Non-obstructive azoospermia (NOA) is the most severe form of male infertility and typically incurable. Defining the genetic basis of NOA has proven challenging, and the most advanced classification of NOA subforms is not based on genetics, but simple description of testis histology. In this study, we exome-sequenced over 1000 clinically diagnosed NOA cases and identified a plausible recessive Mendelian cause in 20%. We find further support for 21 genes in a 2-stage burden test with 2072 cases and 11,587 fertile controls. The disrupted genes are primarily on the autosomes, enriched for undescribed human “knockouts”, and, for the most part, have yet to be linked to a Mendelian trait. Integration with single-cell RNA sequencing data shows that azoospermia genes can be grouped into molecular subforms with synchronized expression patterns, and analogs of these subforms exist in mice. This analysis framework identifies groups of genes with known roles in spermatogenesis but also reveals unrecognized subforms, such as a set of genes expressed across mitotic divisions of differentiating spermatogonia. Our findings highlight NOA as an understudied Mendelian disorder and provide a conceptual structure for organizing the complex genetics of male infertility, which may provide a rational basis for disease classification.

The development and function of male gametes is critically dependent on a dynamic microtubule network, yet how this is regulated remains poorly understood. We have recently shown that microtubule severing, via the action of the meiotic AAA ATPase protein clade, plays a critical role in this process. Here, we sought to elucidate the roles of spastin, an as yet unexplored member of this clade in spermatogenesis. Using a SpastKO/KO mouse model, we reveal that spastin loss resulted in a complete loss of functional germ cells. Spastin plays a critical role in the assembly and function of the male meiotic spindle, and in its absence, apoptosis is significantly increased. Consistent with meiotic failure, round spermatid nuclei were enlarged, indicating aneuploidy, but were still able to enter spermiogenesis. During spermiogenesis, we observed extreme abnormalities in manchette structure, supernumerary acrosome formation, and commonly, a loss of nuclear integrity. This work defines a novel and essential role for spastin in regulating microtubule dynamics during spermatogenesis and is of potential relevance to patients carrying Spastin variants and to the medically assisted reproductive technology industry.

Non-obstructive azoospermia (NOA) is the most severe form of male infertility and typically incurable with current medicine. Due to the biological complexity of sperm production, defining the genetic basis of NOA has proven challenging, and to date, the most advanced classification of NOA subforms is based on simple description of testis histology. In this study, we exome-sequenced over 1,000 clinically diagnosed NOA cases and identified a plausible recessive Mendelian cause in 20%. Population-based testing against fertile controls identified 27 genes as significantly associated with azoospermia. The disrupted genes are primarily on the autosomes, enriched for undescribed human "knockouts", and, for the most part, have yet to be linked to a Mendelian trait. Integration with single-cell RNA sequencing of adult testes shows that, rather than affecting a single cell type or pathway, azoospermia genes can be grouped into molecular subforms with highly synchronized expression patterns, and analogs of these subforms exist in mice. This analysis framework identifies groups of genes with known roles in spermatogenesis but also reveals unrecognized subforms, such as a set of genes expressed specifically in mitotic divisions of type B spermatogonia. Our findings highlight NOA as an understudied Mendelian disorder and provide a conceptual structure for organizing the complex genetics of male infertility, which may serve as a basis for disease classification more advanced than histology.

The development and function of male gametes is critically dependent on a dynamic microtubule network, yet how this is regulated remains poorly understood. We have recently shown that microtubule severing, via the action of the meiotic AAA ATPase protein clade, plays a critical role in this process. Here, we sought to elucidate the roles of spastin, an as yet unexplored member of this clade in spermatogenesis. Using a SpastKO/KO mouse model, we reveal that spastin loss resulted in a complete loss of functional germ cells. Spastin plays a critical role in the assembly and function of the male meiotic spindle. Consistent with meiotic failure, round spermatid nuclei were enlarged, indicating aneuploidy, but were still able to enter spermiogenesis. During spermiogenesis, we observed extreme abnormalities in manchette structure, acrosome biogenesis, and commonly, a catastrophic loss of nuclear integrity. This work defines a novel and essential role for spastin in regulating microtubule dynamics during spermatogenesis and is of potential relevance to patients carrying spastin variants and the medically assisted reproductive technology industry.