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1.
Mol Genet Genomics ; 299(1): 69, 2024 Jul 12.
Artigo em Inglês | MEDLINE | ID: mdl-38992144

RESUMO

TTC12 is a cytoplasmic and centromere-localized protein that plays a role in the proper assembly of dynein arm complexes in motile cilia in both respiratory cells and sperm flagella. This finding underscores its significance in cellular motility and function. However, the wide role of TTC12 in human spermatogenesis-associated primary ciliary dyskinesia (PCD) still needs to be elucidated. Whole-exome sequencing (WES) and Sanger sequencing were performed to identify potentially pathogenic variants causing PCD and multiple morphological abnormalities of sperm flagella (MMAF) in an infertile Pakistani man. Diagnostic imaging techniques were used for PCD screening in the patient. Real-time polymerase chain reaction (RT‒PCR) was performed to detect the effect of mutations on the mRNA abundance of the affected genes. Papanicolaou staining and scanning electron microscopy (SEM) were carried out to examine sperm morphology. Transmission electron microscopy (TEM) was performed to examine the ultrastructure of the sperm flagella, and the results were confirmed by immunofluorescence staining. Using WES and Sanger sequencing, a novel homozygous missense variant (c.C1069T; p.Arg357Trp) in TTC12 was identified in a patient from a consanguineous family. A computed tomography scan of the paranasal sinuses confirmed the symptoms of the PCD. RT-PCR showed a decrease in TTC12 mRNA in the patient's sperm sample. Papanicolaou staining, SEM, and TEM analysis revealed a significant change in shape and a disorganized axonemal structure in the sperm flagella of the patient. Immunostaining assays revealed that TTC12 is distributed throughout the flagella and is predominantly concentrated in the midpiece in normal spermatozoa. In contrast, spermatozoa from patient deficient in TTC12 showed minimal staining intensity for TTC12 or DNAH17 (outer dynein arms components). This could lead to MMAF and result in male infertility. This novel TTC12 variant not only illuminates the underlying genetic causes of male infertility but also paves the way for potential treatments targeting these genetic factors. This study represents a significant advancement in understanding the genetic basis of PCD-related infertility.


Assuntos
Homozigoto , Infertilidade Masculina , Mutação de Sentido Incorreto , Cauda do Espermatozoide , Humanos , Masculino , Mutação de Sentido Incorreto/genética , Paquistão , Infertilidade Masculina/genética , Infertilidade Masculina/patologia , Cauda do Espermatozoide/patologia , Cauda do Espermatozoide/ultraestrutura , Cauda do Espermatozoide/metabolismo , Adulto , Linhagem , Astenozoospermia/genética , Astenozoospermia/patologia , Transtornos da Motilidade Ciliar/genética , Transtornos da Motilidade Ciliar/patologia , Sequenciamento do Exoma , Oligospermia/genética , Oligospermia/patologia , Síndrome de Kartagener/genética , Síndrome de Kartagener/patologia
2.
BMC Genomics ; 25(1): 602, 2024 Jun 17.
Artigo em Inglês | MEDLINE | ID: mdl-38886667

RESUMO

BACKGROUND: Spermatogenesis is a highly regulated and complex process in which DNA methylation plays a crucial role. This study aimed to explore the differential methylation profiles in sperm DNA between patients with asthenospermia (AS) and healthy controls (HCs), those with oligoasthenospermia (OAS) and HCs, and patients with AS and those with OAS. RESULTS: Semen samples and clinical data were collected from five patients with AS, five patients with OAS, and six age-matched HCs. Reduced representation bisulfite sequencing (RRBS) was performed to identify differentially methylated regions (DMRs) in sperm cells among the different types of patients and HCs. A total of 6520, 28,019, and 16,432 DMRs were detected between AS and HC, OAS and HC, and AS and OAS groups, respectively. These DMRs were predominantly located within gene bodies and mapped to 2868, 9296, and 9090 genes in the respective groups. Of note, 12, 9, and 8 DMRs in each group were closely associated with spermatogenesis and male infertility. Furthermore, BDNF, SMARCB1, PIK3CA, and DDX27; RBMX and SPATA17; ASZ1, CDH1, and CHDH were identified as strong differentially methylated candidate genes in each group, respectively. Meanwhile, the GO analysis of DMR-associated genes in the AS vs. HC groups revealed that protein binding, cytoplasm, and transcription (DNA-templated) were the most enriched terms in the biological process (BP), cellular component (CC), and molecular function (MF), respectively. Likewise, in both the OAS vs. HC and AS vs. OAS groups, GO analysis revealed protein binding, nucleus, and transcription (DNA-templated) as the most enriched terms in BP, CC, and MF, respectively. Finally, the KEGG analysis of DMR-annotated genes and these genes at promoters suggested that metabolic pathways were the most significantly associated across all three groups. CONCLUSIONS: The current study results revealed distinctive sperm DNA methylation patterns in the AS vs. HC and OAS vs. HC groups, particularly between patients with AS and those with OAS. The identification of key genes associated with spermatogenesis and male infertility in addition to the differentially enriched metabolic pathways may contribute to uncovering the potential pathogenesis in different types of abnormal sperm parameters.


Assuntos
Astenozoospermia , Metilação de DNA , Oligospermia , Humanos , Masculino , Astenozoospermia/genética , Adulto , Oligospermia/genética , Espermatozoides/metabolismo , Espermatogênese/genética , Estudos de Casos e Controles , Epigênese Genética
3.
Front Endocrinol (Lausanne) ; 15: 1373774, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38863929

RESUMO

Background: Asthenozoospermia, a type of male infertility, is primarily caused by dysfunctional sperm mitochondria. Despite previous bioinformatics analysis identifying potential key lncRNAs, miRNAs, hub genes, and pathways associated with asthenospermia, there is still a need to explore additional molecular mechanisms and potential biomarkers for this condition. Methods: We integrated data from Gene Expression Omnibus (GEO) (GSE22331, GSE34514, and GSE160749) and performed bioinformatics analysis to identify differentially expressed genes (DEGs) between normozoospermia and asthenozoospermia. Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were conducted to gain insights into biological processes and signaling pathways. Weighted Gene Co-expression Network Analysis (WGCNA) identified gene modules associated with asthenozoospermia. Expression levels of key genes were assessed using datasets and experimental data. Gene Set Enrichment Analysis (GSEA) and correlation analysis identified pathways associated with the hub gene and explore the relationship between the ZNF764 and COQ9 and mitochondrial autophagy-related genes. Competitive endogenous RNA (ceRNA) networks were constructed, and in vitro experiments using exosome samples were conducted to validate this finding. Results: COQ9 was identified as a marker gene in asthenozoospermia, involved in autophagy, ATP-dependent chromatin remodeling, endocytosis, and cell cycle, etc. The ceRNA regulatory network (LINC00893/miR-125a-5p/COQ9) was constructed, and PCR demonstrated that LINC00893 and COQ9 were downregulated in asthenozoospermia, while miR-125a-5p and m6A methylation level of LINC00893 were upregulated in asthenozoospermia compared to normozoospermic individuals. Conclusion: The ceRNA regulatory network (LINC00893/miR-125a-5p/COQ9) likely plays a crucial role in the mechanism of asthenozoospermia. However, further functional experiments are needed to fully understand its significance.


Assuntos
Astenozoospermia , Biomarcadores , Biologia Computacional , Redes Reguladoras de Genes , Humanos , Masculino , Astenozoospermia/genética , Astenozoospermia/metabolismo , Biologia Computacional/métodos , Biomarcadores/metabolismo , Perfilação da Expressão Gênica , MicroRNAs/genética , MicroRNAs/metabolismo , Ontologia Genética , Transdução de Sinais/genética , Espermatozoides/metabolismo
4.
Genes (Basel) ; 15(5)2024 05 08.
Artigo em Inglês | MEDLINE | ID: mdl-38790229

RESUMO

Several genes are implicated in spermatogenesis and fertility regulation, and these genes are presently being analysed in clinical practice due to their involvement in male factor infertility (MFI). However, there are still few genetic analyses that are currently recommended for use in clinical practice. In this manuscript, we reviewed the genetic causes of qualitative sperm defects. We distinguished between alterations causing reduced sperm motility (asthenozoospermia) and alterations causing changes in the typical morphology of sperm (teratozoospermia). In detail, the genetic causes of reduced sperm motility may be found in the alteration of genes associated with sperm mitochondrial DNA, mitochondrial proteins, ion transport and channels, and flagellar proteins. On the other hand, the genetic causes of changes in typical sperm morphology are related to conditions with a strong genetic basis, such as macrozoospermia, globozoospermia, and acephalic spermatozoa syndrome. We tried to distinguish alterations approved for routine clinical application from those still unsupported by adequate clinical studies. The most important aspect of the study was related to the correct identification of subjects to be tested and the correct application of genetic tests based on clear clinical data. The correct application of available genetic tests in a scenario where reduced sperm motility and changes in sperm morphology have been observed enables the delivery of a defined diagnosis and plays an important role in clinical decision-making. Finally, clarifying the genetic causes of MFI might, in future, contribute to reducing the proportion of so-called idiopathic MFI, which might indeed be defined as a subtype of MFI whose cause has not yet been revealed.


Assuntos
Motilidade dos Espermatozoides , Espermatozoides , Humanos , Masculino , Espermatozoides/metabolismo , Espermatozoides/patologia , Motilidade dos Espermatozoides/genética , Astenozoospermia/genética , Astenozoospermia/patologia , Infertilidade Masculina/genética , Infertilidade Masculina/patologia , Teratozoospermia/genética , Teratozoospermia/patologia , DNA Mitocondrial/genética , Testes Genéticos
5.
Front Endocrinol (Lausanne) ; 15: 1377780, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38745955

RESUMO

Objective: Multiple morphological abnormalities of the sperm flagella (MMAF) is characterized by abnormal flagellar phenotypes, which is a particular kind of asthenoteratozoospermia. Previous studies have reported a comparable intracytoplasmic sperm injection (ICSI) outcome in terms of fertilization rate and clinical pregnancy rate in patients with MMAF compared with those with no MMAF; however, others have conflicting opinions. Assisted reproductive technology (ART) outcomes in individuals with MMAF are still controversial and open to debate. Methods: A total of 38 patients with MMAF treated at an academic reproductive center between January 2014 and July 2022 were evaluated in the current retrospective cohort study and followed up until January 2023. Propensity score matching was used to adjust for the baseline clinical characteristics of the patients and to create a comparable control group. The genetic pathogenesis of MMAF was confirmed by whole exome sequencing. The main outcomes were the embryo developmental potential, the cumulative pregnancy rate (CLPR), and the cumulative live birth rate (CLBR). Results: Pathogenic variants in known genes of DNAH1, DNAH11, CFAP43, FSIP2, and SPEF2 were identified in patients with MMAF. Laboratory outcomes, including the fertilization rate, 2PN cleavage rate, blastocyst formation rate, and available blastocyst rate, followed a trend of decline in the MMAF group (p < 0.05). Moreover, according to the embryo transfer times and complete cycles, the CLPR in the cohort of MMAF was lower compared with the oligoasthenospermia pool (p = 0.033 and p = 0.020, respectively), while no statistical differences were observed in the neonatal outcomes. Conclusion: The current study presented decreased embryo developmental potential and compromised clinical outcomes in the MMAF cohort. These findings may provide clinicians with evidence to support genetic counseling and clinical guidance in specific patients with MMAF.


Assuntos
Desenvolvimento Embrionário , Taxa de Gravidez , Injeções de Esperma Intracitoplásmicas , Cauda do Espermatozoide , Humanos , Masculino , Feminino , Gravidez , Adulto , Estudos Retrospectivos , Cauda do Espermatozoide/patologia , Desenvolvimento Embrionário/fisiologia , Astenozoospermia/genética , Astenozoospermia/patologia , Infertilidade Masculina/genética , Infertilidade Masculina/patologia , Espermatozoides/patologia
6.
Int J Mol Sci ; 25(7)2024 Apr 08.
Artigo em Inglês | MEDLINE | ID: mdl-38612930

RESUMO

Infertility is a global health challenge that affects an estimated 72.4 million people worldwide. Between 30 and 50% of these cases involve male factors, showcasing the complex nature of male infertility, which can be attributed to both environmental and genetic determinants. Asthenozoospermia, a condition characterized by reduced sperm motility, stands out as a significant contributor to male infertility. This study explores the involvement of the mitochondrial oxidative phosphorylation (OXPHOS) system, crucial for ATP production and sperm motility, in asthenozoospermia. Through whole-genome sequencing and in silico analysis, our aim was to identify and characterize OXPHOS gene variants specific to individuals with asthenozoospermia. Our analysis identified 680,099 unique variants, with 309 located within OXPHOS genes. Nine of these variants were prioritized due to their significant implications, such as potential associations with diseases, effects on gene expression, protein function, etc. Interestingly, none of these variants had been previously associated with male infertility, opening up new avenues for research. Thus, through our comprehensive approach, we provide valuable insights into the genetic factors that influence sperm motility, laying the foundation for future research in the field of male infertility.


Assuntos
Astenozoospermia , Infertilidade Masculina , Masculino , Humanos , Astenozoospermia/genética , Fosforilação Oxidativa , Motilidade dos Espermatozoides/genética , Infertilidade Masculina/genética , Sequenciamento Completo do Genoma
7.
Reprod Sci ; 31(7): 2001-2008, 2024 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-38499948

RESUMO

The methylation pattern of non-imprinting genes was little studied, although it is widely known that the abnormal methylation levels of imprinting genes are associated with different forms of male infertility. The purpose of this research was to assess the CREM gene's methylation status and seminal characteristics in infertile individuals who were potential intracytoplasmic sperm injection (ICSI) candidates. A total of 45 semen samples (15 normospermia, 15 asthenospermia, and 15 oligoasthenoteratospermia) were examined. Using aniline blue (AB) staining, we carried out conventional semen analysis, chromatin quality, and sperm maturity testing. DNA was taken from semen samples, and all isolated DNA was assessed using Nanodrop and gel electrophoresis. A quantitative methylation-specific polymerase chain reaction (Q-MSP) approach was used to quantify the methylation at the DMRs of the CREM gene. According to our findings, sperm count (P=0.012), concentration (P= 0.019), motility (P=0.006), progression (P=0.006), and normal morphology (P=0.004) were all inversely correlated with abnormal sperm chromatin condensation. Additionally, we noted that the methylation level of the CREM gene was considerably more significant in the oligoasthenoteratospermia group compared to the asthenospermia and normospermia groups (P<0.05). Additionally, sperm count (P=0.043), progression (P=0.026), and normal morphology (P=0.024) were all inversely linked with CREM methylation. Overall, the abnormal CREM methylation patterns have a negative impact on sperm parameters. Additionally, the CREM gene's DNA methylation status may serve as an epigenetic indicator of male infertility.


Assuntos
Modulador de Elemento de Resposta do AMP Cíclico , Metilação de DNA , Infertilidade Masculina , Espermatozoides , Humanos , Masculino , Infertilidade Masculina/genética , Infertilidade Masculina/metabolismo , Espermatozoides/metabolismo , Modulador de Elemento de Resposta do AMP Cíclico/genética , Modulador de Elemento de Resposta do AMP Cíclico/metabolismo , Adulto , Motilidade dos Espermatozoides/genética , Análise do Sêmen , Contagem de Espermatozoides , Astenozoospermia/genética
8.
J Cell Mol Med ; 28(7): e18215, 2024 04.
Artigo em Inglês | MEDLINE | ID: mdl-38509755

RESUMO

Oligoasthenoteratospermia (OAT), characterized by abnormally low sperm count, poor sperm motility, and abnormally high number of deformed spermatozoa, is an important cause of male infertility. Its genetic basis in many affected individuals remains unknown. Here, we found that CCDC157 variants are associated with OAT. In two cohorts, a 21-bp (g.30768132_30768152del21) and/or 24-bp (g.30772543_30772566del24) deletion of CCDC157 were identified in five sporadic OAT patients, and 2 cases within one pedigree. In a mouse model, loss of Ccdc157 led to male sterility with OAT-like phenotypes. Electron microscopy revealed misstructured acrosome and abnormal head-tail coupling apparatus in the sperm of Ccdc157-null mice. Comparative transcriptome analysis showed that the Ccdc157 mutation alters the expressions of genes involved in cell migration/motility and Golgi components. Abnormal Golgi apparatus and decreased expressions of genes involved in acrosome formation and lipid metabolism were detected in Ccdc157-deprived mouse germ cells. Interestingly, we attempted to treat infertile patients and Ccdc157 mutant mice with a Chinese medicine, Huangjin Zanyu, which improved the fertility in one patient and most mice that carried the heterozygous mutation in CCDC157. Healthy offspring were produced. Our study reveals CCDC157 is essential for sperm maturation and may serve as a marker for diagnosis of OAT.


Assuntos
Astenozoospermia , Infertilidade Masculina , Proteínas de Membrana , Oligospermia , Animais , Humanos , Masculino , Camundongos , Astenozoospermia/genética , Astenozoospermia/metabolismo , Infertilidade Masculina/genética , Infertilidade Masculina/metabolismo , Camundongos Knockout , Mutação/genética , Oligospermia/genética , Oligospermia/metabolismo , Sêmen/metabolismo , Motilidade dos Espermatozoides/genética , Espermatozoides/metabolismo , Proteínas de Membrana/metabolismo
9.
Reprod Biomed Online ; 48(5): 103765, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38492416

RESUMO

RESEARCH QUESTION: Is the novel homozygous nonsense variant of AK7 associated with multiple morphological abnormalities of the sperm flagella (MMAF), a specific type of oligoasthenoteratozoospermia leading to male infertility? DESIGN: Whole-exome sequencing and Sanger sequencing were performed to identify potential gene variants. Immunoblotting and immunofluorescence were applied to confirm the relationship between mutated genes and disease phenotypes. The concentration of reactive oxygen species and the rate of apoptosis were measured to evaluate the mitochondrial function of spermatozoa. Transmission electron microscopy and scanning electron microscopy were employed to observe sperm ultrastructure. RESULTS: A novel homozygous nonsense variant of AK7, c.1153A>T (p. Lys385*), was identified in two infertile siblings with asthenoteratozoospermia through whole-exome sequencing. Both immunoblotting and immunofluorescence assays showed practically complete absence of AK7 in the patient's spermatozoa. Additionally, the individual with the novel AK7 variant exhibited a phenotype characterized by severe oxidative stress and apoptosis caused by mitochondrial metabolic dysfunction of spermatozoa. Notably, remarkable flagellar defects with multiple axonemes in uniflagellate spermatozoa, accompanied by mitochondrial vacuolization, were observed; this has not been reported previously in patients with other AK7 variants. CONCLUSIONS: This study found that a novel identified homozygous nonsense variant of AK7 may be associated with MMAF-related asthenoteratozoospermia. The observed functional associations between mitochondria and sperm flagellar assembly provide evidence for potential mutual regulation between AK7 and flagella-associated proteins during spermatogenesis.


Assuntos
Códon sem Sentido , Homozigoto , Cauda do Espermatozoide , Humanos , Masculino , Cauda do Espermatozoide/patologia , Cauda do Espermatozoide/ultraestrutura , Infertilidade Masculina/genética , Infertilidade Masculina/patologia , Astenozoospermia/genética , Astenozoospermia/patologia , Adulto , Espermatozoides/ultraestrutura , Espermatozoides/anormalidades , Sequenciamento do Exoma , Mitocôndrias/ultraestrutura , Mitocôndrias/genética , Mitocôndrias/patologia , Linhagem
10.
J Assist Reprod Genet ; 41(5): 1297-1306, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38492154

RESUMO

PURPOSE: To identify the genetic causes of multiple morphological abnormalities in sperm flagella (MMAF) and male infertility in patients from two unrelated Han Chinese families. METHODS: Whole-exome sequencing was conducted using blood samples from the two individuals with MMAF and male infertility. Hematoxylin and eosin staining and scanning electron microscopy were performed to evaluate sperm morphology. Ultrastructural and immunostaining analyses of the spermatozoa were performed. The HEK293T cells were used to confirm the pathogenicity of the variants. RESULTS: We identified two novel homozygous missense ARMC2 variants: c.314C > T: p.P105L and c.2227A > G: p.N743D. Both variants are absent or rare in the human population genome data and are predicted to be deleterious. In vitro experiments indicated that both ARMC2 variants caused a slightly increased protein expression. ARMC2-mutant spermatozoa showed multiple morphological abnormalities (bent, short, coiled, absent, and irregular) in the flagella. In addition, the spermatozoa of the patients revealed a frequent absence of the central pair complex and disrupted axonemal ultrastructure. CONCLUSION: We identified two novel ARMC2 variants that caused male infertility and MMAF in Han Chinese patients. These findings expand the mutational spectrum of ARMC2 and provide insights into the complex causes and pathogenesis of MMAF.


Assuntos
Astenozoospermia , Sequenciamento do Exoma , Homozigoto , Infertilidade Masculina , Cauda do Espermatozoide , Espermatozoides , Humanos , Masculino , Cauda do Espermatozoide/patologia , Cauda do Espermatozoide/ultraestrutura , Cauda do Espermatozoide/metabolismo , Infertilidade Masculina/genética , Infertilidade Masculina/patologia , Astenozoospermia/genética , Astenozoospermia/patologia , Adulto , Espermatozoides/patologia , Espermatozoides/ultraestrutura , Mutação/genética , Linhagem , Células HEK293 , Povo Asiático/genética
11.
J Med Genet ; 61(6): 553-565, 2024 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-38341271

RESUMO

BACKGROUND: The association between the TDRD6 variants and human infertility remains unclear, as only one homozygous missense variant of TDRD6 was found to be associated with oligoasthenoteratozoospermia (OAT). METHODS: Whole-exome sequencing and Sanger sequencing were employed to identify potential pathogenic variants of TDRD6 in infertile men. Histology, immunofluorescence, immunoblotting and ultrastructural analyses were conducted to clarify the structural and functional abnormalities of sperm in mutated patients. Tdrd6-knockout mice were generated using the CRISPR-Cas9 system. Total RNA-seq and single-cell RNA-seq (scRNA-seq) analyses were used to elucidate the underlying molecular mechanisms, followed by validation through quantitative RT-PCR and immunostaining. Intracytoplasmic sperm injection (ICSI) was also used to assess the efficacy of clinical treatment. RESULTS: Bi-allelic TDRD6 variants were identified in five unrelated Chinese individuals with OAT, including homozygous loss-of-function variants in two consanguineous families. Notably, besides reduced concentrations and impaired motility, a significant occurrence of acrosomal hypoplasia was detected in multiple spermatozoa among five patients. Using the Tdrd6-deficient mice, we further elucidate the pivotal role of TDRD6 in spermiogenesis and acrosome identified. In addition, the mislocalisation of crucial chromatoid body components DDX4 (MVH) and UPF1 was also observed in round spermatids from patients harbouring TDRD6 variants. ScRNA-seq analysis of germ cells from a patient with TDRD6 variants revealed that TDRD6 regulates mRNA metabolism processes involved in spermatid differentiation and cytoplasmic translation. CONCLUSION: Our findings strongly suggest that TDRD6 plays a conserved role in spermiogenesis and confirms the causal relationship between TDRD6 variants and human OAT. Additionally, this study highlights the unfavourable ICSI outcomes in individuals with bi-allelic TDRD6 variants, providing insights for potential clinical treatment strategies.


Assuntos
Alelos , Astenozoospermia , Sequenciamento do Exoma , Camundongos Knockout , Espermatogênese , Adulto , Animais , Humanos , Masculino , Camundongos , Acrossomo/patologia , Astenozoospermia/genética , Astenozoospermia/patologia , Infertilidade Masculina/genética , Infertilidade Masculina/patologia , Oligospermia/genética , Oligospermia/patologia , Linhagem , Injeções de Esperma Intracitoplásmicas , Espermatogênese/genética , Espermatozoides/patologia , Espermatozoides/metabolismo
12.
Reprod Sci ; 31(6): 1610-1616, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38212584

RESUMO

Oligo-astheno-teratozoospermia (OAT), which is a common cause of male infertility, can be caused by genetic factors. This study reports on a case of a male patient suffering from infertility concomitant with OAT. Whole-exome sequencing (WES) confirmed the presence of a homozygous variant (NM_003462: c.464-1G > A) in the DNALI1 gene via Sanger sequencing. Immunofluorescence staining demonstrated that the DNALI1 signal was nearly undetectable in the patient's sperm. Bioinformatics analysis revealed that this mutation could reverse the splicing of the exon 4 acceptor splice site. A minigene experiment was performed to verify the mutation and the results confirmed that the mutation disrupted the splicing. Our findings show that this rare mutation in DNALI1 contributes to male infertility and OAT in humans, thereby expanding our understanding of the causes and pathogenesis of male infertility. This knowledge facilitates genetic counseling, clinical diagnosis, and therapeutic development of male infertility.


Assuntos
Astenozoospermia , Infertilidade Masculina , Mutação , Oligospermia , Humanos , Masculino , Infertilidade Masculina/genética , Infertilidade Masculina/diagnóstico , Astenozoospermia/genética , Astenozoospermia/diagnóstico , Oligospermia/genética , Oligospermia/diagnóstico , Adulto , Teratozoospermia/genética , Splicing de RNA , Sequenciamento do Exoma
13.
J Gene Med ; 26(1): e3583, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-37640479

RESUMO

BACKGROUND: Although defects in sperm morphology and physiology lead to male infertility, in many instances, the exact disruption of molecular pathways in a given patient is often unknown. The glycolytic pathway is an essential process to supply energy in sperm cell motility. Enolase 4 (ENO4) is crucial for the glycolytic process, which provides the energy for sperm cells in motility. ENO4 is located in the sperm principal piece and is essential for the motility and organization of the sperm flagellum. In the present study, we characterized a family with asthenozoospermia and abnormal sperm morphology as a result of a variant in the enolase 4 (ENO4) gene. METHODS: Computer-assisted semen analysis, papanicolaou smear staining and scanning electron microscopy were used to examine sperm motility and morphology for semen analysis in patients. For genetic analysis, whole-exome sequencing followed by Sanger sequencing was performed. RESULTS: Two brothers in a consanguineous family were being clinically investigated for sperm motility and morphology issues. Genetic analysis by whole-exome sequencing revealed a homozygous variant [c.293A>G, p.(Lys98Arg)] in the ENO4 gene that segregated with infertility in the family, shared by affected but not controls. CONCLUSIONS: In view of the association of asthenozoospermia and abnormal sperm morphology in Eno4 knockout mice, we consider this to be the first report describing the involvement of ENO4 gene in human male infertility. We also explore the possible involvement of another variant in explaining other phenotypic features in this family.


Assuntos
Astenozoospermia , Infertilidade Masculina , Camundongos , Animais , Humanos , Masculino , Astenozoospermia/genética , Astenozoospermia/metabolismo , Sêmen/metabolismo , Motilidade dos Espermatozoides/genética , Espermatozoides/fisiologia , Infertilidade Masculina/genética , Infertilidade Masculina/metabolismo , Camundongos Knockout , Fosfopiruvato Hidratase/genética , Fosfopiruvato Hidratase/metabolismo
14.
Andrology ; 12(2): 349-364, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-37302001

RESUMO

BACKGROUND: Multiple morphological abnormalities of sperm flagella is an idiopathic asthenoteratozoospermia characterized by absent, short, coiled, angulation, and irregular-caliber flagella. Genetic variants of DNAH1 gene have been identified as a causative factor of multiple morphological abnormalities of sperm flagella and intracytoplasmic sperm injection is an available strategy for infertile males with dynein axonemal heavy chain 1 defects to conceive. OBJECTIVES: To identify novel variants and candidate mutant hotspots of DNAH1 gene related to multiple morphological abnormalities of sperm flagella and male infertility in humans. MATERIALS AND METHODS: The DNAH1 variants were identified by whole exome sequencing and confirmed with Sanger sequencing. Papanicolaou staining, scanning and transmission electron microscopy, and immunostaining were performed to investigate the morphological and ultrastructural characteristics of spermatozoa. Intracytoplasmic sperm injection was applied for the assisted reproductive therapy of males harboring biallelic DNAH1 variants. RESULTS: We identified 18 different DNAH1 variants in 11 unrelated families, including nine missense variants (p.A2564T, p.T3657R, p.G1862R, p.L2296P, p.T4041I, p.L611P, p.A913D, p.R1932Q, p.R2356W) and nine loss-of-function variants (c.2301-1G>T, p.Q1518*, p.R1702*, p.D2845Mfs*2, p.P3909Rfs*33, p.Q4040Dfs*33, p.Q4058*, p.E4060Pfs*61, p.V4071Cfs*54). A total of 66.7% (12/18) of the identified variants were novel. Morphological analysis based on Papanicolaou staining and scanning electron microscopy demonstrated the typical multiple morphological abnormalities of sperm flagella characteristics of dynein axonemal heavy chain 1-deficient spermatozoa. Immunostaining further revealed the absence of inner dynein arms but not outer dynein arms, which induced a general ultrastructural disorganization, such as the loss of central pair and mis-localization of the microtubule doublets and outer dense fibers. To date, seven affected couples have accepted the intracytoplasmic sperm injection treatment, and three of them have given birth to five healthy babies. DISCUSSION AND CONCLUSION: These findings further expand the variant spectrum of DNAH1 gene related to multiple morphological abnormalities of sperm flagella and male infertility in humans, thus providing new information for the molecular diagnosis of asthenoteratozoospermia. The favorable fertility outcomes of intracytoplasmic sperm injection will facilitate the genetic counseling and clinical treatment of infertile males with multiple morphological abnormalities of sperm flagella in the future.


Assuntos
Astenozoospermia , Infertilidade Masculina , Masculino , Humanos , Injeções de Esperma Intracitoplásmicas , Astenozoospermia/genética , Mutação , Sêmen , Cauda do Espermatozoide , Espermatozoides , Infertilidade Masculina/genética , Infertilidade Masculina/terapia , Fertilidade , Dineínas/genética , China , Flagelos/genética
15.
Reprod Sci ; 31(3): 704-713, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-37957468

RESUMO

CatSper affects sperm function and male fertilization capacity markers, including sperm motility and egg penetration. The study has aimed to evaluate the mRNA expression of CatSper1, and CatSper3 in the spermatozoa of men with normozoospermia and Asthenoteratozoospermia, and to assess the correlation between genes expression and sperm parameters, fertilization rate, and embryo quality in intracytoplasmic sperm injection (ICSI). Reverse transcription-polymerase chain reaction was utilized to evaluate the mRNA expression of CatSper1 and CatSper3 in sperm in two patient groups: Normozoospermia (NOR; n = 32), and Asthenoteratozoospermia (AT; n = 22). In all patients receiving intracytoplasmic sperm injection, the fertilization rate and embryo quality were evaluated. CatSper1, and CatSper3 mRNA expression in sperm was significantly lower in AT males than in NOR (P < 0.05). Levels of these genes demonstrated a significant positive correlation with sperm motility, mitochondrial membrane potential (MMP), capacitation, fertilization rate, cleavage rate, and embryo quality (P < 0.05) following ICSI. However, a negative correlation was found between mRNA expression of CatSper1, 3 and sperm DNA fragmentation (P < 0.05). Findings indicate low levels of CatSper1 and CatSper3 mRNA expression in men with Asthenoteratozoospermia, which resulted in poor sperm quality and impaired embryo development following ICSI therapy.


Assuntos
Astenozoospermia , Humanos , Masculino , Astenozoospermia/genética , Astenozoospermia/metabolismo , Sêmen/metabolismo , Motilidade dos Espermatozoides , Espermatozoides/metabolismo , Fertilização , RNA Mensageiro/metabolismo , Fertilização in vitro
16.
Mol Med Rep ; 29(2)2024 02.
Artigo em Inglês | MEDLINE | ID: mdl-38099337

RESUMO

The role of long intergenic noncoding RNA 00893 (Linc00893) in asthenozoospermia (AS) and its impact on sperm motility remains unclear The present study explored the effect of Linc00893 on AS, specifically its effect on sperm motility and its relationship with spermatogonial stem cell (SSC) vitality and myosin heavy chain 9 (MYH9) protein expression. Linc00893 expression was analyzed in semen samples using reverse transcription­quantitative PCR, revealing a significant downregulation in samples from individuals with AS compared with those from healthy subjects. This downregulation was found to be negatively correlated with parameters of sperm motility. To further understand the role of Linc00893, small interfering RNA was used to knockdown its expression in SSCs. This knockdown led to a marked decrease in cell vitality and an increase in apoptosis. Notably, Linc00893 knockdown was shown to inhibit MYH9 expression by competitively binding with microRNA­107, a finding verified by dual­luciferase reporter and RNA immunoprecipitation assays. Furthermore, using the GSE160749 dataset from the Gene Expression Omnibus database, it was revealed that MYH9 protein expression was downregulated in AS samples. Subsequently, lentiviral vectors were constructed to induce overexpression of MYH9, which in turn reduced SSC apoptosis and counteracted the apoptosis triggered by Linc00893 knockdown. In conclusion, the present study identified the role of Linc00893 in AS, particularly its regulatory impact on sperm motility, SSC vitality and MYH9 expression. These findings may provide information on the potential regulatory mechanisms in AS development, and identify Linc00893 and MYH9 as possible targets for diagnosing and treating AS­related disorders.


Assuntos
Astenozoospermia , MicroRNAs , Humanos , Masculino , Astenozoospermia/genética , Astenozoospermia/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , RNA/metabolismo , Análise do Sêmen , Motilidade dos Espermatozoides/genética , Espermatozoides/metabolismo , RNA não Traduzido/genética
17.
Mol Biol Rep ; 50(12): 10131-10136, 2023 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-37921983

RESUMO

BACKGROUND: The mitochondrial genome is substantially susceptible to mutations and has high polymorphism due to structural features, location, and lack of recombinant variability, as its inheritance is strictly maternal. All of these events can be accompanied by the accumulation of mitochondrial single nucleotide polymorphisms (mtSNPs) in the sperm. The aim of this research was to analyze the influence of mutations in the MT-CYB gene on sperm quality. METHODS AND RESULTS: We conducted a case‒control study to identify mutations in the mitochondrial cytochrome B (MT-CYB) gene in men with asthenoteratozoospermia (89 cases) and oligoasthenoteratozoospermia (65 cases). The comparison group consisted of 164 fertile men. Somatic cell lysis followed by mtDNA extraction was conducted to analyze three mtDNA polymorphisms, rs28357373 (T15629C (Leu295=), rs527236194 (T15784C (p.Pro346=), rs2853506 (A15218G, p.Thr158Ala). Detection and genotyping of polymorphic loci in the MT-CYB gene was performed using the TaqMan allelic discrimination assay. To verify mutations in the MT-CYB gene, automated Sanger DNA sequencing was used. We found that rs527236194 was associated with asthenoteratozoospermia. rs28357373 in the MT-CYB gene did not show any polymorphism in the analyzed groups, which indicates a rare frequency of the TT genotype in our region. Rs28357373 and rs2853506 are not associated with male sperm abnormalities in the Volga-Ural region. CONCLUSION: The association of the rs527236194 polymorphic variant with sperm parameter alterations suggests its role in the pathophysiology of male infertility and requires further investigation in larger samples.


Assuntos
Astenozoospermia , Citocromos b , Masculino , Humanos , Citocromos b/genética , Polimorfismo de Nucleotídeo Único/genética , Estudos de Casos e Controles , Astenozoospermia/genética , Sêmen , DNA Mitocondrial/genética , Espermatozoides
18.
Clin Genet ; 104(6): 694-699, 2023 12.
Artigo em Inglês | MEDLINE | ID: mdl-37804054

RESUMO

Asthenozoospermia (AZS) is the primary cause of infertility in males. The radial spoke (RS) is an axonemal structure, connecting the peripheral doublet microtubules with the central pair of microtubules. This T-shaped multiprotein complex functions as a mechanochemical sensor to promote sperm motility. LRRC23 is a novel subunit of the RS complex that is necessary for flagellar assembly and movement in mice. However, the importance of LRRC23 in modulating RS formation in humans remains unclear. Here, we identified a homozygous nonsense mutation in LRRC23 (c.376C>T:p. Arg126X) in an infertile AZS patient whose parents were consanguineous. We verified the adversity of this novel mutation because of its ability to disrupt LRRC23 synthesis and impair RSs integrity. Furthermore, we demonstrated an interaction between LRRC23 and RSPH3 in vitro, indicating that LCCR23 is associated with RS in humans. Meanwhile, the LRRC23-mutant patient had a good prognosis following intracytoplasmic sperm injection. This study provides strong preliminary evidence that LRRC23 defects are potential causative factors of AZS in humans, which expands our knowledge for improved genetic counseling and better reproductive recommendations for patients with AZS.


Assuntos
Astenozoospermia , Infertilidade Masculina , Masculino , Humanos , Animais , Camundongos , Astenozoospermia/genética , Motilidade dos Espermatozoides , Sêmen , Infertilidade Masculina/genética , Axonema/genética , Espermatozoides
20.
EBioMedicine ; 96: 104798, 2023 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-37713809

RESUMO

BACKGROUND: Asthenozoospermia is the primary cause of male infertility; however, its genetic aetiology remains poorly understood. Adenylate kinase 9 (AK9) is highly expressed in the testes of humans and mice and encodes a type of adenosine kinase that is functionally involved in cellular nucleotide homeostasis and energy metabolism. We aimed to assess whether AK9 is involved in asthenozoospermia. METHODS: One-hundred-and-sixty-five Chinese men with idiopathic asthenozoospermia were recruited. Whole-exome sequencing (WES) and Sanger sequencing were performed for genetic analyses. Papanicolaou staining, Haematoxylin and eosin staining, scanning electron microscopy, and transmission electron microscopy were used to observe the sperm morphology and structure. Ak9-knockout mice were generated using CRISPR-Cas9. Sperm adenosine was detected by liquid chromatography-mass spectrometry. Targeted sperm metabolomics was performed. Intracytoplasmic sperm injection (ICSI) was used to treat patients. FINDINGS: We identified five patients harbouring bi-allelic AK9 mutations. Spermatozoa from men harbouring bi-allelic AK9 mutations have a decreased ability to sustain nucleotide homeostasis. Moreover, bi-allelic AK9 mutations inhibit glycolysis in sperm. Ak9-knockout male mice also presented similar phenotypes of asthenozoospermia. Interestingly, ICSI was effective in bi-allelic AK9 mutant patients in achieving good pregnancy outcomes. INTERPRETATION: Defects in AK9 induce asthenozoospermia with defects in nucleotide homeostasis and energy metabolism. This sterile phenotype could be rescued by ICSI. FUNDING: The National Natural Science Foundation of China (82071697), Medical Innovation Project of Fujian Province (2020-CXB-051), open project of the NHC Key Laboratory of Male Reproduction and Genetics in Guangzhou (KF202004), Medical Research Foundation of Guangdong Province (A2021269), Guangdong Provincial Reproductive Science Institute Innovation Team grants (C-03), and Outstanding Young Talents Program of Capital Medical University (B2205).


Assuntos
Astenozoospermia , Infertilidade Masculina , Humanos , Gravidez , Feminino , Masculino , Animais , Camundongos , Astenozoospermia/genética , Astenozoospermia/metabolismo , Nucleotídeos/metabolismo , Sêmen , Espermatozoides , Infertilidade Masculina/genética , Infertilidade Masculina/metabolismo
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