Don is the lead investigator on Project I of the National Center for Translational Research in Reproduction & Infertility (NCTRI) grant entitled Genetics of Infertility: Accelerating the pace of discovery. The grant was originally awarded in 2019 and has now been renewed for another 5 years. Congratulations!

Overall Abstract: Overall 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. 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 develop targeted therapies. Project 1 will discover genetic variants in men with unexplained nonobstructive azoospermia (NOA) to identify targets for development of clinical diagnostics or therapy. In addition, Project 1 assemble an international team of experts to develop criteria for review and interpretation of infertility- associated variants to be included in ClinGen, which will facilitate inclusion in clinical diagnostic screens. Project 2 will establish high throughput in vitro gametogenesis platforms and a 100% chimeric mouse strategy for rapid validation of azoospermia-associated variants discovered in Project 1. Project 2 will also evaluate the impact of epigenetic modifiers on primordial germ cell development. Project 3 will provide “gold standard” reproductive phenotyping in mouse models with human NOA associated variants discovered in Project 1 and validated in Project 2. In mouse models of NOA, Project 3 will test the safety and feasibility of Sertoli cell and germ cell gene therapies. Results of all interlinked projects will help justify the use of whole genome sequencing as a diagnostic screen for male infertility and inform the public dialogue on the risks and benefits of gene therapy in and around the germline. Core A will provide 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-Outreach Core will maximize the public impact of this P50 by developing Saturday Academy hands-on laboratory experiences in reproduction and genetics for middle school and high school students in underserved communities in Pittsburgh, Portland and Ithaca. P50 trainees from all sites will also contribute teaching modules to The DataJam, a national nonprofit that runs academic education programs and competitions to engage middle school, high school and community college students in data analytics and critical thinking.

Project I Summary: Since the beginning of our National Center for Translational Research in Reproduction & Infertility in 2019, our knowledge of genetic causes of infertility has expanded dramatically. In a recent (2022) study of whole-exome sequencing (WES) from 1,000 cases of unexplained male infertility, we identified a plausible monogenic cause in 20% of cases. Similar diagnostic yields have been reported for other male infertility cohorts. Despite these impressive research advances, there are two major shortcomings of our work. First, we are not identifying all genetic causes in our patients. Second, even though we understand the genetics much better than 5 years ago, we have not made any impact on clinical diagnosis. In this proposal, our goal is to improve the diagnostic yield of clinical genome sequencing for male infertility in two ways. First, we aim to improve diagnostic yield with new approaches to sequencing and interpretation of genomes of male infertility. We will transition from WES to whole genome sequencing (WGS), and in the process, expand our analysis methods to new types of variants detectable by WGS. We will expand our analysis to new disease models for which we have been historically underpowered to capture, such as autosomal dominant and digenic, and to interrogate genes involved in testis development which have been previously overlooked. These new approaches will be applied to our existing database of >1,600 WES data from patients, >80% of which are still unsolved, and to new patients that we continue to enroll. Second, we aim to improve translation of our research findings by organizing an international clinical genetics community to generate and share gene and variant interpretations. In the modern clinical genetics landscape, most diagnostic workflows rely heavily on published guidelines for the interpretation of genetic variants. Notably, there are no official expert panels reviewing genes or variants for infertility. We will reactivate our existing international team, which has performed gene and variant interpretation in 2018 and 2020, to fill this gap. We will build open our expertise and accelerate our curation by creating a shared online working environment that records and reconciles interpretations. This review environment will also provide reference information on testis gene expression, fertility phenotypes of mutations across multiple model organisms, and an allele frequency database built from over 1,000 genomes of male infertility cases and 600 genomes from normozoospermic controls. At the end of this 5-year project, we believe we will have achieved a milestone that we targeted over 10 years ago. We will have built a strong foundation of solid research in male infertility genetics, summarized and packaged this knowledge for clinical use. We aspire that, in 2029, genome sequencing will finally become a routine clinical test for the diagnosis of male infertility.