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The Journal of Clinical Endocrinology & Metabolism Vol. 89, No. 9 4338-4342
Copyright © 2004 by The Endocrine Society

A Single Luteinizing Hormone Determination 2 Hours after Depot Leuprolide Is Useful for Therapy Monitoring of Gonadotropin-Dependent Precocious Puberty in Girls

Vinicius N. Brito, Ana C. Latronico, Ivo J. P. Arnhold and Berenice B. Mendonca

Unidade de Endocrinologia do Desenvolvimento, Laboratório de Hormônios e Genética Molecular LIM/42, Disciplina de Endocrinologia, Hospital das Clinicas da Faculdade de Medicina da Universidade de São Paulo, 05403-900 Sao Paulo, Brazil

Address all correspondence and requests for reprints to: Dr. Vinicius N. Brito, Hospital das Clínicas Faculdade de Medicina da Universidade de São Paulo, Disciplina de Endocrinologia e Metabologia, Avenue Dr. Eneas de Carvalho Aguiar, 155 2°andar Bloco 6, 05403900, Sao Paulo SP, Brazil. E-mail: vinbrito{at}uol.com.br or beremen{at}usp.br.


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
Long-acting GnRH analogs represent the standard treatment for gonadotropin-dependent precocious puberty. The aim of this study was to determine the hormonal parameters for monitoring the adequacy of depot leuprolide acetate treatment in girls with clinical and hormonal diagnosis of gonadotropin-dependent precocious puberty. Eighteen girls were treated monthly with 3.75 mg depot leuprolide acetate. Adequate hypothalamic-pituitary-gonadal axis suppression during treatment was achieved in 16 of the 18 girls according to the clinical parameters and prepubertal LH levels. In these 16 well-controlled girls, the LH peak after a classical GnRH test was compared with a single LH measurement obtained 2 h after depot leuprolide acetate administration before and during GnRH analog treatment. Before therapy, the mean ± SD LH peak after a classical GnRH test was 18.4 ± 11.2 IU/liter (ranging from 7–41.5 IU/liter), and it was 22.6 ± 8.3 IU/liter 2 h after the first depot leuprolide dose (ranging from 10–35.3 IU/liter). During therapy, the mean ± SD of LH peak after classical GnRH test was 1.4 ± 0.6 IU/liter (ranging from <0.6 to 2.3 IU/liter), and it was 2.7 ± 1.9 IU/liter (ranging from 0.7–6.6 IU/liter) 2 h after depot leuprolide. The LH peak after a classical GnRH test and that 2 h after depot leuprolide administration correlate significantly before and during treatment. In conclusion, we established the LH cut-off values for an adequate depot leuprolide therapy as an LH peak below 2.3 IU/liter after a classical GnRH test or below 6.6 IU/liter 2 h after depot leuprolide. The latter measurement may replace the classical GnRH test as a reliable and convenient tool for monitoring therapy in female gonadotropin-dependent precocious puberty.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
THE CLASSICAL GnRH stimulation test has been often used for the differential diagnosis between gonadotropin-dependent and -independent precocious puberty in children of both sexes as well as in monitoring the efficacy of depot GnRH analog treatment in patients with gonadotropin-dependent precocious puberty (GDPP). However, this test requires a clinical investigation unit or out-patient testing area and multiple blood sampling, which may be cumbersome, especially in young children, besides being painful and expensive (1, 2, 3, 4, 5). In addition, the lack of commercial availability of synthetic GnRH has made this test infeasible (6). Therefore, other reliable tools for diagnosing and evaluating therapy efficacy that could be easily performed on an out-patient clinic basis are desirable in clinical practice.

Long-acting GnRH analogs represent the standard treatment for GDPP. The depot leuprolide acetate, a GnRH analog frequently used in the treatment of precocious puberty, consists of leuprolide encased in microspheres of a glycolic and lactic acid copolymer, which are responsible for its slow release. Free leuprolide is also present in the preparation and is absorbed into the circulation within minutes after the injection (1, 7). Based on this last feature, Bhatia et al. (6) recently demonstrated that LH peak levels were sustained from 30 min to 2 h after depot leuprolide injection, indicating that a single sample obtained at any point within this time range is a convenient and accurate tool to assess treatment efficiency.

In the present study we determined whether a single LH determination, 2 h after administration of depot leuprolide, can replace the classical GnRH stimulation test in the monitoring of GDPP therapy. Additionally, we compared the LH response to the pretreatment classical GnRH test with that 2 h after the first depot leuprolide injection.


   Subjects and Methods
Top
 Abstract
 Introduction
 Subjects and Methods
Results
 Discussion
 References
 
This protocol was approved by the ethical committee of Hospital das Clinicas da Faculdade de Medicina da Universidade de São Paulo. Written consent was obtained from the patients or their parents. Eighteen girls were recruited for this study. The mean age at onset of treatment of precocious puberty was 5.6 ± 2.6 yr (ranging from 0.9–8.8 yr). All patients had breast and pubic hair development, accelerated growth velocity, advanced skeletal maturation, and pubertal LH responses after classical GnRH stimulation test according to criteria established in our laboratory (8), confirming the diagnosis of GDPP. Bone age was determined by the Greulich and Pyle method (9). Sixteen patients had idiopathic precocious puberty, whereas two had hypothalamic hamartomas (cases 9 and 16). Patients were treated with a standard dose of 3.75 mg depot leuprolide acetate (Lupron, Abbott Laboratories, Chicago, IL), sc, every 28 d; the doses per weight ranged from 89–340 µg/kg.

All girls had a classical GnRH stimulation test performed before treatment by infusing 100 µg GnRH (gonadorelin, Relisorm) iv, and blood samples were drawn at 15 min before and 0, 15, 30, 45, and 60 min after GnRH administration for serum LH measurements. Twelve subjects also had a single LH determination 2 h after the first administration of 3.75 mg depot leuprolide acetate. Clinical parameters monitored at 3-month intervals included Tanner staging, growth velocity, and occurrence of menses, with bone age determined every 6 months. A classical GnRH stimulation test was performed in all patients during treatment on the 28th day after depot leuprolide injection. In the following month, after 28 d, another dose of depot leuprolide was administered, and a single LH determination 2 h after the injection was performed in all patients.

Serum LH concentrations were determined by immunofluorometric kits (AutoDELFIA, Wallac Oy, Turku, Finland). The minimal detectable concentration was set at 0.6 IU/liter for LH (8). The inter- and intraassay variabilities were 4% and 6%, respectively.

The criteria for hypothalamic-pituitary-gonadal axis activation obtained from normal prepubertal girls were basal LH greater than 0.6 IU/liter and/or LH peak after classical GnRH test greater than 6.9 IU/liter (8). Adequate hypothalamic-pituitary-gonadal axis suppression during treatment was established by clinical parameters such as regression or arrest of breast development, normalization of growth velocity and rate of bone maturation, and laboratory parameters characterized by prepubertal basal and GnRH-stimulated LH levels.

Statistical analysis

The data are presented as the mean ± SD and range. The correlation between LH peak after classical GnRH stimulation test and single LH determination 2 h after depot leuprolide, at the first administration and during therapy, was performed by Spearman’s method. P < 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
Sixteen of the 18 girls presented adequate control of breast development, growth velocity, and bone maturation (group A). In contrast, two girls presented evidence of pubertal progression and were not included in the statistical analysis (group B).

In group A (n = 16), basal LH levels were at pubertal levels (>0.6 IU/liter) in 13 of the 16 girls (81.25%) before the administration of leuprolide depot. All girls presented LH peak greater than 6.9 IU/liter demonstrating a pubertal response in the classical GnRH stimulation test (Table 1Go).


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  		TABLE 1. Hormonal data for 16 girls with GDPP, before and during treatment with 3.75 mg of depot leuprolide monthly (group A)

 
Before therapy, the mean ± SD LH peak was 18.4 ± 11.2 IU/liter (ranging from 7–41.5 IU/liter) after a classical GnRH stimulation test, consistent with elevated LH secretion due to GDPP; the LH peak was 22.6 ± 8.3 IU/liter (ranging from 10–35 IU/liter) 2 h after the first depot leuprolide injection. Therefore, an LH level higher than 10 IU/liter 2 h after the first depot leuprolide injection is considered a pubertal response (Table 2Go). LH values obtained 2 h after the first injection of depot leuprolide presented a positive correlation coefficient with the LH peak after a classical GnRH test (r = 0.6; P = 0.03).


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  		TABLE 2. LH cut-off values at initial diagnosis and during therapy with leuprolide depot in girls with GDPP

 
During therapy with depot leuprolide, all girls presented basal LH at prepubertal levels and suppressed LH peak after classical GnRH stimulation test, i.e. LH peak below 6.9 IU/liter according to previously described criteria (8). The mean ± SD LH peak was 1.4 ± 0.6 IU/liter (range, <0.6 to 2.3 IU/liter) after a classical GnRH test and 2.7 ± 1.9 IU/liter (range, 0.7–6.6 IU/liter) 2 h after leuprolide depot, with a positive correlation coefficient (r = 0.62; P = 0.009) between the two LH peaks. The highest LH peaks were 2.3 and 6.6 IU/liter after a classical GnRH stimulation test and 2 h after depot leuprolide, respectively. These values were used as cut-offs for the LH peak after a classical GnRH test and for the LH peak 2 h after leuprolide depot as indicative of adequate hypothalamic-pituitary-gonadal axis suppression (Table 2Go).

In group B, basal LH was at pubertal levels (>0.6 IU/liter) in one girl and at prepubertal levels in the other patient before the administration of leuprolide depot (Table 3Go). Both girls showed LH peak greater than 6.9 IU/liter after a classical GnRH stimulation test, indicating a pubertal response. Before therapy, their LH peaks were 20 and 7.5 IU/liter after a classical GnRH stimulation test, and only one patient had an LH peak of 23 IU/liter 2 h after the first depot leuprolide injection, consistent with elevated LH secretion. During therapy with depot leuprolide, these two girls had basal LH at pubertal levels and LH peaks after a classical GnRH stimulation test of 4.3 and 5.7 IU/liter, respectively. These values were the highest LH concentrations observed among patients from the two groups. In addition, their LH peaks 2 h after leuprolide injection were 11 and 7.5 IU/liter, respectively. Based on pubertal progression and high levels of basal and stimulated LH during treatment, the leuprolide acetate dosage was increased to 7.5 mg, sc, every 28 d in both patients. Three months later, they were retested, and their basal and stimulated LH levels had decreased to prepubertal levels (Table 3Go).


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  		TABLE 3. Hormonal data for two girls with GDPP before and during treatment with 3.75 mg depot leuprolide monthly, presenting evidence of pubertal progression (group B)

 

   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
The LH response to a classical GnRH stimulation test is considered the gold standard for biochemical evaluation of hypothalamic-pituitary-gonadal axis activation in children with precocious puberty (10). The new third-generation LH assays allow the diagnosis of GDPP through LH determination under basal conditions. We have previously reported that a basal LH level greater than 0.6 IU/liter diagnosed GDPP in 63% of girls with precocious puberty, and an LH peak greater than 6.9 IU/liter after classical GnRH administration diagnosed GDPP in 92.2% of them (8). In the present study, basal LH was at pubertal levels in 14 of the 18 girls (78%) before the administration of depot leuprolide, and the LH peak after the classical GnRH test was greater than 6.9 IU/liter in all girls.

The design of the present study included a single LH measurement 2 h after the first dose of leuprolide depot to obtain data to compare with the LH peak after a classical GnRH test in girls with GDPP. Before treatment, the lowest LH peak obtained after the first depot leuprolide administration was 10 IU/liter, which is higher than the LH values obtained after classical GnRH stimulation (8). These data could be an alternative to diagnose GDPP in girls with a high clinical suspicion when gonadorelin or nondepot leuprolide is not available.

Long-acting GnRH analogs represent the standard treatment for GDPP. A fixed dose of 3.75 mg every 28 d ranging from 89–340 µg/kg was used initially in all patients studied. In the majority of girls (16 of 18), this dose resulted in adequate clinical and hormonal control. In the two girls who showed evidence of pubertal progression and pubertal LH levels in basal and stimulated conditions during treatment, the leuprolide acetate dosage was increased to 7.5 mg monthly, achieving adequate clinical and hormonal control. The usually recommended dose for the 1-month depot leuprolide is lower in the European Union (80–120 µg/kg) than in the United States (200–300 µg/kg). However, the lower doses usually result in the same efficacy as the larger ones, as demonstrated by 1-month treatment with 3.75 mg depot leuprolide in 49 patients (11) and more recently with 3-month 11.25 mg depot leuprolide in 44 patients in children with GDPP (12).

The criteria for adequate LH suppression during GnRH analog therapy are controversial. In our view, the main laboratory aim is to achieve basal and stimulated LH levels similar to those observed in normal prepubertal children. Assessment of the adequacy of GnRH analog therapy involves evaluation of clinical parameters and gonadotropin secretion (7, 10). The advantage of laboratorial parameters over clinical ones during GDPP therapy with GnRH analog is that they allow a prompt correction of the depot leuprolide dose, avoiding pubertal advancement. The LH response to a GnRH test is the most conclusive way to determine adequate hypothalamic-pituitary-gonadal axis suppression. It has been demonstrated that a complete standard test may not always be necessary. Several studies have sought alternative means of evaluating LH suppression in children receiving GnRH analog therapy. A single LH sample 40 min after sc GnRH (13), overnight LH values (14), 24-h urinary gonadotropin excretion (4), and a single plasma estradiol measurement 12 h after an im injection of a GnRH agonist (15) are some procedures that simplify this evaluation. However, a single LH measurement in blood collected 2 h after the depot leuprolide injection used for treatment itself is a more simple, comfortable, and inexpensive way to monitor therapy in girls with GDPP. There is no consensus regarding the criteria for LH suppression after classical GnRH stimulation test in patients using a GnRH analog (13). Using specific assays for intact LH, such as the immunofluorometric assay, LH suppression has been defined as an LH peak after iv GnRH stimulation of less than 5 IU/liter (16), less than 2 IU/liter (13, 17), or less than 1.75 IU/liter (4). In our series, we defined the cut-off LH peak based on 16 clinically well controlled girls with GDPP. We determined adequate suppression of LH levels during depot leuprolide treatment as LH less than 2.3 IU/liter after classical GnRH stimulation and LH less than 6.6 IU/liter 2 h after depot leuprolide injection. Although the two girls who showed evidence of pubertal progression presented basal LH at pubertal levels, we cannot conclude that basal LH measurement alone is enough to evaluate hypothalamic-pituitary-gonadal axis suppression, because in normal pubertal children basal LH levels may still be in the prepubertal range (8, 18). However, it is noteworthy that basal pubertal LH levels (>0.6 IU/liter) can eliminate the need for a classical GnRH test (8).

In conclusion, we established the LH cut-off values for an adequate depot leuprolide therapy as an LH peak less than 2.3 IU/liter after classical GnRH test or less than 6.6 IU/liter 2 h after depot leuprolide. The latter measurement may replace the classical GnRH test as a reliable and convenient tool for monitoring therapy in girls with GDPP.


   Acknowledgments
 
We gratefully acknowledge the collaboration of the children in this study as well as laboratory staff for hormonal measurements, and Ms. Sonia Strong for the English review.


   Footnotes
 
Abbreviation: GDPP, Gonadotropin-dependent precocious puberty.

Received September 3, 2003.

Accepted May 27, 2004.


   References
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

  1. Toguchi H 1990 Pharmaceutical manipulation of leuprorelin acetate to improve clinical performance. J Int Med Res 18(Suppl 1):35–41
  2. Parker KL, Baens-Bailon RG, Lee PA 1991 Depot leuprolide acetate dosage for sexual precocity. J Clin Endocrinol Metab 73:50–52[Abstract/Free Full Text]
  3. Manasco PK, Pescovitz OH, Hill SC, Jones JM, Barnes KM, Hench KD, Loriaux DL, Cutler Jr GB 1989 Six year results of luteinizing hormone releasing (LHRH) agonist treatment in children with LHRH-dependent precocious puberty. J Pediatr 114:321–324[CrossRef][Medline]
  4. Witchel SF, Baens-Bailon RG, Lee PA 1996 Treatment of central precocious puberty: comparison of urinary gonadotropin excretion and gonadotropin-releasing hormone (GnRH) stimulation tests in monitoring GnRH analog therapy. J Clin Endocrinol Metab 81:1353–1356[Abstract]
  5. Cavallo A, Richards E, Busey S, Michaels SE 1995 A simplified gonadotrophin-releasing hormone test for precocious puberty. Clin Endocrinol (Oxf) 42:641–646[Medline]
  6. Bhatia S, Neely EK, Wilson DM 2002 Serum luteinizing hormone rises within minutes after depot leuprolide injection: implications for monitoring therapy. Pediatrics 109:E30
  7. Lahlou N, Carel JC, Chaussain JL, Roger M 2000 Pharmacokinetics and pharmacodynamics of GnRH agonists: clinical implications in pediatrics. J Pediatr Endocrinol Metab 13:723–737
  8. Brito VN, Batista MC, Borges MF, Latronico AC, Kohek MBF, Thirone ACP, Jorge BH, Arnhold IJP, Mendonca BB 1999 Diagnostic value of fluorometric assays in the evaluation of precocious puberty. J Clin Endocrinol Metab 84:3539–3544[Abstract/Free Full Text]
  9. Greulich WW, Pyle SI 1959 Radiographic atlas of skeletal development of the hand and wrist. Stanford: Stanford University Press
  10. Lee PA 1994 Laboratory monitoring of children with precocious puberty. Arch Pediatr Adolesc Med 148:369–376[Abstract/Free Full Text]
  11. Carel JC, Lahlou N, Guazzarotti L, Joubert-Collin M, Roger M, Colle M, Chaussain JL 1995 Treatment of central precocious puberty with depot leuprolide acetate. Eur J Endocrinol 132:699–704[Abstract/Free Full Text]
  12. Carel JC, Lahlou N, Jaramillo O, Montauban V, Teinturier C, Colle M, Lucas C, French Leuprorelin Trial Group, Chaussain JL 2002 Treatment of central precocious puberty by subcutaneous injections of leuprorelin 3-month depot (11.25 mg). J Clin Endocrinol Metab 87:4111–4116[Abstract/Free Full Text]
  13. Lawson ML, Cohen N 1999 A single sample subcutaneous luteinizing hormone (LH)-releasing hormone (LHRH) stimulation test for monitoring LH suppression in children with central precocious puberty receiving LHRH agonists. J Clin Endocrinol Metab 84:4536–4540[Abstract/Free Full Text]
  14. Cook JS, Doty KL, Conn PM, Hansen JR 1992 Assesment of depot Lupron dose-adequacy for central precocious puberty. J Clin Endocrinol Metab 74:1206–1209[Abstract]
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