Human Reproduction Update Advance Access originally published online on July 18, 2006
Human Reproduction Update 2006 12(5):519-535; doi:10.1093/humupd/dml032
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Ovarian tissue cryopreservation and transplantation: a review
Department of Gynecology, Université Catholique de Louvain, Brussels, Belgium
1 To whom correspondence should be addressed: Department of Gynecology, Université Catholique de Louvain, Cliniques Universitaires St Luc, Avenue Hippocrate 10, 1200 Brussels, Belgium. E-mail: donnez{at}gyne.ucl.ac.be
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Abstract |
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 TOP Abstract IntroductionFertility preservation in cancer...Embryo cryopreservationOocyte cryopreservationOvarian tissue cryopreservationSafety and ethical issuesConclusionReferences |
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The review covers current options for ovarian tissue cryopreservation and transplantation and provides a systematic review of the existing literature from the last 10 years, taking into account all previously published reviews on the subject. The different cryopreservation options available for fertility preservation in cancer patients are embryo cryopreservation, oocyte cryopreservation and ovarian tissue cryopreservation. The choice depends on various parameters: the type and timing of chemotherapy, the type of cancer, the patient’s age and the partner status. The different options and their results are discussed, as well as their putative indications and efficacy. The review concludes that advances in reproductive technology have made fertility preservation techniques a real possibility for patients whose gonadal function is threatened by premature menopause, or by treatments such as radiotherapy, chemotherapy or surgical castration.
Key words: fertility preservation / ovarian tissue cryopreservation / transplantation
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Introduction |
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TOPAbstractIntroductionFertility preservation in cancer...Embryo cryopreservationOocyte cryopreservationOvarian tissue cryopreservationSafety and ethical issuesConclusionReferences |
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In 2006, 1 399 790 new cancer cases are expected in the United States, of which 679 540 will be women (Jemal et al., 2006
). According to previous reports, 8% of these women will be under the age of 40. By 2010, it is estimated that one in every 250 people in the adult population will be childhood cancer survivors (Blatt, 1999). Advances in the diagnosis and treatment of childhood, adolescent and adult cancer have greatly increased the life expectancy of premenopausal women with cancer but have resulted in a growing population of adolescent and adult long-term survivors of childhood malignancies (Blatt, 1999), who may experience infertility problems due to induced premature ovarian failure (POF).
Aggressive chemotherapy and radiotherapy, and bone marrow transplantation (BMT), can cure >90% of girls and young women affected by such disorders (Ries et al., 1999). However, the ovaries are very sensitive to cytotoxic treatment, especially to alkylating agents, which are classified as high risk for gonadal dysfunction (e.g. cyclophosphamide, busulfan, melphalan, chlorambucil, dacarbazine, procarbazine, ifosfamide, thiotepa and nitrogen mustard) (Warne et al., 1973; Koyama et al., 1977; Fisher et al., 1979; Viviani et al., 1985; Mackie et al., 1996; Teinturier et al., 1998; Legault and Bonny, 1999; Meirow et al., 1999; Blumenfeld et al., 2000; Kenney et al., 2001; Tauchmanova et al., 2002). Doxorubicin and the alkylating-like agents cisplatin and carboplatin fall into the medium-risk category (Hortobagyi et al., 1986; Wallace et al., 1989; Maneschi et al., 1994; Tangir et al., 2003), whereas methotrexate, bleomycin, 5-fluorouracil, actinomycin-D, mercaptopurine and vincristine are considered as low risk for gonadal dysfunction (Van Thiel et al., 1970; Shamberger et al., 1981; Stillman et al., 1981; Sudman et al., 1992; Bines et al., 1996; Bower et al., 1998; Sonmezer and Oktay, 2004; Wallace et al., 2005a) (Table I). The type and dose of chemotherapeutic agent influence the progression to ovarian failure, with alkylating agents increasing the risk of POF by a factor of 9 (Byrne et al., 1992).
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Cyclophosphamide is the agent most commonly implicated in causing damage to oocytes and granulosa cells in a dose-dependent manner (Warne et al., 1973; Sanders et al., 1996; Meirow et al., 1999; Kenney et al., 2001). Since Meirow et al. (1999) demonstrated primordial follicular depletion in mice treated with cyclophosphamide, tremendous progress has been made towards elucidating the cellular and molecular mechanisms responsible for mediating oocyte death and follicular depletion under normal and pathological conditions. Perez et al. (2000) demonstrated that doxorubicin induces apoptosis in pregranulosa cells, and Takai et al. (2003) showed that bax and caspase-2 and caspase-3 are functionally important mediators of vinylcyclohexene diepoxide-induced ootoxicity in mice.
This follicular destruction generally results in the loss of both endocrine and reproductive functions, depending on the dose and the age of the patient. Indeed, Larsen et al. (2003) reported a four-fold increased risk of POF in teenagers treated for cancer, and a risk increased by a factor of 27 in women between 21 and 25 years of age. Complete amenorrhoea was reported after a dose of 5 g of cyclophosphamide in women over 40 years of age, and after doses of 9 and 20 g in women of 30–40 and 20–30 years of age, respectively (Shalet, 1980). A combination of various chemotherapeutic agents further increases gonadal toxicity. After chemotherapy with MOPP/ABV (chlorethamine, vincristine, procarbazine, prednisone, doxorubicin, bleomycin, vinblastine) hybrid chemotherapy, Schilsky et al. (1981) found that amenorrhoea developed in 89 and 20% of patients >25 and <25 years of age at the time of treatment, respectively. The median age of patients who became amenorrhoeic after therapy was significantly higher than that of patients who maintained normal menses (26 versus 20 years; P = 0.008).
Abdominal ionizing radiation associated with alkylating agents often induces POF, rendering patients infertile in almost 100% of cases. Indeed, for radiotherapy, it has been stated that a dose of 5–20 Gy administered to the ovary is sufficient to completely impair gonadal function (Wallace et al., 2005b), whatever the age of the patient. The dose of radiation required to destroy 50% of the oocyte reserve has been found to be <2 Gy (Wallace et al., 2003). Moreover, uterine irradiation at a young age reduces adult uterine volume (Larsen et al., 2004). Radiation doses between 14 and 30 Gy have been reported to result in uterine dysfunction (Critchley et al., 1992; Bath et al., 1999; Critchley and Wallace, 2005). The practitioner should be aware of this effect of radiotherapy on the uterus, which could interfere with the implantation capacity of embryos.
In the field of oncological indications for ovarian tissue cryopreservation, there have been no major modifications since our review published in 1998 (Donnez and Bassil, 1998) (Table II). In countries where surrogacy is not legal, a conservative fertility approach in case of gynaecological malignancy is only valuable if the uterus can be spared during surgery. This includes cases of early cervical carcinoma (Dargent et al., 2000; Burnett et al., 2003; Tanguay et al., 2004), early vaginal carcinoma (Hicks and Piver, 1992), early endometrial adenocarcinoma (Jadoul and Donnez, 2003), ovarian tumours of low malignancy (Donnez et al., 2003; Boran et al., 2005; Fauvet et al., 2005; Tinelli et al., 2005) and some selected cases of unilateral ovarian carcinoma (stage IA) (Donnez and Bassil, 1998; Kleine, 1996). The choice of a possible conservative surgical approach in these patients and the question of implementing such treatment alone remain controversial, as all the published results were obtained on the basis of retrospective studies and/or case reports. The fertility outcome is conditioned by the adjuvant therapy, i.e. local radiotherapy and/or chemotherapy.
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Intensive chemotherapy and/or total body irradiation required before BMT constitute the treatment combination presenting the greatest risk of POF. Indeed, such high doses of chemotherapy (commonly using the highly cytotoxic cyclophosphamide/busulfan regimen) and/or radiotherapy lead to subsequent ovarian failure in almost all cases, children and adults alike (Sanders et al., 1996; Meirow and Nugent, 2001; Lobo, 2005). The risk of POF was estimated to be 92% in the study by Meirow and Nugent (2001), while Teinturier et al. (1998) actually reported 0% of ovarian recovery after busulfan treatment before BMT. A large retrospective survey of pregnancy outcomes after haematopoietic stem cell transplantation (HSCT) (peripheral blood or BMT) involving 37 362 patients revealed that only 0.6% of patients conceived after autologous or allogenic SCT (Salooja et al., 2001; Lutchman Singh et al., 2005). It is thus obvious that high doses of alkylating agents, irradiation and advancing age increase the risk of gonadal damage.
Cryopreservation should not be reserved solely for women with malignant disease (Donnez et al., 2006). Indeed, HSCT has been increasingly used in recent decades for non-cancerous diseases, such as benign haematological disease (sickle cell anaemia, thalassaemia major and aplastic anaemia) and autoimmune diseases previously unresponsive to immunosuppressive therapy (systemic lupus erythematosus and autoimmune thrombocytopenia) (Slavin et al., 2001; Mattle et al., 2005; Wolner-Hanssen et al., 2005; Donnez et al., 2006). Other benign diseases, such as recurrent ovarian endometriosis or recurrent ovarian mucinous cysts, are also indications for ovarian cryopreservation. Patients undergoing oophorectomy for prophylaxis may potentially benefit from ovarian cryopreservation too. The indications for cryopreservation of ovarian tissue in case of non-malignant disease are summarized in Table II.
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Fertility preservation in cancer patients: different cryopreservation options |
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TOPAbstractIntroductionFertility preservation in cancer...Embryo cryopreservationOocyte cryopreservationOvarian tissue cryopreservationSafety and ethical issuesConclusionReferences |
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Several options are currently available to preserve fertility in cancer patients and give them the opportunity to become mothers when they have overcome their disease: embryo cryopreservation, oocyte cryopreservation or ovarian tissue cryopreservation (Gosden et al., 1997
; Donnez and Bassil, 1998; Oktay et al., 1998a, 2004; Donnez et al., 2000, 2005; Oktay and Karlikaya, 2000; Meirow and Nugent, 2001; Torrents et al., 2003; Sonmezer and Oktay, 2004; Gosden, 2005; Lobo, 2005; Meirow et al., 2005; Kim, 2006). The choice of the most suitable strategy for preserving fertility depends on different parameters: the type and timing of chemotherapy, the type of cancer, the patient’s age and the partner status.
The only established method of fertility preservation is embryo cryopreservation, according to the Ethics Committee of the American Society for Reproductive Medicine (2005), but this option requires the patient to be of pubertal age, have a partner or use donor sperm, and be able to undergo a cycle of ovarian stimulation, which is not possible when the chemotherapy has to be initiated immediately or when stimulation is contraindicated according to the type of cancer.
Cryopreservation of oocytes can be performed in single women who can undergo a stimulation cycle, although the effectiveness of this technique is very low, with pregnancy and delivery rates ranging from 1 to 5% per frozen oocyte (Stachecki and Cohen, 2004; Borini et al., 2006; Levi Setti et al., 2006).
Cryopreservation of ovarian tissue is the only option available for prepubertal girls, and for woman who cannot delay the start of chemotherapy. Ovarian tissue can theoretically be frozen using three different approaches: as fragments of ovarian cortex, as entire ovary with its vascular pedicle or as isolated follicles. Human ovarian cryopreservation and transplantation procedures have so far been almost exclusively limited to avascular cortical fragments, both in experimental and in clinical studies, and, for now, this is the only procedure that has yielded live births in humans after autologous transplantation (Donnez et al., 2004; Meirow et al., 2005).
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Embryo cryopreservation |
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TOPAbstractIntroductionFertility preservation in cancer...Embryo cryopreservationOocyte cryopreservationOvarian tissue cryopreservationSafety and ethical issuesConclusionReferences |
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Embryo cryopreservation has become a routine technique in all IVF centres and has proven its efficacy in terms of pregnancy and ‘take-home-baby’ rates. Although this method has already been used for young cancer patients (Winkel and Fossum, 1993
), there are significant drawbacks to its use.
First, medical reasons might impede its application (a) if the beginning of cancer treatment cannot be delayed and there is no time to complete ovarian stimulation or (b) when the stimulation procedure may be theoretically harmful to patients with hormone-sensitive tumours, such as breast cancer. Even if IVF can theoretically be undertaken on the basis of a spontaneous ovarian cycle (Brown et al., 1996), the small number of obtainable oocytes (and subsequently viable embryos for transfer) makes it extremely unlikely that any live births will be achieved in these conditions.
Ginsburg et al. (2001) showed that women undergoing IVF after chemotherapy had a poor response to gonadotrophins and, more recently, Dolmans et al. (2005) clearly demonstrated the poor results of IVF and embryo cryopreservation, even if the attempt is carried out after only one or two regimens of chemotherapy. Recently, tamoxifen and letrozole were employed to stimulate the ovaries for IVF and embryo cryopreservation with some success, whilst possibly providing a safe alternative to traditional ovarian stimulation methods in these patients (Oktay et al., 2005).
The partner status of the patient may also impede embryo cryopreservation. If the patient has no partner or is an adolescent, the only available solution is using donor sperm to ensure fertilization of her oocytes. Finally, this technique is inappropriate for children, who have not reached puberty.
In conclusion, embryo cryopreservation is an efficient technique, but only an option for patients from whom mature oocytes can be collected and who have a partner (or are willing to use donor sperm).
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Oocyte cryopreservation |
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TOPAbstractIntroductionFertility preservation in cancer...Embryo cryopreservationOocyte cryopreservationOvarian tissue cryopreservationSafety and ethical issuesConclusionReferences |
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Oocyte cryopreservation is an alternative option for patients with the same characteristics as those described above for embryo cryopreservation but who are not with a partner and do not wish to use donated sperm. In this case, IVF of their oocytes to produce embryos to be frozen for future implantation is not possible. Thus, the oocytes themselves must be cryopreserved either as mature or as immature oocytes. Human oocyte cryopreservation has been rapidly incorporated into clinical practice in several centres (Porcu et al., 1997
, 2004; Fabbri et al., 2001; Porcu, 2005). Mature oocyte cryopreservation
Mature oocyte freezing appears, at least in theory, to be the most logical way of storing female germ cells, comparable to the routinely performed sperm banking. It is an attractive option for women without a partner, if they have time to complete ovarian stimulation before cancer therapy. However, ovarian stimulation and oocyte collection are not applicable for children (Oktay et al., 1998a; Torrents et al., 2003; Akar and Oktay, 2005).
Still, this procedure has proved largely disappointing. Since the first report of a live birth from a frozen oocyte (Chen, 1986), the results of this procedure worldwide have been variable, with a reported success rate of <2% per thawed oocyte, despite an improved success rate when combined with ICSI (Porcu et al., 1997, 2004; Fabbri et al., 2001; Lutchman Singh et al., 2005; Porcu, 2005). Techniques developed in recent years to improve survival rates of mature oocytes after slow-freezing protocols include increased sucrose concentrations (Fabbri et al., 2001), longer pre-equilibration and thawing times (Yang et al., 2002) and the use of sodium-depleted media (Boldt et al., 2003; Stachecki and Cohen, 2004). Data on frozen-thawed mature oocytes from 21 studies in peer-reviewed journals were examined by Sonmezer and Oktay (2004), who reported a mean survival rate of 47%, a mean fertilization rate of 52.5% and a mean pregnancy rate per thawed oocyte of 1.52%. Very recent data indicate that, although the combination of slow cooling and high sucrose concentrations ensures high rates of oocyte survival, it is not sufficient to guarantee a high standard of clinical efficiency (Borini et al., 2006). Indeed, 18 clinical pregnancies were obtained from 927 oocytes in Borini’s study. In another very recent study, 2900 oocytes were cryopreserved, of which 1087 were subsequently thawed. In this series, 18 pregnancies were also obtained (Levi Setti et al., 2006). Even in the very latest studies, the mean pregnancy rate per thawed oocyte does not exceed 1.8% (Borini et al., 2006; Levi Setti et al., 2006).
The metaphase II (MII) oocyte is a large and highly specialized cell that is extremely fragile. Oocyte freezing is accompanied by various types of cell injury, which may explain the low survival rate (Van der Elst, 2003). There are two main reasons for these poor results. First, the zona pellucida hardens during the freezing process, probably as a consequence of premature exocytosis of the cortical granules. It could then act as a fence, impairing sperm penetration and normal fertilization, although micromanipulation techniques (ICSI) can, to a certain extent, bypass this problem (Porcu et al., 1997; Fabbri et al., 2001; Gook et al., 1993, 1995). Second, in the mature oocyte, the metaphase chromosomes are lined up by the meiotic spindle along the equatorial plate, but the spindle apparatus is easily damaged by intracellular ice formation during the freezing or thawing process (Pickering et al., 1990) (for review, see Mandelbaum et al., 2004). The cellular cooling process induces depolymerization of the meiotic spindle, which is a dynamic structure (microtubules being continually assembled at one of its ends and separated at the other). The cell is thus at risk of losing chromosomes and suffering aneuploidy.
There are two main steps in the process of cryopreservation: (i) chilling, i.e. lowering the temperature from the physiological temperature to the point of freezing and (ii) freezing, i.e. further reducing the temperature to the storage temperature (liquid nitrogen at –196°C). Chilling injury can modify the structure of membranes and therefore their integrity (Ghetler et al., 2005). It also affects oocyte microtubules (Albertini and Eppig, 1995), cytoskeletal organization (Overstrom et al., 1990) and the zona pellucida (Vajta et al., 1998).
Chilling injury is temperature dependent; it is caused by changes in membrane properties and integrity and is responsible for the extensive cell damage that occurs during the process of cryopreservation. Inappropriate handling of oocytes at room temperature appears to pose the greatest threat (Mandelbaum et al., 2004).
In conclusion, mature oocyte banking is still limited by its low success rate; oocytes are sensitive to chilling, often fail to survive freeze-thawing processes, and are susceptible to cytoskeletal damage and aneuploidy. Currently, even the most optimistic success rates offer patients only a slim chance of pregnancy, if few oocytes are available (Gosden, 2005). Ultrarapid freezing with vitrification may offer advantages over conventional slow cooling protocols by improving post-thawing survival rates but needs to be investigated further. Despite the few promising studies on vitrification (Yoon et al., 2000; Katayama et al., 2003), even less is known about the potentially detrimental effects of this process compared with conventional cryopreservation techniques; to date, slow freezing and rapid thawing is the protocol of choice for freezing human oocytes (Falcone et al., 2004). Better evaluation of sucrose concentrations in the freezing medium could lead to improved results in the future (Porcu, 2005; Borini et al., 2006).
Immature oocyte cryopreservation and in vitro maturation
Oocytes at the diplotene stage of prophase I, or germinal vesicle (GV) stage, survive the cryopreservation procedure better than those frozen at the MII stage (Boiso et al., 2002). These cells have reached full size and complete meiotic competence but have not yet resumed their maturation process and initiated their second metaphase. Although the risk of hardening of the zona pellucida or damage to the cytoskeleton cannot be avoided, it is probable that the absence of a meiotic spindle and the presence of a nuclear membrane protecting the chromatin guarantees the absence of cytogenetic anomalies during further cellular divisions.
Freezing immature oocytes followed by in vitro maturation thus offers practical and theoretical advantages (Gosden, 2005), but this method is still suboptimal. Frozen–thawed immature oocytes have to follow a process of in vitro maturation before they are ready to be fertilized. Oocyte maturation is considered as the reinitiation and completion of the first meiotic division from the GV stage to the MII stage, and the accompanying cytoplasmic maturation phase for fertilization and early embryonic development (Cha and Chian, 1998). The co-ordination of nuclear and cytoplasmic maturation in vitro has proved very difficult to achieve.
Although there are several reports of pregnancies achieved after in vitro maturation of fresh GV-stage oocytes (Cha et al., 1991; Trounson et al., 1994), only one live birth has resulted from an immature oocyte cryopreserved at the GV stage, with subsequent in vitro maturation (Tucker et al., 1998). Two other teams have described non-ongoing pregnancies. Kan et al. (2004) reported one pregnancy from an immature frozen oocyte which ended in a blighted ovum at 12 weeks, and Wu et al. (2001) reported one biochemical pregnancy. According to a recent review by Kim, the cryopreservation of GV-stage oocytes will not be a practicable strategy until in vitro maturation of these oocytes becomes more reliable (Kim, 2006).
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Ovarian tissue cryopreservation |
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TOPAbstractIntroductionFertility preservation in cancer...Embryo cryopreservationOocyte cryopreservationOvarian tissue cryopreservationSafety and ethical issuesConclusionReferences |
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For patients who need immediate chemotherapy, ovarian tissue cryopreservation is the only possible alternative (Gosden et al., 1994
; Donnez and Bassil, 1998; Meirow et al., 1998; Oktay et al., 1998a; Donnez et al., 2000, 2005). The main aim of this strategy is to reimplant cortical ovarian tissue into the pelvic cavity (orthotopic site) or a heterotopic site like the forearm or the abdominal wall once treatment is completed and the patient is disease-free (Donnez and Bassil, 1998; Oktay et al., 1998a, 2004; Oktay and Karlikaya, 2000; Radford et al., 2001; Donnez et al., 2004, 2005, 2006; Kim et al., 2004a; Meirow et al., 2005; Schmidt et al., 2005; Demeestere et al., 2006) (Table III).
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Other strategies, such as the transplantation of isolated cryopreserved primordial follicles and the transplantation of cryopreserved whole ovary, will also be discussed here.
Fragments of cortical ovarian tissue
Lessons learned from auto- and xenografting of cryopreserved animal ovarian tissue
To date, ovarian tissue has been successfully cryopreserved and transplanted into rodents, rabbits, sheep, and marmoset monkeys (Candy et al., 1995, 2000; Salle et al., 2002; Almodin et al., 2004). Successful fertilization and pregnancy after oocyte collection from fresh transplanted ovarian tissue have been described in a primate (Lee et al., 2004); the grafted tissue functioned without any surgical connection to major blood vessels.
The generation of live young from xenografted mouse ovarian tissue into a rat recipient was described by Snow et al. (2002). Fresh mouse ovarian tissue was xenografted under the kidney capsule of rat recipients. Mature oocytes were produced from the xenografted tissue and subsequently fertilized; they developed into fertile adult mice. The authors state that xenotransplantation may thus prove to be a potentially promising method for the conservation of rare and endangered species of animals. The use of this strategy in human assisted reproduction should be considered with caution, however (Snow et al., 2002).
In a recent review of their experimental studies, Baird et al. (2004) observed a significant increase in the proportion of growing follicles from <20% in ovaries before grafting to >70% at 7, 30 and 60 days after grafting. According to the authors, the massive recruitment of primordial follicles, which also occurs in cultured fragments, suggests the removal of some inhibitory mechanisms regulating FSH. Following autotransplantation, the number of antral follicles and the secretion of inhibin A are reduced, resulting in raised basal levels of FSH, which could account for the massive recruitment, although the early stage of folliculogenesis can occur in the absence of FSH and LH.
Experimental studies have indicated that the fall in the number of primordial follicles in grafted tissue is due to hypoxia and the delay that occurs before reimplanted cortical tissue becomes revascularized. The loss of primordial follicles in cryopreserved ovarian tissue after transplantation is estimated to be 50–65% in some studies (Baird et al., 1999; Nisolle et al., 2000) and >90% in one study (Aubard et al., 1999).
A very recent study in mice concluded that the graft site affects the number and quality of oocytes produced from ovarian grafts (Yang et al., 2006). The study used a mouse ovarian grafting model to investigate whether the graft site (bursal cavity, kidney capsule or site) influences the number, fertilization rate and developmental potential of oocytes recovered from grafts. Graft retrieval and the number of oocytes found in each graft were lowest from the s.c. graft site. The number of 2-cell embryos produced was significantly higher with oocytes from grafts to the bursa, compared with the other sites.
In a recent article, Israely et al. (2004) analyzed angiogenic events following ovary xenotransplantation. Rat ovaries were transplanted into the muscle of castrated nude mice. The characterization of the neovasculature by dynamic contrast-enhanced magnetic resonance imaging (MRI) confirmed that the graft was devoid of any blood supply. Functional vessels within the graft were detected by MRI and histology from day 7 onwards. By 2–3 weeks, both the blood volume fraction and the permeability were measured using albumin-based MR contrast material and were found to be higher in the graft than in adjacent muscle. The same team recently demonstrated that transplantation into angiogenic granulation tissue created during wound healing shortened the ischaemic interval (Israely et al., 2006). This confirms the benefits of the technique of induced angiogenesis and neovascularization that was used in the first case of successful orthotopic transplantation of cryopreserved ovarian tissue (Donnez et al., 2004).
The assessment of tumour oxygenation by electro-paramagnetic resonance (EPR) was recently reviewed by Gallez et al. (2004). In this review, different methods were used to estimate the oxygenation of tumours and compare EPR oximetry with nuclear magnetic resonance and MRI. The evaluation of oximetry in human ovarian xenografts was initiated to improve the revascularization process, which remains one of the main limitations of ovarian cortical strip use (Donnez et al., 2005).
Lessons learned from xenografting cryopreserved human ovarian tissue
Human ovarian tissue can be successfully cryopreserved, showing good survival and function after thawing. Hovatta (2005) arrived at this conclusion after reviewing all relevant studies since 1996, when the first case of cryopreservation of human ovarian tissue was described. In these studies, survival and morphological and functional recovery of ovarian tissue after thawing were proved by live/dead assays, organ culture, xenotransplantation to severe combined immunodeficient (SCID) mice and normal light and transmission electron microscopy (TEM) analysis.
Adequate penetration of cryoprotectant through the stroma and granulosa cells to the oocytes is necessary, however, while at the same time avoiding possible cryoprotectant toxicity. Indeed, Newton et al. (1996, 1998) demonstrated the importance of the diffusion rate and the diffusion temperature. Ice crystal formation must also 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 (Fuller and Paynter, 2004). Thus, in the ovary, it is a compromise between the stroma, the follicular cells and the oocytes (Hovatta, 2005). On the basis of current knowledge, the standard method for human ovarian cryopreservation is slow-programmed freezing, using human serum albumin-containing medium, and propanediol, dimethylsulphoxide or ethylene glycol as a cryoprotectant, combined or not with sucrose (Hovatta, 2005).
Kim et al. (2004b) showed that a correlation exists between ischaemic tissue damage and the duration of ischaemia. It has been demonstrated that vascular endothelial growth factor does not accelerate revascularization, either after s.c. (Schnorr et al., 2002) or peritoneal (Donnez et al., personal data) implantation of ovarian tissue. According to Kim et al. (2004b), the ovarian cortex can tolerate ischaemia for at least 3 h at 4°C. They also demonstrated that an antioxidant (ascorbic acid) reduces apoptosis in ovarian cortex by up to 24 h in case of incubation in vitro. In their study, significant ischaemic damage was evidenced by decreased oxygen consumption and increased apoptosis. Moreover, it appeared that stromal cells were more vulnerable to ischaemia than primordial follicles. The apoptosis rate of stromal cells was higher in the frozen-thawed group than in the fresh group, regardless of the duration of incubation, which may reflect a degree of freeze-thawing injury to ovarian cortex.
Several immunodeficient animal strains can be used as xenograft recipients, with SCID and non-obese diabetic-SCID mice best suited to xenografting to obviate potential rejection problems, especially if the graft is to be borne for a long period (Weissman et al., 1999; Aubard, 2003). All reports on human ovarian tissue grafting to mice (for review, see Aubard, 2003) that have studied the implantation site have shown peritoneal transplantation, either under the peritoneum (Nisolle et al., 2000) or under the kidney capsule (Abir et al., 2003; Hernandez-Fonseca et al., 2004) to be better than s.c. transplantation in terms of follicular survival and development. After xenografting cryopreserved human ovarian tissue into the kidney capsule of SCID mice, Oktay et al. (1998b) achieved follicular development up to the antral stage, and (Gook et al. 2003, 2005) obtained MII-stage oocytes. The ovulatory capacity of frozen-thawed human follicles in xenografts was evidenced by the formation of morphologically normal corpora lutea and elevated progesterone levels in immunodeficient mice (Kim et al., 2002; Gook et al., 2003).
Very few data are available on the final maturation of follicles in xenografts and therefore the quality of oocytes obtained. In a recent study, Kim et al. (2005) assessed the integrity of human oocytes obtained after s.c. xenografting of cryopreserved ovarian tissue into SCID mice and in vitro maturation of retrieved cumulus–oocyte complexes. By immunocytochemical analysis of microtubules and DNA, some oocytes showed abnormal nuclear and cytoplasmic maturation. Nevertheless, the authors could not determine whether these alterations were due to freeze–thawing injury, the lack of optimal ovarian stimulation protocols, suboptimal conditions of animal hosts for the growth of human follicles, inadequate in vitro maturation techniques or the choice of a s.c. transplantation site. It has therefore not been established whether human oocytes matured in a xenograft are ultrastructurally normal and functionally competent.
Autotransplantation of cryopreserved human ovarian tissue
Reported cases of autotransplantation of cryopreserved ovarian tissue, either to an orthotopic or to a heterotopic site, are summarized in Table III, detailing in each case the age of the patient before freezing, whether the patient received chemotherapy before freezing, the indications for cryopreservation, the graft site and size, the interval before recovery of ovarian function after grafting and the outcome of transplantation (Oktay and Karlikaya, 2000; Callejo et al., 2001; Radford et al., 2001; Kim et al., 2004a; Oktay et al., 2004; Oktay, 2006; Donnez et al., 2004, 2006; unpublished data; Meirow et al., 2005; Schmidt et al., 2005; Wolner-Hanssen et al., 2005; Demeestere et al., 2006).
Orthotopic autotransplantation of cryopreserved human ovarian tissue
In theory, natural pregnancy may be achieved via orthotopic tissue transplantation if the fallopian tubes remain intact.
In 2000, Oktay and Karlikaya (2000) reported laparoscopic transplantation of frozen–thawed ovarian tissue to the pelvic side wall in a 29-year-old patient, who had undergone bilateral oophorectomy for a non-malignant disease. Pieces of cryopreserved ovarian tissue were thawed and transplanted. The patient was stimulated by gonadotrophins once after 15 weeks and then again after 10 months. Follicular development was demonstrated by ultrasonography and ovulation occurred in response to HCG administration.
Radford et al. (2001) reported a patient with a history of Hodgkin’s disease treated by chemotherapy, whose ovarian tissue had been biopsied and cryopreserved 4 years after chemotherapy and later reimplanted. In this case, histological section of the ovarian cortical tissue revealed only a few primordial follicles because of the previous chemotherapy. Estradiol (E2) was detected and the FSH and LH levels decreased 8 months after reimplantation. The patient had one menstrual period, but 9 months after reimplantation, her LH and FSH concentrations returned definitively to menopausal levels.
We reported the first successful transplantation of cryopreserved ovarian tissue (Donnez et al., 2004) resulting in a pregnancy and live birth. In 1997, a 25-year-old woman presented with clinical stage IV Hodgkin’s lymphoma. Ovarian tissue cryopreservation was undertaken before chemotherapy. By laparoscopy, we took five cortical biopsies, about 12–15 mm long and 5 mm wide, from the left ovary. The removal of the whole ovary was not an option because one can never completely exclude recovery of ovarian function after chemotherapy.
After laparoscopy, the patient received hybrid chemotherapy from August 1997 to February 1998, followed by supradiaphragmatic radiotherapy (38 Gy).
According to Schilsky et al. (1981), the risk of POF after such a regimen in a woman of 26 years of age is >90%, whereas according to Wallace et al. (2005a) and Lobo (2005), the risk of subfertility after Hodgkin’s treatment with alkylating agents is >80%. Indeed, not only the type of drug and dose but also the age are important factors when evaluating the risk of POF after chemotherapy.
In 2003, once the patient had been declared completely disease-free, transplantation went ahead. A large strip and 35 small cubes of frozen–thawed ovarian tissue were implanted into a furrow created by the peritoneal window very close to the ovarian vessels and fimbria on the right side. Four months after transplantation, a laparoscopy was carried out to check the viability of the orthotopic graft and to reimplant the remaining 32 ovarian cortical cubes. A follicular structure was visible in the area where the tissue had been reimplanted, clearly outside the native ovary. Biopsy and analysis by vital fluorescent probe staining and histology revealed the presence of viable primordial follicles and a follicular structure with inhibin A-marked cells (Donnez et al., 2004). From 5 to 9 months after reimplantation, concentrations of FSH, E2 and progesterone showed the occurrence of ovulatory cycles. At 11 months, the patient became pregnant and subsequently delivered a healthy baby.
Several lines of evidence lend support to our assertion that the origin of the pregnancy was indeed the autotransplanted cryopreserved tissue. The possibility that the egg was derived from the native ovary is highly unlikely, because vaginal echography demonstrated the development of a follicle of 18 x 22 mm in size outside the native ovary during the cycle which led to the pregnancy. The same day, the E2 level was 156 pg/ml and progesterone 0 ng/ml. This was extensively explained in the publication itself (Donnez et al., 2004), as well as in a letter later published by Donnez and Dolmans (2004) in response to comments by Oktay and Tilly (2004). Another of our main arguments was that laparoscopy performed 4 months after reimplantation proved, by direct visualization, the development of a follicle from the grafted tissue and, on histological examination, the biopsy samples indicated not only the survival of primordial follicles in the grafted tissue but also the maturation of a follicle (granulosa cells marked by inhibin A). It was the first histological proof of follicular maturation from reimplanted cryopreserved ovarian tissue. After delivery, the patient experienced ovulatory cycles every 5 to 6 weeks. Each time, an increase in FSH levels was observed (Figure 1). Figure 1 demonstrates that the ovarian graft is still functioning almost 3 years after transplantation.
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In 2006, the restoration of ovarian function after orthotopic (intraovarian and paraovarian) transplantation of cryopreserved ovarian tissue was reported in a woman treated by BMT for a non-cancerous disease (sickle cell anaemia) (Donnez et al., 2006). Thirty-nine small cryopreserved cubes were thawed and grafted into the ovary itself (24 cubes) and a peritoneal window (15 cubes). Vaginal echography and sequential measurement of FSH, LH, E2 and progesterone concentrations revealed the onset of an ovulatory cycle 4 months after the reimplantation of ovarian tissue, demonstrating the efficacy of orthotopic transplantation and confirming, once again, the time interval between reimplantation and the onset of ovulation. The patient experienced 3 cycles in total, evidenced by the development of a follicle and raised E2 levels. It should be noted that the E2 peaks never exceeded 55 pg/ml and that FSH rose to 40 mIU/ml between the cycles. However, after these 3 cycles, LH and FSH concentrations returned to castrated levels. We then decided to reimplant the 30 remaining cubes into the ovary, the patient being under GnRH agonist to decrease LH and FSH levels. She experienced a first ovulatory cycle 4 months after reimplantation. The pre-ovulatory E2 level was 120 pg/ml. The follicle measured 20 mm before the LH peak and progesterone was at 14.7 ng/ml in the mid-luteal phase (Figure 2).
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Very recently, we applied a technique, similar to that used by Silber et al. (2005) for the transplantation of fresh ovarian cortex between monozygotic twins, in a woman who had also undergone BMT and two regimens of alkylating agents in 2000 for non-Hodgkin’s lymphoma (Table III). Ovarian tissue cryopreservation was carried out 1 year after first-line chemotherapy. One ovary was removed and biopsies of cortical ovarian tissue revealed the presence of histologically normal primordial follicles. Six ovarian cortical pieces measuring 10 x 4–5 mm were then grafted onto the remaining ovary after the cortex of this ovary had been removed (Figure 3). It was 5 months before a mature follicle (21 mm) developed and an increase in E2 levels (194 pg/ml) was noted. The patient experienced an ovulatory cycle every 5 weeks, the pre-ovulatory E2 level reaching values between 210 and 356 pg/ml.
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The analysis of these cases raises some important points for discussion. First, in all three cases, it took between 4
and 5 months after reimplantation before a follicle could be seen. The process of folliculogenesis takes 
4–6 months, during which time the oocyte and surrounding somatic cells undergo a series of changes that eventually result in the development of a large antral follicle, capable of producing a mature oocyte (Gougeon, 1996). Thus, the appearance of the first follicle originating from the grafted tissue 5 months after reimplantation, proved by laparoscopy in one case, is totally consistent with the expected time course. This time interval between implantation of cortical tissue and the first E2 peak is also consistent with data obtained from sheep (Baird et al., 1999, 2004) and human beings (Table III), although some variations may be observed. Indeed, as summarized in Table III, the delay between transplantation and follicular development was found varying from 8 weeks to 8 months. Such a variation could be explained by a difference in follicular reserve at the time of cryopreservation.
Another very interesting finding is the persistence of relatively high FSH levels during the follicular phase. FSH levels remained as high as 25 mIU/ml during the follicular phase until ovulation and then decreased to <15 mIU/ml during the luteal phase. This may constitute an argument against the use of gonadotrophin injections. The relatively high FSH levels may be explained by the relatively low number of surviving primordial follicles in the graft. The patient should be considered a poor responder, with reduced inhibin B secretion. These results are in agreement with those obtained in sheep by Campbell et al. (2000).
A further significant observation is the return to an FSH level of >35 mIU/ml immediately after each menstrual bleed, which supports the theory suggested by Baird et al. (2004) that some inhibitory mechanisms, such as inhibin or anti-Müllerian hormones (AMH) normally produced by developing follicles in intact human ovaries, are probably almost non-existent in transplanted tissue. After transplantation, the patient would have been regarded a poor responder because, of the 500–1000 primordial follicles that would have been transplanted, >50% would have been lost owing to hypoxia (Donnez et al., 2004). This raises the question of the evaluation of the ovarian reserve. There is a lack of data on the ovarian reserve in cancer. Qu et al. (2000), Gook et al. (2005) and Schmidt et al. (2003) have all demonstrated an unequal distribution of primordial follicles in ovarian cortex.
In 2005, Meirow et al. also published a live birth after orthotopic autotransplantation of cryopreserved ovarian tissue in a patient with POF after chemotherapy. Eight months after orthotopic transplantation, the patient spontaneously menstruated. The rise in AMH and increased inhibin B levels were consistent with the presence of early growing follicles and ovulation, respectively. Nine months after transplantation, the patient experienced a second spontaneous menstrual period. After a modified natural cycle, a single mature oocyte was retrieved and fertilized. Two days later, a 4-cell embryo was transferred. The patient became pregnant from this embryo transfer and delivered a healthy infant weighing 3000 g. The possibility that the oocyte was derived from the native ovary is highly unlikely given the consistent evidence of POF after high-dose chemotherapy in this patient, from whom ovarian tissue was harvested after administration of a first-line conventional chemotherapy regimen, before second-line high-dose chemotherapy.
Schmidt et al. (2005) recently reported the results of three cases of ovarian tissue transplantation. All three patients with autotransplanted ovarian tissue regained ovarian function, as confirmed by the recovery of menses, follicles visible on ultrasonography and normal hormone levels. Two embryos were obtained from three MII oocytes and one GV oocyte, but no pregnancy resulted from embryo transfer.
Demeestere et al. (2006) very recently reported a pregnancy after natural conception in a woman who had undergone orthotopic and heterotopic transplantation of cryopreserved ovarian tissue. They observed follicular development in all three transplantation sites: large follicles in the ovarian site, only one dominant follicle in the peritoneal site and follicles <13 mm in size in the heterotopic site. Detectable HCG levels and ultrasonography confirmed the presence of a viable intrauterine pregnancy. Unfortunately, this pregnancy, obtained by natural conception, ended in miscarriage at 7 weeks due to aneuploidy. Interestingly, Demeestere et al. (2006) observed normal FSH values after orthotopic and with heterotopic transplantation of cryopreserved ovarian tissue. As stressed by the authors, this may have been due to the young age of the patient and the large number of tissue fragments transplanted (Demeestere et al., 2006), which could have yielded a rich follicular reserve in the graft.
Heterotopic autotransplantation of cryopreserved human ovarian tissue: a better option than orthotopic transplantation?
There are only six existing reports on this subject (Table III). Callejo et al. (2001) evaluated the long-term function of cryopreserved heterotopic grafts, but no conclusions could be drawn because the patient was perimenopausal at the time of ovarian biopsy for cryopreservation.
In 2004, Kim et al. (2004a) reported a case of a 37-year-old woman who underwent heterotopic (rectus and pectoralis muscle) transplantation of cryopreserved ovarian tissue. By 14 weeks of transplantation, the restoration of endocrine function was demonstrated but, 28 weeks after transplantation, the cessation of ovarian function was evidenced by very high FSH levels (62–99 IU/l) and very low E2 levels.
The same year, Oktay et al. (2004) reported transplantation of frozen–thawed ovarian tissue beneath the skin of the abdomen. A 4-cell embryo was obtained from 20 oocytes retrieved from an ovarian graft, but no pregnancy occurred after transfer. Oocyte quality might have been compromised by transplantation to a heterotopic site.
In 2005, Schmidt et al. reported two cases of mixed (heterotopic and orthotopic) transplantation, as did Demeestere et al. in 2006. These cases are discussed in the section on orthotopic transplantation.
Wolner-Hanssen et al. (2005) reported s.c. transplantation of frozen–thawed tissue to the forearm. Two follicles developed, but only to a maximum diameter of 12.6 and 6.7 mm, respectively, and the tissue survived 7 months. The authors suggested that the risk of graft exposure to suboptimal temperatures or mechanical stress may depend on transplantation site, and thus tissue transplanted under the skin of the forearm will probably be exposed to both higher pressure and lower temperature than ovaries in their normal location.
Very recently, Oktay (2006) reported a pregnancy after heterotopic transplantation of cryopreserved ovarian tissue, but ovulation occurred from the native ovary.
Papers describing heterotopic transplantation have all reported follicular development, but with follicles always <15 mm in size. As stressed by Wolner-Hanssen et al. (2005) and Oktay et al. (2004), differences in temperature and pressure could interfere with follicular development in heterotopic sites.
The primordial follicle is resistant to cryoinjury, because the oocyte it contains has a relatively inactive metabolism, as well as a lack of meiotic spindle, zona pellucida and cortical granules. The small size of primordial follicles also greatly facilitates penetration of cryoprotectant. Oktay et al. (1997) developed an isolation technique for human primordial follicles using enzymatic digestion and microdissection and obtained high follicular viability rates with both fresh and frozen ovarian tissues.
Nevertheless, the procedure to isolate primordial follicles remains difficult (Martinez-Madrid et al., 2004a); to date, it has not been possible to grow human isolated primordial follicles in vitro to the mature oocyte stage (Torrents et al., 2003).
Although safe xenotransplantation of ovarian tissue from lymphoma patients has been reported in SCID mice (Kim et al., 2001), the possibility of reintroducing tumour cells into cancer patients by autografting of ovarian tissue cannot be excluded. To avoid transferring malignant cells, ovarian tissue culture with in vitro follicle maturation could be performed. Culturing isolated follicles from the primordial stage is another particularly attractive proposition because they represent >90% of the total follicular reserve and show high cryotolerance (Smitz and Cortvrindt, 2002). However, isolated primordial follicles do not grow properly in culture (Hovatta et al., 1999; Abir et al., 2001), and further studies are clearly needed to identify factors sustaining follicular growth and maturation in humans (Smitz and Cortvrindt, 2002) and to assess the contribution of stromal cells to these processes. Encouraging results were achieved by Hovatta (2004) when human primordial follicles were grown in organ culture. Follicle isolation, or partial follicle isolation, severely impairs follicular viability in culture and, after isolation, primordial and primary follicles degenerate within the first 24 h of culture (Hovatta et al., 1999; Abir et al., 2001). Only more advanced, multilaminar pre-antral follicular stages can survive in short-term culture, a few reaching the early antral stage (Roy and Treacy, 1993; Abir et al., 1997).
Another approach could be to transplant a suspension of isolated follicles. As the follicular basal lamina encapsulating the membrana granulosa excludes capillaries, white blood cells and nerve processes from the granulosa compartment (Motta et al., 2003; Rodgers et al., 2003), grafting fully isolated follicles could be considered safer. Moreover, this would allow the introduction of a high and known number of follicles into the host, obtaining faster angiogenesis and minimizing ischaemic and reperfusion damage (Laschke et al., 2002). The transplantation of frozen–thawed isolated primordial follicles has indeed been successfully achieved in mice (Carroll and Gosden, 1993), yielding normal offspring. For human primordial follicles, however, mechanical isolation is not possible due to their size (30–40 µm) and their fibrous and dense ovarian stroma, and therefore enzymatic digestion with collagenase or Liberase has to be used (Dolmans et al., 2006). To enhance the chances of follicular survival and reproductive function restoration, enzymatic digestion procedures for human ovarian tissue need to be optimized and standardized.
As we recently demonstrated (Dolmans et al., 2006), Liberase treatment allows the isolation of highly viable follicles with an unaltered morphology and ultrastructure (Figure 4). This purified endotoxin-free enzyme preparation is a prom