**2. Historical perspectives**

Since the discovery of the tissue preserving effect of low temperatures, it has been an aspiration to maintain the vitality of human tissues by freezing. Soon after early attempts at tissue cryopreservation had failed, the main hurdle in achieving this goal became apparent; water crystallizes upon freezing and the sharp edges of the crystals disrupt cell membranes and destroy the cells. From that point onward the history of the study of cryopreservation is a description of the relentless attempts to prevent intracellular crystallization at subzero temperatures. This journey has been even more elusive for those trying to cryopreserve oocytes.

It has been known for many centuries that subzero temperatures can preserve tissue, mostly following the accidental discoveries of intact ancient animals frozen in ice for many years. However, from studying patients inflicted by frostbite it was clear that freezing may also cause tissue destruction [11].

#### **2.1. Early days**

Additional application has unfolded to address needs associated with convenience and transport for the purposes of using gestational carriers, family planning, travel, and using donor oocytes/embryos in support of non-fertile couples in conception and familial continuity.

Advances in cryopreservation are paralleled and even rooted in key developments in assisted reproductive technologies (ARTs). Historically, advanced culture techniques, complex culture media, and supplements specific to the support of late stage embryonic development attempted to mimic in vivo conditions [3]. Together with carefully controlled culture environment,

In addition to these supportive measures, newer technologies including time lapse morphological assessment allow for the development of observation-based algorithms as prognostic indicators of embryonic competence. Collectively these factors form the paradigm in increased opportunities for cryopreservation and subsequent improved selection criteria for single

The goal of healthy ART outcome should not be clouded by commercial success rates, and while not a mandate, single embryo transfer (SET) is now widely accepted as the default position in good prognosis patients. According to a meta-analysis of randomized controlled trials, SET versus double embryo transfer (DET) in a fresh In Vitro Fertilization (IVF) treatment cycle resulted in a lower pregnancy rate, lower rate of multiple births and preterm birth, and better odds of delivering a term singleton live birth. The reported SET versus DET pregnancy rate disparity is virtually eliminated with an additional frozen SET cycle [6]. However, the immediate consequence is that in order to achieve similar results, the patient may require/need

Approximately 30–50% of embryos make it to a blastocyst stage. The average number of embryos frozen per IVF cycle is age dependent: women of age >35 have fewer than two embryos frozen, while younger women responding better to ovarian stimulation and producing more eggs, result in a higher likelihood of having excess embryos available for freezing [7]. The Department of Health and Human Services estimates that in 2015 more than 600K frozen embryos were stored nationwide in the USA [8]. Figures for the year 2012 released by the Human Fertilization and Embryo Authority (UK) report that of the >3.5 million embryos created since 1991, 840K (24%) were cryopreserved for clinical use. In Canada, it is estimated that >60K frozen embryos are in storage [9]. The current trend of freezing all the embryos with no fresh embryo transfer [10] in IVF treatment would suggest these numbers will likely grow much faster. Despite this uncertainty, these values underscore the importance of cryopreser-

Since the discovery of the tissue preserving effect of low temperatures, it has been an aspiration to maintain the vitality of human tissues by freezing. Soon after early attempts at tissue cryopreservation had failed, the main hurdle in achieving this goal became apparent; water

blastulation rates have increased exponentially from decades prior [4, 5].

embryo transfer. In fact, this is the current trend.

multiple cycles of embryo transfers.

vation technologies.

140 Cryopreservation in Eukaryotes

**2. Historical perspectives**

Interestingly, the cells that were chosen for the early studies on the effects of freezing and thawing on cell viability were gametes. Spermatozoa were chosen due to their availability, small size, and their motility, which was a simple marker of viability. Oocytes were chosen since their size is large enough to allow for simple morphological evaluation.

Spallanzani, in 1776, was the first to study the effect of subzero temperatures on stallion semen and silkworm eggs [12]. He discovered that when thawed the sperm regained its motility. This was the first report of a successful sperm freeze thaw. However, it was not until 1938 that Jahnel, while searching for a remedy for syphilis, found that sperm cooled to −79°C for 40 days regained some of its motility upon thaw and reinvigorated efforts to devise an efficient freezing method [13].

Very early in the study of cryopreservation two opposing schools of thought had been developed in parallel; slow freezing with gradual desiccation of the cell and ultra-rapid freezing of small volumes also known as vitrification.

#### **2.2. Vitrification**

The term vitrification originates from the Latin word "vitreum" (glass) that describes the transformation of a substance into a non-crystalline amorphous solid. The process commonly involves rapid cooling of a liquid so that it passes through the glass transition to form a vitrified solid.

The French, Joseph Luis Gay-Lusac, in 1804 ascended in a hot air balloon and noticed that the water drops (size around 8–10 μm) in the clouds are not frozen despite the sub-zero temperatures (−5°C) [14]. He later went on to find that water can be subcooled to −12°C when contained in small tubes [15]. In 1858, Albert J.R. Mousson, a Swiss physicist, had found that the smaller the sprayed water droplets (diameter < 0.5 mm), the longer they can stay subcooled [16]. The liquid state of the water droplets in subzero temperatures is attributed to rapid cooling forming a non-crystalized solid. Luyet in his book coined the term "crystallization zone", which relates to the range of temperatures in which crystals form. He concluded that in order to avoid crystallization one must traverse this zone faster than the time it takes to form crystals [14]. It later became apparent that the small volumes are essential for achieving the high cooling velocity since it is proportional to the ratio of surface area to volume. It was also noticed that different solutions of similar volumes cool at different rates. The concentration of the solutes was shown to affect the "thermal mass (heat capacity)", which represents the ability of a substance to store thermal energy and is inversely proportional to the velocity of cooling. Pure water has a very high heat capacity and therefore is almost impossible to cool fast enough to exceed crystal growth unless very small volumes are used [17]. Walton and Judd measured the velocity of ice crystal growth in undercooled water and found it to be 65 mm/s, thereby providing the basis for calculation of the necessary speed of cooling to avoid crystallization [18]. Fahy and Rall found that in order to vitrify pure water a cooling rate of 100 × 106oC/min is necessary. Since such cooling velocities are not feasible, to achieve vitrification one needs to increase to solute concentration (cryoprotectants) and reduce the solutions volume. This is the current basis for clinically applied vitrification [19].

The work done by Luyet and Hodapp with colloid solutions (gelatin or agar) had led to the first successful vitrification of sperm [20]. They were able to show that the water content of the solutions was the determining factor on whether vitrification was achievable. With a 50% gelatin solution they were able to vitrify layers of 0.3 mm; however, when using a 10% gelatin solution, they could only vitrify a layer a few microns thick [15]. The drawback of these concentrated solutions was their cell toxicity. Therefore, there is a need to balance the solutions' cooling velocity on one hand and the solutes' cell toxicity on the other. It was not until 1985 that an ice-free cryoprotectant system was developed that could attain vitrification and achieve live birth for vitrified thawed mouse embryos [21–23]. Others were able to achieve high postthaw survival rates with vitrified hamster oocytes, as well as with immature and mature murine oocytes [24–26].

Attempts to simplify the vitrification solution using a high concentration of a single cryoprotectant (dimethyl sulphoxide, DMSO) were initially successful for mouse and hamster oocytes, but later proven to be toxic causing aneuploidy, malformations and a high rate of miscarriage [27–30]. These publications halted further attempts to vitrify oocytes and focused the attention on the alternative, slow freezing.

#### **2.3. Slow freezing**

Parkes et al., in 1945 discovered, accidentally, that the rate of cooling is associated with postthaw survival rate. They found that large containers used for freezing semen, in which, due to the large volume, the rate of cooling is slower, gave the best post-thaw motilities [31]. Hence, opposite to vitrification, slower cooling rates were associated with better cell vitality. The explanation for this observation was the physical principle of osmotic dehydration; as ice crystals formed in the suspending solution, the relative concentration of solutes in the unfrozen fraction of the solution increased and thereby increasing its osmolality. The cells suspended in the solution will respond to the higher osmolality by losing water. Therefore, slower cooling rates are associated with greater cellular dehydration and reduced risk of intracellular ice crystals formation, leading to a better post-thaw viability. Further work by Chang on rabbit ova recognized the importance of cooling rate on the maintenance of viability, the artificial activation of oocytes by rapid cooling and the achievement of litters from embryos stored at 0°C [32, 33].

Mazur was the first to describe cell‐specific optimal cooling rates [34]. He was able to formulate an equation that was based on the rate at which the cells responded to osmotic pressure (hydraulic conductivity) and the effect of temperature on the movement of water across the cell membrane (temperature coefficient of water permeability) and could therefore predict cell‐specific optimal cooling rates. Leibo et al. constructed a graph describing cooling rate against survival rate [35]. He showed over a 1000‐fold difference in the optimal rate of cooling between oocytes (0.3°C/min) and erythrocytes (1000°C/min) due to the oocytes low hydraulic conductivity and high temperature coefficient of water permeability.

In order to guarantee ice crystal formation in the cryo‐solution that will ensure the increase in its osmolality and cell desiccation, a process of ice crystal seeding was developed [36].

Two groups worked in the early 1970s independently on slow freezing of embryos. Both groups had published in 1972 the first survival of murine embryos after slow freezing [1, 2] and live offspring [1]. Both groups used slow freezing and a cryosolution containing 1 mol/l of DMSO. Wilmut and Rowson published in 1973 on the first farm animal (a calf) to be born after a transfer of a frozen thawed embryo [37].

With the advent of clinical use of IVF at the beginning of the 1980s a significant effort was made to optimize human embryo freezing in order to increase the efficiency of IVF by storing excess oocytes and embryos. This came to fruition with the first pregnancies and birth from frozen thawed embryos that were frozen using slow freezing and DMSO [38, 39]. Soon after, these were followed by publications reporting on human live births subsequent to the use of other cryoprotectants such as propanediol and sucrose. These methods proved to be more reliable and more widely adopted [40–42]. The success of human embryo freezing ignited a public debate on the ethics of embryo freezing. These ethical dilemmas prompted research on the possibility of clinical application of oocyte freezing, which was deemed to be more ethically acceptable.

In 1986, Chen reported a twin pregnancy following slow freezing of human oocytes with DMSO [43]. Chen reported high post‐thaw survival, fertilization and development rates of oocytes frozen with this technique, however, attempts to replicate his success by others failed [44–46]. Furthermore, in line with the observation in animals, a high proportion of thawed human oocytes resulted in polyploid embryos [44, 47]. The poor results of oocyte cryopreser‐ vation relative to the success with embryo freezing brought clinical oocyte freezing to a halt.

#### **2.4. Cryoprotectants**

velocity since it is proportional to the ratio of surface area to volume. It was also noticed that different solutions of similar volumes cool at different rates. The concentration of the solutes was shown to affect the "thermal mass (heat capacity)", which represents the ability of a substance to store thermal energy and is inversely proportional to the velocity of cooling. Pure water has a very high heat capacity and therefore is almost impossible to cool fast enough to exceed crystal growth unless very small volumes are used [17]. Walton and Judd measured the velocity of ice crystal growth in undercooled water and found it to be 65 mm/s, thereby providing the basis for calculation of the necessary speed of cooling to avoid crystallization [18]. Fahy and Rall found that in order to vitrify pure water a cooling rate of 100 × 106oC/min is necessary. Since such cooling velocities are not feasible, to achieve vitrification one needs to increase to solute concentration (cryoprotectants) and reduce the solutions volume. This is the

The work done by Luyet and Hodapp with colloid solutions (gelatin or agar) had led to the first successful vitrification of sperm [20]. They were able to show that the water content of the solutions was the determining factor on whether vitrification was achievable. With a 50% gelatin solution they were able to vitrify layers of 0.3 mm; however, when using a 10% gelatin solution, they could only vitrify a layer a few microns thick [15]. The drawback of these concentrated solutions was their cell toxicity. Therefore, there is a need to balance the solutions' cooling velocity on one hand and the solutes' cell toxicity on the other. It was not until 1985 that an ice-free cryoprotectant system was developed that could attain vitrification and achieve live birth for vitrified thawed mouse embryos [21–23]. Others were able to achieve high postthaw survival rates with vitrified hamster oocytes, as well as with immature and mature

Attempts to simplify the vitrification solution using a high concentration of a single cryoprotectant (dimethyl sulphoxide, DMSO) were initially successful for mouse and hamster oocytes, but later proven to be toxic causing aneuploidy, malformations and a high rate of miscarriage [27–30]. These publications halted further attempts to vitrify oocytes and focused the attention

Parkes et al., in 1945 discovered, accidentally, that the rate of cooling is associated with postthaw survival rate. They found that large containers used for freezing semen, in which, due to the large volume, the rate of cooling is slower, gave the best post-thaw motilities [31]. Hence, opposite to vitrification, slower cooling rates were associated with better cell vitality. The explanation for this observation was the physical principle of osmotic dehydration; as ice crystals formed in the suspending solution, the relative concentration of solutes in the unfrozen fraction of the solution increased and thereby increasing its osmolality. The cells suspended in the solution will respond to the higher osmolality by losing water. Therefore, slower cooling rates are associated with greater cellular dehydration and reduced risk of intracellular ice crystals formation, leading to a better post-thaw viability. Further work by Chang on rabbit ova recognized the importance of cooling rate on the maintenance of viabil-

current basis for clinically applied vitrification [19].

murine oocytes [24–26].

142 Cryopreservation in Eukaryotes

**2.3. Slow freezing**

on the alternative, slow freezing.

A cryoprotectant is a substance used to protect biological tissue from freezing damage. Arctic and Antarctic insects, fish and amphibians create cryoprotectants (antifreeze compounds and antifreeze proteins) in their bodies to minimize freezing damage during cold winter periods. Their exact mechanism of action is yet not fully understood. 1949, Polge et al., once again by accident, discovered the cryoprotective effects of glycerol [48]. They found that the glycerol solution protects from crystal formation during freezing by cellular dehydration. This discovery had led to successful semen storage of farm animals in 1953 and human sperm in 1964 [49]. Cryoprotectants are divided into two groups: intracellular (such as DMSO, glycerol and propylene glycol) and extracellular (such as sucrose, polyvinyl pyrrolidone, hydroxyethyl starch and dextran). One of their modes of action is lowering of the freezing point of the solution. Use of an intracellular cryoprotectant such as DMSO will prevent intracellular ice formation, while the seeding drives extracellular crystallization and the resulting increase in the osmolality of the cryosolution leading to cellular dehydration [50]. Cryoprotectants may also protect the cell membrane from the drastic changes occurring during the transition between fluid and solid states. Cryoprotectants may, however, be toxic to the cells, therefore over the years a relentless search for less toxic and efficient cryoprotectants ensued as well as for protocols combining several cryoprotectants in order to reduce individual solute concentration and the associated cell toxicity.

#### **2.5. The return of vitrification**

In 1985, Rall and Fahy were able to successfully vitrify a strew of a relatively large volume (0.25 ml) containing mouse embryos with a mixture of DMSO, acetamide and polyethylene glycol that was snap frozen in liquid nitrogen [22]. Shortly after the publication on the first births from slow-frozen oocytes, the first pregnancy and live birth from vitrified oocytes was published [51]. Developments that led to this breakthrough included the understanding that the length of exposure of the cells to the vitrification solution should be minimized to reduce toxicity [52], as well as replacing DMSO with ethylene glycol and mixtures of several cryoprotectants [53]. These changes brought about successful vitrification of bovine, murine as well as human oocytes with multiple live births [54–56]. These advancements were accompanied by the development of appropriate carriers to facilitate rapid cooling such as open-pulled straws [57], electron microscopy grids [55] and nylon loops [58]. By the end of the 1990s, vitrification was applied to human embryos achieving live births with both blastocyst and cleavage stage embryos [59, 60]. The vitrification of oocytes, despite these developments, was lagging until the introduction of appropriate carriers. The development of Cryotop in Japan was the breakthrough that allowed the adoption of oocyte vitrification into routine clinical practice. It allowed for an extremely rapid cooling rate that was facilitated by a minimal volume and resulted in a very high survival rate and live births [61–63]. A few methodological modifications that were made to the kit simplified its use and supported its wide spread distribution. Two large comparative studies established its lead role in oocyte cryopreservation [64, 65].
