Cancer and fertility: strategies to preserve fertility

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1 Reproductive BioMedicine Online (2011) 22, REVIEW Cancer and fertility: strategies to preserve fertility K Diedrich a, *, BCJM Fauser b, P Devroey c, on behalf of the Evian Annual Reproduction (EVAR) Workshop Group a Department of Obstetrics and Gynecology, University Clinic of Schleswig-Holstein, Campus Lübeck, Lübeck, Germany; b Department of Reproductive Medicine and Gynaecology, University Medical Center, Heidelberglaan 100, 3584 CX Utrecht, The Netherlands; c Centre for Reproductive Medicine, Dutch-speaking Brussels Free University, Laarbeeklaan 101, 1090 Brussels, Belgium * Corresponding author. address: Klaus.Diedrich@uk-sh.de (K Diedrich). 1 Safaa Al-Hasani, Richard A Anderson, Pedro N Barri, Maryse Bonduelle, Curt Burger, Jehoshua Dor, Georg Griesinger and Outi Hovatta. Professor Klaus Diedrich, MD, PhD, graduated from the University of Hamburg in After gaining his PhD, he worked in obstetrics and gynaecology, progressing from Head of Department at the University Clinic of Lübeck in 1993 to Medical Director of University Hospital of Lübeck in He has been President of European Society of Human Reproduction and Embryology and German Society of Obstetrics and Gynecology and is Vice-President of German Greek Society of Obstetrics and Gynecology. He is a Fellow of the Royal College of Obstetrics and Gynaecology, a member of 18 editorial boards and has published 480 articles. Abstract Fertility preservation is a key component of cancer management in young people. The Fourth Evian Annual Reproduction Workshop Meeting was held in April 2009 to discuss cancer and fertility in young adults. Specialists in oncology, assisted reproduction, embryology and clinical genetics presented published data and ongoing research on cancer and fertility, with particular focus on strategies to preserve fertility. This report is based on the expert presentations and group discussions, supplemented with publications from literature searches and the authors knowledge. Fertility preservation should be considered for all young people undergoing potentially gonadotoxic cancer treatment. A variety of options are required to facilitate safe and effective fertility preservation for individual patients. Sperm banking is a simple and low-cost intervention. Embryo cryopreservation is the only established method of female fertility preservation. Oocyte cryopreservation offers a useful option for women without a male partner. Emergency ovarian stimulation and cryopreservation of ovarian tissue (followed by tissue transplantation or in-vitro maturation of oocytes) are experimental techniques for women who require urgent cancer treatment. Further prospective studies are required to validate cryopreservation of oocytes and ovarian tissue, in-vitro maturation of oocytes and new vitrification techniques and to identify any long-term sequelae of slow freezing of embryos. RBMOnline ª 2010, Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved. KEYWORDS: assisted reproductive technology, chemotherapy, cryopreservation, premature ovarian failure, radiotherapy /$ - see front matter ª 2010, Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved. doi: /j.rbmo

2 Cancer and fertility 233 Introduction As a result of treatment innovations, the survival rates of young people with cancer have improved markedly. The most commonly occurring cancers among young people aged years in Europe include Hodgkin s lymphoma, testicular cancer and malignant melanoma (Cancer Research UK, 2009; Gatta et al., 2009). Five-year survival rates of over 90% for these malignancies are now reported in young people in Europe (Gatta et al., 2009). The cancers most frequently diagnosed in adults aged years include breast, colorectal and cervical cancer and malignant melanoma (Cancer Research UK, 2009). Breast cancer is the most commonly diagnosed malignancy among women aged under 35 years in the UK (Cancer Research UK, 2009). Since the introduction of polychemotherapy regimens, the annualized mortality rate for early breast cancer has decreased by 38% in women aged under 50 years at diagnosis (Early Breast Cancer Trialists Collaborative Group, 2005). However, the management of many common malignancies involves aggressive radiotherapy or chemotherapy, which may permanently impair reproductive function (Donnez et al., 2006). Iatrogenic infertility is a distressing consequence for many cancer survivors and their families (Schover, 2009). Furthermore, the permanent cessation of ovarian function following cancer treatment has other implications for women s health and quality of life, including increased risks of osteoporosis, depression, cardiovascular disease and cognitive and sexual dysfunction (Brydoy et al., 2007). Accordingly, there is increasing interest among oncologists and reproductive specialists alike to consider fertility preservation as an essential component of a comprehensive approach to cancer treatment. This demand has driven the recent development of a number of novel strategies to preserve fertility. The Fourth Evian Annual Reproduction (EVAR) Workshop Meeting was held on April 2009 to evaluate existing information on cancer and fertility. The Fourth EVAR Workshop Group comprised specialists in oncology, assisted reproduction, embryology and clinical genetics. This paper reports the presentations and discussions of the Fourth EVAR Workshop Meeting. The meeting was structured to discuss the effects of cancer and its treatment on fertility, followed by options for fertility preservation for young adults with cancer. Established and experimental strategies for fertility preservation for both men and women were debated, including options for oocyte and/or ovarian tissue collection and cryostorage. Fertility preservation among children with malignancies was not discussed and is, therefore, beyond the scope of this report. Materials and methods Prior to the Fourth EVAR Workshop Meeting, expert speakers prepared presentations based on published literature and ongoing research. Presenters were asked to include comprehensive information using systematic literature search criteria. Following each presentation, a group discussion to reach joint conclusions on the topics covered was facilitated by the chairmen, Professors Devroey, Diedrich and Fauser. The content of this report is based on the expert presentations and subsequent group discussions that took place during the workshop meeting. Thus, the content of this article reflects the discussions and opinions of the Fourth EVAR Workshop Group. The discussions relating to each topic were complemented as necessary with electronic literature searches and key publications that were known to the authors. Combinations of the following keywords were used for PubMed searches: cancer, chemotherapy, cryopreservation, embryo, fertility preservation, in-vitro maturation (IVM), oocyte, outcomes, radiotherapy, sperm and vitrification. Articles of any type and published in English were permitted and were unlimited by the date of publication. Effects of cancer and its treatment on fertility Male reproductive system Almost 30% of men with testicular cancer have semen abnormalities at the time of diagnosis. Interestingly, semen abnormalities are also common among young men at the time of diagnosis with other cancers. For example, in a study of 158 untreated men (aged years) recently diagnosed with Hodgkin s lymphoma, 111 (70%) had semen abnormalities prior to treatment (Rueffer et al., 2001). Spermatogenesis is a highly prolific process (Orwig and Schlatt, 2005). Therefore, the germinal epithelium is extremely sensitive to the effects of chemotherapy and radiotherapy, which target rapidly dividing cells (Orwig and Schlatt, 2005). Subfertility is observed in the majority of men who receive alkylating agents plus radiotherapy (above or below the diaphragm) for the treatment of cancer. Radiation doses of only Gray (Gy) can impair spermatogenesis and doses of more than 4 Gy may cause permanent damage (Wallace et al., 2005). The mechanism of disruption of spermatogenesis is uncertain, but is believed to involve depletion of both stem cells and differentiating spermatogonia (Meistrich et al., 1982; Wallace et al., 2005). Spermatogenesis declines during the 3 6 months following chemotherapy or radiotherapy for testicular cancer, but steadily recovers thereafter (Gandini et al., 2006). Two years after treatment, 97% and 94% of men treated with chemotherapy or radiotherapy (with shielding of the contralateral testicle), respectively, show good recovery of spermatogenesis (Gandini et al., 2006). In contrast, the prevalence of azoospermia among men treated for lymphoma is reported to be as high as 59% and a long recovery period (45 months) is needed to achieve the highest sperm concentration (Bahadur et al., 2005). There is compelling evidence of short-term DNA damage to sperm following cancer treatment (O Donovan, 2005; Spermon et al., 2006) but a reduction in DNA integrity among long-term survivors has yet to be proven (Thomson et al., 2002). The impact of chemotherapy regimens on spermatogenesis depends on the type of chemotherapeutic agents used and the cumulative dose given (Dohle, 2010). Chemotherapeutic agents may disrupt spermatogenesis by targeting various testicular cell types (Leydig cells, Sertoli cells and germ cells) and by activating numerous molecular

3 234 K Diedrich et al. pathways involved in germ-cell metabolism. In particular, p53 and the Fas system have been shown to have roles as modulators of proapoptotic activity in the testis (Boekelheide, 2005). Alkylating agents, such as cyclophosphamide and procarbazine, and alkylating-like agents, such as cisplatin, carry the greatest risks of infertility (Lee et al., 2006). A high incidence of aneuploidy in sperm has been reported following some chemotherapy regimens (De Mas et al., 2001). Animal studies suggest that combination treatment with bleomycin, etoposide and cisplatinum, a chemotherapy cocktail used to treat testicular cancer, alters sperm chromatin quality, which may have adverse effects on progeny outcome (Delbes et al., 2007). However, there is no evidence to date of an increased incidence of congenital abnormalities among the children of male cancer survivors (Chow et al., 2009; Winther et al., 2009). The somatic (Sertoli and Leydig) cells of the testis are more resistant than germ cells to the effects of cancer treatments. Doses of 30 Gy are required to cause Leydig cell dysfunction in adults (Shalet et al., 1989). Therefore, steroidogenesis in males is usually maintained after radiotherapy treatment, albeit by compensatory elevation of luteinizing hormone concentrations (Wallace et al., 2005). Nonetheless, men should be informed of the potential gonadotoxicity of cancer treatment. Female reproductive system There is no evidence of a direct effect of cancer on the female reproductive system; however, its treatment may adversely affect various sites in the reproductive tract. Doses of radiation of Gy administered in childhood to the whole body or abdomen compromise growth and development of the uterus (Bath et al., 1999; Critchley and Wallace, 2005; Critchley et al., 1992; Green et al., 2009; Larsen et al., 2003). Uterine radiotherapy in childhood or adolescence is associated with an increased incidence of spontaneous miscarriage and intrauterine growth retardation in subsequent pregnancies (Wallace et al., 2005). These effects are believed to be secondary to vascular damage and reduced elasticity of the myometrium (Critchley et al., 1992; Wallace et al., 2005). Data from the Childhood Cancer Survivor Study showed that women treated with pelvic irradiation and/or increasing alkylating agent doses were at risk for acute ovarian failure, premature menopause and having offspring born small for gestational age (Green et al., 2009). Small ovarian volume in female survivors of childhood cancer has also been reported (Larsen et al., 2003). Exposure to chemotherapy or radiotherapy profoundly depletes the number of follicles present in the ovary. Follicular damage in females affects both endocrine and reproductive functions. As the size of the ovarian follicular pool is pre-defined, follicular destruction may cause premature ovarian failure or advanced menopause (Donnez et al., 2006; Wallace et al., 2005). The lethal dose of radiation required to kill half of the primordial follicles in the ovaries is estimated to be less than 2 Gy (Wallace et al., 2003). Treatment with alkylating agents such as cyclophosphamide during adolescence increases the risk of premature ovarian failure by a factor of nine (Byrne et al., 1992) and taxanes may also contribute to germ cell damage (Anderson et al., 2006). Cyclophosphamide treatment appears to exert its ovarian toxicity by significantly reducing primordial follicle count; this has been demonstrated in animals (Plowchalk and Mattison, 1991) and a real-time quantitative evidence of such an effect has been described in a human ovarian xenograft model (Oktem and Oktay, 2007). It has been suggested that gonadotoxic treatments induce a vicious pathophysiological circle of follicular destruction (Blumenfeld and von Wolff, 2008). Depletion of follicles reduces the secretion of oestradiol and inhibin, which causes serum FSH concentrations to rise, and may lead to enhanced recruitment of other follicles, which are further destroyed by chemotherapy (Blumenfeld and von Wolff, 2008). Premature ovarian failure following chemotherapy occurs more frequently in older than younger women (Petrek et al., 2006). Persistent amenorrhoea is used as a surrogate marker of ovarian failure (Petrek et al., 2006) and occurs in almost half of all women aged over 25 years when treated with mechlorethamine, vincristine, procarbazine and prednisolone for Hodgkin s lymphoma (Schilsky et al., 1981). In a multivariable logistic model, age at diagnosis was significantly associated with the likelihood of ongoing menstrual bleeding (P < 0.01); the odds of bleeding were reduced by 24% for each additional year of age (Petrek et al., 2006). This association is likely to be a reflection of the already depleted follicular pool in older women (Petrek et al., 2006). Mathematical modelling based on natural history data can be used to estimate the number of follicles remaining in the human ovary after radiotherapy (Wallace et al., 2003). Models suggest that exposure to gonadotoxic treatment at a younger age leads to the loss of more follicles than at an older age (Wallace et al., 2003). Thus, younger women treated for cancer lose more years of normal ovarian function than do their older counterparts (Wallace et al., 2003). However, as noted above, older patients have lower ovarian reserve than younger women and, therefore, have a higher risk of ovarian failure during or after chemotherapy or radiation therapy. Although younger women will have greater follicle loss because they have a greater number of follicles, they are more likely to retain or regain menstrual function than older patients. It is hoped that mathematical models could be used in future clinical practice to assess a woman s fertility potential prior to, or following, radiotherapy. Further research is needed to elucidate the short- and long-term effects of chemotherapy on ovarian follicles. Markers of ovarian age, such as the antral follicle count and anti-müllerian hormone (AMH), before, during and after chemotherapy may help to evaluate the extent of ovarian damage due to cancer treatment (Anderson et al., 2006; Broekmans et al., 2009). A recent study of 17 women undergoing chemotherapy showed that AMH and inhibin B concentrations immediately declined in response to chemotherapy and that the follicular target of chemotherapy appeared to be growing follicles (Rosendahl et al., 2010a). High pretreatment AMH concentrations were predictive of a higher post-treatment AMH concentration.

4 Cancer and fertility 235 The risk of subfertility in cancer survivors Both chemotherapy and radiotherapy to the pelvis or spinal cord are potentially gonadotoxic (Wallace et al., 2005). The effects of treatment depend on the regimen administered (the combination of therapeutic agents and total dose) and age of the individual patient (Meirow and Nugent, 2001; Royal College of Physicians et al., 2007). Chemotherapeutic agents are often used in combination because of their additive or synergistic antineoplastic effects (Meirow and Nugent, 2001); however, this complicates assessment of the gonadotoxic effects of individual agents (Anderson et al., 2006). Alkylating agents, including cyclophosphamide, are known to be severely gonadotoxic (Meirow and Nugent, 2001; Rosendahl et al., 2008). There is also early evidence that the use of taxanes as adjuvant chemotherapy may increase the toxicity of cyclophosphamide (Anderson et al., 2006). The extent of damage induced by radiotherapy depends on the field of treatment, total dose and fractionation schedule (Wallace et al., 2005). Low-dose cranial irradiation for central nervous system tumours may impair fertility by disruption of the hypothalamic pituitary ovarian axis (Bath et al., 2001; Hall et al., 1994; Littley et al., 1989), although this effect may become apparent only many years after cancer treatment (Wallace et al., 2005). The risk of subfertility can be graded according to the type of cancer and treatment required (Wallace et al., 2005). The greatest threat to fertility is the combination of intensive chemotherapy and total body irradiation that is required prior to bone marrow transplantation (Donnez et al., 2006). Metastatic Ewing s and soft-tissue sarcomas and Hodgkin s lymphoma with alkylating agent treatment are associated with high (>80%) risks of subfertility (Wallace et al., 2005). Localized testicular or pelvic radiotherapy also confers a high risk of subfertility (Wallace et al., 2005). Thus, the cancers that most frequently require fertility preservation are not necessarily the most commonly occurring types. Preservation of fertility for young adults with cancer Preservation of fertility should be offered to all young people who receive potentially curative gonadotoxic cancer therapies (Wallace and Walker, 2001). Experts agree that fertility preservation should be considered as early as possible in the course of cancer treatment (Lee et al., 2006; Wallace and Walker, 2001). However, evidence suggests that cancer treatment often commences without adequate consideration of fertility preservation (Chian et al., 2009b; Partridge et al., 2004; Schover et al., 2002b) and that current services could be much improved (Anderson et al., 2008). Infertility is a major concern for young people with cancer (Partridge et al., 2004). In a multicentre questionnaire-based study (n = 228), consultation with a fertility specialist was reported to be the preferred method for women with early breast cancer to obtain fertility information (Thewes et al., 2005). Oncologists are, therefore, encouraged to provide information on the risk of infertility conferred by their cancer treatment and offer a specialist referral to discuss options for fertility preservation (Ethics Committee of the American Society for Reproductive Medicine, 2005). Fertility-sparing cancer treatment should be considered whenever possible. Conservative options include medical management of early endometrial cancer and radical trachelectomy for cervical cancer (Ethics Committee of the American Society for Reproductive Medicine, 2005). In addition, shielding of the gonads from pelvic irradiation may help to preserve fertility (Ethics Committee of the American Society for Reproductive Medicine, 2005; Lass et al., 2001; Wallace et al., 2005). Storage of gametes, embryos, blastocysts or gonadal tissue prior to cancer treatment should also be considered for young people who require aggressive chemotherapy or radiotherapy. Cryopreservation techniques Cryopreservation of gametes and embryos is being offered with increasing frequency following guidelines to limit the number of fresh embryos transferred per cycle of assisted reproductive technology (ART) and improvements in laboratory techniques and clinical outcomes. Cryostorage is now a valuable component of standard IVF and intracytoplasmic sperm injection (ICSI) regimens. All cryopreservation protocols involve equilibration of cells in cryoprotectant followed by chilling, freezing and storage in liquid nitrogen at 196 C (Donnez et al., 2006). Cryoprotectants, such as ethylene glycol and dimethylsulphoxide, partially replace intracellular fluid and prevent the formation of intracellular ice crystals during the freezing process. The samples are subsequently thawed and the cryoprotectant is removed. The type, concentration and volume of cryoprotectant used in the solution have an impact on the outcome of cryopreservation (Fernandez-Santos et al., 2006; Nowshari and Brem, 1998). Both open and closed cryopreservation systems are commercially available. In closed systems, specimens are sealed inside carriers before being plunged into liquid nitrogen. Open systems involve direct contact of the specimen with liquid nitrogen and carry a theoretical risk of contamination and cross-infection. Cryopreservation techniques include conventional slow freezing (0.5 C/min; Whittingham et al., 1972) and ultrarapid vitrification (25,000 50,000 C/min; Rall and Fahy, 1985). The first live births after slow freezing of embryos were reported in 1984 (Zeilmaker et al., 1984). Conventional slow freezing involves the use of low concentrations (1.5 mol/l) but relatively high volumes ( ml) of cryoprotectants. The slow freezing and thawing stages required are time-consuming laboratory procedures. The first live birth following vitrification of blastocysts was reported in 2001 (Mukaida et al., 2001). Vitrification involves the use of high concentrations ( mol/l) of cryoprotectants to prevent the formation of intra- or extracellular ice crystals and allows amorphous freezing (Hovatta, 2005). Laboratory protocols are simple and less labour intensive than slow-cooling techniques and no expensive equipment is required. Moreover, new vitrification techniques result in significantly higher post-thaw oocyte, embryo and blastocyst survival and pregnancy rates than

5 236 K Diedrich et al. slow-freezing methods (Loutradi et al., 2008). However, there have been concerns related to the offspring over the high concentrations of potentially cytotoxic cryoprotectants used in the vitrification process. Male fertility preservation Cryopreservation of sperm Sperm banking is a simple and low-cost intervention for men who require fertility preservation (Foley et al., 1996). If men are unable to ejaculate semen for storage, sperm can be obtained by epididymal aspiration or testicular biopsy (Ethics Committee of the American Society for Reproductive Medicine, 2005). The use of ICSI largely circumvents the issues of low sperm counts or poor sperm motility that are common in patients with cancer (Wallace et al., 2005). Therefore, cryopreservation of sperm is recommended for all men prior to potentially gonadotoxic cancer treatment (Foley et al., 1996; Lass et al., 2001; Schover et al., 2002b). Despite the advantages of sperm banking, evidence suggests that it is not universally offered prior to cancer treatment (Schover et al., 2002a,b; Wallace et al., 2005). In a large postal survey of men aged years who were recently diagnosed with cancer (n = 201), only half of the respondents (51%) claimed to have been offered sperm banking and less than a quarter (24%) actually banked spermatozoa (Schover et al., 2002a). In a parallel survey, barriers to the provision of information by oncologists included inadequate clinic time and the limited availability of local services (Schover et al., 2002b). Although utilization rates vary, it seems that few cancer survivors (2 27%) proceed to use cryopreserved sperm to attempt a pregnancy (Blackhall et al., 2002; Menon et al., 2009). The reasons for the low rates of utilization are unknown, but it has been suggested that patients either do not have access to ART or fear transmitting the disease to their children (Blackhall et al., 2002). Despite the low utilization rates of cryopreserved sperm, discussion of fertility preservation provides reassurance of the curative intent of cancer treatment and is, therefore, beneficial for many patients and their families (Wallace et al., 2005). Other options Alternative techniques are needed to obtain and preserve spermatogenic tissue from prepubertal boys. Experimental options include autografting or xenografting of testicular tissue or transplanting stem cells (Geens et al., 2008; Goossens et al., 2008). More research is needed to assess whether these complex techniques offer a feasible clinical approach for the restoration of fertility following cancer treatment. Although hormonal manipulations could theoretically protect testes from injury during cancer treatment, gonadoprotection through hormonal manipulation appears to be ineffective (Lee et al., 2006). Several studies have shown no fertility protection benefit when hormonal therapy was given during chemotherapy (Brennemann et al., 1994; Johnson et al., 1985; Kreuser et al., 1990; Waxman et al., 1987). Female fertility preservation Preservation of fertility is more complicated for women than men (Ethics Committee of the American Society for Reproductive Medicine, 2005). The established and experimental options for female fertility preservation are described below. Embryo cryopreservation Cryopreservation of cleavage-stage embryos or blastocysts has become a routine procedure at all ART centres since the first live births from frozen thawed embryos were reported in 1984 (Zeilmaker et al., 1984). Embryo cryopreservation is currently the only established method of fertility preservation for women with cancer (Ethics Committee of the American Society for Reproductive Medicine, 2005; Lee et al., 2006; Royal College of Physicians et al., 2007). This option is available for women with a male partner or those willing to use donor sperm for fertilization (Donnez et al., 2006; Ethics Committee of the American Society for Reproductive Medicine, 2005; Wallace et al., 2005). Results from the 10th European IVF monitoring report show a 19.1% pregnancy rate with frozen-embryo transfer, compared with 29.0% per IVF aspiration and 29.9% per ICSI aspiration in 2006 (de Mouzon et al., 2010). A similar clinical pregnancy rate following frozen-embryo transfer was reported in Australia and New Zealand in 2006 (21.5%; Australian Institute of Health and Welfare (National Perinatal Statistical Unit) and Fertility Society of Australia, 2008), but these rates are lower than those reported in the USA where 35% of non-donor cycles resulted in a clinical pregnancy according to Centres for Disease Control and Prevention (2006) or 32.1% according to Society for Assisted Reproductive Technology (2008). Oocyte cryopreservation The first pregnancy resulting from a frozen thawed oocyte was reported in 1986 (Chen, 1986). Mature, metaphase II (MII) oocytes are fragile cells that are extremely sensitive to cytoskeletal and meiotic spindle damage during chilling (Donnez et al., 2006). Recent improvements in cryopreservation techniques have improved follicular survival. The use of vitrification avoids meiotic spindle damage and results in post-thaw survival rates of mature oocytes of over 80% (Cao et al., 2009; Chian et al., 2009b; Kuwayama et al., 2005). This compares favourably with survival rates of 50 65% that are achieved with slow freezing (Cao et al., 2009; Chian et al., 2009b). Although cryopreservation of oocytes is considered experimental, this technique provides a useful option for fertility preservation for young women who do not have a long-term male partner and do not wish to use donor spermatozoa to create embryos (Donnez et al., 2006). Ongoing initiatives such as the multicentre, observational HOPE Registry study (ClinicalTrials.gov identifier NCT ) is expected to provide valuable efficacy and safety data on oocyte cryopreservation in clinical practice (Ezcurra et al., 2008).

6 Cancer and fertility 237 Table 1 Milestone Historical clinical milestones of IVM of human oocytes. The first full-term, live birth from an immature human oocyte that was matured and fertilized in vitro The first pregnancy and live birth (triplets; in a donor oocyte programme) using IVM oocytes with conventional IVF techniques The first pregnancy and live birth reported following ICSI and in-vitro development of a day-5 blastocyst The first pregnancy and live birth after cryopreservation of immature (germinal-vesicle-stage) oocytes followed by IVM The first healthy live birth from an immature oocyte retrieved during a natural menstrual cycle, followed by IVM and vitrification of a mature oocyte ICSI, intracytoplasmic sperm injection; IVM, in-vitro maturation. Reference Veeck et al. (1983) Cha et al. (1991) Barnes et al. (1995) Tucker et al. (1998) Chian et al. (2009a) Oocyte collection Cryostorage of either oocytes or embryos initially requires oocyte collection. Ovarian stimulation with exogenous gonadotrophins leads to supraphysiological serum concentrations of FSH and oestradiol. Oestrogen stimulates the growth of hormone-sensitive tumours, including breast and endometrial cancers (Sonmezer and Oktay, 2006; West et al., 2009). Thus, the use of conventional ovarian stimulation protocols is considered appropriate only for women who do not have hormone-sensitive tumours and do not require urgent initiation of cancer treatment (Blumenfeld and von Wolff, 2008). Alternative strategies are required when conventional ovarian stimulation and oocyte retrieval is not recommended or is impractical. Modified ovarian stimulation regimens have been proposed to prevent the potentially deleterious effects of elevated oestradiol concentrations during conventional stimulation regimens. Studies of the use of aromatase inhibitors or anti-oestrogens (alone or in combination with recombinant human FSH and a gonadotrophin-releasing hormone (GnRH) agonist) to trigger final oocyte maturation have produced promising early results (Oktay et al., 2003a, 2005; Sonmezer and Oktay, 2006; von Otte et al., 2009). A number of other experimental options for fertility preservation have been proposed for women who require urgent cancer treatment. Suggested strategies include the administration of short ovarian stimulation protocols to produce mature oocytes, collection and IVM of immature oocytes and resection of ovarian tissue. Emergency oocyte collection Emergency ovarian stimulation protocols allow aspiration of cumulus oocyte complexes and could be useful for women who need urgent cancer treatment and for whom hormonal stimulation is not contraindicated. Although standard stimulation protocols can be used when the patient is in the early follicular phase of her menstrual cycle, alternative regimens are required for those who need a luteal phase start. Various cycle-independent ovarian stimulation protocols using GnRH antagonists for pituitary down-regulation have been proposed (Anderson et al., 1999; von Wolff et al., 2009). In one study, co-treatment with a GnRH antagonist and recombinant human FSH stimulation was initiated in the luteal phase of the menstrual cycle (von Wolff et al., 2009). A mean number of 10 oocytes were retrieved per patient (n = 12) and 61% of oocytes were successfully fertilized using ICSI. However, a high total dose (2720 IU) of recombinant human FSH was required per patient (von Wolff et al., 2009). Importantly, it has been shown that cycleindependent ovarian stimulation protocols can be integrated successfully into breast cancer treatment work-up (Madrigrano et al., 2007). However, as the efficacy of IVF is dramatically reduced after even one round of chemotherapy, it has been recommended that IVF should be performed before chemotherapy (Dolmans et al., 2005). Aspiration of immature oocytes during a natural menstrual cycle could provide a useful alternative when gonadotrophin stimulation is contraindicated. Early studies of immature oocyte retrieval in the late follicular phase of the natural menstrual cycle followed by IVM and vitrification of mature oocytes have produced promising outcomes in non-cancer populations (Chian et al., 2009a; Elizur et al., 2008). Emergency collection of immature oocytes in the luteal phase of a natural menstrual cycle followed by IVM to produce MII oocytes has also been described (Demirtas et al., 2008). In-vitro maturation of oocytes Collection of immature germinal vesicle-stage oocytes followed by IVM to complete the first stage of meiotic division circumvents the need for ovarian stimulation protocols to obtain in-vivo-matured oocytes. Indeed, IVM of oocytes is already offered to women with polycystic ovary syndrome as an alternative to conventional ART. As immature oocytes can be collected without delay or hormone stimulation, this technique could provide a useful method of fertility preservation prior to urgent cancer treatment (Chian et al., 2009b; Siristatidis et al., 2009). Clinical milestones of IVM of human oocytes are summarized in Table 1. Since 1983 (Veeck et al., 1983), the number of children born from in-vitro-matured oocytes has increased greatly. Some centres now report pregnancy rates per started cycle after IVM that approach outcomes achieved with conventional IVF (Chian et al., 2009b). However, the optimum protocols for oocyte retrieval and embryo transfer are yet to be established and efficacy rates

7 238 K Diedrich et al. are highly variable between different centres. Miscarriage rates of up to 37% have been reported in pregnancies conceived following IVM of oocytes, but this high rate of pregnancy loss is believed to be related to the population (predominantly women with polycystic ovary syndrome) rather than IVM per se (Benkhalifa et al., 2009; Buckett et al., 2008; Cha et al., 2005). Nonetheless, further research is advised to refine the maturation process and investigate the gene-expression profile of germinal-vesicle- and MIIstage oocytes. Live births have been reported following vitrification of in-vitro-matured oocytes (Cao and Chian, 2009; Chian et al., 2009a). However, significantly lower rates of oocyte survival (P < 0.001), fertilization (P < 0.05) and implantation (P = 0.07) are observed for in-vitro- versus in-vivo-matured oocytes (Cao and Chian, 2009). In theory, oocytes may be cryopreserved before or after IVM. Immature oocytes survive the freeze thaw process better than MII-stage oocytes (Donnez et al., 2006), but poor subsequent maturation, fertilization and cleavage rates are reported (Cao and Chian, 2009; Son et al., 1996). Therefore, it is currently recommended that oocytes are vitrified after IVM rather than at the germinal-vesicle stage (Cao and Chian, 2009). gastrointestinal cancers. In a recent study, potentially malignant cells were identified in ovarian tissue from patients with leukaemia cryopreserved before chemotherapy (Rosendahl et al., 2010b). Meticulous clinical and laboratory work-up for evidence of malignant infiltration of the ovarian cortex is required before cryopreservation and/or tissue transplantation (Kim et al., 2009; Meirow et al., 2008; Sanchez-Serrano et al., 2009). Sonography, computed tomography, computed tomography/positron emission tomography and direct laparoscopic visualization can be used to identify any ovarian, pelvic or abdominal metastases (Meirow et al., 2008). Cytomorphology, immunohistochemistry and polymerase chain reaction can be used to detect malignant cells in tissue fragments (Meirow et al., 2008). A recent study using a combination of cytomorphology and immunohistochemistry showed no evidence of micrometastases in cryopreserved ovarian tissue from 63 women with stage I IIIa infiltrating ductal breast carcinomas (Sanchez-Serrano et al., 2009). However, the laboratory techniques are not yet validated, so close clinical consideration of the safety of ovarian tissue transplantation is required for each individual patient (Meirow et al., 2008). Ovarian tissue cryopreservation Cryopreservation of ovarian tissue could be beneficial when there is insufficient time for ovarian stimulation prior to initiation of cancer treatment, if the ovary fails to respond to ovarian stimulation because of temporary inactivation induced by gonadotoxic chemotherapy and for prepubertal girls undergoing cancer treatment. Cryostorage of human ovarian tissue has proved challenging because of the size and structural complexity of follicles (Ethics Committee of the American Society for Reproductive Medicine, 2005). The choice of the freeze thaw schedule and cryoprotectant must offer a compromise between the most suitable option for the ovarian stroma and follicles (Fuller and Paynter, 2004). Fortunately, primordial and primary follicles comprise the vast majority of follicles in the human ovary and both tolerate cryopreservation regimens relatively well (Hovatta, 2005). Autologous ovarian tissue transplantation aims to restore fertility (Wallace et al., 2005). Ovarian tissue may be harvested and frozen as isolated primordial follicles, cortical strips or an entire ovary with its vascular pedicle (Donnez et al., 2006). Thawed ovarian tissue can be autografted to an orthotopic (Donnez et al., 2006) or heterotopic site, such as the forearm or abdominal wall (Oktay and Oktem, 2010; Oktay et al., 2003b). The development of mature human MII oocytes after xenografting of cryopreserved ovarian tissue into severe combined immunodeficient mice has also been reported (Gook et al., 2003). The risk of reintroducing malignant cells from stored ovarian tissue must always be considered (Chian et al., 2009b; Shaw and Trounson, 1997; Shaw et al., 1996). The likelihood of ovarian metastases depends on the characteristics of each type of cancer and its stage at the time of tissue harvesting. The risk of reseeding with malignant cells is particularly high in cases of leukaemia, non-hodgkin s lymphoma and ovarian cancer, as well as some breast and Ovarian cortical tissue The majority of primordial and primary follicles are located within 2 3 mm of the ovarian capsule so cortical strips are an excellent source of tissue for storage. Avascular ovarian cortical strips can be harvested laparoscopically. This procedure requires minimal cauterization to control bleeding as the major ovarian vessels are avoided. The resected cortical strips are then divided into small fragments for pathological examination and cryopreservation. Tissue fragments are transplanted after thawing. A number of ovarian grafting procedures leading to the restoration of endocrine function have been described (Demeestere et al., 2007; Kim et al., 2004; Oktay and Karlikaya, 2000; Radford et al., 2001). The growth and maturation of primordial follicles following transplantation takes 2 4 months. Exposure to chemotherapy prior to harvesting of ovarian tissue prolongs the development of follicles to 6 8 months. The functional lifespan of an ovarian tissue transplant is variable but is likely to be up to approximately 3 years (Kim et al., 2009; Meirow et al., 2007; Silber et al., 2008a). Thus, multiple transplantations may be required to extend the duration of ovarian function (Kim et al., 2009). The feasibility of cryopreservation of human ovarian cortical tissue was first demonstrated in 1996 (Hovatta et al., 1996; Newton et al., 1996). The first live birth following orthotopic transplantation of cryopreserved ovarian tissue was reported in 2004 (Donnez et al., 2004). To date, nine live births have been reported following orthotopic transplantation of cortical ovarian tissue autotransplants in patients with cancer (Table 2); the babies were conceived either spontaneously or with IVF/ICSI. In two cases, second pregnancies achieved spontaneously with subsequent live births were reported following cryopreserved ovarian grafts, confirming that long-term fertility restoration can be obtained after this procedure (Demeestere et al., 2010; Ernst et al., 2010). A recent case report demonstrated that

8 Cancer and fertility 239 Table 2 Live births reported following orthotopic autotransplantation of cryopreserved ovarian tissue in patients with cancer. Case details Reference Spontaneous pregnancy after (second) orthotopic transplantation of cryopreserved ovarian cortical tissue to a 32-year-old woman. Pretransplant hormone profile consistent with likely ovarian failure following chemotherapy and radiotherapy for Hodgkin s lymphoma Modified natural cycle IVF pregnancy after orthotopic transplantation of cryopreserved ovarian cortical tissue to a 28-year-old woman. Pretransplant hormone profile consistent with ovarian failure following high-dose chemotherapy for non-hodgkin s lymphoma Spontaneous pregnancy after (second) transplantation of cryopreserved ovarian cortical tissue to a 31-year-old woman. Both orthotopic (ovarian and peritoneal) and heterotopic (subcutaneous abdominal) sites were used. Pre-transplant hormone profile and ovarian biopsy consistent with ovarian failure after conditioning therapy for autologous bone marrow transplantation in the treatment of Hodgkin s lymphoma Two assisted reproductive technology pregnancies (in two women) after orthotopic transplantation of cryopreserved ovarian tissue. Both women received chemotherapy; one for Hodgkin s lymphoma and one for Ewing s sarcoma. Prior to transplants, both patients were amenorrhoeic and hormone profiles were consistent with ovarian failure Second pregnancy (spontaneous) 19 months after the first pregnancy following orthotopic transplantation of cryopreserved ovarian tissue. The woman had previously received chemotherapy for Ewing s sarcoma Twin assisted reproductive technology pregnancy after orthotopic transplantation of cryopreserved ovarian cortical tissue to a 38-yearold woman, followed by controlled ovarian stimulation and vitrification of oocytes. Pre-transplant hormone profile was consistent with ovarian failure after chemo- and radiotherapy for atypical medullary carcinoma of the breast Second pregnancy (spontaneous) 29 months after the first pregnancy in a woman who had transplantation of cryopreserved ovarian cortical tissue following treatment for Hodgkin s lymphoma Donnez et al. (2004) Meirow et al. (2005, 2007) Demeestere et al. (2007) Andersen et al. (2008) Andersen et al. (2008), Ernst et al. (2010) Sanchez-Serrano et al. (2010) Demeestere et al. (2007, 2010) a combination of preservation techniques (cryopreservation of ovarian tissue followed by oocyte banking by vitrification) can be used for fertility preservation when the functional lifespan of the ovarian tissue is believed to be short (Sanchez-Serrano et al., 2010). In addition to these successful cases, Silber et al. (2008a) reported two deliveries in two women with premature ovarian failure after isografting of cryopreserved ovarian tissue from monozygotic twins. Oktay (2006) also reported a spontaneous pregnancy and live birth following heterotopic (subcutaneous suprapubic) transplantation of cryopreserved ovarian cortical tissue to a 32-year-old woman whose pre-transplant hormone profile was consistent with ovarian failure following conditioning therapy for autologous haematopoietic stem cell transplantation for Hodgkin s lymphoma. The occurrence of a spontaneous pregnancy after heterotopic grafting highlights the difficulty in establishing the source of oocytes following ovarian tissue transplantation. Indeed, it has yet to be proven beyond doubt that the babies listed in Table 2 were conceived from oocytes originating from the transplanted tissue, as all women had residual ovarian tissue in situ. Viable oocytes have been obtained from heterotopic ovarian tissue transplants (Kim et al., 2009; Oktay et al., 2004). Although a biochemical pregnancy has been reported (Rosendahl et al., 2006), no clinical pregnancies have yet resulted from retrieved oocytes. It has been suggested that exposure of the graft to suboptimal pressure/temperature or mechanical stress at such sites could negatively affect oocyte quality (Donnez et al., 2006; Oktay et al., 2004; Wolner-Hanssen et al., 2005). More research is needed to identify the optimal graft site and, thus, improve the survival of follicles, quality of oocytes from grafted tissue and lifespan of the graft (Kim et al., 2009). Whole ovary Ischaemia after grafting of ovarian tissue causes follicular destruction (Baird et al., 1999). Transplantation of an intact ovary with anastomosis of its vascular pedicle would, theoretically, allow immediate revascularization and thereby would limit the period of ischaemic follicular damage (Donnez et al., 2006). Whole ovary transplantation is technically challenging but could result in a longer duration of function than does cortical grafting (Silber et al., 2008b).

9 240 K Diedrich et al. Restoration of fertility has been demonstrated in rats (Wang et al., 2002) and sheep (Imhof et al., 2006) following autotransplantation of frozen thawed whole ovaries. The size and density of human ovaries prevents adequate diffusion of cryoprotective agents and leaves the tissue susceptible to intravascular ice formation during freezing (Donnez et al., 2006). Early studies following a novel cryopreservation protocol for intact human ovaries and vascular pedicle suggest promising results (Martinez-Madrid et al., 2004, 2007). The histological ovarian structure is well preserved and the vast majority (96.7%, 29/30) of primordial and primary follicles appear healthy after thawing (Martinez-Madrid et al., 2004, 2007). Unilateral oophorectomy and cryopreservation is strongly advocated by some investigators. The EVAR Workshop Group considers this to currently represent an unnecessarily aggressive option in most cases, as spontaneous recovery of ovarian function after cancer treatment is possible (Donnez et al., 2006; Wallace et al., 2005). Although Lass (1999) has shown that, in general, women with one ovary do not have reduced fertility potential to conceive, either naturally or via IVF, such women may have a shorter reproductive life span and the possession of one ovary may be of greater consequence in women who already have diminished ovarian reserve, such as those in their late thirties or early forties. The group supports research to elucidate the optimum cryopreservation protocols and surgical transplantation techniques for intact ovaries. In-vitro follicle growth and maturation Cryopreservation of ovarian tissue preserves primordial and primary follicles but destroys the antral follicles that contain immature oocytes (Gosden et al., 2002). Accordingly, the in-vitro growth of primordial follicles and maturation of oocytes from thawed ovarian tissue is an attractive experimental option for fertility preservation. Isolation of viable primordial and primary follicles has several potential applications, including in-vitro culture and in-vivo grafting (Dolmans et al., 2006). The isolation of follicles from stored tissue could provide a particularly valuable option for women whose ovaries are likely to contain malignant cells (Dolmans et al., 2006; Sanchez-Serrano et al., 2010). Rapid improvements in the growth and maturation of primordial follicles from cryopreserved ovarian tissue have been made in recent years. To date, transplantation of frozen thawed isolated primordial follicles has been achieved successfully in mice (Carroll and Gosden, 1993). Live pups have also been born from mouse oocytes matured in vitro from primordial follicles (Eppig and O Brien, 1996; O Brien et al., 2003). However, the success in murine systems has been difficult to replicate in other species (Picton et al., 2008). Gradual improvements to culture processes have facilitated the in-vitro growth and maturation of human ovarian follicles (Hovatta et al., 1997; Wright et al., 1999), but no pregnancies have yet been reported. Two approaches to in-vitro follicular maturation have been proposed: in follicle maturation (IFM) and ovarian cortical tissue culture (West et al., 2009). In an IFM system, follicles are isolated mechanically or enzymically from ovarian tissue (Telfer et al., 2008; West et al., 2009). The immature follicles are grown in a biomaterial, such as alginate, and mature follicles subsequently undergo IVM to produce MII-stage oocytes (West et al., 2007, 2009). However, no meiotically-competent human oocytes have yet been produced by IFM culture systems (West et al., 2009). Culture of intact ovarian tissue maintains its structural integrity and the cellular interactions between follicles and the surrounding stroma (Hovatta et al., 1997). Thin slices of ovarian cortical tissue ( mm) are cut and excess stroma is removed. The remaining tissue is manually divided into small cubes and placed in a three-dimensional culture system (Figure 1) (Hovatta et al., 1997). Hormones and growth factors, including insulin-like growth factor (IGF) I, IGF II, growth differentiation factor-9 and FSH, are added to the culture medium to promote the growth and development of follicles (Hreinsson et al., 2002; Louhio et al., 2000; Telfer et al., 2008; Wright et al., 1999). A high follicular growth rate is observed during the first few days of ovarian tissue culture; this is believed to be due to the removal of the surrounding excess ovarian stroma, which contains inhibitory growth factors. Progressive atresia of follicles is subsequently observed, in a process that is similar to normal in-vivo development. After days in culture, the percentages of primordial, primary, secondary and tertiary follicles were 21%, 48%, 28% and 3%, respectively (Hovatta et al., 1997). Figure 2 shows maturation of follicles in ovarian tissue culture over 3 weeks. Combined approaches Cryopreservation of either embryos or oocytes obtained by rapid ovarian stimulation and ovarian cortical tissue could offer maximal emergency fertility protection for women who require urgent cancer treatment. The Fourth EVAR Workshop Group fully supports further work to investigate this twofold approach. The cryopreservation of oocytes retrieved during dissection of resected ovarian tissue has also been reported as a potential strategy for fertility preservation for patients with cancer (Huang et al., 2008; Isachenko et al., 2004; Revel et al., 2003). IVM may be attempted for immature oocytes (Huang et al., 2008; Isachenko et al., 2004; Revel et al., 2003). Storage of both ovarian tissue and in-vitro-matured oocytes could provide a useful combined approach to fertility preservation prior to cancer treatment (Huang et al., 2008; Isachenko et al., 2004). Other options A medical approach to fertility preservation during cancer treatment involves the induction of a temporary prepubertal hormonal milieu to protect the ovaries (Revel and Schenker, 2004). It has been suggested that suppression of serum FSH concentrations could break the vicious circle of accelerated follicular recruitment and destruction during gonadotoxic treatment (Blumenfeld and von Wolff, 2008). As such, concomitant treatment with GnRH analogues or oral contraceptives during chemotherapy could provide a simple method of female fertility preservation (Beck-Fruchter et al., 2008; Blumenfeld and von Wolff, 2008). Other agents also shown to protect the ovary from chemotherapeutic or radiation therapy in animals include sphingosine-1- phosphate and the c-abl kinase inhibitor imatinib (Gonfloni et al., 2009; Morita et al., 2000; Paris et al., 2002).

10 Cancer and fertility 241 Slice of ovarian cortex Cut into small cubes Cubes placed in designated well Ovarian cortical tissue Insert ECM Culture media Figure 1 In-vitro ovarian follicle culture system. Published with permission from Dr Inger Britt Carlsson. ECM, extracellular matrix. Preliminary data from animal studies (Ataya et al., 1995; Glode et al., 1981; Meirow et al., 2004) and small controlled trials (Badawy et al., 2009; Blumenfeld and von Wolff, 2008) suggest that gonadal protection from alkylating drugs can be achieved, but the results of large, prospective, randomized clinical trials are awaited (Blumenfeld and von Wolff, 2008; Wallace et al., 2005). Long-term outcomes of children following cryopreservation Cryopreservation of embryos or blastocysts Data from controlled studies indicate that the obstetric outcomes from slow freezing of early-cleavage-stage embryos are generally similar to those from fresh cycles (Kallen et al., 2005; Shih et al., 2008; Wang et al., 2005; Wennerholm et al., 2009). Indeed, some data suggest that, compared with fresh embryos, cryopreserved IVF/ICSI embryos are associated with lower perinatal mortality (P < 0.001) (Shih et al., 2008), higher mean birthweight (Belva et al., 2008) and a lower incidence of low birthweight neonates (<2500 g; P < 0.001) (Kallen et al., 2005; Shih et al., 2008; Wang et al., 2005). No significant differences have been reported in the incidence of de-novo chromosomal anomalies in children born from ICSI in cycles of fresh IVF or after embryo cryopreservation (odds ratio (OR) 1.96, 95% confidence interval (CI) ; Belva et al., 2008). However, the same study indicated a higher rate of major malformations among ICSI children born after transfer of fresh IVF versus embryo cryopreservation (OR 1.96, 95% CI ; Belva et al., 2008). No effects of cryopreservation of embryos on post-natal development have been demonstrated in the few published studies (Nakajo et al., 2004; Sutcliffe et al., 1995; Wennerholm et al., 1998). Methodological differences between published studies on the outcomes of fresh and frozen IVF/ICSI cycles and natural conception prohibit direct comparisons of data sets (Wennerholm et al., 2009). For example, the method of cryopreservation is often unreported, definitions of major congenital malformations are inconsistent, neonatal outcomes are incompletely described and often there is no adjustment for singleton versus multiple pregnancies (Wennerholm et al., 2009). In addition, data on fresh versus

11 242 K Diedrich et al. Figure 2 In-vitro follicle growth and maturation over 3 weeks in cultured intact ovarian tissue. Bar = 100 lm. frozen embryos from the same study are confounded because the best-quality embryos are usually chosen for fresh transfer and women who achieve a pregnancy in the first (fresh) cycle have better pregnancy outcomes than those who require multiple-embryo transfers. As vitrification is a relatively new technique, there are limited available data on clinical outcomes (Wennerholm et al., 2009). Only four retrospective studies and four case reports presenting data on 252 children born from vitrified blastocysts were identified in a recent systematic review (Wennerholm et al., 2009). These data provide no evidence of adverse effects of vitrification on gestational age, birthweight or the incidence of congenital abnormalities (Wennerholm et al., 2009). Cryopreservation of oocytes Similarly, there are few published safety data on children born after slow freezing or vitrification of oocytes. Limited neonatal data from 22 publications (retrospective studies and case reports) were identified on 148 children born after slow freezing of oocytes. The only neonatal information provided in many publications was a comment that the children were healthy. Birthweights were available from 12 publications and were consistently within the normal range. Data on 221 infants born from vitrified oocytes are available from case reports and uncontrolled studies (Wennerholm et al., 2009). This includes data on 200 children reported in a single publication (Chian et al., 2008). Reported birthweights were within the normal range (Wennerholm et al., 2009). However, Chian et al. (2008) reported a preterm singleton delivery rate of 26% (36/137), which is at least double the preterm delivery rate after slow freezing of embryos. In summary, despite the presence of confounding variables, 25 years of experience with slow freezing of embryos has provided reassuring safety data (Wennerholm et al., 2009). However, large registry-based and child follow-up studies are still required to assess the incidence of infrequent treatment outcomes (such as birth defects) and identify any long-term sequelae of slow freezing of embryos (Wennerholm et al., 2009). Limited safety data are available on vitrification of blastocysts and oocytes. Large, well-controlled studies with an adequate duration of follow up are needed to prospectively validate the long-term safety of vitrification techniques (Wennerholm et al., 2009). The future of fertility preservation The EVAR Workshop Group welcomed all efforts to improve and expand the options available for fertility preservation prior to cancer treatment. A multidisciplinary approach to cancer management was strongly recommended, with early referral of patients to a fertility specialist if appropriate. The group encouraged the use of fertility-sparing cancer treatment regimens whenever possible. New alkylating agents and milder treatment regimens have less profound effects on fertility and are being used with increasing frequency in clinical practice. There are also ongoing efforts to clarify the relative gonadotoxicity of treatment regimens and to identify patients who would benefit most from lowor high-dose chemotherapy regimens. Inhibin B and AMH may provide useful markers of gonadotoxicity in men and women, respectively, and warrant further investigation. Although techniques for in-vitro development of spermatogonia are still at an early stage, this option represents an exciting prospect for fertility preservation in prepubertal