Freeze-thaw-induced changes of the zona pellucida explains decreased rates of fertilization in frozen-thawed mouse oocytes

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Abstract
Summary. Frozen–thawed oocytes have a reduced rate of fertilization (48·8%) when compared with unfrozen controls (97%). In this study we have used zona-drilling to bypass the zona pellucida and investigate whether the decreased rate of fertilization is due to freezing-induced changes in the zona pellucida which prevent sperm penetration. After zona drilling the fertilization rate of frozen–thawed oocytes (87·8%) was the same as for zona-intact unfrozen controls (88%), indicating that freeze–thaw-induced changes at the level of the zona pellucida were responsible for the decreased rate of fertilization. To determine whether the changes were occurring during the manipulations before and after freezing or the complete freeze–thaw cycle, oocytes were exposed to the complete set of manipulations normally experienced during cryopreservation and appropriate control groups. A small but significant decrease in the rate of fertilization (82·8%) was apparent in oocytes exposed to the manipulations before and after freezing compared with controls (92·2%). The freeze–thaw-induced changes in the zona pellucida therefore occur primarily during the complete freeze–thaw cycle itself and not the manipulations before and after freezing and are responsible for the decreased rate of fertilization observed in frozen–thawed oocytes. Keywords: cryopreservation; oocyte; zona pellucida; in-vitro fertilization; mouse