Changing the start temperature and cooling rate in a slow-freezing protocol increases human blastocyst viability

Abstract

OBJECTIVE:To determine the effect of start temperature and cooling rate of a slow freezing protocol on human blastocyst viability. DESIGN:Controlled-rate freezing of human blastocysts using different start temperatures and cooling rates. SETTING:Private assisted reproductive technology unit. PATIENT(S):Patients donated with consent cryopreserved pronucleate embryos. INTERVENTION(S):Culture of thawed pronucleate embryos in G III series media, containing hyaluronan, followed by cryopreservation of 36 blastocysts with subsequent noninvasive analysis of embryo metabolism. MAIN OUTCOME MEASURE(S):Pyruvate and glucose consumption and blastocyst reexpansion and quality. RESULT(S):Glucose consumption and blastocyst reexpansion after thaw were significantly higher when a start temperature of -6 degrees C and a cooling rate of 0.5 degrees C/min to -32 degrees C were used compared with a start temperature of 20 degrees C and a cooling rate of 2 degrees C to -6 degrees C, followed by cooling at 0.3 degrees C to -35 degrees C. Pyruvate uptake after thaw was not affected by the freezing procedure. Clinical use of the lower start temperature and quicker cooling rate, combined with culture in hyaluronan-based media, has led to the establishment of a 30% implantation rate. CONCLUSION(S):Human embryos cultured to the blastocyst stage in hyaluronan-based sequential media are readily cryopreserved and maintain their viability after thaw.