Awardee Institutions

Abstract: n the U.S., about 12% of women have impaired fecundity and 7% of couples have infertility. In women, the leading causes are increasing age, ovulatory disorders, endometriosis, and tubal factor, and in couples, about 1/3 of infertility is due to female factors, 1/3 to male factors, and 1/3 to both. As mechanisms underlying the causes of infertility are largely wanting, treatments are mostly empiric. Infertile women who conceive spontaneously or with fertility therapies are at increased risk of adverse pregnancy outcomes due mainly to implantation and placenta disorders, with life-long effects on the health of children and adults born from these pregnancies. Thus, understanding mechanisms underlying reproductive success and compromise at the genomic, molecular, and cellular levels is critical to fertility and the health and well-being of this and future generations. Moreover, building a sustainable pipeline of junior investigators in this field and engaging investigators from multiple disciplines with diverse expertise are essential components to unravel the complexities of successful reproduction with translation to improving reproductive health more broadly. These are core principles of our NIH National Center for Translational Research in Reproduction and Infertility (NCTRI) at the University of California San Francisco (UCSF), funded since 2007 and for which this renewal proposal is submitted. The Specific Aims of our renewal application (overall) are: 1. to advance research in reproductive science and medicine through transdisciplinary collaboration and scientific and technologic innovation with the goal of improving human reproductive health and fertility 2. to serve as a national resource to inspire, mentor and train students, fellows, and junior scientists in reproduction and infertility research and to nurture their career development long-term 3. to communicate and be a national resource regarding the importance of reproductive research and its relevance to reproductive health and fertility for the public, health care professionals, and patients Our NCTRI Center for Research, Innovation and Training in Reproduction and Infertility at UCSF has 4 projects led by an experienced team of investigators/mentors and brings together expertise in clinical medicine, basic and translational science, precision medicine, genomics/eipgenomics, and advanced technologies focused on reproductive biology, oocyte aging, implantation, and placental development. Moreover, it provides a rich environment for trainees and outreach to the community about innovations in reproductive science and medicine. Our projects use advanced technologies and “omics” approaches, animal models and human tissues and cells, and integrate well-annotated, relevant human phenotypic and clinical data to inform our studies. Our immediate goals are to determine epigenetic regulation of processes resulting in successful reproduction or infertility. Our long-term goals are to develop diagnostics and targeted therapies to alleviate infertility and poor reproductive outcomes and enhance the well-being of those with infertility and reproductive compromise. 

Project I: Oocyte Developmental Competence and Aging (M. Conti)

Project Summary: With the progressive delaying of childbearing age in western society, understanding and treating the decline in women’s fertility associated with aging is becoming increasingly important. While the causes of premature aging of the ovary compared to other organs are largely unknown, it is widely accepted that a decline in oocyte competence to develop as an embryo is central to the decline in fertility in women over thirty-five. Several causes are thought to be at the basis of this decreased oocyte quality, the most widely accepted being an increase in age-related aneuploidy. Oocyte competence to complete meiosis and to develop as an embryo depends on complex programs of gene expression. At the end of its growth, oocyte’s transcription is silenced and the control of gene expression is transferred to the cytoplasm, where a program of selective translation of maternal mRNA is executed. Thus, translational regulation is essential for the oocyte to become developmentally competent. Our laboratory has shown that this translation program activated in the final stages of oocyte maturation plays a critical role for the progression through the meiotic cell cycle, for chromosome trafficking and for preimplantation embryo development. Here we propose to test the hypothesis that this oocyte translation program becomes progressively defective during female aging. Preliminary data exploring the translation of candidate maternal mRNAs in oocytes from aging mouse females support this hypothesis. We propose to further these studies by exploring the functionality of the translation program during aging in mice and humans. The first Specific Aim will compare the pattern of maternal mRNA translation in oocytes from young and old females. We will use a mouse model where ribosomes are tagged in the oocyte, and translating mRNAs will be isolated by immunoprecipitation of ribosome/mRNA complexes followed by RNA-Seq. This genome-wide approach will define the extent of translational defects that develop with oocyte aging. The second Specific Aim will be devoted to understanding the molecular mechanisms at the basis of this defective translation. On the basis of genome wide data suggesting defective expression of RNA binding protein networks and polyadenylation machinery, we will investigate the functionality of these components in aging oocytes. With the third Specific Aim, we will test the hypothesis that disruption of the translational program recapitulates the phenotype associated with aging. We will use genetic models of haploinsufficiency and SiRNA mediated knockdown to destabilize the translational program in young oocytes and determine whether oocyte developmental competence is compromised as it is during aging. To consolidate these findings, we will relate the observations made in a rodent model to human oocytes. We will explore whether translation is defective in oocytes from aging women and explore whether defects in biomarkers of translation are present using single cell assays. If confirmed, the hypothesis of compromised translation will open new avenues for understanding the decline in fertility in aging women and for developing new therapies.

Project II: The Human Cytotrophoblast Epigenome: Dramatic Shifts and Functional Consequences (S. Fisher)

Project Summary: We propose testing the hypothesis that shifts in the human cytotrophoblast (CTB) epigenome early in pregnancy are integral to their normal differentiation, formation of the placenta and pregnancy success. CTB fate decisions establish placental structure and thereby function. In one pathway, CTBs fuse to form the syncytiotrophoblasts (STBs) that cover the surface of chorionic villi. These cells produce hormones and exchange myriad substances between the mother and the embryo/fetus. In the other differentiation pathway, CTBs emigrate from the chorionic villi and invade the uterus, anchoring the placenta to the mother. In the process, they tap into the resident arteries and veins, establishing blood flow to the intervillous space. Recently we employed sequencing approaches to profile the CTB epigenome in late 2nd trimester and at term. The data revealed a unique pattern of global hypomethylation punctuated by megabase domains of even deeper valleys of hypomethylation. In comparison, other human embryonic, fetal, and adult genomes were highly methylated. Unexpectedly, H3K9me3 occupancy precisely overlapped domains with more pronounced DNA hypomethylation and repressed transcription. Also, we discovered significant gestational age-related alterations in the CTB epigenome. During the late 2nd trimester-to-term interval, substantial genome-wide increases in DNA methylation were accompanied by depletion of H3K9me3 and H3K4me. Surprisingly, we also found changes in histone abundance at the end of the 1st trimester, i.e., a substantial decrease in H3K27me3 and H3K4me1 signals. Work from other investigators suggests parallel increases in DNA methylation of placental promoters over the same period. Thus, the CTB epigenome appears to be evolving throughout pregnancy with significant changes early as well as later in gestation. To understand the functional consequences of these shifts, we propose two Specific Aims. First, we will investigate the relationship between alterations in the CTB epigenome and transcriptome at the end of the 1st trimester of pregnancy. Changes in DNA methylation and key histone modification profiles will be intersected with expression data to identify pathways and their drivers that are modulated. Second, we will test the functional significance of the drivers in our in vitro models of CTB differentiation, which enable interrogating formation of STBs or invasive extravillous CTBs. The most innovative aspect of this project is the intent to build on our recent discovery of the unique nature of the human CTB epigenome, which exhibits substantial shifts over gestation. The significance lies in the importance of CTB differentiation to placental development and function. Our goal is to achieve a new level of understanding about the mechanisms that are involved, information that has translational value in terms of gaining new insights into failures in these processes. We reason that they may be associated with some cases of infertility and 1st trimester losses or lie at the root of pregnancy complications associated with faulty placentation, e.g., preeclampsia ± intrauterine growth restriction.

Project III: Post-transcriptional Regulation of Trophoblast Differentiation (R. Blelloch)

Project Summary: The placenta is central to embryonic/fetal development of all eutherian species including humans. However, much remains unknown about the diversity, the ontogeny, and the molecular nature of the cells that make up the placenta. The placenta also represents one of the most rapidly evolving organs as exemplified by the its highly distinct structure in mouse and human. The changes in structure arose from a combination of divergent and convergent evolution that cannot be understood by histology alone. Understanding the analogous cell types and molecular programs between species will greatly improve the ability to translate findings from the mouse model system to humans. A long-term goal of the lab is to understand the pathways that regulate placental development from placentation to delivery and determine how these pathways become misregulated in placental diseases such as pre-eclampsia, intrauterine growth retardation, and pre-term birth. Our immediate goal is to better understand the diversity of cell types and cross-species relationships in function between those cell types in mouse and human. Our particular focus in this grant is on the cells that give rise to and constitute the exchange membrane existing between maternal and fetal blood. Across this surface, there is the essential exchange of nutrients, gases, wastes, and signaling molecules. Defects in this membrane are highly detrimental to the developing embryo. During the previous cycle of this center grant, our laboratory discovered that an eutherian-specific microRNA cluster is essential for the normal development of the exchange membrane within the labyrinth of the mouse placenta. Here, we will expand on these findings by first molecularly defining the cell types that exist within the labyrinth, determining how those cell types are impacted at the molecular level by the loss of the miRNA cluster, and how those findings can be translated to the analogous structure in the human placenta. We address these questions in three aims. In aim 1, we will use single nucleus RNA sequencing to determine the cell types and lineage relationships of the cells within the mouse labyrinth including the individual nuclei of the syncytiotrophoblast layer. In aim 2, we will use combination of sorting, single cell sequencing, and in vitro experiments to uncover the impact of miR-290 on the cellular composition and transcriptional heterogeneity of labyrinth trophoblasts. In aim 3, we will use single cell nucleus RNA sequencing, cutting edge bioinformatics approaches, and in vitro manipulation of isolated cells of the human placenta to translate the findings in mouse to human. In addition to dissecting the role of a eutherian-specific microRNA cluster in placental development, successful completion of the proposed experiments will impact our understanding of cellular composition and ontogeny of the key exchange structures of the mouse and human placenta and provide a paradigm on how to translate mouse genetic/molecular findings to human placentation and placental diseases.

Project 4: Human Endometrial Programming for Successful Implantation (L. Giudice)

Project Summary: Events in early implantation are key to fertility and pregnancy success, and the uterine lining (endometrium) plays a critical role in assuring acceptance and sustenance of the conceptus at the beginning and throughout gestation. Essential for pregnancy establishment and maintenance is the progesterone (P4)-induced differentiation (decidualization) of the endometrial stromal fibroblast (eSF) to the “decidualized” phenotype, characterized by major genomic, transcriptomic, morphologic and functional transformations. We found that human eSF derive from the endometrial mesenchymal stem cell (eMSC), and in women with endometriosis, an inflammatory disorder associated with infertility, miscarriage and other poor pregnancy outcomes, eSF have an abnormal response to P4 that is inherited from eMSC, compared to controls. We also found that steroid hormones regulate the eSF DNA methylome and women with endometriosis have pre-existing eSF genome wide aberrant epigenetic marks associated with gene expression changes and that their eSF have a proinflammatory phenotype likely acquired in the endometrial niche. Our overall hypothesis is that epigenetic mechanisms regulate eMSC differentiation and the decidualization of their eSF progeny and that inflammation in endometriosis alters these processes and compromises eSF function and the steroid hormone response essential for successful implantation. We propose 3 specific aims: Aim 1: Test the hypothesis that genome wide epigenetic programming of eSF is inherited from its progenitor and changes in response to steroid hormones and affects eSF functionality. Aim 2. Determine endometrial immune features of the inflammatory milieu in endometriosis and their effects on eMSC lineage differentiation and eSF decidualization. Aim 3. Test the hypothesis that endometriosis results in an aberrant eSF response to soluble signals from placental cytotrophoblasts (CTB) important in implantation. We propose advanced molecular and epigenomic approaches, functional studies to determine roles of candidate genes in decidualization, innovative CyTOF technology to deeply phenotype specific immune populations in the endometrium, advanced computational methods for data integration, and deep learning tools combined with high-throughput imaging to map changes in cell morphology space onto transcriptome changes in epigenetically regulated cells. Understanding mechanisms underlying eSF lineage specification from eMSC, eSF response to steroid hormones, communication with the invasive CTB, and effects of inflammation on these processes is highly significant to reproductive success and the health and well-being of those who conceive and their offspring. The expected results are to derive an in-depth understanding of the role of P4-resistance and inflammation in endometriosis that can also inform infertility and poor pregnancy outcomes in women with other inflammatory disorders. Also, reprogramming eSF and eMSC could lead to novel therapies to maximize functionality of these cells in normal implantation and endometrial homeostasis more broadly.

Multidimensional Quantitative Imaging Core (J. Fung)

Abstract: The Multidimensional Quantitative Imaging Core will provide quantitative image, genomic and statistical analysis for microscopy and sequencing projects generated by the Center’s research investigators. Formerly, the Core operated as a Computational Biology Core and focused only on providing expertise for genomic and statistical analysis for the P50 investigators’ projects. We have expanded the scope of the Core to include image analysis since many of the P50 investigators’ projects will examine cellular and subcellular morphological changes that accompany changes in differentiation, maturation and cell lineage specification. One of the goals of the core is to apply new advances in deep learning to image recognition of cellular structures. Since applying deep learning techniques requires significant computational resources as well as expertise in image acquisition and analysis, the Core facility is better suited and equipped to handle these types of analyses than individual investigators or teams. The Core is partnering with UCSF’s Center of Cellular Construction to develop general deep learning algorithms for image analysis of cell biology. The Core will also integrate “omics” data with imaging data by aiding in the development of de novo mapping analyses to integrate the disparate datasets. Finally, the Core will participate in training and educating P50 students, interns and fellows in the best practices of bioinformatics and image analysis. 

Administrative Core (L. Giudice)

Abstract: Our NIH NCTRI Center is well-integrated within and administered through the Center for Reproductive Sciences in the Department of Obstetrics, Gynecology and Reproductive Sciences at the University of California, San Francisco. The purpose of the Administrative Core is and will be to provide administrative, financial, and organizational support for the investigators and technical staff engaged in our NIH P50 NCTRI Center's research projects and supporting cores. Day-to-day operation of the Administrative Core involves and will continue to involve coordinating all administrative, financial, and compliance aspects of the award. In conjunction with the NCTRI Center Director and the Program Administrator, the Department, and the Center for Reproductive Sciences, the Core will continue to implement an operating framework to ensure that expenditures are consistent with the intent of the award and the guidelines and regulations that govern the use of funds, as well as any institutional and federal requirements. In addition, the Core will continue to organize our monthly P50 NCTRI Steering Committee meetings of the PI/Center Director, Associate Director and all Project and Core Directors, as well as the annual Center for Reproductive Sciences Research Retreat, research meetings, and presentations of our P50 investigators and trainees. The Administrative Core personnel will continue to provide support to the Pl, Project and Core Directors, trainees, technical staff, and all Cores in our NIH P50 NCTRI Center at the University of California, San Francisco. 

Outreach/Education Core (S. Mellon)

Abstract: The goal of the Education and Community Outreach Core (Core C) in our UCSF NCTRI Center renewal proposal is to continue our structured educational outreach and mentoring program that promotes research in reproductive sciences, increases scientific literacy, and engages the San Francisco Bay Area community. Core C has formalized its partnership with four established programs at UCSF: the Science and Educational Partnership (SEP; high school interns), our departmental Undergraduate Research Internship (URI; undergraduate interns), the San Francisco State Bridges Program (undergraduate interns) and the San Francisco State-UCSF NIH BUILD Program (Building Infrastructure Leading to Diversity; undergraduate interns). We will continue to provide an innovative, formal, intensive summer research and didactic experience that educates and trains students in various aspects of reproduction research. To promote community involvement in NCTRI activities and to promote reproductive science literacy in the Bay Area, we highlight student research and other UCSF NCTRI Center research through public presentations by interns and NCTRI members, and through participation in activities hosted by the Bay Area Science Festival. All projects and cores will participate in all Core C activities. Coordination of these teaching and community outreach programs under the aegis of Core C will provide a rich cross-generational and cross-educational mentoring and teaching experience, will highlight our passion for basic and translational reproduction research, and will engage the Bay Area community to participate directly in NCTRI Center activities.

Abstract: The Penn Center for the Study of Epigenetics in Reproduction (PennCSER) will elucidate epigenetic mechanisms that govern male and female reproduction, contribute to male infertility and impact development of mouse and human concepti conceived through assisted reproductive technologies (ART). The PennCSER centerpiece is 4 integrated, innovative research projects, spearheaded by experienced leaders in the areas of epigenetics and reproduction. The Center also features an Outreach program that has been in place for more than 5 years; the Penn Academy of Reproductive Sciences uses hands on laboratory experiences and interactive lectures to educate high school students, largely from the Philadelphia area schools, in the reproductive sciences. The clinical project (Project 1, Coutifaris, Sapienza, Mainigi and Senapati) will assess the impact of the periconceptional maternal environment on DNA methylation and gene expression in embryonic (placental vessels and endothelial cells) and extra-embryonic (trophoblasts) tissues in IVF pregnancies following fresh or frozen transfer, pregnancies resulting from unassisted conception and pregnancies following trophectoderm biopsy for pre-implantation genetic testing for aneuploidy (PGT-A). This project will also determine the stability of epigenetic signatures determined at birth into childhood and correlate them with the children’s growth phenotype. Project 2 (Bartolomei, Mainigi, Schultz) will closely parallel Project 1 using a validated mouse model to study the effect of ART laboratory manipulations on epigenetic gene regulation and physiological outcomes in term conceptuses and adults. Project 2 will address the question of whether embryo freezing and transfer to an unstimulated uterus is optimal and evaluate the safety and outcomes associated with trophectoderm biopsy. Project 2 will also test whether decreased expression of Grb10, a growth-regulatory imprinted gene overexpressed in human and mouse ART-derived conceptuses, can rescue ART-associated phenotypes in a mouse model. Project 3 (Berger) will investigate histone modifications during mouse spermatogenesis and determine their conservation in normal human sperm and disruption in abnormal human sperm, as well as in mouse models exhibiting abnormal histone retention. Project 4 (Wang and Masson) will examine the function of TEX15, a protein that is required for meiosis and male fertility, and is a novel epigenetic regulator essential for retrotransposon silencing. Project 4 will also determine whether aberrant retrotransposon activity is associated with male infertility. PennCSER will not only provide training to clinicians, physician scientists, and basic research fellows in the area of epigenetics but also provide PennCSER’s expertise to the NCTRI and associated program members.

Project I: Epigenetic Regulation of Placental and Fetal Gene Expression in Human Pregnancy (C. Coutifaris)

Project Summary: A fundamental question relevant to clinical IVF is whether and how the peri-conceptional milieu affects embryo implantation and placentation and how clinical manipulations of this environment influence perinatal outcomes. Changes in trophoblast differentiation and function, perturbations of placental vascular development and/or sub-optimal endometrial regeneration during clinical or laboratory manipulations such as trophectoderm biopsy could all contribute to abnormal implantation and placentation and lead to an abnormal fetal growth phenotype. We hypothesize that epigenetic changes in specific placental cellular compartments (embryonic or extraembryonic) and/or the endometrium contribute to the significant adverse outcomes associated with IVF and, specifically, following fresh (hyperstimulated) vs. frozen/thawed embryo transfer or natural conception or following trophectoderm biopsy. We suggest that these epigenetic alterations lead to abnormal gene expression at critical times during development, implantation and placentation and result in abnormal growth and other complications. In Specific Aim 1A we will identify site- and gene-specific epigenetic differences in placentas, in isolated trophoblasts, placental vasculature and endothelial cells from pregnancies following fresh/hyperstimulated vs. frozen/thawed vs. control pregnancies. In Specific Aim 1C, we will begin exploring whether there is an endometrial contribution to the observed peri-conceptional milieu growth-related differences as well. In Specific Aim 2 we will identify specific epigenetic differences in placentas and in isolated trophoblast cells, placental vasculature and endothelial cells in pregnancies following trophectoderm biopsy. In this "clinical" project, we focus on identifying the epigenetic signature(s) related to abnormal placental function and an abnormal birth weight phenotype. In Specific Aim 3 we will determine whether the epigenetic signature determined at birth persists into childhood and may be associated with long term health consequences such as obesity. This initiative continues to be discovery-driven, has and will continue to inform mechanistic studies for our mouse model (Project 2) and should contribute to our understanding of optimal human embryo development, implantation and placentation.

Project II: Epigenetic, Behavioral, and Physiological Outcomes in a Mouse ART Model (M. Bartolomei and M. Mainigi)

Project Summary: Assisted Reproductive Technologies (ART) are invaluable for the increasing number of women who require interventions to treat their infertility. Nevertheless, ART-conceived children are at increased risk for loss-of-imprinting disorders resulting from epigenetic errors, abnormal growth, congenital malformations, and postnatal cardiac and metabolic disorders. Such problems likely arise because ART procedures take place when the mammalian embryo is being epigenetically reprogrammed. Because it is difficult to conduct studies using human embryos, a mouse model system, which anticipated some risks associated with ART, will be used to assess the effect of ART interventions on placental morphology, imprinted gene regulation, growth, metabolic and cardiac phenotypes of the offspring, and epigenetic gene regulation, including DNA methylation and chromatin structure genome-wide. Presently, preliminary evidence suggests that frozen embryos transferred into unstimulated women have less perinatal morbidity than fresh cycles but conflicting data suggest adverse outcomes associated with frozen embryos. Specific Aim 1 will determine the effects of embryo vitrification and maternal hormonal environment on offspring outcome of IVF-generated embryos. Moreover, preimplantation genetic screening (PGS) is being increasingly employed in the absence of research assessing the consequences of blastomere biopsy. Specific Aim 2 will investigate the phenotypes and epigenetic profiles of the placenta and ART offspring derived when PGS is used. Finally, data from our human placental studies demonstrate alterations in DNA methylation in genes critical to early placentation, fetal growth, and adult metabolism. Specific Aim 3 will translate these data to our animal ART model to determine the role of specific genes in adverse outcomes associated with IVF. The role of Grb10 in IVF-associated changes in fetal growth, placentation, and vasculogenesis using a mouse model will be initially assessed. Results of these experiments will provide information regarding the linkage between epigenetic changes and health of offspring conceived by ART. These findings may also suggest experimental modifications to ART procedures that can improve offspring outcomes.

Project III: Epigenetic Regulation of Retrotransposons in the Mouse and Human Germline (J. Wang)

Project Summary: Germ cells undergo extensive epigenetic reprogramming during development. During this critical period, retrotransposons are reactivated due to genome-wide erasure of DNA methylation and are re-silenced by de novo DNA methylation. Retrotransposons, mainly LINEs, SINEs, and endogenous retroviruses (collectively referred to as junk DNA), occupy 40% of the mammalian genome. Although retrotransposons play an important role in genome evolution, their mobilization could be detrimental to genome integrity. Multiple epigenetic mechanisms are responsible for silencing retrotransposons in the germline: DNA methylation, repressive histone modification, small RNAs, and heterochromatinization. Given the extreme abundance of the retrotransposons and the paramount importance of germline genome integrity, novel mechanisms for retrotransposon silencing may exist. In support, we have found that TEX15, a germ cell-specific 3059-aa protein with a newly identified functional domain, is required for meiosis and male fertility, and is a novel epigenetic regulator essential for retrotransposon silencing. Based on these data, we hypothesize that TEX15 is a novel germ cell-specific regulator of retrotransposon activation and that meiotic collapse is the ultimate “fail-safe” mechanism for preventing transmission of male germ cells in which retrotransposons are inordinately activated. In this project, we will 1) determine the TEX15-mediated epigenetic landscape during male germ cell development in mouse using genomic approaches, 2) elucidate molecular mechanisms underlying TEX15 function, and 3) screen for retrotransposon activation and utilize whole exome sequencing to identify genetic mutations that compromise epigenetic silencing of retrotransposons in testis biopsies from azoospermic men. Together, these studies will identify novel factors in the silencing of retrotransposons and provide essential insights into the etiology of male infertility in humans.

Administrative Core (M. Bartolomei and C. Coutifaris)

Abstract: The overall goal of the Penn Center for the Study of Epigenetics in Reproduction (PennCSER) is to investigate the role of epigenetic gene regulation in reproduction, with a specific emphasis on gametogenesis and early development as they pertain to normal development, implantation and placentation, infertility and Assisted Reproductive Technologies (ART). The Administrative Core will facilitate the successful pursuit of each of the individual projects and enable collaboration among projects. The Core will also organize the solicitation and selection of pilot projects that complement the existing Center program. Finally, the Core will support the Outreach Activities, facilitate training, and assist interactions with the NCTRI network, as well as the internal and external review process.

Outreach/Education Core (J. Shuda)

Abstract: Over the last ten years, local under-served and impoverished school systems have increasingly felt the burden of lack of funding and very limited resources in science education. The School District of Philadelphia, our city’s schools, currently has tremendous budget cuts that directly result in under-resourced science classrooms. In addition, attracting the next generation into science, technology, engineering and math (STEM) careers is a fundamental issue facing today’s educators. It is because of these barriers throughout a student’s educational career that the outreach core will build a biological science pipeline for local, under-served high school youth to gain the research skills to be successful in other rigorous science programs, college courses, and the workforce. Our outreach core will include the continuation of two successful outreach projects funded by the P50-HD06817 grant - Project BioEYES, the week-long biology experiment which occurs in classrooms during the school day with the addition of epigenetic presentations, and the Penn Academy for Reproductive Sciences (PARS), the Saturday program at UPenn. In addition, we will expand the outreach core to include a formalized 8-week summer internship for PARS alumnae in co-Investigators’ laboratories. The result of our outreach core is a series of science exposures that first target city biology classrooms and then offer specialized opportunities for high school women who have a genuine interest in science and research.

Abstract: The Oregon National Primate Research Center (ONPRC) at the Oregon Health & Science University (OHSU) proposes to renew its P50 National Center for Translational Research in Reproduction and Infertility (NCTRI) that addresses the effects of hyperandrogenemia and obesity on female reproductive health. Progress in years 01-05 identified metabolic, adipose tissue, ovarian and uterine lesions, as well as subfertility, following chronic testosterone and/or a western-style diet (WSD) treatment of female macaques beginning at puberty through young adulthood. Further studies are proposed to determine if: (1) the effects become more pronounced as treatment continues into adulthood, and (2) the effects are, at least in part, reversed by removal of treatment. Three research projects use the nonhuman primate model at ONPRC, and one project focuses on the specific population of normal weight women with polycystic ovary syndrome (PCOS) at UCLA. Project I, “Metabolic and Adipose Responses to Hyperandrogenemia and Diet” is a collaboration between Drs. C. Roberts, C. True and O. Varlamov. Project II, “Ovarian Structure-Function: Influence of Androgen and Diet”, includes Drs. J. Hennebold, R. Stouffer and S, Chavez. Project III, “Effects of Androgen and Diet on Uterine-Placental Function”, involves Drs. O. Slayden, A. Frias and L. Myatt. Project IV, “Androgen Excess Causes Adipogenic Dysfunction in PCOS Women”, incorporates a consortium with Drs. D. Dumesic and G. Chazenbalk in the Department of Ob-Gyn, UCLA. Projects I-III will be supported by a nonhuman primate (NHP) Core (O. Slayden, Supervisor) operating as a closed resource. This Core will maintain four treatment groups of female rhesus monkeys (control, testosterone or T-treated, WSD-treated and T+WSD) for two additional years, including a fertility trial. Then procedures testing the effects of removal of T and WSD will be supported, including another fertility trial. The Administrative Core (Drs. R. Stouffer and J. Hennebold) will direct the NCTRI activities and promote interactions with other centers and NICHD officers. The Outreach Core (D. Gordon and Dr. M. Zelinski) will increase public awareness and understanding of reproductive health research. Important new information will accrue on the actions of androgen and diet-related factors, individually and in combination, relevant to the etiology of fertility disorders, such as PCOS. Also, the reversibility data will provide insight on the possible efficacy of novel treatments, including epigenetic changes that may limit therapies. The estimated prevalence of infertility in the human population is 9%, with mounting evidence that hyperandrogenemia or obesity alone lead to reproductive dysfunction, and combine to further impair fertility. However, the causes versus effects of androgen, particularly as related to reproductive dysfunction, are controversial. Mechanistic studies in primates and normal-weight PCOS women will discern between the roles of chronic androgen exposure and diet, and offer insight into improving therapy for infertility.

Project I: Metabolic and Adipose Responses to Hyperandrogenemia and Diet (C. Roberts)

Project Summary: Increased levels of androgens, such as testosterone (T), in women are associated with infertility, as well as an increased risk of metabolic dysfunction in endocrine disorders such as polycystic ovary syndrome (PCOS) and congenital adrenal hyperplasia. PCOS in particular is the most common reproductive disorder, affecting up to 12% of women in the US. PCOS is also associated with an increased risk for gestational diabetes and subsequent type-2 diabetes. While elevated androgen levels alone contribute to metabolic disease, many women with PCOS are also obese, which independently increases the risk for gestational and type-2 diabetes. The relationship between androgens and obesity in PCOS is further complicated by studies demonstrating that obesity also affects fertility, so that obese women with PCOS may have an increased degree of infertility. Obesity is driven in most cases by reduced physical activity and, to an even greater degree, by consumption of high-fat Western-style diet (WSD). To investigate the interaction between androgen and obesity in inducing metabolic disease and infertility in women, we developed in the current NCTRI a nonhuman primate model of chronic hyperandrogenemia (elevated T) in the presence or absence of a WSD, beginning at puberty. Over the first 3 years of treatment, we found that the combination of T and WSD worsened systemic measures of metabolism (obesity and insulin resistance) and produced specific changes in adipose tissue such as enlarged visceral adipocytes, increased insulin-stimulated fatty acid uptake, and decreased lipolysis. Notably, animals that were exposed to the WSD with or without T exhibited elevated insulin levels with normal glucose levels, suggesting that, at this stage, the increased insulin was still sufficient to overcome insulin resistance. However, this subclinical insulin resistance was associated with decreased fertility and viable pregnancies by year 4 of treatment, indicating that even mild subclinical insulin resistance can reduce fertility. These data lead to our hypothesis that: a) worsening metabolic status due to continued chronic T and/or WSD will exert adverse effects on maternal gestational metabolic health; b) that some aspects of poor metabolic status will remain after removal of T and WSD due to epigenetic changes; and c) that these persistent effects will compromise maternal metabolic health during subsequent pregnancy. To address this hypothesis, we propose the following specific aims in this NCTRI renewal application: 1. Compare the effects of hyperandrogenemia and/or WSD on metabolic health during and after pregnancy. 2. Characterize how removal of hyperandrogenemia and/or WSD alters metabolic health and epigenetic programming of gene expression in adipose tissue. 3. Examine how reversal of hyperandrogenemia and/or WSD alters metabolic health during pregnancy.

Project II: Ovarian Structure-Function: Influence of Androgen and Diet (J. Hennebold)

Project Summary: Elevated circulating androgen (testosterone; T) and a typical high-fat western-style diet (WSD) are associated with the development of female reproductive disorders, such as polycystic ovary syndrome (PCOS) and reduced fertility. Although the underlying etiology of reproductive dysfunction in PCOS likely includes altered in utero programming and genetic contributions, the pathophysiology of exposure to elevated T at PCOS levels of hyperandrogenemia, WSD, or their combined effects on female fertility is poorly understood, particularly in primates. The current NCTRI established that chronic T and/or WSD treatment beginning just prior to puberty in a nonhuman primate model negatively affects reproductive processes critical for fertility. Through 4 years of treatment, significant effects of T and/or WSD were noted relative to untreated controls in terms of ovarian function, including: the appearance and persistence of numerous small antral follicles typical of a “polycystic” ovarian morphology, reduced ovarian vascular function and luteal progesterone synthesis, altered perivoulatory follicle gene expression, and the presence of degenerated oocytes within the naturally-selected, single ovulatory follicle. Even if the oocyte from T and/or WSD-treated animals appeared healthy and meiotically mature, it often showed abnormal cell division kinetics after fertilization. These ovarian defects correlated with fertility outcomes since exposure to T delayed the time to pregnancy, whereas WSD, particularly in combination with T, reduced overall fertility and led to abnormal, nonviable pregnancies. WSD treated animals that failed to become pregnant exhibited the greatest level of pre-pregnancy and pregnancy associated metabolic abnormalities, including increased insulin resistance. Although prepubertal exposure to T and/or WSD significantly affected ovarian processes and fertility relative to controls, treatment did not impact all animals equally and many continued menstrual cyclicity. Thus, studies are proposed in this NCTRI renewal (Project II) to determine whether continued T and/or WSD treatment worsens ovarian dysfunction and if treatment removal restores ovarian physiology, oocyte/embryo quality and fertility. Experiments will continue during 7 years of T and/or WSD treatment to assess their effects on ovarian follicle growth and development, intrafollicular events necessary for ovulation, luteal progesterone synthesis, and ovarian vascular function, relative to controls (Aim 1). The impact of continued T and/or WSD treatment on oocyte health, fertilization, expression of certain epigenetic factors important for the oocyte-to-embryo transition, as well as embryonic development and chromosomal integrity will also be assessed (Aim 2). After 7 years, T and WSD treatments will cease to determine if ovarian function and oocyte/embryo health is restored (Aim 3) similar to what is observed in age-matched controls. Continued treatment and cessation on ovarian function and oocyte competency will be correlated with changes in metabolic function (Project I), uterine and placental function (Project III), which will be compared to overall fertility and pregnancy outcomes (Nonhuman Primate Core).

Project III: Effects of Androgen and Diet on Uterine-Placental Function (Ov Slayden)

Project Summary: Hyperandrogenism combined with obesity are clinical hallmarks of polycystic ovary syndrome (PCOS), a complex disorder affecting ~10% of reproductive-aged women. Many women with androgen excess (AE) also display “metabolic syndrome” with insulin resistance, hyperinsulinemia, and impaired glucose tolerance. These women often have subfertility associated with menstrual cycle irregularities. Even in women with normal cycles, AE and metabolic syndrome correlate with impaired endometrial and placental function leading to poor reproductive outcome. In this NCTRI center, we will interrogate the actions and interactions of AE and diet on reproductive health with the goal of improving fertility in women with AE and metabolic syndrome. Our premise is that AE and/or exposure to an obesogenic diet, disrupts endometrial and placental physiology. To discern the actions and interactions of AE and diet on reproductive function, we are chronically treating young female macaques with mildly elevated testosterone (T), in the presence or absence of an obesogenic western-style diet (WSD). Our intent is to mimic the conditions in adolescent girls at risk for developing PCOS. We report that after 3-4 years of treatment, the T and T + WSD groups exhibit impaired ovarian and endometrial function, attenuated placental vascular perfusion, and reduced pregnancy rate. These animals are now being treated for 5 years. Our hypothesis is that continued exposure to T and/or WSD will further deteriorate uterine and placental function and exacerbate the effect on reproductive success. To test this hypothesis, we will continue treatment for an additional year and conduct longitudinal studies on endometrial/placental function and fertility. The treatments will then be stopped, and we will test the hypothesis that treatment removal restores uterine/placental phenotype and fertility. Our specific aims are: 1) To characterize the long-term effect of T and/or WSD on endometrial morphology, markers of endometrial receptivity, placental function, and metabolism; 2) To utilize contrast-enhanced ultrasound imaging (CEUS) to identify vascular compromise in the endometrium and placenta associated with T and/or WSD; 3) To determine whether T and/or WSD exacerbates inflammation in the endometrium and placenta using in vivo molecular detection of inflammation by CEUS, and correlating it to immunohistological markers; and, 4) To determine if withdrawal of T and WSD treatments returns the endometrium to a normal pre-implantation phenotype, and restores placental physiology leading to normal reproductive outcome. Results of this research will be correlated with metabolic and ovarian effects identified in NCTRI projects I and II.
 

Project 4: Androgen Excess Causes Adipogenic Dysfunction in PCOS Women (D. Dumesic)

Project Summary: Polycystic ovary syndrome (PCOS) is a complex endocrine disorder of women characterized by androgen excess, menstrual irregularity and polycystic ovaries. Affecting 6-10% of reproductive-aged women, 60–95% of PCOS women have insulin resistance that is exaggerated by increased total and abdominal fat deposition, predisposing to metabolic syndrome and diabetes as risk factors for cardiovascular disease (CVD). Of concern, androgen excess in PCOS women who are normal-weight is closely linked with preferential abdominal fat deposition and increased intra-abdominal fat mass that is positively correlated with serum fasting insulin and lipid levels. Moreover, androgen excess in these PCOS women also is accompanied by a greater proportion of small adipocytes (fat cells) in subcutaneous (SC) abdominal adipose where fat is normally stored. Therefore, normal-weight PCOS women may have a reduced capacity of SC adipose to safely store fat. When energy intake exceeds that capacity, SC abdominal adipocytes may overfill with lipid and promote excess lipid deposition in abnormal (ectopic) locations, where increased oxidative stress underlies metabolic dysfunction (i.e., lipotoxicity). We hypothesize that androgen excess in normal-weight PCOS women alters SC abdominal adipogenesis, defined as the ability of adipose stem cells to develop into mature adipocytes and, in doing so, impairs metabolic function. Further, these androgen actions may originate from heritable changes in the genes of these stem cells or their developing adipocytes without affecting the DNA sequence of the genes themselves (i.e. epigenetic). In this NCTRI Project IV renewal, we will 1) examine molecular mechanisms of SC abdominal adipogenesis in normal-weight PCOS women compared to weight- and age-matched normo-androgenic ovulatory women (controls), 2) determine the role of androgen action in SC abdominal stem cell dysfunction and its relationship to metabolism in these PCOS women receiving the antiandrogen, flutamide or placebo through a clinical trial and 3) identify crucial epigenetic changes that distinguish SC abdominal stem cells in such PCOS women compared to those of weight- and age-matched control women. Understanding androgen action during SC abdominal adipogenesis in normal-weight PCOS women and its adverse effects on metabolic function through stem cell differentiation allows development of new and personalized clinical strategies, including stem cell therapies and pharmacological interventions, that could improve metabolic function in PCOS women and decrease their susceptibility to CVD.

Nonhuman Primate Core (Ov Slayden)

Abstract: The Nonhuman Primate Core (NHP Core) will function as a “closed core” to provide NHP resources and technical support for three projects proposed in this NCTRI renewal. These projects are: Project I, Metabolic & Adipose Response (Roberts, Project Lead); Project II, Ovarian Structure & Function (Hennebold and Chavez, Co-Project Leads); Project III, Uterine & Placental Function (Slayden, Project Lead). The broad goal of this P50 NCTRI Center is to assess the independent and combined effects of testosterone (T) and a high-fat diet (typical of the “Western-style diet”; WSD) on reproductive function and fertility. The specific goal of the NHP Core is to provide significant cost savings to the NCTRI Center projects. As part of the current NCTRI (years 01-05), the NHP Core leased and treated 39 peripubertal rhesus macaques with a controlled regimen of T, WSD and T+WSD for 5 years. The NHP core will continue to oversee treatments (years 06-07) for these animals and assist Projects I-III in sample collection. Shared use of the animals assigned to the core will eliminate repeated lease fees, and setup charges for the individual projects. The NHP Core will coordinate WSD treatment, thereby reducing per diem charges associated with the preparation of special diets. The NHP Core will provide a dedicated set of technical staff and management that would otherwise be cost-prohibitive for studies of this size. The NHP Core staff is trained to perform routine surgical and other procedures (e.g. T implant placement) that would otherwise require scheduling with the DCM Surgical Unit, greatly saving on procedural costs. After 7 years of WSD and T treatment, the animals will undergo treatment reversal in year 08. To fulfill the ultimate goal of the NCTRI center, the NHP Core will conduct fertility trials during treatment (year 06-07) and after treatment reversal (year 09). There will be no fee structure or chargebacks for Projects IIII. The NHP Core will provide general supplies (syringes, blood tubes, storage vials, swabs, etc.) leaving individual investigators responsible only for costs for procedures/assays unique to their particular projects. Researchers will plan studies with the NHP Core during regularly scheduled meetings between the core supervisor, Dr. Slayden, and NHP Core staff. The NHP Core will coordinate veterinary care and husbandry of the animals through the Oregon National Primate Research Center (ONPRC), Division of Comparative Medicine (DCM). NHP Core oversight will be provided by an Oversight Committee consisting of Dr. P. Kievit, Supervisor of the ONPRC Obese Animal Resource (Chair); Dr. H. Sidener; DCM clinical veterinarian, Dr. T. Hobbs, Head, DCM Surgery Unit, and Dr. C. True, NCTRI Co-investigator (Project I).

Administrative Core (J. Hennebold)

Abstract: The goals of the Administrative (Admin) Core are: (1) to coordinate the timely generation and exchange of research ideas and information among ONPRC/OHSU NCTRI project investigators, including the seamless interaction with consortium activities at the University of California, Los Angeles (Project IV, Drs. D. Dumesic and G. Chazenbalk), (2) to facilitate the maintenance and efficient use of female rhesus monkeys within the Nonhuman Primate (NHP) Core, including the assignment and monitoring of the four experimental treatment groups as used by Projects I-III, plus completion of the fertility trials, (3) to promote the training and career development of basic/clinical scientists electing to pursue research in reproduction and infertility research, including the leverage of pilot funds from the NCTRI and local entities, (4) to support outreach activities that increase community and student awareness of the values of research and advances in reproductive health, and (5) to integrate the activities of the ONPRC/OHSU NCTRI with those of other NICHD-funded P50 Centers and scientists through interactions with the NICHD Research Coordinator and various focus groups. Richard L. Stouffer, Ph.D., Professor, Division of Reproductive & Developmental Sciences, ONPRC, and Department of Obstetrics & Gynecology, OHSU, continues as Project Lead of the Admin Core, and P.I. of the ONPRC/OHSU NCTRI. Since Dr. Stouffer will retire within this 5-year award, Dr. Jon Hennebold, Professor and Chief, Division of Reproductive & Developmental Sciences, and Project Lead of Project II, will serve as Co-I providing day-today oversight of NCTRI operations. To provide assistance in resolution of problems, review of progress and research planning, two types of committees will operate: (1) an External Advisory Board comprised of four prominent scientists from outside OHSU that will annually provide research review and visioning, and recommendations for improvement, and (2) an Internal Advisory Board of scientists within OHSU that will provide advice on day-to-day operations and annual review of Core activities. Both EAB and IAB members will assist in selection of pilot projects, based on their areas of expertise. The Project Leads of the Admin Core will interact regularly with ONPRC, OHSU and NICHD officials to promote NCTRI programs locally, nationally and internationally.

Outreach/Education Core (D. Gordon)

Abstract: The primary goal of the NCTRI Outreach Core is to increase public understanding of and support for basic and clinical research in the reproductive sciences. This Core will continue to expand our reach into the adult population (while also providing enhanced opportunities for high school students) to promote better understanding of issues concerning reproductive health and infertility in these two groups. We will achieve this goal through the design and implementation of the following objectives: 1) continuation of our popular Beaverton-area "Science Cafe" series, marketed to adults and high school students, which features evening lectures given by reproductive scientists from ONPRC and OHSU on topics of basic science concepts and reproductive health issues; 2) expanding the Science Café series into a strategic new geographical venue, in Salem, OR; 3) offering a free Summer Teacher Workshop to demonstrate the “ART of Reproduction” curriculum and encourage high school teachers to incorporate reproductive biology into their classes; 4) continuing our "Infertility Saturday Academy" for high school students, consisting of six Saturdays of lecture and hands-on activities covering the topics of infertility and fertility control; 5) maintaining a strong on-line presence to provide curricular materials for educators, information for website visitors, and promote our programs to the general public; 6) developing a series of discrete science lessons for adults, consisting of 3- hour classes that will feature cutting-edge reproductive science topics taught by research scientists and supported by hands-on laboratory activities, and; 7) offering a series of "Mother-Daughter Science Saturday" events, similar in content to the "Science Saturdays," but marketed exclusively to women and their daughters/mothers. The impact of our programs on attendees will be evaluated using pre- and post-tests. These data will be analyzed by a professional evaluator who will report to the NCTRI PD/PI (Dr. R. Stouffer), to assess the effectiveness of the NCTRI outreach programs.

Abstract: Uterine leiomyoma (LM, fibroids) are the most common tumor in women, disproportionately affect African Americans, and cause irregular uterine bleeding and anemia, necessitating more than 200,000 hysterectomies annually in the US. Our long-term objective is to understand novel clinically relevant and medically targetable mechanisms responsible for the pathogenesis and growth of uterine LM in order to reduce associated morbidity. We propose and request funds to support the Northwestern Uterine Leiomyoma Research Center, comprising three highly coordinated and synergistic Research Projects, an Administrative Core and an Education and Outreach Core. Two mutually exclusive key driver somatic mutations (mut-) or rearrangements (-ra) affecting the MED12 and HMGA2 genes have been found in 85% of all LM, but the underlying mechanisms that cause tumorigenesis and tumor growth remain unknown. We propose to ascertain the effects of mut-MED12 and HMGA2-ra on epigenomic programming of LM cells, LM stem cell (LSC) function, development of heterogenic cell populations in these tumors, and genome-wide progesterone (P4) action. We will use cutting-edge in vivo models and high-throughput technologies to uncover novel mechanisms and identify genotype-specific therapeutic targets for developing precision medical treatments for LM. Project 1 (Bulun/Yin/Dai) will test the hypothesis that mut-MED12 alter genome-wide progesterone receptor (PR)- chromatin interaction signatures and associated histone modifications, thereby enhancing P4 action in the LM intermediate cell population (LICs), which provides a support niche for LSC survival and proliferation. Project 2 (Rajkovic) will test the hypothesis that distinct driver mutations affecting MED12 and HMGA2 determine cellular and molecular heterogeneity during LM tumorigenesis. Through cell fate tracing studies, we will determine whether mut-MED12 cells give rise to different cell populations in the myometrium that drive the formation of LM. Project 3 (Chakravarti/Wei) will test the hypothesis that overexpression of HMGA2 in LM alters 3D chromatin interactions and the epigenome to modify the development, progression, and therapeutic response of LM. As model systems, we will use LM tissues, antibody-sorted human LM cell populations and a human equivalent mouse model of LM with mut-MED12 in uterine tissue. The Education and Outreach Core will support research activities performed within and across the Center by developing communication, outreach, and education strategies to promote health equity and eliminate disparities in LM, engaging the general public, students in the Chicago area, and the scientific and medical community. The Administrative Core will ensure that the Center achieves its aims and will synergize the individual Research Projects with the work of the Education-Outreach Core and other institutional cores; it will also solicit and coordinate the review of Pilot Projects. We anticipate that our synergistic approach will lead to the development of mutation- or epigenetic signature-selective therapeutic approaches to LM, moving the field into the realm of personalized medicine.

Project I: Epigenome, MED12 and Progesterone Action in Uterine Leiomyomas (S. Bulun)

Project Summary: Uterine leiomyoma (LM, fibroid) is the most common tumor in women. No long-term medical treatment is available. Each LM seems to originate from the clonal expansion of a single mutated LM stem cell (LSC) in the myometrium (MYO). LSC comprise 5% of tumor mass and differentiate into an intermediate cell population (LIC, 7%), which then become terminally differentiated cells (LDC) comprising 88% of the tumor bulk. Driver mutations of mediator complex subunit 12 (mut-MED12) occur in 70% of all LM. Progesterone (P4) and its receptor PR are essential for LM growth. PR-rich LIC transduce P4 signaling to PR-deficient LSC via paracrine factors to activate their proliferation. Ulipristal acetate (UPA), a PR-selective P4 antagonist, shrank LM and reduced its symptoms, but its use was halted because of risk of liver injury. Our overall goal is to define the role of genome wide P4 action in the etiology of mut-MED12‒associated LM tumorigenesis and identify novel therapeutic targets. We found that mut-MED12 physically interacts with PR and genome wide PR-chromatin interaction landscapes are dramatically dysregulated in LM expressing mut-MED12 vs. normal MYO tissue carrying wild type MED12. Mut-MED12 enhances PR recruitment to cis-regulatory elements of P4 target genes encoding paracrine growth factors, cytokines and extracellular matrix proteins critical for LSC proliferation and LM tumorigenesis. Furthermore, the uteri of mice with a human-equivalent gain-of-function mutation in Med12 develop LM in response to P4, whereas Med12 knockout blocks P4/PR signaling in mouse uterus. We hypothesize that mut-MED12 alters PR-chromatin interaction signatures and enhances P4 action in LIC, providing a support niche for LSC survival and proliferation. Using ChIP-seq, RNA-seq, STARR-seq, and CRISPR/Cas9-gene editing strategies, and in vivo PDX and mut-Med12 knock-in mouse models, we propose the following Aims: (1) Determine whether PR-chromatin interaction loci specifically associated with mutMED12 stimulate P4 action in a distinct stem-support cell population (LIC), which then send tumorigenic paracrine signals to increase LSC activity and tumor growth. We will test the hypothesis that mut-MED12 interacts with PR and alters its interaction with chromatin, thereby enhancing P4 responsiveness of LICs to activate gene transcription and paracrine signaling that support the function of adjacent LSC. (2) Define whether tumorigenic activity of mut-Med12 is mediated via altering PR-chromatin interaction landscapes in a human-equivalent mut-Med12 mouse model. We will test the hypothesis that mut-Med12 disrupts chromatin features surrounding PR-binding sites, thereby supporting gene transcription and pathways critical for LM tumorigenesis, whereas UPA shrinks LM via altering the aberrant PR-chromatin-epigenomic interactions and reversing inappropriate expression of disease-associated genes. Deciphering the genome wide mechanisms at a defined cell population level will help us identify genotype-specific novel targets associated with mut-MED12 for pharmacogenomics and precision medicine in the treatment of LM. 

Project II: The Origin and Cellular Heterogeneity of Uterine Leiomyomas (A. Rajkovic)

Project Summary: Pharmacologic therapies for uterine leiomyomas are hampered by our limited knowledge regarding the origin and evolution of these tumors, as well as the degree of molecular heterogeneity. Leiomyomas, also known as fibroids, are the most common benign tumors of uterine smooth muscle cells and are a major cause of morbidity among American women. Previous studies have suggested that fibroids are monoclonal in origin. However, histological and cell sorting analyses of leiomyomas have shown cellular heterogeneity, with presence of fibroblasts in addition to the smooth muscle cells in leiomyoma tumours. Whole exome approaches from our group and others have identified mutations in the mediator complex subunit 12 (MED12) in approximately 70% of LM patients, indicating that MED12 mutant cells might give rise to leiomyomas. We generated a mouse model that showed leiomyoma tumor formation in uteri that express Med12 mutation. We will utilize our mouse models to begin the study of leiomyoma early origins and interact with other Projects of this P50 application, to define molecular heterogeneity of leiomyomas and their relation to the tumor genotype. Our studies will focus to: 1) understand the onset and progression of Med12 mutation positive leiomyomas, 2) the evolution of genomic instability in uterine leiomyomas, and 3) understand the relationship between leiomyoma genotype and molecular heterogeneity that may impact leiomyoma recurrence rates and non-responsiveness to therapy. Our mouse model and preliminary findings will complement studies of other investigators in this P50 application. We will provide Dr. Bulun’s Project 1 with our mouse model and in vivo preliminary data that Med12 interacts with the progesterone pathway, as well as complement his studies on different human leiomyoma cell types with our own studies in mice and humans. Dr. Chakravarti’s Project 3 will benefit from our single cell sequencing on MED12 positive, HMGA2 positive, and MED12/HMGA2 negative leiomyomas and will complement his epigenetic studies on HMGA2 positive leiomyomas. Together, the studies proposed by us and our collaborators will provide great insights into the pathophysiology of uterine LM. Our studies will identify origin of Med12 positive leiomyomas, determine if genotype drives molecular phenotype and cellular heterogeneity, with a goal of driving targeted therapy for leiomyomas.

Project III: Integrative Genome wide Analyses of HMGA2 Impact on Uterine Leiomyomas (D. Chakravarti)

Project Summary: Uterine leiomyoma (LM), also known as uterine fibroids, are benign, smooth muscle tumors of the uterus characterized by extensive cellular alteration and stiffness of the extracellular matrix (ECM). Alarmingly, almost 75% of all women will develop some form of fibroids in their lifetimes, with some experiencing significant symptoms. Current medical treatments for LM have off-target side effects that limit their long-term use; surgical options are the only definitive treatment. Consequently, LM is a major gynecologic healthcare problem, and its treatment costs billions of dollars annually. Unfortunately, the molecular mechanisms underlying LM tumorigenesis and progression are poorly understood, and this has posed a significant barrier to the development of new treatment options. The proposed studies are highly significant because they will fill a gap in knowledge about this major gynecologic disease, identifying the mechanisms that drive LM formation as well as potential targets for new LM therapies. Although estrogen and progesterone signaling and TGFβ signaling have been implicated in LM, the search for potential driver mutations in LM led to the identification of genes that encode two major proteins that regulate transcription and 3D chromatin topology. Overexpression via chromosomal translocation of the gene encoding chromatin-binding protein high mobility group protein HMGA2 (HMGA2-ra) and mutations in the transcriptional mediator complex subunit Med12 gene (mut-MED12) have been identified as mutually exclusive driver mutations in LM. They together contribute to almost 85% of all LM. Previous cytogenetic, IHC, molecular and whole genome sequencing studies clearly established a role for HMGA2 overexpression in leiomyoma pathogenesis. In this proposal, utilizing these previous observations, we hypothesize that HMGA2 overexpression alters its association with chromatin, thereby changing epigenetic signatures and strongly influencing 3D chromatin topology, which alters gene expression compared to normal myometrial tissues. To test our hypothesis, the following two specific aims will be pursued. Specific Aim 1, we will define the cistrome, transcriptome, and epigenome in HMGA2-ra LM. In Specific Aim 2, we will define chromosomal 3D-topology in HMGA2-ra LM. We will use state-of-the-art genome-wide technology and bioinformatic analysis to achieve these aims. Once completed, the proposed studies will advance our knowledge of this highly prevalent public health challenge in gynecology that affects half of the world population. The proposed work is scientifically, translationally, and clinically significant because these studies will establish a rational pre-clinical framework to assess existing treatments and develop novel therapies for targeting LM, a major gynecologic disease.

Administrative Core (S. Bulun)

Abstract: The overall goal of the Administrative Core is to provide a robust centralized administrative structure that will coordinate the activities of the proposed NCTRI Northwestern Uterine Leiomyoma Center researchers and provide operational support that will reduce the burden on its investigators, allowing them to focus on their research. The Core will ensure that the proposed Center achieves its aims and will synergize the efforts of the Research Project investigators with the work of the Education and Outreach Core and the Tissue Procurement and Cell Culture Core. The Administrative Core will coordinate the participation of the Research Project investigators in the community engagement and research training activities of the Education and Outreach Core. We have developed an optimally organized Tissue/Cell Core, run by an experienced gynecologic pathologist to support LM research at Northwestern; this will provide an efficient facility for procuring and banking tissue samples and for cell preparation and storage. This facility is entirely supported by intramural funds from Northwestern. The Administrative Core will ensure and coordinate the distribution of human tissues and cells to all Center investigators. The Core will also solicit and coordinate a rigorous review process for Pilot Project applications. Below are the aims of the Administrative Core, which capture the overall objectives. AIM 1. Reduce administrative barriers within and between Center Research Projects by providing funds and resource management services. AIM 2. Foster team science through established communication methodologies to integrate cross-disciplinary research and coordinate scientific efforts within the Center and externally. AIM 3. Provide an administrative framework to evaluate the progress of the Research Projects and the Cores to ensure a rapid pace of high-quality research and to solicit Pilot Project applications and rigorously select the best Pilot Project. All aims will be achieved through the efforts of the Core Director, Dr. Bulun and the Research Administrator, Erin Simpson, who will provide overall and comprehensive administrative support. Aim 1 is designed to provide support for financial and human resources management for the investigators, projects and cores; the Bulun and Simpson team will serve as the liaison to NIH and to central offices at Northwestern. Aim 2 will be accomplished through a series of communication efforts, including recurring meetings for research progress and communication updates and the coordination of the Research Projects and Core functions. Aim 3 will support the efforts of the Internal and External Advisory Committees that will review each project’s progress and provide feedback on the overall Center and help review the Pilot Project applications and select the best Pilot Project. By aligning the Center through these activities, the Administrative Core will ensure the uninhibited progress of the Center’s research, leverage team science and cross-disciplinary efforts to accelerate the pace of discovery, and evaluate progress and trajectory so the Center is consistently on target.

Outreach/Education Core (M. Simon)

Abstract: The Education and Outreach Core of the proposed NCTRI Northwestern Leiomyoma Research Center will support research activities performed within and across the Center by developing communication, outreach, and education strategies to promote health equity and eliminate disparities in uterine fibroids (leiomyoma, LM). These strategies will be designed to engage three important communities: the general public, including women most impacted by fibroids; students in the Chicago area; and the scientific and medical community. This Core will: (i) raise awareness, education, and outreach regarding LM and disparities around LM; (ii) promote diverse participation in LM and reproductive research; and (iii) increase the number of bench and translational science research opportunities in LM with a special focus on minority and underserved students by partnering with key local pipeline programs to connect students to bench science opportunities within our Center. LM are highly prevalent, present in up to 80% of all women. However, awareness and knowledge regarding this highly morbid condition remain underdeveloped, especially amongst minority populations such as African American and Latina women, who also have a higher prevalence of fibroids than their white counterparts. Moreover, biospecimen donation and clinical research participation among racial/ethnic minority and medically underserved women continues to fall short, including in the realm of LM research. Without samples from diverse populations, discoveries in LM research will have limited applicability to diverse populations, perpetuating disparities in diagnosis, treatment, and outcomes. Few reproductive health research centers aim to connect education and outreach to building trust and increasing research participation from diverse populations in addition to increasing a diverse, future scientific workforce. Inadequate community engagement and empowerment of diverse communities has limited their awareness, education, and participation in clinical research and biospecimen donation. There is a need for development and implementation of sustainable, equitable partnerships between communities and researchers through collaborative research opportunities and reciprocal transfer of knowledge to reduce health disparities in LM. Through a community-engaged approach (e.g., community-based participatory design), this Core seeks to increase awareness, education, and participation in LM research amongst women, especially those from populations bearing the greatest burden of the disease. Through bridging the information gap and establishing stronger trust with communities, we will be able to better improve communication around and participation in research that directly connects with the work and investigative teams in our proposed Center. In addition, this Core aims to support the engagement of underrepresented students in the scientific research workforce. Building on decades of experience with community-engaged research, outreach, and workforce development, the Education and Outreach Core team will provide lab experiences to support this pipeline. We will first focus on communities across Chicago and then scale up to the US.

Abstract: Azoospermia impacts 1% of men globally, which translates to 645,000 men between the ages of 20 and 50 in the United States. It is estimated that genetic causes explain 50% of infertility. Epidemiological data suggest that fertility status may be a marker of overall health, but the genetic underpinnings are just beginning to be understood. Improved knowledge about the genetic basis of infertility and associated overall health comorbidities will aid in the counseling of infertile couples; justify the development of diagnostic screens; and may lead to patient-specific treatment options. In the current personalized medicine era with reduced cost whole genome sequencing and facile genome editing technologies, it is feasible to discover genetic underpinnings of infertility and overall health comorbidities and develop targeted therapies. Project I will discover genetic variants in men with azoospermia to identify targets for development of clinical diagnostics or therapy. Project I will use Charlson Comorbidity Index questionnaires to determine whether infertile patients or their family members have histories of other overall health comorbidities. Project II will validate infertility associated genetic variants identified in Project I and characterize the fertility and overall health phenotypes. Project II will also work collaborate with Project III to determine the impact of epigenetic perturbations on spermatogonial stem cell function. Project III will develop Sertoli cell and germ cell gene therapies in mouse models of azoospermia and will produce critical safety and feasibility data to inform the public dialogue on the risks and benefits of gene therapy in and around the germline. The Pilot project will discover the epigenetic landscape of germ cells and somatic cells from the testes of fertile versus infertile men. Core A will provide the administrative oversight and facilitate communications and data exchange among projects and cores to ensure that this P50 program achieves an impact that is greater than the sum of its parts. The Education and Outreach Core will maximize the public impact of this P50 by developing hands-on teaching modules to educate middle school and high school students as well as adults in underserved communities about reproductive medicine and the genetics of infertility. Modules will be tested and validated in the Pitt Mobile Science lab before deploying to teacher workshops in St. Louis, Pittsburgh, Ithaca and New York allowing teachers to implement the modules in their classrooms. Male focused teaching modules will also be shared with P50 centers in Oregon and at Northwestern to complement their existing female-focused hands-on modules.

Project I: Discovery and Annotation of Targets for Gene Therapy of Infertile Men (D. Conrad)

Project Summary: Infertility affects 15% of reproductive-age couples in the US, leading to more than 108,000 new visits to reproductive endocrinology and infertility (REI) clinics per year. Nearly all such clinics offer pre-implantation genetic diagnosis of embryos and genetic testing for known mutations associated with endocrine dysfunction, primary gonadal failure, and recurrent pregnancy loss. However, reproductive medicine specialists rely on old technologies like cytogenetics, sequence-tagged site PCR, and Sanger sequencing for these tests and are not taking advantage of modern whole-genome and RNA sequencing technologies common in clinical genetics of other disease states. New genomic tools, both computational and experimental, promise to revolutionize the way we diagnose and treat infertility. An overall goal of this P50 application is to create a roadmap for how these tools can be used to (a) identify mutations contributing to male infertility, (b) characterize how these mutations may contribute to pathology in somatic tissues, and (c) how gene therapy can be used to treat these pathologies in a safe and targeted manner. In this project, we are recruiting patients of male infertility from three primary sites: Washington University, Weill Cornell Medical School, and Magee-Womens Hospital. We will apply whole genome sequencing and high-resolution array CGH to map the location of genetic variation in 500 total cases, including 21 large, multiplex families with heritable forms of azoospermia. As part of the patient phenotyping, we will deploy a specialized instrument known as the Charlson Comorbidity index to specifically document the evidence for comorbidity in each case of infertility. We will develop and apply highly sensitive statistical methods, which draw upon the information in massive population control databases, to identify statistically unusual mutations that are likely to confer risk for spermatogenic impairment. We will develop knowledge bases that summarize the evidence from model organisms that these mutations can cause pathology in both gonadal and somatic tissues. And we will attempt to infer the testicular cell type(s) that are the primary sites of pathology for each mutation, to help guide the targeting of gene therapy. The most important long-term outcome of this work will be the publication of analysis tools and knowledge bases that will facilitate the use of genome sequencing in the treatment of infertility. Combined with the results from Project II and Project III, our results will give reproductive medicine specialists a roadmap for the use of modern genetic technologies to investigate and treat infertility.

Project II: Epigenetics and Genetics of Infertility and Associated Comorbidities (J. Schimenti)

Project Summary: Despite extensive knowledge of genes required for gametogenesis, the causes of most human infertilities are unknown. It is thought that about half of cases have a genetic basis, but it remains problematic to definitively identify the exact genetic lesion(s) that may be responsible in a given person, even with genome sequence information. Given the indications that certain genetically-based causes of infertility are associated with comorbidities, and the high incidence of infertility in the population, it would be of substantial clinical impact to make progress towards reliably identifying molecular causes of infertility. The roots of non-genetic causes are even more difficult to ascertain, but epigenetic alterations may underlie certain types of infertility, particularly in males where such alterations may occur in spermatogonial stem cells (SSCs). Project II has 3 Specific Aims that seek to address these important issues in the field. Aim 1 is to functionally validate candidate genetic variants identified from infertility patient cohorts and families in Project 1. This will be done by modeling the variants in CRISPR-modified mice, then phenotyping the mice for not only reproductive phenotypes, but also comorbidities that may exist in the corresponding patients, such as obesity, cancer susceptibility, or cardiovascular disease. Aim 2 is to characterize mutations that cause infertility or subfertility phenotypes in mice, and which may have comorbidities that have yet to be recognized. An emphasis will be on genes that are expressed in somatic tissues in addition to the testis. Aim 3 employs a novel approach to identify epigenetic alterations that impact SSCs, potentially leading to Sertoli Cell Only syndrome (SCOS) and non-reproductive phenotypes. This approach involves CRISPR inhibition and activation technologies to conduct a screen of known epigenetic modification genes that impact the efficiency somatic cell reprogramming and SSC maintenance. In sum, this project uses diverse, cutting-edge strategies to improve the accuracy and discovery of genetic and epigenetic causes of human male infertility and related comorbidities.

Project III: Gene Therapy for Male Infertility (K. Orwig)

Project Summary: Infertility impacts 10-15% of couples in the United States and a male factor is implicated alone or in combination with female factors in about 50% of cases. Infertility can be caused by hormonal, anatomical, immunological or chromosomal deficiencies, disease or medical treatments, but is frequently of unknown origin (idiopathic). Idiopathic infertility is difficult to counsel and treatment options are empirical. Improved knowledge about the genetic basis of infertility obtained in this program project will aid in the counseling of infertile couples; justify the development of diagnostic screens; and may lead to the development of patient-specific treatment options. Project III will test the hypotheses that: 1) Sertoli cell and germ cell gene therapies can be used to treat nonobstructive azoospermia with maturation arrest (NOA-MA); 2) gene therapy in and around the germline can be achieved with or without germline transmission and 3) germline gene therapy for NOA-MA can eliminate infertility and comorbid somatic diseases from the family lineage. Project I will discover the genetic basis of idiopathic NOA-MA and investigate personal or family histories of overall health problems. Project II will validate genetic variants identified in project I and characterize fertility and overall health comorbidities in mouse models of human NOA-MA. This project will prove the principle that in vivo or ex vivo gene therapies can be used to treat infertility in mouse models of human NOA-MA. For Sertoli cell defects, Aim 1 will prove the principle that in vivo Sertoli cell gene therapy can restore fertility in SCARKO and other mouse models of human NOA-MA with or without germline transmission. For germ cell defects, Aim 2 will prove the principle that ex vivo gene therapy followed by transplantation of spermatogonial stem cells can restore spermatogenesis and fertility in Sohlh1 and Tex11 mouse models of NOA-MA without germline transmission. Aim 3 will test germline gene therapy in Hormad1 and Mcm8 mouse models of human NOA-MA that are associated with overall health comorbidities. We hypothesize that germline gene therapy will restore fertility to the infertile male and reduce or eliminate infertility and associated overall health comorbidities from his family lineage. We will test this hypothesis by collaborating with Project II to examine overall health phenotypes in F1 progeny of gene therapy-treated males. This project will establish the safety and feasibility of gene therapies for male infertility in mouse models to support future translation to the human clinic.

Pilot Project: Epigenetics Landscape in the Testes of Fertile and Infertile Men (M. Mann) 

Project Summary: In humans, 10-20% of infertile men are azoospermic, meaning that they lack spermatozoa. Of the two types, non-obstructive (no blockage) azoospermia, in many cases, is idiopathic. Production of mature sperm involves complex developmental processes, ultimately generating specialized cells for conception. Amongst these processes includes epigenetic programming. While prenatal prospermatogonial genome-scale DNA methylation programming has been well studied, the epigenetic landscape of postnatal testicular cells has not. Current evidence suggests that aberrant epigenetic modifications may be one mechanism that leads to idiopathic, non-obstructive azoospermia. However, this may relate to cell composition rather than etiologies of spermatogenic failure. To distinguish between these possibilities, studies are required to determine whether the epigenetic landscape of specific spermatogonial and Sertoli cells is altered in non-obstructive azoospermia. In Aim 1, we hypothesize that aberrant epigenetic landscapes in postnatal spermatogonia play a role in azoospermia due to etiology rather than whole testes cell composition. To test this hypothesis, we will use two genetic mouse models with azoospermia to delineate whether DNA methylation and chromatin accessibility are compromised. The first model will eliminate all de novo methyltransferase activity using conditional Dnmt3a/Dnmt3b double mutants targeting the germ cell compartment, while the second model will employ the androgen receptor knockout (SCARKOtm2.1) targeting the Sertoli compartment. In Aim 2, we hypothesis that men with non-obstructive azoospermia will harbor aberrant DNA methylation and chromatin accessibility within either the germ cell or Sertoli compartments. To test these hypothesis, we will produce DNA methylation, chromatin accessibility and expression reference sequences from normal undifferentiated and differentiating spermatogonia as well as Sertoli cells, following which we will compare the profile of these cells to those from mutant mice, or men with maturation arrest or Sertoli cell only syndrome. For both aims, cells will be isolated using the same enrichment strategy, and DNA methylation, chromatin accessibility and expression landscapes for undifferentiated spermatogonia, differentiating spermatogonia and Sertoli cells will be generated using NMT-seq (nucleosome, methylation and transcription sequencing), allowing comparative analysis between mouse and human. Overall, we will produce foundational data about the epigenetic landscape in germ cells and somatic cells of the testes and determine whether this landscape is altered in infertile mice and men.

Administrative Core (K. Orwig)

Abstract: The Administrative Core A will provide the organization and oversight to facilitate overall program coordination and maximize the impact of the combined program. Core A will 1) facilitate internal and external communication and coordinate regular meetings among program investigators, staff, trainees, scientific advisors and NIH, 2) Foster intra- and inter-NCTRI P50 collaborations, and 3) provide regulatory and fiscal oversight to the program. Core A will schedule monthly meetings among all project investigators and core directors to review progress, address challenges, troubleshoot, coordinate collaborative experiments and plan future experiments. In conjunction with the monthly progress meetings, Core A will schedule monthly journal club meetings that are open to all program investigators, staff and trainees. The Scientific Advisory Committee(s) will join all program investigators for an annual program progress meeting (1/2 day) to be held in Pittsburgh or at the Male Focus Group Meeting site. Core A will also coordinate an annual NCTRI P50 full day symposium to be held in conjunction with the annual progress meeting. The symposium will be organized by a committee of trainees at the participating P50 institutions and feature a keynote speaker, platform presentations from P50 trainees and leaders in reproductive biology and medicine (e.g., internal/external advisory committee members) and a poster session/social. Core A will help foster intra and inter-P50 interactions by supporting collaborative pilot projects and by helping to disseminate education/outreach teaching modules through project sites in this Reproductive Genetics and Therapy NCTRI P50 center and to other P50 centers. The Core A Program Coordinator will assist the director by helping with the submission of regulatory protocols and ensuring that all program sites and staff have the appropriate training. Core A will also assist the Program Director with maintaining sub-contractual agreements with all project sites, ordering, inventory, and monitoring the budget. Finally, Core A will work with all program investigators to ensure that data are deposited in a timely fashion to a central repository that is accessible to all project investigators. This will help to integrate data collected at different program sites; accelerate the pace of research in individual projects and ensure that the program achieves an impact that is greater than the sum of its parts. Cost effectiveness will be maximized by sharing data, knowledge and resources between projects. If disputes arise, the Program Director (Dr. Orwig) will solicit input from the Scientific Advisory Committee. 

Outreach/Education Core (J. Cameron)

Abstract: The primary goals of the Education and Outreach Core are to promote a better understanding of issues concerning reproductive health and infertility in low income, underserved communities and by middle school and high school students, as well as by adults. To develop a strong relationship with the community we will work with an advocate for community health in African American and other underserved groups to organize community meetings in health clinics in underserved areas of Pittsburgh. The P50 scientists will make presentations and answer questions in these community meetings. This core will also make use of a unique resource at the University of Pittsburgh, Pitt Science Outreach (www.pittscienceoutreach.com), that works with faculty across the university to develop novel science lectures and laboratories to translate an understanding of the research happening at the university into engaging learning experiences for the public. We will develop 3 new laboratory experiences to teach about male fertility and infertility treatment. These labs will be tested and optimized in Pittsburgh schools. We will also develop teacher workshops to facilitate the transfer of these laboratory activities to schools throughout Pittsburgh, Ithaca, New York, St. Louis. In each location we will train post-docs, graduate students and undergraduate students working with the P50 scientists to facilitate these labs when they are given in local classrooms in each community. We will make the new science activities available to all NCHD NCTRI investigators, as well as publish these protocols in education journals to maximize their availability. The aims of this Core are: (1) to hold community meetings in each of three health clinics in underserved communities in Pittsburgh to provide information about the causes and treatments of male infertility, (2) to develop three new hands-on science activities that teach about male reproductive biology and the genetic causes of infertility including a testis histology lab, a mock male fertility work-up lab, and a DNA isolation, sequencing and finger-printing lab, as well as a new lecture on the effects of environmental exposures on the epigenome and potential consequences for fertility and offspring, (3) develop a Teacher’s Workshop curriculum to educate middle and high school teachers about male reproductive biology and the genetic causes of infertility and to provide them with adequate background to run these activities in their classrooms in Pittsburgh, Ithaca, New York and St. Louis, and (4) to deliver labs to reach an average of 1,000 students/year and an average of 150 adults/year in Pittsburgh in collaboration with Pitt Science Outreach, and facilitate delivery of labs to students in Pittsburgh, Ithaca, New York and St. Louis through teacher training with a goal of reaching an additional 400 students/year in underserved communities in all regions associated with this P50.

Abstract: We propose to establish a “Center for Male Reproductive Epigenomics” (herein called the “Center”) in response to the NICHD RFA-HD-19-017. The Center aims to study three key aspects of intergenerational epigenetic inheritance of environmentally-induced epimutations: 1) the impact of lifestyle (diet/activity) on the integrity of the sperm epigenome, 2) the molecular mechanisms underlying transmission of life style induced sperm epimutations to offspring, and 3) the mechanisms by which inherited epimutations can predispose disease states in offspring. By studying the sperm epigenome of male mice maintained on either a high fat diet without exercise or a normal diet + exercise, or transitioned from the former to the latter, in parallel with that of obese, inactive men maintaining an unhealthy lifestyle or transitioned to a healthy diet + exercise lifestyle, we will test our central hypothesis, i.e., paternal epigenetic inheritance of lifestyle-induced metabolic disorders is achieved through a combinatorial molecular mechanism involving sncRNAs, DNA methylation and histone modifications, which relay epimutations from somatic cells to sperm and from a father’s sperm to his offspring where they predispose development of disease-related traits. Our studies in men (Project 1) will establish the extent of the deleterious effects of an unhealthy lifestyle (high fat/caloric diet and physical inactivity) on the human sperm epigenome and will determine whether the incidence of these epimutations can be reduced if an obese/inactive man adopts a healthy lifestyle (low fat/caloric diet + exercise). Our studies in mice (Projects 2 and 3) will reveal the mechanisms by which an unhealthy lifestyle leads to formation of epimutations in spermatozoa that are subsequently transmitted to, propagated within, and deleterious to male offspring – based on mechanistic studies that cannot be done in men. In the short term, our studies will elucidate the underlying mechanisms by which an unhealthy lifestyle in men can predispose development of unhealthy phenotypes or disease in their offspring and the extent to which transition from an obese/inactive lifestyle to a healthy diet + exercise lifestyle can mitigate these effects. In the long term, a thorough understanding of how lifestyle-induced epimutations initially occur and are transmitted from a father to his sons (= intergenerational epigenetic inheritance) will form the basis for future investigations into mechanisms underlying the subsequent transmission of lifestyle-induced epimutations to multiple subsequent generations (= transgenerational epigenetic inheritance).

Project I: Diet and Exercise Modulate the Sperm Epigenome in Men (R. Swerdloff)

Project Summary: It is well known that unhealthy diet and physical inactivity in young men and women are major contributors to later-life development of metabolic syndrome, diabetes and hyperlipidemia, leading to increased cardiovascular disease risk. Genome wide association studies have identified single nucleotide polymorphisms that can only explain about 20% of the heritability of these metabolic diseases. Preclinical models provide clear evidence that dietary or exercise modifications before conception result in metabolic and phenotypic changes in the offspring through intergenerational disruption of normal epigenetic regulations of gene expression. This occurs via alterations in i) DNA methylation, ii) histone modifications, and iii) non-coding RNAs (ncRNAs) both in animal studies and in men. Our central hypotheses are that overweight and inactive lifestyle results in epimutations in the sperm epigenome relative to the normal epigenetic programming in lean and active men and that diet and exercise modulation leads to reversal of these epimutations resulting in both a healthier “phenotype” and “epigenotype” which may persist after stopping the interventions. We propose three aims: Aim 1. Determines the differences in sperm epigenome (DNA methylation, histone modifications and non-coding RNAs) in a cross sectional study in obese inactive vs. healthy active Hispanic men. We will recruit 20 healthy, active men and 80 obese and inactive Hispanic men between 18 and 40 years for this Aim. Only Hispanic men will be studied because of the high prevalence of obesity and inactivity in Hispanic younger men and to reduce the genetic variability influencing the epigenome. Aim 2. Characterize the plasticity of the sperm epigenome in response to 12-week diet and/or exercise training interventions in obese and inactive Hispanic men. 80 obese and inactive men will be randomized to 4 groups of 20 men: 1) No intervention (control); 2) Low fat, low caloric diet; 3) Supervised, periodized endurance and resistance training without modification of diet; and 4) Both exercise and diet modification. Sperm epimutations will be compared before and after intervention within each group and between groups. Aim 3. Identify the persistent effects of diet and exercise training at 12 and 36 weeks after cessation of interventions on the sperm epigenome after stopping the interventions. Project aligns seamlessly with the goals of the Center of Male Reproductive Epigenetics and with studies in mice in Project 2 and 3 that will reveal the mechanisms by which an unhealthy lifestyle leads to formation of epimutations in spermatozoa that are subsequently transmitted to, propagated within, and deleterious to male offspring – based on mechanistic studies that cannot be done in men.

Project II: - Mechanism Underlying the Transduction of Epimutations from the Soma to the Male Germline (W. Yan)

Project Summary: Most of the studies on epigenetic inheritance focus on the identification of sperm-borne factors that carry the epigenetic memory and how they act on the epigenome/genome of the embryos so that the specific epigenetic memory can be recalled and manifested as a specific phenotype in offspring. However, one fundamental question remains unanswered: given that the effects of exposures, either environmental or dietary, are presumably initially manifested as epigenetic changes in directly exposed somatic cells (e.g. pancreatic islet cells, adipocytes, hepatocytes, etc.), how do the phenotype-specific epimutations in somatic cells get transduced into spermatozoa? Using a highly reproducible mouse model for intergenerational epigenetic inheritance of a high fat diet (HFD)-induced metabolic disorders, we here propose a series of experiments to tackle this critical question. Our central hypothesis is that HFD induced epimutations in somatic cells can lead to production of specific sncRNAs that are either encapsulated in extracellular vesicles (EVs), or present as mobile RNAs, which act as the carrier of epigenetic memory once internalized by spermatozoa through either 1) the intra-testicular mechanism (i.e., Sertoli cell HDF-specific epigenetic information transmitted to all developing male germ cells or directly to spermatozoa during spermatogenesis in the testis), or 2) the post-testicular pathway (i.e., HFD-specific epigenetic information transmitted from male reproductive tract epithelial cells to spermatozoa), or 3) a combination of both. To test our hypothesis, we propose to identify when and where male germ cells gain the ability to transmit the HDF-induced metabolic disorder phenotype (Aim1), to study how the intra-testicular pathway contributes to the HFD-specific sperm epigenome (Aim2), to study how the post-testicular pathway influences HFD-specific sperm epigenome (Aim3). Data to be obtained will help fill the knowledge gap in our understanding of the molecular mechanisms underlying the intergenerational epigenetic inheritance of paternally acquired traits in general.

Project III: - Germline-mediated Transgenerational Epigenetic Inheritance of Paternal Epimutations Induced by a High Fat Diet (J. McCarrey)

Project Summary: Abundant data from many labs has established that environmental exposures can predispose development of disease characteristics in an exposed male and also in that male’s offspring, even if the offspring are never, themselves, directly exposed to the original disruptive influence. In addition to chemical exposures, deleterious lifestyle choices such as consumption of an unhealthy diet, lack of exercise, smoking, etc., can predispose disruptions of the epigenome (epimutations) that can be subsequently propagated to many cells or organs in the exposed male’s body, including to his sperm. Once in the exposed male’s sperm, lifestyle-induced epimutations can then be transmitted to the male’s offspring on the basis of epigenetic inheritance, where they can predispose development of similar disease characteristics. Though this phenomenon has now been studied for >10 years, there remains very little information about the underlying molecular mechanisms. We propose a novel, comprehensive, mechanism-focused set of experiments to be conducted using a mouse model subjected to two effects mimicking deleterious lifestyle choices in humans – i) consumption of a high-fat diet and ii) lack of a structured exercise regime. Specifically, we propose experiments designed to 1) identify the specific combination of epigenetic parameters involved in transmission of lifestyle-induced epimutations from sperm to the ensuing fetus, 2) reveal the extent to, and mechanisms by, which resulting epimutations in the F1 fetus are propagated to developing somatic and germ cell lineages and on into the immature and adult offspring, 3) determine the extent of intercellular homo- versus hetero-geneity of life style induced epimutations in spermatogenic cells of the sire and in relevant germ and somatic cell types in his offspring, 4) discern the mechanisms by which inherited epimutations contribute to dysregulated gene expression in tissues relevant to aberrant/disease phenotypes in the offspring, 5) elucidate dysregulated pathways responsible for defective or disease states among offspring of sires transmitting lifestyle-induced epimutations, and 6) determine the extent to which the incidence of all of these deleterious effects can be reduced by transition of males from an unhealthy to a healthy life style, including a normal diet and exercise. A broad range of analyses of epigenomic parameters is proposed as a means to investigate mechanisms underlying the etiology and paternal transmission of lifestyle-induced, intergenerational epimutations. Results of the proposed research will inform future efforts to prevent, diagnose, treat or reverse the deleterious epimutagenic effects of siring offspring while engaged in an unhealthy lifestyle.

Technical Service Core: Animal and Bioinformatics Core (H. Zheng)

Project Summary: The Animal Core (Core C) will provide centralized services by generating all the mice needed for the Center projects. A centralized supply of mice by the Core will ensure that mice with the same genetic background are used to minimize potential variations in phenotypic, genomic or epigenomic analyses. The fact that Core personnel are well trained for HFD treatment, skillful in monitoring development and health status, and measurement of GTT and ITT will ensure consistent data quality and reproducibility. Specifically, Core C will generate high-fat diet (HFD)- and normal diet (ND)-fed wild-type and transgenic mice, collect phenotypic data and prepare sperm and tissue samples for the two basic projects. Both projects 2 and 3 will work on the effects of HFD and/or inactivity on sperm epigenome and offspring health. Both WT or transgenic (TU tracer and KitcopGFP) mice will be on a HFD or a ND for 3 months, and Project 3 also needs to reverse the HFD effect by feeding HFD-treated mice with ND and introducing to a running wheel. To analyze the effects of altered sperm epigenome in response to HFD and/or exercise, the treated male mice need to be bred with WT female mice to generate F1 progeny. Core C will maintain the mouse colonies, perform all the treatments, collect phenotypic data and provide sperm and tissue samples needed for the experiments proposed in Projects 2 and 3. 

Technical Service Core: Center for Male Reproductive Epigenomics (T. Zhou)

Project Summary: While traditional genetic studies to investigate DNA or RNA nucleotide information, epigenetic analyses aim to characterize chemical modifications within both DNA/RNA sequences and histone proteins. Epigenetic changes are preserved during cell division and potentially modify the expression of certain genes instead of altering the genetic code of nucleotide sequences, which is not only due to regular and natural occurrence but also influenced by several factors including age, the environment/lifestyle, and disease status. More importantly, some epigenetic modifications can potentially be transmitted to offspring by transgenerational epigenetic inheritance. Our more recent study suggests that this intergenerational transmission of paternally acquired metabolic disorders is mediated by a nucleic acid modification enzyme called DNA (cytosine-5)-methyltransferase 2 (Dnmt2). Inspired by these findings, this P50 will focus on the relationship between epigenetic profiles and metabolic disorders in both animal models and human subjects. To study the epigenetic regulation, it is critical to know not only what kinds of modifications are present but also their genomic loci. Therefore, high-throughput sequencing will be applied to profile epigenetic alterations in both animal and human samples. This Epigenomics Core (Core D) will provide essential, cutting edge, bioinformatical expertise to facilitate the translational impact of this highly integrated P50. Firstly, Core D will coordinate outsource library sequencing for individual projects (Specific Aim #1). Secondly, Core D will provide centralized computational service to store, process, and analyze the sequencing data, using our in-house well-characterized pipelines (Specific Aims #2). Finally, Core D will be responsible for hosting an in-house web-based server to share the omics data (Specific Aim #3), which provides both the internal and external researchers an easy access to the data generated from individual projects.

Administrative Core (W. Yan)

Abstract: The Administrative Core (Core A) is the principal management component of the Center for Male Reproductive Epigenomics and thus, is critical to the success of the entire Center. Core A will serve as an interface between the Center Project Leaders (PLs), Core Directors, and the Internal (IAC) and External (EAC) Advisory Committees, as well as the three participating institutions. Core A will provide administrative support for the three projects and two technical core labs, as well as the Community Outreach and Education core, including scheduling meetings, coordination of the usage of existing core facilities, managing the budget, helping seminar speakers with their travel and itinerary, depositing data for sharing, facilitating recruitment activities, dealing with public relations, etc. Core A will also maintain the Center’s website that will serve as a window to the public and information source for other NCTRI programs around the nation by displaying relevant information about the Center, including announcements of Center meetings, seminars, workshops, event calendar, noteworthy news, research highlights, publications of Center participants, etc. To accomplish these administrative functions, we will provide centralized administrative and scientific support and oversight to all projects and cores (Aim1), communicate with both the Internal Advisory Committee (IAC) and the External Advisory Committee (EAC) and coordinate the IAC and EAC meetings to enhance the performance of the Center (Aim2), provide financial and managerial support to ensure the Center’s financial health and to minimize the managerial burden of Project Leaders and Core Directors (Aim3), and assist the Internal Advisory Committee in resolving issues of conflict of interest, financial disputes or other matters that are not resolved by the Executive Committee (Aim 4).

Outreach/Education Core (C. Wang)

Abstract: The Community Outreach and Education Core of the Center for Male Reproductive Epigenomics will be located and operated though Los Angeles Biomedical Research Institute (LA Biomed) at Harbor-UCLA Medical Center located in South West Los Angeles. This Core will develop outreach and education programs designed to disseminate information on the effects of life styles on the epigenetic regulation of sperm gene expression that may be transmitted to the next generation. Our theme is to promote “Healthy life style, Healthy sperm epigenome, Healthy babies”. Building on our prior collaboration and community-academic partnership, we plan to achieve our goals by engaging youths, high school and college students, and younger adults from our communities to disseminate information on reproductive epigenomics and their effects on the next generation. We have four aims: Aim 1. Promote and sustain bidirectional knowledge sharing between the community and academic institutions by providing opportunities for networking and developing a community specific dissemination plan for evidence based information on how obesity and inactivity change the sperm epigenome transmitting unhealthy phenotype and epigenotype to the next generation. Aim 2. Create educational programs, workshops and conferences to engage younger adults, college and high school students (future parents) to disseminate information on “healthy life styles, healthy reproductive epigenome; healthy babies”. With our partners we will conduct community-partnered research conferences and workshops for younger adults, and summer program for high school and college students to provide knowledge transfer on the impact of environment on epigenetics? how improvement in the environment may reverse maladaptive epigenetic changes? and how epigenetics are linked to intergenerational transmission of disease phenotype? Aim 3. Develop a community focused website for the Center for Male Reproductive Genomics. Working with our community partners, academic partners at the University of Nevada Reno and the University of Texas San Antonio, we will develop a culturally and racially sensitive website to provide information on the role of epigenomics in reproductive health in general and impact of sperm epigenomics on next generation’s health. Aim 4. Establish the governance and operation structure of the Community Core Advisory Council that strengthens existing partnerships and builds new bridges between community and academia for education and research.

Abstract: The Cornell Center for Reproductive Genomics (CRG) was founded in 2007 with the goal of leveraging state-of the-art genomics technologies for understanding the biology of the mammalian germ cell. More specifically, our goal has been to understand the genetic, epigenetic, and epitranscriptomic basis for the generation of viable healthy gametes and to explore how alterations in these events could contribute to human infertility. It is well known that disruption of genes required for regulating all aspects of gene expression, including chromatin modifiers, the transcription machinery, and components of post-transcriptional regulatory pathways, leads to the formation of spermatozoa with abnormal head morphology in the mouse, while sperm from men with increased abnormal sperm morphology significantly higher rates of chromosomal aneuploidy, chromatin compaction defects, and altered transcriptome profiles compared to sperm from fertile men. Thus, in this application, we seek to understand how transcriptional, post-transcriptional, and epitranscriptomic regulation of gene expression and chromatin state contributes to the differentiation of haploid germ cells into mature spermatozoa. Three projects are proposed and three cores are proposed. PROJECT I (Danko and Cohen) will focus on the importance of transcriptional regulation of gene expression at the exit from meiosis and entry into spermiogenesis, with a focus on the role of the bromodomain protein, BRDT in facilitating transcriptional shutdown and thus permitting appropriate histone-to-protamine replacement and nuclear compaction. PROJECT II (Grimson, Schimenti, Hwang) will focus on mechanisms and functions of post-transcriptional processing and regulation of mRNAs during spermiogenesis and whether defects in these processes can underlie defects in sperm morphology in patients seeking assisted reproductive technologies. PROJECT III (Jaffrey) will explore the dynamics of N6 -methyladenosine (M6A) and N6 , 2’-O-dimethyladenosine (m6Am) modifications on RNA through spermatogenesis in mice and in men, and the importance of these epitranscriptomic changes for the production of healthy sperm in mice and men. These studies will be supported by a well-established ADMINISTRATIVE CORE (Cohen) that will facilitate close interactions through regular meetings, trainee events, pilot and seed grants, and our popular “Tri-Repro” Annual Symposium. Our state-of the-art GENOME INNOVATION CORE (Grenier) will serve as an Innovation Hub for exploring all aspects of gene regulation in reproduction, specializing in a range of next generation sequencing technologies to support the projects. Finally, our OUTREACH CORE (Lin) will provide lab opportunities for nearby, and traditionally underserved, school districts throughout upstate New York, at the same time sending our trainees and faculty out to these communities as role models for young budding scientists. Our center will benefit from the strong research and clinical integration we have established over the past 13 years, by robust and unequivocal institutional support, and by the outstanding scientific environment provided by Cornell University.

Project I: Investigating the role of bromodomain-containing proteins in the production of viable spermatozoa and male fertility (P. Cohen and C. Danko)

Project Summary: Spermatogenesis is a highly unique differentiation process that involves complex mechanisms of gene regulation, particularly at the level of the transcriptional machinery. The complexity of this process is underscored by the fact that 30% of male infertility is attributable to sperm morphology defects and/or poor semen quality. Bromodomain-containing (BD) proteins are critical regulators of transcription, which act by binding acetylated histone residues at their target loci and recruiting the appropriate transcriptional regulators. Mutations in the genes encoding three BD proteins (BRDT, BRD4 and BRWD1), or that of their interacting proteins, result in morphologically abnormal sperm in mice, and have been associated with poor semen quality and infertility in men. We hypothesize that BRDT, BRD4, and BRWD1 play interconnected roles during meiotic prophase I and spermiogenesis, two key stages of spermatogenesis during which stringent transcriptional regulation is exerted, to ensure appropriate transcriptional control and chromatin compaction leading to the production of morphologically normal sperm. We propose the following model: (1) during meiotic prophase I, BRDT ensures appropriate temporo-spatial control of transcriptional repression to allow for the events of recombination and synapsis; (2) then, upon entry into spermiogenesis, BRDT aids in chromatin compaction during histone-to-protamine exchange, by shutting down transcription across the genome; (3) at the same time, BRD4 and BRWD1 are required to overcome the transcriptional silencing imposed by BRDT specifically at genes essential for spermatid development. Studies herein will test this integrated model of BD action in mice and men. In Aim 1, we will elucidate the role of BRDT in mediating progressive transcriptional shut down during meiotic prophase I using a mouse mutant lacking Brdt. We will examine meiotic progression in wildtype and mutant spermatocytes, and will define the genome-wide distribution of BD proteins and components of the transcriptional machinery. We will ask whether BRDT function is dependent on synapsis or recombination. In Aim 2, we will explore the role of BRWD1 and BRD4 in overcoming BRDT-mediated repression and ensuring expression of critical spermatid differentiation genes. We will investigate how BD proteins co-operate to ensure the correct chromatin environment is in place to allow for the progressive nuclear compaction that arises due to the histone-to-protamine exchange. In Aim 3, we will map the genome-wide distribution of BRDT and BRWD1, histone acetylation, and transcription in human testis, with matched analysis of sperm morphology. By combining these data with analysis of sperm chromatin compaction in wildtype and BD knockout mice, we will develop machine learning tools that permit stratification of sperm from infertile men based on BD protein function and chromatin compaction. These studies are the first to elucidate the coordinated roles of BD proteins in ensuring normal sperm morphology and we will apply our understanding of this transcriptional regulation to defining the causes for sperm defects in infertile men.

Project II: Impact of 3' untranslated region sequence variants in spermiogenic gene expression and infertility (A. Grimson and J. Schimenti)

Project Summary: Male infertility is well-recognized as a major health problem in the United States. Clinically, the major cause is poor semen quality, that is, oligozoospermia, asthenozoospermia, and/or teratozoospermia. About half of infertility cases are thought to be genetic in nature, although the molecular bases are mostly unknown. Remarkably, these sperm defects are not limited to infertile men; ~2% of all men exhibit suboptimal sperm parameters, and the sperm counts among men in many developed countries have more than halved in the past 40 years. Again, the underlying causes are unknown. Thus, it is a priority to understand the molecular basis of these disturbing increases in reproductive problems in men. With respect to genetic and epigenetic contributions to infertility, there are many stages and molecular processes that can be affected, including all stages of spermatogenesis, and roles for thousands of genes required for fertility. This project focuses on posttranscriptional gene regulation during spermiogenesis, the elaborate process by which round haploid spermatids undergo dramatic morphological development to become mature, flagellated spermatozoa with a highly compacted, inert genome. Post-transcriptional gene regulation plays a substantial role in spermiogenesis. The 3′UTR (3′ untranslated region) is central in this respect, affecting mRNA stability, translation and localization by influencing polyadenylation, access to regulatory proteins and noncoding RNAs, and loading onto ribosomes or ribonuclear protein particles (RNPs). This project seeks to understand if and how variation/mutation in the 3′UTRs of infertility patients plays a role in haploid gene expression and infertility. Aim I will determine if there are systematic or specific alterations in the post-transcriptional regulatory function of 3′UTRs in fertile versus infertile men, using 3′RNA-seq of round spermatids purified from ejaculates, and public genome sequence data. Based on these data, we will identify and prioritize variants that reside within motifs predicted bioinformatically to impact mRNA stability or other relevant attributes, focusing on genes implicated in spermiogenesis. In Aim II, candidate variants will be screened via a high-throughput in vitro system to identify those that indeed have an impact on mRNA stability and/or translation. Finally, Aim III will create mouse models of these putative pathogenic variants, and assess molecular and phenotypic effects upon reproductive parameters such as sperm number, morphology, motility, and fertility. Overall, if successful, this will be the largest effort to date to identify genetic lesions that cause infertility via disruption of posttranscriptional gene expression.

Project III: Epitranscriptomic control of mRNA and noncoding RNAs in spermatogenesis (S. Jaffrey)

Project Summary: Spermatogenesis is a carefully orchestrated process in which spermatogonial stem cells differentiate into spermatids and eventually motile sperm. This process requires a series of changes in epigenetic changes, chromatin reorganization, dynamic changes in mRNA 3’UTR length, and temporally regulated patterns of translation. Emerging evidence suggests that precise stage-specific alterations in the epitranscriptome are also required for proper spermatogenesis. For example, spermatocyte-specific depletion of “readers,” “writers,” or “erasers,” of N6 -methyladenosine, a modified nucleotide that impacts long noncoding RNA (lncRNA) function and mRNA stability, translation, and splicing, are all associated with stage-specific arrests in spermatogenesis. Additional data suggests that spermatogenesis is also affected by N6 , 2’-Odimethyladenosine (m6Am), a modified adenosine that is found exclusively at the first transcribed nucleotide position of certain mRNAs. Based on these studies, it is clear that epitranscriptomic modifications are required for spermatogenesis. However the mechanisms by which m6A and m6Am regulate spermatogenesis remain unclear. In order to decipher the role of the epitranscriptome in spermatogenesis, the specific aims of this project are: (1) To map m6A in a cell-type specific and quantitative manner during spermatogenesis. We will develop new methods to selectively map m6A in animals, and determine if dynamic changes in m6A control mRNA 3’UTR length. These methods will reveal the dynamics of m6A levels throughout spermatogenesis and if m6A function is involved in orchestrating 3’UTR length dynamics that is characteristic of spermatogenesis. (2) To determine the role of m6A in controlling the epigenome during spermatogenesis. m6A is often enriched in lncRNAs, and it can affect their ability to induce epigenetic gene silencing. We will identify chromatin associated lncRNAs that contain m6A and determine how m6A affects epigenetic dynamics during spermatogenesis. (3) To determine how YTHDC2 and m6Am regulate spermatogenesis. YTHDC2 is required for meiotic progression by binding and regulating m6A mRNAs. However, YTHDC2 shows relatively weak binding to m6A. We find that YTHDC2 shows high-affinity binding to m6Am. We will map m6Am during spermatogenesis, and determine the function of m6Am by depleting its biosynthetic methyltransferase. We will also determine if the function of YTHDC2 is to regulate the stability or translation of m6Am mRNAs during spermatogenesis. Together, these experiments will reveal the cell-type specific dynamics of the epitranscriptome and will reveal how these epitranscriptomic modifications affect epigenetic states, mRNA stability, mRNA 3’UTR processing and mRNA translation during spermatogenesis. 

Genomics Innovation Core (J. Grenier)

Abstract: The primary objective of the Core is to provide high quality, state-of-the-art, efficient and cost-effective genomics services and resources to enable research on gene regulation. For the past six years, the Core has offered RNAseq (transcriptome) and small RNA sequencing services to the Center for Reproductive Genomics and the wider Cornell and NCTRI research communities under the name “RNA Sequencing Core”, recently updated to the “Transcriptional Regulation and Expression” (TREx) Facility to reflect a widening scope of services. All services include library generation, sequencing, and standard analysis, as well as pre- and post project consultation on experimental design and custom data analysis respectively. In addition to the end-to end, fee-for-service genomics services provided by TREx and with significant financial support from the Cornell Provost, we have recently launched the “Genomics Innovation Hub” as an independent, collaborative technology development lab. The Genomics Innovation Hub provides expertise and shared resources for cutting-edge genomics applications with a focus on combinatorial indexing strategies for specialized or single cell readouts of epigenomics and gene expression, and will enable the use of these technologies for reproductive research. Together, these operations will represent the Genomics Innovation Technology Core for the Center for Reproductive Genomics.

Administrative Core (P. Cohen)

Abstract: The goals of the Administrative Core are to provide structure and support for the research and outreach activities within the Cornell Center for Reproductive Genomics (CRG), to facilitate interactions across the two Cornell campuses (Ithaca and Weill Cornell Medicine) and with our clinical research partners at the University of Pittsburgh, and to encourage new research interactions with members of the Cornell community and with NCTRI centers nationwide. This application seeks to build on the firm foundations established over the past 13 years which include: (a) a robust Administrative team consisting of Dr. Paula Cohen (PI and Core Leader), James Bender (CRG Administrator), together with a strong support team; (b) frequent opportunities for intellectual interactions, including monthly work-in-progress seminars, informal weekly video conferences, CRG-sponsored seminars throughout the year, and a trainee-led journal club series; (c) a well-established infrastructure for ongoing interactions through frequent videoconferencing, the Cornell-hosted bus service, and accommodation available on all campuses; and (d) a highly popular Annual Symposium that is open to all members of the Cornell community and to our neighbors in the North East. Since 2019, our symposium has been named The Tri-Institutional Reproductive Sciences Symposium (“Tri-Repro”) since it is now co-hosted with colleagues at the University of Pennsylvania and the Magee Womens Research Institute (MWRI) in Pittsburgh. Tri-Repro focuses on the achievements of our trainees and serves to further enforce our collaborations with Dr. Kathleen Hwang (co-leader on Project II, collaborator on Project III), while fostering collaborations throughout the North East. The Core will oversee our Pilot Project program in which at least one pilot project will be funded each year. We will also continue the CRG’s long-standing Trainee Seed Grant Initiative which seeks to provide trainees with grant writing experience, and to establish innovative projects relating to the missions of the CRG, focusing on gene regulation in the reproductive axis. In addition to weekly informal videoconferences, the Core Leader meets regularly with the Steering Committee, consisting of all Core/Project leaders, and Trainee Executive Committee to discuss progress, cohesion between the group, and to improve our collaboration and mentorship. We have assembled an exceptional Internal Advisory Committee to whom the Core Leader will report frequently, and who will meet with all Center members on a biannual basis. An equally strong External Advisory Committee will monitor the Center’s progress, provide input on direction and productivity, advise on any conflict issues, and attend the annual Tri-Repro meeting. The Core will provide robust support for the P50 core/research components, and with strong institutional support and funding, we will continue to prioritize the highest quality research matched by frequent opportunities to interact and discuss science. These avenues for communication and collaboration will allow the collective Center to become far greater, and have far more impact, than its individual components would do alone.
 

Outreach/Education Core (D. Lin)

Abstract: The goal of this outreach Core is to enhance and expand upon Fall into Science, a K-12 outreach program established by the Director. More than 120 students have participated in Fall into Science since its founding six years ago, and the group has made presentations to more than 1800 area students. Funding from this Core will allow us to recruit students from other graduate Fields on campus, enabling us to expand our ongoing outreach activities and develop new programming. We will further leverage funding for the Core by working with existing groups on campus with established records of performing K-12 outreach. In addition, we will develop new relationships with schools in more rural communities to reach out to new audiences. Finally, we will develop new, reproductive biology-oriented activities to teach fundamental principles of reproductive biology to middle school students.

Abstract: Infertility affects up to 13% of reproductive age couples across the globe but the underlying mechanistic basis of infertility and the role of infertility as an overall marker of general health is unclear. These knowledge gaps hinder the diagnosis, treatment and prevention of infertility leading to physical, psychological and financial burden to couples with infertility. To address these challenges, The Massachusetts General Hospital Harvard Center for Reproductive Medicine has assembled an integrative team of investigators with expertise in reproductive medicine, genomics, population genetics and genetic literacy who will conduct clinical translational investigation in humans with infertility: Aim 1: To elucidate the genetic and phenomic architecture of infertility through the lens of specific rare diseases and common traits; Aim 2: To catalyze a collaborative think-tank focused on reducing the suffering and costs of infertility, in a manner that is outward looking, forward thinking and integrates global perspectives; Aim 3: To nucleate a vibrant hub for outreach, training and community engagement that brings the scientific team closer to the patients they serve, the trainees they want to mentor, and the larger community of scientists and clinicians who are invested in reducing the burdens caused by infertility. These aims will be achieved using two Clinical Research projects which will be supported by a Genomics and Functional Core, Outreach Core and Administrative Core. Project 1 will perform next-generation sequencing and targeted genotype-driven phenotyping studies in clinical cohorts enriched for genetically driven infertility from admixed and consanguineous populations characterized by both hypogonadotropic and hypergonadotropic hypogonadism in both sexes. Project 2 will bring together >1,800,000 multi-ethnic population biobank samples to perform genome-wide association studies, phenome-wide association studies, and Mendelian randomization studies to implicate key biologic pathways determining fertility and synthesize the genetic results across infertility and related traits to characterize the effects of the identified genes and pathways on reproductive health and overall morbidity. The Genomics and Functional Core will provide genomic technologies, data analytics, computational and statistical support, and will generate genetically engineered human induced pluripotent cells for validation of Project 1 & 2 genetic discoveries. The Outreach Core will buoy Project 1 & 2 activities by engaging key stakeholders through patient group meetings, creating clinician-facing materials to enable communication of genetic results and launch a website inspired by design-thinking for broad dissemination to patients, families and clinicians. The Administrative Core will advance the Center’s scientific goals by providing timely support to foster engagement and communication between investigators, patients, research trainees and the broader academic community. Through these interdigitating activities, the Center will train the next generation of reproductive biologists and the results emanating from its activities will help inform clinical care and alleviate the suffering of patients with infertility. 

Project I: Deciphering the Molecular Drivers of Rare Forms of Human Infertility Using Integrative Genomic, Cellular, and Phenomic Approaches (S. Seminara)

Project Summary: Infertility affects up to 13% of childless couples yet the biological mechanisms underlying infertility and the effects of infertility on overall health remains poorly understood. The goal of Project 1 within The Massachusetts General Hospital Harvard Center for Reproductive Medicine will be to apply human genomics, transcriptomics and phenomics to understand the mechanistic biological drivers of rare disorders of human infertility. A pervasive methodologic theme of 'omics’ technologies runs through Project 1’s three specific aims. In Aim 1, infertility will be viewed through the prism of rare hypogonadotropic and hypergonadotropic conditions that cause infertility. Clinical investigators will apply contemporary genomic techniques to define the underlying genetic architecture of infertility and to identify master regulatory pathways and networks that determine fertility. Three unique clinical cohorts will be utilized to achieve this aim: US-based admixed cohorts of patients with idiopathic hypogonadotropic hypogonadism (IHH)/Kallmann Syndrome [Massachusetts General Hospital] and primary ovarian insufficiency [University of Utah]; and, a Saudi Arabia-based consanguineous cohort of patients with a spectrum of rare Mendelian forms of infertility. The full spectrum of genetic variation (coding and non-coding single nucleotide variants, insertion/deletion variants and structural variants) that confer substantial relative risk for infertility will be determined and causal genes identified will be coalesced into common, final pathways elucidating the predominant drivers of rare forms of infertility. In Aim 2, genetic variants identified from Aim 1 and variants identified in Project 2 of the Center that relate to hypothalamic-hypogonadotropic forms of infertility will be validated in CRISPR-engineered GnRH neurons derived from induced pluripotent stem cells generated by the Genomics & Functional Core of the Center. Specifically, the cellular and molecular consequences of genetic variation on GnRH neurons will be defined by comparing and contrasting the GnRH transcriptome, morphology, migratory capability and secretory function between wild-type and edited GnRH neurons. Similarly, genetic variants relating to hypergonadotropic forms of infertility leading to primary ovarian insufficiency will be studied in a Drosophila model system by ovary-specific RNAi knockdown (or overexpression) experiments. Finally, in Aim 3, a hospital-based human biobank (Partners Biobank) will be utilized to perform a recall-by-genotype based targeted phenotypic evaluation in individuals harboring pathogenic variants in infertility-associated genes. The full reproductive phenotype (“reproductome”) will be defined using deep phenotyping studies that will define the effects of harboring genetic risk variants on GnRH-induced pituitary LH pulse profiles and hypothalamic-pituitary responsiveness to exogenous kisspeptin administration. Through these coordinated studies, this project will aid in the diagnosis and management of infertility, inform genetic risk prediction for infertility conditions, and facilitate the development of novel therapeutic options for infertility.

Project II: - Deciphering the Molecular Drivers of Common Forms of Human Infertility Using Integrative Genomic, Cellular, and Phenomic Approaches (C. Lindgren)

Project Summary: Infertility is a common condition that affects over one-tenth of couples attempting to conceive, and the costs of managing infertility exceed 18 billion US dollars worldwide. There are significant gaps in our knowledge of the underlying mechanisms and risk factors for infertility, and these gaps impede the development of targeted treatments that address the root causes of infertility. Project 2 will fill these gaps by conducting human genetic studies of unprecedented scale to study infertility, and related reproductive traits. Through collaborations with large biobanks worldwide, we will access data from over 18 million individuals to identify genetic variants associated with risk for female and male infertility as well as those associated with a number of fertility-related traits, including sex-hormone concentrations, pubertal onset, and pituitary gland and olfactory bulb size (Aim 1). We will then use state-of-the-art computational approaches to pinpoint the specific variants, genes, pathways, and cell types that influence these conditions and traits (Aim 2). These approaches include fine-mapping, pathway analyses, and intersection of genetic association data across studies, and they will be fueled by data from Project 2, Aim 1, as well as data on genetic variants associated with rare infertility conditions identified in Project 1, data on gene expression generated by the Core, as well as extensive publicly available data on genetic linkage patterns, gene expression, protein-protein interactions, and other experimental findings. We will then integrate all these findings to understand how infertility and the reproductive traits studied in Aim 1 relate to each other, with the rare infertility conditions in Project 1, and with general reproductive and non-reproductive health (Aim 3). Phenome-wide association studies will analyze a large number of phenotypes to reveal novel phenotypic associations for the genetic variants studied, investigations of pleiotropy will determine the extent to which the genetic causes of these conditions and traits overlap, and the technique of Mendelian randomization will be used to infer causal relationships. By leveraging the power of large-scale human genetics, this project will lead to a comprehensive understanding of the biology of fertility and infertility, identify targets for fertility treatments, and reveal the pathways by which reproductive health influences overall health.

Technical Service Core: Genomics Core (M. Talkowski)

Project Summary: The Center for Reproductive Medicine (CRM) represents a multi-disciplinary program to explore the genetic architecture of infertility. The CRM Genomics Core (GC) described herein envisages providing a centralized and catalytic resource for genomic variation, statistical association, and functional perturbations for all components of this overarching CRM program. This genomics hub will specifically catalyze discoveries by providing expertise in the data and methods that will be leveraged for deeper insights into rare and common forms of infertility in Projects 1 and 2, respectively. The GC will also serve as the focal point for cross-fertilization of data, analyses, and functional modeling across the program. Over the last several years, we have developed a compendium of computational tools, statistical approaches, and functional genomics methods to interrogate the mutational spectrum of variation in human diseases. Our methods incorporate joint analyses of short variants (SNVs, indels) and structural variants (SVs), including canonical balanced SVs and copy number variants (CNVs), as well as a diverse catalog of complex SVs that are surprisingly abundant and associated with an array of human disease. These studies have required methods to uniformly generate, process, and rigorously analyze genomics datasets for association studies. In the GC, we will discover and annotate variation, interpret association against population-scale datasets in excess of 1,000,000 genomes from our related studies, and perform scalable engineering to generate an allelic series of perturbations in genes associated with rare and common forms of infertility using human induced pluripotent stem cell (hiPSC) derived GnRH models. Overall, we will support the CRM by completing three objectives related to providing datasets, methods, and functional resources. Objective 1 will develop a comprehensive genomics resource from exome, genome and long-read sequencing, and uniform data processing of the CRM cohorts. Objective 2 will perform integrated rare variant association and interpretation of these datasets by jointly analyzing CRM cohorts with population-scale datasets generated in our genome aggregation database (gnomAD) project and complex disease consortia studies. Objective 3 will then perform scalable CRISPR perturbation of infertility genes in GnRH neuronal models by engineering loss-of-function mutations and an allelic series for select infertility genes. Transcriptional profiling in the genomics core will identify signatures associated with perturbation of these infertility genes, and will seek convergence of these signatures on a small number of infertility relevant pathways. All CRISPR-engineered models will be distributed relevant projects for further functional assays, and all data and models will be made openly available for distribution to the community. These objectives in the genomics hub of the CRM will thus provide datasets, gene discoveries, and CRISPR-engineered isogenic models to facilitate new insights into infertility within the CRM and the broader research community.

Administrative Core (S. Seminara)

Abstract: The primary function of the Administrative Core within The Massachusetts General Hospital Harvard Center for Reproductive Medicine will be to provide an organizational platform that will seamlessly integrate the Center's scientific, regulatory, human resource, and financial interfaces across the two individual research Projects, two supporting Cores and collaborating institutions. These functions will be achieved through three aims. Aim 1 is to support the primary scientific goal of the P50 in elucidating the genetic and phenomic architecture of rare and common infertility traits. To achieve this aim, the Core will provide administrative support to: (a) facilitate patient recruitment for phenotyping protocols; (b) coordinate scientific meetings to enhance efficiency and productivity of individual Projects & Cores; (c) coordinate space management; (d) facilitate scientific reporting and, (e) ensure proper regulatory compliance. Aim 2 is to 'un-burden" P50 investigators, by providing infrastructure and timely support to catalyze a collaborative think-tank comprised of Center personnel from across the globe. To achieve this aim, the Core will provide administrative support to: (a) coordinate and disseminate meeting agendas/materials to participants; (b) coordinate travel arrangements for investigators, fellows and staff within the two scientific projects and supporting cores; (c) oversee optimal functioning of information technology (IT) services; and, (d) facilitate post-award grant management. Aim 3 is to serve as a “nuclear hub" for engagement and communication between P50 investigators, the patients they serve, mentees and the broader academic community. To achieve this objective, the Core will provide administrative support to: (a) Education/Outreach Core patient meetings and focus groups; (b) orient new personnel and staff to the Center; (c) coordinate the Visiting Professor series through the academic year; (d) coordinate a Harvard-wide Annual Symposium celebrating reproductive medicine and biology across the Massachusetts General Hospital, Brigham & Women’s hospital and Harvard Chan School of public health; and, (e) maintain a social media presence to disseminate scientific findings, advertise research positions and publicize activities (i.e. patient meetings, Speaker Series, Annual Symposium). The Administrative Core will provide end-to-end support for Projects 1 and 2 as well as Technical Service and Education/Outreach cores to effectively carry out their planned activities. Coordinated Administrative Core activities will amplify the Center’s productivity and dissemination of results that will in turn benefit clinical care of patients with infertility and alleviate the physical and psychological burden of infertility for patients and their families.

Outreach/Education Core (A. Dwyer)

Abstract: The Education/Outreach Core will engage with key stakeholders to magnify impact and dissemination of P50 Center activities and exert sustained impact on clinical practice. To achieve these broad goals, the Core has three specific aims. First, the Core will maintain engagement with patient groups and build on our lengthy history of patient collaboration and co-creation. We will build new relationships and strengthen existing partnerships with patient groups to foster bi-directional exchange. Such engagement helps ensure that research and clinical care are responsive to patient-identified priorities. Engagement with patient communities is essential for ensuring that research findings can be effectively translated into meaningful improvement in health and wellbeing. Deliverables from this aim will provide an evidence base informing best practices/approaches for engaging geographically dispersed patient populations. Second, the Core will develop concise, evidence-based guidance for referring providers to return genetic test results to patients. We will build infrastructure and create resources supporting clinicians in returning research findings to research participants. We will draw on principles of neuroscience, cognitive psychology and therapeutic education to identify the optimal visual presentation of numerical genetic concepts to facilitate effective communication with patients/families with limited genetic literacy/numeracy. Resulting deliverables will deepen clinician knowledge of best practices and accepted reporting guidelines and will support provider confidence in using person-centered communication practices. Using ‘design thinking’ (user-centered/human-centered design) to develop reference materials for clinicians will ensure that resources are accessible, relevant and responsive to clinician and patient needs. Activities will enable discoveries to reach patients/families in ways that empower individuals to use genetic information for improved health and wellbeing. Last, the Core will curate and co-create patient materials and disseminate to patients, families and clinicians via a user-friendly website built on ‘design thinking’ (user-centered design). We will collaborate with patient organizations to respond to patients’ desire for reliable, curated information. We will co-create solutions to unmet patient-identified informational needs and use ‘design thinking’ to develop and launch a user-friendly website for electronic dissemination across multiple online platforms (smartphone, tablet or computer). Deliverables from this aim will enable patients/families to learn about infertility, be informed of emerging research findings, participate in research and find links to peer-to-peer support. The web-enabled platform will also serve as an enduring, supplemental resource for clinicians (i.e. physicians, genetic counselors) in providing infertility care. Providers can direct patients to the website to learn about conditions causing infertility, find resources and support for improving health and wellbeing.