OBJECTIVE:To characterize the expression of adhesion G protein-coupled receptors (ADGR) in the human endometrium and early mouse pregnancy. DESIGN:An in silico analysis was performed using a retrospective data set comprised endometrial samples across normo-ovulatory menstrual cycles. Gene expression was then validated using quantitative reverse transcription polymerase chain reaction and mRNA sequencing (mRNA-seq) in prospectively collected endometrial biopsies in the periovulatory and midsecretory stages of natural cycles. Gene expression was also investigated under ovarian stimulation (OS) conditions using mRNA-seq. Early pregnancy mouse models were used to investigate whether trends of dynamic ADGR expression are also conserved in the mouse. SUBJECTS:Twenty-four women aged 21-42 years. EXPOSURE:Ovulatory menstrual cycle or OS cycle. MAIN OUTCOME MEASURES:Gene expression in endometrial biopsies and pregnant mouse uterus. RESULTS:Fifteen women, aged 21-33 years, were recruited in natural cycles during the proliferative phase (cycle days 10-13; n = 4), periovulatory (luteinizing hormone + 12-24 hours; n = 6) period, and midsecretory (luteinizing hormone + 8-9 days; n = 5) phase. Nine women aged 31-42 years old undergoing in vitro fertilization (without fresh embryo transfer) or oocyte cryopreservation using a gonadotropin releasing hormone antagonist protocol were recruited for the OS cohort in either the periovulatory phase (human chorionic gonadotropin + 2; n = 5) or midsecretory phase (human chorionic gonadotropin + 9; n = 4). The in silico analysis revealed dynamic expression for many ADGRs across the menstrual cycle. Differential gene expression was also seen in the prospective analysis within the menstrual cycle phases and between natural cycle and OS conditions. Within early mouse pregnancy, expression was also found to be altered across several Adgr subfamilies. CONCLUSION:The differential gene expression observed between the proliferative and secretory phases of the menstrual cycle, along with changes in expression seen in OS and early mouse pregnancy suggest that ADGR expression is hormonally regulated by estradiol and progesterone.

At the maternal-fetal interface, tightly regulated levels of retinoic acid (RA), the physiologically active metabolite of vitamin A, are required for embryo implantation and pregnancy success. Herein, we utilize mouse models, primary human cells, and pharmacological tools to demonstrate how depletion of RA signaling via RA receptor (RAR) disrupts implantation and progression of early pregnancy. To inhibit RAR signaling during early pregnancy, BMS493, an inverse pan-RAR agonist that prevents RA-induced differentiation, was administered to pregnant mice during the peri-implantation period. Attenuation of RA/RAR signaling prior to embryo implantation results in implantation failure, whereas attenuation of RA/RAR signaling after embryo implantation disrupts the post-implantation decidual vasculature and results in pregnancy failure by mid-gestation. To inhibit RAR signaling during human endometrial stromal cell (HESC) decidualization, primary HESCs and decidualized primary HESCs were transfected with silencing RNA specific for human RARA. Inhibition of RA/RARA signaling prevents initiation of HESC decidualization, but not maintenance of the decidualized HESC phenotype. These data show that RA/RAR signaling is required for maintenance of the decidual vasculature that supports early pregnancy in mice, and distinct RAR signaling is required for initiation, but not maintenance of primary HESC decidualization in vitro.

Infertility affects one in six couples, with in vitro fertilization (IVF) offering many the chance of conception. Compared to the solitary oocyte produced during the natural menstrual cycle, the supraphysiological ovarian stimulation needed to produce multiple oocytes during IVF results in a dysfunctional luteal phase that can be insufficient to support implantation and maintain pregnancy. Consequently, hormonal supplementation with luteal phase support, principally exogenous progesterone, is used to optimize pregnancy rates; however, luteal phase support remains largely 'black-box' with insufficient clarity regarding the optimal timing, dosing, route and duration of treatment. Herein, we review the evidence on luteal phase support and highlight remaining uncertainties and future research directions. Specifically, we outline the physiological luteal phase, which is regulated by progesterone from the corpus luteum, and evaluate how it is altered by the supraphysiological ovarian stimulation used during IVF. Additionally, we describe the effects of the hormonal triggers used to mature oocytes on the degree of luteal phase support required. We explain the histological transformation of the endometrium during the luteal phase and evaluate markers of endometrial receptivity that attempt to identify the 'window of implantation'. We also cover progesterone receptor signalling, circulating progesterone levels associated with implantation, and the pharmacokinetics of available progesterone formulations to inform the design of luteal phase support regimens.

Abstract Disclosure: Q. Zhao: None. P. Timmins: None. M. Saad-Naguib: None. C. Samuels: None. R. Loia: None. T. Wu: None. A. Chemerinski: None. S.S. Morelli: None. A.V. Babwah: None. N. Douglas: None. Ovarian stimulation (OS) is employed to recruit multiple dominant follicles and mature oocytes for in vitro fertilization (IVF). OS followed by a fresh embryo transfer (ET) is associated with decreased pregnancy rates. By better understanding the processes which prepare the endometrium for implantation, we hope to improve outcomes following OS. To understand the impact of OS on the endometrium, we performed an in-depth comparative analysis of the human endometrium under natural vs OS conditions during the periovulatory (PO) and mid-secretory (MSE) phase of the menstrual cycle. Our data showed that OS, which significantly elevated serum E2 and P4 levels, was associated with dyssynchronous glandular-stromal development, increased glandular epithelial volume, and increased macrophage and B cell abundance. We then performed bulk and single-cell RNA sequencing (RNA-seq) to identify the molecular pathways and cell-type specific transcriptomes associated with these changes. We found that OS significantly changed the expression of numerous genes in the retinoic acid (RA) signaling pathway, a pathway known to be crucial for a successful pregnancy. Our data showed that OS affected the fibroblast and epithelial cell-specific expression of genes that regulate RA signaling, with increased expression of RA synthesis genes and RA target genes, suggesting that OS increases endometrial RA signaling. RA is the metabolically active form of vitamin A which regulates the transcription of over 500 genes and adversely affects pregnancy when deficient or in excess. Thus, these findings led us to hypothesize that OS disrupts normal endometrial RA signaling and thereby endometrial differentiation that is required for implantation. We investigated whether RA is required for endometrial differentiation in humans and the establishment of pregnancy in mice. Using siRNAs against the RA receptor RARA, we observed that reduced expression of RARA mRNA in human endometrial stromal cells reduced their ability to undergo decidualization, an essential requirement for successful embryo implantation. We found that administration of BMS93, a pan RAR receptor antagonist, to pregnant dams prior to implantation from E0.5 or E2.5 until E7.5 resulted in pregnancy failure. Whereas BMS493 administration to pregnant dams from E4.5 to E7.5, resulted in pregnancies with reduced decidual vascular development in the anti-mesometrial region. Together, these data suggest that RA is required for the proper preparation of the endometrium for embryo implantation and loss of RA signaling in the endometrium, not the embryo, is a cause of pregnancy failure. Importantly, OS induced disruptions of RA signaling might contribute to reduced implantation rates in fresh ET, uncovering a novel pharmacologic target for improving pregnancy outcomes in fresh ET following OS. Presentation: Friday, June 16, 2023

Supplementary Data from β-Arrestin/Ral Signaling Regulates Lysophosphatidic Acid–Mediated Migration and Invasion of Human Breast Tumor Cells

Abstract Disclosure: M. Saad-Naguib: None. P. Timmins: None. L. George: None. C. Samuels: None. N. Douglas: None. A.V. Babwah: None. Every successful pregnancy relies on implantation of an embryo into a receptive uterine endometrium. While implantation failure accounts for 50% of pregnancy failures, it remains the least understood aspect of pregnancy. In the mid-secretory (MS) phase of the menstrual cycle, under the influence of progesterone and cAMP, fibroblasts decidualize, a critical requirement for implantation. Decidualization involves the differentiation of stromal fibroblasts into secretory epithelioid cells that contribute to the formation of the decidua, the support of the pregnancy prior to placentation. A defective decidua can lead to implantation failure, but if implantation occurs, it could compromise placenta formation and function. Several studies have demonstrated the importance of G protein-coupled receptors (GPCRs) in mouse endometrial receptivity and decidualization. GPCRs are transmembrane signaling molecules that are ubiquitously expressed in the body and regulate almost every cellular and physiological process. They are major pharmaceutical targets, with 35% of all the drugs approved by the US FDA targeting GPCRs. To identify novel GPCR regulators of embryo implantation, we conducted bulk RNA sequencing of the human endometrium at the periovulatory (PO) and mid-secretory (MS) phase in natural menstrual cycles in healthy women of reproductive age and observed that genes encoding 11 GPCRs, not previously reported to regulate implantation, were differentially expressed between these two time points. Among these was the gene encoding the G protein-coupled bile acid receptor 1 (GPBAR1), and this was upregulated 4.6-fold in the MS vs PO phase. Single cell RNA sequence analysis showed that within the human endometrium, GPBAR1 mRNA expression is mostly and strongly localized to stromal fibroblasts. Upon activation by secondary bile acids, GPBAR1 couples to Gαs leading to an increase in intracellular cAMP levels. We hypothesize that in the MS endometrium, upon ligand binding, GPBAR1 produces cAMP and induces human endometrial stromal cell (HESC) decidualization. This hypothesis is being tested in vitro using primary HESCs. Interestingly, in the pregnant mouse, GPBAR1 mRNA is barely expressed at the implantation site on E7.5, and therefore unlikely plays a role in stromal cell decidualization in the mouse. Whether Gpbar1 is expressed in the pregnant mouse endometrium prior to implantation and plays a role in regulating endometrial receptivity is being investigated. Overall, our studies have uncovered a GPCR, that to our knowledge, has not been previously described in the human endometrium. Our data show that the expression of this GPCR is upregulated at the time of endometrial stromal cell decidualization, and therefore it might regulate decidualization and/or be required for decidual function in women. These possibilities are being examined. Presentation: Friday, June 16, 2023

Nonalcoholic fatty liver disease (NAFLD), the most common liver disease, has become a silent worldwide pandemic. The incidence of NAFLD correlates with the rise in obesity, type 2 diabetes, and metabolic syndrome. A hallmark featureof NAFLD is excessive hepatic fat accumulation or steatosis, due to dysregulated hepatic fat metabolism, which can progress to nonalcoholic steatohepatitis (NASH), fibrosis, and cirrhosis. Currently, there are no approved pharmacotherapies to treat this disease. Here, we have found that activation of the kisspeptin 1 receptor (KISS1R) signaling pathway has therapeutic effects in NAFLD. Using high-fat diet–fed mice, we demonstrated that a deletion of hepatic Kiss1r exacerbated hepatic steatosis. In contrast, enhanced stimulation of KISS1R protected against steatosis in wild-type C57BL/6J mice and decreased fibrosis using a diet-induced mouse model of NASH. Mechanistically, we found that hepatic KISS1R signaling activates the master energy regulator, AMPK, to thereby decrease lipogenesis and progression to NASH. In patients with NAFLD and in high-fat diet–fed mice, hepatic KISS1/KISS1R expression and plasma kisspeptin levels were elevated, suggesting a compensatory mechanism to reduce triglyceride synthesis. These findings establish KISS1R as a therapeutic target to treat NASH.

Ovarian stimulation (OS) is associated with altered endometrial histological development and gene expression which may impact receptivity in fresh embryo transfers. We sought to determine the effect of OS on the expression of endometrial genes encoding adhesion G protein-coupled receptors (ADGRs). While ADGRG2 has been identified as a regulator of human endometrial decidualization and receptivity, less is known regarding the roles of other ADGRs in the human endometrium. We hypothesized that in a natural cycle (NC) ADGRs are expressed in the secretory endometrium but following OS their expression is altered.

Benign disorders of the human female reproductive system, such primary ovarian insufficiency and polycystic ovary syndrome are associated with infertility and recurrent miscarriage, as well as increased risk of adverse health outcomes, including cardiovascular disease and type 2 diabetes. For many of these conditions, the contributing molecular and cellular processes are poorly understood. The overarching similarities between mice and humans have rendered mouse models irreplaceable in understanding normal physiology and elucidating pathological processes that underlie disorders of the female reproductive system. The utilization of Cre-LoxP recombination technology, which allows for spatial and temporal control of gene expression, has identified the role of numerous genes in development of the female reproductive system and in processes, such as ovulation and endometrial decidualization, that are required for the establishment and maintenance of pregnancy in mammals. In this comprehensive review, we provide a detailed overview of Cre drivers with activity in the neuroendocrine-reproductive axis that have been used to study disruptions in key intracellular signaling pathways. We first summarize normal development of the hypothalamus, pituitary, ovary, and uterus, highlighting similarities and differences between mice and humans. We then describe human conditions resulting from abnormal development and/or function of the organ. Finally, we describe loss-of-function models for each Cre driver that elegantly recapitulate some key features of the human condition and are associated with impaired fertility. The examples we provide illustrate use of each Cre driver as a tool for elucidating genetic and molecular underpinnings of reproductive dysfunction.

ABSTRACT Ovarian stimulation (OS), utilized for the development of multiple ovarian follicles for IVF, induces supraphysiologic levels of E2 and an early rise in P4 that disrupt endometrial differentiation and decreases implantation rates or result in placental insufficiency and pregnancy complications. To improve pregnancy rates and reduce the risk of pregnancy complications associated with IVF, it is crucial to advance our molecular understanding of the molecular regulation of endometrial differentiation. Previous studies from our laboratory suggest G protein-coupled receptors (GPCRs) are important regulators of endometrial differentiation. To investigate this further, using a retrospective dataset, we identified all GPCRs expressed across the proliferative and secretory phase of the menstrual cycle and found that many members of the adhesion G protein-coupled receptor (ADGR) family are dynamically expressed. For each ADGR subfamily exhibiting differentially-expressed genes across the cycle, their expression was investigated by RT-PCR in the non-pregnant mouse uterus and decidua on E7.5 of pregnancy. For those genes expressed in the E7.5 decidua, their expression was further quantified by qPCR across early mouse pregnancy. The RT-PCR screen revealed expression of 13 ADGRs (4 of the 9 subfamilies) in E7.5 decidua and among these genes, many were differentially expressed between E0.5 and E5.5 or 6.5 and between E5.5 and E6.5. The dynamic expression of the ADGRs across the menstrual cycle and in early mouse pregnancy, suggests these ADGRs are E2- and/or P4-regulated genes. We therefore hypothesized that for these ADGR genes, mRNA expression would be disrupted in an OS cycle. This hypothesis was tested on endometrial biopsies collected in the secretory phase from prospective cohorts of women in natural and OS cycles. Consistent with the retrospective dataset, our data revealed that members of the ADGR gene family are expressed in the secretory phase of the natural menstrual cycle and for the first time, we show that their expression is altered by ovarian stimulation.

Ovarian stimulation is an indispensable part of IVF and is employed to produce multiple ovarian follicles. In women who undergo ovarian stimulation with gonadotropins, supraphysiological levels of estradiol, as well as a premature rise in progesterone levels, can be seen on the day of hCG administration. These alterations in hormone levels are associated with reduced embryo implantation and pregnancy rates in IVF cycles with a fresh embryo transfer. This article aims to improve the reader’s understanding of the effects of elevated progesterone levels on human endometrial receptivity and oocyte/embryo quality. Based on current clinical data, it appears that the premature rise in progesterone levels exerts minimal or no effects on oocyte/embryo quality, while advancing the histological development of the secretory endometrium and displacing the window of implantation. These clinical findings strongly suggest that reduced implantation and pregnancy rates are the result of a negatively affected endometrium rather than poor oocyte/embryo quality. Understanding the potential negative impact of elevated progesterone levels on the endometrium is crucial to improving implantation rates following a fresh embryo transfer. Clinical studies conducted over the past three decades, many of which have been reviewed here, have greatly advanced our knowledge in this important area.

ABSTRACT Defective endometrial stromal cell decidualization is a major cause of recurrent implantation failure (RIF), a condition with a prevalence of ∼15%. To treat RIF, a stronger understanding of the endometrial factors that regulate decidualization is required. Here we studied the role of the kisspeptin receptor (KISS1R) in regulating human endometrial stromal cell (HESC) decidualization. Our data revealed KISS1R inhibits HESC decidualization in vitro in a manner associated with a striking reduction in ESR1 protein levels. To determine whether KISSR inhibition of decidualization results from reduced ESR1 levels we expressed the dominant negative ESR1-46 isoform in decidualizing HESCs. We found that expression of ESR1-46 in decidualizing HESCs ablated the expression of ESR1-66 and ESR1-54 isomers, and blocked decidualization. Interestingly, when ESR1-64 was co-expressed with ESR1-46, ESR1-66 and ESR1-54 expression was restored and decidualization was rescued. Taken together, these results suggest that KISS1R inhibits HESC decidualization by downregulating ESR1 levels. Based on our findings, we suggest that by inhibiting HESC decidualization, KISS1R regulates the depth of embryo invasion of the stroma, a requirement for a successful pregnancy.

Embryo implantation failure is a major cause of infertility in women of reproductive age and a better understanding of uterine factors that regulate implantation is required for developing effective treatments for female infertility. This study investigated the role of the uterine kisspeptin receptor (KISS1R) in the molecular regulation of implantation in a mouse model. To conduct this study, a conditional uterine knockout (KO) of Kiss1r was created using the Pgr-Cre (progesterone receptor-CRE recombinase) driver. Reproductive profiling revealed that while KO females exhibited normal ovarian function and mated successfully to stud males, they exhibited significantly fewer implantation sites, reduced litter size and increased neonatal mortality demonstrating that uterine KISS1R is required for embryo implantation and a healthy pregnancy. Strikingly, in the uterus of Kiss1r KO mice on day 4 (D4) of pregnancy, the day of embryo implantation, KO females exhibited aberrantly elevated epithelial ERα (estrogen receptor α) transcriptional activity. This led to the temporal misexpression of several epithelial genes [Cftr (Cystic fibrosis transmembrane conductance regulator), Aqp5 (aquaporin 5), Aqp8 (aquaporin 8) and Cldn7 (claudin 7)] that mediate luminal fluid secretion and luminal opening. As a result, on D4 of pregnancy, the lumen remained open disrupting the final acquisition of endometrial receptivity and likely accounting for the reduction in implantation events. Our data clearly show that uterine KISS1R negatively regulates ERα signaling at the time of implantation, in part by inhibiting ERα overexpression and preventing detrimentally high ERα activity. To date, there are no reports on the regulation of ERα by KISS1R; therefore, this study has uncovered an important and powerful regulator of uterine ERα during early pregnancy.

Triple-negative breast cancer (TNBC) is a highly metastatic and deadly disease. TNBC tumors lack estrogen receptor (ERα), progesterone receptor (PR), and HER2 (ErbB2) and exhibit increased glutamine metabolism, a requirement for tumor growth. The G protein-coupled kisspeptin receptor (KISS1R) is highly expressed in patient TNBC tumors and promotes malignant transformation of breast epithelial cells. This study found that TNBC patients displayed elevated plasma kisspeptin levels compared with healthy subjects. It also provides the first evidence that in addition to promoting tumor growth and metastasis in vivo, KISS1R-induced glutamine dependence of tumors. In addition, tracer-based metabolomics analyses revealed that KISS1R promoted glutaminolysis and nucleotide biosynthesis by increasing c-Myc and glutaminase levels, key regulators of glutamine metabolism. Overall, this study establishes KISS1R as a novel regulator of TNBC metabolism and metastasis, suggesting that targeting KISS1R could have therapeutic potential in the treatment of TNBC.

Human GnRH deficiency, both clinically and genetically, is a heterogeneous disorder comprising of congenital GnRH deficiency with anosmia (Kallmann syndrome), or with normal olfaction [normosmic idiopathic hypogonadotropic hypogonadism (IHH)], and adult-onset hypogonadotropic hypogonadism. Our understanding of the neural mechanisms underlying GnRH secretion and GnRH signaling continues to increase at a rapid rate and strikingly, the heterotrimeric guanine nucleotide–binding protein (G protein)-coupled receptors (GPCRs) continue to emerge as essential players in these processes. GPCRs were once viewed as binary on-off switches, where in the "on" state they are bound to their Gα protein, but now we understand that view is overly simplistic and does not adequately characterize GPCRs. Instead, GPCRs have emerged as masterful signaling molecules exploiting different physical conformational states of itself to elicit an array of downstream signaling events via their G proteins and the β-arrestins. The "one receptor-multiple signaling conformations" model is likely an evolved strategy that can be used to our advantage as researchers have shown that targeting specific receptor conformations via biased ligands is proving to be a powerful tool in the effective treatment of human diseases. Can biased ligands be used to selectively modulate signaling by GPCR regulators of the neuroendocrine axis in the treatment of IHH? As discussed in this review, the grand possibility exists. However, while we are still very far from developing these treatments, this exciting likelihood can happen through a much greater mechanistic understanding of how GPCRs signal within the cell.