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Lung adenocarcinoma (LUAD) is a leading cause of cancer mortality, with many patients facing poor prognosis, particularly those with metastatic or drug-resistant tumors. Homologous recombination genes (HRGs) are crucial in tumor progression and therapy resistance, but their clinical significance in LUAD is not well understood. In this study, we systematically characterize key HRGs in LUAD patients, identifying two distinct HR subtypes associated with different outcomes and biological functions. We establish a 5-gene scoring system (XRCC2, RAD51, BRCA1, FANCA, and CHEK1) that reliably predicts patient outcomes and immunotherapy responses in LUAD. Bioinformatics analysis and clinical validation highlight XRCC2 as a crucial biomarker in LUAD. Functional investigations through in vivo and in vitro experiments reveal the role of XRCC2 in promoting lung cancer migration and invasion. Mechanistically, XRCC2 stabilizes vimentin (VIM) protein expression through deubiquitylation. We predict c-MYC as a potential regulator of XRCC2 and demonstrate that inhibiting c-MYC with compound 10058-F4 reduces XRCC2 and VIM expression. Preclinical studies show the synergistic inhibition of metastasis in vivo when combining 10058-F4 with doxorubicin (Dox). Our findings present a potential personalized predictive tool for LUAD prognosis, identifying XRCC2 as a critical biomarker. The c-Myc-XRCC2-VIM axis emerges as a promising therapeutic target for overcoming lung metastasis. This study provides valuable insights into LUAD, proposing a prognostic tool for further clinical validation and unveiling a potential therapeutic strategy for combating lung metastasis by targeting c-Myc-XRCC2-VIM.
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Lung cancer represents one of the most prevalent malignant neoplasms, commanding an alarming incidence and mortality rate globally. Non-small cell lung cancer (NSCLC), constituting approximately 80â¯%-90â¯% of all lung cancer cases, is the predominant pathological manifestation of this disease, with a disconcerting 5-year survival rate scarcely reaching 10â¯%. Extensive prior investigations have elucidated that the aberrant expression of X-ray repair cross-complementing gene 2 (XRCC2), a critical meiotic gene intricately involved in the DNA damage repair process, is intimately associated with tumorigenesis. Nevertheless, the precise roles and underlying mechanistic pathways of XRCC2 in NSCLC remain largely elusive. In the present study, we discerned an overexpression of XRCC2 within NSCLC patient tissues, particularly in high-grade samples, when juxtaposed with normal tissues. Targeted knockdown of XRCC2 notably impeded the proliferation of NSCLC both in vitro and in vivo. Comprehensive RNA sequencing and flow rescue assays unveiled that XRCC2 augments the proliferation of NSCLC cells through the down-regulation of FOS expression. Moreover, the c-Myc gene was definitively identified as an XRCC2 transcriptional factor by means of chromatin immunoprecipitation (ChIP) and luciferase reporter assays, whereby pharmacological attenuation of c-Myc expression, in conjunction with Doxorubicin, synergistically curtailed NSCLC cell growth both in vitro and in vivo. Collectively, our findings proffer critical insights into the novel c-Myc-XRCC2-FOS axis in promoting both proliferation and resistance to Doxorubicin in NSCLC cells, thereby extending a promising avenue for potential new diagnostic strategies and therapeutic interventions in NSCLC.
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Background: Preimplantation genetic testing (PGT) can reduce the risk of familial genetic diseases, chromosome abnormalities, and recurrent abortions. It is unclear whether genetic counselees with PGT indications understand and accept the implications of PGT. A well-developed and validated tool is needed to evaluate the knowledge, attitude, and practice (KAP) levels of genetic counselees with PGT indications. The purpose of this study was to develop and validate a PGT KAP questionnaire (PGT-KAP-Q) for genetic counselees with PGT indications. Methods: First, we established an item pool based on a literature review and qualitative interviews. Second, we developed the PGT-KAP-Q using the Delphi method. Third, we evaluated the quality of the questionnaire using item analysis and psychometric evaluation. The item analysis included extreme value comparison, application of the correlation and Cronbach's alpha (α) coefficient methods, and factor analysis. We also evaluated the content and structural validity of the questionnaire, as well as the internal consistency, test-retest reliability, and split-half reliability. Findings: After the literature review and interviews, and based on three rounds of expert consultations, we formed a 43-item questionnaire. In the validity analysis, the item's content validity index (I-CVI) and the average scale level CVI (S-CVI/Ave) values (>0.78 and >0.95, respectively) confirmed the questionnaire's content validity. Exploratory factor analysis showed that all 43 items had strong factor loadings (>0.4), and the three factors of the PGT-KAP-Q explained 51.97 % of the total variance. The Cronbach's α coefficient for the questionnaire was 0.95 (p < 0.05), the split-half reliability was 0.76 (p < 0.05) and the test-retest reliability coefficient was 0.78 (p < 0.05). Interpretation: The 43-item PGT-KAP-Q for genetic counselees with PGT indications is reliable and valid. It contains a moderate number of items, is easy for patients to understand and accept, and can be used for clinical research and applications.
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PURPOSE: This study aimed to identify the genetic causes of male infertility and primary ciliary dyskinesia (PCD)/PCD-like phenotypes in three unrelated Han Chinese families. METHODS: We conducted whole-exome sequencing of three patients with male infertility and PCD/PCD-like phenotypes from three unrelated Chinese families. Ultrastructural and immunostaining analyses of patient spermatozoa and respiratory cilia and in vitro analyses were performed to analyze the effects of SPEF2 variants. Intracytoplasmic sperm injection (ICSI) was administered to three affected patients. RESULTS: We identified four novel SPEF2 variants, including one novel homozygous splicing site variant [NC_000005.10(NM_024867.4): c.4447 + 1G > A] of the SPEF2 gene in family 1, novel compound heterozygous nonsense variants [NC_000005.10(NM_024867.4): c.1339C > T (p.R447*) and NC_000005.10(NM_024867.4): c.1645G > T (p.E549*)] in family 2, and one novel homozygous missense variant [NC_000005.10(NM_024867.4): c.2524G > A (p.D842N)] in family 3. All the patients presented with male infertility and PCD/likely PCD. All variants were present at very low levels in public databases, predicted to be deleterious in silico prediction tools, and were further confirmed deleterious by in vitro analyses. Ultrastructural analyses of the spermatozoa of the patients revealed the absence of the central pair complex in the sperm flagella. Immunostaining of the spermatozoa and respiratory cilia of the patients validated the pathogenicity of the SPEF2 variants. All patients carrying SPEF2 variants underwent one ICSI cycle and delivered healthy infants. CONCLUSION: Our study reported four novel pathogenic variants of SPEF2 in three male patients with infertility and PCD/PCD-like phenotypes, which not only extend the spectrum of SPEF2 mutations but also provide information for genetic counseling and treatment of such conditions.
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Infertilidad Masculina , Linaje , Inyecciones de Esperma Intracitoplasmáticas , Espermatozoides , Adulto , Humanos , Masculino , China , Cilios/genética , Cilios/patología , Cilios/ultraestructura , Trastornos de la Motilidad Ciliar/genética , Trastornos de la Motilidad Ciliar/patología , Secuenciación del Exoma , Homocigoto , Infertilidad Masculina/genética , Infertilidad Masculina/patología , Mutación/genética , Fenotipo , Espermatozoides/patología , Espermatozoides/ultraestructura , Espermatozoides/metabolismoRESUMEN
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.
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Astenozoospermia , Secuenciación del Exoma , Homocigoto , Infertilidad Masculina , Cola del Espermatozoide , Espermatozoides , Humanos , Masculino , Cola del Espermatozoide/patología , Cola del Espermatozoide/ultraestructura , Cola del Espermatozoide/metabolismo , Infertilidad Masculina/genética , Infertilidad Masculina/patología , Astenozoospermia/genética , Astenozoospermia/patología , Adulto , Espermatozoides/patología , Espermatozoides/ultraestructura , Mutación/genética , Linaje , Células HEK293 , Pueblo Asiatico/genéticaRESUMEN
STUDY QUESTION: Are there other pathogenic genes for asthenoteratozoospermia (AT)? SUMMARY ANSWER: DNAH3 is a novel candidate gene for AT in humans and mice. WHAT IS KNOWN ALREADY: AT is a major cause of male infertility. Several genes underlying AT have been reported; however, the genetic aetiology remains unknown in a majority of affected men. STUDY DESIGN SIZE DURATION: A total of 432 patients with AT were recruited in this study. DNAH3 mutations were identified by whole-exome sequencing (WES). Dnah3 knockout mice were generated using the genome editing tool. The morphology and motility of sperm from Dnah3 knockout mice were investigated. The entire study was conducted over 3 years. PARTICIPANTS/MATERIALS SETTING METHODS: WES was performed on 432 infertile patients with AT. In addition, two lines of Dnah3 knockout mice were generated. Haematoxylin and eosin (H&E) staining, transmission electron microscopy (TEM), immunostaining, and computer-aided sperm analysis (CASA) were performed to investigate the morphology and motility of the spermatozoa. ICSI was used to overcome the infertility of one patient and of the Dnah3 knockout mice. MAIN RESULTS AND THE ROLE OF CHANCE: DNAH3 biallelic variants were identified in three patients from three unrelated families. H&E staining revealed various morphological abnormalities in the flagella of sperm from the patients, and TEM and immunostaining further showed the loss of the central pair of microtubules, a dislocated mitochondrial sheath and fibrous sheath, as well as a partial absence of the inner dynein arms. In addition, the two Dnah3 knockout mouse lines demonstrated AT. One patient and the Dnah3 knockout mice showed good treatment outcomes after ICSI. LARGE SCALE DATA: N/A. LIMITATIONS REASONS FOR CAUTION: This is a preliminary report suggesting that defects in DNAH3 can lead to asthenoteratozoospermia in humans and mice. The pathogenic mechanism needs to be further examined in a future study. WIDER IMPLICATIONS OF THE FINDINGS: Our findings show that DNAH3 is a novel candidate gene for AT in humans and mice and provide crucial insights into the biological underpinnings of this disorder. The findings may also be beneficial for counselling affected individuals. STUDY FUNDING/COMPETING INTERESTS: This work was supported by grants from National Natural Science Foundation of China (82201773, 82101961, 82171608, 32322017, 82071697, and 81971447), National Key Research and Development Program of China (2022YFC2702604), Scientific Research Foundation of the Health Committee of Hunan Province (B202301039323, B202301039518), Hunan Provincial Natural Science Foundation (2023JJ30716), the Medical Innovation Project of Fujian Province (2020-CXB-051), the Science and Technology Project of Fujian Province (2023D017), China Postdoctoral Science Foundation (2022M711119), and Guilin technology project for people's benefit (20180106-4-7). The authors declare no competing interests.
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Oligoasthenoteratozoospermia (OAT) is a common type of male infertility; however, its genetic causes remain largely unknown. Some of the genetic determinants of OAT are gene defects affecting spermatogenesis. BCORL1 (BCL6 corepressor like 1) is a transcriptional corepressor that exhibits the OAT phenotype in a knockout mouse model. A hemizygous missense variant of BCORL1 (c.2615T > G:p.Val872Gly) was reported in an infertile male patient with non-obstructive azoospermia (NOA). Nevertheless, the correlation between BCORL1 variants and OAT in humans remains unknown. In this study, we used whole-exome sequencing to identify a novel hemizygous nonsense variant of BCORL1 (c.1564G > T:p.Glu522*) in a male patient with OAT from a Han Chinese family. Functional analysis showed that the variant produced a truncated protein with altered cellular localization and a dysfunctional interaction with SKP1 (S-phase kinase-associated protein 1). Further population screening identified four BCORL1 missense variants in subjects with both OAT (1 of 325, 0.31%) and NOA (4 of 355, 1.13%), but no pathogenic BCORL1 variants among 362 fertile subjects. In conclusion, our findings indicate that BCORL1 is a potential candidate gene in the pathogenesis of OAT and NOA, expanded its disease spectrum and suggested that BCORL1 may play a role in spermatogenesis by interacting with SKP1.
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Secuenciación del Exoma , Infertilidad Masculina , Proteínas Represoras , Masculino , Humanos , Proteínas Represoras/genética , Infertilidad Masculina/genética , Infertilidad Masculina/patología , Oligospermia/genética , Oligospermia/patología , Adulto , Linaje , Azoospermia/genética , Azoospermia/patología , Mutación con Pérdida de Función/genética , Predisposición Genética a la Enfermedad , Proteína-Arginina N-Metiltransferasas/genética , Mutación Missense/genética , Espermatogénesis/genéticaRESUMEN
Oligozoospermia and azoospermia are two common phenotypes of male infertility characterized by massive sperm defects owing to failure of spermatogenesis. The deleterious impact of candidate variants with male infertility is to be explored. In our study, we identified three hemizygous missense variants (c.388G>A: p.V130M, c.272C>T: p.A91V, and c.467C>T: p.A156V) and one hemizygous nonsense variant (c.478C>T: p.R160X) in the Rhox homeobox family member 1 gene (RHOXF1) in four unrelated cases from a cohort of 1201 infertile Chinese men with oligo- and azoospermia using whole-exome sequencing and Sanger sequencing. RHOXF1 was absent in the testicular biopsy of one patient (c.388G>A: p.V130M) whose histological analysis showed a phenotype of Sertoli cell-only syndrome. In vitro experiments indicated that RHOXF1 mutations significantly reduced the content of RHOXF1 protein in HEK293T cells. Specifically, the p.V130M, p.A156V, and p.R160X mutants of RHOXF1 also led to increased RHOXF1 accumulation in cytoplasmic particles. Luciferase assays revealed that p.V130M and p.R160X mutants may disrupt downstream spermatogenesis by perturbing the regulation of doublesex and mab-3 related transcription factor 1 (DMRT1) promoter activity. Furthermore, ICSI treatment could be beneficial in the context of oligozoospermia caused by RHOXF1 mutations. In conclusion, our findings collectively identified mutated RHOXF1 to be a disease-causing X-linked gene in human oligo- and azoospermia.
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Azoospermia , Infertilidad Masculina , Oligospermia , Humanos , Masculino , Azoospermia/genética , Azoospermia/patología , Genes Ligados a X , Células HEK293 , Infertilidad Masculina/genética , Oligospermia/genética , SemenRESUMEN
Propensity score approach is a popular technique for estimating the population based on volunteer web survey samples. Various models have been used to estimate propensity scores and produce different population estimates. To obtain more accurate population estimators, we propose a model-averaging estimation approach based on propensity score estimates from a parametric logistic regression model and a nonparametric generalized boosted model. Consistency and asymptotic normality of the proposed estimators are established. A computation algorithm is also developed to implement the proposed method. Simulation studies are conducted to compare the performance of the proposed method with the other methods. A survey data from the Netizen Social Awareness Survey (NSAS) is used to illustrate the proposed methodology.
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Primary ciliary dyskinesia (PCD) is a congenital, motile ciliopathy with pleiotropic symptoms. Although nearly 50 causative genes have been identified, they only account for approximately 70% of definitive PCD cases. Dynein axonemal heavy chain 10 (DNAH10) encodes a subunit of the inner arm dynein heavy chain in motile cilia and sperm flagella. Based on the common axoneme structure of motile cilia and sperm flagella, DNAH10 variants are likely to cause PCD. Using exome sequencing, we identified a novel DNAH10 homozygous variant (c.589C > T, p.R197W) in a patient with PCD from a consanguineous family. The patient manifested sinusitis, bronchiectasis, situs inversus, and asthenoteratozoospermia. Immunostaining analysis showed the absence of DNAH10 and DNALI1 in the respiratory cilia, and transmission electron microscopy revealed strikingly disordered axoneme 9+2 architecture and inner dynein arm defects in the respiratory cilia and sperm flagella. Subsequently, animal models of Dnah10-knockin mice harboring missense variants and Dnah10-knockout mice recapitulated the phenotypes of PCD, including chronic respiratory infection, male infertility, and hydrocephalus. To the best of our knowledge, this study is the first to report DNAH10 deficiency related to PCD in human and mouse models, which suggests that DNAH10 recessive mutation is causative of PCD.
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Trastornos de la Motilidad Ciliar , Semen , Humanos , Masculino , Animales , Ratones , Semen/metabolismo , Dineínas/genética , Dineínas/metabolismo , Cilios/genética , Cilios/metabolismo , Mutación , Trastornos de la Motilidad Ciliar/genéticaRESUMEN
Introduction: Tracing the genetic causes for male infertility due to asthenoteratozoospermia has revealed at least 40 causative genes, which provides valuable reference for the genetic testing of asthenoteratozoospermia in clinical practice. To identify deleterious variants in the human tetratricopeptide repeat domain 12 (TTC12) gene in a large cohort of infertile Chinese males with asthenoteratozoospermia. Methods: A total of 314 unrelated asthenoteratozoospermia-affected men were recruited for whole exome sequencing. The effects of the identified variants were evaluated by in silico analysis, and confirmed by in vitro experiments. Intracytoplasmic sperm injection (ICSI) was used to evaluate the efficiency of assisted reproduction technique therapy. Results and Discussion: Novel homozygous TTC12 variants (c.1467_1467delG (p.Asp490Thrfs*14), c.1139_1139delA (p.His380Profs*4), and c.1117G>A (p.Gly373Arg)) were identified in three (0.96%) of the 314 cases. Three mutants were indicated to be damaging using in silico prediction tools, and were further confirmed by in vitro functional analysis. Hematoxylin and eosin staining and ultrastructural observation of the spermatozoa revealed multiple morphological abnormalities of flagella, with the absence of outer and inner dynein arms. Notably, significant mitochondrial sheath malformations were also observed in the sperm flagella. Immunostaining assays indicated that TTC12 is present throughout the flagella, and was strongly concentrated in the mid-piece in control spermatozoa. However, spermatozoa from TTC12-mutated individuals exhibited almost no staining intensity of TTC12 and outer and inner dynein arms components. The three men accepted ICSI treatment using their ejaculated spermatozoa, and two female partners successfully delivered healthy babies. Our findings provide direct genetic evidence that homozygous variants in TTC12 cause male infertility with asthenoteratozoospermia by causing dynein arm complex defects and mitochondrial sheath malformations in the flagellar. We also demonstrated that TTC12 deficiency-mediated infertility could be overcome by ICSI technology.
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STUDY QUESTION: Can whole-exome sequencing (WES) reveal new genetic factors responsible for male infertility characterized by oligozoospermia? SUMMARY ANSWER: We identified biallelic missense variants in the Potassium Channel Tetramerization Domain Containing 19 gene (KCTD19) and confirmed it to be a novel pathogenic gene for male infertility. WHAT IS KNOWN ALREADY: KCTD19 is a key transcriptional regulator that plays an indispensable role in male fertility by regulating meiotic progression. Kctd19 gene-disrupted male mice exhibit infertility due to meiotic arrest. STUDY DESIGN, SIZE, DURATION: We recruited a cohort of 536 individuals with idiopathic oligozoospermia from 2014 to 2022 and focused on five infertile males from three unrelated families. Semen analysis data and ICSI outcomes were collected. WES and homozygosity mapping were performed to identify potential pathogenic variants. The pathogenicity of the identified variants was investigated in silico and in vitro. PARTICIPANTS/MATERIALS, SETTING, METHODS: Male patients diagnosed with primary infertility were recruited from the Reproductive and Genetic Hospital of CITIC-Xiangya. Genomic DNA extracted from affected individuals was used for WES and Sanger sequencing. Sperm phenotype, sperm nuclear maturity, chromosome aneuploidy, and sperm ultrastructure were assessed using hematoxylin and eosin staining and toluidine blue staining, FISH and transmission electron microscopy. The functional effects of the identified variants in HEK293T cells were investigated via western blotting and immunofluorescence. MAIN RESULTS AND THE ROLE OF CHANCE: We identified three homozygous missense variants (NM_001100915, c.G628A:p.E210K, c.C893T:p.P298L, and c.G2309A:p.G770D) in KCTD19 in five infertile males from three unrelated families. Abnormal morphology of the sperm heads with immature nuclei and/or nuclear aneuploidy were frequently observed in individuals with biallelic KCTD19 variants, and ICSI was unable to rescue these deficiencies. These variants reduced the abundance of KCTD19 due to increased ubiquitination and impaired its nuclear colocalization with its functional partner, zinc finger protein 541 (ZFP541), in HEK293T cells. LIMITATIONS, REASONS FOR CAUTION: The exact pathogenic mechanism remains unclear, and warrants further studies using knock-in mice that mimic the missense mutations found in individuals with biallelic KCTD19 variants. WIDER IMPLICATIONS OF THE FINDINGS: Our study is the first to report a likely causal relationship between KCTD19 deficiency and male infertility, confirming the critical role of KCTD19 in human reproduction. Additionally, this study provided evidence for the poor ICSI clinical outcomes in individuals with biallelic KCTD19 variants, which may guide clinical treatment strategies. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the National Key Research and Developmental Program of China (2022YFC2702604 to Y.-Q.T.), the National Natural Science Foundation of China (81971447 and 82171608 to Y.-Q.T., 82101961 to C.T.), a key grant from the Prevention and Treatment of Birth Defects from Hunan Province (2019SK1012 to Y.-Q.T.), a Hunan Provincial Grant for Innovative Province Construction (2019SK4012), and the China Postdoctoral Science Foundation (2022M721124 to W.W.). The authors declare no conflicts of interest. TRIAL REGISTRATION NUMBER: N/A.
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Astenozoospermia , Infertilidad Masculina , Proteínas Nucleares , Oligospermia , Animales , Humanos , Masculino , Ratones , Astenozoospermia/genética , Proteínas Cromosómicas no Histona , Células HEK293 , Infertilidad Masculina/genética , Oligospermia/genética , Semen , Factores de Transcripción , Proteínas Nucleares/genéticaRESUMEN
Male infertility affects approximately 7% of childbearing couples and is a major health issue. Although nearly 50% idiopathic infertile men are assumed to have a genetic basis, the underlying causes remain largely unknown in most infertility cases. Here, we report two rare homozygous variants in two previously uncharacterized genes, C9orf131 and C10orf120, identified in two unrelated men with asthenozoospermia. Both genes were predominantly expressed in the testes. Furthermore, C9orf131 and C10orf120 knockout mice were successfully generated using the CRISPR-Cas9 technology. However, both C9orf131-/- and C10orf120-/- adult male mice were fertile, with testis-to-body weight ratios comparable to those of wild-type mice. No overt differences were found between wild-type, C9orf131-/-, and C10orf120-/- mice regarding testicular/epididymal tissue morphology, sperm count, sperm motility, or sperm morphology. Moreover, TUNEL assays indicated that the number of apoptotic germ cells in testes was not significantly different between the three groups. In summary, these findings suggest that C9orf131 and C10orf120 are redundant genes in male infertility.
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Astenozoospermia , Fertilidad , Fertilidad/genética , Humanos , Ratones , Astenozoospermia/genética , Ratones Noqueados , Testículo/anatomía & histología , Masculino , Motilidad Espermática , Recuento de Espermatozoides , Espermatozoides/citología , Etiquetado Corte-Fin in Situ , AnimalesRESUMEN
Primate-specific genes (PSGs) tend to be expressed in the brain and testis. This phenomenon is consistent with brain evolution in primates but is seemingly contradictory to the similarity of spermatogenesis among mammals. Here, using whole-exome sequencing, we identified deleterious variants of X-linked SSX1 in six unrelated men with asthenoteratozoospermia. SSX1 is a PSG expressed predominantly in the testis, and the SSX family evolutionarily expanded independently in rodents and primates. As the mouse model could not be used for studying SSX1, we used a non-human primate model and tree shrews, which are phylogenetically similar to primates, to knock down (KD) Ssx1 expression in the testes. Consistent with the phenotype observed in humans, both Ssx1-KD models exhibited a reduced sperm motility and abnormal sperm morphology. Further, RNA sequencing indicated that Ssx1 deficiency influenced multiple biological processes during spermatogenesis. Collectively, our experimental observations in humans and cynomolgus monkey and tree shrew models highlight the crucial role of SSX1 in spermatogenesis. Notably, three of the five couples who underwent intra-cytoplasmic sperm injection treatment achieved a successful pregnancy. This study provides important guidance for genetic counseling and clinical diagnosis and, significantly, describes the approaches for elucidating the functions of testis-enriched PSGs in spermatogenesis.
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Astenozoospermia , Tupaia , Animales , Masculino , Macaca fascicularis , Primates , Semen , Motilidad Espermática , TupaiidaeRESUMEN
BACKGROUND: Spermatogenic impairments can lead to male infertility by different pathological conditions, such as multiple morphological abnormalities of the sperm flagella (MMAF) and non-obstructive azoospermia (NOA). Genetic factors are involved in impaired spermatogenesis. METHODS AND RESULTS: Here, we performed genetic analyses through whole-exome sequencing in a cohort of 334 Han Chinese probands with severe MMAF or NOA. Biallelic variants of CFAP54 were identified in three unrelated men, including one homozygous frameshift variant (c.3317del, p.Phe1106Serfs*19) and two compound heterozygous variants (c.878G>A, p.Arg293His; c.955C>T, p.Arg319Cys and c.4885C>T, p.Arg1629Cys; c.937G>A, p.Gly313Arg). All of the identified variants were absent or extremely rare in the public human genome databases and predicted to be damaging by bioinformatic tools. The men harbouring CFAP54 mutations exhibited abnormal sperm morphology, reduced sperm concentration and motility in ejaculated semen. Significant axoneme disorganisation and other ultrastructure abnormities were also detected inside the sperm cells from men harbouring CFAP54 mutations. Furthermore, immunofluorescence assays showed remarkably reduced staining of four flagellar assembly-associated proteins (IFT20, IFT52, IFT122 and SPEF2) in the spermatozoa of CFAP54-deficient men. Notably, favourable clinical pregnancy outcomes were achieved with sperm from men carrying CFAP54 mutations after intracytoplasmic sperm injection treatment. CONCLUSION: Our genetic analyses and experimental observations revealed that biallelic deleterious mutations of CFAP54 can induce severe MMAF and NOA in humans.
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Azoospermia , Proteínas del Citoesqueleto , Infertilidad Masculina , Femenino , Humanos , Masculino , Embarazo , Azoospermia/patología , Infertilidad Masculina/patología , Mutación , Cola del Espermatozoide/patología , Espermatozoides/patología , Proteínas del Citoesqueleto/genéticaRESUMEN
PURPOSE: The aim of this study is to identify the genetic cause of primary ciliary dyskinesia (PCD) and male infertility in two unrelated Han Chinese families. METHODS: We performed whole-exome sequencing in two unrelated male Han Chinese patients suffering from infertility and PCD to identify the pathogenic variants. Ultrastructural and immunostaining analyses of patient's spermatozoa were performed to characterize the effect of the variants. The pathogenicity of the variants was validated using patient's spermatozoa by western blotting and immunostaining analysis. Intracytoplasmic sperm injection (ICSI) was conducted in the affected families. RESULTS: Three variants in leucine-rich repeat containing 6 (LRRC6) [patient 1(compound heterozygote): NM_012472: c.538C > T, (p.R180*) and c.64dupT, (p.S22Ffs*19); patient 2 (homozygote): c.863C > A, (p.P288H)] were identified in two unrelated patients with PCD and male infertility. These variants were predicated deleterious and were absent or rare in human population genome data. LRRC6-mutant spermatozoa showed a highly aberrant morphology and ultrastructure with lacked inner and outer dynein arms. The LRRC6 protein was present along the normal sperm flagella, and was significantly decreased in the mutated spermatozoa. Interestingly, both patients were able to conceive through ICSI and birthed a healthy baby. CONCLUSION: Our results extend the LRRC6 variant spectrum and provide reproductive guidance to families suffering from PCD-linked infertility caused by LRRC6 variants.
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Trastornos de la Motilidad Ciliar , Infertilidad Masculina , Humanos , Masculino , Trastornos de la Motilidad Ciliar/genética , Proteínas del Citoesqueleto/genética , Pueblos del Este de Asia , Infertilidad Masculina/genética , Mutación/genética , Proteínas/genética , Semen/metabolismo , Espermatozoides/metabolismo , ChinaRESUMEN
(A) Characteristics of spermatozoa in asthenoteratozoospermia affected man. (B) Pedigree and Sanger sequencing analysis of the family. (C) The effect of the missense variant in the CCIN gene.
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Infertilidad Masculina , Semen , Masculino , Humanos , Espermatozoides , Infertilidad Masculina/genética , Mutación Missense , Cabeza del EspermatozoideRESUMEN
BACKGROUND: The genetic causes for most male infertility due to severe oligoasthenoteratozoospermia (OAT) remain unclear. OBJECTIVE: To identify the genetic cause of male infertility characterised by OAT. METHODS: Variant screening was performed by whole-exome sequencing from 325 infertile patients with OAT and 392 fertile individuals. In silico and in vitro analyses were performed to evaluate the impacts of candidate disease-causing variants. A knockout mouse model was generated to confirm the candidate disease-causing gene, and intracytoplasmic sperm injection (ICSI) was used to evaluate the efficiency of clinical treatment. RESULTS: We identified biallelic CFAP61 variants (NM_015585.4: c.1654C>T (p.R552C) and c.2911G>A (p.D971N), c.144-2A>G and c.1666G>A (p.G556R)) in two (0.62%) of the 325 OAT-affected men. In silico bioinformatics analysis predicted that all four variants were deleterious, and in vitro functional analysis confirmed the deleterious effects of the mutants. Notably, H&E staining and electron microscopy analyses of the spermatozoa revealed multiple morphological abnormalities of sperm flagella, the absence of central pair microtubules and mitochondrial sheath malformation in sperm flagella from man with CFAP61 variants. Further immunofluorescence assays revealed markedly reduced CFAP61 staining in the sperm flagella. In addition, Cfap61-deficient mice showed the OAT phenotype, suggesting that loss of function of CFAP61 was the cause of OAT. Two individuals accepted ICSI therapy using their own ejaculated sperm, and one of them succeeded in fathering a healthy baby. CONCLUSIONS: Our findings indicate that CFAP61 is essential for spermatogenesis and that biallelic CFAP61 variants lead to male infertility in humans and mice with OAT.
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Anomalías Múltiples , Astenozoospermia , Infertilidad Masculina , Oligospermia , Humanos , Masculino , Animales , Ratones , Infertilidad Masculina/genética , Oligospermia/genética , Astenozoospermia/genética , Semen , Espermatozoides , Anomalías Múltiples/genéticaRESUMEN
BACKGROUND: Meiosis is an essential stage in the life cycle of sexually reproducing species, underlying formation of haploid gametes and serving as the basis of genetic diversity. A central mechanism of meiosis is recombination between homologous chromosomes, during which programmed DNA double-strand breaks (DSBs) are sequentially repaired to form the crossovers essential for faithful chromosomal segregation. Aberrant meiotic recombination often leads to gametogenic failure or produces aneuploid gametes resulting in subfertility or infertility, miscarriage or birth defects. OBJECTIVE AND RATIONALE: The goal of this review was to characterize the molecular mechanisms of meiotic recombination and related human infertility disorders, particularly male infertility caused by non-obstructive azoospermia (NOA). SEARCH METHODS: Our search included PubMed database articles, focusing mainly on English-language publications dated between January 2016 and February 2022. The search term 'meiosis' was combined with the following keywords: meiotic initiation, chromosome pairing, homologous recombination, chromosome axis, DSB, DSB repair, crossover, meiotic sex chromosome inactivation, meiotic checkpoints, meiotic arrest, NOA, premature ovarian insufficiency (POI) or premature ovarian failure, treatment and cancer. In addition, references within these articles were used to identify additional studies. OUTCOMES: The preliminary search generated â¼3500 records. The majority of articles were identified as meeting abstracts or duplicates, contained non-English text or provided insufficient data and were therefore eliminated. A total of 271 articles associated with meiotic recombination were included in the final analysis. This review provides an overview of molecules and mechanisms involved in meiotic recombination processes, specifically meiosis-specific chromosome structures, DSB formation, homology search, formation of recombination intermediates and crossover formation. The cumulative results suggest that meiosis is regulated sequentially by a series of meiotic recombination genes and proteins. Importantly, mutations in these genes often affect meiotic progression, activating meiotic checkpoints, causing germ cell arrest and leading to subfertility or infertility. At least 26 meiotic recombination-related genes have been reported to be mutated in NOA in men, and 10 of these genes are mutated in POI in women. This suggests that variants of meiotic recombination-related genes can cause human subfertility or infertility, especially NOA. WIDER IMPLICATIONS: Understanding the processes of homologous chromosome pairing, recombination and timely resolution of homologous chromosomes may provide guidance for the analysis of potential monogenetic causes of human subfertility or infertility and the development of personalized treatments. In clinical practice, we can develop a meiotic recombination-related gene panel to screen for gene mutations in individuals with subfertility or infertility. Testicular sperm extraction should not be recommended when an NOA-affected individual carries definite disease-causing mutations of a meiotic gene, so as to avoid the unnecessary invasive diagnosis. Risk of ovarian dysfunction should be evaluated if a woman carries meiotic recombination-related gene mutations. It may be possible to improve or restore fertility through manipulation of meiotic recombination-related genes in the future.