**4. Changing demographics of reproductive-aged women**

In the United States, there has been a notable shift in the demographics of reproductiveaged women. Many women are delaying childbearing in the setting of career pursuits and shifting societal expectations of gender roles. An analysis of Danish fertility rates from 1980- 2001 showed an increase in the mean age of childbearing of 3 years over the 21 year period (Hvidtfeldt et al., 2010). There appears to be a global shift in reproduction leading to delayed childbearing and increased maternal age.

Reasons for delaying childbearing are multifaceted and complex. Perceived career threats are a very real and prevalent issue in young women of reproductive age (Willett et al., 2010). For women in professional programs, fear about extension of training, loss of future career

In our NYC-based infertility clinic, women presenting for new oocyte cryopreservation consultations were retrospectively evaluated. Of the 519 women presenting for new patient consultation, approximately 1/3 initiated oocyte cryopreservation cycles. The best predictors of successful oocyte cryopreservation cycles were (in order) BAFC, day 3 FSH, and age (all p<0.05) (Barritt et al., 2010). Importantly, providers must remember that all of these tests and models attempt to predict the *quantity* of oocytes available for future reproduction. Unfortunately, tests to predict oocyte *quality* are still lacking. Models incorporating multiple variables may end up being the best predictor of ovarian reserve and ART cycle success, though many still consider age the best predictor of ovarian reserve and

The risk of aneuploidy is increased in older oocytes, which leads to higher rates of chromosomally abnormal fetuses and spontaneous abortion. Approximately 15-20% of pregnancies end in spontaneous abortion, or miscarriage (Barron, 1968). Maternal age has long been recognized as a risk factor for pregnancy loss. Risk of chromosomal abnormalities, decreased fecundity, and prevalence of comorbid medical illnesses rise with increasing age – all of which may lead to spontaneous abortion (Barron, 1968). Aneuploidy is thought to affect around 20% of human oocytes (Jones, 2008). Some hypothesize that rates of aneuploidy increase with age through a "two-hit" pathway: nondisjunction followed by an inability of the oocyte to detect the chromosomal abnormality. Nondisjunction, or inappropriate chromosomal separation during meiosis I, is a leading cause of aneuploidy and increases with maternal age. Oocytes from older women may have decreased cohesive bonds between chromosomes, further predisposing them to meiotic errors (Jones, 2008). Additionally, as oocytes age, they may be unable to detect errors in recombination and sister

It has been well-documented that infertility rates increase with age and that reproductive aging is primarily related to oocyte age. One prospective study demonstrated infertility rates increasing from 8% in women aged 19-26 years to 13-14% in women aged 27-34 years, and ultimately to 18% for women aged 35-39 years (Dunson, 2004). Similarly, there is a decline in success rates of fresh-cycle, non-donor oocyte IVF as a woman ages. Live birth rates per embryo transfer have been documented around 47.5% for women <35 years old, with a progressive decline to 17.0% in women 41-42 years of age, according to 2009 data from the Society of Assisted Reproductive Technologies (SART) (SART, 2009). In light of this

In the United States, there has been a notable shift in the demographics of reproductiveaged women. Many women are delaying childbearing in the setting of career pursuits and shifting societal expectations of gender roles. An analysis of Danish fertility rates from 1980- 2001 showed an increase in the mean age of childbearing of 3 years over the 21 year period (Hvidtfeldt et al., 2010). There appears to be a global shift in reproduction leading to

Reasons for delaying childbearing are multifaceted and complex. Perceived career threats are a very real and prevalent issue in young women of reproductive age (Willett et al., 2010). For women in professional programs, fear about extension of training, loss of future career

data, strategies to preserve fertility for young women are paramount.

**4. Changing demographics of reproductive-aged women** 

delayed childbearing and increased maternal age.

reproductive potential.

chromatid separation.

opportunities and concern for pregnancy complications are all significantly higher than in men. These concerns lead to choosing between career training and childbearing, thus risking subfertility by delaying reproduction for the sake of a woman's profession. Studies at our center have evaluated motivations for and trends in elective preservation of fertility in women seeking care at a New York City infertility clinic. Women seeking elective egg freezing were likely to have a high level of education, with all women having at least a bachelor's degree and 75% holding a master's or professional degree. These women were all single, nulliparous, and the majority expressed a desire to be sure they had taken advantage of all reproductive opportunities (Gold et al., 2006). Half of women interviewed described being pressured by their "biological clock" and many wanted to freeze eggs as an "insurance policy," though did not anticipate needing to use them. Interestingly, the mean patient age was 39 years old and 65% of women had reported only recently learning about egg freezing technology. In a multicenter analysis, more than 3000 women called to inquire about fertility preservation. Of these women, those who actually completed a cycle had a significantly higher average age of 37.1 years; patients who were older than 35 had fewer cycles that resulted in the recommended number of metaphase II oocytes for cryopreservation (Frank Sage et al., 2008). This may suggest an inadequate awareness of the age-related decline in fertility that occurs as part of normal human aging. Most studies on reproductive outcomes after oocyte cryopreservation (including oocyte survival rates, fertilization rates, and number of pregnancies) have analyzed women under the age of 35 (Jain & Paulson, 2006). Because of this limitation in the body of literature on oocyte cryopreservation, providers should ideally cryopreserve oocytes in women <35 years of age. As oocyte cryopreservation becomes more publicized and accurate information about declining female fertility is disseminated, the mean age of cryopreservation may decrease.

Trends in the local and national economy have been studied in relation to elective medical procedures, including oocyte cryopreservation. Costs of oocyte and embryo cryopreservation have been evaluated through the LIVESTRONG database of 154 participating reproductive centers. For the average patient, the cost of oocyte cryopreservation is around \$7,800, compared to an average of \$9,300 for embryo cryopreservation (Beck et al., 2010). The costs of fertility preservation are variable based on geography and center. In a New York City private IVF program, annual per capita income showed significant positive correlation with new consults for oocyte cryopreservation. Additionally, as annual unemployment rates increased, the number of new consults significantly decreased (Flisser et al., 2009).

Oocyte cryopreservation has many social and ethical advantages over embryo cryopreservation. Embryo cryopreservation remains the standard recommendation for fertility preservation according to ASRM guidelines, mainly due to the amount of literature studying this technique. Single women, however, may encounter social issues with freezing embryos. The option to extend fertility without the need for a male partner or sperm donor is frequently appealing to women who are not in a long-term relationship. The discomfort of anonymity associated with sperm donors is eliminated with egg freezing. Other potential issues include decisions regarding paternity and legal obligations for patients who undergo directed sperm donation, strategies for disposing of embryos if a woman gets married later in life, and how to handle the disposition of embryos if the egg donor dies and does not have explicit advanced directives in place (Jain & Paulson, 2006). These dilemmas are all circumvented with oocyte cryopreservation. Additionally, infertility centers avoid the often difficult task of synchronizing cycles between oocyte donors and recipients, in the case of

Oocyte Cryopreservation for the Elective Preservation of Reproductive Potential 193

levels of non-liquid solutes and electrolytes, known as *solution effect*. Further damage to intracellular proteins can occur in the presence of these toxic levels of intracellular substances during cryopreservation. Finally, *osmotic shock* can occur in the setting of rapid rewarming, during which rapid free water shifts lead to cell shrinking and swelling to accommodate alterations in extracellular osmotic pressure. These three goals are achieved through the use of different cryoprotectant chemicals. Cryoprotectants facilitate oocyte cryopreservation by generating an osmotic gradient by which water can exit the oocyte. Permeating cryoprotectants are able to enter the oocyte, thereby preventing cell shrinkage

Two protocols for oocyte cryopreservation exist, slow-freeze methods and vitrification. While slow-freezing is the most widely used and has been studied more in the literature, recent studies in embryos suggest that vitrification may have improved post-thaw survival rates, though it is still not clear whether there are significant differences in clinical pregnancy rates. These methods are discussed here and are analyzed in light of recent evidence of comparative efficacy. In addition, methods for ovarian tissue 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

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

live birth after mature oocyte cryopreservation in 1986 (Chen, 1986).

near constant temperature for the frozen oocytes during storage.

during osmosis of water to the extracellular space.

are briefly discussed.

**5.1 Slow freeze** 

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 cryopreservation for egg donors.

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

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 advantages of this emerging reproductive technology.
