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O-007 Preimplantation testing for common chromosomal aneuploidies in IVF patients of advanced maternal age
M o n d a y , O c t o b e r 20, 1997 2:00 P.M. to 5:00 P.M. A. T h e S o c i e t y f o r A s s i s t e d R e p r o d u c t i v e Technology
0-006 Recycling of a Single Human Blastomere Fixed on a Microscopic Slide for Sexing and Diagnosis of Specific Mutations by Various Types of PCR. Z.Y. He, H. C. Liu, C. A. Mele, L. L. Veeck, O. K. Davis, Z. Rosenwaks. Department of OB/GYN, The New York HospitalCornell Medical Center, New York, NY. Objective: To investigate the suitability of recycling single blastomeres biopsied from h u m a n embryos in order to obtain multiple genetic information for preimplantation genetic diagnosis. Design: Biopsied blastomeres were fixed on slides. Conventional polymerase chain reaction (PCR), primed in-situ labeling (PRINS) and degenerate oligonucleotide primer (DOP) were performed sequentially on the blastomeres. The rate of allele specific drop out (ADO) was monitored to evaluate the amplification efficiency. Materials and Methods: Sixty blastomeres were individually fixed on slides. Two were lost due to fixation. The remaining 58 blastomeres were separated into 4 groups to amplify the gene sequence by specific primers for sexing, W1282X and AF508 mutation in cystic fibrosis, sickle cell mutation in h u m a n beta-globin, and steroid 21 hydroxylase gene (CYP21) in various conditions. Group I: Blastomeres (n=30) were used to perform 5 sequential PCRs. Group II: Blastomeres (n=10) were used to perform PRINS prior to 5 sequential PCRs. Group III: Blastomeres ( n = l l ) were stained with hematoxylin before performing 5 sequential PCRs. Group IV: Blastomeres (n=7) were used to pre-amplify whole DNA by DOP-PCR before performing PCR. Results: A high percentage (96.7%) of blastomeres were fixed before PCR was performed. The amplification efficiency of 4 experimental groups were shown in the following table:
PCR
Group
Fixation Efficiency
I
30/30
II
10/11
III
11/11
IV
7/8
1st Round Sexing (%)
2nd Round W1282X (%)
3rd Round AF508 (%)
4th Round fl-Globin
5th Round CYP21 (%)
30/30 (100%) 10/10 (100%) 6/11 (54.5%) 7/7 (100%)
30/30 (100%) 10/10 (100%) 4/11 (36.4%) 7/7 (100%)
29/30 (96.6%) 10/10 (100%) 2/11 (18.2%) 7/7 (100%)
25/30 (83.3%) 8/10 (80%) 2/11 (18.2%) 7/7 (100%)
17/30 (56.7%) 4/10 (40%) Ull (9.1%) 7/7 (100%)
In groups I and II, the gene products were successfully amplified from the blastomeres fixed on the slide. Only after the 4th round did the ADO rate increase substantially. A poor amplification efficiency was noticed in group S4
Abstracts
III even in the 1st round of PCR. Group IV demonstrates the best efficiency among the four groups. The consistency of sexing between pairs of sibling blastomeres was 100%. Conclusion: 1. Blastomeres fixed for PRINS and presumably FISH can be recycled for PCR to obtain more genetic information. 2. The process of hematoxylin staining appears to increase the incidence of ADO. 3. With DOP-PCR, the whole DNA of single blastomeres was uniformly and randomly amplified. This technique appears to facilitate simultaneous multiple preimplantation genetic diagnosis with high efficiency. In addition, it also has m a n y applications such as coupling with comparative genomic hybridization (CGH) and with DNA fingerprinting for the analysis of chromosomal imbalance in entire genomes as well as detection of contamination.
0-007 Preimplantation Testing for Common Chromosomal Aneuploidies in IVF Patients of Advanced Maternal Age. Y. Verlinsky, J. Cieslak, V. Ivakhnenko, M. White, A. Lifchez, B. Kaplan, J. Moise, J. Valle, N. Ginsberg, C. Strom and A. Kuliev. Reproductive Genetics Institute, Illinois Masonic Medical Center, Chicago, IL. Objectives: Because most age-related aneuploidies originate mainly from nondisjunction in the first or second maternal meiotic divisions, preimplantation aneuploidy testing of oocytes will make it possible to prevent common chromosomal aneuploidies before pregnancy, and improve the efficiency of IVF in patients of advanced maternal age. Design: The first and second polar bodies (PB) were removed following their extrusion from the oocytes and studied by fluorescent in situ hybridization (FISH), to avoid fertilization and transfer of aneuploid oocytes. Materials and Methods: A total of 2839 oocytes were biopsied from 337 patients of advanced maternal age (484 IVF cycles) and analyzed by FISH, using probes specific for chromosomes 13, 18 and 21. Embryos. resulting from oocytes free from chromosomal abnormalities were transferred back to patients, while those resulting from abnormal oocytes were studied by FISH to confirm the PB diagnosis. Results: Of 2376 (83.7%) oocytes with FISH results, 946 (39.8%) were predicted to be aneuploid and excluded from the transfer. Of 1430 aneuploidy free oocytes (60.2%), 1070 fertilized, developed normally to the 8-cell stage, and were transferred in a total of 418 cycles, resulting in 89 clinical pregnancies. As high as 26% pregnancy rate was observed when the oocytes were tested by both the first and second PB, compared to 18.2% if preimplantation diagnosis was based on the first or second PB analysis only. Overall, 53 healthy children have been born, following confirmation of preimplantation PB diagnosis by CVS or amniocentesis. Conclusion: The study suggests a clinical value ofpreimplantation PB diagnosis for avoiding the age-related risk for common aneuploidies in IVF.
0-008 In Vitro Maturation of H u m a n Oocytes for the Patients w h o are at the Risk of Hyperstimulation Dur-
0-006 Recycling of a Single Human Blastomere Fixed on a Microscopic Slide for Sexing and Diagnosis of Specific Mutations by Various Types of PCR. Z.Y. He, H. C. Liu, C. A. Mele, L. L. Veeck, O. K. Davis, Z. Rosenwaks. Department of OB/GYN, The New York HospitalCornell Medical Center, New York, NY. Objective: To investigate the suitability of recycling single blastomeres biopsied from h u m a n embryos in order to obtain multiple genetic information for preimplantation genetic diagnosis. Design: Biopsied blastomeres were fixed on slides. Conventional polymerase chain reaction (PCR), primed in-situ labeling (PRINS) and degenerate oligonucleotide primer (DOP) were performed sequentially on the blastomeres. The rate of allele specific drop out (ADO) was monitored to evaluate the amplification efficiency. Materials and Methods: Sixty blastomeres were individually fixed on slides. Two were lost due to fixation. The remaining 58 blastomeres were separated into 4 groups to amplify the gene sequence by specific primers for sexing, W1282X and AF508 mutation in cystic fibrosis, sickle cell mutation in h u m a n beta-globin, and steroid 21 hydroxylase gene (CYP21) in various conditions. Group I: Blastomeres (n=30) were used to perform 5 sequential PCRs. Group II: Blastomeres (n=10) were used to perform PRINS prior to 5 sequential PCRs. Group III: Blastomeres ( n = l l ) were stained with hematoxylin before performing 5 sequential PCRs. Group IV: Blastomeres (n=7) were used to pre-amplify whole DNA by DOP-PCR before performing PCR. Results: A high percentage (96.7%) of blastomeres were fixed before PCR was performed. The amplification efficiency of 4 experimental groups were shown in the following table:
PCR
Group
Fixation Efficiency
I
30/30
II
10/11
III
11/11
IV
7/8
1st Round Sexing (%)
2nd Round W1282X (%)
3rd Round AF508 (%)
4th Round fl-Globin
5th Round CYP21 (%)
30/30 (100%) 10/10 (100%) 6/11 (54.5%) 7/7 (100%)
30/30 (100%) 10/10 (100%) 4/11 (36.4%) 7/7 (100%)
29/30 (96.6%) 10/10 (100%) 2/11 (18.2%) 7/7 (100%)
25/30 (83.3%) 8/10 (80%) 2/11 (18.2%) 7/7 (100%)
17/30 (56.7%) 4/10 (40%) Ull (9.1%) 7/7 (100%)
In groups I and II, the gene products were successfully amplified from the blastomeres fixed on the slide. Only after the 4th round did the ADO rate increase substantially. A poor amplification efficiency was noticed in group S4
Abstracts
III even in the 1st round of PCR. Group IV demonstrates the best efficiency among the four groups. The consistency of sexing between pairs of sibling blastomeres was 100%. Conclusion: 1. Blastomeres fixed for PRINS and presumably FISH can be recycled for PCR to obtain more genetic information. 2. The process of hematoxylin staining appears to increase the incidence of ADO. 3. With DOP-PCR, the whole DNA of single blastomeres was uniformly and randomly amplified. This technique appears to facilitate simultaneous multiple preimplantation genetic diagnosis with high efficiency. In addition, it also has m a n y applications such as coupling with comparative genomic hybridization (CGH) and with DNA fingerprinting for the analysis of chromosomal imbalance in entire genomes as well as detection of contamination.
0-007 Preimplantation Testing for Common Chromosomal Aneuploidies in IVF Patients of Advanced Maternal Age. Y. Verlinsky, J. Cieslak, V. Ivakhnenko, M. White, A. Lifchez, B. Kaplan, J. Moise, J. Valle, N. Ginsberg, C. Strom and A. Kuliev. Reproductive Genetics Institute, Illinois Masonic Medical Center, Chicago, IL. Objectives: Because most age-related aneuploidies originate mainly from nondisjunction in the first or second maternal meiotic divisions, preimplantation aneuploidy testing of oocytes will make it possible to prevent common chromosomal aneuploidies before pregnancy, and improve the efficiency of IVF in patients of advanced maternal age. Design: The first and second polar bodies (PB) were removed following their extrusion from the oocytes and studied by fluorescent in situ hybridization (FISH), to avoid fertilization and transfer of aneuploid oocytes. Materials and Methods: A total of 2839 oocytes were biopsied from 337 patients of advanced maternal age (484 IVF cycles) and analyzed by FISH, using probes specific for chromosomes 13, 18 and 21. Embryos. resulting from oocytes free from chromosomal abnormalities were transferred back to patients, while those resulting from abnormal oocytes were studied by FISH to confirm the PB diagnosis. Results: Of 2376 (83.7%) oocytes with FISH results, 946 (39.8%) were predicted to be aneuploid and excluded from the transfer. Of 1430 aneuploidy free oocytes (60.2%), 1070 fertilized, developed normally to the 8-cell stage, and were transferred in a total of 418 cycles, resulting in 89 clinical pregnancies. As high as 26% pregnancy rate was observed when the oocytes were tested by both the first and second PB, compared to 18.2% if preimplantation diagnosis was based on the first or second PB analysis only. Overall, 53 healthy children have been born, following confirmation of preimplantation PB diagnosis by CVS or amniocentesis. Conclusion: The study suggests a clinical value ofpreimplantation PB diagnosis for avoiding the age-related risk for common aneuploidies in IVF.
0-008 In Vitro Maturation of H u m a n Oocytes for the Patients w h o are at the Risk of Hyperstimulation Dur-