**5.4 Ovarian tissue cryopreservation**

196 Current Frontiers in Cryobiology

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

Slow freezing reports first began 13 years before literature on vitrification emerged. Both of these methods have demonstrated increasing efficiency over time, with continually improving live birth and ongoing pregnancy rates per transfer (Oktay et al., 2006). Although there is a lag in the data for vitrification outcomes, the number of babies born after vitrification is approaching that of slow freeze methods for oocyte cryopreservation (Noyes et al., 2009).

In a recent meta-analysis of randomized controlled trials (RCTs) comparing these two methods, vitrification was found to have better post-thawing survival rates for cleavage stage embryos (odds ratio [OR] 6.35, 95% confidence interval [CI] 1.14, 35.26) and for blastocysts (OR 4.09, 95% CI 2.45, 6.84) (Kolibianakis et al., 2009). A significantly higher number of embryos cryopreserved in the cleavage stage developed into blastocysts following vitrification. Clinical pregnancy rates, however, demonstrated no significant difference between slow freeze and vitrification protocols. This meta-analysis was undertaken to evaluate and summarize the available evidence for cryopreservation of human embryos, not oocytes. Additionally, the data amassed for this meta-analysis came from only 6 RCTs, only one of which commented on live birth rates. The authors, in light of this limited data, call for well-designed randomized controlled trials to further study differences between and advantages or disadvantages of these cryopreservation techniques. Recently, a prospective randomized comparison of slow freeze versus vitrification for mature human oocyte cryopreservation was performed in Brazil (Smith et al., 2010). In this study, women with supernumerary oocytes retrieved (more than nine) were consented and randomized to either slow freeze or vitrification of these supernumerary oocytes. Demographic characteristics between the two groups of women were similar, including patient age, baseline laboratory values, and number of oocytes collected. Semen parameters were also similar between the groups and all oocytes were inseminated by intracytoplasmic sperm injection (ICSI). Oocyte survival after thawing was significantly higher in those having undergone vitrification. Additionally, a higher percentage of vitrified oocytes were fertilized (77% vs. 67% of slow freeze oocytes; p<0.03) and more of these zygotes underwent cleavage from day 1 to day 2 (84% vs. 71%, respectively; p<0.01). Perhaps the most important outcome for any assisted reproductive technology, however, is the rate of pregnancy. Biochemical and clinical pregnancy rates per thaw cycle were significantly higher in the vitrification group compared to the slow freeze group (46% vs. 17% and 38% vs. 13%, respectively; p<0.01 and p<0.02) (Table 1). Additionally, the two groups had similar rates of spontaneous abortion following embryo transfer. Perinatal outcomes were not evaluated by these authors. From case reports evaluating live births following oocyte cryopreservation, the average gestational age at delivery for slow freeze was 36.9 weeks, compared to 39 weeks' gestational age at delivery after vitrification (Noyes et al., 2009). This

**5.3 Slow freeze versus vitrification for oocyte cryopreservation** 

protocols worldwide.

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 preliminary studies of this technique in humans are ongoing.
