Human Reproduction Archives
Human Reproduction Archives
Original Article Assisted Reproduction

Vitrification of blastocysts at various degrees of blastocoele expansion using different exposure times to the equilibration solution

Tamara Lamin, Rodrigo Sant’Ana, Alfred Paul Senn, Rafael Alonso Salvador, David Til, Larissa Benvenutti, Vera Lucia Langaro Amaral

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Objectives: To evaluate the efficiency of a vitrification protocol of murine blastocysts, varying the exposure time to the equilibrium solution, at different degrees of blastocoel expansion. Methods: Sixty female mice were superovulated with 10 IU of equine chorionic gonadotropin (eCG, Novormon, Syntex, Argentina) and after 48 hours with 10 IU of human chorionic gonadotropin (hCG, Vetecor, Calier, Barcelona, Spain). Females were then coupled with males overnight, and the presence of vaginal plug indicated that mating had occurred. Three days later, females were euthanized and the embryos at the morula stage (n = 925) were recovered by flushing the uterine tubes with GV-Hepes medium (Ingámed, Maringá, Brazil). Embryos were then cultivated until the blastocyst stage and classified, at the time the vitrification process was initiated, into 3 groups (G1, G2, G4) according to the degree of blastocoele expansion. A control group remained in culture until hatching. All embryos, after warming, were put back into culture for measurement of the rehydration (RR) and hatching (HR) rates. Results: For G1 group, the mean RR and HR obtained over a range of exposure times to the equilibration solution of 94.0 and 84.4%, respectively. For G2, these rates were 95.2 and 87.1%, respectively. For G1 and G2, RR and HR obtained after an equilibration time of 9 min were statistically higher than those obtained after 10, 11, 12, 13 and 14 min. For G4, RR and HR were 76.4 and 69.9%, respectively, but in this case, an equilibration time of 15 min presented statistically higher survival rates compared to shorter exposition times. Conclusions: The results of this study indicate that the variation in the exposure time of the embryos to the equilibration solution influences significantly the rehydration and hatching of the blastocysts, and the degree of blastocoel expansion is inversely correlated with the survival and development potential after warming.


vitrification; blastocysts; blastocoel; cryoprotectant.


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