Whole genome sequencing identifies a homozygous splicing variant in TDRKH segregating with non-obstructive azoospermia in an Iranian family.

Non-obstructive azoospermia (NOA) resulting from primary spermatogenic failure represents one of the most severe forms of male infertility, largely because therapeutic options are very limited. Beyond their diagnostic value, genetic tests for NOA also hold prognostic potential. Specifically, genetic diagnosis enables the establishment of genotype-testicular phenotype correlations, which, in some cases, provide a negative predictive value for testicular sperm extraction (TESE), thereby preventing unnecessary surgical procedures. In this study, we employed whole-genome sequencing (WGS) to investigate two generations of an Iranian family with NOA and identified a homozygous splicing variant in TDRKH (NM_001083965.2: c.562-2A>T). TDRKH encodes a conserved mitochondrial membrane-anchored factor essential for piRNA biogenesis in germ cells. In Tdrkh knockout mice, de-repression of retrotransposons in germ cells leads to spermatogenic arrest and male infertility. Previously, our team reported TDRKH involvement in human NOA cases through the investigation of a North African cohort. This current study marks the second report of TDRKH's role in NOA and human male infertility, underscoring the significance of the piRNA pathway in spermatogenesis. Furthermore, across both studies, we demonstrated that men carrying TDRKH variants, similar to knockout mice, exhibit complete spermatogenic arrest, correlating with failed testicular sperm retrieval.

Phenotypic continuum and poor intracytoplasmic sperm injection intracytoplasmic sperm injection prognosis in patients harboring HENMT1 variants.

BACKGROUND: Small RNAs interacting with PIWI (piRNAs) play a crucial role in regulating transposable elements and translation during spermatogenesis and are essential in male germ cell development. Disruptions in the piRNA pathway typically lead to severe spermatogenic defects and thus male infertility. The HENMT1 gene is a key player in piRNAs primary biogenesis and dysfunction of HENMT1 protein in meiotic and haploid germ cells resulted in the loss of piRNA methylation, piRNA instability, and TE de-repression. Henmt1-knockout mice exhibit a severe oligo-astheno-teratozoospermia (OAT) phenotype, whereas patients with HENMT1 variants display more severe azoospermia phenotypes, ranging from meiotic arrest to hypospermatogenesis. Through whole-exome sequencing (WES) of infertile patient cohorts, we identified two new patients with variants in the HENMT1 gene presenting spermatozoa in their ejcaulate, providing us the opportunity to study spermatozoa from these patients. OBJECTIVES: Investigate the spermatozoa of two patients harboring an HENMT1 variant to determine whether or not these scarce spermatozoa could be used with assisted reproductive technologies. MATERIALS AND METHODS: HENMT1 variants identified by WES were validated through Sanger sequencing. Comprehensive semen analysis was conducted, and sperm cells were subjected to transmission electron microscopy for structural examination, in situ hybridization for aneuploidy assessment, and aniline blue staining for DNA compaction status. Subsequently, we assessed their suitability for in vitro fertilization using intracytoplasmic sperm injection (IVF-ICSI). RESULTS: Our investigations revealed a severe OAT phenotype similar to knockout mice, revealing altered sperm concentration, mobility, morphology, aneuploidy and nuclear compaction defects. Multiple IVF-ICSI attempts were also performed, but no live births were achieved. DISCUSSION: We confirm the crucial role of HENMT1 in spermatogenesis and highlight a phenotypic continuum associated with HENMT1 variants. Unfortunately, the clinical outcome of these genetic predispositions remains unfavorable, regardless of the patient's phenotype. CONCLUSION: The presence of spermatozoa is insufficient to anticipate ICSI pregnancy success in HENMT1 patients.

Lack of CCDC146, a ubiquitous centriole and microtubule-associated protein, leads to non-syndromic male infertility in human and mouse.

From a cohort of 167 infertile patients suffering from multiple morphological abnormalities of the flagellum (MMAF), pathogenic bi-allelic mutations were identified in the CCDC146 gene. In somatic cells, CCDC146 is located at the centrosome and at multiple microtubule-related organelles during mitotic division, suggesting that it is a microtubule-associated protein (MAP). To decipher the molecular pathogenesis of infertility associated with CCDC146 mutations, a Ccdc146 knock-out (KO) mouse line was created. KO male mice were infertile, and sperm exhibited a phenotype identical to CCDC146 mutated patients. CCDC146 expression starts during late spermiogenesis. In the spermatozoon, the protein is conserved but is not localized to centrioles, unlike in somatic cells, rather it is present in the axoneme at the level of microtubule doublets. Expansion microscopy associated with the use of the detergent sarkosyl to solubilize microtubule doublets suggests that the protein may be a microtubule inner protein (MIP). At the subcellular level, the absence of CCDC146 impacted all microtubule-based organelles such as the manchette, the head-tail coupling apparatus (HTCA), and the axoneme. Through this study, a new genetic cause of infertility and a new factor in the formation and/or structure of the sperm axoneme were characterized.