Thus, the beneficial effect of cell membrane stabilization by MβCD could protect the oocytes’ structures, which allows them to reach metaphase II. As expected [12], [21], [22], [38] and [42], vitrification negatively affected the developmental ability of oocytes, and no effect was observed after the MβCD treatment in terms of cleavage and blastocyst rates. Although Horvarth and Seidel [10] found significant differences in cleavage and eight cell embryos when loaded MβCD was used, these variations

gradually disappeared by the learn more blastocyst stage. While day 8 blastocyst rates were similar among vitrified oocytes, higher blastocyst rates at D7 were observed in oocytes exposed to MβCD. It is well established that the speed of development is related to embryo quality; thus, it is possible that the quality of embryos was better. Since

there was no significant difference in D8 blastocysts rates, developmental delay indicates a lower embryonic viability [15]. One approach to confirm the quality of the embryos would be to perform other evaluations, such as embryo cell counting [10], differential staining and gene expression assays [2], [7] and [32]. While the nuclear maturation of vitrified RAD001 oocytes was improved by MβCD, there was no change in blastocyst rate. It is difficult to understand the full impact of this data because there is scarce precedent in the available literature on MβCD pretreatment. However, rationales can be constructed to explain the lack of a beneficial effect. One possibility is that we used a alternate approach for loading MβCD with cholesterol by incubating it with FCS, while previous Tacrolimus (FK506) groups used MβCD that was already

loaded with cholesterol [10]. Potentially, our FCS incubation did not effectively load MβCD with cholesterol; thus, no cholesterol was incorporated into the membrane. The direct isolation of cholesterol incorporation sites in oocytes could answer these questions. An alternative explanation is that MβCD decreased damage to the plasma membrane, possibly supported by the lower degeneration rate, but did not prevent damage to other regions that have a higher impact on oocyte viability. During oocyte maturation, cytoplasmic organelles undergo various remodeling and redistribution processes [8] and [36]. Vitrification has been reported to affect some of those events. Among organelles, cortical granules are seriously affected [11] and [21]. Normally after IVM, cortical granules exhibit a peripheral distribution, while vitrified oocytes display a clustered distribution. This alteration could impair fertilization and compromise embryonic development. In addition, studies show that cryopreservation of mouse oocytes can cause zone hardening [14], which can also impair fertilization.