Biallelic variants in IQCN cause sperm flagellar assembly defects and male infertility.

What is the effect of defects in the manchette protein IQ motif-containing N (IQCN) on sperm flagellar assembly? Deficiency in IQCN causes sperm flagellar assembly defects and male infertility. The manchette is a transient structure that is involved in the shaping of the human spermatid nucleus and protein transport within flagella. Our group recently reported that the manchette protein IQCN is essential for fertilization. Variants in IQCN lead to total fertilization failure and defective acrosome structure phenotypes. However, the function of IQCN in sperm flagellar assembly is still unknown. Fifty men with infertility were recruited from a university-affiliated center from January 2014 to October 2022. Genomic DNA was extracted from the peripheral blood samples of all 50 individuals for whole-exome sequencing. The ultrastructure of the spermatozoa was assessed by transmission electron microscopy. Computer-assisted sperm analysis (CASA) was used to test the parameters of curvilinear velocity (VCL), straight-line velocity (VSL), and average path velocity (VAP). An Iqcn knockout (Iqcn-/-) mouse model was generated by CRISPR-Cas9 technology to evaluate sperm motility and the ultrastructure of the flagellum. Hyperactivation and sperm fertilizing ability were assessed in a mouse model. Immunoprecipitation followed by liquid chromatography-mass spectrometry was used to detect IQCN-binding proteins. Immunofluorescence was used to validate the localization of IQCN-binding proteins. Biallelic variants in IQCN (c.3913A>T and c.3040A>G; c.2453_2454del) were identified in our cohort of infertile men. The sperm from the affected individuals showed an irregular '9 + 2' structure of the flagellum, which resulted in abnormal CASA parameters. Similar phenotypes were observed in Iqcn-/- male mice. VSL, VCL, and VAP in the sperm of Iqcn-/- male mice were significantly lower than those in Iqcn+/+ male mice. Partial peripheral doublet microtubules (DMTs) and outer dense fibers (ODFs) were absent, or a chaotic arrangement of DMTs was observed in the principal piece and end piece of the sperm flagellum. Hyperactivation and IVF ability were impaired in Iqcn-/- male mice. In addition, we investigated the causes of motility defects and identified IQCN-binding proteins including CDC42 and the intraflagellar transport protein families that regulate flagellar assembly during spermiogenesis. More cases are needed to demonstrate the relation between IQCN variants and phenotypes. Our findings expand the genetic and phenotypic spectrum of IQCN variants in causing male infertility, providing a genetic marker for sperm motility deficiency and male infertility. This work was supported by the National Natural Science Foundation of China (81974230 and 82202053), the Changsha Municipal Natural Science Foundation (kq2202072), the Hunan Provincial Natural Science Foundation (2022JJ40658), and the Scientific Research Foundation of Reproductive and Genetic Hospital of CITIC-Xiangya (YNXM-202114 and YNXM-202201). No conflicts of interest were declared. N/A.

Li Q ，Wang Y ，Zheng W ，Guo J ，Zhang S ，Gong F ，Lu GX ，Lin G ，Dai J ... -  《-》

Loss-of-function mutations in IQCN cause male infertility in humans and mice owing to total fertilization failure.

Fertilization failure is a significant manifestation of unexplained male infertility. Previous work has suggested a genetic origin. In this study, we report on a man with unexplained infertility from a large consanguineous marriage family. Whole-exome sequencing and Sanger sequencing identified a homozygous frameshift variation of the IQ motif containing N (IQCN; GenBank: NM_001145304.1; c.1061_1062delAT; p.Y354Sfs*13) in the proband and one of his two brothers, who also remained infertile. Analyses of spermatozoa by quantitative RT-PCR indicated that the level of IQCN mRNA was significantly reduced compared to fertile men and the protein could not be detected by western blotting and immunofluorescent staining in the proband. Immunofluorescent staining of spermatozoa from fertile men showed that IQCN was located in the acrosomal region and translocated to the equatorial segment after the acrosome reaction. The proband spermatozoa had abnormal morphology and function. Finally, the proband couple underwent IVF with donor sperm and a healthy baby was born. Furthermore, we developed an Iqcn-KO mouse model using the CRISPR/Cas9 technique. Sperm quality, except for sperm motility, and the fertility of male Iqcn-/- mice were consistent with those of the proband. In conclusion, the findings in humans and mice demonstrate that the homozygous frameshift variant of IQCN causes male infertility owing to autosomal-recessive fertilization failure.

Wang Y ，Chen G ，Tang Z ，Mei X ，Lin C ，Kang J ，Lian J ，Lu J ，Liu Y ，Lan F ，Huang W ，Zhang D ... -  《-》

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 ... -  《-》

IQCN disruption causes fertilization failure and male infertility due to manchette assembly defect.

Total fertilization failure (TFF) is an important cause of infertility; however, the genetic basis of TFF caused by male factors remains to be clarified. In this study, whole-exome sequencing was firstly used to screen for genetic causes of TFF after intracytoplasmic sperm injection (ICSI), and homozygous variants in the novel gene IQ motif-containing N (IQCN) were identified in two affected individuals with abnormal acrosome structures. Then, Iqcn-knockout mice were generated by CRISPR-Cas9 technology and showed that the knockout male mice resembled the human phenotypes. Additionally, we found that IQCN regulates microtubule nucleation during manchette assembly via calmodulin and related calmodulin-binding proteins, which resulted in head deformity with aberrant oocyte activation factor PLCζ. Fortunately, ICSI with assisted oocyte activation can overcome IQCN-associate TFF and male infertility. Thus, our study firstly identified the function of IQCN, highlights the relationship between the manchette assembly and fertilization, and provides a genetic marker and a therapeutic option for male-source TFF.

Dai J ，Li Q ，Zhou Q ，Zhang S ，Chen J ，Wang Y ，Guo J ，Gu Y ，Gong F ，Tan Y ，Lu G ，Zheng W ，Lin G ... -  《-》

A novel copy number variation in CATSPER2 causes idiopathic male infertility with normal semen parameters.

Are genetic abnormalities in CATSPER (cation channel of sperm) genes associated with idiopathic male infertility with normal semen parameters and, if so, how do they affect male fertility? A novel copy number variation (CNV) in CATSPER2 causes idiopathic male infertility with normal semen parameters by disrupting the ability of sperm to penetrate viscous media, undergo hyperactivation and respond to progesterone. CATSPER is the principle Ca2+ channel mediating extracellular Ca2+ influx into spermatozoa. Although several case reports have suggested a causal relationship between CATSPER disruption and human male infertility, whether genetic abnormalities in CATSPER genes are associated with idiopathic male infertility with normal semen parameters remains unclear. Spermatozoa were obtained from men attending the reproductive medical center at Jiangxi Provincial Maternal and Child Health Hospital, Nanchang, Jiangxi, China between January 2014 and June 2016. In total, 120 men from infertile couples and 20 healthy male donors were selected to take part in the study, based on their normal semen parameters. CATSPER and KSPER currents were assessed using the whole-cell patch-clamp technique. Whole-genome sequencing and TaqMan® CNV assays were performed to identify genetic variations. The expression levels of genes encoding the CATSPER complex were measured by quantitative real-time PCR and Western blot. Sperm motion characteristics and hyperactivation were examined with a computer-aided sperm analysis (CASA) system. Sperm responses to progesterone, assessed as increases in CATSPER current and intercellular Ca2+ concentrations ([Ca2+]i), as well as inducement of penetration ability and acrosome reaction, were examined by means of whole-cell patch-clamp technique, single-sperm [Ca2+]i imaging, penetration into methylcellulose assay and chlortetracycline staining, respectively. An infertile man with complete disruption of CATSPER current was identified. This individual has a novel CNV which disrupts one gene copy in the region 43894500-43950000 in chromosome 15 (GRCh37.p13 Primary Assembly, nsv3067119), containing the whole DNA sequence of CATSPER2. This CNV affected the expression of CATSPER2, resulting in dramatically reduced levels of CATSPER2 proteins in the individual's spermatozoa. Although this individual exhibited normal semen parameters, his spermatozoa showed impaired penetration ability, deficient hyperactivation, and did not respond to progesterone, in terms of monovalent current potentiation, [Ca2+]i increase, penetration ability enhancement and acrosome reaction inducement, which may explain the individual's idiopathic infertility. N/A. Our novel findings require more cases to support the CATSPER2 CNV identified in this study as a common cause of idiopathic male infertility in patients with normal semen parameters. Therefore, caution must be taken when extrapolating the use of this CNV as a potential biomarker for idiopathic male infertility. The findings from the unique human CATSPER 'knockout' model in this study not only confirm the essential roles of CATSPER in mediating progesterone response and regulating hyperactivation in human spermatozoa but also reveal that disruption of CATSPER current is a significant factor causing idiopathic male infertility. This study was funded by National Natural Science Foundation of China (81771644 and 31400996 to T.L.; 31230034 to X.Z.); National Basic Research Program of China (973 Program, 2015CB943003 to X.Z.); National Key Research and Development Program of China (2016YFC1000905 to T.L.); Natural Science Foundation of Jiangxi, China (20121BBG70021 and GJJ12015 to X.Z.; 20161BAB204167 and 20171ACB21006 to T.L.) and the open project of National Population and Family Planning Key Laboratory of Contraceptives and Devices Research (No. 2016KF07 to T.L.). The authors have no conflicts of interest to declare.

Luo T ，Chen HY ，Zou QX ，Wang T ，Cheng YM ，Wang HF ，Wang F ，Jin ZL ，Chen Y ，Weng SQ ，Zeng XH ... -  《-》