Outcome of preimplantation genetic diagnosis in frozen-thawed embryos

Abstract

Cryopreservation of embryos is widely applied in ART. And it is also useful for efficient preimplantation genetic diagnosis (PGD) in cases with supernumerary embryos or with collected embryos through several cycles in poor responders. The aim of this study is to evaluate the feasibility of FISH or PCR in the blastomeres biopsied from frozen-thawed embryos and the outcome of PGD, and to determine whether frozen- thawing process affect the embryo development after blastomere biopsy. Retrospective clinical study. In 86 patients, 319 PGD cycles were performed between Jan. 2000 and Dec. 2003. Patients were divided into 2 groups: thawed PGD group − PGD cycle was performed in frozen-thawed embryos (33 patients, 40 cycles, age = 34.2±4.7yrs. (mean±SD)), fresh PGD group − PGD cycle was performed in fresh embryos (patients=53, cycles=279, mean age = 31.8±4.3yrs.). In 20 cycles, supernumerary embryos were frozen and thawed, and in 19 cycles, embryos were collected through several cycles in poor responders and then thawed altogether. PGD was performed for translocation carriers or aneuploidy screening and some single gene diseases were included. Ovarian stimulation was done using GnRH agonist/FSH long protocol or clomifen citrate/GnRH antagonist protocol in poor responders. Oocytes were inseminated by ICSI and the fertilized embryos were cryopreserved at PN stage or cleaving embryo stage. In thawed embryo transfer cycles, estradiol was administrated from day 2 of menstrual cycle and progesterone injection was started from 1 day before 2PN thawing. The cryopreserved embryos were thawed and blastomeres were biopsied in survived embryos developed to 6–10 cell stage. PGD was performed by using FISH or PCR and the embryos were replaced 3 days after PN thawing. The survival rate of the thawed embryos, the rate of successful diagnosis for FISH or PCR, embryo development and pregnancy rate were compared between thawed and fresh PGD cycles. The survival rate of thawed embryos was 71.2±29.8% (mean±SD). The FISH or PCR analysis was successful in 88.1±22.0 % in thawed PGD group and 95.2±10.3% in fresh PGD group (P>0.05). The proportion of transferable embryos in thawed PGD group was similar to fresh PGD group (21.6±19.7% vs. 27.4±18.8%; P>0.05). The rate of embryos developed cleavage after biopsy was also similar in both groups (62.0±26.5% vs. 66.4±26.1%; P>0.05). The clinical pregnancy rate per transfer was 11.4 % (4/35) in thawed PGD group and 21.3% (57/267) in fresh PGD group. Although it was not statistically significant, the pregnancy rate tends to be lower in thawed PGD group. Our data suggest that blastomere biopsy and analysis with FISH or PCR is feasible in frozen-thawed embryos if the embryos survive well. The embryo development was not affected by biopsy in thawed embryos, but the pregnancy rate was lower in thawed PGD cycles. Therefore PGD in thawed embryos could be applied usefully in cycles with supernumerary embryos or collected cyropreserved embryos, and optimization of freezing-thawing condition might be important.