Leuteinizing hormone responses to leuprolide acetate discriminate between hypogonadotropic hypogonadism and constitutional delay of puberty

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1 FERTILITY AND STERILITY VOL. 77, NO. 3, MARCH 2002 Copyright 2002 American Society for Reproductive Medicine Published by Elsevier Science Inc. Printed on acid-free paper in U.S.A. Leuteinizing hormone responses to leuprolide acetate discriminate between hypogonadotropic hypogonadism and constitutional delay of puberty Maria Elisabeth Street, M.D., a Maria Antonietta Bandello, M.D., a Cesare Terzi, M.D., a Lourdes Ibañez, M.D., b Lucia Ghizzoni, M.D., a Cecilia Volta, M.D., a Candida Tripodi, M.D., a and Raffaele Virdis, M.D. a Department of Paediatrics, University of Parma, Parma, Italy Objective: To assess if leuprolide acetate stimulation discriminates between hypogonadotropic hypogonadism (HH) and constitutional delay of puberty (CDP) in males. Design: Case-control study. Setting: Patients attending an academic research environment. Patients: Only male patients were studied: 10 with HH (group 1, age years), 8 prepubertal with CDP (group 2, age years), 6 healthy prepubertal (group 3, age years), and 8 healthy late-pubertal (group 4, age years). Intervention(s): Blood samples were obtained after an overnight fast. Leuprolide acetate was then administered SC, and blood samples were drawn at 0, 30, 60, 120, 180 minutes, and 6 and 24 hours after stimulation. Main Outcome Measure(s): Clinical follow-up evaluations of data and serum levels of LH, FSH, 17- hydroxyprogesterone, and testosterone. Result(s): Basal LH levels were similar in groups 1 through 3 and differed significantly from those in group 4. Peak serum LH levels were significantly higher in CDP compared with HH ( vs IU/L). Baseline FSH levels were significantly higher only in pubertal boys (versus the HH group); peak levels did not differ among the groups. Basal and peak testosterone levels were significantly higher only in the control pubertal group when compared to the other groups; peak 17-hydroxyprogesterone concentrations were significantly higher in pubertal controls compared with HH and CDP. Conclusion(s): Peak LH responses clearly discriminate HH from CDP. Timing for blood sampling should be fixed at 0, 60, 120, 180 minutes after stimulation. (Fertil Steril 2002;77: by American Society for Reproductive Medicine.) Key Words: GnRH agonist, leuprolide acetate, constitutional delay of puberty, delayed puberty, hypogonadotropic hypogonadism, diagnosis of delayed puberty Received March 20, 2001; revised and accepted September 12, Reprint requests: Raffaele Virdis, M.D., Dipartimento Materno-Infantile, Clinica Pediatrica, Via Gramsci, 14, Parma, Italy (FAX: ; ravirdis@unipr.it). a Department of Paediatrics. b Hospital Sant Joan de Déu, Barcelona, Spain /02/$22.00 PII S (01) Discriminating between constitutional delay of puberty (CDP) and hypogonadotropic hypogonadism (HH) remains problematic in the absence of signs of pubertal development. Constitutional delay of puberty is the commonest cause of sexual immaturity in males, and is a benign condition characterized by delayed but otherwise normal puberty. In contrast, HH is usually a permanent condition; it is associated with infertility and requires specific treatment. The sleep-related increase in LH concentrations that characterizes the onset of normal puberty has been reported to discriminate between HH and CDP (1). Most HH males do not experience an increase in LH during sleep, whereas CDP children usually do (1, 2). However, the sleep test is cumbersome and expensive, and thus is not easily applicable in clinical practice. Previous studies assessing the usefulness of measuring basal and stimulated testosterone and gonadotropin levels via ultrasensitive assay methods have had poor results (3). Considering that the congenital lack of GnRH in HH would be characterized by a reduced stimulation of the pituitary gland with respect to both 555

2 CDP and normal individuals who have intact hypothalamic function, other investigators (4, 5) have hypothesized that exogenous administration of GnRH would elicit a greater pituitary response in HH. Consequently, a series of different stimulation tests were designed to study these patients. First, the circadian variation in gonadotropin levels, followed by a stimulation test with GnRH alone or preceded by small doses of the releasing factor administered in a pulsatile fashion were studied (6). Although the results were satisfactory, the tests lasted approximately 36 hours and were not easily reproducible unless performed in highly specialized environments. Metoclopramide and thyroid-stimulating releasing hormone stimulation of prolactin have also been used (7, 8) without any significant improvement in the ability to discriminate between HH and CDP. More recently, Ehrmann et al. (5) showed that the GnRH agonist nafarelin was more potent than native GnRH in eliciting first a pituitary and subsequently a gonadal response, allowing the assessment of both pituitary and gonadal function. Results were promising, and further studies with this (9, 10) and other GnRH agonists (11, 12) confirmed the initial results. The aim of the present study was to assess the usefulness of the GnRH agonist leuprolide acetate to discriminate between HH and CDP in males. MATERIALS AND METHODS Patients The study population consisted of 32 males who were subdivided into four groups. Group 1 consisted of 11 boys with HH (age years; range: 13 to 29.3 years). Group 2 was seven boys with CDP (age years; range: 13.5 to 15.3 years). Individual baseline clinical characteristics of these patients are reported in Table 1. Pubertal stage was classified according to Tanner s criteria (13); bone age was delayed in all by 2 years on average (mean years). Two comparison groups were tested. Group 3 was six prepubertal healthy boys (age, years; range: 7.5 to 12.5 years, Tanner stage I). Group 4 was eight pubertal healthy boys (age years; range: 12.9 to 16.8 years, Tanner stage III and IV). Body mass indexes were similar in the four groups: group 1, kg/m 2 ; group 2, kg/m 2 ; group 3, kg/m 2 ; and group 4, Kg/m 2. A 4-year follow-up study of the patients confirmed the correct diagnosis of HH or CDP. Study Protocol Blood samples were obtained in the supine position and after an overnight fast, starting between 8:00 and 9:00 AM. Leuprolide acetate (500 g of Procrin; Abbott, Madrid, Spain) was then administered SC, and blood samples were TABLE 1 Chronological age, pubic hair stage, and testicular volume according to Tanner s criteria, presence or absence of anosmia, and family history of delayed puberty in patients with hypogonadotropic hypogonadism (numbers 1 to 11) and with constitutional delay of puberty (numbers 12 to 18). HH CA Pubic hair Testicular volume (ml) Anosmia Family history 1 29,33 PH2 3 No Negative 2 14,75 PH2 2 No Positive 3 13,00 PH1 2 No Negative 4 15,44 PH1 2 No Negative 5 13,83 PH2 1 No Negative 6 15,00 PH2 2 No Negative 7 17,83 PH2 3 No Negative 8 16,50 PH1 1 No Negative 9 14,18 PH1 2 Yes Negative 10 16,25 PH1 3 No Negative 11 14,58 PH1 2 No Unknown CDP 12 13,50 PH2 2 No Negative 13 14,08 PH2 3 No Positive 14 14,77 PH2 2 No Negative 15 15,33 PH2 3 No Positive 16 13,50 PH2 3 No Negative 17 14,70 PH2 2 No Negative 18 14,18 PH1 3 No Negative CA: chronological age, HH: hypogonadotropic hypogonadism, CDP: constitutional delay of puberty. drawn from an indwelling catheter positioned in an antecubital vein at 0, 30, 60, 120, 180 minutes, and 6 and 24 hours after stimulation. The timing of blood sampling was based on previously published data showing that the gonadotropin peak occurs 1 to 6 hours after stimulation and the steroid peak 20 to 24 hours later (14, 16). The blood samples were immediately centrifuged, and serum was kept at 20 C until assayed. The protocol was approved by the ethics committee at the Department of Paediatrics in Parma. Institutional review board approval was also obtained. The leuprolide acetate test has been used in our department since 1992 to diagnose functional ovarian hyperandrogenism (15). Hormonal Assays We measured LH and FSH levels by the use of standard commercial RIA kits (Biomérieux, France). The mean intraassay and interassay coefficients of variation (CV) for LH were 4.8% and 6.1% (calculated at 18.2 and 57 IU/L), respectively; for FSH they were 3.9% and 5.7% (calculated at 10.6 and 24.2 IU/L), respectively. Testosterone (T) and 17-hydroxyprogesterone (17-OHP) 556 Street et al. Stimulated LH discriminates delayed puberty Vol. 77, No. 3, March 2002

3 TABLE 2 Baseline hormonal levels, and peak and incremental ( ) responses to leuprolide acetate stimulation in the study population. Group 1 (HH) (n 10) Group 2 (CDP) (n 8) Group 3 (prepubertal) (n 6) Group 4 (pubertal) (n 8) FIGURE 1 Peak serum LH levels after leuprolide acetate stimulation in patients with hypogonadotropic hypogonadism (HH), constitutional delay of puberty (CDP), and in normal prepubertal and pubertal boys. Values are clearly different in HH and CDP: 2.8 IU/L is the upper limit in HH and 6.1 IU/L is the lower limit in CDP. LH (IU/L) Baseline a Peak b c e d FSH (IU/L) Baseline a Peak T (ng/ml) Baseline a Peak a OHP (ng/ml) Baseline Peak f d T: testosterone, 17-OHP: 17 -hydroxyprogesterone, HH: hypogonadotropic hypogonadism, CDP: constitutional delay of puberty. a P.05 versus groups 1, 2, and 3. b P.05 versus HH. c P.05 versus CDP. d P.05 versus HH. e P.05 versus groups 1 and 4. f P.05 versus groups 1 and 2. were measured by RIA as previously described (16, 17). The intra-assay and interassay coefficients of variation were 3.8% and 8.7% for testosterone, and 1.8% and 6.4% for 17-OHP, respectively. Statistical Analysis Auxological data are expressed as mean SD. Hormonal data are reported as mean SEM. The differences within each group were assessed using the t-test, whereas differences among independent groups were studied using an ANOVA test, followed by Bonferroni s test. Correlation analysis was done using Pearson s linear regression analysis. P.05 was considered statistically significant. RESULTS Baseline hormonal levels, peak gonadotropin and steroid responses to leuprolide acetate, and the differences between baseline and peak hormonal levels ( values) after leuprolide acetate stimulation are depicted in Table 2. Baseline LH levels were discriminative only in pubertal controls (group 4), who in turn showed the highest peak LH responses after leuprolide acetate stimulation (Fig. 1). The CDP group showed intermediate peak LH responses between those found in HH boys and in pubertal controls. Peak serum LH levels occurred between 120 and 180 minutes after stimulation in all four groups. Basal LH values were significantly different from peak values in each subgroup; the increase after leuprolide acetate stimulation was modest in HH and prepubertal boys, and greater in CDP and pubertal boys (Fig. 2). The analysis of the values showed that group 4 (pubertal controls) and group 1 (HH) had the highest and lowest values, respec- FERTILITY & STERILITY 557

4 FIGURE 2 Individual baseline (b) and stimulated LH (p) levels in boys with hypogonadotropic hypogonadism (HH) and constitutional delay of puberty (CDP) compared to mean basal and peak serum LH levels in normal prepubertal and pubertal males. were only significant in pubertal boys; similar values were found among the groups. Basal 17-OHP levels were similar in the four groups. Peak values were significantly higher in pubertal boys compared to boys with HH and CDP, whereas the values were significantly higher in pubertal controls compared to HH boys. Correlation analysis confirmed that basal, peak, and values of each hormone were correlated, as expected. No other statistically significant correlations were detected between basal, peak, and values. DISCUSSION tively, whereas group 2 (CDP) showed intermediate increases. As expected, prepubertal boys showed a smaller increase in LH levels after stimulation compared to pubertal boys. Baseline FSH levels were only discriminative in pubertal controls (group 4), whereas peak FSH levels did not differ among groups. Basal FSH values were significantly different from peak values in HH, CDP, and prepubertal boys, but not in pubertal controls; values did not differ among the groups. Basal and peak testosterone levels were significantly higher in the control pubertal boys (group 4) compared to the other groups. Differences between basal and peak levels Peak LH responses to acute leuprolide acetate stimulation clearly discriminated between HH and CDP, with no overlap in the hormonal levels. In contrast, neither baseline hormonal levels nor 24-hour testosterone concentrations were capable of differentiating these two clinical conditions. Both CDP and HH have clinical characteristics that, if present, may be useful for diagnosis. A positive family history, a slow prepubertal growth with skeletal delay, or the presence of chronic diseases are among the hallmarks of CDP. However, HH is characterized by the absence of previous symptoms and by the frequent association with genetic syndromes. A history of delayed puberty has been also reported in HH; in our series, pubertal delay could only be evidenced in one patient (18). Moreover, considered individually, all these data were inconsistent in our patient population. Similarly, basal hormonal values have been previously shown not to discriminate between HH and CDP, even if assayed with ultrasensitive methods (3). The leuprolide acetate stimulation test relies on the hypothesis that patients with CDP have intact hypothalamicpituitary-gonadal function, and thus a greater capacity to produce gonadotropins due to the priming effect exercised by GnRH in the perinatal period on gonadotropin-producing cells (4, 5). However, although native GnRH is generally not capable of eliciting a discriminatory hormonal response, GnRH agonists can (5, 9, 10, 12), because they are more potent and specific agents. These agonists have a longer half-life in the circulation compared to native GnRH (5). This capacity determines a greater and more persistent response of gonadotropins, in particular of LH, which is sequentially followed by gonadal stimulation and production of sex steroids. In the present study, testosterone responses to GnRH analog stimulation proved to be less reliable than LH responses for discriminating CDP from HH. As expected, HH individuals did not increase testosterone levels after GnRH agonist stimulation, whereas most of those with CDP did. However, some boys with CDP failed to respond, and 558 Street et al. Stimulated LH discriminates delayed puberty Vol. 77, No. 3, March 2002

5 showed testosterone levels overlapping with those of HH patients. As a group, CDP boys showed testosterone responses that were lower than those reported by other investigators who had used different agonists (10, 12). Several reasons could account for these differences. The method used to assay serum testosterone could have not been sufficiently discriminating, as a RIA method calibrated on the concentrations generally found in adults was employed. However, in CDP boys and in prepubertal controls we should have observed concentrations at the lower limit of the normal range. This occurred only in two patients who had basal levels that were already higher than those of prepubertal boys. It is more likely that the lack of response of testosterone to leuprolide acetate could be due to a lower potency of this agonist. In addition, the protocol employed for the test in particular the timing of blood sampling could also have accounted for the differences observed. Blood sampling for testosterone was performed 24 hours after stimulation, according to previous reports (16, 18). In prebubertal boys, the response to leuprolide, a less potent agonist, could have been delayed to 36 or 48 hours, and thus could have been lost. In this setting, Kletter et al. (9) observed that nafarelin determined a maximal increase in testosterone levels after 48 hours, although serum levels at 24 hours were already significantly different from baseline. The LH response alone was capable of discriminating HH from CDP patients. The 4-year follow-up study of the HH boys who were subsequently treated with gonadotropins followed by replacement therapy with testosterone enanthate confirmed the absence of spontaneous puberty. With regard to LH, the cut-off level between a normal response and a response suggestive of HH was between 2.8 and 6.1 IU/L; the former is the upper level of the peak LH response in HH patients, and the latter is the lowest level detected in CDP (Fig. 1). The HH patient with the highest peak LH response started puberty at age 16.5 years, after 3 months of treatment with testosterone. Testicular volume increased initially to 8 ml but did not progress any further, thus the patient was classified as having partial gonadotropin deficiency. All CDP patients started puberty spontaneously. Peak LH levels in HH and in normal prepubertal patients were similar, probably due to the younger age of the latter group, and subsequently to the low circulating androgen levels. The gap between 2.8 and 6.1 IU/L for peak LH levels in HH and CDP leaves the possibility in practice for diagnostic doubt (possibly rarely). In patients with these biochemical findings, only the effects of treatment will allow a definitive diagnosis. Levels of FSH did not increase significantly after GnRH agonist stimulation; however, the value between peak and baseline levels was greater in normal pubertal and CDP boys. The small increase observed in the normal prepubertal boys could be ascribed to the low circulating androgen levels due to their younger age. In addition, peak FSH levels showed a greater variability in pubertal boys compared with peak LH levels, which could also account for the lack of statistical significance among groups. In HH and CDP patients, peak FSH and LH levels after leuprolide acetate stimulation were not significantly different. Thus, it is unlikely that this agonist exerts a more exclusive regulation of LH than of FSH as previously reported with GnRH by other investigators (10). Regarding other GnRH agonists employed in previous studies, leuprolide acetate is less potent than nafarelin in stimulating LH (5, 9). In contrast, triptorelin elicits a greater LH response than leuprolide acetate, but similar FSH responses compared with leuprolide acetate and nafarelin (11). In conclusion, this study confirms the validity and specificity of acute GnRH agonist stimulation in the differential diagnosis between HH and CDP. Our data show a clear discrimination between the two groups of patients based solely on the LH responses. Leuprolide acetate was confirmed as safe, simple to use, and devoid of side effects so far. We suggest that the timing of blood sampling should be fixed at 0, 60, 120, and 180 minutes after stimulation, allowing for the pituitary response alone, if used to discriminate between HH and CDP in male patients. Acknowledgment: The authors thank Dr. Maria Angela Ziveri for careful performance of laboratory assays. References 1. Ghai K, Cara JF, Rosenfield RL. Gonadotropin releasing hormone agonist (nafarelin) test to differentiate gonadotropin deficiency from constitutional delayed puberty in teen-age boys: a clinical research study. J Clin Endocrinol Metab 1995;80: Hayes FS, Seminara SB, Crowley WF. Hypogonadotropic hypogonadism. Endocrinol Metab Clin North Am 1998;27: Goji K, Tanikaze S. Comparison between gonadotropin concentration profiles and response to low-dose gonadotropin releasing hormone in prepubertal and early pubertal boys and patient with hypogonadotropic hypogonadism: assessment by using ultrasensitive, time-resolved immunofluorometric assay. Pedriatr Res 1992;31: Crowley WF Jr, Beitins IZ, Vale W, Kliman B, Rivier J, Rivier C, et al. The biologic activity of a potent analogue of gonadotropin-releasing hormone in normal and hypogonadotropic man. N Engl J Med 1980; 302: Ehrmann DA, Rosenfield RL, Cuttler L, Burstein S, Cara JF, Levitsky LL. A new test of combined pituitary-testicular function using the gonadotropin-releasing hormone agonist nafarelin in the differentiation of gonadotropin deficiency from delayed puberty: pilot studies. J Clin Endocrinol Metab 1989;69: Partsch CJ, Hermanussen M, Sippel WG. Differentiation of male hypogonadotropic hypogonadism and constitutional delay of puberty by pulsatile administration of gonadotropin-releasing hormone. J Clin Endocrinol Metab 1985;60: Moshang T, Marx BS, Cara JF, Snyder PJ. The prolactin response to thyrotropin-releasing hormone does not distinguish teenaged males with hypogonadotropic hypogonadism from those with constitutional delay of growth and development. J Clin Endocrinol Metab 1985;61: Lanes R, Palacios A, Avendano E, Moncada G, Chique GL. The metoclopramide test: a useful tool with the luteinizing hormone-releas- FERTILITY & STERILITY 559

6 ing hormone test in distinguishing between constitutional delay puberty and hypogonadotropic hypogonadism. Fertil Steril 1989;52: Kletter GB, Rolsef-Carl A, Goodpasture JC, Solish SB, Scott L, Henzl MR, et al. Gonadotropin-releasing hormone agonist analog (nafarelin): a useful diagnostic agent for the distinction of constitutional growth delay from hypogonadotropic hypogonadism. J Pediatr Endocrinol Metab 1996;9: Ibañez L, Potau N, Zampolli M, Virdis R, Guissinyé M, Carrascosa A, et al. Use of leuprolide acetate response patterns in the early diagnosis of pubertal disorders: comparison with the gonadotropin releasing hormone test. J Clin Endocrinol Metab 1994;8: Zamboni G, Antoniazzi F, Tatò L. Use of the gonadotropin-releasing hormone agonist triptorelin in the diagnosis of delay puberty in boys. J Pediatr 1995;126: Tanner JM. Physical growth and development. In: Forfar JO, Arneil GC, eds. Textbook of paediatrics. 3rd ed. Edinburgh: Churchill Livingstone, 1984: Greulich WW, Pyle SI. Radiographic atlas of skeletal development of the hand and wrist. 2nd ed. Stanford, CA: Stanford University Press, Ibañez L, Potau N, Virdis R, Zampolli M, Terzi C, Gussinyé M, et al. Postpubertal outcome in girls diagnosed of premature pubarche during childhood: increased frequency of functional ovarian hyperandrogenism. J Clin Endocrinol Metab 1993;76: Barnes SB, Rosenfield RL, Burstein S, Ehrmann DA. Pituitary-ovarian responses to nafarelin testing in polycystic ovary syndrome. N Engl J Med 1989;320: Ghizzoni L, Virdis R, Ziveri M, Lamborghini A, Alberini A, Volta C, et al. Adrenal steroid, cortisol, adrenocorticotropin and -endorphin responses to human corticotropin-releasing hormone stimulation test in normal children and children with premature pubarche. J Clin Endocrinol Metab 1989;69: Ghizzoni L, Vanelli M, Virdis R, Alberini A, Volta C, Bernasconi S. Adrenal steroid and adrenocorticotropin responses to human corticotropin-releasing hormone stimulation test in adolescents with type 1 diabetes mellitus. Metabolism 1993;42: Waldstreicher J, Seminara SB, Jameson JL, Geyer A, Nachtigall LB, Boepple PA, et al. The genetic and clinical heterogeneity of gonadotropin-releasing hormone deficiency in the human. J Clin Endocrinol Metab 1996;81: Street et al. Stimulated LH discriminates delayed puberty Vol. 77, No. 3, March 2002

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