Changing the start temperature and cooling rate in a slow-freezing protocol increases human blastocyst viability.
OBJECTIVE
To determine the effect of start temperature and cooling rate of a slow freezing protocol on human blastocyst viability.


METHODS
Controlled-rate freezing of human blastocysts using different start temperatures and cooling rates.


METHODS
Private assisted reproductive technology unit.


METHODS
Patients donated with consent cryopreserved pronucleate embryos.


METHODS
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.


METHODS
Pyruvate and glucose consumption and blastocyst reexpansion and quality.


RESULTS
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.


CONCLUSIONS
Human embryos cultured to the blastocyst stage in hyaluronan-based sequential media are readily cryopreserved and maintain their viability after thaw.
Gardner
David K
DK
Colorado Center for Reproductive Medicine, Englewood 80110, USA. dgardner@colocrm.com
Lane
Michelle
M
Stevens
John
J
Schoolcraft
William B
WB
D001755:Blastocyst / Q000166:cytology* / Q000502:physiology; D018929:Cell Culture Techniques / Q000379:methods; D002478:Cells, Cultured; D015925:Cryopreservation / Q000379:methods*; D017077:Culture Media, Conditioned; D005260:Female; D006019:Glycolysis; D006801:Humans; D011773:Pyruvates / Q000378:metabolism; D027724:Reproductive Techniques, Assisted; D013696:Temperature
D017077:Culture Media, Conditioned; D011773:Pyruvates