Progress with Cryostorage of Female Gametes
Shady Grove Fertility Reproductive Science Center, Rockville, MD, and Georgia Reproductive Specialists, Atlanta, GA, U.S.A.
A brief overview of human oocyte/ovarian tissue cryopreservation is made, with a discussion of the merits of conventional protocols. Practical issues of cryopreservation such as which oocyte maturity stage is used for cryo-storage, and which post-thaw strategies are most optimal are considered. Alternative cryopreservation technologies are put into perspective with special attention being paid to vitrification. Benefits of female gamete cryostorage are: formation of donor “egg banks” to facilitate and lessen the cost of oocyte donation for women unable to produce their own oocytes; provision of egg cryostorage for women wishing to delay their reproductive choices; and cryopreservation of ovarian tissue taken from women about to undergo therapy deleterious to such tissue, which may threaten their future reproductive health.
Conventional Cryopreservation of the Human Oocyte
The technology so far applied clinically has been based directly on traditional human embryo cryopreservation protocols, and has produced greater than 80 offspring Worldwide. No major abnormalities have been reported from these pregnancies, regardless of the persistent concerns that cryopreservation of mature oocytes may disrupt the meiotic spindle and thus increase the potential for aneuploidy and structural defects in embryos arising from such eggs. Nevertheless a concerted effort to monitor long-term outcomes of such offspring is essential. Cryostorage of donated oocytes has given rise to several reports of pregnancies 1-3. Even use of frozen donor oocytes post-thaw not for whole egg donation, but for ooplasmic transfer has also been reported 4.
Cryostorage of women’s own oocytes was originally reported in the case of three births in the 1980’s by two centers 5,6. More recently, this success has been reproduced by others 3,7-9. Generally these pregnancies have arisen from the freezing of oocytes that have been collected for purposes of infertility therapy where couples may have had religious or ethical concerns with embryo cryopreservation; when couples have consented to research studies, or where sperm are unexpectedly unavailable after the oocytes have been retrieved.
Most pregnancies have arisen from frozen-thawed mature oocytes, but pregnancies have also been generated from cryostored immature germinal vesicle (GV) stage eggs 8. This stage of egg development might prove to be a more successful approach for cryopreservation because its oolemma is more permeable to cryoprotectant, and its chromatin is more conveniently and safely packaged in the nucleus 10. Such eggs, however, still have to undergo GV breakdown and maturation to the MII stage before fertilization, and therefore their developmental competency is not so established as with cryostored fully mature oocytes. Source of the GV eggs, and whether they have been exposed to any exogenous gonadotropins may play a key role in the competency of these eggs 11.
Whether mature or not, standard cryopreservation technologies appear to have their ultimate limitations not only in terms of cryosurvival, but also in their lack of consistency. Consequently, radically different types of protocols may provide the answer to increased consistent success such as vitrification. This has been successfully applied in the mouse 12, bovine 13, and recently in the human 9. While the mouse can be a useful model, it must be remembered that the murine oocyte is only just over half the volume of a human oocyte; this can have a major impact on permeability and perfusion in the two types of eggs 14. ICSI has become the accepted norm for insemination of oocytes post-thaw, to avoid any reduction in sperm penetration of the zona following premature cortical granule release 2 or general hardening of the zona.
Cryostorage of Ovarian Tissue
The most plentiful source of oocytes potentially is ovarian tissue itself, containing as it does many thousands of primordial follicles in healthy cortical tissue. Approaches here, however, must take into consideration that tissue is being cryostored, not individual cells necessarily, unless individual follicles are isolated. Earlier successful work with cryopreservation of rodent ovarian tissue  has led the way to successful cryostorage of both sheep and human tissue 16,17. Over 80% survival of follicles has been reported, but a major issue is how to handle this tissue following its thaw. Tissue that has been removed, for example, from a woman about to undergo cancer therapy may contain malignant cells, and therefore may not be safely used for auto-grafting into such a woman at a later date. This is dependent perhaps on the nature of the malignancy, and certain cancers are thought to be less problematic for autologous grafting when a patient is in remission from the disease . In other cases, the tissue might be screened before or after thawing for the presence of malignant cells. This will enable some assessment of the safety of such an approach, or the tissue may be xeno-grafted into a host animal; e.g., SCID mouse  until such time as in vitro maturation could be undertaken more effectively.
Extended culture of primordial follicles to full oocyte maturity, with subsequent embryonic development and birth has only been recorded in the mouse, and this was not from cryopreserved tissue 19. Studies to establish the in vitro requirements for follicular development are being undertaken in the human 20,21 with much remaining to be done. Fertility has been restored in sheep, in a good model for the human ovary, following cryostorage of ovarian cortex and auto-grafting 16, and this seems the most likely successful clinical model for restoration of fertility of women who are at risk of losing their ovarian function. This may include not only women about to undergo cancer therapy, but also women who have a family history of early menopause, and those with non-malignant diseases such as thalassemia or certain auto-immune conditions which may be treated by high-dose chemotherapy. Ovarian function was restored by such means in a 29year old patient suffering from hypothalamic amenorrhea subsequent to removal of both her ovaries at age 17 22. Heterotopic transplantation of ovarian tissue in the forearm has also enabled follicular growth to be restored with a view to convenient oocyte retrieval . It may be more appropriate in the short term to study these surgical approaches using a non-human primate model however .
Vitrification of the Human Oocyte
Cryopreservation of human oocytes, zygotes, cleavage stage embryos, and blastocysts has become an integral part of human ART. Since the first report of human pregnancy following cryopreservation and transfer of an 8-cell embryo , IVF centers have been using traditional slow-rate or equilibrium freezing protocols routinely. The time to complete these freezing procedures for human embryos ranges from 1.5 to 5hrs. Slow rate cooling attempts to maintain a very delicate balance between several factors which may result in damage, mostly by ice crystallization, but also by osmotic and chilling injury, zona and blastomere fracture, and alterations of the cytoskeleton.
Many studies have been undertaken to reduce the time of the freezing procedure and also to try and eliminate the cost of expensive programmable freezing equipment. One way to avoid crystallization damage is to use a vitrification approach. Vitrification as an ultra-rapid cooling technique is based on direct contact between the vitrification solution containing the cryoprotectant agents and liquid nitrogen (LN2). Vitrification is a result of high cooling rates associated with high concentrations of cryoprotectant. The physical definition of vitrification is the solidification of a solution, such that liquid is so rapidly cooled that it forms into a glassy, vitrified solid state from the liquid phase at low temperature, not by ice crystallization, but by extreme elevation in viscosity during cooling .
There are two ways to achieve vitrification of water inside cells: firstly, increase the speed of thermal conduction, and secondly increase the concentration of cryoprotectant. Also, by using a small volume of high concentration cryoprotectant (<1l), very rapid cooling rates from –15,000 to –30,000°C/min can be achieved. The strategy of vitrification results in the total elimination of ice crystal formation, both intra- and extra-cellularly. Protocols for vitrification are very simple, and involve placing oocytes into the cryoprotectant and then plunging directly into LN2. To minimize the volume of the vitrification solution, special carriers are used during the vitrification process. These include the open pulled straws [13,27], the flexipet-denuding pipette , micro-drops , electron microscopic copper grids , hemi-straw system , nylon mesh , or the cryoloop . These have all been used as carriers to achieve higher cooling rates. The actual cooling rate during vitrification, and therefore the efficiency, may still vary extremely depending on the device used, technical proficiency, and even the specific movement at immersion of the carrier into the LN2.
The convenience of vitrification if properly applied, eliminates the use of expensive controlled rate freezers, but is still awaiting full cross over from use in other species, requiring validation from more extensive experimental study in humans. Despite this, it is likely that the consistency of vitrification will soon see its use for routine oocyte cryostorage. Generally the various possibilities for cryostorage of the female gamete can make for a confusing vision of where clinical applications may occur. However, different clinical needs may actually be met by differing technological approaches, whether they incorporate whole tissue freezing, separate follicle storage, or cryopreservation of immature or mature oocytes themselves. Conventional cryopreservation will probably remain for storage of ovarian cortical tissue, but if primordial and primary follicles can ever be successfully matured subsequent to isolation, then vitrification of these smaller entities will be possible and more appropriate, similar to the trend with the cryostorage of more mature oocytes. There still remains no clear consensus on the best use of thawed ovarian cortical tissue. With respect to salvaging the reproductive integrity of women post-cancer therapy, greater attention to minimizing the risks of creating sterility by ovarian transposition and more effective cocktails of anticancer agents may in any event reduce the need for ovarian tissue cryostorage.
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