CCDC181 is required for sperm flagellum biogenesis and male fertility in mice.

The structural integrity of the sperm flagellum is essential for proper sperm function. Flagellar defects can result in male infertility, yet the precise mechanisms underlying this relationship are not fully understood. CCDC181, a coiled-coil domain-containing protein, is known to localize on sperm flagella and at the basal regions of motile cilia. Despite this knowledge, the specific functions of CCDC181 in flagellum biogenesis remain unclear. In this study, Ccdc181 knockout mice were generated. The absence of CCDC181 led to defective sperm head shaping and flagellum formation. Furthermore, the Ccdc181 knockout mice exhibited extremely low sperm counts, grossly aberrant sperm morphologies, markedly diminished sperm motility, and typical multiple morphological abnormalities of the flagella (MMAF). Additionally, an interaction between CCDC181 and the MMAF-related protein LRRC46 was identified, with CCDC181 regulating the localization of LRRC46 within sperm flagella. These findings suggest that CCDC181 plays a crucial role in both manchette formation and sperm flagellum biogenesis.

Homozygous CCDC146 mutation causes oligoasthenoteratozoospermia in humans and mice.

Infertility represents a significant health concern, with sperm quantity and quality being crucial determinants of male fertility. Oligoasthenoteratozoospermia (OAT) is characterized by reduced sperm motility, lower sperm concentration, and morphological abnormalities in sperm heads and flagella. Although variants in several genes have been implicated in OAT, its genetic etiologies and pathogenetic mechanisms remain inadequately understood. In this study, we identified a homozygous nonsense mutation (c.916C>T, p.Arg306*) in the coiled-coil domain containing 146 ( CCDC146) gene in an infertile male patient with OAT. This mutation resulted in the production of a truncated CCDC146 protein (amino acids 1-305), retaining only two out of five coiled-coil domains. To validate the pathogenicity of the CCDC146 mutation, we generated a mouse model ( Ccdc146 mut/mut ) with a similar mutation to that of the patient. Consistently, the Ccdc146 mut/mut mice exhibited infertility, characterized by significantly reduced sperm counts, diminished motility, and multiple defects in sperm heads and flagella. Furthermore, the levels of axonemal proteins, including DNAH17, DNAH1, and SPAG6, were significantly reduced in the sperm of Ccdc146 mut/mut mice. Additionally, both human and mouse CCDC146 interacted with intraflagellar transport protein 20 (IFT20), but this interaction was lost in the mutated versions, leading to the degradation of IFT20. This study identified a novel deleterious homozygous nonsense mutation in CCDC146 that causes male infertility, potentially by disrupting axonemal protein transportation. These findings offer valuable insights for genetic counseling and understanding the mechanisms underlying CCDC146 mutant-associated infertility in human males.

A homozygous nonsense variant in HENMT1 causes male infertility in humans and mice.

BACKGROUND: HENMT1 encodes a small RNA methyltransferase that plays a crucial role in mouse spermatogenesis through the methylation of the 3' end of PIWI-interacting RNAs. OBJECTIVES: Our study aims to elucidate the relationship between HENMT1 and male infertility in humans. MATERIALS AND METHODS: A consanguineous family, having a single non-obstructive azoospermia patient was recruited for pathogenic variants screening. The research includes genetic analysis and experimental validation using mouse models. The patient was diagnosed with non-obstructive azoospermia. Whole-exome sequencing and subsequent bioinformatic analyses were performed to screen for candidate pathogenic variants. The pathogenicity of the identified variant was assessed and studied in vivo using a mouse model that mimicked the patient's mutation. RESULTS: Through whole-exome sequencing, we identified a homozygous nonsense variant (c.555G > A, p.Trp185*) in HENMT1 in the patient. The presence of the mutant HENMT1 mRNA was detected in the patient's blood, and the truncated HENMT1 protein was observed in transfected HEK293T cells. The mutant mice modeling this HENMT1 variant displayed an infertile phenotype similar to that of the patient, characterized by spermiogenesis arrest. Further analysis revealed a significant derepression of retrotransposon LINE1 in the testes of the Henmt1 mutant mice, and increased apoptosis of spermatids. DISCUSSION AND CONCLUSION: Our findings provide the evidence of pathogenicity of the identified HENMT1 variant, thus shedding light on the indispensable role of HENMT1 in human spermatogenesis.

Pathogenic relationship between phenotypes of ARPKD and novel compound heterozygous mutations of PKHD1.

Background: To investigate whether the novel mutation of PKHD1 could cause polycystic kidney disease by affecting splicing with a recessive inheritance pattern. Methods: A nonconsanguineous Chinese couple with two recurrent pregnancies showed fetal enlarged echogenic polycystic kidney and oligoamnios were recruited. Pedigree WES, minigene splicing assay experiment and following bioinformatics analysis were performed to verify the effects, and inheritance pattern of diseasing-causing mutations. Results: WES revealed that both fetuses were identified as carrying the same novel mutation c.3592_3628 + 45del, p.? and c.11207 T>C, p.(Ile3736Thr) in the PKHD1 gene (NM_138694.4), which inherited from the father and mother respectively. Both bioinformatic method prediction and minigene splicing assay experience results supported the mutation c.3592_3628 + 45del, p.? affects the splicing of the PKHD1 transcript, resulting in exon 31 skipping. Another missense mutation c.11207 T>C, p.(Ile3736Thr) has a low frequency in populations and is predicted to be deleterious by bioinformatic methods. Conclusion: These findings provide a direct clinical and functional evidence that the truncating mutations of the PKHD1 gene could lead to more severe phenotypes, and cause ARPKD as a homozygous or compound heterozygous pattern. Our study broadens the variant spectrum of the PKHD1 gene and provides a basis for genetic counseling and diagnosis of ARPKD.

A novel missense mutation of CCDC34 causes male infertility with oligoasthenoteratozoospermia in a consanguineous Pakistani family.

ABSTRACT: Male infertility is a worldwide health issue, affecting 8%-12% of the global population. Oligoasthenoteratozoospermia (OAT) represents a severe type of male infertility, characterized by reduced sperm count and motility and an increased frequency of sperm with aberrant morphology. Using whole-exome sequencing, this study identified a novel missense mutation (c.848C>A, p.A283E) in the coiled-coil domain-containing 34 gene (CCDC34) in a consanguineous Pakistani family. This rare mutation was predicted to be deleterious and to affect the protein stability. Hematoxylin and eosin staining of spermatozoa from the patient with OAT revealed multiple morphological abnormalities of the flagella and transmission electron microscopy indicated axonemal ultrastructural defects with a lack of outer dynein arms. These findings indicated that CCDC34 plays a role in maintaining the axonemal ultrastructure and the assembly or stability of the outer dynein arms, thus expanding the phenotypic spectrum of CCDC34 missense mutations.

CCDC157 is essential for sperm differentiation and shows oligoasthenoteratozoospermia-related mutations in men.

Oligoasthenoteratospermia (OAT), characterized by abnormally low sperm count, poor sperm motility, and abnormally high number of deformed spermatozoa, is an important cause of male infertility. Its genetic basis in many affected individuals remains unknown. Here, we found that CCDC157 variants are associated with OAT. In two cohorts, a 21-bp (g.30768132_30768152del21) and/or 24-bp (g.30772543_30772566del24) deletion of CCDC157 were identified in five sporadic OAT patients, and 2 cases within one pedigree. In a mouse model, loss of Ccdc157 led to male sterility with OAT-like phenotypes. Electron microscopy revealed misstructured acrosome and abnormal head-tail coupling apparatus in the sperm of Ccdc157-null mice. Comparative transcriptome analysis showed that the Ccdc157 mutation alters the expressions of genes involved in cell migration/motility and Golgi components. Abnormal Golgi apparatus and decreased expressions of genes involved in acrosome formation and lipid metabolism were detected in Ccdc157-deprived mouse germ cells. Interestingly, we attempted to treat infertile patients and Ccdc157 mutant mice with a Chinese medicine, Huangjin Zanyu, which improved the fertility in one patient and most mice that carried the heterozygous mutation in CCDC157. Healthy offspring were produced. Our study reveals CCDC157 is essential for sperm maturation and may serve as a marker for diagnosis of OAT.

A novel recombination protein C12ORF40/REDIC1 is required for meiotic crossover formation.

During meiosis, at least one crossover must occur per homologous chromosome pair to ensure normal progression of meiotic division and accurate chromosome segregation. However, the mechanism of crossover formation is not fully understood. Here, we report a novel recombination protein, C12ORF40/REDIC1, essential for meiotic crossover formation in mammals. A homozygous frameshift mutation in C12orf40 (c.232_233insTT, p.Met78Ilefs*2) was identified in two infertile men with meiotic arrest. Spread mouse spermatocyte fluorescence immunostaining showed that REDIC1 forms discrete foci between the paired regions of homologous chromosomes depending on strand invasion and colocalizes with MSH4 and later with MLH1 at the crossover sites. Redic1 knock-in (KI) mice homozygous for mutation c.232_233insTT are infertile in both sexes due to insufficient crossovers and consequent meiotic arrest, which is also observed in our patients. The foci of MSH4 and TEX11, markers of recombination intermediates, are significantly reduced numerically in the spermatocytes of Redic1 KI mice. More importantly, our biochemical results show that the N-terminus of REDIC1 binds branched DNAs present in recombination intermediates, while the identified mutation impairs this interaction. Thus, our findings reveal a crucial role for C12ORF40/REDIC1 in meiotic crossover formation by stabilizing the recombination intermediates, providing prospective molecular targets for the clinical diagnosis and therapy of infertility.

Loss-of-function variants in KCTD19 cause non-obstructive azoospermia in humans.

Azoospermia is a significant cause of male infertility, with non-obstructive azoospermia (NOA) being the most severe type of spermatogenic failure. NOA is mostly caused by congenital factors, but our understanding of its genetic causes is very limited. Here, we identified a frameshift variant (c.201_202insAC, p.Tyr68Thrfs∗17) and two nonsense variants (c.1897C>T, p.Gln633∗; c.2005C>T, p.Gln669∗) in KCTD19 (potassium channel tetramerization domain containing 19) from two unrelated infertile Chinese men and a consanguineous Pakistani family with three infertile brothers. Testicular histological analyses revealed meiotic metaphase I (MMI) arrest in the affected individuals. Mice modeling KCTD19 variants recapitulated the same MMI arrest phenotype due to severe disrupted individualization of MMI chromosomes. Further analysis showed a complete loss of KCTD19 protein in both Kctd19 mutant mouse testes and affected individual testes. Collectively, our findings demonstrate the pathogenicity of the identified KCTD19 variants and highlight an essential role of KCTD19 in MMI chromosome individualization.

Biallelic mutations in RNA-binding protein ADAD2 cause spermiogenic failure and non-obstructive azoospermia in humans.

STUDY QUESTION: What are some pathogenic mutations for non-obstructive azoospermia (NOA) and their effects on spermatogenesis? SUMMARY ANSWER: Biallelic missense and frameshift mutations in ADAD2 disrupt the differentiation of round spermatids to spermatozoa causing azoospermia in humans and mice. WHAT IS KNOWN ALREADY: NOA is the most severe cause of male infertility characterized by an absence of sperm in the ejaculate due to impairment of spermatogenesis. In mice, the lack of the RNA-binding protein ADAD2 leads to a complete absence of sperm in epididymides due to failure of spemiogenesis, but the spermatogenic effects of ADAD2 mutations in human NOA-associated infertility require functional verification. STUDY DESIGN SIZE DURATION: Six infertile male patients from three unrelated families were diagnosed with NOA at local hospitals in Pakistan based on infertility history, sex hormone levels, two semen analyses and scrotal ultrasound. Testicular biopsies were performed in two of the six patients. Adad2 mutant mice (Adad2Mut/Mut) carrying mutations similar to those found in NOA patients were generated using the CRISPR/Cas9 genome editing tool. Reproductive phenotypes of Adad2Mut/Mut mice were verified at 2 months of age. Round spermatids from the littermates of wild-type (WT) and Adad2Mut/Mut mice were randomly selected and injected into stimulated WT oocytes. This round spermatid injection (ROSI) procedure was conducted with three biological replicates and >400 ROSI-derived zygotes were evaluated. The fertility of the ROSI-derived progeny was evaluated for three months in four Adad2WT/Mut male mice and six Adad2WT/Mut female mice. A total of 120 Adad2Mut/Mut, Adad2WT/Mut, and WT mice were used in this study. The entire study was conducted over 3 years. PARTICIPANTS/MATERIALS SETTING METHODS: Whole-exome sequencing was performed to detect potentially pathogenic mutations in the six NOA-affected patients. The pathogenicity of the identified ADAD2 mutations was assessed and validated in human testicular tissues and in mouse models recapitulating the mutations in the NOA patients using quantitative PCR, western blotting, hematoxylin-eosin staining, Periodic acid-Schiff staining, and immunofluorescence. Round spermatids of WT and Adad2Mut/Mut mice were collected by fluorescence-activated cell sorting and injected into stimulated WT oocytes. The development of ROSI-derived offspring was evaluated in the embryonic and postnatal stages. MAIN RESULTS AND THE ROLE OF CHANCE: Three recessive mutations were identified in ADAD2 (MT1: c.G829T, p.G277C; MT2: c.G1192A, p.D398N; MT3: c.917_918del, p.Q306Rfs*43) in patients from three unrelated Pakistani families. MT1 and MT2 dramatically reduced the testicular expression of ADAD2, likely causing spermiogenesis failure in the NOA patients. Immunofluorescence analysis of the Adad2Mut/Mut male mice with the corresponding MT3 mutation showed instability and premature degradation of the ADAD2 protein, resulting in the spermiogenesis deficiency phenotype. Through ROSI, the Adad2Mut/Mut mice could produce pups with comparable embryonic development (46.7% in Adad2Mut/Mut versus 50% in WT) and birth rates (21.45 ± 10.43% in Adad2Mut/Mut versus 27.5 ± 3.536% in WT, P = 0.5044) to WT mice. The Adad2WT/Mut progeny from ROSI (17 pups in total via three ROSI replicates) did not show overt developmental defects and had normal fertility. LARGE SCALE DATA: N/A. LIMITATIONS REASONS FOR CAUTION: This is a preliminary report suggesting that ROSI can be an effective treatment for infertile Adad2Mut/Mut mice. Further assisted reproductive attempts need to be carefully examined in humans during clinical trials. WIDER IMPLICATIONS OF THE FINDINGS: Our work provides functional evidence that mutations in the ADAD2 gene are deleterious and cause consistent spermiogenic defects in both humans and mice. In addition, preliminary results show that ROSI can help Adad2Mut/Mut to produce biological progeny. These findings provide valuable clues for genetic counselling on the ADAD2 mutants-associated infertility in human males. STUDY FUNDING/COMPETING INTERESTS: This work was supported by the National Natural Science Foundation of China (32000587, U21A20204, and 32061143006), and the National Key Research and Developmental Program of China (2019YFA0802600 and 2021YFC2700202). This work was also supported by Institute of Health and Medicine, Hefei Comprehensive National Science Center, Hefei, China. The authors declare no competing interests.

A recessive ACTL7A founder variant leads to male infertility due to acrosome detachment in Pakistani Pashtuns.

Male infertility affects more than 20 million men worldwide and is a major public health concern. Male infertility has a strong genetic basis, particularly for those unexplained cases. Here, through genetic analysis of three Pakistani families having eight infertile men with normal parameters in routine semen analysis, we identified a novel ACTL7A variant (c.149_150del, p.E50Afs*6), recessively co-segregating with infertility in these three families. This variant leads to the loss of ACTL7A proteins in spermatozoa from patients. Transmission EM analyses revealed acrosome detachment from nuclei in 98.9% spermatozoa of patients. Interestingly, this ACTL7A variant was frequently detected in our sequenced Pakistani Pashtuns with a minor allele frequency of ~0.021 and all the carriers shared a common haplotype of about 240 kb flanking ACTL7A, indicating that it is likely originated from a single founder. Our findings reveal that a founder ACTL7A pathogenic variant confers a high genetic susceptibility for male infertility with normal routine semen parameters but acrosomal ultrastructural defects in Pakistani Pashtun descendants, and highlight that variants not rare should also be considered when trying to identify disease-causing variants in ethnic groups with the tradition of intra-ethnic marriages.

A homozygous KASH5 frameshift mutation causes diminished ovarian reserve, recurrent miscarriage, and non-obstructive azoospermia in humans.

The meiosis-specific LINC complex, composed of the KASH5 and SUN1 proteins, tethers the moving chromosomes to the nuclear envelope to facilitate homolog pairing and is essential for gametogenesis. Here, we applied whole-exome sequencing for a consanguineous family with five siblings suffering from reproductive failure, and identified a homozygous frameshift mutation in KASH5 (c.1270_1273del, p.Arg424Thrfs*20). This mutation leads to the absence of KASH5 protein expression in testes and non-obstructive azoospermia (NOA) due to meiotic arrest before the pachytene stage in the affected brother. The four sisters displayed diminished ovarian reserve (DOR), with one sister never being pregnant but still having dominant follicle at 35 years old and three sisters suffering from at least 3 miscarriages occurring within the third month of gestation. The truncated KASH5 mutant protein, when expressed in cultured cells, displays a similar localization encircling the nucleus and a weakened interaction with SUN1, as compared with the full-length KASH5 proteins, which provides a potential explanation for the phenotypes in the affected females. This study reported sexual dimorphism for influence of the KASH5 mutation on human germ cell development, and extends the clinical manifestations associated with KASH5 mutations, providing genetic basis for the molecular diagnosis of NOA, DOR, and recurrent miscarriage.

Loss-of-function mutations in CFAP57 cause multiple morphological abnormalities of the flagella in humans and mice.

Multiple morphological abnormalities of the sperm flagella (MMAF) are the most severe form of asthenozoospermia due to impaired axoneme structure in sperm flagella. Dynein arms are necessary components of the sperm flagellar axoneme. In this study, we recruited 3 unrelated consanguineous Pakistani families with multiple MMAF-affected individuals, who had no overt ciliary symptoms. Whole-exome sequencing and Sanger sequencing identified 2 cilia and flagella associated protein 57 (CFAP57) loss-of-function mutations (c.2872C>T, p. R958*; and c.2737C>T, p. R913*) recessively segregating with male infertility. A mouse model mimicking the mutation (c.2872C>T) was generated and recapitulated the typical MMAF phenotype of CFAP57-mutated individuals. Both CFAP57 mutations caused loss of the long transcript-encoded CFAP57 protein in spermatozoa from MMAF-affected individuals or from the Cfap57-mutant mouse model while the short transcript was not affected. Subsequent examinations of the spermatozoa from Cfap57-mutant mice revealed that CFAP57 deficiency disrupted the inner dynein arm (IDA) assembly in sperm flagella and that single-headed IDAs were more likely to be affected. Thus, our study identified 2 pathogenic mutations in CFAP57 in MMAF-affected individuals and reported a conserved and pivotal role for the long transcript-encoded CFAP57 in IDAs' assembly and male fertility.

A novel homozygous frameshift variant in DNAH8 causes multiple morphological abnormalities of the sperm flagella in a consanguineous Pakistani family.

Multiple morphological abnormalities of the sperm flagella (MMAF) is a severe form of asthenozoospermia categorized by immotile spermatozoa with abnormal flagella in ejaculate. Whole-exome sequencing (WES) is used to detect pathogenic variants in patients with MMAF. In this study, a novel homozygous frameshift variant (c.6158_6159insT) in dynein axonemal heavy chain 8 (DNAH8) from two infertile brothers with MMAF in a consanguineous Pakistani family was identified by WES. Reverse transcription-polymerase chain reaction (RT-PCR) confirmed DNAH8 mRNA decay in these patients with the DNAH8 mutation. Hematoxylin-eosin staining and transmission electron microscopy revealed highly divergent morphology and ultrastructure of sperm flagella in these patients. Furthermore, an immunofluorescence assay showed the absence of DNAH8 and a reduction in its associated protein DNAH17 in the patients' spermatozoa. Collectively, our study expands the phenotypic spectrum of patients with DNAH8-related MMAF worldwide.

M1AP interacts with the mammalian ZZS complex and promotes male meiotic recombination.

Following meiotic recombination, each pair of homologous chromosomes acquires at least one crossover, which ensures accurate chromosome segregation and allows reciprocal exchange of genetic information. Recombination failure often leads to meiotic arrest, impairing fertility, but the molecular basis of recombination remains elusive. Here, we report a homozygous M1AP splicing mutation (c.1074 + 2T > C) in patients with severe oligozoospermia owing to meiotic metaphase I arrest. The mutation abolishes M1AP foci on the chromosome axes, resulting in decreased recombination intermediates and crossovers in male mouse models. M1AP interacts with the mammalian ZZS (an acronym for yeast proteins Zip2-Zip4-Spo16) complex components, SHOC1, TEX11, and SPO16. M1AP localizes to chromosomal axes in a SPO16-dependent manner and colocalizes with TEX11. Ablation of M1AP does not alter SHOC1 localization but reduces the recruitment of TEX11 to recombination intermediates. M1AP shows cytoplasmic localization in fetal oocytes and is dispensable for fertility and crossover formation in female mice. Our study provides the first evidence that M1AP acts as a copartner of the ZZS complex to promote crossover formation and meiotic progression in males.

A homozygous missense variant in DND1 causes non-obstructive azoospermia in humans.

Non-obstructive azoospermia (NOA) is a severe factor of male infertility; it affects approximately 1% of the global male population and accounts for 40% of male infertility cases. However, the majority of NOA cases remain idiopathic. This is the first study using whole-exome sequencing (WES) to identify a novel missense mutation in the DND1 gene (c.212A>C, p. E71A) from a Pakistani family, that includes three males with NOA. This mutation is predicted to cause DND1 protein misfolding and weaken the DND1 interaction with NANOS2, a significant regulator in primordial germ cell development. Our study identified a DND1 pathogenic mutation in NOA patients and highlighted its critical role in male fertility in humans.

A Homozygous Loss-of-Function Mutation in MSH5 Abolishes MutSγ Axial Loading and Causes Meiotic Arrest in NOA-Affected Individuals.

Non-obstructive azoospermia (NOA), characterized by spermatogenesis failure and the absence of sperm in ejaculation, is the most severe form of male infertility. However, the etiology and pathology between meiosis-associated monogenic alterations and human NOA remain largely unknown. A homozygous MSH5 mutation (c.1126del) was identified from two idiopathic NOA patients in the consanguineous family. This mutation led to the degradation of MSH5 mRNA and abolished chromosome axial localization of MutSγ in spermatocytes from the affected males. Chromosomal spreading analysis of the patient's meiotic prophase I revealed that the meiosis progression was arrested at a zygotene-like stage with extensive failure of homologous synapsis and DSB repair. Therefore, our study demonstrates that the MSH5 c.1126del could cause meiotic recombination failure and lead to human infertility, improving the genetic diagnosis of NOA clinically. Furthermore, the study of human spermatocytes elucidates the meiosis defects caused by MSH5 variant, and reveals a conserved and indispensable role of MutSγ in human synapsis and meiotic recombination, which have not previously been well-described.

Biallelic HFM1 variants cause non-obstructive azoospermia with meiotic arrest in humans by impairing crossover formation to varying degrees.

STUDY QUESTION: Do variants in helicase for meiosis 1 (HFM1) account for male infertility in humans? SUMMARY ANSWER: Biallelic variants in HFM1 cause human male infertility owing to non-obstructive azoospermia (NOA) with impaired crossover formation and meiotic metaphase I (MMI) arrest. WHAT IS KNOWN ALREADY: HFM1 encodes an evolutionarily conserved DNA helicase that is essential for crossover formation and completion of meiosis. The null mutants of Hfm1 or its ortholog in multiple organisms displayed spermatogenic arrest at the MMI owing to deficiencies in synapsis and severe defects in crossover formation. Although HFM1 variants were found in infertile men with azoospermia or oligozoospermia, the causal relationship has not yet been established with functional evidence. STUDY DESIGN, SIZE, DURATION: A Pakistani family, having two infertile brothers born to consanguineous parents, and three unrelated Chinese men diagnosed with NOA were recruited for pathogenic variants screening. PARTICIPANTS/MATERIALS, SETTING, METHODS: All the patients were diagnosed with idiopathic NOA and, for the Chinese patients, meiotic defects were confirmed by histological analyses and/or immunofluorescence staining on testicular sections. Exome sequencing and subsequent bioinformatic analyses were performed to screen for candidate pathogenic variants. The pathogenicity of identified variants was assessed and studied in vivo in mice carrying the equivalent mutations. MAIN RESULTS AND THE ROLE OF CHANCE: Six variants (homozygous or compound heterozygous) in HFM1 were identified in the three Chinese patients with NOA and two brothers with NOA from the Pakistani family. Testicular histological analysis revealed that spermatogenesis is arrested at MMI in patients carrying the variants. Mice modeling the HFM1 variants identified in patients recapitulated the meiotic defects of patients, confirming the pathogenicity of the identified variants. These Hfm1 variants led to various reductions of HFM1 foci on chromosome axes and resulted in varying degrees of synapsis and crossover formation defects in the mutant male mice. In addition, Hfm1 mutant female mice displayed infertility or subfertility with oogenesis variously affected. LIMITATIONS, REASONS FOR CAUTION: A limitation of the current study is the small sample size. Owing to the unavailability of fresh testicular samples, the defects of synapsis and crossover formation could not be detected in spermatocytes of patients. Owing to the unavailability of antibodies, we could not quantify the impact of these variants on HFM1 protein levels. WIDER IMPLICATIONS OF THE FINDINGS: Our findings provide direct clinical and in vivo functional evidence that HFM1 variants cause male infertility in humans and also suggest that HFM1 may regulate meiotic crossover formation in a dose-dependent manner. Noticeably, our findings from mouse models showed that HFM1 variants could impair spermatogenesis and oogenesis with a varying degree of severity and might also be compatible with the production of a few spermatozoa in men and subfertility in women, extending the phenotypic spectrum of patients with HFM1 variants. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the National Natural Science Foundation of China (31890780, 32070850, 32061143006, 32000587 and 31900398) and the Fundamental Research Funds for the Central Universities (YD2070002007 and YD2070002012). The authors declare no potential conflicts of interest. TRIAL REGISTRATION NUMBER: N/A.

A recurrent homozygous missense mutation in CCDC103 causes asthenoteratozoospermia due to disorganized dynein arms.

Asthenoteratozoospermia is one of the most severe types of qualitative sperm defects. Most cases are due to mutations in genes encoding the components of sperm flagella, which have an ultrastructure similar to that of motile cilia. Coiled-coil domain containing 103 (CCDC103) is an outer dynein arm assembly factor, and pathogenic variants of CCDC103 cause primary ciliary dyskinesia (PCD). However, whether CCDC103 pathogenic variants cause severe asthenoteratozoospermia has yet to be determined. Whole-exome sequencing (WES) was performed for two individuals with nonsyndromic asthenoteratozoospermia in a consanguineous family. A homozygous CCDC103 variant segregating recessively with an infertility phenotype was identified (ENST00000035776.2, c.461A>C, p.His154Pro). CCDC103 p.His154Pro was previously reported as a high prevalence mutation causing PCD, though the reproductive phenotype of these PCD individuals is unknown. Transmission electron microscopy (TEM) of affected individuals' spermatozoa showed that the mid-piece was severely damaged with disorganized dynein arms, similar to the abnormal ultrastructure of respiratory ciliary of PCD individuals with the same mutation. Thus, our findings expand the phenotype spectrum of CCDC103 p.His154Pro as a novel pathogenic gene for nonsyndromic asthenospermia.

RAD51AP2 is required for efficient meiotic recombination between X and Y chromosomes.

Faithful segregation of X and Y chromosomes requires meiotic recombination to form a crossover between them in the pseudoautosomal region (PAR). Unlike autosomes that have approximately 10-fold more double-strand breaks (DSBs) than crossovers, one crossover must be formed from the one or two DSBs in PARs, implying the existence of a sex chromosome­specific recombination mechanism. Here, we found that RAD51AP2, a meiosis-specific partner of RAD51, is specifically required for the crossover formation on the XY chromosomes, but not autosomes. The decreased crossover formation between X and Y chromosomes in Rad51ap2 mutant mice results from compromised DSB repair in PARs due to destabilization of recombination intermediates rather than defects in DSB generation or synapsis. Our findings provide direct experimental evidence that XY recombination may use a PAR-specific DSB repair mechanism mediated by factors that are not essential for recombination on autosomes.