Ovarian tissue banking for cancer patients: is ovarian cortex cryopreservation presently justified?

Ariel Revel1 and Joseph Schenker

Department of Gynecology, Hadassah University Hospital, Kiryat hadassah, POB 12000 Jerusalem, Israel

1 To whom correspondence should be addressed. e-mail: revel{at}md.huji.ac.il


    Abstract
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The effect of chemotherapy and radiotherapy on future fertility is of concern to patients and their families. Whereas sperm banking is commonly performed, female gametes are not so amenable to cryopreservation. One alternative includes postponing cancer treatment to enable ovulation induction and oocyte aspiration. Whenever possible, retrieved oocytes should be fertilized in vitro prior to cryopreservation. Frozen embryos could serve to produce pregnancies if ovarian failure occurs. Donor sperm can be offered to single patients, as frozen–thawed unfertilized oocytes yield poor pregnancy rates. Ovarian cortex cryopreservation should still be considered an experimental technology as no pregnancies have been obtained in humans. Therefore, ovarian cortex banking should be used only for young girls, adolescents and when IVF is contraindicated. Reattachment of ovarian vasculature could prevent ischaemic follicular loss and enable ovarian transplantation in the future. This procedure is currently under investigation in animals. At the present time, we recommend urgent IVF in most patients requesting fertility preservation. Ovarian cryopreservation should be offered when emergency IVF is not possible.

Key words: cancer therapy/cryopreservation/oocyte/ovarian tissue/primordial follicle


   
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New advances in medicine usually bring with them hope. However, they also bring new risks. The history of medicine has demonstrated that with almost every new treatment, whether medical or surgical, new complications arise. Thus, a ‘pendulum effect’ can be observed. At first, a problem is addressed by an original solution. At this stage, the idea’s pioneers are its advocates and present it with enthusiasm to patients and colleagues. The next phase is the widespread acceptance of this new idea, followed by a rapid increase in the demand for this procedure or treatment. After a while, one can look back and reflect whether this innovation has actually helped patients or not. Objective parameters of clinical results are reported. As often is the case, flaws and complications are observed and the preliminary enthusiasm subsides.

The enormous advances in oncology, haematology and bone marrow transplantation (BMT) have resulted in a 90% remission rate in various diseases (Green et al., 1998Go; Patte et al., 2001Go; Nachman et al., 2002Go). A common concern of female patients and their families is the effect of chemotherapy and radiotherapy on future fertility (Levitt and Jenney, 1998Go). It should be stressed that some cancer treatments result in 99% ovarian failure (Mertens et al., 1998Go). An 8-fold increase in the risk of premature ovarian failure (POF) has been observed in cancer survivors (Byrne et al., 1992Go). Whereas menopausal symptoms and signs can be treated medically, no solution is available to preserve female gametes.

This situation is the basis for the current enthusiasm and interest in ovarian cortex banking, especially since it could be applied to children and teenagers. We observe that ovarian cryopreservation gives some hope to young patients and their families during a very stressful time. Although ovarian cortex preservation is fraught with practical, ethical, moral and legal dilemmas (Robertson, 2000Go), we hope that the continuing research in this field will enable us to apply this technique clinically within 10–15 years, when young patients will have been cured and express a desire for pregnancy. Surprisingly, ovarian tissue banking is accepted worldwide although we are still awaiting the first human baby to be born from an oocyte matured from frozen ovarian cortex. Although engaging in ovarian banking may be a personal choice for patients with no other option who desperately want to preserve fertility, it is the responsibility of the medical and scientific communities to prevent misunderstanding and misuse of this emerging technology. The ethical question regarding the justification of surgery for harvesting and freezing ovarian tissue without a reasonable assurance of success in transplantation should be discussed (Kim, 2003Go).

Ovarian banking is now at the high point of the pendulum trajectory. We would like to express our opinion that unless an efficient technique is reported for the use of frozen ovarian cortex, we would expect the pendulum to swing back. This opinion is presented following our experience in ovarian banking. At our institution, we have banked ovarian tissue since 1996 from 54 patients. All these patients were carefully selected following haematological and gynaecological assessment. This method has been applied to prepubertal adolescents and adult females. Our series includes female patients 10–37 years old. Sixteen of our patients were under 20 years old. Indications for ovarian banking in our series included sarcoma, carcinoma, lymphoma, leukaemia and patients prior to BMTs.

POF is a common long-term consequence of chemotherapy (Meirow, 2000Go). Whereas the cytotoxic-induced damage is reversible in other tissues of rapidly dividing cells such as bone marrow, gastrointestinal tract and thymus, it appears to be progressive and irreversible in the ovary, where the number of germ cells is limited, fixed since fetal life, and cannot be regenerated. The medical approach to female fertility preservation entails temporary induction of a prepubertal hormonal milieu, such as the use of GnRH analogues or the use of anti-apoptotic agents to hinder loss of ovarian follicles.

The rationale for the use of GnRH analogues for fertility preservation is based on follicles being functionally deprived of gonadotropins. This halts follicular recruitment from the quiescent to the chemotherapy-sensitive pool. Animal studies show that GnRH analogues inhibit cyclophosphamide-induced ovarian follicular depletion in rats (Bokser et al., 1990Go; Ataya and Ramahi-Ataya, 1993Go) and rhesus monkeys (Ataya et al., 1995Go). Although previous data showed an absence of GnRH receptors in human gonadal tissue (Clayton and Huhtaniemi, 1982Go), newer data show that GnRH receptors expressed by human ovarian cancer cell lines mediate the anti-proliferative effects of GnRH (Volker et al., 2002Go). Protective effects of GnRH analogues in patients treated by cyclophosphamide-based chemotherapy for lymphoma (Blumenfeld et al., 1996Go) and systemic lupus erythematosus (Blumenfeld et al., 2000Go) have been reported. In contrast, a randomized controlled study, using intranasal buserelin before chemotherapy, failed to preserve fertility in patients with Hodgkin’s disease (Waxman et al., 1987Go). At present, there is no consensus about the efficacy of GnRH analogues in preventing chemotherapy-induced POF. Adequately controlled studies are still needed to define the efficacy of GnRH analogues in preventing ovarian failure in cancer patients exposed to chemotherapy.

Innovative treatment against oocyte destruction by cancer therapy includes a report on in-vivo therapy with sphingosine-1-phosphate (S1P), an inhibitor of apoptosis, which completely prevented radiation-induced oocyte loss in mice (Morita et al., 2000Go). Currently, no other animal studies or human clinical trials have been reported using anti-apoptotic therapy to protect ovaries during cancer therapy. Moreover, an anti-apoptotic approach will face the difficulty that apoptosis is necessary in the process of cancer treatment.

Every hospital or clinic treating male cancer patients should propose sperm banking. Combined with ICSI, sperm freezing ensures preservation of fertility in practically all male cancer patients. Investigators treating female cancer patients have attempted cryopreservation of embryos, unfertilized oocytes and ovarian cortex, and, recently, intact ovary cryopreservation.

Women may be offered IVF for embryo cryopreservation prior to chemotherapy. This approach is the most successful one as pregnancy rates following embryo cryopreservation are reasonable, although lower by one-third when compared with fresh IVF embryo transfer (American Society for Reproductive Medicine/Society for Assisted Reproductive Technology, 2002Go) and are standard in IVF units since 1983 (Trounson and Mohr, 1983Go). IVF should be offered prior to chemotherapy and not afterwards, since diminished ovarian response was reported in women who underwent IVF after systemic cancer treatment (Ginsburg et al., 2001Go). Although women with significant systemic disease are reported to have poor ovarian response to ovulation induction (Pal et al., 1998Go), most cancer patients can expect an average of 11 embryos cryopreserved (Ginsburg et al., 2001Go). Since a male partner is required for IVF, this option is inappropriate for single women who reject the use of donor sperm. In addition, ovarian stimulation requires deferment of chemotherapy and could be hazardous to estrogen-sensitive malignancies (e.g. breast carcinoma). A sufficient number of viable embryos cannot be guaranteed, and a repeat IVF cycle may be contraindicated. Finally, ethical issues such as the fate of stored embryos, should the patient die, must be resolved at the outset of treatment.

Oocyte cryopreservation would be an ideal technique for fertility preservation in single patients. Its advantages include no need for a partner, which avoids legal, regulatory and religious dilemmas. Nevertheless, this method has a very low success rate. Although attempted for more than a decade (Chen, 1986Go), only a few pregnancies have been achieved from frozen–thawed human oocytes. While the introduction of new cryoprotective agents (Boldt et al., 2003Go) and ICSI, as a means of bypassing the zona pellucida, has improved pregnancy rates of cryopreserved oocytes, pregnancies and deliveries derived from cryopreserved human oocytes have only resulted in a success rate averaging 2% per frozen oocyte (Porcu et al., 1997Go, 2000Go; Fabbri et al., 2001Go). The disappointing success rates can be attributed, in part, to the human oocyte’s biological properties (Fabbri et al., 2001Go).

Three major alternatives have been proposed in order to prevent chilling damage to oocytes: cryopreservation of immature oocytes, oocyte vitrification and ovarian cryopreservation. Banking of germinal vesicle (GV) stage oocytes involves cryopreservation at prophase I before oocytes resume nuclear maturation and progress to metaphase II. At this stage, a reduced risk of cytogenetic errors is theoretically expected since the chromosomes are not aligned along the spindle. Membranes of oocytes at the GV stage are, however, more sensitive to chilling injury (Arav et al., 1996Go). Vitrification refers to a form of cryopreservation where cooling rates are so rapid (>20 000°/min) that ice does not have a chance to form, and the mixture of cryoprotectant and oocyte forms a ‘glass-like’ gel. From a practical standpoint, vitrification is simple and removes the need for expensive programmable controlled-rate freezers (Fahy et al., 1984Go; Arav, 1992Go). Following the first case report of pregnancy after oocyte vitrification (Kuleshova et al., 1999Go), eight more pregnancies were reported (Katayama et al., 2003Go; Yoon et al., 2003Go). The need for high concentrations of cryoprotectants and the paucity of human clinical data require caution in the use of vitrification in clinical practice. A 2–4 month delay in cancer treatment would be required to obtain enough oocytes to enable a single pregnancy. Thus, this method cannot, at present, be offered to cancer patients.

Human ovarian tissue banking is proposed as a method of preserving female fertility and offers the potential of restoring normal ovarian function and natural fertility. This procedure is applied in several medical centres worldwide. Indications for ovarian tissue cryobanking include premenarcheal girls as well as young women facing POF (Donnez and Bassil, 1998Go; Donnez et al., 2000Go). Cryopreservation of ovarian cortex rich in primordial and primary follicles is a strategy for oocyte banking. The rationale is to cryopreserve immature follicles within the ovarian tissue, before oocytes resume nuclear maturation. The main advantage of this technique is that no ovarian stimulation is required and thus the procedure can be performed on an urgent basis. Moreover, small immature follicles in the ovarian cortex probably withstand cryopreservation better than mature ones (Hovatta et al., 1996Go, 1997Go; Newton et al., 1996Go; Oktay et al., 1997Go). This has led to interest in the procedure as a potential strategy for preserving the fecundity of patients at risk of POF (Newton, 1998Go; Oktay et al., 1998Go). In order to obtain ovarian cortex, laparoscopic biopsies (Meirow et al., 1999Go) or unilateral oophorectomy (Radford et al., 2001Go) can be carried out at any stage of the menstrual cycle. Women electing ovarian preservation at the time of abdominal surgery for other indications will incur no further risk from the operative procedure. In most cancer patients, laparoscopic oophorectomy does not incur a special surgical risk. Moreover, in many cases, this general anaesthesia is used as an opportunity to perform a bone marrow aspiration or to insert a porthacath for the administration of chemotherapy. The technical method of ovarian cortex cryopreservation has been well described (Newton et al., 1998Go).

Contraindications for ovarian cryopreservatrion include patients at high surgical risk and those >40 years old (Oktay, 2002Go). In order to prevent possible transmission of disease by ovarian grafts, we routinely send samples for pathological and immunohistochemical analysis. In patients with cancer involving the ovary, transplantation should not be performed since this may result in transferring malignant cells back to the patient (Shaw et al., 1996Go; Meirow et al., 1998Go). Xenotransplantation of human ovarian cortex transmitted cancer in Hodgkin’s disease and leukaemia but not in non-Hodgkin’s lymphoma (Kim et al., 2001Go). Frozen–thawed human ovarian tissue xenotransplanted into nude mice enabled follicular maturation and oocyte retrieval (Revel and Laufer, 2002Go).

Ideally, frozen–thawed ovarian cortex should be used for obtaining oocytes by laboratory methods. In-vitro folliculogenesis entails harvesting mature oocytes in vitro from frozen–thawed ovarian cortex by isolating small follicles (Smitz and Cortvrindt, 1999Go) or oocytes (Abir et al., 1997Go) from the surrounding stroma and growing them to maturity. Oocyte in-vitro maturation (IVM) is currently feasible only in the latest stages of follicular development and requires a lot of optimization before widespread clinical implementation. Freshly aspirated GV stage (prophase I) oocytes can be matured successfully in the laboratory to re-initiate and complete the first meiotic division to metaphase II, including accompanying cytoplasmic maturation and fertilization. This has resulted in the achievement of pregnancies and live births in polycystic ovary syndrome (PCOS) patients (Cha and Chian, 1998Go). It should be remembered, however, that the capacity for oocyte maturation will be significantly lower when cryopreserved immature (GV stage) oocytes are used. With the advancement in IVM of primordial follicles (Vand Hur et al., 2000Go), one could envisage the possibility of safely obtaining oocytes from cryopreserved ovarian cortex. Oocytes competent for meiotic maturation, fertilization and implantation could develop in vitro from primordial follicles. However, oocyte development in vitro beginning with the primordial follicles is a complex and prolonged process which includes follicular recruitment, tonic gonadotropin growth and gonadotropin-dependent stages. The first live offspring produced proved that development of oocytes in vitro from the primordial follicle stage is possible (Eppig and O’Brien, 1996Go). A recent revised protocol presents a significant advance in oocyte culture technology (O’Brien et al., 2003Go).

Although human ovarian banking has been performed for several years, no ensuing pregnancy has been reported. However, the vast majority of patients who have banked frozen ovarian cortex are either still receiving active treatment for their disease, or have just recently completed it. Other patients are still too young or they have not met the man with whom they wish to have children, or they may sadly have died. None of the patients who has cryopreserved ovarian cortex at our institution has asked to transplant it. Thus, the potential of human ovarian cortex to enable resumption of fertility is based on animal data and only few human reports.

Fresh orthotopic autologous ovarian transplantation in humans was described almost a century ago. More recently, however, ovarian autotransplantation to its original site (orthotopic) or a different site (heterotopic) has been reported using pieces of human frozen–thawed ovarian cortex. Orthotopic autotransplantation of frozen–thawed ovarian cortex was reported in two patients, with temporary function resulting (Oktay and Karlikaya, 2000Go; Radford et al., 2001Go). Hetero-transplantation entails transplanting the ovarian grafts to a site other than the ovarian pedicle. The main advantage is the ease of transplantation and oocyte retrieval. Two patients (cervical carcinoma and benign ovarian cysts) were reported to have been treated by ovarian transplantation to the forearm (Oktay et al., 2001Go). The grafts appeared to be functional for a few months. Short-term reproductive function was also reported after fresh (three patients) or cryopreserved (one patient) heterotopic ovarian transplantation in women (Callejo et al., 2001Go).

Although human data do not substantiate fertility resumption after ovarian transplantation, animal data have demonstrated more success. Ovarian transplantation has been tested with various animal models (Parrot, 1960Go; Gosden et al., 1994Go; Harp et al., 1994Go; Aubard et al., 1996Go, 1998Go; Cox et al., 1996Go; Gunasena et al., 1997Go; von Eye Corleta et al., 1998Go). Xenotransplantation of mouse ovarian cortex has enabled pregnancy (Snow et al., 2002Go). The main obstacle to the wide application of ovarian cortex cryopreservation is that the majority of transplanted follicles are lost by ischaemic damage by the time sufficient neoangiogenesis has supplied the graft. A significant fraction of the follicles are lost, however, during the ischaemic phase present until neovascularization takes place (Newton et al., 1996Go; Candy et al., 1997Go; Gunasena et al., 1997Go). Ischaemic damage is more pronounced in frozen–thawed than in fresh grafts (Nisolle et al., 2000Go). However, the majority of ischaemic damage observed in frozen–thawed avascular grafts occurs after transplantation, confirming that grafting procedures are more deleterious for follicle survival than cryopreservation (Aubard et al., 1999Go). Thus, optimal conditions have not yet been established and the success of ovarian tissue transplantation depends on the ability of the graft to support oocyte maturation and ovulation.

It appears that ovarian slices can convey only short-term function due to loss of most follicles by ischaemia. Ovarian transplantation preserving blood vessels could prevent ischaemic follicular loss. Lessons from nature demonstrate the feasibility of intact ovary freezing. One such example is the Canadian wood-frog (Rana sylvatica) which undergoes slow cooling and freezes for 6 months of winter (Storey and Storey, 1986Go). Allografting vascularized rabbit ovaries with their oviducts by microsurgical techniques resulted in a vascular failure rate of only 5% of 40 grafts (Green et al., 1982Go). Intact ovarian freezing and transplantation preserving blood vessels has been reported recently in rats (Wang et al., 2002Go) and sheep (Revel et al., 2001Go; Jeremias et al., 2002Go). Until intact ovaries are successfully transplanted in humans, this technique should also be considered an experimental one.

Since the mature follicles present on the ovarian cortex will be lost by chilling damage, we have recently proposed aspirating them for oocyte cryopreservation (Revel et al., 2003Go). The aspirated oocytes are matured in vitro and fertilized, or are frozen unfertilized.

Extending the application of the technology beyond fertility preservation in cancer patients presents an intriguing question. The changes in lifestyle in Western culture have resulted in women postponing their first pregnancy attempts to beyond age 35, leading to difficulties in some cases as the number and quality of oocytes are reduced at this age. It would be an ethical dilemma if a woman requested cryopreservation of ovarian tissue at a young age to serve as a backup should she face difficulty in conceiving at a later age.

Future potential technologies of cloning could be able to support fertility treatment in female patients with POF by somatic cell nuclear transfer to enucleated oocytes. Syngamy between a gamete nucleus from one parent and a somatic cell nucleus from another could be obtained (Tesarik, 2002Go). The possibility of future application of this technique to human assisted reproduction termed reproductive cloning is currently under moratorium and is subject to intensive public debate (Revel, 2000Go).

We thus propose the following clinical approach. Young female patients from age 5 to 35 should consider fertility preservation before ovarian surgical removal, BMT and chemotherapy. Women of reproductive age should be counselled that IVF is their best approach to ensure future fertility. If a large number of oocytes is obtained, single patients could opt to cryopreserve a few unfertilized oocytes. Ovarian cryopreservation should be offered to children and teenagers, for whom IVF is inapplicable.

Conclusions
There are many reasons to proceed with care in developing ovarian cryopreservation and transplantation. The major concern is the application of ovarian cortex banking without the foreseeable use of this tissue. IVF for embryo freezing should be the first option offered to suitable patients of reproductive age. In children and when IVF is contraindicated, ovarian cryopreservation and transplantation with or without vascular anastomosis may well become the technique of choice in the future after further research and development of the techniques. In order to offer the best chances for fertility preservation, an extensive collaboration between haemato-oncologists, fertility specialists, biologists and psychologists is recommended for each individual patient.


    Acknowledgement
 
The authors would like to thank Mrs June Scher for her help with English correction.


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