Re-Freezing Blastocysts: A Prospective Randomized Study Comparing Slow Freezing and Vitrification.

OBJECTIVE: The potential to re-freeze blastocysts would be useful when surplus high quality blastocysts develop after thawing for frozen embryo transfer (FET). The objective was to compare the efficiency of slow freezing and vitrification for re-freezing blastocysts on survival rates and quality scores. DESIGN: Blastocysts (n=203) donated for research from patients ≤37 years old were used in this study. Quality scores were assessed as: Poor=1, Fair=2, Good=3, Excellent=4. Blastocysts that survived thawing exhibiting quality scores of 3 and 4 (n=122) were randomly divided by expansion stage and score and frozen for a second time using either slow freezing or vitrification. Survival rates and quality scores were assessed after overnight culture. Laboratory end points were analyzed using t-tests and Fisher's exact test. MATERIALS AND METHODS: Slow freeze was carried out in 0.25 ml straws in 9% glycerol and 0.2 M sucrose media. Vitrification was performed using a closed double-straw device after a 2-step loading of cryoprotectants. Blastocysts were artificially collapsed and exposed to equilibration medium containing 7.5% (v/v) DMSO + 7.5% ethylene glycol (EG) for 10 min, then transferred to vitrification medium containing 15% (v/v) DMSO + 15% EG + 0.5 M sucrose for no more than 90 seconds. Thawing and warming for removal of cryoprotectants was performed in an 8- or 2-step sucrose protocol, respectively. RESULTS: There was a significantly higher survival of re-frozen blastocysts after vitrification than after slow freezing (Table 1). Vitrified blastocysts had superior morphology after re-expansion as compared to slow frozen blastocysts.Table 1nSurvival (%)Quality Score ± SDSlow freeze6136 (59)aDifferent superscripts within columns indicate significant differences (P<0.05).2.0 ± 0.9aDifferent superscripts within columns indicate significant differences (P<0.05).Vitrification6158 (95)bDifferent superscripts within columns indicate significant differences (P<0.05).3.1 ± 0.8bDifferent superscripts within columns indicate significant differences (P<0.05).a,b Different superscripts within columns indicate significant differences (P<0.05). Open table in a new tab CONCLUSION: These results suggest that vitrification is a superior method than standard slow freezing for re-freezing supernumerary blastocysts available on FET cycles.