**2. Oocytes**

The cryopreservation of human oocytes constitutes a important step forward in Assisted Reproductive Technology (ART) despite the fact that for more than 2 decades oocyte cryopreservation has long been the focus of unsuccessful efforts to perfect its clinical application. More recently, vitrification as an alternative to traditional slow freezing prootcols has been shown to provide high degrees of success in vitrified metaphase-II

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;

The cryopreservation of human oocytes constitutes a important step forward in Assisted Reproductive Technology (ART) despite the fact that for more than 2 decades oocyte cryopreservation has long been the focus of unsuccessful efforts to perfect its clinical application. More recently, vitrification as an alternative to traditional slow freezing prootcols has been shown to provide high degrees of success in vitrified metaphase-II

2003).

**2. Oocytes** 

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 forward in ART.

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

Vitrification of Oocytes and Embryos 175

2007; Balaban *et al*., 2008). So clearly vitrification appears to have a positive impact on overall embryo utilization. A study on the neonatal outcome of 907 vitrified/warmed cleavage stage embryos found no significant increase in the congenital birth defect rate when compared with pregnancies using fresh cleavage stage embryos (Rama Raju *et al*.,

Vitrification of human blastocysts using different carriers shows survival rates of 70% to 90%, with clinical pregnancy rates of 37% to 53% and implantation rates of 20% to 30% ((Yokota *et al*., 2000, 2001; Reed *et al*., 2002; Mukaida *et al*., 2001; 2003; Hiraoka *et al*., 2004; Vanderzwalmen *et al*., 2002; 2003; Huang *et al.,* 2005; Liebermann & Tucker, 2006;

Activation of the embryonic genome occurs after the 8-cell stage (3 days postoocyte retrieval) is reached (Braude *et al.,* 1988). If the activation does not occur, the embryo will not survive further. Therefore, the improvement of human IVF outcomes requires identification of embryos that will progress beyond the 8-cell stage. Blastocyst culture (5 days postoocyte retrieval) allows for the transfer of embryos that clearly have an activated embryonic genome. This requires that the elimination of embryos in extended culture from day 3 to day 5 should depend solely on their inherited survival potential and not be a consequence of an adverse effect exerted by the sequential media used for culture beyond day 3. Additional advantages in cryopreserving at the blastocyst stage are: 1) At this stage a lower numbers of embryos can be transferred in fresh cycles, resulting in less high order multiple pregnancies, 2) The same is true for cryopreserved blastocysts showing higher pregnancy rates and implantation per thawed embryo transferred, 3) Approximately 120 hours (day five) into development the healthy human embryo should be at the blastocyst stage comprised of some 50 to 150 cells, of which about 20 to 30% make up the inner cell mass (ICM), the remainder making up the trophectoderm (TE), 4) the higher cell number allows better compensation for cryo-injuries, which results in greater viability and faster recovery, 5) the cytoplasmatic volume of the cells is lower, thus the surface-volume ratio is higher, and that in turn makes the penetration of the cryoprotectant faster, and 6) on average fewer embryos per patient were frozen-stored, but

Both natural and hormone replacement cycles seem to provide comparable levels of receptivity in naturally cycling women, though they differ in level of convenience. Regardless of the day of cryopreservation of the embryo (whether day 5, 6 or 7), at thawing/warming blastocysts should be treated as if they had been frozen on the fifth day of development. Vitrification of blastocysts has been undertaken utilizing an "open system" (Cryotop; Kitazato Bio Pharma Co. Ltd., Fuji-shi, Japan), and since 2007 on a "closed system" (HSV [High Security Vitrification Kit]; CryoBio System, L'Aigle, France) after a two-step loading with cryoprotectant agents at 24°C. Briefly, blastocysts were placed in equilibration solution, which is the base medium (Hepes-buffered HTF with 20% Serum Supplement Substition (SSS) containing 7.5% (v/v) ethylene glycol (EG) and 7.5% (v/v) dimethyl sulfoxide (DMSO). After 5-7 min, the blastocysts were washed quickly in vitrification solution, which is the base medium containing 15% (v/v) DMSO, 15% (v/v) EG, and 0.5M sucrose, for 45-60sec and transferred onto the Cryotop or HSV using a micropipette. Immediately after the loading of

2009).

**5. Blastocyst stage** 

Liebermann, 2009, 2011).

**6. The advantage of blastocyst cryopreservation** 

each one when thawed has a greater potential for implantation.

or vitrification technology (Chen *et al*., 2004; Bianchi *et al.,* 2005; Larman *et al*., 2007). The scientific literature on oocyte cryopreservation grows daily it seems. Most reports focus on clinical pregnancy rates (Boldt *et al.*, 2003; Boldt *et al*., 2006), and as such while this data is helpful to increase our confidence in the technology, it does little to research new directions for oocyte cryopreservation.
