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Use of an Ultrasensitive Recombinant Cell Bioassay to Determine Estrogen Levels in Girls with Precocious Puberty Treated with a Luteinizing Hormone-Releasing Hormone Agonist

A. I. duPont Hospital for Children (K.O.K.), Wilmington, Delaware 19899; and the Developmental Endocrinology Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892

Address all correspondence and requests for reprints to: Dr. Karen Oerter Klein, Department of Clinical Science, A. I. duPont Hospital for Children, P.O. Box 269, 1600 Rockland Road, Wilmington, Delaware 19899.

	Abstract

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Although treatment of girls with precocious puberty should ideally restore estradiol levels to the normal prepubertal range, treatment effectiveness has usually been monitored by gonadotropin levels because estradiol RIAs have lacked sufficient sensitivity to monitor treatment effectiveness. We hypothesized that a recently developed ultrasensitive recombinant cell bioassay for estradiol would have sufficient sensitivity to demonstrate a dose-dependent suppression of estradiol during LH-releasing hormone agonist treatment and to determine whether currently used doses are able to suppress estradiol levels to the normal prepubertal range.

Twenty girls with central precocious puberty were assigned randomly to receive deslorelin for 9 months at a dose of 1, 2, or 4 µg/kg·day. A significant dose-response relationship was observed, with mean ± SD estradiol levels of 16.7 ± 6.1, 7.9 ± 1.6, and 6.5 ± 0.7 pmol/L at the doses of 1, 2, and 4 µg/kg·day, respectively (P < 0.01). The highest dose suppressed estradiol levels to just above the 95% confidence limits for normal prepubertal girls (<0.07–6.3 pmol/L).

We conclude that the ultrasensitive bioassay for estradiol has sufficient sensitivity for monitoring the response to LH-releasing hormone agonist treatment of central precocious puberty. Additionally, the observation that the deslorelin dose of 4 µg/kg·day did not fully restore estradiol levels to the normal prepubertal range suggests that some girls with precocious puberty may require higher doses to receive the maximal benefit of treatment. We suggest that restoration of estradiol levels to the normal prepubertal range should be the ultimate biochemical measure of efficacy, as estradiol is the key hormone that accelerates growth rate, bone maturation rate, and breast development in girls with precocious puberty.

	Introduction

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CENTRAL precocious puberty is treated with LH-releasing hormone (LHRH) agonists, which suppress gonadotropins, sex steroids, linear growth, and skeletal maturation. Most studies that have evaluated the degree of suppression have relied on gonadotropin measurements. Estradiol levels in girls with precocious puberty treated with LHRH agonists could not be assessed accurately because of the lack of sensitivity of previously available estradiol assays. Estradiol levels are elevated for age before treatment and are suppressed during treatment to levels below the detection limit of available assays (1, 2, 3, 4).

We hypothesized that an ultrasensitive recombinant cell bioassay for estrogen could be used to define the dose-response relationship between LHRH agonist and serum estradiol levels and to determine whether a given dose of LHRH agonist can suppress estradiol levels into the normal range for prepubertal girls. To test this hypothesis, we measured estradiol levels in 20 girls with precocious puberty during treatment with one of three doses of deslorelin (1, 2, or 4 µg/kg·day).

	Materials and Methods

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Study design

We studied estradiol levels in 20 girls with central precocious puberty. These 20 girls were a subset of 29 patients from a previous study of the dose-response effect of deslorelin on gonadotropin levels, which was published before the ultrasensitive assay for estradiol had been developed (5). Four of the patients from the original study were not included in the current study because there was not enough serum remaining for the estradiol measurement. The 3 male patients in the original study were not included in the current study. Two other girls from the previous study were omitted post-hoc because their estradiol levels determined by the ultrasensitive assay were extreme outliers, greater than 27 SD above the mean for the remaining subjects, which indicated probable contamination of the sample during collection or handling (6). Girls with LHRH-dependent precocious puberty, defined by the presence of advanced pubertal stage and a pubertal response to a LHRH test, were admitted to the Clinical Center of the NIH. After informed consent and assent were obtained, girls were treated with 4 µg/kg·day deslorelin, sc, for 3 months. After 3 months, the girls were randomized into 1 of 3 groups. Group 1 continued to receive 4 µg/kg·day deslorelin (n = 7); group 2 received 2 µg/kg·day deslorelin (n = 7); group 3 received 1 µg/kg·day deslorelin (n = 6). After 9 months of treatment on these doses, the girls underwent testing to determine basal and LHRH-stimulated serum gonadotropin levels and basal estradiol levels. LHRH testing was performed on the morning after the previous evening dose of LHRHa. Gonadotropin and estradiol levels determined by RIA were described in the original report.

Bone age was measured before randomization and 9 months after randomization. Growth velocity was calculated from start of randomization to 9 months of treatment with one of the three doses.

The present study reexamined stored frozen serum from 20 of the 29 girls in the original study for estradiol levels, as determined by the ultrasensitive recombinant cell bioassay (6). Estradiol levels were measured in the serum samples obtained at baseline (before any treatment was initiated) and after 9 months of treatment with deslorelin at a dose of 1, 2, or 4 µg/kg·day. All serum samples were obtained first thing in the morning (between 0700–0900 h) after the previous evening dose of LHRHa.

Estrogen assay

The bioassay for estrogen was performed as described previously (5). Briefly, the assay uses a strain of Saccharomyces cerevisiae that was transformed with two plasmids, one containing the human estrogen receptor complementary DNA and one containing an estrogen response element upstream from the structural gene for ß-galactosidase. After an 8-h incubation of yeast with extracts of 0.8 mL serum or estradiol standard, ß-galactosidase activity was measured using ortho-nitro-phenol-galactopyranoside as substrate. Estradiol equivalent units were calculated by linear interpolation from a standard curve. The sensitivity of the bioassay was 0.07 pmol/L. The intra- and interassay coefficients of variation at 7 pmol/L were approximately 15%.

Statistics

All data are expressed as the mean ± SD. Comparisons between groups were made using two-tailed Student’s t test. Comparisons among groups were made using ANOVA. Differences were considered significant at P < 0.05.

	Results

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The girls in the 3 groups had similar chronological age, bone age, pretreatment growth rate, and pubertal stage. The 3 dose groups did not differ significantly in terms of baseline estradiol level measured by RIA, mean basal LH or FSH, or peak LHRH-stimulated FSH or LH. However, in the original report, the peak LHRH-stimulated LH was significantly higher in group 3 (girls receiving the lowest dose, 1 µg/kg·day deslorelin). In the subset of 20 patients studied in the current report, the peak LHRH-stimulated LH was also highest in group 3 at 9 months after randomization of doses, but this difference was no longer statistically significant (Fig. 1).

View larger version (20K): [in this window] [in a new window]   Figure 1. LHRH-stimulated peak LH (top panel) and peak FSH (lower panel) in each of the three dose groups. Note that 12 months of treatment on the x-axis is equivalent to 9 months of treatment with the randomized doses.

View larger version (21K): [in this window] [in a new window]   Figure 2. Estradiol levels determined by ultrasensitive recombinant cell bioassay in girls with precocious puberty assigned randomly to each of the three dose groups.

View larger version (19K): [in this window] [in a new window]   Figure 3. Correlations between estradiol and peak LH (A) and peak FSH (B).

	Discussion

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The estradiol levels in these deslorelin-treated girls with precocious puberty were considerably lower by the ultrasensitive assay than by the previously reported RIA. In fact, the estradiol levels during treatment reported here were all lower than the detection limit (37 pmol/L) of the estradiol RIA used in the original report. The strong correlation between the original baseline estradiol levels determined by RIA and the baseline estradiol levels determined by the ultrasensitive recombinant cell bioassay confirms the validity of the recombinant cell bioassay. Estradiol levels were significantly lower in girls with precocious puberty receiving the higher doses of 2 or 4 µg/kg·day deslorelin than in girls receiving only 1 µg/kg·day. At the 4 µg/kg·day dose, two of the six girls had estradiol levels within the normal prepubertal range (<0.07 to 6.3 pmol/L); the remainder had levels that were only slightly above normal (maximum, 7.1 pmol/L).

Previous studies have relied upon gonadotropin measurements (usually after LHRH stimulation) and upon clinical measures (such as growth velocity, bone maturation rate, and pubertal progression) to judge the adequacy of pubertal suppression by LHRH agonist. However, we suggest that restoration of estradiol levels to the normal prepubertal range should be the ultimate biochemical measure of efficacy, because estradiol is the key hormone that accelerates growth rate, bone maturation rate, and breast development in girls with precocious puberty. None of the three deslorelin doses employed in the current study achieved this goal for all girls, which suggests that at least some girls may benefit further from the higher dose of 8 µg/kg·day that has been used by some investigators (1, 3).

Estradiol levels in the original report decreased from approximately 200–400 pmol/L before treatment to levels close to the detection limit of 37 pmol/L during treatment. Other reports describe estradiol levels decreasing from 103 ± 18 to 59 ± 7 pmol/L during deslorelin treatment (4 µg/kg/day) (7), from 104 ± 14 pmol/L to less than 73 pmol/L during deslorelin treatment (4–8 µg/kg·day) (1), from 306 pmol/L to 53 pmol/L during deslorelin treatment (4 µg/kg·day) (2), from measurable levels to universally undetectable levels (<72 pmol/L) during deslorelin treatment (8 µg/kg·day) (3), and from 171 ± 25 to 35 ± 7 to 81 ± 18 pmol/L during deslorelin treatment (4 µg/kg·day) (4). None of these earlier reports employed assays sufficiently sensitive to accurately measure the low levels of estrogen that are present in normal prepubertal girls (<0.07 to 6.3 pmol/L by the ultrasensitive assay) or in LHRH agonist-treated girls. A more recent report using a more sensitive estradiol assay (detection limit, 7.8 pmol/L) concluded that morning estradiol levels may be useful in determining the initiation of puberty (8). Estradiol levels before puberty were still usually less than the detection limit, and LH and FSH were not measured. Estradiol did correlate with bone age, which supports the possibility of using estradiol levels to monitor suppression by LHRH agonist therapy, but the ultrasensitive recombinant cell bioassay is still needed to evaluate prepubertal levels.

We conclude that estradiol levels during deslorelin treatment are significantly lower than those previously reported in girls with precocious puberty and are significantly lower in girls receiving 4 µg/kg·day deslorelin than in girls receiving 1 or 2 µg/kg·day. These studies suggest the feasibility of monitoring LHRH agonist treatment by measuring estradiol levels rather than by relying solely upon measurement of serum gonadotropins. Thus, the ultrasensitive assay for estradiol provides a new means for determining the dose of LHRH agonist needed to restore estrogen secretion to the normal prepubertal range and for determining the relative suppression of estradiol by different LHRH agonists, different LHRH agonist formulations (daily vs. depot), and different routes of administration.

	Footnotes

 
¹ Current address: Indiana University Medical Center, Indianapolis, Indiana 46202.

² Current address: Eli Lilly Co., Lilly Research Laboratories, Indianapolis, Indiana 46285.

Received December 15, 1997.

Revised March 2, 1998.

Accepted March 23, 1998.

	References

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1. Mansfield MJ, Beardsworth DE, Loughlin JS, et al. 1983 Long-term treatment of central precocious puberty with a long-acting analogue of luteinizing hormone-releasing hormone: effects on somatic growth and skeletal maturation. N Engl J Med. 309:1286–1290.[Abstract]
2. Styne DM, Harris DA, Egli CA, et al. 1985 Treatment of true precocious puberty with a potent luteinizing hormone-releasing factor agonist: effect on growth, sexual maturation, pelvic sonography, and the hypothalamic-pituitary-gonadal axis. J Clin Endocrinol Metab. 61:142–151.[Abstract]
3. Boepple PA, Mansfield MJ, Wierman ME, et al. 1986 Use of a potent, long acting agonist of gonadotropin-releasing hormone in the treatment of precocious puberty. Endocr Rev. 7:24–33.[Abstract]
4. Manasco PK, Pescovitz OH, Hill SC, et al. 1989 Six-year results of luteinizing hormone releasing hormone (LHRH) agonist treatment in children with LHRH-dependent precocious puberty. J Pediatr. 115:105–108.[CrossRef][Medline]
5. Pescovitz OH, Barnes KM, Cutler Jr GB. 1991 Effect of Deslorelin dose in the treatment of central precocious puberty. J Clin Endocrinol Metab. 72:60–64.[Abstract]
6. Klein K Oerter, Baron J, Colli MJ, McDonnell DP, Cutler Jr GB. 1994 Estrogen levels in childhood determined by an ultra-sensitive recombinant cell bioassay. J Clin Invest. 94:2475–2480.
7. Comite F, Cutler Jr GB, Rivier J, Vale WW, Loriaux DL, Crowley WF. 1981 Short-term treatment of idiopathic precocious puberty with a long-acting analogue of luteinizing hormone-releasing hormone. N Engl J Med. 305:1546–1550.[Abstract]
8. Norjavaara E, Ankarberg C, Albertsson-Wikland K. 1996, Diurnal rhythm of 17ß-estradiol secretion thoughout pubertal development in healthy girls: evaluation by a sensitive readioimmunoassay, J Clin Endocrinol Metab. 81:4095–4102.

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