Bi-allelic human TEKT3 mutations cause male infertility with oligoasthenoteratozoospermia owing to acrosomal hypoplasia and reduced progressive motility.

Oligoasthenoteratozoospermia (OAT) can result in male infertility owing to reduced sperm motility and abnormal spermatozoan morphology. The Tektins are a family of highly conserved filamentous proteins expressed in the axoneme and associated structures in many different metazoan species. Earlier studies on mice identified Tektin3 (Tekt3) as a testis-enriched gene, and knockout of Tekt3 resulted in asthenozoospermia in the mice. Here, whole-exome sequencing of 100 males with asthenozoospermia from unrelated families was performed, followed by Sanger sequencing, leading to the identification of TEKT3 as a candidate gene in two of these patients and their associated family members. In total, three mutations in the TEKT3 gene were identified in both these patients, including one homozygous deletion-insertion mutation (c.543_547delinsTTGAT: p.Glu182*) and one compound heterozygous mutation (c.[548G > A]; [752A > C], p.[Arg183Gln]; [Gln251Pro]). Both of these mutations resulted in the complete loss of TEKT3 expression. The patients were both found to produce sperm that, although those showed no apparent defects in the flagellar structure, had reduced progressive motility. In contrast to mice, most sperm from these two patients exhibited acrosomal hypoplasia, although this did not prevent the use of the sperm for in vitro fertilization through an ICSI approach. TEKT3 was found to bind to other TEKT proteins, suggesting that these proteins form a complex within human spermatozoa. Overall, these results suggest that a loss of TEKT3 function can contribute to OAT incidence in humans. TEKT3 deficiencies can reduce sperm motility and contribute to severe acrosomal hypoplasia in spermatozoa, compromising their normal function.

Bi-allelic variants in chromatoid body protein TDRD6 cause spermiogenesis defects and severe oligoasthenoteratozoospermia in humans.

BACKGROUND: The association between the TDRD6 variants and human infertility remains unclear, as only one homozygous missense variant of TDRD6 was found to be associated with oligoasthenoteratozoospermia (OAT). METHODS: Whole-exome sequencing and Sanger sequencing were employed to identify potential pathogenic variants of TDRD6 in infertile men. Histology, immunofluorescence, immunoblotting and ultrastructural analyses were conducted to clarify the structural and functional abnormalities of sperm in mutated patients. Tdrd6-knockout mice were generated using the CRISPR-Cas9 system. Total RNA-seq and single-cell RNA-seq (scRNA-seq) analyses were used to elucidate the underlying molecular mechanisms, followed by validation through quantitative RT-PCR and immunostaining. Intracytoplasmic sperm injection (ICSI) was also used to assess the efficacy of clinical treatment. RESULTS: Bi-allelic TDRD6 variants were identified in five unrelated Chinese individuals with OAT, including homozygous loss-of-function variants in two consanguineous families. Notably, besides reduced concentrations and impaired motility, a significant occurrence of acrosomal hypoplasia was detected in multiple spermatozoa among five patients. Using the Tdrd6-deficient mice, we further elucidate the pivotal role of TDRD6 in spermiogenesis and acrosome identified. In addition, the mislocalisation of crucial chromatoid body components DDX4 (MVH) and UPF1 was also observed in round spermatids from patients harbouring TDRD6 variants. ScRNA-seq analysis of germ cells from a patient with TDRD6 variants revealed that TDRD6 regulates mRNA metabolism processes involved in spermatid differentiation and cytoplasmic translation. CONCLUSION: Our findings strongly suggest that TDRD6 plays a conserved role in spermiogenesis and confirms the causal relationship between TDRD6 variants and human OAT. Additionally, this study highlights the unfavourable ICSI outcomes in individuals with bi-allelic TDRD6 variants, providing insights for potential clinical treatment strategies.

Novel MEIOB pathogenic variants including a homozygous non-canonical splicing variant, cause meiotic arrest and human non-obstructive azoospermia.

Non-obstructive azoospermia (NOA) is the most severe form of human male infertility, and the genetic causes of NOA with meiotic arrest remain largely unclear. In this study, we identified novel compound heterozygous MEIOB variants (c.814C > T: p.R272X and c.976G > A: p.A326T) and a previously undescribed homozygous non-canonical splicing variant of MEIOB (c.528 + 3A > C) in two NOA-affected individuals from two irrelevant Chinese families. MEIOB missense variant (p.A326T) significantly reduced protein abundance and nonsense variant (p.R272X) produced a truncated protein. Both of two variants impaired the MEIOB-SPATA22 interaction. The MEIOB non-canonical splicing variant resulted in whole Exon 6 skipping by minigene assay, which was predicted to produce a frameshift truncated protein (p.S111Rfs*32). Histological and immunostaining analysis indicated that both patients exhibited a similar phenotype as we previously reported in Meiob mutant mice, that is, absence of spermatids in seminiferous tubules and meiotic arrest. Our study identified three novel pathogenic variants of MEIOB in NOA patients, extending the mutation spectrum of the MEIOB and highlighting the contribution of meiotic recombination related genes in human fertility.

Performance evaluation of differential splicing analysis methods and splicing analytics platform construction.

A proportion of previously defined benign variants or variants of uncertain significance in humans, which are challenging to identify, may induce an abnormal splicing process. An increasing number of methods have been developed to predict splicing variants, but their performance has not been completely evaluated using independent benchmarks. Here, we manually sourced ∼50 000 positive/negative splicing variants from > 8000 studies and selected the independent splicing variants to evaluate the performance of prediction methods. These methods showed different performances in recognizing splicing variants in donor and acceptor regions, reminiscent of different weight coefficient applications to predict novel splicing variants. Of these methods, 66.67% exhibited higher specificities than sensitivities, suggesting that more moderate cut-off values are necessary to distinguish splicing variants. Moreover, the high correlation and consistent prediction ratio validated the feasibility of integration of the splicing prediction method in identifying splicing variants. We developed a splicing analytics platform called SPCards, which curates splicing variants from publications and predicts splicing scores of variants in genomes. SPCards also offers variant-level and gene-level annotation information, including allele frequency, non-synonymous prediction and comprehensive functional information. SPCards is suitable for high-throughput genetic identification of splicing variants, particularly those located in non-canonical splicing regions.

Achieving an optimal pregnancy outcome through the combined utilization of micro-TESE and ICSI in cryptorchidism associated with a non-canonical splicing variant in RXFP2.

PURPOSE: To identify the genetic cause of a cryptorchidism patient carrying a non-canonical splicing variant highlighted by SPCards platform in RXFP2 and to provide a comprehensive overview of RXFP2 variants with cryptorchidism correlation. METHODS: We identified a homozygous non-canonical splicing variant by whole-exome sequencing and Sanger sequencing in a case with cryptorchidism and non-obstructive azoospermia (NOA). As the pathogenicity of this non-canonical splicing variant remained unclear, we initially utilized the SPCards platform to predict its pathogenicity. Subsequently, we employed a minigene splicing assay to further evaluate the influence of the identified splicing variant. Microdissection testicular sperm extraction (micro-TESE) combined with intracytoplasmic sperm injection (ICSI) was performed. PubMed and Human Genome Variant Database (HGMD) were queried to search for RXFP2 variants. RESULTS: We identified a homozygous non-canonical splicing variant (NM_130806: c.1376-12A > G) in RXFP2, and confirmed this variant caused aberrant splicing of exons 15 and 16 of the RXFP2 gene: 11 bases were added in front of exon 16, leading to an abnormal transcript initiation and a frameshift. Fortunately, the patient successfully obtained his biological offspring through micro-TESE combined with ICSI. Four cryptorchidism-associated variants in RXFP2 from 90 patients with cryptorchidism were identified through a literature search in PubMed and HGMD, with different inheritance patterns. CONCLUSION: This is the first cryptorchidism case carrying a novel causative non-canonical splicing RXFP2 variant. The combined approach of micro-TESE and ICSI contributed to an optimal pregnancy outcome. Our literature review demonstrated that RXFP2 variants caused cryptorchidism in a recessive inheritance pattern, rather than a dominant pattern.

ACROSIN deficiency causes total fertilization failure in humans by preventing the sperm from penetrating the zona pellucida.

STUDY QUESTION: Does a homozygous nonsense mutation in ACR lead to total fertilization failure (TFF) resulting in male infertility in humans? SUMMARY ANSWER: A novel homozygous nonsense mutation of ACR (c.167G>A, p.Trp56X) was identified in two infertile brothers and shown to cause human TFF. WHAT IS KNOWN ALREADY: ACROSIN, encoded by ACR, is a major acrosomal enzyme expressed only in the acrosome of the sperm head. Inhibition of acrosin prevents sperm penetration of the zona pellucida (ZP) in several species, including humans. Acr-knockout in hamsters causes male infertility with completely blocked fertilization. Of note, there are no reports of ACR mutations associated with TFF in humans. STUDY DESIGN, SIZE, DURATION: Whole-exome sequencing (WES) was used for the identification of pathogenic genes for male factor TFF in eight involved couples. PARTICIPANTS/MATERIALS, SETTING, METHODS: Data from eight infertile couples who had experienced TFF during their IVF or ICSI attempts were collected. Functional assays were used to verify the pathogenicity of the potential genetic factors identified by WES. Subzonal insemination (SUZI) and IVF assays were performed to determine the exact pathogenesis of TFF caused by deficiencies in ACROSIN. MAIN RESULTS AND THE ROLE OF CHANCE: A novel homozygous nonsense mutation in ACR, c.167G>A, p.Trp56X, was identified in two additional primary infertile brothers whose parents were first cousins. This rare mutation caused ACROSIN deficiency and acrosomal ultrastructural defects in the affected sperm. Spermatozoa lacking ACROSIN were unable to penetrate the ZP, rather than hampering sperm binding, disrupting gamete fusion, or preventing oocyte activation. These findings were supported by the fertilization success of SUZI and ICSI attempts, as well as the normal expression of ACTL7A and PLCζ in the mutant sperm, suggesting that ICSI without remedial assisted oocyte activation is an optimal treatment for ARCOSIN-deficient TFF. LIMITATIONS, REASONS FOR CAUTION: The absence of another independent pedigree to support our argument is a limitation of this study. WIDER IMPLICATIONS OF THE FINDINGS: The findings expand our understanding of the genes involved in human TFF, providing information for appropriate genetic counseling and fertility guidance for these patients. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by the National Natural Science Foundation of China (grant no. 82201803, 81901541, 82271639, and 32000584), University Synergy Innovation Program of Anhui Province (GXXT-2019-044), and the Nonprofit Central Research Institute Fund of the Chinese Academy of Medical Sciences (grant no. 2019PT310002). The authors declare no conflicts of interest. TRIAL REGISTRATION NUMBER: N/A.

Hsp90 is a potential risk factor for ovarian cancer prognosis: an evidence of a Chinese clinical center.

BACKGROUND: The potential treatment effects of heat shock protein 90 (Hsp90) inhibitors in ovarian cancer (OC) are controversial. This research aims to investigate the relationship between the level of Hsp90 in peripheral blood and the prognosis of OC patients, as well as the clinicopathological indicators. MATERIALS AND METHODS: We retrospectively collected the clinicopathological indicators of OC patients who were admitted to the Department of Obstetrics and Gynecology of the First Affiliated Hospital of Anhui Medical University from 2017 to 2022. Hsp90 level in patient blood was detected by enzyme-linked immunosorbent assay, and the correlation between Hsp90 level and OC prognosis was systematically investigated. Kaplan-Meier method was used to draw the survival curve, and the average survival time and survival rate were calculated. The log-rank test and Cox model were used for univariate survival analysis, and the Cox proportional hazards model was applied for multivariate survival analysis. Based on the TCGA dataset of OC obtained by cBioPortal, Pearson's correlation coefficients between Hsp90 level values and other mRNA expression values were calculated to further conduct bioinformatics analysis. GSEA and GSVA analysis were also conducted for gene functional enrichment. The expression of Hsp90 in OC tissues were evaluated and compared by Immunohistochemical staining. RESULTS: According to the established screening criteria, 106 patients were selected. The enzyme-linked immunosorbent assay results showed that 50.94% OC patients with abnormal Hsp90 level. According to the outcome of Kaplan-Meier curves, the results revealed that the abnormal level of Hsp90 was suggested to poor prognosis (P = 0.001) of OC patients. Furthermore, the result of multivariate Cox proportional hazards regression model analysis also predicted that abnormal Hsp90 level (HR = 2.838, 95%CI = 1.139-7.069, P = 0.025) was linked to poor prognosis, which could be an independent prognostic factor for the prognosis of OC patients. Moreover, top 100 genes screened by Pearson's value associated with Hsp90, indicating that Hsp90 participated in the regulation of ATF5 target genes, PRAGC1A target genes and BANP target genes and also enriched in the metabolic processes of cell response to DNA damage stimulus, response to heat and protein folding. CONCLUSION: Hsp90 level is positively associated with OC mortality and is a potential prognostic indicator of OC.

Distributed Li-Ion Flux Enabled by Sulfonated Covalent Organic Frameworks for High-Performance Lithium Metal Anodes.

Metallic Li is considered the most promising anode material for high-energy-density batteries owing to its high theoretical capacity and low electrochemical potential. However, inhomogeneous lithium deposition and uncontrollable growth of lithium dendrites result in low lithium utilization, rapid capacity fading, and poor cycling performance. Herein, two sulfonated covalent organic frameworks (COFs) with different sulfonated group contents are synthesized as the multifunctional interlayers in lithium metal batteries. The sulfonic acid groups in the pore channels can serve as Li-anchoring sites that effectively coordinate Li ions. These periodically arranged subunits significantly guide uniform Li-ion flux distribution, guarantee smooth Li deposition, and reduce lithium dendrite formation. Consequently, these characteristics afford an excellent quasi-solid-state electrolyte with a high ionic conductivity of 1.9 × 10-3  S  cm-1 at room temperature and a superior Li++ transference number of 0.91. A Li/LiFePO4 battery with the COF-based electrolyte exhibited dendrite-free Li deposition during the charge process, accompanied by no capacity decay after 100 cycles at 0.1 C.

Novel deleterious splicing variant in HFM1 causes gametogenesis defect and recurrent implantation failure: concerning the risk of chromosomal abnormalities in embryos.

PURPOSE: Poor ovarian response (POR) affects approximately 9% to 24% of women undergoing in vitro fertilization (IVF) cycles, resulting in fewer eggs obtained and increasing clinical cycle cancellation rates. The pathogenesis of POR is related to gene variations. Our study included a Chinese family comprising two siblings with infertility born to consanguineous parents. Poor ovarian response (POR) was identified in the female patient who had multiple embryo implantation failures occurring in subsequent assisted reproductive technology cycles. Meanwhile, the male patient was diagnosed with non-obstructive azoospermia (NOA). METHODS: Whole-exome sequencing and rigorous bioinformatics analyses were conducted to identify the underlying genetic causes. Moreover, the pathogenicity of the identified splicing variant was assessed using a minigene assay in vitro. The remaining poor-quality blastocyst and abortion tissues from the female patient were detected for copy number variations. RESULTS: We identified a novel homozygous splicing variant in HFM1 (NM_001017975.6: c.1730-1G > T) in two siblings. Apart from NOA and POI, biallelic variants in HFM1 were also associated with recurrent implantation failure (RIF). Additionally, we demonstrated that splicing variants caused abnormal alternative splicing of HFM1. Using copy number variation sequencing, we found that the embryos of the female patients had either euploidy or aneuploidy; however, both harbored chromosomal microduplications of maternal origin. CONCLUSION: Our results reveal the different effects of HFM1 on reproductive injury in males and females, extend the phenotypic and mutational spectrum of HFM1, and show the potential risk of chromosomal abnormalities under the RIF phenotype. Moreover, our study provides new diagnostic markers for the genetic counseling of POR patients.

Novel biallelic mutations in SLC26A8 cause severe asthenozoospermia in humans owing to midpiece defects: Insights into a putative dominant genetic disease.

To investigate the genetic cause of male infertility characterized by severe asthenozoospermia, two unrelated infertile men with severe asthenozoospermia from nonconsanguineous Chinese families were enrolled, and whole exome sequencing were performed to identify the potential pathogenic mutations. Novel compound heterozygous mutations (NK062 III-1: c.290T>C, p.Leu97Pro; c.1664delT, p.Ile555Thrfs*11/NK038 III-1: c.212G>T, p.Arg71Leu; c.290T>C, p.Leu97Pro) in SLC26A8 were identified. All mutations were inherited from their heterozygous parents and are predicted to be disease-causing by sorts intolerant from tolerant, PolyPhen-2, Mutation Taster, and Combined Annotation Dependent Depletion. In silico mutant SLC26A8 models predict that mutations p.Leu97Pro and p.Arg71Leu cause changes in the α-helix, which may result in functional defects in the protein. Notably, heterozygous male carriers of each mutation in both families were able to reproduce naturally, which is inconsistent with previous reports. Ultrastructural analysis revealed severe asthenozoospermia associated with absence of the mitochondrial sheath and annulus in spermatozoa from both the probands, and both structural defects were verified by HSP60 and SEPT4 immunofluorescence analysis. SLC26A8 levels were significantly reduced in spermatozoa from patients harboring biallelic SLC26A8 mutations, and both patients achieved good prognosis following intracytoplasmic sperm injection. Our findings indicate that mutations in SLC26A8 could manifest as a recessive genetic cause of severe asthenozoospermia and male infertility.

Biallelic loss-of-function mutations in SEPTIN4 (C17ORF47), encoding a conserved annulus protein, cause thin midpiece spermatozoa and male infertility in humans.

Asthenoteratozoospermia is the primary cause of infertility in humans. However, the genetic etiology remains largely unknown for those suffering from severe asthenoteratozoospermia caused by thin midpiece defects. In this study, we identified two biallelic loss-of-function variants of SEPTIN4 (previously SEPT4) (Patient 1: c.A721T, p.R241* and Patient 2: c.C205T, p.R69*) in two unrelated individuals from two consanguineous Chinese families. SEPT4 is a conserved annulus protein that is critical for male fertility and the structural integrity of the sperm midpiece in mice. SEPT4 mutations disrupted the formation of SEPT-based annulus and localization of SEPTIN subunits in sperms from patients. The ultrastructural analysis demonstrated striking thin midpiece spermatozoa defects owing to annulus loss and disorganized mitochondrial sheath. Immunofluorescence and immunoblotting analyses of the mitochondrial sheath proteins TOMM20 and HSP60 further indicated that the distribution and abundance of mitochondria were impaired in men harboring biallelic SEPT4 variants. Additionally, we found that the precise localization of SLC26A8, a testis-specific anion transporter that colocalizes with SEPT4 at the sperm annulus, was missing without SEPT4. Moreover, the patient achieved a good pregnancy outcome following intracytoplasmic sperm injection. Overall, our study demonstrated for the first time that SEPT4 variants that induced thin midpiece spermatozoa defects were directly associated with human asthenoteratozoospermia.

Homozygous missense mutation in CCDC155 disrupts the transmembrane distribution of CCDC155 and SUN1, resulting in non-obstructive azoospermia and premature ovarian insufficiency in humans.

Non-obstructive azoospermia (NOA) and premature ovarian insufficiency (POI) represent the most serious forms of human infertility caused by gametogenic failure. Although whole-exome sequencing (WES) has uncovered multiple monogenic causes of human infertility, our knowledge of the genetic basis of human gametogenesis defects remains at a rudimentary stage. Coiled-coil-domain-containing protein 155 (CCDC155) encodes a core component of the linker of the nucleoskeleton and cytoskeleton complex that is essential for modulating telomere-led chromosome movements during the meiotic prophase of mice. Additionally, Ccdc155 deficiency in mice causes infertility in both sexes with meiotic arrest. In this study, we applied WES to identify the pathogenic genes for 15 NOA and POI patients whose parents were consanguineous and identified a novel homozygous missense mutation in CCDC155 [c.590T>C (p.Leu197Pro)] in a pair of familial NOA and POI patients whose parents were first cousins. The affected spermatocytes were unable to complete meiotic division coupled with unresolved repair of the DNA double-strand break. This rare missense mutation with lesions in the conserved CC domain of CCDC155 blocked nuclear envelope (NE) distribution and subsequently prevented NE-specific enrichment of Sad1- and UNC84-domain-containing 1 either ex vivo or in vitro, eventually leading to disruptive NE anchoring of chromosome-induced meiotic arrest in both sexes. This study presents the first evidence of the necessity of the SUN1-CCDC155 complex during human meiosis and provides insight into the CCDC155 CC domain, thereby expanding the genetic spectrum of human NOA and POI and promoting adequate genetic counselling and appropriate fertility guidance for these patients.

Non-invasive molecular biomarkers for predicting outcomes of micro-TESE in patients with idiopathic non-obstructive azoospermia.

Non-obstructive azoospermia (NOA), the most severe type of male infertility, affects approximately 1% of men worldwide. However, the aetiology of most NOA cases is not definite, that is defined as idiopathic NOA (INOA), posing a clinical conundrum worldwide. Most of these patients must receive donor sperm treatment until the emergence of microdissection testicular sperm extraction (micro-TESE). Although this procedure has recently become a promising treatment for INOA, the low sperm retrieval rate and testicular trauma have prompted us to explore appropriate non-invasive molecular biomarkers to predict the outcomes of sperm recovery preoperatively. Previous studies have identified a spectrum of biomarkers to address this challenging issue at various levels in different tissues, such as DNAs, RNAs, protein and steroid levels in the blood and seminal fluid. To better understand and assess the predictive values of diverse molecular biomarkers from different tissues on the outcome of sperm retrieval by micro-TESE in patients with INOA, we summarised recent findings and discussed the potential applications of these methods. The ultimate goal of this study was to provide references for further studies and clinical management.

Loss of function mutation in DNAH7 induces male infertility associated with abnormalities of the sperm flagella and mitochondria in human.

Male infertility is an increasingly serious health problem affecting couples of reproductive age. Mutations in axoneme-associated genes cause male infertility. Dynein arm proteins are essential in sustaining normal axonemes and promote flagellar motility. However, the function of DNAH7 in male fertility in vivo remains unclear. Herein, we showed that DNAH7 disruption in humans results in male infertility, which was characterised by multiple morphological abnormalities of sperm flagella. The axoneme structure of the sperm from a DNAH7-deficient patient revealed the loss of inner dynein arms. Moreover, the mitochondria of the sperm flagella detached and dispersed outside the axoneme, leading to abnormalities in the mitochondrial sheath in the mid-piece region. Live birth was achieved via intracytoplasmic sperm injection. Thus, DNAH7 is critical for axoneme and mitochondrial development in human sperm. These findings further clarify the spectrum of DNAH7 biology and provide new insights for diagnosing infertility and treating patients harbouring DNAH7 mutations.

Identification of deleterious variants in patients with male infertility due to idiopathic non-obstructive azoospermia.

BACKGROUND: Non-obstructive azoospermia (NOA) is the most severe type of male infertility, affecting 1% of men worldwide. Most of its etiologies remain idiopathic. Although genetic studies have identified dozens of NOA genes, monogenic mutations can also account for a small proportion of idiopathic NOA cases. Hence, this genetic study was conducted to explore the causes of monogenic variants of NOA in a cohort of Chinese patients. METHODS: Following the screening using chromosomal karyotyping, Y chromosome microdeletion analyses, and sex hormone assessments, subsequent whole-exome sequencing analysis was performed in 55 unrelated idiopathic NOA patients with male infertility to explore potential deleterious variants associated with spermatogenesis. We also performed Sanger sequencing to demonstrate the variants. Testicular biopsy or microsurgical testicular sperm extraction was also performed to confirm the diagnosis of NOA and identify spermatozoa. Hematoxylin and eosin staining was performed to assess the histopathology of spermatogenesis. RESULTS: Abnormal testicular pathological phenotypes included Sertoli cell-only syndrome, maturation arrest, and hypospermatogenesis. Using bioinformatics analysis, we detected novel variants in two recessive genes, FANCA (NM_000135, c.3263C > T, c.1729C > G) and SYCE1 (NM_001143763, c.689_690del); one X-linked gene, TEX11 (NM_031276, c.466A > G, c.559_560del); and two dominant genes, DMRT1 (NM_021951, c.425C > T, c.340G > A) and PLK4 (NM_001190799, c.2785A > G), in eight patients, which corresponded to 14.55% (8/55) of the patients. CONCLUSION: This study presented some novel variants of known pathogenic genes for NOA. Further, it expanded the variant spectrum of NOA patients, which might advance clinical genetic counseling in the future.

Homozygous SPAG6 variants can induce nonsyndromic asthenoteratozoospermia with severe MMAF.

BACKGROUND: Multiple morphological abnormalities of the sperm flagella (MMAF) is a subtype of severe asthenoteratozoospermia with poorly understood genetic etiology. SPAG6 is a core axonemal component that plays a critical role in the formation of cilia and sperm flagella. Previous studies have reported that mutations in SPAG6 cause primary ciliary dyskinesia (PCD), but the association between SPAG6 gene variants and the MMAF phenotype has not yet been described. METHODS: We performed whole-exome sequencing (WES) in two unrelated Han Chinese men with MMAF. Sanger sequencing was used to validate the candidate variants. Routine semen analysis was carried out according to the WHO guidelines (5th Edition). Sperm morphology was assessed using modified Papanicolaou staining. Scanning and transmission electron microscopy (S/TEM) was performed to observe the ultrastructural defects of the sperm flagella. Western blot analysis and immunofluorescence (IF) of spermatozoa were performed to examine the expression of SPAG6 protein. Assisted fertilization with intracytoplasmic sperm injection (ICSI) was applied. RESULTS: Two homozygous SPAG6 variants were identified by WES and Sanger validation in two patients with MMAF phenotype (F1 II-1: c.308C > A, p. A103D; F2 II-1: c. 585delA, p. K196Sfs*6). Semen analysis showed progressive rates of less than 1%, and most of the spermatozoa presented MMAF by Papanicolaou staining. TEM revealed that the overall axonemal ultrastructure was disrupted and primarily presented an abnormal "9 + 0" configuration. No other PCD-related symptoms were found on physical examination and medical consultations, as well as lung CT screening. The level of SPAG6 protein was significantly decreased in the spermatozoa, and IF analysis revealed that SPAG6 staining was extremely weak and discontinuous in the sperm flagella of the two patients. Notably, F1 II-1 and his wife conceived successfully after undergoing ICSI. CONCLUSIONS: Our research provides new evidence for a potential correlation between SPAG6 variants and the MMAF phenotype.

Homozygous mutation in SLO3 leads to severe asthenoteratozoospermia due to acrosome hypoplasia and mitochondrial sheath malformations.

BACKGROUND: Potassium channels are important for the structure and function of the spermatozoa. As a potassium transporter, the mSlo3 is essential for male fertility as Slo3 knockout male mice were infertile with the series of functional defects in sperm cells. However, no pathogenic variant has been detected in human SLO3 to date. Here we reported a human case with homozygous SLO3 mutation. The function of SLO3 in human sperm and the corresponding assisted reproductive strategy are also investigated. METHODS: We performed whole-exome sequencing analysis from a large cohort of 105 patients with asthenoteratozoospermia. The effects of the variant were investigated by quantitative RT-PCR, western blotting, and immunofluorescence assays using the patient spermatozoa. Sperm morphological and ultrastructural studies were conducted using haematoxylin and eosin staining, scanning and transmission electron microscopy. RESULTS: We identified a homozygous missense variant (c.1237A > T: p.Ile413Phe) in the sperm-specific SLO3 in one Chinese patient with male infertility. This SLO3 variant was rare in human control populations and predicted to be deleterious by multiple bioinformatic tools. Sperm from the individual harbouring the homozygous SLO3 variant exhibited severe morphological abnormalities, such as acrosome hypoplasia, disruption of the mitochondrial sheath, coiled tails, and motility defects. The levels of SLO3 mRNA and protein in spermatozoa from the affected individual were reduced. Furthermore, the acrosome reaction, mitochondrial membrane potential, and membrane potential during capacitation were also afflicted. The levels of acrosome marker glycoproteins and PLCζ1 as well as the mitochondrial sheath protein HSP60 and SLO3 auxiliary subunit LRRC52, were significantly reduced in the spermatozoa from the affected individual. The affected man was sterile due to acrosome and mitochondrial dysfunction; however, intra-cytoplasmic sperm injection successfully rescued this infertile condition. CONCLUSIONS: SLO3 deficiency seriously impact acrosome formation, mitochondrial sheath assembly, and the function of K+ channels. Our findings provided clinical implications for the genetic and reproductive counselling of affected families.

Gene4Denovo: an integrated database and analytic platform for de novo mutations in humans.

De novo mutations (DNMs) significantly contribute to sporadic diseases, particularly in neuropsychiatric disorders. Whole-exome sequencing (WES) and whole-genome sequencing (WGS) provide effective methods for detecting DNMs and prioritizing candidate genes. However, it remains a challenge for scientists, clinicians, and biologists to conveniently access and analyse data regarding DNMs and candidate genes from scattered publications. To fill the unmet need, we integrated 580 799 DNMs, including 30 060 coding DNMs detected by WES/WGS from 23 951 individuals across 24 phenotypes and prioritized a list of candidate genes with different degrees of statistical evidence, including 346 genes with false discovery rates <0.05. We then developed a database called Gene4Denovo (http://www.genemed.tech/gene4denovo/), which allowed these genetic data to be conveniently catalogued, searched, browsed, and analysed. In addition, Gene4Denovo integrated data from >60 genomic sources to provide comprehensive variant-level and gene-level annotation and information regarding the DNMs and candidate genes. Furthermore, Gene4Denovo provides end-users with limited bioinformatics skills to analyse their own genetic data, perform comprehensive annotation, and prioritize candidate genes using custom parameters. In conclusion, Gene4Denovo conveniently allows for the accelerated interpretation of DNM pathogenicity and the clinical implication of DNMs in humans.

Bi-allelic variants in DNAH10 cause asthenoteratozoospermia and male infertility.

PURPOSE: Multiple morphological abnormalities in the sperm flagella (MMAF) comprise a severe phenotype of asthenoteratozoospermia with reduced or absent spermatozoa motility. Whereas dozens of candidate pathogenic genes for MMAF have been identified, the genetic cause in a large proportion of patients is unknown. We attempted to identify novel genetic explanations for MMAF. METHODS: We performed whole-exome sequencing of patients with MMAF to identify pathogenic variants. The phenotypes of spermatozoa in patients carrying DNAH10 variants were investigated using haematoxylin and eosin staining, scanning electron microscopy, and transmission electron microscopy. The expression and location of DNAH10 and other spermatozoa structure-related proteins were analyzed using immunofluorescence assays. RESULTS: We found one homozygous frameshift DNAH10 variant (NM_207437: c.2514delG:p.L839*) and one compound heterozygous DNAH10 variant (NM_207437: c.10820 T > C:p.M3607T; c.12692C > T:p.T4231I) in two patients with MMAF. These variants were absent or rare in the general population. Haematoxylin and eosin staining and scanning electron microscopy revealed the significant disruption of sperm flagella in the patients. In addition, ultrastructural analysis by transmission electron microscopy showed significant inner dynein arm (IDA) deficiency in sperm flagella. Using immunofluorescence assays, we found a significant reduction in IDA-related proteins including DNAH10 and DNAH1. CONCLUSIONS: We identified putative novel pathogenic variants in DNAH10 for MMAF, which might advance the genetic diagnosis and clinical genetic counselling for male infertility.

Bi-allelic truncating variants in CFAP206 cause male infertility in human and mouse.

Spermatozoa are polarized cells with a head and a flagellum joined together by the connecting piece. Flagellum integrity is critical for normal sperm function, and flagellum defects consistently lead to male infertility. Multiple morphological abnormalities of the flagella (MMAF) is a distinct sperm phenotype consistently leading to male infertility due to a reduced or absent sperm motility associated with severe morphological and ultrastructural flagellum defects. Despite numerous genes recently described to be recurrently associated with MMAF, more than half of the cases analyzed remain unresolved, suggesting that many yet uncharacterized gene defects account for this phenotype. By performing a retrospective exome analysis of the unsolved cases from our initial cohort of 167 infertile men with a MMAF phenotype, we identified one individual carrying a homozygous frameshift variant in CFAP206, a gene encoding a microtubule-docking adapter for radial spoke and inner dynein arm. Immunostaining experiments in the patient's sperm cells demonstrated the absence of WDR66 and RSPH1 proteins suggesting severe radial spokes and calmodulin and spoke-associated complex defects. Using the CRISPR-Cas9 technique, we generated homozygous Cfap206 knockout (KO) mice which presented with male infertility due to functional, structural and ultrastructural sperm flagellum defects associated with a very low rate of embryo development using ICSI. Overall, we showed that CFAP206 is essential for normal sperm flagellum structure and function in human and mouse and that bi-allelic mutations in CFAP206 cause male infertility in man and mouse by inducing morphological and functional defects of the sperm flagellum that may also cause ICSI failures.