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A Single Sample Subcutaneous Luteinizing Hormone (LH)-Releasing Hormone (LHRH) Stimulation Test for Monitoring LH Suppression in Children with Central Precocious Puberty Receiving LHRH Agonists¹

Division of Endocrinology and Metabolism, Department of Pediatrics, Children’s Hospital of Eastern Ontario, University of Ottawa, Ottawa, Ontario, Canada K1H 8L1

Address all correspondence and requests for reprints to: Dr. Margaret Lawson, Division of Endocrinology and Metabolism, Children’s Hospital of Eastern Ontario, 401 Smyth Road, Ottawa, Ontario, Canada, K1H 8L1. E-mail: lawson{at}cheo.on.ca

	Abstract

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The effectiveness of LHRH agonist therapy in central precocious puberty depends upon suppression of LH secretion. The iv LHRH stimulation test is the gold standard for evaluating LH suppression, but is difficult to administer because it requires an iv line and multiple blood samples. We hypothesized that a sc LHRH test followed by a single LH measurement 40 min later would be as accurate in the assessment of LH suppression in children receiving LHRH analogs. Eleven children received the sc test 1 month before or after their regularly scheduled iv LHRH treatment. Each child was receiving Lupron to suppress central puberty. Twenty-five comparisons of the iv and sc LHRH tests were completed over 14 months. We developed a clinical score for pubertal suppression using Tanner staging, skeletal maturation, and growth velocity. The best correlation between this clinical score and the iv LHRH test was achieved when biochemical suppression was defined as peak LH less than 2 IU/L (100% sensitivity, 95% specificity). Using this definition, the sc LHRH test was 96% accurate (in 24 of 25 subjects), with a sensitivity of 75% and a specificity of 100% compared to the iv LHRH test. We conclude that the single sample sc LHRH test can accurately determine LH suppression and adequacy of LHRH agonist therapy in central precocious puberty.

	Introduction

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LHRH AGONIST therapy is given to suppress pubertal development in children with central precocious puberty (CPP). The effectiveness of LHRH agonist therapy depends upon the adequacy of LH suppression by the pituitary gland (1). Although clinical indicators of pubertal suppression may be helpful in this regard, it is recommended that these children also be assessed regularly for biochemical evidence of pubertal suppression (2, 3).

The gold standard for biochemical evaluation of LH suppression is the iv LHRH stimulation test (2, 3). This test requires the availability of a clinical investigation unit or out-patient testing area where an iv line can be inserted that is used for LHRH administration and drawing of multiple blood samples over the next 60–120 min. As such, this test is comparatively expensive, painful, and difficult to administer. It is recommended that LHRH stimulation tests be performed within 1–3 months of starting therapy and every 6 months thereafter during treatment (2, 3).

It has recently been demonstrated that a sc LHRH stimulation test with a single LH measurement 40 min post-LHRH correlates well with the standard iv LHRH test when used in the diagnosis of children with CPP (4). In children who present with symptoms suggestive of CPP, the goal is to distinguish those children with CPP who have elevated LH levels in response to LHRH administration from those children with benign variant or peripheral precocious puberty who have low, but not necessarily suppressed, LH levels in response to LHRH. We hypothesized that the single sample sc LHRH test could also be used effectively in children receiving LHRH agonist therapy, in whom the goal is to distinguish those children with suppressed LH levels from those with measurable or unsuppressed levels.

	Subjects and Methods

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The protocol was approved by the human ethics committee of the Children’s Hospital of Eastern Ontario (CHEO; Ottawa, Canada). Each child and a parent provided informed consent, and test results were discussed with each family.

Subjects

Eleven children (9 females and 2 males) participated in the study. All were receiving LHRH agonist therapy (depot Lupron or sc aqueous Lupron) to suppress pubertal development. Nine of the children (all girls) had CPP, with onset of secondary sexual characteristics before age 8 yr, increased growth velocity, advanced bone age, and pubertal LH levels (>10 IU/L) after iv or sc LHRH stimulation tests. Eight of these girls had idiopathic CPP, as evidenced by a normal magnetic resonance imaging scan. One child had a porencephalic cyst that was known to be present since infancy and was attributed to a neonatal intracerebral hemorrhage secondary to thrombocytopenia. Both boys had idiopathic GH deficiency and had been receiving recombinant human GH therapy for 1.5 and 2 yr, respectively, but had recently gone into central puberty, which was rapidly progressing and advancing their bone ages. As a result, LHRH agonist therapy was initiated in an effort to improve final adult height, even though both of these boys were within the normal age range for the onset of puberty. Patient characteristics at the onset of LHRH agonist therapy are shown in Table 1.

In our institution, it is standard practice for children receiving LHRH agonist therapy to undergo an iv LHRH test 3 months after initiation of therapy and every 6 months during treatment. Each study subject also underwent the single sample sc LHRH test 1 month before or 1 month after his/her regularly scheduled, multiple sample, iv LHRH test. The order of tests was randomly determined for each individual in each series of tests. The LHRH tests were performed immediately before receipt of the monthly depot Lupron injection or the daily sc Lupron. Neither dose nor frequency of LHRH agonist therapy was changed between the iv and sc tests.

The iv tests were performed in the Medical Day Unit of CHEO. An iv saline lock was inserted, and LHRH (100 µg/m²; Factrel, Wyeth-Ayerst Laboratories, Inc., Philadelphia, PA) was administered iv, with blood sampling at 0, 30, and 60 min for measurement of LH. The sc test used a standard dose of 100 µg LHRH (Factrel) given sc by our endocrine nurse (N.C.) in CHEO’s out-patient endocrine clinic. Forty minutes later, the children went to CHEO’s out-patient laboratory for a single venipuncture for measurement of LH. All patients had the blood drawn between 35–45 min after the sc injection. All samples were analyzed using the LH chemiluminescent immunoassay (Access Analyzer, Beckman Coulter, Inc., Palo Alto, CA). This assay is specific for the ß-subunit of LH. The lower limit of detection for this assay is 1 IU/L. Inter- and intraassay coefficients of variation at 4 IU/L were 6.4% and 3.8%, respectively.

At 3-month intervals, height was measured using a Harpenden stadiometer by the same pediatric endocrine nurse (N.C.). Each subject had pubertal development evaluated by the same pediatric endocrinologist at each visit, using Tanner genital staging for boys and breast development for girls (5). Bone age was determined at yearly intervals using an x-ray of the left wrist according to Greulich and Pyle tables (6). The films were read independently by a pediatric radiologist and a pediatric endocrinologist (M.L.L.), with disagreements resolved by consensus. Predicted adult height was calculated using the Bayley-Pinneu method (7).

To determine the LH level after LHRH treatment that correlates best with clinical parameters of pubertal suppression, each child was assessed retrospectively by chart review for clinical evidence of pubertal suppression at the time each LHRH test was performed. A child was classified as clinically suppressed if 1) there was regression or no progression in Tanner staging (breast development in girls, testicular enlargement in boys), 2) skeletal maturation index was 1 or less (i.e. change in bone age/change in chronological age, 1), and 3) growth velocity was less than the mean + 2 SD for chronological age (8).

A seven-item questionnaire was developed to assess child and parent satisfaction with the two types of LHRH tests. A five-point Likert scale examined test discomfort, inconvenience, time, and overall level of satisfaction and was administered immediately after each test. A score of 35 indicated the highest possible satisfaction with the test. The patients and their parents were also asked to indicate which test they would prefer to have if additional LHRH tests were required.

Statistical analysis

The peak LH level after iv LHRH administration was compared to the single LH level after sc LHRH. Patients with LH levels less than the lower limit of detectability (<1 IU/L) were assigned a value of 0.5 IU/L. Results are expressed as the mean ± SD. Pearson’s correlation coefficient was calculated using the Spearman rank order method for comparisons between two series of observations obtained in pairs. Sample size calculation determined that 20 comparisons were required to demonstrate a correlation of 0.9 with = 0.01 (two-tailed) and ß = 0.05.

Contingency tables (2 x 2) were formed, using variable definitions of LH suppression (<1, <2, or <3 IU/L) to compare 1) peak LH level after iv LHRH to evidence of clinical suppression, and 2) LH level after sc LHRH to peak LH level after iv LHRH. These tables were used to calculate sensitivity, specificity, and positive and negative predictive values with 95% confidence intervals. We defined a positive test as one that showed pubertal LH levels (i.e. LH level not suppressed). The Wilcoxon signed rank test for paired data was used to analyze the results of the patient/parent questionnaires.

	Results

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Over a 14-month period, 25 iv and sc LHRH test comparisons were performed on 11 children. At the time of the tests, their mean age was 9.0 ± 3.8 (SD) yr (range, 4.7–17.7 yr), and the children had been receiving Lupron therapy for 18 ± 11 months (range, 2–36 months). Ten of the children were receiving depot Lupron (7.5 mg monthly) by im injection, and 1 child was receiving daily sc Lupron (30–40 µg/kg·day) due to the development of a sterile abcess when she had received depot Lupron previously.

Correlation of clinical score with iv LHRH stimulation test

Using the different cut-offs (LH, <1, <2, or <3 IU/L) for the definition of LH suppression, we determined that a peak LH less than 2 IU/L after iv LHRH correlated best with clinical evidence of pubertal suppression, with 100% sensitivity and 95% specificity (Table 2). In our study population, in whom 88% (22 of 25) were suppressed clinically, the positive predictive value for the iv LHRH test with a cut-off of peak LH less than 2 IU/L was 75%, indicating that 75% (3 of 4) of children with a positive iv LHRH test (peak LH, 2 IU/L) had clinical evidence of pubertal progression (Table 3). The negative predictive value was 100%, meaning that all 21 children with a negative iv LHRH test (peak LH, <2 IU/L) had pubertal suppression on clinical grounds. There was a discrepancy between clinical evidence of suppression and the iv LHRH test in 1 patient who was receiving daily sc Lupron. Although the clinical score indicated suppression, it was determined retrospectively, and thus the iv and sc LHRH test results (LH, 2 IU/L for both) were used as the basis for an increase in the Lupron dose; subsequent sc and iv LHRH comparisons in this patient showed clinical and biochemical suppression, with complete agreement among the clinical score and iv and sc LHRH tests.

There was good correlation between LH levels after sc and iv LHRH testing (r = 0.88; 95% confidence interval, 0.74–0.94; P < 0.0001; Fig. 1). Agreement between the iv LHRH stimulation test, evidence of clinical suppression, and the sc LHRH stimulation test are shown in Table 3. With LH suppression defined as LH less than 2 IU/L after LHRH administration, the sc test had an accuracy of 96% (agreement with the iv LHRH test in 24 of 25 comparisons). One child had a positive iv LHRH test (peak LH, 2 IU/L) performed 2 months after initiation of LHRH agonist therapy, with a negative sc LHRH test (LH, 1 IU/L) 1 month later. At the time of the iv LHRH test, she was not suppressed clinically, whereas 1 month later when the sc LHRH test was performed, there was evidence of clinical suppression. Thus, both tests correlated with the clinical score at that time. Neither the dose nor the frequency of Lupron administration was altered, and 6 months later, there was clinical and biochemical suppression after both the iv and sc LHRH tests (LH, <1 IU/L). This suggests that the first iv LHRH test was performed before achievement of the full treatment effect from the LHRH analog. With LH suppression defined as LH less than 2 IU/L after LHRH, sensitivity of the sc LHRH test was 75%, with 100% specificity (Table 4). The positive predictive value for the sc LHRH test was 100%, and the negative predictive value was 95%, with 84% prevalence of LH suppression according to the iv LHRH test.

View larger version (12K): [in this window] [in a new window]   Figure 1. Correlation between peak LH after iv LHRH and LH levels after sc LHRH (r = 0.88; P < 0.0001).

Patient and parent perceptions of the sc and iv LHRH tests

Patient and parent perceptions were significantly more favourable for the sc LHRH test than for the iv LHRH test (mean difference in score, 6.3; 95% confidence interval, 3.7–10.1; P = 0.006). All 11 children and their parents expressed a preference for the sc test.

	Discussion

Top Abstract Introduction Subjects and Methods Results Discussion References

 
LHRH agonist therapy is given to children with CPP to suppress the hypothalamic-pituitary-gonadal axis and prevent further pubertal development and accelerated skeletal maturation. It is currently recommended that the effectiveness of LHRH agonist therapy be regularly assessed using biochemical evaluation of LH suppression (2, 3).

Biochemical evaluation of LH suppression is recommended because clinical evidence of pubertal suppression can only be provided by longitudinal observations demonstrating normalization of growth velocity and bone age advancement as well as stabilization or regression of secondary sexual characteristics. Cook et al.(9) suggested that evidence of regression of breast or testicular size combined with a decrease in the skeletal maturation index may indicate adequate pubertal suppression in the absence of biochemical confirmation. However, the imprecision of bone age assessments means that they are best performed at yearly intervals, which is clearly too prolonged a period in a condition where more frequent dosage adjustments may be required, particularly after initiation of LHRH agonist therapy. Therefore, in practice, clinical parameters are less useful than biochemical parameters in evaluating the response to therapy. However, a retrospective review of clinical progress can provide a useful means of comparing clinical status with biochemical parameters.

Basal LH levels are not useful in evaluating LH suppression. In the child in whom puberty is progressing, basal LH levels may still be in the prepubertal range (10). The gold standard for evaluating LH suppression is the iv LHRH test (2, 3). Although effective, this test is expensive, time consuming, and painful, given that it requires an iv line through which the LHRH is given and multiple blood samples drawn. As a result, many centers do not perform the iv LHRH test as frequently as recommended (i.e. every 6 months during LHRH agonist therapy) (2, 3).

In addition, there is currently no consensus regarding the definition of LH suppression after the iv LHRH test. It is recommended that an assay specific for intact LH (i.e. LHß) be used to avoid the potential confounding effects of measurement of the free biologically inactive -subunit or other LH fragments that remain elevated despite appropriate LHRH agonist therapy (2). Using assays specific for intact LH, LH suppression has been defined as a peak LH after iv LHRH stimulation of less than 5 IU/L (11) (Endocrine Sciences, Inc., Tarzana, CA), less than 2 IU/L (12) (fluorimetric enzyme immunoassay), or less than 1.75 IU/L (2, 13) (LH ß-subunit assay, DELFIA, Wallac, Inc., Turku, Finland).

To determine the optimal level of LH suppression in children receiving LHRH agonist therapy, we developed a clinical score for pubertal suppression based on growth velocity, bone age advancement, and secondary sexual characteristics and compared this clinical score to the gold standard iv LHRH test. The best correlation between this clinical score and the iv LHRH test was achieved when biochemical suppression was defined as peak LH less than 2 IU/L (100% sensitivity, 95% specificity, and 96% accuracy). All but 1 of the 22 cases who were suppressed clinically had LH suppression after iv LHRH, whereas all 3 cases who had clinical evidence of pubertal progression had elevated LH levels after iv LHRH. Furthermore, the child who was suppressed clinically but had a nonsuppressed iv LHRH test also had an elevated LH after the sc test. An increase in the Lupron dose was followed by suppressed LH levels in response to both sc and iv LHRH stimulation. This provides further evidence that it is difficult to determine adequate suppression from LHRH analog therapy on clinical grounds alone. From these data, we conclude that a peak LH less than 2 IU/L after iv LHRH is consistent with suppression of the hypothalamic-pituitary-gonadal axis, indicating adequate dosage of and frequency of treatment with the LHRH analog.

Because of the difficulties inherent in performing regular iv LHRH stimulation tests, several researchers have examined alternative means of evaluating LH suppression in children receiving LHRH agonist therapy. The correlation between overnight LH values and clinical evidence of pubertal suppression has been shown to be better than that observed between the iv LHRH stimulation test and clinical parameters (9). However, nocturnal sampling is too complicated and expensive for use in general practice in all children receiving LHRH agonists. Twenty-four-hour urinary gonadotropin excretion correlates well with serum gonadotropin levels after iv LHRH stimulation in children receiving LHRH agonist therapy (13). However, urinary gonadotropin levels lack the sensitivity and specificity required to evaluate LH suppression (13). Salerno et al. (14) found that a single plasma estradiol measurement 12 h after an im injection of a LHRH agonist correlated better with incomplete LH suppression than the estradiol measurement after the standard iv LHRH stimulation test. This 12-h estradiol test requires a return visit after administration of the im injection. Furthermore, correlation of estradiol levels with LH suppression has not been established. Recent data suggest, however, that the ultrasensitive recombinant cell estradiol bioassay may be able to demonstrate estradiol suppression during LHRH agonist therapy (15), although this assay is not yet widely available.

We have demonstrated that a single LH measurement 40 min after a sc injection of LHRH with LH suppression defined as LH less than 2 IU/L is very accurate (96%), with good specificity (100%) and sensitivity (75%) compared to those of the gold standard iv LHRH stimulation test. The sensitivity may, in fact, be higher than that determined in this study, as our data included one child in whom the iv LHRH test was performed 2 months after initiation of LHRH analog therapy rather than the recommended 3 months. At 2 months, the iv LHRH test was not suppressed, whereas the sc LHRH test showed good LH suppression at 3 months, even though there was no change in the Lupron dose or the frequency of administration. We acknowledge, however, that our data are limited by the small number of subjects who did not have LH suppression during LHRH analog therapy. We also found the sc LHRH test to be easier to administer and less expensive than the iv LHRH test, and it was the preferred test of children receiving LHRH agonists as well as their parents. These results suggest that the single sample sc LHRH stimulation test can easily and accurately determine LH suppression and adequacy of LHRH agonist therapy for suppression of pubertal development. Although further study is required, we suggest that the single sample sc LHRH test should be considered as a replacement for the multiple sample iv LHRH test in the monitoring of children receiving LHRH agonist therapy.

	Acknowledgments

 
We gratefully acknowledge the assistance of the children and parents who participated in this study, and the statistical support provided by Ba’ Pham of the Children’s Hospital of Eastern Ontario Research Institute.

	Footnotes

 
¹ This work was published in abstract form (Pediatr Res 43:79A, 1998) and was presented as a poster at the Annual Meeting of the Society for Pediatric Research, New Orleans, LA, May 1998.

Received May 6, 1999.

Revised August 12, 1999.

Accepted August 19, 1999.

	References

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