Whole-exome sequencing of a cohort of infertile men reveals novel causative genes in teratozoospermia that are chiefly related to sperm head defects.

Can whole-exome sequencing (WES) and in vitro validation studies identify new causative genes associated with teratozoospermia, particularly for sperm head defect? We investigated a core group of infertile patients, including 82 cases with unexplained abnormal sperm head and 67 individuals with multiple morphological abnormalities of the sperm flagella (MMAF), and revealed rare and novel deleterious gene variants correlated with morphological abnormalities of the sperm head or tail defects. Teratozoospermia is one of the most common factors causing male infertility. Owing to high phenotypic variability, currently known genetic causes of teratozoospermia can only explain a rather minor component for patients with anomalous sperm-head shapes, and the agents responsible for atypical sperm head shapes remain largely unknown. We executed WES analysis of a Chinese cohort of patients (N = 149) with teratozoospermia to identify novel genetic causes particularly for defective sperm head. We also sought to reveal the influence of different abnormalities of sperm morphology on ICSI outcome. In this study, a cohort of 149 infertile men (82 with abnormal sperm head and 67 with MMAF) were recruited. We implemented WES on infertile patients and analyzed the negative effects of the mutations of candidate genes on their protein conformations and/or expression. We also investigated the candidate genes' spatiotemporal expression/localization during spermatogenesis in both humans and mice, and explored their interactions with proteins that are known to be involved in sperm development. We also compared the ICSI outcomes of the affected individuals with various aberrations in sperm morphology. We identified rare and deleterious variants of piwi like RNA-mediated gene silencing 4 (PIWIL4: 1/82 patients, 1.21%), coiled-coil and C2 domain containing 1B (CC2D1B: 1/82 patients, 1.21%), cyclin B3 (CCNB3: 1/82 patients, 1.21%), KIAA1210 (KIAA1210: 2/82 patients, 2.43%) and choline phosphotransferase 1 (CHPT1: 1/82 patients, 1.21%), which are novel correlates of morphological abnormalities of the sperm head; functional evidence supports roles for all of these genes in sperm head formation. The mutations of septin 12 (SEPTIN12: 2/82 patients, 2.43%) are suggested to be associated with acrosome defects. We additionally observed novel causative mutations of dynein axonemal heavy chain 2 (DNAH2: 1/67 patients, 1.49%), dynein axonemal heavy chain 10 (DNAH10: 1/67 patients, 1.49%) and dynein axonemal heavy chain 12 (DNAH12: 1/67 patients, 1.49%) in patients with MMAF, and revealed a significantly lower fertilization rate of the abnormal sperm-head group compared to the MMAF group following ICSI. Consequently, our study also suggests that the mutations of PIWIL4 and CC2D1B might be circumvented by ICSI to a degree, and that CHPT1 and KIAA1210 loss-of-function variants might be associated with failed ICSI treatment. In this study, we discovered the relationship between the genotype and phenotype of the novel causative genes of sperm head deformities in humans. However, the molecular mechanism of the relevant genes involved in sperm head development needs to be further illuminated in future research. Furthermore, evidence should be provided using knockout/knock-in mouse models for additional confirmation of the roles of these novel genes in spermatogenesis. This cohort study of 149 Chinese infertile men documents novel genetic factors involved in teratozoospermia, particularly in anomalous sperm head formation. For the first time, we suggest that SEPTIN12 is related to human acrosomal hypoplasia, and that CCNB3 is a novel causative gene for globozoospermia in humans. We also uncovered variants in two genes-KIAA1210 and CHPT1associated with acrosomal biogenesis in patients with small or absent acrosomes. Additionally, it is postulated that loss-of-function mutations of PIWIL4 and CC2D1B have a contribution to the abnormal sperm-head formation. Furthermore, we are first to demonstrate the influence of different sperm morphologies on ICSI outcomes and indicates that the abnormal sperm head may play a significant role in fertilization failure. Our findings therefore provide valuable information for the diagnosis of teratozoospermia, particularly with respect to abnormalities of the sperm head. This will allow clinicians to adopt the optimal treatment strategy and to develop personalized medicine directly targeting these effects. This work was financed by the West China Second University Hospital of Sichuan University (KS369 and KL042). The authors declare that they do not have any conflicts of interests. N/A.

Li Y ，Wang Y ，Wen Y ，Zhang T ，Wang X ，Jiang C ，Zheng R ，Zhou F ，Chen D ，Yang Y ，Shen Y ... -  《-》

Whole-exome sequencing identifies mutations in FSIP2 as a recurrent cause of multiple morphological abnormalities of the sperm flagella.

Martinez G ，Kherraf ZE ，Zouari R ，Fourati Ben Mustapha S ，Saut A ，Pernet-Gallay K ，Bertrand A ，Bidart M ，Hograindleur JP ，Amiri-Yekta A ，Kharouf M ，Karaouzène T ，Thierry-Mieg N ，Dacheux-Deschamps D ，Satre V ，Bonhivers M ，Touré A ，Arnoult C ，Ray PF ，Coutton C ... -  《-》

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

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? 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. 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. This was a retrospective genetics study of patients presenting a MMAF phenotype. Patients were recruited in Iran and Italy between 2008 and 2015. 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. 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. N/A. 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. 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. 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.

Amiri-Yekta A ，Coutton C ，Kherraf ZE ，Karaouzène T ，Le Tanno P ，Sanati MH ，Sabbaghian M ，Almadani N ，Sadighi Gilani MA ，Hosseini SH ，Bahrami S ，Daneshipour A ，Bini M ，Arnoult C ，Colombo R ，Gourabi H ，Ray PF ... -  《-》

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

Does DNAH1 status influence intracytoplasmic sperm injection (ICSI) outcomes for patients with multiple morphological abnormalities of the sperm flagella (MMAF)? 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. 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. 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. 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. 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). 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. 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. 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).

Wambergue C ，Zouari R ，Fourati Ben Mustapha S ，Martinez G ，Devillard F ，Hennebicq S ，Satre V ，Brouillet S ，Halouani L ，Marrakchi O ，Makni M ，Latrous H ，Kharouf M ，Amblard F ，Arnoult C ，Ray PF ，Coutton C ... -  《-》

Genetic diagnosis, sperm phenotype and ICSI outcome in case of severe asthenozoospermia with multiple morphological abnormalities of the flagellum.

Are ICSI outcomes impaired in cases of severe asthenozoospermia with multiple morphological abnormalities of the flagellum (MMAF phenotype)? Despite occasional technical difficulties, ICSI outcomes for couples with MMAF do not differ from those of other couples requiring ICSI, irrespective of the genetic defect. Severe asthenozoospermia, especially when associated with the MMAF phenotype, results in male infertility. Recent findings have confirmed that a genetic aetiology is frequently responsible for this phenotype. In such situations, pregnancies can be achieved using ICSI. However, few studies to date have provided detailed analyses regarding the flagellar ultrastructural defects underlying this phenotype, its genetic aetiologies, and the results of ICSI in such cases of male infertility. We performed a retrospective study of 25 infertile men exhibiting severe asthenozoospermia associated with the MMAF phenotype identified through standard semen analysis. They were recruited at an academic centre for assisted reproduction in Paris (France) between 2009 and 2017. Transmission electron microscopy (TEM) and whole exome sequencing (WES) were performed in order to determine the sperm ultrastructural phenotype and the causal mutations, respectively. Finally 20 couples with MMAF were treated by assisted reproductive technologies based on ICSI. Patients with MMAF were recruited based on reduced sperm progressive motility and increased frequencies of absent, short, coiled or irregular flagella compared with those in sperm from fertile control men. A quantitative analysis of the several ultrastructural defects was performed for the MMAF patients and for fertile men. The ICSI results obtained for 20 couples with MMAF were compared to those of 378 men with oligoasthenoteratozoospermia but no MMAF as an ICSI control group. TEM analysis and categorisation of the flagellar anomalies found in these patients provided important information regarding the structural defects underlying asthenozoospermia and sperm tail abnormalities. In particular, the absence of the central pair of axonemal microtubules was the predominant anomaly observed more frequently than in control sperm (P < 0.01). Exome sequencing, performed for 24 of the 25 patients, identified homozygous or compound heterozygous pathogenic mutations in CFAP43, CFAP44, CFAP69, DNAH1, DNAH8, AK7, TTC29 and MAATS1 in 13 patients (54.2%) (11 affecting MMAF genes and 2 affecting primary ciliary dyskinesia (PCD)-associated genes). A total of 40 ICSI cycles were undertaken for 20 MMAF couples, including 13 cycles (for 5 couples) where a hypo-osmotic swelling (HOS) test was required due to absolute asthenozoospermia. The fertilisation rate was not statistically different between the MMAF (65.7%) and the non-MMAF (66.0%) couples and it did not differ according to the genotype or the flagellar phenotype of the subjects or use of the HOS test. The clinical pregnancy rate per embryo transfer did not differ significantly between the MMAF (23.3%) and the non-MMAF (37.1%) groups. To date, 7 of the 20 MMAF couples have achieved a live birth from the ICSI attempts, with 11 babies born without any birth defects. The ICSI procedure outcomes were assessed retrospectively on a small number of affected subjects and should be confirmed on a larger cohort. Moreover, TEM analysis could not be performed for all patients due to low sperm concentrations, and WES results are not yet available for all of the included men. An early and extensive phenotypic and genetic investigation should be considered for all men requiring ICSI for severe asthenozoospermia. Although our study did not reveal any adverse ICSI outcomes associated with MMAF, we cannot rule out that some rare genetic causes could result in low fertilisation or pregnancy rates. No external funding was used for this study and there are no competing interests. N/A.

Ferreux L ，Bourdon M ，Chargui A ，Schmitt A ，Stouvenel L ，Lorès P ，Ray P ，Lousqui J ，Pocate-Cheriet K ，Santulli P ，Dulioust E ，Toure A ，Patrat C ... -  《-》