Lack of CCDC146, a ubiquitous centriole and microtubule-associated protein, leads to non-syndromic male infertility in human and mouse.

From a cohort of 167 infertile patients suffering from multiple morphological abnormalities of the flagellum (MMAF), pathogenic bi-allelic mutations were identified in the CCDC146 gene. In somatic cells, CCDC146 is located at the centrosome and at multiple microtubule-related organelles during mitotic division, suggesting that it is a microtubule-associated protein (MAP). To decipher the molecular pathogenesis of infertility associated with CCDC146 mutations, a Ccdc146 knock-out (KO) mouse line was created. KO male mice were infertile, and sperm exhibited a phenotype identical to CCDC146 mutated patients. CCDC146 expression starts during late spermiogenesis. In the spermatozoon, the protein is conserved but is not localized to centrioles, unlike in somatic cells, rather it is present in the axoneme at the level of microtubule doublets. Expansion microscopy associated with the use of the detergent sarkosyl to solubilize microtubule doublets suggests that the protein may be a microtubule inner protein (MIP). At the subcellular level, the absence of CCDC146 impacted all microtubule-based organelles such as the manchette, the head-tail coupling apparatus (HTCA), and the axoneme. Through this study, a new genetic cause of infertility and a new factor in the formation and/or structure of the sperm axoneme were characterized.

CCDC65, encoding a component of the axonemal Nexin-Dynein regulatory complex, is required for sperm flagellum structure in humans.

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.

Novel axonemal protein ZMYND12 interacts with TTC29 and DNAH1, and is required for male fertility and flagellum function.

Male infertility is common and complex, presenting a wide range of heterogeneous phenotypes. Although about 50% of cases are estimated to have a genetic component, the underlying cause often remains undetermined. Here, from whole-exome sequencing on samples from 168 infertile men with asthenoteratozoospermia due to severe sperm flagellum, we identified homozygous ZMYND12 variants in four unrelated patients. In sperm cells from these individuals, immunofluorescence revealed altered localization of DNAH1, DNALI1, WDR66, and TTC29. Axonemal localization of ZMYND12 ortholog TbTAX-1 was confirmed using the Trypanosoma brucei model. RNAi knock-down of TbTAX-1 dramatically affected flagellar motility, with a phenotype similar to the sperm from men bearing homozygous ZMYND12 variants. Co-immunoprecipitation and ultrastructure expansion microscopy in T. brucei revealed TbTAX-1 to form a complex with TTC29. Comparative proteomics with samples from Trypanosoma and Ttc29 KO mice identified a third member of this complex: DNAH1. The data presented revealed that ZMYND12 is part of the same axonemal complex as TTC29 and DNAH1, which is critical for flagellum function and assembly in humans, and Trypanosoma. ZMYND12 is thus a new asthenoteratozoospermia-associated gene, bi-allelic variants of which cause severe flagellum malformations and primary male infertility.

New Mutations in DNHD1 Cause Multiple Morphological Abnormalities of the Sperm Flagella.

Male infertility is a common and complex disease and presents as a wide range of heterogeneous phenotypes. Multiple morphological abnormalities of the sperm flagellum (MMAF) phenotype is a peculiar condition of extreme morphological sperm defects characterized by a mosaic of sperm flagellum defects to a total asthenozoospermia. At this time, about 40 genes were associated with the MMAF phenotype. However, mutation prevalence for most genes remains individually low and about half of individuals remain without diagnosis, encouraging us to pursue the effort to identify new mutations and genes. In the present study, an a cohort of 167 MMAF patients was analyzed using whole-exome sequencing, and we identified three unrelated patients with new pathogenic mutations in DNHD1, a new gene recently associated with MMAF. Immunofluorescence experiments showed that DNHD1 was totally absent from sperm cells from DNHD1 patients, supporting the deleterious effect of the identified mutations. Transmission electron microscopy reveals severe flagellum abnormalities of sperm cells from one mutated patient, which appeared completely disorganized with the absence of the central pair and midpiece defects with a shortened and misshapen mitochondrial sheath. Immunostaining of IFT20 was not altered in mutated patients, suggesting that IFT may be not affected by DNHD1 mutations. Our data confirmed the importance of DNHD1 for the function and structural integrity of the sperm flagellum. Overall, this study definitively consolidated its involvement in MMAF phenotype on a second independent cohort and enriched the mutational spectrum of the DNHD1 gene.

A heterozygous missense variant in DLX3 leads to uterine leiomyomas and pregnancy losses in a consanguineous Iranian family.

Uterine leiomyomas (ULs) are benign solid tumors arising from the uterine myometrium. They are the most common pelvic tumors among females of reproductive age. Despite the universal prevalence of ULs and its huge impact on women's lives, the exact etiology and pathophysiologic mechanisms have not been fully understood. Numerous studies indicate that genetic factors play a crucial role in ULs development. This study aims to identify the probable genetic causes of ULs in a consanguineous Iranian family. Whole-exome sequencing (WES) on five family members with ULs revealed a likely pathogenic missense variant encoding for Y88C in the transactivation (TA) domain of DLX3 gene (c.263A > G; p.Y88C). Sanger sequencing of a total of 9 affected and non-affected family members indicated a segregation with disease with autosomal dominant inheritance. Moreover, targeted Sanger sequencing on 32 additional non-related patients with ULs showed none was heterozygous for this variant. MutPred2 predicted the pathogenicity of candidate variant by both phosphorylation and sulfation loss as actionable hypotheses. Project HOPE revealed that the identified variant residue is smaller and more hydrophobic comparing to the wild-type residue. I-TASSER and UCSF Chimera were also used for modeling and visualizing the predicted variant, respectively. This WES analysis is the first to report a variant in DLX3 variation associated with ULs pathogenicity in Iranian population highlighting the effectiveness of WES as a strong diagnostic method. However, further functional studies on this variant are needed to confirm the potential pathogenicity of this mutation.

CYP19A1 Promoters Activity in Human Granulosa Cells: A Comparison between PCOS and Normal Subjects.

Objective: Estrogen, a female hormone maintaining several critical functions in women's physiology, e.g., folliculogenesis and fertility, is predominantly produced by ovarian granulosa cells where aromatase enzyme converts androgen to estrogen. The principal enzyme responsible for this catalytic reaction is encoded by the CYP19A1 gene, with a long regulatory region. Abnormalities in this process cause metabolic disorders in women, one of the most common of which is polycystic ovary syndrome (PCOS). The main purpose of this research was to determine the effect of the promoters on aromatase expression in cells with normal and PCOS characteristics. Materials and Methods: In this experimental study, four promoters of the CYP19A1 gene, including PII, I.3, I.4, and PII/ I .3 promoter fragments, were cloned upstream of the luciferase gene and transfected into normal and PCOS granulosa cells. Subsequently, the effect of follicle-stimulating hormone (FSH) on the activity of these regulatory regions was examined in the presence and absence of FSH. Western blotting was used to confirm aromatase expression in all groups. Data analysis was performed using ANOVA and paired sample t test, compared by post-hoc least significant difference (LSD) test. Results: Luciferase results confirmed the intense activity of PII promoter in the presence of FSH. Moreover, the study demonstrated reduced activity of PII promoter in normal granulosa cells, possibly due to the regulatory region of I.3 next to PII. Conclusion: FSH stimulates transcription of aromatase enzyme by affecting PII promoter, a process regulated by the inhibitory role of the I.3 region in PII activity in granulosa cells. Given the distinct role of these promoters in normal and PCOS granulosa cells, the importance of nuclear factors residing in these regions can be discerned.

Direct visualization of pre-protamine 2 detects protamine assembly failures and predicts ICSI success.

Histone-to-protamine transition is an essential step in the generation of fully functional spermatozoa in various mammalian species. In human and mouse, one of the two protamine-encoding genes produces a precursor pre-protamine 2 (pre-PRM2) protein, which is then processed and assembled. Here, we design an original approach based on the generation of pre-PRM2-specific antibodies to visualize the unprocessed pre-PRM2 by microscopy, flow cytometry and immunoblotting. Using mouse models with characterized failures in histone-to-protamine replacement, we show that pre-PRM2 retention is tightly linked to impaired nucleosome disassembly. Additionally, in elongating/condensing spermatids, we observe that pre-PRM2 and transition protein are co-expressed spatiotemporally, and their physical interaction suggests that these proteins act simultaneously rather than successively during histone replacement. By using our anti-human pre-PRM2 antibody, we also measured pre-PRM2 retention rates in the spermatozoa from 49 men of a series of infertile couples undergoing ICSI, which shed new light on the debated relation between pre-PRM2 retention and sperm parameters. Finally, by monitoring 2-pronuclei embryo formation following ICSI, we evaluated the fertilization ability of the sperm in these 49 patients. Our results suggest that the extent of pre-PRM2 retention in sperm, rather than pre-PRM2 accumulation per se, is associated with fertilization failure. Hence, anti-pre-PRM2 antibodies are valuable tools that could be used in routine monitoring of sperm parameters in fertility clinics, as well as in experimental research programmes to better understand the obscure process of histone-to-protamine transition.

Homozygous mutations in CCDC34 cause male infertility with oligoasthenoteratozoospermia in humans and mice.

BACKGROUND: Oligoasthenoteratozoospermia is a typical feature of sperm malformations leading to male infertility. Only a few genes have been clearly identified as pathogenic genes of oligoasthenoteratozoospermia. METHODS AND RESULTS: Here, we identified a homozygous frameshift variant (c.731dup, p.Asn244Lysfs*3) in CCDC34, which is preferentially expressed in the human testis, using whole-exome sequencing in a cohort of 100 Chinese men with multiple morphological abnormalities of the sperm flagella (MMAF). In an additional cohort of 167 MMAF-affected men from North Africa, Iran and France, we identified a second subject harbouring a homozygous CCDC34 frameshift variant (c.799_817del, p.Glu267Lysfs*72). Both affected men presented a typical MMAF phenotype with an abnormally low sperm concentration (ie, oligoasthenoteratozoospermia). Transmission electron microscopy analysis of the sperm flagella affected by CCDC34 deficiency further revealed dramatic disorganisation of the axoneme. Immunofluorescence assays of the spermatozoa showed that CCDC34 deficiency resulted in almost absent staining of CCDC34 and intraflagellar transport-B complex-associated proteins (such as IFT20 and IFT52). Furthermore, we generated a mouse Ccdc34 frameshift mutant using CRISPR-Cas9 technology. Ccdc34-mutated (Ccdc34mut/mut ) male mice were sterile and presented oligoasthenoteratozoospermia with typical MMAF anomalies. Intracytoplasmic sperm injection has good pregnancy outcomes in both humans and mice. CONCLUSIONS: Our findings support that CCDC34 is crucial to the formation of sperm flagella and that biallelic deleterious mutations in CCDC34/Ccdc34 cause male infertility with oligoasthenoteratozoospermia in humans and mice.

Pathophysiologic Mechanisms of Insulin Secretion and Signaling-Related Genes in Etiology of Polycystic Ovary Syndrome.

Polycystic ovary syndrome (PCOS) is a common endocrinopathy in women. PCOS is characterized by anovulation, hyperandrogenism, polycystic ovaries, insulin resistance, and obesity. Despite the finding that the genetic origin of PCOS is well demonstrated in previous twin and familial clustering studies, genes and factors that can exactly explain the PCOS pathophysiology are not known. Objective(s). In this review, we attempted to identify genes related to secretion and signaling of insulin aspects of PCOS and their physiological functions in order to explain the pathways that are regulated by these genes which can be a prominent function in PCOS predisposition. Materials and Methods. For this purpose, published articles and reviews dealing with genetic evaluation of PCOS in women from peer-reviewed journals in PubMed and Google Scholar databases were included in this review. Results. The genomic investigations in women of different populations identified many candidate genes and loci that are associated with PCOS. The most important of them are INSR, IRS1-2, MTNR1A, MTNR1B, THADA, PPAR-γ2, ADIPOQ, and CAPN10. These are mainly associated with metabolic aspects of PCOS. Conclusions. In this review, we proposed that each of these genes may interrupt specific physiological pathways by affecting them and contribute to PCOS initiation. It is clear that the role of genes involved in insulin secretion and signaling is more critical than other pathways.

Integration and gene co-expression network analysis of scRNA-seq transcriptomes reveal heterogeneity and key functional genes in human spermatogenesis.

Spermatogenesis is a complex process of cellular division and differentiation that begins with spermatogonia stem cells and leads to functional spermatozoa production. However, many of the molecular mechanisms underlying this process remain unclear. Single-cell RNA sequencing (scRNA-seq) is used to sequence the entire transcriptome at the single-cell level to assess cell-to-cell variability. In this study, more than 33,000 testicular cells from different scRNA-seq datasets with normal spermatogenesis were integrated to identify single-cell heterogeneity on a more comprehensive scale. Clustering, cell type assignments, differential expressed genes and pseudotime analysis characterized 5 spermatogonia, 4 spermatocyte, and 4 spermatid cell types during the spermatogenesis process. The UTF1 and ID4 genes were introduced as the most specific markers that can differentiate two undifferentiated spermatogonia stem cell sub-cellules. The C7orf61 and TNP can differentiate two round spermatid sub-cellules. The topological analysis of the weighted gene co-expression network along with the integrated scRNA-seq data revealed some bridge genes between spermatogenesis's main stages such as DNAJC5B, C1orf194, HSP90AB1, BST2, EEF1A1, CRISP2, PTMS, NFKBIA, CDKN3, and HLA-DRA. The importance of these key genes is confirmed by their role in male infertility in previous studies. It can be stated that, this integrated scRNA-seq of spermatogenic cells offers novel insights into cell-to-cell heterogeneity and suggests a list of key players with a pivotal role in male infertility from the fertile spermatogenesis datasets. These key functional genes can be introduced as candidates for filtering and prioritizing genotype-to-phenotype association in male infertility.

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

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

A missense mutation in IFT74, encoding for an essential component for intraflagellar transport of Tubulin, causes asthenozoospermia and male infertility without clinical signs of Bardet-Biedl syndrome.

Cilia and flagella are formed around an evolutionary conserved microtubule-based axoneme and are required for fluid and mucus clearance, tissue homeostasis, cell differentiation and movement. The formation and maintenance of cilia and flagella require bidirectional transit of proteins along the axonemal microtubules, a process called intraflagellar transport (IFT). In humans, IFT defects contribute to a large group of systemic diseases, called ciliopathies, which often display overlapping phenotypes. By performing exome sequencing of a cohort of 167 non-syndromic infertile men displaying multiple morphological abnormalities of the sperm flagellum (MMAF) we identified two unrelated patients carrying a homozygous missense variant adjacent to a splice donor consensus site of IFT74 (c.256G > A;p.Gly86Ser). IFT74 encodes for a core component of the IFT machinery that is essential for the anterograde transport of tubulin. We demonstrate that this missense variant affects IFT74 mRNA splicing and induces the production of at least two distinct mutant proteins with abnormal subcellular localization along the sperm flagellum. Importantly, while IFT74 deficiency was previously implicated in two cases of Bardet-Biedl syndrome, a pleiotropic ciliopathy with variable expressivity, our data indicate that this missense mutation only results in primary male infertility due to MMAF, with no other clinical features. Taken together, our data indicate that the nature of the mutation adds a level of complexity to the clinical manifestations of ciliary dysfunction, thus contributing to the expanding phenotypical spectrum of ciliopathies.

KH domain containing 3 like (KHDC3L) frame-shift mutation causes both recurrent pregnancy loss and hydatidiform mole.

OBJECTIVE: Recurrent pregnancy loss (RPL) is a common infertility-related complication that affects approximately 1-3 % of women worldwide. Known causes of etiology are found in approximately half the cases but the other half remain unexplained. It is estimated that several thousands of genes contribute to reproductive success in mammals and the genetic causes of RPL cannot be fully addressed through targeted genetic tests. In recent years, massive parallel sequencing technologies has helped discovering many causal mutations in hereditary diseases such as RPL. STUDY DESIGN: Using whole-exome sequencing (WES), we studied a large multiplex consanguineous family with multiple cases of RPL and hydatidiform moles (HM). In addition, targeted Sanger sequencing was applied to 40 additional non-related individuals with RPL. RESULTS: The use of WES permitted to identify the pathogenic variant in KHDC3L (c.322_325delGACT) in related who experienced RPL with or without HM. Sanger sequencing confirmed the segregation of the mutation throughout the pedigree and permitted to establish this variant as the genetic cause responsible for RPL and HM in this family. CONCLUSION: KHDC3L is well established as a susceptibility gene for HM but we confirmed here that KHDC3L deleterious variants can also induce RPL. In addition, we observed a genotype-phenotype correlation, demonstrating that women with a truncating KHDC3L homozygous variant could not sustain a pregnancy and often had pregnancy losses mainly due to HM while those with the same heterozygous variant could have children but often endured RPL with no HM.

Profiling of Initial Available SARS-CoV-2 Sequences from Iranian Related COVID-19 Patients.

The etiologic agent SARS-CoV-2 has caused the outbreak of COVID-19 which is spread widely around the world. It is vital to uncover and investigate the full genome sequence of SARS-CoV-2 throughout the world to track changes in this virus. To this purpose, SARS-CoV-2 full genome sequence profiling of 20 patients in Iran and different countries that already had a travel history to Iran or contacts with Iranian cases were provided from the GISAID database. The bioinformatics analysis showed 44 different nucleotide mutations that caused 26 nonsynonymous mutations in protein sequences with regard to the reference full genome of the SARS-CoV-2 sequence (NC_045512.2). R207C, V378I, M2796I, L3606F, and A6407V in ORF1ab were common mutations in these sequences. Also, some of the detected mutations only were found in Iranian data in comparison with all the available sequences of SARS-CoV-2. The position of S protein mutations showed they were far from the binding site of this protein with angiotensin-converting enzyme-2 (ACE2) as the host cell receptor. These results can be helpful to design specific diagnostic tests, trace the SARS-CoV-2 sequence changes in Iran, and explore therapeutic drugs and vaccines.

Regulated Acyl-CoA Synthetase Short-Chain Family Member 2 Accumulation during Spermatogenesis.

OBJECTIVE: Acyl-CoA synthetase short-chain family member 2 (ACSS2) activity provides a major source of acetyl-CoA to drive histone acetylation. This study aimed to unravel the ACSS2 expression during mouse spermatogenesis, where a dynamic and stage-specific genome-wide histone hyperacetylation occurs before histone eviction. MATERIALS AND METHODS: In this experimental study, ACSS2 expression levels during spermatogenesis were verified by Immunodetection. Testis paraffin-embedded sections were used for IHC staining with anti-H4 pan ac and anti-ACSS2. Co-detection of ACSS2 and H4K5ac was performed on testis tubular sections by immunofluorescence. Proteins extracts from fractionated male germ cells were subjected to western-blotting and immunoblot was probed with anti- ACSS2 and anti-actin. RESULTS: The resulting data showed that the commitment of progenitor cells into meiotic divisions leads to a robust accumulation of ACSS2 in the cell nucleus, especially in pachytene spermatocytes (P). However, ACSS2 protein drastically declines during post-meiotic stages, when a genome-wide histone hyperacetylation is known to occur. CONCLUSION: The results of this study are in agreement with the idea that the major function of ACSS2 is to recycle acetate generated after histone deacetylation to regenerate acetyl-CoA which is required to maintain the steady state of histone acetylation. Thus, it is suggested that in spermatogenic cells, nuclear activity of ACSS2 maintains the acetate recycling until histone hyperacetylation, but disappears before the acetylation-dependent histone degradation.

Biallelic mutations in CFAP65 cause male infertility with multiple morphological abnormalities of the sperm flagella in humans and mice.

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.

Designing A Transgenic Chicken: Applying New Approaches toward A Promising Bioreactor.

Specific developmental characteristics of the chicken make it an attractive model for the generation of transgenic organisms. Chicken possess a strong potential for recombinant protein production and can be used as a powerful bioreactor to produce pharmaceutical and nutritional proteins. Several transgenic chickens have been generated during the last two decades via viral and non-viral transfection. Culturing chicken primordial germ cells (PGCs) and their ability for germline transmission ushered in a new stage in this regard. With the advent of CRISPR/Cas9 system, a new phase of studies for manipulating genomes has begun. It is feasible to integrate a desired gene in a predetermined position of the genome using CRISPR/Cas9 system. In this review, we discuss the new approaches and technologies that can be applied to generate a transgenic chicken with regards to recombinant protein productions.

Homozygous mutations in SPEF2 induce multiple morphological abnormalities of the sperm flagella and male infertility.

BACKGROUND: Male infertility due to multiple morphological abnormalities of the sperm flagella (MMAF) is a genetically heterogeneous disorder. Previous studies revealed several MMAF-associated genes, which account for approximately 60% of human MMAF cases. The pathogenic mechanisms of MMAF remain to be illuminated. METHODS AND RESULTS: We conducted genetic analyses using whole-exome sequencing in 50 Han Chinese probands with MMAF. Two homozygous stop-gain variants (c.910C>T (p.Arg304*) and c.3400delA (p.Ile1134Serfs*13)) of the SPEF2 (sperm flagellar 2) gene were identified in two unrelated consanguineous families. Consistently, an Iranian subject from another cohort also carried a homozygous SPEF2 stop-gain variant (c.3240delT (p.Phe1080Leufs*2)). All these variants affected the long SPEF2 transcripts that are expressed in the testis and encode the IFT20 (intraflagellar transport 20) binding domain, important for sperm tail development. Notably, previous animal studies reported spontaneous mutations of SPEF2 causing sperm tail defects in bulls and pigs. Our further functional studies using immunofluorescence assays showed the absence or a remarkably reduced staining of SPEF2 and of the MMAF-associated CFAP69 protein in the spermatozoa from SPEF2-affected subjects. CONCLUSIONS: We identified SPEF2 as a novel gene for human MMAF across the populations. Functional analyses suggested that the deficiency of SPEF2 in the mutated subjects could alter the localisation of other axonemal proteins.