**2.3 Cryoprotectant toxicity to mammalian embryos**

The higher the concentration of cryoprotecting agent is in a solution, the lower is the likelihood water crystals would be formed in the solution in a rapid-cooling process. However, most cryoprotecting molecules are toxic to embryos with toxicity positively correlating to their concentrations and the exposure time. When choosing the appropriate cryoprotectants, toxicity must be considered. Among five common permeating cryoprotecting agents to be tested, toxicity was determined to be dimethylformamide> erythritol > DMSO > glycerol and ethylene glycol on mouse morulae (Kasai et al., 1981). By electron probe microanalysis, Pogorelov et al. (2006 & 2007a) detected a dramatic decrease in intracellular potassium and sodium content in two-cell embryos treated with procedures mimicking vitrification in ethylene glycol, demonstrating a potential stress exerted on the

Cryopreservation of Embryos from Model Animals and Human 267

processes to facilitate the optimization of vitrification protocols in future (Katkov and

The first mammalian embryo that survived cryopreservation was the mouse embryo 40 years ago by slow-cooling (Whittingham et al., 1972). More than 50% of preimplantation embryos survived after thawing and about 40% of the surviving embryos developed to full-term after being transferred to foster females. A similar protocol was applied successfully on cow embryos (Wilmut & Rowson, 1973), sheep embryos (Willadsen, 1977) and many other domestic mammals (Saragusty & Arav, 2011), suggesting that slow cooling is basically applicable to other mammalian embryos, provided the cooling rate can be optimized and well controlled. By using 1.5M DMSO as a cryoprotectant, sheep morulae and blastocysts were cooled at a rate of 0.1°C per minute. A survival rate of 80% can be achieved. With a similar protocol developed from mouse experiments, the first pregnancy after transfer of a cryopreserved human 8-cell embryo was recorded in 1983 (Trounson & Mohr, 1983). The pregnancy terminated at 24-weeks due to premature rupture of the membrane and *Streptomyces agalactiae* infection (Trounson & Mohr, 1983). A year later, Zeilmaker et al. (1984) described the first live birth after the transfer of cryopreserved human embryos. More reports of live births of cryopreserved human embryos were reported in the following year (Cohen et al., 1985; Downing et al., 1985) and

With the extensive characterization of embryo-cryoprotectant interactions, the feasibility of embryo cryopreservation by vitrification was demonstrated by Rall and Fahy (1985). The next challenges were to further lower the concentration of the cryoprotecting agent to be used and to increase the cooling rate. Increasing the cooling rate not only guarantees the absence of ice formation but also allows a further decrease in the amount of cryoprotecting agents being used, thus minimizing the potential toxic effects on the embryos. It can be illustrated by a recent report describing the use of a highly conductive micro-capillary to increase the cooling rate up to about 4,000C per second. The cryoprotectant propylene glycol concentration can be reduced to 1.5M, compared to the normally used 4M or above (Lee et al., 2010). Over the past decade, a variety of holding devices have been developed to allow fast transmission of heat (thus a high cooling rate) from the sample to the coolants (reviewed in detail in Tsang and Chow, 2010). Some researchers have made use of containers with thinner walls, such as pulled-straw (Vajta et al., 1998). Others have made use of an open property of the devices to hold the sample on a surface to allow direct heat transfer between the samples and the coolant. These include the electronic microscopic metal grid (Martino et al., 1996), cryoloop (Lane et al., 1999), nylon mesh (Matsumoto et al., 2001), hemi-straw (Vanderzwalmen et al., 2003), cryotop (Kuwayama et al., 2005), vitrification spatula (Tsang & Chow, 2009) and plastic blade (Sugiyama et al., 2010). Using most of these open-systems, cryo-survival rates of above 80% are usually obtained. The remaining challenge is to select the right tool by considering the microbial surveillance requirement (a closed-storage system versus an open-storage system), the convenience factor and economic considerations in a routine

**2.5 The success of cryopreservation on mammalian embryos** 

Pogorelov, 2007).

in the years after that.

facility operation.

embryos. When the mouse morulae were stored in 1.5M ethylene glycol or glycerol for 6 hours, the majority (>75%) of the embryos retained the capacity to develop into expanded blastocysts (Kasai et al., 1981). To minimize the toxic effect of the cryoprotecting solution to the embryos while retaining the cryoprotecting function, a mixture of two or more cryoprotecting agents could be used to decrease the relative concentration of each chemical. Macromolecules such as polyethylene glycol, ficoll and polyvinylpyrrolidone, which increase the viscosity of a solution, thus slowing down water molecules associating to form ice crystals when cooling, can also lower the concentration of cryoprotecting agents to be used in vitrification.
