Severe asthenozoospermia is a common cause of male infertility. Recent studies have revealed that
SPEF2
mutations lead to multiple morphological abnormalities of the sperm flagella (MMAF) without ...primary ciliary dyskinesia (PCD) symptoms in males, but PCD phenotype was also found in one female individual. Therefore, whether there is a phenotypic continuum ranging from infertile patients with PCD to MMAF patients with no or low noise PCD manifestations remains elusive. Here, we performed whole-exome sequencing in 47 patients with severe asthenozoospermia from 45 unrelated Chinese families. We identified four novel biallelic mutations in
SPEF2
(8.9%, 4/45) in six affected individuals (12.8%, 6/47), while no deleterious biallelic variants in
SPEF2
were detected in 637 controls, including 219 with oligoasthenospermia, 195 with non-obstructive azoospermia, and 223 fertile controls. Notably, all six patients exhibited PCD-like symptoms, including recurrent airway infections, bronchitis, and rhinosinusitis. Ultrastructural analysis revealed normal 9 + 2 axonemes of respiratory cilia but consistently abnormal 9 + 0 axoneme or disordered accessory structures of sperm flagella, indicating different roles of SPEF2 in sperm flagella and respiratory cilia. Subsequently, a
Spef2
knockout mouse model was used to validate the PCD-like phenotype and male infertility, where the subfertility of female
Spef2
−/−
mice was found unexpectedly. Overall, our data bridge the link between MMAF and PCD based on the association of
SPEF2
mutations with both infertility and PCD in males and provide basis for further exploring the molecular mechanism of SPEF2 during spermiogenesis and ciliogenesis.
Objective To provide information of semen quality among young Chinese men in the past 15 years. Design Retrospective cross-sectional study. Setting Sperm bank. Patient(s) A total of 30,636 young ...adult men who applied to be sperm donors at the Hunan Province Human Sperm Bank of China in 2001–2015 were included in the study. Intervention(s) Physical examination and analysis of blood and semen samples. Main Outcome Measure(s) Semen parameters, such as semen volume, sperm concentration, total sperm count, progressively motile sperm count, sperm progressive motility, sperm morphology, and round cells. Result(s) Many of the semen parameters showed a decreasing trend over the 15-year observation period. The sperm concentration and percentage of sperm with normal morphology decreased from 68 × 106 /mL to 47 × 106 /mL and from 31.8% to 10.8%, respectively. Although sperm progressive motility showed irregular variation, the progressively motile sperm count decreased from 34 × 106 to 21 × 106 over the 15-year period. Furthermore, the rate of qualified donors fell from 55.78% in 2001 to 17.80% in 2015, and the rate for 2015 was approximately threefold lower than the corresponding rates in 2001. Conclusion(s) The semen quality among young Chinese men has declined over a period of 15 years, especially in terms of sperm concentration, total sperm count, sperm progressive motility, and normal morphology.
Purpose
To identify the genetic cause of patients with primary ciliary dyskinesia (PCD) and male infertility from two unrelated Han Chinese families.
Methods
We conducted whole-exome sequencing of ...three individuals with PCD and male infertility from two unrelated Chinese families, and performed a targeted look-up for
DNAAF6
variants in our previously reported cohort of 442 individuals (219 with isolated oligoasthenospermia and 223 fertile controls). Ultrastructural and immunostaining analyses of patients’ spermatozoa were performed. The pathogenicity of the variants was validated using patient’s spermatozoa and HEK293T cells. Intracytoplasmic sperm injection (ICSI) treatment was conducted in two patients.
Results
We identified one novel hemizygous frameshift variant (NM_173494, c.319_329del: p.R107fs) of
DNAAF6
gene (previously named
PIH1D3
) in family 1 and one novel hemizygous missense variant (c.290G>T: p.G97V) in family 2. No hemizygous deleterious variants in
DNAAF6
were detected in the control cohort of 442 individuals. Ultrastructural and immunostaining analyses of patients’ spermatozoa showed the absence of outer and inner dynein arms in sperm flagella. Both variants were proven to lead to DNAAF6 protein degradation in HEK293T cells. Both patients carrying
DNAAF6
variants underwent one ICSI cycle and delivered one healthy child each.
Conclusion
We identified novel
DNAAF6
variants causing male infertility and PCD in Han Chinese patients. This finding extended the spectrum of variants in
DNAAF6
and revealed new light on the impact of
DNAAF6
variants in sperm flagella.
Background:
Infertility is a global health concern.
MEIOB
has been found to be associated with premature ovarian insufficiency (POI) and non-obstructive azoospermia (NOA), but its variants have not ...been reported in Chinese patients. The aim of this study was to identify the genetic aetiology of POI or NOA in three Han Chinese families.
Methods:
Whole-exome sequencing (WES) was used to identify candidate pathogenic variants in three consanguineous Chinese infertile families with POI or NOA. Sanger sequencing was performed to validate these variants in the proband of family I and her affected family members.
In vitro
functional analyses were performed to confirm the effects of these variants.
Results:
Two novel homozygous frameshift variants (c.258_259del and c.1072_1073del) and one novel homozygous nonsense variant (c.814C > T) in the
MEIOB
gene were identified in three consanguineous Han Chinese families.
In vitro
functional analyses revealed that these variants produced truncated proteins and affected their function.
Conclusion:
We identified three novel
MEIOB
loss-of-function variants in local Chinese patients for the first time and confirmed their pathogenicity using
in vitro
functional analyses. These results extend the mutation spectrum of the
MEIOB
gene and have important significance for genetic counselling in these families.
Tracing the genetic causes for male infertility due to asthenoteratozoospermia has revealed at least 40 causative genes, which provides valuable reference for the genetic testing of ...asthenoteratozoospermia in clinical practice. To identify deleterious variants in the human tetratricopeptide repeat domain 12 (TTC12) gene in a large cohort of infertile Chinese males with asthenoteratozoospermia.
A total of 314 unrelated asthenoteratozoospermia-affected men were recruited for whole exome sequencing. The effects of the identified variants were evaluated by
analysis, and confirmed by
experiments. Intracytoplasmic sperm injection (ICSI) was used to evaluate the efficiency of assisted reproduction technique therapy.
Novel homozygous
variants (c.1467_1467delG (p.Asp490Thrfs*14), c.1139_1139delA (p.His380Profs*4), and c.1117G>A (p.Gly373Arg)) were identified in three (0.96%) of the 314 cases. Three mutants were indicated to be damaging using
prediction tools, and were further confirmed by
functional analysis. Hematoxylin and eosin staining and ultrastructural observation of the spermatozoa revealed multiple morphological abnormalities of flagella, with the absence of outer and inner dynein arms. Notably, significant mitochondrial sheath malformations were also observed in the sperm flagella. Immunostaining assays indicated that TTC12 is present throughout the flagella, and was strongly concentrated in the mid-piece in control spermatozoa. However, spermatozoa from
-mutated individuals exhibited almost no staining intensity of TTC12 and outer and inner dynein arms components. The three men accepted ICSI treatment using their ejaculated spermatozoa, and two female partners successfully delivered healthy babies. Our findings provide direct genetic evidence that homozygous variants in
cause male infertility with asthenoteratozoospermia by causing dynein arm complex defects and mitochondrial sheath malformations in the flagellar. We also demonstrated that TTC12 deficiency-mediated infertility could be overcome by ICSI technology.
Spermatogonial stem cells (SSCs) are the initial cells for the spermatogenesis. Although much progress has been made on uncovering a number of modulators for the SSC fate decisions in rodents, the ...genes mediating human SSCs remain largely unclear. Here we report, for the first time, that TCF3, a member of the basic helix-loop-helix family of transcriptional modulator proteins, can stimulate proliferation and suppress the apoptosis of human SSCs through targeting podocalyxin-like protein (PODXL). TCF3 was expressed primarily in GFRA1-positive spermatogonia, and EGF (epidermal growth factor) elevated TCF3 expression level. Notably, TCF3 enhanced the growth and DNA synthesis of human SSCs, whereas it repressed the apoptosis of human SSCs. RNA sequencing and chromatin immunoprecipitation (ChIP) assays revealed that TCF3 protein regulated the transcription of several genes, including
WNT2B
,
TGFB3
,
CCN4
,
MEGF6
, and
PODXL
, while PODXL silencing compromised the stem cell activity of SSCs. Moreover, the level of TCF3 protein was remarkably lower in patients with spermatogenesis failure when compared to individuals with obstructive azoospermia with normal spermatogenesis. Collectively, these results implicate that TCF3 modulates human SSC proliferation and apoptosis through PODXL. This study is of great significance since it would provide a novel molecular mechanism underlying the fate determinations of human SSCs and it could offer new targets for gene therapy of male infertility.
Multiple morphological abnormalities of the sperm flagella (MMAF)-induced asthenoteratozoospermia is a common cause of male infertility. Previous studies have identified several MMAF-associated ...genes, highlighting the condition’s genetic heterogeneity. To further define the genetic causes underlying MMAF, we performed whole-exome sequencing in a cohort of 643 Chinese MMAF-affected men. Bi-allelic DNAH10 variants were identified in five individuals with MMAF from four unrelated families. These variants were either rare or absent in public population genome databases and were predicted to be deleterious by multiple bioinformatics tools. Morphological and ultrastructural analyses of the spermatozoa obtained from men harboring bi-allelic DNAH10 variants revealed striking flagellar defects with the absence of inner dynein arms (IDAs). DNAH10 encodes an axonemal IDA heavy chain component that is predominantly expressed in the testes. Immunostaining analysis indicated that DNAH10 localized to the entire sperm flagellum of control spermatozoa. In contrast, spermatozoa from the men harboring bi-allelic DNAH10 variants exhibited an absence or markedly reduced staining intensity of DNAH10 and other IDA components, including DNAH2 and DNAH6. Furthermore, the phenotypes were recapitulated in mouse models lacking Dnah10 or expressing a disease-associated variant, confirming the involvement of DNAH10 in human MMAF. Altogether, our findings in humans and mice demonstrate that DNAH10 is essential for sperm flagellar assembly and that deleterious bi-allelic DNAH10 variants can cause male infertility with MMAF. These findings will provide guidance for genetic counseling and insights into the diagnosis of MMAF-associated asthenoteratozoospermia.
Abstract
Asthenoteratospermia is a common cause of male infertility. Recent studies have revealed that CFAP65 mutations lead to severe asthenoteratospermia due to acrosome hypoplasia and flagellum ...malformations. However, the molecular mechanism underlying CFAP65-associated sperm malformation is largely unclear. Here, we initially examined the role of CFAP65 during spermiogenesis using Cfap65 knockout (Cfap65−/−) mice. The results showed that Cfap65−/− male mice exhibited severe asthenoteratospermia characterized by morphologically defective sperm heads and flagella. In Cfap65−/− mouse testes, hyper-constricted sperm heads were apparent in step 9 spermatids accompanied by abnormal manchette development, and acrosome biogenesis was abnormal in the maturation phase. Moreover, subsequent flagellar elongation was also severely affected and characterized by disrupted assembly of the mitochondrial sheath (MS) in Cfap65−/− male mice. Furthermore, the proteomic analysis revealed that the proteostatic system during acrosome formation, manchette organization and MS assembly was disrupted when CFAP65 was lost. Importantly, endogenous immunoprecipitation and immunostaining experiments revealed that CFAP65 may form a cytoplasmic protein network comprising MNS1, RSPH1, TPPP2, ZPBP1 and SPACA1. Overall, these findings provide insights into the complex molecular mechanisms of spermiogenesis by uncovering the essential roles of CFAP65 during sperm head shaping, acrosome biogenesis and MS assembly.
Male infertility due to spermatogenesis defects affects millions of men worldwide. However, the genetic etiology of the vast majority remains unclear. Here we describe three men with primary ...infertility due to multiple morphological abnormalities of the sperm flagella (MMAF) from two unrelated Han Chinese families. We performed whole-exome sequencing (WES) and Sanger sequencing on the proband of family 1, and found that he carried novel compound heterozygous missense mutations in dynein axonemal heavy chain 6 (DNAH6) that resulted in the substitution of a conserved amino acid residue and co-segregated with the MMAF phenotype in this family. Papanicolaou staining and transmission electron microscopy analysis revealed morphological and ultrastructural abnormalities in the sperm flagella in carriers of these genetic variants. Immunostaining experiments showed that DNAH6 was localized in the sperm tail. This is the first report identifying novel recessive mutations in DNAH6 as a cause of MMAF. These findings expand the spectrum of known MMAF mutations and phenotypes and provide information that can be useful for genetic and reproductive counseling of MMAF patients.
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