Homozygous mutations in CCDC34 cause male infertility with oligoasthenoteratozoospermia in humans and mice

Novel homozygous variants in piRNA pathway factors lead to male infertility in humans.

Harnessing the full potential of reproductive genetics and epigenetics for male infertility in the era of “big data”

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).

BackgroundSpermatogenic impairments can lead to male infertility by different pathological conditions, such as multiple morphological abnormalities of the sperm flagella (MMAF) and non-obstructive azoospermia (NOA). Genetic factors are involved in...

Asthenozoospermia, characterized by reduced sperm motility, is a common cause of male infertility. Multiple morphological abnormalities of the sperm flagella (MMAF) represent a severe and genetically heterogeneous form of asthenozoospermia. Over 50 genes have been associated, but approximately half of MMAF cases remain unexplained. DRC1, a gene involved in the nexin-dynein regulatory complex (N-DRC), has been linked to MMAF and primary ciliary dyskinesia (PCD), often with significant variability in clinical presentation. His study aimed to identify novel pathogenic DRC1 variants in MMAF patients, assess their impact on sperm flagellar structure, and evaluate intracytoplasmic sperm injection (ICSI) and pregnancy outcomes. A cohort of 196 non-syndromic MMAF patients was analyzed using whole exome sequencing (WES). Functional validation of candidate variants included immunofluorescence to assess protein expression and transmission electron microscopy (TEM) to identify ultrastructural abnormalities. Assisted reproductive therapy outcomes were also evaluated. WES identified a recurrent homozygous frameshift variant in DRC1 NM_145038.5: c.109dup; p.(Gln37ProfsTer30) in four patients (2%), all of North African origin, none of whom suffer from PCD-related symptoms. The variant caused a complete absence of DRC1 protein in spermatozoa. TEM showed flagellar abnormalities, with 10% of axonemal sections revealing peripheral doublet dissociation, suggesting N-DRC instability. ICSI resulted in a 68.5% fertilization rate, with three out of four couples successfully delivering healthy children. The identification of a novel and recurrent pathogenic DRC1 variant broadens the mutation spectrum associated with MMAF. The absence of systemic PCD symptoms suggests that DRC1 deficiency may primarily affect spermatogenesis. Notably, the phenotypic spectrum might be influenced by the genetic background, varying across populations. Favorable ICSI outcomes, with a 68.5% fertilization rate and successful pregnancies in three out of four couples, highlight the effectiveness of assisted reproductive techniques for patients with this genetic defect.

Study question Is there any association between the multiple morphological abnormalities of the sperm flagella (MMAF) patients and the comprehensive outcomes of assisted reproductive technology (ART) cycles? Summary answer Patients with MMAF presented compromised ART outcomes, including lower embryo developmental potential and impaired clinical outcomes, compared with the cohort of oligoasthenospermia. What is known already Several studies have reported a positive intracytoplasmic sperm injection (ICSI) outcome in the MMAF cohort, and others have opposite opinions. The association between MMAF and ART outcomes is still controversial and open for debate. Furthermore, the neonatal outcomes and the information regarding the mutations of MMAF patients were incomplete. Study design, size, duration A retrospective cohort study of MMAF patients was performed at a reproductive medicine center in China between January 2014 and July 2022. Participants/materials, setting, methods Thirty-eight MMAF individuals were recruited based on a typical MMAF phenotype (abnormal sperm flagella, including short, absent, coiled, bent, and/or irregular flagella) and 131 couples with male infertility undergoing in vitro fertilization (IVF)/ICSI treatments over the same period were included as the control pool. Whole exome sequencing (WES) was performed to confirm the genetic pathogenesis of MMAF. Main results and the role of chance Pathogenic variants in known genes of DNAH1, DNAH11, CFAP43, FSIP2, and SPEF2 were identified in MMAF patients. The ART outcomes, including the fertilization rate, 2PN cleavage rate, blastocyst formation rate, and available blastocyte rate, were all significantly lower in the MMAF group than those in the control group (P<0.05). Moreover, the CLPR in the MMAF group was lower than that in the control group according to the embryo transfer times (68.6% vs. 86.5%, P=0.033) and complete cycles (66.7% vs. 84.2%, P=0.020), while no significant differences were found in the neonatal outcomes. Limitations, reasons for caution The sample size was limited due to the low incidence of MMAF and WES was not performed in all MMAF patients. Further exploration of the potential genetic mutations or underlying mechanisms in patients without identified mutations was needed. Moreover, long-term follow-up of neonates needs to be addressed in the future. Wider implications of the findings The current study identified the causative genetic mutations which were partly obligated to the pathogenesis of MMAF and presented the relationship between MMAF and compromised ART outcomes. These results may provide evidence support for clinicians in genetic counseling and clinical instruction in specific MMAF patients. Trial registration number the National Key Research & Development Program of China (2021YFC2700603)

Multiple morphological abnormalities of the sperm flagella (MMAF) is a severe form of asthenozoospermia categorized by immotile spermatozoa with abnormal flagella in ejaculate. Whole-exome sequencing (WES) is used to detect pathogenic variants in patients with MMAF. In this study, a novel homozygous frameshift variant (c.6158_6159insT) in dynein axonemal heavy chain 8 (DNAH8) from two infertile brothers with MMAF in a consanguineous Pakistani family was identified by WES. Reverse transcription-polymerase chain reaction (RT-PCR) confirmed DNAH8 mRNA decay in these patients with the DNAH8 mutation. Hematoxylin–eosin staining and transmission electron microscopy revealed highly divergent morphology and ultrastructure of sperm flagella in these patients. Furthermore, an immunofluorescence assay showed the absence of DNAH8 and a reduction in its associated protein DNAH17 in the patients’ spermatozoa. Collectively, our study expands the phenotypic spectrum of patients with DNAH8-related MMAF worldwide.

BackgroundAs a common type of asthenoteratozoospermia, multiple morphological abnormalities of the sperm flagella (MMAF) can cause male infertility. Previous studies have revealed genetic factors as a major cause of MMAF....

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.

To determine whether the social class differences in duodenal ulcer frequency may be explained by differences in physical activity at work, the energy expenditure during work, smoking habits, and social class were compared in 76 recently diagnosed duodenal ulcer patients and in age and sex matched community controls. As anticipated, the relative risk of duodenal ulcer showed significant associations with smoking and social class. Social class and physical activity at work were associated with one another. After adjusting for age, sex, smoking, and social class, physically active work was still associated with duodenal ulcer, with relative risks for moderate and high activity compared with sedentary work being 1.3 (0.6-3.0) and 3.6 (1.3-7.8) respectively. Within each social class stratum, the relative risk of having a duodenal ulcer was greater in those with a high level of occupational activity than in those undertaking sedentary work.

BackgroundMale infertility is a prevalent issue worldwide, mostly due to the impaired sperm motility. Multiple morphological abnormalities of the sperm flagella (MMAF) present aberrant spermatozoa with absent, short, coiled, bent...

Bi-allelic Mutations in TTC21A Induce Asthenoteratospermia in Humans and Mice

Objective To evaluate the relationship between sperm aneuploidy rate and clinical outcome of intracytoplasmic sperm injection (ICSI) in multiple morphological abnormalities of the sperm flagella (MMAF) patients. Methods A total of 5 MMAF patients and 10 normal fertile individuals with normal parameters from Reproductive Center of the Second Affiliated Hospital of Zhengzhou University were collected from January 2017 to June 2018. Papanicolaou staining, scanning electron microscopy and transmission electron microscopy were performed to observe the morphological characteristics of sperm. Fluorescence in situ hybridization (FISH) was used to detect the patients’ sperm aneuploidy rate. Finally, ICSI was performed to observe the clinical outcome. Results The results of the light microscope and scanning electron microscope showed morphologically abnormal spermatozoa in the ejaculated semen of patients, including absent, short, bent, coiled, and irregular flagella, and the central pair complex was absent in flagellum axoneme using transmission electron microscopy. There was no significant increase in patients’ sperm aneuploidy rate compared with normal control. Furthermore, in 7 ICSI cycles of 5 MMAF couples, all of them achieved clinical pregnancy, including 3 cases of live birth and 2 cases of spontaneous abortion. Conclusion There are serious morphological and ultrastructural abnormalities in the sperm flagella of MMAF patients. The low aneuploidy rates suggest that ICSI of MMAF patients will likely be of good prognosis for future pregnancies. Key words: Male infertility; Sperm aneuploidy rate; Multiple morphological abnormalities of the flagella; Intracytoplasmic sperm injection

A novel CCDC39 mutation causes multiple morphological abnormalities of the flagella in a primary ciliary dyskinesia patient

A novel mutation in DNAH17 is present in a patient with multiple morphological abnormalities of the flagella