Bi-allelic Mutations in ARMC2 Lead to Severe Astheno-Teratozoospermia Due to Sperm Flagellum Malformations in Humans and Mice.

Male infertility is a major health concern. Among its different causes, multiple morphological abnormalities of the flagella (MMAF) induces asthenozoospermia and is one of the most severe forms of qualitative sperm defects. Sperm of affected men display short, coiled, absent, and/or irregular flagella. To date, six genes (DNAH1, CFAP43, CFAP44, CFAP69, FSIP2, and WDR66) have been found to be recurrently associated with MMAF, but more than half of the cases analyzed remain unresolved, suggesting that many yet-uncharacterized gene defects account for this phenotype. Here, whole-exome sequencing (WES) was performed on 168 infertile men who had a typical MMAF phenotype. Five unrelated affected individuals carried a homozygous deleterious mutation in ARMC2, a gene not previously linked to the MMAF phenotype. Using the CRISPR-Cas9 technique, we generated homozygous Armc2 mutant mice, which also presented an MMAF phenotype, thus confirming the involvement of ARMC2 in human MMAF. Immunostaining experiments in AMRC2-mutated individuals and mutant mice evidenced the absence of the axonemal central pair complex (CPC) proteins SPAG6 and SPEF2, whereas the other tested axonemal and peri-axonemal components were present, suggesting that ARMC2 is involved in CPC assembly and/or stability. Overall, we showed that bi-allelic mutations in ARMC2 cause male infertility in humans and mice by inducing a typical MMAF phenotype, indicating that this gene is necessary for sperm flagellum structure and assembly.

Mutations in TTC29, Encoding an Evolutionarily Conserved Axonemal Protein, Result in Asthenozoospermia and Male Infertility.

In humans, structural or functional defects of the sperm flagellum induce asthenozoospermia, which accounts for the main sperm defect encountered in infertile men. Herein we focused on morphological abnormalities of the sperm flagellum (MMAF), a phenotype also termed "short tails," which constitutes one of the most severe sperm morphological defects resulting in asthenozoospermia. In previous work based on whole-exome sequencing of a cohort of 167 MMAF-affected individuals, we identified bi-allelic loss-of-function mutations in more than 30% of the tested subjects. In this study, we further analyzed this cohort and identified five individuals with homozygous truncating variants in TTC29, a gene preferentially and highly expressed in the testis, and encoding a tetratricopeptide repeat-containing protein related to the intraflagellar transport (IFT). One individual carried a frameshift variant, another one carried a homozygous stop-gain variant, and three carried the same splicing variant affecting a consensus donor site. The deleterious effect of this last variant was confirmed on the corresponding transcript and protein product. In addition, we produced and analyzed TTC29 loss-of-function models in the flagellated protist T. brucei and in M. musculus. Both models confirmed the importance of TTC29 for flagellar beating. We showed that in T. brucei the TPR structural motifs, highly conserved between the studied orthologs, are critical for TTC29 axonemal localization and flagellar beating. Overall our work demonstrates that TTC29 is a conserved axonemal protein required for flagellar structure and beating and that TTC29 mutations are a cause of male sterility due to MMAF.

A Homozygous Ancestral SVA-Insertion-Mediated Deletion in WDR66 Induces Multiple Morphological Abnormalities of the Sperm Flagellum and Male Infertility.

Multiple morphological abnormalities of the sperm flagellum (MMAF) is a severe form of male infertility defined by the presence of a mosaic of anomalies, including short, bent, curled, thick, or absent flagella, resulting from a severe disorganization of the axoneme and of the peri-axonemal structures. Mutations in DNAH1, CFAP43, and CFAP44, three genes encoding axoneme-related proteins, have been described to account for approximately 30% of the MMAF cases reported so far. Here, we searched for pathological copy-number variants in whole-exome sequencing data from a cohort of 78 MMAF-affected subjects to identify additional genes associated with MMAF. In 7 of 78 affected individuals, we identified a homozygous deletion that removes the two penultimate exons of WDR66 (also named CFAP251), a gene coding for an axonemal protein preferentially localized in the testis and described to localize to the calmodulin- and spoke-associated complex at the base of radial spoke 3. Sequence analysis of the breakpoint region revealed in all deleted subjects the presence of a single chimeric SVA (SINE-VNTR-Alu) at the breakpoint site, suggesting that the initial deletion event was potentially mediated by an SVA insertion-recombination mechanism. Study of Trypanosoma WDR66's ortholog (TbWDR66) highlighted high sequence and structural analogy with the human protein and confirmed axonemal localization of the protein. Reproduction of the human deletion in TbWDR66 impaired flagellar movement, thus confirming WDR66 as a gene associated with the MMAF phenotype and highlighting the importance of the WDR66 C-terminal region.

Patients with multiple morphological abnormalities of the sperm flagella due to DNAH1 mutations have a good prognosis following intracytoplasmic sperm injection.

STUDY QUESTION: Does DNAH1 status influence intracytoplasmic sperm injection (ICSI) outcomes for patients with multiple morphological abnormalities of the sperm flagella (MMAF)? SUMMARY ANSWER: Despite a highly abnormal morphology, sperm from MMAF patients with DNAH1 mutations have a low aneuploidy rate and good nuclear quality, leading to good embryonic development following ICSI and a high pregnancy rate. WHAT IS KNOWN ALREADY: Teratozoospermia represents a heterogeneous group including a wide range of phenotypes. Among all these qualitative defects, a flagellar phenotype called MMAF is characterized by a mosaic of morphological abnormalities of the flagellum, including coiled, bent, irregular, short or/and absent flagella, mainly due to the absence of the axonemal central pair microtubules. We previously demonstrated that homozygous mutations in the DNAH1 gene, encoding an inner arm heavy chain dynein, are frequently found in patients with MMAF (28% of the patients from the initial cohort). Numerous studies have reported an increased rate of aneuploidy and a poor sperm nuclear quality related to sperm flagellar abnormalities, which could impede ICSI outcome. Moreover, success rates after ICSI may be influenced by the type of ultrastructural flagellar defects and/or by the gene defects carried by the patients. STUDY DESIGN, SIZE, DURATION: This retrospective cohort study included 6 infertile males with MMAF due to deleterious homozygous DNAH1 mutations and their respective spouses, who underwent 9 ISCI cycles, with 16 embryos being transferred. ICSI results were compared with two control populations of 13 MMAF men without DNAH1 mutations and an aged-matched control group of 1431 non-MMAF couples. All ICSI attempts took place between 2000 and 2012. PARTICIPANTS/MATERIALS, SETTING, METHODS: Clinical and biological data were collected from patients treated for infertility at the CPSR les Jasmins in Tunis (Tunisia). We compared the ICSI outcomes obtained with couples including DNAH1 mutated and nonmutated patients and non-MMAF couples. For the analysis of the chromosomal status, fluorescence in situ hybridization (FISH) analyses were performed on sperm cells from 3 DNAH1-mutated patients and from 29 fertile control subjects. Sperm chromatin condensation and DNA fragmentation were evaluated using aniline blue staining and TUNEL assays, respectively, on sperm cells from 3 DNAH1-mutated men and 6 fertile controls. MAIN RESULTS AND THE ROLE OF CHANCE: There was a significantly increased proportion of disomy XY and 18 in sperm from DNAH1 mutated patients compared with fertile controls (1.52 versus 0.28%, P = 0.0001 and 0.64 versus 0.09%, P = 0.0001). However, there were no statistically significant differences among sperm from the two groups in their frequencies of either 13, 21, XX or YY disomy or diploidy. Measures of DNA compaction and fragmentation demonstrated a good nuclear sperm quality among DNAH1 mutated men. The overall fertilization, pregnancy and delivery rates of couples including DNAH1 mutated men were of 70.8, 50.0 and 37.5%, respectively. There were no statistically significant differences in any of these parameters compared with the two control groups (P > 0.05). LIMITATIONS, REASONS FOR CAUTION: A limitation of this study is the small number of DNAH1-mutated patients available and the low number of genes identified in MMAF. Further genetic studies are warranted to identify other MMAF-inducing genes to better characterize the genetic etiology of the MMAF phenotype and to improve the management of patients diagnosed with flagellar defects. WIDER IMPLICATIONS OF THE FINDINGS: MMAF patients with DNAH1 mutations have low aneuploidy rates and good nuclear sperm quality, explaining the high pregnancy rate obtained with these patients. Good ICSI results were obtained for both MMAF groups (DNAH1 mutated and nonmutated), suggesting that patients presenting with asthenozoospermia due to flagellar defects have a good ICSI prognosis irrespective of their genotype. The majority of MMAF cases currently remain idiopathic with no genetic cause yet identified. In depth genetic analysis of these patients using next generation sequencing should reveal new causal genes. Subsequent genotype phenotype analyses could improve advice and care provided to MMAF patients. STUDY FUNDING/COMPETING INTERESTS: None of the authors have any competing interest. This work is part of the project 'Identification and Characterization of Genes Involved in Infertility (ICG2I)', funded by the program GENOPAT 2009 from the French Research Agency (ANR) and the MAS-Flagella project, financed by the French ANR and the Direction Générale de l'Offre de Soins (DGOS).

Step-by-step loupes-mTESE in non-obstructive azoospermic men, a retrospective study.

BACKGROUND: Men with non-obstructive azoospermia (NOA) may have sperm in their testes and a procedure of sperm retrieval and assisted reproduction is required in them to allow fertility. Standard procedures such as fine needle aspiration (FNA) and conventional testicular sperm extraction (cTESE) harvest random samples with a sperm retrieval rate (SRR) of 45%. Microdissection testicular sperm extraction (mTESE) is nowadays considered to be the most accurate technique to retrieve sperm in men with NOA. This procedure can identify dilated tubules that are more likely to contain viable sperm with a SRR of 60%. RESULTS: In our center, testicular biopsy was conducted in a standard fashion in 321 patients with NOA until March 2003. From then to December 2017, due to the lack of an operating microscope, we used 6 fold magnifying loupes to perform a step-by-step macro- mTESE in 1050 patients. Sperm was found in the first testis in 61% of the cases, leading to stop the procedure with less testicular damage. We increased our SRR from 43 to 51.8% in an acceptable operating time of 75mn for both sides. CONCLUSIONS: In institutions where surgeons cannot afford an operating microscope, this modified mTESE technique using × 6 magnifying loupes is reliable, especially in patients with low testicular volumes and high FSH, in whom dilated tubules can be easily identified from the surrounding tissue.

CONTEXTE: Les patients ayant une azoospermie non obstructive confirmée peuvent néanmoins présenter des spermatozoïdes intratesticulaires nécessitant un prélèvement chirurgical en vue d'une injection intra cytoplasmique d'un spermatozoïde (ICSI). L'aspiration à l'aiguille ainsi que la biopsie classique à ciel ouvert ne permettent qu'un prélèvement aléatoire à l'aveugle assorti d'un taux de positivité de 45%. La biopsie avec microdissection sous microscope est. désormais considérée comme le « gold standard ¼ et permet d'identifier les foyers de tubes séminifères dilatés qui sont le plus à même de contenir des spermatozoïdes mobiles. RÉSULTATS: Dans notre centre d'Assistance Médicale à la Procréation (AMP), jusqu'en février 2003, le recueil de spermatozoïdes pour ICSI a été réalisé par une biopsie classique chez 321 patients avec une positivité de 43%. De mars 2003 à décembre 2017, du fait de l'absence de microscope opératoire, nous avons adapté le prélèvement microchirurgical à des loupes de fort grossissement (× 6) et pratiqué cette technique simplifiée chez 1050 patients. Les fragments sont examinés en extemporané par les embryologistes et chez 61% des patients, la positivité de la biopsie dans le premier testicule prélevé permet de sursoir à l'exploration du côté controlatéral, évitant ainsi une dissection inutile et potentiellement délétère. Grâce à cette modification, nous sommes passés de 43% à 51,8% de positivité avec un temps opératoire moyen de 75mn pour les 2 côtés. CONCLUSION: Dans les centres d'AMP où l'on ne dispose pas de microscope opératoire ou lorsque le programme ne permet pas d'allouer une longue durée opératoire à la biopsie testiculaire sans compromettre le reste de l'activité chirurgicale, l'utilisation de loupes à fort grossissement (× 6) permet l'amélioration des résultats de la biopsie, particulièrement chez les patients présentant un petit volume testiculaire et une FSH élevée.

PATL2 is a key actor of oocyte maturation whose invalidation causes infertility in women and mice.

The genetic causes of oocyte meiotic deficiency (OMD), a form of primary infertility characterised by the production of immature oocytes, remain largely unexplored. Using whole exome sequencing, we found that 26% of a cohort of 23 subjects with OMD harboured the same homozygous nonsense pathogenic mutation in PATL2, a gene encoding a putative RNA-binding protein. Using Patl2 knockout mice, we confirmed that PATL2 deficiency disturbs oocyte maturation, since oocytes and zygotes exhibit morphological and developmental defects, respectively. PATL2's amphibian orthologue is involved in the regulation of oocyte mRNA as a partner of CPEB However, Patl2's expression profile throughout oocyte development in mice, alongside colocalisation experiments with Cpeb1, Msy2 and Ddx6 (three oocyte RNA regulators) suggest an original role for Patl2 in mammals. Accordingly, transcriptomic analysis of oocytes from WT and Patl2-/- animals demonstrated that in the absence of Patl2, expression levels of a select number of highly relevant genes involved in oocyte maturation and early embryonic development are deregulated. In conclusion, PATL2 is a novel actor of mammalian oocyte maturation whose invalidation causes OMD in humans.

Mutations in CFAP43 and CFAP44 cause male infertility and flagellum defects in Trypanosoma and human.

Spermatogenesis defects concern millions of men worldwide, yet the vast majority remains undiagnosed. Here we report men with primary infertility due to multiple morphological abnormalities of the sperm flagella with severe disorganization of the sperm axoneme, a microtubule-based structure highly conserved throughout evolution. Whole-exome sequencing was performed on 78 patients allowing the identification of 22 men with bi-allelic mutations in DNAH1 (n = 6), CFAP43 (n = 10), and CFAP44 (n = 6). CRISPR/Cas9 created homozygous CFAP43/44 male mice that were infertile and presented severe flagellar defects confirming the human genetic results. Immunoelectron and stimulated-emission-depletion microscopy performed on CFAP43 and CFAP44 orthologs in Trypanosoma brucei evidenced that both proteins are located between the doublet microtubules 5 and 6 and the paraflagellar rod. Overall, we demonstrate that CFAP43 and CFAP44 have a similar structure with a unique axonemal localization and are necessary to produce functional flagella in species ranging from Trypanosoma to human.