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1.
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.

2.
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.

3.
Clin Genet ; 2021 Sep 27.
Artigo em Inglês | MEDLINE | ID: mdl-34569065

RESUMO

Reduced generation of multiple motile cilia (RGMC) and the consequent primary ciliary dyskinesia (PCD) cause infertility due to a substantial reduction in the number of multiciliated cells (MCCs) in the efferent ducts (EDs)/oviducts. MCIDAS acts upstream of CCNO to regulate the biogenesis of basal bodies (BBs); therefore, both genes play a vital role in the multiciliogenesis of the reproductive tract epithelium. In this study, whole-exome sequencing was performed to identify the causative genes in ten unrelated infertile patients with PCD: seven males and three females. Notably, homozygous frameshift mutations in MCIDAS (c.186dupT, p.Pro63Serfs*22) and CCNO (c.262_263insGGCCC, p.Gln88Argfs*8) were identified in one male and one female participant from two unrelated consanguineous families. Haematoxylin-eosin staining/scanning electron microscopy revealed abnormal MCCs in the mutated EDs/oviducts. Furthermore, transmission electron microscopy revealed significantly reduced BBs. Immunofluorescence staining showed the absence of MCIDAS and CCNO signals in the affected tissues and confirmed that MCIDAS acts upstream of CCNO in the context of multiciliogenesis in the reproductive tract epithelium. In vitro fertilisation (IVF)/intracytoplasmic sperm injection (ICSI) was successful, with a positive pregnancy outcome in both MCIDAS- and CCNO-mutated patients. Our results support the use of IVF/ICSI interventions to treat infertility due to RGMC in couples.

4.
Ecotoxicol Environ Saf ; 225: 112736, 2021 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-34481356

RESUMO

BACKGROUND: Based on a medical record or questionnaire survey approach, previous epidemiological studies have investigated associations between maternal antibiotic exposure during pregnancy and childhood allergic diseases. However, biomonitoring studies on the prenatal low-dose antibiotic exposure, mainly from the environment and contaminated food, and in relation to children allergic diseases, are missing. OBJECTIVES: This research aimed to examine the associations between prenatal low-dose antibiotic exposure measured at multiple time points and children current allergic diseases at 4 years of age. METHODS: The current study including 2453 mother-child pairs was based on the Ma'anshan Birth Cohort study. Selected 41 antibiotics and their two metabolites, which including human antibiotics (HAs), preferred as human antibiotics (PHAs), veterinary antibiotics (VAs) and preferred as veterinary antibiotics (PVAs), in urine samples from 2453 pregnant women were biomonitored through liquid chromatography-triple quadrupole tandem mass spectrometry. Information on children current allergic diseases were collected via validated questionnaires. Generalized estimating equation were used to explore the associations between the repeated measurements of maternal urinary antibiotic over three trimesters and current allergic diseases in children. RESULTS: The detection rates of nine individual antibiotics in the three trimester during pregnancy are greater than 10%, and the 90th percentile concentration of the detected antibiotics ranges from 0.07 to 22.34 µg/g, and the 95th percentile concentration ranges from 0.17 to 59.57 µg/g. Among the participants, each one-unit concentration increment of sulfamethazine (adjusted OR=1.28, 95% CI: 1.10, 1.49, P-FDR=0.014) in the first trimester and ciprofloxacin (adjusted OR=1.17, 95% CI: 1.07, 1.28, P-FDR=0.008) in the second trimester were associated with an increased risk of current eczema in children. In the third trimester, each one-unit concentration increment of oxytetracycline (adjusted OR=1.90, 95% CI: 1.30, 2.78, P-FDR=0.014) was associated with an increased risk of current asthma in children. Gender-stratified analyses demonstrated that no gender differences were observed in the associations between prenatal antibiotic exposure and current allergic diseases in children. CONCLUSIONS: Maternal exposure to certain specific VAs or PVAs (sulfamethazine, ciprofloxacin and oxytetracycline) in different trimesters was associated with an increased risk of current asthma and current eczema in 4-year-old children. No gender differences were found in these associations. Further studies are warranted to confirm our findings and explore the potential mechanisms.


Assuntos
Antibacterianos , Exposição Materna , Criança , Pré-Escolar , Estudos de Coortes , Feminino , Humanos , Gravidez , Trimestres da Gravidez , Estudos Prospectivos
5.
J Med Genet ; 2021 Aug 04.
Artigo em Inglês | MEDLINE | ID: mdl-34348960

RESUMO

BACKGROUND: Oligoasthenoteratozoospermia is a typical feature of sperm malformations leading to male infertility. Only a few genes have been clearly identified as pathogenic genes of oligoasthenoteratozoospermia. METHODS AND RESULTS: Here, we identified a homozygous frameshift variant (c.731dup, p.Asn244Lysfs*3) in CCDC34, which is preferentially expressed in the human testis, using whole-exome sequencing in a cohort of 100 Chinese men with multiple morphological abnormalities of the sperm flagella (MMAF). In an additional cohort of 167 MMAF-affected men from North Africa, Iran and France, we identified a second subject harbouring a homozygous CCDC34 frameshift variant (c.799_817del, p.Glu267Lysfs*72). Both affected men presented a typical MMAF phenotype with an abnormally low sperm concentration (ie, oligoasthenoteratozoospermia). Transmission electron microscopy analysis of the sperm flagella affected by CCDC34 deficiency further revealed dramatic disorganisation of the axoneme. Immunofluorescence assays of the spermatozoa showed that CCDC34 deficiency resulted in almost absent staining of CCDC34 and intraflagellar transport-B complex-associated proteins (such as IFT20 and IFT52). Furthermore, we generated a mouse Ccdc34 frameshift mutant using CRISPR-Cas9 technology. Ccdc34-mutated (Ccdc34mut/mut ) male mice were sterile and presented oligoasthenoteratozoospermia with typical MMAF anomalies. Intracytoplasmic sperm injection has good pregnancy outcomes in both humans and mice. CONCLUSIONS: Our findings support that CCDC34 is crucial to the formation of sperm flagella and that biallelic deleterious mutations in CCDC34/Ccdc34 cause male infertility with oligoasthenoteratozoospermia in humans and mice.

6.
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.

7.
Artigo em Inglês | MEDLINE | ID: mdl-34409526

RESUMO

PURPOSE: To identify the genetic causes for acephalic spermatozoa syndrome. METHODS: Whole-exome sequencing was performed on the proband from a non-consanguineous to identify pathogenic mutations for acephalic spermatozoa syndrome. Quantitative real-time polymerase chain reaction and whole genome sequencing were subjected to detect deletion. The functional effect of the identified splicing mutation was investigated by minigene assay. Western blot and immunofluorescence were performed to detect the expression level and localization of mutant TSGA10 protein. RESULTS: Here, we identified a novel heterozygous splicing mutation in TSGA10 (NM_025244: c.1108-1G > T), while we confirmed that there was a de novo large deletion in the proband. The splicing mutation led to the skipping of the exon15 of TSGA10, which resulted in a truncated protein (p. A370Efs*293). Therefore, we speculated that the splicing mutation might affect transcription and translation without the dosage compensation of a normal allele, which possesses a large deletion including intact TSGA10. Western blot and immunofluorescence demonstrated that the very low expression level of truncated TSGA10 protein led the proband to present the acephalic spermatozoa phenotype. CONCLUSION: Our finding expands the spectrum of pathogenic TSGA10 mutations that are responsible for ASS and male infertility. It is also important to remind us of paying attention to the compound heterozygous deletion in patients from non-consanguineous families, so that we can provide more precise genetic counseling for patients.

8.
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
9.
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
10.
Cells ; 10(7)2021 06 25.
Artigo em Inglês | MEDLINE | ID: mdl-34202084

RESUMO

Male infertility is a multifactorial disease with a strong genetic background. Abnormal sperm morphologies have been found to be closely related to male infertility. Here, we conducted whole-exome sequencing in a cohort of 150 Han Chinese men with asthenoteratozoospermia. Two novel hemizygous mutations were identified in USP26, an X-linked gene preferentially expressed in the testis and encoding a deubiquitinating enzyme. These USP26 variants are extremely rare in human population genome databases and have been predicted to be deleterious by multiple bioinformatics tools. Hematoxylin-eosin staining and electron microscopy analyses of the spermatozoa from men harboring hemizygous USP26 variants showed a highly aberrant morphology and ultrastructure of the sperm heads and flagella. Real-time quantitative PCR and immunoblotting assays revealed obviously reduced levels of USP26 mRNA and protein in the spermatozoa from men harboring hemizygous deleterious variants of USP26. Furthermore, intracytoplasmic sperm injections performed on infertile men harboring hemizygous USP26 variants achieved satisfactory outcomes. Overall, our study demonstrates that USP26 is essential for normal sperm morphogenesis, and hemizygous USP26 mutations can induce X-linked asthenoteratozoospermia. These findings will provide effective guidance for the genetic and reproductive counseling of infertile men with asthenoteratozoospermia.

11.
Medicine (Baltimore) ; 100(26): e26488, 2021 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-34190175

RESUMO

BACKGROUND: The influence of pre-treatment controlling nutritional status (CONUT) score on the prognosis of non-small cell lung cancer (NSCLC) patients is inconclusive. We performed this meta-analysis to evaluate the prognostic significance of CONUT score in NSCLC patients. METHODS: A systematic literature review was conducted using PubMed, Embase, and the Cochrane Library databases. The hazard ratio (HR) and 95% confidence interval (CI) were extracted to assess the correlation between the CONUT score and the overall survival (OS), disease-free survival (DFS), recurrence-free survival (RFS), as well as the cancer-specific survival. RESULTS: A total of 11 studies with 3029 patients were included in the analysis. Pooled results indicated that a high CONUT score was positively correlated with poor OS (HR: 1.63, 95%CI: 1.40-1.88, P < .001) and shortened DFS/RFS (HR: 1.65, 95%CI: 1.35-2.01, P < .001), but no significant relationship with the cancer-specific survival (HR: 1.28, 95%CI: 0.60-2.73, P = .517) was identified. The negative effect of high CONUT score on the OS and DFS/RFS was detected in every subgroup with varying treatment methods, cancer stage, CONUT cut-off values, sample size, and analysis methods of HR. Additionally, preoperative high CONUT score was an independent predictor of postoperative complications (odds ratio: 1.58, 95%CI: 1.21-2.06, P = .001) in NSCLC. Last but not least, high CONUT score was not significantly correlated with the patients' sex, smoking status, cancer stage, lymphatic invasion, vascular invasion, pleural invasion, and pathological cancer type. CONCLUSION: These results demonstrate that high CONUT score is positively related to poor prognoses. The CONUT score may therefore be considered as an effective prognostic marker in NSCLC patients.


Assuntos
Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Estado Nutricional , Complicações Pós-Operatórias/diagnóstico , Carcinoma Pulmonar de Células não Pequenas/patologia , Carcinoma Pulmonar de Células não Pequenas/fisiopatologia , Carcinoma Pulmonar de Células não Pequenas/terapia , Humanos , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/fisiopatologia , Neoplasias Pulmonares/terapia , Estadiamento de Neoplasias , Prognóstico , Fatores de Risco
12.
Orphanet J Rare Dis ; 16(1): 258, 2021 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-34099025

RESUMO

BACKGROUND: To investigate the genetic contribution of copy number variations (CNVs) in Wingless-type MMTV integration site family, member 4 (WNT4), in a Chinese population with Müllerian anomalies (MA), copy number analysis of WNT4 by Multiplex ligation-dependent probe amplification (MLPA) was performed on 248 female patients. Some studies have shown that heterozygous missense mutation of WNT4 can lead to MA. However, few studies on the relationship between WNT4 CNVs and MA have been performed. RESULTS: Among the 248 Chinese women affected by MA in this study, heterozygous deletion of WNT4 was detected in a single patient. CONCLUSIONS: MLPA identified one heterozygous deletion in WNT4 in a single female patient among 248 Chinese women affected by MA. This study firstly reports CNVs of WNT4 in a large sample of MA patients from the Chinese population, which suggests that CNVs of WNT4 cannot be excluded in the occurrence of MA. This provides a genetic basis for precise treatment in the future.


Assuntos
Variações do Número de Cópias de DNA , Reação em Cadeia da Polimerase Multiplex , China , Variações do Número de Cópias de DNA/genética , Feminino , Heterozigoto , Humanos , Proteína Wnt4/genética
13.
Stem Cell Res ; 53: 102372, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-34088001

RESUMO

Primary ciliary dyskinesia (PCD) is an autosomal recessive hereditary disorder affecting motile cilia structure and function, which leads to respiratory diseases and infertility. Here, an induced pluripotent stem cell (iPSC) line of PCD was generated from peripheral blood mononuclear cells of a female patient carrying biallelic mutations in Cyclin O (CCNO) gene. Reprogramming was performed with the non-integrated episomal vectors. The obtained transgene-free iPSCs had normal karyotypes, expressed pluripotency genes, and differentiated into three germ layers. This iPSC line could be a useful guide for studying the pathogenic mechanism, establishing a disease model of PCD, and screening potential therapeutic targets.


Assuntos
Transtornos da Motilidade Ciliar , Células-Tronco Pluripotentes Induzidas , Cílios , Feminino , Humanos , Leucócitos Mononucleares , Mutação
14.
Hum Mol Genet ; 30(21): 1996-2011, 2021 Oct 13.
Artigo em Inglês | MEDLINE | ID: mdl-34169321

RESUMO

Motile cilia and flagellar defects can result in primary ciliary dyskinesia, which is a multisystemic genetic disorder that affects roughly 1:10 000 individuals. The nexin-dynein regulatory complex (N-DRC) links neighboring doublet microtubules within flagella, serving as a central regulatory hub for motility in Chlamydomonas. Herein, we identified two homozygous DRC1 variants in human patients that were associated with multiple morphological abnormalities of the sperm flagella (MMAF) and male infertility. Drc1-/-, Drc1R554X/R554X and Drc1W244X/W244X mice on the C57BL/6 background suffered from pre-pubertal mortality. However, when the ICR background was introduced, some of these mice were able to survive and recapitulate the MMAF phenotypes detected in human patients. By analyzing these animals, we determined that DRC1 is an essential regulator of N-DRC assembly in cilia and flagella. When DRC1 is absent, this results in the shortening of cilia and consequent impairment of their motility. Damage associated with DRC1 deficiency in sperm flagella was more pronounced than in cilia, as manifested by complete axoneme structural disorder in addition to the loss of the DRC structure. Altogether, these findings suggest that DRC1 is required for the structural stability of flagella but not cilia, emphasizing the key role of this protein in mammalian species.

15.
Reprod Sci ; 2021 Jun 22.
Artigo em Inglês | MEDLINE | ID: mdl-34159570

RESUMO

Acephalic spermatozoa syndrome (ASS) is a severe form of teratozoospermia, previous studies have shown that SUN5 mutations are the major cause of acephalic spermatozoa syndrome. This study is to identify the pathogenic mutations in SUN5 leading to ASS. PCR and Sanger sequence were performed to define the breakpoints and mutations in SUN5. Whole genome sequencing (WGS) was performed to detect heterozygous deletion. Western blotting and immunofluorescence analysis detected the expression level and localization of SUN5. Furthermore, the pathogenicity of the mutant SUN5 was predicted in silico and was verified by the experiments in vitro. We identified one novel homozygous missense mutation (c.775G>A; p.G259S) and one compound heterozygous including one reported missense mutation (c.1043A>T; p.N348I) and a large deletion that contains partial EFCAB8 ( NM_001143967 .1) and BPIFB2 ( NM_025227 ) and complete SUN5 ( NM_080675 ), and one recurrent homozygous splice-site mutation (c.340G>A; p.G114R) in SUN5 in three patients with ASS. Our results showed that SUN5 could not be detected in the patients' spermatozoa and the exogenous expression level of the mutant protein was decreased in transfected HEK-293T cells. This study expands the mutational spectrum of SUN5. We recommended a clinical diagnostic strategy for SUN5 genomic deletion to screen heterozygous deletions and indicated that the diagnostic value of screening for SUN5 mutations and deletions in infertile men with ASS.

16.
Hum Reprod ; 36(7): 2020-2034, 2021 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-33974705

RESUMO

STUDY QUESTION: What is the potential of applying non-invasive preimplantation genetic testing (niPGT) for chromosome abnormalities in blastocysts reported with a mosaic trophectoderm (TE) biopsy? SUMMARY ANSWER: niPGT of cell-free DNA in blastocyst culture medium exhibited a good diagnostic performance in putative mosaic blastocysts. WHAT IS KNOWN ALREADY: Advances in niPGT have demonstrated the potential reliability of cell-free DNA as a resource for genetic assessment, but information on mosaic embryos is scarce because the mosaicism may interfere with niPGT. In addition, the high incidence of mosaicism reported in the context of PGT and the viability of mosaic blastocysts raise questions about whether mosaicism really exists. STUDY DESIGN, SIZE, DURATION: The study was performed between May 2020 and July 2020. First, clinical data collected by a single-center over a 6-year period on PGT for chromosome aneuploidies (PGT-A) or chromosomal structural rearrangements (PGT-SR) were analyzed. After confirming the reliability of niPGT, 41 blastocysts classified as mosaics by trophectoderm (TE) biopsy were re-cultured. The chromosomal copy number of the blastocyst embryo (BE, the gold standard), TE re-biopsy, and corresponding cell-free DNA in the culture medium was assessed. PARTICIPANTS/MATERIALS, SETTING, METHODS: Data on patients enrolled for PGT at a single center from 2014 to 2019 were collected and the cycles with available putative mosaic blastocysts were evaluated. To verify the diagnostic validity of niPGT, eight aneuploid blastocysts were thawed and re-cultured for 14-18 h. The concordance of the niPGT diagnosis results and the whole blastocyst testing results was analyzed. Forty-one blastocysts reported as mosaics from 22 patients were included and re-cultured for 14-18 h. The genetic material of the BE, TE re-biopsy, and corresponding cell-free DNA in the culture medium was amplified using multiple annealing and looping-based amplification cycles. The karyotype data from niPGT and TE re-biopsy were compared with that from the whole blastocyst, and the efficiency of niPGT was assessed. MAIN RESULTS AND THE ROLE OF CHANCE: Data on 3738 blastocysts from 785 PGT-A or PGT-SR cycles of 677 patients were collected. According to the TE biopsy report, of the 3662 (98%) successfully amplified samples, 24 (0.6%) yielded no results, 849 (23.2%) were euploid, 2245 (61.3%) were aneuploid, and 544 (14.9%) were mosaic. Sixty patients without euploid blastocysts opted for a single mosaic blastocyst transfer, and 30 (50%) of them obtained a clinical pregnancy. With the BE chromosome copy number as the gold standard, niPGT and TE re-biopsy showed reliable detection ability and diagnostic efficiency in eight putative aneuploid blastocysts. Of the 41 putative mosaic blastocysts re-cultured and re-tested, 35 (85.4%) showed euploid BE results. All but two of the blastocysts previously diagnosed with segmental chromosomal mosaic were actually euploid. In addition, all blastocysts previously classified as low degree (20-50%) mosaics were identified as euploid by BE PGT, whereas four of the six putative high degree (50-80%) mosaic blastocysts showed chromosomal abnormalities. The raw concordance rates of spent culture medium (SCM) and TE re-biopsies compared with BE were 74.4% and 82%, respectively, in terms of overall ploidy and 96.2% and 97.6%, respectively, per single chromosome when considering all degree mosaic results as true positives. However, when we set a mosaicism identification threshold of 50%, the concordance rates of SCM and TE re-biopsies compared with BE were 87.2% and 85% at the overall ploidy level and 98.8% and 98.3% at the chromosomal level, respectively. At the full ploidy level, the sensitivity and false negative rates for niPGT were 100% and 0, respectively. After adjustment of the threshold for mosaicism, the specificity of niPGT increased from 69.7% to 84.8% in terms of overall ploidy and from 96.1% to 98.9% at the chromosomal level. LIMITATIONS, REASONS FOR CAUTION: The primary limitation of this study is the small sample size, which decreases the strength of our conclusions. If possible, identifying the clinical outcome of niPGT on reassessed mosaic blastocysts would be further progress in this field. WIDER IMPLICATIONS OF THE FINDINGS: This study is the first to explore the practicability of niPGT in diagnostic reassessment of putative mosaicism. The present study provides a novel opportunity for patients with only mosaic blastocysts and no euploid blastocysts, regardless of the technical or biological basis of mosaicism. Employing niPGT after 14-18 h of re-culturing might be a superior option for the best use of blastocysts because of its minimally invasive nature. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by grants from National Key Technology Research and Development Program of China (No. 2017YFC1002004), the Central Guiding the Science and Technology Development of the Local (2018080802D0081) and College Natural Science Project of Anhui Province (KJ2019A0287). There are no competing interests to declare. TRIAL REGISTRATION NUMBER: N/A.


Assuntos
Diagnóstico Pré-Implantação , Aneuploidia , Blastocisto , China , Feminino , Testes Genéticos , Humanos , Projetos Piloto , Gravidez , Reprodutibilidade dos Testes
17.
Reprod Sci ; 28(10): 2878-2886, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-33978954

RESUMO

Second-hand smoke (SHS) has been shown to be associated with psychiatric distress in pregnant women spontaneously conceived (SC), but this has never been investigated in pregnant women with assisted reproductive technology (ART) treatment. This study aimed to investigate and compare the associations of SHS with psychiatric distress among SC and ART pregnant women. Participants (1467 SC and 857 ART women) were from the sub-study of Chinese National Birth Cohort (CNBC) in Anhui Province. SHS was assessed by the self-reported questionnaire. The symptoms of depression, anxiety, stress, and poor sleep quality were assessed using CES-D, SAS, CPSS, and PSQI questionnaire. Multivariable linear regression was used to determine the association between SHS and psychiatric distress in each trimester. In SC women, SHS (yes or no) was associated with depression and anxiety symptoms in the 3rd trimester (ß = 0.90, 95% CI 0.07-1.73 for depression and ß = 1.21, 95% CI 0.39-2.04 for anxiety) and stress symptom and poor sleep quality in both the 2nd and 3rd trimesters (ß = 0.85, 95% CI 0.20-1.49 in the 2nd trimester and ß = 0.69, 95% CI 0.07-1.32 in the 3rd trimester for stress, and ß = 1.32, 95% CI 0.68-1.96 in the 2nd trimester and ß = 1.38, 95% CI 0.64-2.11 in the 3rd trimester for poor sleep quality). By contrast, in ART women, SHS was associated with depression and stress symptoms in the 1st trimester (ß = 1.97, 95% CI 0.59-3.35 for depression and ß = 1.18, 95% CI 0.24-2.12 for stress) and poor sleep quality throughout the pregnancy (ß = 0.64, 95% CI 0.22-1.06 in the 1st trimester, ß = 0.77, 95% CI 0.35-1.18 in the 2nd trimester, and ß = 0.99, 95% CI 0.50-1.48 in the 3rd trimester, respectively). Our findings indicate a universal and detrimental effect of SHS on psychiatric health among both SC and ART pregnant women. However, the SHS impact may be more substantial at the early stage of pregnancy for ART women and at later stages for SC women. This implies the importance of reducing SHS exposure during pregnancy and the necessary to be aware of the difference in the effect of SHS on psychiatric distress between SC and ART women.

18.
Reproduction ; 162(1): 73-82, 2021 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-33989172

RESUMO

Emerging evidence has demonstrated that melatonin (MT) plays a crucial role in regulating mammalian reproductive functions. It has been reported that MT has a protective effect on polycystic ovary syndrome (PCOS). However, the protective mechanisms of MT remain poorly understood. This study aims to explore the effect of MT on ovarian function in PCOS and to elucidate the relevant molecular mechanisms in vivo and in vitro. We first analysed MT expression levels in the follicular fluid of PCOS patients. A significant reduction in MT expression levels was noted in PCOS patients. Intriguingly, reduced MT levels correlated with serum testosterone and inflammatory cytokine levels in follicular fluid. Moreover, we confirmed the protective function of MT through regulating autophagy in a DHEA-induced PCOS rat model. Autophagy was activated in the ovarian tissue of the PCOS rat model, whereas additional MT inhibited autophagy by increasing PI3K--Akt pathway expression. In addition, serum-free testosterone, inflammatory and apoptosis indexes were reduced after MT supplementation. Furthermore, we also found that MT suppressed autophagy and apoptosis by activating the PI3K-Akt pathway in the DHEA-exposed human granulosa cell line KGN. Our study showed that MT ameliorated ovarian dysfunction by regulating autophagy in DHEA-induced PCOS via the PI3K-Akt pathway, revealing a potential therapeutic drug target for PCOS.

19.
Clin Genet ; 100(3): 324-328, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-33966269

RESUMO

The genetic causes in most of patients with oocyte maturation arrest remain largely unknown. In this study, we identified a homozygous missense mutation (c.895T>C; p.C299R) in TBPL2 (TATA box binding protein like 2) in two infertile sisters with oocyte maturation arrest and degeneration from a consanguineous family by whole-exome sequencing. The TBPL2 mutation is rare and pathogenic, and impaired the transcription initiation function of the protein. Our results showed that TBPL2 mutation might be associated with female infertility due to oocyte maturation arrest and degeneration.

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