Fertility preservation of boys undergoing anti-cancer therapy: a review of the existing situation and prospects for the future: Opinion

Irfan Aslam1,3, Simon Fishel1, Harry Moore2, Ken Dowell1 and Simon Thornton1

1 CARE (Centres for Assisted Reproduction), The Park Hospital, Sherwood Lodge Drive, Burntstump Country Park, Arnold, Nottingham, NG5 8RX and 2 Department of Obstetrics and Gynaecology, The University of Sheffield, Sheffield, UK

Abstract

With the advancement of medical science, most cancers in children are now treatable, the cure rate being almost 85%. In boys, one side effect of treatment (chemotherapy and radiotherapy) is destruction of the sperm precursor cells in the testis, resulting in the failure of sperm formation after puberty, and consequent infertility. At the time of anti-cancer treatment, future fertility of the boy patient is at the very bottom of the relative quality of life (RQL) parameters list; however, in adults infertility is an important issue following cure from cancer. In this article we have first reviewed the existing situation with regard to the state of the art of fertility preservation in young boys with cancer, and have then raised clinical and ethical issues and suggested a way forward. The review concludes with the emphasis that certain important issues still need to be resolved and, until these are, then the different techniques available should be confined to approved, ethical clinical trials where efficacy and safety can be fully evaluated.

Key words: anti-cancer treatment/cancer/fertility preservation/germ cell transplantation/in-vitro spermatogenesis

Introduction

Data obtained from the National Registry of Childhood Tumours (NRCT) and Childhood Cancer Research Group (CCRG) show that between 1978 and 1987, the cumulative incidence of childhood malignancy was 1720 per million, which was equivalent to a risk of 1 in 581 for the first 15 years of life. Boys were affected 1.2 times more frequently than girls (Stiller et al., 1995Go). For some diagnostic groups, the incidence rates during the study period were somewhat higher than those published previously, while the trends in registration rates for all childhood malignancies reflected a true underlying rise in incidence (Draper et al., 1994Go).

As a result of chemo- and radiotherapeutic intervention, about 70% of children with oncological diseases survive their malignancies (Robison, 1996Go). This marked improvement in the survival of childhood cancer generally began during the early 1970s, and there are now over 10 000 adult survivors of childhood cancer in Britain alone (Stiller, 1994Go). Moreover, this group of survivors is expanding very rapidly each year. While the late effects of aggressive therapy on different organ systems including thyroid gland, heart, lung and nervous and skeletal systems are currently under investigation, it is not difficult to foresee the implications for reproductive organs (Pizzo and Poplack, 1993Go; Sherins, 1993Go). Most cancer therapies affect gonadal function at the time of treatment (Bramswig et al., 1990Go); therefore, survival is often associated with infertility during adulthood (Sherins, 1993Go). At the time of anti-cancer treatment, the future fertility of the boy patient is at the very bottom of the relative quality of life (RQL) parameters list; however, in adult life, following cure from cancer, infertility is an important issue.

Gonadal functions and their relationship to puberty in boys

In boys, no data are available on semen variables characterizing the development of gonadal function, and thus spermatogenesis. By evaluation of the relationship between gonadotrophin excretion, the increase in testicular volume, secondary sex characteristics and the determination of spermatozoa in urine, the median age of spermarche is estimated to be 13–14 (range 11 to 17) years of age (Hirsch et al., 1979Go; Schaefer et al., 1990Go). The onset of puberty in boys is marked by an increase in gonadotrophins and testicular growth. A significant increase in testosterone serum concentrations occurs after the onset of testis growth at the age of 10–12 years (Burr et al., 1970Go), and therefore this age can be assumed as a mean reference for the beginning of puberty.

A variety of spermatogenic cells have been described in histological studies on testicular tissue isolated from pre-pubertal boys. The seminiferous epithelium of normal infant and child testes generally consists of immature Sertoli cells and different types of spermatogonia. These include stem cell spermatogonia and differentiating spermatogonia type A (pale and dark types) and type B spermatogonia (from age 4 years onwards) (Nistal and Paniagua, 1984Go). Primary spermatocytes appear largely at the beginning of puberty, shortly after the onset of spermatogenesis. The development of these early spermatocytes is not normal as they undergo degeneration or progress to abnormal spermatids which in turn degenerate (Muller and Skakkebaek, 1983Go).

The effects of anti-cancer treatment on spermatogenesis

Effect of radiotherapy
Irradiation is cytotoxic to differentiating spermatogonia. Studies on spermatogenic cell depletion have revealed that a dose of 1 Gy or more of X-irradiation destroyed all type B spermatogonia in monkeys, and consequently all spermatocytes disappeared 17 days later and all spermatids 31 days thereafter (de Rooij et al., 1986Go). It was also found (Rowley et al., 1974Go) that type B spermatogonia were more sensitive than type A spermatogonia in humans. Quantitative and autoradiographic analysis of adult male mice exposed to 0.33 Gy X-radiation showed that the number of single and paired type A spermatogonia were minimal 2 days later (Huckins and Oakberg, 1978Go). These cells (90–95%) could be labelled by multiple injections of [3H]deoxythymidine at 2–3.5 days post-irradiation, and differentiated into type B spermatogonia by 11 days. A direct comparison of quantitative data also suggests that irradiation is less destructive than chemotherapeutic drugs (busulfan) for type A spermatogonia (Dunn, 1974Go). Therefore, although irradiation is a potent killer of spermatogonia (Meistrich, 1993Go), it is less damaging than chemotherapy.

Effect of chemotherapeutic agents
There are several types of chemotherapeutic drugs which show stage-specific cytotoxicity to spermatogonia (Meistrich, 1984Go). In general, the rapidly dividing spermatogonia seem to be most sensitive to many of these drugs. The relative sensitivity of differentiating and stem spermatogonia in mice to various cytotoxic agents can be determined by counting sperm head numbers at 29 and 56 days after treatment. With busulfan, there is a low ratio of differentiating spermatogonia to stem cells, which indicates that the effect of the compound is directed more against stem spermatogonia than other cell types, with a resultant disruption of spermatogenesis in adult life (Meistrich, 1984Go). Busulfan belongs to the alkylating group of chemotherapy drugs which generally interfere at the early stages of spermatogenesis (Cooper and Jackson, 1970Go).

In the case of chemotherapeutic agents, the type and cumulative dose of the drug is important in anti-fertility outcome (Ridley et al., 1985Go). A review of the literature since 1980, which includes data on semen variables after childhood cancer, showed that among 31 patients treated with non-alkylating agents (such as adriamycin, vincristine, methotrexate and 6-mercaptopurine), 84% had a recovery of spermatogenesis ranging from oligozoospermia to normozoospermia, while 16% remained azoospermic for between 1 and 11 years after the end of treatment. If cisplatin was used for therapy—either alone or in combination—37% of 57 patients remained azoospermic. If alkylating agents such as cyclophosphamide or procarbazine were used in different combinations and in different cumulative doses, then 68% of a total of 93 reported cases remained azoospermic between 1 and 20 years after cessation of therapy (for a review, see Kliesch et al., 1996).

Childhood malignancies

Among the 1 in 581 children who develop some sort of childhood malignancy by the age of 15 years, between 70% and 80% will survive. This means that by the end of year 2000, one in 1000 young adults will be a survivor of childhood cancer, and nearly half of them will be male. Unfortunately, 16–68% of these young men will be infertile with azoospermia as the outcome (Kliesch et al., 1996Go).

Investigations on adult patients have shown that chemotherapy causes gonadal damage (Chatterjee and Goldstone, 1996Go), but much less information is available about the impact of chemotherapy on gonadal functions in children. Being pre-pubertal during therapy was believed to confer protection against chemotherapy-induced gonadal damage. However, when 12 patients who had survived childhood malignancy were examined between 2 and 16.5 years after chemotherapy, it was revealed that although puberty had progressed apparently normally in all of them, seven patients had exaggerated LH response to gonadotrophin-releasing hormone (GnRH), indicative of Leydig cell dysfunction. Eight patients were azoospermic, and only one patient had normal semen analysis (Mustieles et al., 1995Go). There is no direct evidence of gonadal protection in the pre-pubertal male. This is supported by the finding that, following treatment of Hodgkin's disease in childhood, there is a high degree of germinal epithelium damage, with elevated FSH concentrations detected even 17 years after chemotherapy (Shafford et al., 1993Go). In another study on 19 paediatric Hodgkin's disease cases, severe germ cell damage was seen in all patients (Heikens et al., 1996Go).

Preservation of fertility

Usually, the news that a young man has malignancy is devastating, and treatment quickly follows. There is thus little time for the impact of possible future sterility to be absorbed. However, due to the marked improvement in the treatment for childhood malignancies consideration should now be given to the consequential effects on future fertility.

Different options are available for the adult male, including sperm banking. Pre-pubertal boys cannot benefit from this option due to absence of haploid gametes (spermatozoa and spermatids) in the testis. However, with the advances in intracytoplasmic sperm injection (ICSI), mature and immature (spermatid) sperm extraction and maturation (Fishel et al., 1995Go; Hovatta et al., 1996Go; Antinori et al., 1997Go; Tesarik et al., 1999Go), focus has turned towards preserving gonadal tissue for future use (Karrow and Crister, 1997Go) before the start of the anti-cancer treatment in such boys. These isolated cells can be stored frozen for many years. Once the boy is cured from the cancer his frozen testicular precursor cells can either be considered for transplantation back into his own testes (ipsigeneic germ cell transplantation), or these cells might be further matured in vitro or in vivo in another host (xenogeneic germ cell transplantation) until the stage at which they will be competent to procure normal fertilization with the ICSI technique.

Existing situation and options available for pre-pubertal boys

On average, the total number of germ cells in boys varies from 13x106 to 83x106 during childhood (0–10 years) (Muller and Skakkebaek, 1983Go). From birth to puberty, Type Ap and Ad spermatogonia are the most abundant type of germ cells in the testicular tissue. Type B spermatogonia start appearing at 4–5 years of age, after which their number slowly increases until the age of 8–9 years. This is followed by a period of marked spermatogonial proliferation. Finally, primary spermatocytes—and even spermatids—are present in some seminiferous tubules, coinciding with the period of spermatogonial proliferation. Such spermatocytes never seem to give rise to spermatozoa, but instead the cells degenerate. This may be interpreted as the abortive attempts at spermatogenesis (Paniagua and Nistal, 1984Go).

The above description of the histological picture of the pre-pubertal testis shows that not many options are available for pre-pubertal boys. In the late pre-pubertal period, perhaps few spermatids might be present which could be cryopreserved for later use. However, the fertilization potential of these early spermatids has not been evaluated so far.

A review of the literature shows that until recently it has not been possible to develop a culture system to support the in-vitro differentiation of male germ cells. Recently, transmeiotic differentiation of immature male germ cells was reported in two studies (Rassoulzadegan et al., 1993Go; Hue et al., 1998Go) in which cells were explanted from pubertal animals. The effects of these in-vitro culture conditions on pre-pubertal spermatogonia are still to be evaluated before considering this approach for clinical purposes.

Two recent transplantation studies have created a new option for fertility preservation in pre-pubertal boys, however. In the first study, when mouse testes-derived cells were transplanted into the testis of another mouse (ipsigeneic transplantation), donor cells colonized the seminiferous tubules of the recipient and initiated spermatogenesis in more than 70% of recipients. Testes-derived cells from new-born mice were less effective in colonizing recipient testes than cells from 5- to 15-day-old (sexually immature) or 21- to 28-day-old (sexually mature) mice. With recipients that maintained endogenous spermatogenesis, testis cell transplantation yielded mice in which up to 80% of their progeny were sired by donor-derived spermatozoa (Brinster and Avarbock, 1994Go). In the second study, similar results were obtained when frozen–thawed testes-derived cells were transplanted (Avarbock et al., 1996Go). The feasibility of transplanting spermatogonial stem cells from other species to the mouse (xenogeneic transplantation) was examined. Following the transplantation of rat testes cells to the testes of immunodeficient mice, rat spermatogenesis occurred in all recipient mice (Clouthier et al., 1996Go). The generation of rat spermatogenesis in mouse testes suggests that spermatogonial stem cells of different species can be transplanted, and opens the possibility of xenogeneic spermatogenesis for humans.

Male germ cell transplantation: a new approach for fertility preservation in pre-pubertal boys

Considering the high efficiency of sperm production, and the fact that this process is active from puberty to old age, spermatogenesis is a remarkable phenomenon. This unique process is based entirely on the stem cell spermatogonia.

Stem cell spermatogonia
Spermatogonia from the basal layers within the seminiferous tubules have been classified as three types: stem cell spermatogonia; proliferative spermatogonia; and differentiating spermatogonia. Stem cell spermatogonia undergo self-renewal throughout life, and because of their ability to transmit genes from one generation to the next they are the genuine totipotent population of the cells in the adult body. The stem cell spermatogonia are most resistant to a variety of agents which damage the testis, and often these cells survive when other germ cells are destroyed (Russell et al., 1990Go).

These properties render stem cells most suitable for transplantation purposes. These cells are present in the pre-pubertal testicular tissue, and can be isolated, successfully cryopreserved and transplanted back into seminiferous tubules, where they can implant, multiply and differentiate into mature spermatozoa after both syngeneic and xenogeneic transplantation (Brinster and Avarbock, 1994Go; Brinster and Zimmermann, 1994Go; Avarbock et al., 1996Go; Clouthier et al., 1996Go).

Scheme for the fertility preservation with stem cell spermatogonia transplantation
The fertility potential of pre-pubertal boys undergoing anti-cancer therapy can be preserved by isolating their stem cell spermatogonia and cryofreezing them until the individual is recovered from the malignancy. Subsequently, these frozen–thawed stem cells can be transplanted back either into the same individual or into another host to encourage spermatogenesis. However, before embarking upon such a clinical programme there are certain ethical, legal and clinical concerns which need to be evaluated carefully.

Ethical and legal issues regarding pre-pubertal testicular tissue cryopreservation
Children provide a `double-edged sword' when considering issues of testicular tissue freezing. These partly relate to obtaining proper informed consent for taking, storing and using gonadal tissue (McLean, 1997Go).

In the UK, the Human Fertilisation and Embryology Authority (HFEA) Act requires that gametes must be stored on licensed premises, and consent to storage must be given by the person who provides the gametes. The HFEA defines the `gamete' to be `a reproductive cell, such as an ovum or spermatozoa, which has a haploid set of chromosomes and which is able to take part in fertilization with another of the opposite sex to form a zygote' (Deech, 1998Go). Using this definition in conjunction with the six Tanner grades of puberty (Tanner, 1989Go), storage of testicular tissue from boys who have reached at least Tanner stage 2 will require a licence. Consent to storage cannot be given on behalf of any child who has reached Tanner stage 2 and whose testicular tissue is to be stored. However, a child under the age of 16 years can give an effective consent in accordance with the 1990 Act's requirement if he is capable of understanding the proposed course of action. If a boy is pre-pubertal (under Tanner stage 2), then his testicular tissue can be removed with parental consent and stored on unlicensed (by the HFEA) premises. If, however, such material were subsequently developed in vitro in some way as to create `gametes', the storage and use of that material would require a license (Deech, 1998Go). Consent to the removal of testicular tissue from any male of whatever age is covered by common law. Therefore, in the UK, there are now boys who are caught out by the HFEA regulations; viz. those boys who are Tanner stage 2 but too young themselves to give written, informed consent as required by the HFEA.

Against this histological and legal background of pre-pubertal testicular tissue freezing we need justification for subjecting a sick child to the trauma of an invasive testicular biopsy. Secondly, we need to evaluate the risk (if any) of biopsy trauma inducing testicular cancer. In cryptorchid patients, testicular biopsy appeared to be a strong risk factor for testicular cancer, the most common malignancy in men aged 15–34 years (Swerdlow et al., 1997Go); however, the relevance of this finding to pubertal boys biopsied before anti-cancer therapy is unknown.

Clinical concerns for stem cell spermatogonia transplantation
Before considering stem cell spermatogonia transplantation for fertility preservation there are certain concerns which require very careful evaluation.

Low number of stem cell spermatogonia
In boys, the volume of both testes increases from 1.1 to 3.0 cm3 during the first 10 years of life, while the total number of germ cells increases from 13x106 to 83x106 (Muller and Skakkebaek, 1983Go). Since 104 germ cells are thought to contain about two stem cells (Meistrich and van Beek, 1993Go), the low number of stem cell spermatogonia can be problematical during isolation of these cells from pre-pubertal testis. For obvious reasons the whole of the testis cannot be removed; therefore we need to find an in-vitro culture system to increase the number of stem cells after isolation from the pre-pubertal testes. Recently, a study has shown that neonatal mouse gonocytes can be replicated successfully during in-vitro culture (Hasthorpe et al., 1999Go). Studies in our laboratory have shown that stem cell spermatogonia can also be replicated (unpublished results); however, whether these replicated stem cell spermatogonia can be transplanted successfully or not still needs to be evaluated.

Isolation of stem cell spermatogonia
Because of low numbers of spermatogonial stem cells in the pre-pubertal testis it is unlikely that their purification can be achieved by sedimentation or density separation techniques (Aslam et al., 1998Go) alone. Effective purification will likely require development of specific antibody probes to distinguish stem cell spermatogonia from other cells. The surface of stem cell spermatogonia contains certain antigen markers which they share with other progenitor cells including hematopoietic stem cells; ß1, {alpha}6 integrins and c-kit receptor antigens are three common shared antigens on the surface of stem cell spermatogonia (Shinohara et al., 1999Go). By taking advantage of these antigens, mouse stem cell spermatogonia can be isolated using magnetic cell sorting (Schonfeldt et al., 1999Go). We have used commercially available antibodies raised against ß1 integrin antigens to carry out successful isolation of stem cell spermatogonia from neonatal mice testicular tissue (data in preparation).

Cryopreservation of spermatogenic stem cells
Reports published to date have described only the isolation and cryopreservation of murine spermatogenic stem cells (Avarbock et al., 1996Go). The cryoprotectant used in these studies was dimethylsulphoxide (DMSO), which is itself potentially carcinogenic and not suitable for human clinical studies. Therefore, there is a need to develop a safe cryopreservation protocol for the spermatogenic stem cells, similar to freezing of the human spermatozoa and embryos. In our laboratory we have developed a freezing protocol which is based on a glycerol-based cryoprotectant, can provide equally good cryopreservation as DMSO (data in preparation), and would be a safe alternative in clinical situations.

Tumour cell contamination
Contamination of isolated spermatogonia stem cells with tumour cells is an important concern. We should not try to preserve the fertility potential of an individual if there is a risk of re-exposing him to the same problem later on. Currently, no such technology is available to scan all the cryopreserved cells to select the healthy ones. Random cell sampling can be carried out, but this does not ensure contamination-free cell aliquots.

In malignancies which metastasize through blood—for example, sarcomas, leukaemia and lymphomas—heterologous transplantation, i.e. transplantation into another host (xenogeneic) should be considered. On the other hand, for malignancies which do not metastasize through blood (for example, Hodgkin's lymphoma), homologous (ipsigeneic) transplantation should be considered with caution.

Exposure of humans to animal diseases
In xenogeneic transplantation, i.e. transplantation of human spermatogonial stem cells into lower animals including mice or rats, there is concern for the introduction of unknown animal infectious agents to humans when these matured germ cells will be used to procure conception. It has been shown that human kidney cells can be infected by endogenous retroviruses of pigs (Patience et al., 1997Go). An infection of germ cells after xenogeneic transplantation would lead to an accumulation of retroviruses in the human germ line. This concern needs to be examined very carefully before embarking upon clinical trials of xenogeneic spermatogonial transplantation.

Future prospects

Spermatogonial transplantation offers the prospect of several applications. Although most of these applications are not feasible today, in the near future a major impact of germ cell transplantation in areas of human and veterinary medicine does not seem unrealistic.

In man, autologous germ cell transplantation might become a clinically important technique to protect male germ cells during anti-cancer treatment to allow these patients to conserve their germ cell line in vitro. For pre-pubertal boys, this technology has the potential to allow them paternity as an adult. However, to develop a clinically applicable autologous germ cell transplantation programme, techniques must be developed by which human spermatogonial stem cells can be isolated, stored and re-infused into the testis. Recently, a technique was described for germ cell transfer into the primate testis, using ultrasound-guided injections into the rete testis (Schlatt et al., 1999Go). This technique allowed an efficient filling of nearly 70% of all seminiferous tubules. The apparent detection of transplanted cells at 4 weeks after autologous germ cell transfer into monkey testis indicates the potential of this technique for clinical use.

The xenogeneic production of spermatozoa in immunotolerant mice might be developed into a method for male fertility protection in cases of bilateral testicular damage, anti-cancer treatment or bilateral orchidectomy performed for other reasons. In such cases, transplantation of spermatogonial stem cells into immunodeficient mice can be used either for storage of the germ cell line or for xenogeneic production of spermatozoa for insemination in assisted fertilization procedures (Aldhous, 1998Go).

These developments, and work in progress, suggest that it may soon be possible to preserve the fertility of pre-pubertal boys requiring treatment for cancer which ordinarily would lead to their permanent sterility. However, several important issues still need to be resolved, and until this has been achieved the various techniques should be confined to approved, ethical clinical trials where efficacy and safety can be fully evaluated. We should proceed cautiously until we have a clear view of the possible advantages and disadvantages. Alternatively, uncontrolled testicular biopsies, tissue cryopreservation and transplantation of testicular tissue in a wide range of circumstances may—at best—be ineffective or unnecessary, and—at worst—be life-threatening if such tissue were to be retransplanted without appropriate evaluation.

Notes

3 To whom correspondence should be addressed at: CARE (Centres for Assisted Reproduction), The Park Hospital, Sherwood Lodge Drive, Burntstump Country Park, Arnold, Nottingham, NG5 8RX, UK. E-mail: irfan.aslam{at}care-ivf.com Back

References

Aldhous, P. (1998) Surrogate fathers. New Scientist, 157, 4.

Antinori, S., Versaci, C., Dani, G. et al. (1997) Successful fertilisation and pregnancy after injection of frozen-thawed round spermatids into human oocytes. Hum. Reprod., 12, 554–556.[ISI][Medline]

Aslam, I., Robins, R.A., Dowell, K. and Fishel, S. (1998) Isolation, purification and assessment of viability of spermatogenic cells from testicular biopsies of azoospermic men. Hum. Reprod., 13, 639–645.[Abstract]

Avarbock, M.R., Brinster, C.J. and Brinster, R.L. (1996) Reconstitution of spermatogenesis from frozen spermatogonial stem cells. Nature Med., 2, 693–696.[ISI][Medline]

Bramswig, J.H., Heimes, U., Heiermann, E. et al. (1990) The effects of different cumulative doses of chemotherapy on testicular function. Cancer, 65, 1298–1302.[ISI][Medline]

Brinster, R.L. and Avarbock, M.R. (1994) Germ line transmission of donor haplotype following spermatogonial transplantation. Proc. Natl Acad. Sci. USA, 91, 11303–11307.[Abstract/Free Full Text]

Brinster, R.L. and Zimmermann, J.W. (1994) Spermatogenesis following male germ-cell transplantation. Proc. Natl Acad. Sci. USA, 91, 11298–11302.[Abstract/Free Full Text]

Burr, I.M., Sizonenko, P.C., Kaplan, S.L. and Grumbach, M.M. (1970) Hormonal changes in puberty. 1. Correlation of serum luteinizing hormone and follicle stimulating hormone with stages of puberty, testicular size and bone age in normal boys. Pediatr. Res., 4, 25–35.[ISI][Medline]

Chatterjee, R. and Goldstone, A.H. (1996) Gonadal damage and effects on fertility in adult patients with haematological malignancy undergoing stem cell transplantation. Bone Marrow Transplant., 17, 5–11.[ISI][Medline]

Clouthier, D., Avarbock, M.R., Malika, S.D. et al. (1996) Rat spermatogenesis in mouse testis. Nature, 381, 418–421.[ISI][Medline]

Cooper, E.R. and Jackson, H. (1970) Comparative effects of methylene, ethylene and propylene dimethenesulphonates on the male rat reproductive system. J. Reprod. Fertil., 23, 103–108.[Medline]

Deech, R. (1998) Human Fertilisation and Embryology Authority. Br. Med. J., 316, 1095.[Free Full Text]

De Rooij, D.G., van Alphen, M.M. and van de Kant, H.J. (1986) Duration of the cycle of the seminiferous epithelium and its stages in the rhesus monkey (Macaca mulatta). Biol. Reprod., 35, 587–591.[Abstract]

Draper, G.J., Kroll, M.E. and Stiller, C.A. (1994) Childhood cancer. Cancer Surv., 19/20, 493–517.

Dunn, C.D.R. (1974) The chemical and biological properties of busulfan (myleran). Exp. Hematol., 2, 101–117.[ISI][Medline]

Fishel, S., Green, S., Bishop, M. et al. (1995) Pregnancy after intracytoplasmic injection of spermatid. Lancet, 345, 1641–1642.

Hasthorpe, S., Barbic, S., Farmer, P.J. and Huston, J.M. (1999) Neonatal mouse gonocyte proliferation assayed by an in vitro clonogenic method. J. Reprod. Fertil., 116, 335–344.[Abstract]

Heikens, J., Behrendt, H., Adriaanse, R. and Breghout, A. (1996) Irreversible gonadal damage in male survivors of paediatric Hodgkin's disease. Cancer, 78, 2020–2024.[ISI][Medline]

Hirsch, M., Shemesh, J., Modan, M. and Lunenfeld, B. (1979) Emission of spermatozoa. Age of onset. Int. J. Androl., 2, 289–298.[ISI]

Hovatta, O., Foudila, T., Siegberg, R. et al. (1996) Pregnancy resulting from intracytoplasmic injection of spermatozoa from a frozen-thawed testicular biopsy specimen. Hum. Reprod., 11, 2472–2473.[Abstract]

Huckins, C. and Oakberg, E.F. (1978) Morphological and quantitative analysis of spermatogonia in mouse testes using whole mounted seminiferous tubules. II. The irradiated testes. Anat. Rec., 192, 529–542.[ISI][Medline]

Hue, D., Staub, C., Perrard-Sapori, M.H. et al. (1998) Meiotic differentiation of germinal cells in three-week culture of whole cell population from rat seminiferous tubules. Biol. Reprod., 59, 379–387.[Abstract/Free Full Text]

Karrow, A.M. and Crister, J.K. (1997) Reproductive Tissue Banking: Scientific Principles. Academic Press, San Diego.

Kliesch, S., Behre, H.M., Jurgens, H. and Nieschlag, E. (1996) Cryopreservation of semen from adolescent patients with malignancies. Med. Pediatr. Oncol., 26, 20–27.[ISI][Medline]

McLean, S. (1997) Consent and the law: review of the current provisions in the Human Fertilisation and Embryology Act 1990 for the UK health ministers, consultation document and questionnaire. Department of Health, London.

Meistrich, M.L. (1984) Stage-specific sensitivity of spermatogonia to different chemotherapeutic drugs. Biomed. Pharmacother., 38, 137–142.[ISI][Medline]

Meistrich, M.L. (1993) Effects of chemotherapy and radiotherapy on spermatogenesis. Eur. Urol., 23, 136–142.[ISI][Medline]

Meistrich, M.L. and van Beek, M.E.A.B. (1993) Spermatogonial stem cells. In Desjardins, C. and Ewing, L.L. (eds), Cell and Molecular Biology of the Testis. Oxford University Press, New York, pp. 266–295.

Muller, J. and Skakkebaek, N.E. (1983) Quantification of germ cells and seminiferous tubules by stereological examination of testicles from 50 boys who suffered from sudden death. Int. J. Androl., 6, 143–156.[ISI][Medline]

Mustieles, C., Munoz, A., Alonso, M. et al. (1995) Male gonadal function after chemotherapy in survivors of childhood malignancy. Med. Pediatr. Oncol., 24, 347–351.[ISI][Medline]

Nistal, M. and Paniagua, R. (1984) Occurrence of primary spermatocytes in the infant and child testis. Andrologia, 16, 532–536.[ISI][Medline]

Paniagua, R. and Nistal, M. (1984) Morphological and histometric study of human spermatogonia from birth to the onset of puberty. J. Anat., 139, 535–552.[ISI][Medline]

Patience, C., Takeuchi, Y. and Weiss, R.A. (1997) Infection of human cells by an endogenous retrovirus of pigs. Nature Med., 3, 282–286.[ISI][Medline]

Pizzo, P.A. and Poplack, D.G. (eds) (1993) Principles and Practice of Pediatric Oncology. J.B. Lippincott, Philadelphia.

Rassoulzadegan, M., Paquis-Flucklinger, V., Bertino, B. et al. (1993) Transmeiotic differentiation of male germ cells in culture. Cell, 75, 997–1006.[ISI][Medline]

Ridley, M., Nicosia, S. and Meadows, A. (1985) Gonadal effects of cancer therapy in boys. Cancer, 55, 2355–2363.

Robison, L.L. (1996) Methodologic issues in the study of second malignant neoplasms and pregnancy outcomes. Med. Pediatr. Oncol. Suppl., 1, 41–44.[Medline]

Rowley, M.J., Leach, D.R., Warner, G.A. and Heller, C.G. (1974) Effect of graded doses of ionising radiation on the human testis. Radiat. Res., 59, 665–678.[ISI][Medline]

Russell, L.D., Ettlin, R.A., Hakim, A.P. and Clegg, E.D. (1990) Mammalian spermatogenesis. In Histological and Histopathological Evaluation of the Testis. Cache River Press, Clearwater, FL, pp. 1–40.

Schaefer, F., Marr, J., Seidel, C. et al. (1990) Assessment of gonadal maturation by evaluation of spermaturia. Arch. Dis. Child., 65, 1205–1207.[Abstract]

Schlatt, S., Rosiepen, G., Weinbauer, G.F. et al. (1999) Germ cell transfer into rat, bovine, monkey and human testes. Hum. Reprod., 14, 144–150.[Abstract/Free Full Text]

Schonfeldt, V.V., Krishnamurthy, H., Foppiani, L. and Schlatt, S. (1999) Magnetic cell sorting is a fast and effective method of enriching viable spermatogonia from djungarian hamster, mouse and marmoset monkey testes. Biol. Reprod., 61, 582–589.[Abstract/Free Full Text]

Shafford, E.A., Kingston, J.E., Malpas, J.S. et al. (1993) Testicular function following the treatment of Hodgkin's disease in childhood. Br. J. Cancer, 68, 1199–1204.[ISI][Medline]

Sherins, R.J. (1993) Gonadal dysfunction. In De Vita, V.T., Hellman, S. and Rosenburg, S.A. (eds), Cancer. Principles and Practice of Pediatric Oncology. J.B. Lippincott, Philadelphia, pp. 2395–2406.

Shinohara, T., Avarbock, M.A. and Brinster, R.L. (1999) ß1- and {alpha}6-integrins are surface markers on mouse spermatogonial stem cells. Proc. Natl Acad. Sci. USA, 96, 5504–5509.[Abstract/Free Full Text]

Stiller, C.A. (1994) Population based survival rates for childhood cancer in Britain. Br. Med. J., 309, 1612–1616.[Abstract/Free Full Text]

Stiller, C.A., Allen, M.B. and Eatock, E.M. (1995) Childhood cancer in Britain: the national registry of childhood tumours and incidence rates 1978–1987. Eur. J. Cancer, 31A, 2028–2034.

Swerdlow, A.J., Higgins, C.D. and Pike, M.C. (1997) Risk of testicular cancer in cohort of boys with cryptorchidism. Br. Med. J., 314, 1507–1511.[Abstract/Free Full Text]

Tanner, J.M. (1989) Foetus into Man: Physical Growth from Conception to Maturity, 2nd edn. Castlemead, London.

Tesarik, J., Bahceci, M., Ozcan, C. et al. (1999) Restoration of fertility by in-vitro spermatogenesis. Lancet, 353, 555–556.[ISI][Medline]

Submitted on December 8, 1999; accepted on June 15, 2000.