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Original Studies |
Reproductive Endocrine Unit, National Center for Infertility Research, Massachusetts General Hospital; Boston, Massachusetts 02114; Department of Obstetrics/Gynecology, Brigham and Women’s Hospital (J.F.), Boston, Massachusetts 02114; and Department of Pathology and Laboratory Medicine, Women and Infants Hospital (G.M.M.), Providence, Rhode Island 02903
Address all correspondence and requests for reprints to: Alan Schneyer, Ph.D., Reproductive Endocrine Unit, BHX-5, Massachusetts General Hospital, Boston, Massachusetts 02114. E-mail: schneyer.alan{at}mgh.harvard.edu
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Abstract |
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 Top Abstract IntroductionSubjects and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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The biosynthesis of inhibin, activin, and the activin-binding protein
FS has been studied in numerous species using a variety of techniques
(8, 9). In humans, anatomical studies, including immunocytochemistry
and in situ hybridization, have demonstrated that production
of the inhibin/activin 
-, ßA-, and ßB-subunits as well as both
alternatively spliced forms of FS messenger ribonucleic acid (mRNA) are
differentially regulated as ovarian follicles mature. For example, in
preantral and small antral follicles, ßB is the most highly expressed
inhibin/activin subunit, although - and ßA-subunits and FS can
also be identified to a lesser extent. By the time follicles reach
the preovulatory stage, , ßA, and FS become the dominant mRNAs
observed, whereas ßB-subunit expression is low or undetectable
(10, 11, 12). These results suggest that during human follicular
maturation, the intraovarian microenvironment changes from a
primarily activin B-dominant to an inhibin A/FS-dominant
environment.
Although anatomical approaches are useful for characterizing the site and timing of inhibin/activin subunit biosynthesis, they do not elucidate which dimeric hormones are actually secreted. In human follicular fluid (hFF) aspirated from size-differentiated normal follicles identified in ovarian tissue obtained at surgery, inhibin A concentrations were greater in larger follicles, whereas inhibin B and activin A concentrations did not vary consistently with follicle size (13). However, FS concentrations were not investigated in this study. Furthermore, neither the aspiration studies nor the anatomical studies, because of their reliance on surgical specimens, were able to associate observed follicle size- or morphology-related changes in hormone biosynthesis to the maturational status of the follicle with respect to the normal menstrual cycle, nor was it possible to document the developmental history of each analyzed follicle to insure that it was indeed the dominant follicle.
To address these critical issues and to further investigate the hypothesis that follicular development involves a coordinated evolution from an activin-dominant to an inhibin/FS-dominant microenvironment, we examined hFF hormone concentrations in follicles aspirated from unstimulated normal women across the follicular phase. Daily blood samples and frequent ultrasounds were used to characterize follicular development and anchor the results to circulating hormone concentrations. Our results document the differential biosynthesis of steroids, inhibins A and B, activin, and FS as the dominant follicle develops from a cohort of small antral follicles and also demonstrate differences in hormone content between dominant and nondominant follicles in the same patient. These observations suggest that this carefully orchestrated pattern of inhibin/activin/FS biosynthesis may be critical for dominant follicle development.
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Subjects and Methods |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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The study population consisted of 26 normal women with an average age of 30.7 yr (range, 19–42 yr) and a history of regular menstrual cycles of 26–32 days. Subjects had been taking no medication for 3 months before this study, received no medication during the study, and had normal physical examinations and TSH and PRL levels. Daily blood samples were obtained across 1 full menstrual cycle, continuing until the day of transvaginal follicle aspiration in the subsequent cycle. In addition, frequent pelvic ultrasonography was performed in the follicular phase of both cycles to document dominant follicle growth. All subjects gave their informed consent, and the protocol was approved by the human studies internal review board for the Massachusetts General and Brigham and Women’s Hospitals.
Follicle aspirations were performed in the in vitro fertilization (IVF) suite of Brigham and Women’s Hospital. Subjects were offered an oral sedative 1 h before the procedure, but no other general or local anesthesia was used. The dominant follicle was identified by transvaginal ultrasonography as the largest follicle from among the cohort of antral follicles and was aspirated into a sterile needle and tubing (1.8 mL total deadspace), after which the follicle contents were flushed into a sterile tube with 2 mL Dulbecco’s phosphate-buffered saline containing 20 IU heparin/mL (Sage Biopharma, Bedminster, NJ) using standard oocyte retrieval techniques developed for IVF. The difference between the recovered volume and the 2 mL flush volume was considered to be the follicle volume, and a dilution factor was then calculated and used to correct all hormone analyses.
In each subject, the largest visible follicle on either ovary was aspirated. For simplicity, these follicles are referred to as dominant (see Discussion). A total of 28 dominant follicles were analyzed (2 women participated a second time), ranging in size from 6.5–23.5 mm. In 12 of the women, the next largest follicle on the same ovary as the dominant follicle was also aspirated. Only 8 of these smaller follicle aspirates actually contained hFF allowing analysis in this study and are referred to as nondominant to indicate their subordinate status to a larger follicle regardless of whether final follicle selection had actually taken place. In 1 of these 8 patients, the largest (dominant) follicle was not recovered, so the nondominant follicle was deleted from hormonal analyses, leaving a total of 7 matched samples for comparative analysis.
Characterization of developing follicles
The size of each follicle was estimated by ultrasound at the
time of aspiration by averaging two cross-sectional diameters. Maturity
of the aspirated follicle was estimated by determining the day of
ovulation in the first cycle from the daily serum LH and estradiol
levels and follicle size changes as monitored by ultrasound, according
to previously established criteria (14). Serum hormone levels and
follicle development pattern for the second cycle were then
superimposed on the first cycle, thereby allowing estimation of the
number of days before predicted ovulation when follicle aspiration
occurred (see Fig. 1
). This parameter,
termed days before predicted ovulation, represents an index of follicle
maturity.
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Daily blood samples were analyzed for estradiol and LH, whereas
samples from 1–2 days before ovulation until menses were also analyzed
for progesterone. A subset of daily samples was also analyzed for FSH,
inhibin A, and inhibin B (e.g. see Fig. 1
). Follicular fluid
aspirates were analyzed for estradiol, progesterone, androstenedione,
inhibins A and B, activin A, and free FS.
Assays
FSH, LH, estradiol, and progesterone concentrations in serum were determined using AxSYM (Abbott Laboratories, Abbott Park, IL) assays according to the manufacturer’s directions as previously described (15, 16). Androstenedione in hFF was measured by RIA as previously described (17). Dimeric inhibins A and B and activin A levels were determined using commercial enzyme-linked immunosorbent assay kits (Serotec, Cambridge, UK) according to the manufacturer’s instructions, with limits of detection of 0.6 IU/mL, 15 pg/mL, and 0.2 ng/mL, intraassay coefficients of variation (CVs) of less than 10%, and interassay CVs of less than 20%, respectively. Inhibin A results are reported as international units per mL of the WHO International Reference Preparation 91/624 (1 IU I International Reference Preparation = 150 pg inhibin A against Serotec calibrators). Free FS was determined using a two-monoclonal SPICA immunoassay (18) in which FS bound to activin was undetectable. The limit of detection was 2 ng/mL, and the intra- and interassay CVs were less than 6% and less than 17%, respectively. The FS assay standard was recombinant FS288 obtained from the National Hormone and Pituitary Program.
Statistical analysis
The concentrations of inhibin B, progesterone, and the androstenedione/estradiol (A:E) ratio in hFF were not normally distributed and were therefore log transformed before analysis. Hormonal results were then analyzed by follicle size as well as by maturity (days before predicted ovulation) using least squares linear regression analysis over the entire range of samples. Mean hormone levels in dominant and nondominant follicles were compared using two-tailed t testing. To compensate for multiple testing, P < 0.01 was considered significant.
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Results |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Daily blood samples obtained from each subject during one complete
menstrual cycle and the subsequent aspiration cycle were analyzed for
LH, FSH, estradiol, progesterone, inhibin A, and inhibin B (Fig. 1). As
the results for the aspiration cycle were nearly identical to those for
the preceding observation cycle, the number of days before predicted
ovulation that the aspiration took place could be estimated from the
first cycle (i.e. follicle maturity), which was day 0 for
the example in Fig. 1. In addition, frequent ultrasound observations
during each cycle allowed documentation of dominant follicle
development, the patterns of which, like serum hormone results, were
nearly identical for successive cycles within individuals. As we also
determined the size of each aspirated follicle, results for each
analyte were examined using both follicle size and maturity.
Because the uncertainty associated with estimating follicle size from
ultrasound images will be independent from variance associated with
estimating the day before ovulation (i.e. maturity), a
direct comparison of these two parameters provides an indication of how
well each estimation procedure performed. Thus, the strong, significant
linear relationship (r = 0.79; P < 0.0001)
between follicle size and maturity (Fig. 2) indicates that these parameters
coordinately characterize the aspirated follicles.
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In dominant follicles estradiol concentrations increased, and
androstenedione concentrations decreased with both follicle size (Fig. 3, A and C; r = 0.80;
P < 0.0001 and r = -0.51; P <
0.01 respectively) and follicle maturity (Figs. 3B and 3D; r =
0.72, P < 0.001 and r = -0.67, P
< 0.01, respectively), producing a significant inverse relationship
for the A:E ratio with follicle size or maturity (Fig. 3, E and F;
r = -0.74; P < 0.0001 and r = -0.72;
P = 0.01 respectively). Both the increase in estradiol
and the decrease in androstenedione (presumably reflecting the
aromatization of androgen precursor) began at the 8–10 mm stage, which
corresponds to approximately 7–8 days before ovulation in the normal
menstrual cycle. In addition, all small (<8 mm) antral follicles in
these carefully characterized normal women had high A:E ratios.
Interestingly, progesterone was easily detectable in the smallest
follicles and increased linearly with both follicle size and maturity
(r = 0.83; P < 0.0001 and r = 0.77;
P < 0.0001, respectively; Fig. 3, G and H).
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).
However, unlike dominant follicles, the hormone levels in these
nondominant follicles were not significantly related to changes in size
or maturity (Fig. 3, open circles). In addition, when these
nondominant follicles were compared to a group of size-matched dominant
follicles, the steroid hormone concentrations were not significantly
different (Table 2).
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The inhibin A concentration in hFF of dominant follicles increased
linearly with both size and maturity (Fig. 4, A and B; r = 0.73;
P < 0.0001 and r = 0.63; P <
0.001, respectively). This developmental increase in inhibin A was
greater than 10-fold, with inhibin A being easily detectable in the
smallest aspirated follicles (6 mm), even before significant increases
in estradiol became detectable (compare Figs. 3A and 4A). In
nondominant follicles, inhibin A concentrations were significantly
lower than in dominant follicles from the same women (Table 1). On the
other hand, compared to size-matched dominant follicles, the inhibin A
levels in nondominant follicles were not statistically different (Table 2).
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, C and D). Inhibin B
concentrations in nondominant follicles were about one third those in
the dominant follicle, but were not significantly different
(P = 0.03) due to the corrections for multiple
comparisons and to the large interfollicle variation (Table 1).
Similarly, inhibin B concentrations in nondominant follicles tended to
be lower than those in the group of size-matched dominant follicles,
but the differences were not significant (Table 2).
The patterns for free FS and activin A concentrations in dominant
follicles were distinct from those of the previous hormones, but
similar to each other. When all dominant follicles were analyzed
together, no relationship was observed with respect to size or maturity
(Fig. 5) for either hormone. The mean
concentrations of free FS and activin did not differ between
nondominant and dominant follicles from the same ovary (Table 1), nor
were they different between nondominant follicles and size-matched
dominant follicles (Table 2). However, a significant positive
correlation was observed between free FS and activin over the entire
range of follicles studied (Fig. 6), and
with the exception of three follicles, free FS levels were in excess of
total immunoreactive activin, suggesting that the activin in hFF is
completely neutralized by the 6-mm stage (8 days before predicted
ovulation).
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Thus, relative to serum concentrations, estradiol levels in hFF are
about 10,000-fold greater during the early follicular phase and
approximately 25,000-fold greater by day 0. In contrast, although both
serum and hFF levels of inhibin A increase as follicles develop, the
increment in serum levels of inhibin A is greater than that in hFF
(Fig. 7B). Thus, the differential hFF concentrations of inhibin A
decrease from 1,000-fold in the early follicular phase to approximately
300-fold by day 0. Inhibin B concentrations are approximately 1,000- to
3,000-fold greater in hFF compared to serum across the follicular
phase. When directly compared, only serum and hFF estradiol
concentrations were significantly correlated (r = 0.72;
P < 0.0001).
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Intrafollicular inhibin A concentrations increased dramatically and significantly with follicle size over the entire range of dominant follicles, confirming and extending previous studies (13). In addition, we found that hFF inhibin A concentrations increased significantly with follicle maturity in a similar fashion as circulating inhibin A. Thus, relative to inhibin B, activin, and FS, inhibin A levels increase as follicles develop, consistent with our hypothesis of evolution toward an inhibin-dominant microenvironment in preovulatory follicles. Furthermore, although inhibin A concentrations increased in a similar fashion as estradiol when calibrated by size or maturity, inhibin A concentrations were detectable in smaller, less mature follicles than estradiol and did not undergo a dramatic increase at the 8–10 mm stage as seen for estradiol, suggesting that the concentrations of these two hormones are under different regulatory influences in developing follicles.
In contrast to inhibin A, there were no discernable relationships for inhibin B concentrations in dominant follicles with size or maturity, although inhibin B levels appeared to be greatest for 13- to 14-mm follicles, in agreement with previous observations from surgically derived specimens (13). Interestingly, this maximum at 14 mm occurs at 5–6 days before predicted ovulation, or at about days 8–9 of a prototypic menstrual cycle, a time when circulating inhibin B concentrations are also maximal (19, 20), suggesting that the growing dominant follicle may be the source of this circulating inhibin B. On the other hand, it is also possible that the serum peak is due to the presence of a larger number of inhibin B-secreting follicles per ovary in the midfollicular phase compared to the late follicular phase (13). Support for this hypothesis can be found in our observation that serum inhibin B levels in normal women did not correlate with changes in intrafollicular inhibin B levels in dominant follicles. Thus, it would appear that circulating inhibin B may not be reflective of inhibin B production in the dominant follicle per se, but more likely represents the total output of all antral follicles in both ovaries.
When analyzed over the entire spectrum of follicle sizes or maturational stages, activin concentrations did not correlate with size or maturity. Similarly, Magoffin and Jakimiuk (13) did not observe any consistent changes in activin concentrations with size in their surgically derived sample population. However, closer inspection of our results show a high degree of variability in follicles less than 10 mm or more than 8 days before predicted ovulation (day 6 of menstrual cycle), whereas in larger or more mature follicles, this variability vastly decreased. Thus, when follicles more than 10 mm are examined separately, activin concentrations appear to increase with follicle size or maturity, suggesting that activin concentrations increase after aromatase is induced in dominant follicles. The pattern of activin B biosynthesis could not be analyzed, because there are currently no valid activin B assays available. However, given that the ßB-subunit is expressed in early antral follicles (10), it is likely that activin B is the predominant activin in hFF from small follicles.
FS is the primary extracellular regulator of activin action in many tissues, including the ovary, through its ability to nearly irreversibly bind and inactivate activin (21, 22). Like activin, we found no relationship between free FS concentrations and follicle size or maturity when all follicles were analyzed. However, like activin, when only follicles more than 10 mm were examined, free FS concentrations appeared to increase with both follicle size and maturity, suggesting that increasing free FS might be a characteristic of developing dominant follicles. Thus, the 8–10 mm transition, where follicles acquire a dominant phenotype in terms of steroid hormone biosynthesis, may also demarcate a change in the production and/or activity of free FS and activin, such that their concentrations become more tightly regulated as follicles continue to mature.
Interestingly, free FS was detectable in all but three follicles, indicating that hFF activin in antral follicles is completely neutralized by FS. Although this may preclude activin from having an intrafollicular paracrine role, activin may still be acting as an autocrine growth factor in granulosa cells, which is then neutralized when it leaves the immediate environment of the secreting cell. In fact, we recently demonstrated in a cell line model (PA-1 ovarian carcinoma cells) that free FS bound to the cell surface prevent paracrine, but not autocrine, actions of activin (23). Thus, the secretory dynamics and autocrine biological activities of both activin and FS need to be examined in granulosa cells before the implications of our hFF results on intraovarian regulation of follicular development can be fully appreciated. Nevertheless, our analysis of the inhibin/activin/FS axis in developing antral follicles, including the dramatic increase in inhibin A levels, the relatively constant activin concentrations, and excess free FS, is consistent with our hypothesis that the intrafollicular hormonal milieu evolves from an activin-dominant to an inhibin/FS-dominant microenvironment as antral follicles mature.
In seven subjects with dominant follicles more than 10 mm, the next largest follicle from the same ovary was also aspirated. Estradiol and progesterone were significantly lower in the nondominant follicles compared to those in dominant follicles from the same ovary, a finding that agrees with previous observations comparing a large numbers of follicles (reviewed in Ref. 24). In addition, inhibin A and B levels were also lower in nondominant follicles, whereas no difference was observed for free FS or activin in agreement with a previous study comparing total FS in large and small follicles (25). Interestingly, when our nondominant follicles were compared to a group of size-matched dominant follicles, all hormone concentrations were similar, except for inhibin B, which were one third those in the dominant follicle group, although this difference did not reach significance. Furthermore, except for androstenedione and the A:E ratio, hormone levels in nondominant follicles did not vary with size or maturity as seen for dominant follicles, suggesting that these nondominant follicles are biosynthetically active but are not following the same developmental trajectory as the dominant follicle. Thus, although hormone levels are lower in nondominant follicles relative to the dominant follicle of the same ovary, hormone biosynthesis is not necessarily abnormal for follicle size.
To assess whether our sample collection and analysis methods were sufficiently robust and precise to test our hypotheses, we analyzed changes in steroid hormone concentrations with increasing follicle size and/or maturity, which could then be compared to earlier studies. Although there is considerable evidence (reviewed in Ref. 24) that estradiol concentrations increase as follicles mature from the midantral (e.g. >10 mm) to the preovulatory stage, correlation of estradiol levels with size or maturity examined as a continuous variable is less common. An additional complication arises from the variety of criteria used to define dominant and nondominant follicles in the different studies, including follicle size, histochemical analyses, numbers of granulosa cells, estradiol concentrations (alone or relative to androstenedione), and quality of the oocyte (26, 27, 28, 29). In our study the dominant follicle was identified as the largest growing follicle on either ovary based on multiple consecutive ultrasound observations during the aspiration cycle. As follicle aspiration does not remove all granulosa cells, we cannot compare our recovered granulosa cell content with previous studies using granulosa cell number to categorize follicles (26). In addition, we did not routinely recover oocytes, thereby further limiting direct comparisons (27).
Nevertheless, we observed significant positive correlations between follicle size or maturity and estradiol and progesterone concentrations in dominant follicle aspirates as well as inverse correlations with androstenedione and A:E ratio, extending previously reported relationships in 45 dominant (>9 mm) follicles obtained from surgical specimens (30). Our results additionally document that estradiol increases rapidly after the 8–10 mm stage, which is equivalent in our samples to 8 days before presumed ovulation (approximately day 6 of a normal menstrual cycle). This observation extends and refines previous studies describing increases in estradiol in follicles larger than 10 mm, but low estradiol levels in smaller follicles (27, 30, 31). Our results are also compatible with the estimate of day 6.3 and 9-mm diameter for the appearance of a dominant follicle (32). Taken together, our steroid hormone results are consistent with the concept that in humans, the dominant follicle becomes apparent approximately 8 days before ovulation at the 8–10 mm stage (30). Interestingly, increasing progesterone levels were detected in follicles as small as 6 mm and as early as 14 days before ovulation, well before estradiol begins to increase and earlier than previously reported (26, 30). If this progesterone is produced by maturing granulosa cells, then functional LH receptors may also be present, and thus the luteinization process may already be commencing by the 6-mm follicle stage. Alternatively, the progesterone in these small follicles may be derived from thecal cells and may be diffusing into the follicle along with androstenedione.
One unique aspect of our results is the comparison of circulating and intrafollicular hormone levels. Although the overall patterns for estradiol and inhibin were similar in that both hormones increased during the follicular phase as follicles matured, the relative changes in the serum and follicle compartments were opposite. Thus, estradiol increased from 10,000- to 25,000 fold difference between hFF and serum, whereas inhibin A decreased from 1,000-fold to approximately 300-fold. Inhibin B varied from 1,000- to 3,000-fold greater hFF hormone concentrations, with no discernable pattern across the follicular phase. If steroid hormones simply diffuse out of the follicular compartment to the circulation, one might expect estradiol to have a lower concentration difference than either inhibin. Thus, our results suggest that some steroid hormone is actively retained within the follicle, that inhibins are actively removed from the follicle, and/or that the circulating half-life of inhibin A is longer than those of both inhibin B and estradiol, allowing it to accumulate in the circulation during the follicular phase. In addition, regulation of hormone transport across the follicle barrier may change during follicle development, as suggested by the changing concentration ratios for estradiol and inhibin A.
If dominance is established by the 8–10 mm stage, what is the optimal way to characterize smaller follicles whose ultimate fate is unknown at the time of aspiration? In our study, all follicles smaller than 8 mm had high androstenedione and low estradiol levels, with a consequent high A:E ratio. Some investigators would consider all of these follicles atretic based on an A:E ratio greater than 4 (13). In addition, it has been estimated that up to 92% of these small follicles are atretic based on granulosa cell content, estradiol synthesis in vitro, and the absence of a healthy, GV stage oocyte (27). However, there appears to be a large degree of heterogeneity in this ratio among follicles grouped by granulosa cell number per follicle, oocyte quality, granulosa cell secretory content, or other estimates of follicle health (26). Furthermore, as aromatase transcription is not induced until the 7-mm stage (33), it is also possible that all follicles smaller than 8 mm have high androgen and low estrogen concentrations, and that the A:E ratio is not useful for predicting the health or ultimate fate of these small antral follicles. This analysis is consistent with the low estradiol concentrations previously reported for these small follicles (31) as well as with the mean A:E ratio of 5.4 for follicles less than 8 mm in the early follicular phase (34). Thus, there would appear to be no rigorous scientific foundation for classifying these small antral follicles on the basis of steroid concentrations alone. Given the fact that we aspirated the largest of these small antral follicles in each subject we consider them to represent the lower portion of a size/maturation continuum which is contiguous with the clearly dominant (>10-mm) follicle group.
Taken together with previous anatomical studies, our results, in providing a picture of hormone biosynthesis from small antral to preovulatory stages, are consistent with the hypothesis that human follicular development from the preantral to the preovulatory stage is accompanied by a carefully orchestrated transition from an activin-dominant to an inhibin/FS-dominant microenvironment. In addition, these studies provide a significantly more detailed description of intrafollicular changes in hormone concentrations as dominant follicles develop in the normal human ovary. Comparison of hFF and serum hormone levels has uncovered some interesting new insights into potential differences in hormone pharmacodynamics within follicles that need to be investigated further to fully understand intrafollicular hormone dynamics. Finally, establishing a comprehensive normative database for the inhibin/activin/FS axis in developing human follicles will now facilitate detailed investigation of the potential involvement of these hormones in pathophysiological states where normal follicular development is disturbed.
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Acknowledgments |
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Footnotes |
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Received December 22, 1999.
Revised May 15, 2000.
Accepted May 19, 2000.
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References |
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