**1. Introduction**

Currently, controlled ovarian hyperstimulation protocols commonly provide embryos in excess of those needed for fresh transfer. Therefore, techniques have been developed to store these surplus embryos in liquid nitrogen (referred to as cryopreservation) for an indefinite period of time without significant compromise of their quality. Based on data from the Centers for Disease Control and Prevention (CDC) from 2001 to 2004, about 18% of all IVF cycles in the USA used frozen embryos for transfer. In addition, data from the same registry compared live births per transfer using frozen and fresh embryos (25% versus 34% respectively) clearly showing that cryopreservation is an important adjunct to maximize the efficiency of every single patient's oocyte retrieval. The fundamental objectives for successful cryostorage of cells in liquid nitrogen at -196°C can be summarized as follows: **1)** arresting the metabolism reversibly, **2)** maintaining structural and genetic integrity, **3)** achieving acceptable survival rates after thawing, **4)** maintain of developmental competence post thaw and, **5)** the technique has to be reliable and repeatable.

Furthermore, all methods and protocols for cryopreservation should be developed such that ice crystals formation and growth inside the cells or tissues must either be eliminated or massively suppressed. One recent hotly debated topic in the area of reproductive cryobiology is whether slow-cooling or rapid-cooling protocols both satisfy the fundamental cryo-biological principles for reduction of damage by ice crystal formation during cooling and warming, and which approach is better. It is the case nonetheless, that both methods of cryopreservation of biological material include six principal steps: **1)** initial exposure to the cryoprotectant (intracellular water has to be removed by gradual dehydration, **2)** cooling (slow/rapid) to subzero temperatures (-196°C), **3)** storage at low temperature, **4)** thawing/warming by gradual rehydration, **5)** dilution and removal of the cryoprotectant agents and replacement of the cellular and intracellular fluid at precise rate and, **6)** recovery and return to a physiological environment.

Although initially reported in 1985 as a successful cryopreservation approach for mouse embryos, vitrification has taken a backseat in human assisted reproduction. However, the practical advantages of this cryopreservation method have more recently caught the attention of many ART laboratories as a feasible alternative to traditional slow freezing methods. Since 1985 more than 2,100 publications can be found referring to the topic of "vitrification", which is further evidence of the burgeoning growth of interest in this cryopreservation technology. One "drawback" considered by embryologists who are not

Vitrification of Oocytes and Embryos 171

warmed successfully a few years later (Vajta *et al*. 1998). In 1999 and 2000 successful pregnancies and deliveries after vitrification and warming of human oocytes were reported (Kuleshova *et al*., 1999; Yoon *et al*., 2000). Since that time, and because it seems to be that both entities appear to be especially chill-sensitive cells in ART, oocytes and blastocysts seem to receive a potentially significant boost in survival rates by avoiding icecrystallization using vitrification (Walker *et al*., 2004). In general, vitrification solutions are aqueous cryoprotectant solutions that do not freeze when cooled at high cooling rates to very low temperature. Interest in vitrification has clearly risen as evinced by the almost exponential growth of scientific publications about vitrification. Vitrification is very simple, requires no expensive programmable freezing equipment, and relies especially on the placement of the embryo in a very small volume of vitrification medium (refered also as "minimal volume approach") that must be cooled at extreme rates not obtainable in traditional enclosed cryo-storage devices such as straws and vials. The importance of the use of a small volume, also referred to "minimal volume approach" was described and published in 2005 (Kuwayama *et al.,* 2005; Kuwayama, 2007). In general, the rate of cooling/warming and the concentration of the cryoprotectant required to achieve vitrification are in inversely related. In addition, recent publications have shown the dominance of warming rate over cooling rates in the survival of oocytes subjected to a

During vitrification, by using a cooling rate in the range of 2,500 to 30,000ºC/min or greater, water is transformed directly from the liquid phase to a glassy vitrified state. The physical definition of vitrification is the solidification of a solution at low temperature, not by icecrystallization but by extreme elevation in viscosity during cooling (Fahy *et al*., 1984; Fahy 1986). Vitrification of the aqueous solution inside cells can be achieved by increasing the speed of temperature change, and by increasing the concentration of the cryoprotectant used. However, a major potential drawback of vitrification is the use of high concentration of cryoprotectant, and an unintentional negative impact of these cryoprotectants in turn can be their toxicity, which may affect the embryo and subsequent development in utero. It is therefore essential to achieve a fine balance between the speed of cooling and the concentration of the vitrifying cryoprotectants. This is necessitated by the practical limit for the rate of cooling, and the biological limit of tolerance of the cells for the concentration of toxic cryoprotectants being used to achieve the cryopreserved state. It is important to note that recently published papers (Takahashi *et al*., 2005; Liebermann & Tucker, 2006; Liebermann, 2009, 2011) have shown that the use of relatively high concentration of cryoprotectants such as 15% (vol/vol) ethylene glycol (EG) used in an equimolar mixture with dimethyl sulphoxide (DMSO) had no negative effect on the perinatal outcomes from blastocyst transfers following vitrification when compared with those from fresh blastocyst

Vitrification in principle is a simple technology, that is potentially faster to apply, and relatively inexpensive; furthermore, it is becoming clinically established, and is seemingly more reliable and consistent than conventional cryopreservation when carried out

Cryoprotectant agents are essential for the cryopreservation of cells. Basically two groups of cryoprotectants exist: **1)** permeating (*glycerol, ethylene glycol, dimethyl sulphoxide*); and **2)** nonpermeating (*saccharides, protein, polymers*) agents. The essential component of a vitrification

vitrification procedure (Serki & Mazur, 2009; Mazur & Seki, 2011).

appropriately (Tucker *et al*., 2003; Liebermann & Tucker, 2004).

transfers.

familiar with the vitrification technique, is the use of higher concentration of cryoprotectants, which does potentially mean that the vitrification solutions are more toxic than their counterpart solutions used for conventional slow freezing. However, with better understanding of the physical and biological principles of vitrification this has lead to numerous successful clinical applications of this technique within the field of assisted reproduction. As of today, all developmental stages of human embryos cultured in vitro have been successfully vitrified and warmed, with resulting offspring. Today, slow freezing technology still has the longest clinical track record, and greater 'comfort level' amongst embryologists. Nevertheless, vitrification with its increasing clinical application is showing a trend of greater consistency and better outcomes when compared to slow freezing technology. Therefore, when (not if) IVF programs overcome the fear of the 'unknown', and take on the challenge of the short learning curve with vitrification, then at that point vitrification will become the clinical standard for human embryo cryopreservation.

Cryopreservation at low temperature slows or totally prevents unwanted physical and chemical change. The major disadvantage to using low temperature cryostorage is that it can lead to the crystallization of water, and thereby this approach can create new and unwanted physical and chemical events that may injure the cells that are being preserved. Although the results achieved by slow freezing in many cases seem quite successful (Gardner *et al*., 2003; Van den Abbeel *et al*., 2005), ice crystal formation still renders traditional slow-freezing programs generally less consistent in their clinical outcomes. Another downside to the slow freezing approach is the time to complete such freezing procedures for human embryos, which can range from 1.5 to 5hrs. This is due to the fact that the slow rate of cooling attempts to maintain a very delicate balance between multiple factors that may result in cellular damage by ice crystallization and osmotic toxicity. Traditionally slow-freeze embryo cryopreservation has been a positive contributor to cumulative patient pregnancy rates, but ultimately the limitations of current slow-rate freezing methods in ART have become more evident in the shootout with vitrification-based cryostorage.

Vitrification is one of the more exciting developments in ART in recent years that attempts to avoid ice formation altogether during the cooling process by establishing a glassy or vitreous state rather than an ice crystalline state, wherein molecular translational motions are arrested without structural reorganisation of the liquid in which the reproductive cells are suspended. To achieve this glass-like solidification of living cells for cryostorage, high cooling rates in combination with high concentrations of cryoprotectants are used. A primary strategy for vitrifying cells and tissue is to increase the speed of thermal conductivity, while decreasing the concentration of the vitrificants to reduce their potential toxicity. There are two main ways to achieve the vitrification of water inside cells efficiently: **a)** to increase the cooling rate by using special carriers that allow very small volume sizes containing the cells to be very rapidly cooled; and **b)** to find materials with rapid heat transfer. However, one has to take into account that every cell seems to require its own optimal cooling rate, e.g., mature unfertilized oocytes are much more sensitive to chilling injury than any of the cell stages of the pre-implantation embryo.The earliest attempts using vitrification as an ice-free cryopreservation method for embryos were first reported in 1985 (Rall & Fahy, 1985). In 1993 successful vitrification of mouse embryos was demonstrated (Ali & Shelton, 1993). Furthermore, bovine oocytes and cleavage-stages were vitrified and

familiar with the vitrification technique, is the use of higher concentration of cryoprotectants, which does potentially mean that the vitrification solutions are more toxic than their counterpart solutions used for conventional slow freezing. However, with better understanding of the physical and biological principles of vitrification this has lead to numerous successful clinical applications of this technique within the field of assisted reproduction. As of today, all developmental stages of human embryos cultured in vitro have been successfully vitrified and warmed, with resulting offspring. Today, slow freezing technology still has the longest clinical track record, and greater 'comfort level' amongst embryologists. Nevertheless, vitrification with its increasing clinical application is showing a trend of greater consistency and better outcomes when compared to slow freezing technology. Therefore, when (not if) IVF programs overcome the fear of the 'unknown', and take on the challenge of the short learning curve with vitrification, then at that point

vitrification will become the clinical standard for human embryo cryopreservation.

cryostorage.

Cryopreservation at low temperature slows or totally prevents unwanted physical and chemical change. The major disadvantage to using low temperature cryostorage is that it can lead to the crystallization of water, and thereby this approach can create new and unwanted physical and chemical events that may injure the cells that are being preserved. Although the results achieved by slow freezing in many cases seem quite successful (Gardner *et al*., 2003; Van den Abbeel *et al*., 2005), ice crystal formation still renders traditional slow-freezing programs generally less consistent in their clinical outcomes. Another downside to the slow freezing approach is the time to complete such freezing procedures for human embryos, which can range from 1.5 to 5hrs. This is due to the fact that the slow rate of cooling attempts to maintain a very delicate balance between multiple factors that may result in cellular damage by ice crystallization and osmotic toxicity. Traditionally slow-freeze embryo cryopreservation has been a positive contributor to cumulative patient pregnancy rates, but ultimately the limitations of current slow-rate freezing methods in ART have become more evident in the shootout with vitrification-based

Vitrification is one of the more exciting developments in ART in recent years that attempts to avoid ice formation altogether during the cooling process by establishing a glassy or vitreous state rather than an ice crystalline state, wherein molecular translational motions are arrested without structural reorganisation of the liquid in which the reproductive cells are suspended. To achieve this glass-like solidification of living cells for cryostorage, high cooling rates in combination with high concentrations of cryoprotectants are used. A primary strategy for vitrifying cells and tissue is to increase the speed of thermal conductivity, while decreasing the concentration of the vitrificants to reduce their potential toxicity. There are two main ways to achieve the vitrification of water inside cells efficiently: **a)** to increase the cooling rate by using special carriers that allow very small volume sizes containing the cells to be very rapidly cooled; and **b)** to find materials with rapid heat transfer. However, one has to take into account that every cell seems to require its own optimal cooling rate, e.g., mature unfertilized oocytes are much more sensitive to chilling injury than any of the cell stages of the pre-implantation embryo.The earliest attempts using vitrification as an ice-free cryopreservation method for embryos were first reported in 1985 (Rall & Fahy, 1985). In 1993 successful vitrification of mouse embryos was demonstrated (Ali & Shelton, 1993). Furthermore, bovine oocytes and cleavage-stages were vitrified and

warmed successfully a few years later (Vajta *et al*. 1998). In 1999 and 2000 successful pregnancies and deliveries after vitrification and warming of human oocytes were reported (Kuleshova *et al*., 1999; Yoon *et al*., 2000). Since that time, and because it seems to be that both entities appear to be especially chill-sensitive cells in ART, oocytes and blastocysts seem to receive a potentially significant boost in survival rates by avoiding icecrystallization using vitrification (Walker *et al*., 2004). In general, vitrification solutions are aqueous cryoprotectant solutions that do not freeze when cooled at high cooling rates to very low temperature. Interest in vitrification has clearly risen as evinced by the almost exponential growth of scientific publications about vitrification. Vitrification is very simple, requires no expensive programmable freezing equipment, and relies especially on the placement of the embryo in a very small volume of vitrification medium (refered also as "minimal volume approach") that must be cooled at extreme rates not obtainable in traditional enclosed cryo-storage devices such as straws and vials. The importance of the use of a small volume, also referred to "minimal volume approach" was described and published in 2005 (Kuwayama *et al.,* 2005; Kuwayama, 2007). In general, the rate of cooling/warming and the concentration of the cryoprotectant required to achieve vitrification are in inversely related. In addition, recent publications have shown the dominance of warming rate over cooling rates in the survival of oocytes subjected to a vitrification procedure (Serki & Mazur, 2009; Mazur & Seki, 2011).

During vitrification, by using a cooling rate in the range of 2,500 to 30,000ºC/min or greater, water is transformed directly from the liquid phase to a glassy vitrified state. The physical definition of vitrification is the solidification of a solution at low temperature, not by icecrystallization but by extreme elevation in viscosity during cooling (Fahy *et al*., 1984; Fahy 1986). Vitrification of the aqueous solution inside cells can be achieved by increasing the speed of temperature change, and by increasing the concentration of the cryoprotectant used. However, a major potential drawback of vitrification is the use of high concentration of cryoprotectant, and an unintentional negative impact of these cryoprotectants in turn can be their toxicity, which may affect the embryo and subsequent development in utero. It is therefore essential to achieve a fine balance between the speed of cooling and the concentration of the vitrifying cryoprotectants. This is necessitated by the practical limit for the rate of cooling, and the biological limit of tolerance of the cells for the concentration of toxic cryoprotectants being used to achieve the cryopreserved state. It is important to note that recently published papers (Takahashi *et al*., 2005; Liebermann & Tucker, 2006; Liebermann, 2009, 2011) have shown that the use of relatively high concentration of cryoprotectants such as 15% (vol/vol) ethylene glycol (EG) used in an equimolar mixture with dimethyl sulphoxide (DMSO) had no negative effect on the perinatal outcomes from blastocyst transfers following vitrification when compared with those from fresh blastocyst transfers.

Vitrification in principle is a simple technology, that is potentially faster to apply, and relatively inexpensive; furthermore, it is becoming clinically established, and is seemingly more reliable and consistent than conventional cryopreservation when carried out appropriately (Tucker *et al*., 2003; Liebermann & Tucker, 2004).

Cryoprotectant agents are essential for the cryopreservation of cells. Basically two groups of cryoprotectants exist: **1)** permeating (*glycerol, ethylene glycol, dimethyl sulphoxide*); and **2)** nonpermeating (*saccharides, protein, polymers*) agents. The essential component of a vitrification

Vitrification of Oocytes and Embryos 173

human oocytes. Although oocyte cryopreservation historically has low efficiency mainly because of low rates of survival, fertilization, and cleavage, data on 2000 "frozen oocyte" babies born worldwide since 1986 exists. The question arises as to what makes oocytes so unique compared to embryos, besides differences in cell size and membrane permeability? Oocytes have a low volume-to-surface ratio; hence they are less efficient at taking up cryoprotectant and at loosing water. Other differences to be considered are **a)** that the maternal DNA is held suspended in the cytoplasm on the meiotic spindle & not within the protective confines of the nuclear membrane, therefore damage in the DNA and microtubules could explain the limited success of oocytes, **b)** the oocyte is arrested in a state primed for activation, and **c)** the changes in its environment can cause parthenogenetic activation. What are the applications then for oocyte cryopreservation in the US? One application would be to preserve fertility in women with malignant/premalignant conditions who would have to undergo treatment that might negatively impact their future ability to have children (50,000 per year <40 yr old), also in women who may want to delay childbearing ('clock-tickers') because of their careers, partnership status or psychological/ emotional reasons. A very interesting approach is donor oocyte banking, which makes the donor-recipient cycle more convenient by facilitating the "egg donation" and allows quarantining of the oocytes, which provides a unique advantage in economy as well as feasibility. Other applications are if a male is unable to produce a semen sample on the day of egg retrieval and or it could also eliminate ethical/moral questions of producing extra embryos. Overall, oocyte cryostorage offers an opportunity to reduce number of embryos generated per IVF cycle, and therefore lessening the pressure on the patient to increase the number of fresh embryos transferred. In addition, while also reducing embryo cryostorage it has the benefit of helping women "retain ownership" of their ability to be genetic parents at a time of their choosing, a time of greater convenience & health. The live born babies from cryopreserved oocytes have shown no apparent increase in congenital anomalies. Although 13 years later after the first slow-freeze birth, the number of reported babies born as a result of vitrified oocytes is now approaching that of slow-frozen oocytes without any increasing risk in congenital abnormalities (Noyes *et al.,* 2009). Vitrification of oocytes does not appear to increase risks of abnormal imprinting or disturbances in spindle formation or chromosome segregation (Trapphoff *et al.,* 2010). It has the greatest potential for successful oocyte cryopreservation and with its increased clinical application is showing a trend to greater consistency and better outcomes (similar to outcomes between fresh or warmed oocytes). Vitrification of oocytes, when applied to properly screened patients, will be a useful technology in reproductive medicine practice and will constitute a major step

Fortunately to date, no significant increase in abnormalities has been reported from these cryostored oocyte pregnancies (Chian *et al.,* 2009), regardless of the historical concerns that cryopreservation of mature oocytes might disrupt the meiotic spindle and thus increase the potential for aneuploidy in the embryos arising from such eggs. These concerns have mostly been allayed by publications that show no abnormal or stray chromosomes from previously frozen oocytes (Gook & Edgar, 1999), and FISH comparison of embryos from fresh and thawed oocytes show no increase in anomalies (Cobo *et al*., 2001). There also appears to be adequate recovery of the meiotic spindle post-cryopreservation whether using conventional

forward in ART.

solution is the permeating agent. These compounds are hydrophilic non-electrolytes with a strong dehydrating effect. Furthermore, these CPAs are able to depress the "freezing point" of the solution. Regarding the high concentration of cryoprotectant used for vitrification, and in view of the known biological and physiochemical effects of cryoprotectants, it is suggested that the toxicity of these agents is a key limiting factor in cryobiology. Not only does this toxicity prevent the use of fully protective levels of these additives, but it may also be manifested in the form of cryo-injury above and beyond that seen occurring due to classical causes of cell damage (osmotic toxicity and ice formation) during cryopreservation. In spite of this, the permeating CPA should be chosen firstly by their permeating property, and secondly on the basis of their potential toxicity. Because the permeating CPA is responsible for the toxicity (*the key limiting factor in cryobiology*), different cryoprotectants have been tested for their relative toxicity, and the results indicate that ethylene glycol (EG; MW 62.02) is the least toxic followed by glycerol. Additionally, these highly permeating cryoprotectants are also more likely to diffuse out of the cells rapidly and the cells regained their original volume more quickly upon warming, thus preventing osmotic injury. Therefore, the most common and accepted cryoprotectant for vitrification procedures is ethylene glycol (EG). Today EG is more commonly used in an equimolar mixture with DMSO. Often additives are added to the vitrification solution such as disaccharides. Disaccharides, for example sucrose, do not penetrate the cell membrane, but they help to draw out more water from cells by osmosis, and therefore lessen the exposure time of the cells to the toxic effects of the cryoprotectants. The non-permeating sucrose also acts as an osmotic buffer to reduce the osmotic shock that might otherwise result from the dilution of the cryoprotectant after cryostorage. In addition, permeating agents are able to compound with intracellular water and therefore water is very slowly removed from the cell. Hence the critical intracellular salt concentration is reached at a lower temperature. Removal of the cryoprotectant agent during warming can present a very real problem in terms of trying to reduce toxicity to the cells. Firstly, because of the toxicity of the vitrification solutions, quick dilution of them after warming is necessary; and secondly, during dilution water permeates more rapidly in to the cell than the cryoprotective additive diffuses out. As a consequence of the excess water inflow the cells are threatened by injury from osmotic swelling. In this situation the non-permeating sucrose acts as an osmotic buffer to reduce the osmotic shock. During warming using a high extracellular concentration of sucrose (e.g., 1.0M) counterbalances the high concentration of the cryoprotectant agents in the cell, as it reduces the difference in osmolarity between the intra- and extracellular compartments. The high sucrose concentration cannot totally prevent the cell from swelling, but it can reduce the speed and magnitude of swelling (Liebermann and Tucker, 2002; Liebermann *et al*., 2002a; 2003).
