SUMMARY To date, the etiology of idiopathic male infertility is not fully understood, and hormonal male contraceptives have not been effective. Through novel approaches to sequencing and sequencing analysis of male infertile patients and controls, this proposal seeks to identify new coding and non-coding genes and genomic regions required for male fertility and to validate the functional requirement of newly identified genes through mouse models. Past limited sequencing-based approaches have so far only been able to provide diagnostic variants to 1.5% of infertile men who present to the clinic, a yield that will improve with identification of more genes underlying infertility in men. One limitation to past progress has been the inability of conventional short-read sequencing to resolve haplotype-specific information: whether two or more mutations in each gene are on the same or separate alleles. Since gene loss-of-function typically requires damage to both alleles, it is of paramount importance to be able to resolve this information. Through long-read sequencing (a newly established and validated technology) we will identify novel damaging mutations at haplotype-specific resolution, correlating multiple novel damaging mutations with potential loss-of-function in our newly sequenced patients. Additionally, since sperm are haploid and previous studies have demonstrated a requirement for newly synthesized genes after meiosis, we will identify mutations lethal to sperm development through sequencing of both whole blood and sperm. Mutations present in blood (representing both maternal and paternal genomes) that are absent in sperm [with haploid genomes representing 8 million (2^23) chromosome combinations] demonstrate a requirement for those genes in the later stages of sperm development. Thus, through our proposed work we will identify for the first-time genes required for male fertility in men who may in fact be fertile. Lastly, we will apply CRISPR/Cas9 in mice to validate the functional requirement of candidate genes that we identify through our sequencing studies. Novel male infertility associated genes that are found to be expressed in sperm or epididymal cells, are evolutionarily conserved in vertebrates, including mice and humans, and rank among the highest in our patients will be prioritized for validation. We will assess the fertility phenotype of mutant mice and further explore molecular pathophysiology, which for some genes may determine candidacy as a contraceptive target.

NARRATIVE To date, the etiology of idiopathic male infertility is not fully understood, and there are no FDA-approved hormonal or non-hormonal male contraceptives. Through novel approaches to sequencing and sequencing analysis of male infertile patients and controls, this proposal seeks to identify new coding and non-coding genes and genomic regions required for male fertility. By applying CRISPR/Cas9 in mice to validate the functional requirement and identify the molecular pathophysiology of genes that we identify through our sequencing studies, we will clarify the roles of these new genes in male fertility and infertility and validate targets for future contraceptive studies.