William Crowley - US grants

This research proposal plans to investigate the physiology of GnRH in the normal human menstrual cycle; to explore the range of pathophysiologic defects of GnRH in anovulatory infertility; to attempt to induce ovulation in ovulatory disorders with pulsatile GnRH; and to explore a GnRH antagonist across the human menstrual cycle. In continuation of our previous studies on the human menstrual cycle we plan to complete the study of the normal range of frequency and amplitude changes of GnRH-induced gonadotropin pulsations which occur in 60 normal females throughout the course of an ovulatory menstrual cycle. In addition, we will document the spectrum and nature of the abnormalities of GnRH which exist in specific subsets of anovulatory infertility including, a) hypothalamic amenorrhea, b) hyperprolactinemia in the absence of a pituitary tumor, c) polycystic ovarian disease, and d) inadequate luteal phase. Following a comprehensive baseline assessment of their GnRH program we plan to induce ovulation in them using intravenous administration of GnRH at hourly intervals throughout the follicular phase at a dose of 25 ng/kg. When ovulation has been documented by ultrasound, estradiols, and basal body temperature charts, the frequency of GnRH administration will be progressively slowed at a schedule compatible with that observed in the normal human menstrual cycle. Finally, a GnRH antagonist will be explored in three phases of the menstrual cycle in a log order of doses and the optimized dose used at six separate phases of the menstrual cycle to induce a temporary blockade of GnRH function and employ this physiologic probe to examine intra- and inter-cycle relationships mediated by GnRH's induction of pulsatile gonadotropin release.

This proposal plans to explore the inter-relationships between growth, gonadarche and adrenarche during puberty. To this end, two patient populations will be studied: 1) subjects with central precocious puberty, and 2) patients with monotropic growth hormone deficiency undergoing puberty at a normal age. These groups have been selected since they would profit from a delay of their pubertal process. Each group will be examined intensively to determine the hormonal and clinical characteristics of their condition prior to and following administration of a long-acting analogue of the gonadotropin releasing hormone. This agonist, D-Trp6-Pro9-NET-LHRH (LHRHa), has been previously demonstrated to produce a selective chemical desensitization of pituitary gonadotropin release and now permits the study of the effects of selective withdrawal of gonadarche from the pubertal process for the first time. Thus the inter-relationships of gonadarche and adrenarche can be explored in a controlled manner and studied in a longitudinal fashion. In addition when the constraints of epiphyseal closure attendant upon the appearance of sex steroids is removed, considerable new information regarding growth in puberty will be forthcoming. This line of investigation should provide basic information regarding the relationships of several aspects of the pubertal process, have implications for both human and animal science, and potentially translate to therapies of several human disorders.

It is the purpose of this proposal to continue to study the physiology of the gonadotropin releasing hormone, LHRH, in the male utilizing both in vivo and in vitro approaches. LHRH deficient males with idiopathic hypogonadotropic hypogonadism are able to synthesize and release gonadotropins which can elicit physiologic gonadal responses when administered exogenous LHRH. Long-term, out-patient LHRH administration via portable infusion pumps has been possible in these males with IHH permitting systematic variations in the dose and frequency of LHRH administration throughout sexual development. The effects of variations in the frequency of LHRH upon the ratio of gonadotropins secreted and the effects of these, in turn, upon gonadal secretion, can be undertaken with this mode. In addition, the Modulatory role of gonadal steroids and systematic alterations in LHRH frequency upon the biologic to immunologic ratios of gonadotropins can be approached in a human mode. As an adjunct to these studies dispersed, perifused rat pituiticyte systems have also been developed which will permit examination of LHRH stimulated gonadotropin release in vitro in the absence of endogenous gonadal steroid secretions. This experimental model system can be utilized in a parallel series of experiments designed to repeat the observations discovered in vivo and determine their precise mechanisms at the cellular level including study of LHRH receptors in messenger RNA for LH. Employing a combination of these two approaches, each with its unique experimental advantages, should provide insight into the role of LHRH's control of gonadotropin secretion, its modification, and physiologic regulation.

This research proposal plans to investigate the physiology of GnRH in the normal human menstrual cycle; to explore the range of pathophysiologic defects of GnRH in anovulatory infertility; to attempt to induce ovulation in ovulatory disorders with pulsatile GnRH; and to explore a GnRH antagonist across the human menstrual cycle. In continuation of our previous studies on the human menstrual cycle we plan to complete the study of the normal range of frequency and amplitude changes of GnRH-induced gonadotropin pulsations which occur in 60 normal females throughout the course of an ovulatory menstrual cycle. In addition, we will document the spectrum and nature of the abnormalities of GnRH which exist in specific subsets of anovulatory infertility including, a) hypothalamic amenorrhea, b) hyperprolactinemia in the absence of a pituitary tumor, c) polycystic ovarian disease, and d) inadequate luteal phase. Following a comprehensive baseline assessment of their GnRH program we plan to induce ovulation in them using intravenous administration of GnRH at hourly intervals throughout the follicular phase at a dose of 25 ng/kg. When ovulation has been documented by ultrasound, estradiols, and basal body temperature charts, the frequency of GnRH administration will be progressively slowed at a schedule compatible with that observed in the normal human menstrual cycle. Finally, a GnRH antagonist will be explored in three phases of the menstrual cycle in a log order of doses and the optimized dose used at six separate phases of the menstrual cycle to induce a temporary blockade of GnRH function and employ this physiologic probe to examine intra- and inter-cycle relationships mediated by GnRH's induction of pulsatile gonadotropin release.

It is the broad goal of this project to investigate the physiology of GnRH deficient male by using GnRH deficient males undergoing long-term administration of GnRH for induction of sexual maturation and normal men. By contrasting their responses to a variety of modulatory influences known to impact upon the hypothalamic- pituitary-gonadal-axis of the male, we will attempt to determine the site of action and relative contribution of testosterone and estradiol feedback upon the hypothalamic-pituitary-axis in the male. In addition, we will determine the role of physiologic variations in the interpulse interval and contour of the GnRH pulse upon the quantitative and qualitative nature of gonadotropin secretion in the male. Finally, we will measure inhibin levels by radioimmunoassay in GnRH-deficient men during induction of sexual maturation with GnRH and in adult males. These levels will be correlated with gonadal size, the onset of spermatogenesis, and FSH secretion. With the combined use of normal males with an intact and free- running hypothalamic-pituitary-gonadal axis and GnRH deficient men in whom the hypothalamic input can be experimentally defined, it should be possible to provide new and useful information in the physiology of GnRH in the human. Such information is a critical step for the understanding and definition of abnormalities of the male reproductive system.

The Massachusetts General Hospital has a longstanding tradition of excellence in clinical and basic investigations in Reproductive Endocrinology. This tradition remains quite alive to date and is currently embodied by a group of actively-collaborating investigators whose research in reproduction covers a spectrum from basic molecular biology to clinical investigation. Four individual Units (Reproductive Endocrine, General Endocrine, Thyroid, and Developmental Biology) represent the center of these activities. Each has developed a critical mass of staff and an increasing network of informal collaborations over the past decade that capitalizes upon their individual strengths. In recognition of this growing interdependence, we propose to formalize these interactions with the establishment of a Reproductive Endocrine Sciences Center via the P30 Center mechanism consisting of 6 Core facilities: Administrative, Molecular Biology, Peptide Synthesis/Protein Sequencing, Histology/Organ Culture, Cell Culture, and Radioimmunoassay. The Cores will serve 15 investigators with 20 grants from 4 separate departments. This Center structure will dramatically widen the scope of scientific interactions among the center investigators and should translate to unusually swift transfer of basic advances into the clinical arena in this setting. The investigators responsible for the Core facilities are each mid-career scientists with considerable institutional stability; are recognized leaders in the areas of their proposed Core responsibilities; employ state-of-the-art technologies; and provide the Center both competent supervision and considerable economies of scale and improved cost effectiveness. The institutional setting for this proposal is unique in its traditions of commitment to basic and clinical biomedical-investigations, its current composition of productive investigators, and its commitment to the present proposal. The proposed Reproductive Endocrine Sciences Center therefore offers the proper constellation of excellence, efficiency, and collaborative science to result in a marked augmentation in research in Reproductive Endocrinology in this setting, given support by the establishment of a P30 Center.

DESCRIPTION (provided by applicant): Broad Goals: Polycystic Ovary Syndrome (PCOS) is one of the most common endocrine disorders that causes infertility in women. Its marked phenotypic variability, puzzling list of potentially associated conditions, and hence potential risk to a women's health over a lifetime remain a clinical investigational challenge. To a great extent, this variability can be attributed to the marked genetic and environmental heterogeneity of the populations studied. We will address many of these long-term obstacles by studying PCOS in Iceland where we can take advantage of the country's relative genetic and environmental homogeneity, the remarkably rich genealogic database, and their centralized healthcare information. There, aggregation of large numbers of PCOS families whose homogeneity reduces genetic variance can be used to map the genetic basis for this condition. Finally, as the genes responsible for PCOS become evident, determining their consequences and phenotype/genotype correlations in Iceland and contrasting them with US women will be possible for the first time. Specific Aim 1 is to determine the intermediary phenotypes of women with PCOS in Iceland and contrast them to US women with PCOS Specific Aim 2 is to combine Iceland's genealogic database that can identify relationships between affected individuals and construct extended pedigrees with high-density, genomewide scans to determine the genetic basis of PCOS. Specific Aim 3 is to contrast the association of loci, at risk haplotypes, and/or mutations in candidate genes in Icelandic women with PCOS to their U.S. counterparts. Specific Aim 4 is to identify the genotype/phenotype correlations between the identified genes and the multiple organ system defects observed in women with PCOS Specific Aim 5 is to use genealogic information to evaluate the long-term health risks associated with PCOS.

The purpose of this study is to determine the relative roles of the testicular steroid and peptide hormones in the control of secretion of the pituitary hormones LH and FSH in the human male.

human therapy evaluation; hypogonadism; gonadotropins; hormone therapy; synthetic peptide; gonadotropin releasing factor; peptide hormone analog; endocrine disorder chemotherapy; clinical trial phase I; injection /infusion; secretion; longitudinal human study; clinical research; human subject; male;

The aim of this study is to learn more about the regulation of reproductive hormones in adult men, and specifically to determine whether it is testosterone (the main sex hormone produced by the testis) or estradiol (another sex hormone) which is more important in controlling the secretion of leutinizing hormone (LH) and follicle stimulating hormone (FSH) from the pituitary gland. To do this a medication will be administered which blocks the body's ability to convert testosterone to estradiol. Our hypothesis is that estradiol has a greater inhibitory effect on LH and FSH secretion than testosterone and that consequently blocking the production of estradiol will result in an increase in LH and FSH levels.

This project, which has been active on the MGH GCRC since the early 1980's, now focuses on detailed characterization of the growth, final height, and pubertal outcomes of children with central precocious puberty (CPP) who have undergone long-term pituitary-gonadal suppression induced by chronic administration of a GnRH agonist (GnRHa). Evaluations continue to be performed at 6-12 month intervals in children with CPP who have undergone long-term pituitary-gonadal suppression induced by GnRHa administration and who have not yet completed their linear growth and pubertal development. Results in the initial cohort of children with CPP who completed their growth defined a statistically significant increase in adult stature of 4-6 cm compared to pretherapy predictions. However, recent analyses which have included more patients who initiated GnRHa treatment at younger ages and earlier stages of puberty demonstrate a more substantial effect on final height. In the cohort of patients enrolled before CA 6 years, final HTs exceed pretherapy predictions by 8-10 cm. As our final group of young patients complete their therapy with GnRHa and complete their growth post-GnRHa, the impact of long-term gonadal sex steroid suppression on growth and final height may be viewed across the full developmental spectrum of our patient population. The investigators continue to monitor the return of pubertal pituitary function following the discontinuation of GnRHa administration and the eventual development of mature reproductive endocrine function. Follow-up is underway to characterize reproductive function (including fertility) in patients in whom therapy has been discontinued for several years. Most recently, we have utilized the ability of GnRHa administration to reversibly suppress pituitary-gonadal function to probe further the physiology of the non-steroidal FSH regulators, inhibin A & B and follistatin, and leptin, the product of the ob gene.

Understanding the genetic and molecular determinants of the neuroendocrine control of reproduction has been an important but elusive goal. Idiopathic hypogonadotropic hypogonadism (IHH) is a human disorder in which selective failure of the neuroendocrine components of sexual maturation occurs in the face of low levels of gonadotropins without any demonstrable anatomic cause. The considerable genetic and phenotypic heterogeneity of this condition suggests it is a rich source of unique information, not only about the neuroendocrine control of reproduction, but also about the development of several other organs. Thus, patients with IHH can be viewed as 'experiments of nature/ in which a series of mutations in genes both specific for the human and key to reproduction can be discovered. Utilizing clinical investigation and phenotyping, biochemical profiling, classical genetic studies, and new genomic tools, this proposal plans to address this issue using several unique resources. A broad spectrum of carefully profiled patients with these disorders has been assembled by the PI over the past 25 years and an evolving database of their phenotypic and genotypic features as well as a growing repository of their serum and DNA samples has been established. In our 1st aim, the 3 genes already known to cause this condition in a minority of IHH cases (KAL, DAX, and GnRH/rec) will be screened in this population. Subsequently, using iterative phenotyping, their full biologic spectrum will be defined in the 2nd aim. High throughput screening technologies will then be used to examine a wide spectrum of new candidate genes in this population and their phenotypes similarly identified in the 3rd aim. Finally, where appropriate, linkage analysis and genome mismatch scanning (GMS) will be employed in IHH families with known autosomal recessive inheritance. Together, this combination of resources and approaches should permit a unique opportunity to gain biologic insights into the genetic control of both GnRH and gonadotropin secretion in the human.

DESCRIPTION: (Adapted from the applicant's description) This application for the Harvard-wide Specialized Center for Reproduction proposes to bring the neuroendocrine and gonadal control of reproduction in the male. Two of the component of reproduction while the other two will explore the gonadal level as Leydig cell control of steroidogenesis. In Project I, will employ tandem phenotyping and genotyping approaches in patients with idiopathic hypogonadotropic hypogonadism (IHH). This is a human model in which a series of genetic defects result in failure of the neuroendocrine component of sexual maturation. In Project II, Dr. Ursula Kaiser will explore the GnRH of gonadotropic genes using LH-T2 cells which are of gonadotrope origin. In Inhibiting Substance (MIS) upon steroidogenesis using the CYP17 gene as well MIS receptor. In addition, she will chart the developmental expression of this receptor and correlate it with steroidogenesis. In Project IV, Dr. Joel Habener will continue his explorations of the role of cAMP-activated pathways of alternative splicing of the gene encoding of the transcription factor, CREB. He will also explore the role of pituitary adenyl cyclase activating peptide; PACAP, and its alternatively spliced type 1 receptor in the autocrine control of spermatogenesis.

male; inhibin; follicle stimulating hormone; hormone regulation /control mechanism; pituitary gonadal axis; biofeedback; luteinizing hormone; clinical research; human subject; cryoscience;

[unreadable] DESCRIPTION (provided by applicant): Using the human disease model of men with idiopathic hypogonadotropic hypogonadism (IHH), it is possible to: a) control the timing and pace of their testicular maturation via administration of either exogenous gonadotropins or pulsatile GnRH; b) manipulate their gonadal sex steroid hormonal milieu by administration of steroid hormone inhibitors; and c) vary the dose and frequency of their hypothalamic input of GnRH. This ability to clamp the hypothalamic input to their pituitary via GnRH and contrast the responses of IHH men with those of normal and castrate men (whose hypothalamic-pituitary-axis is unconstrained) allows us to dissect the hypothalamic from pituitary sites of action and address several physiologic issues that are not otherwise approachable in the human. Thus, In Specific Aim #1, we will thus explore the relative roles of gonadal sex steroids and Inhibin B (IB) in restraining FSH secretion across the full spectrum of gonadal development were serum IB levels vary from prepubertal levels to those of normal adult men. Our preliminary results suggest that their relative roles appear to change during gonadal maturation. It also permits us to understand the role of the frequency of GnRH in FSH feedback and thus place it in context with both sex steroids and lB. In Specific Aims # 2 & 3, this model allows us to test the ability of FSH to increase Sertoli and germ cell number, testicular size, and sperm counts when administered to IHH men with immature gonads and compare these results with those obtained when FSH stimulation is accompanied by that of LH via pulsatile GnRH therapy. During such studies we will also have access to testicular tissue in these men and thus can validate both MIS and IB as markers of Sertoli cell numbers, proliferation, and maturation in a quantitative and statistically valid fashion that has not been previously possible. In collaboration with Dr. Martin Dym, we will perform quantitative histomorphometry on these maturing gonads via serial testicular biopsies prior to and following 4 months of therapy and correlate these histological findings with serial measurements of IB and MIS as well as other clinical and biochemical aspects of gonadal maturation. Finally, in Specific Aim #4, the availability of serial testicular tissue in these men during Sertoli and early germ cell development will allow us to gain insight into the specific genes activated in the human during early testicular development. Thus, these studies offer a unique approach to gain new insights into the physiology and developmental biopsy of the male reproductive axis. They also lay the groundwork that provides a context for the exploration of new tools to gain further insights into the complexities of reproductive dysfunction in the male.

pituitary gonadal axis; follicle stimulating hormone; hormone regulation /control mechanism; secretion; clinical research; human subject; male;

pituitary gonadal axis; gonadotropin releasing factor; human therapy evaluation; hormone therapy; neuroendocrine system; hormone regulation /control mechanism; secretion; drug administration rate /duration; patient oriented research; male; human subject; clinical research;

Administrative Core Abstract The Administrative Core of our Center provides oversight for the 3 Research Projects as well as the Education& Outreach Core and the Technical Core (Genotyping & Phenotyping Core). Its role is to promote coordination of all intramural administrative and research activities and external collaborations. From an intramural perspective, these tasks involve coordination of all administrative functions of several collaborating institutions including the MGH (including our Clinical Research Center), the Partners Healthcare System (including its Clinical & Translational Science Award), The Broad Institute, Oxford University, and Duke University as well as hundreds of collaborating physicians from around the globe who either donate material to our genetic studies or come to us for specialized training within our Center. From an extramural perspective, these tasks include administrative interfaces with NICHD, its other National Centers for Translational Research in Reproduction and Infertility (NCTRIs) P50 Programs, and the FDA. The Administrative Core oversees all financial activities and human resource needs related to the Center. It also supports the activities of the External Advisory Board which is critical to the functioning of the Center.

Project I: Understanding the developmental biology of GnRH in the human is complex but critical to gaining insight into several human reproductive disorders. A few key genes/proteins have been identified in developmental pathways of GnRH neuronal migration, several of them using the unique human model of idiopathic hypogonadotropic hypogonadism (IHH) or Kallmann Syndrome (KS). KAL1 was the first to be discovered 10 years ago through very careful clinical and genetic investigations. This gene has shed considerable light on the physiology of GnRH neuronal migration. Three new genes have now been discovered (2 by this Project's team) during the last cycle of this Center grant - GPR54 and its ligand, KISS1, and FGFR1. Project I will now focus on FGFR1. Having the largest IHH/KS population in the world, assembled a talented Core of well-trained individuals to obtain quantitative phenotyping and perform human genetic studies, and now having the laboratory capabilities to screen all of the genes in this area, we are now well positioned: 1) to use genetic techniques to discover new genes important for reproduction; 2) to perform detailed genotype-phenotype correlations in a new gene FGFR1 causing IHH; and 3) to contrast the phentypes of FGFR1 and KAL1 mutations. The confluence of these resources will now allow us to develop clinical predictors for KAL1 and FGFR1 as well as define the clinical outcomes to treatment for each genotype. Finally, we will examine the biologic impact of the mutant receptors in vitro. This line of investigation has proven to be a fruitful area of research during our previous Grant cycle and will hopefully continue to elucidate what is increasingly clear as a 'GnRH neuronal migration pathway', all in the human.

ABSTRACT NOT PROVIDED

DESCRIPTION (provided by applicant): During the past 15 years, The Harvard Reproductive Endocrine Sciences Center has been dedicated to translational research in reproduction in the human. Its multidisciplinary investigative team and Human Genotyping and Phenotyping Core has now focused on the genetics of the neuroendocrine control of reproduction using unique human populations available to it. During the past cycle, this team identified three new genes that clearly control the migration and/or functioning of GnRH neurons in the human and cause hypogonadotropic hypogonadism in both men and women when mutated. These results indicate that all three are clearly key gatekeepers of reproductive competency in the human. Hence, Project I (KI = W. Crowley) now proposes to characterize the full phenotype/genotype spectrum of FGFR1 mutations in Idiopathic Hypogonadotropic Hypogonadisms (IHH) and Kallmann Syndrome (KS); contrast them with mutations in KAL1; continue to search for new genes in this evolving "GnRH neuronal migratory pathway"; and examine their in vitro biology. Project II (KI = S. Seminara) will now expand the phenotype of mutations in the GPR54 and KISS-1 genes causing IHH; examine the physiology of metastin, the gene product of KiSS-1, in the human; and examine the biologic effects of metastin administration on GnRH secretion in humans; and contrast the phenotype of the kisspeptin1 knock-out in the mouse with that of our GPR54 knockout. Finally, Project III (KI = U. Kaiser) will examine the molecular mechanisms of GPR54's cellular action in the hypothalamus (using GT1-7 cells); define its regulation by sex steroids; and examine the biologic activity of mutations identified in Project lI, in vitro. Together, this ensemble of synergistic approaches should provide swifter transfer of these truths into the human, one of the central goals of our Center.

[unreadable] DESCRIPTION (provided by applicant): Using the human disease model of men with idiopathic hypogonadotropic hypogonadism (IHH), it is possible to: a) control the timing and pace of their testicular maturation via administration of either exogenous gonadotropins or pulsatile GnRH; b) manipulate their gonadal sex steroid hormonal milieu by administration of steroid hormone inhibitors; and c) vary the dose and frequency of their hypothalamic input of GnRH. This ability to clamp the hypothalamic input to their pituitary via GnRH and contrast the responses of IHH men with those of normal and castrate men (whose hypothalamic-pituitary-axis is unconstrained) allows us to dissect the hypothalamic from pituitary sites of action and address several physiologic issues that are not otherwise approachable in the human. Thus, In Specific Aim #1, we will thus explore the relative roles of gonadal sex steroids and Inhibin B (IB) in restraining FSH secretion across the full spectrum of gonadal development were serum IB levels vary from prepubertal levels to those of normal adult men. Our preliminary results suggest that their relative roles appear to change during gonadal maturation. It also permits us to understand the role of the frequency of GnRH in FSH feedback and thus place it in context with both sex steroids and lB. In Specific Aims # 2 & 3, this model allows us to test the ability of FSH to increase Sertoli and germ cell number, testicular size, and sperm counts when administered to IHH men with immature gonads and compare these results with those obtained when FSH stimulation is accompanied by that of LH via pulsatile GnRH therapy. During such studies we will also have access to testicular tissue in these men and thus can validate both MIS and IB as markers of Sertoli cell numbers, proliferation, and maturation in a quantitative and statistically valid fashion that has not been previously possible. In collaboration with Dr. Martin Dym, we will perform quantitative histomorphometry on these maturing gonads via serial testicular biopsies prior to and following 4 months of therapy and correlate these histological findings with serial measurements of IB and MIS as well as other clinical and biochemical aspects of gonadal maturation. Finally, in Specific Aim #4, the availability of serial testicular tissue in these men during Sertoli and early germ cell development will allow us to gain insight into the specific genes activated in the human during early testicular development. Thus, these studies offer a unique approach to gain new insights into the physiology and developmental biopsy of the male reproductive axis. They also lay the groundwork that provides a context for the exploration of new tools to gain further insights into the complexities of reproductive dysfunction in the male.

CRISP; Computer Retrieval of Information on Scientific Projects Database; D-His-6-Pro-8-NEt-LHRH; Dysfunction; FSH-Releasing Hormone; Functional disorder; Funding; GnRH (gonadotropin releasing hormone); Goals; Gonadoliberin; Gonadorelinum; Gonadotropin Hormone Releasing Hormone; Gonadotropin-Releasing Hormone; Gonadotropins; Grant; Hoe- 471; Human; Human, General; Hypogonadotropic Hypogonadism; Institution; Investigators; Klinefelter syndrome (KS); Klinefelter's Syndrome; Klinefelter-Reifenstein syndrome; Klinefelter-Reifenstein-Albright syndrome; LH-FSH Releasing Hormone; LH-Releasing Hormone; LHFSH Releasing Hormone; Luliberin; Luteinizing Hormone-Releasing Factor; Luteinizing Hormone-Releasing Hormone; Luteinizing hormone-releasing factor (pig); Man (Taxonomy); Man, Modern; NIH; National Institutes of Health; National Institutes of Health (U.S.); Neuroendocrine; Neuroendocrine System; Neurosecretory Systems; Physiology; Physiopathology; Protocol; Protocols documentation; Pulsti; Recombinant Gonadorelin; Reproduction; Research; Research Personnel; Research Resources; Researchers; Resources; Role; Source; Therapeutic GRH; United States National Institutes of Health; Woman; XXY syndrome; XXY trisomy; Xq Klinefelter syndrome; chromosome XXY syndrome; follicle stimulating hormone-releasing factor; gonadotropin releasing factor; pathophysiology; primary hypogonadism; seminiferous tubule dysgenesis; social role

I. Objectives and Background of this Core Conducting genetic investigations and performing associated quantitative phenotyping in humans is a demanding and multidisciplinary task. However, the key to all subsequent utility and success of genetic inquiries hinge upon the successful accomplishment of these functions. Therefore, accomplishing this effectively involves the seamless integration of several complex tasks, each of which is essential to obtaining information that can be used to inform the biology of genetic investigations. It involves access to a genetically informative population in combination with a dedicated multidisciplinary team, both of which we have been fortunate to assemble in our Center over the past 5 years. The disciplines required involve the integration of quantitative human phenotyping, database management, genetic analyses, bioinformatics, and biostatistical genetics that have been key to our team's success in the past grant cycle.

Project I: Understanding the developmental biology of GnRH in the human is complex but critical to gaining insight into several human reproductive disorders. A few key genes/proteins have been identified in developmental pathways of GnRH neuronal migration, several of them using the unique human model of idiopathic hypogonadotropic hypogonadism (IHH) or Kallmann Syndrome (KS). KAL1 was the first to be discovered 10 years ago through very careful clinical and genetic investigations. This gene has shed considerable light on the physiology of GnRH neuronal migration. Three new genes have now been discovered (2 by this Project's team) during the last cycle of this Center grant - GPR54 and its ligand, KISS1, and FGFR1. Project I will now focus on FGFR1. Having the largest IHH/KS population in the world, assembled a talented Core of well-trained individuals to obtain quantitative phenotyping and perform human genetic studies, and now having the laboratory capabilities to screen all of the genes in this area, we are now well positioned: 1) to use genetic techniques to discover new genes important for reproduction;2) to perform detailed genotype-phenotype correlations in a new gene FGFR1 causing IHH;and 3) to contrast the phentypes of FGFR1 and KAL1 mutations. The confluence of these resources will now allow us to develop clinical predictors for KAL1 and FGFR1 as well as define the clinical outcomes to treatment for each genotype. Finally, we will examine the biologic impact of the mutant receptors in vitro. This line of investigation has proven to be a fruitful area of research during our previous Grant cycle and will hopefully continue to elucidate what is increasingly clear as a 'GnRH neuronal migration pathway', all in the human.

DESCRIPTION (as provided by applicant): The Harvard Reproductive Endocrine Sciences Center has been dedicated to translational research in reproduction for the past 20 years. The Center uses interdisciplinary approaches to define the genetic control of puberty and reproduction in the human incorporating techniques from clinical investigation, human genetics, molecular biology, and bioinformatics. Broadly, the Center aims to continue its discovery program to find novel genes that control reproductive function and elucidate their biology to enhance the diagnostics, treatment, and counseling of patients with infertility and reproductive disorders. PROJECT 1 (Pl: Crowley) will utilize modern genetic and genomic tools to identify genes involved in the biology of GnRH neuronal development capitalizing on our unique cohort of >1,300 patients/families with isolated GnRH deficiency that has been assembled by our Phenotyping, Genotyping, &Bioinformatics Core (Core B). It will then chart the genotype-phenotype spectrum of these new genes and determine their role in both congenital GnRH deficiency and more common reproductive disorders. PROJECT 2 (Pl: Seminara) will utilize human genetics and murine studies to elucidate the interplay of the Neurokinin B and KISS1/KISS1R pathways. These studies will also draw on our unique cohort of GnRH deficient patients as well as targeted gene deletions of these systems in mice. PROJECT 3 (Pl: Kaiser) will explore the molecular biology of G-coupled protein receptors (GPCRs) and the mechanisms by which they impact human reproduction. Using mutations in both ligands and their cognate GPCRs, including those from Project 1, Project 3 will chart their biologic activity and use in vitro techniques to elucidate their cellular actions and the effects of mutations on identified reproductive pathways. CORE A: will provide organizational, logistical, and administrative support for the center investigators;CORE B: will recruit patients for genetic studies/detailed phenotyping and manage the Progeny database;CORE C: will provide web based patient centered information on reproductive disorders and will link other investigators to our Progeny database.

Overview - This Core lies at the very heart of our Center which is relatively unique in its use of various human disease models involving abnormalities of GnRH secretion. Our Center's central vision is that patients with isolated GnRH deficiency [nIHH or KS] and other more common reproductive disorders such as delayed and precocious puberty and hypothalamic amenorrhea represent unique opportunities to define the genetic control of GnRH in the human. Consequently, the recruitment, phenotyping, genotyping, and cataloguing of data from individuals with these disorders are chronicled and entered into our relational database (Progeny) for ongoing investigations foundational to our Center. Consequently, this Core's skilled, stable, well-trained, and multidisciplinary staff are all crucial and dedicated to these longterm programmatic goals. Over the past grant cycle, there has also been a marked increase in regulatory compliance that has fallen to this Core and the Administrative Core's joint responsibilities. Consequently, rising IRB maintenance of approvals/reapprovals, data encryption, and HIPAA compliance have increased the transactional burdens that fall to our Center. Nonetheless, given the relatively high impact of the results of these approaches on the field of reproduction and our unique ability to provide insights directly relevant to the human, we remain committed to the importance of this approach and the efficient functioning of this Core. Clearly, as the number of our DNAs, informed consents, families, and phenotyping data rise, this Core's logistical functions become more important. In addition, the amount of sequencing data, particularly as we enter into the era of full exonic and the genomic sequence, having such a well organized and smoothly running Core is foundational to our Center's vision and function. This is why we have been extraordinarily fortunate to have recruited and retained talented interdisciplinary personnel to its staffing.

PROJECT I (Kl: CROWLEY) The developmental biology of GnRH in humans involves a complex interplay of biochemical and genetic systems all interacting over time and space. Notably, defects in GnRH neuronal ontogeny and function underlie a range of reproductive disorders which account for significant health care expenditures related to its consequent infertility. In the past cycle, strong collaborations between Project I and our Administrative and Phenotyping, Genotyping, and Bioinformatics Cores (Cores A & B), have allowed our Center to assemble a population of >1,300 patients/families with GnRH deficiency. We have successfully used this cohort to identify novel genes affecting the biology of the GnRH neurons and define patient phenotypes. This expanding database; its unique phenotypes and increasing genotypes; our global network of referring physicians and our increasing bioinformatic infrastructure provides a unique 'platform for human gene discovery' that will permit us to use state-of-the art genetic and genomic tools to augment the power, speed, and resolution of our gene discovery program. In Specific Aim 1, we will use contemporary genetic and genomic tools including endogamous populations; copy number variations and methylation pattern analysis; and whole exomic sequencing to identify novel genes underlying human isolated GnRH deficiency. Specific Aim 1 will also map the oligogenic interactions between the genes in the pathogenesis of isolated GnRH deficiency in humans. In Specific Aim 2, we will characterize the full genotypic-phenotypic spectrum of these new genes in the human and use the identified mutated regions of the genes to define their domain-specific biologic interactions. Finally, in Specific Aim 3, we will define the rotes played by these new genes causing isolated GnRH deficiency in common reproductive disorders such as hypothalamic amenorrhea (HA), delayed and precocious puberty.

Outreach &Education Core (Core C) Background In our previous grant cycles, this Center has assembled a considerable amount of valuable information regarding the genotypes and phenotypes of GnRH deficient patients including those with isolated GnRH deficiency both with anosmia, i.e. Kallmann Syndrome (KS) and with normosmic Idiopathic Hypogonadotropic Hypogonadism (nIHH). We have also: a) described novel clinical variations on this theme such as reversal of GnRH deficiency (PI 9) and adult onset variety (Nachtigall et. al. N Engl J Med 1997 &P42); b) documented that digenicity/oligogenicity is evident in a significant proportion of cases (PI 4, P45); c) begun exploring the role of mutations in these genes in more common reproductive disorders such as precocious puberty (P22), and hypothalamic amenorrhea (P46). Thus, through the incremental work of this Center and other investigators around the world, the collective understanding of the genetics of this condition has advanced considerably. From completely lacking a genetic explanation in 1990 to now accounting for 33% of patients having an identifiable genetic defect (cf. Introductory Overview/ Fig 1 page 93) this striking change represents a rather remarkable gain in information. These findings have not only propelled several areas of neuroendocrine research but have also made significant contributions to the care and genetic counseling of patients and families with these disorders. As a direct consequence of this rapid progress, we have now amassed the world[unreadable]s largest and most thoroughly phenotyped and genotyped cohort of patients with GnRH deficiency and their family members (cf. Introductory Overview, pg. 93). Over 400 of these probands now undergone complete sequencing of all known genes that are causative for this disease (publication P45). In addition, we also have assembled DNA samples from large and growing populations of the more common reproductive conditions in which some abnormality of GnRH secretion has been documented as described in the introductory Overview

Overview - This Core lies at the very heart of our Center which is relatively unique in its use of various human disease models involving abnormalities of GnRH secretion. Our Center's central vision is that patients with isolated GnRH deficiency [nIHH or KS] and other more common reproductive disorders such as delayed and precocious puberty and hypothalamic amenorrhea represent unique opportunities to define the genetic control of GnRH in the human. Consequently, the recruitment, phenotyping, genotyping, and cataloguing of data from individuals with these disorders are chronicled and entered into our relational database (Progeny) for ongoing investigations foundational to our Center. Consequently, this Core's skilled, stable, well-trained, and multidisciplinary staff are all crucial and dedicated to these longterm programmatic goals. Over the past grant cycle, there has also been a marked increase in regulatory compliance that has fallen to this Core and the Administrative Core's joint responsibilities. Consequently, rising IRB maintenance of approvals/reapprovals, data encryption, and HIPAA compliance have increased the transactional burdens that fall to our Center. Nonetheless, given the relatively high impact of the results of these approaches on the field of reproduction and our unique ability to provide insights directly relevant to the human, we remain committed to the importance of this approach and the efficient functioning of this Core. Clearly, as the number of our DNAs, informed consents, families, and phenotyping data rise, this Core's logistical functions become more important. In addition, the amount of sequencing data, particularly as we enter into the era of full exonic and the genomic sequence, having such a well organized and smoothly running Core is foundational to our Center's vision and function. This is why we have been extraordinarily fortunate to have recruited and retained talented interdisciplinary personnel to its staffing.

Outreach & Education Core (Core C) Background In our previous grant cycles, this Center has assembled a considerable amount of valuable information regarding the genotypes and phenotypes of GnRH deficient patients including those with isolated GnRH deficiency both with anosmia, i.e. Kallmann Syndrome (KS) and with normosmic Idiopathic Hypogonadotropic Hypogonadism (nIHH). We have also: a) described novel clinical variations on this theme such as reversal of GnRH deficiency (PI 9) and adult onset variety (Nachtigall et. al. N Engl J Med 1997 & P42); b) documented that digenicity/oligogenicity is evident in a significant proportion of cases (PI 4, P45); c) begun exploring the role of mutations in these genes in more common reproductive disorders such as precocious puberty (P22), and hypothalamic amenorrhea (P46). Thus, through the incremental work of this Center and other investigators around the world, the collective understanding of the genetics of this condition has advanced considerably. From completely lacking a genetic explanation in 1990 to now accounting for 33% of patients having an identifiable genetic defect (cf. Introductory Overview/ Fig 1 page 93) this striking change represents a rather remarkable gain in information. These findings have not only propelled several areas of neuroendocrine research but have also made significant contributions to the care and genetic counseling of patients and families with these disorders. As a direct consequence of this rapid progress, we have now amassed the world¿s largest and most thoroughly phenotyped and genotyped cohort of patients with GnRH deficiency and their family members (cf. Introductory Overview, pg. 93). Over 400 of these probands now undergone complete sequencing of all known genes that are causative for this disease (publication P45). In addition, we also have assembled DNA samples from large and growing populations of the more common reproductive conditions in which some abnormality of GnRH secretion has been documented as described in the introductory Overview

PROJECT SUMMARY & ABSTRACT The long term goals of this T32 post-doctoral Training Program are twofold: 1). To recruit the best and brightest young physicians and basic science trainees interested in successful academic careers in biomedical research operating at the translational interface between bench and bedside using the contemporary tools of Reproduction, Development, and Genetics; and 2). To provide these high quality trainees with a secure period of stable funding so they can be exposed to: a) a rich spectrum of competitively funded and challenging biomedical problems; b) the most contemporary tools with which to attack them; c) optimal didactic experiences; and d) carefully mentored research experiences during periods of increasing autonomy. Taken together, this tight matrix of interwoven experiences equips our trainees with the foundational experiences necessary to achieve independence in research as is attested to by this program's track record of previous trainees. The key elements of success in meeting these goals are a rich pool of talented applicants and an exciting context within an Academic Medical Center. Our parent institution, the Massachusetts General Hospital (MGH), has a strong focus on human disease/disease models with a rich tradition of inter-disciplinary collaboration across a broad mix of talented training faculty (Tables 1 & 2). Each faculty member has been carefully selected by the Program Directors (PDs) on the basis of their demonstrated commitment to the independence of their trainees, exciting and important biologic problems on which to work, and stable funding. The spectrum of our program's planned research projects occurs in the laboratories of 11 mid- and senior level faculty members (Tables 1-4), each focused on key questions in human development, reproduction, systems and molecular biology, and/or genetics. Our Training Program taps into an exciting blend of basic and clinical researchers who have chosen to focus their interdisciplinary skills on human disease targets, i.e. the genetics of Mendelian development disorders like Isolated GnRH Deficiency, fetal congenital lung and diaphragmatic anomalies, neurologic disorders, as well as complex genetic disorders such as polycystic ovarian syndrome (PCOS). Given our nation's pressing need and expressed priorities for training translational researchers pointed out by the Institute of Medicine's Clinical Research Roundtable (References Cited: 1 & 2) as well as the powerful synergies emerging in reproduction, development and genetics, we propose to continue our current level of training 4 physicians and/or basic scientists for the next 5 years of this competing renewal application.

Project 1 Abstract Using the human disease model of Isolated GnRH Deficiency (IGD), ~35 genes that control human reproduction have been discovered, several from our Center. We now propose three new Specific Aims (SAs) to accelerate these new genes discovery efforts dramatically. SA #1 will select subsets of our IGD cohort of 2,000+ IGD patients who: a) share a second rare phenotype; or b) are from multiplex IGD families, and provide them facilitated access to our whole exome sequencing (WES) pipeline in which we can define new coding sequence mutations. SA #2 will then use novel statistical enrichment analysis that will allow novel gene discovery using WES in our full IGD cohort. Similarly, this aim will also employ novel copy number variation (CNV) analysis pipelines to identify the role of CNVs in IGD. Finally, SA #3 will utilize whole genome sequencing (WGS) combined with RNA sequencing in IGD patients who: a) harbor ?balanced? chromosomal rearrangements; or b) are from multiplex families or trios whose WES/CNV analysis were negative in order to define the full range of structural and non-coding variation in our IGD patients. Taken together, this combination of contemporary approaches, unique populations, and advances in next generation (NGS) should provide us with a fuller understanding of the mendelian genes that control human reproduction.