In designing our study, we assumed that the use of testosterone to inhibit germinal cell activity might reduce the sensitivity of these cells to the effects of cyclophosphamide. We administered cyclophosphamide as an immunosuppressive agent to manage glomerulonephritis, either orally or in an intravenous bolus [11, 12]. We therefore could evaluate the effects of the two methods of administration on germinal cell function.

Methods

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We studied 15 men who had the nephrotic syndrome, (age range, 23 to 35 years). Each patient was told of the possible risks of treatment and gave informed consent.

Patients were randomly divided into three groups. The five patients in group A (three with membranous nephritis, one with mesangial nephritis, and one with lupus nephritis) were given oral cyclophosphamide at an average dosage of 150 mg daily for 6 to 8 months; the total dose was 27 to 36 g. The five patients in group B (two with membranous nephritis, two with mesangial nephritis, and one with focal glomerulosclerosis) and the five patients in group C (two with membranous nephritis, one with mesangial nephritis, and two with lupus nephritis) received cyclophosphamide as a monthly intravenous bolus, 15 mg/kg of body weight, according to the protocol reported elsewhere [11, 12]. The average duration of treatment was 8 months; the total dose was 10.4 g. Patients in group C also received testosterone, 100 mg intramuscularly every 15 days, in addition to cyclophosphamide. Patients began receiving testosterone (a commercial blend of enanthate and propionate esthers of testosterone) 30 days before starting cyclophosphamide therapy, and they continued to receive it during the course of immunosuppressive therapy.

Because ours was a pilot study, the testosterone dose that we used was lower than that currently used to treat hypogonadism; however, it proved to be effective in inhibiting gonadotropin secretion and spermatogenesis [13]. To ensure the possibility of future paternity, sperm samples from each patient were collected before the start of therapy and stored in liquid nitrogen. Additional sperm samples were collected from each patient before therapy, during the third and sixth months of cyclophosphamide therapy, and 3 and 6 months after the end of the therapy. All sperm samples were analyzed in the same laboratory by the same technician. Serum samples for luteinizing hormone and follicle-stimulating hormone assays were collected at the same intervals as the sperm samples. Hormone levels were measured in duplicate by specific immunoradiometric methods using commercial kits (Serono Diagnostic, Milan Italy). Both of the preceding assays are sensitive to about 0.15 IU/L. Levels are reported in IU/L in accordance with the First International Reference Preparation 68/40 and Second International Reference Preparation 78/549 guidelines for reporting luteinizing hormone levels and follicle-stimulating hormone levels, respectively. Statistical analysis was done using the Student t-test. All results are given as the mean ± SE.

Results

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Under control conditions, all patients had normal sperm counts; the means were 43.70 ± 6.56 x 10^6/mL in group A, 45.52 ± 7.04 x 10^6/mL in group B, and 42.91 ± 5.27 x 10^6/mL in group C. During immunosuppressive therapy, sperm counts abruptly decreased in all patients in groups A and B (Table 1), and all patients were azoospermic 6 months after starting immunosuppressive therapy. Three and 6 months after completing therapy, all patients in group A and four of the five patients in group B were azoospermic. During the third and sixth months of immunosuppressive therapy, three patients in group C had azoospermia or severe oligospermia; sperm counts were lower than 1 x 10^6/mL. After therapy was discontinued, sperm counts increased progressively in group C; mean counts were 28.64 ± 3.47 x 10^6/mL 3 months after therapy ended and 45.78 ± 3.89 x 10^6/mL 6 months after therapy ended. In these patients, mean percentages of sperm motility (88%; range, 63% to 92%) and sperm that showed no structural abnormalities (80%; range, 73% to 89%) were in the normal range.

In groups A and B, serum luteinizing hormone levels did not change throughout the observation period (Table 2) but follicle-stimulating hormone levels progressively increased. The following are the mean follicle-stimulating hormone levels: group A, 8.54 ± 0.69 IU/L and 14.82 ± 2.15 IU/L during the third and sixth months of cyclophosphamide therapy and 18.40 ± 1.79 IU/L and 19.20 ± 1.28 IU/L 3 and 6 months after discontinuation of therapy; group B, 9.30 ± 0.87 IU/L and 18.48 ± 2.81 IU/L during the third and sixth months of cyclophosphamide therapy and 16.80 ± 2.15 IU/L and 16.04 ± 2.22 IU/L 3 and 6 months after discontinuation of therapy. All of these values were significantly higher than those seen under control conditions (P = 0.002). Luteinizing hormone and follicle-stimulating hormone levels decreased in group C during cyclophosphamide therapy because of the inhibitory effect of testosterone (Table 2). In particular, luteinizing hormone levels during the third and sixth months of immunosuppressive therapy were 1.62 ± 0.24 IU/L and 1.06 ± 0.19 IU/L, respectively; follicle-stimulating hormone levels at the same time points were 2.12 ± 0.38 IU/L and 1.56 ± 0.33 IU/L, respectively. The differences with respect to baseline values were statistically significant (P < 0.005). In these patients, luteinizing hormone levels measured 3 and 6 months after discontinuation of therapy were 6.44 ± 0.87 IU/L and 4.80 ± 0.40 IU/L, respectively; follicle-stimulating hormone levels at the same time points were 6.60 ± 0.86 IU/L and 5.08 ± 0.56 IU/L, respectively. Differences between the follicle-stimulating hormone levels in group C and those in groups A and B were statistically significant (P < 0.002). All 15 patients in the study had clinical remission of their underlying diseases. No side effects related to testosterone administration, such as liver enlargement or gynecomastia, were seen in group C.

Discussion

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Our findings confirm that cyclophosphamide therapy can affect germinal cell function in humans [1-36, 7]. All patients who received daily oral cyclophosphamide and four of five who received a monthly intravenous bolus of cyclophosphamide were azoospermic for as long as 6 months after the end of immunosuppressive therapy. Serum follicle-stimulating hormone levels were elevated in all patients who had become azoospermic, a finding that reflects a severe alteration of the germinal epithelium. In contrast, spermatogenesis returned to normal in the five patients who received testosterone before and during immunosuppressive therapy; follicle-stimulating hormone levels for all patients were within the normal range. This finding indicates that the use of testosterone to inhibit germinal cell activity may protect against the effects of cyclophosphamide. Leydig cell function was not affected in any study patient, as shown by normal levels of luteinizing hormone; previous reports have shown that Leydig cells are more resistant than germinal cells to the action of alkylating agents [6, 10].

Testosterone has been used as a contraceptive in men; it induces azoospermia or severe oligospermia by reducing levels of gonadotropins and intragonadal testosterone [13, 14]. In accordance with this observation, our data show a substantial reduction in gonadotropin levels during testosterone administration. Moreover, other studies [13, 14] have shown that testosterone can be used safely at high doses and for long periods.

No data are available on the effects of testosterone in nephrotic patients receiving cyclophosphamide, and previous reports on gonadal protection during therapy with cytotoxic drugs are conflicting. Experimental studies in rats have shown that the use of triptorelin to suppress gonadal function may protect the testes from the adverse effects of cyclophosphamide therapy [15]. In contrast, nafarelin given to dogs was shown to potentiate the damaging effects of the alkylating agents [16]. Oral contraceptives and luteinizing hormone-releasing hormone agonists have also been used in humans in attempts to preserve gonadal function during chemotherapy. However, results of previous studies are conflicting and inconclusive [3, 17-20].

Our data were obtained from a small number of patients observed for only 6 months after the discontinuation of immunosuppressive therapy. However, our findings may indicate a safe way to preserve gonadal function in nephrotic patients who are treated with cyclophosphamide.

Dr. Bartoli: Medicina Interna, University of Udine, Piazzale S. Maria della Misericordia 1, 33100 Udine, Italy.