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.

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.

Evaluation of sperm DNA fragmentation and chromatin structure in infertile men with immotile short-tail sperm defect.

Teratozoospermia is characterised by the presence of spermatozoa with abnormal morphology. One of the morphological disorders that lead to male infertility is immotile short-tail sperm (ISTS) defect. In this study, we evaluated the levels of chromatin packing and DNA fragmentation in patients with immotile short-tail sperm defect. Semen samples were obtained from 31 infertile men with ISTS as case group and 31 normozoospermic men as a control group. Protamine status was evaluated using chromomycin A3 (CMA3) staining and sperm DNA fragmentation assessed by sperm chromatin structure assay (SCSA) and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate biotin nick-end labelling (TUNEL). The percentage of positive CMA3 spermatozoa was significantly higher in patients' samples (22.6 ± 6.9) compared with controls (16.3 ± 4.2) (p < .05) and also mean (±SD) of sperm DNA fragmentation was significantly higher in patients compared with controls, as measured by TUNEL assay (10.45 ± 4.60 vs. 7.03 ± 2.86, p < .05) and SCSA (24.80 ± 13.1 vs. 15.2 ± 7.2, p < .05). According to our study, sperm DNA fragmentation and chromatin packing abnormality are significantly higher in the ISTS samples compared with normal samples. A possible explanation for this relationship is that sperm chromatin condensation and sperm flagellum formation occur during the same phase of spermatogenesis.

Whole-exome sequencing of familial cases of multiple morphological abnormalities of the sperm flagella (MMAF) reveals new DNAH1 mutations.

STUDY QUESTION: Can whole-exome sequencing (WES) of patients with multiple morphological abnormalities of the sperm flagella (MMAF) identify causal mutations in new genes or mutations in the previously identified dynein axonemal heavy chain 1 (DNAH1) gene? SUMMARY ANSWER: WES for six families with men affected by MMAF syndrome allowed the identification of DNAH1 mutations in four affected men distributed in two out of the six families but no new candidate genes were identified. WHAT IS KNOWN ALREADY: Mutations in DNAH1, an axonemal inner dynein arm heavy chain gene, have been shown to be responsible for male infertility due to a characteristic form of asthenozoospermia called MMAF, defined by the presence in the ejaculate of spermatozoa with a mosaic of flagellar abnormalities including absent, coiled, bent, angulated, irregular and short flagella. STUDY DESIGN, SIZE, DURATION: This was a retrospective genetics study of patients presenting a MMAF phenotype. Patients were recruited in Iran and Italy between 2008 and 2015. PARTICIPANTS/MATERIALS, SETTING, METHODS: WES was performed for a total of 10 subjects. All identified variants were confirmed by Sanger sequencing. Two additional affected family members were analyzed by direct Sanger sequencing. To establish the prevalence of the DNAH1 mutation identified in an Iranian family, we carried out targeted sequencing on 38 additional MMAF patients of the same geographical origin. RT-PCR and immunochemistry were performed on sperm samples to assess the effect of the identified mutation on RNA and protein. MAIN RESULTS AND THE ROLE OF CHANCE: WES in six families identified a causal mutations in two families. Two additional affected family members were confirmed to hold the same homozygous mutation as their sibling. In total, DNAH1 mutations were identified in 5 out of 12 analyzed subjects (41.7%). If we only include index cases, we detected two mutated subjects out of six (33%) tested MMAF individuals. Furthermore we sequenced one DNAH1 exon found to be mutated (c.8626-1G > A) in an Iranian family in an additional 38 MMAF patients from Iran. One of these patients carried the variant confirming that this variant is relatively frequent in the Iranian population. The effect of the c.8626-1G > A variant was confirmed by RT-PCR and immunochemistry as no RNA or protein could be observed in sperm from the affected men. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: WES allows the amplification of 80-90% of all coding exons. It is possible that some DNAH1 exons may not have been sequenced and that we may have missed some additional mutations. Also, WES cannot identify deep intronic mutations and it is not efficient for detection of large genomic events (deletions, insertions, inversions). We did not identify any causal mutations in DNAH1 or in other candidate genes in four out of the six tested families. This indicates that the technique and/or the analysis of our data can be improved to increase the diagnosis efficiency. WIDER IMPLICATIONS OF THE FINDINGS: Our findings confirm that DNAH1 is one of the main genes involved in MMAF syndrome. It is a large gene with 78 exons making it challenging and expensive to sequence using the traditional Sanger sequencing methods. We show that WES sequencing is good alternative to Sanger sequencing to reach a genetic diagnosis in patients with severe male infertility phenotypes. STUDY FUNDING/COMPETING INTERESTS: This work was supported by following grants: the 'MAS-Flagella' project financed by the French ANR and the DGOS for the program PRTS 2014 and the 'Whole genome sequencing of patients with Flagellar Growth Defects (FGD)' project financed by the Fondation Maladies Rares for the program Séquençage à haut débit 2012. The authors have no conflict of interest.