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.

A splice donor variant of GAS8 induces structural disorganization of the axoneme in sperm flagella and leads to nonsyndromic male infertility.

Motile cilia and flagella are closely related organelles structured around a highly conserved axoneme whose formation and maintenance involve proteins from hundreds of genes. Defects in many of these genes have been described to induce primary ciliary dyskinesia (PCD) mainly characterized by chronic respiratory infections, situs inversus and/or infertility. In men, cilia/flagella-related infertility is usually caused by asthenozoospermia due to multiple morphological abnormalities of the sperm flagella (MMAF). Here, we investigated a cohort of 196 infertile men displaying a typical MMAF phenotype without any other PCD symptoms. Analysis of WES data identified a single case carrying a deleterious homozygous GAS8 variant altering a splice donor consensus site. This gene, also known as DRC4, encodes a subunit of the Nexin-Dynein Regulatory Complex (N-DRC), and has been already associated to male infertility and mild PCD. Confirming the deleterious effect of the candidate variant, GAS8 staining by immunofluorescence did not evidence any signal from the patient's spermatozoa whereas a strong signal was present along the whole flagella length in control cells. Concordant with its role in the N-DRC, transmission electron microscopy evidenced peripheral microtubule doublets misalignments. We confirm here the importance of GAS8 in the N-DRC and observed that its absence induces a typical MMAF phenotype not necessarily accompanied by other PCD symptoms.

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.

Identification of IQCH as a calmodulin-associated protein required for sperm motility in humans.

Sperm fertilization ability mainly relies on proper sperm progression through the female genital tract and capacitation, which involves phosphorylation signaling pathways triggered by calcium and bicarbonate. We performed exome sequencing of an infertile asthenozoospermic patient and identified truncating variants in MAP7D3, encoding a microtubule-associated protein, and IQCH, encoding a protein of unknown function with enzymatic and signaling features. We demonstrate the deleterious impact of both variants on sperm transcripts and proteins from the patient. We show that, in vitro, patient spermatozoa could not induce the phosphorylation cascades associated with capacitation. We also provide evidence for IQCH association with calmodulin, a well-established calcium-binding protein that regulates the calmodulin kinase. Notably, we describe IQCH spatial distribution around the sperm axoneme, supporting its function within flagella. Overall, our work highlights the cumulative pathological impact of gene mutations and identifies IQCH as a key protein required for sperm motility and capacitation.

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.

The [PSI+] prion modulates cytochrome c oxidase deficiency caused by deletion of COX12.

Cytochrome c oxidase (CcO) is a pivotal enzyme of the mitochondrial respiratory chain, which sustains bioenergetics of eukaryotic cells. Cox12, a peripheral subunit of CcO oxidase, is required for full activity of the enzyme, but its exact function is unknown. Here experimental evolution of a Saccharomyces cerevisiae Δcox12 strain for ∼300 generations allowed to restore the activity of CcO oxidase. In one population, the enhanced bioenergetics was caused by a A375V mutation in the cytosolic AAA+ disaggregase Hsp104. Deletion or overexpression of HSP104 also increased respiration of the Δcox12 ancestor strain. This beneficial effect of Hsp104 was related to the loss of the [PSI+] prion, which forms cytosolic amyloid aggregates of the Sup35 protein. Overall, our data demonstrate that cytosolic aggregation of a prion impairs the mitochondrial metabolism of cells defective for Cox12. These findings identify a new functional connection between cytosolic proteostasis and biogenesis of the mitochondrial respiratory chain.

A recurrent ZP1 variant is responsible for oocyte maturation defect with degenerated oocytes in infertile females.

A female factor is present in approximately 70% of couple infertility, often due to ovulatory disorders. In oocyte maturation defect (OMD), affected patients have a primary infertility with normal menstrual cycles but produce no oocyte, degenerated (atretic) or abnormal oocytes blocked at different stages of maturation. Four genes have so far been associated with OMD: PATL2, TUBB8, WEE2, and ZP1. In our initial study, 6 out of 23 OMD subjects were shown to carry the same PATL2 homozygous loss of function variant and one patient had a TUBB8 truncating variant. Here, we included four additional OMD patients and reanalyzed all 27 subjects. In addition to the seven patients with a previously identified defect, five carried the same deleterious homozygous ZP1 variant (c.1097G>A; p.Arg366Gln). All the oocytes from ZP1-associated patients appeared shriveled and dark indicating that the abnormal ZP1 protein induced oocyte death and degeneration. Overall ZP1-associated patients had degenerated or absent oocytes contrary to PATL2-associated subjects who had immature oocytes blocked mainly at the germinal vesicle stage. In this cohort of North African OMD patients, whole exome sequencing permitted to diagnose 44% of the patients studied and to identify a new frequent ZP1 variant.

Whole-exome sequencing improves the diagnosis and care of men with non-obstructive azoospermia.

Non-obstructive azoospermia (NOA) is a severe and frequent cause of male infertility, often treated by testicular sperm extraction followed by intracytoplasmic sperm injection. The aim of this study is to improve the genetic diagnosis of NOA, by identifying new genes involved in human NOA and to better assess the chances of successful sperm extraction according to the individual's genotype. Exome sequencing was performed on 96 NOA-affected individuals negative for routine genetic tests. Bioinformatics analysis was limited to a panel of 151 genes selected as known causal or candidate genes for NOA. Only highly deleterious homozygous or hemizygous variants were retained as candidates. A likely causal defect was identified in 16 genes in a total of 22 individuals (23%). Six genes had not been described in man (DDX25, HENMT1, MCMDC2, MSH5, REC8, TDRKH) and 10 were previously reported (C14orf39, DMC1, FANCM, GCNA, HFM1, MCM8, MEIOB, PDHA2, TDRD9, TERB1). Seven individuals had defects in genes from piwi or DNA repair pathways, three in genes involved in post-meiotic maturation, and 12 in meiotic processes. Interestingly, all individuals with defects in meiotic genes had an unsuccessful sperm retrieval, indicating that genetic diagnosis prior to TESE could help identify individuals with low or null chances of successful sperm retrieval and thus avoid unsuccessful surgeries.

From azoospermia to macrozoospermia, a phenotypic continuum due to mutations in the ZMYND15 gene.

Thanks to tremendous advances in sequencing technologies and in particular to whole exome sequencing (WES), many genes have now been linked to severe sperm defects. A precise genetic diagnosis is obtained for a minority of patients and only for the most severe defects like azoospermia or macrozoospermia which is very often due to defects in the aurora kinase C (AURKC gene. Here, we studied a subject with a severe oligozoospermia and a phenotypic diagnosis of macrozoospermia. AURKC analysis did not reveal any deleterious variant. WES was then initiated which permitted to identify a homozygous loss of function variant in the zinc finger MYND-type containing 15 (ZMYND15 gene. ZMYND15 has been described to serve as a switch for haploid gene expression, and mice devoid of ZMYND15 were shown to be sterile due to nonobstructive azoospermia (NOA). In man, ZMYND15 has been associated with NOA and severe oligozoospermia. We confirm here that the presence of a bi-allelic ZMYND15 variant induces a severe oligozoospermia. In addition, we show that severe oligozoospermia can be associated macrozoospermia, and that a phenotypic misdiagnosis is possible, potentially delaying the genetic diagnosis. In conclusion, genetic defects in ZMYND15 can induce complete NOA or severe oligozoospermia associated with a very severe teratozoospermia. In our experience, severe oligozoospermia is often associated with severe teratozoospermia and can sometimes be misinterpreted as macrozoospermia or globozoospermia. In these instances, specific AURKC or dpy-19 like 2 (DPY19L2) diagnosis is usually negative and we recommend the direct use of a pan-genomic techniques such as WES.

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.

Identification and Characterization of the Most Common Genetic Variant Responsible for Acephalic Spermatozoa Syndrome in Men Originating from North Africa.

Acephalic spermatozoa syndrome (ASS) is a rare but extremely severe type of teratozoospermia, defined by the presence of a majority of headless flagella and a minority of tail-less sperm heads in the ejaculate. Like the other severe monomorphic teratozoospermias, ASS has a strong genetic basis and is most often caused by bi-allelic variants in SUN5 (Sad1 and UNC84 domain-containing 5). Using whole exome sequencing (WES), we investigated a cohort of nine infertile subjects displaying ASS. These subjects were recruited in three centers located in France and Tunisia, but all originated from North Africa. Sperm from subjects carrying candidate genetic variants were subjected to immunofluorescence analysis and transmission electron microscopy. Moreover, fluorescent in situ hybridization (FISH) was performed on sperm nuclei to assess their chromosomal content. Variant filtering permitted us to identify the same SUN5 homozygous frameshift variant (c.211+1_211+2dup) in 7/9 individuals (78%). SUN5 encodes a protein localized on the posterior part of the nuclear envelope that is necessary for the attachment of the tail to the sperm head. Immunofluorescence assays performed on sperm cells from three mutated subjects revealed a total absence of SUN5, thus demonstrating the deleterious impact of the identified variant on protein expression. Transmission electron microscopy showed a conserved flagellar structure and a slightly decondensed chromatin. FISH did not highlight a higher rate of chromosome aneuploidy in spermatozoa from SUN5 patients compared to controls, indicating that intra-cytoplasmic sperm injection (ICSI) can be proposed for patients carrying the c.211+1_211+2dup variant. These results suggest that the identified SUN5 variant is the main cause of ASS in the North African population. Consequently, a simple and inexpensive genotyping of the 211+1_211+2dup variant could be beneficial for affected men of North African origin before resorting to more exhaustive genetic analyses.

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.

The sodium/proton exchanger SLC9C1 (sNHE) is essential for human sperm motility and fertility.

Asthenozoospermia, defined by the absence or reduction of sperm motility, constitutes the most frequent cause of human male infertility. This pathological condition is caused by morphological and/or functional defects of the sperm flagellum, which preclude proper sperm progression. While in the last decade many causal genes were identified for asthenozoospermia associated with severe sperm flagellar defects, the causes of purely functional asthenozoospermia are still poorly defined. We describe here the case of an infertile man, displaying asthenozoospermia without major morphological flagellar anomalies and carrying a homozygous splicing mutation in SLC9C1 (sNHE), which we identified by whole-exome sequencing. SLC9C1 encodes a sperm-specific sodium/proton exchanger, which in mouse regulates pH homeostasis and interacts with the soluble adenylyl cyclase (sAC), a key regulator of the signalling pathways involved in sperm motility and capacitation. We demonstrate by means of RT-PCR, immunodetection and immunofluorescence assays on patient's semen samples that the homozygous splicing mutation (c.2748 + 2 T > C) leads to in-frame exon skipping resulting in a deletion in the cyclic nucleotide-binding domain of the protein. Our work shows that in human, similar to mouse, SLC9C1 is required for sperm motility. Overall, we establish a homozygous truncating mutation in SLC9C1 as a novel cause of human asthenozoospermia and infertility.

Combined Use of Whole Exome Sequencing and CRISPR/Cas9 to Study the Etiology of Non-Obstructive Azoospermia: Demonstration of the Dispensable Role of the Testis-Specific Genes C1orf185 and CCT6B.

The genetic landscape of male infertility is highly complex. It is estimated that at least 4000 genes are involved in human spermatogenesis, but only few have so far been extensively studied. In this study, we investigated by whole exome sequencing two cases of idiopathic non-obstructive azoospermia (NOA) due to severe hypospermatogenesis. After variant filtering and prioritizing, we retained for each patient a homozygous loss-of-function (LoF) variant in a testis-specific gene, C1orf185 (c.250C>T; p.Gln84Ter) and CCT6B (c.615-2A>G), respectively. Both variants are rare according to the gnomAD database and absent from our local control cohort (n = 445). To verify the implication of these candidate genes in NOA, we used the CRISPR/Cas9 system to invalidate the mouse orthologs 4930522H14Rik and Cct6b and produced two knockout (KO) mouse lines. Sperm and testis parameters of homozygous KO adult male mice were analyzed and compared with those of wild-type animals. We showed that homozygous KO males were fertile and displayed normal sperm parameters and a functional spermatogenesis. Overall, these results demonstrate that not all genes highly and specifically expressed in the testes are essential for spermatogenesis, and in particular, we conclude that bi-allelic variants of C1orf185 and CCT6B are most likely not to be involved in NOA and male fertility.

Genetic analyses of a large cohort of infertile patients with globozoospermia, DPY19L2 still the main actor, GGN confirmed as a guest player.

Globozoospermia is a rare phenotype of primary male infertility inducing the production of round-headed spermatozoa without acrosome. Anomalies of DPY19L2 account for 50-70% of all cases and the entire deletion of the gene is by far the most frequent defect identified. Here, we present a large cohort of 69 patients with 20-100% of globozoospermia. Genetic analyses including multiplex ligation-dependent probe amplification, Sanger sequencing and whole-exome sequencing identified 25 subjects with a homozygous DPY19L2 deletion (36%) and 14 carrying other DPY19L2 defects (20%). Overall, 11 deleterious single-nucleotide variants were identified including eight novel and three already published mutations. Patients with a higher rate of round-headed spermatozoa were more often diagnosed and had a higher proportion of loss of function anomalies, highlighting a good genotype phenotype correlation. No gene defects were identified in patients carrying < 50% of globozoospermia while diagnosis efficiency rose to 77% for patients with > 50% of globozoospermia. In addition, results from whole-exome sequencing were scrutinized for 23 patients with a DPY19L2 negative diagnosis, searching for deleterious variants in the nine other genes described to be associated with globozoospermia in human (C2CD6, C7orf61, CCDC62, CCIN, DNAH17, GGN, PICK1, SPATA16, and ZPBP1). Only one homozygous novel truncating variant was identified in the GGN gene in one patient, confirming the association of GGN with globozoospermia. In view of these results, we propose a novel diagnostic strategy focusing on patients with at least 50% of globozoospermia and based on a classical qualitative PCR to detect DPY19L2 homozygous deletions. In the absence of the latter, we recommend to perform whole-exome sequencing to search for defects in DPY19L2 as well as in the other previously described candidate genes.

Defect in the nuclear pore membrane glycoprotein 210-like gene is associated with extreme uncondensed sperm nuclear chromatin and male infertility: a case report.

After the two meiotic divisions, haploid round spermatids undergo dramatic changes to become mature spermatozoa. One of the main transformations consists of compacting the cell nucleus to confer the sperm its remarkable hydrodynamic property and to protect its DNA from the oxidative stress it will encounter during its reproductive journey. Here, we studied an infertile subject with low sperm count, poor motility and highly abnormal spermatozoa with strikingly large heads due to highly uncondensed nuclear sperm DNA. Whole-exome sequencing was performed on the subject's DNA to identify the genetic defect responsible for this severe sperm anomaly. Bioinformatics analysis of exome sequence data uncovered a homozygous loss of function variant, ENST00000368559.7:c.718-1G>A, altering a consensus splice site expected to prevent the synthesis of the nucleoporin 210 like (NUP210L) protein. High-resolution mass spectrometry of sperm protein extracts did not reveal any NUP210L peptide sequence in the patient's sperm, contrary to what was observed in control donors, thus confirming the absence of NUP210L in the patient's sperm. Interestingly, homozygous Nup210l knock-out mice have been shown to be infertile due to a reduced sperm count, a high proportion of round-headed sperm, other head and flagella defects and a poor motility. NUP210L is almost exclusively expressed in the testis and sequence analogy suggests that it encodes a nuclear pore membrane glycoprotein. The protein might be crucial to regulate nuclear trafficking during and/or before spermiogenesis, its absence potentially impeding adequate nuclear compaction by preventing the entry of histone variants/transition proteins/protamines into the nucleus and/or by preventing the adequate replacement of core histones. This work describes a new gene necessary for male fertility, potentially improving the efficiency of the genetic diagnosis of male infertility. The function of NUP210L still remains to be resolved and its future investigation will help to understand the complex mechanisms necessary for sperm compaction.

Biallelic variants in MAATS1 encoding CFAP91, a calmodulin-associated and spoke-associated complex protein, cause severe astheno-teratozoospermia and male infertility.

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.