Before the cystine-depleting agent cysteamine became widely available [7, 8], more than 70 patients with cystinosis in North America underwent renal transplantation, which allowed them to survive into adulthood [9, 10]. Patients with cystinosis who receive renal transplants experience the same relief of uremic symptoms and have the same complications as other renal allograft recipients, but otherwise the clinical course of the disease continues [9, 10]. Although donor kidneys do not accumulate cystine, the effects of the long-standing extrarenal accumulation of cystine become increasingly evident. These include decreased visual acuity, recurrent corneal erosions, central nervous system involvement, swallowing dysfunction, and severe myopathy [9-15].

Hormonal insufficiencies resulting from damage to endocrine organs have also been reported in patients with cystinosis [16-21]. The best characterized are thyroid dysfunction and pancreatic endocrine insufficiency [16-20]. Whether patients with cystinosis also have other endocrine impairment, such as pituitary-testicular dysfunction, however, has not been investigated in an organized fashion. For example, when gonadotropin levels were measured in a group of prepubertal patients with cystinosis, one male patient had an elevated luteinizing hormone (LH) level, suggesting Leydig cell dysfunction [17]. Normal fertility has also been reported in a female patient [21], but primary ovarian failure was noted in a second patient [12]. To determine the extent of pituitary-testicular dysfunction in men with cystinosis, we evaluated reproductive function in 10 patients who received renal allografts and who are currently being followed at the National Institutes of Health.

Methods

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Patients

Nephropathic cystinosis was diagnosed in all patients based on elevated leukocyte cystine levels and the presence of corneal crystals on slit-lamp examination [1-3]. All 10 patients had received a renal transplant 3 to 10 years before the study. Patients were receiving maintenance immunosuppressive therapy consisting of prednisone and either azathioprine or cyclosporine. Nine patients were receiving thyroid replacement as treatment for cystinosis-related hypothyroidism. All patients were seen as part of an institutional review board-approved protocol of the National Institute of Child Health and Human Development to study the complications of cystinosis after transplantation. All patients or their parents gave written informed consent for these studies. Blood was also collected from 11 renal transplant recipients with a primary renal disorder other than cystinosis. They were receiving similar immunosuppressive therapy and were matched with the study group for age and renal function. Tanner stages [22] were defined as follows: Patients were considered stage 1 if they were prepubertal, had no pubic hair, and had a testicular length less than 2.5 cm; stage 2 patients were defined by a sparse growth of slightly pigmented pubic hair at the base of the penis and a testicular length between 2.5 and 3.2 cm; stage 3 patients had thicker, curlier hair that had spread to the mons pubis and a testicular length between 3.3 and 4 cm; stage 4 patients had adult-type hair that had not spread to the medial thighs and a testicular length between 4.1 and 4.5 cm; stage 5 patients were defined by the presence of adult-type hair that had spread to the medial thighs and a testicular length greater than 4.5 cm.

Hormone Assays

Plasma follicle-stimulating hormone (FSH) and LH levels were determined by specific double-antibody radioimmunoassays as described previously [23, 24]. For FSH, the intra- and interassay variations were both 7.5%. For LH, the intra- and interassay variations were 3.7% and 8.4%, respectively. Plasma testosterone was measured by radioimmunoassay after extraction and chromatography [25]. The intra-and interassay variations were 7.1% and 9.5%, respectively.

Serum -subunit, LH-ß, and FSH-ß were determined by specific double-antibody radioimmunoassays as described previously [26, 27]. The cross-reactivities of LH and FSH in the -subunit assay and of -subunit in LH and FSH assays were less than 5%. In contrast, the cross-reactivities of LH in the LH-ß assay and of FSH in the FSH-ß assay were 17% and 37.5%, respectively. The detection limits for the -subunit, LH-ß, and FSH-ß assays were 0.4 mg/L, 0.7 mg/L, and 1.5 mg/L, respectively. A mouse Leydig cell assay was used to assess the bioactivity of LH in the 10 patients with cystinosis. The bioassay used was adapted from that described by Dufau and colleagues [28].

Luteinizing Hormone Pulsatility

Seven patients underwent testing to determine LH pulsatility. Patients were admitted to the ward on the afternoon before the study day to allow acclimatization to the unit. An indwelling catheter was placed in a forearm vein at admission, and patency was maintained by intermittent injections of a dilute solution of heparin. Patients were allowed to ambulate freely until 2200 h, at which time they retired to their beds and room lights were turned off. Sleep was monitored visually by trained nursing personnel. On the study day, blood samples were obtained every 20 minutes for 12 to 24 hours. The duration of the sampling periods was determined by the patient's body weight and renal function; those weighing less than 30 kg or having a creatinine clearance of less than 25 mL/min per 1.73 m² underwent 12-hour sampling and the remaining patients underwent 24-hour sampling.

Stimulation Tests

Four patients had the human chronic gonadotropin (HCG) stimulation test. Daily intramuscular injections of 4000 IU of chorionic gonadotropin (Pregnyl, Organon, West Orange, New Jersey) were given for 3 consecutive days. Testosterone was measured in blood samples collected before the injections and daily for the subsequent 4 days. Five patients had the gonadotropin-releasing hormone (GnRH) stimulation test. After three basal estimates of FSH and LH over consecutive 15-minute intervals, a 100-mg bolus of GnRH was administered intravenously. Levels of LH and FSH were measured in blood samples collected every 15 minutes for the next hour and then every 30 minutes for the following hour.

Histologic Study of Testicular Tissue

Testicular specimens for histologic examination were obtained in one patient who died of aspiration pneumonia [14]. Samples were fixed and placed in neutral buffered formalin and in absolute alcohol before examination by light microscopy.

Statistical Analysis

Data are expressed as mean ± SE. Group comparisons were analyzed using the Student t-test, with statistical significance set at P < 0.05. Pearson correlation coefficients were calculated to evaluate the degree of linear association between measured variables. The LH pulse analysis was done using the Pulsar pulse detection computer program [29].

Results

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Growth and Pubertal Development

The 10 patients with cystinosis had a mean age of 20.8 ± 1.6 years and a mean serum creatinine clearance of 54.1 ± 12.4 mL/min per 1.73 m² (Table 1). These values did not differ significantly from those in the control patients with other renal diseases (Table 2). However, patients with cystinosis were markedly smaller in terms of height (P < 0.001) and weight (P < 0.001). Bone age was delayed by 2 to 10 years in patients with cystinosis; only 2 of 11 control patients had delayed bone age, 1 patient by 1 year and the other by 5 years. No patients with cystinosis reached Tanner stage 5, but only 2 of 11 control patients (17 and 21 years old) failed to reach Tanner stage 5. In the study group, patient 5 (20 years old) was prepubertal and had a 10-year bone age delay; the other 9 patients all gave a history of onset of puberty with a growth spurt and increased libido, and 3 also gave histories of increased shaving. Puberty, however, failed to progress in most patients. Although 9 of 10 patients gave a history of erections, only 3 reported being able to ejaculate (patients 4, 8, and 9). Semen analyses were done in these 3 patients, and azoospermia was noted in all three. A reduced testis volume was also noted in most patients (Table 1).

Hormone Studies

Patient 5 was prepubertal with low testosterone and low LH levels (Figure 1). Of the remaining nine study patients, two (patients 2 and 10) had low testosterone and high LH levels and four (patients 4, 6, 7, and 9) had normal testosterone and high LH levels, consistent with primary hypogonadism. Patient 8 was azoospermic with normal testosterone, high FSH, and normal LH levels. Patients 1 and 3 had normal testosterone and normal LH levels. They were probably peripubertal because both were at Tanner stage 3 and had a bone age of 13 years, and patient 1 also had an increased LH pulse amplitude at night (Figure 2). Five of seven patients with normal testosterone levels had increased levels of testosterone-binding globulin (TeBG). The mean TeBG level was 13.2 ± 3.0 mg/L (normal, 3.7 to 7.0 mg/L). In comparison, only 2 of 11 control patients had an elevated LH level, 3 had an elevated FSH level, and all had normal testosterone levels (Figure 1). The mean LH, FSH, and testosterone levels were 85.4 ± 27.6 IU/L, 51.6 ± 14.4 IU/L, and 11.5 ± 2.0 nmol/L, respectively, in patients with cystinosis compared with 19.4 ± 1.9 IU/L, 17.8 ± 3.5 IU/L, and 24.2 ± 3.0 nmol/L in control patients (P < 0.05 for LH and FSH and P < 0.005 for testosterone).

View larger version (17K): [in this window] [in a new window]   Figure 1. Serum testosterone, luteinizing hormone, and follicle-stimulating hormone levels in patients with cystinosis and in control patients. Top. Bivariate plot of the serum testosterone level against the luteinizing hormone level. Bottom. Bivariate plot of the serum testosterone level against the follicle-stimulating hormone level. The widths of the rectangles represent the ranges of values between the 5th and 95th percentiles of luteinizing hormone or follicle-stimulating hormone values in normal men, and the heights of the rectangles represent a similar range of testosterone values in normal men.

View larger version (30K): [in this window] [in a new window]   Figure 2. Episodic secretion of luteinizing hormone in seven patients with cystinosis. Serum luteinizing hormone (LH) concentrations were determined at 20-minute intervals.

Four patients had 24-hour sampling for LH pulsatility and three had 12-hour sampling (Figure 2). Patients with cystinosis had an increased LH pulse frequency (15.5/24 h) Table 3 when compared with normal men (12.0/24 h and 12.9/24 h) in two previous studies [30, 31].

Patients 1, 4, 9, and 10 underwent HCG stimulation tests. Basal testosterone levels were 13.0 ± 1.2 nmol/L. Testosterone levels measured after one injection of HCG and again after three daily injections were 14.2 ± 1.0 nmol/L (109.3% ± 7.7%) and 21.9 ± 1.4 nmol/L (168.5% ± 10.8%), respectively. These increases in testosterone levels after HCG stimulation were reduced compared with the findings of previous investigators, who reported at least doubling of testosterone levels after multiple HCG injections in normal adolescent and adult males [32-34].

A GnRH stimulation test was done in patients 1, 4, 5, 8, and 9. Basal LH and FSH levels were elevated at 54.1 ± 13.6 IU/L (normal, 6 to 26 IU/L) and 44.2 ± 9.1 IU/L (normal 5 to 25 IU/L), respectively. Peak LH and FSH responses, which occurred between 30 and 60 minutes after GnRH stimulation, were 153.9 ± 59.0 IU/L (284.5% ± 109.0%) and 57.8 ± 22.2 IU/L (130.8% ± 50.2%), respectively. These increases in LH and FSH levels after GnRH stimulation were similar to those reported previously in normal men [35, 36].

Patient 10, who had untreated hypothyroidism after his second renal transplant and a creatinine clearance of 5 mL/min per 1.73 m², showed a grossly elevated TSH level (>1000 IU/L) and an elevated -subunit level (21.7 mg/L). (These data are not shown in Figure 3.) The mean -subunit level for the remaining nine patients was 3.0 ± 0.6 mg/L (normal, <3 mg/L), and the mean LH-ß and FSH-ß levels were 1.1 ± 0.2 mg/L (normal, 1.0 to 2.1 mg/L) and 2.4 ± 0.4 mg/L [normal 1.6 to 3.8 mg/L], respectively Figure 3, top panel). The LH-ß and FSH-ß levels were below the detection limits of the assays in four patients. Patients with elevated LH levels had higher -subunit, LH-ß, and FSH-ß measurements. Correlations between LH level and LH-ß, FSH-ß, and -subunit levels were 0.91 (P < 0.001), 0.85 (P < 0.001), and 0.60 (P = 0.09), respectively. Correlations between subunits of glycoprotein and creatinine clearance were not significant (P > 0.2).

View larger version (16K): [in this window] [in a new window]   Figure 3. Gonadotropin subunit and bioactive luteinizing hormone measurements in patients with cystinosis. Top. -Subunit (LH ), luteinizing hormone-ß (LH ß), and follicle-stimulating hormone-ß (FSH ß) levels. Bottom. Bioactive and immunoreactive luteinizing hormone (LH) levels in three patients with cystinosis who had normal LH levels and in seven patients with elevated LH levels (closed circle). The line in the bottom panel represents the best least-squares fit to the data (r = 0.89).

Patients with elevated immunoreactive LH levels also had elevated bioactive LH levels Figure 3, bottom panel). The mean ratio of bioactive LH to immunoreactive LH was 3.18 ± 0.32 (normal, 2.5 ± 0.4 [see reference 28]), and the correlation between the bioactive LH level and the immunoreactive LH level was 0.89 (P < 0.001).

Histologic Study

Histologic examination of testicular tissue was done in patient 7, who died of aspiration pneumonia [14]. Light microscopic examination showed germinal dysplasia, increased fibrosis, and Leydig cell hyperplasia Figure 4, top panel]. Under polarized light, many cystine crystals were noted Figure 4, bottom panel).

View larger version (139K): [in this window] [in a new window]   Figure 4. Light microscopic examination of testis specimen from patient 7. Top. Hematoxylin and eosin stain of a formalin-fixed specimen showing increased fibrosis (top arrow), residual seminiferous tubules (bottom arrow), and Leydig cell hyperplasia (upper right). Bottom. Polarized light-bright field examination of absolute alcohol-fixed specimen showing numerous cystine crystals.

Discussion

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Although abnormalities in growth have been well characterized in patients with cystinosis who receive renal allografts [7, 8], reproductive function has not been investigated in this group. We found a high incidence of hypogonadism in 10 men with cystinosis (15 to 28 years old) who received renal transplants. Testis volume was reduced in all patients, and most gave a history suggesting failure of progression of puberty. The bone age was commonly delayed, ranging from 2 to 10 years behind chronologic age.

In one case (patient 5), central hypogonadism was associated with markedly delayed puberty. Whether this was secondary to the underlying disease or chronic rejection remains unclear. Seven of 10 patients had primary testicular failure in association with various reproductive abnormalities (ranging from markedly elevated gonadotropin levels and reduced testosterone levels to azoospermia with normal hormone levels). Only two patients had normal gonadotropin and testosterone levels; both were peripubertal. Two sets of brothers (patients 1 and 5 and patients 3 and 6) were included in the study, and, in each case, reproductive abnormalities were less severe in the younger sibling. Whether this finding was related to earlier detection and treatment of the underlying disease remains to be determined.

The diagnosis of primary hypogonadism was based on several observations in addition to elevated basal gonadotropin levels. The mean LH pulse frequency was increased in the seven patients who underwent frequent sampling [37], both LH and FSH levels increased further with GnRH stimulation [38], and the testosterone responses to HCG stimulation were reduced [39]. Control patients did not undergo stimulation tests, because all 11 had normal testosterone levels and only 3 had minimally elevated gonadotropin levels.

Although our study was not designed to elucidate the mechanisms underlying hypogonadism, the finding of a normal ratio of bioactive LH to immunoreactive LH excluded the possibility that the elevated LH level was secondary to reduced clearance or biologically inactive LH. One patient with severely impaired renal function and untreated hypothyroidism had a markedly elevated -subunit level. Among the remaining nine patients, those with elevated LH levels also had higher -subunit, LH-ß, and FSH-ß levels, and these subunit concentrations were similar to those reported previously in patients with elevated gonadotropin levels [40].

The abnormal reproductive findings were unlikely to be secondary to previous chronic renal insufficiency or caused by the multiple medications administered after transplantation. This was apparent because control patients who were receiving similar immunosuppressive agents exhibited only minimally impaired testicular function. The control group, however, was not perfectly matched to the study group: None of the control patients had a creatinine clearance level less than 38 mL/min per 1.73 m², the youngest control patient was 17 years old, and controls had a greater mean body size than the study patients. Furthermore, similar stimulation tests were not done in the control group.

One factor that may contribute to the primary hypogonadism is the accumulation of cystine in testicular tissue. Histologic study of the testis obtained from one patient showed the presence of many cystine crystals (see Figure 4). The morphology of the testis was consistent with the diagnosis of primary hypogonadism characterized by germinal dysplasia and Leydig cell hyperplasia. Even though cystine deposits have been found in the pituitary gland of a patient with cystinosis and primary ovarian failure, LH and FSH secretion appeared to be unaffected [12].

Hypogonadism, therefore, is another extrarenal manifestation of cystinosis. As more patients with cystinosis reach adulthood, this problem will be encountered more frequently. Affected patients may benefit from testosterone therapy, but prevention, if it is possible, will require long-term cystine depletion with cysteamine, a drug with proven efficacy in preserving renal function and enhancing growth in patients with this systemic disorder [8].