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                  Progress with Cryostorage of Female Gametes
M.J Tucker
Shady Grove Fertility Reproductive Science Center, Rockville, MD, and Georgia Reproductive Specialists, Atlanta,
GA, U.S.A.
                                              Summary
        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


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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 [15] 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 [18]. 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 [17]
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 [23]. It may be more appropriate in the short term to study these surgical
approaches using a non-human primate model however [24].
                                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 [25], 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


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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 [26].
         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 (<1l), 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
[28], micro-drops [29], electron microscopic copper grids [30], hemi-straw system [31], nylon
mesh [32], or the cryoloop [33]. 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.
                                             Where Next?
         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.
                                              References
1. Polak de Fried E, Notrica J, Rubinstein M, et al. Pregnancy after human donor oocyte
    cryopreservation and thawing in association with intracytoplasmic sperm injection in a
    patient with ovarian failure. Fertility and Sterility, 1998;69:555-557.
2. Tucker MJ, Morton PC, Wright G, et al. Clinical application of human egg
    cryopreservation. Human Reproduction, 1998;13:3156-159.
3. Yang DS, Winslow KL, Blohm PL. Improved survival rate after cryopreservation of
    human fresh and aged unfertilized oocytes using a specially developed oocyte
    cryopreservation regime. Fertility and Sterility, 1998;70(suppl 1):S86. Abstract O-232.




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4. Lanzendorf SE, Mayer JF, Toner J, et al. Pregnancy following transfer of ooplasm from
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5. Chen C. Pregnancies after human oocyte cryopreservation. Annals of the New York
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6. Van Uem JFHM, Siebzehnruebl ER, Schuh B, et al. Birth after cryopreservation of
    unfertilized oocytes. Lancet, 1987;i:752-753.
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11. Cortvrindt RG, Hu Y, Liu J, et al. Timed analysis of the nuclear maturation of oocytes in
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14. Paynter SJ, Cooper A, Gregory L, et al. Permeability characteristics of human oocytes in
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15. Gunasena KT, Villines PM, Critser ES, Critser JK. Live births after autologous transplant
    of cryopreserved mouse ovaries. Human Reproduction 1997;12:101-6.
16. Gosden RG, Baird DT, Wade JC, Webb R. Restoration of fertility to oophorectomized
    sheep by ovarian autografts stored at –196oC. Human Reproduction 1994;9:597-603.
17. Gook DA, McCully BA, Edgar DH, McBain JC. Development of antral follicles in
    human cryopreserved ovarian tissue following xenografting. Human Reproduction,
    2001;16:417-22.
18. Kim SS, Radford J, Harris M, et al. Ovarian tissue harvested from lymphoma patients to
    preserve fertility may be safe for auto-transplantation. Human Reproduction
    2001;16:2056-60.
19. Eppig JJ, O’Brien MJ. Development in mouse oocytes from primordial follicles. Biology
    of Reproduction, 1996;54:197-201.
20. Abir R, Roizman P, Fisch B, et al. Pilot study of isolated early human follicles cultured in
    collagen gels for 24 hours. Human Reproduction, 1999;14:1299-301.
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    factors I and II on human ovarian follicles in long-term culture. Molecular Human
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22. Oktay K, Karlikaya G. Ovarian function after transplantation of frozen, banked
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    forearm. JAMA, 2001;286:1490-1493.
24. Schnorr J, Oehninger S, Toner J, et al. Functional studies of subcutaneous ovarian
    transplants in non-human primates: steroidogenesis, endometrial development, ovulation
    menstrual patterns and gamete morphology. Human Reproduction 2002;17:612-9.
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    of an eight-cell embryo. Nature, 1983;305:707-709.
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    GV-oocytes: positive effect of rapid cooling or rapid thawing or both? Cryo-Letters,
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28. Liebermann J, Tucker M, Graham J, et al. Blastocyst development after vitrification of
    multipronucleate zygotes using the flexipet denuding pipette (FDP). Reproductive
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29. Papis K, Shimizu M, Izaike Y. Factors affecting the survivability of bovine oocytes
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31. Kuwayama M, Kato O. Successful vitrification of human oocytes. Fertility and Sterility,
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33. Lane M, Bavister BD, Lyons EA, et al. Container-less vitrification of mammalian oocytes
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