2005 © Oxford University Press
Hormonal Approaches to Preservation and Restoration of Male Fertility After Cancer Treatment
Affiliation of authors: Department of Experimental Radiation Oncology, The University of Texas M.D. Anderson Cancer Center, Houston
Correspondence to: Gunapala Shetty, Department of Experimental Radiation Oncology, The University of Texas M.D. Anderson Cancer Center, Houston, TX 77030 (e-mail: sgunapal{at}mdanderson.org).
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ABSTRACT |
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 Top Abstract IntroductionEFFECTS OF CYTOTOXIC AGENTS...HORMONAL ATTEMPTS AT PRESERVING...References |
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It is important to develop methods to prevent or reverse the sterility caused by chemotherapy or radiation therapy for cancer in men. Using a rat model, we have shown that infertility after testicular exposure to moderate doses of radiation and some chemotherapeutic agents occurs as a result of the inability of spermatogonia to differentiate. There is evidence that this phenomenon also occurs in men. Spermatogenesis and fertility can be restored in rats following treatment with radiation or some chemotherapeutic agents by suppressing testosterone with gonadotropin releasing hormone (GnRH) agonists or antagonists either before or after the cytotoxic insult. However, species differences exist in the testicular response to radiation or GnRH antagonist therapy, so rescue protocols that work in rodents do not work in nonhuman primates. The applicability of this procedure to humans is still largely unknown. In rodents, suppression of testosterone with GnRH analog treatment also appears to enhance the success of spermatogonial transplantation—an option when all stem cells are killed by cytotoxic therapy.
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INTRODUCTION |
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TopAbstractIntroductionEFFECTS OF CYTOTOXIC AGENTS...HORMONAL ATTEMPTS AT PRESERVING...References |
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For young men who have cancer, the success of treatment with regimens that are toxic to testicular function has made infertility an important problem. When the cancer is controlled, quality of life, which often includes the ability to have a normal child, becomes a major issue. In the United States, 17 000 men ages 15–45 years old are diagnosed each year with Hodgkin disease, lymphoma, bone and soft tissue sarcomas, testicular cancer, or leukemia (1). Of these men, over 3000 are treated with doses of alkylating agents, platinum drugs, or radiation that are sufficient to induce prolonged azoospermia.
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EFFECTS OF CYTOTOXIC AGENTS ON TESTICULAR FUNCTION IN MEN |
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TopAbstractIntroductionEFFECTS OF CYTOTOXIC AGENTS...HORMONAL ATTEMPTS AT PRESERVING...References |
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The testis consists of the seminiferous (or germinal) epithelium arranged in tubules and endocrine components (testosterone-producing Leydig cells) in the interstitial region between the tubules. The seminiferous tubules contain the germ cells, which consist of stem and differentiating spermatogonia, spermatocytes, spermatids, and sperm, and the Sertoli cells, which support and regulate germ cell differentiation. Among the germ cells, the differentiating spermatogonia proliferate most actively and are extremely susceptible to cytotoxic agents. In contrast, the Leydig and Sertoli cells, which do not proliferate in adults, survive most cytotoxic therapies. These cells may, however, suffer functional damage. Frequently, following cytotoxic therapies, germ cells appear to be absent, and the tubules contain only Sertoli cells. This could be a result of the killing the spermatogenic stem cells, the loss of the ability of the somatic cells to support the differentiation of a few surviving stem cells, or a combination of the two.
The eventual recovery of sperm production depends on the survival of the spermatogonial stem cells and their ability to differentiate. If treatment is limited to the cytotoxic agents that do not kill stem spermatogonia or block their differentiation, normospermia is usually restored within 3 months after the cytotoxic therapy. However, if agents that kill stem spermatogonia or affect differentiation are used, longer periods of azoospermia ensue. Surviving stem cells can remain in the testis but fail to differentiate into sperm for several years after cytotoxic insult, so delayed recovery is possible (2). At lower doses of these agents, recovery to normospermic levels can occur within 1–3 years, but at higher doses, azoospermia can be more prolonged or even permanent.
The loss of germ cells has secondary effects on the hypothalamic-pituitary-gonadal axis. Inhibin secretion by the Sertoli cells declines and, consequently, serum follicle-stimulating hormone (FSH) levels rise. Testicular blood flow is reduced, resulting in less testosterone being distributed into the circulation (3). Therefore luteinizing hormone (LH) levels increase to maintain constant serum testosterone levels. The reduction in the testis size and increased LH levels also contribute to an increased concentration of testosterone within the testis.
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HORMONAL ATTEMPTS AT PRESERVING OR RESTORING FERTILITY |
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TopAbstractIntroductionEFFECTS OF CYTOTOXIC AGENTS...HORMONAL ATTEMPTS AT PRESERVING...References |
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Preservation and Restoration of Spermatogenesis and Fertility by Gonadotropin Releasing Hormone Analog Treatment in Rats
Spermatogenesis in Lewis, Brown-Norway F1 hybrid rats was found to be as sensitive to cancer therapeutic agents as it was humans and appeared to be a good model to study sensitive targets of loss of spermatogenesis after cancer treatment. In these rats, even moderate doses of radiation (4) or procarbazine (5) depleted all the germ cells except stem spermatogonia. These surviving spermatogonia continuously proliferated after cytotoxic exposure but underwent apoptosis when they were ready to differentiate (6)—a situation that is sometimes observed in humans. In contrast, in mice, surviving spermatogonia immediately repopulate the testis after cytotoxic exposure; hence, mice are not good models to study the effect of cytotoxic agents on stem spermatogonia and their differentiation in human. However, there are genetic models for defects of spermatogenesis in mice that mimic the phenotype observed in the rats exposed to cytotoxic agents and that could be used to investigate the mechanisms of the block in spermatogonial differentiation (7).
Examination of the hormonal status of rats after cytotoxic treatment indicated that the failure of spermatogonial differentiation is not a result of insufficient stimulation by gonadotropins or testosterone. Both gonadotropin levels were significantly elevated after the rats were treated with radiation (4) or procarbazine (5). Whereas serum testosterone levels were unchanged, intratesticular concentration were two- to fourfold normal (5,6). Androgen receptors remained present in the somatic cells (O. U. Bolden-Tiller, unpublished data), and FSH receptor messenger RNA (mRNA) expression in the Sertoli cells was unchanged (8).
We hypothesized that in these situations, testosterone or FSH might actually be inhibiting spermatogonial differentiation. On the basis of this hypothesis, we suppressed testosterone and FSH by treating the rats, after cytotoxic insult, with gonadotropin releasing hormone (GnRH) agonists or antagonists. When GnRH-analog treatment (9) was started immediately after irradiation, or procarbazine administration (5), the numbers of differentiated germ cells dramatically increased. However, because GnRH-analog treatments suppress testosterone, which is required for spermatid differentiation, spermatogenesis proceeded only to the round spermatid stage—no sperm were produced. Nevertheless, when additional time without further GnRH-analog treatment was allowed before the rats were killed, all tubules showed almost complete spermatogenic recovery, sperm counts increased, and there was a significant increase in the fertility of the GnRH-treated rats (10). Other researchers have shown that GnRH-agonist administration after busulfan treatment also stimulated the recovery of spermatogenesis, supporting the generality of this phenomenon (11).
Even after the block to spermatogonial differentiation had developed, 10–20 weeks after irradiation, administration of GnRH analogs still overcame the block and restored spermatogenesis (6,10), although there were indications that delayed treatment might not be as effective as immediate treatment.
The maintenance of spermatogenesis in irradiated rats after GnRH analog treatment is stopped depends on the toxicant dose and time of initiation and duration of the hormone treatment. Normal fertility can be restored by GnRH treatment after irradiation, although that may depend on the initiation of the GnRH analog treatment soon after a toxicant exposure that is not too severe.
The above studies involved suppression of testosterone and FSH after the cytotoxic insult. Earlier studies had employed suppression of hormones before and during the cytotoxic insult, with the idea that these treatments would protect the survival of stem cells. In fact, such pretreatment of rats does enhance the recovery of spermatogenesis after irradiation (12) or procarbazine treatment (13). Hormonal treatment given before the cytotoxic treatment does not protect the survival of stem cells but, rather, enhances the ability of the testis to maintain the differentiation of type A spermatogonia after the exposure (14).
Testosterone Inhibits Spermatogenesis by Blocking Spermatogonial Differentiation
It was surprising that testosterone or FSH, which are required to support normal spermatogenesis, appeared to inhibit this process after cytotoxic treatment. Therefore, we performed studies confirming that testosterone was indeed inhibitory.
In irradiated rats treated with GnRH antagonist, testosterone dose-dependently reduced the GnRH antagonist–stimulated spermatogonial differentiation (15,16). Further, the stimulatory action of low-dose testosterone alone, which reduces intratesticular testosterone concentrations, was also reduced with increasing doses of testosterone, which increased both intratesticular and serum testosterone concentrations. The inhibition of spermatogonial differentiation by testosterone was further confirmed by showing that flutamide reversed the inhibition induced by exogenous testosterone in GnRH antagonist–treated, irradiated rats (15). These results show that GnRH analogs are restoring spermatogonial differentiation primarily by suppressing testosterone, ruling out possibility of a major direct effect of the GnRH analogs on spermatogenesis. Further support for our hypothesis that it is indeed testosterone acting through the androgen receptor, and not a nonandrogenic metabolite of testosterone, that inhibits spermatogonial differentiation was obtained by showing that various androgens, including 5
-dihydrotestosterone (a nonaromatizable androgen), 7-methyl-19-nortestosterone (a non-5-reducible androgen), and methyltrienolone (a nonmetabolizable androgen) also suppressed spermatogonial differentiation in GnRH antagonist–treated irradiated rats (16).
Because FSH was also suppressed during recovery of spermatogenesis, we tested whether FSH directly inhibited spermatogenic recovery in irradiated rats by giving exogenous recombinant hFSH to irradiated rats after suppressing levels and action of testosterone with a combination of GnRH antagonist and flutamide. FSH was also found to have a small inhibitory effect on spermatogenic recovery compared to the large inhibitory effect of testosterone. Interestingly, estradiol did not inhibit recovery (16) but, rather, stimulated it, in part by suppressing testosterone levels (17).
Regimens for Spermatogenic Recovery
Although suppression of intratesticular testosterone is required for spermatogenic recovery after irradiation, maintenance of peripheral testosterone levels is important for maintenance of male functions. We compared the spermatogenic stimulatory effects in irradiated rats of the testosterone suppressors, such as GnRH antagonist, medroxyprogesterone acetate (MPA), and estradiol, each given in combination with testosterone (Fig. 1). MPA was a less effective stimulator of spermatogenic recovery than estradiol or GnRH-antagonist alone, as MPA also acts as a weak androgen. When given in combination with maintenance doses of testosterone, estrogen appears to be most effective to stimulate such recovery (17). We suggest that progestins with estrogenic but minimal androgenic activity may be optimal for stimulating recovery of spermatogenesis when given with maintenance doses of testosterone.
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Studies in Human and Nonhuman Primates
Because stem spermatogonia are sometimes present in regions of the testes of some cancer patients during prolonged periods of iatrogenic azoospermia, their recovery could possibly be stimulated. However, only one (18) out of seven clinical trials (19–24)(Table 1) has been able to demonstrate protection of spermatogenesis in humans by hormone treatment both before and during cytotoxic therapy (25). The failure of some of the human trials, in which testosterone was combined with a GnRH analog or MPA, could be explained by the counteractive effect of testosterone supplementation observed in the studies on rats and the low effectiveness of MPA in stimulating recovery (Fig. 1). However, in view of the studies on rats (Fig. 1), it was surprising that the one successful study was done using testosterone alone to protect the ability to recover spermatogenesis after therapy (18). One of the factors that might have contributed to the successful outcome was that the chemotherapy was cyclophosphamide treatment for nephrotic disorders, which may have produced a specific amount of differentiation block and only moderate stem cell kill, whereas the other studies on cancer patients included almost none that were treated with cyclophosphamide, despite the fact that it is used widely in cancer treatment. One attempt to restore spermatogenesis by steroid hormone treatment after cytotoxic therapy was unsuccessful (26); however, the doses of cytotoxic therapy were very high, and the hormonal suppression was given many years after the anticancer treatment. Moreover, MPA combined with testosterone, used in that study as the hormone suppressor, was found to be a relatively ineffective regimen in rats (Fig. 1).
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Recently, two studies were conducted in nonhuman primates to test whether hormone suppression by GnRH antagonist can protect from radiation-induced spermatogenic damage. In the first study (27), the macaques were exposed to testicular irradiation (4 Gy) on day 29 of GnRH treatment, and the hormone treatment continued for 1 week more. In the second study (28), GnRH-antagonist treatment was started immediately after testicular irradiation (7 Gy) and continued for 12 weeks. It was discouraging that in both cases GnRH-antagonist treatment failed to enhance spermatogenic recovery, indicating that there are important differences in this process between rats and primates. Despite negative results in monkeys, it is possible that the sensitivity of testis to radiation is different between monkeys and humans, as a radiation-induced block in spermatogenic cell differentiation was not apparent in monkeys (29) but has been observed in humans (30).
Thus, despite the success in restoring fertility by hormone treatment of rats rendered azoospermic by chemotherapy and radiation, the application of these procedures to humans is uncertain. It is important to understand the molecular mechanisms and the specific cells involved in the androgen inhibition of spermatogonial differentiation after cytotoxic exposure. This will be useful in formulating treatment regimens that may be successful in humans. Such a pursuit will also have relevance in the restoration of fertility in men rendered infertile as a result of occupational cytotoxic exposures. Hormonal suppression facilitated spermatogenic recovery in rats treated with a pesticide dibromochloropropane (31) that has caused prolonged oligo- and azoospermia in men (32).
Application to Spermatogonial Transplantation
Because some anticancer agents may kill all the stem cells, alternative strategies are required to restore spermatogenesis. One of the options would be transplantation of spermatogonia cryopreserved before the cytotoxic treatment; clinical trials are underway (33). In rodents, suppression of testosterone with GnRH analogs also appears to enhance the success of such transplantation (34,35). The GnRH analogs might be acting on the supporting (primarily Sertoli) cells to enhance the survival and differentiation of the transplanted cells. Thus, hormonal treatment might be an important, and possibly necessary, adjunct to the attempts at spermatogonial transplantation in humans.
It is hoped that further research will bridge the gap between species and permit development of methods to preserve or restore fertility in men receiving cancer treatment or similar cytotoxic exposures.
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