Background
Currently, the cryopreservation of embryos and oocytes is essential for assisted reproductive technology (ART) laboratories worldwide. To date, several vitrification methods based on ultra-rapid cooling have been developed for the cryopreservation of animal embryos [
1‐
8]. The most important concept of these vitrification methods is to minimize the volume of the extracellular vitrification solution in order to obtain a higher cooling rate. However, to our knowledge, even if the volume of the extracellular solution is decreased, the vitrification solution remains present around the cells. In order to address this issue, we previously developed a novel vitrification system for the cryopreservation of bovine embryos [
9]. This simple method vitrifies embryos on a membrane filter, which absorbs excess vitrification solution around the embryos. Accordingly, we believe that this vitrification system can achieve greater and more rapid rates of cooling and warming, while also causing less cryodamage. Additionally, a key feature of this system is that the absorption of excess vitrification solution is easy and stable. However, it is difficult to observe embryos that are deposited on the membrane filter under a stereomicroscope. During the cryopreservation of human embryos, an operator has to observe the embryos on the cryodevice under a stereomicroscope at the time of vitrification. However, we have developed a new vitrification device in which the embryo can easily be observed under a stereomicroscope after it and the vitrification solution are placed on the device absorber. We designated our vitrification system the Kitasato Vitrification System (KVS) and further developed it as a vitrification device for the cryopreservation of embryos. In this study, we examined the efficacy of the KVS in the vitrification of mouse embryos by analyzing the viability of embryos and their development to live offspring after vitrified warming to determine whether this novel device can be adapted to the field of ART. In addition, the direct measurement of cooling and warming for vitrification procedures via a physical set-up has been reported [
10,
11]. These previous studies have indicated the importance of the warming rate, rather than the cooling rate, with regard to the survival of mouse oocytes subjected to vitrification. Recently, we developed a measurement system for cooling and warming rates during vitrification procedures through the analysis of thermal transfer using a numerical simulation. Therefore, in this study, we also evaluated the relationship of viability with cooling and warming rates during vitrification procedures using the KVS.
Discussion
In the present study, it was demonstrated for the first time that mouse embryos can be successfully vitrified using the KVS. To date, many cryodevices for vitrification, such as the Cryoloop [
3], nylon loop [
7], open pulled straws [
2], the Cryotop [
5], and gel-loading tips [
6], have been developed. The basic concept of these devices for vitrification is to minimize the volume of the vitrification solution around the embryos to increase the rates of cooling and warming during the vitrification and devitrification processes. In order to address this issue, we previously reported the effectiveness of the KVS as a simple method that vitrifies embryos or oocytes on a membrane filter, which absorbs extracellular vitrification solution [
9,
15,
16]. However, because the total light transmittance of the membrane filter is low, it is difficult to observe the embryo deposited on a membrane filter under a stereomicroscope. Therefore, it is almost impossible to check the deposition of the embryo in a reliable manner. In contrast, a notable feature of the KVS in this study is that after the embryo and vitrification solution are placed on the absorber of the device, the embryo can easily be observed under a stereomicroscope (Fig.
2). In the cryopreservation of human embryos, an operator has to observe the embryos on a cryodevice under a stereomicroscope at the time of vitrification. Another feature of the KVS in this study was that the process for absorbing excess vitrification solution around the embryo is simple and stable. In addition, it is easy to control the volume of vitrification solution in the KVS. Therefore, we believe that the KVS is applicable to the cryopreservation of human embryos in ART.
The most notable feature of vitrification using the KVS is the absorption of excess vitrification solution around the embryos to achieve more rapid cooling and warming rates. In the first experiment, we examined the effect of absorbing excess vitrification solution around the embryos to confirm whether this feature of the KVS is effective for the viability of vitrified embryos. As a result, although a high survival rate (100%) was achieved with or without the absorption of excess vitrification solution, the subsequent development rate of embryos, such as the re-expand and hatching rates, were higher than those of embryos in which the excess vitrification solution was not absorbed (Table
2). It must be noted that blastocyst hatching is a manifestation of good-quality embryos and high developmental competence [
17]. A possible explanation for the high viability and developmental competence of vitrified embryos achieved using the KVS was that the more rapid cooling and warming rates achieved by absorbing the excess vitrification solution did not result in cryodamage to the embryo during the vitrification and devitrification processes. In fact, in Experiment 4, we confirmed the cooling and warming rates of cells vitrified using the KVS device using a simulation analysis (Figs.
3 and
4).
To date, it is known that the cryotolerance of embryos depends on the developmental stage in many mammalian species. Dhali et al. [
18] reported that the modified droplet vitrification (MDV) procedure resulted in better survival at the morula stage than that achieved for zygotes and 2-cell-stage mouse embryos. Accordingly, in Experiment 2, we compared the viability of mouse embryos at the 2-cell stage vitrified or not vitrified. As shown in Table
3, there were no significant differences in the survival or blastocyst formation rates between vitrified or not vitrified embryos. Zhang et al. [
19] reported that the survival and blastocyst formation rates of vitrified-warmed mouse embryos vitrified using the Cryotop at the 2-cell stage were 96.0 and 69.4%, respectively. Furthermore, An et al. [
20] reported that the blastocyst formation rate (85.7%) of vitrified-warmed mouse embryos using the MDV method at the 2-cell stage was significantly lower than that of non-vitrified embryos. In contrast, the blastocyst formation rate for the KVS in this study was higher than that measured in the previous studies [
19,
20] of vitrified mouse embryos. Our results indicate that vitrification using the KVS is effective for the cryopreservation of mouse 2-cell-stage embryos as well as blastocysts.
As shown in Table
4, this is the first report of the ability of mouse embryos, vitrified using the KVS, to develop into live offspring. An et al. [
20] reported that when mouse embryos at the 4-cell stage were vitrified using several vitrification methods (e.g., MDV, Cryoloop, and Cryotech) and transferred into recipients, the birth rates ranged between 35.4 and 43.8%, with no significant differences between these rates and those for fresh embryos (45.1%). Similarly, in this study, the rates of development to live offspring for vitrified mouse embryos were not different from those of non-vitrified embryos. These results prove the effectiveness of the KVS.
In Experiment 4, we assessed the cooling and warming rates in the KVS and the control device via a thermal transfer-based simulation method using numerical modeling. This simulation method was previously reported by Tarakanov et al. [
14] in a manner similar to this analysis. Indirect methods to estimate the cooling and warming rates of vitrification devices are important tools because it is difficult for direct measuring methods to assess real phenomena in closed spaces, such as closed-type vitrification devices, or small spaces, such as nano-order droplets, as described in this work. On the contrary, Mazur and Seki [
21] reported direct measurement methods using thermoelectric coupling and the Cryotop, with a vitrification solution volume of 100 nL and cooling and warming rates of 69,000 and 118,000 °C/min, respectively. The Cryotop, consisting of a polypropylene film with a width and thickness of 0.7 and 0.1 mm, respectively [
22], is currently said to be one of the most excellent devices for enabling rapid freezing. To the best of our knowledge, the embryologists used the vitrification solution volume by applying the Cryotop that was within the specified ranges, although it differs greatly between individuals. The cooling and warming rates of the Cryotop device calculated by the indirect measurement method in this study, using previous report parameters, obtained almost the same results as those reported from the direct measurement method by Mazur and Seki [
21] (data not shown). In this analysis, the cooling and warming rates of the KVS device, which can be automatically achieved to 1.3 nL, were estimated at 683,000 and 612,000 °C/min, respectively. These rates are extremely high compared to those reported for other vitrification devices [
14,
21,
23]. The results of our simulation analysis indicate that decreasing the volume of the vitrification solution around the embryo is critical for increasing the cooling and warming rates. This may be because of the low thermal conductivity of the vitrification solution surrounding the embryo. The thermal distribution using the KVS during the vitrification procedure indicates that thermal transfer to the embryo may exert a dominant influence via a direct pathway from LN
2 through the vitrification solution surrounding the embryo, as opposed to an indirect pathway through the support under the embryo. Thus, the KVS, which minimizes the vitrification solution surrounding the embryo, may prove to be the preferred device for achieving rapid cooling and warming rates.
Acknowledgements
The authors thank Ms. Nagisa Ohi of the Department of Obstetrics and Gynecology, Faculty of Medicine, Tokyo University and Dr. Noritaka Fukunaga of the Asada Ladies Clinic for providing valuable advice. The authors issue special thanks to Dr. Miyuki Harada of the Department of Obstetrics and Gynecology, Faculty of Medicine, Tokyo University for providing valuable suggestions and critically reading the manuscript.