**5.1 Slow freeze**

192 Current Frontiers in Cryobiology

third party reproduction (Oktay et al., 2010). Improvement in coordination of care, costs, and the ability to quarantine oocytes for infectious disease testing are benefits of oocyte

Fertility preservation for cancer patients undergoing potentially sterilizing chemotherapy and radiation has been a widely accepted application of oocyte cryopreservation. Management of all of the gynecologic cancers has the potential to affect ovarian reserve. Cervical cancer often requires pelvic radiation and endometrial cancer is frequently treated with hysterectomy and bilateral salpingoophorectomy. Therapy for breast cancer, the most common cancer in women in the United States, commonly utilizes cyclophosphamide, which has well-known ovary-toxic effects and leads to premature ovarian failure (Oktay & Sönmezer, 2007). Ovarian stimulation is necessary for both oocyte and embryo cryopreservation for these patients; stimulation protocols have been developed to avoid excessive estrogen exposure in women with estrogen-responsive cancers. For patients who do not need to immediately initiate chemotherapy (or other therapies that may affect the ovary), cryopreservation is a viable option for fertility preservation. In a retrospective data analysis of a NYC infertility clinic from 2005-2007, women presenting for pre-cancer treatment oocyte cryopreservation cycles were evaluated. The average time between initial consultation and completion of the cryopreservation cycle was 37.2 ± 22.5 days, and a mean number of 17.8 oocytes were retrieved across the 4 patients studied (Barritt et al., 2008). Early referral to a fertility center is vital, as patients will require 2 weeks of stimulation after menses in order to retrieve oocytes for cryopreservation. Many oncologists are supportive of their patients' desire to preserve fertility, even in light of the potential delay of chemotherapy and need for gonadotropin stimulation. Women who require immediate initiation of chemotherapy or pediatric cancer patients may benefit from ovarian tissue cryopreservation, though studies of this technique are still quite small and this strategy has

Reproductive endocrinologists approaching the patient interested in elective fertility preservation need to recognize the demographic shifts and societal attitudes toward oocyte cryopreservation. The wide variety of applications of oocyte cryopreservation, including delayed childbearing, ethical opposition to embryo cryopreservation, improvement in third party oocyte donation and fertility preservation for cancer patients, all highlight the

Oocyte cryopreservation is a delicate and complex process. Mammalian cells are generally stored at a temperature of -196°C, at which no biological activity takes place. Cryopreservation must transform human oocytes from a biologically active system at 37°C to an inert structure at -196°C; oocytes are most vulnerable during this temperature transition. Membrane permeability and kinetics vary throughout the developmental cycle of the oocyte; metaphase II oocytes have demonstrated higher post-cryopreservation survival in a mouse model (Gook & Edgar, 2007). There are three main goals of oocyte cryopreservation: avoidance of ice crystal formation, avoidance of solution effect, and avoidance of osmotic shock (Jain & Paulson, 2006). As water freezes and expands to form ice, *crystal formation* causes shearing forces on organelles and increases intracellular pressure. Additionally, as water transitions from its liquid to solid form, any solutes dissolved in liquid water are excluded from the ice. This can lead to very high, if not toxic,

cryopreservation for egg donors.

not yet been widely used (Oktay & Sönmezer, 2007).

advantages of this emerging reproductive technology.

**5. Technical aspects of oocyte cryopreservation** 

Slow freezing has traditionally been the more widely-used technique for mature oocyte cryopreservation. This technique was first described in 1972 by Whittingham et al. after successful slow freeze and post-thaw survival of mouse embryos (Whittingham et al., 1972). The technology was first applied to human embryos in 1983, and resulted in successful postthaw survival and pregnancy after cryopreservation (Trounson & Mohr, 1983), followed by live birth after mature oocyte cryopreservation in 1986 (Chen, 1986).

Slow freeze cryopreservation is achieved using initial low cryoprotectant concentrations to reduce toxicity while the oocyte is still metabolically active (Jain & Paulson, 2006). The temperature is lowered gradually, at rates between 0.3-2°C/minute. This slow rate of cooling allows retardation of the metabolic rate in the oocyte without accumulating toxic levels of cryoprotectant. Propanediol (PROH) and dimethylsulfoxide (DMSO) are permeating cryoprotectants which form hydrogen bonds with intracellular water molecules and prevent ice crystal formation, thus achieving the first goal of successful cryopreservation. Additionally, the presence of PROH dilutes electrolyte concentrations by remaining in solution (due to its low freezing point); this prevents solution effect, which is the second goal of cryopreservation. PROH is preferred to DMSO as a cryoprotectant, as it is thought to be less toxic to the oocyte (Renard & Babinet, 1984). Additionally, using 0.2-0.3M sucrose as a nonpermeating cryoprotectant during oocyte dehydration seems to improve post-thaw survival (Fabbri et al., 2001). "Seeding" the extracellular solution with an ice crystal occurs around -6°C, during which an ice front grows and excludes solutes, thereby increasing their concentration around the oocyte. This ice front can potentially cause intracellular damage to the oocyte if it comes in contact with the cell or can lead to gas bubble formation (Ashwood et al., 1988). The oocyte is maintained at -6°C for 10 to 30 minutes before being further cooled to -32°C. At this point, metabolic activity in the oocyte is extremely low and the cell is plunged into a Dewar vessel of liquid nitrogen to vitrify any remaining cryoprotectant solution (Figure 4). The Dewar vessel is capable of maintaining a near constant temperature for the frozen oocytes during storage.

Oocyte Cryopreservation for the Elective Preservation of Reproductive Potential 195

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

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.*

**5.2 Vitrification** 

a glassy, vitrified state.

Fig. 4. Liquid nitrogen Dewar vessel, used for freezing and storing cryopreserved oocytes. *Courtesy of Reproductive Medicine Associates of New York, LLP.*

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 (Figure 5).

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 Herrero et al., 2011.*

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 about tested protocols and outcomes.
