**2. The history of human oocyte cryopreservation**

The first human pregnancy after cryopreservation and thaw of an 8-cell embryo occurred in 1983 (Trounson & Mohr, 1983). The first reports of mature human oocyte cryopreservation also occurred in the 1980s, with the first live birth after oocyte cryopreservation using a slow-freeze method being reported in 1986 (Chen, 1986). In this early case report, mature oocytes were cryopreserved using a slow-freeze, rapid-thaw method and DMSO was used as a cryopreservant (Figure 1). Chen achieved an egg survival rate of 80%, with an 83% fertilization rate in the thawed surviving oocyte population. This cohort of 40 oocytes ultimately resulted in one viable twin gestation. Despite this promising early work, significant advances in the field of oocyte cryopreservation did not occur until decades later.

Since the first reports of successful egg freezing, there have been many changes and advances in the protocols and techniques utilized to maximize post-thaw success rates. Alterations in cryopreservants and media have been tested and improved in the past three decades. Replacement of sodium with choline in the cryopreservation media has been shown to improve cryopreservation outcomes (Quintans et al., 2002; Stachecki et al., 1998). Alternative strategies, including trehalose injection have also been introduced in attempts to improve survival of cryopreserved oocytes (Eroglu et al., 2000; Jain & Paulson, 2006).

Oocyte Cryopreservation for the Elective Preservation of Reproductive Potential 187

ovarian tissue. Minimal residual disease (MRD) in cryopreserved ovarian tissue of patients with leukemia has been demonstrated in humans, with the prevalence of MRD in chronic myeloid leukemia and acute lymphoblastic leukemia as high as 33% and 70%, respectively (Dolmans et al., 2010). Given these risks, strategies to effectively test cryopreserved ovarian tissue for evidence of MRD are required before this technique can be widely utilized in clinical practice. In light of these uncertainties, ovarian tissue cryopreservation is still in its infancy with regard to fertility preservation. Additionally, immature oocyte cryopreservation is being studied but is also in early experimental stages, according to the American Society for Reproductive Medicine (ASRM) (ASRM Practice Committee, 2008). This potentially new frontier is still being studied in primate models and preliminary human studies are

These changes in cryopreservants, rates of freezing, fertilization, and protocols for cryopreservation have improved outcomes. As research funding, referring provider knowledge, and patient interest in oocyte cryopreservation increase, we can anticipate

Human aging has been well-studied and is a known contributor to the decline in fertility experienced by women. Female fecundity, or the ability to produce offspring, declines with advancing age. This is partially due to decreased numbers of oogonia, which have a steady rate of atresia from birth, with a more rapid decline around the age of 37.5 years. Numbers of oogonia, or primordial fetal oocytes, are maximal at 20 weeks' gestation, totaling between six and seven million. At birth, this number has already declined to one to two million; a mere 400,000 oocytes remain at the beginning of puberty. While this number still seems rather high, only around 500 of these oocytes are destined for maturation and ovulation. The remainder will be lost through a highly controlled system of follicular atresia and apoptosis (Williams Gynecology, 2008 Ed.), until around 1000 oocytes remain at the time of menopause (Figure 2). Since women now live longer, a larger portion of their lives are spent in reproductive senescence, and the need for reproductive assistance due to challenges

Recent research has suggested that there may be a population of oogonial stem cells, similar to that seen in males for lifelong spermatocyte production. Several studies have pointed toward the presence of mitotically-active germline stem cells in the mammalian ovary (Johnson et al., 2004; Pacchiarotti et al., 2010; Parte et al., 2011; Zou et al., 2009). Many groups have conducted experiments which have isolated stem cells capable of sustaining oocyte and follicle production *in vitro*. While these results are controversial and disputed by some (Byskov et al., 2005), the potential for regeneration of oocytes and follicular development throughout the

Mathematical models have been developed in order to generate prediction rules for numbers of remaining oocytes and reproductive capacity. Oocyte atresia appears to follow a biexponential pattern, with a more rapid decline in oocyte number occurring after a critical number of 25,000 follicles remain around the age of 37.5 years (Faddy et al., 1992). According to this model, around 1000 follicles remain at the age of 51, which corresponds to the median age of menopause in the general population. Other authors have studied histological samples to identify the rate of recruitment of non-growing follicles (NGF) in human ovaries from

associated with diminished ovarian reserve has increased (Faddy et al., 1992).

female life span is an exciting and promising future area in assisted reproduction.

continued advancements in the field of fertility preservation.

**3. The significance of human aging** 

ongoing.

*Courtesy of Reproductive Medicine Associates of New York, LLP.* Fig. 1. Mature human oocyte.

The introduction of vitrification led to the first live birth after this technique in 1999 (Kuleshova et al., 1999). Vitrification of embryos has been shown to be reliable (Kolibianakis et al., 2009) and vitrification is now routinely applied to oocytes. The efficiency of both oocyte cryopreservation methods has been studied and shows similar trends in improvement. While more data exists for slow-freeze cryopreservation, due solely to the number of years that this method has been available, vitrification data is equally promising. In a 2006 meta-analysis, Oktay et al. demonstrated live birth rates increasing after slowfreezing from 21.6% per transfer (from 1996 to 2004) to 32.4% (from 2002 to 2004). The vitrification data showed a similar trend, with a live birth rate of 29.4% before 2005 and 39% after 2005 (Oktay et al., 2006). Through the use of intracytoplasmic sperm injection (ICSI), many of the concerns about zona pellucida hardening from cryopreservation were bypassed. This ability to augment fertilization of thawed oocytes altered the outlook on oocyte cryopreservation and made this a more viable option for fertility preservation.

More recently, experimentation with cryopreservation of ovarian tissue through orthotopic or heterotopic transplantation has been attempted. The first child born after ovarian tissue cryopreservation was documented in 2004, to a woman who had a history of chemotherapy and radiation treatment for lymphoma (Donnez et al., 2004). Due to fewer studies on optimal cryopreservation protocols and methods of tissue selection, this technique has been considered more experimental than oocyte cryopreservation. In addition, optimal strategies for enhancing graft revascularization are limited in the literature and ideal tissue size has not been established. In a case series of 13 patients who underwent ovarian tissue cryopreservation due to various diseases requiring chemotherapy, large strips (8-10mm x 5mm) and small cubes (2mm x 2mm) of ovarian tissue were both effective in restoring ovarian function (Donnez et al., 2011). However, due to small numbers of human patients having undergone this procedure and a lack of standardized protocols, these outcomes are difficult to interpret. Furthermore, appropriate candidate selection for ovarian tissue cryopreservation has note been defined. Due to waning primordial follicle counts as women age, it has been suggested that ovarian tissue cryopreservation should be limited, at the very least, to women <40 years of age (Oktay, 2002). Some concern also exists about the risk of ovarian metastasis and the reintroduction of malignant cells upon transplantation of thawed ovarian tissue. Minimal residual disease (MRD) in cryopreserved ovarian tissue of patients with leukemia has been demonstrated in humans, with the prevalence of MRD in chronic myeloid leukemia and acute lymphoblastic leukemia as high as 33% and 70%, respectively (Dolmans et al., 2010). Given these risks, strategies to effectively test cryopreserved ovarian tissue for evidence of MRD are required before this technique can be widely utilized in clinical practice. In light of these uncertainties, ovarian tissue cryopreservation is still in its infancy with regard to fertility preservation. Additionally, immature oocyte cryopreservation is being studied but is also in early experimental stages, according to the American Society for Reproductive Medicine (ASRM) (ASRM Practice Committee, 2008). This potentially new frontier is still being studied in primate models and preliminary human studies are ongoing.

These changes in cryopreservants, rates of freezing, fertilization, and protocols for cryopreservation have improved outcomes. As research funding, referring provider knowledge, and patient interest in oocyte cryopreservation increase, we can anticipate continued advancements in the field of fertility preservation.
