RESUMEN
OBJECTIVE: To evaluate the applicability of the polymorphic marker closely linked with beta-globin gene for the preimplantation genetic diagnosis (PGD) in couples at risk of having child with beta-thalassemia. METHODS: Single cell multiplex nested PCR which coamplifies the beta-globin gene and the closely linked polymorphic marker, HumTHO1 gene, was applied in six clinical PGD cycles for four couples with beta-thalassemia. RESULTS: In six clinical PGD cycles, a total of 44 embryos were biopsied and 44 blastomeres were obtained. Forty-one blastomeres were amplified and thirty-five embryos were given definite diagnoses. Fourteen embryos were transferred back to the uterus of the patients and one pregnancy went on well and ended with one live healthy birth, which confirmed the results of PGD. The average amplification efficiency of single blastomere was 89.7% and the average allele drop-out(ADO) rate was 14.4%. The coamplification of HumTHO1 could help to detect the existence of ADO and contamination. CONCLUSION: This is the first report on unaffected pregnancy resulting from PGD using multiplex nested PCR in China. The simultaneous amplification of polymorphic marker closely linked to beta-globin gene(HumTHO1) could help to resist the risk of misdiagnosis in PGD caused by ADO and contamination.
Asunto(s)
Diagnóstico Preimplantación/métodos , Talasemia beta/genética , Adulto , Femenino , Humanos , Masculino , Reacción en Cadena de la Polimerasa , Embarazo , Globinas beta/genética , Talasemia beta/diagnósticoRESUMEN
OBJECTIVE: To develop single-cell fluorescent gap polymerase chain reaction (PCR) assay for preimplantation genetic diagnosis (PGD) in couples at risk of having child with alpha-thalassemia. METHODS: Single cell fluorescent gap PCR which can detect the alpha-thalassemia Southeast Asia deletion (-SEA deletion), was applied to single lymphocytes and blastomeres which coming from four clinical PGD cycles. RESULTS: The Single cell fluorescent gap PCR can detect the alpha-thalassemia -SEA deletion, which account for 94% of hydrop fetalis in Chinese population with an amplification efficiency of 90.0% (72/80) and allele drop-out (ADO) rate of 8.3% (6/72) in lymphocytes. In four clinical PGD cycles, a total 38 embryos were detected and 38 blastomeres were obtained. Thirty-four blastomeres were amplified with the amplification efficiency of 89.5% (34/38) and ADO rate of 5.9% (2/34). Eleven embryos were shown to be normal homozygous, eight embryos were shown to be heterozygous and 15 embryos were shown to be affected homozygous. Eleven embryos were transferred back to the uterus of the patients. Two pregnancy achieved, resulting in two live healthy births, which confirmed the results of PGD. CONCLUSION: This first reported unaffected pregnancy resulting from PGD using fluorescent gap PCR for alpha-thalassemia demonstrates that this technique, as an alternative to prenatal diagnosis, is a reliable and effective way to help those carrier couples to get a healthy baby.
Asunto(s)
Reacción en Cadena de la Polimerasa/métodos , Diagnóstico Preimplantación/métodos , Talasemia alfa/diagnóstico , Adulto , Pueblo Asiatico/genética , Femenino , Humanos , Diagnóstico Prenatal , Talasemia alfa/genéticaRESUMEN
OBJECTIVE: To develop single-cell multiplex nested polymerase chain reaction (PCR) assays for preimplantation genetic diagnosis (PGD) in couples at risk of having child with beta-thalassemia. METHODS: Primers were designed and synthesized according to the documented mutation sites common among Chinese. Venous blood was collected from 4 pairs of husband and wife, all heterozygotes for beta-thalassemia, and underwent multiple nested PCR. Intraooplasmic sperm injection and mechanical bio psy was used to obtain single blastomere. Multiplex nested PCR was used to detect the CD41-42 mutation and the closely linked polymorphic marker, HumTHO1 gene or CD41-42, CD41-28, IVSII654 mutation and HumTHO1 gene in the single blastomeres from four clinical PGD cycles. The normal embryos with high scores capable of continuing to divide were transplanted into the uteri. The process of gestation was observed. RESULTS: 200 lymphocytes were amplified by nested PCR. The average amplification rate of the most common 16 beta-thalassemia mutations in Chinese population was 91.3% and the average rate of allele drop out for different sites was 17.0% without differences between any 2 sites. During the 4 PGD cycles 33 embryos underwent bioassay with a success rate of 100%. 33 blastomeres were obtained to undergo PCR, of which 30 were successfully amplified with an amplification rate of 90.9%. Explicit diagnosis was obtained in 26 of the 30 embryos: 7 normal homozygotes, 11 heterozygotes, and 8 abnormal or complex heterozygotes. One or more embryos were transferred back into the uteri of the 4 women and clinical pregnancy occurred in one woman. Five weeks after the implantation B-mode ultrasonography showed monocyesis, and in the 17th week of gestational period paracentesis of cord blood showed normal homozygote. At last a normal female infant confirming the PGD result had been born, which was the first reported unaffected pregnancy resulting from PGD using multiplex nested PCR for couples as beta-thalassemia gene carriers. The results of diagnosis for embryo all corresponded to those for blastomere. The average ADO rate of blastomere was 13.3% (4/30). CONCLUSION: PGD using multiplex nested PCR, as an alternative to prenatal diagnosis, is a reliable and effective way to help couples-carriers of pathogenetic genes to get a healthy baby.
Asunto(s)
Reacción en Cadena de la Polimerasa/métodos , Diagnóstico Preimplantación/métodos , Talasemia beta/diagnóstico , Adulto , Biopsia , Transferencia de Embrión , Embrión de Mamíferos/patología , Femenino , Humanos , Masculino , Mutación , Talasemia beta/genética , Talasemia beta/prevención & controlRESUMEN
OBJECTIVE: To assess the clinical value of pronuclear stage scoring system in human conventional in vitro fertilization-embryo transfer (IVF-ET) program. METHODS: A modified pronuclear scoring system was used to score zygotes 16 - 18 hours after insemination during conventional IVF. RESULTS: A total of 178 IVF cycles were recruited in this study. Cycles with mean pronuclear score (sum of pronuclear scores of all transferred embryos divided by total number of transferred embryos) > or = 13 had significant high pregnancy and implantation rate, as compared to those with less than 13 (49.1% and 26.5% Vs 29.4% and 15.0% respectively). Although the pregnancy, implantation rate and multiple pregnancy rate tend to increase with the increasing number of zygotes with full pronuclear score (= 15) among transferred embryos, no significant difference could be found (P > 0.05). When all embryos were analyzed, 77.2% zygotes with pronuclear score > or = 11 could be transferred or cryopreserved, while those with pronuclear score less than 11 decreased to 46.1%. CONCLUSIONS: Pronuclear stage scoring system provides additional parameter for embryo selection. However, pronuclear morphology could only partly represent embryonic viability. Combination with the scoring of both pronuclear stage and cleavage stage embryos for selecting transferred embryos is indispensable.
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Implantación del Embrión , Transferencia de Embrión , Fertilización In Vitro , Adulto , Fase de Segmentación del Huevo , Embrión de Mamíferos/ultraestructura , Humanos , Valor Predictivo de las Pruebas , Cigoto/ultraestructuraRESUMEN
OBJECTIVES: To review the retrospective treatment results of the azoospermia patients during January 2001 to January 2002 in the fertility center. METHODS: One hundred males attempted intracytoplasmic sperm injection (ICSI) cycle for treatment of azoospermia. All patients were undergone sperm retrieval by percutaneous epididymal sperm aspiration (PESA) or testicular sperm extraction (TESE) while their wives received conventional ovarian hyperstimulation. The hormone levels, testicular histology, the rates of sperm retrieval, fertilization, implantation and pregnancy were analysed and evaluated. RESULTS: Sperm were retrieved by PESA in 76 of 100 (76%) and by TESE in 23 of 100 (23%) men of azoospermia. The fertilization rate, implantation rate and clinical pregnancy rate were 71.3%, 20.35% and 42.11% respectively in PESA group, and 75.18%, 22.05% and 41.60% respectively in TESA group. Thirty-two clinical pregnancies were achieved with 15 ongoing pregnancies and subsequent live delivery for 15 cases in PESA group, and 2 cases of miscarriage, while 10 clinical pregnancies were achieved with 6 ongoing pregnancies, 2 cases of live delivery and 2 cases of miscarriage in TESA group. One case failed to retrieve sperm by TESE and canceled. CONCLUSIONS: Hormonal levels and testicular histology are unable to predict which men with azoospermia will have sperm retrieved by PESA and TESE. PESA and TESE with ICSI are effective methods to treat azoospermia. There were no significant differences in fertilization, implantation and clinical pregnancy rate between two groups.
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Oligospermia/terapia , Inyecciones de Esperma Intracitoplasmáticas/métodos , Adulto , Femenino , Hormona Folículo Estimulante/sangre , Humanos , Hormona Luteinizante/sangre , Masculino , Persona de Mediana Edad , Oligospermia/sangre , Embarazo , Índice de Embarazo , Estudios RetrospectivosRESUMEN
OBJECTIVE: To review the outcome of repeated percutaneous sperm aspiration (PESA) and testicular sperm extraction (TESE) for intracytoplasmic sperm injection (ICSI). METHODS: Forty-three cycles of 31 cases of azoospermic patients which underwent at least two PESA or TESE for ICSI from January 2001 to December 2002 were collected. The sperm retrieval, fertilization, implantation and clinical pregnancy were analyzed. RESULTS: Twenty-four cases underwent PESA and 7 cases underwent TESE. There were not any complications in these patients. Compared with the first cycle of 154 cases, the fertilization rate, implantation rate and clinical pregnancy rate were 78.39% vs 73.64%, 19.68% vs 18.38% and 34.88% vs 37.91%, respectively(P > 0.05). CONCLUSIONS: Repeated PESA or TESE is safe and well tolerated in azoospermic patients. Compared with the first cycle, the differences of repeated PESA or TESE cycles in fertilization rate, implantation rate and clinical pregnancy rate were not statistically significant.
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Azoospermia/terapia , Inyecciones de Esperma Intracitoplasmáticas/métodos , Recolección de Tejidos y Órganos/métodos , Adulto , Femenino , Humanos , Masculino , Persona de Mediana Edad , Embarazo , Resultado del Embarazo , Índice de EmbarazoRESUMEN
PURPOSE: The aim of this study is to investigate the relationship between spindle location and embryonic development of in vivo and in vitro matured human oocytes. METHODS: The spindles of 134 in vivo matured, 105 in vitro matured oocytes were examined by Polscope at the time of ICSI. RESULTS: The spindles were visualized in 83.6 and 77.1% of in vivo and in vitro matured oocytes respectively. The rate of fertilization of in vivo matured oocytes with spindles beneath or adjacent to the first polar body (angle of 0-5 degrees) was significantly higher (93.3%) than all other groups. The proportions of various spindle positions did not differ statistically in in vivo and in vitro matured oocytes. CONCLUSIONS: Meiotic spindle location with regard to the first polar body appears to influence fertilization rate.
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Desarrollo Embrionario , Oocitos/ultraestructura , Huso Acromático/ultraestructura , Adulto , Embrión de Mamíferos/citología , Embrión de Mamíferos/ultraestructura , Fertilización/fisiología , Fertilización In Vitro , Humanos , Meiosis , Oocitos/crecimiento & desarrolloRESUMEN
METHODS & RESULTS: In southern China, the average carrier rates of alpha-thalassemia and beta-thalassemia in the population are as high as 10.3% and 2.8%, respectively. Because of the high rates, they are known as 'social diseases' in some regions. In this study, the fluorescent gap PCR, which can detect the alpha-thalassemia Southeast Asia deletion (SEA deletion), was applied in four clinical applications of preimplantation genetic diagnosis (PGD) on four couples, among whom both partners were alpha-thalassemia carriers. Two patients became pregnant and two healthy babies were born, which confirmed the PGD results. The single cell multiplex nested PCR followed by reverse dot blot (RDB), which can simultaneously detect the 16 beta-thalassemia mutations in the Chinese population, was applied in four clinical PGD cycles on four couples among whom both partners were beta-thalassemia carriers. One pregnancy was achieved and it resulted in a live healthy birth, which confirmed the results of PGD. The amplification efficiencies of the two protocols described above were 89.5% and 93.9%, respectively. The allele drop-out (ADO) rates of these two protocols were 5.9% and 10.9%, respectively. CONCLUSION: These studies represent the successful applications of PGD protocols that can detect more than 95% of alpha- and beta -thalassemia mutations in the Chinese population.