In the present study, we demonstrated that by optimizing a vitrification protocol, cryopreserved and fresh human eggs have similar developmental competence with respect to blastocyst formation. Transfer of embryos resulting from frozen eggs produced similar clinical pregnancy and implantation rates as fresh eggs. Our results indicate that post warming survival, fertilization and cleavage are not good indicators for frozen egg survival, but blastocyst development is a better indicator for egg survival after freezing/thawing. These results suggest that frozen egg banks can be established after considering the efficiency of production of high quality embryos and ongoing pregnancy.
Gradual exposure of eggs to ES during equilibration is important for egg vitrification. A few years ago, Kuwayama developed a gradual exposure method for egg vitrification using a specifically designed culture dish and reported a high post warming survival rate
. However, a special culture dish is required for vitrification of eggs with Kuwayama’s method, and also a particular handling is necessary during vitrification, which makes it difficult to be followed by others. In some other reports, oocytes were vitrified after being equilibrated in different concentrations of ES made in-house
[22, 35]. However, such multiple step solutions are not commercially available. In the protocol designed in the present study, eggs were first placed in a drop of BS and then merged to an adjacent drop of ES twice, finally placed in a new drop of ES. Through this method, the eggs can also be equilibrated from low concentration of CPAs to high concentration CPAs with the standard commercial embryo cryopreservation solutions.
Exposure time of eggs in different ES and VS before vitrification varies among protocols. These steps appear to be key components for successful cryopreservation of eggs. Some researchers have used a maximum of 15 minutes in the ES depending on egg shape recovery
[20, 34, 38]. It may be difficult for embryologists to evaluate the optimal equilibration status of an egg. To simplify the protocol, a fixed time in each step may be necessary for all laboratory technicians to follow. Therefore, in the present study, we used a fixed time for a total of 9 minutes in the ES and found that it was appropriate for vitrification of human eggs if the time in VS was appropriate. This time period is obviously shorter than other reports, which may be better for the egg’s viability by reducing time of exposure eggs to the toxic CPAs. We found that this method is very practical and easy to use and learn in the laboratory. Importantly, this method yielded high egg survival rates and favorable clinical outcomes.
Another major key for egg vitrification is the exposure time of eggs in the VS. It was reported that the short time was essential for vitrification of human eggs and embryos, because high concentrations of CPAs were added in the VS
. Many early reports suggested that the time should be less than 10 seconds
. However, based on the studies in the mouse and human, short exposure of eggs to VS was unfavorable to egg survival and their competence to develop to blastocysts after vitrification
[23, 29, 41]. In some early reports, it was found that vitrified mouse and human oocytes could survive and form embryos by using brief exposure to VS, but the survival rates and blastocyst formation rates were quite low
[41–43]. In the present study, we used two different exposure times for vitrification, one was 45 seconds and another was 90 seconds. We found that post warming survival, fertilization and cleavage rates were not statistically different between both groups. However blastocyst development rates were significantly lower in the short protocol group. This suggests that intracellular water may not be completely replaced by CPAs in the eggs exposed to VS for a shorter time. This could result in damage to some organelles inside the eggs, which escape detection via microscopic evaluation of general oocyte morphology in the present study.
Outcomes from vitrified donor eggs and sibling fresh donor eggs derived from the same stimulation cycles were compared in the present study. There were no significant differences in fertilization rates and blastocyst development rates except that the cleavage rate was lower in the frozen eggs than that in fresh eggs. Our results are in contradistinction to a previous study that indicated that embryo quality was affected during the procedure of cryopreservation when frozen-thawed oocytes were compared with fresh oocytes
 but comparable to those reported by others
[20, 25, 44]. Our results indicate that not only fertilization and blastocyst development rates, but also the implantation rates were similar between frozen and fresh eggs.
Creation of cryo-stored oocyte banks for oocyte donation has been advocated in recent years. Accumulated data from different clinics indicate that oocyte cryo-banking using vitrification represents advancement in oocyte donation
[20, 25, 44]. Egg banks have the potential to lower cost and also overcome the obstacle of synchronization of the recipients’ cycles with that of the donor
[20, 21, 23, 26, 45, 46]. The sharing of cryo-donated oocytes to several infertile recipients is a means of reducing waiting time and cost for recipients. Most importantly, the use of frozen donated eggs can produce satisfactory pregnancy rates
[14, 20, 25, 47]. In the present study, we also obtained similar rates of blastocyst development, embryo implantation and clinical pregnancy by using frozen and fresh oocytes obtained from the same donors. These results indicate that our optimized protocol for egg cryopreservation does not disturb eggs’ structure and function during freezing and thawing.
Most studies on egg cryopreservation were focused on donated eggs from young women
[20, 21, 23, 25, 26, 46]. It would be possible that eggs from young women or egg donors can tolerate cryo-injuring better than eggs from women with advanced maternal age or infertile women. In the present study, with limited data, we observed high survival, fertilization, and embryo implantation rates when the eggs from infertility patients were vitrified/warmed. These preliminary results are encouraging and suggest that the protocol used in the present study is not only appropriate for eggs from young donors, but also for eggs from infertile patients.
Although the survival rate of egg vitrification can exceed 90% after warming, some eggs may not be able to develop further after insemination. The survival rate immediately after egg warming may not be a good indicator for egg competence after cryopreservation. For example, in the present study, we found that the egg survival rate was high but blastocyst development rate was low if the short protocol was used. These results indicate that blastocyst development would be a better indicator for egg cryopreservation and should be used as the primary indicator of egg survival or egg competence after cryopreservation.