Ryan M. Marquardt

The oviduct (fallopian tube) houses the site of fertilization and transports embryos to the uterus for on‐time implantation. Hormone‐regulated contractions of the oviductal smooth muscle (myosalpinx) are thought to participate in this process. Serum response factor (SRF) is a widely expressed transcription factor shown to facilitate smooth muscle development as well as murine oviductal function. Our past work in the uterus has suggested that SRF transcriptional activity is associated with the master pregnancy regulator progesterone receptor (PGR). We therefore hypothesized that SRF and PGR work together to influence oviductal gene expression. Ablation of Srf in the Pgr ‐expressing cells of the mouse oviduct resulted in extensive transcriptomic consequences. Of 1670 differentially expressed genes in the Srf ‐deficient oviduct, 306 also contained an SRF binding region within 10 kb of the TSS, including cytoskeletal, contractile, and extracellular matrix genes important for muscle structure and function. Histological analysis confirmed disruption of the smooth muscle layer in the Srf ‐deficient oviduct, and functional contractility analyses demonstrated that contractile activity was deficient and irregular. Comparative transcriptomic analysis of oviducts deficient in smooth muscle Pgr expression uncovered parallel downregulation of extracellular matrix organization pathways and smooth muscle contraction processes. Integration of SRF and PGR cistromic analyses identified a set of structural and contractile smooth muscle genes co‐occupied and co‐regulated by SRF and PGR including Myh11 , Actg2 , and Des . Our findings highlight a prominent regulatory mechanism for SRF and PGR in the oviduct, disruption of which contributes to impaired myosalpinx function and infertility.

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.

Estrogen is a critical regulator of endometrial health. Aberrant estrogen stimulation can result in infertility, endometrial cancer, and endometriosis. Here, we identified Zinc Finger MIZ-Type Containing 1 ( Zmiz1 ) as a coregulator of uterine estrogen signaling. ZMIZ1 is colocalized with an estrogen receptor α–binding (ESR1-binding) super enhancer. ZMIZ1 mutations are found in endometrial cancer and its RNA levels trend toward reduction in endometrium of patients with endometriosis. ZMIZ1 is dynamically expressed in human endometrial tissues during the menstrual cycle. Disrupting ZMIZ1 in cultured human endometrial stromal cells resulted in impaired cell proliferation and decidual differentiation. Ablation of Zmiz1 using the Pgr ^Cre mouse (Zmiz1 ^d/d ) resulted in infertility and accelerated age-dependent uterine fibrosis. Zmiz1 ^d/d mice showed reduced ovulation and progesterone levels while maintaining normal serum prolactin during pregnancy. Uteri of Zmiz1 ^d/d mice were unable to undergo a hormonally induced decidual response, had decreased expression of stromal progesterone receptor (PGR) and decreased stromal and epithelial cell proliferation. Analysis of the transcriptome of Zmiz1 ^d/d mouse uteri showed decreased E2F, CCNA2, and FOXM1 signaling. Challenging ovariectomized Zmiz1 ^d/d mice with estrogen resulted in a decreased amplitude of some estrogen-regulated gene responses. Our findings demonstrate the importance of ZMIZ1 as an ESR1 coregulator in uterine biology and pathology.

More than 60% of pregnancy losses occur during the first trimester, highlighting the need to understand the role of the oviduct in early pregnancy. In this study, we conditionally ablated the classical progesterone receptor ( Pgr ) in oviductal epithelial cells, called the Pgr ^d/d mouse model. We found that 40% of embryos collected from Pgr ^d/d females were nonviable or developmentally delayed, indicating that epithelial PGR expression is crucial for embryonic development. Single-cell RNA sequencing revealed up-regulation of proinflammatory genes, including interleukin-22 (IL-22), in the epithelial cells of Pgr ^d/d females. Pharmacological inhibition of inflammation using nonsteroidal anti-inflammatory drugs significantly reduced IL-22 levels in the oviducts and rescued embryonic developmental rates in Pgr ^d/d females. Coculture of wild-type zygotes with IL-22 significantly decreased the number of expanded blastocysts. Our findings suggest that progesterone signaling is vital for immunoregulation and normal preimplantation development, potentially providing insights for developing diagnostic tools and therapeutic strategies to address pregnancy failures.

Over 75% of failed pregnancies involve implantation defects. Growth factor receptor-bound protein 2 (GRB2) is an adaptor protein involved in signal transduction and cell communication. Here we show that the expression of GRB2 protein is lower in endometrium of infertile women with endometriosis compared to controls. Our mouse endometriosis model revealed that endometriosis development results to GRB2 loss in the eutopic endometrium. To understand the role of GRB2 in the uterus, we generated mice with conditional ablation of Grb2 in the Pgr positive cells (Grb2^d/d). Grb2^d/d mice were infertile due to implantation failure. Although ovarian functions were normal, Grb2^d/d mice had a non-receptive endometrium due to progesterone resistance and dysregulation of steroid hormone and FOXA2 signaling pathways. Furthermore, our results were supported by findings of GRB2 attenuation in primary human endometrial stromal cells from women with endometriosis. Our results demonstrate that GRB2 is critical for endometrial receptivity and decidualization.

Estrogen and progesterone, acting through their cognate receptors the estrogen receptor α (ERα) and the progesterone receptor (PR) respectively, regulate uterine biology. Using rapid immunoprecipitation and mass spectrometry (RIME) and co-immunoprecipitation, we identified TRIM28 (Tripartite motif containing 28) as a protein which complexes with ERα and PR in the regulation of uterine function. Impairment of TRIM28 expression results in the inability of the uterus to support early pregnancy through altered PR and ERα action in the uterine epithelium and stroma by suppressing PR and ERα chromatin binding. Furthermore, TRIM28 ablation in PR-expressing uterine cells results in the enrichment of a subset of TRIM28 positive and PR negative pericytes and epithelial cells with progenitor potential. In summary, our study reveals the important roles of TRIM28 in regulating endometrial cell composition and function in women, and also implies its critical functions in other hormone regulated systems.

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.

Endometriosis is a condition of the female reproductive tract characterized by endometrium-like tissue growing outside the uterus. Though it is a common cause of pelvic pain and infertility, there is currently no reliable noninvasive method to diagnose the presence of endometriosis without surgery, and the pathophysiological mechanisms that lead to the occurrence of symptoms require further inquiry. Due to patient heterogeneity and delayed diagnosis, animal models are commonly used to study the development of endometriosis, but these are costly due to the large number of animals needed to test various treatments and experimental conditions at multiple endpoints. Here, we describe a method for synthesis of multimodal imaging gold-fluorescein isothiocyanate (FITC) nanoparticles with preclinical application via induction of nanoparticle-labeled endometriosis-like lesions in mice. Labeling donor endometrial tissue fragments with gold-FITC nanoparticles prior to induction of endometriosis in recipients enables in vivo detection of the gold-labeled lesions with photoacoustic imaging. The same imaging method can be used to visualize embryos noninvasively in pregnant mice. Furthermore, the conjugated FITC dye on the gold nanoparticles allows easy isolation of labeled lesion tissue under a fluorescence dissection microscope. After dissection, the presence of gold-FITC nanoparticles and endometrium-like histology of lesions can be verified through fluorescence imaging, gold enhancement, and immunostaining. This method for in vivo imaging of endometriosis-like lesions and fluorescence-guided dissection will permit new experimental possibilities for the longitudinal study of endometriosis development and progression as well as endometriosis-related infertility.

A growing body of work suggests epigenetic dysregulation contributes to endometriosis pathophysiology and female infertility. The chromatin remodeling complex subunit AT-rich interaction domain 1A (ARID1A) must be properly expressed to maintain normal uterine function. Endometrial epithelial ARID1A is indispensable for pregnancy establishment in mice through regulation of endometrial gland function; however, ARID1A expression is decreased in infertile women with endometriosis. We hypothesized that ARID1A performs critical operations in the endometrial epithelium necessary for fertility besides maintaining gland function. To identify alterations in uterine gene expression resulting from loss of epithelial ARID1A, we performed RNA-sequencing analysis on pre-implantation uteri from Ltf^iCre/+Arid1a^f/f and control mice. Differential expression analysis identified 4181 differentially expressed genes enriched for immune-related ingenuity canonical pathways including agranulocyte adhesion and diapedesis and natural killer cell signaling. RT-qPCR confirmed an increase in pro-inflammatory cytokine and macrophage-related gene expression but a decrease in natural killer cell signaling. Immunostaining confirmed a uterus-specific increase in macrophage infiltration. Flow cytometry delineated an increase in inflammatory macrophages and a decrease in uterine dendritic cells in Ltf^iCre/+Arid1a^f/f uteri. These findings demonstrate a role for endometrial epithelial ARID1A in suppressing inflammation and maintaining uterine immune homeostasis, which are required for successful pregnancy and gynecological health.

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 (Pgr^cre/+Mig-6^f/f; Mig-6^d/d). To investigate the effect of ERBB2 targeting on endometrial progesterone resistance, fertility, and endometriosis, we introduce Erbb2 ablation in Mig-6^d/d mice (Mig-6^d/dErbb2^d/d mice). The additional knockout of Erbb2 rescues all phenotypes seen in Mig-6^d/d mice. Transcriptomic analysis shows that genes differentially expressed in Mig-6^d/d mice revert to their normal expression in Mig-6^d/dErbb2^d/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.