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P6 Combination of biopsy and cryopreservation on cleavage stage human embryos
POSTERS and FISH for translocation on a second blastomere without success. The overall ongoing pregnancy rate per embryo transfer was 35/161 (21.7%), ranging from 0% in 2003 to 55.6% in 2010. The annual cancellation rate ranged from 0% up to 50%, on average 57/218 (26.1%) (28 cases with no normal embryo for replacement, 12 cases due to risk of ovarian hyperstimulation syndrome, and 8 cases due to poor embryo quality). By univariate logistic regression, only the number of top quality embryos on Day 2 (OR 1.166, 95% CI 1.035 1.312, p = 0.011) was the significant confounder on the ongoing pregnancy rate, but not women age (OR 0.908, 95% CI 0.822 1.004, p = 0.060). Conclusion: There is an increase in the number of PGD cases in the recent few years, especially for single gene diseases. The ongoing pregnancy rate increased as well and is compatible with the European data [2]. Only the number of top quality embryos is the significant confounder on the ongoing pregnancy rates. Further development of using comparative genomic hybridization (CGH) in PGD should be explored, especially in couples with multiple genetic mutations. Keywords: review; PGD service; FISH; PCR Reference(s) [1] Veeck L. An atlas of human gametes and conception. 1999. [2] Harper JC, Coonen E, De Rycke M, Harton G, Moutou C, Pehlivan T, et al. ESHRE PGD consortium data collection X: cycles from January to December 2007 with pregnancy follow-up to October 2008. Human Reproduction. 2010; 25(11): 2685 707. P6 Combination of biopsy and cryopreservation on cleavage stage human embryos O. Okutman-Emonts1 , M. Gultomruk1 , T. Aksoy1 , C. Goktas1 , H.N. Ciray1 , M. Bahceci1 . 1 Bahceci Fulya IVF Center, ˙Istanbul, Turkey Introduction: The pregnancy and live birth rates after frozenthawed embryo transfers have been shown to be comparable to those obtained from fresh cycles. Cryopreservation may be required in PGD cycles where conditions do not favour fresh embryo transfers or to increase the number of embryos to analyse, especially for patients with a high risk of genetically unbalanced conceptions. However the information obtained from the limited number of studies stated that the outcomes of cycles in which thawing and blastomere biopsy have been performed concomitantly were poor, regardless of sequence of procedures. The aim of the present study was to investigate the outcome of PGD cycles in which biopsy procedure has been applied on day 3 embryos after the cryopreservation procedure. Methods: Embryos from 24 patients have been frozen on day 3 via vitrification protocols, indications were RIF (n = 18), karyotypically abnormal abort history (n = 1) and translocation (n = 5). The mean age of the women was 33.4±5.5 years (mean±SD). 265 frozen D3 embryos were thawed and biopsied. Biopsied blastomeres were fixed separately on the slide with tween/HCl method. 9-probe set for aneuploidy screening was used in case no other indication has been pointed while telomeric and locus specific probes in combination with centromeric probes were used for translocation analysis. Normal (or balanced) embryos were transferred on the fifth day of development. Remaining good quality spare normal embryos have been frozen after transfer. Results: 258 embryos qualified for the biopsy procedure after thawing (97.4%). FISH was not successful in 6 blastomeres, either because of anuclear blastomere or ambiguous signal. Six cycles were cancelled because no normal embryo was obtained. For the analysed chromosomes 60 embryos were identified as normal. Twenty-five embryos were transferred to 18 patients resulting in 12 clinical pregnancies (66.6%, mean number of transferred
S47 embryos per patient: 1.38±0.75). Two resulted in abortion while 2 delivered and 8 are ongoing. 27 good quality normal embryos were re-frozen on day 5. Until today, 10 embryos have been thawed in 3 cycles and embryos which survived were transferred to two patients at same day. One pregnancy was obtained and is continuing (>20 weeks). Conclusions: According to the results of the present study, blastomere biopsy can be performed effectively on vitrifiedthawed day 3 embryos. Endometrial-embryonic synchronization is important and the natural uterine environment in thaw cycles, which is similar to spontaneous pregnancy, may also explain good outcomes in our study group. Thaw-PGD is a feasible method, which can be suggested especially those who have poor endometrium in the fresh cycle, to poor responder patients with PGD indication but also it gives the financial advantage to the patients. Keywords: thaw PGD, cryopreservation, FISH P7 Embryo rebiopsy in PGD program 1
2
a case report 1
G. Biran , R. Tomashov-Matar , K. Naomi , G. Roni1 , O. Sapir1 , M. Shohat2,3 , B. Fisch1,3 . 1 Infertility and IVF Unit, 2 Raphael Recanati Genetic Institute, Rabin Medical Center, Petah-Tikva, Israel, 3 Sackler Faculty of Medicine, Tel-Aviv University, Israel During the process of PGD, the eligible number of embryos for transfer declines. To overcome the problem of small number of transferable embryos, several strategies are available: raising the oocyte number in the retrieved cohort, rebiopsy of embryos in which the genetic test did not reveal an answer, and vitrifing healthy embryos to use in the following cycles. Case report: A 31-year-old patient and her 32-year-old husband came to our IVF clinic to participate in our IVF-PGD program. The woman is known to have genetic distal arthrogryposis type I since childhood and the mutation (R174Q on TNN12 gene) was established. After getting married, the couple had genetic disease screening according to their parent’s countries of origin, and both were found to be carriers of a mutation that causes Fanconi A anemia (2172 2713insG on the FANCA gene). The aim of their IVF-PGD treatment was to eliminate the possibility of a child with arthrogryposis and or Fanconi anemia. The first stage was to prepare the linkage analysis system. The distal arthrogryposis mutation (R174Q) in the TNNI2 gene together with the Fanconi a mutation (2172 2173 insertion G) in the FANCA gene were analyzed in association with closely linked polymorphic markers (short tandem repeat). In each gene/mutation, we determined the phase of the polymorphic markers regarding the mutation by linkage analysis of the patient, the first-degree relative known to be a carrier (mother, father, and CVS from previous pregnancy), and the partner of the patient. We chose the informative markers for the PGD. On the fifth IVF cycle we achieved a dizygotic twin pregnancy from transfer of a fresh healthy embryo together with an embryo that was TAF (total fertilization failure) on the previous cycle, had a second biopsy of two blastomeres, and was vitrified and thawed on the next cycle. Conclusion: What to do with those embryos one does not have an answer for to do a second biopsy (rebiopsy) or to discard the embryo? The answer to this question is what happened to the embryo since the biopsy. If the embryo continued dividing (compacted, morula, and blastocyst), it is worse while taking another biopsy. If the second biopsy provides a result of a healthy embryo, then one can either transfer or vitrify the embryo.
S47 embryos per patient: 1.38±0.75). Two resulted in abortion while 2 delivered and 8 are ongoing. 27 good quality normal embryos were re-frozen on day 5. Until today, 10 embryos have been thawed in 3 cycles and embryos which survived were transferred to two patients at same day. One pregnancy was obtained and is continuing (>20 weeks). Conclusions: According to the results of the present study, blastomere biopsy can be performed effectively on vitrifiedthawed day 3 embryos. Endometrial-embryonic synchronization is important and the natural uterine environment in thaw cycles, which is similar to spontaneous pregnancy, may also explain good outcomes in our study group. Thaw-PGD is a feasible method, which can be suggested especially those who have poor endometrium in the fresh cycle, to poor responder patients with PGD indication but also it gives the financial advantage to the patients. Keywords: thaw PGD, cryopreservation, FISH P7 Embryo rebiopsy in PGD program 1
2
a case report 1
G. Biran , R. Tomashov-Matar , K. Naomi , G. Roni1 , O. Sapir1 , M. Shohat2,3 , B. Fisch1,3 . 1 Infertility and IVF Unit, 2 Raphael Recanati Genetic Institute, Rabin Medical Center, Petah-Tikva, Israel, 3 Sackler Faculty of Medicine, Tel-Aviv University, Israel During the process of PGD, the eligible number of embryos for transfer declines. To overcome the problem of small number of transferable embryos, several strategies are available: raising the oocyte number in the retrieved cohort, rebiopsy of embryos in which the genetic test did not reveal an answer, and vitrifing healthy embryos to use in the following cycles. Case report: A 31-year-old patient and her 32-year-old husband came to our IVF clinic to participate in our IVF-PGD program. The woman is known to have genetic distal arthrogryposis type I since childhood and the mutation (R174Q on TNN12 gene) was established. After getting married, the couple had genetic disease screening according to their parent’s countries of origin, and both were found to be carriers of a mutation that causes Fanconi A anemia (2172 2713insG on the FANCA gene). The aim of their IVF-PGD treatment was to eliminate the possibility of a child with arthrogryposis and or Fanconi anemia. The first stage was to prepare the linkage analysis system. The distal arthrogryposis mutation (R174Q) in the TNNI2 gene together with the Fanconi a mutation (2172 2173 insertion G) in the FANCA gene were analyzed in association with closely linked polymorphic markers (short tandem repeat). In each gene/mutation, we determined the phase of the polymorphic markers regarding the mutation by linkage analysis of the patient, the first-degree relative known to be a carrier (mother, father, and CVS from previous pregnancy), and the partner of the patient. We chose the informative markers for the PGD. On the fifth IVF cycle we achieved a dizygotic twin pregnancy from transfer of a fresh healthy embryo together with an embryo that was TAF (total fertilization failure) on the previous cycle, had a second biopsy of two blastomeres, and was vitrified and thawed on the next cycle. Conclusion: What to do with those embryos one does not have an answer for to do a second biopsy (rebiopsy) or to discard the embryo? The answer to this question is what happened to the embryo since the biopsy. If the embryo continued dividing (compacted, morula, and blastocyst), it is worse while taking another biopsy. If the second biopsy provides a result of a healthy embryo, then one can either transfer or vitrify the embryo.