The Dysregulated IL-23/TH17 Axis in Endometriosis Pathophysiology.

Endometriosis is a chronic inflammatory disease in which endometrial-like tissue grows ectopically, resulting in pelvic pain and infertility. IL-23 is a key contributor in the development and differentiation of TH17 cells, driving TH17 cells toward a pathogenic profile. In a variety of inflammatory and autoimmune disorders, TH17 cells secrete proinflammatory cytokines, including IL-17, contributing to disease pathophysiology. Our studies and others have implicated IL-17 and TH17 cell dysregulation in endometriosis, which is associated with disease severity. In this article, we address whether IL-23-driven TH17 cells contribute to cardinal features of lesion proliferation, vascularization, and inflammation in endometriosis using patient samples, representative cell lines, and our established mouse model of endometriosis. The results indicated dysregulated expression of key genes in the IL-23/TH17 axis in patient ectopic and eutopic endometrial samples and increased IL-23 protein in patient plasma compared with controls. In vitro studies using primary human TH cells determined that rIL-23 mixture treatment increased pathogenic TH17 cell frequency. Similarly, rIL-23 treatment of cell lines (12Z cells, EECCs, HUVECs, and hESCs) representative of the endometriotic lesion microenvironment increased cytokines and growth factors, which play a role in lesion establishment and maintenance. In a syngeneic mouse model of endometriosis, rIL-23 treatment altered numbers of myeloid and T cell subsets in peritoneal fluid and increased giant cells within the lesion. Lesions from rIL-23-treated mice did not reveal significant alterations in proliferation/vascularization, although trends of increased proliferation and vascularization were observed. Collectively, these findings provide insights into the impact of the IL-23/TH17 axis on local immune dysfunction and broadly on endometriosis pathophysiology.

Transcriptomic changes in eutopic endometrium and ectopic lesions during endometriosis progression in a mouse model.

OBJECTIVE: To identify the transcriptomic changes of ectopic lesions and eutopic endometrial tissues during the progression of endometriosis, we performed transcriptomic analysis in the eutopic endometrium and ectopic lesions. DESIGN: Laboratory study. SETTING: Academic medical center. ANIMALS: Four fertile and 4 subfertile Pgrcre/+Rosa26mTmG/+ mice with endometriosis, and 4 sham mice for each group of endometriosis mice as control. These mice underwent either surgery to induce endometriosis or sham surgery. Fertile sham and mice with endometriosis were used 1 month after surgery, whereas subfertile ones were used 3 months after surgery. INTERVENTIONS: Early and chronic effects of endometriosis on transcriptomics of ectopic lesions and eutopic endometrium. MAIN OUTCOME MEASURES: RNA-sequencing analysis and identification of differentially expressed genes and pathways in the ectopic lesions and eutopic uteri from mice with endometriosis and sham mice at day 3.5 of pregnancy. RESULTS: Our mouse model recapitulates the transcriptomic changes of ectopic lesions in humans. RNA-sequencing analysis was performed in ectopic lesions and eutopic uteri from mice with or without endometriosis during the progression of the disease. Estrogen activity, inflammation, angiogenesis, and fibrosis pathways were consistently elevated in all the ectopic lesions compared with eutopic endometrium. Cholesterol/glucose synthesis and stem cell pluripotency pathways were more enhanced in ectopic lesions from subfertile mice compared with their eutopic endometrium. Dysregulation of infiltration of macrophage, dendritic, T and B cells was validated with the use of immunohistochemistry in ectopic lesions. Multiple ligand-receptor pairs between the ectopic and eutopic endometrium were altered compared with the sham endometrium. Suppressed WNT and EGF pathways were only found in the eutopic endometrium from subfertile not fertile mice compared with sham. CONCLUSIONS: Our mouse endometriosis model recapitulates the transcriptomics of ectopic lesions in humans. Our transcriptomic analysis during endometriosis progression in our mouse model will help us understand the pathophysiology of endometriosis.

A Plain Language Summary to learn about relugolix combination therapy for the treatment of pain associated with endometriosis.

WHAT IS THIS SUMMARY ABOUT?: This is a summary of research studies (known as clinical trials) called SPIRIT 1 and SPIRIT 2. The SPIRIT 1 and SPIRIT 2 studies compared how well a medicine called relugolix combination therapy worked in relieving pain in women with moderate to severe endometriosis compared to a placebo, a pill with no active medication. Endometriosis occurs when tissue similar to what normally lines the uterus grows in other places, such as the ovaries, fallopian tubes, and bowels. WHAT WERE THE RESULTS?: Researchers looked at 1261 adult women with moderate to severe endometriosis. Randomly, 420 (33%) of these women were assigned to relugolix combination therapy, 420 (33%) were assigned to delayed relugolix combination therapy (relugolix alone first and then relugolix combination therapy for the remainder of the study), and 421 (33%) were assigned to placebo. The SPIRIT 1 and SPIRIT 2 studies showed that more women taking relugolix combination therapy (75% from SPIRIT 1 and 75% from SPIRIT 2) for 24 weeks had both less pelvic or groin pain during menstrual periods from endometriosis and no need for more pain medicines than women who took placebo (27% from SPIRIT 1 and 30% from SPIRIT 2). The SPIRIT 1 and SPIRIT 2 studies also showed that more women taking relugolix combination therapy (59% from SPIRIT 1 and 66% from SPIRIT 2) for 24 weeks had both less pelvic or groin pain between menstrual periods from endometriosis and no need for more pain medicines than women who took placebo (40% from SPIRIT 1 and 43% from SPIRIT 2). Women taking relugolix combination therapy had less pelvic or groin pain during and between menstrual periods within 4 weeks of starting the medicine. The most common side effects were headaches, the common cold, and hot flushes or feeling hot among women taking relugolix combination therapy, delayed relugolix combination therapy, and placebo. Relugolix combination therapy was considered safe for those with no major medical problems. Women taking relugolix combination therapy had little to no loss of bone mineral density (a way of knowing how strong bones are) after 24 weeks of treatment. WHAT DO THE RESULTS OF THESE STUDIES TELL US?: Women with moderate to severe endometriosis taking relugolix combination therapy had much less pain from endometriosis than women taking placebo. Clinical Trial Registration: NCT03204318 (SPIRIT-1); NCT03204331 (SPIRIT-2) (ClinicalTrials.gov).

Epigenetic Dysregulation in Endometriosis: Implications for Pathophysiology and Therapeutics.

Endometriosis is a prevalent gynecological condition associated with pelvic pain and infertility. Despite more than a century of research, the etiology of endometriosis still eludes scientific consensus. This lack of clarity has resulted in suboptimal prevention, diagnosis, and treatment options. Evidence of genetic contributors to endometriosis is interesting but limited; however, significant progress has been made in recent years in identifying an epigenetic role in the pathogenesis of endometriosis through clinical studies, in vitro cell culture experiments, and in vivo animal models. The predominant findings include endometriosis-related differential expression of DNA methyltransferases and demethylases, histone deacetylases, methyltransferases, and demethylases, and regulators of chromatin architecture. There is also an emerging role for miRNAs in controlling epigenetic regulators in the endometrium and endometriosis. Changes in these epigenetic regulators result in differential chromatin organization and DNA methylation, with consequences for gene expression independent of a genetic sequence. Epigenetically altered expression of genes related to steroid hormone production and signaling, immune regulation, and endometrial cell identity and function have all been identified and appear to play into the pathophysiological mechanisms of endometriosis and resulting infertility. This review summarizes and critically discusses early seminal findings, the ever-growing recent evidence of epigenetic contributions to the pathophysiology of endometriosis, and implications for proposed epigenetically targeted therapeutics.

The dysregulation of leukemia inhibitory factor and its implications for endometriosis pathophysiology.

Endometriosis is an estrogen dominant, chronic inflammatory disease characterized by the growth of endometrial-like tissue outside of the uterus. The most common symptoms experienced by patients include manifestations of chronic pelvic pain- such as pain with urination, menstruation, or defecation, and infertility. Alterations to Leukemia Inhibitory Factor (LIF), a cytokine produced by the luminal and glandular epithelium of the endometrium that is imperative for successful pregnancy, have been postulated to contribute to infertility. Conditions such as recurrent implantation failure, unexplained infertility, and infertility associated diseases such as adenomyosis and endometriosis, have demonstrated reduced LIF production in the endometrium of infertile patients compared to fertile counterparts. While this highlights the potential involvement of LIF in infertility, LIF is a multifaceted cytokine which plays additional roles in the maintenance of cell stemness and immunomodulation. Thus, we sought to explore the implications of LIF production within ectopic lesions on endometriosis pathophysiology. Through immunohistochemistry of an endometrioma tissue microarray and ELISA of tissue protein extract and peritoneal fluid samples, we identify LIF protein expression in the ectopic lesion microenvironment. Targeted RT qPCR for LIF and associated signaling transcripts, identify LIF to be significantly downregulated in the ectopic tissue compared to eutopic and control while its receptor, LIFR, is upregulated, highlighting a discordance in ectopic protein and mRNA LIF expression. In vitro treatment of endometriosis representative cell lines (12Z and hESC) with LIF increased production of immune-recruiting cytokines (MCP-1, MCP-3) and the angiogenic factor, VEGF, as well as stimulated tube formation in human umbilical vein endothelial cells (HUVECs). Finally, LIF treatment in a syngeneic mouse model of endometriosis induced both local and peripheral alterations to immune cell phenotypes, ultimately reducing immunoregulatory CD206+ small peritoneal macrophages and T regulatory cells. These findings suggest that LIF is present in the ectopic lesions of endometriosis patients and could be contributing to lesion vascularization and immunomodulation.

MIG-6 Is Critical for Progesterone Responsiveness in Human Complex Atypical Hyperplasia and Early-Stage Endometrial Cancer.

Women with complex atypical hyperplasia (CAH) or early-stage endometrioid endometrial cancer (EEC) are candidates for fertility preservation. The most common approach is progesterone (P4) therapy and deferral of hysterectomy until after completion of childbearing. However, P4 therapy response rates vary, and molecular mechanisms behind P4 resistance are poorly understood. One potential molecular cause of P4 resistance is a loss or attenuation of PGR expression. Mitogen-inducible gene 6 (MIG-6) is critical for P4 responsiveness. MIG-6 protein expression in the endometrial epithelial and stromal cells from women with CAH and EEC was significantly lower compared to women without CAH or EEC. The P4-responsive women (10/15) exhibited an increase of MIG-6 expression in epithelial and stromal cells compared to P4-resistant women (5/15). In addition, immunohistochemical analysis for PGR results showed that stromal PGR levels are significantly higher in P4-responsive women compared to P4-resistant women, whereas epithelial PGR expression was not different. A reverse correlation of MIG-6 and pAKT levels was observed in early-stage EEC patients. Studies strongly suggest that loss of MIG-6 and PGR and activation of pAKT lead to P4 resistance in CAH and EEC. These results will help to elucidate the molecular mechanism leading to P4 resistance in CAH and EEC.

Estrogen mediates inflammatory role of mast cells in endometriosis pathophysiology.

Endometriosis is an estrogen dependent, chronic inflammatory disease characterized by the growth of endometrial lining outside of the uterus. Mast cells have emerged as key players in regulating not only allergic responses but also other mechanisms such as angiogenesis, fibrosis, and pain. The influence of estrogen on mast cell function has also been recognized as a potential factor driving disease pathophysiology in number of allergic and chronic inflammatory conditions. However, precise information is lacking on the cross talk between endocrine and immune factors within the endometriotic lesions and whether that contributes to the involvement of mast cells with disease pathophysiology. In this study, we observed a significant increase in mast cell numbers within endometriotic lesions compared to matched eutopic endometrium from the same patients. Compared to eutopic endometrium, endometriotic lesions had significantly higher levels of stem cell factor (SCF), a potent growth factor critical for mast cell expansion, differentiation, and survival for tissue resident mast cells. Targeted mRNA Q-PCR array revealed that the endometriotic lesions harbour microenvironment (upregulation of CPA3, VCAM1, CCL2, CMA1, CCR1, and KITLG) that is conducive to mast cells recruitment and subsequent differentiation. To examine cross-talk of mast cells within the endometriotic lesion microenvironment, endometriotic epithelial cells (12Z) and endometrial stromal cells (hESC) incubated with mast cell-conditioned media showed significantly increased production of pro-inflammatory and chemokinetic cytokines. To further understand the impact of estrogen on mast cells in endometriosis, we induced endometriosis in C57BL/6 mice. Mature mast cells were significantly higher in peritoneal fluid of estrogen-treated mice compared to untreated mice within the sham operated groups. Mouse endometriotic lesion tissue revealed several genes (qRT-PCR) relevant in mast cell biology significantly upregulated in the estrogen treated, endometriosis-induced group compared to control endometrium. The endometriotic lesions from estrogen treated mice also had significantly higher density of Alcian blue stained mast cells compared to untreated lesions or control endometrium. Collectively, these findings suggest that endometriotic lesions provide a microenvironment necessary for recruitment and differentiation of mast cells. In turn, mast cells potentially release pro-inflammatory mediators that contribute to chronic pelvic pain and endometriosis disease progression.

Once daily oral relugolix combination therapy versus placebo in patients with endometriosis-associated pain: two replicate phase 3, randomised, double-blind, studies (SPIRIT 1 and 2).

BACKGROUND: Endometriosis is a common cause of pelvic pain in women, for which current treatment options are suboptimal. Relugolix, an oral gonadotropin-releasing hormone receptor antagonist, combined with estradiol and a progestin, was evaluated for treatment of endometriosis-associated pain. METHODS: In these two replicate, phase 3, multicentre, randomised, double-blind, placebo-controlled trials at 219 community and hospital research centres in Africa, Australasia, Europe, North America, and South America, we randomly assigned women aged 18-50 years with surgically or directly visualised endometriosis with or without histological confirmation, or with histological diagnosis alone. Participants were eligible if they had moderate to severe endometriosis-associated pain and, during the 35-day run-in period, a dysmenorrhoea Numerical Rating Scale (NRS) score of 4·0 or higher on two or more days and a mean non-menstrual pelvic pain NRS score of 2·5 or higher, or a mean score of 1·25 or higher that included a score of 5 or more on 4 or more days. Women received (1:1:1) once-daily oral placebo, relugolix combination therapy (relugolix 40 mg, estradiol 1 mg, norethisterone acetate 0·5 mg), or delayed relugolix combination therapy (relugolix 40 mg monotherapy followed by relugolix combination therapy, each for 12 weeks) for 24 weeks. During the double-blind randomised treatment and follow-up period, all patients, investigators, and sponsor staff or representatives involved in the conduct of the study were masked to treatment assignment. The co-primary endpoints were responder rates at week 24 for dysmenorrhoea and non-menstrual pelvic pain, both based on NRS scores and analgesic use. Efficacy and safety were analysed in the modified intent-to-treat population (randomised patients who received ≥1 study drug dose). The studies are registered at ClinicalTrials.gov (SPIRIT 1 [NCT03204318] and SPIRIT 2 [NCT03204331]) and EudraCT (SPIRIT 1 [2017-001588-19] and SPIRIT 2 [2017-001632-19]). Eligible patients who completed the SPIRIT studies could enrol in a currently ongoing 80-week open-label extension study (SPIRIT EXTENSION [NCT03654274, EudraCT 2017-004066-10]). Database lock for the on-treatment duration has occurred, and post-treatment follow-up for safety, specificially for bone mineral density and menses recovery, is ongoing at the time of publication. FINDINGS: 638 patients were enrolled into SPIRIT 1 and randomly assigned between Dec 7, 2017, and Dec 4, 2019, to receive relugolix combination therapy (212 [33%]), placebo (213 [33%]), or relugolix delayed combination therapy (213 [33%]). 623 patients were enrolled into SPIRIT 2 and were randomly assigned between Nov 1, 2017 and Oct 4, 2019, to receive relugolix combination therapy (208 [33%]), placebo (208 [33%]), or relugolix delayed combination therapy (207 [33%]). 98 (15%) patients terminated study participation early in SPIRIT 1 and 115 (18%) in SPIRIT 2. In SPIRIT 1, 158 (75%) of 212 patients in the relugolix combination therapy group met the dysmenorrhoea responder criteria compared with 57 (27%) of 212 patients in the placebo group (treatment difference 47·6% [95% CI 39·3-56·0]; p<0·0001). In SPIRIT 2, 155 (75%) of 206 patients in the relugolix combination therapy group were dysmenorrhoea responders compared with 62 (30%) of 204 patients in the placebo group (treatment difference 44·9% [95% CI 36·2-53·5]; p<0·0001). In SPIRIT 1, 124 (58%) of 212 patients in the relugolix combination therapy group met the non-menstrual pelvic pain responder criteria versus 84 (40%) patients in the placebo group (treatment difference 18·9% [9·5-28·2]; p<0·0001). In SPIRIT 2, 136 (66%) of 206 patients were non-menstrual pelvic pain responders in the relugolix combination therapy group compared with 87 (43%) of 204 patients in the placebo group (treatment difference 23·4% [95% CI 13·9-32·8]; p<0·0001). The most common adverse events were headache, nasopharyngitis, and hot flushes. There were nine reports of suicidal ideation across both studies (two in the placebo run-in, two in the placebo group, two in the relugolix combination therapy group, and three in the delayed relugolix combination therapy group). No deaths were reported. Least squares mean percentage change in lumbar spine bone mineral density in the relugolix combination therapy versus placebo groups was -0·70% versus 0·21% in SPIRIT 1 and -0·78% versus 0·02% in SPIRIT 2, and in the delayed relugolix combination group was -2·0% in SPIRIT 1 and -1·9% in SPIRIT 2. Decreases in opioid use were seen in treated patients as compared with placebo. INTERPRETATION: Once-daily relugolix combination therapy significantly improved endometriosis-associated pain and was well tolerated. This oral therapy has the potential to address the unmet clinical need for long-term medical treatment for endometriosis, reducing the need for opioid use or repeated surgical treatment. FUNDING: Myovant Sciences.

Loss of MIG-6 results in endometrial progesterone resistance via ERBB2.

Female subfertility is highly associated with endometriosis. Endometrial progesterone resistance is suggested as a crucial element in the development of endometrial diseases. We report that MIG-6 is downregulated in the endometrium of infertile women with endometriosis and in a non-human primate model of endometriosis. We find ERBB2 overexpression in the endometrium of uterine-specific Mig-6 knockout mice (Pgrcre/+Mig-6f/f; Mig-6d/d). To investigate the effect of ERBB2 targeting on endometrial progesterone resistance, fertility, and endometriosis, we introduce Erbb2 ablation in Mig-6d/d mice (Mig-6d/dErbb2d/d mice). The additional knockout of Erbb2 rescues all phenotypes seen in Mig-6d/d mice. Transcriptomic analysis shows that genes differentially expressed in Mig-6d/d mice revert to their normal expression in Mig-6d/dErbb2d/d mice. Together, our results demonstrate that ERBB2 overexpression in endometrium with MIG-6 deficiency causes endometrial progesterone resistance and a nonreceptive endometrium in endometriosis-related infertility, and ERBB2 targeting reverses these effects.

SIRT1 plays an important role in implantation and decidualization during mouse early pregnancy.

Sirtuin 1 (SIRT1) is a member of the sirtuin family that functions to deacetylate both histones and non-histone proteins. Previous studies have identified significant SIRT1 upregulation in eutopic endometrium from infertile women with endometriosis. However, SIRT1 function in the uterus has not been directly studied. Using immunochemistry analysis, we found SIRT1 to be most strongly expressed at GD4.5 and GD5.5 in decidualized cells and at GD7.5 in secondary decidual cells in mouse. To assess the role of SIRT1 in uterine function, we generated uterine Sirt1 conditional knockout mice (Pgrcre/+Sirt1f/f; Sirt1d/d). A 6-month fertility trial revealed that Sirt1d/d females were subfertile. Implantation site numbers were significantly decreased in Sirt1d/d mice compared with controls at GD5.5. Sirt1d/d implantation sites at GD4.5 could be divided into two groups, Group #1 with luminal closure and nonspecific COX2 expression compared with controls (14/20) and Group #2 with an open lumen and no COX2 (6/20). In Sirt1d/d Group #1, nuclear FOXO1 expression in luminal epithelial cells was significantly decreased. In Sirt1d/d Group #2, nuclear FOXO1 expression was almost completely absent, and there was strong PGR expression in epithelial cells. At GD5.5, stromal PGR and COX2 were significantly decreased in Sirt1d/d uterine in the areas surrounding the embryo compared with controls, indicating defective decidualization. An artificially induced decidualization test revealed that Sirt1d/d females showed defects in decidualization response. All together, these data suggest that SIRT1 is important for decidualization and contributes to preparing a receptive endometrium for successful implantation.

Role of SIRT1 and Progesterone Resistance in Normal and Abnormal Endometrium.

CONTEXT: Progesterone resistance, a known pathologic condition associated with a reduced cellular response to progesterone and heightened estrogen responses, appears to have a normal physiologic role in mammalian reproduction. The molecular mechanism responsible for progesterone resistance in normal and abnormal endometrium remains unclear. OBJECTIVE: To examine the roles of sirtuin-1 (SIRT1) in normal endometrium as well as endometrium associated with infertility and endometriosis, as an epigenetic modulator associated with progesterone resistance. METHODS: SIRT1 expression was examined by Western blot, quantitative real-time polymerase chain reaction, and immunohistochemistry in mouse uterus and human endometrium. Mice with uterine specific Sirt1 overexpression were developed to examine SIRT1's role in endometrial function and endometriosis development. EX-527, a SIRT1 inhibitor, and SRT1720, a SIRT1 agonist, were also used to evaluate SIRT1 effect on endometriosis. RESULTS: In normal healthy women, endometrial SIRT1 is expressed only during menses. SIRT1 was dramatically overexpressed in the endometrium from women with endometriosis in both the epithelium and stroma. In mice, SIRT1 is expressed at the time of implantation between day 4.5 and 5.5 of pregnancy. Overexpression of SIRT1 in the mouse uterus leads to subfertility due to implantation failure, decidualization defects and progesterone resistance. SIRT1 overexpression in endometriotic lesions promotes worsening endometriosis development. EX-527 significantly reduced the number of endometriotic lesions in the mouse endometriosis model. CONCLUSIONS: SIRT1 expression and progesterone resistance appears to play roles in normal endometrial functions. Aberrant SIRT1 expression contributes to progesterone resistance and may participate in the pathophysiology of endometriosis. SIRT1 is a novel and targetable protein for the diagnosis as well as treatment of endometriosis and the associated infertility seen in this disease.

Evaluation of BCL6 and SIRT1 as Non-Invasive Diagnostic Markers of Endometriosis.

(1) Background: Endometriosis is characterized by the presence of endometrial glands and stroma outside of the uterus and is often associated with severe pelvic pain and infertility. Our study explored the utilization of B-Cell Lymphoma 6 (BCL6) and Sirtuin 1 (SIRT1) as potential biomarkers in serum, plasma, urine, and cervical mucus for a non-invasive diagnostic test for endometriosis. BCL6 was chosen based on its previously reported elevated expression in endometrial biopsies, and SIRT1 is co-expressed and upregulated in the endometrium of women with endometriosis. (2) Methods: BCL6 and SIRT1 levels were measured using enzyme-linked immunoassay (ELISA) in samples from 20 women with endometriosis (ten with stages I/II and ten with stages III/IV) and ten women without endometriosis. (3) Results: Levels of SIRT1 in sera showed a statistically significant elevation in advanced stages III/IV compared to controls and stages I/II. No significant differences were found in other bodily fluids for SIRT1 or any bodily fluids tested for BCL6. (4) Conclusions: These results suggest some potential of SIRT1 expression within serum as a predictor of advanced asymptomatic stages of endometriosis. Using immunohistochemistry (IHC) staining and H-SCORE values for the elevated BCL6 (and potentially SIRT1) levels in endometrial biopsy samples seems to have higher diagnostic potential based on the previously published studies.

IL-33 activates group 2 innate lymphoid cell expansion and modulates endometriosis.

Chronic inflammation and localized alterations in immune cell function are suspected to contribute to the progression of endometriosis and its associated symptoms. In particular, the alarmin IL-33 is elevated in the plasma, peritoneal fluid, and endometriotic lesions from patients with endometriosis; however, the exact role of IL-33 in the pathophysiology of endometriosis is not well understood. In this study, we demonstrate, in both humans and a murine model, that IL-33 contributes to the expansion of group 2 innate lymphoid cells (ILC2s), and this IL-33-induced ILC2 expansion modulates the endometriosis lesion microenvironment. Importantly, we show that IL-33 drives hallmarks of severe endometriosis, including elevated inflammation, lesion proliferation, and fibrosis, and that this IL-33-induced aggravation is mediated by ILC2s. Finally, we demonstrate the functionality of IL-33 neutralization as a promising and potentially novel therapeutic avenue for treating the debilitating symptoms of endometriosis.

Altered eutopic endometrial T-regulatory and T-helper 17 lymphocyte ratio in women with unexplained subfertility.

PROBLEM: Perturbations in T-helper lymphocyte profiles have previously been associated with endometriosis related subfertility and conception failure. Hence a retrospective in vitro study was conducted to evaluate the relationship between T-regulatory (Treg) and T-helper 17 (Th17) lymphocytes in the eutopic endometrium of women with unexplained subfertility and correlate these profiles to their conception status. METHOD OF STUDY: Eutopic endometrial biopsies were collected during the mid-secretory phase of the menstrual cycle, from women with unexplained subfertility. These samples were evaluated immunohistochemically for Treg and Th17 lymphocytes as well as the related proinflammatory cytokine, Interleukin-17 (IL-17). These eutopic endometrial T lymphocyte subpopulations were compared to the patients' conception status in subsequent cycles. RESULTS: Though Treg cells were not indicative of conception success in subsequent cycles, patients who maintained their subfertile (no conception) status were observed to have a higher Th17 cell count in their eutopic endometrium. The ratio of Treg:Th17 cell counts was significantly correlated to patient conception status as well. These trends stayed consistent irrespective of concurrent endometriosis. CONCLUSION: Patients with a high proinflammatory Th17 lymphocyte profile and low Treg:Th17 ratio in their eutopic endometrium during the secretory phase of their menstrual cycle are more likely to not conceive in subsequent cycles.

Podocalyxin is a key negative regulator of human endometrial epithelial receptivity for embryo implantation.

STUDY QUESTION: How is endometrial epithelial receptivity, particularly adhesiveness, regulated at the luminal epithelial surface for embryo implantation in the human? SUMMARY ANSWER: Podocalyxin (PCX), a transmembrane protein, was identified as a key negative regulator of endometrial epithelial receptivity; specific downregulation of PCX in the luminal epithelium in the mid-secretory phase, likely mediated by progesterone, may act as a critical step in converting endometrial surface from a non-receptive to an implantation-permitting state. WHAT IS KNOWN ALREADY: The human endometrium must undergo major molecular and cellular changes to transform from a non-receptive to a receptive state to accommodate embryo implantation. However, the fundamental mechanisms governing receptivity, particularly at the luminal surface where the embryo first interacts with, are not well understood. A widely held view is that upregulation of adhesion-promoting molecules is important, but the details are not well characterized. STUDY DESIGN, SIZE, DURATION: This study first aimed to identify novel adhesion-related membrane proteins with potential roles in receptivity in primary human endometrial epithelial cells (HEECs). Further experiments were then conducted to determine candidates' in vivo expression pattern in the human endometrium across the menstrual cycle, regulation by progesterone using cell culture, and functional importance in receptivity using in vitro human embryo attachment and invasion models. PARTICIPANTS/MATERIALS, SETTING, METHODS: Primary HEECs (n = 9) were isolated from the proliferative phase endometrial tissue, combined into three pools, subjected to plasma membrane protein enrichment by ultracentrifugation followed by proteomics analysis, which led to the discovery of PCX as a novel candidate of interest. Immunohistochemical analysis determined the in vivo expression pattern and cellular localization of PCX in the human endometrium across the menstrual cycle (n = 23). To investigate whether PCX is regulated by progesterone, the master driver of endometrial differentiation, primary HEECs were treated in culture with estradiol and progesterone and analyzed by RT-PCR (n = 5) and western blot (n = 4). To demonstrate that PCX acts as a negative regulator of receptivity, PCX was overexpressed in Ishikawa cells (a receptive line) and the impact on receptivity was determined using in vitro attachment (n = 3-5) and invasion models (n = 4-6), in which an Ishikawa monolayer mimicked the endometrial surface and primary human trophoblast spheroids mimicked embryos. Mann-Whitney U-test and ANOVA analyses established statistical significance at *P ≤ 0.05 and **P ≤ 0.01. MAIN RESULTS AND THE ROLE OF CHANCE: PCX was expressed on the apical surface of all epithelial and endothelial cells in the non-receptive endometrium, but selectively downregulated in the luminal epithelium from the mid-secretory phase coinciding with the establishment of receptivity. Progesterone was confirmed to be able to suppress PCX in primary HEECs, suggesting this hormone likely mediates the downregulation of luminal PCX in vivo for receptivity. Overexpression of PCX in Ishikawa monolayer inhibited not only the attachment but also the penetration of human embryo surrogates, demonstrating that PCX acts as an important negative regulator of epithelial receptivity for implantation. LIMITATIONS, REASONS FOR CAUTION: Primary HEECs isolated from the human endometrial tissue contained a mixture of luminal and glandular epithelial cells, as further purification into subtypes was not possible due to the lack of specific markers. Future study would need to investigate how progesterone differentially regulates PCX in endometrial epithelial subtypes. In addition, this study used primary human trophoblast spheroids as human embryo mimics and Ishikawa as endometrial epithelial cells in functional models, future studies with human blastocysts and primary epithelial cells would further validate the findings. WIDER IMPLICATIONS OF THE FINDINGS: The findings of this study add important new knowledge to the understanding of human endometrial remodeling for receptivity. The identification of PCX as a negative regulator of epithelial receptivity and the knowledge that its specific downregulation in the luminal epithelium coincides with receptivity development may provide new avenues to assess endometrial receptivity and individualize endometrial preparation protocols in assisted reproductive technology (ART). The study also discovered PCX as progesterone target in HEECs, identifying a potentially useful functional biomarker to monitor progesterone action, such as in the optimization of progesterone type/dose/route of administration for luteal support. STUDY FUNDING/COMPETING INTEREST(S): Study funding was obtained from ESHRE, Monash IVF and NHMRC. LR reports potential conflict of interests (received grants from Ferring Australia; personal fees from Monash IVF Group and Ferring Australia; and non-financial support from Merck Serono, MSD, and Guerbet outside the submitted work. LR is also a minority shareholder and the Group Medical Director for Monash IVF Group, a provider of fertility preservation services). The remaining authors have no potential conflict of interest to declare. TRIAL REGISTRATION NUMBER: NA.

ARID1A and PGR proteins interact in the endometrium and reveal a positive correlation in endometriosis.

Endometriosis is a disorder in which endometrial cells normally limited to the lining of the uterus proliferate outside the uterine cavity and can cause pelvic pain and infertility. ARID1A levels are significantly reduced in the eutopic endometrium from women with endometriosis. Uterine specific Arid1a knock-out mice were infertile due to loss of epithelial progesterone receptor (PGR) signaling. However, the functional association of ARID1A and PGR in endometriosis has not been studied. We examined the expression patterns and co-localization of ARID1A and PGR in eutopic endometrium from women with and without endometriosis using immunostaining and Western blot analysis. ARID1A and PGR proteins co-localized in the epithelium during the proliferative and the early secretory phases. Our immunoprecipitation analysis and proximity ligation assay (PLA) revealed physical interaction between ARID1A and PGR-A but not PGR-B in the mouse and human endometrium. ARID1A levels positively correlated with PGR levels in the eutopic endometrium of women with endometriosis. Our results bring new perspectives on the molecular mechanisms involved in endometrial receptivity and progesterone resistance in endometriosis. The interrelationship between ARID1A and PGR may contribute to explaining the non-receptive endometrium in endometriosis-related infertility.

Genetic and epigenetic changes in the eutopic endometrium of women with endometriosis: association with decreased endometrial αvß3 integrin expression.

About 40% of women with infertility and 70% of women with pelvic pain suffer from endometriosis. The pregnancy rate in women undergoing IVF with low endometrial integrin αvß3 (LEI) expression is significantly lower compared to the women with high endometrial integrin αvß3 (HEI). Mid-secretory eutopic endometrial biopsies were obtained from healthy controls (C; n=3), and women with HEI (n=4) and LEI (n=4) and endometriosis. Changes in gene expression were assessed using human gene arrays and DNA methylation data were derived using 385 K Two-Array Promoter Arrays. Transcriptional analysis revealed that LEI and C groups clustered separately with 396 differentially expressed genes (DEGs) (P<0.01: 275 up and 121 down) demonstrating that transcriptional and epigenetic changes are distinct in the LEI eutopic endometrium compared to the C and HEI group. In contrast, HEI vs C and HEI vs LEI comparisons only identified 83 and 45 DEGs, respectively. The methylation promoter array identified 1304 differentially methylated regions in the LEI vs C comparison. The overlap of gene and methylation array data identified 14 epigenetically dysregulated genes and quantitative RT-PCR analysis validated the transcriptomic findings. The analysis also revealed that aryl hydrocarbon receptor (AHR) was hypomethylated and significantly overexpressed in LEI samples compared to C. Further analysis validated that AHR transcript and protein expression are significantly (P<0.05) increased in LEI women compared to C. The increase in AHR, together with the altered methylation status of the 14 additional genes, may provide a diagnostic tool to identify the subset of women who have endometriosis-associated infertility.

Implications of dysregulated endogenous cannabinoid family members in the pathophysiology of endometriosis.

OBJECTIVE: To determine the involvement of the endocannabinoid (EC) family member in the pathophysiology of endometriosis (EMS). DESIGN: Mass spectrometry analysis of plasma and tissue samples from patients with EMS, controls, and a mouse model of EMS and messenger RNA and immunohistochemistry analysis of the samples from patients with EMS and controls. SETTING: Academic teaching hospital and university. PATIENT(S): Patients with EMS and healthy fertile control subjects. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Endocannabinoid analysis in patient plasma, EMS lesions, and healthy endometrial samples. RESULT(S): Circulating ECs were detected in the plasma samples, whereas no significant changes were observed in patients with EMS compared with healthy fertile controls. However, the palmitoylethanolamide levels were significantly higher in the EMS lesions than in the endometrium from patients with EMS. Similarly, genes involved in the EC signaling pathways were differentially expressed in the EMS lesions. Analysis of cannabinoid 1 and 2 receptors in the EMS lesions revealed a significantly lower cannabinoid 2 receptor expression, whereas no significant changes were observed in cannabinoid 1 receptor expression compared with those in the endometrium from both patients with EMS and healthy fertile controls. The palmitoylethanolamide levels were significantly elevated in plasma from EMS mice compared with that from sham controls and in EMS lesions compared with uterine samples. CONCLUSION(S): Together, we provide evidence toward dysregulation of members of the ECs in both patients with EMS and the mouse model of EMS. These findings will advance the knowledge of the role of ECs in EMS and their potential implications as therapeutic targets.

Endometrial epithelial ARID1A is critical for uterine gland function in early pregnancy establishment.

Though endometriosis and infertility are clearly associated, the pathophysiological mechanism remains unclear. Previous work has linked endometrial ARID1A loss to endometriosis-related endometrial non-receptivity. Here, we show in mice that ARID1A binds and regulates transcription of the Foxa2 gene required for endometrial gland function. Uterine-specific deletion of Arid1a compromises gland development and diminishes Foxa2 and Lif expression. Deletion of Arid1a with Ltf-iCre in the adult mouse endometrial epithelium preserves the gland development while still compromising the gland function. Mice lacking endometrial epithelial Arid1a are severely sub-fertile due to defects in implantation, decidualization, and endometrial receptivity from disruption of the LIF-STAT3-EGR1 pathway. FOXA2 is also reduced in the endometrium of women with endometriosis in correlation with diminished ARID1A, and both ARID1A and FOXA2 are reduced in nonhuman primates induced with endometriosis. Our findings describe a role for ARID1A in the endometrial epithelium supporting early pregnancy establishment through the maintenance of gland function.