ABSTRACT
Cryopreservation of human embryos has become a routine procedure to increase cumulative pregnancy rates, to help avoid the risk of multiple pregnancies after the transfer of many embryos, and to avoid unnecessary additional stimulation procedures. Recently, blastocyst transfer based on an improved culture system has been proven effective for increasing the pregnancy rate in assisted reproductive technology (ART).1,2 Therefore, a reliable procedure for the cryopreservation of supernumerary blastocysts is needed because usually only a small number of blastocysts after transfer are likely to be available for cryopreservation. Freezing of human blastocysts has been carried out with the slow-cooling method, but clinically satisfactory results have not been obtained.3,4 In addition, the slow-freezing method requires expensive equipment, and is time-consuming. Therefore, it is essential to establish a simple, fast, and reliable procedure to optimize clinical outcomes of cryopreservation at the blastocyst stage. Vitrification has become more widely used, and is now regarded as a potential alternative to conservative slow freezing. Vitrification takes only a few seconds to cool embryos, and there is no extracellular crystallization, which is one of the major causes of cell injury.5 It also offers lower osmotic and toxic effects, and less severe chilling injury resulting from the rapid passage through the ‘dangerous’ temperature zone.6 Furthermore, it does not require specialized programmable
freezers. Initially, most vitrification methods used standard French mini-straws for holding the embryos during cooling, storage, and thawing. Recently, to overcome the disadvantages of straws that have low cooling and warming rates, techniques using either electron microscope (EM) grids,7,8 Cryotop,9
hemi-straw,10 Cryoloop,11,12 or cryotip13 that substantially increase the cooling rate have been applied, and significant improvements in the success rate of human blastocyst vitrification have been reported.7-13 Among them, it has been shown that an ultrarapid freezing method using EM grids was efficient for the cryopreservation of bovine oocytes.14 Since then, several researchers have applied EM grids successfully for the vitrification of human oocytes15 and blastocysts.7,8,16
Embryo cryopreservation using EM grids was originally designedfor the vitrification of exceedingly chill-sensitive Drosophila embryos.17,18 The EM grid has about 3-fold higher cooling rates than those obtained with straws. It is known that the increased rate of cooling and thawing may considerably decrease the chilling injury of embryos. In addition, the EM grid is much cheaper than another apparatus that is being used regularly for vitrification.
