Plasma Adrenal and Gonadal Sex Steroids in Human Pubertal Development JACQUES-R. DUCHARME, MAGUELONE G. FOREST, EVELINE DE PERETTI, MICHEL SEMPfi, ROBERT COLLU, AND JEAN BERTRAND Unite de Recherches Endocriniennes et Metaboliques chez I'Enfant, INSERM, U. 34, Hopital Debrousse, 69005 Lyon, France; Centre de Recherche Pediatrique, Hopital Sainte-Justine and Department of Pediatrics, Universite de Montreal, Montreal, Quebec, H3T 1C5, Canada ABSTRACT. Plasma free dehydroepiandrosterone (DHA), androstenedione (A), testosterone (T), dihydrotestosterone (DHT), estrone (E,), and estradiol (E2) were measured by radioimmunoassay in 55 boys and 54 girls 3.5 to 16.3 years of age. Plasma DHA increased significantly between 6 and 8 years of age in girls and between 8 and 10 years of age in boys. A further significant increase was noted between 10 and 12 years of age in both sexes. A rose significantly between 8 and 10 years of age in girls and between 10 and 12 years in boys. In contrast,
T
HE hypothalamic-pituitary-gonadal relationships during the course of pubertal development in man have been extensively studied and the hormonal changes characteristic of puberty attributed to modifications of the sensitivity of the hypothalamic gonadostatic threshold to circulating androgens and estrogens (2,3). However, the mechanisms responsible for this decrease in the sensitivity of the hypothalamus to the feedback effect of sex steroids remain unclear. Ducharme et at. (1) and Collu and Ducharme (4) have presented evidence that some adrenal steroids may play a role in the activation of the hypothalamic-pituitary-gonadal axis at puberty. Recently, Gorski and Lawton (5) have shown that adrenalectomy of immature female rats at 18 and 25 days of age, but not at 35 days, significantly delayed the advent of puberty compared with normal or sham-operated Received July 30, 1975. This work was presented in part at the Canadian Society for Clinical Investigation, Montreal, Canada, January 1974 and appeared in abstract form (1). Requests for reprints should be addressed to: Jacques-Raymond Ducharme, Centre de Recherche Pediatrique, Hopital Sainte-Justine, Montreal, Quebec, H3T 1C5, Canada.
no significant increase in T, DHT, or E, was noted prior to 12 years of age in both sexes. However, E2 showed a significant increase between 10 and 12 years of age in girls. This early rise in the course of pubertal development of the two sex steroids predominantly of adrenal origin, DHA and A, and its occurrence 1 to 2 years earlier in girls than in boys, as does puberty itself, suggest a possible role for these steroids in the mechanisms involved in triggering the hypothalamic-pituitary-gonadal axis at puberty. (J Clin Endocrinol Metab 42: 468, 1976)
controls and that the autotransplantation of the removed adrenals at 18 days prevented this delay. These data were interpreted as evidence that, during a certain period, the adrenals play a role in the maturation of the brain-pituitary-ovarian axis. In human beings, the activation of the adrenal cortex prior to puberty is characterized by a significant increase in plasma dehydroepiandrosterone (DHA) and its sulfate (DHAS) (6) and estrone (EJ (7,8). However, the role of these or other sex hormones in the initiation of puberty is at best speculative. There is clinical evidence that exposure of the prepubertal child to sex steroids of either adrenal or gonadal origin may hasten puberty. This effect is closely related with advanced bone maturation to a bone age compatible with physiological onset of puberty. Indeed, boys and girls with virilizing adrenal hyperplasia, either untreated or treated with inadequate suppressive therapy, may undergo rapid pubertal development once adequate glucocorticoid treatment is instituted, provided their skeletal maturation reaches the pubertal range. Treatment before puberty of children with
468
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469
PLASMA SEX STEROIDS AND PUBERTY TABLE 1. Clinical material Age group (years)
N*
C.A.|
H.A.§
P
W.A."
B.A.H
15.4 db 1.2 13.4 dt4.4 10.7 dt 1.7
15.0 dt 1.1 13.4 dt 1.7 11.6 dt 1.1 9.4:t 1.1 7.9:t 1.0 5.7:t 1.1
4.2 db0.4 3.1 dt 1.1 2.3 dt 0.7 1.1 dt 0.2 1.0 dtO.O 1.0 dbO.O
14.7 ± 12.1 ± 10.3 ± 8.9: t 7.2 :t 5.5:t
4.6 db0.5 2.9 db0.8 1.5 db0.5 1.0 db0.3 1.0 dbO.O 1.0 db 0.0
Girls 12-14 10-12 8-10 6-8 14 12-14 10-12 8-10 6-8 14
1.2 0.8 2.1 1.7 1.3 1.0
9.7:t2.5 7.7 :t 1.1 4.7:t 1.2
Boys 15.4 dt 1.6 13.2 dt 1.5 11.5 dt 1.5
8.7 :t 1.6 7.3 dt 1.1 4.8 dt 1.0
8.6: t 1.6 7.4 :t 1.1 4.8:t 1.4
1.1 1.1 0.8 0.9 1.0 2.1
* Number of subjects \ Chronological age ± SEM. § Height age ± SEM. 11 Weight age ± SEM. H Bone age ±SEM. ** Stage of pubertal development according to Tanner (13) ± SEM.
sex steroids may lead to similar events if the exposure has been of sufficient magnitude and duration to advance sufficiently their bone age. The advent of radioimmunoassay techniques for gonadotropins has made it possible to delineate the pattern of their secretion throughout infancy, childhood, and adolescence and to show that gonadotropin levels begin to rise prior to any evidence of secondary sex characteristics (9-12). In addition, this rise in plasma gonadotropins occurs some two years earlier in female than male subjects, (9-13) which is consistent with the earlier onset of puberty in girls. The present study was undertaken in an attempt to clarify further the relationships between adrenal sex steroids, gonadotropin secretion, and indices of pubertal development and to verify whether adrenal steroids play a role in the triggering of the hypothalamic-pituitary-gonadal axis at puberty. If such an effect does take place, it could result either from a direct and specific action of these steroids at the hypothalamic level or, more likely, be mediated through hastening the onset of maturation of the
central nervous system mechanisms which lead to puberty. Materials and Methods Subjects. One hundred and nine children, 3.5 to 16.3 years of age, 55 boys and 54 girls, were the subjects of this study (Table 1). Each subject was submitted to physical examination, weighed, measured, and ascribed to a stage of genital or breast development according to Tanner (14). Bone age (15) and height and weight ages (16) were estimated according to Sempe. Each subject was free of acute or chronic illness at the time of study and was within the normal range for his age in height and weight. Plasma was obtained by antecubital vein puncture in an heparinized disposable syringe between 9 and 11 AM in most instances, and the plasma was immediately separated and frozen at - 2 0 C until assayed. Informed consent was obtained in each instance. Free steroid analysis. Plasma DHA,* A, T, and DHT
were
measured
by
radioimmunoassay
* Abbreviations and trivial names used in this paper: A4-androstenedione (A), androst-4-ene-3,17 dione; dehydroepiandrosterone (DHA), A5-androsten-3/3-ol-
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470
DUCHARME ET AL.
essentially as described by Forest (17,18). Each steroid extract was eluted on celite with gradients of benzene and ethyl acetate in isooctane. DHA and DHT were eluted with 15% benzene in isooctane while pure isooctane and 3% benzene in isooctane yielded the androstenedione fraction. The latter was then reduced to T with sodium borohydride and eluted with 25% ethyl acetate in isooctane as for free testosterone initially present in the plasma tested (17). The antibody to 3-(O-Carboxy-methyl) oxime of T coupled with BSA was produced in rabbits as previously described (18) and showed a cross-reactivity of the order of 76% between T and DHT but no significant cross reaction with the other steroids herein measured except for A which cross-reacted with T approximately 1%. The anti-DHA antibody was obtained similarly in rabbits immunized with DHA-17-carboxime coupled with BSA and cross-reacted with A and iso-androsterone to a degree of 4% and 8%, respectively. Ej and E2 were also measured by RIA using a sheep antibody to E2-17-/3-hemisuccinate graciously supplied by Dr. Jose Saez. This antibody showed complete cross-reactivity between E2 and Ej but no cross-reactivity with any of the other steroids reported in this study. The RIA was carried out essentially as described by Saez (19). Due to simultaneous initial separation of A, T, and DHA on the same column, 0.5 to 3.0 ml of plasma were used while 10.0 ml of plasma was usually utilized for sequential elution of DHT, Ej, and E2 on the same plasma sample. Free cortisol was measured by competitive protein-binding assay by the method of Murphy (20). One ml of the same adult female plasma pool was used as reference preparation for DHA, A, and T in each assay together with appropriate non-radioactive standards diluted in water. Two ml of the same adult male plasma pool was used similarly together with appropriate standards for DHT, Ei, and E2. The blank value obtained was 17 one; dihydrotestosterone (DHT), 17/3-hydroxy-5aandrostan-3 one; estrone (Et) Ali3j3 (10)-estratrien-3ol-17 one; estradiol (E2), Au,5 (10)estratrien-3,17/3diol; testosterone (T), 17/3-hydroxy-androst-4-ene-3 one; cortisol (F), A4-pregnen-ll/M7a,21-triol-3,20dione. RIA, radioimmunoassay; TeBg, testosteroneestradiol-binding globulin; BSA, bovine serum albumin.
JCE & M 1976 Vol 42 . No 3
less than 15 pg for androgens and 7.0 pg for estrogens. No radioactive DHA was added prior to extraction of the plasma samples to avoid any risk of cross-over of the radioactivity and consequently faulty evaluation of the recovery. However, since in our hands, the recovery of DHA and T eluted from the same celite column was identical, the latter was used to correct for experimental losses. Indeed, in 18 experiments, the mean recovery for DHA and T eluted in their respective fractions from the same column was (mean ± SD) 92.1 ± 1.9% and 93.5 ± 1.6%, respectively. In addition, 30 paired samples of DHA measured either separately or calculated from the recovery of T on a column run simultaneously gave a mean difference of 4.9%, which is equal to or lower than the inter-assay variations. Inter-assay variations for A, T, and DHA were 9%, .11%, and 10%, while those for E,, E2, and DHT were 14%, 17%, and 5%, respectively. The intra-assay variations were in the order of 7% for A and T and 5% for DHA, and 10%, 6%, and 5%, respectively for E,, E2, and DHT. Finally, to assure better comparative homogeneity between the different assays performed, each RIA included samples obtained from children of both sexes in each age group category. Statistical analysis. For the purpose of the statistical analysis, values less than 1 ng/100 ml were taken to be 1 ng/100 ml since the sensitivity of the radioimmunoassay procedure would not permit their more accurate determination. Since Hartley tests (21) revealed raw data for all hormones to be heteroscedastic with variance increasing in proportion to the magnitude of hormone concentration, data were transformed to decimal logarithms, after which they were satisfactorily homoscedastic. Results were then submitted, separately for each hormone, to a twoway analysis of variance with age and sex as factors (22,23). Since there were unequal numbers of subjects in cells (age-sex groups), the analyses were made on a CDC 6400 computer using a multiple regression program. Age effects adjusted for sex differences, sex effects adjusted for age differences, and the sex-age interaction adjusted for both age and sex differences were calculated. The results are presented as mean concentrations (transformed back from logarithms) with 95% confidence intervals based on appropriate error mean squares and degrees of freedom from the above analyses. A set of 2 by 2
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PLASMA SEX STEROIDS AND PUBERTY individual comparisons of age effects within each sex were assessed for statistical significance and are presented.
TABLE 2. Factorial analysis of variance
sv*
Results
The analyses of variance for each steroid appear in Table 2; plasma steroid levels (mean and 95% confidence intervals) and the statistical significance of differences are presented in Table 3. A statistically significant increase in DHA levels was already evident in girls between the group less than 6 years old and the group 6-8 years of age. In boys, this difference becomes apparent between 6-8 and 8-10 years of age. A second and highly significant increase was observed after 8-10 years in both sexes. For A, the interaction age by sex was not significant. In girls, there was a continuous statistically significant increase between the 6-8 year old group and the 12-14 year age group whereas, in boys, this increment was only significant between 8-10 and 1012 years of age. As for the other sex steroids measured, no significant increase was noted prior to the 12-14 year age group with the exception of E2 in girls, which rose significantly prior to 10-12 years of age. Testosterone levels were similar in both sexes until the 12-14 year age group. T levels rose significantly only after the 10-12 year group in girls while this simultaneous increase in boys remained significant thereafter. This finding explains why the interaction age by sex observed was statistically significant whereas the sex difference was not. A similar pattern was found for DHT in boys. In addition, in general a higher mean ratio based on all age groups of DHT/T was noted in girls (.33) compared with boys (.20). No significant change in plasma Ej was observed in boys. In contrast, a significant rise was seen in girls after 10-12 years of age. A significant rise in plasma E2 was observed after 10 years of age in girls, while
471
Dehydroepiandrosterone (DHA) Total Sex Age
Age x sex Error Androstenedione (A) Total Sex Age
Age x sex Error Testosterone
SS|
Exact significance
DF§
23.625160 .203951 16.741766 .780913 6.099704
96 1 5 5 85
20.011041 .713390 11.757628 .143095 7.739889
106 1 5 5 95
27.634025 .073076 16.103985 2.088800 9.154010
106 1
14.153380 .002444 7.017391 2.150817 4.797024
73 1
2.842 46.660 2.176
.09152 .00000 .06364
8.756200 28.862808 .351273
.00398 .00000 NS
.758384 33.425319 4.335498
.00000 .00143
.031587 18.139508 5.559726
.00000 .00030
7.319179 2.090244 1.783443
.00810 .07388 .12404
3.347594 5.782903 3.802524
.06747 .00015 .00388
4.075138 1.726864 1.720472
.04407
(T)
Total Sex Age
Age x sex Error Dihydrotestosterone (DHT) Total Sex Age
Age x sex Error Estrone (E,) Total Sex Age
Age x sex Error Estradiol (E2) Total Sex Age
Age x sex Error Cortisol (F) Total Sex Age
Age x sex Error
9.420546 .625385 .893001 .761929 7.177354
5 5 95
5 5
NS
NS
62 95 1
5 5 84
12.502703 .313172 2.704988 1.778654 7.764751
94 1
5.203000 .193917 .410867 .409346 4.187506
99 1
5 5 83
5 5 88
* Sources of variation. \ Sum of squares. § Degree of freedom. 11 Fisher coefficient F.
no significant difference was seen in boys, although somewhat higher values were found after 14 years of age. This discrepancy of sex pattern for age accounts for
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JCE & M . 1976 Vol 42 • No 3
DUCHARME ET AL.
472
TABLE 3. Plasma free steroid levels in children with increasing age (ng/100 ml) Age group (years)
14
Dehydroepiandrosterone (DHA): Girls 6 9 10 9 N* 8 6 116.4 351.7 370.1 465.9 28.6 109.3 X| CL§ 19.4-42.1 72.6-164.5 74.4-179.6 233.7-529.4 224.3-610.6 282.4-770.5 P < 0.0001 P < 0.0005 Boys N 9 7 11 10 6 6 X 43.4 43.8 84.7 264.8 352.9 505.9 CL 26.3-71.7 29.1-66.0 53.3-134.6 183.0-383.3 239.5-520.3 306.7-834.8 P < 0.05 P P < 0.0005 Androstenedione (A): Girls 11 9 6 N 9 9 9 X 14.8 17.7 32.1 64.5 123.2 131.4 CL 9.6-22.9 11.5-27.4 21.6-47.4 41.7-99.6 79.8-190.4 77.2-223.9 P P < 0.05 P < 0.05 P < 0.05 Boys 9 12 11 9 6 N 7 X 20.2 44.7 66.6 82.0 11.6 16.1 CL 7.1-19.0 13.1-31.3 30.7-65.2 44.9-98.7 48.2-139.7 10.4-24.8 P P < 0.01 Testosterone (T) Girls N 6 9 9 9 9 11 8.4 9.8 X 8.7 15.1 33.0 37.3 CL 5.2-13.4 6.1-15.8 5.7-13.3 9.4-24.2 20.6-53.0 20.9-66.5 P P < 0.05 Boys N 7 7 11 9 9 11 X 6.2 7.2 6.9 13.1 50.7 204.2 CL 4.5-11.5 8.6-20.1 33.1-77.8 119.4-349.1 3.6-10.6 4.3-11.1 P < 0.001 P P < 0.001 Dihydrotestosterone (DHT): Girls N 4 8 7 4 7 5 X 1.4 6.1 11.7 7.1 2.8 7.7 CL 3.9-9.6 4.7-12.4 7.2-19.0 4.0-12.6 0-2.6 1.5-5.4 r Boys 11 N 1 3 7 10 7 13.4 X 3.4 2.6 31.8 2.0 3.9 CL 1.6-4.2 9.1-19.7 19.6-51.6 2.6-5.9 0-7.2 1.6-7.1 P P < 0.0001 P < 0.01 Estrone (E,): Girls N 11 6 10 8 9 9 X 2.7 4.6 4.4 8.9 5.9 3.0 CL 3.1-7.0 3.4-10.2 2.8-6.8 5.7-14.0 1.9-4.6 1.7-4.4 P P < 0.05 Boys N 6 7 7 10 8 5 X 3.0 3.3 3.2 2.7 2.9 3.9 CL 2.0-5.4 1.7-4.4 2.1-7.1 2.1-4.9 1.8-5.3 1.7-4.8 r> r Estradiol (E8): Girls N 10 9 6 10 8 9 6.4 2.1 7.0 1.2 4.9 X 1.9 3.6-11.3 4.4-11.1 CL 1.4-3.3 3.1-7.8 1.3-3.1 0-2.0 P P < 0.05
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473
PLASMA SEX STEROIDS AND PUBERTY TABLE 3. (Continued)
Age group (years) Boys N X CL P Cortisol (F): Girls N X CL P Boys N X CL P
14
6 3.0 1.7-5.3
7 2.3 1.3-3.8
7 2.0 1.2-3.5
10 2.0 1.3-3.1
8 2.3 1.4-3.9
5 4.5 2.4-8.4
7 7.7 5.3-11.2
9 9.3 6.6-12.9
11 10.0 9.4-13.6
8 7.8 5.5-11.2
8 9.8 6.9-14.0
6 12.6 8.4-19.0
6 11.3 7.5-17.1
8 7.4 5.2-10.5
8 6.1 4.3-8.6
11 11 5.7 10.1 4.2-7.6 7.5-13.6 P< 0.01
7 7.6 5.2-11.1
* Number of observations. | Mean concentration transformed back from logarithms. § 95% confidence intervals. 11 Statistical significance.
the significant interaction age by sex found as was also observed for testosterone. Finally, there was no significant difference in plasma free cortisol (F) at any age except between the 10-12 and 12-14 year old groups in boys. When androgens and estrogens were correlated with bone maturation, a significant correlation was found in girls for DHA, A, T, DHT, E1} and E2 while such correlation was seen for DHA, A, T, and DHT, but not for Ej and E2 in boys (Fig. 1).
earlier onset of puberty in girls. In addition, a similar time difference in girls and boys for gonadotropin secretion has been reported (9-12) with a significant rise prior to any evidence of pubertal development and, as this study shows, prior to any significant rise in gonadal sex steroids. Our results for plasma DHA are in general agreement with those reported by Saez et al. (6,7), Hopper and Yen (31), and Sizonenko (30). However, we were unable to confirm the significant difference in estrone levels found by Saez et al. (6,7) between Discussion children of both sexes less than 6 years old and those 7 to 10 years of age. Nevertheless, The values obtained for androgens and in girls higher values were detected after estrogens in this study are in general agree8 years of age (Table 3), compared with ment with those previously published (6,7, younger children, in accord with studies by 13,17,18,24-33). In addition, an interesting time-sequence relationship was found for Bidlingmaier (26). This rise became signifiplasma levels of some sex steroids and cant after 12 years of age (stage P2) in girls, in indices of sexual maturation. Indeed, in agreement with the data of Angsusingha girls plasma levels of DHA started to rise as et al. (32) and of Gupta et al. (33). No such early as 6 to 8 years followed by a significant trend has been observed for boys at any age increase in A by 8 to 10 years, while no in- in this study, at variance with studies by crease in T, E1} or E2 was noted prior to Saez reported by Forest (7). Another interesting observation concerns 10-12 years of age and thereafter. The same time-sequence was also observed in boys the higher DHT/T ratio observed in girls, but with a one to two year delay compared which is also found in data from Gupta et with girls, which is consistent with the al. (33). Thisfindingmay be related to differ-
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DUCHARME ET AL.
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JCE & M • 1976 Vol 42 • No 3
FIG. 1. Linear correlation between steroid levels and bone maturation (BA). The honnone concentrations are expressed as arithmetic mean concentrations in nanograms/100 ml on the vertical axis, and bone maturation (BA) in years on the horizontal axis. Full lines and interrupted lines represent the linear relationship obtained for male and female children respectively, n = number of observation; r = the coefficient of correlation, and P = the statistical significance of the correlation assessed. 16
12-
12-
8-
8-
4-
4-
BA
0 0
8
16 BA
8
ences in the origin and disposal of DHT in females. Indeed, Saez et al. (34) have shown that the metabolic clearance rate of DHT is lower in female than in male adults, and that at least 50% of circulating DHT in females comes from the metabolism of A and not T. In girls, the increase in this ratio is more evident after 8-10 years of age and is concomitant with the increase in E2. However, estrogens are known to increase TeBg but to inhibit the 5a reductase (34). In girls, a positive correlation was found between all sex steroids measured and bone
16 BA
maturation, while in boys such linear relationship was found only with DHA, A, T, and DHT, but not with E t and E2. In addition, an excellent correlation was found in both sexes for DHA and A, and the levels of these sex steroids, presumably reflecting mainly, albeit not exclusively, the activity of the adrenal cortex (34-36), are increased long before any increase in T and DHT in boys and E2 in girls. It must be noted that we have no information on the phase of the menstrual cycle in older girls in which cyclicity was established, and their results
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PLASMA SEX STEROIDS AND PUBERTY were not analyzed separately, although it is well known that important cyclic variations of estrogens do occur (29). Since, prior to puberty, it is likely that T, DHT, and possibly Ej reflect mainly the peripheral metabolism of A (35-37), it is possible that A or one of its metabolites may play an important role in the central nervous system processes of maturation of the hypothalamus, the first significant increase in A being determinant in the activation of the hypothalamic-pituitary-gonadal axis. It would also appear that A is an excellent index of general physical maturation in both sexes from early childhood through adolescence. It is also possible that the hormonal effects that lead to the elevation of the gonadostatic threshold to circulating sex steroids and, consequently, to the release of gonadotropins, may be extended over several years and that DHA may have an earlier and additive influence on this process. Whether these or other adrenal sex hormones may have a direct and specific effect upon the hypothalamus cannot be verified by the results herein reported. However, the time-sequence relationship observed between adrenal and gonadal sex steroids is evidence in favor of a role for adrenal steroids in the maturation processes of the CNS mechanisms which lead to puberty. Since this is a cross-sectional and descriptive study at various ages using measurements of several steroids and does not involve any experimental administration of DHA and A, the exact action of these steroids remains speculative. Comparative studies in precocious, early, or late puberty and in agonadal and hypogonadal children at a chronological age compatible with puberty may help to verify the hypothesis of a permissive role, if not an essential one, of the adrenals in the regulatory processes which control the onset of puberty. However, in agonadal and primary hypogonadal children in whom an early rise in gonadotropins frequently occurs, adrenal sex steroids are presumably secreted in normal quantities, but the absence or defi-
475
cient levels of gonadal sex steroids may be insufficient to exert a negative feedback effect of these steroids upon gonadotropin release. Acknowledgment This work was supported by Le Conseil de la Recherche en Sante de Quebec, Le Ministere de l'Education (FCAC) Quebec, The Medical Research Council (Canada), La Fondation Justine LacosteBeaubien, and l'lnstitut National de la Sante et de la Recherche Medicale (France). The authors are indebted to Dr. J. M. Saez for his advice and interest; to Ms. G. Alberti, E. Romanet, A. M. Morera, and A. Cathiard for skillful technical assistance; and to Ms. M. F. Rocheleau for secretarial help. We are particularly grateful to Drs. M. Jeune, R. Caron, and M. David and the staff of l'Hopital Debrousse and Charney; to Drs. H. Berger and H. Gleispach of Innsbruck, who provided a good part of the clinical material used in this study; and to Drs. J. C. Jequier and M. A. Gagnon; and to Mr. L. Annable, INRS Sante of l'Universite du Quebec, for the statistical analysis.
References 1. Ducharme, J. R., M. G. Forest, E. de Peretti, M. Sempe, and J. Bertrand, Pattern of plasma androgens and estrogens from childhood through adolescence, Clin Res 21: 1025, 1973 (Abstract). 2. Donovan, D. T., and J. J. Van Der Werff Ten Bosch, Physiology of Puberty, E. Arnold, Publishers, London, 1965. 3. Kulin, H. E., M. M. Grumbach, and S. L. Kaplan, Changing sensitivity of the pubertal gonadal hypothalamic feedback mechanism in man, Science 166: 1012, 1969. 4. Collu, R., and J. R. Ducharme, Role of adrenal steroids in the regulation of gonadotropin secretion at pubertyj Steroid Biochem 6: 869, 1975. 5. Gorski, M. E., and I. E. Lawton, Adrenal involvement in determining the time of onset of puberty in the rat, Endocrinology 93: 1232, 1973. 6. Saez, J., and J. Bertrand, Studies on testicular function in children: Plasma concentrations of testosterone, dehydroepiandrosterone, and its sulfate before and after stimulation with human chorionic gonadotrophin, Steroids 12: 749, 1968. 7. Forest, M. G., J. M. Saez, and J. Bertrand, Assessment of gonadal function in children, Pediatrician 2: 102, 1973. 8. Forest, M. G., J. M. Saez, L. Sann, and J. Bertrand, La fonction gonadique chez le nourrisson et 1'enfant, Arch Fr Pediatr 31: 587, 1974. 9. Baghdassarian, A., H. Guyda, A. P. Johanson, C. J. Migeon, and R. M. Blizzard, Urinary excretion of radioimmunoassayable luteinizing hormone (LH)
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20.
21. 22. 23. 24.
DUCHARME ET AL. in normal male children and adults according to age and stage of sexual development, 7 Clin Endocrinol Metab 31: 428, 1970. Burr, I. M., P. Sizonenko, S. L. Kaplan, and M. M. Grumbach, Hormonal changes in puberty: I. Correlation of serum luteinizing hormone and follicle-stimulating hormone with stages of puberty, testicular size, and bone age in normal boys, Pediatr Res 4: 25, 1970. Sizonenko, P. C , I. M. Burr, S. L. Kaplan, and M. M. Grumbach, Hormonal changes in puberty: II. Correlation of serum luteinizing hormone and follicle-stimulating hormone with stages of puberty and bone age in normal girls, Pediatr Res 4: 36, 1970. Winter, J. S. D., and C. Faiman, Pituitary gonadal relations in female children and adolescents, Pediatr Res 7: 948, 1973. Root, A. W. Endocrinology of puberty: I. Normal sexual maturation,/ Pediatr 83: 1, 1973. Tanner, J. M., Growth at adolescence. Blackwell Scientific Publications, Oxford, 1962, p. 32. Sempe, M., Pour une determination "dynamique" de la maturation osseuse, C.R. XIe reunion des equipes chargees des etudes sur la croissance et le developpement de l'enfant normal, Centre International de l'Enfance, Paris 1: 78, 1972. Sempe, M., Croissance et maturation osseuse, Therapix, Paris, 1972, p. 16. Forest, M. G., P. C. Sizonenko, A. M. Cathiard, and J. Bertrand, Evidence for testicular activity in early infancy, J Clin Invest 53: 819, 1974. Forest, M. G., A. M. Cathiard, and J. Bertrand, Total and unbound testosterone levels in the newborn and in normal and hypogonadal children: Use of a sensitive radioimmunoassay for testosterone,; Clin Endocrinol Metab 36: 1132, 1973. Saez, J. M., A. M. Morera, A. Dazord, and J. Bertrand, Adrenal and testicular contribution to plasma estrogens,/ Endocrinol 55: 41, 1972. Murphy, B. E. P., Some studies of the proteinbinding of steroids and their application to the routine micro and ultra-micro measurement of various steroids in body fluid by competitive binding radioassay, / Clin Endocrinol Metab 27: 973, 1967. Winer, B. J., Statistical principles in experimental design, McGraw Hill, New York, 1971, p. 206. Winer, B. J., Statistical principles in experimental design, McGraw Hill, New York, 1971, p. 404. Nelder, J. A., Letter to the Editor, Appl Statist 23: 232, 1974. Saez, J. M., and A. M. Morera, Plasma concentration of estrone (E,) and estradiol (E2) before puberty in humans, Ada Paediatr Scand 62: 84, 1973. (Abstract).
JCE & M • 1976 Vol 42 • No 3
25. Canlorbe, P., J. E. Toublanc, J. O. Job, R. Scholler, M. Roger, M. Castagner, and P. Leymarie, La fonction endocrine du testicule chez l'enfant et l'adolescent, Ann Pediatr 2: 13, 1974. 26. Bidlingmaier, F., M. Wagner-Barnack, O. Butenandt, and D. Knorr, Plasma estrogens in childhood and puberty under physiological and pathological conditions. Pediatr Res 3: 901, 1973. 27. Winter, J. S. D., and C. Faiman, Pituitary-gonadal relationship in male children and adolescents, Pediatr Res 6: 126, 1972. 28. Jenner, M. R., R. P. Kelch, S. L. Kaplan, and M. M. Grumbach, Hormonal changes in puberty: IV. Plasma estradiol, LH and FSH in prepubertal children, pubertal females, and in precocious puberty, premature thelarche, hypogonadism and in child with feminizing ovarian tumor, / Clin Endocrinol Metab 34: 521, 1972. 29. Winter, J. S. D., and C. Faiman, The development of cyclic pituitary-gonadal function in adolescent females,/ Clin Endocrinol Metab 37: 714, 1973. 30. Sizonenko, P. C , and L. Paunier, Hormonal changes in puberty. III. Dehydroepiandrosterone, testosterone, FSH, and LH with stages of puberty and bone age in normal boys and girls and in patients with Addison's disease or hypogonadism or with premature or late adrenarche, / Clin Endocrinol Metab 41: 894, 1975. 31. Hopper, B. R., and S. S. C. Yen, Circulating concentrations of dehydroepiandrosterone and dehydroepiandrosterone sulfate during puberty, / Clin Endocrinol Metab 40: 458, 1975. 32. Angsusingha, K., F. M. Kenny, H. R. Nankin, and F. H. Taylor, Unconjugated estrone, estradiol, and FSH and LH in prepubertal and pubertal males and females, / Clin Endocrinol Metab 39: 63, 1974. 33. Gupta, D., A. Attanasio, and S. Raaf, Plasma estrogen and androgen concentrations in children during adolescence, / Clin Endocrinol Metab 40: 636, 1975. 34. Saez, J. M., M. G. Forest, A. M. Morera, and J. Bertrand, Metabolic clearance rate and blood production rate of testosterone and dihydrotestosterone in normal subjects, during pregnancy, and in hyperthyroidism,/ Clin Invest 51: 1226, 1972. 35. Baird, D. T., R. Horton, C. Longcope, and J. F. Tait, Steroid prehormones, Perspect Biol Med 11: 384, 1968. 36. Baird, D. T., R. Horton, C. Longcope, and J. F. Tait, Steroid dynamics under steady state conditions, Recent Prog Horm Res 25: 611, 1969. 37. Van de Wiele, R., P. McDonald, E. Gurpide, and S. Lieberman, Studies on the secretion and interconversion of the androgens, Recent Prog Horm Res 19: 275, 1963.
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T
HE hypothalamic-pituitary-gonadal relationships during the course of pubertal development in man have been extensively studied and the hormonal changes characteristic of puberty attributed to modifications of the sensitivity of the hypothalamic gonadostatic threshold to circulating androgens and estrogens (2,3). However, the mechanisms responsible for this decrease in the sensitivity of the hypothalamus to the feedback effect of sex steroids remain unclear. Ducharme et at. (1) and Collu and Ducharme (4) have presented evidence that some adrenal steroids may play a role in the activation of the hypothalamic-pituitary-gonadal axis at puberty. Recently, Gorski and Lawton (5) have shown that adrenalectomy of immature female rats at 18 and 25 days of age, but not at 35 days, significantly delayed the advent of puberty compared with normal or sham-operated Received July 30, 1975. This work was presented in part at the Canadian Society for Clinical Investigation, Montreal, Canada, January 1974 and appeared in abstract form (1). Requests for reprints should be addressed to: Jacques-Raymond Ducharme, Centre de Recherche Pediatrique, Hopital Sainte-Justine, Montreal, Quebec, H3T 1C5, Canada.
no significant increase in T, DHT, or E, was noted prior to 12 years of age in both sexes. However, E2 showed a significant increase between 10 and 12 years of age in girls. This early rise in the course of pubertal development of the two sex steroids predominantly of adrenal origin, DHA and A, and its occurrence 1 to 2 years earlier in girls than in boys, as does puberty itself, suggest a possible role for these steroids in the mechanisms involved in triggering the hypothalamic-pituitary-gonadal axis at puberty. (J Clin Endocrinol Metab 42: 468, 1976)
controls and that the autotransplantation of the removed adrenals at 18 days prevented this delay. These data were interpreted as evidence that, during a certain period, the adrenals play a role in the maturation of the brain-pituitary-ovarian axis. In human beings, the activation of the adrenal cortex prior to puberty is characterized by a significant increase in plasma dehydroepiandrosterone (DHA) and its sulfate (DHAS) (6) and estrone (EJ (7,8). However, the role of these or other sex hormones in the initiation of puberty is at best speculative. There is clinical evidence that exposure of the prepubertal child to sex steroids of either adrenal or gonadal origin may hasten puberty. This effect is closely related with advanced bone maturation to a bone age compatible with physiological onset of puberty. Indeed, boys and girls with virilizing adrenal hyperplasia, either untreated or treated with inadequate suppressive therapy, may undergo rapid pubertal development once adequate glucocorticoid treatment is instituted, provided their skeletal maturation reaches the pubertal range. Treatment before puberty of children with
468
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469
PLASMA SEX STEROIDS AND PUBERTY TABLE 1. Clinical material Age group (years)
N*
C.A.|
H.A.§
P
W.A."
B.A.H
15.4 db 1.2 13.4 dt4.4 10.7 dt 1.7
15.0 dt 1.1 13.4 dt 1.7 11.6 dt 1.1 9.4:t 1.1 7.9:t 1.0 5.7:t 1.1
4.2 db0.4 3.1 dt 1.1 2.3 dt 0.7 1.1 dt 0.2 1.0 dtO.O 1.0 dbO.O
14.7 ± 12.1 ± 10.3 ± 8.9: t 7.2 :t 5.5:t
4.6 db0.5 2.9 db0.8 1.5 db0.5 1.0 db0.3 1.0 dbO.O 1.0 db 0.0
Girls 12-14 10-12 8-10 6-8 14 12-14 10-12 8-10 6-8 14
1.2 0.8 2.1 1.7 1.3 1.0
9.7:t2.5 7.7 :t 1.1 4.7:t 1.2
Boys 15.4 dt 1.6 13.2 dt 1.5 11.5 dt 1.5
8.7 :t 1.6 7.3 dt 1.1 4.8 dt 1.0
8.6: t 1.6 7.4 :t 1.1 4.8:t 1.4
1.1 1.1 0.8 0.9 1.0 2.1
* Number of subjects \ Chronological age ± SEM. § Height age ± SEM. 11 Weight age ± SEM. H Bone age ±SEM. ** Stage of pubertal development according to Tanner (13) ± SEM.
sex steroids may lead to similar events if the exposure has been of sufficient magnitude and duration to advance sufficiently their bone age. The advent of radioimmunoassay techniques for gonadotropins has made it possible to delineate the pattern of their secretion throughout infancy, childhood, and adolescence and to show that gonadotropin levels begin to rise prior to any evidence of secondary sex characteristics (9-12). In addition, this rise in plasma gonadotropins occurs some two years earlier in female than male subjects, (9-13) which is consistent with the earlier onset of puberty in girls. The present study was undertaken in an attempt to clarify further the relationships between adrenal sex steroids, gonadotropin secretion, and indices of pubertal development and to verify whether adrenal steroids play a role in the triggering of the hypothalamic-pituitary-gonadal axis at puberty. If such an effect does take place, it could result either from a direct and specific action of these steroids at the hypothalamic level or, more likely, be mediated through hastening the onset of maturation of the
central nervous system mechanisms which lead to puberty. Materials and Methods Subjects. One hundred and nine children, 3.5 to 16.3 years of age, 55 boys and 54 girls, were the subjects of this study (Table 1). Each subject was submitted to physical examination, weighed, measured, and ascribed to a stage of genital or breast development according to Tanner (14). Bone age (15) and height and weight ages (16) were estimated according to Sempe. Each subject was free of acute or chronic illness at the time of study and was within the normal range for his age in height and weight. Plasma was obtained by antecubital vein puncture in an heparinized disposable syringe between 9 and 11 AM in most instances, and the plasma was immediately separated and frozen at - 2 0 C until assayed. Informed consent was obtained in each instance. Free steroid analysis. Plasma DHA,* A, T, and DHT
were
measured
by
radioimmunoassay
* Abbreviations and trivial names used in this paper: A4-androstenedione (A), androst-4-ene-3,17 dione; dehydroepiandrosterone (DHA), A5-androsten-3/3-ol-
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470
DUCHARME ET AL.
essentially as described by Forest (17,18). Each steroid extract was eluted on celite with gradients of benzene and ethyl acetate in isooctane. DHA and DHT were eluted with 15% benzene in isooctane while pure isooctane and 3% benzene in isooctane yielded the androstenedione fraction. The latter was then reduced to T with sodium borohydride and eluted with 25% ethyl acetate in isooctane as for free testosterone initially present in the plasma tested (17). The antibody to 3-(O-Carboxy-methyl) oxime of T coupled with BSA was produced in rabbits as previously described (18) and showed a cross-reactivity of the order of 76% between T and DHT but no significant cross reaction with the other steroids herein measured except for A which cross-reacted with T approximately 1%. The anti-DHA antibody was obtained similarly in rabbits immunized with DHA-17-carboxime coupled with BSA and cross-reacted with A and iso-androsterone to a degree of 4% and 8%, respectively. Ej and E2 were also measured by RIA using a sheep antibody to E2-17-/3-hemisuccinate graciously supplied by Dr. Jose Saez. This antibody showed complete cross-reactivity between E2 and Ej but no cross-reactivity with any of the other steroids reported in this study. The RIA was carried out essentially as described by Saez (19). Due to simultaneous initial separation of A, T, and DHA on the same column, 0.5 to 3.0 ml of plasma were used while 10.0 ml of plasma was usually utilized for sequential elution of DHT, Ej, and E2 on the same plasma sample. Free cortisol was measured by competitive protein-binding assay by the method of Murphy (20). One ml of the same adult female plasma pool was used as reference preparation for DHA, A, and T in each assay together with appropriate non-radioactive standards diluted in water. Two ml of the same adult male plasma pool was used similarly together with appropriate standards for DHT, Ei, and E2. The blank value obtained was 17 one; dihydrotestosterone (DHT), 17/3-hydroxy-5aandrostan-3 one; estrone (Et) Ali3j3 (10)-estratrien-3ol-17 one; estradiol (E2), Au,5 (10)estratrien-3,17/3diol; testosterone (T), 17/3-hydroxy-androst-4-ene-3 one; cortisol (F), A4-pregnen-ll/M7a,21-triol-3,20dione. RIA, radioimmunoassay; TeBg, testosteroneestradiol-binding globulin; BSA, bovine serum albumin.
JCE & M 1976 Vol 42 . No 3
less than 15 pg for androgens and 7.0 pg for estrogens. No radioactive DHA was added prior to extraction of the plasma samples to avoid any risk of cross-over of the radioactivity and consequently faulty evaluation of the recovery. However, since in our hands, the recovery of DHA and T eluted from the same celite column was identical, the latter was used to correct for experimental losses. Indeed, in 18 experiments, the mean recovery for DHA and T eluted in their respective fractions from the same column was (mean ± SD) 92.1 ± 1.9% and 93.5 ± 1.6%, respectively. In addition, 30 paired samples of DHA measured either separately or calculated from the recovery of T on a column run simultaneously gave a mean difference of 4.9%, which is equal to or lower than the inter-assay variations. Inter-assay variations for A, T, and DHA were 9%, .11%, and 10%, while those for E,, E2, and DHT were 14%, 17%, and 5%, respectively. The intra-assay variations were in the order of 7% for A and T and 5% for DHA, and 10%, 6%, and 5%, respectively for E,, E2, and DHT. Finally, to assure better comparative homogeneity between the different assays performed, each RIA included samples obtained from children of both sexes in each age group category. Statistical analysis. For the purpose of the statistical analysis, values less than 1 ng/100 ml were taken to be 1 ng/100 ml since the sensitivity of the radioimmunoassay procedure would not permit their more accurate determination. Since Hartley tests (21) revealed raw data for all hormones to be heteroscedastic with variance increasing in proportion to the magnitude of hormone concentration, data were transformed to decimal logarithms, after which they were satisfactorily homoscedastic. Results were then submitted, separately for each hormone, to a twoway analysis of variance with age and sex as factors (22,23). Since there were unequal numbers of subjects in cells (age-sex groups), the analyses were made on a CDC 6400 computer using a multiple regression program. Age effects adjusted for sex differences, sex effects adjusted for age differences, and the sex-age interaction adjusted for both age and sex differences were calculated. The results are presented as mean concentrations (transformed back from logarithms) with 95% confidence intervals based on appropriate error mean squares and degrees of freedom from the above analyses. A set of 2 by 2
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PLASMA SEX STEROIDS AND PUBERTY individual comparisons of age effects within each sex were assessed for statistical significance and are presented.
TABLE 2. Factorial analysis of variance
sv*
Results
The analyses of variance for each steroid appear in Table 2; plasma steroid levels (mean and 95% confidence intervals) and the statistical significance of differences are presented in Table 3. A statistically significant increase in DHA levels was already evident in girls between the group less than 6 years old and the group 6-8 years of age. In boys, this difference becomes apparent between 6-8 and 8-10 years of age. A second and highly significant increase was observed after 8-10 years in both sexes. For A, the interaction age by sex was not significant. In girls, there was a continuous statistically significant increase between the 6-8 year old group and the 12-14 year age group whereas, in boys, this increment was only significant between 8-10 and 1012 years of age. As for the other sex steroids measured, no significant increase was noted prior to the 12-14 year age group with the exception of E2 in girls, which rose significantly prior to 10-12 years of age. Testosterone levels were similar in both sexes until the 12-14 year age group. T levels rose significantly only after the 10-12 year group in girls while this simultaneous increase in boys remained significant thereafter. This finding explains why the interaction age by sex observed was statistically significant whereas the sex difference was not. A similar pattern was found for DHT in boys. In addition, in general a higher mean ratio based on all age groups of DHT/T was noted in girls (.33) compared with boys (.20). No significant change in plasma Ej was observed in boys. In contrast, a significant rise was seen in girls after 10-12 years of age. A significant rise in plasma E2 was observed after 10 years of age in girls, while
471
Dehydroepiandrosterone (DHA) Total Sex Age
Age x sex Error Androstenedione (A) Total Sex Age
Age x sex Error Testosterone
SS|
Exact significance
DF§
23.625160 .203951 16.741766 .780913 6.099704
96 1 5 5 85
20.011041 .713390 11.757628 .143095 7.739889
106 1 5 5 95
27.634025 .073076 16.103985 2.088800 9.154010
106 1
14.153380 .002444 7.017391 2.150817 4.797024
73 1
2.842 46.660 2.176
.09152 .00000 .06364
8.756200 28.862808 .351273
.00398 .00000 NS
.758384 33.425319 4.335498
.00000 .00143
.031587 18.139508 5.559726
.00000 .00030
7.319179 2.090244 1.783443
.00810 .07388 .12404
3.347594 5.782903 3.802524
.06747 .00015 .00388
4.075138 1.726864 1.720472
.04407
(T)
Total Sex Age
Age x sex Error Dihydrotestosterone (DHT) Total Sex Age
Age x sex Error Estrone (E,) Total Sex Age
Age x sex Error Estradiol (E2) Total Sex Age
Age x sex Error Cortisol (F) Total Sex Age
Age x sex Error
9.420546 .625385 .893001 .761929 7.177354
5 5 95
5 5
NS
NS
62 95 1
5 5 84
12.502703 .313172 2.704988 1.778654 7.764751
94 1
5.203000 .193917 .410867 .409346 4.187506
99 1
5 5 83
5 5 88
* Sources of variation. \ Sum of squares. § Degree of freedom. 11 Fisher coefficient F.
no significant difference was seen in boys, although somewhat higher values were found after 14 years of age. This discrepancy of sex pattern for age accounts for
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JCE & M . 1976 Vol 42 • No 3
DUCHARME ET AL.
472
TABLE 3. Plasma free steroid levels in children with increasing age (ng/100 ml) Age group (years)
14
Dehydroepiandrosterone (DHA): Girls 6 9 10 9 N* 8 6 116.4 351.7 370.1 465.9 28.6 109.3 X| CL§ 19.4-42.1 72.6-164.5 74.4-179.6 233.7-529.4 224.3-610.6 282.4-770.5 P < 0.0001 P < 0.0005 Boys N 9 7 11 10 6 6 X 43.4 43.8 84.7 264.8 352.9 505.9 CL 26.3-71.7 29.1-66.0 53.3-134.6 183.0-383.3 239.5-520.3 306.7-834.8 P < 0.05 P P < 0.0005 Androstenedione (A): Girls 11 9 6 N 9 9 9 X 14.8 17.7 32.1 64.5 123.2 131.4 CL 9.6-22.9 11.5-27.4 21.6-47.4 41.7-99.6 79.8-190.4 77.2-223.9 P P < 0.05 P < 0.05 P < 0.05 Boys 9 12 11 9 6 N 7 X 20.2 44.7 66.6 82.0 11.6 16.1 CL 7.1-19.0 13.1-31.3 30.7-65.2 44.9-98.7 48.2-139.7 10.4-24.8 P P < 0.01 Testosterone (T) Girls N 6 9 9 9 9 11 8.4 9.8 X 8.7 15.1 33.0 37.3 CL 5.2-13.4 6.1-15.8 5.7-13.3 9.4-24.2 20.6-53.0 20.9-66.5 P P < 0.05 Boys N 7 7 11 9 9 11 X 6.2 7.2 6.9 13.1 50.7 204.2 CL 4.5-11.5 8.6-20.1 33.1-77.8 119.4-349.1 3.6-10.6 4.3-11.1 P < 0.001 P P < 0.001 Dihydrotestosterone (DHT): Girls N 4 8 7 4 7 5 X 1.4 6.1 11.7 7.1 2.8 7.7 CL 3.9-9.6 4.7-12.4 7.2-19.0 4.0-12.6 0-2.6 1.5-5.4 r Boys 11 N 1 3 7 10 7 13.4 X 3.4 2.6 31.8 2.0 3.9 CL 1.6-4.2 9.1-19.7 19.6-51.6 2.6-5.9 0-7.2 1.6-7.1 P P < 0.0001 P < 0.01 Estrone (E,): Girls N 11 6 10 8 9 9 X 2.7 4.6 4.4 8.9 5.9 3.0 CL 3.1-7.0 3.4-10.2 2.8-6.8 5.7-14.0 1.9-4.6 1.7-4.4 P P < 0.05 Boys N 6 7 7 10 8 5 X 3.0 3.3 3.2 2.7 2.9 3.9 CL 2.0-5.4 1.7-4.4 2.1-7.1 2.1-4.9 1.8-5.3 1.7-4.8 r> r Estradiol (E8): Girls N 10 9 6 10 8 9 6.4 2.1 7.0 1.2 4.9 X 1.9 3.6-11.3 4.4-11.1 CL 1.4-3.3 3.1-7.8 1.3-3.1 0-2.0 P P < 0.05
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473
PLASMA SEX STEROIDS AND PUBERTY TABLE 3. (Continued)
Age group (years) Boys N X CL P Cortisol (F): Girls N X CL P Boys N X CL P
14
6 3.0 1.7-5.3
7 2.3 1.3-3.8
7 2.0 1.2-3.5
10 2.0 1.3-3.1
8 2.3 1.4-3.9
5 4.5 2.4-8.4
7 7.7 5.3-11.2
9 9.3 6.6-12.9
11 10.0 9.4-13.6
8 7.8 5.5-11.2
8 9.8 6.9-14.0
6 12.6 8.4-19.0
6 11.3 7.5-17.1
8 7.4 5.2-10.5
8 6.1 4.3-8.6
11 11 5.7 10.1 4.2-7.6 7.5-13.6 P< 0.01
7 7.6 5.2-11.1
* Number of observations. | Mean concentration transformed back from logarithms. § 95% confidence intervals. 11 Statistical significance.
the significant interaction age by sex found as was also observed for testosterone. Finally, there was no significant difference in plasma free cortisol (F) at any age except between the 10-12 and 12-14 year old groups in boys. When androgens and estrogens were correlated with bone maturation, a significant correlation was found in girls for DHA, A, T, DHT, E1} and E2 while such correlation was seen for DHA, A, T, and DHT, but not for Ej and E2 in boys (Fig. 1).
earlier onset of puberty in girls. In addition, a similar time difference in girls and boys for gonadotropin secretion has been reported (9-12) with a significant rise prior to any evidence of pubertal development and, as this study shows, prior to any significant rise in gonadal sex steroids. Our results for plasma DHA are in general agreement with those reported by Saez et al. (6,7), Hopper and Yen (31), and Sizonenko (30). However, we were unable to confirm the significant difference in estrone levels found by Saez et al. (6,7) between Discussion children of both sexes less than 6 years old and those 7 to 10 years of age. Nevertheless, The values obtained for androgens and in girls higher values were detected after estrogens in this study are in general agree8 years of age (Table 3), compared with ment with those previously published (6,7, younger children, in accord with studies by 13,17,18,24-33). In addition, an interesting time-sequence relationship was found for Bidlingmaier (26). This rise became signifiplasma levels of some sex steroids and cant after 12 years of age (stage P2) in girls, in indices of sexual maturation. Indeed, in agreement with the data of Angsusingha girls plasma levels of DHA started to rise as et al. (32) and of Gupta et al. (33). No such early as 6 to 8 years followed by a significant trend has been observed for boys at any age increase in A by 8 to 10 years, while no in- in this study, at variance with studies by crease in T, E1} or E2 was noted prior to Saez reported by Forest (7). Another interesting observation concerns 10-12 years of age and thereafter. The same time-sequence was also observed in boys the higher DHT/T ratio observed in girls, but with a one to two year delay compared which is also found in data from Gupta et with girls, which is consistent with the al. (33). Thisfindingmay be related to differ-
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DUCHARME ET AL.
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JCE & M • 1976 Vol 42 • No 3
FIG. 1. Linear correlation between steroid levels and bone maturation (BA). The honnone concentrations are expressed as arithmetic mean concentrations in nanograms/100 ml on the vertical axis, and bone maturation (BA) in years on the horizontal axis. Full lines and interrupted lines represent the linear relationship obtained for male and female children respectively, n = number of observation; r = the coefficient of correlation, and P = the statistical significance of the correlation assessed. 16
12-
12-
8-
8-
4-
4-
BA
0 0
8
16 BA
8
ences in the origin and disposal of DHT in females. Indeed, Saez et al. (34) have shown that the metabolic clearance rate of DHT is lower in female than in male adults, and that at least 50% of circulating DHT in females comes from the metabolism of A and not T. In girls, the increase in this ratio is more evident after 8-10 years of age and is concomitant with the increase in E2. However, estrogens are known to increase TeBg but to inhibit the 5a reductase (34). In girls, a positive correlation was found between all sex steroids measured and bone
16 BA
maturation, while in boys such linear relationship was found only with DHA, A, T, and DHT, but not with E t and E2. In addition, an excellent correlation was found in both sexes for DHA and A, and the levels of these sex steroids, presumably reflecting mainly, albeit not exclusively, the activity of the adrenal cortex (34-36), are increased long before any increase in T and DHT in boys and E2 in girls. It must be noted that we have no information on the phase of the menstrual cycle in older girls in which cyclicity was established, and their results
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PLASMA SEX STEROIDS AND PUBERTY were not analyzed separately, although it is well known that important cyclic variations of estrogens do occur (29). Since, prior to puberty, it is likely that T, DHT, and possibly Ej reflect mainly the peripheral metabolism of A (35-37), it is possible that A or one of its metabolites may play an important role in the central nervous system processes of maturation of the hypothalamus, the first significant increase in A being determinant in the activation of the hypothalamic-pituitary-gonadal axis. It would also appear that A is an excellent index of general physical maturation in both sexes from early childhood through adolescence. It is also possible that the hormonal effects that lead to the elevation of the gonadostatic threshold to circulating sex steroids and, consequently, to the release of gonadotropins, may be extended over several years and that DHA may have an earlier and additive influence on this process. Whether these or other adrenal sex hormones may have a direct and specific effect upon the hypothalamus cannot be verified by the results herein reported. However, the time-sequence relationship observed between adrenal and gonadal sex steroids is evidence in favor of a role for adrenal steroids in the maturation processes of the CNS mechanisms which lead to puberty. Since this is a cross-sectional and descriptive study at various ages using measurements of several steroids and does not involve any experimental administration of DHA and A, the exact action of these steroids remains speculative. Comparative studies in precocious, early, or late puberty and in agonadal and hypogonadal children at a chronological age compatible with puberty may help to verify the hypothesis of a permissive role, if not an essential one, of the adrenals in the regulatory processes which control the onset of puberty. However, in agonadal and primary hypogonadal children in whom an early rise in gonadotropins frequently occurs, adrenal sex steroids are presumably secreted in normal quantities, but the absence or defi-
475
cient levels of gonadal sex steroids may be insufficient to exert a negative feedback effect of these steroids upon gonadotropin release. Acknowledgment This work was supported by Le Conseil de la Recherche en Sante de Quebec, Le Ministere de l'Education (FCAC) Quebec, The Medical Research Council (Canada), La Fondation Justine LacosteBeaubien, and l'lnstitut National de la Sante et de la Recherche Medicale (France). The authors are indebted to Dr. J. M. Saez for his advice and interest; to Ms. G. Alberti, E. Romanet, A. M. Morera, and A. Cathiard for skillful technical assistance; and to Ms. M. F. Rocheleau for secretarial help. We are particularly grateful to Drs. M. Jeune, R. Caron, and M. David and the staff of l'Hopital Debrousse and Charney; to Drs. H. Berger and H. Gleispach of Innsbruck, who provided a good part of the clinical material used in this study; and to Drs. J. C. Jequier and M. A. Gagnon; and to Mr. L. Annable, INRS Sante of l'Universite du Quebec, for the statistical analysis.
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