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Preimplantation genetic diagnosis (PGD) as both a diagnostic and therapeutic tool in assisted reproductive technology (ART)
be shipped to a reference laboratory for analysis, allowing 48 hours for reporting results before embryo transfer.
P-205 Preimplantation genetic diagnosis (PGD) as both a diagnostic and therapeutic tool in assisted reproductive technology (ART). L. B. Werlin, A. DeCherney, I. Rodi, D. Hill, E. Marello, S. Munne. Genesis Network for Reproductive Health, Irvine, CA; ART Reproductive Ctr, Beverly Hills, CA; Coastal Fertility Medical Ctr, Irvine, CA; St Barnabas Medical Ctr, Livingston, NJ. Objective: To evaluate the role of PGD as both a diagnostic and therapeutic tool in ART. Design: An IRB approved, multicenter, randomized prospective study was initiated with 60 patients. Three high-risk groups were identified: 1. Group I: Women with recurrent pregnancy loss (RPL) 2. Group II: Women with advanced maternal age (AMA) 3. Group III: Women who have failed ⬎2 IVF cycles (FC) Results RPL
AMA
FC
TOTAL
# of patients enrolled 11 16 14 41 # of patients PGD 7 7 6 20 # of embryos biopsied 33 41 18 92 # of abnormal embryos 23/33 (69.7%) 22/41 (53.6%) 15/18 (83.3%) 60/92 (65.2%) # of no transfer 2/7 (28.6%) 1/7 (14.3%) 3/6 (50%) 6/20 (30%) # of pregnancies PGD 5/7 (71.4%) 3/7 (42.8%) 0 8/20 (40%) # of pregnancies control 2/4 (50%) 3/9 (33%) 0 5/21 (23.8%)
Materials/Methods: All patients entered into the study met all inclusion criteria and were then randomized into PGD or control in one of the 3 groups. All stimulation protocols utilized Follistim/Antagon and Pregnyl. Oocyte recovery was performed by ultrasound guided transvaginal approach. On day #3 post oocyte recovery all 6 – 8 cell embryos in the PGD group underwent blastomere biopsy and fixation. Slides were then federal expressed to St. Barnabas Hospital in Livingston, New Jersey. FISH was performed utilizing the following probes: x, y, 13, 15, 16, 17, 18, 21, 22. On day #5 post oocyte recovery results were obtained and embryo transfer of no more than 3-chromosomally normal embryos was performed. Medications including corticosteroids, antibiotics, low dose aspirin (80 – 81mg) and progestational supplementation were utilized. Serum B-HCG levels were obtained 12 days post embryo transfer. Results: See table Conclusions: A number of preliminary comments can be made: 1. PGD does appear to be beneficial in the RPL group. 2. PGD does appear to confirm that aneuploidy is a common cause of RPL. 3. PGD does not appear to be beneficial in either the AMA or FC groups. 4. In view of the large number of abnormal embryos in each group, couples may consider alternative options earlier such as donor oocytes or donor embryos. Supported by: Education Grant funded by Organon Inc. West Orange, New Jersey.
P-206 The effect of oocyte morphology on aneuploidy rates and blastocyst formation in patients with repeated implantation failures following IVF/ICSI. Basak Balaban, Nesrin Ercelen, Aycan Isiklar, Cengiz Alatas, Bulent Urman. VKV American Hosp Istanbul, Istanbul, Turkey. Objective: PGD and aneuploidy assessment have been advocated in couples with repeated implantation failures in ART. PGD may uncover certain chromosomal defects that may be associated with unsuccessful embryo transfer attempts. The aim of this study was correlate the multicolour FISH findings of the day 3 embryo with oocyte morphology and subsequent blastocyst formation in couples with 2 or more previous implantation failures. Design: Propsectively designed case study.
S184
Abstracts
Materials/Methods: A total of 65 couples were included in the study. At OPU morphologically abnormal oocytes were retrieved from 55 and normal oocytes from 10. Oocyte abnormalities were grouped as extracytoplasmic (dark zona and large PVS), cytoplasmic abnormalities (dark cytoplasm, granular cytoplasm, vacuolar cytoplasm, and refractile body), shape abnormalities, double anomaly, and triple anomaly. The oocytes were fertilized with ICSI. On day 3-embryo biopsy and subsequent multicolor FISH analysis for 13, 16, 18, 21, 22, X, and Y-chromosomes was performed. The embryos were transferred on day 5. The correlation between oocyte morphology, PGD results, blastocyst formation, and pregnancy and implantation rates was studied. Results: Mean age was similar in cycles where normal and abnormal oocytes were retrieved. Oocytes in the cytoplasmic anomaly group showed the lowest fertlization (64.7%) and highest aneuploidy (59.1%) rates. The rate of blastocyst formation from aneuploidic embryos was significantly lower compared with euploidic embryos (22.7% vs 60% for embryos derived from normal oocytes). The same rates were 11.9% and 50% when oocytes with cytoplasmic abnormalities were considered. Karyotypically normal blastocysts derived from oocytes with cytoplasmic abnormalities were of inferior quality (30.7% G1⫹G2) and implanted at a lower rate when compared to karyotypically normal blastocysts derived from normal oocytes (61.1% G1⫹G2). The two clinical pregnancies attained after blastocysts transfer in the cytoplasmic anomaly group ended in spontaneous abortions. Conclusions: Cytoplasmic abnormalities of the oocyte are associated with higher aneuploidy rates of the resulting embryo. Following PGD, blastocysts formed from chromosomally normal embryos implant at a lower rate compared with chromosomally normal blastocysts formed from normal oocytes and oocytes with extracytoplasmic and shape anomalies. Oocyte morphology should be incorporated into the predictors of success followingG PGD and blastocyst transfer. Supported by: None.
P-208 Preimplantation genetic screening of human embryos by PCR using an alternative non commercial method. Beatriz Xoconostle, Roberto Ruiz, Alexandra Bermudez, Silvio Cuneo, Concepcion Yerena, Alfonso Gutierrez Najar. Biotech & Bioengineering Dept of Ctr for Research and Advanced Study, National Polytechnic, Mexico City, Mexico; Reproduccion y Genetica, Mexico City, Mexico. Objective: to screen the genetic status of morphologically normal blastomeres of arrested human embryos by PCR using a method developed in our molecular biology laboratory. Design: prospective and experimental study Materials/Methods: we studied a total of 47 arrested embryos from consent patients of our ART program. Morphologically normal blastomeres were used to amplified by PCR the following genes: ZFX (chromosome X and Y), SOD (chromosome 21) and DCK (chromosome12). For cell lysis a buffer containing non-ionic detergent and Pronase P was used. Further amplification was performed using 24mers designed to posses high annealing temperature. PCR products were obtained using a Robocycler (Stratagen) and then resolved in agarose-gels. A single sharp band of the expected size was obtained in each case. In order to asses gene identity automated DNA sequencing was performed. Results: it was observed that morphologically normal blastomeres from arrested embryos had chromosomic alterations varying from 56% when 2 oligonucleotides were used to 73% when 4 oligonucleotides were tested. The most common chromosomic alteration detected were: aneuploids (41.5%), haploids (7%), poliploids (6%), mosaisisms (32.5%). The X/Y relation was 1.28, being 23 male embryos (56.1%) and 18 (43.9%) females. Conclusions: A highly reproducible and cost-effective PCR method was established to amplified genes from single cell that permitted the detection of high chromosomic abnormalities in morphologically normal blastomeres of non viable human embryos. Currently we are developing new oligonucleotides for detections of others genetic markers. Supported by: Reproduccion y Genetica, Hospital Angeles del Pedregal, Universidad La Salle Biotechnology & Bioengineering Department of Center for Research and Advanced Studies - National Polytechnic Institute (CINVESTAV-IPN), Mexico City.
Vol. 78, No. 3, Suppl. 1, September 2002
P-205 Preimplantation genetic diagnosis (PGD) as both a diagnostic and therapeutic tool in assisted reproductive technology (ART). L. B. Werlin, A. DeCherney, I. Rodi, D. Hill, E. Marello, S. Munne. Genesis Network for Reproductive Health, Irvine, CA; ART Reproductive Ctr, Beverly Hills, CA; Coastal Fertility Medical Ctr, Irvine, CA; St Barnabas Medical Ctr, Livingston, NJ. Objective: To evaluate the role of PGD as both a diagnostic and therapeutic tool in ART. Design: An IRB approved, multicenter, randomized prospective study was initiated with 60 patients. Three high-risk groups were identified: 1. Group I: Women with recurrent pregnancy loss (RPL) 2. Group II: Women with advanced maternal age (AMA) 3. Group III: Women who have failed ⬎2 IVF cycles (FC) Results RPL
AMA
FC
TOTAL
# of patients enrolled 11 16 14 41 # of patients PGD 7 7 6 20 # of embryos biopsied 33 41 18 92 # of abnormal embryos 23/33 (69.7%) 22/41 (53.6%) 15/18 (83.3%) 60/92 (65.2%) # of no transfer 2/7 (28.6%) 1/7 (14.3%) 3/6 (50%) 6/20 (30%) # of pregnancies PGD 5/7 (71.4%) 3/7 (42.8%) 0 8/20 (40%) # of pregnancies control 2/4 (50%) 3/9 (33%) 0 5/21 (23.8%)
Materials/Methods: All patients entered into the study met all inclusion criteria and were then randomized into PGD or control in one of the 3 groups. All stimulation protocols utilized Follistim/Antagon and Pregnyl. Oocyte recovery was performed by ultrasound guided transvaginal approach. On day #3 post oocyte recovery all 6 – 8 cell embryos in the PGD group underwent blastomere biopsy and fixation. Slides were then federal expressed to St. Barnabas Hospital in Livingston, New Jersey. FISH was performed utilizing the following probes: x, y, 13, 15, 16, 17, 18, 21, 22. On day #5 post oocyte recovery results were obtained and embryo transfer of no more than 3-chromosomally normal embryos was performed. Medications including corticosteroids, antibiotics, low dose aspirin (80 – 81mg) and progestational supplementation were utilized. Serum B-HCG levels were obtained 12 days post embryo transfer. Results: See table Conclusions: A number of preliminary comments can be made: 1. PGD does appear to be beneficial in the RPL group. 2. PGD does appear to confirm that aneuploidy is a common cause of RPL. 3. PGD does not appear to be beneficial in either the AMA or FC groups. 4. In view of the large number of abnormal embryos in each group, couples may consider alternative options earlier such as donor oocytes or donor embryos. Supported by: Education Grant funded by Organon Inc. West Orange, New Jersey.
P-206 The effect of oocyte morphology on aneuploidy rates and blastocyst formation in patients with repeated implantation failures following IVF/ICSI. Basak Balaban, Nesrin Ercelen, Aycan Isiklar, Cengiz Alatas, Bulent Urman. VKV American Hosp Istanbul, Istanbul, Turkey. Objective: PGD and aneuploidy assessment have been advocated in couples with repeated implantation failures in ART. PGD may uncover certain chromosomal defects that may be associated with unsuccessful embryo transfer attempts. The aim of this study was correlate the multicolour FISH findings of the day 3 embryo with oocyte morphology and subsequent blastocyst formation in couples with 2 or more previous implantation failures. Design: Propsectively designed case study.
S184
Abstracts
Materials/Methods: A total of 65 couples were included in the study. At OPU morphologically abnormal oocytes were retrieved from 55 and normal oocytes from 10. Oocyte abnormalities were grouped as extracytoplasmic (dark zona and large PVS), cytoplasmic abnormalities (dark cytoplasm, granular cytoplasm, vacuolar cytoplasm, and refractile body), shape abnormalities, double anomaly, and triple anomaly. The oocytes were fertilized with ICSI. On day 3-embryo biopsy and subsequent multicolor FISH analysis for 13, 16, 18, 21, 22, X, and Y-chromosomes was performed. The embryos were transferred on day 5. The correlation between oocyte morphology, PGD results, blastocyst formation, and pregnancy and implantation rates was studied. Results: Mean age was similar in cycles where normal and abnormal oocytes were retrieved. Oocytes in the cytoplasmic anomaly group showed the lowest fertlization (64.7%) and highest aneuploidy (59.1%) rates. The rate of blastocyst formation from aneuploidic embryos was significantly lower compared with euploidic embryos (22.7% vs 60% for embryos derived from normal oocytes). The same rates were 11.9% and 50% when oocytes with cytoplasmic abnormalities were considered. Karyotypically normal blastocysts derived from oocytes with cytoplasmic abnormalities were of inferior quality (30.7% G1⫹G2) and implanted at a lower rate when compared to karyotypically normal blastocysts derived from normal oocytes (61.1% G1⫹G2). The two clinical pregnancies attained after blastocysts transfer in the cytoplasmic anomaly group ended in spontaneous abortions. Conclusions: Cytoplasmic abnormalities of the oocyte are associated with higher aneuploidy rates of the resulting embryo. Following PGD, blastocysts formed from chromosomally normal embryos implant at a lower rate compared with chromosomally normal blastocysts formed from normal oocytes and oocytes with extracytoplasmic and shape anomalies. Oocyte morphology should be incorporated into the predictors of success followingG PGD and blastocyst transfer. Supported by: None.
P-208 Preimplantation genetic screening of human embryos by PCR using an alternative non commercial method. Beatriz Xoconostle, Roberto Ruiz, Alexandra Bermudez, Silvio Cuneo, Concepcion Yerena, Alfonso Gutierrez Najar. Biotech & Bioengineering Dept of Ctr for Research and Advanced Study, National Polytechnic, Mexico City, Mexico; Reproduccion y Genetica, Mexico City, Mexico. Objective: to screen the genetic status of morphologically normal blastomeres of arrested human embryos by PCR using a method developed in our molecular biology laboratory. Design: prospective and experimental study Materials/Methods: we studied a total of 47 arrested embryos from consent patients of our ART program. Morphologically normal blastomeres were used to amplified by PCR the following genes: ZFX (chromosome X and Y), SOD (chromosome 21) and DCK (chromosome12). For cell lysis a buffer containing non-ionic detergent and Pronase P was used. Further amplification was performed using 24mers designed to posses high annealing temperature. PCR products were obtained using a Robocycler (Stratagen) and then resolved in agarose-gels. A single sharp band of the expected size was obtained in each case. In order to asses gene identity automated DNA sequencing was performed. Results: it was observed that morphologically normal blastomeres from arrested embryos had chromosomic alterations varying from 56% when 2 oligonucleotides were used to 73% when 4 oligonucleotides were tested. The most common chromosomic alteration detected were: aneuploids (41.5%), haploids (7%), poliploids (6%), mosaisisms (32.5%). The X/Y relation was 1.28, being 23 male embryos (56.1%) and 18 (43.9%) females. Conclusions: A highly reproducible and cost-effective PCR method was established to amplified genes from single cell that permitted the detection of high chromosomic abnormalities in morphologically normal blastomeres of non viable human embryos. Currently we are developing new oligonucleotides for detections of others genetic markers. Supported by: Reproduccion y Genetica, Hospital Angeles del Pedregal, Universidad La Salle Biotechnology & Bioengineering Department of Center for Research and Advanced Studies - National Polytechnic Institute (CINVESTAV-IPN), Mexico City.
Vol. 78, No. 3, Suppl. 1, September 2002