Ovarian tissue and follicle transplantation as an option for fertility preservation

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1 Ovarian tissue and follicle transplantation as an option for fertility preservation Michael Grynberg, M.D., Ph.D., a,b Marine Poulain, Pharm.D., b,c Sarah Sebag-Peyrelevade, M.D., a,b Soizic le Parco, M.D., a,b Renato Fanchin, M.D., Ph.D., a,b and Nelly Frydman, Pharm.D., Ph.D. b,c a Department of Obstetrics and Gynecology and Reproductive Medicine, H^opital Antoine Beclere, b Univ Paris-Sud, and c Service de Biologie de la Reproduction, H^opital Antoine Beclere, Clamart, France Objective: To review and summarize data from the scientific literature on ovarian tissue and follicle transplantation as an option for fertility preservation. Design: Review of pertinent literature. Setting: University hospital. Patient(s): Women having undergone ovarian tissue transplantation. Intervention(s): None. Main Outcome Measure(s): Review of the literature. Result(s): Over the last decade, the field of ovarian transplantation and cryopreservation has significantly progressed, becoming applicable in humans. Indeed, fresh and frozen cortical ovarian tissue transplantations have been successfully reported worldwide, resulting in around 28 healthy babies. Although ovarian-tissue harvesting seems to be safe, the risk of reimplantation of cancer from ovarian cortical transplants cannot be estimated at this time. As a consequence, auto-transplantation of ovarian tissue in women having suffered from systemic hematological malignancies is not recommended. In these situations, reimplantation of isolated ovarian follicles might represent an interesting option in the future. Conclusion(s): Although the clinical experience is limited, the robust results obtained open new perspectives for the management of premature ovarian failure resulting or not from gonadotoxic treatments. (Fertil Steril Ò 2012;97: Ó2012 by American Society for Reproductive Medicine.) Key Words: Auto-transplantation, cryopreservation, gonadotoxic treatments, ovarian transplantation Over the past 25 years, major advances in the diagnosis and treatment of many childhood and adult cancers have markedly improved rates of cure for many childhood and adult cancers (1). As a consequence, the number of long-term survivors is increasing and their future quality of life has become a major concern (2). However, loss of ovarian function and therefore fertility is one of the most common long-term adverse effects affecting premenopausal patients treated with chemotherapy and/or radiation therapy (3). For these patients, ensuring their reproductive capacity after oncologic treatment has become a major concern. Therefore, a number of strategies have been developed in recent years to enable these women to preserve their fertility and their ability to become genetic mothers. The field of fertility preservation has more recently been extended to nononcological diseases, such as autoimmune diseases and premature ovarian insufficiency. Cryopreservation of oocytes and/or embryos is currently the most established fertility preservation technique. However, the delay, which is often required for controlled ovarian hyperstimulation prior to oocyte collection, excludes this option in some cases since it may postpone the initiation of cancer treatments (4). Other pharmacological approaches for fertility preservation are Received February 29, 2012; revised and accepted April 26, M.G. has nothing to disclose. M.P. has nothing to disclose. S.S.-P. has nothing to disclose. S.l.P. has nothing to disclose. R.F. has nothing to disclose. N.F. has nothing to disclose. Reprint requests: Michael Grynberg, M.D., Ph.D., Department of Obstetrics and Gynecology and Reproductive Medicine, H^opital Antoine Beclere, 157, rue de la Porte de Trivaux, 92141, Clamart, France ( michael.grynberg@abc.aphp.fr). Fertility and Sterility Vol. 97, No. 6, June /$36.00 Copyright 2012 American Society for Reproductive Medicine, Published by Elsevier Inc. doi: /j.fertnstert currently studied: use of GnRH agonists, apoptosis blockers, and drugs that limit follicle depletion (5, 6). In addition, protocols combining aromatase inhibitors with exogenous gonadotropin administration have also been proposed in patients suffering from breast cancer (7). Over the last decade, the field of ovarian transplantation and cryopreservation has significantly progressed, becoming applicable in humans. Indeed, fresh and frozen cortical ovarian tissue transplantations have been successfully reported worldwide, resulting in around 28 healthy babies (8). Although clinical experience is limited, these robust results open new perspectives for the management of premature ovarian failure, whether or not it is a result of gonadotoxic treatment. The present review summarizes the state of the art of ovarian cryopreservation and transplantation, as well as the limits and future prospects of these new techniques VOL. 97 NO. 6 / JUNE 2012

2 Fertility and Sterility CRYOPRESERVATION AND TRANSPLANTATION OF OVARIAN CORTEX Cryopreservation of ovarian cortex combined with orthotopic transplantation into an irradiated ovary restored fertility in rodents 50 years ago (9). However, more than 3 decades were necessary to improve the freezing protocols and transplantation procedures in larger animals, namely, in species that could be considered applicable to humans such as sheep (10, 11). In this species, ovarian tissue was frozen in slices and the thawed slices were later transplanted orthotopically. In the lamb, orthotopic transplantation has led to recovery of cyclic ovarian activity with long-term ovarian function, together with the achievement of live births (12). Since successful ovarian cortex transplantation has been described in human, new perspectives in terms of fertility preservation have been opened, in combination with more established technologies such as embryo and oocytes cryopreservation (13 19). Although it is still considered an experimental method for fertility preservation, freezing/ thawing of ovarian cortex may be indicated for adult women who cannot delay the start of chemotherapy, making controlled ovarian stimulation for oocyte or embryo freezing unrealistic. In addition, it is the only option available to spare eggs for prepubertal girls (20 30). As most oocytes are within primordial follicles in the ovarian cortex, a large number of eggs may be preserved by freezing pieces of ovarian cortex, irrespective of their volume. Ovarian tissue may be harvested by minimally invasive surgery, such as laparoscopy. Two techniques have been reported. The first option is to extract around 50% of one ovary by removing a block of cortical tissue or by removing 5 10 ovarian cortex biopsies with an average volume of around 5 mm 3 per biopsy. As consensus is lacking about how much ovarian tissue should be harvested for cryopreservation, for some, unilateral oophorectomy should be proposed (31, 32), whereas for others, this procedure should be reserved for cases with pelvic radiation, bone marrow transplantation, or high risk of complete ovarian destruction. When the estimated rate of chemotherapy-related amenorrhea is <50%, partial oophorectomy should be preferred as total oophorectomy could possibly increase the risk of secondary amenorrhea due to the decrease of the ovarian reserve (33). Ideally, cryopreservation of ovarian cortex should be carried out before the start of gonadotoxic treatment to cryopreserve undamaged ovarian follicles (34 37). Nevertheless, it may still be worth harvesting and freezing ovarian tissue after cancer treatments in girls and young women previously unable to undergo the procedure, as these patients normally have high follicle counts in their ovaries (38). In addition, chemotherapy may help to eliminate malignant cells from ovarian tissue that represent a risk of recurrence of the primary disease after reimplantation. Techniques of Cryopreservation of Ovarian Cortex Cryopreservation procedures must ensure follicular viability and integrity of tissue compartments and cell-to-cell contacts (39, 40). Thus, studies investigating the most favorable cooling rates and dehydration times have been conducted. It is now well established that adequate penetration of cryoprotectant through the stroma and granulosa cells to the oocytes is required for obtaining satisfactory results (40). Ice-crystal formation must be minimized by choosing optimal freezing and thawing rates. The choice of cryoprotectant with maximum permeation capacity but minimum toxicity and ice crystal formation potential is specific to each cell and tissue type. In the ovary, this choice requires the adequate compromise among the stroma, the follicular cells, and the oocytes (40). On the basis of current knowledge, the standard method for human ovarian cryopreservation is slowprogrammed freezing using a combination of permeating cryoprotectants such as propanediol dimethylsulphoxide (DMSO) or ethylene glycol, with non-permeating substances such as human serum albumin-containing medium or sucrose (40). At 196 C, ovarian tissue maintains viability for many years, as demonstrated by the live birth of children after transplantation of tissue that was stored for up to 6 years (41). A concern of slow-programmed freezing has been the relatively poor survival of the ovarian stroma (42). This has been demonstrated by transmission-electron microscopy, a method that accurately evaluates cryoinjury of membranes, mitochondria, and other organelles (43). Vitrification is a promising cryopreservation technique, in which the tissue is first exposed to high concentrations of permeating cryoprotectants for a short interval and then plunged directly into liquid nitrogen. This induces a glass-like state in the cells and avoids the formation of destructive ice crystals (44). Vitrification technique for cryopreservation of ovarian tissue has been shown to improve the viability of all tissue compartments. While survival rate of follicles with vitrification was similar to that obtained after slow freezing, integrity of ovarian stroma and blood vessels was largely improved (43). Successful orthotopic transplantation of ovarian tissue after vitrification has already been reported in rodents (45) and sheep (46). Human studies comparing slow-freezing protocols with vitrification of ovarian tissue have produced conflicting results, which may be explained by differences in the protocols and the medium used (47, 48). However, transmissionelectron microscopy data suggest that vitrification could be more effective than slow-programmed freezing when cryopreserving ovarian tissue (43). This hypothesis has recently been reinforced by the results published by Silber et al., after evaluation of the impact of slow-freezing and vitrification procedures on oocyte survival rates (13). A total of 16 cancer patients requesting fertility preservation by ovarian banking were prospectively studied. In 8 cases, the tissue has been preserved by vitrification, in 6 by a slow-freezing protocol, and in 2 only fresh tissue was analyzed. The high viability (92%) of oocytes in both fresh and vitrified specimens indicated that cryopreservation technique only causes minimal damage to oocytes. Among the 2,301 oocytes studied, no significant difference was noted between fresh and vitrified tissue, while the viability of slow freeze-cryopreserved was less than onehalf (42%, P<.01). Finally, given these recent in vitro and in vivo results, evidence strongly favors vitrification (8). The increasing number of women who are offered cryopreservation of ovarian tissue and the necessity for proper quality control in view of future transplantation require VOL. 97 NO. 6 / JUNE

3 VIEWS AND REVIEWS appropriate structures as well as proper equipment to fulfill clinical, legal, and scientific standards. Therefore, centralized cryobanking is an economic and effective way to reach this goal. Indeed, the creation of highly specialized cryopreservation platforms capable of performing specific investigations and research, will probably have the best benefit for the development of ovarian transplantation, as well as for patients. Results of Autotransplantation of Cryopreserved Human Ovarian Cortex The choice of the transplantation sites constitutes a determining factor for future graft viability and subsequent oocyte competence. Ovarian tissue can be grafted back to the original site (orthotopic) or to alternative sites (heterotopic). Orthotopic transplantation. Two different sites can be considered for orthotopic transplantation, and both have proved to be successful. The tissue is either transplanted in or onto the remaining ovary or into a peritoneal pocket in the pelvic peritoneum of the fossa ovarica. In many cases, transplantation in or onto the ovary was preferred to heterotopic graft as it allows natural conception. The first case of orthotopic auto-transplantation of cryopreserved and thawed ovarian tissue by laparoscopy was reported in a 29-year-old patient, who had previously undergone bilateral oophorectomy for intractable dysfunctional bleeding (49). The ovarian tissue was frozen by using a slow-freezing protocol and re-transplanted 8 months later to overcome severe menopausal symptoms. Controlled ovarian hyperstimulation with daily administration of exogenous gonadotropins 15 weeks after transplantation resulted in follicle development and ovulation. Unfortunately, no pregnancy occurred. The first pregnancy and live birth in humans after ovarian tissue transplantation was reported in 2004, in a 25-year-old woman whose ovary was taken before administration of MOPP/ABV hybrid chemotherapy (mechlorethamine, vincristine, procarbazine, prednisone, doxorubicin, bleomycin, vinblastine) for clinical stage IV Hodgkin s lymphoma (41). Chemotherapy was followed by radiotherapy (38 Gy). Amenorrhea occurred shortly after initiation of chemotherapy, combined with serum FSH, LH and estradiol levels at 91.1 miu/ml, 85 miu/ml and 17 pg/ml, respectively. This premature ovarian failure was confirmed 3 months later. Six years after chemotherapy and radiotherapy, a laparoscopy was performed to create a peritoneal window to induce neovascularization in this area. A second laparoscopy was carried out 7 days later and frozen-thawed ovarian tissue was pushed into the furrow created by the peritoneal window. A third laparoscopy was performed 4 months later to reimplant the remaining ovarian cortical tissue. Resumption of menstrual cycle was obtained from 5 to 9 months after transplantation. Eleven months after grafting, the patient became naturally pregnant, resulting in the live birth of a healthy girl, weighing 3,720 kg. Since 2004, several centers worldwide have reported cases of orthotopic transplantation of cryopreserved ovarian tissue in cancer survivors, resulting in live births (50 55). A recent review of successful orthotopic transplantation indicates that age at cryopreservation may be an important prognostic factor, as all patients except one woman were younger than 30 years, and most patients were younger than 25 years (19). In addition, as noted by the American Society of Clinical Oncology recommendations on fertility preservation, the benefit of ovarian cryopreservation for women older than 40 years of age is uncertain because of their age-reduced reserve of primordial follicles (15). In reports of successful ovarian transplantation, slowprogrammed freezing was used to cryopreserve the ovarian tissue and both large strips of 8 10 mm 5 mm or small pieces of 2 2 mm of tissue were shown to restore ovarian endocrine function effectively. Overall, follicular development and restoration of ovarian function usually occur 4 5 months after transplantation (37), which is consistent with the 120 days necessary to initiate follicular growth and the approximately 85 days to reach final maturation stage from a pre-antral follicle (56). The time interval between implantation of cortical tissue and the first rise of estradiol levels is also consistent with data obtained in sheep (11, 57) and in humans, although some variations may be observed. Indeed, the delay between transplantation and follicular development was found to vary from 6 weeks to more than 5 years (37, 58). Such a difference could be explained by variations in follicular reserve at the time of cryopreservation. Despite the restoration of regular menstrual cycles, high basal FSH and low antim ullerian hormone levels during the early follicular phase are usually observed after ovarian tissue transplantation, reflecting the relatively low number of surviving primordial follicles in the graft (59, 60). Another interesting point is the systematic return to serum FSH levels > 35 UI/L immediately after each menstrual cycle. This observation suggests that some inhibitory mechanisms, such as inhibin B hormone normally produced by developing follicles in intact ovaries are probably insufficient in transplanted tissue (57, 61), thereby reflecting the paucity of viable follicles. Taken together, these data illustrate the importance of achieving an optimal hormonal environment by improving the vascularization process and by grafting sufficient amounts of ovarian tissue. At the time of this writing, 18 healthy babies have been born from frozen-thawed ovarian tissue grafting orthotopically in 13 patients (Table 1). In addition, the first successful induction of puberty after autograft of cryopreserved ovarian tissue, recovered before allogenic hematopoietic stem-cell transplantation for severe homozygous sickle-cell disease, was recently reported (62). Heterotopic transplantation. Heterotopic sites for transplantation of ovarian tissue include the subcutaneous tissue of the abdominal wall, forearm, or chest wall. Heterotopic grafts have been associated with reports of restoration of hormonal function, follicle development, and oocyte retrieval (63, 64). In addition, embryos were obtained after in vitro fertilization of oocytes recovered from heterotopic transplants (64 66). Follicles in heterotopic locations generally do not grow to more than mm in diameter, so judging the optimum time for oocyte retrieval has proved to be difficult. Some investigators have speculated 1262 VOL. 97 NO. 6 / JUNE 2012

4 Fertility and Sterility TABLE 1 Worldwide frozen ovarian cortical tissue transplantations live births. Case no. Diagnosis Age at cryopreservation (y) Chemotherapy before cryopreservation Conception Babies Authors 1 Hodgkin s lymphoma 25 No Natural 1 Donnez et al. 2 Neurotumor 19 No Natural 1 Donnez et al. 3 Non-Hodgkin s lymphoma 28 Yes IVF-ET 1 Meirow et al. 4 Hodgkin s lymphoma 24 Yes Natural 2 Demeestere et al. 5 Ewing sarcoma 27 No IVF-ET and natural 2 Andersen et al. 6 Hodgkin s lymphoma 25 Yes IVF-ET 1 Andersen et al. 7 Premature ovarian failure 25 No Natural 1 Silber et al. 8 Hodgkin s lymphoma 20 No Natural 2 Silber et al. 9 Polyangiitis 27 Yes IVF-ET 1 Piver et al. 10 Breast cancer 36 No IVF-ET 2 Pellicer et al. 11 Sickle cells 27 No Natural 1 Piver et al. 12 Thalassemia 19 No IVF-ET 2 Revel et al. 13 Hodgkin s lymphoma 27 Yes Ovulation induction 1 Dittrich et al. Note: Total, 13 patients and 18 babies. Grynberg. Ovarian and follicle transplantation. Fertil Steril that temperature changes or even mechanical stresses hamper follicle development in these areas. In addition, decreased revascularization of ovarian cortical tissue grafts in the subcutaneous location in mice (compared with in the intraperitoneal location) has been reported (67, 68). Despite the achievement of initial results, no live births have yet been reported. Several cases have reported spontaneous pregnancies resulting in women who have undergone ovarian graft transplants simultaneously at orthotopic and heterotopic locations (51, 69, 70). Oktay et al., reported the unexpected reactivation of the pelvic ovary after having grafted ovarian cortex in suprapubic area, in a patient treated for Hodgkin s lymphoma (71). He raised the intriguing possibility that germ line stem cells in a heterotopic location had somehow been activated by the autotransplant. This hypothesis is supported by findings by other investigators (72, 73) that germ line stem cells may replenish the sterilized ovary after bone marrow transplantation in mice. Yet, this idea has been refuted by other investigators (74, 75). The advantages of a subcutaneous heterotopic transplantation include the simplicity and non-invasiveness of the surgical technique, as well as the potential benefit of closely monitoring the tissue for an eventual recurrence of malignancy in the graft. The disadvantages of heterotopic transplantation include cosmetic issues, potential adverse effects of an environment different from the pelvic intraabdominal to the growing follicles, and the presumed requirement for assisted reproduction to achieve a pregnancy. This last point may be a major limit of the reproductive success of heterotopic locations as in vitro fertilization-embryo transfer outcomes have been shown to be poor even when oocytes are retrieved from orthotopic transplants (36). WHOLE-ORGAN FREEZING AND TRANSPLANTATION Despite the fact that ovarian cortical tissue strips have been cryopreserved, thawed, and autotransplanted with successful reproductive function, the technique is limited by the ischemia that occurs at the time of reimplantation. Because these small cortical pieces are grafted without any vascular anastomoses, they are completely dependent for their survival on the time necessary for the establishment of neovascularization after grafting. As this process may require a minimum of 7 days (76, 77), the cells in the graft may undergo significant ischemic damage, which results in massive loss of primordial follicles (49, 78 81) and drastically reduces the lifespan of the graft (82). Thus, to overcome this problem for patients who desire long-term resumption of endocrine function, whole-ovary freezing has also been investigated, with the aim of achieving vascular anastomoses and a functioning organ after transplantation (83). Ovarian vascular transplantation using intact fresh ovaries has been shown to restore fertility in rats (84), sheep (85, 86), and monkeys (87), but a high rate of follicle loss is still a concern (88). A limited number of human studies with transplantation of fresh whole ovaries in orthotopic (88, 89) and heterotopic (90, 91) sites have been attempted with some success. Silber et al. reported the first live birth after transplantation of the whole fresh ovary between monozygotic twins discordant for premature ovarian failure (88). The first case of restoration of fertility after whole frozenthawed ovary transplantation was described by Wang et al. in rats (92). In addition, Imhof et al. recently demonstrated that autotransplantation of whole cryopreserved sheep ovaries with microanastomosis of the ovarian vascular pedicle could lead to pregnancy and delivery (93). However, the retransplantation process still represents a major limit due to the small diameter of the ovarian artery. The difficulty is further exacerbated by inadequate length of the vascular pedicle. If the microvascular anastomosis fails, then the whole organ survival is irreversibly compromised, thus preventing a second attempt of transplantation. This contrasts with failure of transplanted cortical strip, which allows multiple attempts according to the availability of ovarian cortex. Another challenge of whole ovary cryopreservation and transplantation is to find the optimal cryopreservation protocol for an entire organ. Indeed, in large mammals and humans, cryopreserving such a large-sized intact ovary may prove more problematic VOL. 97 NO. 6 / JUNE

5 VIEWS AND REVIEWS than in small animals, due to the difficulty of adequate diffusion of cryoprotectant agents into large tissue masses and vascular injuries caused by intravascular ice formation. Although no human cases of whole ovary retransplantation after cryopreservation have been performed to date, the preliminary studies have been encouraging, and it is likely that this option for fertility preservation will be a viable approach in the future. FRESH CORTICAL OVARIAN TISSUE TRANSPLANTATION The first successful fresh human ovary transplantation was reported in In this first experiment, donor and recipient were monozygotic twins discordant for premature ovarian failure (94). Subsequently, the same team performed 8 other fresh ovary transplants, all successful, resulting in 11 healthy babies in 7 patients (13, 95, 96). Technically, the first step of the procedure is to remove the whole cortical tissue from both the donor's and the recipient's ovaries. It is noteworthy that ovarian medulla in the recipient ovary must be thoroughly exposed. Then, donor cortex slices are transplanted onto the exposed medulla using 9.0 interrupted sutures. It seems important to prepare cortical tissue slices no thicker z 1.5 mm to facilitate rapid revascularization while keeping the tissue constantly irrigated with the icecold Leibovitz L-15 medium. In addition, avoidance of adhesion formation and micro-hematomas between donor and recipient tissues remains a major issue. Usually, one third of the ovarian cortex is grafted fresh and two-thirds are frozen (8). Resumption of ovulatory menstrual cycles with normal serum FSH levels was observed by 4.5 months. In addition, the duration of function of fresh ovarian grafts, which have lasted more than 7 years, indicates minimal follicle loss due to ischemia. SAFETY CONCERNS WITH OVARIAN TISSUE TRANSPLANTATION Most of the women who would be candidates for ovarian cryopreservation have hematologic malignancies or widespread or aggressive malignancies, thereby requiring systemic chemotherapy or pelvic irradiation. The magnitude of the risk of reimplanting malignant cells by autotransplantation of ovarian tissue is completely unknown. In a mouse model, the potential for re-introduction of malignant cells has been shown by grafting fresh and frozen ovarian tissue from mice with an aggressive type of lymphoma (97). The transplant of human ovarian tissue however, from women with Hodgkin s lymphoma into immunodeficient mice, did not transfer the disease to the recipients (98). In addition, Seshadri et al. reviewed the histopathology and immunohistochemistry of 26 specimens of ovary from women with Hodgkin s lymphoma and found no evidence of lymphoma (99). Cases have been reported, however, of diagnosis and treatment of a donor-derived lymphoma in two renal allograft recipients (100). In addition, Harbell et al. reported a case of anaplastic lymphoma of pancreas, liver, and both kidneys from a donor with a central nervous system lymphoma (101). While some investigators have tried to clarify which malignancies carry a significant risk of metastasis to the ovaries (102), it is not clear that these theoretical guidelines are valid. In a Japanese retrospective study performed on autopsy specimens, 22.4% of cancer patients under the age of 40 had ovarian metastases (103). Most of the metastases affecting ovaries are derived from the gastrointestinal tract, breast cancer, or endometrial cancer (104). Methods for detecting cancer cells in the ovarian tissue of women having suffered from hematological malignancies are under development, including immunohistochemistry or the polymerase chain reaction applied to the tissue (105). The investigation of residual malignant cells in the ovarian tissue, with the aim of grafting, may also be carried out by xenotransplantation to an immunodeficient SCID mouse before transplant (98). Currently, autotransplantation of ovarian tissue in women who have suffered from systemic hematological malignancies is not recommended because of the high risk of retransmission of malignancy. Only women with cancer diagnosis associated with a negligible or no risk of ovarian compromise should be considered for future autotransplantation (106). To avoid possible reimplantation of malignant cells, two approaches have been suggested, such as grafting of isolated follicles in an artificial ovary (19, 107, 108) and in vitro maturation of primordial follicles ( ). In this setting, a whole ovary cryopreservation has an advantage over cortical strips. Patients with malignancies at high risk of ovarian metastasis could have an ovary removed, perfused in vitro to stimulate folliculogenesis and then frozen for future in vivo use. TRANSPLANTATION OF ISOLATED HUMAN FOLLICLES Reimplantation of isolated follicles in an artificial ovary or scaffold (19, 107, 108) is an emerging technology with a view to potential application as an alternative to cortical fragment transplantation in patients at risk of ovarian metastasis. However, isolation of human follicles still remains a challenge due to the fibrous and dense nature of the ovarian stroma. Several protocols using enzymatic digestion have been described to successfully isolate ovarian follicles (109, ). However, frequent morphological and functional alterations of isolated follicles were reported (108, 118). Studies in mice have demonstrated that ovarian tissue enzymatically digested and subsequently grafted to the bursa of sterile mice could lead to the reorganization of stroma and follicles into a functioning organ that allowed restoration of fertility (119). Recent advances have been reported using the xenografting model. Indeed, Dolmans et al. showed that isolated human primordial follicles were able to survive and grow in vivo one week after xenografting in nude mice (120). In addition, massive in vivo follicular activation was observed after transplantation of isolated follicles. Further investigations, performed by the same group, revealed the possible long-term survival of isolated primordial follicles after xenografting, and development into antral follicles after FSH administration (121) VOL. 97 NO. 6 / JUNE 2012

6 Fertility and Sterility Another interesting point was the presence, 7 days after grafting, of a well-structured stroma-like tissue of human origin around isolated follicles. The possible origin of this stroma remains unclear, since only follicles isolated from their surrounding stoma were grafted. This tissue may arise either from contaminating stromal cells grafted together with the isolated follicles, or from the differentiation of granulosa cells into a stromal cell type. As a consequence, this approach warrants further investigation, with a view to potential clinical application as an alternative to cortical fragment transplantation, in patients at risk of ovarian metastasis. Currently, the use of xenotransplantation to mature follicles in vivo is not ready to be used in clinical applications since its safety and ethical issues have yet to be discussed (122). Therefore the creation of an artificial ovary (19, 107, 108) represents a major issue for patients having contraindication to ovarian tissue or whole ovary transplantation due to the risk of reintroducing malignant cells present in the cryopreserved tissue. Finding the optimal matrix to support in vitro culture of isolated human follicles constitutes an important challenge on the way to the artificial ovary. Some ethical questions are specifically raised by ovarian tissue cryopreservation and transplantation. Indeed, as these procedures are still considered experimental, informed consent is essential. This consent, which has to be obtained from patients in vulnerable situations, may require involving a surrogate decision-maker as in the case of a child. The most important ethical concern is to ensure the surgical procedure does not harm the patient by delaying cancer treatment and that no remnant malignant cells are reintroduced at the time of ovarian tissue graft. Regarding the health of children born from frozen/retransplanted human ovarian tissue, the preliminary results are encouraging but more and larger follow-ups are needed. CONCLUSIONS Recent available data in the literature indicates that ovarian tissue transplantation is feasible and effective for preserving fertility. Therefore, cryopreservation of ovarian tissue should be proposed to all adolescents and young women having to undergo highly gonadotoxic chemotherapy. However, the rate of survival of follicles after transplantation is still low and has to be improved. Although ovarian-tissue harvesting seems to be safe, the risk of reimplantation of cancer from ovarian cortical transplants cannot be estimated at this time. As a consequence, auto-transplantation of ovarian tissue in women having suffered from systemic hematological malignancies is not recommended. In these situations, reimplantation of isolated ovarian follicles might represent an interesting option in the future. 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