(223) Variant Identified in Male Patient with Idiopathic Non-Obstructive Azoospermia Leads to Infertile Mouse Model

Georgia Rae Atkins^1,2, Rachel Hvasta-Gloria², Yi Sheng², Alexander N. Yatsenko², Kyle E. Orwig²

1. Molecular Genetics and Developmental Biology Program, University of Pittsburgh, United States

2. Department of Obstetrics, Gynecology, and Reproductive Sciences, Magee-Womens Research Institute, University of Pittsburgh School of Medicine, United States

Abstract Text:

Non-obstructive azoospermia (NOA) is defined by the absence of sperm in the ejaculate due to a spermatogenic failure. NOA is prevalent, impacting 1% of men worldwide. Although there are known causes of NOA, approximately 50% of cases are idiopathic, leaving many patients without a clear diagnosis for their infertility or a definitive treatment plan. However, researchers believe many of these idiopathic cases are due to novel variants in uncharacterized genes involved in spermatogenesis. We can model these variants in mice to determine if these mutations lead to loss of function and result in an azoospermia phenotype. We hypothesized that a missense mutation in TOPAZ1 identified in a familial case of NOA would lead to azoospermia when replicated in mice. Whole exome sequencing and subsequent variant filtering identified a homozygous TOPAZ1 missense variant (c.4679C >T; p.A1560V) in the proband and two infertile brothers from a Pakistani family. We used CRISPR/Cas9 to introduce the missense variant into the orthologous region of the mouse genome. After line expansion, Topaz1^1560V+/- and Topaz1^1560V-/- male mice underwent fertility analysis by breeding studies, morphological assessment of male reproductive tract, and histological analyses of the testes.  Male Topaz1^1560V-/- failed to produce a litter (0±0 pups/breeding) and had no sperm present in the tail of the epididymis (0±0 mil/mL). Heterozygous male Topaz1^1560V+/- mice produced litters (4.32±0.90 pups/breeding;) comparable to Topaz1^+/+ littermate controls (3.77±0.78 pups/breeding), but did have an increased sperm count (8.06±1.42 mil/mL) compared to Topaz1^+/+ males (5.88±1.60 mil/mL). Furthermore, Topaz1^1560V-/- had reduced testis (40.3±2.29 mg) and epididymis weight (38±2.74 mg) compared to Topaz1^1560V+/- (121.7±8.84 mg testis; 64.5±5.56 mg epididymis) and Topaz1^+/+ (113.9±6.62 mg testis; 57.1±7.09 mg epididymis). There was no difference in body weight or seminal vesicle weight in any genotype, suggesting that testosterone production was not impacted. Histological data showed a maturation arrest phenotype in the seminiferous tubules of Topaz1^1560V-/-mice; there were a few layers of germ cells, tubule size was reduced and there were no post-meiotic spermatids. Overall, our study used a mouse model to validate a human variant in TOPAZ1 as causal in an NOA phenotype. This identifies a new gene that could be screened for in male patients suffering from idiopathic NOA and, in the future, may serve as a target for therapy. This work was supported by the Eunice Kennedy Shriver Institute for Child Health and Human Development grants HD114406 to GRA and HD096723 to KEO as well as the Magee-Womens Research Institute and Foundation.