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.

Sperm Meiotic Segregation Analysis of Reciprocal Translocations Carriers: We Have Bigger FISH to Fry.

Reciprocal translocation (RT) carriers produce a proportion of unbalanced gametes that expose them to a higher risk of infertility, recurrent miscarriage, and fetus or children with congenital anomalies and developmental delay. To reduce these risks, RT carriers can benefit from prenatal diagnosis (PND) or preimplantation genetic diagnosis (PGD). Sperm fluorescence in situ hybridization (spermFISH) has been used for decades to investigate the sperm meiotic segregation of RT carriers, but a recent report indicates a very low correlation between spermFISH and PGD outcomes, raising the question of the usefulness of spermFISH for these patients. To address this point, we report here the meiotic segregation of 41 RT carriers, the largest cohort reported to date, and conduct a review of the literature to investigate global segregation rates and look for factors that may or may not influence them. We confirm that the involvement of acrocentric chromosomes in the translocation leads to more unbalanced gamete proportions, in contrast to sperm parameters or patient age. In view of the dispersion of balanced sperm rates, we conclude that routine implementation of spermFISH is not beneficial for RT carriers.

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.

Treatment of Mouse Sperm with a Non-Catalytic Mutant of PLA2G10 Reveals That PLA2G10 Improves In Vitro Fertilization through Both Its Enzymatic Activity and as Ligand of PLA2R1.

The group X secreted phospholipase A2 (PLA2G10) is present at high levels in mouse sperm acrosome. The enzyme is secreted during capacitation and amplifies the acrosome reaction and its own secretion via an autocrine loop. PLA2G10 also improves the rate of fertilization. In in vitro fertilization (IVF) experiments, sperm from Pla2g10-deficient mice produces fewer two-cell embryos, and the absence of PLA2G10 is rescued by adding recombinant enzymes. Moreover, wild-type (WT) sperm treated with recombinant PLA2G10 produces more two-cell embryos. The effects of PLA2G10 on mouse fertility are inhibited by sPLA2 inhibitors and rescued by products of the enzymatic reaction such as free fatty acids, suggesting a role of catalytic activity. However, PLA2G10 also binds to mouse PLA2R1, which may play a role in fertility. To determine the relative contribution of enzymatic activity and PLA2R1 binding in the profertility effect of PLA2G10, we tested H48Q-PLA2G10, a catalytically-inactive mutant of PLA2G10 with low enzymatic activity but high binding properties to PLA2R1. Its effect was tested in various mouse strains, including Pla2r1-deficient mice. H48Q-PLA2G10 did not trigger the acrosome reaction but was as potent as WT-PLA2G10 to improve IVF in inbred C57Bl/6 mice; however, this was not the case in OF1 outbred mice. Using gametes from these mouse strains, the effect of H48Q-PLA2G10 appeared dependent on both spermatozoa and oocytes. Moreover, sperm from C57Bl/6 Pla2r1-deficient mice were less fertile and lowered the profertility effects of H48Q-PLA2G10, which were completely suppressed when sperm and oocytes were collected from Pla2r1-deficient mice. Conversely, the effect of WT-PLA2G10 was not or less sensitive to the absence of PLA2R1, suggesting that the effect of PLA2G10 is polymodal and complex, acting both as an enzyme and a ligand of PLA2R1. This study shows that the action of PLA2G10 on gametes is complex and can simultaneously activate the catalytic pathway and the PLA2R1-dependent receptor pathway. This work also shows for the first time that PLA2G10 binding to gametes' PLA2R1 participates in fertilization optimization.

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.

[AFU and SALF recommendations for the evaluation of male infertility]. / Recommandations de l'AFU et de la SALF concernant l'évaluation de l'homme infertile.

BACKGROUND: The aim of these Association Française d'Urologie (AFU) and Société d'Andrologie de Langue Française (SALF) common recommendations are to provide practice guidelines for the French Urological and Andrological community regarding the evaluation of infertile men. MATERIAL AND METHODS: Literature search in PubMed using the keywords "male infertility", "diagnosis", "management" and "evaluation" limited to clinical articles in English and French prior to 1/01/2020. To inform the level of evidence, the HAS grading system (2013) was applied. RESULTS: Concerning the evaluation of infertile men, the AFU and the SALF recommend : (1) a systematic interview exploring the family history, the fertility history of the man outside the couple, the patient's personal history that may have an impact on his fertility, lifestyle habits, treatments, symptoms and possible sexual difficulties of the couple; (2) a general physical examination to assess signs of hypogonadism and secondary sexual characters; (3) a scrotal physical examination performed by an urologist or andrologist to assess (i) the testes for volume and consistency, (ii) vas deferens and epididymes for total or partial absence or nodules, and (iii) presence of varicoceles; (4) Performing two semen analyses, according to World Health Organization guidelines, if the first one has at least one abnormaly; (5) a scrotal ultrasound as part of routine investigation, that can be completed with an endorectal pelvic ultrasound according to the clinic; (6) an endocrine evaluation with at least a Testosterone and FSH serum determination; (7) Karyotype analysis in infertile men with a sperm concentration ≤10 106/mL; (8) assessment of Yq microdeletions in infertile men with a sperm concentration ≤1 106/mL; (9) Cystic fibrosis transmembrane conductance regulator gene evaluation in case of suspicion for bilateral or unilateral congenital agenesis of vas deferens and seminal vesicles. The interest of tests analyzing DNA fragmentation (TUNEL, SCSA) is still under investigation. CONCLUSION: These guidelines can be applied in routine clinical practice in all infertile men.

Prokineticin 1 is a new biomarker of human oocyte competence: expression and hormonal regulation throughout late folliculogenesis.

CONTEXT: Prokineticin 1 (PROK1) quantification in global follicular fluid (FF) has been recently reported as a predictive biomarker of in vitro fertilization (IVF) outcome. It is now necessary to evaluate its clinical usefulness in individual follicles. OBJECTIVES: To evaluate the clinical value of PROK1 secretion in individual FF to predict oocyte competence. To determine the impact of follicular size, oocyte maturity, and gonadotropin treatments on PROK1 secretion. DESIGN AND SETTING: Prospective cohort study from May 2015 to May 2017 at the University Hospital of Grenoble. PATIENTS: A total of 69 infertile couples underwent IVF. INTERVENTION(S): Collection of 298 individual FF from 44 women undergoing IVF; 52 individual cumulus cell (CC) samples and 15 CC primary cultures from 25 women undergoing IVF-intracytoplasmic sperm injection (ICSI). MAIN OUTCOME MEASURE(S): Oocyte competence was defined as the ability to sustain embryo development to the blastocyst stage. Follicular size was measured by 2D-sonography. PROK1 concentration was quantified by ELISA assay. RESULTS: PROK1 concentration was correlated to follicular size (r = 0.85, P = 2.2 × 10-16). Normalized PROK1 concentration in FF was predictive of subsequent oocyte competence (AUROC curve = 0.76 [95% CI, 0.69-0.83]; P = 1.7 × 10-9), irrespectively of day-2 embryo morphokinetic parameters. The expression and secretion of PROK1 were increased in FF and CC of mature oocytes (P < 0.01). Follicle Stimulating Hormone and hCG up-regulated PROK1 secretion in CC primary cultures (P < 0.01; P < 0.05), probably through the cAMP pathway (P < 0.01). CONCLUSIONS: PROK1 quantification in individual FF could constitute a new predictive biomarker of oocyte competence in addition with embryo morphokinetic parameters. TRIAL REGISTRATION NUMBER: none.

Progesterone-induced Acrosome Exocytosis Requires Sequential Involvement of Calcium-independent Phospholipase A2ß (iPLA2ß) and Group X Secreted Phospholipase A2 (sPLA2).

Phospholipase A2 (PLA2) activity has been shown to be involved in the sperm acrosome reaction (AR), but the molecular identity of PLA2 isoforms has remained elusive. Here, we have tested the role of two intracellular (iPLA2ß and cytosolic PLA2α) and one secreted (group X) PLA2s in spontaneous and progesterone (P4)-induced AR by using a set of specific inhibitors and knock-out mice. iPLA2ß is critical for spontaneous AR, whereas both iPLA2ß and group X secreted PLA2 are involved in P4-induced AR. Cytosolic PLA2α is dispensable in both types of AR. P4-induced AR spreads over 30 min in the mouse, and kinetic analyses suggest the presence of different sperm subpopulations, using distinct PLA2 pathways to achieve AR. At low P4 concentration (2 µm), sperm undergoing early AR (0-5 min post-P4) rely on iPLA2ß, whereas sperm undergoing late AR (20-30 min post-P4) rely on group X secreted PLA2. Moreover, the role of PLA2s in AR depends on P4 concentration, with the PLA2s being key actors at low physiological P4 concentrations (≤2 µm) but not at higher P4 concentrations (~10 µm).

Mutations in DNAH1, which encodes an inner arm heavy chain dynein, lead to male infertility from multiple morphological abnormalities of the sperm flagella.

Ten to fifteen percent of couples are confronted with infertility and a male factor is involved in approximately half the cases. A genetic etiology is likely in most cases yet only few genes have been formally correlated with male infertility. Homozygosity mapping was carried out on a cohort of 20 North African individuals, including 18 index cases, presenting with primary infertility resulting from impaired sperm motility caused by a mosaic of multiple morphological abnormalities of the flagella (MMAF) including absent, short, coiled, bent, and irregular flagella. Five unrelated subjects out of 18 (28%) carried a homozygous variant in DNAH1, which encodes an inner dynein heavy chain and is expressed in testis. RT-PCR, immunostaining, and electronic microscopy were carried out on samples from one of the subjects with a mutation located on a donor splice site. Neither the transcript nor the protein was observed in this individual, confirming the pathogenicity of this variant. A general axonemal disorganization including mislocalization of the microtubule doublets and loss of the inner dynein arms was observed. Although DNAH1 is also expressed in other ciliated cells, infertility was the only symptom of primary ciliary dyskinesia observed in affected subjects, suggesting that DNAH1 function in cilium is not as critical as in sperm flagellum.

Spermaurin, an La1-like peptide from the venom of the scorpion Scorpio maurus palmatus, improves sperm motility and fertilization in different mammalian species.

STUDY QUESTION: Is it possible to identify original compounds that are able to enhance sperm motility from the venom of the scorpion Scorpio maurus palmatus? SUMMARY ANSWER: We identified a potent disulfide-rich peptide (DRP) of 73 amino acids that significantly improved the motility of fresh and frozen-thawed sperm in different mammalian species, including human, and improved fertilization outcome in mouse IVF experiments. WHAT IS KNOWN ALREADY: Any disturbance of sperm motility has a strong impact on fertilization and can lead to subfertility or infertility. Significant efforts have, therefore,  been made to identify pharmacological drugs that might improve sperm motility. Such compounds are particularly useful in azoospermia to improve testicular sperm extraction and in the domain of cryopreservation because the motility of frozen-thawed sperm is reduced. STUDY DESIGN, SIZE, DURATION: This was a basic science/medical research study aimed at identifying original compounds from a library of venoms able to enhance mammalian sperm motility, including human. We first identified in the venom of a scorpion S. m. palmatus a fraction able to potently activate sperm motility. We next purified and characterized the compound by liquid chromatography, mass spectrometry and peptide synthesis. Finally, the potency and toxicity of both purified and synthetic versions of the identified compound on sperm motility were assessed using different in vitro tests in different mammalian species. PARTICIPANTS/MATERIALS, SETTING, METHODS: For human sperm, biological samples were collected from normozoospermic donors and subfertile patients attending a reproduction department for diagnostic semen analysis. Testicular sperm was collected from cynomolgus monkeys (Macaca fascicularis) euthanized for the needs of specific authorized research projects. The peptide was also tested on bovine and mouse epidydimal sperm. We measured different sperm motility parameters with a computer-assisted sperm analysis system in the presence or absence of the peptide. MAIN RESULTS AND THE ROLE OF CHANCE: Size exclusion chromatography enabled us to isolate a fraction of the venom of S. m. palmatus able to increase sperm motility. By liquid chromatography and mass spectrometry, a peptide comprising 73 amino acids with 4 disulfide bridges was identified as responsible for the biological activity and called 'spermaurin'. The identity of spermaurin was confirmed by chemical synthesis. We showed that the peptide increased the motility of fresh and frozen-thawed human sperm. We observed that the potency of the peptide was higher on fresh ejaculated spermatozoa with a low motility, achieving a 100% increase of curvilinear velocity in poorly performing sperm. We also demonstrated that peptide is effective on bovine and mouse fresh epididymal, bovine frozen-thawed ejaculated and fresh non-human primate testicular sperm. Finally, in mouse IVF, the production of 2-cell embryos was increased by 24% when sperm were treated with the peptide. LIMITATIONS, REASONS FOR CAUTION: This work is an in vitro evaluation of the ability of spermaurin to improve sperm motility parameters. Another limitation of this study is the small number of human sperm samples tested with the natural (n = 36) and synthetic (n = 12) peptides. Moreover, the effect of the peptide on IVF outcome was only tested in mouse and further tests with human and bovine gametes are required to confirm and extend this result in other mammalian species. WIDER IMPLICATIONS OF THE FINDINGS: This work confirms our initial study showing that venoms represent an interesting source of molecules that are able to modify sperm physiology. Moreover, this work presents the first demonstrated biological action of a venom peptide from the scorpion S. m. palmatus with sequence similarities to La1 peptide from Liocheles australasiae (Wood scorpion), a widespread family of DRPs. LARGE SCALE DATA: Not applicable. STUDY FUNDING/COMPETING INTEREST(S): This work is part of the project 'LAB COM-14 LAB7 0004 01-LIPAV', funded by the program LabCom 2014 from the French Research Agency (ANR). Dr Arnoult reports grants from IMV Technologies during the conduct of the study. In addition, Drs Arnoult, Martinez, Ray and Schmitt have a patent EP16305642.7 pending containing some of the information presented in this manuscript.

Abnormal retention of nuclear lamina and disorganization of chromatin-related proteins in spermatozoa from DPY19L2-deleted globozoospermic patients.

The aim of this study was to characterize the nuclear lamina (NL) and lamin chromatin-partners in spermatozoa from four DPY19L2-deleted globozoospermic patients. We tested for spermatid transcripts encoding lamins and their chromatin-partners emerin, LAP2α, BAF and BAF-L, by reverse transcriptase-PCR using spermatozoa RNA. We also determined the localization of lamin B1, BAF and BAF-L by immunofluorescent analysis of spermatozoa from all patients. In RNA from globozoospermic and control spermatozoa we detected transcripts encoding lamin B1, lamin B3, emerin, LAP2α and BAF-L, but not A-type lamins. In contrast, BAF transcripts were detected in globozoospermic but not control spermatozoa. The NL was immature in human globozoospermic spermatozoa: lamin B1 signal was detected in the nuclei of globozoospermic spermatozoa in significantly higher proportions than the control (P < 0.05; 56-91% versus 40%) and was predominantly observed at the whole nuclear periphery, not polarized as in control spermatozoa. Conversely, BAF and BAF-L were detected in control, but not globozoospermic spermatozoa. Our results strongly emphasize the importance of the NL and associated proteins during human spermiogenesis. In globozoospermia, the lack of maturation of the NL, and the modifications in expression and location of chromatin-partners, could explain the chromatin defects observed in this rare phenotype.

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).

Homozygous mutation of AURKC yields large-headed polyploid spermatozoa and causes male infertility.

The World Health Organization conservatively estimates that 80 million people suffer from infertility worldwide. Male factors are believed to be responsible for 20-50% of all infertility cases, but microdeletions of the Y chromosome are the only genetic defects altering human spermatogenesis that have been reported repeatedly. We focused our work on infertile men with a normal somatic karyotype but typical spermatozoa mainly characterized by large heads, a variable number of tails and an increased chromosomal content (OMIM 243060). We performed a genome-wide microsatellite scan on ten infertile men presenting this characteristic phenotype. In all of these men, we identified a common region of homozygosity harboring the aurora kinase C gene (AURKC) with a single nucleotide deletion in the AURKC coding sequence. In addition, we show that this founder mutation results in premature termination of translation, yielding a truncated protein that lacks the kinase domain. We conclude that the absence of AURKC causes male infertility owing to the production of large-headed multiflagellar polyploid spermatozoa.

Absence of Dpy19l2, a new inner nuclear membrane protein, causes globozoospermia in mice by preventing the anchoring of the acrosome to the nucleus.

Sperm-head elongation and acrosome formation, which take place during the last stages of spermatogenesis, are essential to produce competent spermatozoa that are able to cross the oocyte zona pellucida and to achieve fertilization. During acrosome biogenesis, acrosome attachment and spreading over the nucleus are still poorly understood and to date no proteins have been described to link the acrosome to the nucleus. We recently demonstrated that a deletion of DPY19L2, a gene coding for an uncharacterized protein, was responsible for a majority of cases of type I globozoospermia, a rare cause of male infertility that is characterized by the exclusive production of round-headed acrosomeless spermatozoa. Here, using Dpy19l2 knockout mice, we describe the cellular function of the Dpy19l2 protein. We demonstrate that the protein is expressed predominantly in spermatids with a very specific localization restricted to the inner nuclear membrane facing the acrosomal vesicle. We show that the absence of Dpy19l2 leads to the destabilization of both the nuclear dense lamina (NDL) and the junction between the acroplaxome and the nuclear envelope. Consequently, the acrosome and the manchette fail to be linked to the nucleus leading to the disruption of vesicular trafficking, failure of sperm nuclear shaping and eventually to the elimination of the unbound acrosomal vesicle. Finally, we show for the first time that Dpy19l3 proteins are also located in the inner nuclear envelope, therefore implying that the Dpy19 proteins constitute a new family of structural transmembrane proteins of the nuclear envelope.

Dpy19l2-deficient globozoospermic sperm display altered genome packaging and DNA damage that compromises the initiation of embryo development.

We recently identified the DPY19L2 gene as the main genetic cause of human globozoospermia. Non-genetically characterized cases of globozoospermia were associated with DNA alterations, suggesting that DPY19L2-dependent globozoospermia may be associated with poor DNA quality. However the origins of such defects have not yet been characterized and the consequences on the quality of embryos generated with globozoospermic sperm remain to be determined. Using the mouse model lacking Dpy19l2, we compared several key steps of nuclear compaction. We show that the kinetics of appearance and disappearance of the histone H4 acetylation waves and of transition proteins are defective. More importantly, the nuclear invasion by protamines does not occur. As a consequence, we showed that globozoospermic sperm presented with poor sperm chromatin compaction and sperm DNA integrity breakdown. We next assessed the developmental consequences of using such faulty sperm by performing ICSI. We showed in the companion article that oocyte activation (OA) with globozoospermic sperm is very poor and due to the absence of phospholipase Cζ; therefore artificial OA (AOA) was used to bypass defective OA. Herein, we evaluated the developmental potential of embryos generated by ICSI + AOA in mice. We demonstrate that although OA was fully rescued, preimplantation development was impaired when using globozoospermic sperm. In human, a small number of embryos could be generated with sperm from DPY19L2-deleted patients in the absence of AOA and these embryos also showed a poor developmental potential. In conclusion, we show that chromatin compaction during spermiogenesis in Dpy19l2 KO mouse is defective and leads to sperm DNA damage. Most of the DNA breaks were already present when the sperm reached the epididymis, indicating that they occurred inside the testis. This result thus suggests that testicular sperm extraction in Dpy19l2-dependent globozoospermia is not recommended. These defects may largely explain the poor embryonic development of most mouse and human embryos obtained with globozoospermic sperm.

Subcellular localization of phospholipase Cζ in human sperm and its absence in DPY19L2-deficient sperm are consistent with its role in oocyte activation.

We recently identified the DPY19L2 gene as the main genetic cause of human globozoospermia (70%) and described that Dpy19l2 knockout (KO) mice faithfully reproduce the human phenotype of globozoospermia making it an excellent model to characterize the molecular physiopathology of globozoospermia. Recent case studies on non-genetically characterized men with globozoospermia showed that phospholipase C, zeta (PLCζ), the sperm factor thought to induce the Ca(2+) oscillations at fertilization, was absent from their sperm, explaining the poor fertilization potential of these spermatozoa. Since 30% of globozoospermic men remain genetically uncharacterized, the absence of PLCζ in DPY19L2 globozoospermic men remains to be formally established. Moreover, the precise localization of PLCζ and the reasons underlying its loss during spermatogenesis in globozoospermic patients are still not understood. Herein, we show that PLCζ is absent, or its presence highly reduced, in human and mouse sperm with DPY19L2-associated globozoospermia. As a consequence, fertilization with sperm from Dpy19l2 KO mice failed to initiate Ca(2+) oscillations and injected oocytes remained arrested at the metaphase II stage, although a few human oocytes injected with DPY19L2-defective sperm showed formation of 2-pronuclei embryos. We report for the first time the subcellular localization of PLCζ in control human sperm, which is along the inner acrosomal membrane and in the perinuclear theca, in the area corresponding to the equatorial region. Because these cellular components are absent in globozoospermic sperm, the loss of PLCζ in globozoospermic sperm is thus consistent and reinforces the role of PLCζ as an oocyte activation factor necessary for oocyte activation. In our companion article, we showed that chromatin compaction during spermiogenesis in Dpy19l2 KO mouse is defective and leads to sperm DNA damage. Together, these defects explain the poor fertilization potential of DPY19L2-globozoospermic sperm and the compromised developmental potential of embryos obtained using sperm from patients with a deletion of the DPY19L2 gene.

A recurrent deletion of DPY19L2 causes infertility in man by blocking sperm head elongation and acrosome formation.

An increasing number of couples require medical assistance to achieve a pregnancy, and more than 2% of the births in Western countries now result from assisted reproductive technologies. To identify genetic variants responsible for male infertility, we performed a whole-genome SNP scan on patients presenting with total globozoospermia, a primary infertility phenotype characterized by the presence of 100% round acrosomeless spermatozoa in the ejaculate. This strategy allowed us to identify in most patients (15/20) a 200 kb homozygous deletion encompassing only DPY19L2, which is highly expressed in the testis. Although there was no known function for DPY19L2 in humans, previous work indicated that its ortholog in C. elegans is involved in cell polarity. In man, the DPY19L2 region has been described as a copy-number variant (CNV) found to be duplicated and heterozygously deleted in healthy individuals. We show here that the breakpoints of the deletions are located on a highly homologous 28 kb low copy repeat (LCR) sequence present on each side of DPY19L2, indicating that the identified deletions were probably produced by nonallelic homologous recombination (NAHR) between these two regions. We demonstrate that patients with globozoospermia have a homozygous deletion of DPY19L2, thus indicating that DPY19L2 is necessary in men for sperm head elongation and acrosome formation. A molecular diagnosis can now be proposed to affected men; the presence of the deletion confirms the diagnosis of globozoospermia and assigns a poor prognosis for the success of in vitro fertilization.

MLPA and sequence analysis of DPY19L2 reveals point mutations causing globozoospermia.

STUDY QUESTION: Do DPY19L2 heterozygous deletions and point mutations account for some cases of globozoospermia? SUMMARY ANSWER: Two DPY19L2 heterozygous deletions and three point mutations were identified, thus further confirming that genetic alterations of the DPY19L2 gene are the main cause of globozoospermia and indicating that DPY19L2 molecular diagnostics should not be stopped in the absence of a homozygous gene deletion. WHAT IS KNOWN ALREADY: Globozoospermia is a rare phenotype of primary male infertility characterized by the production of a majority of round-headed spermatozoa without acrosome. We demonstrated previously that most cases in man were caused by a recurrent homozygous deletion of the totality of the DPY19L2 gene, preventing sperm head elongation and acrosome formation. In mammals, DPY19L2 has three paralogs of yet unknown function and one highly homologous pseudogene showing >95% sequence identity with DPY19L2. Specific amplification and sequencing of DPY19L2 have so far been hampered by the presence of this pseudogene which has greatly complicated specific amplification and sequencing. STUDY DESIGN, SIZE, DURATION: In this cohort study, 34 patients presenting with globozoospermia were recruited during routine infertility treatment in infertility centers in France and Tunisia between January 2008 and December 2011. The molecular variants identified in patients were screened in 200 individuals from the general population to exclude frequent non-pathological polymorphisms. PARTICIPANTS/MATERIALS, SETTING, METHODS: We developed a Multiplex Ligation-dependent Probe Amplification test to detect the presence of heterozygous deletions and identified the conditions to specifically amplify and sequence the 22 exons and intronic boundaries of the DPY19L2 gene. The pathogenicity of the identified mutations and their action on the protein were evaluated in silico. MAIN RESULTS AND THE ROLE OF CHANCE: There were 23 patients who were homozygous for the DPY19L2 deletion (67.6%). Only eight of the eleven non-homozygously deleted patients could be sequenced due to poor DNA quality of three patients. Two patients were compound heterozygous carrying one DPY19L2 deleted allele associated respectively with a nonsense (p.Q342*) and a missense mutation (p.R290H). One patient was homozygous for p.M358K, another missense mutation affecting a highly conserved amino acid. Due to the localization of this mutation and the physicochemical properties of the substituted amino acids, we believe that this variant is likely to disrupt one of the protein transmembrane domains and destabilize the protein. Overall, 84% of the fully analysed patients (n = 31) had a molecular alteration of DPY19L2. There was no clear phenotypic difference between the homozygous deleted individual, patients carrying a point mutation and undiagnosed patients. LIMITATIONS, REASONS FOR CAUTION: Globally poor fertilization rates are observed after intracytoplasmic sperm injection of round spermatozoa. Further work is needed to assess whether DPY19L2 mutated patients present a better or worse prognostic than the non-diagnosed patients. Evaluation of the potential benefit of treatment with a calcium ionophore, described to improve fertilization, should be evaluated in these two groups. WIDER IMPLICATIONS OF THE FINDINGS: In previous work, deletions of DPY19L2 had only been identified in North African patients. Here we have identified DPY19L2 deletions and point mutations in European patients, indicating that globozoospemia caused by a molecular defect of DPY19L2 can be expected in individuals from any ethnic background. STUDY FUNDING/COMPETING INTEREST(S): 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).

Identification of a new recurrent aurora kinase C mutation in both European and African men with macrozoospermia.

STUDY QUESTION: Can we identify new sequence variants in the aurora kinase C gene (AURKC) of patients with macrozoospermia and establish a genotype-phenotype correlation? SUMMARY ANSWER: We identified a new non-sense mutation, p.Y248*, that represents 13% of all mutant alleles. There was no difference in the phenotype of individuals carrying this new mutation versus the initially described and main mutation c.144delC. WHAT IS KNOWN ALREADY: The absence of a functional AURKC gene causes primary infertility in men by blocking the first meiotic division and leading to the production of tetraploid large-headed spermatozoa. We previously demonstrated that most affected men were of North African origin and carried a homozygous truncating mutation (c.144delC). STUDY DESIGN, SIZE, DURATION: This is a retrospective study carried out on patients consulting for infertility and described as having >5% large-headed spermatozoa. A total of 87 patients are presented here, 43 patients were published previously and 44 are new patients recruited between January 2008 and December 2011. PARTICIPANTS/MATERIALS, SETTING, METHODS: All patients consulted for primary infertility in fertility clinics in France (n = 44), Tunisia (n = 30), Morocco (n = 9) or Algeria (n = 4). Sperm analysis was carried out in the recruiting fertility clinics and all molecular analyses were performed at Grenoble teaching hospital. DNA was extracted from blood or saliva and the seven AURKC exons were sequenced. RT-PCR was carried out on transcripts extracted from leukocytes from one patient homozygous for p.Y248*. Microsatellite analysis was performed on all p.Y248* patients to evaluate the age of this new mutation. MAIN RESULTS AND THE ROLE OF CHANCE: We identified a new non-sense mutation, p.Y248*, in 10 unrelated individuals of European (n = 4) and North African origin (n = 6). We show that this new variant represents 13% of all mutant alleles and that the initially described c.144delC variant accounts for almost all of the remaining mutated alleles (85.5%). No mutated transcripts could be detected by RT-PCR suggesting a specific degradation of the mutant transcripts by non-sense mediated mRNA decay. A rare variant located in the 3' untranslated region was found to strictly co-segregate with p.Y248*, demonstrating a founding effect. Microsatellite analysis confirmed this linkage and allowed us to estimate a mutational age of between 925 and 1325 years, predating the c.144delC variant predicted by the same method to have arisen 250-650 years ago. Patients with no identified AURKC mutation (n = 15) have significantly improved parameters in terms of vitality and concentration of normal spermatozoa, and a decreased rate of spermatozoa with a large head and multiple flagella (P < 0.001). LIMITATIONS, REASONS FOR CAUTION: Despite adherence to the World Health Organization guidelines, large variations in most characteristic sperm parameters were observed, even for patients with the same homozygous mutation. We believe that is mainly related to inter-laboratory variability in sperm parameter scoring. This prevented us from establishing clear-cut values to indicate a need for molecular analysis of patients with macrozoospermia. WIDER IMPLICATIONS OF THE FINDINGS: This study confirms yet again the importance of AURKC mutations in the aetiology of macrozoospermia. Although a large majority of patients are of North African origin, we have now identified European patients carrying a new non-sense mutation indicating that a diagnosis of absence of a functional AURKC gene should not be ruled out for non-Magrebian individuals. Indirect evidence indicates that AURKC might be playing a role in the meiotic spindle assembly checkpoint (SAC) during meiosis. We postulate that heterozygous men might have a more relaxed SAC leading to a more abundant sperm production and a reproductive advantage. This could be the reason for the rapid accumulation of the two AURKC mutations we observe in North African individuals. STUDY FUNDING/COMPETING INTEREST(S): 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 programme GENOPAT 2009 from the French Research Agency (ANR).

[Do phospholipases, key enzymes in sperm physiology, represent therapeutic challenges?]. / Les phospholipases, enzymes clés de la physiologie spermatique: quels enjeux thérapeutiques ?

The spermatozoon is one of the most differentiated cells in mammals and its production requires an extremely complex machinery. Subtle but critical molecular changes take place during capacitation, which comprises the last series of maturation steps that naturally occur between the cauda epididymidis where spermatozoa are stored and their ultimate destination inside the oocyte. Phospholipases, by hydrolyzing various phospholipids, have been found to be critical in sperm processes such as 1) the control of flagellum beats, 2) capacitation - the molecular transformations preparing the sperm for fertilization, 3) acrosome reaction and 4) oocyte activation by eliciting calcium oscillations. The emerging important role of phospholipases is also emphasized by the fact that alterations of sperm lipids can lead to infertility. Phospholipases may represent valuable targets to develop anti- and pro-fertility drugs. Results obtained in mice are encouraging, since treatment of sperm with recombinant sPLA(2) of group X, known to be involved in capacitation, improves fertilization in vitro, while co-injection of PLCζ RNA with infertile sperm restores oocyte activation.