Fertility issues in survivors from adolescent cancers

Cancer Treatment Reviews (2007) 33, 646– 655

available at www.sciencedirect.com

journal homepage: www.elsevierhealth.com/journals/ctrv

LABORATORY–CLINICAL INTERFACE

Fertility issues in survivors from adolescent cancers A.A. Pacey

*

Academic Unit of Reproductive and Developmental Medicine, The University of Sheffield, Level 4, The Jessop Wing, Royal Hallamshire Hospital, Sheffield S10 2SF, UK Received 1 February 2007; accepted 6 February 2007

KEYWORDS

Summary Infertility is a common and distressing late-effect of cancer treatment. Whist sperm banking for post-pubertal males and embryo freezing for women (who are in a stable relationship at the time of treatment) are highly successful fertility preservation strategies, for females without a partner (including young and pre-pubescent girls) and pre-pubescent boys (or azoospermic men), there remain no effective approaches. Whilst the biological effects of cancer treatments on the reproductive system are well described, there are few data on the relative incidence of infertility (failure to conceive after one year of trying) in cancer survivors. This makes it difficult to advise survivors about their future fertility prospects. Whilst some will undoubtedly conceive naturally with their partner, others will require assisted conception treatment of which in vitro fertilisation (IVF) and intra-cytoplasmic sperm injection (ICSI) are the most common. Pregnancy outcomes of cancer survivors are generally good, although there is increased risk of pre-term birth and low birth-weight in the offspring of women who have received pelvic irradiation. There is no increased incidence of genetic disease or cancer incidence in the offspring of cancer survivors. Current research directions are focussing on alternative fertility preservation strategies including in vitro maturation techniques, xenotransplantation and the development of technology to create artificial gametes in the laboratory. Finally, although the reproductive techniques discussed are highly effective, country specific differences in the legal framework means that cancer survivors may be denied access to certain treatments (e.g. embryo cryopreservation) because they are forbidden by specific national legislation. c 2007 Elsevier Ltd. All rights reserved.

Infertility; Fertility; Sperm; Oocyte; IVF; ICSI; Assisted conception



* Tel.: +44 0114 226 8290; fax: +44 0114 226 1074. E-mail address: [email protected]



0305-7372/$ - see front matter c 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.ctrv.2007.02.001

Fertility issues in survivors from adolescent cancers

Introduction Since infertility is one of the major late effects of cancer treatment it represents a number of unique challenges for reproductive medicine. This is either in the attempt to provide adequate fertility preservation strategies to young people at the time of treatment, or in dealing with the consequences of any reduced fertility later in life by providing assisted conception. This review will examine the current state of the art in both these issues and will highlight current areas of success as well of those with future promise.

Reproductive biology of males and females Males and females are obviously very different in their reproductive biology and this has been the major reason that underlies the difference in effectiveness of current approaches to fertility preservation and treatment between the sexes. Whilst in males testicular development results in boys being born with gonads that do not normally start to produce sperm until puberty, the differentiation of eggs in females is already underway at birth or shortly thereafter. Therefore, whilst the ovaries of female infant contain the maximum number of oocytes that will see her through her adult life until the menopause, the testicle of a boy contains the sperm production machinery that will make sperm almost continually from the onset of puberty until death. Therefore, even in the normal healthy individual there is a marked difference between the numbers of gametes available for fertility preservation at different ages of life and according to the individual sex. In males, the development of the testicles can lead to post-pubertal sperm counts of up to several hundred million (or more) sperm per ml of semen depending on the adult testicular volume reached.98 Whilst there is a broad correlation between the probability of conception in a given period of time and the number of sperm ejaculated,14 clinical suspicion of infertility would only be raised if the features of a man’s ejaculate was consistently below the thresholds set by the World Health Organisation.97 Although the semen quality of men does not change markedly as a function of age (European Society for Human Reproduction and Embryology33), there is growing evidence that older men find it harder to conceive37 as well as have increased risk of fathering a child a variety of genetically related conditions, such as Schizophrenia,82 autism spectrum disorders70 and congenital malformations.100 Increasing paternal age is also thought to independently contribute to the risk of miscarriage.28 These age related effects are almost certainly as a consequence of new de novo gene mutations that arise as a consequence of the number of mitotic cell divisions of the germ line that occurs throughout a man’s life26 in combination with a reduced efficiency of the apoptotic mechanisms that regulate sperm production as men age.18 Interestingly, there is growing concern that in some countries the incidence of male infertility is increasing79 and may be linked with a suite of syndromes (termed Testicular Dysgenesis Syndrome) characterised by poor testicular development and increased rates of urogenital abnormalities in newborns and increased rates of testicular cancer in adult males.78 It has been recorded that as many as 20%

647 of young men may have sperm concentrations below the WHO97 reference ranges.52 By contrast to the situation in males, females typically release a single gamete (egg) each month throughout their reproductive life, ovulating only about 400 eggs from the onset of puberty to the time of the menopause. Moreover, these monthly ovulations occur against a background of an age-related depletion of the oocyte pool35 which begins to accelerate at about the age of 37 and underpins the age related decline in female fertility that has been described in a number of populations33 and in women undergoing IVF.49 As well as finding it harder to become pregnant, there are additional risks to the pregnancy in older women and the outcome for both the mother and child are poorer.44 Therefore it is generally recommended that the best age for childbearing is between 20 and 35 years old8 and any woman who delays motherhood for non-biological reasons risks disappointment. Rates of biological infertility in women in the general population are difficult to establish reliably because of the masking effects of the use of contraceptives and the social decision to delay parenthood observed in many western countries in recent years.15 However, in a isolated population of Hutterites, where contraception was not allowed, Tietze88 demonstrated that only 2.5% of women were found to be childless at the end of their reproductive life. A more recent study of married women in the United States demonstrated a 12-month infertility rate of 7.4%.84 The reasons for infertility in women are obviously more numerous than in males because although they include a failure to produce gametes (eggs), through premature ovarian failure or endocrine disorders of ovulation, there also exists the opportunity for conception to be blocked because of barriers to fertilisation (through blocked Fallopian (uterine) tubes) or for implantation to fail or gestational issues preventing a pregnancy being carried to term.6

The effects of cancer and its treatment on the reproductive system The current or future fertility of an individual can be affected by cancer and its treatment either by a direct effect on the gonad or by affecting the endocrine support of gonandal function through a deleterious effect to another endocrine organ, such as the pituitary. Sadly, the precise effects of in any one individual are often difficult to predict. In males, sperm production is primarily compromised because the seminiferous epithelium becomes damaged and the population of stem cells that normally differentiate to produce sperm post-puberty either become depleted or are unable to differentiate after treatment has finished.77 This can occur after the testis has been irradiated with doses of radiation as low as 0.1–1.2 Gy76 or in response to some chemotherapeutic drugs. It is more difficult to determine the testicular response to specific doses and agents of chemotherapy, particularly when they are given in combinations, although it is generally considered that the alkylating agents and cisplatin have the most profound effect.51 Normally, because the testosterone producing Leydig cells remain unaffected, then the secondary sexual characteristics of most individuals are normal86 and the testicular damage only become evident at semen analysis. Even then,

648 because following some treatments sperm production can start spontaneously56 the post-treatment diagnosis of azoospermia needs to be interpreted with caution, depending on the follow-up time. For many years, it was considered that the pre-pubertal testicle was largely quiescent and therefore less susceptible to damage. However, more recent studies have shown that even though it may not be producing sperm at the time of treatment, there are significant changes in its structure and organization24 and similar damage to that that seen in adults can be observed after treatment.68 In females, the primary effects of chemo- and radiotherapy is to reduce the size of the follicle pool which in some cases can lead to an early menopause for the individual concerned. For example, Larsen et al.57 reported a fourfold increase in the risk of premature ovarian failure in teenagers treated for cancer, and an increased risk of 24-fold in women treated between the age of 21 and 25 years. Similarly, it is now estimated that a dose of ionising radiation of <2 Gy is sufficient to destroy 50% of the follicle pool92 and a dose of 5–20 Gy sufficient to completely impair gonadal function regardless of age.93 In addition to effects on the ovary, total body irradiation and some radiotherapy can also lead to impaired uterine growth and reduced blood flow.27 This may mean that even if pregnancy is started that it may not be able to reach term or that the health of the infants born may be compromised.

Current fertility preservation strategies Techniques for the fertility preservation in post-pubertal males are well established and rely upon the ability to freeze semen samples from the patient before the start of treatment. Assuming the male is able to masturbate a specimen for analysis, the evidence to date would suggest that in cancer patients the semen quality is broadly similar across disease states4 and is comparable to that of males without malignancies.72 However, there will inevitably be some patients where no (or too few) sperm can be obtained for storage and this may occur for a variety of reasons relating to an inherent underlying genetic condition that is independent of the disease state under treatment, or due to a direct physiological association with it. In younger patients, their level of sexual experience and awareness are additional important parameters in determining whether or not they are able or willing to masturbate and provide a specimen for banking. Sanger et al.74 was the first author to describe the successful storage of sperm in men prior to cancer treatment and its subsequent use in assisted conception some years later. He reported a series of 115 births from treatment by intra-uterine insemination (IUI) or in vitro fertilisation (IVF) using sperm stored in some cases up to 10 years earlier. Since then, further reports have appeared reporting successful pregnancies after sperm have been stored for 21 (Horne et al.47) and 28 (Feldschuh et al.36) years respectively. However, it is interesting to note that the current data suggests that of the males who do successfully bank sperm, only a relatively small proportion ever returned to use the samples in assisted conception procedures. In an audit of 692 Australian men who banked sperm and survived

A.A. Pacey their illness, only 64 men (7% of the total) attempted conception with their frozen sperm within a 10-year follow-up period.54 A similar figure (9%) has been reported by Agarwal et al.1 for patients in the United States with a variety of diagnoses, but for a cohort of 122 men sperm banking following a diagnosis of Hogkin’s disease in the United Kingdom (Blackhall et al.9) an actuarial rate of 27% utilisation was recorded (with a median follow-up time of 10.1 years). Whilst to some these figures may be disappointing, to others they may simply reflect that the follow-up time have been too short given the well described secular trend for couples to delay conception until later in life and generally have fewer children. Moreover, it could also reflect the complexity of the decisions to sperm bank in the first instance as well as the subsequent access and availability of assisted conception services in the years following treatment. In contrast to the situation in males, ‘‘egg banking’’ in females is not widespread. Unlike the collection of sperm for cryopreservation, the harvesting of eggs is a much more involved procedure requiring drug therapy to increase the number of eggs available for banking and then a minor surgical procedure to remove the eggs from the ovary.16 This makes the process less acceptable to young women because it will inevitably lead to delays in the start of cancer treatment. Also, the science of egg freezing has been technically challenging and following the first report of a pregnancy from a frozen-thawed human egg23 there have been only about 100 babies born worldwide using this technique. Success rates have remained disappointingly low, with Gosden39 summarising recent studies that show of 4010 frozen eggs being thawed and used, only 57 pregnancies were reported with 74 live infants born. Therefore, with a less than 2% of frozen/thawed eggs used actually leading to a baby, few reproductive biologists are prepared to currently recommend this as routine procedure. On the horizon are potential improvements to the freezing process in the form of vitrification techniques55,99 but this has yet to be proven in large-scale clinical trials. A more effective strategy to preserve the fertility of young women is to bank embryos created with their partner prior to the onset of treatment. Since the first report of a pregnancy following the replacement into a woman’s uterus of a frozen/thawed embryo89 frozen embryo replacement has become a routine component of assisted conception procedures.21 For the patient diagnosed with cancer, embryo freezing has the same disadvantages as egg banking in terms of the timescale involved and the need for drugs and minor surgery. Furthermore, it has the additional disadvantage that it is only an option for those women who have a partner at the time of diagnosis, with whom they are committed to have a family in the future. The use of donor sperm to fertilise eggs from a female who does not yet have a partner is controversial to many. Moreover the availability of donor sperm is limited in some countries.67 Success rates with frozen embryos are generally lower than those observed when fresh embryos are transferred and in 2002 were in the order of 18.4% vs 29.5% according to registry data from 25 European countries.85 Concerns about the possible detrimental effects of long-term storage of embryos appear at the present time to be unfounded with most studies showing that the perinatal outcome and early infant development of children born from frozen embryos is not

Fertility issues in survivors from adolescent cancers significantly different from those born from fresh embryo transfers.96 However, the obvious lack of long-term follow-up of such individuals into middle and later life means that this issue needs to be kept under surveillance.

Natural fertility in survivors of cancer Research suggests that having children is very important for young cancer survivors.75 However, in general terms the fertility of cancer survivors is significantly lower than that of their sibling controls.43 Sadly, few studies have systematically described the ability of cohorts of cancer survivors to conceive naturally in the years following treatment and therefore it remains difficult to reliably advise patients of their reproductive future without the need for assisted conception. The recovery (or maintenance) of natural fertility is perhaps best observed in the male where measurements of semen quality can be easily made. For example, Bahadur et al.5 found that across all disease states only 37% of patients had permanent post-treatment azoospermia after a mean follow-up period 48.6 months. The type of cancer (or disease) and the initial pre-treatment sperm concentration were the most significant factors governing post-treatment semen quality and the recovery of spermatogenesis. For example, patients with lymphoma and leukaemia had the highest incidence of post-treatment azoospermia and oligozoospermia but whilst men with testicular cancer had the lowest pre-treatment sperm concentrations, they also had the lowest incidence of azoospermia after treatment. This confirms the observation by Herr et al.45 who showed that over a 10-year follow-up period of men with stage 1 testis tumour that 65% of couples that attempted a pregnancy were successful. More recently, a study by Brydoy et al.17 of 1814 men previously diagnosed with testicular cancer found that the 15-year actuarial post-treatment paternity rate was 71% without the use of cryopreserved sperm. Clearly, such studies need to be conducted for males with other types of cancer to establish more reliably the rates of natural paternity after treatment. For example, although following some treatments (e.g. total body irradiation) the rate of spontaneous pregnancy is known to be very low, a handful of pregnancies have been reported64,22 but the true incidence remains unknown. Comparable studies describing the post-treatment fertility of cohorts of female cancer survivors are rare. Indirect assessment of biochemical markers of ovarian function can provide evidence of partial loss of the ovarian reserve7 and data can provide the cumulative incidence of non-surgical premature menopause.80 However, both of these approaches do not assist in the understanding of how many female cancer survivors suffer infertility. In a different approach, Wallace et al.91 studied a cohort of 40 long-term survivors treated for acute lymphoblastic leukaemia and showed that after a median follow-up age of 18.8 years (range 12–34.7) all were having regular menses and there were 14 live births reported between 10 of the patients. Similarly, in a cohort of 6494 women who were diagnosed with cancer below the age of 21 and who returned a pregnancy questionnaire, 1915 reported that they had been pregnant and delivered one or more offspring,41 which re-

649 lates to a 30% pregnancy rate. However, in both of these studies it is not clear if the women who did not report a pregnancy had evidence of infertility or simply had not yet chosen, or had an opportunity, to conceive. Clearly this is a topic that requires greater exploration. As with the situation in males, treatment of women following classically sterilising treatments such as total body irradiation prior to bone marrow transplantation has been known to lead to pregnancies94 albeit very rarely.

Assisted conception in the adult survivor In any couple, should methods of natural family planning fail to achieve a pregnancy in a reasonable length of time, there are currently a number of powerful methods of assisted conception that can be used to help them. Infertility is generally defined as a failure of conception after two years of regular unprotected sexual intercourse in the absence of any known reproductive pathology, although it is acknowledged (National Institute for Clinical Excellence61) that where there is a known condition or reason for infertility (such as prior treatment for cancer) treatment should begin immediately after presentation. The general principles of assisted conception have recently been described73 and can be divided into three main procedures: intra uterine insemination (IUI), in vitro fertilisation (IVF) and intra-cytoplasmic sperm injection (ICSI). Whilst IUI is a relatively simple procedure, in which a sample of sperm is prepared in the laboratory and inseminated into the female partners uterus just prior to ovulation, IVF and ICSI are more complex and require oocytes recovered from the female partner to be fertilised outside the body, with the early stages of embryo development taking place in the laboratory.16 The choice of which technique to use for any couple (cancer survivor or not) is dependent on a range of factors that include any pre-existing infertility issues as well as those related specifically to the cancer treatment. Sadly, there is no single or simple algorithm that can summarise the possible treatment pathway for all cancer survivors (or combinations thereof), but Fig. 1 provides a basic outline of the decision that a reproductive medicine specialist might take in assessing the treatment options for a given survivor. Techniques of Assisted Conception are now commonplace with nearly 325,000 cycles of IVF and ICSI being carried out in Europe in 2002, which is on average 916 cycles of treatment per million inhabitants.85 Moreover, the percentage of the total births being as a consequence of assisted conception treatment ranges from 1.3% to 4.2%. In the same period, and across all age groups, the success rates of IVF and ICSI were 26.0% and 27.2% per egg collection and 29.5% and 29.4% per embryo transfer, respectively. Success rates with IUI (using partner sperm) were significantly lower at 11.6% in women under 40% and 7.8% in women who are over 40. Sadly, such registry data is not broken down to show the success rates of cancer survivors, although it is likely the number receiving treatment each year would be a very small proportion of the total. Moreover, there are few large cohort studies of the outcome of cancer survivors undertaking assisted conception by which meaningful information can be drawn. Perhaps the largest

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Figure 1

A simple flow diagram to illustrate the treatment options available to male and female cancer survivors.

is that published by Brydoy et al.17 which followed-up 1814 men previously diagnosed with testicular cancer and found that 22% had attempted assisted conception, but it was not systematically investigated whether this was for fertility issues related to the cancer treatment or other medical issues of the female partner. However, of the 100 patients who had attempted assisted conception only a minority had used IVF. Whilst the repertoire of current assisted conception techniques are highly effective, it seems intuitive that given the incidence of premature menopause or spermatogenic failure after complex or aggressive cytotoxic therapies (see above) that cancer survivors probably have a greater reliance on the use of donor gametes than other infertile couples. This is particularly true in men who might not have been given the opportunity to bank sperm (or be too young or ill to do so) prior to treatment, or in women who were without a partner at the time of cancer diagnosis and were unable (or did not have time) to freeze embryos. In general terms, the success rates when using donor sperm or eggs are comparable to that when couples use their own ‘fresh’ gametes. Where both male and female problems occur, then embryo donation remains an option58 although in most countries there is a severe shortage of embryos available for donation and in others it is not lawful (see below). In situations where women have difficulty in carrying a baby to term following cancer treatment, Gestational Surrogacy is an option.20

Outcome of pregnancy Whether a pregnancy in the cancer survivor is spontaneous or as a result of assisted conception treatment, there are understandable concerns raised by patients that the children born would be at high risk for birth defects or an increased incidence of cancer themselves.75 Whilst there is some evidence that the children of female cancer survivors treated with radiation to the pelvis are more likely to be born pre-term and have a low birth-weight in comparison to those children from sibling controls,81 there is little evidence that the partners of male survivors are at risk of having an adverse pregnancy outcome or have any signifi-

cant increased risk of malignancy.42 Similarly, Meistrich and Byrne60 reported that there is no increased risk of genetic disease in the children of cancer survivors treated with potentially mutagenic therapies.

Fertility preservation strategies of the future Although significant progress is being made in the repertoire of assisted conception techniques available to the cancer survivor (see above), perhaps the most exiting area of current research is that which is investigating alternative strategies for fertility preservation in cancer patients of the future. Currently there are five main lines of approach being considered by various investigators as summarised briefly below.

Endocrine manipulation There has been a growing body of evidence to suggest that the administration of a variety of drugs to manipulate the endocrine environment of the patient shortly before or at the time of treatment could potentially provide a protective mechanism for the gonad of both males and females. If true, this could markedly reduce the detrimental effects of gonadotoxic agents and potentially lead to the restoration of natural fertility in a larger number of individuals without the need for cryopreservation strategies. In females the use GnRH analogues to temporarily induce the pre-pubertal hormonal environment during treatment has shown in animal models to inhibit the cyclophosphamide-induced depletion of oocytes from the ovary.2,3 However, in humans although the same has been shown for women being treated for lymphoma,10 leukemia12 and lupus erythematosus (Blumenfeld et al.11) it was not seen for women being treated for Hodkin’s disease.95 Therefore, the general consensus appears that the technique remains controversial with the need for further adequately controlled trials in order to establish its efficacy.71 A similar approach has been investigated in males, and although early experiments were attempted with the aim of protecting against the loss of stem cells during the cytotoxic insult, it soon became clear that the hormonal

Fertility issues in survivors from adolescent cancers treatments probably enhanced the ability of the testis to maintain the differentiation of spermatozoa in the population of stem cells that survive treatment but which otherwise fail to differentiate once treatment has ended.77 Whilst most of the experimental work to date has been performed on rats, it is unclear whether it can be adapted for human clinical application. Encouraging results from a small study of 15 patients with nephritic syndrome (being treated with cyclophosphamide) have been obtained.59 But more recent experiments in non-human primates13 have been unable to reproduce the experimental results obtained in rats, raising the possibility that there are important species-specific differences in the testicular response either to the treatment (in this case radiotherapy) or the endocrine rescue protocol, or both. As an approach, therefore, the use of endocrine manipulation to protect the testis from damage (or salvage them from it) is still very much experimental.

Freezing and re-transplantation of gonadal tissue/cells post treatment Unlike the current approaches of freezing mature sperm or eggs as well as developing embryos, many authors have commented on the desirability of being able to remove (before treatment) and potentially replace (after treatment) sufficient gonadal tissue that might allow the patient to subsequently reproduce normally. Clearly, this would require the need for gonadal tissue to be frozen for a period of time (perhaps many years) and retain its viability when transplanted. In the case of males, the most progress has been in the area of Spermatogonial Transplantation. Although this remained theoretical for many years, major progress was made when Brinster and Zimmermann19 published details of experiments where they had successfully removed spermatogonia from one mouse and transplanted them back into the testes of another. This provided proof of concept that the technique was feasible, and the underlying technology for retrieval, storage and transplantation of spermatogonia has been developed in subsequent years (see Johnson et al.50). In progressing to human clinical trials scientists have been notably cautious, although some small-scale trials have been attempted,69 at the time of writing, the results have not yet been published. In females, a variety of strategies have been attempted following initial reports that fertility of mice66 and sheep40 could be restored after the auto-transplantation of frozen– thawed ovarian tissue. Subsequent studies in women first confirmed that endocrine function could be restored after autotransplantation of frozen–thawed ovarian tissue63 before the report of a birth of a live infant in a woman following orthotopic transplantation of ovarian tissue that had been stored before treatment for Hodkin’s Lymphoma.31 Whilst orthotopic transplantation has the theoretical advantage that it provides the best chance of natural conception, heterotopic transplantation (in the forearm or abdominal wall) may be more suitable if repeated transplantation is required or if eggs may need to be recovered for IVF. At the present time, there appears to be no consensus concerning the best approach for surgical transplantation or indeed the laboratory protocols that give maximal survival of ovarian

651 tissue and it is clear that there is much further work to be carried out before this could become a routine procedure.32 A major concern with re-transplantation of gonadal tissue in both sexes is the fear that during the process malignant cells may be re-introduced into the patient who was otherwise cured of their original disease. At the present time, there is too little information about such risks to adequately advise patients and it is clear that only following large-scale studies could this be properly evaluated. As such, many authors are advising caution and are hopeful that the development of methods of in vitro culture or xenotransplantation might allow these risks to be avoided.

In vitro maturation of gametes for future use If post-treatment transplantation of a patient’s gonadal cells/tissue which had been cryopreserved pre-treatment proves to be too risky or technically challenging, then the development of in vitro culture technology might in theory allow both sperm or oocytes to be developed in the laboratory which could then be used assisted conception procedures. For males, the prospects for spermatogenesis in vitro have recently been reviewed by Parks et al.65 who outlined the technical complexity of cell culture systems that would be required to maintain the development of spermatozoa in vitro. Although some progress in animal models has been made (in as much as presumptive spermatids [i.e. early sperm cells] expressing appropriate genetic markers have been identified and have been injected into eggs to produce viable embryos), it is far from the stage of being clinically useful for human applications. By contrast, the prospect of in vitro maturation of oocytes is potentially more achievable after the report by Eppig and O’Brien34 that fertile mouse oocytes could be generated entirely in vitro from primordial germ cells leading to the birth of healthy mouse pups. Whilst further work is required to develop a similar system for human tissues, some progress has been made in the in vitro maturation of later stages of oocytes recovered from the antral follicles of unstimulated ovaries (reviewed by Jurema and Nogueira53). Whilst only about 300 children have been born worldwide as a result of this technique, and its application to frozen/thawed ovarian tissue remains to be shown, in theory at least we are getting close to the possibility of being able to generate mature oocytes in the laboratory for women who have previously had ovarian tissue frozen prior to cancer treatment.

Xenotransplantation of reproductive tissues An alternative area with some promise is the development of techniques to transplant gonadal tissue from males and females into a ‘host’ animal that could act to support the development of eggs or sperm to that point that they might be harvested and used in IVF or ICSI. Proof of principle has been demonstrated for both male and female tissues. In males, experiments dating back to the 1970s have illustrated the potential for testicular tissue or isolated spermatogonial stem cells to be transplanted from one species to another and for spermatogenesis to be supported in

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the host animal (reviewed by Parks et al.65). Such approaches have been pivotal in furthering our understanding of the biology of spermatogenesis30 but also in opening up the avenue for restoring fertility for men following cancer therapies. Whilst primarily this might be seen as a method of generating sperm from tissue taken from post-pubertal individuals before cancer treatment, recently Honaramooz et al.46 demonstrated that this technique could even be used to initiate spermatogenesis in pre-pubertal testicular tissue taken from a variety of species. This gives hope for fertility preservation in pre-pubertal boys where traditional sperm banking is not possible. For ovarian tissues, the birth of live young following xenotransplantation was first described by Snow et al.83 In this instance, fresh ovarian tissue from the mouse was transplanted under the kidney capsule of a rat recipient. Xenografting of human ovarian tissue has been attempted into various host species32 and this has provided valuable data concerning the techniques of grafting and the assessment of follicular development and oocyte quality. However, to date there are no concrete data to show that this approach can generate functionally competent oocytes and so it remains very much an experimental idea, albeit one with much promise. As a cautionary note, it should be recognised that whilst the xenotransplantation of male and female gonadal tissue currently has many research applications, its use as a method of generating gametes for human reproductive application raises many ethical concerns.25 There are obvious safety concerns to address about the potential for non-human DNA (or animal viruses) being transferred to the human genome during the assisted conception procedure and as such much work is also needed to establish the safety and acceptability of this approach.

Creation of artificial gametes The idea that it might one day be possible to create germ cells artificially for patients in the laboratory using a variety of technologies has been discussed.90 But the idea remained largely theoretical until the observations by Toyooka et al.87

Geijsen et al.38 and Hubner et al.48 showed that sperm and egg-like cells could be generated from mouse embryonic stem cells cultured in the laboratory. This has opened the debate as to whether stem cell and cloning technologies could be used to generate artificial gametes from the adult cells of individuals who were otherwise sterile from cancer treatments. Clearly there are ethical and safety concerns to consider before using such an approach and in a more recent report although Nayernia et al.62 showed that these spermlike cells could induce normal development and live pups when injected into mouse eggs, the pups were unhealthy and died prematurely. The concern about the safety aspects of generating artificial gametes for human applications was recently highlighted by the UK Government which proposes to make their use in Assisted Conception illegal in a forthcoming revision of the fertility laws in the UK.29

Legal and ethical aspects Whilst this review has focussed primarily on the biological and medical aspects of how a cancer diagnosis and treatment may affect the male and female reproductive system, and how reproductive tissues can be stored and later used in assisted conception procedures if required, it is also important to consider how the legal framework in which these are delivered can affect the outcome for the cancer survivor. Whilst in some countries there is very little regulation, and reproductive material can be cryopreserved (and later used) within the boundaries of good medical practice with local scrutiny from an ethics committee or an institutional review board (e.g. in the USA), in other countries strict laws are in force. For example, in the United Kingdom or Canada there is specific legislation to allow the storage of sperm, eggs and embryos for young cancer patients (and others) as well as regulating aspects of sperm, egg and embryo donation (see Table 1). By contrast, other countries, such as Switzerland and Italy, have passed much stricter laws that specifically forbid a number of these techniques (e.g. embryo freezing, sperm, embryo donation, surrogacy). Since arguably these are more likely to be needed by the cancer survivor (see above), it is of concern that cancer

Table 1 Examples of differences in the legal framework of fertility preservation and assisted conception techniques from seven countries around the world Cryopreservation strategies

Donor treatments

Gestational surrogacy

Sperm

Egg Embryo

Sperm

Egg

Embryo

Canada France Italy New Zealand

X X X X

X X X X

X X · X

X X · X

X X · X

X X · X

X · · X

Switzerland

X

X

X

X

·

·

·

United Kingdom USA

X X

X X

X X

X X

X X

X X

X X

Assisted Human Reproduction Act (2004) The French Bioethic Law of 29th July 1994 Law 40/2004 on Medically Assisted Reproduction Human Assisted Reproductive Technology Act 2004 Federal Law on Medically-assisted Reproduction (2001) Human Fertilisation and Embryology Act (1990) n/a

A cross indicates that the technique is outlawed in the particular country by specific legislation whereas a tick indicates that it is permitted, albeit it sometimes under specific conditions (e.g. licenses) the terms of which may differ from country to country.

Fertility issues in survivors from adolescent cancers survivors in these countries may have a poorer reproductive prognosis than their equivalents in a neighbouring country where the legislation is more lenient.

Concluding remarks Whilst our understanding of the effects of cancer and its treatment on the reproductive system of males and females had advanced enormously in recent years, there is still much to learn. Similarly, whilst the science of assisted conception has become very sophisticated with the manipulation of individual sperm and eggs to facilitate conception, this has largely been of most benefit for the male survivor. Sadly, the fertility preservation and subsequent treatment options for women remain limited. Further advances in the fields of medicine, science and law are required if we are to optimally secure the reproductive futures of subsequent generations of cancer survivors.

Acknowledgements The author thank the following people for help and assistance in completing this manuscript: Andrea Boggio (Bryant University, USA), James Lawford-Davies (Bevan Brittan), Sue Avery (Birmingham Women’s Hospital), Hany Lashen and William Ledger (University of Sheffield), Marc Van den Bergh (Kantonsspital Baden, Switzerland) and Grace Centola (New England Cryogenic Center, USA).

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