The Freezing of Human Oocytes (Eggs)
Michael J. Tucker PhD FIBiol
Georgia Reproductive Specialists, Atlanta Georgia 30342
Outline of Chapter:
- Overview of human egg (oocyte) freezing (cryopreservation), and what is currently feasible
- Discussion of the merits and limitations of currently available egg cryopreservation protocols
- Practical issues of cryopreservation: egg selection; how the eggs are used after thawing; and who might actually be helped by this technology
- The future and what alternative cryopreservation technologies might be on the horizon in the next few years.
It is worthwhile becoming comfortable with the technical terms for several of the words that will be used interchangeably in this chapter, and in case you wish to make more comprehensive searches of the literature, such explanation of terms will improve your level of understanding. Firstly, the rather loose term egg is usually referred to as an oocyte by biologists, which is the unfertilized female gamete (sex cell). The sperm(atozoon) is the male counterpart gamete. Cryopreservation is the very specific term for all stages involved in the cryostorage of the oocyte, and refers to the freezing, storage and the thawing processes. Cryoprotectant is the term used to refer to the fluid that is used to place the eggs in prior to freezing, and is usually a mixture of sugars and organic chemical liquids that are designed to buffer the egg cell during the stresses of freezing and thawing.
The last few years have seen a significant resurgence of interest in the potential benefits of human egg freezing. In essence these potential benefits are as follows:
To put egg freezing into context, it is interesting to consider that human sperm (spermatozoa) have been successfully frozen for decades, and that the first successful report of human embryo freezing that generated a pregnancy was in 1983. Subsequently both human sperm and embryo cryopreservation have become considered routine and consistent technologies. Frustratingly human egg freezing has not yet reached such apparent levels of acceptance or consistency.
- 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 harmful to such tissue, which may threaten their reproductive health; e.g. prior to cancer treatment by chemotherapy.
The technology so far applied clinically has been based directly on traditional human embryo cryopreservation protocols, and has produced relatively few offspring when compared with human embryo cryopreservation. 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 chromosomal apparatus in these cells (meiotic spindle), and so increase the potential for chromosome abnormalities (aneuploidy) in the embryos that arise from such eggs. With respect to cryostorage of donated oocytes, for use eventually as eggs to donate to recipient women, there have been several reports that have shown some success with this approach. In fact, it has been reported that there have been 10 babies born from frozen-thawed donor oocytes. In another unusual case, frozen donor eggs after thawing have been used, not for whole egg donation, but actually for ooplasmic transfer which gave rise to the successful delivery of a twin following thawed ooplasmic donation. In this procedure the cytoplasm of the egg was injected into the eggs of another woman. The cytoplasm is the part of the egg outside of the nucleus that does not contain the genetic elements (chromosomes), but contains elements of cellular functioning. Transfer of this is thought to bolster the quality of the recipient egg making it healthier, and possibly more likely to give rise to a healthy embryo.
The first successful cryostorage of women’s own oocytes occurred with the reporting of three births over a decade ago by two centers in Australia and Germany. However, at that time reports of egg freezing studies in mice suggested that although eggs could survive freezing and thawing, they might possess higher levels of chromosomal anomalies following this procedure when compared to fresh eggs. Of note, is the fact that these studies were not performed on human tissue, and the procedures used were totally different from that utilized with the successful human egg freezing cases.
Nevertheless, the suspicion of this problem was enough to prompt a sort of voluntary worldwide moratorium on clinical oocyte cryopreservation, until studies prove the fear of chromosomal abnormalities to be unsubstantiated. Unfortunately for oocyte freezing research, human embryo cryopreservation was just starting to be undertaken much more routinely and successfully in the mid-eighties, due to the growing ethical concerns with the fate of surplus embryos following in vitro fertilization (IVF). This presented a pressing clinical problem that drove embryo freezing research, while egg freezing languished without any clear clinical need, worsened still by the worries over its safety.
Sporadic research reports continued to investigate egg freezing principally in animal models, and occasionally in the human. However, these reports tended to underline the complications and lack of consistency between cross-species comparisons. For example, while the mouse can be a useful model it must be remembered that its eggs are only just over half the volume of the human egg, and this can have a major impact on the approach to cryopreservation in these two very different species cell types. Eventually though, driven by a series of papers published by an Australian researcher called Debbie Gook from Melbourne, clinical application of egg cryopreservation began to find favor by the middle of the nineties.
Recently then, the early successes have been reproduced by others in both Italy and the USA giving rise conservatively to 10 babies from the freezing of women’s own eggs. Also at least one other baby has arisen from a clinical circumstance that is not completely unfamiliar to IVF clinics. In this case eggs had been collected during a routine IVF case, but no sperm were retrievable for insemination. So the oocytes were frozen, and donor semen was selected for a later IVF attempt. Ultimately both sets of gametes were thawed and used in a subsequent IVF procedure, which achieved a health delivery. This rather specific area of application is of great potential benefit to infertile couples undergoing IVF therapy where perhaps problems arise unexpectedly necessitating a halt to treatment prior to insemination of the eggs with sperm. For example, in another actual situation where an IVF couple suffered an untimely death in the family at the time of their procedure, they then chose to freeze all the eggs that had been retrieved until such time as they felt able to move forward with their therapy.
All of these pregnancies and reports arose from work with frozen-thawed mature oocytes (see Figure 1 “Two Mature Oocytes”), but for one notable exception, where a pregnancy arose from an immature germinal vesicle (GV) stage egg (see Figure 2 “Germinal Vesicle Immature Egg”). This may not sound to be of such great importance, but it could be that this stage of egg development may prove to be a more successful approach for egg cryopreservation. Such “young” eggs are approximately one to two days away from full maturity, and as such require further growth in the laboratory in culture after thawing. They currently appear as a by-product of less than optimal ovarian stimulation for IVF where not all eggs collected are mature. However, they survive freezing well, and possess certain features that help to maintain their integrity during the rigors of cryopreservation. For example, their membranes are more permeable to the cryoprotectant (“antifreeze”), and their chromosomes are more conveniently and safely packaged in the nucleus protecting them from disruption. Such eggs, however, still have to undergo nuclear breakdown and full maturation before they can be fertilized, and therefore their developmental competency is not so clearly established as with fully mature oocytes that are frozen. The source of these GV eggs, and whether they have been exposed to any external hormones may play a key role in the competency of these eggs. Harvesting of these eggs and the conditions for maturation remains to be resolved fully. But provisional studies in this area are the first to lend credence to the possibility that immature follicles and the immature eggs inside isolated from ovarian tissue, may one day be fully grown in the laboratory outside of the body.
How does Cryopreservation work, and what are the present limitations?
Whether eggs are mature or not, standard cryopreservation technologies appear to have their ultimate limitations not only in terms of cryosurvival (% of eggs that are alive after thawing), but also more importantly in their lack of consistency. 50% cryosurvival may be an adequate overall outcome and is now commonly reported, 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 freezing protocol may provide the answer to increased consistent success. Different approaches have been applied, and include replacing the principal salts in the freezing solutions in an attempt to help reduce the stresses on the egg membranes during cryoprotectant exposure. This has provided significant improvements in mouse egg freezing, though it has yet to be applied clinically in the human.
Traditional freezing protocols aim to achieve several things in a minimal amount of time. Firstly, the human egg is the largest cell in the human body, and as such is full of water. This is problematic as this water will turn to ice upon freezing, and will disrupt the egg’s internal and membrane structure. Therefore cryoprotectants (“antifreezing solutions”) are used to replace this water to reduce ice formation. The issue is that it takes time to replace the water with these liquid chemicals by diffusion across the egg membrane. These membranes, especially in the mature human egg, are notoriously impermeable to such agents, in particular the cryoprotectants with large molecular sizes. Thus the exposure time of eggs to these chemicals before freezing can be long to achieve good levels of water replacement, but the dilemma is that these cryoprotectants tend to be cytotoxic! As such they will tend to kill the egg if exposure is too long at “high” temperatures (4 to 37oC). So cryobiology is all about compromise: how to remove as much water as quickly as possible, without toxifying and damaging the egg. Notably, mature eggs are both the largest and the least permeable of all eggs having the smallest surface to volume ratio, and this explains the appeal of investigating the freezing of less mature eggs that reach equilibrium with cryoprotectants faster having less water to replace.
Alternatively, traditional cryopreservation protocols which impose “slow” cooling rates of about minus one to two degrees Celsius per minute might be replace by more ultrarapid freezing technology. Vitrification refers to a form of cryopreservation where cooling rates are so rapid (>minus 20,000o/minute) that ice does not have a chance to form, and the mixture of cryoprotectant and egg forms a “glass-like” gel. The first reports of success with this approach have very recently come from Italy and South Korea. From a practical standpoint, vitrification is very simple and actually removes the need for the expensive programmable controlled-rate freezers currently used to freeze egg and embryos.
While the overall aim of egg cryopreservation is to get the egg to survive upon thawing, certain damage or consequences of the procedure may not kill the cell but may render it less viable. A major issue is that eggs do not fertilize well after thawing. This is due to the partial disruption of the membrane which causes a block to the conventional fusion and penetration of sperm with the egg surface. So artificial forms of assisted insemination have to be used to achieve acceptable fertilization outcomes with thawed eggs. This procedure is referred to as intracytoplasmic sperm injection (ICSI), and is a very commonplace procedure in all infertility centers worldwide. It involves the direct injection of a single sperm into an egg, thereby avoiding most of the usual barriers to fertilization (see Figure 3 “ICSI of a Mature Egg”). Originally this procedure was designed specifically to treat male infertility where sperm count is low and sperm functioning is poor. It is now applied to many cases of IVF where fertilization needs to be maximized. Indeed, some IVF centers only undertake insemination by ICSI.
Other Forms of Egg Freezing
All the situations discussed so far address egg freezing in the context of retrieval of individual eggs and freezing these as separate units. It can be difficult to isolate and collect individual eggs near full maturity by aspiration of follicles in the ovary without the aid of current IVF technology. This involves the use of relatively large amounts of “fertility drugs” (gonadotropins) to stimulate growth of a whole group of maturing oocytes, which are then collected using a fairly simple surgical procedure while under sedation. It is usual, for example, after a couple of weeks of monitoring and drug injections for say a 32 year old woman to have up to 20 to 30 eggs collected in one go. These eggs can then be processed for cryopreservation, and may allow between one to three attempts at IVF after thawing, assuming the eggs survive adequately, and that they fertilize normally and develop successfully as embryos. Embryos can be and have been frozen following earlier egg cryopreservation, and may improve overall utilization of the original frozen eggs. The majority of investigations to date have used eggs from this source. There is no reason to suspect that such frozen eggs cannot survive in that state for many years without harm, assuming they remain cooled in liquid nitrogen at minus 196oC. However, the most plentiful source of oocytes potentially is the ovary itself, containing as it does many thousands of primordial follicles in its outer layer (cortex). Earlier successful work with cryopreservation of rat ovarian tissue has led the way to successful cryostorage of both sheep and human tissue in the last few years. Up to 80% survival of the egg containing follicles has been reported, but the real 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 cancerous cells, and therefore may not be safely used for grafting back into such a woman if she were to survive post-cancer therapy. So the tissue might be screened before or after thawing for the presence of malignant cancerous cells to allow some assessment of the safety of this approach.
The alternative to grafting thawed ovarian tissue straight back into the body, is to take the tissue and culture it instead in the laboratory for an extended period of time may be even as long a six to eight weeks to grow very young follicles all the way to full maturity when they are ready to ovulate mature eggs. The steps to achieve this are only just now being investigated and may take some years to resolve. A part way step, would be to take the human tissue and grow it in a host animal (e.g., mouse or rabbit) until such time as in vitro maturation of such tissue could be undertaken more effectively in the lab during the last few days/weeks of follicle/egg maturation. So far in research studies, extended culture of the very immature ovarian follicles to get the eggs to full maturity with subsequent embryonic development and birth, has only been recorded in the mouse, and this was not from cryopreserved ovarian tissue. Early studies are being undertaken in the human to achieve this, though there is much to be done. Where grafting post-thaw has been undertaken, fertility has been restored in sheep, which is a good model for the human ovary, 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. Amazingly, it was very recently reported that ovarian function was restored by successfully grafting ovarian tissue in a human. This was in a 29year old American woman suffering from hypothalamic amenorrhea subsequent to removal of both her ovaries for persistent cysts at age 17.
The Future of Egg Cryopreservation
The multiple potential routes for cryostorage of the female gamete makes for a confusing vision of where clinical applications might occur. However, different clinical needs may actually be met by differing technological approaches, whether they incorporate whole ovarian tissue freezing, separate ovarian follicle storage, or cryopreservation of mature or near-mature oocytes themselves.
It is probable that oocyte cryopreservation will slowly enter the mainstream of techniques in Assisted Reproductive Technologies (ART) in humans, most likely in the area of oocyte donation. Here information, in terms of clinical success of protocols, is generated within months not years, as would be the case with freezing of eggs for single women concerned with their future reproductive choices. In accepting that cryopreservation of human eggs and embryos seems here to stay, in remains important that we research the consequences of these therapies carefully to ensure that we truly do no harm. With respect to this, there is increasing proficiency in the area of egg and embryo genetic screening through micro-biopsy procedures that analyze in particular the chromosomal status of these microscopic entities. This is of particular relevance for thawed eggs, and embryos arising from them, ultimately permitting screening of all embryos that come from cryopreserved female gametes. Indeed, extended culture of human embryos in vitro in the lab for up to six days to the blastocyst embryo stage (see Figure 4 “Blastocyst: A Human Six Day Old Embryo”) acts as a form of non-invasive screen of both the embryo’s viability and potentially its chromosomal normality. This is the latest stage to which an embryo can be grown outside of the human body before it is ready actually to implant into the uterus. As human in vitro fertilization in general becomes more consistent, the desire to minimize the production of excess and unwanted human embryos will increase. The need to reduce embryo production initially may prompt the limited insemination of fresh oocytes, with the surplus being stored frozen as eggs for future use, rather than freezing surplus embryos after fertilization. This is already being undertaken by some couples who have ethical and moral objections to embryo freezing.
Intriguingly, the reason why the research and development of egg cryopreservation may have been so retarded to this point is possibly due to the difficulty of finding an easy clinical area of application. As the initial concerns with egg freezing are being eroded by better understanding of the principals of cryopreserving such large and delicate cells, and by better technology, there becomes less and less reason not to start taking this form of tissue storage more seriously. Specific clinical niches are now appearing, such as creating effective donor egg banks to allow responsible quarantine of donated material (a possibility not currently available with fresh human egg donation), and also it is hoped to reduce the costs of egg donation. Additionally, with technologies available for consistently freezing parts of or whole ovaries, it is feasible that routine ovarian biopsy should eventually be available to any woman of reproductive age about to undergo treatment that may be detrimental to her ovarian functioning, or even as an effective option for young women wishing to store some of their “young eggs” for use in later life. This would allow cryopreservation of this tissue for future resuscitation of women’s fertility by either grafting of this tissue directly back into the body, or by extended growth of this tissue in the laboratory to grow the eggs entirely outside of the body.