Recovery of Persistent Hypogonadism by Clomiphene in Males with. Prolactinomas under Dopamine Agonist Treatment

Transcription

1 Page of Accepted Preprint first posted on April 00 as Manuscript EJE-0-00 Recovery of Persistent Hypogonadism by Clomiphene in Males with Prolactinomas under Dopamine Agonist Treatment Rogerio Silicani Ribeiro and Julio Abucham Neuroendocrine Unit, Division of Endocrinology Department of Medicine, Escola Paulista de Medicina Universidade Federal de São Paulo 0 ClinicalTrials.gov number: NCT00 0 Keywords: prolactinoma, hypogonadotropic hypogonadism, clomiphene, dopamine agonist, fertility Running title: Clomiphene in Males with Prolactinoma Word count: 0 words Correspondence to: Julio Abucham, MD, PhD Disciplina de Endocrinologia, Escola Paulista de Medicina Rua Pedro de Toledo, 0. São Paulo, Brasil, Fone / fax 0 julioabucham@uol.com.br Copyright 00 European Society of Endocrinology.

2 Page of 0 0 Abstract Context: Persistence of hypogonadism is common in male patients with prolactinomas under dopamine agonist (DA) treatment. Conventional therapy with testosterone causes undesirable fluctuations in serum testosterone levels and inhibition of spermatogenesis. Objective: To evaluate the use of clomiphene as a treatment for persistent hypogonadism in males with prolactinomas. Design: Open label, single-arm, prospective trial. Patients: Fourteen adult hypogonadal males (testosterone <00ng/dL and low/normal LH) with prolactinomas on DA, including seven with high prolactin (range: - µg/l; median: 0 µg/l) despite maximal doses of DA. Intervention: Clomiphene (0mg/day orally) for weeks. Measures: Testosterone, estradiol, LH, FSH and prolactin were measured before and 0 days,, and weeks after clomiphene. Erectile function, sperm analysis, body composition, and metabolic profiles were evaluated before and after clomiphene. Results: Ten patients (%), hyperprolactinemic and normoprolactinemic, responded to clomiphene (testosterone >00ng/dL). Testosterone levels increased from 0± ng/dl to ± ng/dl, ± ng/dl, and 0± ng/dl at, and weeks, respectively (0.00<P<0.0). Estradiol increased significantly and peaked at weeks. LH increased from.±0. IU/l to.±.0 IU/l,.±0. IU/l, and.±0. IU/l at, and weeks, respectively (0.00<P<0.0). FSH levels increased in a similar fashion. Prolactin levels remained unchanged. Erectile function improved (P<0.0) and sperm motility increased (P<0.0) in all patients with asthenospermia before clomiphene. Conclusions: Clomiphene restores normal testosterone levels and improves sperm motility in most male patients with prolactinomas and persistent hypogonadism under DA therapy. Recovery of gonadal function by clomiphene is independent of prolactin levels.

3 Page of Introduction Prolactinomas cause hypogonadotropic hypogonadism in both sexes. Medical treatment with dopamine agonists corrects hyperprolactinemia, decreases tumor size, and restores gonadal function in most patients. However, 0-0% of male patients with prolactinomas under dopamine agonist treatment, both with normal and high prolactin levels, still remain hypogonadal. - Persistent hypogonadism in these patients is treated with testosterone replacement, most often with intramuscular injections that require frequent applications and induce large fluctuations in serum testosterone levels with corresponding fluctuations in patients energy, libido, sexual performance and mood., In addition, testosterone 0 replacement has an inhibitory effect on spermatogenesis and fertility that is undesirable in patients wishing to have children. 0 Clomiphene citrate is a well known selective estrogen receptor modulator that increases gonadotropin secretion via hypothalamic-pituitary action. Clomiphene has already been extensively used in the evaluation of the gonadotropic axis and in the induction of ovulation. Clomiphene has also been shown to revert hypogonadotropic hypogonadism in several conditions like falciform anemia, uremia, alcohol and steroid abuse, and to stimulate gonadotropin secretion in patients with sulpiride-induced hyperprolactinemia and gonadotropin suppression. 0- We conducted a prospective, open label clinical trial to evaluate the use of clomiphene as a treatment for persistent hypogonadism in male patients with prolactinomas under dopamine agonist therapy (ClinicalTrials.gov number: NCT00). Methods Subjects

4 Page of 0 Sixteen out of (%) consecutive adult male patients with prolactinomas under dopamine agonist therapy, seen between June/00 and May/00 at the Neuroendocrine Unit of the Endocrinology Division, Escola Paulista de Medicina - Universidade Federal de São Paulo, presented persistent hypogonadism and were considered to enter the protocol. Persistent hypogonadism was defined by total testosterone below 00 ng/dl (0. nmol/l) either normal/low LH and FSH levels after at least months of dopamine agonist therapy and normal serum prolactin levels or high serum prolactin levels under maximal dopamine agonist doses (0 mg/day of bromocriptine or. mg/week of cabergoline for at least months). Patients on testosterone replacement were evaluated after discontinuing testosterone for at least months. All patients had normal serum thyroxine (nine on levothyroxine) and were on regular glucocorticoid replacement therapy. One patient with previous noncompliance to dopamine agonist treatment was not included and another one left the study between and weeks and returned to testosterone replacement due to erectile dysfunction. All fourteen patients who completed the study were analyzed. 0 Study design and protocol This study was an interventional, open label, uncontrolled, single-arm, prospective trial of clomiphene citrate (Clomid, Medley, kindly provided by Dr Enrico Repetto) (0 mg/day orally for weeks) designed to assess its effects on serum testosterone levels in male patients with prolactinomas and persistent hypogonadism during dopamine agonist therapy. As secondary aims, the effects of clomiphene on sexual function, sperm analysis, body composition, lipid and glucose metabolism, and quality of life were also evaluated. Physical examination, erectile function and quality of life questionnaires, serum hormone measurements (testosterone, SHBG, estradiol, prolactin, LH and FSH), PSA, lipid (total, LDL and HDL cholesterol, and triglycerides), serum glucose and insulin (before and after oral glucose), body composition, and sperm analysis (after days of sexual abstinence) were all performed before and at the end of weeks of

5 Page of clomiphene treatment. In addition, hormonal evaluations were performed at 0 days and at and weeks of treatment and after discontinuing clomiphene for weeks. The study protocol was approved by the institution s ethical committee and a written informed consent was obtained from all participants. 0 Assays Serum testosterone, SHBG, estradiol, prolactin, LH, FSH, insulin and PSA were measured in fasting serum by automated immunochemoluminescent assays (ADVIA Centaur, Bayer). Free testosterone values were calculated from total testosterone and SHBG results using a fixed albumin level as previously described. Glucose was measured in plasma using an enzymatic method (Hexokinase II). Total cholesterol and triglycerides were measured in plasma by peroxidase colorimetric immunoenzymatic method. HDL cholesterol was measured in serum using immunoenzimatic method. LDL and VLDL were calculated by the Friedwald equation. Body composition Body composition [lean and fat mass and bone mineral content (BMC)] was determined by dual-energy x- ray absorptiometry (QDR 000-Plus, Hologic), according to ICSD recommendations. 0 Questionnaires Erectile function was evaluated using a -question questionnaire (IIEF, international index of erectile function). Quality of life was evaluated using a -question questionnaire (WHOQOL-BREF, World Health Organization - Quality of Life). Both questionnaires have been previously validated in portuguese., Statistical analysis

6 Page of 0 Patients were classified as responders and nonresponders according to their testosterone levels during treatment (>00 ng per deciliter or 0. nmol/l) in at least of measurements between and weeks of clomiphene. Considering baseline testosterone, a sample size of patients has a % power to detect a normalization of mean testosterone. Statistical analyses were carried out using ANOVA or Friedman tests, according to data distribution, followed by Bonferroni or Dunn s post-tests, respectively. Comparisons within the same group were performed by paired t-test or Wilcoxon signed-rank test according to data distribution. Comparisons between two independent groups were performed by unpaired t-test or Mann- Whitney test, as appropriate. Correlations between two variables were calculated by Pearson coefficient (r). Statistical significance was set at P<0.0. Data are presented as mean±se or as median when indicated. Data were analyzed using GraphPad Prism.0 and GraphPad StatMate. Results 0 Baseline characteristics Among the fourteen patients who concluded the study (age: - years; median: years), had macroadenomas and had microadenomas at diagnosis; patients were on bromocriptine and were on cabergoline; patients had normal prolactin levels for at least months before the study (range - months; median. months) and still had high prolactin levels (range: - µg/l; median: 0 µg/l) on maximal doses of dopamine agonist for at least two months before entering the study (range: - months). Testosterone had been withdrawn for at least months (range: - months, median: months) in all patients receiving testosterone replacement before entering the study. Six patients with macroprolactinomas had been previously submitted to pituitary surgery. Effects of clomiphene on serum testosterone, estradiol and gonadotropin levels

7 Page of Ten patients (%) were considered responders to clomiphene, including with hyperprolactinemia (range: µg/l, median: 0 µg/l); patients (%) were considered nonresponders, including patients with hyperprolactinemia ( and, µg/l). 0 As shown in Figure A, serum testosterone levels in responders to clomiphene increased from 0± ng/dl (.0±0. nmol/l), at baseline, to ± ng/dl (.±. nmol/l) at 0 days (P>0.0), ± ng/dl (±. nmol/l) at weeks (P<0.00), 0± ng/dl (±. nmol/l) at weeks (P<0.00) and ± ng/dl (±. nmol/l) at weeks (P<0.00). Testosterone levels remained unchanged between and weeks of clomiphene (P=0.). Serum SHBG and free testosterone levels, evaluated at baseline and at weeks, also increased significantly (P<0.0 and P<0.0, respectively), from ±. nmol/l to ±. nmol/l and from.±0. ng/dl (0.±0.0 nmol/l) to.±.0 ng/dl (0.±0.0 nmol/l), respectively. At weeks of clomiphene, free testosterone levels were within the normal range in all responders. Serum estradiol levels increased from.±. pg/ml (.±. pmol/l) at baseline to. ±. pg/ml (.±0. pmol/l) at 0 days (P>0.0),.±. pg/ml (.±. pmol/l) at weeks (P>0.0),.±. pg/ml (.± pmol/l) at weeks (P<0.0) and.0±. pg/ml (.± pmol/l) at weeks of clomiphene (P<0.0) (Figure B). 0 As shown in Figure C, serum LH levels increased from.±0. IU/L to.0±0. IU/L at 0 days (P<0.0),.±.0 IU/L at weeks (P<0.00),.±0. IU/L at weeks (P<0.0) and.±0. IU/L at weeks (P<0.0). Serum FSH levels increased from.±. IU/L to.±. IU/L at 0 days (P>0.0),.±. IU/L at weeks (P<0.0),.±. IU/L at weeks (P<0.0) and.±. IU/L at weeks (P<0.0) (Figure D). Both LH and FSH levels remained unchanged between and weeks of clomiphene (P=0. and P=0., respectively). After weeks of clomiphene withdrawn, total testosterone, LH and FSH levels decreased significantly to pretreatment levels [± ng/dl (.±0. nmol/l),.±0. IU/L, and ±. IU/L, respectively, P<0.0].

8 Page of Among responders, hyperprolactinemic patients tended to have lower testosterone and LH levels as compared to normoprolactinemic patients before clomiphene [± ng/dl (.± nmol/l) versus ± ng/dl (.±0. nmol/l), P=0.0, and.0±0. IU/L versus.±0. IU/L, P=0.0, respectively], but not after - weeks of clomiphene (P=0. and P=0., respectively). To further analyze the influence of baseline prolactin status on the response to clomiphene, comparisons of testosterone levels at baseline and along clomiphene treatment between all hyperprolactinemic (n=) and all normoprolactinemic patients (n=) were performed, but failed to show any significant differences at any time point (0.<P<0.; unpaired t test). 0 As shown in Table, responders showed significantly higher total and free testosterone levels before treatment as compared to nonresponders, whereas LH and FSH levels tended to be higher, but no significant differences were observed in other characteristics. Both total and free testosterone levels at baseline significantly correlated (r=0., P=0.00, and r=0.0, P=0.00, respectively) with mean serum total testosterone levels during clomiphene treatment (Figure ). 0 Effects of clomiphene on serum prolactin and PSA levels In responders, serum prolactin levels showed no significant differences during clomiphene (P=0.), even when normoprolactinemic and hyperprolactinemic patients were analyzed in separate (P=0. and P=0., respectively), and no patient changed prolactin status. PSA levels also remained unchanged during treatment with clomiphene (P=0.). Effects of clomiphene on erectile function and sperm analysis As shown in Figure, erectile function scores (IIEF) increased significantly in responders, from ± to ± (P<0.0). In all patients with asthenospermia before treatment, sperm motility increased from ±% to ±% after weeks of clomiphene (P<0.0). Sperm count was normal in all but one of these

9 Page of patients and remained unchanged after clomiphene. In the patients not analyzed, two had vasectomy, one had aspermia, and another one had normal sperm analysis before clomiphene. Effects of clomiphene on body composition In responders, a significant increase in bone mineral density was observed after clomiphene (.±0.0 g/cm versus.±0.0 g/cm, P<0.0), but no significant changes were observed in BMI (.±. kg/m versus.±. kg/m, P=0., t test), fat-free mass (±. kg versus 0±. kg, P=0.) or fat mass (.±. kg versus.±. kg, P=0.). However, responders tended to gain and nonresponders tended to loose fat-free mass after clomiphene treatment (.0±0. kg versus -.0±. kg, P=0.0). 0 Metabolic effects In responders, no significant variations in weight, abdominal circumference, blood pressure, fasting glucose, insulin, HOMA-IR, total cholesterol and cholesterol fractions were observed after weeks of clomiphene treatment. Serum triglycerides decreased significantly [±0 mg/dl (±0. mmol/l) versus ±0 mg/dl (.±0. mmol/l) at weeks, P=0.0]. Serum glucose at 0 minutes after OGTT increased significantly at weeks of clomiphene [0± mg/dl (.±0. mmol/l) versus ± mg/dl (.±0. mmol/l), P<0.0], but no patient presented glicemia >00 mg/dl [highest value: mg/dl (. mmol/l)] at 0 min. Serum glucose at 0 minutes of OGTT before and after clomiphene correlated significantly (r=0., P=0.0). 0 Discussion In this study, we have shown that chronic administration of clomiphene citrate was able to recover gonadal function in male patients with prolactinomas and persistent hypogonadotropic hypogonadism

10 Page 0 of under dopamine agonist treatment. Testosterone levels reached the normal range in nearly half of the responders as soon as 0 days after clomiphene was started, but only at weeks had all responders reached normal testosterone levels which remained stable thereafter. Thus, a -week trial of clomiphene is able to identify most responders. Free testosterone levels also increased to the normal range in all responders, in spite of the significant increase in serum SHBG levels. When clomiphene was discontinued for weeks, testosterone levels returned to the hypogonadal range in all responders, supporting both the therapeutic effect of clomiphene and the persistent nature of hypogonadism in those patients. 0 In responders, both erectile function and sperm motility also showed significant improvement during clomiphene treatment. Decreased motility is the most prevalent abnormality observed in sperm analysis of patients with untreated prolactinomas and may persist well beyond normalization of prolactin and testosterone levels with dopamine agonists. Improvement of sperm motility by clomiphene is likely to benefit fertility, in contrast with conventional testosterone replacement, which improves erectile function but usually decreases fertility. As expected, estradiol levels also increased during clomiphene treatment, but only two of the ten responders showed slightly elevated estradiol levels (individual data not shown) and none developed gynecomastia. In these two patients, it is likely that lowering the dose of clomiphene could keep both testosterone and estradiol levels within the normal ranges. 0 At the end of the -week clomiphene treatment, responders also showed increased bone mineral density, reflecting a direct agonistic effect of clomiphene and/or of increased estradiol levels. Although the expected changes in fat and fat-free masses were not significant during this period of time, responders tended to gain fat-free mass as compared to nonresponders. Serum triglycerides decreased, whereas fasting glucose and insulin levels and HOMA-IR were not affected by clomiphene treatment. A slight increase in serum glucose levels at 0 min after glucose was observed in responders, but no patient reached diagnostic criterion for diabetes mellitus. This finding is likely to reflect an antiestrogenic activity of clomiphene impairing glucose uptake. In male patients with inactivating mutations of estrogen receptor

11 Page of or aromatase, insulin resistance is increased and estradiol replacement improves insulin sensitivity in patients with aromatase deficiency. 0, 0 The increase in testosterone levels during clomiphene treatment was brought about by its effects on gonadotropin secretion and not by decreasing prolactin levels as shown by increased LH and FSH and unchanged prolactin levels. In addition, clomiphene was equally effective in recovering gonadal function in both normoprolactinemic and hyperprolactinemic patients under dopamine agonist treatment and no hyperprolactinemic patient became normoprolactinemic during clomiphene. Nevertheless, tamoxifen, another selective estrogen receptor modulator, has been reported to reduce prolactin secretion and tumor size in a few patients with resistant pituitary prolactinomas. Estrogen seems to be physiologically more relevant than testosterone in the negative feed-back control of LH secretion in males. Accordingly, patients with inactivating mutations of the estrogen receptor or aromatase exhibit increased levels of gonadotropins and testosterone. 0, Also, circulating rather than locally-generated estradiol (from testosterone aromatization) inhibits LH secretion in males by acting at both hypothalamus and pituitary, but the major effect is through inhibition of hypothalamic GnRH release. Testosterone, on the other hand, exerts its own negative feed-back on LH secretion through androgenic receptors at the hypothalamus. 0 The ultimate in vivo effects of clomiphene, an estrogen receptor agonist/antagonist, are dependent on dose, target tissue and endogenous estrogenic activity. At low doses ( to 0 mg/day), clomiphene is reportedly ineffective in prepubertal boys, but stimulates gonadotropin secretion in pubertal and adult males. At high doses (00 mg/day), clomiphene suppresses gonadotropin secretion even in normal adult males., In our study, a relatively low dose of clomiphene (0 mg/day) was able to increase and sustain serum gonadotropins and testosterone levels even in face of raising estradiol levels, which indicates an

12 Page of anti-estrogenic effect at the hypothalamus and/or pituitary. These results are completely in agreement with a major role of estrogen in the inhibitory feed-back of gonadotropin secretion in males. 0 The gonadotropin-stimulating effect of clomiphene also indicates the presence of a critical inhibitory estrogen activity at the hypothalamus and/or pituitary in responders that was effectively antagonized by clomiphene. However, no difference in serum estradiol was detected between responders and nonresponders, probably due to the insufficient sensitivity and accuracy of the estradiol assay. Notwithstanding, serum testosterone, the major source of estradiol in males, was significantly higher before treatment in responders as compared to nonresponders and strongly correlated with serum testosterone levels during clomiphene treatment. These data strongly suggest that the recovery of gonadal function by clomiphene in male patients with prolactinomas requires a critical hypothalamic-pituitary reserve. In conclusion, clomiphene is an effective treatment for persistent hypogonadism in male patients with prolactinomas under dopamine agonist treatment irrespective of serum prolactin levels. By restoring endogenous testosterone production and improving sperm motility, clomiphene is potentially beneficial in patients desiring to have children or exhibiting large fluctuations in serum testosterone levels under usual testosterone preparations.

13 Page of Declaration of interest: The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported. This work was supported by a PhD grant from CAPES/PROEX (RSR). Funding: This research did not receive any specific grant from any funding agency in the public, commercial or not-for-profit sector.

14 Page of 0 0 References. Gillam MP, Molitch ME, Lombardi G & Colao A. Advances in the treatment of prolactinomas. Endocrine Reviews Hulting AL, Muhr C, Lundberg PO & Werner S. Prolactinomas in men: clinical characteristics and the effect of bromocriptine treatment. Acta Medica Scandinavica Pinzone JJ, Katznelson L, Danila DC, Pauler DK, Miller CS & Klibanski A. Primary medical therapy of micro- and macroprolactinomas in men. Journal of Clinical Endocrinology and Metabolism Colao A, Vitale G, Cappabianca P, Briganti F, Ciccarelli A, De Rosa M, Zarrili S & Lombardi G. Outcome of cabergoline treatment in men with prolactinoma: effects of a -month treatment on prolactin levels, tumor mass, recovery of pituitary function, and semen analysis. Journal of Clinical Endocrinology and Metabolism Sibal L, Ugwu P, Kendall-Taylor P, Ball SG, James RA, Pearce SH, Hall K & Quinton R. Medical therapy of macroprolactinomas in males: I. Prevalence of hypopituitarism at diagnosis. II. Proportion of cases exhibiting recovery of pituitary function. Pituitary Chattopadhyay A, Bhansali A & Masoodi SR. Long-term efficacy of bromocriptine in macroprolactinomas and giant prolactinomas in men. Pituitary Gillam MP, Middler S, Freed DJ & Molitch ME. The novel use of very high doses of cabergoline and a combination of testosterone and an aromatase inhibitor in the treatment of a giant prolactinoma. Journal of Clinical Endocrinology and Metabolism Bhasin S, Cunningham GR, Hayes FJ, Matsumoto AM, Snyder PJ, Swerdloff RS & Montori VM. Testosterone therapy in adult men with androgen deficiency syndromes: an endocrine society clinical practice guideline Journal of Clinical Endocrinology and Metabolism Rhoden EL & Morgentaler A. Risks of testosterone-replacement therapy and recommendations for monitoring. New England Journal of Medicine

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17 Page of 0 Figure. Response of serum testosterone, estradiol, LH and FSH levels to clomiphene (0mg/day) in male patients with prolactinomas and persistent hypogonadism under dopamine agonist therapy. Legend: Responders (R, solid squares) were defined as patients who showed serum testosterone levels >00 ng/dl during clomiphene. Responders showed significant increases in serum testosterone, estradiol, LH and FSH as compared to baseline levels (*P<0.0, **P<0.0, ***P<0.00). After clomiphene withdrawn, all hormones decreased to pretreatment levels. N=0. Nonresponders (NR, open squares) did not reach testosterone levels >00 ng/dl during clomiphene and showed no significant changes in testosterone, estradiol, LH and FSH levels as compared to baseline levels. To convert the values for testosterone to nmol/l, multiply by 0.0. To convert the values for estradiol to pmol/l, multiply by.. N=. 0 Figure. Correlation between pretreatment (baseline) serum testosterone and mean testosterone levels during clomiphene treatment. Legend: To convert the values for testosterone to nmol/l, multiply by 0.0. Figure. Effect of clomiphene (0 mg/day for weeks) on erectile function and sperm motility* in responders with asthenospermia before treatment. Legend: *sperm motility was analyzed in out of 0 responder patients after exclusion of patients with vasectomy, one with aspermia, and one with normal sperm analysis.

18 Page of Responders (R, solid squares, n= 0) were defined as patients who showed serum testosterone levels >00 ng/dl during clomiphene. Responders showed significant increases in serum testosterone, estradiol, LH and FSH as compared to baseline levels (*P<0.0, **P<0.0, ***P<0.00). After clomiphene withdrawn, all hormones decreased to pretreatment levels. Nonresponders (NR, open squares, n=) did not reach testosterone levels >00 ng/dl during clomiphene and showed no significant changes in testosterone, estradiol, LH and FSH levels as compared to baseline levels. To convert the values for testosterone to nmol/l, multiply by 0.0. To convert the values for estradiol to pmol/l, multiply by.. xmm (00 x 00 DPI)

19 Page of *sperm motility was analyzed in out of 0 responder patients after exclusion of patients with vasectomy, one with aspermia, and one with normal sperm analysis. x0mm (00 x 00 DPI)

20 Legend: To convert the values for testosterone to nmol/l, multiply by 0.0. xmm (00 x 00 DPI) Page 0 of

21 Page of Table. Baseline characteristics of male patients with prolactinomas and persistent hypogonadism under dopamine agonist treatment according to response to clomiphene. Characteristic Responders Non responders (n=0) (n=) P Age (years) +, Hyperprolactinemia /0 /.0 Macroadenomas /0 /.0 Previous apoplexy /0 /.0 Previous surgery /0 / 0. Previous testosterone replacement /0 /.00 Thyroxine replacement /0 / 0. Glucocorticoid replacement /0 / 0. Testosterone (ng per deciliter) 0 ± ± 0.0 Free testosterone (ng per deciliter). ± 0..0 ± LH (IU/L), + 0, 0, ± 0, 0.0 FSH (IU/L), +,, ± 0, 0.0 To convert the values for testosterone to nmol/l, multiply by 0.0.