Deleterious variants in TAF7L cause human oligoasthenoteratozoospermia and its impairing histone to protamine exchange inducing reduced in vitro fertilization.

Oligoasthenoteratozoospermia (OAT) is a major cause of infertility in males. Only a few pathogenic genes of OAT have been clearly identified till now. A large number of OAT-affected cases remain largely unknown. Here, Whole-exome sequencing (WES) in 725 idiopathic OAT patients was performed. Ejaculated spermatozoa by OAT patients were microinjected into mouse oocytes to estimate fertilization potential. Diff-quick staining and transmission electron microscopy were performed to evaluate sperm morphology and ultrastructure. The protein expression level and localization In vitro were detected by Western Blotting and Immunocytochemistry. We identified four X-linked hemizygous deleterious variants of TAF7L-namely, c.1301_1302del;(p.V434Afs*5), c.699G>T;(p.R233S), c.508delA; (p. T170fs), c.719dupA;(p.K240fs) -in five probands. Intracytoplasmic sperm injection (ICSI) were carried out in M1, M2-1and M3 patient's wife. However only M1 patient's wife became pregnant after embryo transfer. In vitro study demonstrated significantly reduced fertilization ability in patient with TAF7L mutation. The TAF7L mutation let to abnormal sperm head and impaired histone-to protamine exchange. Variant 719dupA (p. K240fs) resulted in producing a truncated TAF7L protein and localized massively within the nucleus. In addition, TAF7L expression were not able to be detected due to variants c.1301_1302del (p. V434Afs*5) and c.508delA (p. T170fs) In vitro. Our findings support that TAF7L is one of pathogenic genes of OAT and deleterious mutations in TAF7L may cause impaired histone-to-protamine affected the chromatin compaction of sperm head.

Bai H ，Sha Y ，Tan Y ，Li P ，Zhang Y ，Xu J ，Xu S ，Ji Z ，Wang X ，Chen W ，Zhang J ，Yao C ，Li Z ，Zhi E ... -  《Frontiers in Endocrinology》

TENT5D disruption causes oligoasthenoteratozoospermia and male infertility.

Oligoasthenoteratozoospermia (OAT) is one of the most complex aggregators of male gametic problems. However, the genetic etiology of OAT is still largely unknown. To reveal the new genetic factors responsible for male infertility owning to OAT and reveal the outcomes of the affected patients from intracytoplasmic sperm injection (ICSI). Two infertile men with typical OAT were recruited in 2018 and retrospected a cohort that included 47 patients with OAT from 2013 to 2021. Fifty healthy men with proven fertility served as control subjects. To identify the novel pathogenic variants, whole-exome sequencing and Sanger sequencing were used. In silico analysis revealed the affecting of the variants. Field emission scanning electron microscopy was employed to observe the morphological defects of the spermatozoa. Immunofluorescence was used to analyze the expression and localization of the related protein. CRISPR/Cas9 was used to generate the mouse model. ICSI was used as a treatment for the patients and to assess the effects of the pathogenic variant on fertilization and embryo development. We identified a loss-of-function mutation NM_001170574.2:c.823G > T (p.Glu275*) in X-linked TENT5D from two patients with OAT. This variant is highly deleterious and has not been found in the human population. The count of patients' spermatozoa is dramatically decreased and displays multiple morphologic abnormalities with poor motility. Tent5d knockout mice are infertile and exhibit parallel defects. ICSI could rescue the infertility of the Tent5d knockout male mice. Moreover, the proband was treated with ICSI and achieved a successful pregnancy outcome for the first time. Subsequent mutation screening identified no TENT5D mutations among 47 additional patients with OAT and 50 control subjects. Mutation in TENT5D results in OAT and male infertility, and this terrible situation could be rescued by ICSI.

Sha Y ，Liu W ，Tang S ，Zhang X ，Xiao Z ，Xiao Y ，Deng H ，Zhou H ，Wei X ... -  《-》

Biallelic CFAP61 variants cause male infertility in humans and mice with severe oligoasthenoteratozoospermia.

The genetic causes for most male infertility due to severe oligoasthenoteratozoospermia (OAT) remain unclear. To identify the genetic cause of male infertility characterised by OAT. Variant screening was performed by whole-exome sequencing from 325 infertile patients with OAT and 392 fertile individuals. In silico and in vitro analyses were performed to evaluate the impacts of candidate disease-causing variants. A knockout mouse model was generated to confirm the candidate disease-causing gene, and intracytoplasmic sperm injection (ICSI) was used to evaluate the efficiency of clinical treatment. We identified biallelic CFAP61 variants (NM_015585.4: c.1654C>T (p.R552C) and c.2911G>A (p.D971N), c.144-2A>G and c.1666G>A (p.G556R)) in two (0.62%) of the 325 OAT-affected men. In silico bioinformatics analysis predicted that all four variants were deleterious, and in vitro functional analysis confirmed the deleterious effects of the mutants. Notably, H&E staining and electron microscopy analyses of the spermatozoa revealed multiple morphological abnormalities of sperm flagella, the absence of central pair microtubules and mitochondrial sheath malformation in sperm flagella from man with CFAP61 variants. Further immunofluorescence assays revealed markedly reduced CFAP61 staining in the sperm flagella. In addition, Cfap61-deficient mice showed the OAT phenotype, suggesting that loss of function of CFAP61 was the cause of OAT. Two individuals accepted ICSI therapy using their own ejaculated sperm, and one of them succeeded in fathering a healthy baby. Our findings indicate that CFAP61 is essential for spermatogenesis and that biallelic CFAP61 variants lead to male infertility in humans and mice with OAT.

Hu T ，Meng L ，Tan C ，Luo C ，He WB ，Tu C ，Zhang H ，Du J ，Nie H ，Lu GX ，Lin G ，Tan YQ ... -  《-》

Biallelic variants in KCTD19 associated with male factor infertility and oligoasthenoteratozoospermia.

Can whole-exome sequencing (WES) reveal new genetic factors responsible for male infertility characterized by oligozoospermia? We identified biallelic missense variants in the Potassium Channel Tetramerization Domain Containing 19 gene (KCTD19) and confirmed it to be a novel pathogenic gene for male infertility. KCTD19 is a key transcriptional regulator that plays an indispensable role in male fertility by regulating meiotic progression. Kctd19 gene-disrupted male mice exhibit infertility due to meiotic arrest. We recruited a cohort of 536 individuals with idiopathic oligozoospermia from 2014 to 2022 and focused on five infertile males from three unrelated families. Semen analysis data and ICSI outcomes were collected. WES and homozygosity mapping were performed to identify potential pathogenic variants. The pathogenicity of the identified variants was investigated in silico and in vitro. Male patients diagnosed with primary infertility were recruited from the Reproductive and Genetic Hospital of CITIC-Xiangya. Genomic DNA extracted from affected individuals was used for WES and Sanger sequencing. Sperm phenotype, sperm nuclear maturity, chromosome aneuploidy, and sperm ultrastructure were assessed using hematoxylin and eosin staining and toluidine blue staining, FISH and transmission electron microscopy. The functional effects of the identified variants in HEK293T cells were investigated via western blotting and immunofluorescence. We identified three homozygous missense variants (NM_001100915, c.G628A:p.E210K, c.C893T:p.P298L, and c.G2309A:p.G770D) in KCTD19 in five infertile males from three unrelated families. Abnormal morphology of the sperm heads with immature nuclei and/or nuclear aneuploidy were frequently observed in individuals with biallelic KCTD19 variants, and ICSI was unable to rescue these deficiencies. These variants reduced the abundance of KCTD19 due to increased ubiquitination and impaired its nuclear colocalization with its functional partner, zinc finger protein 541 (ZFP541), in HEK293T cells. The exact pathogenic mechanism remains unclear, and warrants further studies using knock-in mice that mimic the missense mutations found in individuals with biallelic KCTD19 variants. Our study is the first to report a likely causal relationship between KCTD19 deficiency and male infertility, confirming the critical role of KCTD19 in human reproduction. Additionally, this study provided evidence for the poor ICSI clinical outcomes in individuals with biallelic KCTD19 variants, which may guide clinical treatment strategies. This work was supported by the National Key Research and Developmental Program of China (2022YFC2702604 to Y.-Q.T.), the National Natural Science Foundation of China (81971447 and 82171608 to Y.-Q.T., 82101961 to C.T.), a key grant from the Prevention and Treatment of Birth Defects from Hunan Province (2019SK1012 to Y.-Q.T.), a Hunan Provincial Grant for Innovative Province Construction (2019SK4012), and the China Postdoctoral Science Foundation (2022M721124 to W.W.). The authors declare no conflicts of interest. N/A.

Wang W ，Su L ，Meng L ，He J ，Tan C ，Yi D ，Cheng D ，Zhang H ，Lu G ，Du J ，Lin G ，Zhang Q ，Tu C ，Tan YQ ... -  《-》

Genetic characterization of a missense mutation in the X-linked TAF7L gene identified in an oligozoospermic man†.

Although hundreds of knockout mice show infertility as a major phenotype, the causative genic mutations of male infertility in humans remain rather limited. Here, we report the identification of a missense mutation (D136G) in the X-linked TAF7L gene as a potential cause of oligozoospermia in men. The human aspartate (D136) is evolutionally conserved across species, and its change to glycine (G) is predicted to be detrimental. Genetic complementation experiments in budding yeast demonstrate that the conserved aspartate or its analogous asparagine (N) residue in yeast TAF7 is essential for cell viability and thus its mutation to G is lethal. Although the corresponding D144G substitution in the mouse Taf7l gene does not affect male fertility, RNA-seq analyses reveal alterations in transcriptomic profiles in the Taf7l (D144G) mutant testes. These results support TAF7L mutation as a risk factor for oligozoospermia in humans.

Ling L ，Li F ，Yang P ，Oates RD ，Silber S ，Kurischko C ，Luca FC ，Leu NA ，Zhang J ，Yue Q ，Skaletsky H ，Brown LG ，Rozen SG ，Page DC ，Wang PJ ，Zheng K ... -  《-》