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Testicular Function in Poor-Risk Nonseminomatous Germ Cell Tumors Treated With Methotrexate, Paclitaxel, Ifosfamide, and Cisplatin Combination Chemotherapy

A. MACHERAS

From the ^* Second Department of Internal Medicine, Propaedeutic, Oncology Section; and the Third Department of Surgery, University of Athens, Attikon University Hospital, Haidari, Athens, Greece

Correspondence to: Dr D. Pectasides, Second Department of Internal Medicine, Propaedeutic, Oncology Section, Attikon University Hospital, Rimini 1, Haidari, Athens, Greece (e-mail: pectasid{at}otenet.gr).

Received for publication July 26, 2008; accepted for publication December 27, 2008.

	Abstract

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Our objective was to investigate the impact of methotrexate, paclitaxel, ifosfamide, and cisplatin (M-TIP) on long-term fertility in poor-risk nonseminomatous germ cell tumors (NSGCT). Thirty patients with poor-risk NSGCT (median age, 29 years; range, 17–62 years) were treated with methotrexate 250 mg/m² with folinic acid rescue (day 1) and paclitaxel 175 mg/m² (day 1), followed by ifosfamide 1.2 g/m² and cisplatin 20 mg/m² (days 2–6). Treatment consisted of 4 cycles of M-TIP administered every 3 weeks. Twenty-one patients were continuously disease-free at a median follow-up of 5.3 years (range, 0.9–8.4 years). Sperm count and hormonal analyses were examined prechemotherapy (30 patients) and postchemotherapy (21 patients). Counts were classified as follows: lower than 1 x 10⁶/mL, azoospermia; 1–20 x 10⁶/mL, oligospermia (OS); higher than 20 x 10⁶/mL, normospermia (NS). Patients were followed for a median of 2.3 years (range, 0.9–3.8 years) postchemotherapy. The prechemotherapy median luteinizing hormone (LH) serum levels were slightly above the upper normal limit, whereas the serum levels of follicle-stimulating hormone (FSH) and testosterone (T) were within the reference interval. Eleven (52.3%) patients had NS prechemotherapy. Among the patients with NS, 72.7% still had NS following chemotherapy. Overall, 17 of 21 (80.9%; 33.3% OS and 47.6% NS) patients had recovery of spermatogenesis after treatment. The median FSH serum levels were significantly elevated at least 1 year postchemotherapy when compared with the pretreatment levels. Eighteen months after the completion of chemotherapy the median FSH levels had returned to the reference limits. Serum LH and T levels were unaffected by chemotherapy. Prior to chemotherapy 4 of 30 patients had fathered 5 children. Since completion of chemotherapy, 5 patients have fathered 5 children. The majority of men with poor-risk germ cell tumors who were treated with the M-TIP regimen demonstrated recovery spermatogenesis after treatment, and Leydig cell function was unaffected.

     Key words: Fertility, gonadal toxicity

We treated 30 poor-risk NSGCT patients with a methotrexate, paclitaxel, ifosfamide, and cisplatin (M-TIP) regimen for 4 cycles in a phase II study (Pectasides et al, 2008). The primary endpoint of the study was to demonstrate that M-TIP has comparable efficacy to BEP in this poor-risk subset without increasing toxicity. The main toxicity was myelosuppression. Neurotoxicity and nephrotoxicity were recorded in 6.6% and 3.3% of patients, respectively. However, attention has been directed to the long-term complications of this therapy, such as infertility and delayed teratogenesis. Men treated with chemotherapy for GCT are at the peak of their reproductive age and there are good prospects for cure. Therefore, it is important to consider the effect that chemotherapy has on fertility. Apart from a minority of patients who have conditions such as cryptorchidism or testicular atrophy, the majority of men with GCT are fertile prior to the development of the tumor. However, these patients often have low sperm counts after unilateral orchidectomy for testicular cancer, and in some of them the sperm counts remain low (Berthelsen and Skakkebaek, 1983; Jewett et al, 1983). Although the effects on fertility of extended retroperitoneal lymph node dissection and para-aortic and inguinal lymph node irradiation are well known (Rustin et al, 1986; Brydoy et al; 2005), little information is available on the long-term gonadal toxicity of cisplatin-containing regimens (Drasga et al, 1983; Fosså et al, 1985; Nijman et al, 1987; Lampe et al, 1997; Reiter et al, 1998; Spermon et al, 2006). In addition, there is no study in the English literature investigating the effect of M-TIP regimen on spermatogenesis.

The aim of this study was to investigate the long-term gonadal toxicity of M-TIP in poor-risk patients treated with 4 cycles of this combination.

	Materials and Methods

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Patients

Thirty NSGCT patients with poor-risk features were registered into this single-center trial from July 1997 to November 2003 (Pectasides et al, in press). Eligible patients had NSGCT with the following features: histological diagnosis of NSGCT and a primary site in the testis or mediastinal primary; measurable disease by radiographic assessment; raised serum concentration of tumor markers; and poor-risk disease, according to the IGCCCG criteria (alpha-fetoprotein above 10 000 ng/mL, beta human chorionic gonadotropin [β-HCG] above 50 000 U/L, lactate dehydrogenase above 10 times the upper normal limit, nonpulmonary visceral metastases, or mediastinal primary site). Patients were not to have received previous chemotherapy or radiotherapy. Patients were also required to have no history of other malignancy. Chemotherapy consisted of methotrexate 250 mg/m² given as a 4-h infusion with folinic acid rescue on day 1 and paclitaxel 175 mg/m² given as a 3-h infusion on day 1, followed by ifosfamide 1.2 g/m² given as a 2-h infusion and cisplatin 20 mg/m² given as a 2-h infusion on days 2–6. Treatment consisted of 4 cycles of M-TIP administered every 3 weeks. Mesna 400 mg/m² was given intravenously before ifosfamide infusions and every 4 hours thereafter, for a total for 3 doses per day. All patients received prophylactic premedication with 20 mg dexamethasone 12 and 6 hours before paclitaxel infusions and intravenous ranitidine and oral diphenyhdramine (each 50 mg) 30 min prior to paclitaxel administrations. The aim of the trial was to test the efficacy of the chemotherapy regimen, and secondarily to investigate its influence on fertility.

Semen Samples

For successful artificial insemination the minimum requirements for storage are 4 ejaculates with density 10 x 10⁶/mL with at least 30% motility and a volume of at least 1 mL. Patients who obtained complete response and did not relapse were asked to participate in a follow-up examination of fertility after chemotherapy. Sperm counts were classified as follows: lower than 1 x 10⁶/mL, azoospermia (AS); 1–20 x 10⁶/mL, oligospermia (OS); and higher than 20 x 10⁶/mL, normospermia (NS).

Semen samples were analyzed and serum follicle-stimulating hormone (FSH), luteinizing hormone (LH), and testosterone (T) were recorded prior to chemotherapy and at follow-up, on a 6-month basis starting approximately 1 year postchemotherapy. All patients had no evidence of disease at the time of posttreatment fertility evaluation. Semen was obtained by masturbation after at least 3 days of sexual abstinence. The samples were analyzed within 2–4 hours of ejaculation. The sampling continued until sperm counts had reached the pretreatment level for at least 2 years or on the request of patients. The volume and appearance of the semen and the concentration of spermatozoa and their motility and morphology were examined. The methods used for examination of sperm samples were in accordance with World Health Organization (WHO) (1992) criteria. Serum FSH, LH, and T were measured by radioimmunoassay. LH and FSH were expressed in IU/L (LH, WHO first International Reference Preparation [IRP] 68/40, and FSH, WHO second IRP 78/549). The reference values of our laboratory were: FSH, 1.0–8.0 IU/L; LH, 0.6–12 IU/L; and T, 2.8–9.0 ng/mL. Gonadal function was evaluated for a median of 2.3 years (range, 0.9–3.8 years).

Our study was approved by the Hospital Ethics Committee, and informed consent was obtained from all patients before study entry.

Statistics

Statistical analysis was performed using Wilcoxon paired test, paired t test, and Fisher exact test as appropriate. A P value of less than .05 was considered statistically significant.

	Results

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A total of 30 patients were enrolled in this trial. The patient characteristics at the initiation of M-TIP treatment are shown in Tables 1 and 2. Six patients (20%) received 5 cycles of M-TIP. These patients had tumor marker normalization after the third cycle of chemotherapy and received 2 additional courses of M-TIP. The median age was 29 years (range, 17–62 years). The study included 25 patients with primary testicular cancer and 5 patients with primary mediastinal GCT. Five patients (16.6%) achieved clinical complete response with chemotherapy only, 15 patients (50%) achieved pathologic complete response (11 patients had necrosis/fibrosis and 4 patients had mature teratoma), and 3 patients (10%) achieved surgical complete response, for an overall favorable response rate of 76.6%. Twenty-one patients were continuously disease-free at a median follow-up of 5.3 years (range, 0.9–8.4 years), resulting in a 5-year progression-free survival rate of 66.6% and a 5-year survival rate of 70%.

Pretreatment Hormone and Sperm Analysis

All 30 patients were assessed for pretreatment hormone and sperm analysis. The serum LH was slightly elevated (upper normal limit 12 IU/L) because of the cross-reacting with β-HCG in patients with far-advanced disease. FSH and T values were within the normal range, although most of the individual T levels fell within the lower half of the normal range (Table 3). Nine patients succumbed from disease shortly (median survival 5 months; range, 2–21 months) having salvage chemotherapy just prior to death. Among those 21 patients achieving long-term disease-free survival, 3 patients had AS, 7 OS, and 11 NS before chemotherapy.

Posttreatment Hormone and Sperm Analysis

Posttreatment hormone and sperm analysis was performed only in 21 patients who were long-term disease-free. Testosterone levels remained within the normal range after chemotherapy. LH levels returned to normal at the 18-month measurement (LH normalization because of the effective treatment of β-HCG–producing metastases) and remained within the normal limits for the rest of the study. There were not statistically significant differences in T or LH levels measured before chemotherapy and those measured 1 year post-chemotherapy or later. FSH levels increased significantly 1 year postchemotherapy (P = .001). However, there was no significant difference when comparing FSH levels before and 18 months or later after chemotherapy.

Table 4 summarizes the pretreatment sperm count and the effect of chemotherapy on spermatogenesis of patients who were analyzed at least 1 year after chemotherapy. Of the 3 patients who had AS before treatment, 1 recovered to OS and 2 remained azoospermic postchemotherapy. Among the 7 patients who had OS, 4 (57.1%) remained oligospermic, 2 (28.5%) recovered to NS, and 1 became azoospermic postchemotherapy. Of the 11 patients with NS before treatment, 2 (16.5%) patients had OS and 1 (9%) patient AS postchemotherapy. Overall, 19% of patients had AS postchemotherapy. Among the 6 patients who received more than 4 cycles of chemotherapy, 1 had AS, 1 had OS, and 2 others had NS.

The median time between the end of chemotherapy and when spermatogenesis was first noted to have returned was 12 months (range, 10–30 months). The median semen volume was 3.2 mL (range, 0.5–9.6 mL). Semen volume was less than or equal to 1 mL in 2 patients. Fourteen patients (67%) had 50% or greater motile sperm, and the median motility was 48.3% (range, 0%–80%).

Prior to chemotherapy 4 of 30 patients had fathered 5 children. Two of them also fathered children postchemotherapy. In total, since the completion of chemotherapy 5 patients have fathered 5 children. No congenital abnormalities have been detected in any of those children. There was 1 termination of pregnancy when the molecular analysis following amniocentesis revealed a fetus with β-thalassemia. All patients conceived by natural conception. Two of the 5 patients who fathered children after treatment were reported as having OS; these 2 patients fathered a child at 11 and 14 months, respectively. One patient, who had AS at 12 months, repeated a semen analysis at 24 months, when his wife became pregnant, which showed sperm concentration of 17.9 x 10⁶/mL.

	Discussion

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Standard treatment for GCT consists of cisplatin-containing regimens with BEP or, before the mid-1980s, with a cisplatin, vinblastine, and bleomycin (PVB) regimen. Schedules including increasing the dose intensity, substituting bleomycin for ifosfamide, or using high-dose chemotherapy with autologous hematopoietic stem cell support in poor-risk patients have been introduced in the treatment of GCT during the last decade. To the best of our knowledge, this is the first trial that assesses the effects of M-TIP regimen on gonadal function.

Testicular cancer is associated with OS before treatment in approximately 50% of cases (Jewett et al, 1983; Drasga et al, 1983; Nijman et al, 1987; Dearnaley et al, 1991). According to our data, 33.3% of patients had sperm concentration of less than 20 x 10⁶/mL. This study, similarly to most others, demonstrated that pretreatment gonadal dysfunction occurs in patients with poor-risk GCT. According to our definitions, 52.3% of patients were normospermic prior to chemotherapy. Drasga et al (1983) and Nijman et al (1987), using 20 x 10⁶/mL as the lowest limit for NS, reported a 6.6% and 28% occurrence of pretreatment NS, respectively. Several factors have been suggested to contribute to the depression of spermatogenesis, even at the time of diagnosis (Hendry et al, 1983; Petersen et al, 1998). Local tumor effects, including elevation of scrotal temperature, alteration of testicular blood flow, and disruption of the blood-testis barrier may play a role (Petersen et al, 1998). Abnormalities of testicular pathology were apparent on biopsies of the contralateral testis, including significant fibrosis of seminiferous tubules in 24%–60%, Sertoli cells in only 8%, and in situ carcinoma in 8% (Berthelsen and Skakkebæk, 1983). Furthermore, testicular cancer may be hormonally active and local hormone production may also impair spermatogenesis. Finally, antitesticular antibodies have been found in a high proportion of GCT patients (Guazzieri et al, 1985).

BEP is the standard regimen for the treatment of GCT. BEP has been reported to have gonadotoxicity comparable to that seen with the PVB regimen (Dearnaley et al, 1991; Petersen et al, 1994; Spermon et al, 2006). Cisplatin-containing regimens induce gonadal dysfunction in nearly all patients with a simultaneous increase of FSH serum values shortly after treatment. Recovery of spermatogenesis is seen 2–4 years after the completion of chemotherapy in most cases. Most of the studies focusing on the long-term effects of chemotherapy on testicular function showed a poor semen quality with low sperm counts (Drasga et al, 1983; Fosså et al, 1985; Nijman et al, 1987; Dearnaley et al, 1991; Guazzieri et al, 1985). Drasga et al (1983) and Nijman et al (1987) reported AS in 25% and 28% of patients and OS in 75% and 48% of patients, respectively, 2 years after 4 cycles of PVB. Restoration of spermatogenesis following PVB-induced gonadal toxicity was also demonstrated by other investigators (Fosså et al, 1985). Also, Brydoy et al (2005) followed a large patient population for a long period and found that the cumulative paternity rate at 15 years after orchiectomy of patients treated with a cumulative cisplatin dose less than or equal to 850 mg/m² exceeded 60%. It was significantly lower than the paternity rate of patients subjected only to orchiectomy (92%), but did not differ significantly than those treated with retroperitoneal lymph node dissection or radiotherapy.

Gonadal damage following chemotherapy seems to be dose dependent. Our patients received a cumulative cisplatin dose of 400–500 mg/m², which is lower than the 600 mg/m² limit reported to cause severe OS or AS (Stephenson et al, 1995). Similarly, Lampe et al (1997) reported that patients who received more than 4 cycles of cisplatin had severe gonadal dysfunction. Also, in the study of Brydoy et al (2005), patients treated with a cumulative cisplatin dose greater than 850 mg/m² had a significantly lower cumulative paternity rate at 15 years after orchiectomy (48%) than the lower-dose group. Reduced germ cell destruction may therefore follow the administration of fewer treatment cycles and the ensuing decrease of the total dose of cytostatics. Another important factor is the platinum compound used. Lampe et al (1997), using Cox regression analysis, found that carboplatin-treated patients had a 4.4 times better chance to recover to NS and a 2.5 times better chance to recover to OS compared with the cisplatin-treated group.

Methotrexate (MTX)-induced gonadal toxicity has been extensively studied in the laboratory and in the clinic. Investigators observed that MTX is detected in testicular tissues after parenteral administration in animal models (Koehler et al, 1986b), although concentrations were 2- to 4-fold lower in the testicular interstitial fluid and 18- to 50-fold lower in the seminiferous tubules compared with the plasma levels (Riccardi et al, 1982). In addition, β-HCG appears to favor the accumulation of MTX in testes, probably by increasing testicular capillary permeability and lymphatic and blood flow (Bettencourt et al, 1997). Furthermore, MTX-induced spermatogonial damage (Russell and Russell, 1991) and OS following repeated administration of MTX (Johnson et al, 1994) resulted in decrease in diameter of seminiferous tubules, increase in interstitial spaces, and morphology distortion of Leydig cells in a dose-dependent manner (Shrestha et al, 2007). On the contrary, Meistrich et al (1982) did not observe significant effects on spermatogenesis after MTX administration. In addition, it was shown that MTX reduces testicular steroidogenesis, especially after repetitive dosing (Koehler et al, 1986a). However, these effects do not seem to remarkably and irreversibly influence male fertility. Low doses of MTX used to treat psoriatic patients did not affect gonadal function (French et al, 2003), whereas high doses caused transient testicular dysfunction (Shamberger et al, 1981), especially when combined with known gonadotoxic agents (Gaffan et al, 2003), or radiotherapy (Shamberger et al, 1981). In contrast, steroidogenic testicular activity was not significantly influenced by MTX (Shamberger et al, 1981; Gaffan et al, 2003).

Ifosfamide is an alkylating agent with known gonadal toxic action (Ypsilantis et al, 2003a,b). This was shown experimentally with weekly (Ypsilantis et al, 2003a) as well as with single-dose (Ypsilantis et al, 2003b) administration of ifosfamide in animals. Testicular dysfunction was shown to be dose-dependent (Ypsilantis et al, 2003a,b) and could be reversed after long follow-up dependent on the treatment dose (Ypsilantis et al, 2003a). Longhi et al (2003) observed that ifosfamide affected testicular function in a dose-dependent manner. Furthermore, in a recently published study including both pediatric and adult patients (approximately 5–21 years old), Williams et al (2008) reported that gonadal dysfunction occurred only in those treated with ifosfamide doses greater than 60 g/m², which is far higher than the cumulative dose administered in our patients (24–30 g/m²). There was no correlation with age and no gonadal dysfunction was observed in female patients.

FSH and LH levels reflect the state of germinal epithelium and Leydig cells. In accordance with the impaired semen quality, 15% and 18% of our patients had elevated levels of FSH and LH, respectively, before treatment, reflecting the impairment of germinal epithelium. The median levels of FSH, however, were within the reference interval, whereas the median levels of LH were slightly increased, probably because of the cross-reaction with the β-HCG–producing patients. The median T levels were within the reference range. Furthermore, Reiter et al (1998) reported that the prechemotherapy level of FSH was increased (15.5 ± 5.6 IU/L; reference range, 0.8–9.0 IU/L) because of subnormal spermatogenesis. Chemotherapy with the M-TIP regimen was followed by significantly elevated FSH values until at least 1 year after treatment. During follow-up, there was a significant decrease of FSH levels, which returned to reference range 18 months or later postchemotherapy in most of the cases. In the study of Reiter et al (1998), FSH values started to decline towards normalization after 1 year postchemotherapy, in accordance with recovery of spermatogenesis. Some investigators (Drasga et al, 1983; Nijman et al, 1987) have evidence to support that impairment of Leydig cell function may be caused by PVB, whereas others (Fossa et al, 1985) have reported no effect of PVB therapy on LH and T levels. Nord et al (2003) reported that patients with testicular cancer had significantly increased age-adjusted LH values and odds ratio of hypogonadism compared with controls. Also, they mentioned that these values increased significantly with treatment intensity. Except for a temporary increase of serum LH levels noted in a few cases, serum LH and T levels were within the reference intervals in M-TIP patients. Therefore, the M-TIP regimen does not appear to induce any long-term changes in the function of Leydig cells.

Five patients fathered 5 children after receiving chemotherapy, and no birth defects were identified. It should be noted that OS did not preclude some patients from fathering children, because 2 of the 4 patients who had OS at the time of semen analyses have fathered children.

In conclusion, the M-TIP regimen seems to have a relatively beneficial toxicity profile concerning fertility, because the majority of patients with poor-risk GCT regained spermatogenesis following chemotherapy. Furthermore, Leydig cell function was unaffected by the treatment. However, until less gonadotoxic regimens with activity in GCT have been discovered, all patients should be offered sperm cryopreservation prior to chemotherapy.

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