Bruce A. Lessey

Pregnancy requires a supportive uterine environment facilitated by steroid hormone–regulated differentiation of endometrial stromal fibroblasts into decidual cells and tight control of inflammation. Serum response factor (SRF) is a widely expressed transcription factor essential for mesenchymal cell growth and differentiation with noted roles in hormonal regulation of muscle tissues but little characterization in reproductive organs. Here, we reveal that endometrial SRF is dysregulated in human endometriosis and is critical for female reproductive success in mice through regulation of endometrial stromal and epithelial cells. Immunohistochemical analysis identified decreased endometrial SRF expression in infertile endometriosis patient tissues. RNAi-based SRF knockdown in human endometrial stromal cells resulted in disrupted cytoskeletal structure, viability, and decidual response. Conditional Srf knockout mice (Srf ^d/d ) generated using the Pgr ^Cre were infertile with implantation failure, lack of decidual response, and development of severe endometrial fibrosis. Single-cell RNA sequencing identified dramatic changes in Srf ^d/d endometrial cell populations including large-scale myeloid immune cell infiltration. Srf ^d/d stromal fibroblasts displayed aberrant cytoskeletal and extracellular matrix gene expression and downregulation of genes important for decidual growth response. Srf -deficient epithelial cells displayed the most prominent dysregulation, strongly overexpressing estrogenic innate inflammatory genes including C3 and Lcn2 and fibrogenic genes Mmp7 and Fbln1 , all of which paralleled patterns in human endometriosis patient data that we identified through comparative analysis using a published single-cell atlas. These results demonstrate the profound impact of SRF on endometrial homeostasis with relevance to human endometriosis–related infertility.

Ovarian steroid hormones—estrogen and progesterone—play a central role in regulating epithelial‐stromal interactions in the uterus. These interactions are critical for uterine function, including endometrial receptivity, implantation, and decidualization. These interactions involve complex signaling crosstalk between the uterine epithelium and the underlying stroma, with dynamic cell population‐specific roles. Mitogen‐inducible gene 6 (MIG‐6) is a key mediator of progesterone signaling in the endometrium, and loss of MIG‐6 results in implantation failure due to a non‐receptive endometrium. To investigate whether MIG‐6 deficiency disrupts the complex and dynamic cell population‐specific roles of uterine cells, we performed single‐cell RNA sequencing to characterize the cellular composition and functional alterations in the non‐receptive endometrium of uterine‐specific Mig‐6 knockout ( Pgr ^cre/+ Mig‐6 ^f/f ; Mig‐6 ^d/d ) mice. Compared to control mice, Mig‐6 ^d/d mice exhibited distinct gene expression patterns in both endometrial epithelial and stromal cells. We identified dysregulation of Egr1 in the non‐receptive endometrium along with attenuated expression of Foxa2 and Cyp26A1 in epithelial cells of Mig‐6 ^d/d mice. Notably, LRP2 expression was diminished in epithelial cells of both Mig‐6 ^d/d mice and infertile women with endometriosis. These findings demonstrate that MIG‐6 is a critical progesterone receptor (PGR) mediator that maintains endometrial epithelial and stromal cell function essential to uterine receptivity. Our study advances the understanding of the molecular pathways underlying endometrial receptivity and provides a foundation for therapeutic strategies to improve pregnancy outcomes.

Endometriosis is a chronic disorder in which endometrial-like tissue presents outside the uterus. Patients with endometriosis have been shown to exhibit aberrant immune responses within the lesion microenvironment and in circulation which contribute to the development of endometriosis. Thymic stromal lymphopoietin (TSLP) is an alarmin involved in cell proliferation and the induction of T helper 2 (Th2) inflammation in various diseases, such as asthma, atopic dermatitis, and pancreatic and breast cancer. Recent studies have detected TSLP within endometriotic lesions and shown that its concentrations are elevated in the peritoneal fluid of patients compared with control subjects. However, its role in disease pathophysiology remains unclear. Here, we compared TSLP messenger RNA and protein expression between patient eutopic endometrium, endometriotic lesions, and control endometrial samples. We also assessed its effect on the proliferation and apoptosis of human endometriosis-representative cell lines, as well as on lesion development and inflammation in a mouse model of the disease. We demonstrated that TSLP expression was elevated in the stroma of patient endometriotic lesions compared with control endometrial samples. In cell lines, TSLP treatment reduced the apoptosis of endometrial stromal cells and promoted the proliferation of THP-1 cells. In mice induced with endometriosis, TSLP treatment induced a Th2 immune response within the lesion microenvironment, and led to TSLP receptor modulation in macrophages, dendritic cells, and CD4+ T cells. Furthermore, treatment increased murine endometriotic lesion proliferation. Overall, these results suggest that TSLP modulates the endometriotic lesion microenvironment and promotes a Th2 immune response that could support lesion development.

Purpose: To evaluate the effect of endometrial scratching on in vitro fertilization (IVF) success rates in women undergoing frozen embryo transfer with preimplantation genetic testing for aneuploidy (PGT-A). Methods: Biopsy was performed at oocyte retrieval in cases and compared to those who did not undergo scratching (controls). Endpoints included pregnancy loss, total pregnancy, clinical pregnancy rate (CPR), ongoing pregnancy rate/live birth rate (OPR), and the incidence of chronic endometritis (CE). Results: Women in case and control groups had similar characteristics, such as age, body mass index, and parity. Overall total pregnancy rate was 82.43% following single euploid embryo transfer in cases compared to 85.7% in controls (p = 0.57; OR = 1.27; 95% CI = 0.565 to 2.78). CPR was 73.3% and 76.2% (p = 0.62; OR = 1.18; 95% CI = 0.594 to 2.29), and OPR between groups was 64.8% and 72.3% (p = 0.28; OR = 1.42; 95% CI = 0.74 to 2.7). First trimester loss rate was similar at 16.9%% and 15.5%%, respectively (p = 0.82). Subjects who underwent scratching closer to their frozen embryo transfer (≤ 3 months) had a similar overall pregnancy rate to those with a longer delay between retrieval and frozen embryo transfer (> 3 months 81.4% vs.87.5%; p = 0.56). CE incidence was 10.8% (8 out of 74; 95% CI = 5.5% to 19.9%). The odds of having a second trimester loss were significantly higher in the scratch group compared to controls (p = 0.03; OR = infinity; 95% CI = 1.3 to infinity). Conclusions: Endometrial scratching before frozen euploid embryo transfer did not influence pregnancy outcome but increased the chances of second trimester loss. CE was a frequent finding in those undergoing endometrial scratching at the time of retrieval.

Unexplained euploid embryo transfer failure (UEETF) is a frustrating and unanswered conundrum accounting for 30 to 50% of failures in in vitro fertilization using preimplantation genetic testing for aneuploidy (PGT-A). Endometriosis is thought by many to account for most of such losses and menstrual suppression or surgery prior to the next transfer has been reported to be beneficial. In this study, we performed endometrial biopsy in a subset of women with UEETF, testing for the oncogene BCL6 and the histone deacetylase SIRT1. We compared 205 PGT-A cycles outcomes and provide those results following treatment with GnRH agonist versus controls (no treatment). Based on these and previous promising results, we next performed a pilot randomized controlled trial comparing the orally active GnRH antagonist, elagolix, to oral contraceptive pill (OCP) suppression for 2 months before the next euploid embryo transfer, and monitored inflammation and miRNA expression in blood, before and after treatment. These studies support a role for endometriosis in UEETF and suggest that medical suppression of suspected disease with GnRH antagonist prior to the next transfer could improve success rates and address underlying inflammatory and epigenetic changes associated with UEETF.

Endometriosis is a chronic inflammatory disease where endometrial-like tissue grows ectopically, resulting in pelvic pain and infertility. Interleukin (IL)-23 is established as a key contributor in the development and differentiation of a subset of T cells known as T-helper 17 (T _H 17) cells, driving T _H 17 cells towards a pathogenic profile. In a variety of inflammatory and autoimmune disorders, such as psoriasis and rheumatoid arthritis, T _H 17 cells secrete proinflammatory cytokines including IL-17, contributing to the disease pathophysiology. Our studies and others have implicated IL-17 and T _H 17 cell dysregulation in endometriosis, which is associated with disease severity. Here we address whether IL-23 driven T _H 17 cells contribute to the cardinal features of lesion proliferation, vascularization, and inflammation in endometriosis using patient samples, representative cell lines, and our established mouse model of endometriosis. Our results indicate significantly dysregulated expression of key genes in the IL-23/T _H 17 axis in patient ectopic and eutopic endometrial samples and increased IL-23 protein in patient plasma compared to healthy controls. In-vitro studies using primary human T _H cells determined that IL-23 cocktail treatment significantly increased the frequency of pathogenic T _H 17 cells. Similarly, treatment with recombinant human (rh)IL-23 on cell lines (12Z, EECC, HUVEC, and hESC) representative of the endometriotic lesion microenvironment led to a significant increase in cytokines and growth factors known to play a role in lesion establishment and maintenance. In a syngeneic mouse model of endometriosis, treatment with recombinant mouse (rm)IL-23 led to significant alterations in numbers of myeloid and T cell subsets in peritoneal fluid and significantly increased numbers of giant cells within the lesion. Endometriotic lesions from rmIL-23 mice did not reveal significant alterations in proliferation and vascularization, although trends of increased proliferation and vascularization were observed. Collectively, these findings provide insights into the impact of the IL-23/T _H 17 axis on local immune dysfunction and broadly on the pathophysiology of endometriosis.

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.

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.