2005 © Oxford University Press
Prospects for Oocyte Banking and In Vitro Maturation
Affiliation of author: Weill-Cornell Medical College, New York, NY
Correspondence to: Roger G. Gosden, PhD, DSc, Center for Reproductive Medicine and Infertility, Weill Medical College, Cornell University, 505 East 70th Street (HT340), New York, NY 10021 (e-mail: rgg2004{at}med.cornell.edu).
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ABSTRACT |
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 Top Abstract IntroductionPRINCIPLES OF CRYOPRESERVATIONCRYOBIOLOGY OF OOCYTESEXPERIMENTAL AND CLINICAL...References |
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There is little debate about the desirability of human oocyte ("egg") banking but plenty of discussion about its prospects. Egg banking is needed by young cancer patients before they undergo potentially sterilizing treatment and is a desirable alternative to in vitro fertilization and embryo cryopreservation. However, egg banking is inefficient—oocytes are sensitive to chilling, often fail to survive freeze–thawing, and are susceptible to cytoskeletal damage and aneuploidy. Currently, even the most optimistic success rates offer patients only a slim chance of pregnancy if few oocytes are available. Ultra-rapid freezing with vitrification may offer advantages over conventional equilibrium cooling protocols and needs to be investigated further. Likewise, freezing immature oocytes followed by in vitro maturation offers practical and theoretical advantages, but this method is still inefficient. Nevertheless, all these technologies are improving, and egg banking will eventually become an option for patients seeking fertility preservation.
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INTRODUCTION |
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TopAbstractIntroductionPRINCIPLES OF CRYOPRESERVATIONCRYOBIOLOGY OF OOCYTESEXPERIMENTAL AND CLINICAL...References |
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The lifetime production of oocytes has long been assumed to be completed before birth because oogonial stem cells disappear. Although this theory was recently challenged (1), it remains true that the mature oocyte is the rarest cell in the body. A maximum of only about 400 oocytes is ovulated during the menstrual lifespan, although treatment with exogenous gonadotropins can trigger the ripening and superovulation of 10 or more Graafian follicles during a given cycle. The final menstrual cycle, or menopause, occurs in midlife, when depletion of the follicular store is imminent. In addition to quantitative changes, there is a qualitative decline in follicular quality manifested by an increased miscarriage rate and a reduction in success with assisted reproductive technologies, largely because of an increased incidence of aneuploidy and other karyotypic anomalies in oocytes (2). The effects of environmental toxins, including therapeutic doses of alkylating agents and ionizing radiation for cancer, are superimposed on this background of low fertility. In consequence, primary or secondary amenorrhea and either temporary or permanent ovarian failure occur, depending on the treatment protocol and age at the time of treatment (3).
Whether through natural aging or iatrogenic damage, women no longer have the potential for genetic parenthood after menopause. Other parenting options are egg or embryo donation or adoption. As increasing numbers of young patients become long-term survivors of malignant diseases, fertility preservation assumes greater importance. Many options are available for them, but this article will consider only egg banking. This procedure involves the cryopreservation of fully grown oocytes, when they are either at metaphase II stage or are still in prophase I ("germinal vesicle" stage). The record of genetic safety for cryopreserving embryos and sperm has been mostly reassuring, although there are contrary reports based on the mouse oocyte model (4,5). Concerns have been expressed about human oocyte freezing, especially the risks of aneuploidy, but clinical experience is still too limited for a robust assessment.
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PRINCIPLES OF CRYOPRESERVATION |
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TopAbstractIntroductionPRINCIPLES OF CRYOPRESERVATIONCRYOBIOLOGY OF OOCYTESEXPERIMENTAL AND CLINICAL...References |
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The integrity and survival of cells are at risk at every stage of the process. Some cells—notably oocytes—are highly sensitive to chilling and can be injured even at room temperatures. When the oocytes are cooled below the freezing point, the principal danger is intracellular ice crystal formation, which is invariably lethal to cells. Cryoprotective agents (CPAs), such as 1,2-propanediol, ethylene glycol, and dimethylsulfoxide, are needed for cell survival and to replace cell water, although their beneficial effects require the application of potentially toxic concentrations.
To cryopreserve oocytes using conventional "equilibrium" cooling, they are first immersed in a high concentration of CPAs, which include both membrane-permeable compounds (such as ethylene glycol and 1,2-propanediol at approximately 1.5 mol/L) and membrane-impermeable compounds (such as monosaccharides or disaccharides at 0.1–0.5 mol/L) in a buffered salt solution. Maternal serum or human serum albumin is usually added. Cells shrink from osmotic stress and because of the lower permeability of the cell membrane to the CPA than to water, but they gradually recover their normal volume as chemical equilibrium is re-established. A second phase of volume change occurs during cooling, when ice crystals begin to form extracellularly. As a consequence, the extracellular fluid becomes more concentrated, and water moves osmotically out of the cell (6). The rate of cooling of the cells is critical. If it is too fast, there is insufficient dehydration to avoid ice crystallization and promote intracellular vitrification conditions; if it is too slow, on the other hand, the cells may be damaged from prolonged exposure to CPAs at "moderately high" temperatures. Ice crystallization is induced ("seeded") at about –7 °C to avoid supercooling, which is an unstable state. Human oocytes have remarkably low membrane permeability to water and CPAs and must therefore be cooled very slowly (7–9). After the oocytes have been sufficiently dehydrated, the cooling rate can be accelerated and the specimens can be plunged into liquid nitrogen for long-term storage. Rewarming must also be controlled and is normally carried out very rapidly to avoid intracellular ice crystallization during temperature reversal. Further volume excursions occur when the CPAs are washed out, which is normally performed in step dilutions to reduce cellular stress (8). The impermeable CPAs (sugars) serve to reduce cellular volume stress, but they may have other benefits (10). Thus, cells are exposed to a number of stressors during cryopreservation—changes in temperature, volume, and solute concentrations—which they never experience physiologically.
An alternative strategy for low-temperature banking is to vitrify the entire medium (11). If the viscosity of the medium is very high as a result of very high concentrations of CPAs (5–6 mol/L), a glassy solid is formed. Because cooling is performed ultrarapidly, it can minimize the effects of chilling and prevent ice crystal formation. However, the cells are exposed to higher concentrations of solutes and osmotic stress. Vitrifying media usually include more than one permeable CPA to minimize the toxicity of these compounds. To achieve cooling rates of several thousand degrees per minute, the specimen is held in a minimal volume of medium and may be directly exposed to liquid nitrogen (which could potentially transmit pathogenic viruses). Embryos and eggs have been vitrified using the open pulled straw, copper grid, and cryoloop (11–16), and randomized trials are needed to determine optimal methods.
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CRYOBIOLOGY OF OOCYTES |
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TopAbstractIntroductionPRINCIPLES OF CRYOPRESERVATIONCRYOBIOLOGY OF OOCYTESEXPERIMENTAL AND CLINICAL...References |
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In contrast with the relatively high success rates obtained with sperm and embryos, cryopreservation of oocytes is more problematic. The first obstacle is their sensitivity to chilling (especially human and bovine oocytes). This problem is probably due, at least in part, to the delicacy of the spindle apparatus and to the higher lipid content of the cells. When bovine oocytes are chilled to room temperatures, their viability is drastically reduced and the spindle apparatus anchoring the chromosomes disassembles (17–19). Although reliable data for human cells are scarce because of shortages of research material, cooling and exposure to some CPAs evidently affects the cytoskeleton and may aggravate the already high incidence of aneuploidy in human oocytes (20).
Because small cells tend to cryopreserve more successfully than do large ones, because of a more favorable surface area-to-volume ratio, the relatively voluminous human oocyte is at a disadvantage. In the early days of egg cryopreservation, survival rates were rarely greater than 50%, although protocols with higher concentrations of sugars and step changes in CPA concentrations have produced better results (21,22). Exposure to CPAs may increase intracellular Ca2+ and trigger premature exocytosis of cortical granule material, which enzymatically modifies the zona pellucida and renders it harder to bind spermatozoa ("zona hardening"). This problem, however, has been circumvented since the early 1990s by intracytoplasmic sperm injection (ICSI) (23). Nowadays, virtually all protocols for egg cryopreservation involve ICSI as a precaution against zona hardening. But, whatever the method chosen, fertilization must be performed about 3–5 hours after thawing while the oocyte remains fertile. Thus, there is little time for recovery before the cell has to undergo the complex changes of penetration, activation, and completion of the second meiotic division with formation of pronuclei.
An alternative strategy that allows a longer recovery time and avoids depolymerization of the spindle is to cryopreserve oocytes at the germinal vesicle stage, when the chromatin is diffuse and the cell is still at prophase I. This strategy is also attractive because oocytes can be recovered from unstimulated ovaries, perhaps from children too. However, the results to date have not been very encouraging, because germinal vesicle oocytes do not appear to cryopreserve better than do those at the metaphase II stage (24). Furthermore, they have to undergo incubation for 24–48 hours in culture for meiotic maturation, although oocytes that have reached metaphase II are not necessarily fully fertile, and this technology remains inefficient.
Oocytes are recovered for in vitro maturation (IVM) before the dominant follicle emerges during the mid-follicular phase of the menstrual cycle. Normally, the follicles are 8–10 mm in diameter; smaller follicles in the range of 3–7 mm also contain oocytes that are capable of maturation in vitro, but they are much less successful, probably because of cytoplasmic immaturity (25,26). The numbers of oocytes that are recovered is predictive of success with IVM–in vitro fertilization (IVF or ICSI). Patients with polycystic ovaries generally produce harvests of more than 10 oocytes from unstimulated ovaries, and the number of oocytes is a predictor of clinical pregnancy rates after IVM (27). Immature oocytes are aspirated without flushing and at a lower pressure than normal to avoid rapid collapse of the follicles and to increase the rate of egg recovery. The efficiency, however, is lower than that with periovulatory follicles because the cumulus cell mass around the oocyte is unmucified unless gonadotropin stimulation is given.
The cumulus–oocyte complex is normally cultured intact so that the physiological interactions between the oocyte and its somatic cell envelope are preserved (28). However, cryopreservation of the intact cumulus–oocyte complex is problematic because the optimal times for equilibration are likely to be different for the oocyte than for its relatively diminutive cumulus cells. Moreover, immersion in hypertonic CPAs causes cell shrinkage, which may disrupt the granulosa cell processes and gap junction communication with the oocyte, which is believed to be vital for transmitting nutrient and regulatory molecules. It is doubtful whether vitrification, which requires equilibration of the cumulus–oocyte complex in even higher concentrations of CPAs, can improve results. At present, the reported success of IVM in young women with polycystic ovaries is a pregnancy rate of approximately 25%–30% per cycle; the miscarriage rate is high (29). These rates are only half those obtained at the best clinical centers with IVF with fresh oocytes, which are likely to be compromised further if the oocytes are cryopreserved before IVM.
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EXPERIMENTAL AND CLINICAL EXPERIENCE |
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TopAbstractIntroductionPRINCIPLES OF CRYOPRESERVATIONCRYOBIOLOGY OF OOCYTESEXPERIMENTAL AND CLINICAL...References |
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Because only a few clinical pregnancies were reported after the cryopreservation of oocytes in the 1980s, further efforts were voluntarily suspended by the end of the decade. This reaction was not so much in response to the low efficiency of the techniques but to concerns about the effects of CPAs and cooling on the cytoskeleton, aneuploidy rate, and zona hardening. When ICSI was introduced and more reassuring data were published (30,31), efforts to cryopreserve oocytes resumed—first with conventional slow cooling–rapid thawing protocols and later with vitrification. To date, more than 4300 oocytes have been cryopreserved and more than 80 children have been born according to reports in the literature, mostly from the former technique (Tables 1 and 2). Unfortunately, full information is not always available, and negative results sometimes go unpublished. The overall live birth rate per cryopreserved oocyte is about 2%, which is much lower than that with IVF using fresh oocytes. Eleven children have been born after vitrification, but a comparison of success rates is unreliable because of variations in protocols and in the numbers of embryos transferred per cycle. A prospective randomized trial is urgently needed; however, this will likely require a multicenter study to accumulate sufficient cases.
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Most reports have been based on small numbers of patients; therefore, statistical estimates of success rates can be misleading. One of the possible reasons for wide variations between centers, even when similar protocols are used, is differences in the tolerance of oocytes to freezing and thawing between women. Such differences are known to exist for human and donor sperm, and recent evidence from cryopreserved Rhesus monkey oocytes indicates considerable variation in cryosensitivity from female to female (32). Such differences are likely to be related to membrane properties, including membrane permeability, cholesterol content, and phospholipid ordering. Investigation of these factors may help to improve the success of cryopreservation, although no single protocol is likely to be optimal for every oocyte.
Overall, the low clinical success rates with oocyte cryopreservation are discouraging, and the recent data from vitrification studies should be interpreted cautiously until more data are available. Cancer patients may not have more than one opportunity for egg harvesting before undergoing potentially sterilizing treatment. A cycle of controlled stimulation requires several weeks, and there is normally a delay of a few months between treatment cycles. If a patient can produce only the average harvest of 10 oocytes, her prospects for pregnancy are slight. Only six of the 10 cells are likely to survive, and not all will produce viable embryos; embryo quality is further compromised in reproductively aged patients (>35 years). Unfortunately, it is not possible to make a firm estimate of the chances of success from global data. Considering the costs of drugs, the inconvenience, the discomfort, the slight risks, and the annual charges for egg banking, few cancer patients are likely to opt for this procedure in the near future. Those that do should be carefully counseled and fully informed. Nevertheless, we can expect success rates to gradually improve, and patients who are young and produce large numbers of eggs will always have the best chances of success. Efforts to improve existing protocols continue. These efforts include the testing of new strategies, such as intracellular injection of trehalose to protect against intramolecular damage (33) and choline substitution to reduce the potentially harmful build-up of Na+ (34). In the long term, immature oocytes may be the answer, because the harvesting of these cells is less expensive, is less risky, and can be repeated frequently, but this procedure will require further advances in cryotechnology.
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