Objective: @EN When embryo is exposed to a cryoprotectant for cryopreservation, it can be damaged by the initial hyperosmolarity of the extracellular solution and the rapid change of osmolarity in media during the freezing and thawing procedure.
The
purpose of this study was to evaluate and examine freezing and thawing methods to diminish this osmotic shock.
@ES Design: @EN During the period August 1992 to February 1995, 170 thawing cycles were retrospectively analyzed. We evaluated survival, developmental, and pregnancy rates according to freezing and thawing methods.
@ES Materials and Methods: @EN Embryo cryopreservation was performed at pronuclear (PN) or 2- to 4-cell stage using a slow freezing method with a programmed cell freezer (Kryo-10, Planer). The cryoprotectants were 1.5 M 1, 2-propanediol (PROH)
and
0.1 M
sucrose. Embryos were thawed by a rapid method using 1.0 M or 0.5 M PROH and 0.2 M sucrose. Freezing procedures were performed as three different methods. First, drops (50μl) of freezing media were placed on the dish, and then freezing procedure
was
carried out with media exposed to air. Second, we performed freezing procedure after covering the media with oil to prevent a slow increase of osmolarity. Third, Embryos were treated with 0.75 M PROH prior to 1.5 M PROH to diminish high osmotic
shock.
Also, thawing procedures were performed with or without oil covering. In total, 170 patients entered in the program. 619 embryos were cryopreserved at PN stage and 315 embryos at 2- to 4-cell stage.
@ES Results: @EN After thawing, survival rates of embryos frozen at PN stage and 2-4 cell stage were entirely similar as about 75% regardless freezing methods. However, intact blastomere rate after thawing of embryos frozen at 2-4 cell stage was
increased in third freezing method (45%) compared with other freezing methods (25%, 20%). After incubation for 24 hours of embryos frozen and thawed at PN stage, the number of good developmental embryos was increased in first freezing method. In
contrast, fragmentation of embryo was increased in third freezing method. Embryo development rate according to thawing methods showed a good results in group performed thawing procedure with oil covering. No significant difference was found n the
pregnancy rates among the three freezing methods.
@ES Conclusions: @EN These data suggest that to diminish osmotic shock during the cryopreservation increase the survival and development rates of embryos after thawing. We recommend the method 1 for freezing of PN stage embryos and the method 3
for
that
of 2-4 cell stage embryos, and propose to cove media with oil for thawing of both PN and 2-4 cell stage embryos.
|