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O4 First clinical application of karyomapping for PGD of Gaucher disease combined with 24 chromosome screening
S16
10th International Congress on Preimplantation Genetic Diagnosis
women in subsequent pregnancies is controversial and not always effective. Aim: To set up and validate a preimplantation genetic diagnosis protocol based on genotyping the HPA-1a/1b polymorphism from single blastomeres in order to avoid the fetomaternal HPA-1a incompatibility. Materials and Methods: Single blastomeres from previously HPA-1 typed couples were obtained by embryo biopsy. The corresponding genomic DNA was amplified by a modified “Multiple Displacement Amplification” procedure using components of the GenomiPhi v.2 kit. Subsequent analysis of the blastomere’s HPA-1 genotype was carried out by Real-time PCR with allele-specific TaqMan® MGB probes. In parallel, a set of previously selected microsatellite markers were amplified using labeled primers and the allelic profile was determined by capillary electrophoresis. A total of 14 couples participated in the validation study. After obtaining informed consent of these couples, whole embryos not selected for transfer were collected. A total of 70 blastomeres, corresponding to 36 embryos, were tested during this evaluation. Results: We have selected a set of microsatellite markers adjacent to the HPA-1 locus and have assessed their informativity in a study including 10 familial FTNAI cases with previously known HPA-1a incompatibility. A PGD protocol for the detection of HPA1a negative embryos has been set up, which includes the analysis of 3 extragenic (D17S1183, D17S806, D17S1827) and 1 intragenic STR markers. This protocol has been tested in a validation study with single blastomeres (n = 70). Preliminary data analysis indicates an ADO rate of aprox. 15% but we have been able to identify all HPA-1a false negative embryos with the STRs genetic linkage. As an attempt to reduce the incidence of ADO, a modification of the current protocol consisting of the addition of locus-specific primers to the reaction mix during the MDA step is currently evaluated. Conclusions: This PGD approach offers new prospects to HPA1a alloimmunized women with a heterozygous husband and a previous history of an affected NAIT child. The protocol here described allows to avoid the HPA-1a incompatibility through the selection of the embryo(s) to be transfered. O4 First clinical application of karyomapping for PGD of Gaucher disease combined with 24 chromosome screening A. Handyside, J. Grifo, A. Gabriel, A. Thornhill, D. Griffin, e. K. Ketterson4 , R. Prates, S. Tormasi, J. Fischer, S. Munn´ 1 London Bridge Fertility, Gynaecology and Genetics Centre, UK, 2 New York University School of Medicine Fertility Center, New York, USA, 3 School of Biosciences, University of Kent, Canterbury, UK, 4 Reprogenetics LLC, Livingston, NJ, USA Aim: Genome wide SNP genotyping and karyomapping for PGD of Gaucher disease combined with 24 chromosome aneuploidy screening was compared with conventional single gene defect detection and array comparative genomic hybridisation (aCGH). Method: A couple in which both partners carry the N370S mutation of the glucocerebrosidase gene causing Gaucher Disease (inherited from the paternal and maternal grandfathers, respectively) underwent two cycles of PGD supplemented with embryos derived from cryopreserved oocytes. Six embryos were biopsied at the blastocyst stage and vitrified pending diagnosis. Two arrested/abnormal embryos were biopsied at cleavage stages and the single cells and biopsied embryos and 6 other arrested embryos lysed for whole genome amplification (WGA) with patients’ informed consent (IRB#6902 NYU Langone Medical Center). Following WGA (Sureplex), SNP genotyping (Illumina HumanCytoSNP-12) of the samples and parental and grandparental DNA and karyomap analysis was carried out. In
addition, aCGH (BlueGnome) and direct mutation and linked marker (D1S394) analysis was performed on the WGA products. Results: Karyomap analysis of the 6 blastocysts demonstrated that one was unaffected, three were carriers and two were affected for Gaucher disease. All but one had chromosomal abnormalities including maternal trisomy 15, maternal monosomy 10, 20, 21 and 22 and a partial deletion of the paternal chromosome 11p. Multiple chromosome aneuploidies were detected in the arrested embryos including 4 with maternal monosomy 22 and paternal monosomy 2 and 16. Karyomap results were confirmed via mutation analysis and aCGH. Discussion: Karyomapping combines accurate analysis of single gene defects and chromosome abnormalities including whole chromosome aneuploidies and partial deletions with their parental origin. The prevalence of chromosomal abnormalities in this patient’s embryos underlines the importance of combined analysis for PGD. O5 PGS by polar body biopsy High aneuploidy rate independent of age B. Acar-Perk, J. Weimer, A. Salmassi, L. Mettler, N. Arnold, A.G. Schmutzler. University Women’s Hospital, CA University, Kiel, Germany Aim: Postulated indications for PGS are advanced maternal age, repeated implantation failure, and recurrent abortion. As we performed PGS without age indication, we evaluated the biologic and clinical findings. Methods: After approval by the local ethics committee, 104 PGS cycles were performed between 2004 and 2008. PGS was offered to all patients with 8 or more oocytes, independent of age or other indications. It was performed by laser biopsy of the first polar body on day 0, fixation for FISH and hybridization with chromosomes 13, 16, 18, 21, 22 (Vysis). Zygote selection was carried out on day 1, in accordance with the German embryo protection law, and embryo transfer on day 2, with an intended elective double embryo transfer from proven “euploid” (for 5 chromosomes) oocytes. If no two euploid oocytes were available, possibly one or two non-detected oocytes were transferred. Results: The average age of the women was 34 (23 43) years. 1285 oocytes were retrieved and 1150 were mature (89%). From mature oocytes 893 (78%) were biopsied and 778 (87%) polar bodies could be fixed, of these genetic results for 621 (80%) oocytes were obtained. 227 (37%) were euploid, 394 (63%) aneuploid and 157 (20%) oocytes genetically could not be assigned. The fertilisation rate with euploid oocytes was 68% (155/227), with aneuploid oocytes 68% as well (270/ 394). 197 embryos were transferred, 117 of these (59%) were generated from only euploid oocytes and 80 (41%) were with non-assigned oocytes. However, in every transfer minimum one diploid oocyte was used. ET was performed in 100 cycles (96%), resulting in 29 (29%) pregnancies, 20 deliveries and 9 abortions. In 83 women (80%), with an average age of 34 years, more than 50% oocytes showed aneuploidy. According to age distribution the aneuploidy rate grouped as follow: group I (23 29 years / 16 cycles) with 65% aneuploidy rate, group II (30 34 years / 40 cycles) with 55% aneuploidy rate, group III (35 39 years / 32 cycles) with 69% aneuploidy rate and group IV (40 43 years / 16 cycles) with 69% aneuploidy rate. The aneuploidy rates between the groups were statistically not significant (p > 0.05). Conclusions: Aneuploidy does not influence fertilisation and our pregnancy rate does not significantly differ from the national average with ICSI (28%). The aneuploidy rate of human oocytes per patient is high even with non-age related indication and high number of patients observed where the majority of the oocytes were aneuploid. To find the benefits of PGS, indications should be made embryologically and clinically.
10th International Congress on Preimplantation Genetic Diagnosis
women in subsequent pregnancies is controversial and not always effective. Aim: To set up and validate a preimplantation genetic diagnosis protocol based on genotyping the HPA-1a/1b polymorphism from single blastomeres in order to avoid the fetomaternal HPA-1a incompatibility. Materials and Methods: Single blastomeres from previously HPA-1 typed couples were obtained by embryo biopsy. The corresponding genomic DNA was amplified by a modified “Multiple Displacement Amplification” procedure using components of the GenomiPhi v.2 kit. Subsequent analysis of the blastomere’s HPA-1 genotype was carried out by Real-time PCR with allele-specific TaqMan® MGB probes. In parallel, a set of previously selected microsatellite markers were amplified using labeled primers and the allelic profile was determined by capillary electrophoresis. A total of 14 couples participated in the validation study. After obtaining informed consent of these couples, whole embryos not selected for transfer were collected. A total of 70 blastomeres, corresponding to 36 embryos, were tested during this evaluation. Results: We have selected a set of microsatellite markers adjacent to the HPA-1 locus and have assessed their informativity in a study including 10 familial FTNAI cases with previously known HPA-1a incompatibility. A PGD protocol for the detection of HPA1a negative embryos has been set up, which includes the analysis of 3 extragenic (D17S1183, D17S806, D17S1827) and 1 intragenic STR markers. This protocol has been tested in a validation study with single blastomeres (n = 70). Preliminary data analysis indicates an ADO rate of aprox. 15% but we have been able to identify all HPA-1a false negative embryos with the STRs genetic linkage. As an attempt to reduce the incidence of ADO, a modification of the current protocol consisting of the addition of locus-specific primers to the reaction mix during the MDA step is currently evaluated. Conclusions: This PGD approach offers new prospects to HPA1a alloimmunized women with a heterozygous husband and a previous history of an affected NAIT child. The protocol here described allows to avoid the HPA-1a incompatibility through the selection of the embryo(s) to be transfered. O4 First clinical application of karyomapping for PGD of Gaucher disease combined with 24 chromosome screening A. Handyside, J. Grifo, A. Gabriel, A. Thornhill, D. Griffin, e. K. Ketterson4 , R. Prates, S. Tormasi, J. Fischer, S. Munn´ 1 London Bridge Fertility, Gynaecology and Genetics Centre, UK, 2 New York University School of Medicine Fertility Center, New York, USA, 3 School of Biosciences, University of Kent, Canterbury, UK, 4 Reprogenetics LLC, Livingston, NJ, USA Aim: Genome wide SNP genotyping and karyomapping for PGD of Gaucher disease combined with 24 chromosome aneuploidy screening was compared with conventional single gene defect detection and array comparative genomic hybridisation (aCGH). Method: A couple in which both partners carry the N370S mutation of the glucocerebrosidase gene causing Gaucher Disease (inherited from the paternal and maternal grandfathers, respectively) underwent two cycles of PGD supplemented with embryos derived from cryopreserved oocytes. Six embryos were biopsied at the blastocyst stage and vitrified pending diagnosis. Two arrested/abnormal embryos were biopsied at cleavage stages and the single cells and biopsied embryos and 6 other arrested embryos lysed for whole genome amplification (WGA) with patients’ informed consent (IRB#6902 NYU Langone Medical Center). Following WGA (Sureplex), SNP genotyping (Illumina HumanCytoSNP-12) of the samples and parental and grandparental DNA and karyomap analysis was carried out. In
addition, aCGH (BlueGnome) and direct mutation and linked marker (D1S394) analysis was performed on the WGA products. Results: Karyomap analysis of the 6 blastocysts demonstrated that one was unaffected, three were carriers and two were affected for Gaucher disease. All but one had chromosomal abnormalities including maternal trisomy 15, maternal monosomy 10, 20, 21 and 22 and a partial deletion of the paternal chromosome 11p. Multiple chromosome aneuploidies were detected in the arrested embryos including 4 with maternal monosomy 22 and paternal monosomy 2 and 16. Karyomap results were confirmed via mutation analysis and aCGH. Discussion: Karyomapping combines accurate analysis of single gene defects and chromosome abnormalities including whole chromosome aneuploidies and partial deletions with their parental origin. The prevalence of chromosomal abnormalities in this patient’s embryos underlines the importance of combined analysis for PGD. O5 PGS by polar body biopsy High aneuploidy rate independent of age B. Acar-Perk, J. Weimer, A. Salmassi, L. Mettler, N. Arnold, A.G. Schmutzler. University Women’s Hospital, CA University, Kiel, Germany Aim: Postulated indications for PGS are advanced maternal age, repeated implantation failure, and recurrent abortion. As we performed PGS without age indication, we evaluated the biologic and clinical findings. Methods: After approval by the local ethics committee, 104 PGS cycles were performed between 2004 and 2008. PGS was offered to all patients with 8 or more oocytes, independent of age or other indications. It was performed by laser biopsy of the first polar body on day 0, fixation for FISH and hybridization with chromosomes 13, 16, 18, 21, 22 (Vysis). Zygote selection was carried out on day 1, in accordance with the German embryo protection law, and embryo transfer on day 2, with an intended elective double embryo transfer from proven “euploid” (for 5 chromosomes) oocytes. If no two euploid oocytes were available, possibly one or two non-detected oocytes were transferred. Results: The average age of the women was 34 (23 43) years. 1285 oocytes were retrieved and 1150 were mature (89%). From mature oocytes 893 (78%) were biopsied and 778 (87%) polar bodies could be fixed, of these genetic results for 621 (80%) oocytes were obtained. 227 (37%) were euploid, 394 (63%) aneuploid and 157 (20%) oocytes genetically could not be assigned. The fertilisation rate with euploid oocytes was 68% (155/227), with aneuploid oocytes 68% as well (270/ 394). 197 embryos were transferred, 117 of these (59%) were generated from only euploid oocytes and 80 (41%) were with non-assigned oocytes. However, in every transfer minimum one diploid oocyte was used. ET was performed in 100 cycles (96%), resulting in 29 (29%) pregnancies, 20 deliveries and 9 abortions. In 83 women (80%), with an average age of 34 years, more than 50% oocytes showed aneuploidy. According to age distribution the aneuploidy rate grouped as follow: group I (23 29 years / 16 cycles) with 65% aneuploidy rate, group II (30 34 years / 40 cycles) with 55% aneuploidy rate, group III (35 39 years / 32 cycles) with 69% aneuploidy rate and group IV (40 43 years / 16 cycles) with 69% aneuploidy rate. The aneuploidy rates between the groups were statistically not significant (p > 0.05). Conclusions: Aneuploidy does not influence fertilisation and our pregnancy rate does not significantly differ from the national average with ICSI (28%). The aneuploidy rate of human oocytes per patient is high even with non-age related indication and high number of patients observed where the majority of the oocytes were aneuploid. To find the benefits of PGS, indications should be made embryologically and clinically.