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Preembryonic Diagnosis for Genetic Disorders
transfer, difficult or easy; and a pregnancy prediction of ,25%, 25–50% of .50%. Predictions were compared to the actual outcome. Results: When $3 of the 5 infertility specialist/embryologist predicted chance of a pregnancy to be #25% the actual pregnancy rate was 27%. The pregnancy rate was 54% when $3 of them predicted pregnancy rate to be 25–50% and the pregnancy rate was 71% when they predicted the pregnancy rate to be .50%. Conclusions: Embryologists as well as infertility specialists have different perspectives by which they evaluate IVF outcome. Mutual agreement between experienced ART personnel may assist to better predict a particular patient’s chance to conceive after ET. This may help in counseling patients and in the decision making process in planning future ART cycles.
ART: PREIMPLANTATION GENETICS Monday, October 23, 2000 P-053 Preembryonic Diagnosis for Genetic Disorders. Y. Verlinsky, S. Rechitsky, O. Verlinsky, V. Ivakhnenko, C. Strom, A. Kuliev. Reproductive Genetics Institute, Chicago, IL.
into individual blastomeres (n5178 and 175, respectively) by removing the zona pellucida with acid Tyrode’s solution. Individual blastomeres were transferred to a slide, fixed with methanol:acetic acid (3:1) and subjected to FISH using X,Y and 18 CEP direct-labeled probes (Vysis) for 30 min in HYBrite. Signals were analyzed by a multi fluorescence microscope (Olympus). Results: Twenty-three embryos (49%) of the male factor men showed abnormal chromosomal signals (11 aneuploidies and 12 chaotic); of the 178 individual blastomeres, 72 (40%) showed an abnormal chromosomal status (33 aneuploidies and 39 chaotic status) and 4 (2%) showed no signal. Of the 3 control embryos, 13 (25%) were abnormal (10 aneuploidies and 3 chaotic); of the 175 control blastomeres, 35 (20%) displayed a chromosomal abnormality (21 aneuploidies and 14 chaotic status) and 5 (3%) gave no signal. Conclusions: The present findings show a significant high incidence of chromosomal abnormalities, especially chaotic status, using FISH to detect for aneuploidies of chromosomes X,Y, and 18 in the embryos from men with male factor infertility. Thus, the low overall success rates in infertility treatment of these men may be the consequence of genetic alterations that impair chromosomal segregation. Routine screening of embryos of male factor patients by preimplantation genetic diagnosis might increase their chances of fathering a successful and healthy pregnancy.
P-056 Objectives: Embryos found to be abnormal during preimplantation genetic diagnosis (PGD) are discarded or analyzed to confirm the diagnosis. Because destruction of affected embryos is ethically unacceptable in some communities and ethnic groups, we introduced a preembryonic genetic diagnosis that uses sequential first and second polar body (PB1 & PB2) removal. This was applied in a patient at risk of having child with sickle cell disease. Design: PB1 and PB2 were removed and tested before pronuclei fusion, so only mutation free oocytes were cultured into cleaving embryos and transferred, avoiding the development of mutant oocytes beyond pronuclear stage. Metherials and Methods: Fourteen oocytes were obtained and tested for the maternal sickle cell allele by sequential PCR analysis of PB1 and PB2. PB genotyping was performed using multiplex PCR with separate primer sets for the sickle cell mutation and two linked short tandem repeat markers, one located at the 59 end of the beta-globin gene, and the other in the human thyrosin hydroxylase gene. Only those oocytes that were predicted to be mutation-free were cultured further into cleaving 8-cell embryos, and transferred back to patient. The mutant oocytes were not further cultured, but analyzed directly at the pronuclear-stage for the confirmation of PB diagnosis. Results: Of 28 aspirated oocytes, 14 extruded PB1 and were studied for the presence of sickle cell mutation. Following intracytoplasmic sperm injection PB2 were extruded from 13 of them and studied, but both PB1 and PB2 results were available in 12 oocytes. Six oocytes were predicted to contain a normal allele based on heterozygous PB1 and hemizygous mutant PB2. Five of these oocytes developed into cleaving embryos and were transferred in two consecutive cycles, both resulting in a singleton pregnancy and birth of unaffected child in one of them, following confirmation of PB diagnosis by chorionic villus sampling (CVS). Conclusion: The results demonstrate feasibility of preembryonic diagnosis for single gene disorders, avoiding the establishment and destruction of mutant embryos. P-054 Embryos of Male Factor Infertility Patients Display a High Rate of Chromosomal Abnormalities. Y. X. Tang, C. Hansis, L. Chi, L. C. Krey, J. A. Grifo. Program for In Vitro Fertilization, Reproductive Surgery and Infertility, New York University School of Medicine, New York, NY. Objective: Many studies have reported a high incidence of chromosomal aneuploidies in the sperm of male factor infertility patients. In this study individual blastomeres of embryos fertilized by male factor patients with oligo- or azoospermia were examined by FISH for the presence of chromosomal abnormalities. Design: We used FISH to detect for aneuploidies of chromosomes X,Y, and 18 in individual blastomeres from embryos generated by 17 men diagnosed with male factor infertility according to W.H.O. criteria. Blastomeres from 53 ICSI-generated embryos from healthy male patients with no history of male factor infertility were studied as a control group. Materials and Methods: Embryos (n547 male factor and 53 control male) were discarded at the 4 – 8 cell stage with patient consent and dissociated
S112
Abstracts
A Model for Preimplantation Genetic Diagnosis (PGD) of the BRCA Tumor Suppressor Gene-Familial Breast and Ovarian Cancer Prevention Using In-Vitro Vertilization (IVF) Technology. 1M. T. Kan, 1K. Xu, 1 Z. M. Shi, 2K. Nafa, 1Z. Rosenwaks. 1The Center for Reproductive Medicine and Infertility, Weill Medical College of Cornell University, New York, NY. 2Department of Human Genetics-Memorial Sloan-Kettering Cancer Center, New York, NY. Objective: The purpose of this study was to develop a model for PGD of the BRCA1 tumor suppressor gene by detecting the 185delAG mutation at the picogram level of DNA using amplification created restriction enzyme site (ACRES) analysis. One of every eight women develops breast cancer in their lifetime (Cancer Statistics 1999). It is estimated that 5–10% of breast cancer demonstrates a hereditary component while approximately 1% of the US Ashkenazi Jewish population is affected by the BRCA1 185delAG mutation. In women who carry a mutation in the BRCA1 tumor suppressor gene, the risk of developing breast cancer has been estimated to be between 52% and 87% (Struewing 1997, Ford 1994). Effective PGD could eliminate this mutation from the lineage. Design: Lymphoblasts from a patient with a known BRCA1 185delAG mutation were grown in cell culture and isolated. DNA analysis was performed by fluorescent polymerase chain reaction (PCR) and ACRES. Materials and Methods: Lymphoblasts from a known BRCA1 185delAG patient were aspirated using micromanipulation techniques. These cells were lysed in groups ranging from 1 cell to 10 cells and subjected to PCR amplification. Normal DNA as well as DNA from a patient with the known mutation were used as controls. Non-nested PCR was performed with hex-labeled primers created specifically for the induced endonuclease restriction site. PCR products were processed with gel electrophoresis after Taq I endonuclease digestion in addition to fluorescent sizing analysis. Results: The BRCA1 185delAG mutation was detected at the picogram DNA level (1–10 cells) using ACRES. The normal template DNA was cleaved by the restriction enzyme while the mutant allele was not, yielding a difference of 122 and 144 base pairs on polyacrylamide gel electrophoresis. Fluorescent sizing analysis also detected the mutation. Conclusions: The ACRES technique has been used at higher concentrations of DNA for clinical BRCA mutation detection in over 230 patients and has been concordant in each case when compared with direct sequencing (Nafa 1999). ACRES is relatively inexpensive and does not require fluorescent analysis. This study demonstrates the ability to detect the BRCA1 185delAG mutation at the picogram level of DNA using ACRES, which may be applied to preimplantation genetic diagnosis. References Cancer Statistics 1999. CA Cancer J Clin, 49:8 –31. Ford et al. Breast Cancer Linkage Consortium. Risk of cancer in BRCA1mutation carriers. Lancet 1994 Mar 19;343(8899):692–5. Nafa, K. Direct Detection of Common Mutations in the BRCA1 and BRCA2 Genes by Amplification Created Restriction Enzyme Site (ACRES). Am J
Vol. 74, No. 3, Suppl. 1, September 2000
ART: PREIMPLANTATION GENETICS Monday, October 23, 2000 P-053 Preembryonic Diagnosis for Genetic Disorders. Y. Verlinsky, S. Rechitsky, O. Verlinsky, V. Ivakhnenko, C. Strom, A. Kuliev. Reproductive Genetics Institute, Chicago, IL.
into individual blastomeres (n5178 and 175, respectively) by removing the zona pellucida with acid Tyrode’s solution. Individual blastomeres were transferred to a slide, fixed with methanol:acetic acid (3:1) and subjected to FISH using X,Y and 18 CEP direct-labeled probes (Vysis) for 30 min in HYBrite. Signals were analyzed by a multi fluorescence microscope (Olympus). Results: Twenty-three embryos (49%) of the male factor men showed abnormal chromosomal signals (11 aneuploidies and 12 chaotic); of the 178 individual blastomeres, 72 (40%) showed an abnormal chromosomal status (33 aneuploidies and 39 chaotic status) and 4 (2%) showed no signal. Of the 3 control embryos, 13 (25%) were abnormal (10 aneuploidies and 3 chaotic); of the 175 control blastomeres, 35 (20%) displayed a chromosomal abnormality (21 aneuploidies and 14 chaotic status) and 5 (3%) gave no signal. Conclusions: The present findings show a significant high incidence of chromosomal abnormalities, especially chaotic status, using FISH to detect for aneuploidies of chromosomes X,Y, and 18 in the embryos from men with male factor infertility. Thus, the low overall success rates in infertility treatment of these men may be the consequence of genetic alterations that impair chromosomal segregation. Routine screening of embryos of male factor patients by preimplantation genetic diagnosis might increase their chances of fathering a successful and healthy pregnancy.
P-056 Objectives: Embryos found to be abnormal during preimplantation genetic diagnosis (PGD) are discarded or analyzed to confirm the diagnosis. Because destruction of affected embryos is ethically unacceptable in some communities and ethnic groups, we introduced a preembryonic genetic diagnosis that uses sequential first and second polar body (PB1 & PB2) removal. This was applied in a patient at risk of having child with sickle cell disease. Design: PB1 and PB2 were removed and tested before pronuclei fusion, so only mutation free oocytes were cultured into cleaving embryos and transferred, avoiding the development of mutant oocytes beyond pronuclear stage. Metherials and Methods: Fourteen oocytes were obtained and tested for the maternal sickle cell allele by sequential PCR analysis of PB1 and PB2. PB genotyping was performed using multiplex PCR with separate primer sets for the sickle cell mutation and two linked short tandem repeat markers, one located at the 59 end of the beta-globin gene, and the other in the human thyrosin hydroxylase gene. Only those oocytes that were predicted to be mutation-free were cultured further into cleaving 8-cell embryos, and transferred back to patient. The mutant oocytes were not further cultured, but analyzed directly at the pronuclear-stage for the confirmation of PB diagnosis. Results: Of 28 aspirated oocytes, 14 extruded PB1 and were studied for the presence of sickle cell mutation. Following intracytoplasmic sperm injection PB2 were extruded from 13 of them and studied, but both PB1 and PB2 results were available in 12 oocytes. Six oocytes were predicted to contain a normal allele based on heterozygous PB1 and hemizygous mutant PB2. Five of these oocytes developed into cleaving embryos and were transferred in two consecutive cycles, both resulting in a singleton pregnancy and birth of unaffected child in one of them, following confirmation of PB diagnosis by chorionic villus sampling (CVS). Conclusion: The results demonstrate feasibility of preembryonic diagnosis for single gene disorders, avoiding the establishment and destruction of mutant embryos. P-054 Embryos of Male Factor Infertility Patients Display a High Rate of Chromosomal Abnormalities. Y. X. Tang, C. Hansis, L. Chi, L. C. Krey, J. A. Grifo. Program for In Vitro Fertilization, Reproductive Surgery and Infertility, New York University School of Medicine, New York, NY. Objective: Many studies have reported a high incidence of chromosomal aneuploidies in the sperm of male factor infertility patients. In this study individual blastomeres of embryos fertilized by male factor patients with oligo- or azoospermia were examined by FISH for the presence of chromosomal abnormalities. Design: We used FISH to detect for aneuploidies of chromosomes X,Y, and 18 in individual blastomeres from embryos generated by 17 men diagnosed with male factor infertility according to W.H.O. criteria. Blastomeres from 53 ICSI-generated embryos from healthy male patients with no history of male factor infertility were studied as a control group. Materials and Methods: Embryos (n547 male factor and 53 control male) were discarded at the 4 – 8 cell stage with patient consent and dissociated
S112
Abstracts
A Model for Preimplantation Genetic Diagnosis (PGD) of the BRCA Tumor Suppressor Gene-Familial Breast and Ovarian Cancer Prevention Using In-Vitro Vertilization (IVF) Technology. 1M. T. Kan, 1K. Xu, 1 Z. M. Shi, 2K. Nafa, 1Z. Rosenwaks. 1The Center for Reproductive Medicine and Infertility, Weill Medical College of Cornell University, New York, NY. 2Department of Human Genetics-Memorial Sloan-Kettering Cancer Center, New York, NY. Objective: The purpose of this study was to develop a model for PGD of the BRCA1 tumor suppressor gene by detecting the 185delAG mutation at the picogram level of DNA using amplification created restriction enzyme site (ACRES) analysis. One of every eight women develops breast cancer in their lifetime (Cancer Statistics 1999). It is estimated that 5–10% of breast cancer demonstrates a hereditary component while approximately 1% of the US Ashkenazi Jewish population is affected by the BRCA1 185delAG mutation. In women who carry a mutation in the BRCA1 tumor suppressor gene, the risk of developing breast cancer has been estimated to be between 52% and 87% (Struewing 1997, Ford 1994). Effective PGD could eliminate this mutation from the lineage. Design: Lymphoblasts from a patient with a known BRCA1 185delAG mutation were grown in cell culture and isolated. DNA analysis was performed by fluorescent polymerase chain reaction (PCR) and ACRES. Materials and Methods: Lymphoblasts from a known BRCA1 185delAG patient were aspirated using micromanipulation techniques. These cells were lysed in groups ranging from 1 cell to 10 cells and subjected to PCR amplification. Normal DNA as well as DNA from a patient with the known mutation were used as controls. Non-nested PCR was performed with hex-labeled primers created specifically for the induced endonuclease restriction site. PCR products were processed with gel electrophoresis after Taq I endonuclease digestion in addition to fluorescent sizing analysis. Results: The BRCA1 185delAG mutation was detected at the picogram DNA level (1–10 cells) using ACRES. The normal template DNA was cleaved by the restriction enzyme while the mutant allele was not, yielding a difference of 122 and 144 base pairs on polyacrylamide gel electrophoresis. Fluorescent sizing analysis also detected the mutation. Conclusions: The ACRES technique has been used at higher concentrations of DNA for clinical BRCA mutation detection in over 230 patients and has been concordant in each case when compared with direct sequencing (Nafa 1999). ACRES is relatively inexpensive and does not require fluorescent analysis. This study demonstrates the ability to detect the BRCA1 185delAG mutation at the picogram level of DNA using ACRES, which may be applied to preimplantation genetic diagnosis. References Cancer Statistics 1999. CA Cancer J Clin, 49:8 –31. Ford et al. Breast Cancer Linkage Consortium. Risk of cancer in BRCA1mutation carriers. Lancet 1994 Mar 19;343(8899):692–5. Nafa, K. Direct Detection of Common Mutations in the BRCA1 and BRCA2 Genes by Amplification Created Restriction Enzyme Site (ACRES). Am J
Vol. 74, No. 3, Suppl. 1, September 2000