0021-972X/99/$03.00/0
Journal of Clinical Endocrinology and Metabolism
Copyright © 1999 by The Endocrine Society
Vol. 84, No. 1
Printed in U.S.A.
Female Reproductive Aging Is Marked by Decreased
Secretion of Dimeric Inhibin*
CORRINE K. WELT, DENNIS J. MCNICHOLL, ANN E. TAYLOR,
JANET E. HALL
AND
Reproductive Endocrine Unit, Reproductive Endocrine Sciences Center and National Center for
Infertility Research, Department of Medicine, Massachusetts General Hospital, Boston, Massachusetts
02114
ABSTRACT
The increase in serum FSH that accompanies female reproductive
aging occurs before changes in estradiol (E2). A decrease in negative
feedback from inhibin A (a product of the dominant follicle and corpus
luteum) and/or inhibin B (secreted by developing follicles) may explain the rise in FSH with age. To test the hypothesis that decreases
in inhibin A or inhibin B occur at an age at which the first increase
in follicular phase FSH is evident, daily blood samples were obtained
across the menstrual cycle from younger (,35 yr; n 5 23) and older
(35– 46 yr; n 5 21) cycling women. These cross-sectional studies were
complemented by longitudinal data in 3 women studied at a 10-yr
interval.
In the early follicular phase, mean inhibin B was lower in older
cycling women (88 6 7 vs. 112 6 10 pg/mL; P , 0.05) and FSH was
higher (13.0 6 0.5 vs. 11.2 6 0.7 IU/L in older vs. younger, respectively; P , 0.04). In the mid- and late follicular phases, inhibin B was
also lower in the older women (117 6 9 vs. 146 6 10 and 85 6 8 vs.
117 6 11 pg/mL; P , 0.04), whereas E2 was higher (105 6 14 vs. 68 6
5 and 240 6 27 vs. 163 6 9 pg/mL; P , 0.02), and no differences in
FSH were observed in the two groups at these times. In women
studied longitudinally, FSH and inhibin B varied inversely in the
follicular phase.
In the early luteal phase, mean inhibin B was lower in the older
group (64 6 6 vs. 94 6 12 pg/mL; P , 0.03), and FSH was higher
(12.5 6 1.0 vs. 9.7 6 0.6 IU/L; P , 0.03). In the mid- and late luteal
phases, inhibin B was also lower in older subjects (21 6 2 vs. 33 6 5
and 22 6 2 vs. 36 6 6 pg/mL; P , 0.02). No difference in inhibin A,
E2, or progesterone was observed across the luteal phase, between the
two groups. However, in all subjects studied longitudinally, increased
age was associated with a decrease in inhibin A, inhibin B, and
progesterone in the absence of changes in E2.
Our conclusions were: 1) reproductive aging is accompanied by
decreases in both inhibin B and inhibin A; 2) the decrease in inhibin
B precedes the decrease in inhibin A and occurs in concert with an
increase in E2, suggesting that inhibin B negative feedback is the
most important factor controlling the earliest increase in FSH with
aging; 3) these studies suggest that the decrease in inhibin B is the
earliest marker of the decline in follicle number across reproductive
aging. (J Clin Endocrinol Metab 84: 105–111, 1999)
R
EPRODUCTIVE aging is associated with a decline in
fertility, which begins in a moderate and steady fashion starting in the third to fourth decade (1, 2) but accelerates
rapidly after age 35 (1). A gradual diminution of the pool of
ovarian follicles seems to underlie the decline in fertility
(3–5). This age-related decrease in follicle number and fertility is marked by a rise in follicular-phase FSH (6 –9), which
also commences after age 35 (7) and is followed several years
later by an increase in LH (6, 7, 9). Although changes in
estradiol (E2) were initially sought to explain this differential
increase in FSH, E2 has variably been reported to decrease (8,
10, 11), remain constant (7, 12), or even increase (9, 13–15) in
the follicular phase of older women with ovulatory menstrual cycles.
Inhibin, a dimeric glycoprotein composed of an a-subunit
and a bA-subunit (inhibin A) or bB-subunit (inhibin B), was
initially identified based on its ability to suppress FSH (16,
17). Thus, changes in inhibin levels have been examined to
explain the monotropic FSH rise that occurs with age. During
the normal menstrual cycle, inhibin B levels are highest in the
early to midfollicular phases (18) and decrease in the late
follicular phase (19), suggesting secretion by developing follicles, whereas inhibin A is highest in the late follicular and
luteal phases as a product of the preovulatory follicle and,
subsequently, the corpus luteum (20, 21). Therefore, decreased secretion of either inhibin B in the follicular phase or
inhibin A in the luteal phase could account for the early
follicular-phase increase in FSH in older cycling women. In
women over age 45, higher follicular-phase levels of FSH
were associated with a decrease in total inhibin in both follicular and luteal phases (11), as measured by an assay which
uses an antibody directed against the a-subunit and detects
all forms of inhibin (22–24). Recent studies that demonstrated
lower inhibin B in the follicular phase of 40- to 45-yr-old
cycling women with elevated FSH levels provide indirect
evidence that inhibin B may also act as an important regulator of follicular-phase FSH in the human (25, 26). The first
detectable rise in follicular-phase FSH levels occurs much
earlier than age 40 (7), however, indicating the importance of
examining the relationship between inhibin A and inhibin B
and FSH in younger subjects and in subjects whose FSH
levels are not yet outside the normal range.
Daily blood samples were obtained, across an entire menstrual cycle, in a group of 44 women, ranging in age from
22– 46 yr, with regular menstrual cycles and normal early-
Received April 17, 1998. Revision received August 11, 1998. Rerevision received September 15, 1998. Accepted September 24, 1998.
Address all correspondence and requests for reprints to: Dr. Corrine
K. Welt, Reproductive Endocrine Unit, Massachusetts General Hospital,
55 Fruit Street, Boston, Massachusetts 02114-2696.
* This work was supported by National Institutes of Health Grants
U-54-HD-29164, M-01-RR-01066, and P-30-HD-28138.
105
106
JCE & M • 1999
Vol 84 • No 1
WELT ET AL.
follicular-phase FSH levels. Results in women 35 yr old or
older were then compared with those in women less than 35
yr, based on the age at which the first marked decline in
fertility occurs (1) and in which the first rise in follicularphase FSH is detectable (7). To determine whether differences seen in cross-sectional studies were representative of
changes in individual women over time, 3 women were
studied on 2 occasions at least 10 yr apart.
Materials and Methods
Subjects
Two studies were undertaken to evaluate inhibin A and B levels
across reproductive aging. The first was a cross-sectional study in which
23 younger cycling women (20 –34 yr old) and 21 older cycling women
(35– 46 yr old) were studied. All subjects had normal PRL and TSH
levels, were on no medication, had normal body weight (with BMI of
18 –27 kg/m2), had no history of excessive exercise, and showed no
evidence of androgen excess on physical examination, as previously
described (27, 28). All subjects had a history of regular (25–35 days)
menstrual cycles with evidence of ovulation in the preceding cycle, as
indicated by a serum progesterone (P4) level of more than 6 ng/mL.
Blood samples were drawn daily for the duration of 1 menstrual cycle
in all subjects.
In a subset of subjects (,35 yr, n 5 14; and $35 yr, n 5 13), transvaginal ultrasounds (Toshiba SAL 77B, 5 MHz convex array transducer,
Toshiba Corp., Japan) were performed, starting in the midfollicular
phase, to assess follicular development. Subsequent ultrasounds were
performed every 2–5 days until ovulation was documented by collapse
of the dominant follicle, with or without internal echoes. Three younger
subjects and 1 older subject had only 1 ultrasound in the midfollicular
phase. The number and maximum diameter of all follicles of more than
6 mm was recorded.
Three additional women, now aged 37, 42, and 47 yr, had daily blood
samples drawn across a menstrual cycle approximately 10 yr previously,
and repeated daily blood sampling for the present study, to provide
longitudinal data. The 47-yr-old subject obtained blood samples across
two cycles at each age. All blood samples were measured for LH, FSH,
E2, P4, inhibin A, and inhibin B.
The study was approved by the Subcommittee on Human Studies of
the Massachusetts General Hospital. Informed consent was obtained
from each subject before participation.
Assays
Serum LH, FSH, E2, and P4 were measured by RIA, as previously
described (29, 30). The 95% confidence limits for early-follicular-phase
FSH levels (days 1–5, starting at menses) in this assay were 3.2–22.5
IU/L, based on data from 109 normal cycling women, 19 – 42 yr old. All
samples for LH, FSH, E2, and P4 were analyzed in duplicate, and all
samples from an individual were analyzed in the same assay. The interand intraassay coefficients of variation were similar to those previously
described (31). Gonadotropin levels are expressed in IU/L, as equivalents of the Second International Reference Preparation 71/223 of human
menopausal gonadotropins.
Inhibin A was measured in duplicate by enzyme-linked immunosorbent assay (Serotec, Oxford, England), as previously described (20). The
assay uses a lyophilized human follicular fluid calibrator standardized
as equivalents of the World Health Organization recombinant human
inhibin A preparation 91/624, and values are reported as IU/mL. The
limit of detection of the assay was 0.6 IU/mL. The intraassay coefficient
of variation for the dimeric inhibin A assay was 3.9% at the ED20 dose,
and the interassay coefficient of variation was 6.8% at the ED30 dose. All
samples for a given individual were run in the same assay.
Inhibin B was measured as single samples by enzyme-linked immunosorbent assay (Serotec), as previously described (32). The limit of
detection of the inhibin B assay (mean 6 2 sd of multiple zero standard
measurements) was 15.6 pg/mL. The intraassay coefficient of variation
was 4 – 6%, and the interassay coefficient of variation was 15–18% for
serum spiked with 121, 250, and 723 pg/mL inhibin B. All samples with
levels in excess of 500 pg/mL were appropriately diluted. All samples
for a given individual were run in the same assay.
Data analysis and statistics
Data were centered to ovulation for comparison of hormonal dynamics across the follicular and luteal phases of the menstrual cycle
using three of four of the following criteria: 1) day of LH peak; 2) day
of the midcycle FSH peak; 3) day of or after the midcycle E2 peak; and
4) day that the P4 doubled from baseline or reached 0.6 ng/mL (27).
Mean values for each hormone were calculated across the follicular
phase from menses to the day before ovulation and the luteal phase from
the day after ovulation to the day before menses. In addition, menstrual
cycles were standardized to a 28-day cycle length with the day of
ovulation centered to day 0, and mean hormone levels were determined
in the early (days 213 to 29), mid- (days 28 to 25), and late (days 24
to 21) follicular phase and early (days 1– 4), mid- (days 5–9) and late
(days 10 –14) luteal phase, as previously described (33). The mean value
approximates the area under the curve for hormonal values at each cycle
stage, based on the trapezoidal rule. Mean values were compared between older and younger cycling women studied cross-sectionally using
two-tailed, unpaired t tests, whereas comparisons between older and
younger cycles in women studied longitudinally were performed using
paired t tests.
Pearson correlations were used to examine associations between follicle size and inhibin A, inhibin B, and E2 levels obtained on the day of
the ultrasound measurement. The ratio of inhibin A and E2 to the
maximum diameter of the dominant follicle was then determined, and
resulting values were compared between older and younger cycling
subjects by t test.
Results are expressed as mean 6 sem unless otherwise indicated. A
P value of less than 0.05 was considered significant.
Results
Cross-sectional studies
Inhibin B and inhibin A were differentially secreted across
the menstrual cycle in older and younger cycling women
(Fig. 1), following a pattern that is qualitatively similar to
results in previous smaller series (18 –21).
Follicular phase
Follicular-phase length did not differ between older and
younger cycling women (12.9 6 0.5 and 14.0 6 0.7 days,
respectively). Although mean FSH across the entire follicular
phase was not different between older and younger women
selected for regular menstrual cycles, the peak FSH level
from the day of menses to the day before ovulation occurred
earlier in the older women (day 3.5 6 0.5 vs. 6.6 6 0.7; P ,
0.001), and FSH was higher in the early follicular phase
(Table 1, Fig. 2). Inhibin B was lower in the older group in
the early follicular phase, whereas E2 was not different between the two groups (Table 1, Fig. 2). In the mid- and late
follicular phase, inhibin B was lower and E2 was higher in
older subjects, compared with younger subjects, perhaps
accounting for the absence of differences in FSH levels at
these cycle stages (Table 1, Fig. 2). Inhibin A was not different
between the older and younger groups in the follicular
phase, and there were no differences in LH or P4.
Age correlated inversely with inhibin B (r 5 20.4; P , 0.03)
and positively with FSH (r 5 0.3; P , 0.03) across the menstrual cycle. Age correlated with E2 (r 5 0.3; P , 0.05) in the
follicular phase only. Mean follicular-phase FSH correlated
inversely with E2 (r 5 20.402, P , 0.03) and showed a trend
toward an inverse correlation with inhibin B (r 5 20.334, P ,
0.06) when adjusted for age.
DIMERIC INHIBIN IN REPRODUCTIVE AGING
107
FIG. 1. Mean inhibin B, FSH, E2, inhibin A, LH, and P4 levels in younger
(E; 20 –34 yr) and older (F; 35– 46 yr)
cycling women, centered to the day of
ovulation. Notice that FSH is higher
and inhibin B is lower in the early follicular phase of older subjects. In contrast, FSH is not different between
older and younger subjects during the
remainder of the follicular phase, when
E2 is higher and inhibin B is lower in
older subjects. FSH is also higher in the
early luteal phase of older subjects
when inhibin B is lower. Inhibin A is
lower in older subjects only on the day
after the LH peak. (**, P , 0.02; *, P ,
0.04).
TABLE 1. Mean follicular phase FSH, inhibin B, inhibin A, and estradiol levels
FSH (IU/L)
,35 yra
$35 yrb
Inhibin B (pg/mL)
,35 yr
$35 yr
Inhibin A (IU/mL)
,35 yr
$35 yr
Estradiol (pg/mL)
,35 yr
$35 yr
Follicular phase (FP)
Early FP
Mid FP
Late FP
10.6 6 0.6
11.2 6 0.5
11.2 6 0.7
13.0 6 0.5c
11.3 6 0.6
11.4 6 0.8
9.2 6 0.6
8.8 6 0.5
125 6 9
96 6 6d
112 6 10
88 6 7c
146 6 10
117 6 9c
117 6 11
85 6 8c
2.3 6 0.3
2.1 6 0.3
1.2 6 0.3
1.0 6 0.2
1.6 6 0.4
1.3 6 0.2
7.9 6 3.9
4.1 6 0.5
90 6 5
130 6 16d
47 6 4
64 6 10
68 6 5
105 6 14d
163 6 9
240 6 27e
n 5 23.
n 5 21.
c
P , 0.05 between subjects ,35 yr and $35 yr.
d
P , 0.02.
e
P , 0.01.
a
b
Twenty-seven of the subjects studied cross-sectionally underwent ultrasound evaluation of follicular development. In
this subset, older and younger subjects developed a single
preovulatory follicle of similar size (22.9 6 1.0 vs. 23.0 6 0.6
mm in older and younger subjects, respectively). Three older
subjects and two younger subjects had 1–2 additional follicles of #11 mm, whereas one younger subject had an additional follicle of 20 mm, and one older subject had an additional follicle of 14 mm, neither of which seemed to ovulate,
as determined by ultrasound. Maximum follicle diameter
correlated positively with inhibin A and E2 levels drawn on
the same day but not with inhibin B (Fig. 3). When expressed
in relation to size of the dominant follicle, older women had
similar levels of serum inhibin A (0.26 6 0.04 vs. 0.17 6 0.03
IU/mLzmm) but higher levels of E2 (13.8 6 1.8 vs. 9.3 6 1.3
pg/mLzmm; P , 0.05), compared with younger women.
Luteal phase
Luteal phase length did not differ between the two groups
(13.8 6 0.4 vs. 13.5 6 0.3 days for older and younger women,
respectively). FSH levels were higher in the early luteal phase
in older, compared with younger, cycling women (Table 2;
Fig. 1). Inhibin B was lower both in the early luteal phase and
across the entire luteal phase in older women (Table 2; Fig.
1). Inhibin A was also lower on the day after the LH peak in
the older group (4.5 6 0.7 vs. 7.7 6 1.2 IU/mL; P , 0.04; Fig.
1). However, there were no further differences in inhibin A
across the luteal phase, nor were differences observed in LH,
E2, or P4.
Longitudinal cycles
In all three subjects studied longitudinally, FSH was
higher across the menstrual cycle after 10 yr (Fig. 4), although
these differences did not reach statistical significance, because of small numbers and interindividual variation. At the
time of the earlier study, the average inhibin B levels in these
subjects had already reached the levels seen in the older
subjects studied cross-sectionally. Despite these low baseline
levels, the average inhibin B in the early phase (87 6 15 vs.
36 6 20 pg/mL; P 5 0.09) and midfollicular phase (111 6 10
108
WELT ET AL.
FIG. 2. Mean FSH, inhibin B (Inh B), and E2 in younger (open bars;
20 –34 yr) and older (closed bars; 35– 46 yr) women in the early (EFP),
mid- (MFP), and late follicular phase (LFP) of the menstrual cycle (**,
P , 0.05).
vs. 53 6 25 pg/mL; P 5 0.09) showed a trend toward a further
decrease after 10 yr. In the oldest woman, 47 yr old, cycles
had become less regular by the second study. FSH was elevated above the 95% confidence limit of normal women and
demonstrated remarkable cycle-to-cycle variability. The cycle in which FSH was highest in the follicular phase also
showed the lowest inhibin B levels (Fig. 5). These data complement the similar inverse relationship between inhibin B
and FSH observed in the cross-sectional data.
Inhibin A levels were lower in the late follicular phase in
all three subjects after 10 yr, where E2 was not different (Figs.
4 and 5). Interestingly, increased age was associated with a
decrease in luteal phase secretion of inhibin A and P4 in all
three subjects after 10 yr, in the absence of changes in E2 (Fig.
4). This was true, even for the 37-yr-old subject, whose cycles
remained regular and in whom FSH was not elevated.
Discussion
Lower inhibin levels in the follicular and luteal phases of
older cycling women have been demonstrated in some (11),
but not all (10, 13), previous studies, using an assay directed
against the inhibin a-subunit and measuring a combination
of inhibin A, inhibin B, and the nonbiologically active a-sub-
JCE & M • 1999
Vol 84 • No 1
FIG. 3. Correlation between maximum follicle diameter on ultrasound and inhibin A, E2, and inhibin B levels on the same day.
Correlation coefficients and P values are indicated.
unit (22–24). Using assays specific for dimeric inhibin (19 –
21), we have now shown in cross-sectional and longitudinal
studies that reproductive aging is associated with changes in
both dimeric inhibin A and inhibin B. Inhibin B is decreased
in women 35 yr old or older, the age at which an increase in
follicular-phase FSH is first detectable (7). Though less apparent in cross-sectional studies, changes in inhibin A are
clearly seen in patients studied longitudinally. These studies
suggest that inhibin B plays an important role in the early
follicular-phase FSH rise that occurs with reproductive aging, but a role for inhibin A cannot be excluded.
The decrease in inhibin B, demonstrated across the menstrual cycle in older cycling women in this study, confirms
and extends the work of Klein et al., which indicated that
inhibin B levels were lower in the 5 days surrounding the
FSH peak in the follicular phase of women who already had
modestly elevated FSH levels (25). In the current study, FSH
is higher and inhibin B lower in the early follicular phase in
subjects studied both cross-sectionally and longitudinally,
and inhibin B varies inversely with FSH when controlled for
age, thus providing further evidence for a negative feedback
role for inhibin B. The increased FSH in the early follicular
phase is not sustained through the mid- and late follicular
DIMERIC INHIBIN IN REPRODUCTIVE AGING
109
TABLE 2. Mean luteal phase inhibin B, inhibin A, estradiol and progesterone levels
Luteal phase (LP)
FSH (IU/L)
,35 yra
$35 yrb
Inhibin B (pg/mL)
,35 yr
$35 yr
Inhibin A (IU/mL)
,35 yr
$35 yr
Estradiol (pg/mL)
,35 yr
$35 yr
Progesterone (ng/mL)
,35 yr
$35 yr
Early LP
Mid LP
Late LP
7.3 6 0.5
8.6 6 0.6
9.7 6 0.6
12.5 6 1.0*
6.4 6 0.5
6.8 6 0.6
6.2 6 0.4
7.0 6 0.4
51 6 5
37 6 3c
94 6 12
64 6 6c
33 6 5
21 6 2d
36 6 6
22 6 2d
7.2 6 0.9
5.6 6 0.6
8.5 6 1.1
7.2 6 0.7
10.0 6 1.5
7.4 6 0.8
3.5 6 0.5
2.4 6 0.5
119 6 9
143 6 17
110 6 10
133 6 17
142 6 11
160 6 20
102 6 10
110 6 12
10.3 6 0.8
10.6 6 0.6
6.5 6 0.6
8.0 6 0.7
16.5 6 1.4
17.1 6 1.4
6.7 6 0.6
7.7 6 0.7
n 5 23.
n 5 21.
P , 0.05 between subjects ,35 yr and $35 yr.
d
P , 0.02.
a
b
c
FIG. 4. Mean FSH, Inh B, inhibin A (Inh A), P4, and E2 in the
follicular and luteal phase of all three subjects studied longitudinally.
Subjects were 28, 32, and 36 yr old in the initial study (open bars) and
37, 42, and 47 yr old, respectively, in the second study (closed bars).
ELP, Early; MLP, mid-; and LLP, late luteal phase (**, P , 0.05).
phase, when E2 levels are clearly higher in older women,
however, alluding to the important balance between E2 and
inhibin B in the regulation of FSH.
In vitro studies in nonhuman primates (34) and in the
human (35) indicate that inhibin B is a product of small antral
follicles. The rise in inhibin B across the luteal-follicular transition (18) when the pool of small follicles is recruited and the
decrease in the late follicular phase in this study and others
(19) is consistent with these findings. Further, no relationship
was demonstrated between inhibin B and follicle size, in
contrast to inhibin A and E2 which increase with increasing
follicle size, suggesting that in normal cycles inhibin B is
derived from follicles other than the dominant follicle. The
decrease in inhibin B secretion in women $35 yr in this study
occurs concurrent with the first marked decline in fertility
with age (1) suggesting that it reflects a decrease in the
number of follicles available for recruitment. The decrease in
inhibin B is demonstrated even in the luteal phase, when
levels are close to the lower limit of assay sensitivity and the
coefficient of variation is high (32). When FSH is elevated and
cycles have become slightly irregular, as seen in the woman
studied longitudinally, a more marked decrease in inhibin B
is observed in the follicular phase, paralleling the accelerated
decrease in follicle number at the time of the perimenopausal
transition (4). Thus, lower inhibin B levels in older cycling
women may be the earliest marker of the decreased number
of available follicles in reproductive aging.
The changes in inhibin A secretion with age are more
subtle. A decrease in inhibin A was not demonstrated with
aging in the cross-sectional studies, but it was seen in individual subjects studied longitudinally over a 10-yr period.
These changes were seen even in the youngest subject, who
was 37 yr old at the time of the second study. Therefore, the
changes in inhibin A may be detectable only when the large
interindividual variability is eliminated. Alternatively, decreases in inhibin A may occur later than decreases in inhibin
B, as suggested by recent data in women studied across the
perimenopause (36). A decrease in P4 accompanied the decreased inhibin A in the luteal phase of subjects studied
longitudinally in this study, whereas E2 was relatively preserved, as previously described (9, 15). Taken together, the
results suggest that luteinized granulosa cell synthetic function is relatively preserved until late in aging but is disrupted
before changes in aromatase function are manifested.
110
JCE & M • 1999
Vol 84 • No 1
WELT ET AL.
FIG. 5. Inhibin B, FSH, E2, inhibin A,
LH, and P4 in a representative subject
studied across two menstrual cycles at
age 36 (E, M) and again at age 47 (F, f),
when cycles had become slightly irregular (21–28 days) and FSH levels were
elevated. Note the lower levels of inhibin B across the cycle, the decrease in
inhibin A and P4 in the luteal phase,
and the relative preservation of E2 with
age and increasing FSH and LH. Data
are centered to the day of ovulation.
The current data are consistent with studies that uniformly
demonstrate an increase in late follicular-phase E2 in older
reproductive-age women (7, 10, 25), whereas a decrease in E2
across the follicular phase is eventually observed in women
more than 45 yr old (8, 11). The results of the present study
further demonstrate an increased E2 level, in relation to
follicle size and in the absence of multifollicular development. Thus, the increased E2 indicates changes in aromatase
activity, perhaps caused by increased FSH or substrate availability at the level of the dominant follicle. The higher late
follicular-phase E2 levels may also indicate a decrease in
pituitary sensitivity with age, resulting in the need for higher
E2 levels to elicit the LH surge.
The control of FSH is dependent not only on inhibin and
E2 but also on the activin/follistatin system (16, 17). Some
(26, 37), but not all (38), recent studies have demonstrated an
increase in activin A levels across reproductive aging, suggesting that activin A may play an endocrine role in stimulating FSH secretion. However, an increase in follistatin (37)
or similar levels of free follistatin (26), the major activin
binding protein, accompanied the increase in activin A in
these studies, suggesting that circulating activin A was not
biologically active. Further, no correlation was demonstrated
between FSH and activin A in the largest study (38). Previous
work from our group demonstrated no difference in total
activin A or total follistatin among premenopausal, postmenopausal (38 – 40), and castrate women (40). Therefore, it
does not seem that the ovary is the major source of circulating
activin A or follistatin in women or that activin A plays a
major endocrine role in the control of FSH in aging.
In summary, in a large population of regularly cycling
women with normal day 3 FSH levels, we have demonstrated
that reproductive aging is accompanied by changes in both
inhibin B and inhibin A. Decreases in inhibin B precede those
of inhibin A and occur in concert with increases in E2, suggesting that inhibin B negative feedback is the most important factor controlling the rise in FSH across aging. Our data
also suggest that a decrease in inhibin B across the cycle may
be the earliest marker of the decline in follicle number with
reproductive aging.
Acknowledgments
We would like to thank Geralyn Lambert-Messerlian, Ph.D.; Patrick
Sluss, Ph.D.; Rita Khoury, M.D.; and Patty Smith, B.S. for inhibin A and
inhibin B assay development. We also thank the members of the P30 core
laboratory for their technical assistance. We acknowledge Judy Adams,
D.M.U., for her expertise in performing and analyzing the ultrasound
data. We would also like to thank William F. Crowley Jr., M.D., for his
careful review of the manuscript; Brooke Wexler, B.A., for her assistance
in patient recruitment; and Douglas Hayden, Ph.D., and David Schoenfeld, Ph.D., for assistance with statistical analysis.
References
1. Schwartz D, Mayaux MJ. 1982 Female fecundity as a function of age: results
of artificial insemination in 2193 nulliparous women with azoospermic husbands. New Engl J Med. 306:404 – 406.
2. Menken J, Trussell J, Larsen U. 1986 Age and infertility. Science.
233:1389 –1394.
3. Block E. 1952 Quantitative morphological investigations of the follicular system in women. Acta Anat. 14:108 –122.
4. Richardson SJ, Senikas V, Nelson JF. 1987 Follicular depletion during the
menopausal transition: evidence for accelerated loss and ultimate exhaustion.
J Clin Endocrinol Metab. 65:1231–1237.
5. Gougeon A, Echochard R, Thalabard JC. 1994 Age-related changes of the
population of human ovarian follicles: increase in the disappearance rate of
non-growing, and early-growing follicles in aging women. Biol Reprod.
50:653– 663.
6. Lenton EA, Sexton L, Lee S, Cooke ID. 1988 Progressive changes in LH and
FSH and LH:FSH ratio in women throughout reproductive life. Maturitas.
10:35– 43.
7. Lee S, Lenton E, Sexton L, Cooke I. 1988 The effect of age on the cyclical
patterns of plasma LH, FSH, estradiol and progesterone in women with regular
menstrual cycles. Hum Reprod. 3:851– 855.
8. Sherman BM, West JH, Korenman SG. 1976 The menopausal transition:
analysis of LH, FSH, estradiol, and progesterone concentrations during menstrual cycles of older women. J Clin Endocrinol Metab. 42:629 – 636.
9. Reyes FI, Winter JSD, Faiman C. 1977 Pituitary-ovarian relationships preceding the menopause. Am J Obstet Gynecol. 129:557–564.
10. Lenton EA, DeKretser DM, Woodward AJ, Robertson DM. 1991 Inhibin
concentrations throughout the menstrual cycles of normal, infertile, and older
women compared with those during spontaneous conception cycles. J Clin
Endocrinol Metab. 73:1180 –1190.
DIMERIC INHIBIN IN REPRODUCTIVE AGING
11. MacNaughton J, Banah M, McCloud P, Hee J, Burger H. 1992 Age related
changes in follicle stimulating hormone, luteinizing hormone, oestradiol and
immunoreactive inhibin in women of reproductive age. Clin Endocrinol (Oxf).
36:339 –345.
12. Reame NE, Kelch RP, Beitins IZ, Yu MY, Zawacki CM, Padmanabhan V. 1996
Age effects on follicle-stimulating hormone and pulsatile luteinizing hormone
secretion across the menstrual cycle of premenopausal women. J Clin Endocrinol Metab. 81:1512–1518.
13. Klein NA, Battaglia DE, Fujimoto VY, Davis GS, Bremner WJ, Soules MR.
1996 Reproductive aging: accelerated ovarian follicular development associated with a monotropic follicle-stimulating hormone rise in normal older
women. J Clin Endocrinol Metab. 81:1038 –1045.
14. Klein NA, Bataglia DA, Miller PB, Branigan EF, Giudice L, Soules MR. 1996
Ovarian follicular development and the follicular fluid hormones and growth
factors in normal women of advanced reproductive age. J Clin Endocrinol
Metab. 81:1946 –1951.
15. Santoro N, Rosenberg-Brown J, Adel T, Skurnick JH. 1996 Characterization
of reproductive hormonal dynamics in the perimenopause. J Clin Endocrinol
Metab. 81:1495–1501.
16. Vale W, Rivier C, Hsueh A, et al. 1988 Chemical and biological characterization of the inhibin family of protein hormones. Recent Prog Horm Res.
44:1–34.
17. DeKretser DM, Robertson DM. 1989 The isolation and physiology of inhibin
and related proteins. Biol Reprod. 40:33– 47.
18. Welt C, Martin KA, Taylor AE, et al. 1997 Frequency modulation of FSH
during the luteal-follicular transition: evidence for FSH control of inhibin B in
normal women. J Clin Endocrinol Metab. 82:2645–2652.
19. Groome NP, Illingworth PJ, O’Brien M, et al. 1996 Measurement of dimeric
inhibin B throughout the human menstrual cycle. J Clin Endocrinol Metab.
81:1401–1405.
20. Lambert-Messerlian GM, Hall JE, Sluss PM, et al. 1994 Relatively low levels
of dimeric inhibin circulate in men and women with polycystic ovarian syndrome using a specific two-site enzyme-linked immunosorbent assay. J Clin
Endocrinol Metab. 79:45–50.
21. Groome NP, Illingworth PJ, O’Brien M, et al. 1994 Detection of dimeric
inhibin throughout the human menstrual cycle by two-site enzyme immunoassay. Clin Endocrinol (Oxf). 40:717–723.
22. Robertson D, Giacometti M, Foulds L. 1989 Isolation of inhibin a-subunit
precursor proteins from bovine follicular fluid. Endocrinology. 125:2141–2149.
23. Schneyer A, Mason A, Burton L, Ziegner J, Crowley Jr WF. 1990 Immunoreactive inhibin a-subunit in human serum. Implications for radioimmunoassay. J Clin Endocrinol Metab. 70:1208 –1212.
24. Robertson DM, Cahir N, Findlay JK, Burger HG, Groome N. 1997 The
biological and immunological characterization of inhibin A and B forms in
human follicular fluid and plasma. J Clin Endocrinol Metab. 82:889 – 896.
25. Klein NA, Illingworth PJ, Groome NP, McNeilly AS, Battaglia DE, Soules
MR. 1996 Decreased inhibin B secretion is associated with the monotropic FSH
rise in older, ovulatory women: a study of serum and follicular fluid levels of
dimeric inhibin A and B in spontaneous menstrual cycles. J Clin Endocrinol
Metab. 81:2742–2745.
111
26. Reame NE, Wyman TL, Phillips DJ, DeKretser DM, Padmanabhan V. 1998
Net increase in stimulatory tone resulting from a decrease in inhibin B and an
increase in activin A may contribute, in part, to the rise in follicular phase FSH
of aging cycling women. J Clin Endocrinol Metab. 83:3302–3307.
27. Filicori M, Santoro N, Merriam GR, Crowley Jr WF. 1986 Characterization of
the physiological pattern of episodic gonadotropin secretion throughout the
human menstrual cycle. J Clin Endocrinol Metab. 62:1136 –1144.
28. Martin K, Santoro N, Hall J, Filicori M, Wierman M, Crowley Jr WF. 1990
Management of ovulatory disorders with pulsatile gonadotropin-releasing
hormone. J Clin Endocrinol Metab. 71:1081A–1081G.
29. Filicori M, Butler JP, Crowley Jr WF. 1984 Neuroendocrine regulation of the
corpus luteum in the human. J Clin Invest. 73:1638 –1647.
30. Crowley Jr WF, Beitins IZ, Vale W, et al. 1980 The biologic activity of a potent
analogue of gonadotropin releasing hormone in normal and hypogonadotropic men. N Engl J Med. 302:1052–1057.
31. Hall JE, Schoenfeld DA, Martin KA, Crowley Jr WF. 1992 Hypothalamic
gonadotropin-releasing hormone secretion and follicle-stimulating hormone
dynamics during the luteal-follicular transition. J Clin Endocrinol Metab.
74:600 – 607.
32. Seminara SB, Boepple PA, Nachtigall LB, et al. 1996 Inhibin B in males with
gonadotropin-releasing hormone (GnRH) deficiency: changes in serum concentration after short term physiologic GnRH replacement. J Clin Endocrinol
Metab. 81:3692–3696.
33. Lavoie HB, Martin KA, Taylor AE, Crowley WF, Hall JE. 1998 Exaggerated
free a-subunit levels during pulsatile gonadotropin-releasing hormone replacement in women with idiopathic hypogonadotropic hypogonadism. J Clin
Endocrinol Metab. 83:241–247.
34. Schwall RH, Mason AJ, Wilcox JN, Bassett SG, Zeleznik AJ. 1990 Localization of inhibin/activin subunit mRNAs within the primate ovary. Mol Endocrinol. 4:75–79.
35. Roberts VJ, Barth S, El-Roeiy A, Yen SSC. 1993 Expression of inhibin/activin
subunits and follistatin messenger ribonucleic acids and proteins in ovarian
follicles and the corpus luteum during the human menstrual cycle. J Clin
Endocrinol Metab. 77:1402–1410.
36. Burger HG, Cahir N, Robertson DM, et al. 1998 Serum inhibins A and B fall
differentially as FSH rises in perimenopausal women. Clin Endocrinol (Oxf).
48:809 – 813.
37. Shintani Y, Wakatsuki M, Harada K, et al. 1998 Immunoassays for activin and
follistatin: results in normal and diseased subjects. In: Aono T, Sugino H, Vale
WW, eds. Inhibin, activin and follistatin: regulatory function in system and cell
biology. New York: Springer; 130 –140.
38. Loria P, Petraglia F, Concari M, et al. 1998 Influence of age and gender on
serum levels of total dimeric activin A. Eur J Endocrinol. 139:487– 492.
39. Khoury RH, Wang QF, Crowley WF, et al. 1995 Serum follistatin levels in
women: evidence against an endocrine function of ovarian follistatin. J Clin
Endocrinol Metab. 80:1361–1368.
40. Welt CK, Lambert-Messerlian GM, Zheng W, Crowley Jr WF, Schneyer AL.
1997 The presence of activin, inhibin and follistatin in epithelial ovarian carcinoma. J Clin Endocrinol Metab. 82:3720 –3727.