Day 5 blastocyst has higher viability compared to day 6 blastocyst as vitrified: clinical outcome of 1074 cases

Abstract

OBJECTIVE: The purpose of the study was to examine whether the viability of the blastocyst-stage embryos vitrified using EM-grid after artificial shrinkage on day 5 and day 6.DESIGN: Retrospective clinical study.MATERIALS AND METHODS: Approval for this study was obtained from the Institutional Review Board of the Maria Infertility Hospital. This retrospective study was performed for 1074 patients participated in thawing-ET program from January 2004 to October 2006. The embryos derived from conventional IVF or ICSI were cocultured with cumulus cells in a 20ul YS (Yoon Sanhyun) medium containing 20% hFF. After transferring the embryos into the uterus on day 3 or 5, the surplus embryos developed to the expanded blastocyst stage were vitrified on day 5 or 6 using EM-grid following artificial collapse of blastocoel. The solution for vitrification was composed of DPBS containing 20% (v/v) hFF, 40% (v/v) ethylene glycol, 18% (w/v) Ficoll, and 0.3 M sucrose. For thawing of vitrified blastocysts, a two-step cryoprotectant dilution method was used in which the EM grids stored in LN2 were directly transferred into a 500ul drop of 0.5 mol/L sucrose solution (prepared in DPBS containing 20% human FF) at room temperature as soon as possible and then were transferred in to 500ul drops containing 20% human FF in DPBS for 5minutes at room temperature. The embryos were washed three times in culture medium and co-cultured with cumulus cells in 20ul of YS medium containing 10% human FF. Thawed blastocyst were overnight cultured and transferred on the next day. The viability was assessed based on the survival, hatching embryo, implantation, and clinical pregnancy (x2 test).RESULTS: The survival rate of blastosysts that were thawed had no difference between day 5 (95.0%, 1324/1393) and day 6 (91.5% 1409/1539). However the clinical pregnancy rate (56.4%, 288/511) and implantation rate (30.8%, 404/1393) of day 5 blastocysts were significantly higher than those on day 6(41.0%, 231/563; 20.4%, 286/1539).CONCLUSIONS: The survival and hatching rates of the blastocysts vitrified on day 6 were similar to those on day 5. However the clinical pregnancy and implantation rates of day 6 blastocysts were significantly lower than those on day 5. These results suggest that the viability of day 5 blastocysts was superior to that of day 6 blastocysts. Therefore, further efforts to improve culture conditions are necessary so that a great number of embryos can normally develop to the expanded blastocyst stage on day 5. OBJECTIVE: The purpose of the study was to examine whether the viability of the blastocyst-stage embryos vitrified using EM-grid after artificial shrinkage on day 5 and day 6. DESIGN: Retrospective clinical study. MATERIALS AND METHODS: Approval for this study was obtained from the Institutional Review Board of the Maria Infertility Hospital. This retrospective study was performed for 1074 patients participated in thawing-ET program from January 2004 to October 2006. The embryos derived from conventional IVF or ICSI were cocultured with cumulus cells in a 20ul YS (Yoon Sanhyun) medium containing 20% hFF. After transferring the embryos into the uterus on day 3 or 5, the surplus embryos developed to the expanded blastocyst stage were vitrified on day 5 or 6 using EM-grid following artificial collapse of blastocoel. The solution for vitrification was composed of DPBS containing 20% (v/v) hFF, 40% (v/v) ethylene glycol, 18% (w/v) Ficoll, and 0.3 M sucrose. For thawing of vitrified blastocysts, a two-step cryoprotectant dilution method was used in which the EM grids stored in LN2 were directly transferred into a 500ul drop of 0.5 mol/L sucrose solution (prepared in DPBS containing 20% human FF) at room temperature as soon as possible and then were transferred in to 500ul drops containing 20% human FF in DPBS for 5minutes at room temperature. The embryos were washed three times in culture medium and co-cultured with cumulus cells in 20ul of YS medium containing 10% human FF. Thawed blastocyst were overnight cultured and transferred on the next day. The viability was assessed based on the survival, hatching embryo, implantation, and clinical pregnancy (x2 test). RESULTS: The survival rate of blastosysts that were thawed had no difference between day 5 (95.0%, 1324/1393) and day 6 (91.5% 1409/1539). However the clinical pregnancy rate (56.4%, 288/511) and implantation rate (30.8%, 404/1393) of day 5 blastocysts were significantly higher than those on day 6(41.0%, 231/563; 20.4%, 286/1539). CONCLUSIONS: The survival and hatching rates of the blastocysts vitrified on day 6 were similar to those on day 5. However the clinical pregnancy and implantation rates of day 6 blastocysts were significantly lower than those on day 5. These results suggest that the viability of day 5 blastocysts was superior to that of day 6 blastocysts. Therefore, further efforts to improve culture conditions are necessary so that a great number of embryos can normally develop to the expanded blastocyst stage on day 5.