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OBJECTIVE: To investigate the association between a low oocyte maturity ratio from in vitro fertilization cycle and blastocyst euploidy. METHODS: A total of 563 preimplantation genetic testing (PGT) cycles (PGT cycles with chromosomal structural rearrangements were excluded) were performed between January 2021 and November 2022 at our center (average oocyte maturity rate: 86.4% ± 14.6%). Among them, 93 PGT cycles were classified into the low oocyte maturity rate group (group A, < mean - 1 standard deviation [SD]), and 186 PGT cycles were grouped into the average oocyte maturity rate group (group B, mean ± 1 SD). Group B was 2:1 matched with group A. Embryological, blastocyst ploidy, and clinical outcomes were compared between the two groups. RESULTS: The oocyte maturity (metaphase II [MII oocytes]), MII oocyte rate, and two pronuclei (2PN) rates were significantly lower in group A than in group B (5.2 ± 3.0 vs. 8.9 ± 5.0, P = 0.000; 61.6% vs. 93.0%, P = 0.000; 78.7% vs. 84.8%, P = 0.002, respectively). In group A, 106 of 236 blastocysts (44.9%) that underwent PGT for aneuploidy were euploid, which was not significantly different from the rate in group B (336/729, 46.1%, P = 0.753). However, euploid blastocysts were obtained only in 55 cycles in group A (55/93, 59.1%), which was lower than the rate in group B (145/186, 78.0%, P = 0.001). The clinical pregnancy rate in group B (73.9%) was higher than that in group A (58.0%) (P = 0.040). CONCLUSION: Our results suggest that a low oocyte maturity ratio is not associated with blastocyst euploidy but is associated with fewer cycles with euploid blastocysts for transfer, lower 2PN rates, and lower clinical pregnancy rates.
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OBJECTIVE: To determine whether a reduced dose of follicle-stimulating hormone (FSH) before human chorionic gonadotropin (hCG) trigger during ovarian stimulation can affect in vitro fertilization (IVF) outcomes. METHODS: This study included 347 patients with a normal ovarian response who received a reduced dose of FSH before hCG trigger for 2-3 days (Group A) and 671 patients who did not receive a reduced dose (Group B) from a university-affiliated IVF center between January 2021 and December 2022. The primary endpoint was estrogen (E2) and progesterone (P) levels on the day of hCG trigger, fresh embryo transfer cycles, laboratory outcomes, and clinical outcomes between the two groups. RESULTS: On the day of hCG trigger, Group A had significantly lower E2 and P levels than those in Group B (3454.95 ± 1708.14 pg/mL versus 3798.70 ± 1774.26 pg/mL, p = 0.003; and 1.23 ± 0.53 ng/mL versus 1.37 ± 0.59 ng/mL, p < 0.001, respectively). The proportion of patients with P levels ≥ 1.5 ng/mL was 22.48% in Group A compared to 34.58% in Group B (p < 0.001), while the proportion of patients with E2 ≥ 5000 pg/mL was 15.27% in Group A compared to 25.93% in Group B (p < 0.001). The fresh embryo-transfer cycle rate in Group A was higher than that in group B (54.47% and 32.64%, respectively; p < 0.001). Despite the reduction in FSH dosage, there were no significant differences between groups regarding the number of oocytes retrieved, total number of mature oocytes, normal fertilization rate, cleavage rate, Day 3 top-quality rate, implantation rate, pregnancy rate per cycle, and early pregnancy loss rate. CONCLUSION: While a reduced dose of FSH prior to hCG trigger during ovarian stimulation did not significantly affect IVF outcomes, it was associated with lower E2 and P levels, resulting in fewer cycles with E2 ≥ 5000 pg/mL and P ≥ 1.5 ng/mL on the day of the hCG trigger.
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Hormona Folículo Estimulante Humana , Hormona Folículo Estimulante , Femenino , Embarazo , Humanos , Fertilización In Vitro , Transferencia de Embrión , Gonadotropina CoriónicaRESUMEN
X-Linked Alport Syndrome (XLAS) is an X-linked, dominant, hereditary nephropathy mainly caused by mutations in the COL4A5 gene, found on chromosome Xq22. In this study, we reported a pedigree with XLAS caused by a COL4A5 mutation. This family gave birth to a boy with XLAS who developed hematuria and proteinuria at the age of 1 year. We used next-generation sequencing (NGS) to identify mutations in the proband and his parents and confirmed the results using Sanger sequencing. This testing showed there was a single nucleotide missense variation, c.3659G>A (p.Gly1220Asp) (NM_033380.3), in the COL4A5 gene. To prevent the inheritance of the syndrome, we used eight embryos for trophoblast biopsy after assisted reproductive technology treatment, and whole genome amplification (WGA) was performed using multiple annealing and looping-based amplification cycles (MALBAC). Embryos were subjected to Preimplantation Genetic Testing (PGT) procedures, including Sanger sequencing, NGS-based single nucleotide polymorphism (SNP) haplotype linkage analysis, and chromosomal copy number variation (CNV) analysis. The results showed that three embryos (E1, E2, and E4) were free of CNV and genetic variation in the COL4A5 gene. Embryo E1 (4AA) was transferred after consideration of the embryo growth rate, morphology, and PGT results. Prenatal diagnosis in the second trimester showed that the fetus had a normal karyotype and did not carry the COL4A5 mutation (c.3659G>A). Ultimately, a healthy boy was born and did not carry the pathogenic COL4A5 mutation, which indicated that PGT prevented the intergenerational transmission of the causative mutation of XLAS.
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BACKGROUND: The contents of ovarian endometrioma (OMA) such as inflammatory mediators, reactive oxygen species, and iron may disrupt normal folliculogenesis and result in subsequent oocyte apoptosis. Therefore, women with OMA have a potential risk of diminished ovarian reserve (DOR). The purpose of this study is to compare the in vitro fertilization (IVF) outcomes and efficiency between DOR patients with and without current OMA. METHODS: This retrospective case-control study included a total of 493 women with DOR (serum anti-Müllerian hormone level < 1.1 ng/mL). Ninety patients with OMA (Group A) underwent 191 IVF cycles and 403 patients without ovarian OMA (Group B) underwent 888 IVF cycles in our center between January 2014 and December 2018. Basal characteristics and IVF outcomes were compared between Group A and Group B. Time to achieve live birth were compared between patients with live birth in two groups (Group A1, 31 patients; Group B1, 132 patients). RESULTS: Clinical and demographic characteristics of patients were similar respectively between groups (A vs. B, A1 vs. B1). There were no statistically significant differences in implantation rate, live birth rate per OPU and per ET cycle and the cumulative live birth rate per patient and per patient with good-quality embryos between Group A and Group B (P > 0.05). Total time to achieve live birth has no statistically significant difference between Group A1 and Group B1 (P > 0.05). CONCLUSION: For DOR women, presence of endometrioma did not affect the IVF outcomes. Even the time to get live birth was not prolonged by current OMA.
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Endometriosis , Nacimiento Vivo , Tasa de Natalidad , Estudios de Casos y Controles , Transferencia de Embrión , Femenino , Fertilización In Vitro , Humanos , Masculino , Inducción de la Ovulación , Embarazo , Índice de Embarazo , Estudios RetrospectivosRESUMEN
BACKGROUND: This study aimed to evaluate the ability of next-generation sequencing (NGS) to conduct preimplantation genetic testing (PGT) for thalassemia using affected embryos. METHODS: This study included data from 36 couples who underwent PGT for thalassemia without probands and relative pedigrees. NGS results were compared with prenatal diagnosis results. RESULTS: Thirty-six couples (29 α-thalassemia and 7 ß-thalassemia) underwent 41 PGT cycles (31 α-thalassemia and 10 ß-thalassemia). Analysis using NGS produced conclusive results for all biopsied blastocysts (100%, 217/217). One hundred and sixty (73.7%, 160/217) were unaffected by thalassemia. Preimplantation genetic testing for aneuploidy revealed that 112 (70.0%, 112/160) were euploid. Single blastocysts were transferred into the uteri of 34 women (53 frozen embryo transfer [FET] cycles). Thirty-two cycles resulted in clinical pregnancies, with a clinical pregnancy rate of 60.1% (32/53) per FET cycle. Twenty-two cycles (22 couples) resulted in 23 live births, with a live birth rate of 43.4% (23/53; 3 cycles were ongoing pregnancies). All 25 embryos' prenatal diagnosis results and/or thalassemia gene analyses after delivery were concordant with the NGS-PGT results. Seven embryos (21.9%, 7/32) were miscarried before 12 weeks' gestation, and the abortion villus in four showed a normal karyotype and thalassemia results consistent with the NGS-PGT results. Aborted fetus samples from 3 cycles were not available because the pregnancy lasted less than 5 weeks. CONCLUSION: NGS can be used to conduct PGT for thalassemia using affected embryos as a reference. TRIAL REGISTRATION: Retrospectively registered.
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Diagnóstico Preimplantación , Talasemia alfa , Talasemia beta , Transferencia de Embrión , Femenino , Pruebas Genéticas/métodos , Humanos , Masculino , Embarazo , Diagnóstico Preimplantación/métodos , Talasemia alfa/genética , Talasemia beta/diagnóstico , Talasemia beta/genéticaRESUMEN
Marfan syndrome (MFS), an autosomal dominant heritable disease of the connective tissue, is characterized by broad clinical manifestations in the musculoskeletal, cardiovascular, pulmonary, and ocular systems. In this study, a male patient with MFS caused by a heterozygous mutation NM_000138.5(FBN1):c.6037 + 2 T > C in the fibrillin 1 gene (FBN1) underwent preimplantation genetic testing (PGT) by using affected-embryo-based single nucleotide polymorphism (SNP) haplotyping. Multiple displacement amplification was used for whole genome amplification of biopsied trophectoderm cells after controlled ovarian stimulation. Sanger sequencing and next-generation sequencing (NGS) were used to detect the state of FBN1 mutation. A total of 14 blastocysts formed after intracytoplasmic sperm injection were biopsied. After NGS, 60 informative polymorphic SNP markers located upstream and downstream of the FBN1 gene and its pathogenic mutation site were linked to individual alleles. Sanger sequencing further confirmed that 8 blastocysts carried the mutation NM_000138.5(FBN1):c.6037 + 2 T > C, while 6 did not. Four of the non-carriers were euploid verified by copy number variation results. A female infant without MFS was born at 37 weeks gestation after a subsequent frozen embryo transfer. In conclusion, the successful case indicates that SNP haplotyping using sibling embryos as a reference is applicable to PGT in monogenetic diseases.Abbreviations MFS: Marfan syndrome; PGT: preimplantation genetic testing; FBN1: fibrillin 1 gene; NGS: next-generation sequencing; SNP: single nucleotide polymorphism.
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Síndrome de Marfan , Diagnóstico Preimplantación , Variaciones en el Número de Copia de ADN , Femenino , Fibrilina-1/genética , Pruebas Genéticas , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Masculino , Síndrome de Marfan/diagnóstico , Síndrome de Marfan/genética , Polimorfismo de Nucleótido Simple , EmbarazoRESUMEN
ß-thalassemia, an autosomal recessive blood disorder that reduces the production of hemoglobin, is majorly caused by the point mutation of the HBB gene resulting in reduced or absent ß-globin chains of the hemoglobin tetramer. Animal models recapitulating both the phenotype and genotype of human disease are valuable in the exploration of pathophysiology and for in vivo evaluation of novel therapeutic treatments. The docile temperament, short vital cycles, and low cost of rabbits make them an attractive animal model. However, ß-thalassemia rabbit models are currently unavailable. Here, using CRISPR/Cas9-mediated genome editing, we point mutated the rabbit ß-globin gene HBB2 with high efficiency and generated a ß-thalassemia rabbit model. Hematological and histological analyses demonstrated that the genotypic mosaic F0 displayed a mild phenotype of anemia, and the heterozygous F1 exhibited typical characteristics of ß-thalassemia. Whole-blood transcriptome analysis revealed that the gene expression was altered in HBB2-targeted when compared with WT rabbits. And the highly expressed genes in HBB2-targeted rabbits were enriched in lipid and iron metabolism, innate immunity, and hematopoietic processes. In conclusion, using CRISPR-mediated HBB2 knockout, we have created a ß-thalassemia rabbit model that accurately recapitulates the human disease phenotype. We believe this tool will be valuable in advancing the investigation of pathogenesis and novel therapeutic targets of ß-thalassemia and associated complications.
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Modelos Animales de Enfermedad , Globinas beta/genética , Talasemia beta/genética , Animales , Sistemas CRISPR-Cas , Diferenciación Celular/genética , Edición Génica/métodos , Técnicas de Inactivación de Genes/métodos , Ingeniería Genética/métodos , Células Madre Hematopoyéticas/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Mutación/genética , Conejos , Globinas beta/metabolismo , Talasemia beta/metabolismoRESUMEN
Objectives: To compare successful beta-thalassemia (ß-thalassemia) detection rates obtained using spent culture medium and spent culture medium containing blastocoelic fluid (BF). Method: This study involved data from 10 couples who underwent preimplantation genetic testing (PGT) for ß-thalassemia. A total of 26 samples of spent culture medium containing BF (group A) and 33 samples without BF (group B) were collected and analyzed. The DNA concentration and ß-thalassemia detection rates were evaluated. Results: The HBB mutation analysis results of 34 samples were concordant with the biopsy results (34/59, 57.6%). In group A, the HBB mutation analysis results of 19 of 26 samples (73.1%) were concordant with the biopsy results. The concordance rate in group A was higher than that in group B (15/33, 45.5%; P < 0.05). The haplotyping results of 38 samples were concordant with the biopsy results (38/59, 64.4%). The concordance rate in group B was 17/33 (51.5%), which was significantly lower than that in group A (21/26, 80.8%) (P < 0.05). In group A, the mean DNA concentration of samples with <10% fragmentation was 107.3 ± 70.1 ng/µL, which was lower than that of samples with ≥10% fragmentation (194.6 ± 28.0 ng/µL) (P < 0.05). However, the detection rates of <10% and ≥10% fragmentation were not significantly different (P > 0.05). Conclusion: The ß-thalassemia detection rate with non-invasive PGT using the spent culture medium containing BF was higher than that using the spent culture medium alone. Fragmentation is associated with DNA concentration in the spent culture medium containing BF.
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Medios de Cultivo/análisis , Diagnóstico Preimplantación/métodos , Globinas beta/genética , Talasemia beta/diagnóstico , Biopsia , ADN/análisis , Técnicas de Cultivo de Embriones , Fertilización In Vitro , Humanos , Polimorfismo de Nucleótido Simple , Talasemia beta/genéticaRESUMEN
BACKGROUND: ß-Thalassaemia is a clinically common cause of hereditary haemolytic anaemia stemming from mutations in important functional regions of the ß-globin gene. The rapid development of gene editing technology and induced pluripotent stem cell (iPSC)-derived haematopoietic stem cell (HSC) transplantation has provided new methods for curing this disease. METHODS: Genetically corrected ß-thalassaemia (homozygous 41/42 deletion) iPSCs that were previously established in our laboratory were induced to differentiate into HSCs, which were transplanted into a mouse model of IVS2-654 ß-thalassaemia (B6;129P2-Hbbtm2Unc/J mice) after administration of an appropriate nonmyeloablative conditioning regimen. We also investigated the safety of this method by detecting the incidence of tumour formation in these mice after transplantation. RESULTS: The combination of 25 mg/kg busulfan and 50 mg/(kg day) cyclophosphamide is an ideal nonmyeloablative protocol before transplantation. Genetically corrected ß-thalassaemic HSCs survived and differentiated in nonmyeloablated thalassaemia mice. No tumour formation was observed in the mice for 10 weeks after transplantation. CONCLUSION: Our study provides evidence that the transplantation of genetically corrected, patient-specific iPSCs could be used to cure genetic diseases, such as ß-thalassaemia major.
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Células Madre Pluripotentes Inducidas , Talasemia beta , Animales , Edición Génica , Células Madre Hematopoyéticas , Humanos , Ratones , Globinas beta/genética , Talasemia beta/genética , Talasemia beta/terapiaRESUMEN
PURPOSE: The effects of cigarette smoking on male semen quality are controversial, and the molecular mechanisms underlying how cigarette smoking affects semen quality are not clear yet. METHODS: In this study, semen samples from 70 heavy smokers and 75 non-smokers receiving infertility treatment were included. Basic semen parameters in non-smokers and heavy smokers were evaluated. Levels of glutathione (GSH), lipid reactive oxygen species (ROS), iron and GSH-dependent peroxidase 4 (GPX4) protein level were observed in human seminal plasma and in GC-2Spd cells exposed to cigarette smoke condensate (CSC). RESULTS: Heavy smokers had significantly higher abnormalities (sperm viability and sperm progressive motility) than non-smoking counterparts. Comparing non-smokers group, GSH level was reduced in the group of heavy smokers (P < 0.05). However, the level of lipid ROS and iron were significantly increased (P < 0.05). Besides, GSH level was reduced following treatment with CSC for 24 h, while lipid ROS and iron levels were increased (P < 0.05). However, the levels were reduced after being co-cultured with Ferrostatin-1 (Fer-1) (P < 0.05). The level of GPX4 protein was reduced after being treated with CSC in 24 h, and increased after being co-cultured with Fer-1(P < 0.05). CONCLUSION: Cigarette smoking is associated with high level of ferroptosis in seminal plasma and affect semen quality.
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Fumar Cigarrillos/efectos adversos , Ferroptosis , Espermatozoides/fisiología , Adulto , Estudios de Casos y Controles , Células Cultivadas , China , Ferroptosis/efectos de los fármacos , Ferroptosis/fisiología , Glutatión/metabolismo , Humanos , Infertilidad Masculina/etiología , Infertilidad Masculina/metabolismo , Infertilidad Masculina/patología , Masculino , Especies Reactivas de Oxígeno/metabolismo , Semen/efectos de los fármacos , Semen/metabolismo , Semen/fisiología , Análisis de Semen , Humo/efectos adversos , Motilidad Espermática/efectos de los fármacos , Espermatozoides/efectos de los fármacos , Nicotiana/efectos adversosRESUMEN
BACKGROUND: To compare the concordance between trophectoderm (TE) analysis and whole blastocyst analysis of embryos from chromosomal structural rearrangement (SR) carriers. METHOD: Sixty-three abnormal blastocysts identified by preimplantation genetic testing for chromosomal structural rearrangement (PGT-SR) were included. The whole blastocysts were processed through multiple displacement amplification cycle and sequenced for 24-chromosome aneuploidy screening by next-generation sequencing (NGS). The sequencing results were compared with those of TE biopsy from the same blastocysts and the primary chromosomal rearrangement of the couples. RESULTS: Analysis of the 63 blastocysts showed 68% (43/63) complete concordance between TE sequencing analysis and whole blastocyst results. Approximately one third (20/63, 32%) of the sequencing results showed some level of discordance between the two samples. Of these, 14% (9/63) of the embryos were identified as euploid after whole blastocyst sequencing. Among them, seven blastocysts were classified as chromosome mosaicism (five whole chromosomes, two segmental) after TE analysis, while two displayed non-SR related segmental changes in the TE biopsy. Of the original analyses, 70% (44/63) of findings were associated with the primary parental chromosomal rearrangement, while 30% (19/63) had no association. CONCLUSIONS: TE biopsy with NGS for PGT-SR is an efficient strategy to identify embryos suitable for transfer. While there was a high concordance between TE and whole blastocyst chromosome results, some embryos classified as mosaic in the original analysis and therefore unsuitable for transfer were reclassified as chromosomally balanced. To maximize the number of embryos available for PGT-SR patients, we suggest that embryos with mosaic non-SR chromosomal rearrangement should be stored and considered for transfer after appropriate counseling.
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Aneuploidia , Blastocisto/metabolismo , Aberraciones Cromosómicas , Pruebas Genéticas/métodos , Diagnóstico Preimplantación/métodos , Trofoblastos/metabolismo , Biopsia , Blastocisto/citología , Femenino , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Embarazo , Trofoblastos/citologíaRESUMEN
BACKGROUND: In thawed embryo transfer cycles, the most common method is to transfer the embryos after 2 h of culture. Clinical outcomes of frozen-thawed cleavage embryo transfer cycles regarding the embryos status and the culture time of frozen-thawed cleavage embryos were limited and did not elucidate all unclear issues. OBJECTIVES: The objective of this study was to examine the clinical outcomes of frozen-thawed cleavage embryo transfer cycles according to the embryos status and the culture time (2 h or overnight). MATERIAL AND METHODS: In this retrospective study (5-year period), 1,654 frozen-thawed embryos were analyzed. Firstly, frozen-thawed cleavage embryos were divided into 2 groups according to their status as follows: with at least 1 optimal embryo and without optimal embryos. Secondly, both of them were divided into 2 groups according to the culture time (2 h or overnight). Age of the female, infertility factors, clinical pregnancy, implantation rate, and live birth rate were compared. RESULTS: There were no statistically significant differences in the pregnancy rate, the implantation rate, live birth rate, the miscarriage rate, and the ectopic pregnancy rate in each group. However, the implantation rate increased after 2 h of incubation (41.1%) compared to overnight incubation (36.0%) in the group with at least 1 optimal day-3 embryo (p < 0.05). The cancellation rate in the suboptimal day-3 embryos group (9.1%) was higher than in the group containing at least 1 optimal embryo (0.2%) for the long (overnight) culture (p < 0.05). CONCLUSIONS: The implantation rate can be improved in the optimal day-3 embryos transferred after 2 h of culture, but not for suboptimal day-3 embryos. Some unnecessary transfers can be avoided after overnight culture because of no further cleavage of the embryos.
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Criopreservación , Implantación del Embrión , Transferencia de Embrión/métodos , Fertilización In Vitro/métodos , Femenino , Humanos , Embarazo , Índice de Embarazo , Técnicas Reproductivas Asistidas , Estudios Retrospectivos , Factores de Tiempo , VitrificaciónRESUMEN
BACKGROUND: The outcomes of in vitro fertilization and/or intracytoplasmic sperm injection (IVF/ICSI) are contradictory among individuals with chromosomal polymorphisms. OBJECTIVES: To assess whether chromosomal polymorphisms affect the outcomes of assisted reproductive technologies. SEARCH STRATEGY: Four online databases were searched from inception to September 18, 2017, using terms including "chromosomal polymorphisms" and "In vitro fertilization." SELECTION CRITERIA: The meta-analysis included studies published in any language on IVF/ICSI outcomes in relation to male and/or female chromosomal polymorphisms (n=8). DATA COLLECTION AND ANALYSIS: Data were extracted using a predesigned form. The IVF/ICSI outcomes were then pooled and their heterogeneity assessed. MAIN RESULTS: Male chromosomal polymorphisms showed lowered values for fertilization rate (odds ratio [OR] 1.29, 95% confidence interval [CI] 1.09-1.54; P=0.004); cleavage rate (OR 2.65, 95% CI 1.88-3.72; P<0.001); good quality embryos rate (OR 1.26, 95% CI 1.15-1.39; P<0.001); and live birth rate (OR 1.42, 95% CI 1.10-1.83; P=0.007). By contrast, early spontaneous abortion rate, clinical pregnancy rate, and ongoing pregnancy rate were not affected in this group. No relationship was found between IVF/ICSI outcomes and female chromosomal polymorphisms. CONCLUSIONS: Male, but not female, chromosomal polymorphisms were associated with lowered values for some outcomes of IVF/ICSI.
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Transferencia de Embrión/estadística & datos numéricos , Polimorfismo Genético , Inyecciones de Esperma Intracitoplasmáticas/estadística & datos numéricos , Tasa de Natalidad , Estudios de Casos y Controles , Femenino , Humanos , Masculino , Evaluación de Resultado en la Atención de Salud , Embarazo , Factores SexualesRESUMEN
Spinocerebellar ataxia type 3 (SCA3) is caused by an abnormal expansion of the cytosine-adenine-guanine (CAG) triplet in ATXN3, which translates into a polyglutamine (polyQ) tract within ataxin-3 (ATXN3) protein. Although the pathogenic mechanisms remain unclear, it is well established that expression of mutant forms of ATXN3 carrying an expanded polyQ domain are involved in SCA3 pathogenesis, and several strategies to suppress mutant ATXN3 have shown promising potential for SCA3 treatment. In this study, we described successful clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-mediated deletion of the expanded polyQ-encoding region of ATXN3 in induced pluripotent stem cells (iPSCs) derived from a SCA3 patient, and these patient-specific iPSCs retained pluripotency and neural differentiation following expanded polyQ deletion. Furthermore, the ubiquitin-binding capacity of ATXN3 was retained in the neural cells differentiated from the corrected iPSCs. For the first time, this work provides preliminary data for gene editing by CRISPR/Cas9 in SCA3, and demonstrates the feasibility of using a single-guide RNA pair to delete the expanded polyQ-encoding region of ATXN3, suggesting the potential efficacy of this method for future therapeutic application.
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Ataxina-3/genética , Sistemas CRISPR-Cas , Eliminación de Gen , Células Madre Pluripotentes Inducidas/metabolismo , Enfermedad de Machado-Joseph/genética , Péptidos/genética , Proteínas Represoras/genética , Adulto , Diferenciación Celular/genética , Células Cultivadas , Femenino , Edición Génica/métodos , Humanos , Células Madre Pluripotentes Inducidas/citología , Enfermedad de Machado-Joseph/metabolismo , Enfermedad de Machado-Joseph/patología , Células-Madre Neurales/citología , Células-Madre Neurales/metabolismo , Expansión de Repetición de Trinucleótido/genéticaRESUMEN
47, XXX syndrome is one of several sex-chromosomal aneuploidies, and it has an incidence of approximately 1/1000 in newborn females. Because of heterogeneity in X-inactivation, these patients may exhibit a variety of clinical symptoms. Here, we report the generation of an integration-free human induced pluripotent stem cell line (GZHMUi001-A) by using Sendai virus to reprogram peripheral blood mononuclear cells from a 47, XXX syndrome patient with premature ovarian failure. This 47, XXX iPS cell line has characteristics of pluripotent stem cells and is a useful tool for the investigation of this X chromosome aneuploid disease.
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Técnicas de Cultivo de Célula/métodos , Reprogramación Celular , Células Madre Pluripotentes Inducidas/patología , Leucocitos Mononucleares/patología , Trastornos de los Cromosomas Sexuales del Desarrollo Sexual/patología , Trisomía/patología , Cromosomas Humanos X , Femenino , Humanos , Aberraciones Cromosómicas Sexuales , Adulto JovenRESUMEN
OBJECTIVE: To investigate the in vitro effects of different culture systems on hematopoietic differentiation ability of induced pluripotent stem (iPS) cells. METHOD: Two culture systems including E8 and mTESR(freeder-free medium), and the classical ES culture medium were chosen for culture of iPS cells. The iPS cells maintaining in above mentioning culcure systems were co-cultured with OP9 cells(murine bone marrow stromal cells) in vitro to be induced to differentiate into hematopoietic stem/progenitor cells. Flow cytometry and real-time quantitative PCR were used to detect the expression of specific hematopoietic markers and the effects of different culture systems on the differentiation of iPS in vitro. RESULT: iPS cultured in the 3 selected medium could be differentiated into hematopoietic stem cells. Efficiency of hematopoietic differentiation was up to 28.4% in classical ES culture system, which was significantly higher than that in E8 and mTESR system. CONCLUSION: Under the co-culture with OP9, iPS can differentiate into hematopoietic stem/progenitor cells, which shows higher efficiency when iPS maintained in the ES medium.
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Diferenciación Celular , Células Madre Pluripotentes Inducidas , Animales , Células Cultivadas , Técnicas de Cocultivo , Citometría de Flujo , Células Madre Hematopoyéticas , Células Madre Mesenquimatosas , RatonesRESUMEN
Spinocerebellar ataxia-3 (SCA3) is the most common dominant inherited ataxia worldwide and is caused by an unstable CAG trinucleotide expansion mutation within the ATXN3 gene, resulting in an expanded polyglutamine tract within the ATXN3 protein. Many in vitro studies have examined the role of autophagy in neurodegenerative disorders, including SCA3, using transfection models with expression of pathogenic proteins in normal cells. In the current study, we aimed to develop an improved model for studying SCA3 in vitro using patient-derived cells. The patient-derived iPS cells presented a phenotype similar to that of human embryonic stem cells and could be differentiated into neurons. Additionally, these cells expressed abnormal ATXN3 protein without changes in the CAG repeat length during culture for at least 35 passages as iPS cells, up to 3 passages as neural stem cells, and after 4 weeks of neural differentiation. Furthermore, we demonstrated that neural differentiation in these iPS cells was accompanied by autophagy and that rapamycin promoted autophagy through degradation of mutant ATXN3 proteins in neurally differentiated spinocerebellar ataxia-3 human induced pluripotent stem cells (p < 0.05). In conclusion, patient-derived iPS cells are a good model for studying the mechanisms of SCA3 and may provide a tool for drug discovery in vitro.
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Ataxina-3/metabolismo , Autofagia/genética , Células Madre Pluripotentes Inducidas/metabolismo , Enfermedad de Machado-Joseph/metabolismo , Proteínas Represoras/metabolismo , Ataxina-3/genética , Diferenciación Celular/genética , Línea Celular , Regulación de la Expresión Génica , Humanos , Células Madre Pluripotentes Inducidas/patología , Cariotipo , Enfermedad de Machado-Joseph/genética , Enfermedad de Machado-Joseph/patología , Células-Madre Neurales/metabolismo , Células-Madre Neurales/patología , Proteolisis , Proteínas Represoras/genética , Repeticiones de Trinucleótidos/genéticaRESUMEN
ß-thalassemia results from point mutations or small deletions in the ß-globin (HBB) gene that ultimately cause anemia. The generation of induced pluripotent stem cells (iPSCs) from the somatic cells of patients in combination with subsequent homologous recombination-based gene correction provides new approaches to cure this disease. CRISPR/Cas9 is a genome editing tool that is creating a buzz in the scientific community for treating human diseases, especially genetic disorders. Here, we reported that correction of ß-thalassemia mutations in patient-specific iPSCs using the CRISPR/Cas9 tool promotes hematopoietic differentiation in vivo. CRISPR/Cas9-corrected iPSC-derived hematopoietic stem cells (HSCs) were injected into sublethally-irradiated NOD-scid-IL2Rg-/- (NSI) mice. HBB expression was observed in these HSCs after hematopoietic differentiation in the NSI mice. Importantly, no tumor was found in the livers, lungs, kidneys, or bone marrow at 10 weeks in the NSI mice after implantation with these HSCs. Collectively, our findings demonstrated that CRISPR/Cas9 successfully corrects ß-thalassemia mutations in patient-specific iPSCs. These CRISPR/Cas9-corrected iPSC-derived HSCs express normal HBB in mice without tumorigenic potential, suggesting a safe strategy for personalized treatment of ß-thalassemia.
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Sistemas CRISPR-Cas , Terapia Genética/métodos , Células Madre Hematopoyéticas/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Globinas beta/genética , Talasemia beta/terapia , Animales , Diferenciación Celular , Femenino , Edición Génica/métodos , Expresión Génica , Trasplante de Células Madre Hematopoyéticas , Células Madre Hematopoyéticas/citología , Recombinación Homóloga , Humanos , Células Madre Pluripotentes Inducidas/patología , Subunidad gamma Común de Receptores de Interleucina/deficiencia , Subunidad gamma Común de Receptores de Interleucina/genética , Ratones , Ratones Endogámicos NOD , Ratones SCID , Mutación , Irradiación Corporal Total , Globinas beta/deficiencia , Talasemia beta/genética , Talasemia beta/metabolismo , Talasemia beta/patologíaRESUMEN
The CRISPR-Cas9 system is a new targeted nuclease for genome editing, which can directly introduce modifications at the targeted genomic locus. The system utilizes a short single guide RNA (sgRNA) to direct the endonuclease Cas9 in the genome. Upon targeting, Cas9 can generate DNA double-strand breaks (DSBs). As such DSBs are repaired by non-homologous end joining (NHEJ) or homology directed repair (HDR), therefore facilitates introduction of random or specific mutations, repair of endogenous mutations, or insertion of DNA elements. The system has been successfully used to generate gene targeted cell lines including those of human, animals and plants. This article reviews recent advances made in this rapidly evolving technique for the generation of animal models for human diseases.
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Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Modelos Animales de Enfermedad , Edición de ARN/genética , Animales , HumanosRESUMEN
ß-Thalassemia (ß-Thal) is one of the most common genetic diseases in the world. The generation of patient-specific ß-Thal-induced pluripotent stem cells (iPSCs), correction of the disease-causing mutations in those cells, and then differentiation into hematopoietic stem cells offers a new therapeutic strategy for this disease. Here, we designed a CRISPR/Cas9 to specifically target the Homo sapiens hemoglobin ß (HBB) gene CD41/42(-CTTT) mutation. We demonstrated that the combination of single strand oligodeoxynucleotides with CRISPR/Cas9 was capable of correcting the HBB gene CD41/42 mutation in ß-Thal iPSCs. After applying a correction-specific PCR assay to purify the corrected clones followed by sequencing to confirm mutation correction, we verified that the purified clones retained full pluripotency and exhibited normal karyotyping. Additionally, whole-exome sequencing showed that the mutation load to the exomes was minimal after CRISPR/Cas9 targeting. Furthermore, the corrected iPSCs were selected for erythroblast differentiation and restored the expression of HBB protein compared with the parental iPSCs. This method provides an efficient and safe strategy to correct the HBB gene mutation in ß-Thal iPSCs.