Recent Advancements In Oocyte Cryopreservation

Patrick L. Blohm, M.D.
Patrick L. Blohm, M.D. is a Reproductive Endocrinology specialist with
the Florida Institute for Reproductive Medicine in Jacksonville.

Recently, however, advancements in the technology of successful oocyte freezing, thawing, and utilization have appeared to open this previously locked door. Once the efficiency of the oocyte cryopreservation process is established, dramatic clinical applications may be considered which may not only markedly alter the "standard of care" for current infertility patients, but may also extend new treatment options to altogether new groups of patients.

The apparent elusiveness of successful oocyte cryopreservation was not completely unanticipated. Several known physical characteristics of the oocyte predicted its resistance to successful freezing. Firstly, the human egg is a relatively voluminous cell with abundant cytoplasm. Secondly, a metaphase II, or mature oocyte, contains the spindle apparatus; a fragile intracellular superstructure suspending the genetic material which is known to be susceptible to the deleterious effects of hypothermia and ice crystal formation. Thirdly, the membrane of the egg is known to be delicate, and very sensitive to ice crystal formation. Finally, although incompletely understood, the freezing and thawing processes are thought to alter the zona in a way which makes it less receptive to penetration by sperm. Despite these known or suspected roadblocks, attempts at oocyte cryopreservation continued — largely in vain. Current thinking at the time assumed that the physiologic dynamics of the egg should be at least similar to the embryo, and therefore variations of freezing protocols which already had proved successful in freezing embryos were applied to eggs. Unfortunately, this consistently yielded disappointing results. Although reports of isolated cases of successful oocyte cryopreservation,¹ and pregnancies utilizing cryopreserved oocytes,^2,3 began to appear in the literature, the gross inefficiency of these techniques became overwhelmingly clear. Therefore, alternative egg cryopreservation methods were suggested including cocktail combinations of multiple cryoprotectants, physical activation of the oocyte prior to freezing, and cryopreservation of ovarian tissue or immature oocytes with post thaw in vitro maturation.

Ironically, the breakthroughs during the last several years have come as a result of the appreciation not of the similarities, but rather of the differences between the oocytes and the embryos. The oocyte, in contrast to its fertilized counterpart the embryo, has a cellular membrane apparently much less permeable to standard cryoprotectants. Therefore, techniques have been developed which involve osmotically driven oocyte dehydration to further facilitate diffusion of the cryoprotectants into the oocyte. In addition, it was discovered that adequate intracellular diffusion of cryoprotectants at physiologic temperatures actually yielded higher post thaw survival rates than those protocols carried out at room temperature. Once post thaw survival had been enhanced, the next step was to address fertilization issues. Past work had demonstrated suboptimal post thaw fertilization rates of previously cryopreserved oocytes undergoing conventional IVF. Again, this phenomenon was thought to be due to effects of the freezing and thawing process on the zona. Fortunately, by this time, intracytoplasmic sperm injection (ICSI), was a well refined process routinely utilized for treatment of significant male factor infertility. ICSI, therefore, was a ready and waiting answer for this problem.

Most recently, groups⁴ have been reporting their short series of clinical experience with cryopreserved oocytes. Although data sets are small, the overall picture is quite encouraging. Post thaw oocyte survival rates as high as 50-60% are being observed. In addition, fertilization rates of surviving eggs are comparable to those fresh oocytes. Transfers of cleaving embryos from previously frozen eggs have yielded acceptable implantation rates and ongoing pregnancies have frequently been seen. Continued development of these techniques is occurring, and it is hoped that in the very near future oocyte cryopreservation will become as refined as embryo cryopreservation. Finally, as more and more clinical experience accumulates, safety data should become available to allow for accurate discussions of these issues prior to widespread utilization of this technology. Until then, oocyte cryopreservation technology should be used in selected settings with full disclosure to the patients regarding the limited amount of safety data available.

Potential Clinical Utility Of Oocyte Cryopreservation

The potential clinical applications of oocyte cryopreservation technology are many. Emphasis should be placed on "potential" as all of the proposed clinical applications are not currently considered to be standard of care. In addition, some of these potential applications carry with them ethical issues of their own. What follows is a brief, non-exhaustive discussion of some of the clinical scenarios thought to potentially benefit from application of oocyte cryopreservation technology.

Anonymous oocyte donation (AOD) is one such area that may benefit from application of oocyte cryopreservation technology. Currently, oocyte donation usually involves synchronization of the menstrual cycles of the donor and recipient. Controlled ovarian hyperstimulation of the donor frequently yields large numbers of eggs and subsequently numerous embryos. Transfer of very few of these embryos often results in pregnancy and large numbers of additional embryos are cryopreserved. The entire AOD process is logistically difficult to coordinate, results in the inefficient use of the valuable, limited commodity of donor oocytes, lends itself to the chronic stockpiling of excess embryos and requires that the entire cost of the therapy be borne by one couple. Utilization of oocyte cryopreservation techniques would allow for controlled ovarian hyperstimulation of the donor independent of potential recipients. Those recipients could then, at their convenience, utilize cryopreserved oocytes in numbers sufficient to generate individual embryo transfers. These changes would dramatically improve the efficiency of the oocyte donation process, minimize the stockpiling of large numbers of embryos, significantly reduce the per couple cost of therapy and allow more logistical freedom for those parties involved. In addition, cryopreservation of the donated oocytes would also allow for an appropriate quarantine period, prior to utilization, to minimize the risk of transmissible diseases.

In a similar way, conventional IVF may be improved by implementing oocyte cryopreservation techniques. It is now not uncommon for large numbers of eggs to be obtained from patients with high responses to gonadotropins. Fertilization of these eggs often results in embryo numbers greatly in excess of what can be responsibly transferred back to the patient. Given recent trends toward improved per transfer pregnancy rates, the trend of chronic stockpiling of cryopreserved excess embryos needs to be addressed. Ultimately, in most programs, it is the couple's responsibility to make final disposition of these embryos. Unfortunately, this frequently leaves the couple with the hard decision of thawing and discarding their embryos versus releasing them for anonymous embryo donation with no information regarding outcome. All too often, when patients are unwilling, unable, or unavailable to make this decision, fertility programs are left to deal with completely "abandoned" embryos. In addition, not all marriages of fertility couples survive, and disputes over "custody" and "ownership" of cryopreserved embryos can further complicate already painful divorce proceedings. Furthermore, and understandably so, some other couples have significant personal reservations about the entire embryo cryopreservation process. Oocyte cryopreservation, however, would tend to help, if not alleviate these dilemmas. At the time of oocyte retrieval, only those eggs destined for the fresh embryo transfer would undergo fertilization, and excess eggs would be cryopreserved as oocytes. These changes would eliminate, or at least markedly reduce, the freezing of excess embryos, while not drastically sacrificing the overall total reproductive potential of the IVF cycle.

Oocyte cryopreservation techniques also have the potential to help entirely new groups of patients. Many oncology patients, at the onset of their treatment, face uncertainty. Not only is the response of their underlying disease to proposed radiation or chemotherapy unknown, but this potentially lifesaving therapy may induce permanent gonadal damage and sterility. Male patients have long had the option of pretreatment cryopreservation of sperm. Female counterparts, until recently, have had few suboptimal alternatives. One choice was to undergo therapy and resign to utilization of egg donation if necessary. A second option, should the patient be married, was to undergo urgent IVF with cryopreservation of all embryos and subsequent transfer should the patient survive her disease. This practice, however, begs ethical questions such as the prudence of creating embryos in the face of a potentially fatal maternal condition. Oocyte cryopreservation may offer the oncology patient an opportunity to "bank" her eggs prior to initiation of her treatment. Should the patient be cured of her underlying disease but sustain permanent ovarian damage, she may rely on these banked gametes to allow her to bear genetic offspring.

The potential indications for egg "banking" could be expanded to include women considering delaying childbearing. The link between advancing reproductive age and a qualitative and quantitative wane in ovarian function is well appreciated. Frequently patients suffering from age related problems have to make a difficult choice between low pregnancy rates utilizing their own eggs and markedly higher chances of success with donor oocytes. This frequently causes great anguish for the patient over "time lost". With the potential to cryopreserve eggs, patients anticipating a delay in childbearing may choose to store their gametes during their reproductive prime for autologous use in the twilight of their reproductive years.

Summary

Recent advances in oocyte cryopreservation technology have led to a dramatic improvement in the efficiency of this process. Most of these advances have come with the appreciation of the differences in permeability between the oocyte and embryo membranes. Currently, oocyte cryopreservation has reached a level of development where entertainment of potential clinical applications is appropriate. Use of this technology in limited settings with strict adherence to informed consent is prudent until greater data regarding the safety of this technology is available.

REFERENCES

1. Tucker M, Wright G, Morton P, Shanguo L, Massey J, Hilton K. Preliminary experience with human oocyte cryopreservation using 1,2-propanediol and sucrose. Human Reproduction. 1996; II(7): 1513-1515.
2. Chen C. Pregnancy after human oocyte cryopreservation. Lancet. 1986; (1): 884-886.
3. Van Uem J, Siebzehnrubl ER, Schuh B, et al. Birth after cryopreservation of unfertilized oocytes. Lancet. 1987; (1): 752-753.
4. Data to be submitted for publication, Florida Institute for Reproductive Medicine.

May, 2000/ Jacksonville Medicine

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