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Original Studies |
Department of Pediatrics, Asahikawa Medical College, Asahikawa 078-8510, Japan
Address all correspondence and requests for reprints to: Ryo Mitamura, M.D., Shimizu Red Cross Hospital, Minami 2–2, Shimizu-cho, Kamikawa-gun, Hokkaido 089-0195, Japan.
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Abstract |
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Diurnal rhythms of LH, FSH, and testosterone were apparent in all subjects, including those aged 4–5 yr. Serum LH and FSH concentrations showed night-day variation in a pulsatile fashion. The serum testosterone concentration was elevated at early morning in all subjects. Mean 24-h LH, FSH, and testosterone concentrations of prepubertal subjects who did not attain puberty for at least 3 yr were 0.10 U/L, 0.63 U/L, and 0.06 ng/mL, respectively, whereas those of prepubertal subjects who attained puberty within 1 yr (0.54 U/L, 1.68 U/L, and 0.10 ng/mL, respectively) were significantly higher. Furthermore, mean 24-h LH, FSH, and testosterone concentrations increased with developing puberty. All of the 46 subjects showed positive cross-correlation between the LH and testosterone time series. The mean lag time from the LH to the testosterone time series in the prepubertal subjects who attained puberty within 1 yr (4.7 ± 2.4 h, mean ± SD) was shorter than that in the prepubertal subjects who attained puberty after at least 3 yr (7.3 ± 2.2 h). This lag time decreased with developing puberty, plateauing at 1.4 ± 0.9 h at midpuberty.
Thus, the diurnal rhythms of LH, FSH, and testosterone already exist at 4–5 yr of age; serum LH, FSH, and testosterone levels increase before the onset of puberty; and a time delay is observed between the LH and testosterone time series that decreases before the onset of puberty.
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Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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The present study investigates the diurnal profiles of LH, FSH, and testosterone over 1 yr before the onset of puberty, including those in subjects 4–5 yr of age, and the relationship between hormonal changes and the onset of puberty.
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Subjects and Methods |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Forty-six Japanese boys (32 prepubertal and 14 pubertal) of
short stature, aged 4.4–19.3 yr (mean, 10.3 yr), participated in this
study (Table 1
). The height of the
subjects ranged from -1.3 to -4.7 SD of Japanese standard
height (14). Thirty-three boys showed normal GH response to provocative
tests, and 13 boys showed isolated GH deficiency. Informed consent was
obtained from each subject or his parents. The protocol was approved by
the ethics committee of Asahikawa Medical College Hospital.
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All subjects were admitted to Asahikawa Medical College Hospital for the evaluation of pituitary function and were followed up by measuring height and weight and evaluating pubertal development every 1–3 months for 1–10 yr (mean, 4.7 yr). No medicine that could influence pubertal development was used, except GH treatment for isolated GH deficiency. The onset of puberty was defined as the point at which testicular volume attained 3 mL. Testicular volumes were determined by comparison with Prader’s orchidometer (15), and mean testicular volume was considered. The subjects were divided into four groups as follows: group I, prepuberty, testicular volume less than 3 mL (n = 32); group II, onset of puberty, testicular volume 3–4 mL (n = 5); group III, testicular volume 5–7 mL (n = 5); and group IV, testicular volume 10–16 mL (n = 4). Group I was subdivided into three groups as follows: group I-A, prepubertal subjects who did not attain puberty within 3 yr (n = 15); group I-B, prepubertal subjects who attained puberty within 1–3 yr (n = 10); and group I-C, prepubertal subjects who attained puberty within 1 yr (n = 7). All subjects of group I-A attained puberty with 5.0 ± 0.4 yr (mean ± SD), at 11.6 ± 0.3 yr of chronological age. Thereafter, all subjects progressed through pubertal development normally. Blood samples were drawn every 20 min for 24 h using a Kowarski-Cormed pump (16). Bone age was assessed according to the Tanner and Whitehouse (TW2) method (17).
Hormone assays
Serum LH and FSH concentrations were measured every 20 min using a time-resolved immunofluorometric assay (TR-FIA, DELFIA, Pharmacia Wallac, Turku, Finland). All samples of each subject were analyzed in separate assays. The assay standard was the WHO First International Reference Preparation of Pituitary LH Human for Immunoassay (68/40) and the Second International Reference Preparation of Pituitary FSH/LH Human for Bioassay. The detection limits of the LH and FSH assays were 0.03 and 0.05 U/L, respectively. Within- and between-assay coefficients of LH variation were 2.4% and 7.2% (15 U/L), 3.1% and 7.8% (0.6 U/L), and 8.1% and 16.4% (0.03 U/L), respectively. Within- and between-assay coefficients of FSH variation were 5.7% and 2.5% (16 U/L), 6.1% and 2.5% (1 U/L), and 7.0% and 7.3% (0.05 U/L), respectively. LH values below the detection limit were taken to be 0.03 U/L.
Serum testosterone concentrations were measured every 1 h by RIA after n-hexane and diethyl ether extraction (18). Both the serum samples and the assay standard were measured after extraction. All samples from each subject were analyzed in separate assays. The sample volume of the testosterone assay was 300 µL, and the detection limit was 0.01 ng/mL (0.035 nmol/L). Within- and between-assay coefficients of testosterone variation were 6.4% and 3.7% (0.25 ng/mL), 2.7% and 7.0% (0.063 ng/mL), and 4.9% and 19.7% (0.016 ng/mL), respectively.
Identification of hormone pulses
Episodic pulses of LH and FSH were identified by the PULSAR computer program (19). The SD of the pulse detection algorithm was approximated as follows: SD = (2.98x + 2.00)/100, where x is the concentration of LH or FSH. The SDs used in the program (DEFAULT, DAT file) were as follows: G(1) = 3.20, G(2) = 2.50, G(3) = 1.90, G(4) = 1.50, and G(5) = 1.20. There was no false positive pulse from a single specimen in LH concentrations of 0.03, 0.6, and 15 U/L (n = 8) and FSH concentrations of 0.05, 1, and 16 U/L (n = 10).
Statistical analysis
All hormonal measurements were transformed logarithmically. Daytime gonadotropin levels were recorded from 0800–2000 h, and nighttime levels were recorded from 2000–0800 h. Early morning levels of testosterone were recorded from 0400–0800 h, and late evening levels were recorded from 2000–0000 h. The temporal relationship between the LH time series and the testosterone time series was determined using cross-correlation analysis, and lag time was defined as the acrophase of the cross-correlation coefficient curve. Means across all groups were compared using ANOVA. Means between pairs of groups were compared using a t test. Statistical significance was established at P < 0.05.
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Results |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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All subjects showed a pulsatile pattern of serum LH level and
diurnal rhythm (Fig. 1). Serum LH
concentrations during the night (2000–0800 h) were significantly
higher than those during the day (0800–2000 h) in all subjects and all
groups (Table 2). The maximum serum LH
pulse amplitude during the night was significantly higher than that
during the day in all but three subjects (two in group I-A and one in
group III) and all groups. The mean 24-h, daytime, and nighttime serum
LH concentrations and maximum pulse amplitude significantly increased
with pubertal development and bone maturation (Fig. 2). Moreover, the mean 24-h, daytime, and
nighttime serum LH concentrations and maximum pulse amplitude in group
I-C were significantly higher than those in groups I-A and I-B and were
approximately equal to those in group II. The mean serum LH
concentration and maximum pulse amplitude during the night in group I-B
were significantly higher than those in group I-A (Fig. 3). However, no significant differences
in LH pulse frequency were observed between the groups, except for
pulse frequency during the night between groups I-B and IV.
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All subjects showed a pulsatile pattern of serum FSH level
and diurnal rhythm. Serum FSH concentrations during the night
(2000–0800 h) were significantly higher than those during the day
(0800–2000 h) in all but two subjects (one in group I-A and one in
group I-B) and all groups (Table 3). The
maximum serum FSH pulse amplitude during the night was higher than that
during the day in 37 subjects and 2 groups (groups I-C and II). Mean
24-h, daytime and nighttime serum FSH concentrations and maximum pulse
amplitude significantly increased with pubertal development and bone
maturation. Moreover, the mean 24-h, daytime, and nighttime
concentrations and maximum pulse amplitude during daytime in group I-B
were significantly higher than those in group I-A. Those in group I-C
were also significantly higher than those in group I-A and were
approximately equal to those in group II (Fig. 4). Serum FSH concentrations were
significantly higher than serum LH concentrations in groups I-A, I-B,
I-C, and II. Although the maximum serum FSH pulse amplitude during the
night was higher than the maximum serum LH pulse amplitude during the
night in group I-A, the former was lower than the latter in groups II,
III, and IV. However, no significant differences in pulse frequency
were observed between the groups, except pulse frequency during the day
between groups I-A and IV, between groups I-B and I-C, and between
groups I-B and IV. There were 437 LH pulses and 347 FSH pulses in 46
subjects. Seventy-one FSH pulses (20%) of all FSH pulses coincided
with LH pulses. Sixty-one FSH pulses (18%), 44 FSH pulses (13%), and
36 FSH pulses (10%) of all FSH pulses were 20, 40, and 60 min behind
LH pulses, respectively.
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Serum testosterone concentrations during the early morning
(0400–0800 h) were higher than those during the late evening
(2000–0000 h) in all subjects and in all but one (group IV) group
(Table 4). Mean 24-h, early morning, and
late evening serum testosterone concentrations significantly increased
with pubertal development and bone maturation. The mean 24-h and early
morning concentrations in group I-C were significantly higher than
those in group I-A. The mean 24-h, early morning, and late evening
concentrations in group II were significantly higher than those in
groups I-A, I-B, and I-C (Fig. 5).
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The 24-h mean testosterone concentration significantly correlated
with the 24-h mean LH concentration (partial correlation
coefficient = 0.88; P < 0.001), but not with the
24-h mean FSH concentration (partial correlation coefficient =
-0.21; P = 0.498) according to multiple regression
analysis. The temporal relationship between the LH time series and the
testosterone time series was determined using cross-correlation
analysis. All subjects showed positive cross-correlation between the LH
and testosterone time series. The mean lag time between the LH and
testosterone time series in group I-C (4.7 ± 2.4 h,
mean ± SD) was significantly shorter than that in
group I-A (7.3 ± 2.2 h) and decreased with developing
puberty. The mean lag times between the LH and testosterone time series
in groups III and IV were 1.4 ± 0.9 and 1.5 ± 0.6 h,
respectively (Fig. 6).
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Previously, LH and FSH night-day rhythms were thought to begin just before the onset of puberty, triggering its onset. The 24-h mean LH and FSH concentrations in pubertal boys are higher than those in prepubertal boys (3, 4, 7, 8, 9, 10). In the present study, we divided prepubertal boys into three groups to investigate hormonal changes before the onset of puberty. Serum LH levels during the night, and serum FSH levels for 24 h, during the day, and during the night in prepubertal boys who attained puberty within 1–3 yr (group I-B) were significantly higher than those in prepubertal boys who did not attain puberty for at least 3 yr (group I-A). Serum LH and FSH levels in prepubertal boys who attained puberty within 1 yr (group I-C) were approximately equal to those in early pubertal boys (group II). These results indicate that serum gonadotropin levels start to increase over 1 yr before the onset of puberty, not immediately before the onset of puberty.
Serum LH concentrations and LH pulse amplitude dramatically increased from early prepuberty to pubertal onset, but increases in serum FSH concentrations and FSH pulse amplitude from early prepuberty to pubertal onset were less than those for LH, and night-day differences in the serum FSH concentrations and FSH pulse amplitude were also less than those for LH. However, the serum FSH concentrations and FSH pulse amplitude were significantly higher than those for LH in early prepubertal period (group I-A). FSH may play an important role for the onset of puberty in early prepubertal period.
Parker et al. (12), Wu et al. (3), and Goji et al. (9) reported a diurnal rhythm in serum testosterone in late prepubertal boys and pubertal boys. In these previous studies, the existence of a diurnal rhythm in serum testosterone was not investigated in early prepubertal boys. The detection limit of the testosterone assay was 0.028 ng/mL in Parker’s report (12), 0.17 ng/mL in Wu’s report (3), and 0.05 ng/mL in Goji’s report (9). These detection limits are above the serum testosterone concentrations seen in early prepubertal boys. Due to the inadequate sensitivity of the testosterone assay, the previous studies did not identify the diurnal rhythm of serum testosterone in early prepubertal boys. In the present study, the detection limit of the testosterone assay was 0.01 ng/mL, so all of the serum samples were measured within the detectable range. Our results revealed that the diurnal rhythm of testosterone exists in early prepubertal boys as well as in late prepubertal boys and pubertal boys. Serum testosterone peaked in the early morning and reached its minimum value during the late evening.
Serum testosterone levels during the early morning in prepubertal boys who attained puberty within 1 yr (group I-C) were significantly higher than those in prepubertal boys who required 3 yr or longer to attain puberty (group I-A), indicating that serum testosterone levels during the early morning show an increase before the onset of puberty.
Changes in LH and FSH pulse frequency before and during puberty are controversial (1, 2, 3, 4, 5, 7, 9, 10). Recently, very low level fluctuations in gonadotropin were detected using a time-resolved immunofluorometric assay, and pulsatile secretion of gonadotropin in prepubertal boys was observed (5, 7, 9, 10). In the present study, no significant changes were observed in LH and FSH pulse frequency before and during puberty, in agreement with these recently published studies (5, 9). Changes in gonadotropins before and during puberty mainly appear to be due to increases in mean 24-h concentrations and pulse amplitude and not to increases in pulse frequency.
Relationships between testosterone and gonadotropin concentrations were assessed using multiple regression analysis. The serum testosterone concentration correlated with the LH concentration, but not with the FSH concentration. Therefore, the temporal relationship between the LH time series and the testosterone time series was determined using cross-correlation analysis. Previous studies (9, 20) have determined the lag time between the time series of LH and testosterone using cross-correlation analysis. The lag time between LH and testosterone was 120 min in late prepubertal boys (9), 60 min in early pubertal boys (9), and 10–20 min in adult men (20). However, because previous studies did not recognize the diurnal rhythm in serum testosterone in early prepubertal boys, the lag time in early prepubertal boys was not determined. The present study identified a diurnal rhythm in testosterone in all subjects, including early prepubertal boys and determined the lag time using cross-correlation analysis. The mean lag times in midpubertal and late pubertal boys were 1.4 ± 0.9 and 1.5 ± 0.6 h, respectively. These lag times were significantly shorter than that in prepubertal boys. Furthermore, the mean lag time in prepubertal boys who attained puberty within 1 yr (group I-C, 4.7 ± 2.4 h) was significantly shorter than that in prepubertal boys who required 3 yr or longer to attain puberty (group I-A, 7.3 ± 2.2 h). These results indicate that the lag time between the time series of LH and testosterone starts to decrease before the onset of puberty. The synthesis and secretion of testosterone by the testes in response to LH stimulation may increase before the onset of puberty.
We studied subjects of short stature because it was difficult to obtain 24-h blood sampling in normal healthy children. Due to tendencies toward delayed puberty and isolated GH deficiency in our subjects, the present study may not strictly reflect pubertal changes in healthy children. We investigated differences between isolated GH deficiency (n = 5) and idiopathic short stature (n = 10) in group I-A (data not shown), but there were no significant differences in any parameter (chronological age, bone age, age of pubertal onset, testicular volume, LH and FSH concentrations, LH and FSH pulse amplitude, LH and FSH pulse frequency, testosterone concentrations, or lag time between LH and testosterone time series) between the two groups. Moreover, we investigated hormonal changes by the standard of testicular volume, and all subjects attained puberty and progressed through pubertal development in a normal manner. Therefore, the present study is thought to reflect hormonal changes before and during puberty in healthy boys.
In conclusion, the present study revealed that LH and FSH show night-day rhythms, and that testosterone has a diurnal rhythm, with augmentation in early morning. These rhythms already exist in 4- to 5-yr-old boys. Serum LH, FSH, and testosterone levels start to increase before the onset of puberty. A delay is observed between the LH and testosterone time series, which decreases before the onset of puberty. Thus, preparation for the onset of puberty may begin in 4- to 5-yr-old boys.
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Acknowledgments |
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Footnotes |
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Received May 1, 1998.
Revised September 23, 1998.
Accepted October 5, 1998.
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References |
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