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.

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.

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.

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.

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.

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.

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.

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.

Neutrophil recruitment and function in endometriosis patients and a syngeneic murine model.

Endometriosis is a chronic inflammatory, gynecological disease characterized by the presence of endometrial-like tissue lesions outside of the uterus. Neutrophils are elevated in the systemic circulation and peritoneal fluid of endometriosis patients; however, whether and how neutrophils contribute to endometriosis pathophysiology remain poorly understood. With emerging roles for neutrophils in chronic and sterile inflammatory conditions, we sought to provide in-depth characterization of neutrophil involvement in endometriosis. We demonstrate that neutrophils reside within patient endometriotic lesions and that patient lesions possess a microenvironment that may influence neutrophil recruitment and function. We also provide the first evidence that systemic circulating neutrophils from endometriosis patients display distinct transcriptomic differences compared neutrophils from healthy control subjects. Time course characterization of our syngeneic, immunocompetent mouse model of endometriosis revealed that neutrophils are rapidly recruited to the peritoneal environment early after endometriotic lesion establishment and remain present in murine lesions long term. In vivo neutrophil depletion altered the systemic and peritoneal immune microenvironment of mice with endometriosis as demonstrated by changes in pro-inflammatory and angiogenic mediators. Taken together, these findings highlight a novel role for neutrophils in early events such as angiogenesis and modulation of the local inflammatory environment associated with endometriosis pathogenesis.

FOXO1 regulates uterine epithelial integrity and progesterone receptor expression critical for embryo implantation.

Successful embryo implantation requires a receptive endometrium. Poor uterine receptivity can account for implantation failure in women who experience recurrent pregnancy loss or multiple rounds of unsuccessful in vitro fertilization cycles. Here, we demonstrate that the transcription factor Forkhead Box O1 (FOXO1) is a critical regulator of endometrial receptivity in vivo. Uterine ablation of Foxo1 using the progesterone receptor Cre (PgrCre) mouse model resulted in infertility due to altered epithelial cell polarity and apoptosis, preventing the embryo from penetrating the luminal epithelium. Analysis of the uterine transcriptome after Foxo1 ablation identified alterations in gene expression for transcripts involved in the activation of cell invasion, molecular transport, apoptosis, ß-catenin (CTNNB1) signaling pathway, and an increase in PGR signaling. The increase of PGR signaling was due to PGR expression being retained in the uterine epithelium during the window of receptivity. Constitutive expression of epithelial PGR during this receptive period inhibited expression of FOXO1 in the nucleus of the uterine epithelium. The reciprocal expression of PGR and FOXO1 was conserved in human endometrial samples during the proliferative and secretory phase. This demonstrates that expression of FOXO1 and the loss of PGR during the window of receptivity are interrelated and critical for embryo implantation.

Treatment of Endometriosis-Associated Pain with Elagolix, an Oral GnRH Antagonist.

BACKGROUND: Endometriosis is a chronic, estrogen-dependent condition that causes dysmenorrhea and pelvic pain. Elagolix, an oral, nonpeptide, gonadotropin-releasing hormone (GnRH) antagonist, produced partial to nearly full estrogen suppression in previous studies. METHODS: We performed two similar, double-blind, randomized, 6-month phase 3 trials (Elaris Endometriosis I and II [EM-I and EM-II]) to evaluate the effects of two doses of elagolix - 150 mg once daily (lower-dose group) and 200 mg twice daily (higher-dose group) - as compared with placebo in women with surgically diagnosed endometriosis and moderate or severe endometriosis-associated pain. The two primary efficacy end points were the proportion of women who had a clinical response with respect to dysmenorrhea and the proportion who had a clinical response with respect to nonmenstrual pelvic pain at 3 months. Each of these end points was measured as a clinically meaningful reduction in the pain score and a decreased or stable use of rescue analgesic agents, as recorded in a daily electronic diary. RESULTS: A total of 872 women underwent randomization in Elaris EM-I and 817 in Elaris EM-II; of these women, 653 (74.9%) and 632 (77.4%), respectively, completed the intervention. At 3 months, a significantly greater proportion of women who received each elagolix dose met the clinical response criteria for the two primary end points than did those who received placebo. In Elaris EM-I, the percentage of women who had a clinical response with respect to dysmenorrhea was 46.4% in the lower-dose elagolix group and 75.8% in the higher-dose elagolix group, as compared with 19.6% in the placebo group; in Elaris EM-II, the corresponding percentages were 43.4% and 72.4%, as compared with 22.7% (P<0.001 for all comparisons). In Elaris EM-I, the percentage of women who had a clinical response with respect to nonmenstrual pelvic pain was 50.4% in the lower-dose elagolix group and 54.5% in the higher-dose elagolix group, as compared with 36.5% in the placebo group (P<0.001 for all comparisons); in Elaris EM-II, the corresponding percentages were 49.8% and 57.8%, as compared with 36.5% (P=0.003 and P<0.001, respectively). The responses with respect to dysmenorrhea and nonmenstrual pelvic pain were sustained at 6 months. Women who received elagolix had higher rates of hot flushes (mostly mild or moderate), higher levels of serum lipids, and greater decreases from baseline in bone mineral density than did those who received placebo; there were no adverse endometrial findings. CONCLUSIONS: Both higher and lower doses of elagolix were effective in improving dysmenorrhea and nonmenstrual pelvic pain during a 6-month period in women with endometriosis-associated pain. The two doses of elagolix were associated with hypoestrogenic adverse effects. (Funded by AbbVie; Elaris EM-I and EM-II ClinicalTrials.gov numbers, NCT01620528 and NCT01931670 .).

Non-bladder centric interstitial cystitis/bladder pain syndrome phenotype is significantly associated with co-occurring endometriosis.

INTRODUCTION: Interstitial cystitis/bladder pain syndrome (IC/BPS) and endometriosis are coexistent diagnoses in 48%-65% of women with chronic pelvic pain (CPP), suggesting that dual screening may be warranted. To further investigate the clinical relationship and risk factors between these two conditions, we performed a retrospective review of our large IC/BPS patient data registry. MATERIALS AND METHODS: We evaluated IC/BPS patients who were prospectively enrolled into our registry who completed validated questionnaires and underwent therapeutic hydrodistension, during which anesthetic bladder capacity (BC) and Hunner's lesion (HL) status were recorded. Demographic/medical history were reviewed. IC/BPS patients with co-occurring endometriosis diagnosis versus those without were compared using descriptive statistics as well as multivariate regression analyses to determine predictors of co-occurring disease. RESULTS: Of 431 IC/BPS participants, 82 (19%) were also diagnosed with endometriosis. These women were significantly younger, had increased prevalence of non-low BC (> 400 cc), and decreased prevalence of HL (p < 0.05). Patients with co-occurring endometriosis also had increased prevalence of irritable bowel syndrome (IBS), CPP, fibromyalgia, and vulvodynia (p < 0.05). On multivariate analysis, non-low BC (OR 4.53, CI 1.004-20.42, p = 0.049), CPP (OR 1.84, CI 1.04-3.24, p = 0.04), and fibromyalgia (OR 1.80, CI 1.03-3.14, p < 0.04) were significantly associated with a diagnosis of endometriosis. CONCLUSIONS: Patients with IC/BPS and co-occurring endometriosis were significantly more likely to carry a non-bladder centric IC/BPS phenotype as well as several comorbid, systemic pain diagnoses. This study characterizes features of a target IC/BPS phenotype that could potentially benefit from endometriosis and systemic pain syndrome screening.

MDSCs drive the process of endometriosis by enhancing angiogenesis and are a new potential therapeutic target.

Endometriosis affects women of reproductive age via unclear immunological mechanism(s). Myeloid-derived suppressor cells (MDSCs) are a heterogeneous group of myeloid cells with potent immunosuppressive and angiogenic properties. Here, we found MDSCs significantly increased in the peripheral blood of patients with endometriosis and in the peritoneal cavity of a mouse model of surgically induced endometriosis. Majority of MDSCs were granulocytic, produced ROS, and arginase, and suppressed T-cell proliferation. Depletion of MDSCs by antiGr-1 antibody dramatically suppressed development of endometrial lesions in mice. The chemokines CXCL1, 2, and 5 were expressed at sites of lesion while MDSCs expressed CXCR-2. These CXC-chemokines promoted MDSC migration toward endometriotic implants both in vitro and in vivo. Also, CXCR2-deficient mice show significantly decreased MDSC induction, endometrial lesions, and angiogenesis. Importantly, adoptive transfer of MDSCs into CXCR2-KO mice restored endometriotic growth and angiogenesis. Together, this study demonstrates that MDSCs play a role in the pathogenesis of endometriosis and identifies a novel CXC-chemokine and receptor for the recruitment of MDSCs, thereby providing a potential target for endometriosis treatment.

RBPJ mediates uterine repair in the mouse and is reduced in women with recurrent pregnancy loss.

Unexplained recurrent pregnancy loss (uRPL) is associated with repeated embryo loss and endometrial repair with elevated endometrial expression of inflammatory cytokines, including IFN-γ. Notch signaling through its transcription factor recombination signal binding protein Jκ (RBPJ) regulates mechanisms including the immune response and repair after tissue injury. Initially, null mutation of RBPJ in the mouse uterus ( Pgrcre/+Rbpjf/f; Rbpj c-KO) results in subfertility, but we have found that these mice become infertile after pregnancy as a result of dysfunctional postpartum uterine repair, including delayed endometrial epithelial and myometrial regeneration. RNA sequencing of postpartum uterine repair sites revealed global up-regulation of inflammatory pathways, including IFN signaling. Consistent with elevated IFN-γ, macrophages were recruited and polarized toward an M1-cytotoxic phenotype, which is associated with preventing repair and promoting further tissue injury. Through embryo transfer experiments, we show that dysfunctional postpartum repair directly impairs future embryo implantation in Rbpj c-KO mice. Last, we clinically correlated our findings from the Rbpj c-KO mouse in women diagnosed with uRPL. Reduced RBPJ in women with uRPL was associated with increased levels of IFN-γ. The data, taken together, indicate that RBPJ regulates inflammation during endometrial repair, which is essential for future pregnancy potential, and its dysregulation may serve as an unidentified contributor to uRPL in women.-Strug, M. R., Su, R.-W., Kim, T. H., Mauriello, A., Ticconi, C., Lessey, B. A., Young, S. L., Lim, J. M., Jeong, J.-W., Fazleabas, A. T. RBPJ mediates uterine repair in the mouse and is reduced in women with recurrent pregnancy loss.

Medical or surgical treatment before embryo transfer improves outcomes in women with abnormal endometrial BCL6 expression.

PURPOSE: To evaluate the effect of medical or surgical treatment prior to embryo transfer in women with elevated endometrial BCL6 expression and suspected endometriosis in a prospective, cohort study design at a university-associated infertility clinic. METHODS: All subjects had at least 1 year of unexplained infertility (UI) and each prospectively underwent endometrial biopsy and immunostaining for the oncogene BCL6, prior to embryo transfer during an assisted reproductive technology (ART) cycle. To be included, subjects had to have an abnormal BCL6 result, defined by elevated HSCORE ≥ 1.4. Women that were pre-treated with laparoscopy or medical suppression with GnRH agonist (depot leuprolide acetate; Lupron®, Abbvie, Inc., Chicago, IL) for 2 months were compared to a group that went untreated (controls). Endpoints included implantation rate (IR), clinical pregnancy rate (CPR), and live birth rate (LBR), and as well as cycle characteristics. Miscarriage rate were also compared between treatment and control group. RESULTS: Women in each group had similar characteristics. Those treated by medical suppression and those undergoing laparoscopy for endometriosis had a significantly higher LBR, (5/10; 50%; 95%CI 23.7 to 76.3%) and (11/21; 52.4%; 95%CI 32.4 to 71.7), respectively, compared to controls (4/54; 7.4%; 95%CI 2.9 to 17.6). An absolute benefit of 44.2% (16/31; 95%CI 24.6 to 61.2) and a number need to treat of 3 for those that received treatment (medical suppression and laparoscopy), compared to no treatment. Miscarriages were significantly more common in the control group. CONCLUSIONS: Women with suspected endometriosis and aberrant endometrial BCL6 expression have worse reproductive outcomes following embryo transfer, including a high miscarriage rate, poor IR, and low LBR and CPR compared to cycles pre-treated with medical and surgical management.

ARID1A Is Essential for Endometrial Function during Early Pregnancy.

AT-rich interactive domain 1A gene (ARID1A) loss is a frequent event in endometriosis-associated ovarian carcinomas. Endometriosis is a disease in which tissue that normally grows inside the uterus grows outside the uterus, and 50% of women with endometriosis are infertile. ARID1A protein levels were significantly lower in the eutopic endometrium of women with endometriosis compared to women without endometriosis. However, an understanding of the physiological effects of ARID1A loss remains quite poor, and the function of Arid1a in the female reproductive tract has remained elusive. In order to understand the role of Arid1a in the uterus, we have generated mice with conditional ablation of Arid1a in the PGR positive cells (Pgrcre/+Arid1af/f; Arid1ad/d). Ovarian function and uterine development of Arid1ad/d mice were normal. However, Arid1ad/d mice were sterile due to defective embryo implantation and decidualization. The epithelial proliferation was significantly increased in Arid1ad/d mice compared to control mice. Enhanced epithelial estrogen activity and reduced epithelial PGR expression, which impedes maturation of the receptive uterus, was observed in Arid1ad/d mice at the peri-implantation period. The microarray analysis revealed that ARID1A represses the genes related to cell cycle and DNA replication. We showed that ARID1A positively regulates Klf15 expression with PGR to inhibit epithelial proliferation at peri-implantation. Our results suggest that Arid1a has a critical role in modulating epithelial proliferation which is a critical requisite for fertility. This finding provides a new signaling pathway for steroid hormone regulation in female reproductive biology and furthers our understanding of the molecular mechanisms that underlie dysregulation of hormonal signaling in human reproductive disorders such as endometriosis.

Effect of randomized serum progesterone concentration on secretory endometrial histologic development and gene expression.

STUDY QUESTION: What doses of secretory phase progesterone (P) in women are associated with altered endometrial structure and/or function? SUMMARY ANSWER: Consistently delayed histological maturation was seen at the lowest tested daily P dose (2.5 mg), whereas consistently altered functional response, as reflected by microarray analysis of gene expression was seen at both the 5 and 2.5 mg doses. WHAT IS KNOWN ALREADY: Progesterone is absolutely required for normal embryo implantation and pregnancy survival. Progesterone supplementation is beneficial in ART cycles. STUDY DESIGN, SIZE, DURATION: In this case-control experimental trial, 46 healthy young female volunteers (age 19-34) underwent a single modeled endometrial cycle after GnRH down-regulation or monitored in natural cycles. PARTICIPANTS/MATERIALS, SETTING, METHODS: In a university hospital, modeled cycles were obtained by GnRH agonist down-regulation, transdermal estradiol (E2) (0.2 mg/d), and daily injections of P in oil for 10 days: 2.5 mg (n = 6), 5 mg (n = 6), 10 mg (n = 12) or 40 mg (n = 12), after the 10th day of E2. Ten healthy, ovulatory women were used as controls. Endometrial biopsies were obtained on the 10th day of P exposure, or urinary LH surge (in controls). Analysis included histological dating, serum progesterone levels, microarray analysis of the whole genome, RT-PCR, western blot and comparison with the GEO database. MAIN RESULTS AND THE ROLE OF CHANCE: In endometrial biopsies, a morphological delay appears in the 2.5 mg/day of P group. Higher sub-physiological levels of P (≥5 mg/day) resulted in normal histology, but aberrant gene expression. P levels required for consistent histological delay were lower than those in all ovulatory women. Gene expression abnormalities occurred at higher sub-physiological P concentrations, without a change in histology, a functional-morphological disassociation. The expression of some endometrial receptivity-associated genes appeared multiphasic, with peak or nadir of mean or median expression levels between the lowest and highest doses, suggesting sustained supraphysiological doses seen in ART treatment cycles may not be optimal. LARGE SCALE DATA: GEO DataSets ID: 200056980; GSE 56980. LIMITATIONS, REASONS FOR CAUTION: These results were obtained in fertile women, who may respond differently from infertile subjects. WIDER IMPLICATIONS OF THE FINDINGS: The dose of P required for normal endometrial structure (5 mg/day) corresponds to a P concentration well below that seen in ovulatory women, suggesting that persistently delayed mid-secretory histology cannot be solely due to inadequate P concentrations in an ovulatory cycle. Endometrial gene expression is differentially regulated by different doses of progesterone. The apparent multiphasic response of some genes to P dose suggests the possibility that P concentration kinetics may play a role in normal endometrial preparation for receptivity. These findings strongly confirm that histologic development is not a reliable measure of endometrial P action. STUDY FUNDING/COMPETING INTEREST(S): Supported by The Eunice Kennedy Shriver National Institute for Child Health and Disease, National Institute of Health, USA (NICHD/NIH) (R01HD067721 and U54HD30476; SLY and BAL) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) 240239/2012-1 (RFS). All authors have no competing interests.

Luteal phase HCG support for unexplained recurrent pregnancy loss - a low hanging fruit?

Recurrent pregnancy loss (RPL) is defined by two or more failed pregnancies and accounts for only 1-5% of pregnancy failures. Treatment options for unexplained RPL (uRPL) are limited. Previous studies suggest a link between delayed implantation and pregnancy loss. Based on this, a timely signal for rescue of the corpus luteum (CL) using human chorionic gonadotrophin (HCG) could improve outcomes in women with uRPL. This retrospective cohort study included 98 subjects with uRPL: 45 underwent 135 monitored cycles without HCG support; and 53 underwent 142 cycles with a single mid-luteal HCG injection. Based on Log-rank Mantel-Cox survival curves, miscarriage rate and time to pregnancy decreased in the HCG group (P = 0.0005). Women receiving luteal HCG support had an increased chance of an ongoing pregnancy compared with those not receiving it (RR = 2.4; 95% CI 1.4-3.6; number need to treat (NNT) = 7; 95% CI 4-18). Subjects receiving HCG support had a significant absolute risk reduction (ARR) of miscarriage (P < 0.001; ARR = 11.5%; 95% CI 3.6-19.5; NNT = 9(5-27). These data suggest restoration of synchrony and CL support improves outcomes in women with RPL. Further randomized controlled trials of luteal-phase HCG in women with RPL appears warranted.

IL-17A Contributes to the Pathogenesis of Endometriosis by Triggering Proinflammatory Cytokines and Angiogenic Growth Factors.

Endometriosis is a chronic, inflammatory disease characterized by the growth of endometrial tissue in aberrant locations outside the uterus. Neoangiogenesis or establishment of new blood supply is one of the fundamental requirements of endometriotic lesion survival in the peritoneal cavity. IL-17A is emerging as a potent angiogenic and proinflammatory cytokine involved in the pathophysiology of several chronic inflammatory diseases such as rheumatoid arthritis and psoriasis. However, sparse information is available in the context of endometriosis. In this study, we demonstrate the potential importance of IL-17A in the pathogenesis and pathophysiology of endometriosis. The data show a differential expression of IL-17A in human ectopic endometriotic lesions and matched eutopic endometrium from women with endometriosis. Importantly, surgical removal of lesions resulted in significantly reduced plasma IL-17A concentrations. Immunohistochemistry revealed localization of IL-17A primarily in the stroma of matched ectopic and eutopic tissue samples. In vitro stimulation of endometrial epithelial carcinoma cells, Ishikawa cells, and HUVECs with IL-17A revealed significant increase in angiogenic (vascular endothelial growth factor and IL-8), proinflammatory (IL-6 and IL-1ß), and chemotactic cytokines (G-CSF, CXCL12, CXCL1, and CX3CL1). Furthermore, IL-17A promoted tubulogenesis of HUVECs plated on Matrigel in a dose-dependent manner. Thus, we provide the first evidence, to our knowledge, that endometriotic lesions produce IL-17A and that the removal of the lesion via laparoscopic surgery leads to the significant reduction in the systemic levels of IL-17A. Taken together, our data show a likely important role of IL-17A in promoting angiogenesis and proinflammatory environment in the peritoneal cavity for the establishment and maintenance of endometriosis lesions.