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Original Studies |
Department of Growth and Reproduction, Rigshospitalet (A.S., A.J., A.M.A., T.K.J., J.M., N.E.S.), and Department of Biostatistics, Panum Institute (J.H.P.), University of Copenhagen, 2100 Copenhagen, Denmark; and Department of Epidemiology and Public Health (T.K.J.), Imperial College of Medicine at St. Mary’s, London, United Kingdom W21PG
Address all correspondence and requests for reprints to: Dr. Astrid Sehested, Department of Growth and Reproduction GR 5064, Rigshospitalet, Blegdamsvej 9, 2100 Copenhagen Ø, Denmark. E-mail: sehested{at}rh.dk
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Abstract |
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In conclusion, serum inhibin A and inhibin B levels in normal puberty in girls show consistency with our knowledge of the manner in which these hormones are secreted within the menstrual cycle in adult women. The presented reference values may be of use in the clinical evaluation of pubertal development in girls.
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Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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-subunit
disulfide linked to either a ßA-subunit
(inhibin A) or a ßB-subunit (inhibin B) (3).
Earlier serum inhibin RIAs could not distinguish among inhibin A,
inhibin B, and inactive free subunits and precursor forms. New two-site
enzyme-linked immunosorbent assays specific for dimeric inhibin A and
inhibin B have made it possible to begin to elucidate the role these
hormones play in gonadal function and the regulation of the
hypothalamic-pituitary-gonadal axis in both sexes. In infant boys, levels of inhibin B peak in the supra-adult range at 3 months of age and thereafter decline slowly during the first 2 yr of life (4). In boys, inhibin B levels increase sharply from childhood to adult levels between pubertal stages I and II, and there seems to be a switch in the regulation of inhibin B production between early and midpuberty (5); the negative correlation between FSH and inhibin B seen in adult men (6) first appears in midpuberty. Inhibin A is undetectable in male serum.
We have previously shown that inhibin B levels in infant girls also peaked at 3 months of age, although with a lower peak and greater variation than in boys, indicating early activation of the pituitary-gonadal axis. We subsequently found very low or undetectable levels in girls from 6–24 months of age (4). Levels of circulating gonadotropins and sex steroids are low in prepubertal girls and increase through puberty until the adult pattern of cyclical variation in gonadotropin and sex steroid secretion is achieved. Previous studies of total inhibin (i.e. using a RIA assay with overlapping specificity for inhibin A, inhibin B, inactive subunits, and precursor proteins) in girls in puberty have shown that levels increase through puberty (7, 8), but have failed to demonstrate correlations between mean FSH levels and mean inhibin levels. In both studies lower mean inhibin concentrations were found in girls than in boys at all stages of puberty.
Using newly developed specific assays, we here present serum values of inhibin A and inhibin B in relation to age, pubertal stage, and serum FSH, LH, and estradiol in a cross-sectional study of 403 healthy prepubertal, pubertal, and adolescent females, aged 6–20 yr. Hormone values were also measured in 181 women, aged 20–32 yr, as well as in 10 healthy women throughout their menstrual cycles.
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Subjects and Methods |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Girls 6–20 yr. Serum was obtained from 403 healthy schoolgirls. None had acute or chronic disease, and none was taking any medication. Girls taking the contraceptive pill were excluded. Tanner breast stage (9) was assessed by the same two physicians and was available for 402 subjects. The girls were also asked whether they had experienced menarche. All participants and their parents (6–18 yr) gave their informed consent. Other aspects of this study have been presented previously (10).
Adult women. Blood samples were taken from 181 healthy nonpregnant women, aged 20–32 yr. The samples were taken at random within the menstrual cycle; the women were taking no medication. Ascertainment of nonpregnant status at the time of blood sampling was achieved by measurement of urinary hCG during the cycle. Other aspects of this study have been presented previously (11).
Menstrual cycle. Daily blood samples were drawn from 10 healthy nonpregnant women (aged 18–40 yr) for 1 month. Other aspects of this study have been reported previously (12).
All studies had been approved by the local ethics committees.
Methods
Blood samples were drawn from an antecubital vein between
0800–1300 h and centrifuged. Serum was stored at -20°C until
analysis. Serum inhibin A and inhibin B were measured in duplicate in
double antibody enzyme immunometric assays using monoclonal antibodies
raised against either the ßA- or
ßB-subunit, respectively, in combination with a
labeled antibody raised against the inhibin -subunit as previously
described (13, 14). The detection limits were 7 and 20 pg/mL for
inhibin A and inhibin B, respectively. Intra- and interassay
coefficients of variation were, respectively, less than 15% and less
than 16% for inhibin A and less than 15% and less than 18% for
inhibin B.
Serum FSH and LH were measured by time-resolved immunofluorometric assay (DELFIA, Wallac, Inc., Turku, Finland), with detection limits of 0.06 and 0.05 U/L, respectively. Intra- and interassay coefficients of variation were both below 8% in the FSH and LH assays.
Estradiol was measured by RIA [Coat-a-Count, Diagnostic Products (Los Angeles, CA) and ImmunoDiagnostic Systems (Boldon, UK), respectively]. The detection limit was 18 pmol/L, the intraassay coefficient of variation was less than 8%, and the interassay coefficient of variation was less than 13%.
Progesterone was only measured in the 10 women with daily blood samples throughout the menstrual cycle. The analysis was performed by time-resolved immunofluorometric assay, with a detection limit of 0.8 nmol/L, an interassay variation of 10.1%, and an intraassay variation of 7.3%.
Statistics
Reference curves for the highly skewed distribution of the reproductive hormones as a function of age were calculated. The curves represent the median, the 2.5th percentile, and the 97.5th percentile, corresponding to the mean - 2 SD and the mean + 2 SD, respectively. The reference curves were obtained by locally weighted regression quantiles (15). This allowed taking into account both the age-dependent and highly skewed distribution of the hormones as well as the left-censoring, i.e. the measurements that were below the detection limit of the assays.
Hormone values through different stages of puberty in girls and phase of the menstrual cycle in adult women were first analyzed with the Kruskal-Wallis test, and values at each stage or age were thereafter compared with those from the previous stage using the Mann-Whitney U test.
Correlations within stages of puberty corrected for age trend and left-censoring were calculated by first subjecting the reproductive hormones to a cubic root transformation. The transformation corrected the marked right skewness of the data and resulted in the best approximation to a normal distribution. Pearson correlations could now be calculated based on a Tobit analysis (16). The analysis was carried out using the procedure LIFEREG in the statistical package SAS (SAS Institute, Inc., Cary, NC).
Sensitivities and specificities for various cut-off values of inhibin B, LH, and estradiol with respect to whether the child was clinically in puberty (i.e. in Tanner stage I or in Tanner stage II or II/III) were calculated as described by Cavallo et al. (17). The specificity refers to the true negative fraction, i.e. the proportion of girls clinically in Tanner stage I with hormone values below the cut-off level. The sensitivity refers to the true positive fraction, i.e. the proportion of girls in Tanner stage II or in stage II/III with hormone values at or above the cut-off level.
Serum values in adult women. The group of 181 women was subdivided according to phase of menstrual cycle as follows, using the assumption that the luteal phase is of a fixed length of 14 days: early follicular phase, from the first day of menstruation until 22 days before the first day of the next menstruation (=day -22) inclusively; late follicular phase, days -21 to -16 inclusively; periovulatory phase, days -15 to -12 inclusively; midluteal phase, days -11 to -6 inclusively; and end-luteal phase, days -5 to -1 inclusively.
The median cycle length was 29 days, with a range of 20–68 days. Excluding those women with a cycle length of less than 24 days or more than 35 days made no difference to the results, so they were included. Medians and 95% prediction intervals of reproductive hormones were calculated for each cycle phase.
Menstrual cycle. All 10 women had ovulatory cycles, as demonstrated by a LH peak followed by luteal values of serum progesterone. The menstrual cycles were aligned to the LH peak. Mean and SEM were calculated for daily measurements of inhibin A, inhibin B, FSH, and LH. Additionally, the mean and SEM of the hormone values relative to the intercycle FSH peak as well as relative to the first day of menses were calculated to show the luteal-follicular transition.
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Results |
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The median and 2.5th and 97.5th percentiles of values of inhibin
A, inhibin B, FSH, LH, and estradiol in relation to stage of puberty
are given in Table 1
, and the medians are
plotted without percentiles in Fig. 1.
The values in 181 adult women are also shown in relation to phase
within the menstrual cycle in Table 2.
The individual serum inhibin A and inhibin B levels in girls in puberty
are shown in relation to age in Fig. 2.
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Our group of prepubertal (Tanner stage I) girls consisted of 124 girls in the age range 6–12 yr. Inhibin B, FSH, LH, and estradiol all increased significantly within Tanner stage I as a function of age (Kruskal-Wallis P values over age groups: P < 0.0005, P < 0.0005, P = 0.003, and P = 0.004, respectively). Median inhibin B values increased from less than 20 pg/mL in girls younger than 8 yr of age to 38 pg/mL in girls aged 11 yr or older who were still in Tanner stage I.
The distribution of values of inhibin A and inhibin B within Tanner
stages was highly left-skewed for all stages except inhibin B in stage
III, where the distribution was near normal. This was in part due to a
proportion of subjects within each stage having inhibin levels that
were below the assay’s detection limits, as described in Table 3. The percentage of subjects with
undetectable levels of inhibin A fell progressively through Tanner
stages I–V and into adulthood, whereas the percentage of subjects with
undetectable levels of inhibin B fell from 35% in stage I to zero in
stage III and increased to 34% in adult women.
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). Inhibin
B displayed a nonsignificant (P = 0.132) tendency to
fall after the menarche, which was mainly due to an increasingly
left-skewed distribution (only 1 of 34 premenarcheal girls in stages
III–V had undetectable inhibin B, whereas 32 of 201 postmenarcheal
girls had undetectable levels). Correlations
The correlations of serum inhibin A and inhibin B to FSH, LH, and
estradiol levels according to stage of puberty corrected for age and
left-censoring are shown in Table 4.
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Sensitivities and specificities for various cut-off levels of LH,
estradiol, inhibin A, and inhibin B or combinations thereof were
determined in relation to whether the subject was clinically in puberty
(both stage II alone and stage II together with stage III; see
Subjects and Methods) and are presented in Table 5.
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Serum inhibin A and inhibin B levels in 181 adult women in
relation to cycle phase (Table 2, random sample group) and in 10 women
who had serum values measured daily through the menstrual cycle with
values aligned to the LH peak (Fig. 3, menstrual cycle group) showed similar patterns that were in agreement
with previous reports (13, 14, 18, 19). Serum inhibin B levels in the
random sample group were at their highest in the early and late
follicular phases, fell in the periovulatory phase, and were at their
lowest in the mid- and end-luteal phases. Serum inhibin A levels were
at their lowest in the early follicular phase, increased significantly
in the late follicular phase, continued to increase through the
periovulatory phase, were at their highest in the midluteal phase, and
fell significantly in the end-luteal phase. The median and 95%
prediction interval according to cycle phase in the menstrual cycle
group were calculated for comparison (not shown). The numbers were
similar, with two differences: 1) there were no significant differences
in inhibin B levels among the early follicular, late follicular, and
periovulatory phases in the menstrual cycle group; and 2) there were
women with unmeasurable inhibin A levels in the periovulatory and
midluteal phases in the random sample group. This was not the case in
the menstrual cycle group, where the lowest measured serum inhibin A
levels at any time during the periovulatory and midluteal phases were
16 and 23 pg/mL, respectively. In the menstrual cycle group all women
had ovulated and had a functioning corpus luteum, whereas in the
population of adult women some of the cycles may have been anovulatory,
or the luteal phase may have been insufficient.
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), regardless of whether the menstrual
cycle was aligned to the day of the intercycle FSH peak or the first
day of menses, showed changes in inhibin B that followed changes in FSH
with a lag of 1–2 days. Inhibin B concentrations had risen
considerably on the day of the FSH peak and peaked the day after.
Estradiol levels increased more slowly, although an increasing tendency
was evident from the day of the FSH peak, and levels had doubled 3 days
after the FSH peak compared with levels 2 days before the peak.
Estradiol levels were still increasing when both inhibin B levels and
FSH levels were falling.
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Serum inhibin A was undetectable in all girls staged as Tanner breast stage I with one exception; this girl also had the highest stage I levels of inhibin B (180 pg/mL), estradiol (176 pmol/L), FSH (5.04 U/L), and LH (1.5 U/L), indicating that the hormonal changes of puberty had begun. Levels of inhibin A increased progressively from Tanner stage I into adulthood; however, levels corresponding to those seen in the midluteal phase in adult women (>19 pg/mL) were only seen in girls that had experienced menarche. The decrease in inhibin B levels and the increase in inhibin A levels seen from Tanner stage III into adulthood could be explained by an increasing proportion of the subjects at each stage and age having regular, ovulatory menstrual cycles. This would give a progressively greater likelihood of being in the luteal phase on the day of blood sampling and thus having a high serum inhibin A and a low serum inhibin B. Manasco et al. (8), when measuring total inhibin, report a tendency for total inhibin levels to drop in Tanner IV before increasing again in Tanner V. Depending on the specificities of their total inhibin assay for inhibin A and inhibin B, this could be explained by our results as being the consequence of summing the falling tendency of inhibin B in stages IV and V with the rising tendency of inhibin A.
Serum levels of inhibin B were considerably (medians less than half the value) lower in girls than boys at all stages of puberty using our reference values for boys in puberty (5), in agreement with previous findings (7, 8, 21).
For clinical purposes, a basal hormone assay that could satisfactorily distinguish a pre-pubertal from a pubertal girl could be very useful. Unfortunately, like the other reproductive hormones, the overlap between levels of inhibin A and inhibin B in different Tanner stages is so great that measurement of inhibin A or inhibin B alone cannot fulfil this role. The sensitivities and specificities regarding whether a girl is in Tanner stage II or Tanner stage II/III for single measurements of inhibin B are no better than those for estradiol. Combining cut-off values for inhibin B and LH improves sensitivity, but at the cost of specificity, i.e. very few false negatives, but many false positives. Levels of inhibin A of 7 pg/mL (the detection limit) or more make it highly likely that the girl has entered puberty (high specificity); however, levels below 7 pg/mL can in no way exclude puberty (low sensitivity).
Examining the changes in correlations between reproductive hormones through the stages of puberty may shed some light on the maturational processes of puberty. In early puberty, the reproductive hormones are all positively correlated to one another, reflecting the fact that all of the levels increase as puberty progresses. However, interestingly, LH is more strongly correlated with both inhibin A and inhibin B in early puberty than is FSH, supporting observations that increasing secretion of LH is an early and significant event in the onset of puberty. With respect to inhibin A, changes in mid- and late puberty include a negative correlation appearing between inhibin A and inhibin B and between inhibin A and FSH, reflecting that inhibin A is secreted out of phase with inhibin B and FSH within the menstrual cycle. With respect to inhibin B, the positive gonadotropin correlations of early puberty disappear in stage III to reappear in stage IV, now more strongly positively correlated to FSH than to LH, in agreement with FSH and inhibin B being secreted in phase with one another within the menstrual cycle.
We found a specific pattern of inhibin A and inhibin B secretion through the menstrual cycle. Inhibin B is principally secreted in the follicular phase, and inhibin A is principally secreted in the luteal phase; levels fall synchronously with those of progesterone, as previously reported (13, 14, 18, 19). There are some minor discrepancies in timing between our present results and the findings of Groome et al. (14). They report the postovulatory inhibin B peak to occur 2 days after the LH peak, whereas in our data it occurs the day after. This inhibin B peak is probably derived from follicular fluid released during ovulation. With respect to the timing during the luteal-follicular transition, Groome et al. (14) describe inhibin B levels as being unchanged on the day of the FSH peak, but rising thereafter to peak 4 days later. The study by Welt et al. (18) aligns the data to the first day of menses and shows inhibin B concentrations to increase more or less coincidentally with FSH. We found inhibin B concentrations to follow FSH concentrations with a lag of 1–2 days, and inhibin B levels peaked the day after FSH. The fact that inhibin B levels rise before estradiol levels during this period has been taken as suggesting that inhibin B could have a distinct role in modulating the intercycle FSH peak (14, 18). We also found that inhibin B levels peak well before estradiol levels. However, it is worth pointing out that estradiol is a very potent agent, and that levels of estradiol are clearly on the increase from the day of the FSH peak and have doubled by day 3 after the FSH peak. It is an open question as to how great an increase in estradiol is necessary to exert a suppressor influence on FSH secretion.
In conclusion, inhibin A and inhibin B are gonadal hormones that are produced in a specific pattern in response to gonadotropin stimulation in females. Their role during pubertal development in girls is not known; they may have paracrine actions involved in the growth and maturation of the ovary as well as endocrine actions in the maturation of the hypothalamic-pituitary-gonadal axis. The pattern of inhibin secretion seen in girls during pubertal development is consistent with our knowledge of the pattern in adult normally menstruating women and reflects the average degree of ovarian follicular maturation at various stages of puberty. In physiological terms, inhibin B in prepubertal and early pubertal girls can be seen as a marker of gonadotropin-stimulated early follicular activity. The appearance of measurable inhibin A can be seen as a marker for a follicle having at least matured to a stage corresponding to the late follicular stage in adult women. Although measuring serum inhibin A and inhibin B did not offer superior prediction of pubertal onset compared to LH and estradiol, they do give unique information about the level of follicular maturation on the day of the blood test and could thus contribute to the evaluation of puberty in a clinical setting, particularly in concert with ovarian ultrasound counting the number and size of visible follicles.
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Footnotes |
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Received September 2, 1999.
Revised December 3, 1999.
Accepted December 17, 1999.
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