Fresh human orthotopic ovarian cortex transplantation: long-term results

Transcription

1 Human Reproduction Vol.22, No.3 pp , 2007 Advance Access publication November 16, doi: /humrep/del440 Fresh human orthotopic ovarian cortex transplantation: long-term results M.Sánchez 1,2, P.Alamá 1, B.Gadea 2, S.R.Soares 2, C.Simón 2 and A.Pellicer 1,2,3 1 Department of Obstetrics and Gynaecology, Hospital Universitario Dr Peset and 2 Instituto Universitario Valenciano de Infertilidad (IVI), University of Valencia, Valencia, Spain 3 To whom correspondence should be addressed at: Department of Obstetrics and Gynaecology, Hospital Universitario Dr Peset, Av Gaspar Aguilar 90, Valencia, Spain. pellicer_ant@gva.es BACKGROUND: Ovarian orthotopic transplantation in patients with premature ovarian failure is reported to result in full-term pregnancies. Ischaemia and freezing/thawing are potentially injurious for tissues. This study was designed to analyse the effect of ischaemia on long-term ovarian function in humans. METHODS: Prospective case control study. Subjects were 12 premenopausal women undergoing hysterectomy and fresh orthotopic transplantation of the entire ovarian cortex plus a control group of five patients undergoing hysterectomy only. Follow-up lasted 2 years. Serum FSH and anti-müllerian hormone (AMH) were recorded, and ovulatory cycles were determined by vaginal ultrasound and serum progesterone levels. RESULTS: Follow-up showed that ovulation was restored in 11 of the 12 patients who received grafts over the duration of the study (9.3 ± 1.73 ovulations versus 12.0 ± 0.86 in controls, NS), and 9 of 12 patients remained ovulatory after 2 years. We identified four patterns of FSH secretion during the study, 5 of 12 (41.7%) women having the same pattern as controls. There was a trend for serum AMH levels 7 days after surgery (0.16 ± 0.02 mg/l) to be lower than pre-surgery levels (0.38 ± 0.09 mg/l, P = 0.07) and higher in women whose FSH patterns suggested normal ovarian function, but the results did not reach significance. After transplantation, FSH correlated more closely (r = 0.639, P = 0.02) with normal ovarian function than AMH (r = 0.465, P = 0.12). CONCLUSIONS: Fresh orthotopic ovarian cortex transplantation is a viable procedure. It maintains normal ovarian function after 2 years in 75% of cases and preserves ovarian function against ischaemia in 41.7% of patients. Key words: anti-müllerian hormone/fsh/ischaemia/ovarian orthotopic transplantation Introduction The number of young women diagnosed with cancer is increasing. The expected demand for fertility preservation techniques will mainly correspond to breast cancer and Hodgkin disease, the latter being the most common solid tumour seen in adolescent people (Viviani et al., 1985), while breast cancer is the most common tumour in western women, accounting for 30% of all tumours and 20% of all cancer-related deaths. The number of adults with a history of childhood cancer is also increasing because of the rising disease rates among young people and advances in its treatment (Weir et al., 2003; Gatta et al., 2005). Survival rates among young people with malignancies have reached 90 95% (Kim, 2006), but most cancer therapies produce non-reversible consequences for the reproductive system that are age- and dose-dependent (Meirow and Nugent, 2001). Several strategies have been explored to overcome this unfortunate secondary effect. Ovarian stimulation and preservation of oocytes or embryos is an option, with the disadvantage that relatively few oocytes/embryos are preserved. Moreover, this option is far from ideal, given the delay caused to the initiation of cancer treatment while ovarian stimulation is carried out to retrieve oocytes, plus the potential contraindication of hormonal treatment and detrimental effect for some cancers. In addition, cryopreservation of embryos implies the existence of a partner, and the current success rate of oocyte cryopreservation is low (Porcu and Venturoli, 2006). A second strategy for preserving fertility in cancer patients is cryopreservation of ovarian tissue for later autotransplantation, which can be performed at a heterotopic or orthotopic site. Orthotopic transplantation is preferable in that it permits natural fertility using fresh (Silber et al., 2005) or frozen and thawed ovarian cortex (Donnez et al., 2004). When natural conception fails, IVF remains an option (Meirow et al., 2005). The report of the first three full-term pregnancies achieved with ovarian orthotopic transplantation has promoted its application (Donnez et al., 2004; Silber et al., 2005; Meirow et al., 2005). Nevertheless, it must be said that the long-term accumulated experience of ovarian grafting is as yet limited. Ovarian cryopreservation has two drawbacks that limit its successful application: the cryopreservation procedure and ischaemic damage. Experimental evidence suggests that cryopreservation per se does not affect the long-term viability of 786 The Author Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please journals.permissions@oxfordjournals.org

2 Long-term outcome of fresh ovarian grafts ovarian tissue; in fact, a normal reproductive lifespan can be restored in mice through orthotopic grafting of a frozen ovary (Candy et al., 2000; Liu et al., 2002). Thus, the major hurdle represented by this technique is ischaemia and the follicular loss it can induce (Baird et al., 1999; Kim et al., 2004), with the subsequent detrimental effect caused to the pool of primordial follicles (Liu et al., 2002). We hypothesized that, to minimize the risk of ischaemia, the ovarian medulla would be the most appropriate site for orthotopic ovarian cortex transplantation, as maintenance of the blood supply is assured by the ovarian artery and because of the role the medulla seems to play in follicular development (Burden, 1985; Lara et al., 1990) and steroidogenesis (Hsueh et al., 1984; Ojeda et al., 1989). Thus, the aim of this work was to investigate the possible effects of ischaemia in contralateral orthotopic human ovarian grafting, and to do this, we designed an experimental study of autografting in women undergoing gynaecological surgery due to benign indications. We have addressed the short- and longterm results of this technique on ovarian function. We were also interested in exploring the endocrine changes described in the literature following fresh ovarian cortex orthotopic transplantation in humans (Donnez et al., 2005; Silber et al., 2005) and in determining the long-term results, given that the longest follow-up period reported until now has been 11 months (Donnez et al., 2004). In addition to serum FSH, anti- Müllerian hormone (AMH) is a powerful biochemical marker. AMH messenger RNA and protein expression have been detected in follicles that are starting to grow, being more pronounced at the pre-antral and small antral stages ( 4 mm) and declining in larger antral follicles (Weenen et al., 2004). AMH is produced by granulosa cells from around week 36 of gestation until menopause (Lee et al., 1996), and serum AMH levels have proved to be a reliable estimate of the number of follicles to be recruited in a particular menstrual cycle (Pastor et al., 2005). Materials and methods This is a prospective case series study of 12 cases and 5 controls. The study was approved by the ethics committee of the Hospital Universitario Dr Peset, and all patients signed a consent form once the purpose of the study, proposed intervention, risks and the approximate increase in surgery time (20 to 30 minutes) due to the specific procedure had been explained in detail. Patients were offered the possibility of re-transplantation if menopausal symptoms appeared during the course of the study. Twelve premenopausal women, years (mean 40.8 ± 0.7 SEM), who were programmed to undergo an abdominal hysterectomy because of uterine disease, were included in the study. In 10 patients, the indication for surgery was uterine fibroids, whereas the remaining 2 patients suffered menorrhagia and endometrial polyps (Table I). Inclusion criteria were (i) regular cycles and normal endocrinological parameters, confirmed by basal serum FSH levels 10 IU/l and serum progesterone 5 ng/ml on days of the menstrual cycle, and (ii) absence of adnexal pathology. Oral combined contraceptives were administered to patients to maintain their ovaries at quiescent stage, in that way avoiding the growth of follicles in the ovarian cortex before surgery, therefore enabling us to ensure that all the follicles revealed by ultrasound had developed after surgical intervention. The surgical procedure consisted in opening the abdominal wall via a supra-pubic transverse incision. An incision was made with the scalpel close to the left ovarian hilum, and scissors were employed to separate the cortical from the medullar tissue. This manoeuvre was performed all along the cortex inner surface; nevertheless, after the whole cortex removal, electrocautery was applied to ensure haemostasia and the complete destruction of any cortical tissue that could be present afterwards. A small sample of cortex (5 mm 5 mm) was sent to pathology for histological evaluation. The rest of the ovarian cortex was maintained in Ham s F10 culture medium until reimplantation. A right ovarian cortex resection was then performed, and the cortex was prepared for cryopreservation by cutting it into one or two pieces whose thickness did not exceed 2 mm, so as to facilitate the action of cryoprotectants. The left ovarian cortex was then transplanted into the right ovary medullar tissue. Fibrin glue (TISSUCOL DUO 2 ml, Baxter International AG, Vienna, Austria) was used for fixation, and, if necessary, one to three stitches were applied with 4 0 non-absorbable polyglycolic acid suture [ASSUFIL, ASSUT EUROPE SPA, Magliano del Marsi (AQ), Italy]. Electrocautery was not performed in the medulla so that ovarian cortex irrigation could be re-established and the risk of ischaemia thus minimized, but if the presence of any macroscopic cortical tissue near the hilum was suspected, it was cauterized before transplantation. Hysterectomy was then performed following the Masterson technique, with some slight modifications to maintain the correct ovarian blood supply as long as possible and thereby minimize ischaemia. A control group was established to analyse any possible effects of hysterectomy on the pituitary gonadal axis and follicular reserve and the effect of the surgical procedure itself. This group was composed of five premenopausal women, years old (mean 40.8 ± 1.2), who were to undergo a simple abdominal hysterectomy because of uterine fibroids and who fulfilled the same criteria as the study group. Post-operative endocrine follow-up for cases and controls was based on serum hormone determinations and ultrasound scans. Blood was drawn on the day of intervention and 7 days later and subsequently every month during the first year and bimonthly for the second year. Part of the blood was processed to determine FSH, while aliquots were also frozen to 80 C and subsequently analysed in parallel for serum AMH. Serum FSH served as a marker to detect ovarian function and subsequent ovulation, and when it dropped <20 IU/l, an ultrasound was performed every 2 days to determine follicular growth. Blood was drawn 7 9 days after apparent follicular rupture, at which point follow-up visits were planned. Ovulation was confirmed when the corpus luteum was detected by ultrasound and progesterone level was >5 ng/ml. Serum FSH and progesterone were analysed using a commercially available microparticle enzyme immunoassay kit AXSYM SYS- TEM (Abbot, Weisbaden, Germany). Inter-assay and intra-assay coefficients of variation for FSH were 2.3 and 4.5%, respectively, and 2.0 and 5.1% for progesterone, respectively. Serum AMH levels were measured using an ultrasensitive enzymelinked immunoabsorbent assay (Immunotech SA, Marseille, France). Intra-assay and inter-assay coefficients of variation were <12.3 and <14.2%, respectively. Data were expressed as the mean ± SEM. Analysis of variance and Student s t-test were employed to compare the number of ovulatory cycles and serum AMH levels. Linear correlation was employed to determine the reliability of FSH and AMH for predicting ovarian function. Statistical analysis was carried out with the Statistical Package for the Social Sciences 12.0 for Windows. Statistical significance was considered when P < Results Table I summarizes the epidemiologic data of the patients included in the study. Bilateral cortex resection and transplantation 787

3 M.Sánchez et al. Table I. Clinical characteristics of the patients included in the study Case number Age (years) Parity Medical antecedents Surgery indication Surgery 1 40 G0 No Fibroids Total hysterectomy 2 37 G2P2 Malaria, tuberculosis Fibroids Subtotal hysterectomy 3 37 G0 No Fibroids Total hysterectomy 4 41 G3P2A1 No Fibroids Total hysterectomy 5 41 G3P1A1C1 Myopia magna Fibroids Total hysterectomy 6 45 G0 No Fibroids Total hysterectomy 7 43 G2P2 No Fibroids Total hysterectomy 8 41 G2C2 No Menorrhagia Subtotal hysterectomy + left adnexectomy 9 37 G0 No Fibroids Total hysterectomy G1P1 High blood pressure Fibroids Total hysterectomy G2A1C1 No Fibroids Subtotal hysterectomy G2P2 No Endometrial polyps Total hysterectomy were accomplished in 30 min. No complication was associated with the procedure. The pattern of serum FSH levels is shown in Figure 1. A pattern of maintained normal basal FSH (pattern 1) comparable with that of the controls was observed in five patients (41.7%), whereas increased serum FSH after surgery and subsequent decline to levels <20 IU/l after 150 days (pattern 2) was observed in six cases (50%). Unfortunately, in the latter group, serum FSH levels subsequently increased to menopausal levels (pattern 2B) in two patients, although they were maintained <20 IU/l for at least 360 days in the remaining four patients (pattern 2A). Only one patient in the grafted group experienced a cessation of ovarian function after surgery (pattern 3). Longterm follow-up over the next 2 years revealed ovulation in 11 of the 12 patients (91.7%). Table II summarizes the age and number of ovulations in each of the FSH patterns established. Women showing pattern 2B were significantly (P = 0.02) older than women in pattern 1. The number of ovulations in each patient was recorded over the 24-month period of follow-up, noting that during the second year women were monitored every 2 months. A mean of 9.3 ± 1.73 ovulations were detected in the grafted patients compared with 12.0 ± 0.86 in the controls (P = 0.818). When the number of ovulations was evaluated according to the pattern of FSH secretion, pattern 2B showed a significantly lower number of ovulatory cycles than patterns 1 (P = 0.02) and 2A (P = 0.04) (Table II). AMH levels in grafted patients before (0.38 ± 0.09 μg/l) surgery were higher than those 1 week after the intervention (0.16 ± 0.02 μg/l), but the difference was not statistically significant (P = 0.07). When serum AMH levels were analysed according to the pattern of FSH secretion (Table II), no statistically significant differences were observed among groups. At day 30, the pattern 1 patients showed a trend towards recovery of serum AMH, while the remaining patients status did not change. In fact, linear correlation between number of ovulations and serum markers showed an inverse relationship (r = 0.639, P = 0.02) with serum FSH levels and no correlation with serum AMH (r = 0.465, P = 0.12) (Figure 2). The secretion of AMH over time in controls and cases under study is shown in Figure 3. Discussion This study was designed to evaluate the technical feasibility of an orthotopic transplantation in the medulla of the contralateral ovary. This is a well-vascularized tissue that provides a significant number of sympathetic and sensory neurons. Both ves- FSH serum levels (miu/ml) Figure 1. Serum FSH secretion patterns throughout the study period (720 days) in premenopausal patients undergoing hysterectomy and fresh orthotopic transplantation of the entire ovarian cortex plus a control group undergoing hysterectomy only. Hysterectomy and transplantation were performed on day 0. Data are mean ± SEM Time (Days) Pattern 1 Pattern 2A Pattern 2B Pattern 3 Control Table II. Age, number of ovulatory cycles in 24 months and serum anti-müllerian hormone (AMH) levels in patients with ovarian cortex transplant before surgery (0) and 7, 30, 180 and 720 days after surgery, among the different serum FSH patterns established FSH patterns 1 2A 2B 3 N Age* 39.0 a ± ± a ± Ovulations* 10.8 b ± c ± b,c ± AMH (μg/l)* 0 days 0.57 ± ± days 0.17 ± ± ± days 0.36 ± ± ± days 0.15 ± ± ± days 0.25 ± ± ± AMH: no significant differences among groups. a P = 0.02, 2B versus 1; b P = 0.02, 2B versus 1; c P = 0.04, 2B versus 2A. *Mean ± SEM.

4 Long-term outcome of fresh ovarian grafts A FSH (IU/l) B AMH (µg/l) ,8 0,7 0,6 0,5 0,4 0,3 0,2 0, OVULATORY CYCLES r = p = 0.02 r = 0.46 p = 0.12 Figure 2. Linear correlation between the number of ovulatory cycles, documented by vaginal ultrasound and serum progesterone, and (A) serum FSH (r = 0.639; P = 0.02) and (B) serum anti-müllerian hormone (AMH) (r = 0.465, P = 0.12) in patients with ovarian cortex transplant. sels and nerves are important in the control of folliculogenesis (Burden, 1985; Lara et al., 1990) and steroidogenesis (Hsueh et al., 1984; Ojeda et al., 1989). According to this procedure, in the case of a female cancer sufferer, one of her ovaries is maintained, while the contralateral ovary is removed and the cortex cryopreserved. When the patient has overcome the cancer, but has experienced premature ovarian failure, we apply our approach with the aim of restoring ovarian function, because the anatomy of one ovary will have been maintained. A very similar approach has been published by Silber et al. (2005) with fresh tissue and by Donnez et al. (2006) employing a mixed (intraovarian and periovarian) transplantation of frozen tissue in small cubes. The principal limitation of the present study was the age of the population (40.8 ± 0.7 years), whose pool of ovarian follicles was already substantially reduced. Thus, the application of the same procedure in young women may lead to improved results. Nevertheless, despite the subjects age, 91.6% ovulated, 75% of them maintained normal ovulatory cycles after 2 years and 5 of the 12 women (41.7%) showed normal FSH levels after surgery, in other words, had normal ovarian function immediately after surgery. These data provide further evidence that orthotopic transplantation is a feasible approach and follow reports of natural term pregnancies achieved after fresh (Silber et al., 2005) and frozen/thawed (Donnez et al., 2004) ovarian tissue transplant. The fast recovery of ovarian function in 41.7% of our subjects also compares favourably with the findings of previous reports employing fresh tissue that have described a temporary (3 6 months) increase in serum FSH before ovarian function was restored (Callejo et al., 2001; Oktay et al., 2001, 2003; Kiran et al., 2004; Donnez et al., 2005; Silber et al., 2005). A detailed analysis of Table II provides some clues regarding the ovaries capacity to restore their function, either immediately or with some delay. In patients who displayed serum FSH levels similar to those of the controls (pattern 1), serum AMH levels before surgery were 0.57 μg/l. In the other three groups, initial serum AMH levels were considerably lower, despite the detection of normal FSH levels, and remained low throughout the study period. It could be argued that we inadvertently left some of the ovarian cortex in place in some of the patients. However, special care was taken to destroy the entire ovarian cortex before transplantation by means of electrocautery, and no follicular growth was noticed at the left adnexa 1,6 1,4 a Controls Cases 1,2 1 AMH µg/l 0,8 0,6 b 0,4 0,2 c Figure 3. Line graph representing mean anti-müllerian hormone (AMH) values at each time point in both the cases and the controls; a > b, P = 0.017; b > c, P = Time (Days) 789

5 M.Sánchez et al. during the follow-up period. It is reasonable to assume that the percentage of younger patients in whom the pituitary ovarian axis is maintained intact would be greater than that of older women. In our study, differences were observed between patterns 1 and 2B, which partially can explain the results. Due to some reports having observed a detrimental effect of hysterectomy on hormonal status (Derksen et al., 1998), we felt it appropriate to establish a control group. Our data do not support such findings, because all hysterectomized patients with no transplant had normal serum FSH and experienced frequent ovulatory cycles after surgery. Controversy still exists as to whether ischaemia, freezing/ thawing or both are harmful to ovarian tissue. The follicular stage that is most sensitive to these procedures is also a subject of debate. Experiments performed in rodents showed that ischaemia was more detrimental than freezing/thawing, as the total number of recovered follicles was similar in both fresh and frozen/thawed ortho-transplanted ovaries, and both groups of animals displayed significantly less follicles than controls (Liu et al., 2002). Moreover, apoptosis was detected in the ovaries 30 min after the removal of the tissue from the medulla (Liu et al., 2002). These authors found that it was the pool of primordial follicles that was destroyed by these procedures (Liu et al., 2002). Others have shown that cryopreservation per se does not affect the long-term viability of ovarian tissue and that a normal reproductive lifespan can be restored in mice by orthotopic grafting of a frozen ovary (Candy et al., 2000). Therefore, the primary obstacle of the grafting technique would seem to be ischaemia (Baird et al., 1999; Kim et al., 2004). We aimed to address this issue in the present study by evaluating the extent of damage caused by ischaemia and by identifying the population of follicles that is most likely to be affected by ischaemia. The fact that 7 of 12 women displayed elevated serum FSH levels, as reported by several other authors after fresh ovarian tissue transplantation (Callejo et al., 2001; Oktay et al., 2003; Kiran et al., 2004; Silber et al., 2005; Donnez et al., 2005), suggests that the pool of growing follicles that produce negative feedback signals, such as inhibins and estradiol (E 2 ), to the pituitary may be destroyed by the surgical procedure described here. The reports of Callejo et al. (2001), Oktay et al. (2003) and Kiran et al. (2004) refer to a heterotopic site, whereas the report of Donnez et al. (2005) describes the transplantation into a pelvic peritoneal window. Our technique was previously described by Oktay and Karlikaya (2000) but using frozen ovarian tissue. We also measured serum AMH levels, as AMH is a product of pre-antral and small antral follicles (Weenen et al., 2004), and AMH levels are known to provide a reliable estimate of the number of follicles to be recruited in a particular menstrual cycle (Pastor et al., 2005). Although the difference in serum AMH levels before and 7 days after transplantation did not reach statistical significance (P = 0.07), we did detect a decrease, suggesting that the main target of ischaemia is the pool of follicles that express and secrete AMH. As this pool is invariably destroyed by ischaemia, FSH levels increase, and normal folliculogenesis would not be restored until 3 6 months have passed. 790 Whether or not freezing/thawing is damaging to human ovaries is an issue to be addressed in future studies. It has been shown that the ovaries of rodents are not significantly affected by freezing/thawing (Liu et al., 2002), and the few cases reported so far suggest that the same can be said for humans. In fact, ovarian function has been seen to recover following ischaemia, after which both natural (Donnez et al., 2004) and IVF (Meirow et al., 2005) pregnancies have been reported. Moreover, long-term follow-up over 2 years in our study demonstrated ovulation in 11 of 12 patients. Data regarding the long-term results of fresh orthotopic ovarian tissue transplant in premenopausal women are scarce, although it must be said that ovarian function recovery usually appears a few months after transplantation (Silber et al., 2005). The differences in our results may be due to employing the entire ovarian cortex and the availability of a normally developed and functioning medulla. At the same time, it should be emphasized once more that our patients were relatively mature. Either way, future approaches to the transplantation of ovaries with the aim of preserving fertility should include the transplantation of as much ovarian tissue as possible into a vascularized area such as the ovarian medulla. As linear correlation in these subjects shows, FSH was a better predictor of ovarian function and ovulation than AMH. AMH has been employed to predict ovarian reserve (Van Rooij et al., 2002; de Vet et al., 2002; Pastor et al., 2005), ovarian response to stimulation with gonadotrophins (Seifer et al., 2002) and even embryo quality in IVF (Silberstein et al., 2006). However, in our population, AMH showed a marked reduction in the follicular pool that is characteristic of an aged population. On the basis of the experience published by Welt et al. (2005), we decided to closely follow ovulation by ultrasound and serum progesterone when serum FSH levels dropped to <20 IU/l. This led us to consider whether or not ovulatory cycles accompanied by high FSH levels are likely to result in normal offspring. In this respect, transplant patients are comparable to and should be approached in the same way as women who suffer premature ovarian failure, of whom 50% will experience intermittent and variable ovarian function that may continue for many years (Rebar et al., 1982; Conway et al., 1996) and of whom 5 10% will conceive spontaneously, sometimes many years later (van Kasteren and Schoemaker, 1999; Welt et al., 2005). References Baird DT, Webb R, Campbell BK, Harkness LM and Gosden RG (1999) Long-term ovarian function in sheep after ovariectomy and transplantation of autografts stored at 196 C. Endocrinology 140, Burden HW (1985) The adrenergic innervation of mammalian ovaries. In Ben-Jonathan N, Bahr JM and Weiner RI (eds) Catecholamines as Hormone Regulators. Raven Press, New York, pp Callejo J, Salvador C, Miralles A, Vilaseca S, Lailla JM and Balasch J (2001) Long-term ovarian function evaluation after autografting by implantation with fresh and frozen thawed human ovarian tissue. J Clin Endocrinol Metab 86, Candy CJ, Wood MJ and Whittingham DG (2000) Restoration of a normal reproductive lifespan after grafting of cryopreserved mouse ovaries. Hum Reprod 15, Conway GS, Kaltsas G, Patel A, Davies MC and Jacobs HS (1996) Characterization of idiopathic premature ovarian failure. Fertil Steril 65,

6 Long-term outcome of fresh ovarian grafts Derksen JG, Brolmann HA, Wiegerinck MA, Vader HL and Heintz AP (1998) The effect of hysterectomy and endometrial ablation on follicle stimulating hormone (FSH) levels up to 1 year after surgery. Maturitas 29, Donnez J, Dolmans MM, Demylle D, Jadoul P, Pirard C, Squifflet J, Martinez- Madrid B and van Langendonckt A (2004) Livebirth after orthotopic transplantation of cryopreserved ovarian tissue. Lancet 364, Donnez J, Squifflet J, Dolmans MM, Martinez-Madrid B, Jadoul P and Van Langendonckt A (2005) Orthotopic transplantation of fresh ovarian cortex: a report of two cases. Fertil Steril 84, Donnez J, Dolmans MM, Demylle D, Jadoul P, Pirard C, Squifflet J, Martinez-Madrid B and Van Langendonckt A (2006) Restoration of ovarian function after orthotopic (intraovarian and periovarian) transplantation of cryopreserved ovarian tissue in a woman treated by bone marrow transplantation for sickle cell anemia: case report. Hum Reprod 21, Gatta G, Capocaccia R, Stiller C, Kaatsch P, Berrino F, Terenziani M and EUROCARE Working Group (2005) Childhood cancer survival trends in Europe: a EUROCARE Working Group study. J Clin Oncol 23, Hsueh AJW, Adashi EY, Jones PBC and Welsh THJ (1984) Hormonal regulation of the differentiation of cultured ovarian granulosa cells. Endocr Rev 5,76. van Kasteren YM and Schoemaker J (1999) Premature ovarian failure: a systematic review on therapeutic interventions to restore ovarian function and achieve pregnancy. Hum Reprod Update 5, Kim SS (2006) Fertility preservation in female cancer patients: current developments and future directions. Fertil Steril 85,1 11. Kim SS, Yang HW, Kang HG, Lee HH, Lee HC, Ko DS and Gosden RG (2004) Quantitative assessment of ischemic tissue damage in ovarian cortical tissue with or without antioxidant (ascorbic acid) treatment. Fertil Steril 82, Kiran G, Kiran H, Coban YK, Guven AM and Yuksel M (2004) Fresh autologous transplantation of ovarian cortical strips to the anterior abdominal wall at the pfannenstiel incision site. Fertil Steril 82, Lara HE, McDonald JK, Ahmed CE and Ojeda SR (1990) Guanithidinemediated destruction of ovarian sympathetic nerves disrupts ovarian development and function in rats. Endocrinology 127, Lee MM, Donahoe PK, Hasegawa T, Silverman B, Crist GB, Best S, Hasegawa Y, Noto RA, Schoenfeld D and MacLaughlin DT (1996) Mullerian inhibiting substance in humans: normal levels from infancy to adulthood. J Clin Endocrinol Metab 81, Liu J, Van der Elst J, Van den Broecke R and Dhont M (2002) Early massive follicle loss and apoptosis in heterotopically grafted newborn mouse ovaries. Hum Reprod 17, Meirow D and Nugent D (2001) The effects of radiotherapy and chemotherapy on female reproduction. Hum Reprod Update 7, Meirow D, Levron J, Eldar-Geva T, Hardan I, Fridman E, Zalel Y, Schiff E and Dor J (2005) Pregnancy after transplantation of cryopreserved ovarian tissue in a patient with ovarian failure after chemotherapy. N Engl J Med 353, Ojeda SR, Lara H and Ahmed CE (1989) Potential relevance of vasoactive intestinal peptide to ovarian physiology. Semin Reprod Endocrinol 7, Oktay K and Karlikaya G (2000) Ovarian function after transplantation of frozen, banked autologous human ovarian tissue. N Engl J Med 342,1919. Oktay K, Economos K, Kan M, Rucinski J, Veeck L and Rosenwaks Z (2001) Endocrine function and oocyte retrieval after autologous transplantation of ovarian cortical strips in the forearm. JAMA 286, Oktay K, Buyuk E, Rosenwaks Z and Rucinski J (2003) A technique for transplantation of ovarian cortical strips to the forearm. Fertil Steril 80, Pastor CL, Vanderhoof VH, Lim LCL, Calis KA, Premkumar A, Guerrero NT and Nelson LM (2005) Pilot study investigating the age-related decline in ovarian function of regularly menstruating normal women. Fertil Steril 84, Porcu E and Venturoli S (2006) Progress with oocyte cryopreservation. Curr Opin Obstet Gynecol 18, Rebar RW, Erickson GF and Yen SSC (1982) Idiopathic premature ovarian failure: clinical and endocrine characteristics. Fertil Steril 37, Seifer DB, MacLaughlin DT, Christian BP, Feng B and Shelden RM (2002) Early follicular serum mullerian-inhibiting substance levels are associated with ovarian response during assisted reproductive technology cycles. Fertil Steril 77, Silber SJ, Lenahan KM, Levine DJ, Pineda JA, Gorman K, Friez M, Crawford E and Gosden R (2005) Ovarian transplantation between monozygotic twins discordant for premature ovarian failure. N Engl J Med 353, Silberstein T, MacLaughlin DT, Shai I, Trimarchi JR, Lambert-Messerlian G, Seifer DB, Keefe DL and Blazar AS (2006) Müllerian inhibiting substance levels at the time of HCG administration in IVF cycles predict both ovarian reserve and embryo morphology. Hum Reprod 21, Van Rooij IA, Broekmans FJ, Te Velde ER, Fauser BC, Bancsi LF, de Jong FH and Themmen AP (2002) Serum anti-mullerian hormone levels: a novel measure of ovarian reserve. Hum Reprod 17, de Vet A, Laven JS, de Jong FH, Themmen AP and Fauser BC (2002) Antimullerian hormone serum levels: a putative marker for ovarian ageing. Fertil Steril 77, Viviani S, Santoro A, Ragni G, Bonfante V, Bestetti O and Bonadonna G (1985) Gonadal toxicity after combination chemotherapy for Hodgkin s disease. Comparative results of MOPP vs ABVD. Eur J Cancer Clin Oncol 21, Weenen C, Laven JS, Von Bergh AR, Cranfield M, Groome NP, Visser JA, Cramer P, Fauser BC and Themmen AP (2004) Anti-müllerian hormone expression pattern in the human ovary: potential implications for initial and cyclic follicle recruitment. Mol Hum Reprod 10, Weir HK, Thun MJ, Hankey BF, Ries LA, Howe HL, Wingo PA, Jemal A, Ward E, Anderson RN and Edwards BK (2003) Annual report to the nation on the status of cancer, , featuring the uses of surveillance data for cancer prevention and control. J Natl Cancer Inst 95, Welt CK, Hall JE, Adams JM and Taylor AE (2005) Relationship of estradiol and inhibin to the follicle-stimulating hormone variability in hypergonadotropic hypogonadism or premature ovarian failure. J Clin Endocrinol Metab 90, Submitted on April 28, 2006; resubmitted on July 18, 2006; accepted on September 18,