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.

Coutton C ，Martinez G ，Kherraf ZE ，Amiri-Yekta A ，Boguenet M ，Saut A ，He X ，Zhang F ，Cristou-Kent M ，Escoffier J ，Bidart M ，Satre V ，Conne B ，Fourati Ben Mustapha S ，Halouani L ，Marrakchi O ，Makni M ，Latrous H ，Kharouf M ，Pernet-Gallay K ，Bonhivers M ，Hennebicq S ，Rives N ，Dulioust E ，Touré A ，Gourabi H ，Cao Y ，Zouari R ，Hosseini SH ，Nef S ，Thierry-Mieg N ，Arnoult C ，Ray PF ... -  《-》

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 ... -  《-》

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 ... -  《-》

Biallelic Mutations in CFAP43 and CFAP44 Cause Male Infertility with Multiple Morphological Abnormalities of the Sperm Flagella.

Sperm motility is vital to human reproduction. Malformations of sperm flagella can cause male infertility. Men with multiple morphological abnormalities of the flagella (MMAF) have abnormal spermatozoa with absent, short, coiled, bent, and/or irregular-caliber flagella, which impair sperm motility. The known human MMAF-associated genes, such as DNAH1, only account for fewer than 45% of affected individuals. Pathogenic mechanisms in the genetically unexplained MMAF remain to be elucidated. Here, we conducted genetic analyses by using whole-exome sequencing and genome-wide comparative genomic hybridization microarrays in a multi-center cohort of 30 Han Chinese men affected by MMAF. Among them, 12 subjects could not be genetically explained by any known MMAF-associated genes. Intriguingly, we identified compound-heterozygous mutations in CFAP43 in three subjects and a homozygous frameshift mutation in CFAP44 in one subject. All of these recessive mutations were parentally inherited from heterozygous carriers but were absent in 984 individuals from three Han Chinese control populations. CFAP43 and CFAP44, encoding two cilia- and flagella-associated proteins (CFAPs), are specifically or preferentially expressed in the testis. Using CRISPR/Cas9 technology, we generated two knockout models each deficient in mouse ortholog Cfap43 or Cfap44. Notably, both Cfap43- and Cfap44-deficient male mice presented with MMAF phenotypes, whereas the corresponding female mice were fertile. Our experimental observations on human subjects and animal models strongly suggest that biallelic mutations in either CFAP43 or CFAP44 can cause sperm flagellar abnormalities and impair sperm motility. Further investigations on other CFAP-encoding genes in more genetically unexplained MMAF-affected individuals could uncover novel mechanisms underlying sperm flagellar formation.

Tang S ，Wang X ，Li W ，Yang X ，Li Z ，Liu W ，Li C ，Zhu Z ，Wang L ，Wang J ，Zhang L ，Sun X ，Zhi E ，Wang H ，Li H ，Jin L ，Luo Y ，Wang J ，Yang S ，Zhang F ... -  《-》

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 ... -  《-》