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1.
Clin Genet ; 2021 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-34595750

RESUMO

Non-obstructive azoospermia (NOA) represents one of the most serious forms of male infertility caused by spermatogenic failure. Despite multiple genes found to be associated with human NOA, the genetic basis of this idiopathic disease remains largely unknown. FBXO43 is a direct inhibitor of the anaphase-promoting complex/cyclosome (APC/C) E3 ligase and crucially important in mouse spermatogenesis. In this study, for the first time, we identified a homozygous nonsense mutation in FBXO43 c.1747C > T:p.Gln583X in two NOA brothers from a Chinese consanguineous family via whole-exome sequencing. FBXO43 was absent from testicular tissue of the proband, and FBXO43-immunostaining signals were invisible in the affected seminiferous tubules. Furthermore, in humans, FBXO43 defects cause meiotic arrest within early diplotene of prophase I. The results here demonstrate the pathogenicity of this loss-of-function mutation and confirmed that spermatocytes were unable to complete meiotic divisions without FBXO43 in humans. In mouse testicular protein extracts, three subunits of the APC/C, including ANAPC2, ANAPC8 and ANAPC10, were validated to interact directly with FBXO43, whereas no interactions were detected for FBXO43 and SKP1. This study furthers our understanding of the genetic basis of human NOA and provides insights into FBXO43 and male infertility.

2.
Artigo em Inglês | MEDLINE | ID: mdl-34657236

RESUMO

PURPOSE: Multiple morphological abnormalities in the sperm flagella (MMAF) comprise a severe phenotype of asthenoteratozoospermia with reduced or absent spermatozoa motility. Whereas dozens of candidate pathogenic genes for MMAF have been identified, the genetic cause in a large proportion of patients is unknown. We attempted to identify novel genetic explanations for MMAF. METHODS: We performed whole-exome sequencing of patients with MMAF to identify pathogenic variants. The phenotypes of spermatozoa in patients carrying DNAH10 variants were investigated using haematoxylin and eosin staining, scanning electron microscopy, and transmission electron microscopy. The expression and location of DNAH10 and other spermatozoa structure-related proteins were analyzed using immunofluorescence assays. RESULTS: We found one homozygous frameshift DNAH10 variant (NM_207437: c.2514delG:p.L839*) and one compound heterozygous DNAH10 variant (NM_207437: c.10820 T > C:p.M3607T; c.12692C > T:p.T4231I) in two patients with MMAF. These variants were absent or rare in the general population. Haematoxylin and eosin staining and scanning electron microscopy revealed the significant disruption of sperm flagella in the patients. In addition, ultrastructural analysis by transmission electron microscopy showed significant inner dynein arm (IDA) deficiency in sperm flagella. Using immunofluorescence assays, we found a significant reduction in IDA-related proteins including DNAH10 and DNAH1. CONCLUSIONS: We identified putative novel pathogenic variants in DNAH10 for MMAF, which might advance the genetic diagnosis and clinical genetic counselling for male infertility.

3.
Reprod Biol Endocrinol ; 19(1): 129, 2021 Aug 24.
Artigo em Inglês | MEDLINE | ID: mdl-34429122

RESUMO

BACKGROUND: Non-obstructive azoospermia (NOA) is the most severe form of male infertility; more than half of the NOA patients are idiopathic. Although many NOA risk genes have been detected, the genetic factors for NOA in majority of the patients are unknown. In addition, it is difficult to retrieve sperm from these patients despite using the microsurgical testicular sperm extraction (microTESE) method. Therefore, we conducted this genetic study to identify the potential genetic factors responsible for NOA and investigate the sperm retrieval rate of microTESE for genetically deficient NOA patients. METHODS: Semen analyses, sex hormone testing, and testicular biopsy were performed to categorize the patients with NOA. The chromosome karyotypes and Y chromosome microdeletion analyses were used to exclude general genetic factors. Whole exome sequencing and Sanger sequencing were performed to identify potential genetic variants in 51 patients with NOA. Hematoxylin and eosin staining (H&E) and anti-phosphorylated H2AX were used to assess the histopathology of spermatogenesis. Quantitative real time-polymerase chain reaction, western blotting, and immunofluorescence were performed to verify the effects of gene variation on expression. RESULTS: We performed whole exome sequencing in 51 NOA patients and identified homozygous helicase for meiosis 1(HFM1) variants (NM_001017975: c.3490C > T: p.Q1164X; c.3470G > A: p.C1157Y) in two patients (3.9%, 2/51). Histopathology of the testis showed that spermatogenesis was completely blocked at metaphase in these two patients carrying the HFM1 homozygous variants. In comparison with unaffected controls, we found a significant reduction in the levels of HFM1 mRNA and protein expression in the testicular tissues from these two patients. The patients were also subjected to microTESE treatment, but the sperms could not be retrieved. CONCLUSIONS: This study identified novel homozygous variants of HFM1 that are responsible for spermatogenic failure and NOA, and microTESE did not aid in retrieving sperms from these patients.

4.
Front Genet ; 12: 715339, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34394199

RESUMO

Primary ciliary dyskinesia (PCD) is a clinically and genetically heterogeneous ciliopathy affecting the cilia and sperm flagella. Mutations in genes related to the structural and functional defects of respiratory ciliary axoneme have been reported to be the predominant cause of this symptom; however, evidence regarding male infertility and genotype-phenotype associations between some of these genes and flagellar axoneme remains unclear. Here, we reported a male patient from a non-consanguineous Chinese family who exhibited left/right body asymmetry and oligoasthenoterazoospermia factor infertility. Novel compound heterozygous mutations in ARMC4 (NM:018076: c.2095C>T: p. Gln699*; c.1679C>T: p. Ala560Val) were identified in this patient, and his parents were a heterozygous carrier for the mutations. Morphological and ultrastructural analysis of the spermatozoa from the man showed aberrant sperm flagella with axonemal disorganization and outer dynein arm (ODA) loss. In addition, immunofluorescence analysis of the spermatozoa from the proband and a control man revealed a significant lower expression of ARMC4 protein due to pathogenic mutations. Therefore, our findings help to expand the spectrum of ARMC4 pathogenic mutations and linked biallelic ARMC4 mutations to male infertility for the first time.

5.
Am J Hum Genet ; 108(8): 1466-1477, 2021 08 05.
Artigo em Inglês | MEDLINE | ID: mdl-34237282

RESUMO

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.


Assuntos
Astenozoospermia/complicações , Modelos Animais de Doenças , Dineínas/genética , Infertilidade Masculina/patologia , Mutação , Fenótipo , Espermatozoides/patologia , Alelos , Animais , Homozigoto , Humanos , Infertilidade Masculina/etiologia , Infertilidade Masculina/metabolismo , Masculino , Camundongos , Camundongos Knockout , Espermatozoides/metabolismo , Sequenciamento Completo do Exoma
6.
Hum Genet ; 140(9): 1367-1377, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34255152

RESUMO

Spermatozoa are polarized cells with a head and a flagellum joined together by the connecting piece. Flagellum integrity is critical for normal sperm function, and flagellum defects consistently lead to male infertility. Multiple morphological abnormalities of the flagella (MMAF) is a distinct sperm phenotype consistently leading to male infertility due to a reduced or absent sperm motility associated with severe morphological and ultrastructural flagellum defects. Despite numerous genes recently described to be recurrently associated with MMAF, more than half of the cases analyzed remain unresolved, suggesting that many yet uncharacterized gene defects account for this phenotype. By performing a retrospective exome analysis of the unsolved cases from our initial cohort of 167 infertile men with a MMAF phenotype, we identified one individual carrying a homozygous frameshift variant in CFAP206, a gene encoding a microtubule-docking adapter for radial spoke and inner dynein arm. Immunostaining experiments in the patient's sperm cells demonstrated the absence of WDR66 and RSPH1 proteins suggesting severe radial spokes and calmodulin and spoke-associated complex defects. Using the CRISPR-Cas9 technique, we generated homozygous Cfap206 knockout (KO) mice which presented with male infertility due to functional, structural and ultrastructural sperm flagellum defects associated with a very low rate of embryo development using ICSI. Overall, we showed that CFAP206 is essential for normal sperm flagellum structure and function in human and mouse and that bi-allelic mutations in CFAP206 cause male infertility in man and mouse by inducing morphological and functional defects of the sperm flagellum that may also cause ICSI failures.


Assuntos
Proteínas do Citoesqueleto , Mutação da Fase de Leitura , Homozigoto , Infertilidade Masculina , Cauda do Espermatozoide/metabolismo , Animais , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Humanos , Infertilidade Masculina/genética , Infertilidade Masculina/metabolismo , Masculino , Camundongos
7.
Front Genet ; 12: 666136, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34178031

RESUMO

Preimplantation embryonic lethality is a rare cause of primary female infertility. It has been reported that variants in the transducin-like enhancer of split 6 (TLE6) gene can lead to preimplantation embryonic lethality. However, the incidence of TLE6 variants in patients with preimplantation embryonic lethality is not fully understood. In this study, we identified four patients carrying novel biallelic TLE6 variants in a cohort of 28 patients with preimplantation embryonic lethality by whole-exome sequencing and bioinformatics analysis, accounting for 14.29% (4/28) of the cohort. Immunofluorescence showed that the TLE6 levels in oocytes from patients were much lower than in normal control oocytes, suggesting that the variants result in the lower expression of the TLE6 protein in oocytes. In addition, a retrospective analysis showed that the four patients underwent a total of nine failures of in vitro fertilization and intracytoplasmic sperm injection attempts, and one of them became pregnant on the first attempt using donated oocytes. Our study extends the genetic spectrum of female infertility caused by variants in TLE6 and further confirms previously reported findings that TLE6 plays an essential role in early embryonic development. In such case, oocyte donation may be the preferred treatment.

8.
Biol Reprod ; 105(2): 554-566, 2021 08 03.
Artigo em Inglês | MEDLINE | ID: mdl-33907797

RESUMO

Deoxynivalenol (DON) is one of the most prevalent Fusarium mycotoxins, which cause detrimental effects on human and animal reproductive systems by inducing oxidative stress. Increasing evidence has suggested the potential roles of melatonin in protecting granulosa cells from oxidative injury, but the underlying mechanisms remain largely elusive. Here, we demonstrated that suppression of FOXO1 and endoplasmic reticulum (ER) stress was engaged in melatonin-mediated protection against oxidative damage in human granulosa cells upon DON exposure in vitro. DON induced excess reactive oxygen species accumulation, cells viability loss, reduced estradiol-17ß, and progesterone production in human granulosa cells, whereas melatonin ameliorated these phenotypes. Next, we found that the protective effect of melatonin against apoptosis was via reducing ER stress because the inhibition of ER stress displayed similar protective effects during DON treatment. Moreover, melatonin provided no additional protection when ER stress was inhibited. We further found that FOXO1 is a pivotal downstream effector of melatonin and ER stress in regulating DON-induced apoptosis in human granulosa cells. Blocking of FOXO1 reduced DON-induced cells death and FOXO1 activation could be suppressed by melatonin or ER stress inhibitor. However, melatonin failed to further restore cells viability in the presence of FOXO1 inhibitor. Collectively, our results reveal a new mechanism of melatonin in protecting against DON-induced apoptosis and dysfunction by suppressing ER stress and FOXO1 in human granulosa cells.

9.
J Assist Reprod Genet ; 38(8): 2041-2048, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-33786731

RESUMO

PURPOSE: To evaluate the association between the DNA methylation of specific genes and sperm DNA integrity status in human sperm samples. METHODS: A total of 166 semen samples were evaluated (86 controls and 80 cases with impaired sperm DNA integrity). We detected the methylation status of 257 CpG sites among two imprinted genes (H19 and SNRPN) and four non-imprinted genes related to male infertility (MTHFR, GSTM1, DAZL, and CREM) by using a targeted next-generation sequencing method. RESULTS: Differential methylation was found in 43 CpG sites of the promoters of the six candidate genes. H19, SNRPN, MTHFR, DAZL, GSTM1, and CREM contained 22, 12, 1, 4, 0, and 4 differentially methylated CpG sites (P<0.05), respectively. The imprinting genes were associated with relatively higher rates of differentially methylated CpG sites (28.21% in H19 and 41.38% in SNRPN) than the non-imprinting genes. One CpG site in H19 remained significant after performing strict Bonferroni correction. CONCLUSION: In this study, we found that different site-specific DNA methylation signatures were correlated with sperm DNA integrity status. Further studies are needed to investigate the specific mechanisms leading to the epigenetic modifications.

10.
Reprod Biomed Online ; 42(5): 963-972, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33771466

RESUMO

RESEARCH QUESTION: Multiple morphological abnormalities of the flagella (MMAF) is characterized by excessive immotile spermatozoa with severe flagellar abnormalities in the ejaculate. Previous studies have reported a heterogeneous genetic profile associated with MMAF. What other genetic variants might explain the cause of MMAF? DESIGN: Whole-exome sequencing was conducted in a cohort of 90 Chinese patients with MMAF. The pathogenicity of identified mutations was assessed through electron microscopy and immunofluorescent examinations. RESULTS: Three unrelated men with bi-allelic DNAH2 variants were identified. Sanger sequencing verified that the six novel variants originated from every parent. All these variants were located at the conserved domains of DNAH2 and predicted to be deleterious by bioinformatic tools. Haematoxylin and eosin staining and scanning electron microscopy revealed that spermatozoa harbouring DNAH2 variants displayed severely aberrant morphology mainly with absent and short flagella (≥78%). Moreover, transmission electron microscopy revealed the obvious absence of a central pair of microtubules and inner dynein arms in the spermatozoa with mutated DNAH2. Immunofluorescence data further validated these findings, showing reduced DNAH2 protein expression in the spermatozoa with DNAH2 variants, compared with normal spermatozoa. Intracytoplasmic sperm injection using spermatozoa from the three men with mutated DNAH2 resulted in blastocyst formation in all cases. Embryo transfer was carried out in two couples, both resulting in clinical pregnancy. CONCLUSIONS: These experimental and clinical data suggest that bi-allelic DNAH2 variants might induce MMAF-associated asthenoteratozoospermia, which can be overcome through intracytoplasmic sperm injection. These findings contribute to the knowledge of the genetic landscape of asthenoteratozoospermia and clinical counselling of male infertility.

11.
Am J Hum Genet ; 108(2): 309-323, 2021 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-33472045

RESUMO

Asthenoteratozoospermia characterized by multiple morphological abnormalities of the flagella (MMAF) has been identified as a sub-type of male infertility. Recent progress has identified several MMAF-associated genes with an autosomal recessive inheritance in human affected individuals, but the etiology in approximately 40% of affected individuals remains unknown. Here, we conducted whole-exome sequencing (WES) and identified hemizygous missense variants in the X-linked CFAP47 in three unrelated Chinese individuals with MMAF. These three CFAP47 variants were absent in human control population genome databases and were predicted to be deleterious by multiple bioinformatic tools. CFAP47 encodes a cilia- and flagella-associated protein that is highly expressed in testis. Immunoblotting and immunofluorescence assays revealed obviously reduced levels of CFAP47 in spermatozoa from all three men harboring deleterious missense variants of CFAP47. Furthermore, WES data from an additional cohort of severe asthenoteratozoospermic men originating from Australia permitted the identification of a hemizygous Xp21.1 deletion removing the entire CFAP47 gene. All men harboring hemizygous CFAP47 variants displayed typical MMAF phenotypes. We also generated a Cfap47-mutated mouse model, the adult males of which were sterile and presented with reduced sperm motility and abnormal flagellar morphology and movement. However, fertility could be rescued by the use of intra-cytoplasmic sperm injections (ICSIs). Altogether, our experimental observations in humans and mice demonstrate that hemizygous mutations in CFAP47 can induce X-linked MMAF and asthenoteratozoospermia, for which good ICSI prognosis is suggested. These findings will provide important guidance for genetic counseling and assisted reproduction treatments.


Assuntos
Astenozoospermia/genética , Infertilidade Masculina/genética , Animais , Astenozoospermia/patologia , Astenozoospermia/fisiopatologia , Estudos de Coortes , Feminino , Deleção de Genes , Genes Ligados ao Cromossomo X , Hemizigoto , Humanos , Infertilidade Masculina/metabolismo , Infertilidade Masculina/patologia , Infertilidade Masculina/fisiopatologia , Masculino , Camundongos Endogâmicos C57BL , Mutação , Mutação de Sentido Incorreto , Linhagem , Fenótipo , Injeções de Esperma Intracitoplásmicas , Motilidade Espermática , Cauda do Espermatozoide/ultraestrutura , Espermatozoides/patologia , Espermatozoides/fisiologia , Espermatozoides/ultraestrutura , Sequenciamento Completo do Exoma
12.
Am J Hum Genet ; 107(3): 514-526, 2020 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-32791035

RESUMO

Multiple morphological abnormalities of the sperm flagella (MMAF) is a severe form of asthenoteratozoospermia. Although recent studies have revealed several MMAF-associated genes and demonstrated MMAF to be a genetically heterogeneous disease, at least one-third of the cases are still not well understood for their etiology. Here, we identified bi-allelic loss-of-function variants in CFAP58 by using whole-exome sequencing in five (5.6%) unrelated individuals from a cohort of 90 MMAF-affected Chinese men. Each of the men harboring bi-allelic CFAP58 variants presented typical MMAF phenotypes. Transmission electron microscopy demonstrated striking flagellar defects with axonemal and mitochondrial sheath malformations. CFAP58 is predominantly expressed in the testis and encodes a cilia- and flagella-associated protein. Immunofluorescence assays showed that CFAP58 localized at the entire flagella of control sperm and predominantly concentrated in the mid-piece. Immunoblotting and immunofluorescence assays showed that the abundances of axoneme ultrastructure markers SPAG6 and SPEF2 and a mitochondrial sheath protein, HSP60, were significantly reduced in the spermatozoa from men harboring bi-allelic CFAP58 variants. We generated Cfap58-knockout mice via CRISPR/Cas9 technology. The male mice were infertile and presented with severe flagellar defects, consistent with the sperm phenotypes in MMAF-affected men. Overall, our findings in humans and mice strongly suggest that CFAP58 plays a vital role in sperm flagellogenesis and demonstrate that bi-allelic loss-of-function variants in CFAP58 can cause axoneme and peri-axoneme malformations leading to male infertility. This study provides crucial insights for understanding and counseling of MMAF-associated asthenoteratozoospermia.


Assuntos
Anormalidades Múltiplas/genética , Astenozoospermia/genética , Axonema/genética , Infertilidade Masculina/genética , Peptídeos e Proteínas de Sinalização Intercelular/genética , Anormalidades Múltiplas/patologia , Alelos , Animais , Astenozoospermia/fisiopatologia , Axonema/patologia , Sistemas CRISPR-Cas/genética , Proteínas de Ciclo Celular/genética , Homozigoto , Humanos , Infertilidade Masculina/patologia , Mutação com Perda de Função/genética , Perda de Heterozigosidade/genética , Masculino , Camundongos , Camundongos Knockout , Proteínas dos Microtúbulos/genética , Mitocôndrias/genética , Cauda do Espermatozoide/metabolismo , Cauda do Espermatozoide/patologia , Testículo/metabolismo , Testículo/patologia , Sequenciamento Completo do Exoma
13.
J Assist Reprod Genet ; 37(6): 1431-1439, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32323121

RESUMO

BACKGROUND: Asthenoteratospermia with multiple morphological abnormalities in the sperm flagella (MMAF) is a significant cause of male infertility. WDR19 is a core component in the IFT-A complex and has a critical role in intraflagellar transport. However, the role of WDR19 mutations in male infertility has yet to be examined. METHODS AND RESULTS: We performed whole exome sequencing (WES) for 65 asthenoteratospermia individuals and identified a proband who carried a homozygous WDR19 (c.A3811G, p.K1271E) mutation from a consanguineous family. Systematic examinations, including CT scanning and retinal imaging, excluded previous ciliopathic syndromes in the proband. Moreover, semen analysis of this patient showed that the progressive rate decreased to zero, and the sperm flagella showed multiple morphological abnormalities. Scanning and transmission electron microscopy assays indicated that the ultrastructure of sperm flagella in the patient was completely destroyed, while immunofluorescence revealed that WDR19 was absent from the sperm neck and flagella. Moreover, IFT140 and IFT88, predicted to interact with WDR19 directly, were mis-allocated in the WDR19-mutated sperm. Notably, the MMAF subject harboring WDR19 variant and his partner successfully achieved clinical pregnancy through intracytoplasmic sperm injection (ICSI). CONCLUSIONS: We identified WDR19 as a novel pathogenic gene for male infertility caused by asthenoteratospermia in the absence of other ciliopathic phenotypes, and that patients carrying WDR19 variant can have favorable pregnancy outcomes following ICSI.


Assuntos
Astenozoospermia/genética , Proteínas do Citoesqueleto/genética , Infertilidade Masculina/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Cauda do Espermatozoide/metabolismo , Adulto , Astenozoospermia/patologia , Exoma/genética , Feminino , Homozigoto , Humanos , Infertilidade Masculina/patologia , Masculino , Microtúbulos/genética , Microtúbulos/patologia , Mutação/genética , Gravidez , Injeções de Esperma Intracitoplásmicas , Cauda do Espermatozoide/patologia , Espermatozoides/patologia , Sequenciamento Completo do Exoma
14.
J Assist Reprod Genet ; 37(6): 1421-1429, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32314195

RESUMO

PURPOSE: Cystic fibrosis transmembrane conductance regulator (CFTR) and adhesion G protein-coupled receptor G2 (ADGRG2) have been identified as the main pathogenic genes in congenital bilateral absence of the vas deferens (CBAVD), which is an important cause of obstructive azoospermia. This study aimed to identify the disease-causing gene in two brothers with CBAVD from a Chinese consanguineous family and reveal the intracytoplasmic sperm injection (ICSI) outcomes in these patients. METHODS: Whole-exome sequencing and Sanger sequencing were used to identify the candidate pathogenic genes. Real-time polymerase chain reaction, immunohistochemistry, and immunofluorescence were used to assess the expression of the mutant gene. Moreover, the ICSI results from both patients were retrospectively reviewed. RESULTS: A novel hemizygous loss-of-function mutation (c.G118T: p.Glu40*) in ADGRG2 was identified in both patients with CBAVD. This mutation is absent from the human genome databases and causes an early translational termination in the third exon of ADGRG2. Expression analyses showed that both the ADGRG2 mRNA and the corresponding protein were undetectable in the proximal epididymal tissue of ADGRG2-mutated patients. ADGRG2 expression was restricted to the apical membranes of non-ciliated epithelia in human efferent ducts, which was consistent with a previous report in mice. Both ADGRG2-mutated patients had normal spermatogenesis and had successful clinical outcomes following ICSI. CONCLUSIONS: Our study verifies the pathogenic role of ADGRG2 in X-linked CBAVD and broadens the spectrum of ADGRG2 mutations. In addition, we found positive ICSI outcomes in the two ADGRG2-mutated CBAVD patients.


Assuntos
Azoospermia/genética , Infertilidade Masculina/genética , Doenças Urogenitais Masculinas/genética , Receptores Acoplados a Proteínas G/genética , Ducto Deferente/anormalidades , Adulto , Animais , Azoospermia/fisiopatologia , Regulação da Expressão Gênica no Desenvolvimento/genética , Hemizigoto , Humanos , Infertilidade Masculina/patologia , Mutação com Perda de Função/genética , Masculino , Doenças Urogenitais Masculinas/patologia , Camundongos , Injeções de Esperma Intracitoplásmicas/normas , Espermatogênese/genética , Ducto Deferente/patologia , Sequenciamento Completo do Exoma
15.
J Assist Reprod Genet ; 37(4): 829-840, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32124190

RESUMO

PURPOSE: To investigate the relation between mutations in ciliopathy-related SPAG6 and RSPH3 and male infertility with severe asthenoteratospermia characterized by multiple flagellar malformations and reveal the intracytoplasmic sperm injection (ICSI) outcomes of those primary ciliary dyskinesia (PCD) patients. METHODS: Whole-exome sequencing was applied to identify the pathogenic genes for the five PCD patients. The ICSI outcomes of those patients were compared with eight DNAH1-mutated patients and 215 oligo-asthenospermia (OAT) patients. RESULTS: We identified, for the first time, the compound heterozygous SPAG6 mutations (c.143_145del: p.48_49del, c.585delA: p.Lys196Serfs*6) in a sporadic PCD patient. Further, a novel homozygous nonsynonymous RSPH3 mutation (c.C799T: p.Arg267Cys) was identified in another PCD patient with consanguineous parents. The pathogenicity of these mutations in the assembly of sperm flagella was confirmed by flagellar ultrastructure analysis, immunofluorescence, and quantitative real-time PCR. All five patients underwent six ICSI cycles. The fertilization rate, blastocyst development rate, and clinical pregnancy rate were 69.3%, 50.0%, and 66.7%, respectively. Four of the five couples, including the subjects carrying mutations in SPAG6 or RSPH3, got healthy children born after ICSI. Additionally, the ICSI outcomes of the five PCD couples were statistically comparable with those of the eight DNAH1-mutated couples and the 215 OAT couples. CONCLUSIONS: Mutations in ciliopathy-related SPAG6 and RSPH3 cause severe asthenoteratospermia characterized by multiple flagellar malformations, resulting in sterility. ICSI is an optimal management with a positive pregnancy outcome.


Assuntos
Astenozoospermia/genética , Dineínas/genética , Infertilidade Masculina/genética , Proteínas dos Microtúbulos/genética , Proteínas do Tecido Nervoso/genética , Adulto , Astenozoospermia/diagnóstico , Astenozoospermia/patologia , Feminino , Homozigoto , Humanos , Infertilidade Masculina/diagnóstico , Infertilidade Masculina/patologia , Masculino , Mutação/genética , Gravidez , Resultado da Gravidez , Taxa de Gravidez , Injeções de Esperma Intracitoplásmicas , Espermatozoides/patologia , Sequenciamento Completo do Exoma , Adulto Jovem
16.
Reprod Biomed Online ; 40(4): 501-509, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32201115

RESUMO

RESEARCH QUESTION: Does calcium ionophore treatment of oocytes improve fertilization rate, embryo development and outcomes in specific groups of infertile couples? DESIGN: This retrospective cohort study involved 796 couples undergoing oocyte activation with calcium ionophore (A23187) after intracytoplasmic sperm injection (ICSI) between 2016 and 2018. All metaphase II oocytes were exposed to 5 µmol/l ionophore for 15 min immediately after ICSI, cultured in vitro to the blastocyst stage, and transferred to the uteri of recipients on day 5 or cryopreserved for transfer in the next cycle. The previous cycles of the same patients formed the control group. RESULTS: Among 1261 ICSI cycles and 796 ICSI-artificial oocyte activation (ICSI-AOA) cycles, implantation, positive beta-HCG, clinical pregnancy and live birth rates were significantly (P < 0.05 to P < 0.001) improved for all groups, compared with previous cycles, except live birth rate in women with primary ovarian insufficiency (POI). Compared with previous cycles, rates of blastulation (all P < 0.001) and high-quality blastocysts (P < 0.05 to P < 0.001) were increased significantly for couples with male factor (oligoasthenoteratozoospermia [OAT]), unexplained infertility and couples with both factors in the ICSI-AOA cycles. High-quality blastocyst rate was increased in couples with polycystic ovary syndrome (PCOS) (P = 0.0453). Miscarriage rates were decreased significantly (P < 0.05 to P < 0.001) in couples with OAT, PCOS and unexplained infertility in the treatment cycles. No significant differences were found for fertilization rate, embryo development or live birth rate in patients with POI between both groups. CONCLUSIONS: Artificial oocyte activation was able to 'rescue' the poor reproductive outcomes in certain types of infertile couples with history of failure to achieve pregnancy.


Assuntos
Calcimicina/administração & dosagem , Ionóforos de Cálcio/administração & dosagem , Fertilização In Vitro/métodos , Infertilidade/terapia , Oócitos/efeitos dos fármacos , Injeções de Esperma Intracitoplásmicas , Adulto , Coeficiente de Natalidade , Calcimicina/uso terapêutico , Ionóforos de Cálcio/uso terapêutico , Transferência Embrionária , Feminino , Humanos , Infertilidade/tratamento farmacológico , Nascido Vivo , Masculino , Recuperação de Oócitos , Oócitos/citologia , Gravidez , Taxa de Gravidez , Estudos Retrospectivos , Resultado do Tratamento
17.
Exp Ther Med ; 19(2): 956-964, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-32010257

RESUMO

Infantile neuroaxonal dystrophy (INAD) is a rare, lethal, autosomal recessive neurodegenerative disease and leads to progressive impairment of movement and cognition. A couple with a proband child with calcium-independent group VI phospholipase A2 (PLA2G6)-associated INAD and a previous affected pregnancy sought pre-implantation genetic diagnosis (PGD) to bear a healthy child. Intracytoplasmic sperm injection treatment was performed and 15 blastocystic embryos were obtained at days 5 and 6, and these biopsies were amplified. PGD was performed by next-generation sequencing-based linkage analysis in conjunction with aneuploidy screening. Only two embryos were considered for transfer. In the second frozen-thawed embryo transfer cycle, transfer of a mosaic PLA2G6 c.692G>T heterozygous embryo resulted in a singleton ongoing pregnancy. Prenatal diagnosis was performed using amniotic fluid cells, providing results consistent with those of PGD. The aneuploidy screen and karyotype analysis indicated that the chromosomes of the fetus were normal without any mosaicism. The present study reported the first successful PGD for INAD. For parents at risk, this strategy may successfully lead to pregnancies with embryos unlikely to develop INAD, thus providing valuable experience in reproductive management regarding INAD and potentially other single-gene disorders.

18.
J Med Genet ; 57(7): 445-453, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32051257

RESUMO

BACKGROUND: Asthenoteratospermia, one of the most common causes for male infertility, often presents with defective sperm heads and/or flagella. Multiple morphological abnormalities of the sperm flagella (MMAF) is one of the common clinical manifestations of asthenoteratospermia. Variants in several genes including DNAH1, CEP135, CATSPER2 and SUN5 are involved in the genetic pathogenesis of asthenoteratospermia. However, more than half of the asthenoteratospermia cases cannot be explained by the known pathogenic genes. METHODS AND RESULTS: Two asthenoteratospermia-affected men with severe MMAF (absent flagella in >90% spermatozoa) from consanguineous families were subjected to whole-exome sequencing. The first proband had a homozygous missense mutation c.188G>A (p.Arg63Gln) of DZIP1 and the second proband had a homozygous stop-gain mutation c.690T>G (p.Tyr230*). Both of the mutations were neither detected in the human population genome data (1000 Genomes Project, Exome Aggregation Consortium) nor in our own data of a cohort of 875 Han Chinese control populations. DZIP1 encodes a DAZ (a protein deleted in azoospermia) interacting protein, which was associated with centrosomes in mammalian cells. Immunofluorescence staining of the centriolar protein Centrin1 indicated that the spermatozoa of the proband presented with abnormal centrosomes, including no concentrated centriolar dot or more than two centriolar dots. HEK293T cells transfected with two DZIP1-mutated constructs showed reduced DZIP1 level or truncated DZIP1. The Dzip1-knockout mice, generated by the CRSIPR-Cas9, revealed consistent phenotypes of severe MMAF. CONCLUSION: Our study strongly suggests that homozygous DZIP1 mutations can induce asthenoteratospermia with severe MMAF. The deficiency of DZIP1 induces sperm centrioles dysfunction and causes the absence of flagella.


Assuntos
Anormalidades Múltiplas/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Astenozoospermia/genética , Anormalidades Múltiplas/patologia , Animais , Astenozoospermia/patologia , Exoma/genética , Células HEK293 , Homozigoto , Humanos , Infertilidade Masculina , Masculino , Camundongos , Camundongos Knockout , Mutação/genética , Cauda do Espermatozoide/metabolismo , Cauda do Espermatozoide/patologia , Espermatozoides/metabolismo , Espermatozoides/patologia , Sequenciamento Completo do Exoma
19.
J Med Genet ; 57(2): 89-95, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31501240

RESUMO

BACKGROUND: Male 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 and irregular-calibre flagella resulting in severely decreased motility. Previous studies reported several MMAF-associated genes accounting for approximately half of MMAF cases. METHODS AND RESULT: We conducted genetic analysis using whole-exome sequencing in 88 Han Chinese MMAF probands. CFAP65 homozygous mutations were identified in four unrelated consanguineous families, and CFAP65 compound heterozygous mutations were found in two unrelated cases with MMAF. All these CFAP65 mutations were null, including four frameshift mutations (c.1775delC [p.Pro592Leufs*8], c.3072_3079dup [p.Arg1027Profs*41], c.1946delC [p.Pro649Argfs*5] and c.1580delT [p.Leu527Argfs*31]) and three stop-gain mutations (c.4855C>T [p.Arg1619*], c.5270T>A [p.Leu1757*] and c.5341G>T [p.Glu1781*]). Additionally, two homozygous CFAP65 variants likely affecting splicing were identified in two MMAF-affected men of Tunisian and Iranian ancestries, respectively. These biallelic variants of CFAP65 were verified by Sanger sequencing and were absent or very rare in large data sets aggregating sequence information from various human populations. CFAP65, encoding the cilia and flagella associated protein 65, is highly and preferentially expressed in the testis. Here we also generated a frameshift mutation in mouse orthologue Cfap65 using CRISPR-Cas9 technology. Remarkably, the phenotypes of Cfap65-mutated male mice were consistent with human MMAF. CONCLUSIONS: Our experimental observations performed on both human subjects and on Cfap65-mutated mice demonstrate that the presence of biallelic mutations in CFAP65 causes the MMAF phenotype and impairs sperm motility.


Assuntos
Anormalidades Múltiplas/genética , Infertilidade Masculina/genética , Proteínas de Membrana/genética , Proteínas de Ligação a RNA/genética , Cauda do Espermatozoide/metabolismo , Anormalidades Múltiplas/patologia , Adulto , Alelos , Animais , Flagelos/genética , Flagelos/patologia , Humanos , Infertilidade Masculina/patologia , Irã (Geográfico) , Masculino , Camundongos , Mutação/genética , Fenótipo , Motilidade Espermática/genética , Cauda do Espermatozoide/patologia , Espermatozoides/crescimento & desenvolvimento , Espermatozoides/patologia , Testículo/patologia , Sequenciamento Completo do Exoma
20.
J Med Genet ; 57(1): 31-37, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31048344

RESUMO

BACKGROUND: Male infertility due to multiple morphological abnormalities of the sperm flagella (MMAF) is a genetically heterogeneous disorder. Previous studies revealed several MMAF-associated genes, which account for approximately 60% of human MMAF cases. The pathogenic mechanisms of MMAF remain to be illuminated. METHODS AND RESULTS: We conducted genetic analyses using whole-exome sequencing in 50 Han Chinese probands with MMAF. Two homozygous stop-gain variants (c.910C>T (p.Arg304*) and c.3400delA (p.Ile1134Serfs*13)) of the SPEF2 (sperm flagellar 2) gene were identified in two unrelated consanguineous families. Consistently, an Iranian subject from another cohort also carried a homozygous SPEF2 stop-gain variant (c.3240delT (p.Phe1080Leufs*2)). All these variants affected the long SPEF2 transcripts that are expressed in the testis and encode the IFT20 (intraflagellar transport 20) binding domain, important for sperm tail development. Notably, previous animal studies reported spontaneous mutations of SPEF2 causing sperm tail defects in bulls and pigs. Our further functional studies using immunofluorescence assays showed the absence or a remarkably reduced staining of SPEF2 and of the MMAF-associated CFAP69 protein in the spermatozoa from SPEF2-affected subjects. CONCLUSIONS: We identified SPEF2 as a novel gene for human MMAF across the populations. Functional analyses suggested that the deficiency of SPEF2 in the mutated subjects could alter the localisation of other axonemal proteins.


Assuntos
Proteínas de Ciclo Celular/genética , Homozigoto , Infertilidade Masculina/genética , Mutação , Cauda do Espermatozoide/metabolismo , China , Análise Mutacional de DNA , Humanos , Infertilidade Masculina/metabolismo , Infertilidade Masculina/patologia , Irã (Geográfico) , Masculino , Linhagem , Cauda do Espermatozoide/patologia , Cauda do Espermatozoide/ultraestrutura , Sequenciamento Completo do Exoma
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