A. Masala

Use of Testosterone To Prevent Cyclophosphamide-Induced Azoospermia

Several cytotoxic agents used to treat neoplastic or immunologic diseases may alter endocrine function in humans [1-5]. In particular, gonadal failure with azoospermia, amenorrhea, or anovulatory cycles has been reported in patients treated for various diseases [1-3]. Cyclophosphamide, an alkylating drug, is widely used as an antineoplastic or immunosuppressive agent. Severe gonadal failure with transient or permanent azoospermia is found in 50% to 90% of men treated with cyclophosphamide [6, 7]. Prepubertal patients who receive large doses of cyclophosphamide seem to recover gonadal function better than adults do; normal pubertal development and normal spermatogenesis have been reported in many of these patients [3, 8-10]. These findings may indicate that active germinal cells are more sensitive to cyclophosphamide because of their elevated mitotic activity. 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 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 Under control conditions, all patients had normal sperm counts; the means were 43.70 6.56 106/mL in group A, 45.52 7.04 106/mL in group B, and 42.91 5.27 106/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 1 106/mL. After therapy was discontinued, sperm counts increased progressively in group C; mean counts were 28.64 3.47 106/mL 3 months after therapy ended and 45.78 3.89 106/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. Table 1. Sperm Counts under Control Conditions and during and after Cyclophosphamide Therapy* 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. Table 2. Serum Follicle-Stimulating Hormone and Luteinizing Hormone Levels under Control Conditions and during and after Cyclophosphamide Therapy* Discussion 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.

Interleukin 1, Interleukin 6, Interleukin 10, and Tumor Necrosis Factor α in Active and Quiescent Systemic Lupus Erythematosus

Objectives Several studies have investigated the cytokine profile of patients with systemic lupus erythematosus (SLE); however, their role is still controversial, mostly because SLE has a heterogeneous disease manifestation. We measured 4 of the most important cytokines in patients with SLE after dividing them in uniform groups according to disease activity and organ involvement. Materials and Methods Eighty-two adult female patients with SLE were divided into 3 groups according to disease activity and organ involvement: Group A (SLE activity index [SLEDAI] score, 7 ± 0.4) included subjects with newly diagnosed, active SLE, investigated before starting therapy. Group B (SLEDAI score, < 6) included patients without renal involvement, treated with prednisone and azathioprine or hydroxychloroquine. Group C (SLEDAI score, < 6) included patients with lupus nephritis, treated with methylprednisolone and cyclophosphamide, reaching complete remission. Fourteen healthy females served as controls. Results Interleukin-1 levels were 1.0, 0.8, 0.7, and 0.25 pg/mL in groups A, B, C, and D, respectively. Interleukin-6 levels were 3.2, 3.6, 4.0, and 1.4 pg/mL in groups A, B, C, and D, respectively; Il-10 levels, 3.05, 1.1, 1.5, and 1.65; tumor necrosis factor-α levels, 8.75, 5.8, 5.4, and 3.6. Interleukin 1, IL-6, and tumor necrosis factor-α were significantly higher in the patients with SLE than in the healthy controls; IL-1 was significantly higher in group A than in group C. Interleukin 10 showed positive correlation with C-reactive protein, whereas it showed negative correlation with C3. Conclusions Data from our cohort, one of the largest so far reported, add to the evidence that proinflammatory cytokines such as Interleukin-1, Interleukin-6, Interleukin-10 and tumor necrosis factor-α are important in SLE pathogenesis.

The sclerodermic hand : A radiological and clinical study

OBJECTIVE To assess the clinical and radiographic features of hand involvement in patients with systemic sclerosis (SSc). METHODS Forty-one unselected Sardinian SSc patients (32 women, 9 men; mean age 58.9, range 31-81 years; mean disease duration 11.8 years, range 1-36 years) were evaluated in this observational cross-sectional study. Twenty-six patients had diffuse scleroderma (dSSc) and 15 limited scleroderma (lSSc). Radiological examination of the hands was performed and the films were read by two independent rheumatologists blinded to the diagnosis using a classification system of four predefined radiological patterns (normal/minimal changes, articular degenerative, articular inflammatory and periarticular pattern). Correlations between radiological pattern, clinical and serological features were assessed. RESULTS The skeletal and articular involvement of the hand was frequent in SSc, being clinically evident in 30/41 (73%) and radiologically in 33/41 (80%) of patients. The periarticular pattern (defined as the occurrence of bone resorption of ungueal tufts, soft tissue calcifications and/or flexion deformities) was the most frequent pattern detected (14/41, 34.1%) and finger flexion contractures and bone resorptions were significantly associated with interstitial lung disease, reduced FVC, oesophagus involvement and prostacycline therapy. Calcinosis (29.2%) was found to be associated with erosions, suggesting a pathogenic link. An inflammatory pattern was also radiologically frequent (8/41, 19.5%), but erosions, with the exception of those localized at distal interphalangeal joints, were demonstrated mainly in patients with clinical picture of rheumatoid arthritis overlapped with SSc. We found no significant differences in terms of radiographic findings between lSSc and dSSc with the exception of calcinosis, which was more frequent in patients with lSSc. CONCLUSION This cross-sectional study confirms that the skeletal and articular involvement of the hand is frequent in SSc.

EFFECT OF METOCLOPRAMIDE ON SERUM PROLACTIN LEVELS IN HUMANS

The effect of acute and chronic administration of metoclopramide on serum prolactin levels in normal subjects was studied. Metoclopramide 10 mg i.v. induced a prompt rise in serum prolactin levels in all subjects. At 180 min the levels remained high. Prolactin levels were markedly elevated during a 5 day course of treatment with metoclopramide in six subjects. It is suggested that metoclopramide could be used in the functional exploration of the hypothalamic‐pituitary axis.

INHIBITION OF LACTATION AND INHIBITION OF PROLACTIN RELEASE AFTER MECHANICAL BREAST STIMULATION IN PUERPERAL WOMEN GIVEN TAMOXIFEN OR PLACEBO

Tamoxifen was given orally to 60 puerperal women to inhibit lactation. Twenty control puerperal women were given placebo. Fifteen women receiving tamoxifen and 15 women receiving placebo were studied before, during and after the use of a breast pump under basal conditions and after five days of treatment. Tamoxifen was effective in inhibiting lactation; no rebound phenomena were observed. Its administration was free from side effects. This drug was capable of preventing the prolactin release induced by mechanical breast stimulation. Placebo failed to inhibit lactation and had no effect on prolactin release induced by the use of a breast pump.

Hormonal Strategies for Fertility Preservation in Patients Receiving Cyclophosphamide to Treat Glomerulonephritis: A Nonrandomized Trial and Review of the Literature

BACKGROUND Prepubertal patients receiving chemotherapy are relatively resistant to cyclophosphamide-induced germinal cell alterations. We studied the possible protective effect of testosterone and triptorelin to inhibit gonadal activity in men and women receiving cyclophosphamide, respectively. STUDY DESIGN Nonrandomized trial. SETTING & PARTICIPANTS 28 consecutive patients, 11 men and 17 women, from a university medical center with various forms of glomerulonephritis, treated with cyclophosphamide. INTERVENTION Men received cyclophosphamide plus testosterone; women were divided into 2 groups: 13 patients (group A) received cyclophosphamide plus triptorelin; 4 (group B) received only cyclophosphamide. OUTCOMES & MEASUREMENTS Serum follicle-stimulating hormone (FSH) and serum luteinizing hormone levels and, in addition, sperm counts and testosterone levels in men and estradiol levels in women were measured before and after treatment with cyclophosphamide. RESULTS All 10 men became azoospermic or severely oligospermic during treatment; after 12 months, all except 1 had a normal sperm count and FSH levels were normal. In women during cyclophosphamide therapy, amenorrhea occurred in all patients. After cessation of therapy, all women in group A started to menstruate regularly, and at the end of follow-up, ovulatory cycles were demonstrated in all women. Hormone levels showed no significant changes throughout the observation period. Six women conceived, and the pregnancies were brought to term successfully without complications. In group B, all 4 women developed sustained amenorrhea; serum FSH and luteinizing hormone levels at the end of therapy and follow-up were significantly higher with respect to baseline; estradiol levels at the end of follow-up were significantly lower compared with baseline and corresponding values in group A. LIMITATIONS The substudy in men is uncontrolled, the substudy in women is nonrandomized. CONCLUSIONS The study suggests a protective effect of testosterone and triptorelin against cyclophosphamide-induced gonadal damage in men and women with various forms of kidney disease, respectively.

Effect of clomiphene citrate on plasma levels of immunoreactive luteinizing hormone-releasing hormone, gonadotropin, and testosterone in normal subjects and in patients with idiopathic oligospermia.

The effect of clomiphene citrate on plasma immunoreactive luteinizing hormone-releasing hormone (LH-RH), gonadotropin, and testosterone levels was investigated in 10 patients with idiopathic oligospermia and in 10 normal volunteers. A daily 100-mg dose of clomiphene citrate induced a marked, significant increase in plasma immunoreactive LH-RH levels, followed by significant increments in gonadotropin and testosterone values. No significant differences were detected in LH-RH, gonadotropin, and testosterone levels between controls and patients with oligospermia either under basal conditions or during clomiphene treatment.

Effects of 12 months rec-GH therapy on bone and collagen turnover and bone mineral density in GH deficient children with thalassaemia major.

Children suffering from thalassaemia major are reported to have growth delay and bone alterations even when well transfused and chelated. In the present study we evaluated bone and collagen turnover (bone Gla-protein, BGP; carboxyterminal telopeptide of type I collagen, ICTP; aminoterminal propeptide of type III procollagen, PIIINP, respectively) and bone mineral density (BMD) in 5 pre-pubertal GH deficient thalassaemic children before and during rec-GH treatment (0.6 IU/kg/week). Data were compared with those recorded in an age- and sex-matched control group. Before treatment, serum BGP and ICTP levels were significantly lower (p<0.0001) in children with thalassaemia (9.3±0.7 ng/ml and 5.3±0.5 ng/ml, respectively) than in healthy controls (18.9±0.9 ng/ml and 14.4±0.6 ng/ml, respectively), while serum PIIINP levels did not significantly differ in the two groups (6.7±0.7 ng/ml vs 6.7±0.7 ng/ml). Mean lumbar BMD values of patients (0.62±0.05 g/cm2) were significantly lower (p<0.05) than those recorded in healthy controls (0.78±0.01 g/cm2), while femoral BMD values were similar in the two groups (patients: 0.70±0.08 g/cm2vs controls: 0.74±0.01 g/cm2). One-year GH therapy significantly increased height velocity (from 2.3±0.2 cm/year to 6.1±0.4 cm/yr, p<0.0001) and IGF-I levels (from 61.6±15.4 to 342±38.5 ng/ml, p<0.005). Serum BGP (basal: 9.3±0.7 ng/ml, 6th month: 10.8±0.6 ng/ml, 12th month: 14.9±1.4 ng/ml), ICTP (basal: 5.3±0.5 ng/ml, 6th month: 7.9±0.8 ng/ml, 12th month: 10.9±1.7 ng/ml) and PIIINP levels (basal: 6.7±0.7 ng/ml, 6th month: 9.9±1.0 ng/ml, 12th month: 9.6±1.4 ng/ml) significantly increased (p<0.05), while no significant effects were observed on lumbar and femoral BMD values. Although the GH-induced stimulation of bone turnover markedly increased BGP (+60%) and ICTP (+105%) levels, one-year GH therapy was not sufficient to completely normalize these parameters, which remained significantly lower than in healthy controls. In conclusion, our study shows that pre-pubertal GH deficient children with thalassaemia major have reduced bone turnover (both bone formation and resorption) and lumbar BMD values, thus indicating that bone metabolism should be monitored and improved even in well-transfused patients. One-year GH treatment is able to increase, but not normalize, bone turnover, this effect being insufficient to improve BMD values. More prolonged periods of GH therapy are probably requested to positively affect both bone turnover and BMD values in GH deficient thalassaemic patients, as occurs in children and adults with GH deficiency.

Dynamic evaluation of prolactin secretion in patients with oligospermia: effects of treatment with metergoline.

Serum prolactin levels were measured in 50 patients with oligospermia and in 20 control subjects under fasting conditions and following the administration of levodopa, pyridoxine, metoclopramide, and synthetic thyrotropin-releasing hormone. Four patients (8%) under fasting conditions had prolactin levels slightly above the normal range. However, no significant differences in prolactin behavior were detected between patients with hyperprolactinemia, patients with normal prolactin levels, and control subjects. The four patients with hyperprolactinemia were treated with metergoline, an ergoline derivative. Metergoline administration promptly reduced the prolactin levels. Spermatogenesis was restored in three patients after 4 to 5 months of treatment.

Evaluation of serum leptin levels and thyroid function in morbidly obese patients treated with bariatric surgery

Biliopancreatic diversion (BPD), a gastrectomy with a long ROUX en Y reconstruction, reduces intestinal absorption by delaying the mixing of food and biliopancreatic juices, and induces persistent weight loss in obese patients unresponsive to medical treatments. The levels of leptin (a plasma protein synthesised in human adipose tissue) are increased in obese subjects and significantly decrease after a major weight loss. A possible role of thyroid hormones in regulating adipose tissue metabolism in humans has been proposed, but it is not universally accepted and the relationship between thyroid function and leptin levels has not yet been clearly defined. We studied serum leptin, TSH, fT4 and fT3 levels in 38 obese patients (26 women and 12 men), before and 12 months after BPD. There was a significant post-surgical decrease in BMI and circulating leptin levels in all of the treated subjects, but thyroid function did not seem to be affected (TSH and fT4 levels were unchanged). However, fT3 levels significantly decreased after surgery. Our data suggest that BPD-induced malabsorption has no direct effect on thyroid function, but possibly reduces the peripheral conversion of thyroxine to T3. Further studies seem to be necessary to clarify the clinical relevance of these observations.