In assisted reproductive technology, cryopreservation of human oocytes and embryos has been significantly improved by refined slow-cooling and the new vitrification method. The slow-cooling method requires a programmed cryo-machine, and usually takes several hours. It is, however, difficult to eliminate injuries resulting from ice formation completely. Vitrification has become a reliable strategy because it is simple, can lead to high survival rates and viability, and has better clinical outcome. Vitrification transforms cells into an amorphous glassy state inside and outside the vitrified cell with ultra-rapid cooling and warming steps by plunging the oocytes and embryos into liquid nitrogen, instead of ice-crystal formation. Over the past decade, several advances in vitrification technologies have improved clinical efficiency and outcome. In this chapter, we focus on vitrification technologies for cryopreservation in human assisted reproductive technology.
Introduction
In assisted reproductive technology (ART), cryopreservation of embryos has become important for the best use of supernumerary embryos. During the cryopreservation of embryos, various types of injury may occur. Among the most damaging is the formation of intracellular ice. The first strategy to prevent intracellular ice from forming was to use a lower concentration of cryoprotectant and a long slow-cooling stage. This slow-freezing method has proven effective for embryos of a wide range of mammalian species. Unlike embryos of laboratory animals and domestic animals, in which dimethylsulphoxide (DMSO), glycerol or ethylene glycol are commonly used as the cryoprotectant, human embryos at early cleavage stages have most often been frozen in a solution of propanediol supplemented with sucrose ; however, those at the blastocyst stage have more frequently been frozen with glycerol and sucrose. With slow freezing, however, it is difficult to eliminate injuries occurring from ice formation completely. Furthermore, the slow-freezing method requires a long period of time before embryos are stored in liquid nitrogen (LN 2 ).
In 1985, Rall and Fahy applied the innovative approach of vitrification, in which injuries related to ice crystal formation are minimised, by using high concentrations of cryoprotective agent (CPA) together with rapid temperature change. The definition of vitrification is the solidification of a solution at a low temperature without the formation of ice crystals, by increasing the viscosity using high cooling rates. The rapid cooling process can minimise chilling injury and osmotic shock to the embryos. With recent improvements in past decades, vitrification has become the most reliable strategy because it is technically simple, and can lead to high survival and implantation rates. To induce vitrification in LN 2 , or super cooled air, the solution must contain a high concentration of CPA. This approach simplifies the cooling process, because embryos can be rapidly cooled directly in LN 2 . Although embryos subjected to vitrification are potentially liable to be affected by the toxicity of the high concentration of CPA, the method has been refined and proven to be effective for the cryopreservation of embryos at various stages of development in laboratory and domestic species. In 1998, it was shown that vitrification using an ethylene glycol-based vitrification solution (EFS40) with conventional cryo-straws was relatively effective for human embryos at the four- to eight-cell stage. The effectiveness of vitrification was confirmed for human embryos at the eight- to 16-cell stage and the morula stage, also using ethylene glycol-based solutions.
Over the past decade, several advances have taken place in vitrification technologies such that it can provide high clinical efficiency along with better clinical outcome. It is proposed that vitrification will definitely become the most suitable method for cryopreservation of any cells and tissues in the near future. In this chapter, therefore, we will focus on vitrification technologies for cryopreservation in human ART.
Principles of vitrification
The basic procedure for vitrification is simple. Embryos are suspended in a vitrification solution and then plunged in LN 2 , or super-cooled air. Embryos are warmed rapidly and diluted quickly with a sucrose solution. The most important stage is the exposure of embryos to the vitrification solution before rapid cooling. In order to prevent intracellular ice from forming, a longer period of exposure is desirable. If the exposure is too long, however, embryos suffer from the toxicity of the CPA solution. Therefore, the optimal exposure time for successful vitrification must be a compromise between preventing the formation of intracellular ice and preventing toxic injury. Ironically, embryos may be injured by the toxicity of the cryoprotectant before enough cryoprotectant can permeate inside the embryos. To prevent this, a two-step procedure is commonly used, in which embryos are first equilibrated in a dilute (e.g. 10%) CPA solution, followed by a brief (30–60 s) exposure to a vitrification solution before embryos are cooled with LN 2 . The optimal exposure time in the vitrification solution depends on the CPA solution and the temperature, as both the permeability of embryos and the toxicity of the CPA are largely influenced by the temperature.
In vitrification, the selection of CPA requires extreme care because their concentration can be as high as 6 M, which can make the toxicity of these compounds a key limiting factor in cryobiology. The most appropriate characteristics of a penetrating CPA are low toxicity and high permeability. For cryopreservation of human embryos, propanediol and DMSO have been used as the dominant CPAs, although glycerol is used when embryos are frozen at the blastocyst stage. As a less toxic CPA, ethylene glycol is commonly and widely used. A few comparative studies, however, have examined the effect of the CPA on the survival of vitrified embryos.
Principles of vitrification
The basic procedure for vitrification is simple. Embryos are suspended in a vitrification solution and then plunged in LN 2 , or super-cooled air. Embryos are warmed rapidly and diluted quickly with a sucrose solution. The most important stage is the exposure of embryos to the vitrification solution before rapid cooling. In order to prevent intracellular ice from forming, a longer period of exposure is desirable. If the exposure is too long, however, embryos suffer from the toxicity of the CPA solution. Therefore, the optimal exposure time for successful vitrification must be a compromise between preventing the formation of intracellular ice and preventing toxic injury. Ironically, embryos may be injured by the toxicity of the cryoprotectant before enough cryoprotectant can permeate inside the embryos. To prevent this, a two-step procedure is commonly used, in which embryos are first equilibrated in a dilute (e.g. 10%) CPA solution, followed by a brief (30–60 s) exposure to a vitrification solution before embryos are cooled with LN 2 . The optimal exposure time in the vitrification solution depends on the CPA solution and the temperature, as both the permeability of embryos and the toxicity of the CPA are largely influenced by the temperature.
In vitrification, the selection of CPA requires extreme care because their concentration can be as high as 6 M, which can make the toxicity of these compounds a key limiting factor in cryobiology. The most appropriate characteristics of a penetrating CPA are low toxicity and high permeability. For cryopreservation of human embryos, propanediol and DMSO have been used as the dominant CPAs, although glycerol is used when embryos are frozen at the blastocyst stage. As a less toxic CPA, ethylene glycol is commonly and widely used. A few comparative studies, however, have examined the effect of the CPA on the survival of vitrified embryos.
Day two to three embryo vitrification
In 1998, an investigation was conducted to find a suitable CPA and suitable conditions for exposing embryos to a vitrification solution using eight-cell mouse embryos. The survival rates of eight-cell mouse embryos vitrified in various solutions after exposure to the solutions for 0.5 and 2 min at 20 °C and 25 °C were measured. The highest rates of survival were obtained with ethylene glycol-based solutions, regardless of time and temperature. Although none of the vitrified embryos were morphologically normal when embryos were vitrified after 0.5 min exposure to any mixture of 30% CPA, the survival rate was over 90% when embryos were treated for a longer time (2 mins) at a higher temperature (25 °C), or when embryos were treated with a higher concentration of ethylene glycol (EFS40) at a higher temperature (25 °C).
In addition, a small saccharide (e.g. sucrose) and a macromolecule (e.g. Ficoll 70, bovine serum albumin or polyvinyl pyrrolidone) are frequently included in vitrification solutions. These non-permeating agents are much less toxic and are known to promote vitrification of the solution. Therefore, their inclusion can reduce the toxicity of the solution by decreasing the concentration of the permeating agent required for vitrification. In addition, inclusion of a saccharide promotes shrinkage of embryos, and thus reduces the amount of intracellular cryoprotectant, which will also reduce the toxic effect of the permeating CPA. At the same time, the osmotic action of saccharide plays an important role in minimising the swelling of embryos during dilution, as a quick dilution is necessary to prevent the toxic effect of the CPA solution.
Protocols and clinical results of day two to three vitrification
Several protocols have been introduced for human day 2–3 vitrification. In those protocols, however, the basic concept is similar, and the differences between the protocols are related to the type and concentration of CPAs and duration of exposure of CPAs. Protocols and clinical outcomes are presented in Tables 1 and 2 .
| Author and reference | Mukaida et al. | Desai et al. | Rama Raju et al. | Kuwayama et al. |
|---|---|---|---|---|
| Type of container | Cryostraw | Cryoloop a | Cryoloop b | Cryotop |
| Temperature | Room (25–27 °C) | Warm stage (37 °C) | Warm stage (37 °C) | Room (25–27 °C) |
| Equilibration step | Ethylene glycol, Ficoll, sucrose 20 (2 min): 20% EG | 7.5% ethylene glycol + 7.5% DMSO (2 min) | 10%EG (5 min) | 7.5% ethylene glycol + 7.5% DMSO (5-10 min c ) |
| Vitrification step | Ethylene glycol, Ficoll, sucrose 40 (1 min): 40% EG | 15% EG + 15% DMSO + F.+S. (35 s) | 40%EG + S. (30 sec) | 15% ethylene glycol + 15% DMSO + sucrose (1 min) |
| Cooling system | Vapor phase LN 2 (3 min), then plunged into LN 2 | Plunged into LN 2 directly (ultra-rapid cooling) | Plunged into LN 2 directly (Ultra-rapid cooling) | Plunged into LN 2 directly (ultra-rapid cooling) |
| Warming step | One step | Two steps | Four Steps | Two Steps |
| 0.5 M sucrose (5 min) | 0.25 M S. (2 min) | 1 M S. (2.5 min) | 1 M sucrose (1 min) | |
| 0.125 M S. (3 min) | 0.5 M S. (2.5 min) | 0.5 M sucrose (3 min) | ||
| 0.25 M S. (2.5 min) | ||||
| 0.125 M S. (2.5 min) |
a Reported by Desai et al., in 2007.
b Reported by Raju et al, in 2005.
c The duration of equilibrium is adjusted according to the time needed for re-expansion of the vitrified embryos.
| Author and reference | Mukaida et al. | Desai et al. | Ramu Raju et al. | Results of Nagata Clinic |
|---|---|---|---|---|
| Type of container | Cryostraw | Cryoloop a | Cryoloop b | Cryotop |
| Age (years) | Not available | 34.1 ± 4.5 | 31.3 ± 4.5 | 35.0 ± 4.5 |
| Number of cycles | 127 | 77 | 40 | 604; 346 women |
| Survival rate | Not available | 201/236 | 121/127 | 1701 out of 1774 |
| 85% | 95% | 95.9% | ||
| Cleavage rate c | 486 out of 661 | 184/236 | Not available | 1289 out of 1774 |
| 76% | 78% | 72.7% | ||
| Pregnancy rate | 34 out of 127 | 34/77 | 14/40 | 164 out of 604 |
| 26.8% | 44.2% | 35.0% | 27.2% | |
| Implantation rate | Not available | 40/201 | 18/121 | 192 out of 1442 |
| 19.9% | 14.9% | 13.3% | ||
| Delivery rate d | 22 out of 127 | Not available | 13/40 | 118 out of 604 |
| 17% | 32.5% | 19.5% |
a Reported by Desai et al., 2007.
b Reported by Raju et al., 2005.
c Including survival and further cleavage rate.
Vitrification using conventional cryostraws for day two to three embryos
A two-step protocol for vitrification with straw as a container using ethylene glycol-based solutions, EFS20 and EFS40, has been described. The two solutions (EFS20 and EFS40) are used for pre-treatment and vitrification, respectively, and contain ethylene glycol diluted to 20% (v/v) or 40% (v/v) with Ficoll–sucrose solution. This method has been proven suitable for human embryos on day 2–3. (Mukaida et al., unpublished data). In 1998, the effectiveness of this vitrification method for day 2–3 human embryos was confirmed.
Vitrification using the cryoloop for day two to three embryos
An improvement to cleavage stage ultra-rapid vitrification came with the cryoloop ( Tables 1 and 2 ). This method is effective for embryos on day 2–3, for which conventional vitrification using a straw was found to be less effective. The protocol for vitrification using the cryoloop can be found in the following section on vitrification of blastocysts.
In 2008, Balaban et al. reported a two-step protocol of cryoloop vitrification for day 2–3 embryos cryopreservation. Their protocol ( Table 1 ) was originally described by Larman et al. Two steps of dehydration and equilibration of CPA are applied before cooling. The embryos are loaded onto the cryoloop (Hampton Research, Aliso Viejo, CA, USA), transferring as little medium as possible, typically around 50 nl. For warming, multiple steps of rehydration with several different sucrose concentrations are carried out. Clinical outcome was as follows ( Table 2 ); a total of 73 women subsequently underwent vitrified-warmed embryo transfers. A mean number of 3.3 embryos were warmed ( n = 241). The cryosurvival rate was 92.1%. All blastomeres were intact in 72.1% of the embryos after the warming procedure. The mean number of embryos transferred was 2.3 ( n = 168). A clinical pregnancy rate and ongoing pregnancy rate of 49.3 and 45.2% was achieved, respectively. The implantation rate was 29.7% ( n = 50), resulting in a multiple pregnancy rate of 36.1% ( n = 13: 1 triplet, 12 twins). At the time of reporting, eight of the ongoing 33 pregnancies have had successful deliveries of healthy children (two twins, six singletons). Moreover, in this study, they revealed that vitrification was a more effective approach of cryopreserving the human embryo than conventional slow freezing.
In 2007, Desai et al. reported the post-vitrification development, pregnancy outcomes and live births for cryoloop vitrification of human day 3 cleavage-stage embryos. Their protocols and results are presented in Tables 1 and 2 . They include consecutive vitrification-warming cycles carried out over a 2.5-year interval.
In 2005, Rama Raju et al. reported a modified protocol for vitrification of human eight-cell embryos using the cryoloop technique. The protocol, including the type of CPA and duration of exposure, is different from the one reported by Desai et al. ( Table 1 ). Results showing the effectiveness of their protocol are included in Table 2 .
Vitrification using cryotops
As the vitrification approach of a cryotop and a cryoloop is similar to a minimal volume cooling system, the basic concept of the protocol is the same. The following protocol was originally introduced by Kuwayama et al.
For vitrification using a cryotop, the protocol is similar to blastocyst vitrification using a cryoloop technique described at the section of blastocyst vitrification. The differences of the one using a cryotop are duration of equilibration of CPA for cooling steps and concentration of sucrose for warming steps. Embryos with 70% or more intact blastomeres are considered as indicative of survival and kept in culture until transfer on the following day ( Table 1 ).
The results from the use of the cryotops at Nagata Clinic show the effectiveness of clinical application and are presented in Table 2 .
Blastocyst vitrification
Recent advances in culture systems with sequential media have made it possible to develop human in-vitro fertilisation embryos to the blastocyst stage quite easily. Because the blastocyst is better suited to the uterine environment, and blastocyst formation is a form of selection for more viable embryos, blastocyst transfer has become a promising option for raising the overall pregnancy rate. Accordingly, the need to cryopreserve human blastocysts is increasing. Menezo et al. cryopreserved human blastocysts that were developed in a co-culture system using the slow-freezing method with glycerol, and obtained reasonable clinical results (27% pregnancy rate, 17% implantation rate). Results reported by other clinics, however, have not been consistent. Menezo et al. speculated that the cryopreservation outcome might be influenced by the culture conditions, such as a co-culture system.
Recently, human blastocysts were successfully vitrified in straws. Our own attempts to vitrify human blastocysts using straws, however, resulted in only 45% survival (39 out of 86, unpublished data). Vanderzwalmen et al. also reported a low pregnancy rate with human blastocysts vitrified in straws. This is probably because human blastocysts are much less permeable to CPA and water, as it has been observed that they shrink more slowly than mouse and bovine blastocysts in the CPA solution. This suggests that human blastocysts are more likely to be injured by intracellular ice crystal formation.
Increased rates of cooling and warming can help circumvent the problem of intracellular ice formation in less permeable embryos. Faster rates of cooling and warming can be achieved by minimising the volume of the solution with which embryos are vitrified (i.e. by using minute tools such as electron microscopic grids, open pulled straws, cryoloops, or cryotop). We showed that the transfer of human blastocysts vitrified with cryoloops can lead to successful births. Since this original report, we have continued to use this vitrification approach for the cryopreservation of blastocysts on day 5 and day 6.
Currently, several established blastocyst vitrification techniques have been reported. In this chapter, we include our protocol of blastocyst vitrification and a summary of the clinical outcomes for the past 10 years. This confirms the safety and effectiveness of the cryoloop technique for the cryopreservation of human blastocysts.
Protocol for blastocysts vitrification
The protocol for the cryoloop vitrification of blastocysts was adapted from the work of Lane et al. with slight modifications. ( Fig. 1 ). Procedures of vitrification involve equilibration and vitrification steps carried out at 37 °C in 7.5% (DMSO) and 7.5% ethylene glycol for 2 mins, and 15% DMSO, 15% EG, 1% Ficoll 70 and 0.65 M sucrose for 30–45 s in heat transfer fluid (HTF) and human serum albumin (HSB). At the end of 30–45 s, the blastocysts are loaded onto a small nylon loop (Hamilton Research, CA, USA) and are plunged directly into LN 2 . They are warmed by placing the tip of the cryoloop into 0.5 M sucrose in HTF and HAS, and are kept there for 2 mins and then in 0.25 M sucrose in HTF and HSA for 3 mins. With the use of a cryoloop as a container, the vitrified blastocyst almost floats in the thin filmy layer of the droplet on the nylon loop, and heat conduction to the blastocyst becomes homogenous and extremely high. With the high cooling rate, full equilibration of CPA is not necessary to avoid ice-crystal formation, and inside the cell is a so-called ‘meta-stable situation’. That is why around 3 mins of CPA exposure will be enough to reach vitrified status inside the cells. This duration of exposure is shorter than that of other vitrification approaches. Shorter exposure of CPA is more favourable to avoid exposure of the potentially toxic agent to the cells.
