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Changing the start temperature and cooling rate in a slow-freezing protocol increases human blastocyst viability
David K Gardner1, Michelle Lane, John Stevens
1Colorado Center for Reproductive Medicine, Englewood 80110, USA. dgardner@colocrm.com
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.