Mark F. H. Brougham¹, Christopher J. H. Kelnar¹, Richard M. Sharpe², W. Hamish B. Wallace¹

Keywords: infertility; childhood cancer; semen cryopreservation; germ cell transplantation; In vitro germ cell maturation; assisted reproductive techniques

Abstract

1 Introduction

2 Gonadal toxicity following cancer treatment

3 Potential for fertility following cancer treatment

Whilst these techniques, once fully developed, have enormous potential and offer hope to childhood cancer survivors at risk of infertility, these procedures are associated with a number of problems that must be overcome before application in a clinical setting.

Firstly, obtaining the testicular tissue would require an additional surgical procedure following confirmation of the diagnosis and prior to the commencement of any cytotoxic treatment. This would necessitate a further general anaesthetic, although in practice it should be possible to combine the testicular biopsy with other interventions required at this time. In addition, the procedure itself could result in damage to the testis, for example with bleeding and infection of the biopsy site. The prepubertal testis would be particularly vulnerable due to its small size in this age group.

Secondly, autologous germ cell transplantation requires tissue that was removed from a patient with cancer prior to treatment to be returned to the patient following cure. There is, therefore, a genuine risk of reintroducing malignant cells, with potentially fatal consequences. This is unlikely to occur with malignancies such as Hodgkins Disease, which is often localised at presentation, but the risk would be substantial with haematological malignancies [60], where the testes can act as sanctuary sites for leukaemic cells. Indeed, any theoretical risk of returning cancer cells following treatment, however small, would not be acceptable.

In order to circumvent this problem, it is likely that the germ cells, and in particular the stem cells, from the testicular biopsy would need to be isolated prior to transplantation. This would be safer and more acceptable than the use of semi-purified cell populations of germ cells and somatic cells as used in transplantation in the rodent models discussed above. However, the term stem cell?is a functional description as there are no specific morphological, antigenic or biochemical criteria to identify these cells. Although stem cells do express certain surface antigens, such as a-6 and b-1 integrins [61], which would allow purification using magnetic cell sorting [62], other progenitor cells including haemopoietic cells also express these antigens, and thus these markers will not be specific enough to exclude malignant cells. For purification to be effective, specific antibody probes must be developed in order to distinguish stem cells from other cells.

A further issue to be resolved before clinical application of these techniques is that of the cryopreservation process. Cryopreservation of mature, haploid spermatozoa is well established. However, there are substantial biological differences between these cells and undifferen-tiated, diploid stem cell spermatogonia, and thus the requirements of successful freezing and thawing differ considerably. Glycerol is used as the preservative of choice for ejaculated spermatozoa. However earlier germ cells, with proportionately larger amounts of cytoplasm, are more successfully preserved using dimethylsulphoxide (DMSO) as the cryoprotectant [63]. Despite this potential, the safety of DMSO in clinical use requires further evaluation prior to human application. Further studies are therefore needed on the optimal cryopreservation process for human testicular stem cells.

For germ cell transplantation to be successful, sufficient numbers of stem cells must be injected into the testes. It has been estimated that 104 germ cells isolated from an adult rodent testis contain as few as 2 stem cells [64]. Experiments using murine models have demonstrated that the seminiferous tubules of recipient mice testes have a volume of approximately 10 mL. Since cell concentrations of (1-2) ?108 cells per mL can be injected, the recipient mouse will only receive approximately 200~400 stem cells in a transplant [65]. There is thus a need to develop an in vitro enrichment system that will safely augment stem cell numbers following harvesting and isolation, thus improving the cell yield returned to the patient. Nagano et al [58] have demonstrated successful in vitro culture of mouse stem germ cells, and thus enrichment is likely to be feasible. However, because of the low numbers of stem cells that are likely to be returned, it may be that patients in whom germ cell transplantation is successful will still only be oligospermic rather than having normal sperm counts. They will therefore still require assisted reproduction in order to produce offspring.

Following harvest, stem cell isolation, cryopreser-vation and enrichment, the cells then need to be returned to the testes. Although this has been successfully performed in animal models as discussed above, the procedure for transplantation into the human testis remains unclear. The three main routes of re-infusion used previously are multiple microinjections into superficial seminiferous tubules, injection into the rete testis or injection into the efferent ducts. Although all of these techniques have been successful in the mouse [66], the most effective method in larger testes, including humans, is likely to be injection into the rete testis under ultrasound guidance [56]. Injection into this area allows a much larger volume to be infused. This technique is particularly successful in immature or regressed testes because of the lower intratubular fluid pressure, which can impair the injection into fully active testes. As the testes in cancer patients can be regressed secondary to cytotoxic treatment, this technique is certainly promising, although still needs to be refined prior to clinical application.

As can be seen, germ cell transplantation has potential, but there are a number of problems with the technique that need to be resolved. It could be argued that the safest method would be xenografting testicular tissue, for example into nude mice as demonstrated by Honaramooz et al [55]. In particular this eliminates the risk of re-introducing malignant cells. There would, however, be a risk of inter-species transfer of potentially pathogenic microorganisms. In addition, and perhaps more importantly, this process would not be ethically acceptable for widespread clinical use.

The technique of in vitro maturation of stem cells shares many of the problems discussed above but also circumvents the risk of re-introducing malignant cells, thus making this procedure potentially highly beneficial in this patient group. However, as discussed above, the technology to enable this maturation to occur may be some time away. Although ICSI has been successful using nuclear material from a round spermatid [67] and thus the maturation to spermatozoa need not be complete; the ability to artificially mature a diploid stem cell into a suitable haploid cell is not possible at the present time.

Finally, ICSI itself is not without potential compli-cations. Because ICSI involves the injection of nuclear material directly into an oocyte, some of the natural mechanisms that normally prevent sperm with defective DNA being involved in fertilisation are bypassed. In addition, the oocyte fertilized may itself be abnormal. There is, therefore, genuine concern that DNA and chromosomal abnormalities are more likely to be passed on to offspring produced with such techniques. Indeed, a number of large follow up studies have demonstrated an increase in chromosomal abnormalities, particularly of the sex chromosomes, in offspring born using ICSI [68, 69]. However, rather than the technique itself predisposing to these abnormalities, the increased incidence is felt to reflect the higher rate of constitutional chromosomal anomalies observed in infertile couples utilizing ICSI, particularly on the paternal side [69].

Transmission of constitutional chromosomal anomalies is less likely in cancer survivors because their infertility is secondary to gonadotoxic treatment. However, concerns have been raised that the mutagenic potential of cancer therapy could predispose the offspring of these patients to congenital abnormalities, and even cancer themselves. A large epidemiological study has failed to demonstrate any such link [70], except in those with familial malignancies, although these offspring resulted from natural conception. A recent study [18] investigated the integrity of spermatozoal DNA in men who had undergone treatment for childhood cancer. These sperm did not carry a greater burden of damaged DNA as compared to age-matched controls, providing some reassurance with regards to the use of spermatozoa from oligospermic men who have survived childhood cancer.

A further concern of offspring born using ICSI and IVF is the risk of congenital malformations. A number of large follow up studies have demonstrated no difference between ICSI and IVF in this regard, and indeed have suggested a comparable rate of major malformations to that seen following natural conception [71, 72]. Other studies have suggested an increased malformation rate, although have attributed this to the higher rate of multiple births seen following assisted reproduction [73].

However, interpretation of studies such as these can be problematic due to the inherent difficulty in obtaining appropriate control data for comparison, as maternal age, parity, sex of the infant and multiple births will affect the incidence of these abnormalities. In addition, variability exists between definitions of major and minor birth defects. In contrast to the aforementioned studies, Hansen et al [74] suggested that infants conceived using ICSI or IVF are twice as likely to suffer a major birth defect, and that this risk remained significant even allowing for these factors. Infants born of such techniques will be followed up more closely and thus one could argue that defects are more likely to be diagnosed in this cohort. However, the authors included defects diagnosed up to one year of age, at which time the majority of major defects would have been detected in all patient groups. In addition, there is evidence to suggest that babies born following assisted reproduction are at greater risk of low and very low birth weight [75]. Although this can again be explained, in part, by an increase in multiple births, it can also be a feature of singletons born with this technology.

Further long-term prospective studies are required to obtain accurate information on these children. If an increased risk of abnormalities is genuine, consideration must be given to the aetiology of these problems. The difficulty in identifying causative factors is that couples utilizing these techniques are inherently more at risk prior to fertility treatment, and thus the effect of the procedure itself can be difficult to elucidate. As discussed above, these risk factors may be less likely in survivors of childhood cancer.

In summary, both germ cell transplantation and in vitro maturation offer real hope for fertility preservation in males treated for cancer, particularly those in the prepubertal age group. However, there are a number of potential complications with both techniques, which must be overcome before they form part of the routine management in these patients.

Cytotoxic treatment, as mentioned previously, acts principally on rapidly dividing cells. As the testis demonstrates much cellular activity it is therefore prone to this damage and gonadotoxicity may result. In view of this, it has been postulated that inducing testicular quiescence could prevent gonadal toxicity, by making germ cells less vulnerable to the cytotoxic effects.

Studies investigating this technique using rodent models have produced very encouraging results. Ward et al [76] induced suppression of the hypothalamic-pituitary-gonadal axis using a gonadotrophin-releasing hormone (GnRH) agonist. Pulsatile GnRH is required for normal gonadal function, but if administered at a supraphysiological dose in a non-pulsatile, chronic manner, suppression of the hypothalamic-pituitary-gonadal axis will occur via down-regulation of pituitary GnRH receptors. When administered to rats prior to treatment with procarbazine, enhanced recovery of spermatogenesis was demonstrated, as compared to control animals. Similar protection of spermatogenesis has also been demonstrated using other methods of suppressing the reproductive axis, including treatment with testosterone, testosterone and oestradiol [77], GnRH antagonists and anti-androgens such as flutamide [78]. In addition, these methods have been protective against other gonadotoxic agents such as cyclophosphamide [79] and radiotherapy [80], suggesting that the mechanism of protection is not specific to particular modes of cytotoxic therapy.

A number of issues have arisen from these experi-ments. Firstly, the time required to suppress the reproductive axis is an important factor if this procedure is to be utilized in a clinical setting. Many of the studies discussed above involve hormonal manipulation commencing a number of weeks prior to administration of the cytotoxic agent. Cancer treatment should, in general, start as soon as possible following confirmation of the diagnosis, and therefore this delay would not be acceptable in patient management.

Secondly, the timing of suppression of the reproductive axis in relation to the cytotoxic damage has provoked many questions regarding the mechanism of gonadal protection. Meistrich et al [81] demonstrated that hormonal manipulation with a GnRH antagonist or testosterone led to the successful recovery of spermatogenesis in rats even when given after irradiation. Similar results have also been observed with hormonal treatment after the administration of procarbazine [82]. Whilst this would circumvent the concerns highlighted above regarding the delay in cancer treatment, these studies suggest that the original hypothesis of gonadal protection simply involving a reduction in the susceptibility of germ cells is incorrect.

There is evidence, based on rodent studies, to suggest that azoospermia following cytotoxic therapy does not necessarily result from complete depletion of spermatogonia, but is secondary to a failure of surviving spermatogonia to replicate and differentiate [5]. It has therefore been postulated that the recovery of spermatogenesis following hormonal treatment observed in these rodent models is due to stimulation of differentiation of surviving spermatogonia. A consistent feature observed in recovery of spermatogenesis is a reduction in intratesti-cular testosterone [81, 83], and it has been suggested that this relieves the block of differentiation, perhaps by alterations in the endocrine and paracrine environment of the testes. This subsequently allows the restoration of spermatogenesis.

Despite the success of these techniques in rodent models, clinical trials of hormonal manipulation in patients receiving gonadotoxic therapy have thus far failed to demonstrate any benefit. Administration of a GnRH analogue prior to and during treatment for lymphoma has been ineffective in conserving fertility [84, 85]. Subsequent to these earlier trials, and in view of the studies discussed above regarding the optimal timing of hormonal treatment, Thomson et al attempted to restore spermatogenesis in seven men rendered azoospermic following treatment for childhood cancer [86]. However, after suppression of the hypothalamic-pituitary-gonadal axis with testosterone and medroxyprogesterone acetate the men remained azoospermic on reassessment, and their testes, on biopsy, lacked germ cells. Perhaps in this study the hormonal treatment was initiated too long after the cytotoxic therapy. In addition, the lack of success may be because the initial testicular insult was too severe, and that this technique may be more beneficial in patients with less severe gonadal damage in whom some spermatogonial stem cells are preserved following cytotoxic treatment. However, it is difficult to justify further clinical trials in this area until the mechanism of gonadal protection?is better understood. Indeed, extrapolating evidence from rodent models to clinical trials assumes that both the physiology of spermatogenesis and the mechanisms of gonadotoxic damage are similar in both species. However, there is evidence to suggest that significant inter-species differences do exist, and that perhaps other primates would be more appropriate models in which to study spermatogenesis in this regard [87, 88].

The common marmoset (Callithrix jacchus), a New World primate, demonstrates similar phases of testicular development and displays similarities in its organization of spermatogenesis to the human [89]. It therefore represents a useful model for studying prepubertal testicular development. Kelnar et al [90] investigated this phase of development using immunoexpression studies of marmoset testes. Functional development of both Sertoli cells, based on the expression of sulphated glycoprotein-2 and androgen receptor, and of Leydig cell activity, based on the expression of 3b-hydroxysteroid dehydrogenase, was demonstrated pre-pubertally. In addition, proliferation of germ cells was noted at this age, indicated by the immunoexpression of proliferating cell nuclear antigen (PCNA). This provides further evidence that the pre-pubertal testis is not quiescent, and improves our understanding of why the testis is susceptible to cytotoxic damage at this age group.

In addition, this study also investigated the effects of treatment with a GnRH antagonist on the prepubertal marmoset. It was found that this treatment largely prevented many of the changes highlighted above. However, unexpectedly, the proliferation of germ cells, as assessed by the PCNA labeling index of spermatogonia, was unaffected by administration of the GnRH antagonist, when compared to control animals. This suggests that germ cell proliferation, in primates, is in fact gonadotrophin-independent, and therefore hormonal manipulation based on suppression of the gonadotrophin axis is unlikely to be successful in alleviating gonadotoxic damage secondary to cancer treatment. Studies are currently in progress in order to identify what factors do regulate spermatogonial proliferation, with the hope that these may offer novel targets of gonadal protection during cytotoxic therapy.

Thus, hormonal manipulation may in the future become a beneficial therapeutic intervention in boys with cancer, but a greater understanding of the physiology of human spermatogenesis is required before the clinical application of these techniques is feasible.

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Correspondence to: Dr W.H.B. Wallace, Consultant Paediatric Oncologist, Department of Paediatric Haematology and Oncology, Royal Hospital for Sick Children, 17 Millerfield Place, Edinburgh EH9 1LW, UK.
Tel: +44-131 536 0420, Fax: +44-131 536 0430
E-mail: Hamish.Wallace@luht.scot.nhs.uk
Received 2003-09-10 Accepted 2003-09-15