Patients with multiple morphological abnormalities of the sperm flagella harbouring CFAP44 or CFAP43 mutations have a good pregnancy outcome following intracytoplasmic sperm injection.

Multiple morphological abnormalities of the sperm flagella (MMAF) are a rare type of male infertility. Mutations in DNAH1, CFAP43 and CFAP44 are the main aetiology of the disorder. Previously, good intracytoplasmic sperm injection (ICSI) outcomes were reported for MMAF patients with DNAH1 mutations. However, the ICSI prognosis for MMAF patients with CFAP43 or CFAP44 mutations was not known. We designed a retrospective cohort study. Molecular genetic testing identified six MMAF patients with biallelic CFAP44 (CFAP44+ group) or CFAP43 mutations and 12 patients with homozygous or compound heterozygous DNAH1 mutations (DNAH1+ group). A control group consisted of age-matched, non-MMAF men. For MMAF patients carrying CFAP44 mutations, the recorded rates of fertilisation, transferable embryos, pregnancy and delivery after ICSI were 76.47%, 88.46%, 50.0% and 50.0% respectively. The fertilisation rate was significantly higher in the CFAP44+ group than in the DNAH1+ group (76.47% vs. 54.5%, p = 0.0196). There were no statistically significant differences in the rates of transferable embryos, implantation, clinical pregnancy and miscarriage between the CFAP44+ group and either the DNAH1+ group or the age-matched control group. Our results support a good ICSI prognosis for MMAF patients carrying CFAP44 or CFAP43 mutations.

Sha YW ，Wang X ，Su ZY ，Mei LB ，Ji ZY ，Bao H ，Li P ... -  《-》

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 ... -  《-》

NovelCFAP43 andCFAP44 mutations cause male infertility with multiple morphological abnormalities of the sperm flagella (MMAF).

Multiple morphological abnormalities of the sperm flagella (MMAF) comprise a rare congenital disease that can cause primary male infertility. Several pathogenic genes (e.g. AKAP4, DNAH1, CFAP43 and CFAP44) are associated with MMAF but the pathogenic mechanisms have not been elucidated. Whole-exome sequencing (WES) was applied to identify the pathogenic genes in 13 Chinese patients with MMAF; the patients were unrelated but all had consanguineous parents (usually first cousins). Real-time polymerase chain reaction and immunofluorescence staining were employed to assess the pathogenicity of these mutations. Four novel homozygous CFAP43 mutations in four (30.8%) MMAF patients and one novel homozygous CFAP44 mutation in one (7.7%) other case were identified. The four novel homozygous CFAP43 mutations included one frameshift mutation (c.1140_1143del: p.Asn380Lysfs*3), one nonsense mutation (c.739A>T: p.Lys247*) and two missense mutations (c.1474G>C: p.Gln492Arg; c.4600C>G: p.Leu1534Val). The novel mutation in CFAP44 was a homozygous nonsense mutation (c.4963C>T: p.Arg1655*). Co-segregation of the mutations was verified by Sanger sequencing of the families. The relative mRNA expression levels of CFAP43 in patients 1 and 9 and the levels of CFAP44 in patient 5 were significantly lower than those in control sperm samples. Immunofluorescence analysis of CFAP43 showed the protein was absent in the sperm flagella of patients 1 and 9. Furthermore, two previously reported mutations of DNAH1 were also identified in another four (30.8%) patients. This study demonstrated that CFAP43 and CFAP44 mutations are important causes of MMAF in the Chinese population. These novel mutations broaden the spectrum of CFAP43 and CFAP44 mutations.

Wu H ，Li W ，He X ，Liu C ，Fang Y ，Zhu F ，Jiang H ，Liu W ，Song B ，Wang X ，Zhou P ，Wei Z ，Zhang F ，Cao Y ... -  《-》

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 ... -  《-》

Novel Mutations in CFAP44 and CFAP43 Cause Multiple Morphological Abnormalities of the Sperm Flagella (MMAF).

Sha YW ，Wang X ，Xu X ，Su ZY ，Cui Y ，Mei LB ，Huang XJ ，Chen J ，He XM ，Ji ZY ，Bao H ，Yang X ，Li P ，Li L ... -  《-》