High survival of mouse oocytes using an optimized vitrification protocol

Abstract

The method of vitrification has been widely used for cryopreservation. However, the effectiveness of this method for mammalian oocytes could be improved by optimizing each step of the process. In the present study, we tested the effects of varying several key parameters to determine the most effective protocol for mouse oocyte vitrification. We found that cryoprotectant containing ethylene glycol and dimethylsulfoxide plus 20% fetal calf serum produced the highest rates of oocyte survival, fertilization, and blastocyst formation. The duration and temperature of oocyte exposure to vitrification and thawing solutions influenced survival rate. The presence of cumulus cells surrounding oocytes and the incubation of thawed oocytes in Toyoda-Yokoyama-Hosoki medium also increased oocyte survival. Open pulled straw and nylon loop methods were more effective than the mini-drop method. Finally, the combination of these improved methods resulted in better spindle morphology when compared to the unimproved methods. These results demonstrate that the outcomes of mouse oocyte vitrification can be improved by a suitable combination of cryopreservation methods, which could be applied to future clinical research with human oocytes.

Perfusion, cryopreservation, and nanowarming of whole hearts using colloidally stable magnetic cryopreservation agent solutions

“…Vitrification relies on cryoprotecting agent (CPA) solutions that are perfused through the organ to prevent ice formation at practically feasible cooling rates (7). Cryopreservation by vitrification has successfully worked in small samples such as cells, ovaries, and embryos (2,3,8,9). Larger organs, such as rabbit kidneys (10) and rat hindlimb (11,12), have occasionally worked but have had a notable number of failures because of problems with achieving fast and uniform rewarming.…”

DNA damage in cumulus cells generated after the vitrification of in vitro matured porcine oocytes and its impact on fertilization and embryo development

“…Also, CCs are more sensitive to vitri cation than the oocytes due to the fact that their viability was considerably reduced in comparison with that of the oocytes. In this regard, compared to oocytes, the CCs are smaller in size, and are the rst in contact with the CPAs, which implies that high CPAs concentrations are initially received by these cells, making them less cryotolerant [34]. Results obtained in the present study demonstrate that the CCs protected the oocytes during vitri cation.…”

“…Accordingly, the results obtained in the present study indicate that CCs do protect oocytes after vitri cation; however, most of them lose their viability. In this regard, other studies reported that the CCs protect and promote cumulus enclosed MII oocyte survival after vitri cation in equine [13] and mouse oocytes [34]. Therefore, COCs vitri cation preserves oocyte competence.…”

Investigation of methods and factors influencing mouse oocyte quality, in vitro fertilization and embryo development

“…There have been several studies which investigated the cryopreserved MII oocyte subcellular structures including cortical granules in humans (191)(192)(193)(194)(195), meiotic spindles in mice (194,196,197), mitochondria distribution in humans and domestic cats (195,198,199) and F-actin in humans (108). Our study simultaneously characterized the vitrified-warmed MII mouse oocyte subcellular structures including meiotic spindles, Factin, CGs and mitochondria.…”

“…Cryopreservation by vitrification has been widely used for cryopreservation of oocyes and embryos since it is more effective, easier to perform, and less time-consuming than the slow cooling method (134). In order to avoid formation of lethal intracellular ice, several methods have been used to perform cryopreservation by vitrification.…”