Cancer and male infertility

BaillieÁre's Clinical Endocrinology and Metabolism Vol. 14, No. 3, pp. 453±471, 2000

doi:10.1053/beem.2000.0091, available online at http://www.idealibrary.com on

9 Cancer and male infertility Aleksander Giwercman Senior Consultant University Department of Urology, MalmoÈ University Hospital, SE 20502 MalmoÈ, Sweden

Peter Meidahl Petersen Registrar University Department of Oncology, Herlev University Hospital, DK 2730 Herlev, Denmark

An increasing proportion of boys and young men with cancer will survive their disease and desire fertility. Unfortunately, the cancer treatment, and in some cases the malignant disease itself, may have a negative and permanent impact on the individual's fertility potential. This e€ect is highly dependent on the type and dose of therapy as well as the age at which it has been given. Basic knowledge in this ®eld is necessary to enable oncologists and fertility specialists to counsel these patients about their fertility prospects and, if appropriate, advise them to take precautions (e.g. the cryopreservation of semen) to safeguard their fertility. Another aspect of the relationship between cancer and infertility is the possibility that men with testicular dysfunction may have an increased risk of testicular cancer. Screening for early testicular malignancy may therefore be advisable in some groups of men with poor semen quality. Key words: cancer; testicular cancer; carcinoma in situ; testis; fertility; male infertility; semen quality; testosterone; cryopreservation; cytotoxic drugs; radiotherapy; orchidectomy.

An increasing number of men who have survived a malignant disease are being referred because of infertility problems. The main reason for this is the improved prognosis for many childhood cancers and malignancies of young adulthood, for example testicular germ cell cancer (TGCC) and lymphoma. One in 650 children will develop cancer by the age of 15 years, and 50±60% will be cured. Today 1 in 1000 young adults is a survivor of childhood cancer.1 Testicular cancer and malignant lymphomas in young adulthood are even more frequent, the cure rate for TGCC being close to 95%.2 Medical sta€ taking care of these young cancer patients should possess the necessary knowledge to give these men realistic counselling and proper treatment regarding their fertility potential, as well as to ensure that precautions such as semen cryopreservation have been considered prior to cancer therapy. The relationship between cancer and male fertility has, however, some other intriguing aspects. There may be a pathogenetic relationship between male gonadal function and malignancies such as testicular cancer. This relationship may have clinical implications for de®ning high-risk groups for such malignancies and also provide information about the mechanisms behind at least some malignant diseases. The aim of 1521±690X/00/030453‡00 $35.00/00

c 2000 Harcourt Publishers Ltd. *

454 A. Giwercman and P. M. Petersen

this chapter is to summarise the clinical and biological knowledge related to male fertility and cancer.

TESTICULAR CANCER AND MALE INFERTILITY Most attention ± in relation to fertility potential ± has been given to testicular cancer. There seems to be a very speci®c link between TGCC per se and gonadal function. For other forms of cancer in young males, the relationship to fertility potential is, however, less well established3 (Figure 1). 150

Sperm concentration (106/ml)

125

100

75

50

25

0 Before orchidectomy

After orchidectomy

After chemotherapy

Malignant lymphoma

Figure 1. Sperm concentration in testicular cancer patients prior to orchidectomy, after the orchidectomy and after cisplatin-based chemotherapy, as compared to men with newly diagnosed malignant lymphoma and 142 men from general Danish male population (reference group). The boxes indicate 25±75% interval. The thick bars correspond to median value and the thin bars to the range. The shaded area gives the 25±75% interval for the reference group.

Biological implications Origin of TGCC There is increasing evidence indicating that TGCC arises in early fetal life, at about the 8th±10th week of development, which coincides with the di€erentiation of the indi€erent gonad to the testis or ovary.4 The stage of invasive testicular cancer is preceded by so-called carcinoma-in-situ of the testis (CIS), a pre-invasive stage in which the malignant germ cells are limited to the seminiferous tubules.5 It has been suggested that the CIS cells are fetal gonocytes that have undergone malignant transformation. The CIS cells seem to stay dormant in the gonad until the

Cancer and male infertility 455

post-pubertal period, when ± probably under the in¯uence of sex steroids or gonadotrophins ± they divide and develop into an invasive tumour.6 The evidence for the fetal origin of CIS cells comes from di€erent sources. Morphologically, at both the light microscopic and electron microscopic levels, CIS cells have many similarities to fetal germ cells.4 Furthermore, several of the immunohistochemical markers of CIS cells, including the proto-oncogene c-kit and the marker of embryonal carcinoma cell line 2102Ep, TRA-1-60, are expressed in fetal germ cells, with a peak at around fetal weeks 8±12.7,8 The strongest evidence for a prenatal origin of TGCC comes, however, from epidemiological data: 1. Boys born during World War II have been found to have a lower risk of TGCC than birth cohorts before and after this war.9 2. The risk of testicular cancer seems to be strongly correlated to the year of birth.10 3. Several prenatal factors, including birthweight, parity, and the weight as well as the age of the mother, are associated with an increased risk of TGCC.11,12 Relationship between TGCC and other gonadal abnormalities Whereas it seems rather evident that testicular malignancy arises during fetal life, it is still not completely clear which aetiological factors are responsible for the initiation of the malignant process. It has been proposed that environmentally derived so-called `endocrine disrupters' with an oestrogenic or anti-androgenic e€ect are responsible not only for development of testicular malignancy, but also for some cases of male infertility and congenital malformations of the male genital organs, such as cryptorchidism and hypospadias.13 De®nitive evidence for this hypothesis is still lacking, however. Patients with androgen insensitivity, causing a relative predominance of oestrogen action because of a defect of the androgen receptor and the conversion of testosterone to oestradiol, have a risk of testicular malignancy of approximately 25%.14 In animal studies, prenatal exposure to oestrogenic compounds may cause malformation of the male genital organs and an impairment of fertility.15 Unfortunately, the relationship between hormonally active compounds and germ cell malignancy cannot be con®rmed experimentally since good animal models for human testicular cancer are lacking. Studies on gonadal function in men with TGCC have shown the following: 1. There is an impairment of sperm production. Sperm output in men with TGCC is only 25±30% that of a control group of normal men or of patients with newly diagnosed lymphoma16 (Figure 1). 2. Twenty-®ve per cent of contralateral testes in men with unilateral testicular cancer have qualitative disturbances of spermatogenesis, 5±6% harbouring CIS.17 3. Reduced fertility potential being encountered even prior to the diagnosis of cancer. Compared with men in the general population, patients with TGCC have on average 0.7 fewer children, and the proportion of subfertile men is signi®cantly higher in the patient group.18 4. Circulating levels of basal as well as human chorionic gonadotrophin (hCG) stimulated testosterone were found to be signi®cantly lower in men in whom the testis was removed because of cancer than in patients orchiectomized for a benign disease.19 Thus, clinical data con®rm the association between an impairment of spermatogenesis and Leydig cell function in men with TGCC, although proof of a directional causal relationship is still lacking.

456 A. Giwercman and P. M. Petersen

Geographical and racial di€erences in testicular function Epidemiological studies have provided further evidence for the link between fertility potential and germ cell cancer. An inverse correlation has been found between the incidence of TGCC and sperm concentration in a given population. Danish men have a TGCC risk ®ve times higher than that seen in Finland.20 On the other hand, Finnish males were found to have up to an 100% higher sperm concentration compared with corresponding groups of Danish males.21 Although it has been suggested that environmental factors are important in testicular cancer and male infertility, genetic aspects are undoubtedly also of importance. The risk of testicular malignancy is much lower among blacks than in Caucasian men.22 Sex ratio in the o€spring of cancer patients Testicular cancer has not only been found to be associated with a decreased fertility of the patient: the sex ratio of the o€spring is also apparently changed, from the normal 51.3% of boys and 48.7% of girls to 49% to 51% respectively.23 A decreased male-tofemale ratio was also found among the o€spring of men with poor fertility and after the Seveso accident, which was associated with heavy pollution with the hormonally active compound dioxin.24 The sex ratio of the o€spring has been suggested to be an indicator of male reproductive potential.25 In contrast to TGCC, cancer of the prostate has been reported to be more frequent among fertile men than infertile individuals.26 The sex ratio of the o€spring of patients with prostate cancer is remarkable since 57% boys and only 43% girls have apparently been reported.27 These epidemiological data might indicate that the two forms of genital cancer represent endocrinologically opposite scenarios, an oestrogenic predominance predisposing to testicular cancer and a relatively high androgenic level being associated with a higher risk of the cancer of the prostate. However, these associations need to be con®rmed. In summary, testicular germ cell cancer seems to arise during early fetal life as preinvasive CIS. Hormonal factors with an oestrogenic or anti-androgenic e€ect may be important in the aetiology of this malignancy and its link with testicular dysfunction, including infertility. Clinical implications Screening for testicular malignancy among infertile males The only prospective study of men from infertile couples failed to show any increased risk of testicular CIS. In this study, 207 men were selected for biopsy because they had a sperm concentration below 20 million/ml in two consecutive samples or below 10 million/ml in at least one sample. None of them exhibited CIS.28 Males from infertile couples, however, represent a wide range of pathophysiological mechanisms, from di€erent types of dysfunction of the accessory sex glands to the most severe forms of testicular dysfunction.29 From a theoretical point of view, only the latter condition is expected to be associated with an increased risk of testicular malignancy. Impalpable testicular tumours can be disclosed by means of scrotal ultrasound. Since it is well established that men with a history of cryptorchidism have a 5±10 times increased risk of testicular malignancy30, scrotal ultrasound may be recommended in such patients referred for infertility. Some studies have also indicated that a very irregular echo pattern31 or microcalci®cations32 on ultrasound might be indicative of

Cancer and male infertility 457

Figure 2. Carcinoma-in-situ of the testis visualized by immunohistochemical staining with the monoclonal antibody M2A. Tubule with CIS cells marked with `C'. `N' indicates a tubule without malignancy. Note the positive immunohistochemical reaction on the surface of CIS germ cells. Stieve's ®xative. 4 mm section.

testicular CIS. Thus, scrotal ultrasound may be considered in high-risk patients prior to deciding whether or not a biopsy should be performed; it has even been suggested as routine in the management of infertile males.33 Patients with non-obstructive azoospermia referred for the surgical extraction of spermatids prior to intracytoplasmic sperm injection (ISCI) have an increased risk of testicular malignancy, and some of the tissue should be saved for histological evaluation.34 The diagnosis of CIS of the testis can be facilitated by use of any of the known immunohistochemical markers, for example c-kit, TRA-1-60, P1AP, M2A or 43-9F7,30 (Figure 2).

EFFECT OF CANCER TREATMENT ON GONADAL FUNCTION ± GENERAL ASPECTS The question of gonadal function in men treated for cancer diseases has two major issues: fertility potential and androgen production. Gonadal function may be a€ected not only by the impact on the testis itself, but also indirectly via an impairment of hypothalamo-pituitary hormone secretion, for example as a consequence of cranial irradiation or brain surgery. Furthermore, social, psychological and sexual factors, general performance status, and so on may also have an impact on reproductive capacity. The following section focuses on the e€ects of cancer treatment on testicular function.

458 A. Giwercman and P. M. Petersen

E€ect of radiotherapy Spermatogenesis Experimental studies have shown that testicular irradiation is harmful to germinative cells at quite low dose levels, and at higher doses may also cause an impairment of Leydig cell function. The most sensitive cells are the DNA-synthesizing type A spermatogonia. Irreversible damage to stem cells occurs after a single dose of 4 Gy in rats and after 7 Gy in mice. Radiation-induced disruption of spermatogenesis at doses of less than 0.50 Gy is caused by mitotic inhibition among the spermatogonia, whereas doses above 0.50 Gy kill di€erentiating type A spermatogonia.35 Later stages of germ cells are much more resistant to irradiation, the LD50 of elongated spermatids being more than 15 Gy.36 The time to recovery is dose dependent, showing a linear correlation between dose and logarithm of time until the recovery of fertility.35 Fractionated radiation was shown in animal studies to cause more pronounced stem cell killing than single doses.37 The e€ects of single-dose irradiation on spermatogenesis in healthy men are well known. Irreversible azoospermia may result when the testicular dose exceeds 6±8 Gy.38 Histological investigations have shown that spermatogonia are the most radiosensitive germ cells in men, showing a dose-dependent reduction in the range 0.1±6.0 Gy, while no deleterious e€ect on spermatocytes was seen until 8.0 Gy.38 In cancer treatment, however, radiotherapy is given in fractionated schedules, which may be more toxic to the germ cells than an otherwise bio-equivalent dose given as a single dose. In cancer patients, the risk of a persistent impairment of spermatogenesis and the time to recovery of spermatogenesis were found to be dose dependent in the range 0.2±3 Gy.39 Most of the data are based on men with TGCC; extrapolation to other patient groups must be done with caution because patients with testicular cancer may be more vulnerable to the harmful e€ects of irradiation as a result of a pre-irradiation impairment of spermatogenesis. Men treated for testicular cancer with infradiaphragmatic irradiation will receive scattered irradiation (approximately 0.5 Gy) on the residual testis despite a gonadal shield, while the testicular dose has been calculated to be 1.7 Gy in patients treated without a shield but with testis drawn outside the irradiated ®eld.40,41 The probability of and time to recovery were dose-dependent. All patients who received a dose of irradiation below 1.5 Gy had some degree of recovery of spermatogenesis. A complete recovery of spermatogenesis was seen in most patients who received a total dose below 1 Gy. Recovery to the pre-treatment level was also dose dependent, and overall recovery to the pre-treatment level of sperm count was seen in 14% of the patients 5 years after treatment and in 49% 9 years after treatment.40,41 Leydig cell function A dose-dependent impairment of Leydig cell function, with increased luteinizing hormone (LH) values but unchanged testosterone measurements was seen in patients who received a testicular dose above 0.5 Gy.39 This increase in LH values was permanent in patients who had received a dose above 20 Gy but not in patients treated with a lower dose.39 Testicular radiotherapy with doses of 30 Gy given to the contralateral testis of men with TGCC was shown to imply a persistent impairment of Leydig cell function with a

Cancer and male infertility 459

decreased level of testosterone.42 In patients irradiated for CIS in the contralateral testis, the basal as well as the hCG-stimulated testosterone level decreased, and the concentration of LH increased, after 20 Gy radiotherapy given as 10 fractionated doses.43 Leydig cell toxicity was greater in the ®ve patients who were pre-pubertal at the time of treatment than in the adult men who received fractionated radiotherapy with a total dose of 27.5±30.0 Gy.44 E€ect of chemotherapy on spermatogenesis The e€ect of chemotherapy on testicular function ± including the probability of recovery and the length of time preceding the recovery of spermatogenesis ± depends on the type and dosage of the agents used. Cytotoxic drugs exert their e€ect on spermatogenesis by eradicating or reducing the number of germ cells, in some cases even by the ®brosis and hyalinization of the interstitial tissue. Animal studies have shown that the most sensitive stage of germ cells is that of the di€erentiating spermatogonia and that only 5-¯uorouracil killed spermatocytes in doses that did not kill di€erentiating spermatogonia.45 In one study, cisplatinum was the only agent that killed spermatids.45 The mechanisms of cytotoxicity are listed in Table 1, and from our knowledge of the mechanism of cytotoxicity, it is evident that most of these agents are cell cycle speci®c, the cells showing DNA synthesis and mitosis being the most sensitive. Alkylating agents Alkylating agents are among the most gonadotoxic agents, treatment with chemotherapy regimens containing alkylating agents always being associated with some risk of permanent sterility. Cyclophosphamide was shown to cause dose dependent impairment of spermatogenesis when given intravenously in series reporting on the treatment of cancer such as malignant lymphoma.46 Toxicity is clearly correlated to the cumulative dose, while the e€ect of the peak dose is unclear. The risk of permanent infertility is shown to be increased after dosages of more than 7.5±9.5 g/m2, only 10±20% of patients returning to a normozoospermic level even after 7 years of follow-up.46,47 There is, however, much individual variation in susceptibility, and a recovery of spermatogenesis was seen in some patients after a much higher dose, especially when cyclophosphamide was given orally in daily dosage.48 The tumour-active agent is identical in cyclophosphamide and the newer agent iphosphamide, which, because of a di€erence in kinetics, is less haematotoxic than cyclophosphamide. There are, however, no comparative data with respect to testicular toxicity. Another well-known alkylating agent with a gonadotoxic e€ect is nitrogen mustard, which is still commonly used as a part of combination chemotherapy (MOPP ± nitrogen Mustard, Oncovin [vincristine], Procarbazine and Prednisone) in patients with Hodgkin's disease.49 Melphalan was shown to cause severe gonadotoxicity in men treated for malignant lymphomas.50 Melphalan and BCNU exhibited severe acute- and long-term spermatogenetic toxicity when included in high-dose regimens as conditioning for autologous bone marrow transplantation.51 Platinum analogues Cisplatin-based chemotherapy induces azoospermia in all patients during treatment, the recovery of spermatogenesis beginning between the second and fourth year after

460 A. Giwercman and P. M. Petersen Table 1. Examples of gonadal toxicity of cytotoxic agents. Class

Agent

Mechanism of action

Plant derivatives

Vincristine Vindesine Etoposide Teniposide Taxol

Inhibition of formation of microtubules

Antibiotics

Dactinomycin Doxorubicin/ 4-epi-doxorubicin Daunorubicin Idarubicin Mitramycin Mitomycin C Bleomycin

Anti-metabolites

Inhibition of topoisomerase II activity Inhibits function of microtubules

Gonadal toxicity ‡ ‡ ‡ ‡ ?

Binding to DNA causing inhibition of ? RNA synthesis Triggering of topoisomerase II-dependent ‡ DNA Fragmentation/formation of free radicals ‡ ‡ Inhibition of RNA synthesis ? Alkylating and cross-linking of DNA ? Single- and double-strand breaks in DNA ‡

Methotrexate 5-Fluorouracil 5-Fluorodeoxyuridine 6-Mercaptopurine 6-Thioguanine Cytarabin 5-Azacytidine Hydroxurea Fludarabine

Anti-folate Pyrimidine analogues

Alkylating agents

Cyclophosphamide Ifosfamide Melphalan Busulfan CCNU BCNU Streptozotocin Chlorambucil

Adding alkyl groups to DNA altering DNA structure/function

‡‡‡ ‡‡ ? ‡‡ ‡‡ ‡‡ ‡‡ ? ‡‡

Miscellaneous

Dacarbazine

Demethylation of nucleic acids, inhibition of purine incorporation into DNA Produces single- and double-strand breaks in DNA ?Alkylation of DNA Arti®cial anthracycline Inhibition of topoisomerase I Inhibits microtubule function Inhibition of protein synthesis

‡?

Formation of DNA adducts, DNA interstrand cross-links

‡‡‡ ‡‡

mAMSA Procarbazine Mitoxantrone Topotecan Taxotere L-Asparaginase Platinum analogues Cisplatin Carboplatin

Purine analogues Nucleoside 20 -deoxycytidine analogues Ribonucleotidase inhibition Adenosine analogue

‡ ‡? ‡? ‡ ‡ ‡ ‡ ‡? ‡?

? ‡‡‡ ‡ ? ? ‡

‡ ˆ temporary impairment, full recovery; ‡‡ ˆ risk of persistent dysfunction; ‡ ‡‡ ˆ persistent dysfunction. ? ˆ insucient knowledge.

chemotherapy.52 In studies of long-term testicular toxicity, a large interindividual variation in susceptibility has been shown, but persistently poor semen quality with a low sperm count was seen in most cases (see Figure 1). The large variation seems to depend on the dose of chemotherapy, the duration of the follow-up period and the

Cancer and male infertility 461

pre-treatment semen quality. Permanent azoospermia or severe oligozoospermia is seen in most patients who have received a cumulative dose of cisplatin of 600 mg/m2 or more.53,54 Moreover, the time to recovery of spermatogenesis seems to be dose dependent.53 In accordance with the impaired semen quality, a persistent elevation of serum follicle-stimulating hormone (FSH) level is seen in many patients after chemotherapy.52 It should, however, be kept in mind that most of the data derive from TGCC patients, in a large proportion of whom spermatogenesis was already impaired prior to cytotoxic treatment. It is claimed that the substitution of cisplatin with carboplatin in the treatment of patients with testicular cancer reduces the e€ect on spermatogenesis. There are, however, no studies testing whether carboplatin is less gonadotoxic than cisplatin when compared at a dose level with equal non-gonadal activity.55 Miscellaneous cytotoxic agents Some important agents in this group are listed in Table 1. Procarbazine is included in the MOPP regimen and is responsible for a substantial part of the very severe gonadal toxicity in patients treated with that regimen. Other types of cytotoxic drug, such as anti-metabolites and plant derivatives, seem to have a less pronounced e€ect on gonadal function, although the available information is still rather limited. Other gonadal e€ects of cytotoxic drugs Leydig cell function Leydig cell function is in general more resistant than spermatogenesis to cytotoxic agents. Until now, no studies have been able to show a subnormal level of testosterone as a consequence of cytotoxic treatment. Compensated Leydig cell dysfunction with an elevated LH but unchanged testosterone concentration has been reported after various combination chemotherapy regimens such as PEB (Cis-Platinum, Etopside and Bleomycin), CHOP (Cyclophosphamide, adriamycin, Oncovin [vincristine], Prednisone) and MOPP.52 In patients with testicular cancer, an increased LH level, seen in up to 85% of the patients more than 2 years after chemotherapy, indicates persistent Leydig cell dysfunction despite a normal testosterone level in most patients.52 As already indicated, however, the impact of pre-treatment gonadal dysfunction cannot be excluded. Teratogenicity With few exceptions, cytotoxic agents interfere with either cell division or DNA replication. A teratogenetic potential should thus be theoretically expected.56 At least transient chromosome abnormalities in sperms have been shown in both experimental and clinical studies.57,58 An increased risk of inborn errors or cancer in children fathered by men previously treated with chemotherapy has not been shown.58,59 The studies are, however, based on relatively small samples, and the pregnancies arose from natural conception. The introduction of the ICSI procedure into the treatment of infertile men with a low sperm count bypasses the natural selection procedure (see

462 A. Giwercman and P. M. Petersen

Chapters 6 and 7), implying an increased risk of the transmission of genetic defects in sperm caused by the use of cytotoxic drugs. EFFECT OF CANCER TREATMENT IN SPECIFIC GROUPS OF PATIENTS Testicular germ cell cancer The e€ects of radiotherapy and chemotherapy have already been described above. However, surgical procedures such as orchidectomy and retroperitoneal lymph node dissection (RPLND) may also have a negative impact on reproductive function. E€ect of orchidectomy There is only limited information on the e€ect of unilateral orchidectomy on sperm output, but the available data indicate a 50% reduction in sperm concentration during the ®rst months after orchidectomy for TGCC compared with the pre-orchidectomy level (see Figure 1). Moreover, approximately 10% of men with sperm in the ejaculate before orchidectomy become azoospermic after the testis has been removed.60 These observations are supported by histological investigations showing that 8% of patients have no spermatid production in the contralateral testis40 and by hormone investigations showing an increased FSH and a decreased inhibin B level after orchidectomy compared with the pre-orchidectomy level.60 In patients treated with orchidectomy alone for stage I testicular cancer, some compensatory improvement of semen quality ± probably caused by a rising FSH level ± seems to occur during the ®rst 2 years after surgery.61 Whether a long-term deterioration of semen quality takes place is still unclear. The androgen level seems to be unchanged after orchidectomy as the result of an increased LH stimulation of the Leydig cells. Leydig cell dysfunction with an increased level of LH and/or a decreased level of testosterone has been reported in up to 25% of the patients treated with orchidectomy alone.60 The long-term impact of orchidectomy on Leydig cell function has not yet been investigated. E€ect of retroperitoneal lymph node dissection The most common complications following classical RPLND are anejaculation and retrograde ejaculation. During the past 10±15 years, the frequency of these complications has been reduced from more than 75% to less than 33% by changing from radical RPLND to a modi®ed (right or left) RPLND, without any e€ect on the relapse rate.52 More speci®c methods have been developed in selected groups of patients. Nerve-sparing techniques are possible in 20% of patients depending on the extent and localization of the disease. Only 15% of these patients had ejaculatory dysfunction after such speci®c treatment.62 The complications of surgery depend, however, on the experience of the surgeon. Lymphoma The e€ect of treatment on gonadal function in men with Hodgkin's lymphoma depends very much on the type of treatment given. A multimodal approach is often used in these patients, and treatment may include surgery, chemotherapy and/or

Cancer and male infertility 463

radiotherapy. Procarbazine, alkylating agents and testicular irradiation seem to be responsible for most of the impact on fertility, surgery and other cytotoxic agents having a lesser role. Permanent azoospermia with Sertoli cell-only pattern in the testis and a raised serum FSH level were found in the great majority of men receiving procarbazine regimens and regimens containing alkylating agents, such as MOPP, COPP (Cyclophosphamide, Oncovin [vincristine], Procarbazine and Prednisone) and MVPP (nitrogen Mustard, Vinblastine, Procarbazine and Prednisone). All patients become azoospermic during MOPP treatment, fewer than 25% showing any recovery 5 years after six series of MOPP treatment, although the probability of recovery of spermatogenesis increases with the length of follow-up.49,63 The time to recovery may be rather long, in some cases more than 10 years. In patients who receive regimens without alkylating agents, such as ABVD (Adriamycin, Bleomycin, Vinblastine and Dacarbazine) and NOVP (Novantrone, Oncovin [vincristine], Vinblastine and Prednisone), only a minority became azoospermic during treatment, and a full recovery of spermatogenesis was already observed in most patients by 12 months.49,64 In addition to a€ecting spermatogenesis, the combination chemotherapy for Hodgkin's disease caused Leydig cell dysfunction, resulting in an elevated LH level despite a normal serum testosterone level. In patients treated for non-Hodgkin's lymphomas, gonadotoxicity depends on the dose of the alkylating agent(s). In men given the classical CHOP combination chemotherapy, the risk of permanent sterility increases after a cyclophosphamide dosage of more than 9.5 g/m2.46,47 In patients who have been treated with radiotherapy to the abdominal ®eld, scattered irradiation to the testes potentiates the gonadotoxicity induced by chemotherapy.46,47,65 Leukaemia and lymphoproliferative disorders The fertility potential of men treated for leukaemia also depends on the dose of alkylating agents, whereas regimens containing anthracyclines and anti-metabolites show only a limited and transient impairment of gonadal function.37 Intensive treatment with high-dose chemotherapy with or without total body irradiation (TBI) before bone marrow transplantation is necessary in some patients with a haematological malignancy. TBI leads to permanent azoospermia in almost all men. The majority of men have an elevated FSH and LH level and a normal testosterone level.37,66 In patients treated with bone marrow transplantation without TBI, gonadal toxicity also depends on the dose of alkylating agents. Sarcoma In bone sarcoma, treatment with cisplatin- and doxorubicin-based chemotherapy has resulted in azoospermia followed by recovery in most patients. Re-establishing spermatogenesis depends on the cumulative dose of cisplatin, while no relationship to other drugs ± including cyclophosphamide given in doses lower than 4 g/m2 ± is apparent. A sperm density above 10 million/ml was found in 95% of men after cisplatin doses of less than 600 mg/m2 compared with 43% after doses above 600 mg/m2.53 In men with soft tissue sarcomas, the treatment is based on cyclophosphamide or iphosphamide and anthracyclines. Cyclophosphamide is associated with an increased risk of permanent sterility in doses of more than 7.5 g/m2 in these patients.46,47 The knowledge of testicular toxicity induced by iphosphamide is very limited. Dose-dependent toxicity with increased FSH and LH levels has been seen after radiotherapy.67

464 A. Giwercman and P. M. Petersen Table 2. Examples of commonly used combination regimens in young men. Regimen

Clinical use

Gonadal toxicity

MOPP/MVPP/COPP ABVD/NOVP PVB/PEB

Malignant lymphomas Malignant lymphomas Testicular cancer

‡‡‡ ‡ ‡‡

‡ ˆ temporary impairment, full recovery; ‡‡ ˆ risk of persistent dysfunction; ‡ ‡‡ ˆ persistent dysfunction. For constituents of drug regimens, see text.

The gonadotoxic e€ect of some of the most commonly used combination regimens is summarised in Table 2. PRESERVATION AND RECOVERY OF FERTILITY POTENTIAL IN CANCER PATIENTS As indicated, cancer treatment frequently imposes long-term or even permanent azoospermia on men who have survived a malignant disease. A longstanding ± or even reversible ± suppression of spermatogenesis may, in practical terms, be equivalent to infertility, since, for most couples, the wish and ability to reproduce are limited to a relatively narrow window in their life. In each young patient treated for cancer disease, therefore, the possibility of preserving germ cells and/or accelerating the process of regeneration of spermatogenesis needs to be considered. At present, semen cryopreservation is the only proven method for `preserving' fertility potential in cancer patients threatened by a permanent or longstanding impairment of spermatogenesis. Other possible treatment modalities are, however, currently being explored. Semen cryopreservation The cryopreservation of spermatozoa prior to cancer treatment is a relatively easy and ecient way of preserving reproductive capacity in patients faced with a potential risk of permanent or longstanding azoospermia. The introduction of the modern techniques of assisted reproduction has opened up new possibilities of utilizing semen samples of a very poor quality, as are often found in men with testicular cancer.16 A properly designed programme for semen cryopreservation is an indispensable part of the modern treatment of oncological disease.68 Age of patients It is important to realize that pubertal boys, as early as 12±13 years of age, may be able to provide an ejaculate suitable for cryopreservation.69,70 However, because of the variation in the stage of physical and mental development among boys of this age group, an individual assessment should be made for each case. This should be based on an evaluation of the physical signs of puberty, for example testis size and gonadal stage, as well as on an evaluation of psychological maturation. The latter should be performed by a physician with paediatric endocrinological expertise, and it is necessary for the decision to be made in agreement with the boy and his parents. Because of the psychological stress, some boys may be unable to produce an ejaculate by masturbation,

Cancer and male infertility 465

so using a vibrator, or even electroejaculation, may be the only chance of providing material for cryopreservation.71 Timing in relation to cancer treatment In men with TGCC, the cryopreservation of semen is usually limited to those who are going to be treated with cytotoxic drugs, irradiation or RPLND. Recent data, however, indicate that it may also be advantageous to cryopreserve the semen prior to the orchidectomy. A signi®cant decrease in sperm concentration from 17 million/ml to 7.2 million/ml was seen among 39 men who had undergone orchidectomy alone for TGCC. Furthermore, four (10%) of the men who had sperm in their ejaculate prior to surgery became azoospermic.60 These ®ndings indicate that cryopreservation in TGCC men should ideally be performed prior to orchidectomy. Some men are admitted for cryopreservation after they have already started cytotoxic treatment. Although many of these men still have living spermatozoa in their ejaculate, it has been shown that numerous chromosomal aberrations can be seen in the sperm up to 100 days after chemotherapy.57 Therefore, the utilization of spermatozoa cryopreserved after or during cytotoxic therapy implies a potentially increased risk of the transmission of genetic abnormalities, especially if modern techniques of reproduction are applied. Transmission of contagious diseases The transmission of contagious diseases via a liquid nitrogen tank containing semen straws has never been reported. On the other hand, the spread of the hepatitis virus has been reported for cryopreserved bone marrow.72 This report has prompted many sperm banks to introduce routine tests for HIV and hepatitis among cancer patients delivering an ejaculate for cryopreservation. Psychological aspects The cryopreservation of semen should be seen not only as a way to preserve the fertility potential of cancer patients, but also as a psychological boost. The fact that the doctor worries about the patient's future possibilities of fatherhood indicates that, despite the malignant diagnosis, there is a general belief that he will survive. Hormonal treatment for the protection and recovery of spermatogenesis Attempts have been made to develop hormonal treatment modalities that might protect the seminiferous tissue from the deleterious e€ect of cytotoxic drugs or irradiation. Early work explored the use of gonadotrophin-releasing hormone (GnRH) analogues and/or testosterone to suppress pituitary gonadotrophins in the belief that such treatment would induce a dormant pre-pubertal state in which the spermatogonia would be less sensitive to the cancer treatment. Although animal studies provided promising results, this approach proved not to be successful in the clinical situation.73,74 Meistrich and co-workers recently introduced a new concept in hormonal manipulation that aims to stimulate the post-treatment recovery of spermatogenesis.75 This is based on the observation that some spermatogonial stem cells may survive irradiation or cytotoxic treatment but fail to proliferate and di€erentiate. Furthermore, after cancer treatment, the intratesticular level of testosterone was signi®cantly higher

466 A. Giwercman and P. M. Petersen

than in the control, untreated animals. The idea therefore emerged that post-treatment hormone manipulation to prevent the rise of or suppress intratesticular testosterone might be e€ective in stimulating or accelerating the recovery of spermatogenesis. Studies in rats indicated that GnRH agonists stimulated the recovery of spermatogenesis and fertility in procarbazine- or irradiation-treated rats, not only when the hormone was administered immediately after the treatment, but even 20 weeks after irradiation.76 This new and intriguing concept remains to be tested on humans. Autotransplantation of testicular tissue The concept of transplantation seems particularly attractive in the management of prepubertal boys, who are unable to deliver an ejaculate for cryopreservation. In limited animal experiments, germ cell transplantation has been encouraging, but an extrapolation to human application will require extreme caution. In 1996, Russell et al77 reported on germ cell allotransplantation into the lumen of the seminiferous tubules of another mouse. In some of the tubules, apparently normal spermatogenesis was established, whereas other tubules showed impaired spermatogenesis, mainly resulting from abnormalities in the elongation of the spermatids and their subsequent degeneration. The transplantation of primordial germ cells and gonocytes from rat fetuses and neonates via rete testis injection into adult rats resulted in qualitatively normal spermatogenesis in 10 out of the 16 recipients.78 In another study79, germ cells isolated from the testes of donor male mice repopulated the sterile adult recipient testes when injected into the seminiferous tubules. Donor cell spermatogenesis in recipient testes showed normal morphological characteristics and produced mature spermatozoa. Protocols for the autotransplantation of testicular cells in humans have not yet been developed. The concept of transfer of spermatogonial cells recovered from testicular biopsy after isolation and cryostorage is attractive since it may provide another option for rescuing fertility in adult cancer patients, and a much-needed one in children. Currently ongoing research will demonstrate whether extrapolation from these rodent experiments to clinical germ cell autotransplantation treatment in man is realistic. One serious limitation of this technique was, however, demonstrated in a recent study showing that the transmission of as few as 20 malignant cells from a testis containing leukaemic in®ltrates could induce a relapse of the disease after the transplantation of the gonadal tissue (Kainen, personal communication). PRACTICAL ANDROLOGICAL MANAGEMENT OF MEN WITH CANCER The andrological management of cancer patients should begin before cancer treatment. The following aspects need to be considered: 1. fertility; 2. Leydig cell function; 3. general andrological counselling. Management before cancer treatment The cryopreservation of semen prior to cancer therapy should be considered according to the guidelines above. Using ICSI, even ejaculates with very few sperm can

Cancer and male infertility 467

be suitable for fertilization. Therefore, even men with very poor semen quality should be o€ered cryopreservation if some motile sperm are present. Since the sperm count drops signi®cantly after orchidectomy and the risk of azoospermia increases, a patient with TGCC should be o€ered sperm-banking before the testis is removed. Postponing chemotherapy or surgery for a few days does not imply any worsening of the prognosis. At the same time, the patient and his partner should be counselled regarding his future fertility potential. It is important to give a realistic picture of the future possibilities of parenthood, both with and without the use of assisted reproduction techniques. One should be hesitant about cryopreserving sperm during or shortly (56 months) after chemotherapy since cytotoxic drugs may have a deleterious e€ect on sperm DNA.56,57 For the same reason, reliable contraception is recommended to couples during and for 6 months after the male partner has completed cytotoxic treatment because spermatozoa exfoliated from the testis may survive chemotherapy. Serum samples for measurement of testosterone, sex hormone-binding globulin, oestradiol, LH, FSH and ± if available ± inhibin B should also be taken since this information will facilitate the monitoring of future gonadal function in these patients.

Management after cancer treatment Fertility aspects Since the risk of relapse of cancer is highest during the ®rst year after completion of the treatment, patients should not begin any assisted reproduction treatment during this period. The patient who has been cured of his malignant disease should be o€ered andrological counselling if he wishes fatherhood or simply wants to know the status of his reproductive capacity for planning his future life. In the evaluation of fertility potential after cancer treatment, the following aspects should be considered: . the current and, if available, pre-treatment semen analyses and reproductive hormone levels; . a knowledge of the therapy and its gonadotoxic e€ect; . the length of time since completion of the treatment; . pre-treatment testicular histology, which may be available in TGCC patients in whom a contralateral biopsy was performed. Careful andrological examination is necessary in order to exclude any scrotal abnormalities that may have a coincidental in¯uence on fertility.

Androgen substitution Patients with testicular cancer have a higher risk of developing androgen de®ciency. Their symptoms may be atypical or simply overlooked as being psychological reactions following cancer diagnosis and treatment. These men should therefore be carefully interviewed and investigated for potential evidence of androgen de®ciency. A knowledge of the pre-treatment testosterone level may be helpful in identifying susceptible patients.

468 A. Giwercman and P. M. Petersen

SUMMARY In the management of young males with cancer, the issue of quality of life ± including reproductive function ± should be an important consideration during treatment and follow-up. E€orts should be made to develop and select e€ective treatment modalities with the least gonadotoxicity in order to optimize the cryopreservation of semen, and to give the patient realistic and individualized counselling regarding his fertility potential. The autotransplantation of testicular tissue and hormonal treatment may in future become new options in restoring the fertility potential of cancer survivors. The introduction of modern techniques of assisted reproduction can help many cancer survivors to achieve fatherhood, which might otherwise be impossible by natural conception. Such techniques may, however, run an as yet indeterminate risk of transmission of genetic abnormalities via damaged sperm DNA.

Practice points . ultrasound of the testes and even testicular biopsy for CIS should be considered in infertile men with a history of cryptorchidism . all types of cancer treatment imply a potential risk of impairment of sperm production and, in some cases, even longstanding or permanent azoospermia . among the cytotoxic drugs, alkylating agents, cisplatin and procarbazine have the most deleterious e€ect on spermatogenesis, the e€ect being dose dependent . cryopreservation of the semen should be considered in all young men and even pubertal boys prior to cancer treatment . in men with testicular cancer, semen storage should preferably be carried out prior to the orchidectomy . Leydig cells are more resistant but not entirely immune to cytotoxic and radiotherapy; the threshold for the investigation of androgen de®ciency should thus be low following cancer treatment

Research agenda Research is needed to investigate the following: . the role of endocrine and genetic factors in the aetiology and pathogenesis of TGCC and other cancers of the reproductive organs . the impact of non-testicular malignancies on male reproductive function . the development of less gonadotoxic cancer treatment modalities . an evaluation of the e€ects of chemo- and radiotherapy on sperm DNA in view of the use of techniques of assisted reproduction . the improvement of protocols for semen cryopreservation . the development of regimens for the hormonal prevention or rescue of testicular damage caused by cancer treatment . the establishment of protocols for the cryopreservation and autotransplantation of gonadal tissue or germ cells

Cancer and male infertility 469

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