Comparison between slow freezing and vitrification for human embryos

Embryo cryopreservation has several potential advantages in human in vitro fertilization. The goal of the cryopreservation procedure in human assisted reproductive technologies should be to ensure high survival and viability of human embryos after thawing. Two important parameters determine the success of any cryopreservation protocol: the manner in which cells regain equilibrium in response to cooling and the speed of freezing (cooling rate). Traditional slow-freezing protocols have been used to freeze all kinds of human embryos, but clinically satisfactory results have not been obtained; furthermore, the results are not consistent. In addition, the slow-freezing method requires expensive equipment and is time-consuming. Vitrification, in which high cooling rates in combination with a high concentration of cryoprotectant are used, does not produce any ice crystals during cooling and warming. From several recent studies, it appears that the vitrification method is better than slow-cooling procedures in assisted reproductive technologies. Although many of the potential problems, such as toxic effects caused by high concentration of cryoprotectants or liquid nitrogen-mediated contamination, the vitrification method has received increasing interest during the last few years and has achieved significant developments. Therefore, in the near future, the vitrification method will be chosen over the slow freezing method to freeze human embryos at any stage as well as oocytes.

Assisted reproductive technologies have been used to help couples with infertility problems. Recently, many related techniques have been improved in terms of ovarian stimulation, criteria and selection processes to assess the quality and viability of gametes and embryos, as well as culture conditions and culture media. These have led to the ability to obtain significantly more viable and transferable embryos. Moreover, awareness of the risks linked to multiple pregnancies has required the implementation of standards limiting the number of embryos transferred at any one time. Therefore, cryopreservation of residual human embryos between zygotes and blastocyst stages has become a routine procedure to increase cumulative pregnancy rates, to help avoid the risk of multiple pregnancies after the transfer of many embryos, avoid unnecessary additional stimulation procedures and escape the ovarian hyperstimulation syndrome [1].

During various stages of the cryopreservation process, including exposure to cryoprotectants, cooling, storage in liquid nitrogen, rewarming and return to a physiological solution, it is solely the skill of being able to prevent ice-crystal formation that determines the viability of an embryo after rewarming. It is obvious that ice crystallization is incompatible with any developmental stage embryos and must be avoided as far as possible [2]. There are two general approaches to prevent ice crystals: slow-rate freezing and vitrification.

Since Whittingham et al. reported the first successful live offspring after cryopreservation [3], based on the use of slow freezing on mouse embryos, the cryopreservation technique was applied to human early cleavage-stage embryos and resulted in the first report of pregnancy and live birth from human cryopreserved embryos [4,5]. This approach was rapidly applied by using the cryoprotectants 1,2 propanediol and sucrose [6,7], and have become widely used for the cryopreservation of zygotes and early cleavage-stage embryos. For blastocysts, Cohen et al. reported the first pregnancy after slow freezing of blastocysts, using glycerol as a permeable cryoprotectant [8]. This protocol, modified by Menezo et al. via the addition of sucrose as a nonpermeable cryoprotectant, was considered popular for the slow cooling of blastocysts [9]. However, in spite of the vast efforts invested, advances of slow-freezing techniques have been rather slow. The main problem is the lack of consistency, as well as differences in survival and developmental rates after warming between laboratories, developmental stages and quality [10,11].

Vitrification in assisted reproductive technologies has been described for gametes and embryos, as well as for ovarian tissue. In 1985, vitrification was first reported with mouse embryos and was then further developed in animal reproduction. In 1999, the first successful pregnancies and deliveries after vitrification of human oocytes were reported [12]. Since then, scientific interest in vitrification has risen significantly and the greatest advantage of vitrification has been seen in all human embryos. The convincing evidence that has accumulated regarding the huge potential importance of vitrification was reported recently [13–20]. Therefore, vitrification has become a viable and promising alternative to traditional approaches in cryopreservation of mammalian embryos and oocytes.

This editorial will focus on a comparison of embryological and clinical outcomes between traditional slow freezing and vitrification to freeze different developmental stages of human embryos.

Slow cooling versus vitrification technique

Before initiating slow cooling in a programmable controlled system, embryo equilibrium is achieved at a relatively low cryoprotectant concentration (∼1.5 mol/l), and a progressive dehydration, shrinkage and increase in intracellular solute concentration occurs, due to the rising osmolarity in the extracellular solution during the cooling process (0.3–2°C/min). Under slow cooling conditions, it is possible to prevent intracellular ice formation because the concentration of intracellular solutes is slightly higher than outside during dehydration, thus lowering the freezing point within the cell. Even as a result of the presence of extracellular ice, supercooling of the cell interior occurs, but this disrupts equilibrium between the interior and exterior of the cell. To prevent supercooling, the straw containing the embryos is manually or automatically seeded at 6–8°C, which allows for large ice crystals to grow slowly and to maintain a very delicate balance between factors, which may result in damage mostly by ice crystallization. Therefore, to achieve this balance, the time to complete slow-cooling procedures for human oocytes and embryos requires a minimum of 90 min. In the most favorable situation, the intracellular concentration of cryoprotectant becomes high enough so that the remaining intracellular unfrozen fraction will vitrify, preventing intracellular ice formation. However, it is undesirable to remove too much intracellular water, since dehydration can result in an increase in the intracellular solute concentration to toxic concentrations for the cells. The most critical moment for the cells during the freezing and thawing phase is the intermediate temperature zone (-15 to -60°C) [21]. The samples are then stored at -196°C in liquid nitrogen. After storage, the possibility of ice recrystallization while thawing exists and is thought to be the major cause of cryoinjury; this can be avoided by warming the sample quickly.

Whereas in conventional slow freezing the concentration of the cryoprotectant is low and the cooling rate is very slow to avoid ice crystallization, vitrification is an ultrarapid cooling technique that requires a high concentration of cryoprotectant. Therefore, vitrification takes only a few seconds to cool embryos, and it does not require specialized expensive equipment. The physical definition of vitrification is a vitreous, transparent, ice-free solidification of water-based solutions at subzero temperatures, not by ice crystallization but by extreme elevation in viscosity during cooling, such that the cells are placed into the cryoprotectant and then plunged directly into liquid nitrogen. Water is largely replaced by the cryoprotectant. The cooling rate achieved is between 15,000 and 30,000°C/min, and water is transformed directly from the liquid phase to a glassy, vitrified state. No ice crystals form that can damage the cells with this method. A problem for cells can be the high concentration of cryoprotectant that is required for vitrification. A biological limitation exists on the concentration of cryoprotectant tolerated by the cells during vitrification. Therefore, the aim in any vitrification protocol is to increase the speed of temperature change while keeping the concentration of cryoprotectant as low as possible [21].

Initially, most vitrification methods use standard French ministraws for holding the embryos during cooling, storage and thawing. Recently, to overcome the disadvantages of straws, which have low cooling and warming rates, techniques using either a electron microscopy grid [13,14], cryotop [15], hemistraw [16], cryoloop [17,18], cryotip [19] or cryoleaf [20] that substantially increase the cooling rate as a carrier have been applied, significant increases of the success rate of human oocyte or embryo vitrification have been reported [13–20].

Slow freezing versus vitrification of different developmental stages of human embryos

Pronuclear stage embryos

Conventional slow freezing of human fertilized zygotes (2 pronuclei [PN]) has been the most widely used, well-established method for preservation [22,23]. In some countries in particular, the study of cryopreservation of human zygotes is an important topic, as it is illegal to cryopreserve zygotes after fusion of the pronuclei due to ethical concerns. Numerous babies have been born after transfer of embryos developed from frozen pronuclear embryos. However, variable efficiencies on survival or pregnancy have been reported and lower pregnancy rates have been achieved when compared with fresh zygote cycles. Nevertheless, several reports have indicated that the survival rate and clinical outcome of embryos cryopreserved at the 2PN stage using slow freezing are higher than those of early cleaved embryos [11,22,23]. In Germany, the total pregnancy of frozen–thawed PN is approximately 17% [21].

Recently, successful vitrification of PN with high survival (i.e., 81–93%) and pregnancies were reported [24,25]. The PN stage is well able to withstand the vitrification and warming conditions, probably due to processes during and after fertilization, such as the cortical reaction and subsequent zona hardening, which may give the ooplasm more stability to cope with the low temperature and osmotic changes.

There are not many studies published that compare both slow freezing and vitirification. Kuwayama et al. reported that vitrification of 5881 human PN stage embryos resulted in 100% survival, 93% cleavage and 52% blastocyst rates [19]. After slow freezing of 1944 PN stage embryos, survival, cleavage and blastocyst rates were 89, 90 and 41%, respectively. Therefore, vitrification was found to be better for PN embryo cryopreservation, with regard to survival, cleavage and developmental rates. In addition, Al-Hasani et al. reported higher clinical pregnancy rates (36.9%) after vitrification than after slow freezing (10.2%), although a prospective randomized study was not performed [26].

Based on the reports, the vitrification technique with human 2PN embryos yields the same or even better results than slow freezing in terms of survival, implantation and pregnancy rates.

Cleavage-stage embryos

With a traditional slow-freezing cryopreservation, survival rates of 76–80.6% were reported for cleavage-stage embryos [27]. Several studies of human cleavage-stage embryo vitrification have been reported. Most studies on vitrification of cleavage-stage embryos reported high survival rates of over 80%, and pregnancy rates in the range of 22–35%, which were much higher than the rates of slow-freezing procedures [19,28]. More recently, pregnancy rates have been increased to 49% with vitrification of cleavage-stage embryos [29].

There are a few studies that compare survival and clinical outcome after thawing of early cleavage-stage embryos frozen after slow freezing or vitrification. The largest comparative investigation between the effects of slow freezing versus vitrification was published by Kuwayama et al.[19]. The survival rate of four cell-stage human embryos after vitrification (98%) was also significantly higher than that after slow freezing (91%). Pregnancy rates after cryopreservation with the two methods were not significantly different (slow freezing: 51% versus vitrification: 53%). On the other hand, Rama Raju et al. reported a direct comparison of day 3 cryopreservation with slow freezing and vitrification prospectively [28]. The survival rate (95.3%), as well as the pregnancy (35%) and implantation (14%) rates after vitrification, was significantly higher than after slow freezing (60.0, 17.4 and 4.2%, respectively). Recently, Balaban et al. reported the results of the randomized, controlled study of human day 3 embryo cryopreservation by slow freezing or vitrification [29]. They compared survival rate, embryonic metabolism and subsequent development to blastocyst. Significantly, embryos survived the vitrification procedure (222/234, 94.8%) than slow freezing (206/232, 88.7%), pyruvate uptake was significantly greater in the vitrification group, reflecting a higher metabolic rate, and the development to the blastocyst stage was also higher following vitrification (134/222, 60.3%) than following slow freezing (106/206, 49.5%). Based on those results, the authors performed a clinical trial and obtained a 30% implantation rate and 49% clinical pregnancy rate [29]. They suggest that vitrification imparts fewer traumas to cells and is, therefore, a more effective means of cryopreserving the human embryo than conventional slow freezing.

These recent data imply that vitrification is more efficient for freezing human cleavage-stage embryos than slow freezing in terms of the high rates of pregnancy and survival outcome.

Human blastocysts

There is much debate as to the developmental stage at which human embryos are best cryopreserved [30,31]. The disadvantage of zygote-stage embryos is that nothing is known regarding their developmental competence. On the other hand, a major complication of cleavage-stage embryos is that damaged blastomeres often coexist with intact ones after thawing and it has been demonstrated that the implantation potential of such embryos is much lower than that of fully intact ones [32,33]. Compared with early embryos, blastocysts have the advantage of containing numerous small cells and thus, the loss of some cells during cryopreservation is probably less harmful for the further development of the embryo. Recently, blastocyst transfer based on an improved culture system has been proven effective for increasing the pregnancy rate in assisted reproductive technologies [34,35] since blastocysts are much better suited to the uterine milieu, while minimizing multiple gestations. Therefore, a reliable procedure for the cryopreservation of supernumerary blastocysts is needed because, usually, only a small number of blastocysts after transfer are likely to be available for cryopreservation.

Freezing of human blastocysts has been carried out with the slow-freezing method, which led to the first successful human cryopreservation pregnancies, but clinically satisfactory results have not been obtained [9,36]. Throughout the 1990s, reports of clinical pregnancies after blastocyst thaw indicated rates of well under 30% per transfer. The survival rate of blastocysts following the slow freezing method is 60% in general. Due to this, the freezing of blastocysts did not appear to present a better cryostorage option when compared with earlier stages.

Therefore, with respect to its efficiency, vitrification technology has become an encouraging alternative to slow freezing thawing. Since the first pregnancy after vitrification of a human blastocyst was reported [17], more attention has been focused on vitrification due to significantly higher survival and pregnancy rates [37]. Many human pregnancies that originate from the vitrification technique are achieved after cryopreservation of blastocysts using different kinds of carriers such as cryotop [15], the cryoloop [17,18], electron microscope grids [14,38] or the hemistraw [16]. In addition, the survival rate of expanded blastocysts after vitrification increases when the blastocoele is shrunk artificially with a glass microneedle [39], two 29-gauge needles [38], pipetting [40] or a laser pulse [41], which is thought to reduce ice crystal formation. Several studies reported survival rates of 72–90%, clinical pregnancy rates of 37–48% and implantation rates of 22–29% with blastocyst vitrification [38–41]. Takahashi et al. reported that the perinatal outcome of infants delivered after vitrification with a congenital defect rate was similar to fresh blastocyst transfer (1.4%) [42]. Liebermann and Tucker also did not find any defects in the infants delivered after slow freezing (79 infants) or vitrification (54 infants) [10].

Some studies were performed to compare a slow-freezing protocol with a vitrification protocol for the cryopreservation of human blastocysts. Stehlik et al. reported that the survival and pregnancy rates in slow-frozen blastocysts were 83.1 and 16.7% (4/24), respectively [43]. By contrast, 100% survival and 50% pregnancies were obtained after thawing of the vitrified blastocysts. Huang et al. also compared survival rates after warming of blastocyst frozen with slow-freezing or vitrification methods [44]. The survival rate following vitrification (84.0%, 68/81) was significantly higher than that for the slow-freezing group (56.9%, 41/72). Therefore, survival, implantation and pregnancy rates of vitrified blastocysts were significantly higher than slow-frozen blastocysts. However, in the study performed by Leiberman and Tucker, the authors reported that the vitrification technique yielded similar survival (96.5%) and pregnancy (46.1%) rates to slow-frozen blastocyst transfers (92.1%, 42.9%) [10]. Furthermore, the strongest data came from a large study by Kuwayama et al.[19]. In this study, the survival and development of human embryos were compared following slow cooling versus vitrification involving more than 6000 blastocysts. In all, 90.0% (5695/6328) of vitrified blastocysts survived and resulted in a 53.0% (2516/4745) pregnancy rate following transfer compared with 84.0% (131/156) survival and 51.0% (50/98) pregnancy rates following slow cooling. Therefore, comparing the survival rate of blastocysts from slow-freezing protocols and vitrification protocols, these studies showed, in human blastocysts, a post-thaw survival rate between 56.9 and 91.2% after slow freezing, and a post-thaw survival rate of 90–100% after vitrification.

These successful and consistent recent results obtained from vitrification of blastocysts document the fact that vitrification is the perfect alternative to slow-freezing techniques.

Selection of slow freezing versus vitrification of embryos biopsied for preimplantation genetic diagnosis

In consideration of the complexity and labor intensity of preimplantation genetic diagnosis, the availability of an efficient cryopreservation program is especially important in the case of biopsied embryos. Embryos diagnosed as transferable by preimplantation genetic diagnosis are of particular value, and the possibility of storing them prevents the couple from having to repeat all the steps of the preimplantation genetic diagnosis cycle. Unfortunately, the cryopreservation of biopsied embryos has given disappointing results when performed at the cleavage stage using conventional slow-freezing protocols [45,46]. However, Zheng et al. found that the survival rate after vitrification was 94% compared with 16–85% after three versions of slow freezing [47].

Vitrification of embryos derived from in vitro maturation of immature oocytes

In vitro maturation (IVM) is an attractive option to eliminate several problems of controlled ovarian hyperstimulation used for conventional in vitro fertilization (IVF). Recent studies have shown improved pregnancy rates per embryo transfer in small numbers of cycles [48,49]. In addition, several IVF centers have reported that acceptable rates of oocyte maturation and pregnancies were achieved in patients with polycystic ovary syndrome [49]. However, they reported that the implantation rate was less than 15%. Therefore, more embryos in an IVM program have been transferred to obtain acceptable pregnancy rates than in controlled ovarian hyperstimulation cycles. In addition, although cryopreservation is now a routine procedure in human IVF programs, there have been only a few case reports in which embryos generated from an IVM program were frozen at 2PN [50] and cleavage [51] using the slow-cooling method. One of the reasons is that considerable differences of efficiency exist depending on the origin (in vivo or in vitro) of the matured oocytes.

Generally, in vitro-produced oocytes or embryos were much more sensitive to freezing than the in vivo-derived counterparts [52]. Suikkari et al. also found that the cryosurvival of the in vitro-matured zygotes and -cleaved embryos was very poor compared with embryos generated from in vivo-matured oocytes using the slow-freezing method [53]. Therefore, clinically satisfactory results from embryos generated from IVM oocytes have not been obtained up to now [53].

Although no studies have been reported on slow freezing of blastocysts produced from in vitro-matured oocytes, successful pregnancies can be achieved after vitrification of blastocyst stages generated from IVM cycles [54,55]. Recently, Lee et al. reported the clinical outcome after vitrification and thawing of the blastocysts produced from 32 IVM cycles using the vitrification method [56]. A total of 92% of the blastocysts were re-expanded after warming, and 73.9% had hatching or hatched blastocysts stage at the time of transfer. The clinical pregnancy and implantation rates after vitrification were as similar to fresh cycles. This result implies that the vitrification method is a safe cryopreservation method for blastocysts produced from IVM programs and it highlights the use of three advanced assisted reproductive techniques, namely IVM, blastocyst culture and vitrification, to overcome infertility problems.

Conclusion

The vitrification method has received increasing interest during the last few years. Although there is insufficient evidence to determine whether vitrified embryos are more viable than the embryos frozen by slow-freezing methods, comparative studies from a small number of clinics suggest that the clinical outcome using vitrification yields the same or even better results than slow-freezing methods in different development stages of human embryos. Although many vitrification protocols exist, the survival and pregnancy rates of embryos have been consistent compared with slow-freezing methods. Therefore, undoubtedly, the vitrification method is a promising approach to the storage of all stages of human embryos.

Five-year view

The concept that living systems could be cooled so quickly that ice would not have time to form was proposed more than 60 years ago [21], and this proposal now appears more realistic. The initial theoretical postulates and principles on vitrification were replaced by a long period of slow-cooling protocols that were believed to be suitable for low-temperature preservation of cells and tissues. Now the field of cryobiology has progressed by returning to vitrification protocols. The slow-freezing protocols have been replaced by vitrification, which is a very short and more effective procedure, and it is envisaged that the time will come when vitrification will be used more commonly and widely for storage of all kinds of human embryos. During the advances that took place, a variety of new techniques and types of carriers that enhance the cryopreservation of mammalian embryos were developed. However, as yet, more evidence is needed to conclude whether vitrification is the way forward. Prospective comparison of the two freezing approaches is needed focusing on overall outcomes, such as live birth data.

The most widely emphasized concerns in the vitrification process are toxicity and danger of contamination [21,57]. Unfortunately, available vitrification methods still struggle with these problems to date. Regarding the toxic and osmotic effects, the latest cryoprotectant combinations and concentrations cannot be considered to get any higher. Moreover, in most vitrification methods, the time of exposure to the final cryoprotectant concentrations is very limited at temperatures where this toxic effect may still be significant (i.e., above -20 to -40 °C). Regarding contamination, the latest and most successfully applied approach has introduced a problem related to storage. Many recent vitrification techniques separate the cooling and storage phases; in this way the requirement for direct contact is limited to a relatively small amount of liquid nitrogen that can be filtered, UV-sterilized or just purchased as sterile stock. Therefore, the authors think that these negative effects will be minimized or entirely eliminated with the development of new techniques in the future.

Although a standardized vitrification protocol has not yet been defined, vitrification has been used routinely in most IVF laboratories recently and good survival and pregnancy rates of embryos have been achieved consistently. The latest vitrification methods are more efficient and reliable than any version of slow freezing. Application of the proper vitrification methods increases the efficiency of long-term storage of any kinds of human embryos. Therefore, the opinion of the authors is that vitrification is the future for cryopreservation and more significant developments should be expected in the near future.

Acknowledgements

The authors are very grateful to Belen Herrero, PhD and Jin-Tae Chung, MSc, Department of Obstetrics and Gynecology, McGill University, Montreal, Quebec, Canada, for their critical review of this manuscript.

Financial & competing interests disclosure

The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

No writing assistance was utilized in the production of this manuscript.