I-7: Human Ovarian Tissue Cryopreservation:State of The ART

In recent years, advances in the diagnosis and treatment of childhood, adolescent and adult cancer have significantly improved the survival rate and life expectancy of cancer patients. However, chemotherapy and radiotherapy treatments are gonadotoxic and may induce the loss of ovarian function and fertility with consequent premature ovarian failure (POF). A number of factors determine the level of ovarian damage caused by chemotherapy: the type of drug, administration protocol (duration and dosage) and the patient age. Cyclophosphamide and other alkylating agents are the most toxic factors for the ovary, producing a decline in primordial follicle density which is exponential as the dose increases. Moreover the effect of chemotherapy on female gonadal function is related to the patient’s ovarian reserve: younger patients are less likely to experience severe POF. Ovarian damage from radiotherapy depends on the dosage and irradiation field. In particular more than 30 Gy hypothalamic/ pituitary radiation produces impaired secretion of gonadotropins, 2-5 Gy ovarian/uterine radiation results in half of follicles being lost with consequent impaired gonadal function and more than 5 Gy abdominal radiation causes irreversible ovarian failure. Different options are available to preserve fertility in cancer patients and give them the opportunity to restore fertility and also to become mothers when they have recovered from disease. The choice of the most suitable strategy for preserving fertility depends on the type and timing of therapy, the type of cancer, patient age and the partner status. Ovarian tissue cryopreservation is an important strategy for conserving both steroidogenic and gametogenic functions and it is the only option available for pre-pubertal girls, women with hormone-dependent tumours and women who cannot delay the start of chemotherapy. Cryopreservation of ovarian tissue could be applied in different malignant diseases (systemic, extra-pelvic and pelvic) and in benign haematological, autoimmune or genetic diseases. Other benign diseases, such as ovarian endometriosis or recurrent ovarian cysts are also indications for ovarian tissue cryopreservation. Ovarian tissue can theoretically be frozen using two different approaches: as fragments of ovarian cortex or as an entire ovary with its vascular pedicle. To date, human ovarian tissue cryopreservation has so far been almost exclusively limited to avascular cortical fragments. The cryopreservation of ovarian tissue is a complex procedure because the heterogeneous cytological composition (oocytes, granulosa and stromal cells) of the ovarian tissue makes the adaptation of protocols for ovarian tissue cryopreservation difficult. Many factors influence the effect of ovarian cryopreservation, such as cryoprotectant, frozen carrier, cortical sample size and freezing procedure. The standard method for ovarian tissue cryopreservation is slow freezing/rapid thawing. This results in good preservation of all types of follicles and is the method of choice for the cryopreservation of ovarian fragments. Another method, albeit experimental, is the vitrification of cortical fragments. Vitrification is technologically promising. It is simpler and one cryocycle is less time-consuming and cheaper than the conventional freezing method. However, results have shown that vitrification can guarantee the storage of viable follicles after warming, but conventional freezing is more effective. Moreover during the vitrification procedure the tissue is cooled at an extremely rapid rate, because it is brought into direct contact with liquid nitrogen, particularly when specific “open carrier” for ultrarapid cooling are used. There is also a hypothetical risk of disease transmission through contact with accidentally contaminated liquid nitrogen. Therefore, for cryopreservation of human ovarian tissue, conventional freezing is more promising than vitrification at present. The main aim of ovarian tissue cryopreservation is to re-implant cortical ovarian tissue after thawing. Tissue is re-implanted into the pelvic cavity (orthotopic site) or a heterotopic site such as the forearm or the abdominal wall once oncological treatment is completed and the patient is disease-free, in order to restore ovarian function and normalize levels of gonadotrophins. The advantage of orthotopic transplantation is that follicles are in their natural micro-environment and can develop with fewer problems. Moreover, it allows for a natural pregnancy to occur. To date worldwide, 19 children have been born as a result of orthotopic transplantation of frozen/thawed ovarian tissue. Until now all pregnancies obtained after transplantation of cryopreserved ovarian tissue were observed in adult patients at the time of harvesting. However there is no reason to doubt the capacity of pre-pubertal ovarian cortex to develop and function correctly after re-implantation. Animal studies have demonstrated that puberty and cyclic hormonal activity can be restored by re-implantation of fresh and frozenthawed pre-pubertal ovarian tissue in both pre-pubertal and adult mice. In humans only two experiences have been reported. For heterotopic transplantation there are many unresolved issues. One of them is the optimal site for transplantation. In theory, the optimal site should be vascularised, because the rapid revascularization of the graft is crucial for the survival of ovarian follicles. In addition the site of re-implantion should be easily accessible without any invasive procedure, because repeated ovarian transplantation and/or egg retrieval may be necessary if early graft exhaustion is expected. Another concern related to transplanting ovarian tissue to the heterotopic site is the environmental factors that can affect the follicular development such as temperature, pressure, space for follicular growth, peritoneal fluid, cytokines, angiogenic factors, and hormonal milieu, so the oocyte maturation process appears to occur differently than in the orthotopic environment. The main drawback of the cryopreservation of ovarian cortical strips is the ischemia that occurs at the time of ortho/heterotopic transplantation. Because these small cortical pieces are grafted without any vascular anastomoses, they are completely dependent for their survival on the time necessary for establishment of neovascularization after grafting. Therefore, the reduction of the ischemic interval between transplantation and revascularization is essential for maintaining the viability and functional lifetime of the graft. To this end, cryopreservation of the whole ovary with an intact pedicle and vascular supply could potentially overcome this problem because reperfusion will occur immediately upon re-transplantation and anastomosis. This strategy is still experimental and no human studies of whole ovary transplant after cryopreservation have been performed to date. Ovarian tissue cryopreservation offers real hope for fertility preservation to young cancer patients and it should be offered before gonadotoxic chemotherapy in all cases where there is a high risk of POF.