1 Section of Child Life and Health, Department of Reproductive and Developmental Sciences, University of Edinburgh, Edinburgh EH9 1LW and 2 MRC Human Reproductive Sciences Unit, Centre for Reproductive Biology, Edinburgh EH3 9ET, UK
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Abstract |
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Key words: cancer/childhood/hormone suppression/spermatogenesis/testis
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Introduction |
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A number of approaches to this problem have been investigated, based on the idea that suppression of spermatogenesis might protect the normally rapidly dividing germ cell population from damage. Suppression of the rat hypothalamicpituitarygonadal (HPG) axis by administration of the GnRH analogue, goserelin, before and during chemotherapy with procarbazine, enhanced recovery of spermatogenesis (Ward et al., 1990). Similarly, protection of spermatogenesis in rats subjected to treatment with procarbazine, cyclophosphamide and radiotherapy has been demonstrated using a number of hormones including testosterone alone (Delic et al., 1986
) or in combination with estrogen (Kurdoglu et al., 1994
), GnRH analogues in combination with testosterone (Pogach et al., 1988
) or the anti-androgen flutamide (Kangasniemi et al., 1995
; Meistrich et al., 1995
).
Furthermore, recovery from spermatogenic damage in rats induced by radiotherapy or procarbazine treatment has been shown to be enhanced by treatment with GnRH analogues or testosterone even when administered after the gonadotoxic agent (Pogach et al., 1988; Meistrich and Kangasniemi 1997
; Meistrich et al., 1999
). The mechanisms by which such hormonal manipulation offers protection or enhancement of recovery of spermatogenesis are unclear. Hormonal analysis following irradiation in rats has shown a marked increase in intratesticular testosterone levels and it has been postulated that suppression of the HPG axis promotes multiplication and differentiation of spermatogonia by lowering testosterone concentrations within the testis (Meistrich and Kangasniemi, 1997
).
While there is significant evidence for the success of protection/recovery strategies in rats, clinical studies in man have to date been inconclusive (Johnson et al., 1985; Waxman et al., 1987
; Masala et al., 1997
) and there have been no trials investigating the effects of post-gonadotoxic hormonal suppression. The present study has investigated whether suppression of the HPG axis in men rendered azoospermic by treatment for childhood cancer might restore spermatogenesis, using both semen analysis and testicular biopsy as endpoints.
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Materials and methods |
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Patients
A review of the oncology database at the Royal Hospital for Sick Children, Edinburgh for men rendered azoospermic secondary to treatment for childhood cancer, identified seven men aged 22.2 (1825.3) [median (range)] years. All men were invited to participate in the study regardless of the underlying malignancy or cytotoxic therapy and all seven accepted. The median age at original diagnosis was 10.4 (4.413.3) years with a disease-free survival of 8.4 (3.314.7) years. The underlying malignancies were acute lymphoblastic leukaemia (n = 2), Hodgkin's disease (n = 4) and non-Hodgkin's lymphoma (n = 1). A summary of the patients' diagnoses with details of the gonadotoxic chemotherapy and radiotherapy received is given in Table I.
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Assessment of testicular function
Pubertal maturation was assessed according to the Tanner criteria and testicular volume (ml) was measured using a Prader orchidometer (Tanner and Whitehouse, 1976). The mean value of the two testes was taken to represent the subject's testicular volume. Venous blood samples were collected (20 ml), and LH, FSH and testosterone levels determined using an automated immunoassay analyser (Bayer Immuno 1, Bayer plc., Newbury, Berks, UK). Inhibin B was measured as previously described (Groome et al., 1996
), with the limit of assay sensitivity being 7.8 pg/ml. Semen samples were collected in a room adjacent to the laboratory, by masturbation into sterile wide-mouthed non-toxic containers, following an abstinence period of
48 h. Samples were centrifuged at 3000 g for 30 min and the pellet examined to confirm azoospermia (World Health Organization, 1999
). Seminal plasma was stored at 70°C until assayed for inhibin B (Anderson et al., 1998
). Testicular biopsy under general anaesthetic was undertaken on all patients at the start of the study to exclude obstructive azoospermia. The specimens were fixed in Bouin's fixative and after routine processing and paraffin embedding, sections were cut at 5 µm and examined. A second biopsy of the same testis was performed at the end of the study.
HPG axis suppression
All men underwent a period of suppression of the HPG axis, designed to induce hypogonadotrophic hypogonadism with reduced intratesticular testosterone levels for a period of ~24 weeks, followed by a recovery period of 24 weeks. Following testicular biopsy, subjects were administered depot medroxyprogesterone acetate (DMPA, 300 mg i.m.; Pharmacia and Upjohn, Milton Keynes, UK) and testosterone pellets (4x200 mg s.c.; NV Organon, Oss, The Netherlands). Administration of DMPA was repeated 12 weeks later.
Subjects were reviewed at 6 weekly intervals throughout the 48 weeks of the study for clinical assessment, blood sampling and semen analysis.
Immunohistochemistry of testicular tissue
The objective of the immunohistochemical analysis was to investigate whether or not any germ cells, in any developmental stage, were present in the testes of patients before or after HPG suppressive treatment. This was achieved using immunoexpression of the MAGE-57B antigen and androgen receptor (AR). The MAGE-57B antigen is expressed in early germ cells, strongly in the spermatogonial and weakly in early spermatocytes (Aubry et al., 2001). AR is expressed in the nuclei of all Sertoli cells but not in the nuclei of any germ cells that might be present (Saunders et al., 1996
).
Unless otherwise stated, all incubations were undertaken at room temperature. Sections were deparaffinized in xylene, rehydrated in graded ethanols and washed in water. A temperature-induced antigen retrieval step was required for AR only. The sections were pressure-cooked in 0.01 mol/l citrate buffer, pH 6.0 for 5 min at full pressure, allowed to stand for 20 min, cooled in running tap water and washed twice in (5 min each wash) in Tris-buffered saline [TBS: 0.05 mol TrisHCl, pH 7.4, 0.85% (w/v) NaCl]. Endogenous peroxidase activity was then blocked by immersing sections in 3% (v/v) H2O2 in methanol (both from BDH Laboratory Supplies, Poole, UK) for 30 min, followed by two 5 min washes in TBS. Sections were incubated for 30 min with the appropriate normal serum diluted 1:5 in TBS containing 5% bovine serum albumin (BSA; Sigma-Aldrich Co. Ltd., Poole, Dorset, UK) to block non-specific binding sites. Normal swine serum (NSS) and normal rabbit serum (NRS) (both from Diagnostics Scotland, Carluke, UK) were used for AR and MAGE-57B respectively. Primary antibodies were added to the sections at the appropriate dilution in either NSSTBSBSA (for AR 1:2000: AR N-20, Santa Cruz Biotechnology SC0816, Santa Cruz, CA, USA) or NRSTBSBSA (for MAGE-57B: 1:50, source of antibody) and incubated overnight at 4°C in a humidified chamber. The sections were washed twice in TBS and then incubated for 30 min with anti-rabbit or anti-mouse horse-radish peroxidase-labelled polymer (EnVision: Dako, Ely, UK) for AR and MAGE-57B respectively. Sections were washed twice (5 min each) in TBS and immunostaining was developed using liquid diaminobenzidine (Dako) until staining was optimal, when the reaction was stopped by immersing sections in distilled water. The sections were counterstained with haematoxylin, dehydrated in graded ethanols, cleared in xylene and cover-slipped using Pertex mounting medium (CellPath plc, Hemel Hempstead, UK). As negative controls, slides were processed as above except that the appropriate normal serum was substituted for the primary antibody.
Immunostained sections were examined and a mean of 132 (range 40252) tubular cross-sections were counted for each specimen pre- and post-HPG suppression. The sections were photographed using an Olympus Provis microscope (Olympus Optical, Honduras Street, London, UK) fitted with a digital camera (Kodak DCS330: Eastman Kodak, Rochester, NY, USA). Captured images were stored on a computer (G4; Apple Computer Inc., Cupertino, CA, USA) and compiled using Photoshop 5.0 before printing using an Epson Stylus 870 colour printer (Seiko Epson Corp., Nagano, Japan).
Statistical analysis
Statistical analysis was performed by the Statistical Package for Social Science (SPSS Inc., Chicago, III) version 10.0. For each individual patient, hormone concentrations at the beginning and end of the study were compared using paired t-tests and P < 0.05 was considered significant.
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Results |
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FSH was suppressed to undetectable concentrations during MPAtestosterone treatment for 12 weeks, and remained partially suppressed during the subsequent 12 weeks (Figure 1). Thereafter, there was a gradual rise by weeks 4248 to 19.5 ± 3.6 U/l, which was not significantly different from pretreatment concentrations. LH showed a similar pattern to FSH, with suppression to undetectable concentrations for 12 weeks, followed by gradual recovery to 8.9 ± 1.6 U/l at 48 weeks (Figure 1
). There was no statistically significant difference between LH concentrations pretreatment and at 48 weeks.
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Serum inhibin B concentrations increased from 35 to 60.9 ng/l at 12 weeks in the subject with the highest inhibin B pretreatment, and rose from <15 ng/l to low but detectable concentrations (range 1635 ng/l) in the five other subjects (venous serum reference range: mean 257, 95% CI 231284ng/l). Serum inhibin B concentrations fell towards the end of the study, becoming undetectable in all subjects other than the individual with the highest pretreatment concentration at weeks 4248. This same individual was the only subject with readily detectable seminal plasma inhibin B concentration pretreatment (770 versus <20 ng/l in the others, seminal plasma reference range: mean 2279, 95% CI 6983864 ng/l) (Anderson and Sharpe, 2000). Seminal plasma inhibin B was not determined during MPAtestosterone treatment as the volume of the ejaculate was insufficient. In all subjects, seminal plasma inhibin B concentrations were undetectable at the end of the study.
Semen analysis
All men remained azoospermic throughout the study.
Testicular tissue
Light microscopy
Examination of testicular tissue pre- (Figure 2a) and post- (Figure 2b
) HPG axis suppression indicated complete absence of all germ cells, in contrast to the abundant different germ cell types in the normal seminiferous epithelium of a healthy adult man (Figure 2c
). This was representative of all seven cancer survivors.
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Discussion |
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The success of hormonal treatment to aid recovery of spermatogenesis in rats subjected to chemotherapy is believed to be based on lowering intratesticular testosterone levels. While the prepubertal testis is relatively quiescent, there is a steady turnover of early germ cells, which undergo spontaneous degeneration before maturation is reached (Muller and Skakkebaek, 1983). This relatively low activity, compared with the adult, does not protect the prepubertal testis from the deleterious impact of cytotoxic therapy, as the present data confirm. The slow turnover of germ cells and their subsequent degeneration in the prepubertal testis may be partly due to low levels of intratesticular testosterone, which is required to complete the end-stages of spermatogenesis (Chemes, 2001
). The lack of protection afforded to the prepubertal testis, at a time when testosterone levels are low, would suggest that additional environmental factors play a role in the successful recovery of spermatogenesis in rats and the vulnerability of the prepubertal human testis to cytotoxic therapy. In this regard, our studies with the marmoset, a primate surrogate for man, have demonstrated that activation of testicular cell function occurs well before puberty and is largely gonadotrophin-dependent, but that spermatogonial replication appears to be independent of gonadotrophin stimulation (Kelnar et al., 2002
).
In one study in which cyclophosphamide was administered as immunosuppressive therapy for nephrotic syndrome in adult men, preservation of fertility was achieved via supraphysiological testosterone therapy (Masala et al., 1997). Of 15 men treated with cyclophosphamide, five received testosterone to suppress testicular function before and during the 8 month cycle of chemotherapy. All men were azoospermic or severely oligozoospermic within 6 months of commencing cyclophosphamide. Nine of the 10 men who received cyclophosphamide alone remained azoospermic 6 months after the end of immunosuppressive therapy, whereas sperm concentrations returned to normal in all five of the men who received testosterone therapy. High dose cyclophosphamide is known to be associated with impaired spermatogenesis, which is often temporary, and it is probable that in this study the simultaneous administration of testosterone with cyclophosphamide provided some protection or hastened the recovery of spermatogenesis. It would be interesting to have long-term follow-up data on the 10 patients who received cyclophosphamide-only treatment to enable a direct comparison with the natural history of recovery of sperm production. In contrast, other studies have failed to show similar benefits in humans. For example, suppression of testicular function with a GnRH agonist, alone or in combination with testosterone during gonadotoxic chemotherapy treatment for lymphoma, did not confer any protective benefit or enhance recovery of spermatogenesis (Johnson et al., 1985
; Waxman et al., 1987
). A number of reasons may be considered for the lack of successful outcome in the aforementioned studies. The number of patients and controls studied was small and the cancer therapies variable, in contrast to monotherapy with cyclophosphamide for a non-malignant condition. Treatment regimens may not have been sufficiently gonadotoxic to cause sterility, so no recovery effect could be seen or, conversely, the agents were so gonadotoxic that permanent ablation of all germ cells was induced. Waxman et al. studied the protective effects of a GnRH agonist during the treatment of 20 men with cytotoxic chemotherapy for advanced Hodgkin's disease (Waxman et al., 1987
). Following administration of the GnRH agonist, standard GnRH testing demonstrated adequate suppression of LH, but not of FSH, throughout the chemotherapy treatment. Follow-up assessment of the men after a 3 year interval showed that all remained severely oligozoospermic (Waxman et al., 1987
). In another study, the effect of GnRH agonist administration during combination chemotherapy for advanced lymphoma was evaluated in six patients (Johnson et al., 1985
). By 6 years post treatment, only one patient demonstrated any evidence of spermatogenesis. While the present study explored the delayed suppression aspect of this hypothesis, no human studies have combined pre-chemotherapy suppression with continued suppression for a significant length of time following chemotherapy.
A number of steroid hormone combinations have been used to suppress the HPG axis in rats and successfully restore spermatogenesis after chemotherapy, including MPA in combination with testosterone (Velez de la Calle et al., 1990; Jegou et al., 1991
). Low dose testosterone, MPA or GnRH analogues alone have been shown to stimulate recovery of spermatogenesis in rats following sterilization with radiotherapy. However, the addition of testosterone to GnRH analogues may reduce the effectiveness of GnRH analogues (Shetty et al., 2000
). The combination of testosterone with MPA may also have a reduced effect compared with either agent alone, although this combination results in a profound reduction in intratesticular testosterone concentrations in men (McLachlan et al., 2002
). Although further study is warranted, the appropriate choice of hormone suppression will require careful consideration. Long-acting gonadotrophin analogues, such as goserelin, have been shown to be ineffective at suppressing FSH long term in normal men, with recovery of FSH and resumption of spermatogenesis occurring within 23 months (Behre et al., 1992
; Bhasin et al., 1994
).
Inhibin B mediates non-steroidal negative feedback from the testes, reflecting the number of sperm produced and regulating FSH secretion (Andersson et al., 1999; Peterson et al., 1999; Anderson and Sharpe, 2000
; Kolb et al., 2000
). Inhibin B secretion in the adult requires the presence of germ cells (Andersson et al., 1999
). Inhibin B concentrations were barely detectable in the azoospermic patients, despite the preservation of Sertoli cells. This provides further evidence for an essential role of the germ cellSertoli cell interaction in the production of inhibin B and confirms the value of inhibin B as a non-invasive marker of spermatogenesis following cytotoxic therapy. Inhibin B was also undetectable in seminal plasma in most subjects, as previously found in men with azoospermia of other aetiologies (Anderson et al., 1998
).
Although the gonadotoxic effect of chemotherapy depends upon dosage and drugs administered, and radiotherapy-induced damage upon field of irradiation and dose received, it is difficult to reliably predict the extent of testicular damage and which azoospermic patients may show recovery of spermatogenesis. Our study population comprised an unselected group of seven men rendered azoospermic secondary to treatment for childhood cancer. Testicular biopsies from all seven patients demonstrated complete absence of spermatogonia, yet survival of stem cells is a prerequisite for endocrine restoration of spermatogenesis. It was felt to be unethical to exclude men from the trial on the basis of Sertoli cell-only biopsy specimens for several reasons. Testicular volume in these men was markedly reduced and thus it was justified to take only a small piece of testicular tissue, to eliminate any impact which a reduction in testicular tissue may have on Leydig cell numbers. Small islands of spermatogonia may be present but in our study were absent from the biopsied tissue. Survival of germ cells following apparently sterilizing chemotherapy is evident from a number of studies. Temporary azoospermia and late recovery of spermatogenesis following chemotherapy have been reported, indicating the survival of stem cells (Viviani et al., 1985; Wallace et al., 1991
; Pryzant et al., 1993
), although permanent azoospermia tends to follow procarbazine and alkylating agent-based regimens, typical of treatment for Hodgkin's disease (Whitehead et al., 1982
; da Cuhna et al., 1984; Bramswig et al., 1990
). Similar histological findings have been reported in other studies following treatment for Hodgkin's disease with procarbazine-based regimens (Chapman et al., 1979
; Charak et al., 1990
). With advances in assisted reproduction techniques, the development of testicular sperm extraction (TESE) combined with ICSI offers potential for paternity in these young men. Chan and co-workers report the use of TESEICSI to retrieve sperm from men with long-standing azoospermia and achieve a pregnancy (Chan et al 2001
). Seventeen men, median age (range) 37.4 (2854) years, had undergone sterilizing chemotherapy treatment 16.3 (634) years previously. Of the 17 men, 13 demonstrated Sertoli cell-only on biopsy and the remaining four were described as having hypospermatogenesis. Using microdissection TESE techniques, sperm retrieval was achieved in seven subjects, three (43%) of whom demonstrated Sertoli cell-only on testicular histology and four (57%) with hypospermatogenesis. The seven subjects underwent nine TESE combined with ICSI procedures resulting in a clinical pregnancy in three (33%) and a live birth in two (22%). These encouraging results suggest that microscopic visualization of the seminiferous tubules may enable identification of areas of continued spermatogenesis within the testis and sperm retrieval using microdissection techniques. This reiterates the importance of not excluding men from hormone restoration clinical trials or from assisted reproduction techniques on the basis of a Sertoli cell-only testicular biopsy.
Although small islands of germ cell spermatogonia may exist in the sections of testes that were not biopsied, it is likely that in our patients, the severity of the cytotoxic-induced germ cell loss is such that recovery of spermatogenesis is simply impossible. This does not exclude the possibility that earlier intervention and HPG axis suppression might have been beneficial. However, it seems more probable that HPG axis suppression to restore spermatogenesis may be more successful in patients in whom the testicular insult is less severe and in whom there is some preservation of spermatogonial stem cells.
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Acknowledgements |
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Notes |
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References |
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Submitted on November 6, 2001; accepted on March 15, 2002.