Cryopreservation and in vitro maturation of murine germinal vesicle stage oocytes

Cryopreservation of unfertilised oocytes for banking or oocyte donation would be a valuable adjunct to reproductive technology. As the mature oocyte contains a temperature-sensitive meiotic spindle, cryopreservation of immature germinal vesicle (GV) stage oocytes, which do not contain the spindle, may be a practical alternative. However, one of the major obstacles to the application of immature oocyte cryopreservation is the difficulty associated with in vitro maturation (IVM) of the thawed oocytes prior to in vitro fertilisation. The cumulus cells surrounding the oocyte are essential to oocyte maturation. Thus the aim was to assess survival and function of both oocyte and cumulus cells post-cryopreservation. Initially, culture conditions during IVM of murine GV stage cumulus-oocyte complexes (COCs) were modified. In the second part of the study, survival (morphological appearance and membrane integrity) and function (ability, in vitro, to mature, be fertilised and develop into blastocysts) of the oocytes and their associated cumulus cells was assessed following cryopreservation. An attempt was made to determine the stage of the protocol at which damage was incurred. Alterations to culture conditions had little impact on the ability of fresh GV stage oocytes to develop to blastocysts, although IVM in the presence of mixed ovarian cells was found to be detrimental. Treatment with 1.5M dimethyl sulphoxide (Me2SO) without freezing had little effect on the parameters investigated, unlike exposure to a 6M Me2SO solution. Slow-cooled/thawed or cumulus-denuded oocytes had decreased developmental potential when compared with control oocytes. Development was not improved by co-culture with fresh cumulus cells. Much of the damage caused to the cumulus cells occurred during plunging from -60 °C to -196 °C. Damage was reduced by cooling at 10 °C/min from -60 °C to -150 °C prior to plunging to -196 °C. However, embryo development was not improved. Vitrification of COCs led to substantial cumulus cell damage and very poor embryo development

Simplified EM Grid Vitrification Is a Convenient and Efficient Method for Mouse Mature Oocyte Cryopreservation

This study was performed to evaluate the efficiency of simplified EM grid vitrification, skipping the step of removing the cryoprotectant (5.5 M EG + 1.0 M sucrose) droplet on the grid after loading oocytes, compared to conventional cryopreservation protocols for mouse mature oocytes. Firstly, the recovery, survival, fertilization and hatching rates of simplified EM grid vitrification were compared with those of the slow freezing method using 1.5 M DMSO. Then, conventional EM grid vitrification was compared with simplified EM grid vitrification. Simplified EM grid vitrification showed higher survival, fertilization and hatching rates than those of the slow freezing method (85.6% vs. 63.2%; 51.0% vs. 22.3%; 38.7% vs. 12.5%, p < 0.01, respectively). Moreover, simplified EM grid vitrification showed higher recovery, survival and fertilization rates than those of conventional EM grid vitrification (100% vs. 95.0%, p = 0.024; 90.0% vs. 78.9%, p = 0.033; 56.7% vs. 38.7%, p = 0.021, respectively). Hatching rate tended to be higher for simplified EM grid vitrification compared to conventional EM grid vitrification (41.1% vs. 24.1%). In conclusion, simplified EM grid vitrification is a convenient and efficient method for cryopreservation of mouse mature oocytes, compared to conventional EM grid vitrification and slow freezing methods