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Asthenoteratozoospermia is a major cause of male infertility. Thus far, the identified related genes can explain only a small share of asthenoteratozoospermia cases, suggesting the involvement of other genes. The transmembrane protein TMEM232 is highly expressed in mouse testes. In the present study, to determine its function of TMEM232 in testes, we constructed a Tmem232-null mouse model using CRISPR-Cas9 technology. Tmem232 knockout (KO) male mice was completely infertile, and their sperm were immotile, with morphological defects of the flagellum. Electron microscopy revealed an aberrant midpiece-principal junction and the loss of the fourth outer microtubule doublet in the sperm of Tmem232 -/- mice. Sperm cells presented an 8 + 2 conformation and an irregular arrangement of the mitochondrial sheath. Proteomic analysis revealed altered expression of proteins related to flagellar motility, sperm capacitation, the integrity and stability of sperm structure, especially an upregulated expression of multiple ribosome components in TMEM232-deficient spermatids. Additionally, TMEM232 was observed to be involved in autophagy by interacting with autophagy-related proteins, such as ATG14, to regulate ribosome homeostasis during spermiogenesis. These results suggest that TMEM232, as a potential scaffold protein involving in the correct assembly, distribution, and stability maintenance of certain functional complexes by recruiting key intracellular proteins, is essential for the formation of a highly structured flagellum and plays an important role in the autophagic elimination of cytosolic ribosomes to provide energy for sperm motility., (© 2024. The Author(s).)

Axonemal protein complexes, including the outer and inner dynein arms (ODA/IDA), are highly ordered structures of the sperm flagella that drive sperm motility. Deficiencies in several axonemal proteins have been associated with male infertility, which is characterized by asthenozoospermia or asthenoteratozoospermia. Dynein axonemal heavy chain 3 (DNAH3) resides in the IDA and is highly expressed in the testis. However, the relationship between DNAH3 and male infertility is still unclear. Herein, we identified biallelic variants of DNAH3 in four unrelated Han Chinese infertile men with asthenoteratozoospermia through whole-exome sequencing (WES). These variants contributed to deficient DNAH3 expression in the patients' sperm flagella. Importantly, the patients represented the anomalous sperm flagellar morphology, and the flagellar ultrastructure was severely disrupted. Intriguingly, Dnah3 knockout (KO) male mice were also infertile, especially showing the severe reduction in sperm movement with the abnormal IDA and mitochondrion structure. Mechanically, nonfunctional DNAH3 expression resulted in decreased expression of IDA-associated proteins in the spermatozoa flagella of patients and KO mice, including DNAH1, DNAH6, and DNALI1, the deletion of which has been involved in disruption of sperm motility. Moreover, the infertility of patients with DNAH3 variants and Dnah3 KO mice could be rescued by intracytoplasmic sperm injection (ICSI) treatment. Our findings indicated that DNAH3 is a novel pathogenic gene for asthenoteratozoospermia and may further contribute to the diagnosis, genetic counseling, and prognosis of male infertility., Competing Interests: XW, GS, YY, CJ, TR, XY, LZ, YZ, YO, XZ, SL, XT, TL, YS No competing interests declared, (© 2024, Wang, Shen, Yang et al.)

Male infertility presents a substantial challenge in reproductive medicine, often attributed to impaired sperm motility. The present study investigates the role of CCDC28A, a protein expressed specifically in male germ cells, whose paralog CCDC28B has been implicated in ciliogenesis. We identify unique expression patterns for CCDC28A and CCDC28B within the mouse testes, where CCDC28A is expressed in germ cells, whereas CCDC28B is expressed in supporting somatic cells. Through knockout mouse models and histological analyses, we reveal that CCDC28A deficiency results in diminished sperm motility and structural aberrations in sperm tails, notably affecting the head-tail coupling apparatus (HTCA), thereby causing male infertility. Fine structural analyses by transmission electron microscopy reveal disruptions at the capitulum-basal plate junction of the HTCA in the CCDC28A mutants. This results in the bending of the head within the neck region, often accompanied by thickening of the tail midpiece. Our discovery demonstrates that CCDC28A plays an essential role in male fertility and sperm tail morphogenesis through the formation of HTCA., (© 2024. The Author(s).)

Background: Bi-allelic variants in DNAH11 have been identified as causative factors in Primary Ciliary Dyskinesia, leading to abnormal respiratory cilia. Nonetheless, the specific impact of these variants on human sperm flagellar and their involvement in male infertility remain largely unknown., Methods: A collaborative effort involving two Chinese reproductive centers conducted a study with 975 unrelated infertile men. Whole-exome sequencing was employed for variant screening, and Sanger sequencing confirmed the identified variants. Morphological and ultrastructural analyses of sperm were conducted using Scanning Electron Microscopy and Transmission Electron Microscopy. Western Blot Analysis and Immunofluorescence Analysis were utilized to assess protein levels and localization. ICSI was performed to evaluate its efficacy in achieving favorable pregnancy outcomes for individuals with DNAH11 variants., Results: In this study, we identified seven novel variants in the DNAH11 gene in four asthenoteratozoospermia subjects. These variants led the absence of DNAH11 proteins and ultrastructure defects in sperm flagella, particularly affecting the outer dynein arms (ODAs) and adjacent structures. The levels of ODA protein DNAI2 and axoneme related proteins were down regulated, instead of inner dynein arms (IDA) proteins DNAH1 and DNAH6. Two out of four individuals with DNAH11 variants achieved clinical pregnancies through ICSI. The findings confirm the association between male infertility and bi-allelic deleterious variants in DNAH11, resulting in the aberrant assembly of sperm flagella and contributing to asthenoteratozoospermia. Importantly, ICSI emerges as an effective intervention for overcoming reproductive challenges caused by DNAH11 gene variants., (© 2024. The Author(s).)

Purpose: To identify novel variants in ACTL9 and new phenotypes responsible for male infertility., Methods: Genomic DNA was extracted from peripheral blood samples for whole-exome sequencing (WES). Computer-assisted sperm analysis (CASA) was used to test the motility of spermatozoa. The ultrastructure of flagella and the mitochondrial sheath were assessed by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Immunostaining was used to validate the localization and expression of ACTL9 and ACTL7A. An Actl9-mutated mouse model was used to validate the phenotypes by CASA and TEM., Results: We identified novel homozygous variants in ACTL9 in two independent Chinese families. Spermatozoa with ACTL9 mutations showed decreased CASA parameters and a higher proportion of spermatozoa with abnormal morphology, exhibiting coiled flagella and a thickened midpiece. The spermatozoa were characterized by chaotic or irregular '9+2' structures and irregular mitochondrial sheath arrangements in the flagellum. Actl9 knock-in mice also showed abnormal CASA parameters and irregular '9+2' structures in flagella., Conclusions: Our study expands the mutation spectrum and phenotypic spectrum of ACTL9., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Ribonucleoprotein (RNP) granules are membraneless electron-dense structures rich in RNAs and proteins, and involved in various cellular processes. Two RNP granules in male germ cells, intermitochondrial cement and the chromatoid body (CB), are associated with PIWI-interacting RNAs (piRNAs) and are required for transposon silencing and spermatogenesis. Other RNP granules in male germ cells, the reticulated body and CB remnants, are also essential for spermiogenesis. In this study, we disrupted FBXO24, a testis-enriched F-box protein, in mice and found numerous membraneless electron-dense granules accumulated in sperm flagella. Fbxo24 knockout (KO) mice exhibited malformed flagellar structures, impaired sperm motility, and male infertility, likely due to the accumulation of abnormal granules. The amount and localization of known RNP granule-related proteins were not disrupted in Fbxo24 KO mice, suggesting that the accumulated granules were distinct from known RNP granules. Further studies revealed that RNAs and two importins, IPO5 and KPNB1, abnormally accumulated in Fbxo24 KO spermatozoa and that FBXO24 could ubiquitinate IPO5. In addition, IPO5 and KPNB1 were recruited to stress granules, RNP complexes, when cells were treated with oxidative stress or a proteasome inhibitor. These results suggest that FBXO24 is involved in the degradation of IPO5, disruption of which may lead to the accumulation of abnormal RNP granules in sperm flagella., Competing Interests: YK, HM, ZX, KS, MK, TN, CE, MI No competing interests declared, (© 2024, Kaneda et al.)

Human infertility affects 10-15% of couples. Asthenozoospermia accounts for 18% of men with infertility and is a common male infertility phenotype. The nexin-dynein regulatory complex (N-DRC) is a large protein complex in the sperm flagellum that connects adjacent doublets of microtubules. Defects in the N-DRC can disrupt cilia/flagellum movement, resulting in primary ciliary dyskinesia and male infertility. Using whole-exome sequencing, we identified a pathological homozygous variant of the dynein regulatory complex subunit 3 (DRC3) gene, which expresses leucine-rich repeat-containing protein 48, a component of the N-DRC, in a patient with asthenozoospermia. The variant ENST00000313838.12: c.644dup (p. Glu216GlyfsTer36) causes premature translational arrest of DRC3, resulting in a dysfunctional DRC3 protein. The patient's semen count, color, and pH were normal according to the reference values of the World Health Organization guidelines; however, sperm motility and progressive motility were reduced. DRC3 protein was not detected in the patient's sperm and the ultrastructure of the patient's sperm flagella was destroyed. More importantly, the DRC3 variant reduced its interaction with other components of the N-DRC, including dynein regulatory complex subunits 1, 2, 4, 5, 7, and 8. Our data not only revealed the essential biological functions of DRC3 in sperm flagellum movement and structure but also provided a new basis for the clinical genetic diagnosis of male infertility., (© 2024. The Author(s), under exclusive licence to The Japan Society of Human Genetics.)

Aims: Asthenozoospermia is the most common factor of male infertility, mainly caused by multiple morphological abnormalities of the sperm flagella (MMAF) and primary ciliary dyskinesia (PCD). Previous studies have shown that genetic factors may contribute to MMAF and PCD. The study aimed to identify novel potentially pathogenic gene mutations in a Chinese infertile man with MMAF and PCD-like phenotypes. Methods: A Chinese infertile man with MMAF and PCD was enrolled in this study. Whole exome sequencing and Sanger sequencing were performed to identify potential causative genes and mutations. Results: A novel homozygous missense mutation (c.1450G>A; p.E484K) of CCDC40 was finally identified and Sanger sequencing confirmed that the patient carried the homozygous mutation, which was inherited from his parents. We reported the first homozygous missense CCDC40 mutation in infertile men with MMAF but had other milder PCD symptoms. Conclusion: Our findings not only broaden the disease-causing mutation spectrum of CCDC40 but also provide new insight into the correlation between CCDC40 mutations and MMAF.

TTC12 is a cytoplasmic and centromere-localized protein that plays a role in the proper assembly of dynein arm complexes in motile cilia in both respiratory cells and sperm flagella. This finding underscores its significance in cellular motility and function. However, the wide role of TTC12 in human spermatogenesis-associated primary ciliary dyskinesia (PCD) still needs to be elucidated. Whole-exome sequencing (WES) and Sanger sequencing were performed to identify potentially pathogenic variants causing PCD and multiple morphological abnormalities of sperm flagella (MMAF) in an infertile Pakistani man. Diagnostic imaging techniques were used for PCD screening in the patient. Real-time polymerase chain reaction (RT‒PCR) was performed to detect the effect of mutations on the mRNA abundance of the affected genes. Papanicolaou staining and scanning electron microscopy (SEM) were carried out to examine sperm morphology. Transmission electron microscopy (TEM) was performed to examine the ultrastructure of the sperm flagella, and the results were confirmed by immunofluorescence staining. Using WES and Sanger sequencing, a novel homozygous missense variant (c.C1069T; p.Arg357Trp) in TTC12 was identified in a patient from a consanguineous family. A computed tomography scan of the paranasal sinuses confirmed the symptoms of the PCD. RT-PCR showed a decrease in TTC12 mRNA in the patient's sperm sample. Papanicolaou staining, SEM, and TEM analysis revealed a significant change in shape and a disorganized axonemal structure in the sperm flagella of the patient. Immunostaining assays revealed that TTC12 is distributed throughout the flagella and is predominantly concentrated in the midpiece in normal spermatozoa. In contrast, spermatozoa from patient deficient in TTC12 showed minimal staining intensity for TTC12 or DNAH17 (outer dynein arms components). This could lead to MMAF and result in male infertility. This novel TTC12 variant not only illuminates the underlying genetic causes of male infertility but also paves the way for potential treatments targeting these genetic factors. This study represents a significant advancement in understanding the genetic basis of PCD-related infertility., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

In a recent journal article, Chen et al. identified a germ cell-specific cofactor, STYXL1, associated with male fertility function. Deletion of STYXL1 prevents the LEGO player CCT complex from properly folding key microtubule proteins of the sperm flagellum, which affects sperm motility and male fertility function., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

By analyzing a mouse Interspecific Recombinant Congenic Strain (IRCS), we previously identified a quantitative trait locus (QTL), called Mafq1 on mouse chromosome 1, that is associated with male hypofertility and ultrastructural sperm abnormalities. Within this locus, we identified a new candidate gene that could be implicated in a reproductive phenotype: Tex44 (Testis-expressed protein 44). We thus performed a CRISPR/Cas9-mediated complete deletion of this gene in mice in order to study its function. Tex44-KO males were severely hypofertile in vivo and in vitro due to a drastic reduction of sperm motility which itself resulted from important morphological sperm abnormalities. Namely, Tex44-KO sperm showed a disorganized junction between the midpiece and the principal piece of the flagellum, leading to a 180° flagellar bending in this region. In addition, the loss of some axonemal microtubule doublets and outer dense fibers in the flagellum's principal piece has been observed. Our results suggest that, in mice, TEX44 is implicated in the correct set-up of the sperm flagellum during spermiogenesis and its absence leads to flagellar abnormalities and consequently to severe male hypofertility., (© 2024. The Author(s).)

Purpose: To identify the genetic causes of multiple morphological abnormalities in sperm flagella (MMAF) and male infertility in patients from two unrelated Han Chinese families., Methods: Whole-exome sequencing was conducted using blood samples from the two individuals with MMAF and male infertility. Hematoxylin and eosin staining and scanning electron microscopy were performed to evaluate sperm morphology. Ultrastructural and immunostaining analyses of the spermatozoa were performed. The HEK293T cells were used to confirm the pathogenicity of the variants., Results: We identified two novel homozygous missense ARMC2 variants: c.314C > T: p.P105L and c.2227A > G: p.N743D. Both variants are absent or rare in the human population genome data and are predicted to be deleterious. In vitro experiments indicated that both ARMC2 variants caused a slightly increased protein expression. ARMC2-mutant spermatozoa showed multiple morphological abnormalities (bent, short, coiled, absent, and irregular) in the flagella. In addition, the spermatozoa of the patients revealed a frequent absence of the central pair complex and disrupted axonemal ultrastructure., Conclusion: We identified two novel ARMC2 variants that caused male infertility and MMAF in Han Chinese patients. These findings expand the mutational spectrum of ARMC2 and provide insights into the complex causes and pathogenesis of MMAF., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

The transition zone is a specialised gate at the base of cilia/flagella, which separates the ciliary compartment from the cytoplasm and strictly regulates protein entry. We identified a potential new regulator of the male germ cell transition zone, CEP76. We demonstrated that CEP76 was involved in the selective entry and incorporation of key proteins required for sperm function and fertility into the ciliary compartment and ultimately the sperm tail. In the mutant, sperm tails were shorter and immotile as a consequence of deficits in essential sperm motility proteins including DNAH2 and AKAP4, which accumulated at the sperm neck in the mutant. Severe annulus, fibrous sheath, and outer dense fibre abnormalities were also detected in sperm lacking CEP76. Finally, we identified that CEP76 dictates annulus positioning and structure. This study suggests CEP76 as a male germ cell transition zone protein and adds further evidence to the hypothesis that the spermatid transition zone and annulus are part of the same functional structure., (© 2024 Houston et al.)

Sperm flagella share an evolutionary conserved microtubule-based structure with motile cilia expressed at the surface of several cell types, such as the airways epithelial cells. As a result, male infertility can be observed as an isolated condition or a syndromic trait, illustrated by Primary Cilia Dyskinesia (PCD). We report two unrelated patients showing multiple morphological abnormalities of the sperm flagella (MMAF) and carrying distinct homozygous truncating variants in the PCD-associated gene CCDC65. We characterized one of the identified variants (c.1208del; p.Asn403Ilefs*9), which induces the near absence of CCDC65 protein in patient sperm. In Chlamydomonas, CCDC65 ortholog (DRC2, FAP250) is a component of the Nexin-Dynein Regulatory complex (N-DRC), which interconnects microtubule doublets and coordinates dynein arms activity. In sperm cells from the patient, we also show the loss of GAS8, another component of the N-DRC, supporting a structural/functional link between the two proteins. Our work indicates that, similarly to ciliary axoneme, CCDC65 is required for sperm flagellum structure. Importantly, our work provides first evidence that mutations in the PCD-associated gene CCDC65 also cause asthenozoospermia., (© 2023 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Variations in the dynein axonemal heavy chain gene, dynein axonemal heavy chain 6 ( DNAH6 ), lead to multiple morphological abnormalities of the flagella. Recent studies have reported that these deficiencies may result in sperm head deformation. However, whether DNAH6 is also involved in human acrosome biogenesis remains unknown. The purpose of this study was to investigate DNAH6 gene variants and their potential functions in the formation of defective sperm heads and flagella. Whole-exome sequencing was performed on a cohort of 375 patients with asthenoteratozoospermia from the First Affiliated Hospital of Anhui Medical University (Hefei, China). Hematoxylin and eosin staining, scanning electron microscopy, and transmission electron microscopy were performed to analyze the sperm morphology and ultrastructure. Immunofluorescence staining and Western blot analysis were conducted to examine the effects of genetic variants. We identified three novel deleterious variants in DNAH6 among three unrelated families. The absence of inner dynein arms and radial spokes was observed in the sperm of patients with DNAH6 variants. Additionally, deficiencies in the acrosome, abnormal chromatin compaction, and vacuole-containing sperm heads were observed in these patients with DNAH6 variants. The decreased levels of the component proteins in these defective structures were further confirmed in sperm from patients with DNAH6 variants using Western blot. After intracytoplasmic sperm injection (ICSI) treatment, the partner of one patient with a DNAH6 variant achieved successful pregnancy. Overall, novel variants in DNAH6 genes that contribute to defects in the sperm head and flagella were identified, and the findings indicated ICSI as an effective clinical treatment for such patients., (Copyright © 2023 Copyright: © The Author(s)(2023).)

Multiple morphological abnormalities of the flagella (MMAF) is a severe disease of male infertility, while the pathogenetic mechanisms of MMAF are still incompletely understood. Previously, we found that the deficiency of Ccdc38 might be associated with MMAF. To understand the underlying mechanism of this disease, we identified the potential partner of this protein and found that the coiled-coil domain containing 146 (CCDC146) can interact with CCDC38. It is predominantly expressed in the testes, and the knockout of this gene resulted in complete infertility in male mice but not in females. The knockout of Ccdc146 impaired spermiogenesis, mainly due to flagellum and manchette organization defects, finally led to MMAF-like phenotype. Furthermore, we demonstrated that CCDC146 could interact with both CCDC38 and CCDC42. It also interacts with intraflagellar transport (IFT) complexes IFT88 and IFT20. The knockout of this gene led to the decrease of ODF2, IFT88, and IFT20 protein levels, but did not affect CCDC38, CCDC42, or ODF1 expression. Additionally, we predicted and validated the detailed interactions between CCDC146 and CCDC38 or CCDC42, and built the interaction models at the atomic level. Our results suggest that the testis predominantly expressed gene Ccdc146 is essential for sperm flagellum biogenesis and male fertility, and its mutations might be associated with MMAF in some patients., (© 2023. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Study Question: What is the effect of defects in the manchette protein IQ motif-containing N (IQCN) on sperm flagellar assembly?, Summary Answer: Deficiency in IQCN causes sperm flagellar assembly defects and male infertility., What Is Known Already: 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., Study Design, Size, Duration: Fifty men with infertility were recruited from a university-affiliated center from January 2014 to October 2022., Participants/materials, Setting, Methods: 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., Main Results and the Role of Chance: 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., Limitations, Reasons for Caution: More cases are needed to demonstrate the relation between IQCN variants and phenotypes., Wider Implications of the Findings: 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., Study Funding/competing Interest(s): 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., Trial Registration Number: N/A., (© The Author(s) 2023. Published by Oxford University Press on behalf of European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Asthenozoospermia characterized by decreased sperm motility is a major cause of male infertility, but the majority of the etiology remains unknown. Here, we showed that the cilia and flagella associated protein 52 (Cfap52) gene was predominantly expressed in testis and its deletion in a Cfap52 knockout mouse model resulted in decreased sperm motility and male infertility. Cfap52 knockout also led to the disorganization of the midpiece-principal piece junction of the sperm tail but had no effect on the axoneme ultrastructure in spermatozoa. Furthermore, we found that CFAP52 interacted with the cilia and flagella associated protein 45 (CFAP45) and knockout of Cfap52 decreased the expression level of CFAP45 in sperm flagellum, which further disrupted the microtubule sliding produced by dynein ATPase. Together, our studies demonstrate that CFAP52 plays an essential role in sperm motility by interacting with CFAP45 in sperm flagellum, providing insights into the potential pathogenesis of the infertility of the human CFAP52 mutations., Competing Interests: Conflict of interest The authors declare no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Sperm motility is crucial for successful fertilization. Highly decorated doublet microtubules (DMTs) form the sperm tail skeleton, which propels the movement of spermatozoa. Using cryo-electron microscopy (cryo-EM) and artificial intelligence (AI)-based modeling, we determined the structures of mouse and human sperm DMTs and built an atomic model of the 48-nm repeat of the mouse sperm DMT. Our analysis revealed 47 DMT-associated proteins, including 45 microtubule inner proteins (MIPs). We identified 10 sperm-specific MIPs, including seven classes of Tektin5 in the lumen of the A tubule and FAM166 family members that bind the intra-tubulin interfaces. Interestingly, the human sperm DMT lacks some MIPs compared with the mouse sperm DMT. We also discovered variants in 10 distinct MIPs associated with a subtype of asthenozoospermia characterized by impaired sperm motility without evident morphological abnormalities. Our study highlights the conservation and tissue/species specificity of DMTs and expands the genetic spectrum of male infertility., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Elsevier Inc. All rights reserved.)

The axoneme and accessory structures of flagella are critical for sperm motility and male fertilization. Sperm production needs precise and highly ordered gene expression to initiate and sustain the many cellular processes that result in mature spermatozoa. Here, we identified a testis enriched gene transmembrane protein 232 ( Tmem232 ), which is essential for the structural integrity of the spermatozoa flagella axoneme. Tmem232 knockout mice were generated for in vivo analyses of its functions in spermatogenesis. Phenotypic analysis showed that deletion of Tmem232 in mice causes male-specific infertility. Transmission electron microscopy together with scanning electron microscopy were applied to analyze the spermatozoa flagella and it was observed that the lack of TMEM232 caused failure of the cytoplasm removal and the absence of the 7th outer microtubule doublet with its corresponding outer dense fiber (ODF). Co-IP assays further identified that TMEM232 interacts with ODF family protein ODF1, which is essential to maintain sperm motility. In conclusion, our findings indicate that TMEM232 is a critical protein for male fertility and sperm motility by regulating sperm cytoplasm removal and maintaining axoneme integrity.

Spermatogenesis defects are important for male infertility; however, the etiology and pathogenesis are still unknown. Herein, we identified two loss-of-function mutations of STK33 in seven individuals with non-obstructive azoospermia. Further functional studies of these frameshift and nonsense mutations revealed that Stk33 -/KI male mice were sterile, and Stk33 -/KI sperm were abnormal with defects in the mitochondrial sheath, fibrous sheath, outer dense fiber, and axoneme. Stk33 KI/KI male mice were subfertile and had oligoasthenozoospermia. Differential phosphoproteomic analysis and in vitro kinase assay identified novel phosphorylation substrates of STK33, fibrous sheath components A-kinase anchoring protein 3 and A-kinase anchoring protein 4, whose expression levels decreased in testis after deletion of Stk33. STK33 regulated the phosphorylation of A-kinase anchoring protein 3/4, affected the assembly of fibrous sheath in the sperm, and played an essential role in spermiogenesis and male infertility., Competing Interests: Conflict of interest The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

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., Competing Interests: None

The axonemal dynein arms (outer (ODA) and inner dynein arms (IDAs)) are multiprotein structures organized by light, intermediate, light intermediate (LIC), and heavy chain proteins. They hydrolyze ATP to promote ciliary and flagellar movement. Till now, a variety of dynein protein deficiencies have been linked with asthenospermia (ASZ), highlighting the significance of these structures in human sperm motility. Herein, we detected bi-allelic DNALI1 mutations [c.663_666del (p.Glu221fs)], in an ASZ patient, which resulted in the complete loss of the DNALI1 in the patient's sperm. We identified loss of sperm DNAH1 and DNAH7 rather than DNAH10 in both DNALI1 663_666del patient and Dnali1 -/- mice, demonstrating that mammalian DNALI1 is a LIC protein of a partial IDA subspecies. More importantly, we revealed that DNALI1 loss contributed to asymmetries in the most fibrous sheath (FS) of the sperm flagellum in both species. Immunoprecipitation revealed that DNALI1 might interact with the cytoplasmic dynein complex proteins in the testes. Furthermore, DNALI1 loss severely disrupted the transport and assembly of the FS proteins, especially AKAP3 and AKAP4, during flagellogenesis. Hence, DNALI1 may possess a non-classical molecular function, whereby it regulates the cytoplasmic dynein complex that assembles the flagella. We conclude that a DNALI deficiency-induced IDAs injury and an asymmetric FS-driven tail rigid structure alteration may simultaneously cause flagellum immotility. Finally, intracytoplasmic sperm injection (ICSI) can effectively resolve patient infertility. Collectively, we demonstrate that DNALI1 is a newly causative gene for AZS in both humans and mice, which possesses multiple crucial roles in modulating flagellar assembly and motility., (© 2023. The Author(s).)

Background: As a common type of asthenoteratozoospermia, multiple morphological abnormalities of the sperm flagella (MMAF) can cause male infertility. Previous studies have revealed genetic factors as a major cause of MMAF. The known MMAF-associated genes are involved in the mitochondrial sheath, outer dense fibre or axoneme of the sperm flagella. These findings indicate the genetic heterogeneity of MMAF., Methods and Results: Here, we conducted genetic analyses using whole-exome sequencing in a cohort of 150 Han Chinese men with asthenoteratozoospermia. Homozygous deleterious variants of AKAP3 (A-kinase anchoring protein 3) were identified in two MMAF-affected men from unrelated families. One AKAP3 variant was a frameshift (c.2286_2287del, p.His762Glnfs*22) and the other variant was a missense mutation (c.44G>A, p.Cys15Tyr), which was predicted to be damaging by multiple bioinformatics tools. Further western blotting and immunofluorescence assays revealed the absence of AKAP3 in the spermatozoa from the man harbouring the homozygous frameshift variant, whereas the expression of AKAP3 was markedly reduced in the spermatozoa of the man with the AKAP3 missense variant p.Cys15Tyr. Notably, the clinical outcomes after intracytoplasmic sperm injection (ICSI) were divergent between these two cases, suggesting a possibility of AKAP3 dosage-dependent prognosis of ICSI treatment., Conclusions: Our study revealed AKAP3 as a novel gene involved in human asthenoteratozoospermia., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2023. No commercial re-use. See rights and permissions. Published by BMJ.)

This study aimed to identify genetic causes responsible for multiple morphological abnormalities of the sperm flagella (MMAF) in the Han Chinese population. Three primary infertile males with completely immobile sperm and MMAF were enrolled. Whole-exome sequencing and Sanger sequencing were performed to identify disease-causing genes. Subsequently, morphological and ultrastructural analyses of sperm flagella were investigated. The probable impact of genetic variants on protein function was analyzed by online bioinformatic tools and immunofluorescence assay. Three patients with dynein axonemal heavy chain 1 (DNAH1) gene compound heterozygous variations were identified. DNAH1 c.7435C>T, p.R2479X and c.10757T>C, p.F3586S were identified in the patient from Family 1, c.11726_11727delCT, p.P3909fs and c.12154delC, p.L4052fs were found in the patient from Family 2, and c.10627-3C>G and c.11726_11727delCT, p.P3909fs existed in the patient from Family 3. Four of these variations have not been reported, and all the mutations showed pathogenicity by functional effect predictions. The absence of the center pair and disorganization of the fibrous sheath were present in sperm flagella at the ultrastructural level. Moreover, the expression of DNAH1 was absent in spermatozoa from the participants, validating the pathogenicity of the variants. All three couples have undergone intracytoplasmic sperm injection (ICSI), and two couples of them became pregnant after the treatment. In conclusion, the newly identified DNAH1 mutations can expand the mutational and phenotypic spectrum of MMAF genes and provide a theoretical basis for genetic diagnosis in MMAF patients. It is recommended to conduct genetic screening in male infertility patients with MMAF and provide rational genetic counseling, and ICSI might be an optimal strategy to help with fertilization and conception for patients with DNAH1 mutations.

Background: Multiple morphological abnormalities of the sperm flagella (MMAF), which is characterized as asthenoteratospermia involving absent, short, bent, coiled, and/or irregular-caliber flagella, is a rare recessive inherited disorder associated with male infertility. To date, genetic causes of MMAF cases are not fully explored., Methods: Whole-exome sequencing was conducted to identify pathogenic variants in a patient with MMAF. The functional effect of the identified mutations was investigated by immunofluorescence staining and western blotting. Intracytoplasmic sperm injection was used to assist fertilization for the patient with MMAF., Results: We identified novel biallelic mutations, a splicing variant NC_000004.12:g.146937593C>T (c.254+1G>A), and a nonsense mutation NM_001300761.4:c.1185C>G (NP_001287690.1:p.Tyr395*), in TTC29 from an infertile patient. In addition to the typical MMAF phenotype, the patient also presented aberrant morphology of sperm heads. Further functional experiments confirmed the absence of TTC29 expression in the spermatozoa. We also explored the specific expression pattern of TTC29 in human and mouse spermatogenesis. The outcome of intracytoplasmic sperm injection in the patient was unsuccessful, while additional female risk factors should not be excluded., Conclusions: Our study revealed the novel biallelic mutations in TTC29 in a MMAF patient, which findings expand the mutational spectrum of TTC29 and further contribute to the diagnosis, genetic counseling, and prognosis of male infertility., (© 2022 The Authors. Molecular Genetics & Genomic Medicine published by Wiley Periodicals LLC.)

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., (© 2022 Wiley Periodicals LLC.)

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., (© 2022 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

The sperm flagellum is essential for male fertility. Multiple morphological abnormalities of the sperm flagella (MMAF) is a severe form of asthenoteratozoospermia. MMAF phenotypes are understood to result from pathogenic variants of genes from multiple families including AKAP, DANI, DNAH, RSPH, CCDC, CFAP, TTC, and LRRC, among others. The Leucine-rich repeat protein (LRRC) family includes two members reported to cause MMAF phenotypes: Lrrc6 and Lrrc50 . Despite vigorous research towards understanding the pathogenesis of MMAF-related diseases, many genes remain unknown underlying the flagellum biogenesis. Here, we found that Leucine-rich repeat containing 46 (LRRC46) is specifically expressed in the testes of adult mice, and show that LRRC46 is essential for sperm flagellum biogenesis. Both scanning electron microscopy (SEM) and Papanicolaou staining (PS) presents that the knockout of Lrrc46 in mice resulted in typical MMAF phenotypes, including sperm with short, coiled, and irregular flagella. The male KO mice had reduced total sperm counts, impaired sperm motility, and were completely infertile. No reproductive phenotypes were detected in Lrrc46 -/- female mice. Immunofluorescence (IF) assays showed that LRRC46 was present throughout the entire flagella of control sperm, albeit with evident concentration at the mid-piece. Transmission electron microscopy (TEM) demonstrated striking flagellar defects with axonemal and mitochondrial sheath malformations. About the important part of the Materials and Methods, SEM and PS were used to observe the typical MMAF-related irregular flagella morphological phenotypes, TEM was used to further inspect the sperm flagellum defects in ultrastructure, and IF was chosen to confirm the location of protein. Our study suggests that LRRC46 is an essential protein for sperm flagellum biogenesis, and its mutations might be associated with MMAF that causes male infertility. Thus, our study provides insights for understanding developmental processes underlying sperm flagellum formation and contribute to further observe the pathogenic genes that cause male infertility.

The sperm flagellum is essential for male fertility, and defects in flagellum biogenesis are associated with male infertility. Deficiency of coiled-coil domain-containing (CCDC) 42 (CCDC42) is specifically associated with malformation of mouse sperm flagella. Here, we find that the testis-specific protein CCDC38 interacts with CCDC42, localizing on the manchette and sperm tail during spermiogenesis. Inactivation of CCDC38 in male mice results in a distorted manchette, multiple morphological abnormalities of the flagella of spermatozoa and eventually male sterility. Furthermore, we find that CCDC38 interacts with intraflagellar transport protein 88 (IFT88), as well as outer dense fibrous 2 (ODF2), and the knockout of Ccdc38 reduces transport of ODF2 to the flagellum. Altogether, our results uncover the essential role of CCDC38 in sperm flagellum biogenesis, and suggest that some mutations of these genes might be associated with male infertility in humans., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2022. Published by The Company of Biologists Ltd.)

AKAP3 is a member of the A-kinase anchoring proteins and it is a constituent of the sperm fibrous sheath. AKAP3 is needed for the formation of sperm flagellum structure, sperm motility, and male fertility. This study aims to model the AKAP3 tertiary structure and identify the probable impact of four mutations characterized in infertile men on the AKAP3 structure. The T464S, I500T, E525K, and I661T substitutions were analyzed using in silico methods. The secondary structure and three-dimensional model of AKAP3 were determined using PSI-BLAST based secondary structure prediction and Robetta servers. The TM-score was used to quantitatively measure the structural similarities between native and mutated models. All of the desired substitutions were classified as benign. I-Mutant results showed all of the substitutions decreased AKAP3 stability; however, the I500T and I661T were more effective. Superposition and secondary structure comparisons between native and mutants showed no dramatic deviations. Our study provided an appropriate model for AKAP3. Destabilization of AKAP3 caused by these substitutions did not appear to induce structural disturbances. As AKAP3 is involved in male infertility, providing more structural insights and the impact of mutations that cause protein functional diversity could elucidate the etiology of male fertility problems at molecular level., (© 2022. The Author(s).)

The CatSper cation channel is essential for sperm capacitation and male fertility. The multi-subunit CatSper complexes form highly organized calcium signaling nanodomains on flagellar membranes. Here, we report identification of an uncharacterized protein, C2CD6, as a subunit of the mouse CatSper complex. C2CD6 contains a calcium-dependent, membrane-targeting C2 domain. C2CD6 associates with the CatSper calcium-selective, core-forming subunits. Deficiency of C2CD6 depletes the CatSper nanodomains from the flagellum and results in male sterility. C2CD6-deficient sperm are defective in hyperactivation and fail to fertilize oocytes both in vitro and in vivo. CatSper currents are present but at a significantly lower level in C2CD6-deficient sperm. Transient treatments with either Ca2+ ionophore, starvation, or a combination of both restore the fertilization capacity of C2CD6-deficient sperm. C2CD6 interacts with EFCAB9, a pH-dependent calcium sensor in the CatSper complex. We postulate that C2CD6 facilitates incorporation of the CatSper complex into the flagellar plasma membrane and may function as a calcium sensor. The identification of C2CD6 may enable the long-sought reconstitution of the CatSper ion channel complex in a heterologous system for male contraceptive development., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2022. Published by The Company of Biologists Ltd.)

Sperm fibrous sheath (FS) is closely related to sperm maturation, capacitation and motility, and A-kinase anchor protein 4 (AKAP4) is the most abundant protein in sperm FS. Previous studies found incomplete sperm FSs and abnormal flagella in Akap4 knockout mice. Meanwhile, it was reported that the partial deletion in AKAP4 is highly relevant to the dysplasia of the FS in an infertile man, and so far, there is no report about male infertility caused by hemizygous AKAP4 variant. Furthermore, the specific mechanisms of how the variant is relevant to the phenotype remain elusive. In this study, we investigated three multiple morphological abnormalities of the sperm flagella-affected men from three independent families (including one consanguine family) carried hemizygous c.C1285T variant in AKAP4. The patients carried this variant, which showed dysplastic sperm FS, and the protein expression of AKAP4 was decreased in flagella, which was further confirmed in HEK-293T cells in vitro. In addition, the co-localization and interaction between AKAP4 and glutamine-rich protein 2 (QRICH2) on the molecular level were identified by immunofluorescence and co-immunoprecipitation (CO-IP). The hemizygous c.1285C > T variant in AKAP4 induced decreased protein expression of QRICH2 in spermatozoa. These results suggested that the normal expression of AKAP4 is required for maintaining the expression of QRICH2 and the decreased protein expression of AKAP4 and QRICH2，as well as the interaction between them induced by the hemizygous variant of AKAP4 caused dysplastic fibrous sheath, which eventually led to reduced sperm motility and male infertility., (© The Author(s) 2021. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Defects in the structure or motility of cilia and flagella may lead to severe diseases such as primary ciliary dyskinesia (PCD), a multisystemic disorder with heterogeneous manifestations affecting primarily respiratory and reproductive functions. We report that CFAP61 is a conserved component of the calmodulin- and radial spoke-associated complex (CSC) of cilia. We find that a CFAP61 splice variant, c.143+5G>A, causes exon skipping/intron retention in human, inducing a multiple morphological abnormalities of the flagella (MMAF) phenotype. We generated Cfap61 knockout mice that recapitulate the infertility phenotype of the human CFAP61 mutation, but without other symptoms usually observed in PCD. We find that CFAP61 interacts with the CSC, radial spoke stalk and head. During early stages of Cfap61-/- spermatid development, the assembly of radial spoke components is impaired. As spermiogenesis progresses, the axoneme in Cfap61-/- cells becomes unstable and scatters, and the distribution of intraflagellar transport proteins is disrupted. This study reveals an organ-specific mechanism of axoneme stabilization that is related to male infertility., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2021. Published by The Company of Biologists Ltd.)

Motile cilia and flagellar defects can result in primary ciliary dyskinesia, which is a multisystemic genetic disorder that affects roughly 1:10 000 individuals. The nexin-dynein regulatory complex (N-DRC) links neighboring doublet microtubules within flagella, serving as a central regulatory hub for motility in Chlamydomonas. Herein, we identified two homozygous DRC1 variants in human patients that were associated with multiple morphological abnormalities of the sperm flagella (MMAF) and male infertility. Drc1-/-, Drc1R554X/R554X and Drc1W244X/W244X mice on the C57BL/6 background suffered from pre-pubertal mortality. However, when the ICR background was introduced, some of these mice were able to survive and recapitulate the MMAF phenotypes detected in human patients. By analyzing these animals, we determined that DRC1 is an essential regulator of N-DRC assembly in cilia and flagella. When DRC1 is absent, this results in the shortening of cilia and consequent impairment of their motility. Damage associated with DRC1 deficiency in sperm flagella was more pronounced than in cilia, as manifested by complete axoneme structural disorder in addition to the loss of the DRC structure. Altogether, these findings suggest that DRC1 is required for the structural stability of flagella but not cilia, emphasizing the key role of this protein in mammalian species., (© The Author(s) 2021. Published by Oxford University Press.)

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., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Purpose: Multiple morphological abnormalities of the sperm flagella (MMAF) are important causes of male infertility. Mutations in DNAH1 are the main causative factors proven so far. We aim to determine the mutational landscape of DNAH1 in Chinese patients with MMAF., Methods: Forty-one Chinese patients with MMAF were enrolled and underwent a 10-gene next-generation sequencing panel screening., Results: Only the DNAH1 gene was found to have mutations in 12 of these unrelated individuals (29%). Combining published data from two other cohorts of Chinese men with MMAF, we suggest that p.P3909fs*33, p.R868X, p.Q1518X, p.E3284K, and p.R4096L are hotspot mutations. A polymorphism-rs12163565 (G>A)- showed linkage to p.P3909fs*33, suggesting that this involved a founder effect. Four of the 12 patients with DNAH1 mutations were able to use intracytoplasmic sperm injection with their partners and all were successful in obtaining embryos., Conclusions: Hotspot mutations were identified for Chinese patients with MMAF. MMAF sub-phenotypes might be associated with different combinations of DNAH1 mutations., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Purpose: To identify the genetic factors responsible for asthenozoospermia, which is a major cause of male infertility characterized by immotile and malformed spermatozoa., Methods: Whole-exome sequencing was performed in two brothers from a family with asthenozoospermia to identify pathogenic variants. The functional effect of the identified variant was investigated in HEK293T cells using a minigene assay., Results: We identified a novel homozygous splicing variant c.6311-2A>G in DNAH8 from two affected brothers belonging to the same consanguineous family. The splicing variant altered a consensus splice acceptor site of DNAH8 intron 44, which led to the deletion of exon 45 and resulted in a frameshift and a predicted truncated protein (p.G2104Efs*19). Although most spermatozoa from the patients presented with reduced sperm motility, intracytoplasmic sperm injection was able to overcome the inability of the spermatozoa to reach the ovum and thus produce a healthy child for the proband., Conclusions: This finding expands the mutational spectrum of DNAH8, making it a potential genetic diagnostic marker for those suffering from primary male infertility.

Asthenoteratospermia is an important cause of male infertility. Here, we report two infertile patients with severe asthenoteratospermia accompanied by new genetic abnormality. Whole-exome sequencing and bioinformatics analysis suggested that compound heterozygous mutations in DNAH8 (MIM:603337) may be responsible for multiple morphological abnormalities of the sperm flagella (MMAF). Immunofluorescence assay showed that DNAH8 protein expression was significantly decreased in the sperm tail of the patients, and electron microscopy exhibited an abnormal flagellum ultrastructure, while clinical pregnancy could be achieved by intracytoplasmic sperm injection. Therefore, the compound heterozygous mutations in the DNAH8 gene may be responsible for MMAF., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Artificial insemination in pig (Sus scrofa domesticus) breeding involves the evaluation of the semen quality of breeding boars. Ejaculates that fulfill predefined quality requirements are processed, diluted and used for inseminations. Within short time, eight Swiss Large White boars producing immotile sperm that had multiple morphological abnormalities of the sperm flagella were noticed at a semen collection center. The eight boars were inbred on a common ancestor suggesting that the novel sperm flagella defect is a recessive trait. Transmission electron microscopy cross-sections revealed that the immotile sperm had disorganized flagellar axonemes. Haplotype-based association testing involving microarray-derived genotypes at 41,094 SNPs of six affected and 100 fertile boars yielded strong association (P = 4.22 × 10-15) at chromosome 12. Autozygosity mapping enabled us to pinpoint the causal mutation on a 1.11 Mb haplotype located between 3,473,632 and 4,587,759 bp. The haplotype carries an intronic 13-bp deletion (Chr12:3,556,401-3,556,414 bp) that is compatible with recessive inheritance. The 13-bp deletion excises the polypyrimidine tract upstream exon 56 of DNAH17 (XM_021066525.1: c.8510-17_8510-5del) encoding dynein axonemal heavy chain 17. Transcriptome analysis of the testis of two affected boars revealed that the loss of the polypyrimidine tract causes exon skipping which results in the in-frame loss of 89 amino acids from DNAH17. Disruption of DNAH17 impairs the assembly of the flagellar axoneme and manifests in multiple morphological abnormalities of the sperm flagella. Direct gene testing may now be implemented to monitor the defective allele in the Swiss Large White population and prevent the frequent manifestation of a sterilizing sperm tail disorder in breeding boars., (© The Author(s) 2020. Published by Oxford University Press on behalf of Genetics Society of America. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Protein phosphatase 4 (PPP4) is a protein phosphatase that, although highly expressed in the testis, currently has an unclear physiological role in this tissue. Here, we show that deletion of PPP4 catalytic subunit gene Ppp4c in the mouse causes male-specific infertility. Loss of PPP4C, when assessed by light microscopy, did not obviously affect many aspects of the morphology of spermatogenesis, including acrosome formation, nuclear condensation and elongation, mitochondrial sheaths arrangement and '9 + 2' flagellar structure assembly. However, the PPP4C mutant had sperm tail bending defects (head-bent-back), low sperm count, poor sperm motility and had cytoplasmic remnants attached to the middle piece of the tail. The cytoplasmic remnants were further investigated by transmission electron microscopy to reveal that a defect in cytoplasm removal appeared to play a significant role in the observed spermiogenesis failure and resulting male infertility. A lack of PPP4 during spermatogenesis causes defects that are reminiscent of oligoasthenoteratospermia (OAT), which is a common cause of male infertility in humans. Like the lack of functional PPP4 in the mouse model, OAT is characterized by abnormal sperm morphology, low sperm count and poor sperm motility. Although the causes of OAT are probably heterogeneous, including mutation of various genes and environmentally induced defects, the detailed molecular mechanism(s) has remained unclear. Our discovery that the PPP4C-deficient mouse model shares features with human OAT might offer a useful model for further studies of this currently poorly understood disorder., (© The Author(s) 2020. Published by Oxford University Press on behalf of European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Multiple morphological abnormalities of the flagella (MMAF) is a genetically heterogeneous disorder leading to male infertility. Recent studies have revealed that DNAH17 variants are associated with MMAF, yet there is no functional evidence in support of their pathnogenicity. Here, we recruited two consanguineous families of Pakistani and Chinese origins, respectively, diagnosed with MMAF. Whole-exome sequencing identified novel homozygous DNAH17 variants, which led to loss of DNAH17 proteins, in the patients. Transmission electron microscope analyses revealed completely disorganized axonemal structure as the predominant anomaly and increased frequencies of missings of microtubule doublet(s) 4-7 in sperm flagella of patients. Similar to those found in patients, Dnah17 -/- mice also displayed MMAF phenotype along with completely disorganized axonemal structures. Clusters of disorganized microtubules and outer dense fibers were observed in developing spermatids, indicating impaired sperm flagellar assembly. Besides, we also noticed many elongating spermatids with a deformed nuclear shape and abnormal step 16 spermatids that failed to spermiate, which subsequently underwent apoptosis in Dnah17-null mice. These findings present direct evidence establishing that DNAH17 is a MMAF-related gene in humans and mice, extend the clinical interpretations of DNAH17 variants, and highlight an essential and complex role of DNAH17 in spermatogenesis., (© 2020 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Primary ciliary dyskinesia (PCD) is a rare genetic disorder characterized by recurrent respiratory infections, nasosinusitis, tympanitis, and/or male infertility, all of which can severely impair the patient's quality of life. Multiple morphological abnormalities of the sperm flagella (MMAF) is one type of severe teratozoospermia and results from a variety of flagellar defects. In this study, we conducted whole-exome sequencing to identify and evaluate the genetic lesions in two patients with potential PCD and MMAF. Biallelic mutations in exon 10, c.983G>A; p.(Gly328Asp), and exon 29, c.3532G>A; p.(Asp1178Asn), of the CFAP74 (NM_001304360) gene were identified in patient 1 (P1), and biallelic mutations in exon 7, c.652C>T; p.(Arg218Trp), and exon 35, c. 4331G>C; p.(Ser1444Thr), of the same gene were identified in patient 2 (P2). Bioinformatic analysis suggested that these variants may be disease causing. Immunofluorescence confirmed that CFAP74 was absent in these patients' sperm samples. Intracytoplasmic sperm injection (ICSI) was carried out for P1, and his wife became pregnant after embryo transfer and gave birth to a healthy baby. To the best of our knowledge, this study is the first to identify the importance of CFAP74 in potential PCD and MMAF, contributing to the genetic diagnosis of these disorders and helping to predict pregnancy outcomes relevant in in vitro fertilization.

Background: Multiple morphological abnormalities of the flagella (MMAF) consistently lead to male infertility due to a reduced or absent sperm motility defined as asthenozoospermia. Despite numerous genes recently described to be recurrently associated with MMAF, more than half of the cases analysed remain unresolved, suggesting that many yet uncharacterised gene defects account for this phenotype METHODS: Exome sequencing was performed on 167 infertile men with an MMAF phenotype. Immunostaining and transmission electron microscopy (TEM) in sperm cells from affected individuals were performed to characterise the ultrastructural sperm defects. Gene inactivation using RNA interference (RNAi) was subsequently performed in Trypanosoma ., Results: We identified six unrelated affected patients carrying a homozygous deleterious variants in MAATS1, a gene encoding CFAP91, a calmodulin-associated and spoke-associated complex (CSC) protein. TEM and immunostaining experiments in sperm cells showed severe central pair complex (CPC) and radial spokes defects. Moreover, we confirmed that the WDR66 protein is a physical and functional partner of CFAP91 into the CSC. Study of Trypanosoma MAATS1's orthologue (TbCFAP91) highlighted high sequence and structural analogies with the human protein and confirmed the axonemal localisation of the protein. Knockdown of TbCFAP91 using RNAi impaired flagellar movement led to CPC defects in Trypanosoma as observed in humans., Conclusions: We showed that CFAP91 is essential for normal sperm flagellum structure and function in human and Trypanosoma and that biallelic variants in this gene lead to severe flagellum malformations resulting in astheno-teratozoospermia and primary male infertility., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2020. No commercial re-use. See rights and permissions. Published by BMJ.)

Asthenozoospermia is a common cause of male infertility associated with the reduced motility and/or abnormal morphology of spermatozoa, although its etiology remains incompletely understood. Multiple morphological abnormalities of the sperm flagella (MMAF) is one of the main causes of asthenozoospermia. However, the MMAF-associated genes identified to date cannot explain all the human MMAF cases. Herein, a loss-of-function mutation of DNAH8 was identified in an asthenozoospermia patient with MMAF. Moreover, the negative effect of this mutation on DNAH8 expression was confirmed by immunofluorescence staining and western blotting. Remarkably, it is the first time that DNAH8 is suggested to be associated with human MMAF. Our findings provide strong evidence that a loss-of-function mutation in DNAH8 can cause male infertility with MMAF and that DNAH8 is essential for sperm flagellar formation., (© 2020 John Wiley & Sons A/S . Published by John Wiley & Sons Ltd.)

Multiple morphological abnormalities of the sperm flagella (MMAF) is a severe form of asthenoteratozoospermia. Although recent studies have revealed several MMAF-associated genes and demonstrated MMAF to be a genetically heterogeneous disease, at least one-third of the cases are still not well understood for their etiology. Here, we identified bi-allelic loss-of-function variants in CFAP58 by using whole-exome sequencing in five (5.6%) unrelated individuals from a cohort of 90 MMAF-affected Chinese men. Each of the men harboring bi-allelic CFAP58 variants presented typical MMAF phenotypes. Transmission electron microscopy demonstrated striking flagellar defects with axonemal and mitochondrial sheath malformations. CFAP58 is predominantly expressed in the testis and encodes a cilia- and flagella-associated protein. Immunofluorescence assays showed that CFAP58 localized at the entire flagella of control sperm and predominantly concentrated in the mid-piece. Immunoblotting and immunofluorescence assays showed that the abundances of axoneme ultrastructure markers SPAG6 and SPEF2 and a mitochondrial sheath protein, HSP60, were significantly reduced in the spermatozoa from men harboring bi-allelic CFAP58 variants. We generated Cfap58-knockout mice via CRISPR/Cas9 technology. The male mice were infertile and presented with severe flagellar defects, consistent with the sperm phenotypes in MMAF-affected men. Overall, our findings in humans and mice strongly suggest that CFAP58 plays a vital role in sperm flagellogenesis and demonstrate that bi-allelic loss-of-function variants in CFAP58 can cause axoneme and peri-axoneme malformations leading to male infertility. This study provides crucial insights for understanding and counseling of MMAF-associated asthenoteratozoospermia., (Copyright © 2020 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

The flagellum is essential for sperm motility and fertilization in vivo. The axoneme is the main component of the flagella, extending through its entire length. An axoneme is comprised of two central microtubules surrounded by nine doublets, the nexin-dynein regulatory complex, radial spokes, and dynein arms. Failure to properly assemble components of the axoneme in a sperm flagellum, leads to fertility alterations. To understand this process in detail, we have defined the function of an uncharacterized gene, Cfap97 domain containing 1 (Cfap97d1). This gene is evolutionarily conserved in mammals and multiple other species, including Chlamydomonas. We have used two independently generated Cfap97d1 knockout mouse models to study the gene function in vivo. Cfap97d1 is exclusively expressed in testes starting from post-natal day 20 and continuing throughout adulthood. Deletion of the Cfap97d1 gene in both mouse models leads to sperm motility defects (asthenozoospermia) and male subfertility. In vitro fertilization (IVF) of cumulus-intact oocytes with Cfap97d1 deficient sperm yielded few embryos whereas IVF with zona pellucida-free oocytes resulted in embryo numbers comparable to that of the control. Knockout spermatozoa showed abnormal motility characterized by frequent stalling in the anti-hook position. Uniquely, Cfap97d1 loss caused a phenotype associated with axonemal doublet heterogeneity linked with frequent loss of the fourth doublet in the sperm stored in the epididymis. This study demonstrates that Cfap97d1 is required for sperm flagellum ultra-structure maintenance, thereby playing a critical role in sperm function and male fertility in mice., Competing Interests: The authors have declared that no competing interests exist.

Background: Asthenoteratospermia with multiple morphological abnormalities in the sperm flagella (MMAF) is a significant cause of male infertility. WDR19 is a core component in the IFT-A complex and has a critical role in intraflagellar transport. However, the role of WDR19 mutations in male infertility has yet to be examined., Methods and Results: We performed whole exome sequencing (WES) for 65 asthenoteratospermia individuals and identified a proband who carried a homozygous WDR19 (c.A3811G, p.K1271E) mutation from a consanguineous family. Systematic examinations, including CT scanning and retinal imaging, excluded previous ciliopathic syndromes in the proband. Moreover, semen analysis of this patient showed that the progressive rate decreased to zero, and the sperm flagella showed multiple morphological abnormalities. Scanning and transmission electron microscopy assays indicated that the ultrastructure of sperm flagella in the patient was completely destroyed, while immunofluorescence revealed that WDR19 was absent from the sperm neck and flagella. Moreover, IFT140 and IFT88, predicted to interact with WDR19 directly, were mis-allocated in the WDR19-mutated sperm. Notably, the MMAF subject harboring WDR19 variant and his partner successfully achieved clinical pregnancy through intracytoplasmic sperm injection (ICSI)., Conclusions: We identified WDR19 as a novel pathogenic gene for male infertility caused by asthenoteratospermia in the absence of other ciliopathic phenotypes, and that patients carrying WDR19 variant can have favorable pregnancy outcomes following ICSI.

Purpose: To identify the genetic cause of patients with primary ciliary dyskinesia (PCD) and male infertility from two unrelated Han Chinese families., Methods: We conducted whole-exome sequencing of three individuals with PCD and male infertility from two unrelated Chinese families, and performed a targeted look-up for DNAAF6 variants in our previously reported cohort of 442 individuals (219 with isolated oligoasthenospermia and 223 fertile controls). Ultrastructural and immunostaining analyses of patients' spermatozoa were performed. The pathogenicity of the variants was validated using patient's spermatozoa and HEK293T cells. Intracytoplasmic sperm injection (ICSI) treatment was conducted in two patients., Results: We identified one novel hemizygous frameshift variant (NM_173494, c.319_329del: p.R107fs) of DNAAF6 gene (previously named PIH1D3) in family 1 and one novel hemizygous missense variant (c.290G>T: p.G97V) in family 2. No hemizygous deleterious variants in DNAAF6 were detected in the control cohort of 442 individuals. Ultrastructural and immunostaining analyses of patients' spermatozoa showed the absence of outer and inner dynein arms in sperm flagella. Both variants were proven to lead to DNAAF6 protein degradation in HEK293T cells. Both patients carrying DNAAF6 variants underwent one ICSI cycle and delivered one healthy child each., Conclusion: We identified novel DNAAF6 variants causing male infertility and PCD in Han Chinese patients. This finding extended the spectrum of variants in DNAAF6 and revealed new light on the impact of DNAAF6 variants in sperm flagella.

Background: Male infertility is a prevalent issue worldwide, mostly due to the impaired sperm motility. Multiple morphological abnormalities of the sperm flagella (MMAF) present aberrant spermatozoa with absent, short, coiled, bent and irregular-calibre flagella resulting in severely decreased motility. Previous studies reported several MMAF-associated genes accounting for approximately half of MMAF cases., Methods and Result: We conducted genetic analysis using whole-exome sequencing in 88 Han Chinese MMAF probands. CFAP65 homozygous mutations were identified in four unrelated consanguineous families, and CFAP65 compound heterozygous mutations were found in two unrelated cases with MMAF. All these CFAP65 mutations were null, including four frameshift mutations (c.1775delC [p.Pro592Leufs*8], c.3072_3079dup [p.Arg1027Profs*41], c.1946delC [p.Pro649Argfs*5] and c.1580delT [p.Leu527Argfs*31]) and three stop-gain mutations (c.4855C>T [p.Arg1619*], c.5270T>A [p.Leu1757*] and c.5341G>T [p.Glu1781*]). Additionally, two homozygous CFAP65 variants likely affecting splicing were identified in two MMAF-affected men of Tunisian and Iranian ancestries, respectively. These biallelic variants of CFAP65 were verified by Sanger sequencing and were absent or very rare in large data sets aggregating sequence information from various human populations. CFAP65 , encoding the cilia and flagella associated protein 65, is highly and preferentially expressed in the testis. Here we also generated a frameshift mutation in mouse orthologue Cfap65 using CRISPR-Cas9 technology. Remarkably, the phenotypes of Cfap65 -mutated male mice were consistent with human MMAF., Conclusions: Our experimental observations performed on both human subjects and on Cfap65 -mutated mice demonstrate that the presence of biallelic mutations in CFAP65 causes the MMAF phenotype and impairs sperm motility., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2020. No commercial re-use. See rights and permissions. Published by BMJ.)