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The number of available embryos for biopsy is predictive for the outcome of PGD cycles
However, the overall frequency of chromosome anomalies among spontaneous abortions was found to be considerable high. We initiated a prospective study to obtain an insight in fertilization process in PCO as compared with non PCO patients that underwent in vitro fertilization treatment. Design: Prospective consecutive study. Materials/Methods: Sixty one PCO and 86 non PCO patients underwent 104 and 144 IVF cycles respectively, between 1.1.2000 to 31.12.2001. Same protocol of stimulation was applied to both groups. The length of ovarian stimulation, quantity of hMG used, peak E2 levels, mean number of follicles developed, oocytes retrieved, fertilization rate, percentage of triploid fertilization, cleavage rate and embryo quality were compared between the groups. Results: There were no significant differences between the groups in regard the length of stimulation, peak E2 levels, mean number of oocytes retrieved, fertilization and cleavage rate, embryo morphology and pregnancy rate. The mean dose of hMG administered in the PCO group was lower 37.1 plusminus 14.0 vs. 47.8 plusminus 19.9 ampoules, p ⬍0.05). The incidence of polyploid fertilization was 139 out of 832 fertilized oocytes vs. 62 out of 708 in PCO and non PCO patients, accordingly (p ⬍0.005). Conclusions: High incidence of polyploid fertilization occurred in PCO patients, possible either due to dispermic fertilization or failure of second polar body extrusion, as a consequence of more compromised oocytes retrieved. In IVF cycles, those polyploid embryos will be not transferred. However, in natural cycles, polyploid fertilization may occurred and the resulting embryos implant, possibly causing the increase in abortion rate found higher in PCO patients. Supported by: Hillel Yaffe medical center.
P-213 Pregnancy following successful preimplantation genetic diagnosis for alagille syndrome. Kangpu Xu, Steve Spandorfer, Hongmei Yuan, Lucinda Veeck, Zev Rosenwaks. CRMI, Weill Medical Coll of Cornell Univ, New York, NY. Objective: Fluorescent in situ hybridization (FISH) has been widely applied for preimplantation genetic diagnosis (PGD) of X-linked disorders and aneuploidy analysis. Recently, FISH has also been effective in detection of chromosomal imbalance for translocation carriers. FISH for chromosome deletion, however, has not been extensively studied. We report here a successful PGD case analyzed by FISH for Alagille syndrome (MIM# 118450), a dominant inherited developmental disorder. Design: A 26 years old female patient was diagnosed as having a mosaic cytogenetically visible deletion of chromosome band 20p11.23-p12. Because the couple had one affected child, IVF and PGD was recommended as an option to avoid transferring affected embryos. The study was approved by the IRB. Materials/Methods: To confirm the status of the deletion and to test the probes, interphase and metaphase nuclei were obtained from cultured female blood. The DNA probe specific for the deletion (20p12) was kindly provided by Dr. N. Spinner (Philadelphia Children’s Hospital, PA). To test the deletion status, a control probe (TelVysion 20p, Vysis) was also applied to the patient specimen and the lymphocyte nuclei of known to have a normal karyotype. Deletion was clearly confirmed from the patient in 35% (855/2442) of her nuclei examined. No deletion was seen in the control sample. After a standard ovarian stimulation and IVF procedure, Day-3 embryo biopsy was performed and embryo transfer was carried out on Day-5. Results: In the first IVF-PGD attempt, 6 of the 10 oocytes retrieved were mature and these were each injected with a single sperm. Of those, 5 were normally fertilized and 5 embryos were biopsied on Day-3. One embryo showed a missed signal from the specific DNA probe, while two signals from the control probe were clearly visible, indicating an affected embryo. One blastomere from a second embryo appeared to be abnormal but this was not certain. Two multinucleated blastomeres from a third embryo (5-cell) did not give informative signals from either of the two probes. Two blastomeres from two remaining embryos showed both specific and control signals and thus were diagnosed as normal. These two embryos were transferred, one at the blastocyst stage and the other as a morula. Clinical pregnancy was established and the patient delivered a normal healthy boy. Confirmation test is pending. Conclusions: To our knowledge, this is the first PGD case for Alagille
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syndrome. It demonstrates that FISH can be used effectively for preimplantation diagnosis in the case of chromosome deletions. Supported by: Institutional Fund.
P-214 The number of available embryos for biopsy is predictive for the outcome of PGD cycles. Soraya Abdelmassih, Zsolt Peter Nagy, Vicente Abdelmassih, Roger Abdelmassih, Santiago Munne. Clin e Ctr de Pesquisa em Reproducao Humana, Sao Paulo, Brazil; The Institute for Reproductive Medicine and Science of Saint Barnabas, West Orange, NJ. Objective: Removal of a single blastomere for preimplantation genetic diagnosis (PGD) is a well established method, however, it is not known if the number of embryos available for biopsy procedure has an important influence on pregnancy outcome. In the present study, therefore, we investigated the correlation between the number of embryos available for biopsy procedure (in PGD treatment cycles) and the pregnancy outcome and we compared these data to those of the control population (patients without embryo biopsy procedure). Design: Retrospective analysis of data obtained from 87 PGD (embryo biopsy group) cycles and from 222 ICSI-IVF cycles (control group). Patients (from both groups) were divided into two groups according to the number of embryos potentially available for biopsy procedure (4 or less embryos in one group and 5 or more embryos in the second group) and laboratory and clinical data were analyzed respectively. Materials/Methods: Controlled ovarian stimulation and other IVF procedures were standard in all patients. Embryo for biopsy procedure was eligible when there were 5 or more blastomeres present on day 3. Biopsy procedure was performed on day 3 after laser drilling of the zona pellucida by removing a single blastomere in patients of the PGD group. Embryos of the control group were not manipulated, however, assisted hatching was occasionally performed. ET was performed 2–3 hours after micromanipulation using the Wallace catheter (in both groups). Laboratory and clinical data were evaluated and compared by the One-way ANOVA test or by the Fisher exact test whenever applicable. Results: The most important clinical and laboratory data are summarized in the table. Age of women, fertilization and embryo quality (cell number and fragmentation) values were similar between all groups. Implantation rates were 9.1%, 14.4%, 17.2 and 17.4% respectively (NS). Outcome of PGD and Control cycles with ⬍5 or ⫽5 embryos eligible for biopsy. Groups
PGD-Biopsy PGD-Biopsy
Control
Control
Embryos eligible ⬍5 embryos ⫽5 embryos ⬍5 embryos ⫽5 embryos for biopsy Cycles 35 52 85 137 Age of women 37.5 (5.9) 36.5 (5.4) 37.1 (5.7) 36.9 (5.2) (⫽/⫺S.D.) MII oocytes aspirated 5.6 (2.9) 10.3 (3.7) 5.8 (3.1) 10.9 (3.8) (⫹/⫺S.D.)1 Embryos transferred 1.9 (1.1) 3.2 (1.1) 2.1 (0.7) 4.1 (0.9) (⫹/⫺S.D.)2 3 Positive hCG (%) 5 (14.3) 20 (38.5) 29 (34.1) 56 (40.9) Ongoing clinical 4 (11.4) 19 (36.5) 17 (20.0) 41 (30.0) 4 pregnancy (%) 1 P ⬍ 0.05 between column 1 vs. 2 and column 3 vs. 4. 2 P ⬍ 0.05 between column 1 vs. 2 and 4 and 2 vs. 4. 3 P ⬍ 0.05 between column 1 vs. 2. 4 P ⬍ 0.05 between column 1 vs. 2 and 2 vs. 3.
Conclusions: Number of embryos available for biopsy procedure is correlated with the pregnancy outcome in PGD treatment cycles. Fewer number of embryos available for transfer (⬍5) do not decrease significantly the pregnancy rate in ICSI-IVF patients without embryo biopsy, however, in PGD cycles where there are less than 5 embryos for biopsy the initial and ongoing clinical pregnancy rates are impaired. Therefore, it is advisable that embryo biopsy is avoided (if possible) when patients have less than 5 embryos. Supported by: None.
Vol. 78, No. 3, Suppl. 1, September 2002
P-213 Pregnancy following successful preimplantation genetic diagnosis for alagille syndrome. Kangpu Xu, Steve Spandorfer, Hongmei Yuan, Lucinda Veeck, Zev Rosenwaks. CRMI, Weill Medical Coll of Cornell Univ, New York, NY. Objective: Fluorescent in situ hybridization (FISH) has been widely applied for preimplantation genetic diagnosis (PGD) of X-linked disorders and aneuploidy analysis. Recently, FISH has also been effective in detection of chromosomal imbalance for translocation carriers. FISH for chromosome deletion, however, has not been extensively studied. We report here a successful PGD case analyzed by FISH for Alagille syndrome (MIM# 118450), a dominant inherited developmental disorder. Design: A 26 years old female patient was diagnosed as having a mosaic cytogenetically visible deletion of chromosome band 20p11.23-p12. Because the couple had one affected child, IVF and PGD was recommended as an option to avoid transferring affected embryos. The study was approved by the IRB. Materials/Methods: To confirm the status of the deletion and to test the probes, interphase and metaphase nuclei were obtained from cultured female blood. The DNA probe specific for the deletion (20p12) was kindly provided by Dr. N. Spinner (Philadelphia Children’s Hospital, PA). To test the deletion status, a control probe (TelVysion 20p, Vysis) was also applied to the patient specimen and the lymphocyte nuclei of known to have a normal karyotype. Deletion was clearly confirmed from the patient in 35% (855/2442) of her nuclei examined. No deletion was seen in the control sample. After a standard ovarian stimulation and IVF procedure, Day-3 embryo biopsy was performed and embryo transfer was carried out on Day-5. Results: In the first IVF-PGD attempt, 6 of the 10 oocytes retrieved were mature and these were each injected with a single sperm. Of those, 5 were normally fertilized and 5 embryos were biopsied on Day-3. One embryo showed a missed signal from the specific DNA probe, while two signals from the control probe were clearly visible, indicating an affected embryo. One blastomere from a second embryo appeared to be abnormal but this was not certain. Two multinucleated blastomeres from a third embryo (5-cell) did not give informative signals from either of the two probes. Two blastomeres from two remaining embryos showed both specific and control signals and thus were diagnosed as normal. These two embryos were transferred, one at the blastocyst stage and the other as a morula. Clinical pregnancy was established and the patient delivered a normal healthy boy. Confirmation test is pending. Conclusions: To our knowledge, this is the first PGD case for Alagille
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Abstracts
syndrome. It demonstrates that FISH can be used effectively for preimplantation diagnosis in the case of chromosome deletions. Supported by: Institutional Fund.
P-214 The number of available embryos for biopsy is predictive for the outcome of PGD cycles. Soraya Abdelmassih, Zsolt Peter Nagy, Vicente Abdelmassih, Roger Abdelmassih, Santiago Munne. Clin e Ctr de Pesquisa em Reproducao Humana, Sao Paulo, Brazil; The Institute for Reproductive Medicine and Science of Saint Barnabas, West Orange, NJ. Objective: Removal of a single blastomere for preimplantation genetic diagnosis (PGD) is a well established method, however, it is not known if the number of embryos available for biopsy procedure has an important influence on pregnancy outcome. In the present study, therefore, we investigated the correlation between the number of embryos available for biopsy procedure (in PGD treatment cycles) and the pregnancy outcome and we compared these data to those of the control population (patients without embryo biopsy procedure). Design: Retrospective analysis of data obtained from 87 PGD (embryo biopsy group) cycles and from 222 ICSI-IVF cycles (control group). Patients (from both groups) were divided into two groups according to the number of embryos potentially available for biopsy procedure (4 or less embryos in one group and 5 or more embryos in the second group) and laboratory and clinical data were analyzed respectively. Materials/Methods: Controlled ovarian stimulation and other IVF procedures were standard in all patients. Embryo for biopsy procedure was eligible when there were 5 or more blastomeres present on day 3. Biopsy procedure was performed on day 3 after laser drilling of the zona pellucida by removing a single blastomere in patients of the PGD group. Embryos of the control group were not manipulated, however, assisted hatching was occasionally performed. ET was performed 2–3 hours after micromanipulation using the Wallace catheter (in both groups). Laboratory and clinical data were evaluated and compared by the One-way ANOVA test or by the Fisher exact test whenever applicable. Results: The most important clinical and laboratory data are summarized in the table. Age of women, fertilization and embryo quality (cell number and fragmentation) values were similar between all groups. Implantation rates were 9.1%, 14.4%, 17.2 and 17.4% respectively (NS). Outcome of PGD and Control cycles with ⬍5 or ⫽5 embryos eligible for biopsy. Groups
PGD-Biopsy PGD-Biopsy
Control
Control
Embryos eligible ⬍5 embryos ⫽5 embryos ⬍5 embryos ⫽5 embryos for biopsy Cycles 35 52 85 137 Age of women 37.5 (5.9) 36.5 (5.4) 37.1 (5.7) 36.9 (5.2) (⫽/⫺S.D.) MII oocytes aspirated 5.6 (2.9) 10.3 (3.7) 5.8 (3.1) 10.9 (3.8) (⫹/⫺S.D.)1 Embryos transferred 1.9 (1.1) 3.2 (1.1) 2.1 (0.7) 4.1 (0.9) (⫹/⫺S.D.)2 3 Positive hCG (%) 5 (14.3) 20 (38.5) 29 (34.1) 56 (40.9) Ongoing clinical 4 (11.4) 19 (36.5) 17 (20.0) 41 (30.0) 4 pregnancy (%) 1 P ⬍ 0.05 between column 1 vs. 2 and column 3 vs. 4. 2 P ⬍ 0.05 between column 1 vs. 2 and 4 and 2 vs. 4. 3 P ⬍ 0.05 between column 1 vs. 2. 4 P ⬍ 0.05 between column 1 vs. 2 and 2 vs. 3.
Conclusions: Number of embryos available for biopsy procedure is correlated with the pregnancy outcome in PGD treatment cycles. Fewer number of embryos available for transfer (⬍5) do not decrease significantly the pregnancy rate in ICSI-IVF patients without embryo biopsy, however, in PGD cycles where there are less than 5 embryos for biopsy the initial and ongoing clinical pregnancy rates are impaired. Therefore, it is advisable that embryo biopsy is avoided (if possible) when patients have less than 5 embryos. Supported by: None.
Vol. 78, No. 3, Suppl. 1, September 2002