Restoration of ovarian function in orthotopically transplanted cryopreserved ovarian tissue: a pilot experience

RBMOnline - Vol 16. No 5. 2008 694-704 Reproductive BioMedicine Online; www.rbmonline.com/Article/3176 on web 19 March 2008

Article Restoration of ovarian function in orthotopically transplanted cryopreserved ovarian tissue: a pilot experience Jacques Donnez studied medicine and completed his internship in gynecology and obstetrics at the Université Catholique de Louvain (UCL). His subsequent research career there has been devoted to identifying and understanding the molecular and pathophysiological mechanisms underlying female infertility and the development of new therapeutic options to restore or preserve women’s health and fertility. In 1984 he defended his PhD thesis ‘The Fallopian tube: normal and pathological histophysiology’ and then went on to found the Infertility Research Unit at UCL, becoming Head and Chairman of the Department of Gynaecology in 1986. Most recently, he has focused his research on ovarian cryopreservation and transplantation. Dr Jacques Donnez Jacques Donnez1, Jean Squifflet, Anne-Sophie Van Eyck, Dominique Demylle, Pascale Jadoul, Anne Van Langendonckt, Marie-Madeleine Dolmans Department of Gynecology, Cliniques Universitaires St. Luc, Université Catholique de Louvain, Avenue Hippocrate, 10, B-1200 Brussels, Belgium 1 Correspondence: e-mail: [email protected]

Abstract Cryopreservation of ovarian tissue is currently proposed to young cancer patients before chemo- or radiotherapy to preserve their fertility. In this study, ovarian cortex was removed by laparoscopy from five women and cryopreserved before chemotherapy. After chemotherapy, they all experienced amenorrhoea due to premature ovarian failure and requested reimplantation of their cryopreserved ovarian tissue several years later. Thawed fragments were then grafted to an orthotopic site in all five women. Two of them underwent a second reimplantation. Ovarian function recovery was evaluated by hormone concentration measurement, follicular development on ultrasound and menstruation recovery. The first signs of ovarian function restoration (oestradiol peak, decrease in FSH, ultrasound showing follicular development) occurred between 16 and 26 weeks after reimplantation. Elevated FSH concentrations were sometimes observed between series of consecutive ovulatory cycles, demonstrating the presence of a relatively low ovarian reserve. There were no signs of disease recurrence in any patients with malignant disease. In conclusion, restoration of ovarian function was observed in all cases. Grafts remained functional in all the women. Transplantation of cryopreserved ovarian tissue to an orthotopic site appears to restore ovarian endocrine function, without any signs of disease recurrence. Keywords: cryopreservation, fertility, graft, ovarian cortex, preservation, transplantation

Introduction Advances in the diagnosis and treatment of childhood, adolescent and adult cancer have greatly increased the life expectancy of young 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 premature ovarian failure and infertility as a result of the aggressive chemotherapy and radiotherapy treatments indicated for both cancer and bone marrow transplantation (Byrne et al., 1992; Larsen et al., 2003).

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Ovaries are very sensitive to cytotoxic treatment, especially

to radiation and alkylating agents, which are classified as high risk for gonadal dysfunction (for review, see Donnez et al., 2006a). Cyclophosphamide is the agent most commonly implicated in causing damage to oocytes and granulosa cells in a dose-dependent manner (Meirow and Nugent, 2001; Wallace et al., 2005a). This follicular destruction generally results in the loss of both endocrine and reproductive functions, depending on the dose and the age of the patient. Several options are currently available to preserve fertility in cancer patients and allow them to conceive when they

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Article - Orthotopically reimplanted cryopreserved ovarian tissue - J Donnez et al.

have overcome their disease: embryo cryopreservation, oocyte cryopreservation and ovarian tissue cryopreservation (Gosden et al., 1997; Donnez and Bassil, 1998; Oktay et al., 1998, 2004; Donnez et al., 2000, 2005, 2006b; 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; Bedaiwy and Falcone, 2007). According to the Ethics Committee of the American Society for Reproductive Medicine (2005), the only established method of fertility preservation is embryo cryopreservation. However, 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 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 are able to undergo a stimulation cycle, although the effectiveness of this technique is still 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 (Gosden et al., 1994; Donnez and Bassil, 1998; Oktay et al., 1998; Meirow et al., 1998; Donnez et al., 2000, 2005). Ovarian tissue can theoretically be frozen using three different approaches: as fragments of ovarian cortex; as the entire ovary with its vascular pedicle; or as isolated follicles (Donnez et al., 2006a). The indications for cryopreservation of ovarian tissue in cases of malignant and non-malignant disease are summarized in a recent review (Donnez et al., 2006a). The main aim of this strategy is to reimplant ovarian cortical tissue into the pelvic cavity (orthotopic site) or a heterotopic site, such as the forearm or abdominal wall, once treatment is completed and the patient is disease-free (Donnez and Bassil, 1998; Oktay et al., 1998, 2004; Oktay and Karlikaya, 2000; Radford et al., 2001; Donnez et al., 2004, 2005, 2006b; Kim et al., 2004; Schmidt et al., 2005; Demeestere et al., 2006; Rosendahl et al., 2006).

× 2 mm) or strips (± 10 × 3 mm). These fragments of ovarian tissue were suspended in cryoprotective medium and then placed into precooled 2 ml cryogenic vials (Simport, Quebec, Canada) filled with L-15 medium supplemented with 4 mg/ ml of human serum albumin (Red Cross, Brussels, Belgium) and 1.5 mol/l of dimethylsulphoxide (Sigma, St Louis, MO, USA). The cryotubes were cooled in a programmable freezer (Kryo 10, Series III; Planer, Sunbury-on-Thames, UK) with the following programme: cooled from 0°C to –8°C at –2°C/min; seeded manually by touching the cryotubes with forceps pre-chilled in liquid nitrogen; cooled to –40°C at –0.3°C/min; cooled to –150°C at –30°C/min; and transferred to liquid nitrogen (–196°C) immediately for storage. The thawing procedure was as follows: the cryogenic vials were thawed at room temperature (between 21°C and 23°C) for 2 min and immersed in a water bath at 37°C for another 2 min. Ovarian tissue was immediately transferred from the vials to tissue culture dishes (Becton Dickinson, NY, USA) in L-15 medium and subsequently washed three times at room temperature with fresh medium to remove cryoprotectant before further processing. Thawed ovarian cortical tissue was then placed in sterile medium and immediately transferred to the operating theatre.

Results Patient 1 In 1997, a 25-year-old woman presented with clinical stage IV Hodgkin’s lymphoma. Ovarian tissue cryopreservation was undertaken before chemotherapy. This case is described in detail in the Lancet (Donnez et al., 2004). Eleven months after reimplantation, the patient became pregnant and the pregnancy resulted in the live birth of a healthy girl, weighing 3.72 kg. This was the first ever live birth after orthotopic transplantation of cryopreserved ovarian tissue (Donnez et al., 2004).

Materials and methods

Following delivery and breastfeeding for 5 weeks, a first peak in oestradiol, concomitant with the development of a follicular structure, was observed 4 weeks after cessation of breastfeeding. The patient then experienced regular menstrual cycles every 4–6 weeks. She experienced 2 months of amenorrhoea on three occasions, with FSH values >40 mIU/ ml, subsequently returning to normal cycles. Four years after transplantation, the patient still has menstrual cycles every 4–6 weeks. Increased FSH concentrations have sometimes been observed before the follicular phase. Indeed, the patient has shown relatively high basal FSH concentrations during the last 12 months (ranging from 8.1–72 mIU/ml, median 35.9 mIU/ml), while 17β-oestradiol has been between 21 and 178 pg/ml (median 88 pg/ml) (Figure 1).

Freezing and thawing procedures

Patient 2

Freezing of ovarian tissue was undertaken according to the protocol described by Gosden et al. (1994). Biopsy samples were immediately transferred to the laboratory in Leibovitz L-15 medium supplemented with Glutamax (GIBCO, Paisley, UK); in the laboratory, the remaining stromal tissue was gently removed. The cortical samples were cut into small cubes (± 2

In 1999, a 21-year-old woman presented with complications (spleen abscess and cerebral thrombosis) of homozygous sickle cell anaemia. Bone marrow transplantation (BMT) was proposed to the patient, her sister being human leukocyte antigen (HLA)-compatible. Ovarian tissue cryopreservation was undertaken before chemotherapy (with

Here the pattern of ovarian endocrine function restoration after orthotopic autotransplantation of cryopreserved ovarian tissue is reported. To date, seven reimplantations of cryopreserved ovarian cortex in five women have been performed, two of them undergoing reimplantation twice.

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two alkylating agents: busulfan and cyclophosphamide). A right oophorectomy was performed by laparoscopy. It was decided to remove the whole ovary because ovarian failure is almost always induced after chemotherapy given prior to BMT (Teinturier et al., 1998; Meirow and Nugent, 2001; Wallace et al., 2005b). In July 1999, BMT was carried out, with the HLA-compatible sister acting as donor (Donnez et al., 2006b). After treatment, ultrasound evaluation of the remaining ovary revealed an atrophic ovary measuring 1.5 × 1 × 1 cm. The patient underwent a first reimplantation in 2006 (Donnez et al., 2006b). Forty cubes were reimplanted into a peritoneal window and the residual atrophic ovary. She experienced three ovulatory cycles 4 months later, but after these three cycles, FSH and LH concentrations rose to values above 40 mIU/ml (Figure 2) and remained high for more than 6 months. It was therefore decided to reimplant the remaining fragments. The patient received preoperative gonadotrophinreleasing hormone (GnRH) agonist (Zoladex®; AstraZeneca, Brussels, Belgium) and oestro-progestogen therapy in order to decrease FSH and LH concentrations before reimplantation (see Discussion). During the surgical procedure, the ovarian cortex was decorticated over a surface area of 1.5 × 1 cm, the fragments were placed on the medulla, and an absorbable adhesion barrier (Interceed®; Johnson and Johnson, New Brunswick, USA) was used to hold the fragments in place. The Interceed edges were sutured to the remaining ovarian cortex. Transmission electron microscope analysis of the decorticated area revealed a normal ultrastructure in previously grafted follicles (Camboni et al., 2007). After cessation of the GnRH agonist and oestro-progestogen therapy, LH and FSH concentrations returned to castrated levels for 3 months. Subsequently, the patient experienced ovulatory cycles with oestrogen values over 100 pg/ml. Between some cycles, a small rise in FSH and LH was seen. The interval between the second reimplantation and recovery of ovarian function was 4 months. Despite normal follicular maturation, as proved by both echography and steroid and gonadotrophin concentrations, the patient failed to become pregnant. It was thus decided to optimize follicular maturation by adding recombinant FSH (Puregon; Organon, Oss, The Netherlands). GnRH antagonist (Ganerelix; Organon, Oss, The Netherlands) was administered when follicular size reached 14 mm to prevent an LH surge, and human chorionic gonadotrophin (HCG; Pregnyl, Organon) when follicular size reached 18 mm. Oocyte retrieval was performed 35 h after HCG administration. Empty follicles were diagnosed on two attempts as no oocytes were retrieved on puncture. The third time, a metaphase I (MI) oocyte was recovered and matured in vitro. At the MII stage, intracytoplasmic sperm injection (ICSI) was performed, but no fertilization occurred. More than 3 years after the first reimplantation, and 18 months after the second, the graft is still functioning, with progesterone ranging between 0.3 and 14.5 ng/ml.

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Patient 3 In 1997, a 22-year-old woman presented with stage IIIA Hodgkin’s lymphoma. She immediately received ABVD (adriamycin, bleomycin, vinblastine, dacarbazine) chemotherapy. One year later, she suffered a relapse and subsequently underwent ovarian tissue cryopreservation before BMT. A left oophorectomy was performed by laparoscopy and ovarian cortical strips were cryopreserved. A small biopsy was sent for histological analysis in order to evaluate the ovarian follicular reserve. Histology revealed the presence of more than 22 primordial follicles per mm3. The patient then received MOPP-BEAM (mustine, oncovin, procarbazine, prednisone – carmustine, etoposide, cytarabine, melphalan) chemotherapy, as well as total body irradiation (TBI), before peripheral stem cell transplantation. After TBI, FSH and LH concentrations were at menopausal levels and the patient started hormone replacement therapy (HRT). At the beginning of 2005, she wanted to become pregnant. HRT was stopped and FSH and LH immediately returned to postmenopausal levels. Transplantation was then decided upon and carried out in June 2005. The day before transplantation, GnRH antagonists (Ganerelix, Organon, Oss, The Netherlands) and oestro-progestogens were started for a period of 8 weeks in order to decrease FSH and LH concentrations. A laparotomy was performed and five large strips (8 to 10 × 4 to 5 mm) were reimplanted onto the remaining right ovary after removal of the native cortex (Figure 3a). Serial sections of the native cortex failed to reveal the presence of any primordial follicles. Each strip was attached to the medulla with two stitches of 7/0 suture (Prolene). No more than five strips could be reimplanted onto this ovary because of its small size. After 8 weeks of combined GnRH antagonist and oestro-progestogen treatment, FSH, LH and oestradiol concentrations all returned to castrated levels (Figure 3b). Five months after transplantation, ultrasound revealed a preovulatory follicle of 16 × 20 mm and endometrial thickness of 6.5 mm. Oestradiol concentrations rose to 194 pg/ml and FSH dropped to 23 mIU/ml. Three follicles (26, 14 and 10 mm) were detected by vaginal echography during the second ovulatory cycle. The oestradiol concentration then reached 326 pg/ml and the patient menstruated. FSH and LH concentrations rose again for 6 weeks, and the patient began to have regular ovulatory cycles, as proved by the presence of preovulatory follicles of more than 18 mm, progesterone concentrations during the mid-luteal phase, and menstrual bleeding. It should nevertheless be noted that, while the patient experienced regular cycles, some of them (about 70%) were characterized by low mid-luteal phase progesterone concentrations (<5 ng/ml), even with a preovulatory follicular size between 19 and 22 mm. In about 30% of cycles, midluteal progesterone was found to be over 10 ng/ml. More than 20 months after reimplantation of ovarian cortex, follicular maturation is still ongoing. With menstruation occurring every 4 to 6 weeks, it was decided to perform oocyte retrieval 9 months after transplantation, when the oestradiol concentration had risen to 256 pg/ml and an LH peak (132 mIU/ml) was observed. RBMOnline®

Article - Orthotopically reimplanted cryopreserved ovarian tissue - J Donnez et al.

Figure 1. Patient 1. FSH and 17β-oestradiol concentrations after delivery (27 September 2004). Four years after transplantation, the patient shows great variation in oestradiol and FSH concentrations.

Figure 2. Patient 2. FSH and 17β-oestradiol concentrations from before the first reimplantation to date. The first reimplantation was followed by three menstrual cycles, then there was a return of the hormone concentrations to castrated levels. The second reimplantation was carried out under gonadotrophinreleasing hormone agonist and oestroprogestogen therapy. Four and a half months later, recovery of ovarian function was observed.

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Figure 3. Patient 3. (a) After the ovary was decorticated, five large strips (8–10 × 4–5 mm) were sutured to the medulla. (b) FSH and 17β-oestradiol concentrations. Five months after transplantation, restoration of ovarian function was demonstrated by a rise in oestradiol concentration.

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The follicle measured 18 mm but, on puncture, no oocyte could be retrieved. Three months later, a second oocyte retrieval was attempted. The oestradiol concentration had now reached 502 pg/ml, LH 15 mIU/ml and FSH 7.3 mIU/ ml. Two follicles were visible on ultrasound, measuring 21 and 16 mm, respectively. HCG (5000 UI) (Pregnyl; Organon, Oss, The Netherlands) was then given. Only one empty zona pellucida was recovered. During subsequent cycles, progesterone concentrations reached 15.4 ng/ml. During the third attempt, an oocyte was retrieved but sudden lysis of the zona pellucida was observed during the ICSI procedure. Thereafter, the patient continued to experience regular ovulatory cycles (Figure 3b), but has not yet become pregnant.

Patient 4 This patient was 28 years old when she was diagnosed with stage IIB non-Hodgkin’s lymphoma type B. Treatment with ACVBP (doxorubicin, cyclophosphamide, vindesine, bleomycin, prednisone) was initiated. Two months later, it was decided to perform BMT and ovarian tissue was cryopreserved prior to the procedure. By laparoscopy, four ovarian cortical biopsies (10 mm × 5 mm) were retrieved from the right ovary. A small sample (6 × 2 mm) was sent for histological evaluation and revealed eight primordial follicles per mm3. The patient then underwent BMT. Soon after, the concentration of oestradiol (<10 pg/ml) and FSH (89 mIU/ml) were found to be at castrated levels. The patient received HRT for 5 years. It was stopped because she wanted to become pregnant. FSH and LH concentrations returned to castrated levels. Transplantation of frozen–thawed ovarian tissue was then proposed. GnRH agonist injections (Zoladex; AstraZeneca, Brussels, Belgium) and oestro-progestogen therapy were started 2 weeks before laparotomy for a period of 2 months. During laparotomy (Figures 4a and 4b), the cortex of both remaining ovaries was removed and thawed ovarian fragments were placed on the decorticated medulla. The fragments were too small to suture to the remaining ovary, so absorbable adhesion barrier (Interceed) was used to cover and fix them to the ovary. The barrier was itself secured by sutures. Once the down-regulation effect induced by the GnRH agonist ceased, LH and FSH concentrations returned to castrated levels (Figure 4c). Six and a half months after transplantation, a first follicle of 17 mm in size was echographically observed, with an oestradiol concentration of 138 pg/ml. Menstrual bleeding then occurred. Four weeks later, another follicle was observed, the oestradiol concentration was 100 pg/ml and the patient experienced a second menstrual bleed. During the third cycle, the progesterone concentration was 10.1 ng/ml and, after this ovulatory cycle, the patient once again menstruated. During these three cycles, LH and FSH concentrations nevertheless remained quite elevated (FSH between 27 and 42 mIU/ml, LH between 21 and 38 mIU/ml).

observed, concomitant with an oestradiol concentration of 140 pg/ml and FSH remaining at about 20 mIU/ml (between 18 and 23 mIU/ml), even during the following cycle, when oestradiol rose to 421 pg/ml. No progesterone was detected during these two cycles. The patient then had a cycle with a progesterone concentration of more than 10 ng/ml during the luteal phase. Both ovaries demonstrated follicular development, more or less alternately.

Patient 5 A 22-year-old woman presented with severe complications of Wegener’s granulomatosis. The pneumologists decided to initiate cyclophosphamide treatment (100 mg/day). Before chemotherapy was started, the patient underwent ovarian tissue cryopreservation in 2001. Three biopsies were taken from each ovary, cut into small cubes (2 mm) and stored in liquid nitrogen. The patient received 100 mg cyclophosphamide a day for 5 years. Two months after the start of cyclophosphamide treatment, she experienced amenorrhoea. Her FSH concentration was 114 mIU/ml, LH 45 mIU/ml and oestradiol less than 10 pg/ml, so she was given HRT. On stabilization of the disease 5 years later, she was able to stop cyclophosphamide and switch to azathioprine. After obtaining informed consent and reaching a multidisciplinary decision, reimplantation of cryopreserved ovarian cortex was proposed. The patient stopped HRT, and her LH, FSH and 17β-oestradiol concentrations returned to castrated levels. Two weeks before reimplantation, GnRH agonist (Zoladex®; AstraZeneca, Brussels, Belgium) was administered, together with oestro-progestogens, and continued for 2 months. A laparoscopy was carried out and both ovaries appeared atrophic (± 1.5 mm in size). They were both decorticated and small cubes of thawed ovarian tissue were then placed on the medulla and held in place with absorbable adhesion barrier (Interceed), itself sutured to the remaining cortex of the native ovary. Several biopsies of the native ovary, obtained by decortication, failed to demonstrate the presence of any primordial follicles (no primordial follicles at all in the 44 mm3 analysed). During this treatment, the FSH concentration was <0.4 mIU/ml, LH <0.2 mIU/ml and oestradiol <10 pg/ml. When the down-regulation effect induced by the GnRH agonist ceased, LH and FSH concentrations returned to castrated levels (Figure 5). Four and a half months after reimplantation, an increase in the oestradiol concentration (132 pg/ml), concomitant with the development of two follicles (both 18 mm in size, as proved by vaginal ultrasonography), was observed. FSH and LH concentrations decreased to 32.6 and 17.7 mIU/ml, respectively, during this first cycle of ovarian activity. A second cycle followed immediately, with an increase in oestradiol to 188 pg/ml. Progesterone concentrations remained low during these two cycles.

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Thereafter, LH and FSH concentrations rose again for 5 weeks to castrated levels. Subsequently, a follicle was

Human ovarian tissue can be successfully cryopreserved, RBMOnline®

Article - Orthotopically reimplanted cryopreserved ovarian tissue - J Donnez et al.

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Figure 4. Patient 4. (a) After the cortex of the native ovary was removed, thawed ovarian fragments were placed on the decorticated medulla. (b) Absorbable adhesion barrier was used to cover and fix the thawed fragments to the ovary. (c) FSH and 17β-oestradiol concentrations. Gonadotrophin-releasing hormone agonist (GnRHa) and oestro-progestogens (OP) were given for 2 months around the time of reimplantation. Six and a half months after reimplantation, restoration of ovarian function was observed.

Figure 5. Patient 5. FSH and 17β-oestradiol concentrations after reimplantation. Four and a half months after reimplantation, restoration of ovarian function was observed. GnRHa = gonadotrophin-releasing hormone agonist; OP = oestroprogestogens.

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Figure 6. Mean FSH and 17β-oestradiol concentrations (± SD) in the seven cases of frozen-thawed ovarian tissue transplantation. It took between 4 and 6 months after transplantation before a rise in oestradiol and a drop in FSH concentrations were observed. (Note that, in the last four cases, FSH and oestradiol concentrations at 1 month post-transplantation were not taken into account, as the patients were under gonadotrophin-releasing hormone agonist down-regulation).

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. Reported cases of autotransplantation of cryopreserved ovarian tissue to an orthotopic or heterotopic site (Oktay and Karlikaya, 2000; Callejo et al., 2001; Radford et al., 2001; Donnez et al., 2004; Kim et al., 2004; Oktay et al., 2004; Meirow et al., 2005; Schmidt et al., 2005; Wolner-Hanssen et al., 2005; Demeestere et al., 2006; Donnez et al., 2006b; Oktay, 2006) were recently reviewed by Donnez et al. (2006a). To date, ovarian tissue has been successfully cryopreserved and transplanted into rodents, rabbits, sheep, marmoset monkeys and humans (Candy et al., 1995, 2000; Salle et al., 2002; Almodin et al., 2004; Donnez et al., 2004; Meirow et al., 2005; Demeestere et al., 2006; Rosendahl et al., 2006). Successful fertilization and pregnancy from fresh transplanted ovarian tissue have been described in a primate (Lee et al., 2004) and humans (Silber et al., 2005; Silber and Gosden, 2007); in both cases, the grafted tissue functioned without any surgical connection to major blood vessels. In the present study, the restoration of ovarian function after orthotopic frozen–thawed ovarian cortex reimplantation (n = 7) in five women is reported and analysed.

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There are several reasons why cryopreserved ovarian tissue transplantation has been performed in only five patients to date. Although this work was started over 10 years ago, it was often hard to convince oncologists and paediatricians of its worth in the early years. Despite having more than 280

samples of cryobanked ovarian tissue, a marked increase in requests for cryobanking was only seen after 2000–2001. Oncologists recommend a minimum time interval of 5 years between chemotherapy and reimplantation and, very often, the patient waits for at least 6 years before asking for the procedure. Three reimplantations have been refused because of the risk of transmission of malignant cells (leukaemia), which is why the development of isolated follicle transplantation is currently a priority (Dolmans et al., 2008). Analysis of these seven reimplantations performed in our department raises some important points for discussion. First, in all cases, it took between 16 and 26 weeks after reimplantation before a follicle was seen and a rise in oestradiol was detected (Figure 6). 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 ≥18 weeks after reimplantation is consistent with the expected time course. The time interval between implantation of cortical tissue and the first oestradiol peak is also consistent with data obtained from sheep (Baird et al., 1999; 2004) and human beings (Donnez et al., 2004), although some variations may be observed. Indeed, in the literature, the delay between transplantation and follicular development was found to vary from 8 weeks to 8 months (Donnez et al., 2006a). Such a variation could be partially explained by a difference in follicular reserve at the time of cryopreservation, some women having already received a first regimen of chemotherapy before ovarian biopsy and cryopreservation. In our study, RBMOnline®

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an interval of 20 to 26 weeks was observed in women who had chemotherapy prior to cryopreservation, compared with 16–18 weeks in those who underwent tissue cryopreservation before starting chemotherapy. Another very interesting finding was the persistence of relatively high FSH concentrations during the follicular phase. FSH concentrations remained as high as 25 mIU/ml during the follicular phase until ovulation, and then decreased to <15 mIU/ml during the luteal phase if progesterone was more than 5 ng/ml. This may be explained by the relatively low number of surviving primordial follicles in the graft. Such patients should be considered poor responders, with reduced inhibin B secretion. These results are in line with those obtained in sheep by Campbell et al. (2000) and Baird et al. (2004). A further notable observation was the return to FSH concentrations of >35 mIU/ml after some menstrual bleeding, which supports the theory suggested by Baird et al. (1999, 2004) that some inhibitory mechanisms, such as inhibin or antiMüllerian hormone (AMH) normally produced by developing follicles in intact human ovaries, are probably significantly diminished in transplanted tissue. After transplantation, these patients would have been regarded as poor responders because, of the 500–1000 primordial follicles that would have been transplanted, >50% would have been lost owing to hypoxia (Donnez et al., 2004). Moreover, due to the unequal distribution of primordial follicles in ovarian cortex, as demonstrated by Qu et al. (2000), Schmidt et al. (2003) and Gook et al. (2005), it is quite impossible to evaluate their exact number. Patients sometimes experienced three or four consecutive ovulatory cycles, followed by a period of 2–3 weeks of high FSH and LH concentrations, again followed by consecutive menstrual cycles. Such increases in FSH and LH between series of ovulatory cycles were observed in all the cases described and should be considered as activators of maturation of primordial follicle ‘cohorts’. The differences in behaviour of primordial follicles in grafted tissue could either be due to their unequal distribution or an unequal vascular network resulting in unevenly vascularized areas, alternating with areas of fibrosis, as observed in animal models (Nisolle et al., 2000). In the last four cases, a decrease in LH and FSH concentrations was induced before reimplantation by administration of GnRH agonist (or antagonist) and oestro-progestogens, some experimental studies having demonstrated significant follicular activation soon after xenotransplantation (Dolmans et al., 2007). Indeed, 7 days after grafting cryopreserved human ovarian tissue to nude mice, a significant decrease was observed in the proportion of primordial follicles due to activation of follicular development (high mitotic index in granulosa cells), as has been observed by others in animal models (Baird et al., 2004). This process could be even more pronounced in cases of high FSH and LH concentrations. Indeed, it was recently suggested that rising FSH concentrations could accelerate female reproductive failure (McTavish et al., 2007). This is why we propose administering GnRH agonist and oestro-progestogens around the time of ovarian tissue reimplantation, in order to avoid the stimulating effect of high concentrations of gonadotrophins on grafted follicles. The timing of administration of GnRH agonists or antagonists is dependent on their mechanism of action. As GnRH agonists induce an FSH flare-up in the first 2 weeks,

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transplantations were performed 2 weeks after the first injection. With GnRH antagonists, transplantation was performed the day after, as they immediately inhibit the receptor. To date, six attempts at oocyte retrieval have been made (three during natural cycles, three during modified natural cycles adding recombinant FSH and GnRH antagonist). In four of them, no eggs were retrieved from aspirated follicles (empty follicle syndrome), while two attempts yielded eggs, but they failed to fertilize. Similar data were recently obtained by Meirow et al. (2007), who reported recovery of only one egg out of four attempts (25%). In modified natural cycles, Pellinck et al. (2007) recently achieved an oocyte retrieval rate of 73% per attempt. Women with transplanted frozen-thawed ovarian tissue have low ovarian reserves and elevated FSH concentrations, frequently failing to have oocytes in aspirated follicles. This could arise from the difference in activation of granulosa cells and oocytes, due to the presence of persistently high FSH concentrations, which provokes a discrepancy in maturation. It is also possible that asynchrony between granulosa cell maturation and oocyte maturation, as observed in xenografts to nude mice, is responsible for the absence of eggs upon follicle aspiration, the granulosa cells being mature (size, oestradiol concentration >200 pg/ml), while the oocyte is immature. Another hypothesis to explain the prevalence of empty follicles is that the oocyte itself could be damaged by the cryopreservation, thawing and reimplantation procedures. This premature follicular cell activation was observed in xenografts (Nottola et al., 2007). It may result from stimulation of follicular growth by ischaemic and oxidative stress after transplantation via hypoxia-inducible factor-1 (Alam et al., 2004), or be due to the removal of some inhibitory mechanisms that normally operate in an intact ovary (Baird et al., 2004). Alternatively, primary oocyte development may be delayed because the oocyte is not yet developmentally competent when follicular growth is triggered. In the reported series, the peritoneal window created close to the ovarian hilus, as well as the ovarian medulla, were both demonstrated to be equally efficient sites of reimplantation. Large strips (8–10 mm × 5 mm) or small cubes (2 mm) were reimplanted. Both sizes effectively restored ovarian endocrine function. From a microsurgical point of view, however, it is easier to attach large strips to the medulla than small cubes, which cannot be sutured. Since reimplantation of large strips is easier and just as effective as small cube reimplantation, it is strongly suggested that large strips be taken from the ovarian cortex for cryopreservation purposes. The maximum follow-up in our series is now 4 years and the patient is still experiencing follicular development from the grafted tissue.

Discussion Advances in reproductive technology have made fertility preservation a real possibility for patients whose gonadal function is threatened by premature menopause, or by treatments such as radiotherapy, chemotherapy or surgical castration. Cryopreservation of ovarian tissue should be seriously considered

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for any patient undergoing treatment likely to impair future fertility, the indications being pelvic, extra-pelvic and/or systemic malignant diseases, as well as non-malignant diseases. We firmly believe that ovarian cortex banking should be offered before chemotherapy in all cases where emergency IVF is not possible. One of the most important ethical issues is to ensure that the intervention does not harm the patient by dangerously delaying cancer treatment and that no remnant cells are reintroduced by subsequent transplantation (Kim et al., 2001). If this risk exists, other options must be considered in the future, such as transplantation of isolated follicles (Dolmans et al., 2006, 2007, 2008), or even oocytes derived from artificially made embryonic stem cells (Okita et al., 2007). This last option has been discussed by Edwards and Ahuja (2006). It has been demonstrated here that cryopreserved primordial follicles can survive the thawing and grafting process and that restoration of ovarian function occurs in all women undergoing reimplantation of cryopreserved ovarian cortex. Further experimental studies are needed to confirm the benefits of ovarian tissue strip versus cube transplantation, to establish whether hormonal treatment is required before transplantation and to investigate the mechanism of empty follicle syndrome. Ovarian transplants appear to behave more like perimenopausal ovaries, but with young oocytes. In the future, to avoid ischaemia-related damage, cryopreservation and reimplantation of a whole ovary may be considered as an alternative option (Martinez-Madrid et al., 2004, 2007; Jadoul et al., 2007).

Acknowledgements This work was supported by the Fonds National de la Recherche Scientifique de Belgique (grant no. 7.4519.04), Télévie (grant 7.4547.06), the Fondation St Luc, the Belgian Federation Against Cancer (non-profit organization) and donations from A. Frère and Ph. de Spoelberch.

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Received 12 September 2007; refereed 11 October 2007; accepted 7 December 2007.

Declaration: The authors report no financial or commercial conflicts of interest.

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