0021-972X/91/7304-0793$03.00/0 Journal of Clinical Endocrinology and Metabolism Copyright (c) 1991 by The Endocrine Society
Vol. 73, No. 4 Printed in U.S.A.
Serum Melatonin in Central Precocious Puberty Is Lower than in Age-Matched Prepubertal Children* F. WALDHAUSER, P. A. BOEPPLE, M. SCHEMPER, M. J. MANSFIELD, AND W. F. CROWLEY, JR. Department of Pediatrics (F. W.) and Second Department of Surgery (M.S.), University of Vienna, Vienna, Austria; the Reproductive Endocrine Unit, Massachusetts General Hospital (P.A.B., W.F.C.), Boston, Massachusetts 02114; and the Division of Endocrinology, Children's Hospital (M.J.M.), Boston, Massachusetts 02115
to endocrinologically normal children, there was no age-dependent decrease in nocturnal MT in untreated precocious puberty; rather, it appeared that serum MT had already declined in association with the onset of sexual maturation. Although there was a significant difference in weight between patients and agematched controls, the low MT values in patients 1-5 yr old were only partly explained by the weight difference (P < 0.0009); their pubertal status also contributed significantly (P < 0.006). Pituitary-gonadal suppression induced by long term GnRH analog treatment did not result in a return to prepubertal MT levels; rather, nocturnal MT decreased during therapy. The collected data indicate that nocturnal serum MT levels are related to sexual maturation, since serum MT is similar in precocious puberty and normal pubertal children. Since suppression of the pituitary-gonadal axis did not result in increases in nocturnal MT levels in young patients with precocity {i.e. return to age-appropriate levels), the reduction of nocturnal MT with normal puberty is not likely to be dependent on pubertal gonadotropin or sex steroid milieu. (J Clin Endocrinol Metab 73: 793796,1991)
ABSTRACT. In children a progressive decrease in nocturnal serum melatonin (MT) has been shown with advancing age, suggesting a reduction in the amplitude of the circadian MT curve with maturation. Whether this alteration of MT levels is related to human sexual maturation or occurs independently remains to be elucidated. Also, the impact of gonadal steroids on the MT rhythm remains an open question. We examined 56 patients (51 females and 5 males) with central precocious puberty (52 idiopathic and 4 neurogenic). Patients were studied before and 3, 6, and 12 months after initiation of GnRH analog treatment. Three hundred and thirtyseven endocrinologically normal subjects (190 males and 147 females) served as controls. In all subjects nocturnal serum MT (blood collection between 2300 and 0100 h) was measured with a highly specific RIA. In young patients, aged 1-5 yr, we found significantly lower MT levels than in age-matched controls. Pubertal patients, aged 5-9 yr, displayed nocturnal MT levels in the same range as control subjects approaching normal pubertal age. In contrast
I
N THE last 2 decades much information has been gathered on the physiological role of the pineal gland and its principle product, melatonin (MT), in seasonal mammals (1). In these species the pineal acts as a neuroendocrine transducer (2); it secretes MT in a circadian manner, with low levels during the day and high levels at night. The duration and/or position of the nocturnal MT elevation are modulated by the varying duration of the daily light period of the annual seasons. The alterations of the nocturnal MT elevation achieved in this way are interpreted as either progonadal or antigonadal, turning an animal's reproductive capabilities on or off (3-5). In addition to gonadal function, several other seasonal
events, e.g. replacement of antlers, growth of horns, moulting, etc., are regulated by this mechanism (6). Although the human is not considered to be a seasonal species, a potential involvement of MT in human reproductive function and sexual maturation has been discussed for some time (7-9). While the initial efforts to characterize MT levels in children were often confounded by the use of nonspecific assays (for review, see Ref. 10), in recent years a major decline in nocturnal serum MT has been established during childhood (11-17), indicating a reduction in the amplitude of the circadian MT rhythm during development. In light of the above-outlined neuroendocrine transducer hypothesis (3-5), the time course, not the amplitude, of the circadian MT rhythm would be the critical feature for a potential action of MT. However, the importance of the amplitude of the circadian MT signal has not been studied in detail, and it remains to be elucidated whether the age-related alterations of serum MT levels in man occur independently of sexual maturation or whether the decreased MT secre-
Received November 7,1990. Address requests for reprints to: F. Waldhauser, M.D., Department of Pediatrics, University of Vienna, Waehringer Guertel 18-20, A-1090 Vienna, Austria. * This work was supported in part by Grant P-6193 from Fonds zur Foerderung der Wissenschaftlichen Forschung, a donation from the Erste Osterreichische Sparcasse, and NIH Grants HD-18169, RR01066, and RR-02172.
793
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 08 July 2014. at 01:04 For personal use only. No other uses without permission. . All rights reserved.
WALDHAUSER ET AL.
794
tion might have a connection to puberty. One approach to study this is the examination of patients with central precocious puberty (CPP), who represent a faithful model of normal puberty in every way except for the timing of its onset. Thus, they are ideal models in which the timing of their puberty might permit examination of MT changes. In addition, GnRH analog (GnRHa) suppression of their gonadal steroid levels permits a direct examination of the role of sex steroids in the maturation of the MT rhythm. Since data collected to date are fragmentary and inconsistent (18, 19), we examined nocturnal serum MT levels before and after treatment in a sizeable number of such patients and controls. Subjects and Methods Fifty-six children, aged 1.6-9.0 yr (bone age, 2.6-15.1 yr), with CPP were studied. The diagnosis of CPP was made in 51 girls [2 neurogenic (1 septo-optico dysplasia and 1 cerebellar astrocytoma) and 49 idiopathic] and 5 boys [2 neurogenic (2 hypothalamic hamartomas) and 3 idiopathic] based upon the onset of secondary sexual development before age 8 (girls) or 9 (boys) yr associated with a pulsatile pattern of pituitary gonadotropin secretion and a pubertal response to exogenous GnRH. Each patient received daily sc injections of 4-8 MgAg (DTrp6,Pro9-NEt)GnRH (Salk Institute, La Jolla, CA) or 10 fig/ kg (imBzl-DHis6, Pro9-NEt)GnRH (Histrelin, Ortho Pharmaceuticals, Inc., Raritan, NJ). During therapy, pituitary-gonadal suppression was confirmed by the documentation of suppressed LH and FSH secretion during the night and day, the ablation of LH and FSH responses after administration of GnRH (2.5 Mg/kg, iv), and the return of gonadal steroid levels to the prepubertal range, as previously reported (20). Before and after 3, 6, and 12 months of GnRHa therapy a blood specimen was obtained between 2300-0100 h after exposure to darkness for at least 1 h. Three hundred and thirty-seven generally healthy human subjects, aged 1 to 90 yr, who were previously described in detail served as controls (14). The blood specimens were centrifuged, and the serum was stored at -20 C until assay. The study was carried out during all seasons of the year. Before the experiment, the protocol was explained to the participants and/ or their parents, and informed consent was obtained. The MT concentrations in the serum were measured in duplicates by a RIA previously reported in detail (13) and recently updated with respect to performance criteria (14). The highly specific anti-MT serum (R-158) used for hormone detection was previously provided by Dr. G. Brown (McMaster University, Hamilton, Ontario, Canada), but is now distributed by CID-Tech Research, Inc. (Hamilton, Ontario, Canada) (21). Statistical methods The Pearson product-moment correlation was used for estimation of a relationship between nocturnal MT and age in precocious puberty. For comparison of MT within age groups, in patients and controls aged 1-9 yr, the two-sample Wilcoxon
JCE & M • 1991 Vol 73 • No 4
test was used because of nonparametric distribution of MT levels. The significance of each comparison was judged according to Holm's adjustment for multiple tests (Fig. 2) (22). For comparison of the body weight in these subjects, two-way analysis of variance with the factors of age group and disease state was applied using the GLM procedure of SAS (Table 1) (23). Since body weight has been identified as a major determinant of nocturnal serum MT levels in children (14-16), a multiple regression analysis of MT on weight and disease state was performed. For this purpose MT and weight were rank transformed to make inferences more robust in presence of outlying observations. This analysis was performed using type I (sequential) sums of squares tests in the GLM procedure of SAS (23). To assess the impact of pituitary-gonadal suppression in patients with CPP, nocturnal MT values collected before and 3, 6, and 12 months after initiation of GnRHa treatment were compared (Fig. 3). Trends in nocturnal MT values of individual patients over the l-yr observation period were characterized by 1. Body weight (in kilograms) of patients with CPP and controls of comparable age
TABLE
Age (yr) CPP Controls
1-3
3-5
5-7
7-9
18.1 ± 1.5 11.9 ± 0.3
23.8 ± 1.0 16.5 ± 0.5
30.6 ± 1.7 24.6 ± 1.3
36.3 ± 1.9
27.4 ± 1.1
Values are the mean ± SEM. Two-way analysis of variance revealed a significant influence of disease state (P < 0.0001) and age group (P < 0.0001) on weight. CPP • control
400-
1600
•1200
5
200 -800
UJ
3
en
CC LLJ
K/)
100
li
•400
15
20
QduUs
AGE (yrs) FIG. 1. Nocturnal serum MT values (mean ± SEM) of patients with CPP (n = 56; thick line) and controls (n = 337; thin line) grouped according to age.
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 08 July 2014. at 01:04 For personal use only. No other uses without permission. . All rights reserved.
MELATONIN IN PRECOCIOUS PUBERTY
• CPP o control x p< 0.025 »x p < 0.01 3000
700-
e ~ SOOH
2000 I
£300 o I
•1000
8_
100
o •
3-5
1-3
5-7
7-9
AGE(yrs)
FiG. 2. Individual nocturnal serum MT values in patients with CPP (•) and age-matched controls (O). The bar represents the median of each group. •*, P < 0.01; *, P < 0.025.
= 140
- 600 =? T
1120 31
1 -
Vol. 73, No. 4 Printed in U.S.A.
Serum Melatonin in Central Precocious Puberty Is Lower than in Age-Matched Prepubertal Children* F. WALDHAUSER, P. A. BOEPPLE, M. SCHEMPER, M. J. MANSFIELD, AND W. F. CROWLEY, JR. Department of Pediatrics (F. W.) and Second Department of Surgery (M.S.), University of Vienna, Vienna, Austria; the Reproductive Endocrine Unit, Massachusetts General Hospital (P.A.B., W.F.C.), Boston, Massachusetts 02114; and the Division of Endocrinology, Children's Hospital (M.J.M.), Boston, Massachusetts 02115
to endocrinologically normal children, there was no age-dependent decrease in nocturnal MT in untreated precocious puberty; rather, it appeared that serum MT had already declined in association with the onset of sexual maturation. Although there was a significant difference in weight between patients and agematched controls, the low MT values in patients 1-5 yr old were only partly explained by the weight difference (P < 0.0009); their pubertal status also contributed significantly (P < 0.006). Pituitary-gonadal suppression induced by long term GnRH analog treatment did not result in a return to prepubertal MT levels; rather, nocturnal MT decreased during therapy. The collected data indicate that nocturnal serum MT levels are related to sexual maturation, since serum MT is similar in precocious puberty and normal pubertal children. Since suppression of the pituitary-gonadal axis did not result in increases in nocturnal MT levels in young patients with precocity {i.e. return to age-appropriate levels), the reduction of nocturnal MT with normal puberty is not likely to be dependent on pubertal gonadotropin or sex steroid milieu. (J Clin Endocrinol Metab 73: 793796,1991)
ABSTRACT. In children a progressive decrease in nocturnal serum melatonin (MT) has been shown with advancing age, suggesting a reduction in the amplitude of the circadian MT curve with maturation. Whether this alteration of MT levels is related to human sexual maturation or occurs independently remains to be elucidated. Also, the impact of gonadal steroids on the MT rhythm remains an open question. We examined 56 patients (51 females and 5 males) with central precocious puberty (52 idiopathic and 4 neurogenic). Patients were studied before and 3, 6, and 12 months after initiation of GnRH analog treatment. Three hundred and thirtyseven endocrinologically normal subjects (190 males and 147 females) served as controls. In all subjects nocturnal serum MT (blood collection between 2300 and 0100 h) was measured with a highly specific RIA. In young patients, aged 1-5 yr, we found significantly lower MT levels than in age-matched controls. Pubertal patients, aged 5-9 yr, displayed nocturnal MT levels in the same range as control subjects approaching normal pubertal age. In contrast
I
N THE last 2 decades much information has been gathered on the physiological role of the pineal gland and its principle product, melatonin (MT), in seasonal mammals (1). In these species the pineal acts as a neuroendocrine transducer (2); it secretes MT in a circadian manner, with low levels during the day and high levels at night. The duration and/or position of the nocturnal MT elevation are modulated by the varying duration of the daily light period of the annual seasons. The alterations of the nocturnal MT elevation achieved in this way are interpreted as either progonadal or antigonadal, turning an animal's reproductive capabilities on or off (3-5). In addition to gonadal function, several other seasonal
events, e.g. replacement of antlers, growth of horns, moulting, etc., are regulated by this mechanism (6). Although the human is not considered to be a seasonal species, a potential involvement of MT in human reproductive function and sexual maturation has been discussed for some time (7-9). While the initial efforts to characterize MT levels in children were often confounded by the use of nonspecific assays (for review, see Ref. 10), in recent years a major decline in nocturnal serum MT has been established during childhood (11-17), indicating a reduction in the amplitude of the circadian MT rhythm during development. In light of the above-outlined neuroendocrine transducer hypothesis (3-5), the time course, not the amplitude, of the circadian MT rhythm would be the critical feature for a potential action of MT. However, the importance of the amplitude of the circadian MT signal has not been studied in detail, and it remains to be elucidated whether the age-related alterations of serum MT levels in man occur independently of sexual maturation or whether the decreased MT secre-
Received November 7,1990. Address requests for reprints to: F. Waldhauser, M.D., Department of Pediatrics, University of Vienna, Waehringer Guertel 18-20, A-1090 Vienna, Austria. * This work was supported in part by Grant P-6193 from Fonds zur Foerderung der Wissenschaftlichen Forschung, a donation from the Erste Osterreichische Sparcasse, and NIH Grants HD-18169, RR01066, and RR-02172.
793
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 08 July 2014. at 01:04 For personal use only. No other uses without permission. . All rights reserved.
WALDHAUSER ET AL.
794
tion might have a connection to puberty. One approach to study this is the examination of patients with central precocious puberty (CPP), who represent a faithful model of normal puberty in every way except for the timing of its onset. Thus, they are ideal models in which the timing of their puberty might permit examination of MT changes. In addition, GnRH analog (GnRHa) suppression of their gonadal steroid levels permits a direct examination of the role of sex steroids in the maturation of the MT rhythm. Since data collected to date are fragmentary and inconsistent (18, 19), we examined nocturnal serum MT levels before and after treatment in a sizeable number of such patients and controls. Subjects and Methods Fifty-six children, aged 1.6-9.0 yr (bone age, 2.6-15.1 yr), with CPP were studied. The diagnosis of CPP was made in 51 girls [2 neurogenic (1 septo-optico dysplasia and 1 cerebellar astrocytoma) and 49 idiopathic] and 5 boys [2 neurogenic (2 hypothalamic hamartomas) and 3 idiopathic] based upon the onset of secondary sexual development before age 8 (girls) or 9 (boys) yr associated with a pulsatile pattern of pituitary gonadotropin secretion and a pubertal response to exogenous GnRH. Each patient received daily sc injections of 4-8 MgAg (DTrp6,Pro9-NEt)GnRH (Salk Institute, La Jolla, CA) or 10 fig/ kg (imBzl-DHis6, Pro9-NEt)GnRH (Histrelin, Ortho Pharmaceuticals, Inc., Raritan, NJ). During therapy, pituitary-gonadal suppression was confirmed by the documentation of suppressed LH and FSH secretion during the night and day, the ablation of LH and FSH responses after administration of GnRH (2.5 Mg/kg, iv), and the return of gonadal steroid levels to the prepubertal range, as previously reported (20). Before and after 3, 6, and 12 months of GnRHa therapy a blood specimen was obtained between 2300-0100 h after exposure to darkness for at least 1 h. Three hundred and thirty-seven generally healthy human subjects, aged 1 to 90 yr, who were previously described in detail served as controls (14). The blood specimens were centrifuged, and the serum was stored at -20 C until assay. The study was carried out during all seasons of the year. Before the experiment, the protocol was explained to the participants and/ or their parents, and informed consent was obtained. The MT concentrations in the serum were measured in duplicates by a RIA previously reported in detail (13) and recently updated with respect to performance criteria (14). The highly specific anti-MT serum (R-158) used for hormone detection was previously provided by Dr. G. Brown (McMaster University, Hamilton, Ontario, Canada), but is now distributed by CID-Tech Research, Inc. (Hamilton, Ontario, Canada) (21). Statistical methods The Pearson product-moment correlation was used for estimation of a relationship between nocturnal MT and age in precocious puberty. For comparison of MT within age groups, in patients and controls aged 1-9 yr, the two-sample Wilcoxon
JCE & M • 1991 Vol 73 • No 4
test was used because of nonparametric distribution of MT levels. The significance of each comparison was judged according to Holm's adjustment for multiple tests (Fig. 2) (22). For comparison of the body weight in these subjects, two-way analysis of variance with the factors of age group and disease state was applied using the GLM procedure of SAS (Table 1) (23). Since body weight has been identified as a major determinant of nocturnal serum MT levels in children (14-16), a multiple regression analysis of MT on weight and disease state was performed. For this purpose MT and weight were rank transformed to make inferences more robust in presence of outlying observations. This analysis was performed using type I (sequential) sums of squares tests in the GLM procedure of SAS (23). To assess the impact of pituitary-gonadal suppression in patients with CPP, nocturnal MT values collected before and 3, 6, and 12 months after initiation of GnRHa treatment were compared (Fig. 3). Trends in nocturnal MT values of individual patients over the l-yr observation period were characterized by 1. Body weight (in kilograms) of patients with CPP and controls of comparable age
TABLE
Age (yr) CPP Controls
1-3
3-5
5-7
7-9
18.1 ± 1.5 11.9 ± 0.3
23.8 ± 1.0 16.5 ± 0.5
30.6 ± 1.7 24.6 ± 1.3
36.3 ± 1.9
27.4 ± 1.1
Values are the mean ± SEM. Two-way analysis of variance revealed a significant influence of disease state (P < 0.0001) and age group (P < 0.0001) on weight. CPP • control
400-
1600
•1200
5
200 -800
UJ
3
en
CC LLJ
K/)
100
li
•400
15
20
QduUs
AGE (yrs) FIG. 1. Nocturnal serum MT values (mean ± SEM) of patients with CPP (n = 56; thick line) and controls (n = 337; thin line) grouped according to age.
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 08 July 2014. at 01:04 For personal use only. No other uses without permission. . All rights reserved.
MELATONIN IN PRECOCIOUS PUBERTY
• CPP o control x p< 0.025 »x p < 0.01 3000
700-
e ~ SOOH
2000 I
£300 o I
•1000
8_
100
o •
3-5
1-3
5-7
7-9
AGE(yrs)
FiG. 2. Individual nocturnal serum MT values in patients with CPP (•) and age-matched controls (O). The bar represents the median of each group. •*, P < 0.01; *, P < 0.025.
= 140
- 600 =? T
1120 31
1 -
