- Email: [email protected]
P-931
pick-up. On day 1, fertilized zygotes were cultured in IVF medium (Vitrolife, Kungsbacka, Sweden). On day 2, embryos were passed to culture plates with endometrial epithelial cells, and were co-cultured in a mixture of IVF:CCM medium in 1:1 ratio (Vitrolife, Kungsbacka, Sweden). On day 3, in group 1 the medium was changed to CCM in culture plates and embryos were cultured until day 5, the day of transfer. On day 3, in group 2, embryos with more than 5 blastomeres underwent embryo biopsy with laser or acid tyrode⬘s using G-PGD medium (Vitrolife, Kungsbacka, Sweden). 1 or 2 blastomeres were biopsied depending on embryo quality and were later analyzed by fluorescent in situ hybridization (FISH). FISH analysis was done for chromosomes 13, 15, 16, 21, 22, X and Y according to manufacturer⬘s instructions (Vysis Inc., Downers Grove, Il., USA). Biopsied embryos were left in coculture in CCM medium until day 5 when chromosomally and morphologically normal embryos were transferred. The statistical analysis between the two groups was done using Chi-Square and Fisher⬘s exact test where appropriate. RESULTS: The percentage of transfers done per pick-up in group 1 was 97.06% vs. 84.16% in group 2. The mean age was 32.41⫾2.67 in group 1 vs. 33.48⫾2.38 in group 2. The mean number of embryos transferred in group 1 was 2.82⫾0.8 vs. 1.84⫾0.65 in group 2. The implantation rate was significantly higher in group 2 compared to group 1; being 30.77% vs. 17.2% respectively (p⫽ 0.0264).In group 2, the pregnancy rate was higher than group 1, being 40.0% vs. 30.3% respectively. In group 2, miscarriage rates were less than in group 1; being 8.82% vs. 30.0% respectively. The statistical comparison of miscarriage rates and pregnancy rates between the two groups did not show significant differences. CONCLUSION: Use of PGD in implantation failure patients provides significantly higher implantation rates compared to blastocyst transfer without previous aneuploidy screening. Chromosomal abnormalities in embryos might be one of the factors leading to implantation failure, and therefore, by transfer of chromosomally normal embryos the reproductive outcome in these patients can be improved. Supported by: None.
CONCLUSION: This approach does not provide the error rate of PGD since only blastocysts were reanalysed and not arrested embryos. It is well known (Sandalinas et al. 2001) that some abnormalities survive less often to blastocyst than normal embryos, thus the population here has been enriched with normal embryos.These findings indicate that the majority of embryos reaching blastocyst stage previously classified by Day 3 PGD as complex, trisomy or monosomy X or 21 were abnormal. Their average numbers of cells did not differ from normal embryos. In contrast, the few monosomies other than X and 21 reaching blastocyst were not monosomies but extensive mosaics or minimal mosaics. We suggest that for those embryos, a blastocyst biopsy could be considered and if confirmed normal, replaced. Supported by: None.
P-931 P-930 PLOIDY OF DAY 5 AND 6 EMBRYOS REACHING BLASTOCYST AND CLASSIFIED AS CHROMOSOMALLY ABNORMAL AFTER SINGLE CELL BIOPSY AND PGD ON DAY 3. A. Mick, H. F. Rodriguez, P. Colls, M. Sandalinas, J. Cohen, S. Munne. Center for Advanced Reproductive Endocrinology, Plantation, FL; Reprogenetics, West Orange, NJ; Reprogenetics, Barcelona, Spain. OBJECTIVE: To assess D5 and 6 ploidy and cell numbers in some embryos donated to research that reached post-compaction and were diagnosed as abnormal by Day 3 FISH results. The purpose of this study was not to determine the error rate of PGD, which can only be accomplished by reanalysis of all abnormal embryos, and not only those reaching blastocyst, but to determine if morphology can help detect those errors. DESIGN: Cross-sectional analysis. MATERIALS AND METHODS: Some embryos reported as abnormal by Day 3 single blastomere FISH, which reached various post-compaction developmental stages by days 5-6, were frozen. Those which survived freeze-thaw and underwent uneventful nuclear fixation were analysed (N⫽23). FISH analyses involved chromosomes X,Y,13,15,16,17,18,21,22 and were performed at Reprogenetics, N.J. Day 5 or 6 embryos with ⬍28% abnormal cells were classified as minimal mosaics; mean cell number/ embryo was 79.5⫾44.6 (range:10-230 cells). RESULTS: Of the embryos reported by Day 3 PGD as abnormal, 23 reached day 5 or 6 and survived thawing. Of those, 8 developed into blastocysts with ⱖ90cells. For embryos classified by PGD as monosomies other than X and 21, the proportion forming blastocysts with ⱖ90 cells was 2/5. Of those two, one was a minimal mosaic and the other an extensive mosaic with most cells abnormal. Average number of cells for Day 5/6 Extensive Mosaic, Minimal Mosaic, Monosomy X or 21, other monosomies and Trisomy embryos was 78, 87.3, 75, 10, and 64.5, respectively. The frequency of chromosome anomalies and number of Day 5/6 embryos reaching ⱖ90cells as compared to Day 3 FISH findings is shown in the Table.
FERTILITY & STERILITY威
ANEUPLOIDY ANALYSIS OF SINGLE CELLS BY HIGH RESOLUTION COMPARATIVE GENOMIC HYBRIDIZATION AFTER WHOLE GENOME AMPLIFICATION USING A RANDOM FRAGMENTATION APPROACH. B. Levy, O. Nahum, K. Hirschhorn. Columbia Univ Medical Center, New York, NY; Mount Sinai School of Medicine, New York, NY. OBJECTIVE: To use a random fragmentation whole genome amplification (WGA) approach combined with high resolution comparative genomic hybridization (CGH) to detect the chromosomal status of single cells. DESIGN: Molecular cytogenetic analysis of single cells extracted from discarded and de-identified clinical cell cultures after conventional cytogenetic analysis. IRB-approved study. MATERIALS AND METHODS: Single cells were extracted from 20 random specimen cultures that were initially obtained for clinical cytogenetic analysis. All cultures were de-identified and blinded (with respect to the cytogenetic diagnosis) before being transferred from the clinical laboratory to the research laboratory. Single cells were lysed and then subject to WGA using a random fragmentation approach (GenomePlex® - SigmaAldrich). The resulting DNA was labeled using Nick-translation and high resolution CGH was performed to determine DNA copy number and thus the chromosomal status of the single cells. CGH results were compared to the karyotype as determined by conventional cytogenetic analysis. RESULTS: See Table. CONCLUSION: High resolution CGH analysis of single cells following WGA using a random fragmentation approach has yielded extremely promising results. There were no false negatives and all but one of the chromosomal abnormalities was apparent at the 99.99% CI. Certain artifactual abnormalities were observed in addition to the true abnormality in some cases and the impact of this still needs to be determined in a larger test series. This approach offers significant benefits over current FISH-based preimplantation genetic diagnosis as the entire genome is scanned for chromosomal imbalances. Further assessment of sensitivity and specificity is required before such methods can be utilized in a clinical setting.
S479
tablishment of pregnancies after frozen-thaw processes. This reports a successful singleton pregnancy after embryo biopsy - vitrification - FET in a subsequent cycle following HD-free ET in view that international blastomere transport was delayed at the origin customs. DESIGN: Case report. MATERIALS AND METHODS: A 33 yo WMF, G0, daughter of an affected mother sought IVF-PGD alternative. After several consultations and a failed IVF-PGD abroad, the couple consented for the procedure with the understanding of possible unforeseen “roadblocks”. Pre-treatment testing checking all situations was concluded without problems. Ovarian stimulation was accomplished with a standard long protocol employing daily 225 IU of r-FSH. Twelve mature oocytes obtained were inseminated by ICSI; 10 zygotes were cultured in sequential medium. Seven morphologically superior embryos containing at least six-cells were biopsied, 1 or 2 blastomeres were removed, fixed and sent to Genesis Genetics Institute, MI, USA for analysis. Shipment was hold at Brazilian customs for an additional day; therefore we decided to vitrified the embryos by the Vitri-Safe Tip method. Briefly, the biopsied embryos were placed in an equilibrated solution (ES; 7.5% ethylene glycol, 7.5% DMSO and 20% SSS) for 5-10 min followed by introducing in vitrification solution (VS; 15% ethylene glycol, 15% DMSO, 0.5M sucrose and 20% SSS) for 30 sec. At room temperature embryos were loaded into Vitri-Safe tips, the both ends were sealed and immediately submerged in liquid nitrogen. Following the menstrual cycle, uterus priming was carried out with estradiol valerate and micronized progesterone. Three HD-free embryos were thawed in a twosteps method through dilutions of sucrose. After washing the embryos were cultured for 3 hrs and transferred via Sidney catheter. RESULTS: Patient established a viable singleton pregnancy currently at 12 weeks. Ultrasound exam revealed a normal nuchal translucency (1.1 cm) and without the presence of major abnormalities. CONCLUSION: To our knowledge, this is the first reported ongoing pregnancy from EBx-IVF-ICSI-PGD-HD, vitrified embryos with the VitriSafe Tip technique. This case illustrates that meticulous preparedness should be taken before engagement of IVF clinics from under-or-developing countries seek to help their patients. Alternative use of cryopreservation with vitrification of biopsied embryos could lead to a favorable outcome. This adds to the armamentarium of vitrifying all embryos before to international transportation of blastomeres. Supported by: None.
P-933
* Partial Trisomy 22 was apparent at the 99.9% CI but not at the 99.99% CI Supported by: grants from the NIH (K08-HD043112) and MOD (1FY03-102) P-932 EMBRYO VITRIFICATION AFTER BIOPSY (EBX) FOR PREIMPLANTATION GENETIC DIAGNOSIS (PGD) TO ACCOMMODATE FOR OVERSEAS TESTING: ONGOING HUNTINGTON’S DISEASE (HD)-FREE PREGNANCY FOLLOWING FROZENTHAWED TRANSFER (FET). P. C. Serafini, J. Alegretti, P. Hassun, E. Motta, G. Smith, M. Hughes. Centro Reproduc¸ao Huntington Brazil, Sao Paulo, Brazil; Dept. Obstetrics and Gynecology, U. Michigan, Ann Arbour, MI; Genetics Institute, Detroit, MI. OBJECTIVE: PGD for the investigation of monogenetic disorders is currently offered to couples at risk in developed countries. High costs with the setting and running of a center of excellence, hiring of professional experts make these services very difficult to afford in developing countries. Couple’s time out of work, to traveling and accommodation costs make IVF-ICSI-PGD unaffordable to a large needy population worldwide. Although there are few publications on FET followed by EBx for PGD; there were no cases described for embryo freezing after biopsy to accommodate for a safe embryo transfer (ET). World zones time differences, international courier timetables, customs requirements among other issues may prevent a time reporting of a PGD to allow for proper “uterine-embryonic implantation window”. Advances in vitrification have permitted an increasing es-
S480
Abstracts
PREIMPLANTATION GENETIC SCREENING AS AN ALTERNATIVE TO PRENATAL SCREENING FOR DOWN SYNDROME: PREFERENCES OF WOMEN UNDERGOING IVF/ICSI TREATMENT. M. Twisk, M. L. Haadsma, S. Repping, S. Mastenbroek, M. Heineman, J. C. Korevaar. Academic Medical Center, Amsterdam, The Netherlands; Univ. Medical Center Groningen, Groningen, The Netherlands. OBJECTIVE: Although the primary goal of preimplantation genetic screening (PGS) is to increase pregnancy rates in women undergoing IVF/ICSI, it has been suggested that it may also be used as an alternative to prenatal screening for Down syndrome. However, PGS might not be able to prevent all aneuploid pregnancies, mainly due to mosaicism. Furthermore, biopsy of one or more blastomeres might reduce pregnancy rates. We assessed women’s attitudes towards PGS as an alternative to prenatal screening. DESIGN: Trade-off questionnaires MATERIALS AND METHODS: Between May and December 2005 questionnaires were handed out to women in the stimulation phase of an IVF/ICSI cycle in two academic hospitals in the Netherlands. First, information on all forms of prenatal testing was provided and women were asked whether they would consider prenatal testing for Down syndrome if they were pregnant. Subsequently, information on PGS as a possible alternative was provided followed by three scenarios that differed in pregnancy chances achieved after PGS and in sensitivity of PGS in detecting Down syndrome embryos. In the first scenario PGS prevented all Down syndrome pregnancies, without any negative effect on pregnancy chances. In the second scenario PGS prevented all Down syndrome pregnancies, but lowered pregnancy chances from 1/5 to 1/7 per IVF cycle. In the third scenario PGS lowered the chance of having a child with Down syndrome from 1/ 200 to 1/1000, without having any negative effect on pregnancy chances. After each scenario women were asked whether they would choose to have PGS
Vol. 86, Suppl 2, September 2006
CONCLUSION: This approach does not provide the error rate of PGD since only blastocysts were reanalysed and not arrested embryos. It is well known (Sandalinas et al. 2001) that some abnormalities survive less often to blastocyst than normal embryos, thus the population here has been enriched with normal embryos.These findings indicate that the majority of embryos reaching blastocyst stage previously classified by Day 3 PGD as complex, trisomy or monosomy X or 21 were abnormal. Their average numbers of cells did not differ from normal embryos. In contrast, the few monosomies other than X and 21 reaching blastocyst were not monosomies but extensive mosaics or minimal mosaics. We suggest that for those embryos, a blastocyst biopsy could be considered and if confirmed normal, replaced. Supported by: None.
P-931 P-930 PLOIDY OF DAY 5 AND 6 EMBRYOS REACHING BLASTOCYST AND CLASSIFIED AS CHROMOSOMALLY ABNORMAL AFTER SINGLE CELL BIOPSY AND PGD ON DAY 3. A. Mick, H. F. Rodriguez, P. Colls, M. Sandalinas, J. Cohen, S. Munne. Center for Advanced Reproductive Endocrinology, Plantation, FL; Reprogenetics, West Orange, NJ; Reprogenetics, Barcelona, Spain. OBJECTIVE: To assess D5 and 6 ploidy and cell numbers in some embryos donated to research that reached post-compaction and were diagnosed as abnormal by Day 3 FISH results. The purpose of this study was not to determine the error rate of PGD, which can only be accomplished by reanalysis of all abnormal embryos, and not only those reaching blastocyst, but to determine if morphology can help detect those errors. DESIGN: Cross-sectional analysis. MATERIALS AND METHODS: Some embryos reported as abnormal by Day 3 single blastomere FISH, which reached various post-compaction developmental stages by days 5-6, were frozen. Those which survived freeze-thaw and underwent uneventful nuclear fixation were analysed (N⫽23). FISH analyses involved chromosomes X,Y,13,15,16,17,18,21,22 and were performed at Reprogenetics, N.J. Day 5 or 6 embryos with ⬍28% abnormal cells were classified as minimal mosaics; mean cell number/ embryo was 79.5⫾44.6 (range:10-230 cells). RESULTS: Of the embryos reported by Day 3 PGD as abnormal, 23 reached day 5 or 6 and survived thawing. Of those, 8 developed into blastocysts with ⱖ90cells. For embryos classified by PGD as monosomies other than X and 21, the proportion forming blastocysts with ⱖ90 cells was 2/5. Of those two, one was a minimal mosaic and the other an extensive mosaic with most cells abnormal. Average number of cells for Day 5/6 Extensive Mosaic, Minimal Mosaic, Monosomy X or 21, other monosomies and Trisomy embryos was 78, 87.3, 75, 10, and 64.5, respectively. The frequency of chromosome anomalies and number of Day 5/6 embryos reaching ⱖ90cells as compared to Day 3 FISH findings is shown in the Table.
FERTILITY & STERILITY威
ANEUPLOIDY ANALYSIS OF SINGLE CELLS BY HIGH RESOLUTION COMPARATIVE GENOMIC HYBRIDIZATION AFTER WHOLE GENOME AMPLIFICATION USING A RANDOM FRAGMENTATION APPROACH. B. Levy, O. Nahum, K. Hirschhorn. Columbia Univ Medical Center, New York, NY; Mount Sinai School of Medicine, New York, NY. OBJECTIVE: To use a random fragmentation whole genome amplification (WGA) approach combined with high resolution comparative genomic hybridization (CGH) to detect the chromosomal status of single cells. DESIGN: Molecular cytogenetic analysis of single cells extracted from discarded and de-identified clinical cell cultures after conventional cytogenetic analysis. IRB-approved study. MATERIALS AND METHODS: Single cells were extracted from 20 random specimen cultures that were initially obtained for clinical cytogenetic analysis. All cultures were de-identified and blinded (with respect to the cytogenetic diagnosis) before being transferred from the clinical laboratory to the research laboratory. Single cells were lysed and then subject to WGA using a random fragmentation approach (GenomePlex® - SigmaAldrich). The resulting DNA was labeled using Nick-translation and high resolution CGH was performed to determine DNA copy number and thus the chromosomal status of the single cells. CGH results were compared to the karyotype as determined by conventional cytogenetic analysis. RESULTS: See Table. CONCLUSION: High resolution CGH analysis of single cells following WGA using a random fragmentation approach has yielded extremely promising results. There were no false negatives and all but one of the chromosomal abnormalities was apparent at the 99.99% CI. Certain artifactual abnormalities were observed in addition to the true abnormality in some cases and the impact of this still needs to be determined in a larger test series. This approach offers significant benefits over current FISH-based preimplantation genetic diagnosis as the entire genome is scanned for chromosomal imbalances. Further assessment of sensitivity and specificity is required before such methods can be utilized in a clinical setting.
S479
tablishment of pregnancies after frozen-thaw processes. This reports a successful singleton pregnancy after embryo biopsy - vitrification - FET in a subsequent cycle following HD-free ET in view that international blastomere transport was delayed at the origin customs. DESIGN: Case report. MATERIALS AND METHODS: A 33 yo WMF, G0, daughter of an affected mother sought IVF-PGD alternative. After several consultations and a failed IVF-PGD abroad, the couple consented for the procedure with the understanding of possible unforeseen “roadblocks”. Pre-treatment testing checking all situations was concluded without problems. Ovarian stimulation was accomplished with a standard long protocol employing daily 225 IU of r-FSH. Twelve mature oocytes obtained were inseminated by ICSI; 10 zygotes were cultured in sequential medium. Seven morphologically superior embryos containing at least six-cells were biopsied, 1 or 2 blastomeres were removed, fixed and sent to Genesis Genetics Institute, MI, USA for analysis. Shipment was hold at Brazilian customs for an additional day; therefore we decided to vitrified the embryos by the Vitri-Safe Tip method. Briefly, the biopsied embryos were placed in an equilibrated solution (ES; 7.5% ethylene glycol, 7.5% DMSO and 20% SSS) for 5-10 min followed by introducing in vitrification solution (VS; 15% ethylene glycol, 15% DMSO, 0.5M sucrose and 20% SSS) for 30 sec. At room temperature embryos were loaded into Vitri-Safe tips, the both ends were sealed and immediately submerged in liquid nitrogen. Following the menstrual cycle, uterus priming was carried out with estradiol valerate and micronized progesterone. Three HD-free embryos were thawed in a twosteps method through dilutions of sucrose. After washing the embryos were cultured for 3 hrs and transferred via Sidney catheter. RESULTS: Patient established a viable singleton pregnancy currently at 12 weeks. Ultrasound exam revealed a normal nuchal translucency (1.1 cm) and without the presence of major abnormalities. CONCLUSION: To our knowledge, this is the first reported ongoing pregnancy from EBx-IVF-ICSI-PGD-HD, vitrified embryos with the VitriSafe Tip technique. This case illustrates that meticulous preparedness should be taken before engagement of IVF clinics from under-or-developing countries seek to help their patients. Alternative use of cryopreservation with vitrification of biopsied embryos could lead to a favorable outcome. This adds to the armamentarium of vitrifying all embryos before to international transportation of blastomeres. Supported by: None.
P-933
* Partial Trisomy 22 was apparent at the 99.9% CI but not at the 99.99% CI Supported by: grants from the NIH (K08-HD043112) and MOD (1FY03-102) P-932 EMBRYO VITRIFICATION AFTER BIOPSY (EBX) FOR PREIMPLANTATION GENETIC DIAGNOSIS (PGD) TO ACCOMMODATE FOR OVERSEAS TESTING: ONGOING HUNTINGTON’S DISEASE (HD)-FREE PREGNANCY FOLLOWING FROZENTHAWED TRANSFER (FET). P. C. Serafini, J. Alegretti, P. Hassun, E. Motta, G. Smith, M. Hughes. Centro Reproduc¸ao Huntington Brazil, Sao Paulo, Brazil; Dept. Obstetrics and Gynecology, U. Michigan, Ann Arbour, MI; Genetics Institute, Detroit, MI. OBJECTIVE: PGD for the investigation of monogenetic disorders is currently offered to couples at risk in developed countries. High costs with the setting and running of a center of excellence, hiring of professional experts make these services very difficult to afford in developing countries. Couple’s time out of work, to traveling and accommodation costs make IVF-ICSI-PGD unaffordable to a large needy population worldwide. Although there are few publications on FET followed by EBx for PGD; there were no cases described for embryo freezing after biopsy to accommodate for a safe embryo transfer (ET). World zones time differences, international courier timetables, customs requirements among other issues may prevent a time reporting of a PGD to allow for proper “uterine-embryonic implantation window”. Advances in vitrification have permitted an increasing es-
S480
Abstracts
PREIMPLANTATION GENETIC SCREENING AS AN ALTERNATIVE TO PRENATAL SCREENING FOR DOWN SYNDROME: PREFERENCES OF WOMEN UNDERGOING IVF/ICSI TREATMENT. M. Twisk, M. L. Haadsma, S. Repping, S. Mastenbroek, M. Heineman, J. C. Korevaar. Academic Medical Center, Amsterdam, The Netherlands; Univ. Medical Center Groningen, Groningen, The Netherlands. OBJECTIVE: Although the primary goal of preimplantation genetic screening (PGS) is to increase pregnancy rates in women undergoing IVF/ICSI, it has been suggested that it may also be used as an alternative to prenatal screening for Down syndrome. However, PGS might not be able to prevent all aneuploid pregnancies, mainly due to mosaicism. Furthermore, biopsy of one or more blastomeres might reduce pregnancy rates. We assessed women’s attitudes towards PGS as an alternative to prenatal screening. DESIGN: Trade-off questionnaires MATERIALS AND METHODS: Between May and December 2005 questionnaires were handed out to women in the stimulation phase of an IVF/ICSI cycle in two academic hospitals in the Netherlands. First, information on all forms of prenatal testing was provided and women were asked whether they would consider prenatal testing for Down syndrome if they were pregnant. Subsequently, information on PGS as a possible alternative was provided followed by three scenarios that differed in pregnancy chances achieved after PGS and in sensitivity of PGS in detecting Down syndrome embryos. In the first scenario PGS prevented all Down syndrome pregnancies, without any negative effect on pregnancy chances. In the second scenario PGS prevented all Down syndrome pregnancies, but lowered pregnancy chances from 1/5 to 1/7 per IVF cycle. In the third scenario PGS lowered the chance of having a child with Down syndrome from 1/ 200 to 1/1000, without having any negative effect on pregnancy chances. After each scenario women were asked whether they would choose to have PGS
Vol. 86, Suppl 2, September 2006