Human Oocyte And Embryo Cryopreservation

Summary of Syllabus:

• Brief overview of human egg and embryo freezing.
• Discussion of merits of currently utilized cryopreservation protocols.
• Practical issues of cryopreservation with reference to egg or embryo selection, and thaw replacement protocols.
• Alternative cryopreservation technologies.

Cryostorage of the female gamete

The last few years have seen a significant resurgence of interest in the potential benefits of human egg freezing. Essentially, these benefits are:

• Formation of donor “egg banks” to facilitate and lessen the cost of oocyte donation for women that are unable to produce their own oocytes.
• Provision of egg cryostorage for women wishing to delay their reproductive choices.
• Convenient cryopreservation of ovarian tissue taken from women about to undergo therapy deleterious to such tissue, which may threaten their reproductive health.

The technology so far applied clinically has been based directly on traditional human embryo cryopreservation protocols, and has produced relatively few offspring. Fortunately to date, no abnormalities have been reported from these pregnancies, regardless of the persistent concerns that freezing and thawing of mature oocytes may disrupt the meiotic spindle and thus increase the potential for aneuploidy in the embryos arising from such eggs. With respect to cryostorage of donated oocytes there have been several reports that have shown some success with this approach (Polak de Fried et al, 1998; Tucker et al, 1998a; Yang et al, 1998 & personal communication). Six pregnancies have generated 10 babies from cryopreserved donor oocytes in these reports. Use of frozen donor oocytes post-thaw not for whole egg donation, but for ooplasmic transfer has been reported with a successful delivery of a twin following thawed ooplasmic donation (Lanzendorf et al., 1999).

Cryostorage of women's own oocytes was originally reported in the case of three births over a decade ago by two centers (Chen, 1988; Van Uem et al, 1987). More recently these successes have been reproduced by others (Porcu et al, 1998; Tucker et al, 1998b; Yang et al, 1998 & personal communication; Kuleshova et al., 1999), giving rise to 10 babies. One other baby has arisen from a clinical circumstance that is not completely unfamiliar to IVF clinics: oocytes had been collected but no sperm were retrievable for insemination. In this case, the oocytes were frozen, and donor semen was selected for future use. Ultimately both sets of gametes were thawed and used in a subsequent IVF attempt, which achieved a health delivery (Moody & San Roman, personal communication).

All of these pregnancies were from frozen-thawed mature oocytes, but for one notable exception, where a pregnancy arose from an immature germinal vesicle (GV) stage egg (Tucker et al, 1998b). Interestingly, 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 (Van Blerkom & Davis, 1994). 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 clearly established as with fully mature oocytes that are frozen. 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 (Cortvrindt et al, 1998).

Whether mature or not, standard cryopreservation technologies appear to have their ultimate limitations in not only cryosurvival, but also more importantly in their lack of consistency. 50% cryosurvival is an adequate overall outcome, but not if it is a statistic that is arrived at by 90-100% survival in one case, and 0-10% in the next. Consequently, radically different types of protocol may provide the answer to increased consistent success. One approach has been to replace sodium as the principal cation in the cryoprotectant with choline in an attempt to shut down the sodium ion pumps in the oocyte membrane during cryoprotectant exposure, thus minimizing potentially deleterious “solution effects” during cooling (Stachecki et al, 1998). This has provided significant improvements in murine egg freezing, though it has yet to be applied clinically in the human. Alternatively, traditional slow cooling/rapid thaw protocols might be replaced with vitrification. Which again has been successfully applied in the mouse (O'Neil et al, 1997), bovine (Vajta et al., 1998), and very recently in the human (Kuleshova et al., 1999). 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 if the two types of egg (Paynter et al. 1999). ICSI has become the accepted norm for insemination of oocytes post-thaw, to avoid any reduction in sperm penetration of the zona with premature cortical granule release (Gook & Edgar 1999).

The most plentiful source of oocytes potentially is ovarian tissue itself, containing as it does many thousands of primordial follicles in healthy cortical tissue. Earlier successful work with cryopreservation of rodent ovarian tissue has led the way to successful cryostorage of both sheep and human tissue (Gosden et al, 1998; Gook & Edgar 1999). Up to 80% survival of follicles has been reported, but the 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 in to such a woman if she were to survive. The tissue might be screened before or after thawing for the presence of malignant cells to enable some assessment of the safety of such an approach, or it may be grown in 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 (Eppig & O'Brien, 1996). Early studies are being undertaken in the human (Abir et al. 1999) with much 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 (Gosden et al, 1994), 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. Recently it was reported that ovarian function was restored by such means in the human, in a 29 year old patient suffering from hypothalamic amenorrhea subsequent to removal of both her ovaries at age 17 (Oktay et al., 1999).

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