Comparison of slow freezing in the cleavage stage and ultra-rapid vitrification of blastocysts

Abstract

Objective: Freezing technology for blastocysts is an absolute necessity at facilities that perform ART. Ultra-rapid vitrification of blastocysts, which can be conveniently performed in a short period of time, is actively performed at our clinic. The objective of the current research has been comparison of results of slow freezing in the cleavage stage and ultra-rapid vitrification of blastocysts. Design: Retrospective. Materials and Methods: From August 2001 to December 2002, 9 cycles of freeze/thaw ET were performed with slow freezing on Day3 and 38 cycles of freeze/thaw ET were performed with ultra-rapid vitrification of blastocysts on Day 5 and 6 at our clinic. Day 3 Embryos were frozen using PROH and sucrose as cryoprotectants. For thawing of the embryos, the cryoprotectants were then removed by stepwise dilutions with thawing solution (5min or 10min each step). The blastocysts are suspended in solution I, which is HEPES-buffered HTF medium containing 5 mg/ml of HSA(base medium) containing 7.5%(v/v) DMSO and 7.5%(v/v) EG at about 37°C. A cryoloop is dipped into cryoprotectant solution II, which is a base medium containing 15%(v/v) DMSO, 15%(v/v) EG, 10mg/ml Ficoll 70, and 0.65M sucrose, at about 37°C to create a filmy layer of the solution on the cryoloop. 1min and 45sec after suspending the blastocysts in solution I, the blastocysts are washed in solution II and transferred onto the filmy layer on the cryoloop. Within 30 sec of suspension in solution II, the cryoloop is plunged into liquid nitrogen. For thawing of the blastocysts, the loop containing blastocysts is removed from the liquid nitrogen and placed quickly and directly into a well of the base medium containing 0.33M sucrose at 37°C. After 2 min, embryos are transferred to the base medium containing 0.2M sucrose. After an additional 3min, embryos are suspended in base medium for 5min. Results: The survival rate was 81.8% (63/77) with slow freezing and 81.6% (93/114) with vitrification; no significant difference was found. ET was possible in 9 of 10 cycles with slow freezing and in 38 of 45 cycles with vitrification. The mean embryo transfer number was 2.6±0.9 with slow freezing and 1.8±0.7 with vitrification; the mean ET number with vitrification was significantly lower. The implantation rate was 21.7% (5/23) with slow freezing and 26.9% (18/67) with vitrification; significance was not noted. The clinical pregnancy rate (FHM) was 33.3% (3/9) with slow freezing and 36.8% (14/38) with vitrification; no significant difference was found. Conclusion: The fact that a high pregnancy rate and implantation rate can be obtained with freeze/thaw ET via vitrification has been demonstrated. Because vitrification can be conveniently performed in a short period of time, it is also linked to a reduction in laboratory work, which grows in complexity each day. However, the safety of vitrification in aspects like the toxicity of the cryoprotectant has yet to be established, so further improvements are needed.