Ovarian Transplantation and Cryopreservation
Ovarian Transplantation and Cryopreservation in IVF Treatment and Infertility
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As long ago as 1932 Aldous Huxley foretold the use of frozen ovarian tissue explants in human fertility for test-tube reproduction -processes which we now refer to as in-vitro fertilization (IVF) and cloning (Huxley, 1932). Since the 1950's there has been intense interest and research into the field of ovarian transplantation, spurred on by recent advances in the cryopreservation of ovarian tissue, immunosuppression technology and the use of angiogenic factors to promote the neovascularization of newly implanted tissues. Aside from the obvious application of frozen ova in the assisted reproductive technologies there is a need for those suffering from ovarian cancer or from ovarian failure, a common side effect of chemotherapy (Falcone et al., 2004). In this short review we will discuss the development and potential impact of ovarian cryopreservation upon assisted fertility and in the restoration of ovarian function following cancer treatment or menopause.
The advent and development of ovarian cryopreservation and transplantation technologies
The cryopreservation of ovarian tissue is a technique which was developed in order to bank oocytes to counter the loss of all viable eggs following a medical treatment, disease or through the aging process. Ovarian cryopreservation may be used to restore fertility and normal ovarian hormone production without the need for hormone replacement therapy (HRT). The first reported development of this technique was only a decade ago (Gosden et al., 1994), but interest in this new technology is growing rapidly.
A number of medical conditions, other than the aging process threaten to destroy all the viable eggs within a woman’s ovaries. These include ovarian cancer, and the loss of eggs through chemotherapy or radiation treatment. Whilst sperm production in men is continuous throughout life, it is commonly accepted that women are born with a fixed number of eggs which will in part determine their reproductive lifespan. Any treatment that accelerates the loss of eggs may hasten the onset of the menopause. Whilst the cryopreservation of sperm is a technology which has been in use for decades, the development of ovarian cryopreservation technology has been hampered by difficulties in conserving the cytoplasmic and nuclear integrity of unfertilized oocytes (eggs) during the freeze-thaw process, cells which are notoriously perishable. With the advent of assisted reproductive technologies such as IVF, egg or embryo freezing became possible, as eggs could be harvested following hormonal treatment at specific times of the menstrual cycle. By this means excess eggs may be collected for later use (egg banking), which particularly benefits those women who delay child-bearing until their late thirties or beyond.
Cryopreservation was demonstrated to allow long-term functioning of transplanted ovarian tissue for some two years in sheep (Baird et al, 1999), and the fertility rate after ovarian tissue cryopreservation and autotransplantation in mice was found to be approximately 50 - 75% (Nugent et al., 1997). Furthermore human ovarian tissue has been shown to be viable following xenograft transplantation into a murine model (Gunasena et al., 1997). In the next step towards clinical human trials, Virginia researchers reported that a method of transplanting ovarian tissue into the upper arm appears to preserve ovarian function, menstrual cycles and egg production in monkeys whose ovaries had been removed. The team surgically removed the ovaries from 16 monkeys and sliced them into sections. In the first group six monkeys had ovarian tissue transplanted into the inner part of their upper arms. The second group of monkeys had fat transplanted into their arms instead whilst their ovaries were frozen. After a year, this cryopreserved tissue was thawed and retransplanted into their arms. Five of the six monkeys in the first group returned to a normal, regular menstrual cycle and produced normal levels of hormones. More importantly researchers were able to retrieve mature eggs from the arm transplants, suggesting that they could be fertilized in vitro to produce offspring. Around half of the monkeys who had received cryopreserved ovarian tissue demonstrated restored ovarian function after the transplant (Schnorr et al., 2002). Although live offspring have been produced from transplanted ovarian tissue in mice and sheep, until recently this had not been achieved in monkeys or humans, although sex steroid hormones are still secreted from the tissue transplant. Lee and colleagues (2004) have recently reported the first successful transplantation of fresh ovarian tissue to a different site in a monkey, which led to the birth of a healthy female after oocyte production, fertilization and transfer to a surrogate mother. NAME OF MONKEY The ectopically grafted ovarian tissue functions without surgical connection to major blood vessels, and sets the stage for the transfer of ovarian transplantation technology to human trials. Whilst no pregnancies had been reported from a small number of early human trials which have been conducted, the ovarian transplant technology also showed promise as an alternative form of hormone replacement therapy due to continued hormonal output from the ovaries.
Recent trials shown early promise, and an ovarian transplant has produced the first human embryo by this method. Kutluk Oktay and colleagues took a strip of ovarian tissue from a 30-year-old woman with breast cancer before chemotherapy treatment diminished her ovarian function. The strip was cryopreserved using a slow freezing technique and a cryoprotectant. Six years later, after the patient had entered menopause, the tissue was retransplanted back underneath the skin of her abdomen. After some three months the tissue regained normal function and started to produce viable eggs which were fertilised in vitro, resulting in one promising embryo. Whilst the four cell-stage embryo did not produce a pregnancy, this was not deemed unusual, as the chances of a successful implantation of a four-cell stage embryo after routine IVF is at best only one in ten (Oktay et al, March 2004a). This recent clinical development indicates that endocrine function and fertility may be restored by long-term cryopreservation of ovarian tissue retransplanted back into post-menopausal women.
The Science of Ovarian Cryopreservation and Transplantation
The cryopreservation of ovarian tissue begins with laparoscopy or mini-laparotomy. Normally only one ovary is removed to allow normal ovarian hormone production from the other ovary. The egg-containing ovarian cortex is sliced thinly and the tissue slices are cryopreserved at -196°C using specialized cryoprotectants such as dimethylsulphoxide and freezing equipment designed to produce a slow and controlled freezing process.
When clinical circumstances are conducive, retransplantation of the ovarian tissue strips can be attempted, as frozen ovarian slices have a high survival rate after thawing. Several sites may be chosen for the location for ovarian tissue retransplantation, including the abdomen near the fallopian tube to allow natural ovulation and conception, a technique which has allowed natural conception to occur in all successful animal studies to date. The ovarian tissue begins to function again within several months of transplantation, although intra-abdominal transplantation affords only limited access and potentially less viable tissue, as neovascularization occurs primarily from only one side of the tissue and scar tissue forms during the recovery period following surgery. Transplantation of human ovarian tissue into the forearm results in follicle formation and permits egg retrieval with a needle. As the ovarian tissue in the forearm becomes well-vascularized it may recover function more rapidly and show long-term functioning. However transplantation of ovarian tissue into the forearm precludes natural conception, and requires assisted reproduction with IVF technology using eggs retrieved from the arm.
Potentially thousands of follicles may be successfully transplanted within a single slice of ovarian cortex, although the period of time for which the ovarian transplant remains viable remains to be determined. In every cycle of ovarian stimulation the ovarian tissue is first induced with a combination of follicle-stimulating hormone (FSH) and human menopausal gonadotropin, and the final oocyte maturation may be induced by recombinant human chorionic gonadotropin (hCG). Oocytes produced by this technique were retrieved some 36-40 hours later and intracytoplasmic sperm injection was carried out (Oktar et al., 2004b).
Ovarian transplantation and cancer
Ovarian failure is a frequent consequence of high-dose chemotherapy and radiotherapy treatments for cancer, which are more effective but may severely damage the ovarian follicular store, compromising the fertility of young patients surviving cancer. The process involves removing and freezing pieces of the patient’s ovarian tissue prior to chemotherapy. After chemotherapy the ovarian tissue can be transplanted back to help restore ovarian function (Torrents et al., 2003). Thus cryopreservation and transplantation of ovarian tissue has become a promising alternative in the prevention of the loss of fertility in these patients, and slices of animal and human ovarian tissue have been shown to survive the cryopreservation process. After successful transplantation, follicular development and restoration of hormone secretion have been observed in both animal and human studies (Torrents et al., 2003). Studies are ongoing to optimise cryopreservation methods for ovarian tissue, and in the future, patients may also have the ability to "bank" ovarian tissue for future use in assisted reproduction technologies (such as IVF) or for alternative hormone replacement therapy (HRT).
Other techniques
Cryopreservation of unfertilized eggs is an alternative that has gained interest with newer freezing techniques, and there have already been success achieved through this approach. An Italian group has achieved success in the freezing and thawing of oocytes leading to the birth of healthy children (Fabbri et al., 1998).
However many cancer patients begin their cancer treatment before IVF treatment can be completed, and many are young or unmarried and therefore do not have a partner to provide sperm to fertilize the eggs and IVF which uses hormone regimes to produce many follicles is medically inappropriate for many women with hormonally responsive tumors such as breast cancer. In vitro maturation, where the women requires no hormonal stimulation and the developing egg are cultured in the laboratory may be a choice for many.
Future Directions
One potential alternative involves the growth of ovarian tissue from one species within another species (as a xenograft, e.g. Nisolle et al., 2000). This technique has shown early promise, and even patients with ovarian cancers may be able to use this technique to recover normal eggs without the risk the restoration of any associated cancer cells. Furthermore this technique allows the more efficient maturation and retrieval of eggs for IVF. Moreover the limited quantity of ovarian tissue allows strips to be unfrozen gradually over time to allow additional attempts to produce offspring. Even small biopsies of tissue, if proven adequate for IVF, may be taken to create an egg bank for later use.
Soon advances in the cryopreservation of excess eggs harvested from hormonal treatment cycles may be used to develop general use egg banks in the same way as sperm banks are used now. As embryos derived from cryopreserved unfertilized eggs have developed into normal healthy children, and the number of successful births clearly exceeds that currently achieved through ovarian tissue cryopreservation, this may become the preferred technology in assisted fertility treatment. Ovarian hormone suppression using gonadotropin releasing hormone agonists during chemotherapy significantly protected human eggs in one research study. Gonadotropin releasing hormone agonist administration is inexpensive, relatively safe and unlikely to compromise other treatments. As a last resort, donor eggs, for those women who have irreversible ovarian failure, have provided very high rates of successful pregnancies.
There are of course a number of ethical issues raised by these new technologies. For instance the use of egg banks to allow mothers to delay child rearing until middle age, and recent advances in ovarian cryopreservation and transplantation which make it theoretically possible to restore fertility after the menopause, augurs the introduction of an unprecedented generation gap. These technologies raise the moral dilemma of delaying child-bearing for socioeconomic reasons, and the immense economic costs and health risks associated with delaying reproduction until middle age. Further it remains to be established whether the use of frozen eggs from banks is completely safe, due to the chemical treatment of the eggs during the freezing process. Such advances may lead to further advances in ovarian cryopreservation technology so that ovarian tissue transplants may one day function for a reproductive lifetime.
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