et al., 2018). Additionally, several reports have indicated that endometriosis causes inadequacies in endometrial receptivity owing to chronic inflammation without any overt morphological difference in the endometrium (Klemmt et al., 2006; Ulukus et al., 2006; Minici et al., 2008; Bondza et al., 2009; Brosens et al., 2012; Da Broi et al., 2017; Lessey and Kim, 2017).

In several studies, the cytokine composition of the peritoneal fluid from patients with endometriosis was investigated to gain more insight into the basis of its pathophysiology and associated infertility (Kalu et al., 2007; Weiss et al., 2009; Gueye et al., 2017; Jørgensen et al., 2017; Wang et al., 2018). To the best of our knowledge, no study to date has reported on the profile of the known inflammatory cytokines secreted from eutopic endometrial stromal cells during the secretory phase of infertile patients with endometriosis. The main aim of the present study was to examine the concentration profile of 48 inflammation-linked cytokines secreted in vitro by secretory phase endometrial stromal cells (ESC) collected during the implantation window (days 20–24 of the menstrual cycle) from primary infertility patients with severe ovarian endometriosis, and infertile patients without endometriosis, followed by immunochemical and functional validation of the observations. Our aim was to test the hypothesis that ESC from infertile patients collected during the implantation window displayed a differential secretory profile of inflammation-associated cytokines that might provide predictable cues for the occurrence of primary infertility during severe ovarian endometriosis.