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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 5–6 yr. Serum LH and FSH concentrations showed night-day variation in a pulsatile fashion. The serum testosterone concentration was elevated in the early morning in all subjects. The serum estradiol concentration was elevated in the early morning in 4 of 13 prepubertal subjects and all 4 early pubertal subjects. The diurnal pattern of the serum estradiol concentration was similar to that of the serum testosterone concentration. Mean 24-h LH and testosterone concentrations in prepubertal subjects who did not attain puberty for at least 1 yr were 0.07 U/L and 65 pg/mL, respectively, whereas those in prepubertal subjects who attained puberty within 1 yr (0.14 U/L and 106 pg/mL, respectively) were significantly higher. Furthermore, mean 24-h LH, FSH, testosterone, and estradiol concentrations increased with the onset of puberty.
In conclusion, the diurnal rhythms of LH, FSH, and testosterone already exist at 5–6 yr of age, and serum LH and testosterone levels increase before the onset of puberty. These results suggest that preparation for the onset of female puberty may begin in 5- to 6-yr-old girls.
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Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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The present study investigated the diurnal profiles of LH, FSH, testosterone, and estradiol over 1 yr before the onset of female puberty, including those in subjects 5–6 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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Seventeen Japanese girls (13 prepubertal and 4 pubertal) of
short stature, aged 5.1–11.4 yr (mean, 9.0 yr), participated in the
present study (Table 1
). The height of
the subjects ranged from -1.9 to -4.4 SD (mean, -2.9
SD) of Japanese standard height (19). All girls showed
normal GH response to provocative tests. Informed consent was obtained
from each subject or her parents. The protocol for the present study
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 evaluation of pituitary function and were followed up by assessing height, weight, and pubertal development every 1–3 months for 1–8 yr (mean, 4.8 yr). No subjects received medicine that could influence pubertal development. Clinical stages of puberty were assessed by the criteria of Tanner (20). All subjects were stage 1 of pubic hair development and premenarcheal at the time of blood sampling. The onset of puberty was defined as the point at which breast bud appeared. The subjects were divided into two groups as follows: group I, prepuberty, breast stage 1 (n = 13); and group II, pubertal onset, breast stage 2 (n = 4). Group I was subdivided into two groups as follows: group I-1, prepubertal subjects who did not attain puberty within 1 yr of the study (n = 7); and group I-2, prepubertal subjects who attained puberty within 1 yr (n = 6). All subjects of group I-1 attained puberty within 3.5 ± 1.0 yr (mean ± SD), at 10.5 ± 1.6 yr of chronological age. Thereafter, all subjects progressed normally through pubertal development. Eleven subjects reached menarche at 13.5 ± 1.4 yr of chronological age, but six subjects (four in group I-1, one in group I-2, and one in group II) had not reached menarche by the end of their observation period, at 11.3 ± 1.4 yr of chronological age. Blood samples were drawn every 20 min for 24 h using a Kowarski-Cormed pump (21). Bone age was assessed according to the Tanner and Whitehouse (TW2) method (22).
Hormone assays
Serum LH and FSH concentrations were measured every 20 min using a time-resolved immunofluorometric assay (DELFIA, Pharmacia Wallac, Inc., Turku, Finland). All samples from a subject were analyzed separately. The assay standard was the WHO First International Reference Preparation of Pituitary LH Human for immunoassay (68/40) and Second International Reference Preparation of Pituitary FSH/LH Human for bioassay. The detection limits of LH and FSH 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 (23) and estradiol (Double Antibody Estradiol Kit, Diagnostic Products, Los Angeles, CA) concentrations were measured every hour by RIA after n-hexane and diethyl ether extraction. Both serum samples and assay standard were measured after extraction. All samples from a subject were analyzed separately. The sample volumes of the testosterone and estradiol assay were 300 and 200 µL, respectively. The detection limit of the testosterone assay was 10 pg/mL (35 pmol/L). Within- and between-assay coefficients of testosterone variation were 6.4% and 3.7% (250 pg/mL), 2.7% and 7.0% (63 pg/mL), and 4.9% and 19.7% (16 pg/mL), respectively. The detection limit of the estradiol assay was 2 pg/mL (7.3 pmol/L). Within- and between-assay coefficients of estradiol variation were 6.7% and 7.8% (150 pg/mL), 7.5% and 9.1% (20 pg/mL), and 9.8% and 9.8% (5 pg/mL), respectively. Estradiol values below the detection limit were taken to be 2 pg/mL.
Identification of hormone pulses
Episodic pulses of LH and FSH were identified by the PULSAR computer program (24). 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. No false positive pulses were detected from specimens at LH concentrations of 0.03, 0.6, and 15 U/L (n = 8) or at FSH concentrations of 0.05, 1, and 16 U/L (n = 10).
Statistical analysis
All hormonal measurements were transformed logarithmically, but identification of hormone pulses and cross-correlation analysis were performed before values 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 and estradiol were recorded from 0400–0800 h, and late evening levels were recorded from 2000–0000 h. The temporal relationships between LH and testosterone time series, between LH and estradiol time series, and between testosterone and estradiol time series were determined using cross-correlation analysis, where one time series was correlated against the other time series at a distance time (0–23 h) apart. Lag time was defined as the peak 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 levels and
diurnal rhythm (Fig. 1). Serum LH
concentrations during the night (2000–0800 h) were 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 higher than that during the day in all but three
subjects (three in group I-1). The mean 24-h, daytime, and nighttime
serum LH concentrations and maximum pulse amplitude during the night
significantly increased with the onset of puberty (Table 2 and Fig. 2). Moreover, the mean 24-h and nighttime
serum LH concentrations and maximum pulse amplitude during the night in
group I-2 were significantly higher than those in groups I-1. However,
no significant differences in LH pulse frequency were observed between
the groups.
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All subjects showed a pulsatile pattern of serum FSH levels and
diurnal rhythm (Fig. 1
). Serum FSH concentrations during the night
(2000–0800 h) were higher than those during the day (0800–2000 h) in
all subjects and all groups (Table 3).
The maximum serum FSH pulse amplitude during the night was higher than
that during the day in all but one subject (in group II). Mean 24-h,
daytime, and nighttime serum FSH concentrations significantly increased
with the onset of puberty (Table 3 and Fig. 3). However, no significant differences
in pulse frequency were observed between the groups. We detected 182 LH
pulses and 98 FSH pulses in 17 subjects. Thirty-three (34%) of all FSH
pulses coincided with LH pulses. Fifteen (15%), 11 (11%), and 9 (9%)
of all FSH pulses were 20, 40, and 60 min behind LH pulses,
respectively.
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The serum testosterone concentration was elevated in the early
morning in all subjects (Fig. 1). 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 all groups (Table 4). Mean 24-h, early morning, and late
evening serum testosterone concentrations significantly increased with
the onset of puberty (Table 4 and Fig. 4). The mean 24-h and late evening serum
testosterone concentrations in group I-2 were significantly higher than
those in group I-1.
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The serum estradiol concentrations in nine prepubertal subjects
(seven in group I-1 and two in group I-2) were below the detection
limit of the estradiol assay (2 pg/mL) at each sampling time. Serum
estradiol concentrations in four prepubertal subjects (four in group
I-2) and all four early pubertal subjects were above the detection
limit during the early morning (0400–0800 h; Fig. 1). The estradiol
and testosterone diurnal patterns showed similar augmentation during
the early morning (Fig. 4). Mean 24-h and early morning serum estradiol
concentrations significantly increased with the onset of puberty (Table 4).
Relationships between LH, FSH, testosterone, and estradiol concentrations and time series
The 24-h mean testosterone concentration correlated with the 24-h mean LH concentration (r = 0.85; P = 0.0001) and the 24-h mean FSH concentration (r = 0.76; P = 0.0004). The 24-h mean estradiol concentration also correlated with the 24-h mean LH concentration (r = 0.83; P = 0.0001) and the 24-h mean FSH concentration (r = 0.76; P = 0.0004). The 24-h mean estradiol concentration correlated with the 24-h mean testosterone concentration (r = 0.78; P = 0.0002).
The temporal relationship between LH and testosterone time series was
determined using cross-correlation analysis (Fig. 5). All subjects showed positive
cross-correlation between LH and testosterone time series. The mean lag
time between LH and testosterone time series in group I-2 (mean ±
SD, 4.7 ± 1.5 h) and group II (3.8 ±
1.0 h) were significantly shorter than that in group I-1 (8.9
± 2.9 h). The temporal relationships between LH and estradiol
time series and between testosterone and estradiol time series were
determined using cross-correlation analysis in eight subjects in whom
estradiol concentrations were detectable (Fig. 5). These eight subjects
showed positive cross-correlation between LH and estradiol time series
and between testosterone and estradiol time series. The mean lag time
between LH and estradiol time series in groups I-2 and II were 5.0
± 1.4 and 5.3 ± 1.7 h, respectively. The mean lag time
between testosterone and estradiol time series in groups I-2 and II
were 0.3 ± 1.9 and 1.5 ± 2.1 h, respectively. No
significant differences in the mean LH-estradiol lag time and the mean
testosterone-estradiol lag time were observed between the groups.
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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 the onset of puberty. In the present study, we divided prepubertal girls into two groups (groups I-1 and I-2) to investigate hormonal changes that occur before the onset of puberty. Twenty-four-hour and nighttime serum LH levels were significantly higher in prepubertal girls who attained puberty within 1 yr (group I-2) than in prepubertal girls who did not attain puberty for at least 1 yr (group I-1). These results indicate that serum LH levels start to increase before the onset of puberty, not with pubertal onset.
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 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 of LH during the prepubertal period (groups I-1 and I-2). FSH may play an important role in the onset of puberty.
Using one-point sample per day (1, 2, 14), seven samples per day (25), and 24-h pooled samples (9), previous studies found that serum testosterone concentrations increased during puberty in girls. Recently, Ankarberg and Norjavaara (25) reported that 24-h concentration profiles of serum testosterone, based on seven samples per 24 h, in the prepubertal and pubertal girls using a RIA on unextracted serum samples. They found the highest testosterone concentrations during the morning (0600–1000 h) and the lowest concentrations during the late evening in prepubertal and pubertal girls, but no diurnal variation was found in prepubertal girls with bone ages below 6 yr (25). In the present study we measured the testosterone concentrations hourly for 24 h and found a diurnal rhythm that peaked in the early morning and reached its nadir during the late evening, in agreement with Ankarberg’s study (25). Moreover, in the present study this diurnal rhythm was detected in all of the prepubertal and early pubertal girls. The serum testosterone levels in prepubertal 5- to 6-yr-old girls were extremely low, but the diurnal rhythm was still apparent. The diurnal testosterone rhythm found in girls is similar to the rhythm we previously recorded in prepubertal and pubertal boys (18). Serum testosterone levels over 24 h were significantly higher in prepubertal girls who attained puberty within 1 yr (group I-2) than in prepubertal girls who required 1 yr or longer to attain puberty (group I-1), indicating that serum testosterone levels show an increase before the onset of puberty.
Diurnal variation in estradiol concentrations in prepubertal and pubertal girls has been disputed (7, 15, 16, 17). Boyar et al. (16) found the highest estradiol concentrations during the midafternoon (1400–1600 h) in midpubertal girls. Goji (7) also found the highest estradiol concentrations during the daytime (1100–1400 h) in prepubertal and early pubertal girls, but these estradiol assays were less sensitive (10–20 pg/mL). Norjavaara et al. (15) reported that 24-h concentration profiles of serum estradiol, based on seven samples per 24 h, in prepubertal and pubertal girls using a sensitive RIA (2.1 pg/mL) on extracted serum samples. They found the highest estradiol concentrations during the early morning (0400–1000 h) in prepubertal and early pubertal girls before menarche. In the present study we measured estradiol concentrations in prepubertal and early pubertal girls hourly for 24 h using a sensitive RIA (2 pg/mL) on extracted serum samples. The serum estradiol concentration was elevated in the early morning (0400–0800 h) in late prepubertal girls (group I-2) and early pubertal girls, in agreement with Norjavaara’s study (15). However, serum estradiol concentrations in early prepubertal girls (group I-1) were below the detection limit (2 pg/mL) at each sampling time. We recognized that testosterone showed a diurnal rhythm, with augmentation during the early morning in early prepubertal girls (group I-1). Because testosterone is precursor of estradiol, the diurnal rhythm of estradiol in early prepubertal girls may exist in extremely low levels. Recently, Klein et al. (17, 26) developed an ultrasensitive recombinant cell bioassay to measure extremely low levels of estradiol (0.02 pg/mL). They found a diurnal rhythm of estradiol concentrations that peaked in the morning (0900 h) in early pubertal girls (17). In the future, the diurnal variation in estradiol in early prepubertal girls may be clarified by this assay.
Changes in LH and FSH pulse frequency before and during puberty are also controversial (3, 5, 6, 7, 9, 27). Recently, fluctuations in very low levels of gonadotropins were detected using a time-resolved immunofluorometric assay, and pulsatile secretion of gonadotropins in prepubertal girls was observed (7, 9). In the present study no significant changes were observed in LH and FSH pulse frequency before or during pubertal onset. Changes in gonadotropins before and during pubertal onset mainly appear to be due to increases in mean 24-h concentrations and pulse amplitude and not to increases in pulse frequency.
The temporal relationships between LH and testosterone time series, between LH and estradiol time series, and between testosterone and estradiol time series were determined using cross-correlation analysis. Goji’s study (7) has also determined the lag time between LH and estradiol time series using cross-correlation analysis. That study found a lag time of 5.7–9.3 h between LH and estradiol in early pubertal girls (7). He speculated that this long lag time might correspond to the time required for aromatization for estradiol synthesis. In the present study the mean lag time between LH and estradiol was 5.0–5.3 h. We found that the mean lag time between LH and testosterone time series was approximately equal to that between LH and estradiol time series, and the mean lag time between testosterone and estradiol time series ranged from 0.3–1.5 h. The present results indicate that the aromatization for estradiol synthesis is rapid, and a long lag time may correspond to the time required for testosterone synthesis. In the present study the mean lag time between LH and testosterone in prepubertal girls who attained puberty within 1 yr (group I-2; mean ± SD, 4.7 ± 1.5 h) was significantly shorter than that in prepubertal girls, who required 1 yr or longer to reach puberty (group I-1; 8.9 ± 2.9 h). These results indicate that the lag time between LH and testosterone time series starts to decrease before the onset of puberty. The synthesis and secretion of testosterone by the ovaries in response to LH stimulation may increase before the onset of puberty.
Subjects of short stature were studied because it is difficult to obtain 24-h blood sampling in normal healthy children. Due to tendencies toward delayed puberty in our subjects, the present study may not strictly reflect pubertal changes in healthy children. However, we investigated hormonal changes by assessing breast development, 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 girls.
In conclusion, the present study revealed that LH and FSH show night-day rhythms, and that testosterone has a diurnal rhythm with augmentation during early morning. These rhythms are found to already exist in 5- to 6-yr-old girls. Estradiol also has a diurnal rhythm, with augmentation during the early morning in late prepubertal and early pubertal girls. The diurnal patterns of serum estradiol and testosterone were similar. Serum LH and testosterone levels start to increase before the onset of puberty. A delay is observed between LH and testosterone time series, which decreases before the onset of puberty. Thus, preparation for the onset of puberty may begin in girls as early as 5–6 yr of age.
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Acknowledgments |
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Footnotes |
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Received August 4, 1999.
Revised November 4, 1999.
Accepted November 22, 1999.
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References |
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