**5.2 Vitrification**

194 Current Frontiers in Cryobiology

Fig. 4. Liquid nitrogen Dewar vessel, used for freezing and storing cryopreserved oocytes.

Thawing of embryos occurs at a rate of 4-25°C/minute. A relatively rapid temperature transition is needed to prevent recrystallization of water in the cell. Nonpermeating cryoprotectants, such as sucrose or other disaccharides, are utilized to help prevent osmotic shock during thawing, as high levels of permeating cryoprotectants are present intracellularly (Jain & Paulson, 2006). This helps achieve the third goal of cryopreservation

Fig. 5. Oocyte volume changes during freezing and thawing with slow-freeze protocol. (a) De-cumulated oocyte. (b)-(d) Oocyte undergoing freezing stages. (e)-(h) Oocyte undergoing thawing stages of slow-freeze protocol. (i) Oocyte after cryopreservation. *Images courtesy of* 

Slow freezing has limitations. First, this method is expensive and requires programmable freezing equipment that must be purchased by the IVF laboratory. This poses a substantial cost to many centers. Additionally, this method is extremely time-consuming, taking embryologists at least 90 minutes to successfully cryopreserve oocytes. Despite these drawbacks, this technique is still the most widely used and has the most literature available

*Courtesy of Reproductive Medicine Associates of New York, LLP.*

(Figure 5).

*Herrero et al., 2011.*

about tested protocols and outcomes.

Vitrification, which literally means "the act or process of converting into glass," is an alternative method to slow freeze cryopreservation. This technique uses high concentrations of cryoprotectants and a rapid cooling rate to convert liquid intracellular water directly into a glassy, vitrified state.

This method of oocyte cryopreservation was first described in humans in 1986 (Fahy et al., 1986), with the first live birth after vitrification occurring in 1999 (Kuleshova et al., 1999). Oocytes are directly exposed to liquid nitrogen which practically eliminates ice crystal formation, due to the rapid cooling rate, around 20,000°C/minute. This minimizes the risk of physical damage to the oocyte from shearing of organelles or increased intracellular pressure. The oocyte is converted rapidly into an amorphous state (Figure 6). A plastic straw containing cryoprotectants and the oocyte is directly plunged into the liquid nitrogen. While initial studies of these "cryoprotectant cocktails" found them to be incredibly toxic, extensive evaluation has indicated that the combination with minimal toxicity is a combination of a high concentration of ethylene glycol (5.5M) and sucrose (1.0M) (Ali & Shelton, 1993). Further modification of the cryoprotectant protocols has decreased the concentration of ethylene glycol to 5.0M (Kuwayama et al., 2005a). Other groups have had high success with vitrification using 2.5M ethylene glycol, 0.5M sucrose and 2.1M DMSO (Gook & Edgar, 2007). These changes in methodology have led to continued improvement in vitrification outcomes, including improved oocyte post-thaw survival, fertilization rates, and pregnancy outcomes.

Some studies have reported the potential for disease transmission, especially viral illnesses, through direct contact with contaminated liquid nitrogen using open-carrier systems for vitrification (Bielanski et al., 2000, 2003), in which there is direct contact between the cryoprotectant media and liquid nitrogen. Closed-carrier system vitrification, in which oocytes are not in direct contact with liquid nitrogen, have been shown to have similar blastocyst survival, pregnancy rates, and live birth rates as open-carrier systems (Kuwayama et al., 2005b), without the theoretical risk of horizontal viral transmission (Bielanski et al., 2000). Closed-carrier systems cool at a slower rate (around 200°C/minute) but have similar rates of post-thaw embryo development, and may demonstrate similar efficacy (Jain & Paulson, 2006).

Fig. 6. Oocyte volume changes during vitrification and thaw. (a) De-cumulated oocyte before cryopreservation. (b)-(e) Oocyte undergoing vitrification. (f)-(g) Oocyte during warming phase of vitrification protocol. (h) Oocyte after cryopreservation. *Images courtesy of Herrero et al., 2011.*

Oocyte Cryopreservation for the Elective Preservation of Reproductive Potential 197

data suggests improved efficiency and a clinical advantage of oocyte vitrification for elective fertility preservation. Reproductive endocrinologists should be aware of this recent data when considering the implementation of oocyte cryopreservation into their clinical practice

Number of cycles 30 48 NA Oocytes thawed 238 349 NA

(mean ± SE) 7.9 ± 0.5 7.3 ± 0.3 NS Immediate post-thaw survival (%) 159/238 (67%) 281/349 (81%) <0.001 4-hour post-thaw survival (%) 155/238 (65%) 260/349 (75%) <0.01 Fertilization (%) 104/155 (67%) 200/260 (77%) <0.03 Cleavage from Day 1 to Day 2 74/104 (71%) 168/200 (84%) <0.01 Biochemical pregnancies per cycle (%) 5/30 (17%) 22/48 (46%) <0.01 Clinical pregnancies per cycle (%) 4/30 (13%) 18/48 (38%) <0.02

thawed (%) 4/238 (1.7%) 18/349 (5.2%) <0.03

Table 1. Oocyte survival and function following slow-freeze or vitrification for

preliminary studies of this technique in humans are ongoing.

**6. Is oocyte cryopreservation "experimental?"** 

NA = not applicable; NS = not significant. SE = standard error. *Adapted and reproduced with permission* 

Ovarian tissue cryopreservation (oophoropexy) and transplantation can also be considered for female children who will survive childhood cancers but have potentially sterilizing chemotherapy and/or radiation. Ovarian tissue cryopreservation was first described using a sheep model (Gosden et al., 1994). After oophorectomy, strips of ovarian cortex were cryopreserved using a slow-freeze protocol with DMSO. Ovarian tissue was cooled to -140°C before being plunged into liquid nitrogen and stored for 3 weeks. Tissue was thawed and grafted back into the same animal after removal of the remaining ovary, after which animals were returned to the pasture and normal husbandry conditions. This protocol has been followed in human studies of ovarian tissue cryopreservation (Donnez et al., 2011). After thawing, decortication of the patient's atrophic ovaries occurs before transplantation of cryopreserved tissue (Donnez & Dolmans, 2009). Return of ovarian function appears to occur between 3.5-6.5 months after transplantation, as evidenced by an increase in E2 and decreased basal FSH levels. In a small case study, the duration of ovarian activity after transplantation appears to be about 2-5 years (Donnez et al., 2011). Heterotopic transplantation of fresh ovarian tissue to the forearm has been successful in 2 cancer patients with return of ovarian function (Oktay et al., 2001). Forearm heterotopic transplantation of cryopreserved ovarian tissue has been successful in primates (Schnorr et al., 2002), and

The American Society for Reproductive Medicine (ASRM) published a committee opinion in 2008 which stated that "the experimental nature of oocyte cryopreservation suggests

Slow-Freeze Vitrification **p-value** 

and when counseling patients seeking fertility preservation.

Oocytes thawed per treatment

Clinical pregnancy per oocytes

**5.4 Ovarian tissue cryopreservation** 

*from Smith et al., 2010.* 

cryopreservation

Vitrification has its own drawbacks. This method, too, is very expensive for IVF centers to implement in terms of costs of freezing and thawing media. Additionally, this technique has a high learning curve, which must be considered. On the other hand, vitrification does not take as much time as slow freezing due to the rapid cooling procedure and does not require expensive embryology lab equipment. As vitrification continues to be used and data accrued about the success of this method, it is likely to alter the choice of cryopreservation protocols worldwide.
