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Survival of frozen-thawed blastocysts after preimplantation genetic diagnosis: pregnancy and delivery
Abstracts - PGDIS: 8th International Symposium on PGD
to determine aneuploidy for chromosomes X, Y, and 21. The tests were carried out on fresh and processed (by using twolayered density centrifugation, 40%, 80% Percoll+ EHM) sperm samples. Wilcoxon rank test was used to determine the relationship between aneuploidy rates and DNA fragmentation index. When the P-value was less than 0.05 it was considered a statistically significant difference of ratios. Pearson’s correlation with regression analysis at a P-value of less than 0.05 was used to determine the relationship between male age, DNA fragmentation and aneuploidy. Results: An increase of sex chromosome abnormalities in the fragmented spermatozoa was observed when compared with non-fragmented in fresh (P = 0.0012) and processed (P = 0.004) samples. A decreased frequency of aneuploidy (P = 0.027) and DNA fragmentation index (P = 0.032) was observed in DGC compared with neat sperm samples, respectively. A positive relation between age and DNA fragmentation index in fresh samples (P = 0.027 and r = 0.21), which was ruled out after density centrifugation (P = 0.13 and r = 0.1) was also observed. Aneuploidy was not related to age in samples before (P = 0.32 and r = 0.22), and after processing (P = 0.28 and r = 0.17). Conclusion: Sex chromosome abnormalities were increased in the fragmented spermatozoa compared with non-fragmented spermatozoa in neat and processed sperm samples. Sperm samples after DGC had a decreased frequency of aneuploidy and DNA fragmentation index compared with neat sperm samples, possibly because sperm preparation process aids to select mature and functional spermatozoa. Age was not related to sperm aneuploidy in both neat and DGC samples. However, age affected DNA damage in neat sperm samples, which was not confirmed in the processed ones. Survival of frozen-thawed blastocysts after preimplantation genetic diagnosis: pregnancy and delivery Hernández J1, Chinea E1, Sanabria V1, Peña V1, Sandalinas M2, Palumbo A1 1Centro de Asistencia a la Reproducción Humana de Canarias, La Laguna, Tenerife, Spain; 2Reprogenetics Spain, Campus UAB Bellaterra, Barcelona, Spain Objective: Preimplantation genetic diagnosis (PGD) is a labourintensive process that places a burden, both psychologically and economically, on the couple undergoing treatment. When supernumerary healthy embryos are obtained, they can be frozen, thus allowing one more chance for pregnancy with much less effort. However, disappointing results have been reported with the freezing of biopsied embryos. This is a case report of three pregnancies obtained after transferring frozen– thawed blastocysts that had been previously biopsied on day 3. Materials/Methods: Between January 2004 and December 2007 we have performed 24 PGD cycles in 15 patents; in four cycles we were able to freeze supernumerary blastocysts. Embryo biopsy was performed on day 3 and one cell was analysed in embryos with six or more cells. All embyo transfers were performed on day 4 with ultrasound guidance. One to three embryos were transferred. Supernumerary normal embryos were grown to the blastocyst stage (day 5) and then were frozen. The cryopreservation solution was Freeze-kit blast (Vitrolife, Kungsbacka, Sweden) and slow freezing was used for all blastocysts. For thawing we used the Thaw-kit blast (Vitrolife, Kungsbacka, Sweden). Patients received either oral oestradiol valerate or transdermal oestradiol until they had an endometrial thickness ≥8 mm and an oestradiol value between
S-20 Reproductive BioMedicine Online, Vol. 16, Suppl. 3, April 2008
150–300 pg. Vaginal progesterone suppositories were started 6 days before the thaw. All transfers were performed 4 h after the thaw. Results: A total of eight blastocysts were frozen and six of seven were thawed successfully; one blastocysts is still frozen. Two of the patients had one embryo transferred and the other two had two embryos transferred. Pregnancy was achieved in the latter three patients. The first patient was a sex selection case for haemophilia, which was delivered in January 2005. The second patient, who underwent aneuploidy screening for recurrent pregnancy loss, had a 9-week miscarriage. The third patient was monogenetic disease (polycystic kidney) and this pregnancy is currently ongoing. Overall, in this small series, 10 out of 15 (66.6%) patients obtained a pregnancy, and three of these came from cryotransfers. Conclusion: These data indicate that it is always worth freezing supernumerary embryos after performing PGD, since this adds a substantial chance of pregnancy. Human susceptibility to aneuploidy and the link to infertility Mantzouratou A1, Xanthopoulou L1, Mania A1, Tashkandi S1, Harper JC 1, Serhal P 2, Delhanty JDA 1 1Institute of Women’s Health, University College London; 2Assisted Conception Unit, UCLH, UK In preimplantation embryos a wide range of errors has been reported where the incidence of chromosomal aneuploidy is thought to be around 50–80%. A very high proportion of these errors are due to post-zygotic events during mitosis in the embryonic cells and to a much lesser degree during meiosis in the gametes and can have a devastating effect on the fertility of certain couples. Although older females show higher rates of meiotic aneuploidy in prenatal studies, some younger couples going through IVF programmes have an almost equally increased chance of chromosomal abnormalities. In this study, a detailed investigation of embryos from 101 PGS cycles revealed differences in aneuploidy mechanisms in embryos from certain PGS couples. Follow-up results were obtained for 596 of the 787 embryos available for reanalysis (76%). Among these 596 embryos, 53.4% were fully chaotic mosaic and 40.3% were classified as other mosaic types. The most prevalent of the other mosaic types were the aneuploid mosaics (31.9%) followed by those that were diploid/chaotic (26.1%) and aneuploid/chaotic (17.2%). Significant differences were found in the distribution of uniformly abnormal embryos, embryos with meiotic errors and diploid/chaotic mosaic embryos in relation to the referral group. Uniformly abnormal embryos and embryos with meiotic errors were significantly decreased in the RIF group compared with the RM and AMA groups. Diploid chaotic mosaic embryos were found in higher frequency in the RIF group. Within the RIF group significant differences were observed in the distribution of diploid/chaotic mosaic embryos and aneuploid mosaic embryos in relation to insemination method. The RIFICSI group had highly significant increase in the diploid/chaotic mosaic embryos. Differences in susceptibility to aneuploidy of specific chromosomes were found in relation to the mechanism of aneuploidy overall and for each referral group. This study has revealed that susceptibility to embryonic chromosomal abnormalities may differ in certain groups of couples that present with infertility. In general, this study has provided some significant evidence for the following: in a general background of error prone cell divisions
to determine aneuploidy for chromosomes X, Y, and 21. The tests were carried out on fresh and processed (by using twolayered density centrifugation, 40%, 80% Percoll+ EHM) sperm samples. Wilcoxon rank test was used to determine the relationship between aneuploidy rates and DNA fragmentation index. When the P-value was less than 0.05 it was considered a statistically significant difference of ratios. Pearson’s correlation with regression analysis at a P-value of less than 0.05 was used to determine the relationship between male age, DNA fragmentation and aneuploidy. Results: An increase of sex chromosome abnormalities in the fragmented spermatozoa was observed when compared with non-fragmented in fresh (P = 0.0012) and processed (P = 0.004) samples. A decreased frequency of aneuploidy (P = 0.027) and DNA fragmentation index (P = 0.032) was observed in DGC compared with neat sperm samples, respectively. A positive relation between age and DNA fragmentation index in fresh samples (P = 0.027 and r = 0.21), which was ruled out after density centrifugation (P = 0.13 and r = 0.1) was also observed. Aneuploidy was not related to age in samples before (P = 0.32 and r = 0.22), and after processing (P = 0.28 and r = 0.17). Conclusion: Sex chromosome abnormalities were increased in the fragmented spermatozoa compared with non-fragmented spermatozoa in neat and processed sperm samples. Sperm samples after DGC had a decreased frequency of aneuploidy and DNA fragmentation index compared with neat sperm samples, possibly because sperm preparation process aids to select mature and functional spermatozoa. Age was not related to sperm aneuploidy in both neat and DGC samples. However, age affected DNA damage in neat sperm samples, which was not confirmed in the processed ones. Survival of frozen-thawed blastocysts after preimplantation genetic diagnosis: pregnancy and delivery Hernández J1, Chinea E1, Sanabria V1, Peña V1, Sandalinas M2, Palumbo A1 1Centro de Asistencia a la Reproducción Humana de Canarias, La Laguna, Tenerife, Spain; 2Reprogenetics Spain, Campus UAB Bellaterra, Barcelona, Spain Objective: Preimplantation genetic diagnosis (PGD) is a labourintensive process that places a burden, both psychologically and economically, on the couple undergoing treatment. When supernumerary healthy embryos are obtained, they can be frozen, thus allowing one more chance for pregnancy with much less effort. However, disappointing results have been reported with the freezing of biopsied embryos. This is a case report of three pregnancies obtained after transferring frozen– thawed blastocysts that had been previously biopsied on day 3. Materials/Methods: Between January 2004 and December 2007 we have performed 24 PGD cycles in 15 patents; in four cycles we were able to freeze supernumerary blastocysts. Embryo biopsy was performed on day 3 and one cell was analysed in embryos with six or more cells. All embyo transfers were performed on day 4 with ultrasound guidance. One to three embryos were transferred. Supernumerary normal embryos were grown to the blastocyst stage (day 5) and then were frozen. The cryopreservation solution was Freeze-kit blast (Vitrolife, Kungsbacka, Sweden) and slow freezing was used for all blastocysts. For thawing we used the Thaw-kit blast (Vitrolife, Kungsbacka, Sweden). Patients received either oral oestradiol valerate or transdermal oestradiol until they had an endometrial thickness ≥8 mm and an oestradiol value between
S-20 Reproductive BioMedicine Online, Vol. 16, Suppl. 3, April 2008
150–300 pg. Vaginal progesterone suppositories were started 6 days before the thaw. All transfers were performed 4 h after the thaw. Results: A total of eight blastocysts were frozen and six of seven were thawed successfully; one blastocysts is still frozen. Two of the patients had one embryo transferred and the other two had two embryos transferred. Pregnancy was achieved in the latter three patients. The first patient was a sex selection case for haemophilia, which was delivered in January 2005. The second patient, who underwent aneuploidy screening for recurrent pregnancy loss, had a 9-week miscarriage. The third patient was monogenetic disease (polycystic kidney) and this pregnancy is currently ongoing. Overall, in this small series, 10 out of 15 (66.6%) patients obtained a pregnancy, and three of these came from cryotransfers. Conclusion: These data indicate that it is always worth freezing supernumerary embryos after performing PGD, since this adds a substantial chance of pregnancy. Human susceptibility to aneuploidy and the link to infertility Mantzouratou A1, Xanthopoulou L1, Mania A1, Tashkandi S1, Harper JC 1, Serhal P 2, Delhanty JDA 1 1Institute of Women’s Health, University College London; 2Assisted Conception Unit, UCLH, UK In preimplantation embryos a wide range of errors has been reported where the incidence of chromosomal aneuploidy is thought to be around 50–80%. A very high proportion of these errors are due to post-zygotic events during mitosis in the embryonic cells and to a much lesser degree during meiosis in the gametes and can have a devastating effect on the fertility of certain couples. Although older females show higher rates of meiotic aneuploidy in prenatal studies, some younger couples going through IVF programmes have an almost equally increased chance of chromosomal abnormalities. In this study, a detailed investigation of embryos from 101 PGS cycles revealed differences in aneuploidy mechanisms in embryos from certain PGS couples. Follow-up results were obtained for 596 of the 787 embryos available for reanalysis (76%). Among these 596 embryos, 53.4% were fully chaotic mosaic and 40.3% were classified as other mosaic types. The most prevalent of the other mosaic types were the aneuploid mosaics (31.9%) followed by those that were diploid/chaotic (26.1%) and aneuploid/chaotic (17.2%). Significant differences were found in the distribution of uniformly abnormal embryos, embryos with meiotic errors and diploid/chaotic mosaic embryos in relation to the referral group. Uniformly abnormal embryos and embryos with meiotic errors were significantly decreased in the RIF group compared with the RM and AMA groups. Diploid chaotic mosaic embryos were found in higher frequency in the RIF group. Within the RIF group significant differences were observed in the distribution of diploid/chaotic mosaic embryos and aneuploid mosaic embryos in relation to insemination method. The RIFICSI group had highly significant increase in the diploid/chaotic mosaic embryos. Differences in susceptibility to aneuploidy of specific chromosomes were found in relation to the mechanism of aneuploidy overall and for each referral group. This study has revealed that susceptibility to embryonic chromosomal abnormalities may differ in certain groups of couples that present with infertility. In general, this study has provided some significant evidence for the following: in a general background of error prone cell divisions