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Reproductive Endocrinology |
-Inhibin Levels in Polycystic Ovary Syndrome: Relationship to the Serum Androstenedione Level1
Department of Endocrinology and Diabetology (R.D., C.C.R., D.D.R., D.D.) and Laboratory of Endocrinology (P.P., A.R.), Marc Linquette Clinic, Hospital and University Center, F-59037 Lille; and Faculty of Medicine, Centre d’Etude et de Recherche en Informatique Medicale (A.D.), F-59045 Lille, France
Address all correspondence and requests for reprints to: Prof Didier Dewailly, Service d’Endocrinologie et Diabétologie, Clinique Marc Linquette, Centre Hospitalier et Universitaire, F 59037 Lille Cedex, France.
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Abstract |
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 Top Abstract IntroductionSubjects and MethodsResultsDiscussionReferences |
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-inhibin levels were assayed in the serum of 61 healthy women and 72
PCOS patients by means of an - enzyme-linked immunosorbent assay.
Serum -inhibin levels together with LH and androstenedione (A)
levels were significantly increased in PCOS women (mean ±
SD, 1.45 ± 0.55 vs. 0.94 ± 0.36
U/mL in controls; P < 0.001). Moreover, simple and
partial regression analysis demonstrated that serum A levels were
positively and independently correlated to serum -inhibin (r =
0.32; P < 0.01) and LH levels (r =
0.48; P < 0.001) in PCOS. The respective
influences of -inhibin and LH on A variability were 20% and 80%,
as determined by multiple regression analysis. In conclusion, in
agreement with recent in vitro data, our in
vivo results argue for a role of inhibin in the
hyperandrogenism of PCOS together with, but independently from, that of
LH. Further studies are needed to determine whether this effect is
produced by inhibin A and/or B. |
Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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-subunit covalently joined by disulfide links to
either a ßA-subunit (inhibin A) or a
ßB-subunit (inhibin B). Besides these mature bioactive
forms, inhibins also exist in the circulation as either unprocessed or
partially processed high mol wt forms, and free -subunit. The
physiological significance of the precursor forms of inhibin remains a
matter of debate (1). Inhibins are primarily produced by the gonads and
the placenta, and have been initially characterized by their ability to
exert a negative feedback on pituitary FSH synthesis and release (2).
Moreover, numerous recent studies have emphasized the fact that
inhibins may act at the gonadal level as paracrine and/or autocrine
factors (3, 4, 5, 6).
First generation inhibin immunoassays used antibodies raised against
-subunit and measured immunoreactive inhibin levels (or
-inhibin). Among them, the Monash RIA using antiserum 1989 (7) was
the first available and the most widely used assay (8). The lack of
specificity of these assays for the bioactive dimeric forms of inhibin
may explain why a physiological role for inhibin in FSH release could
not be demonstrated in men (9). However, evidence supporting such a
role in women has been reported (10). Recently, new two-site inhibin
immunoassays have been developed, permitting the measurement of either
dimeric inhibin A or inhibin B (11, 12, 13). However, a high degree of
assay specificity does not seem to be required for all clinical
applications of inhibin measures, as previously suggested (14). For
example, -inhibin levels may be used as a valuable index of
granulosa cell (GC) activity or maturity (15, 16), as the majority of
serum immunoreactive inhibin is thought to arise from the GC in
nonpregnant women.
The polycystic ovary syndrome (PCOS) is a heterogeneous entity
characterized by insulin resistance, hyperandrogenemia, a high serum
LH/FSH ratio and chronic anovulation in the presence of bilateral
polycystic ovaries (17, 18, 19). To explain the inappropriately low serum
FSH levels found in this syndrome, it has been suggested that
polycystic ovaries might secrete higher amounts of inhibin, leading to
inhibition of pituitary FSH secretion (20, 21). Furthermore, recent
in vitro data indicate that inhibin enhances both basal and
LH-induced androgen production by human thecal cells (TC) (22, 23, 24).
Some studies have evaluated the hypothesis that the endocrine or
paracrine influence of inhibin might be enhanced in PCOS. However,
these studies were performed by measuring either -inhibin or dimeric
inhibin, and generally did not find a difference in serum inhibin
levels between control women and PCOS patients (25, 26, 27), with one
exception (28). In these studies no relationship between either serum
androgen or FSH and inhibin levels in PCOS women was noted. However,
these reports included small numbers of women with PCOS (between
5–23). As PCOS is a heterogeneous entity involving several
pathophysiological factors, a small sample size could mask such a
relationship. Here we report a large scale study evaluating -inhibin
levels in women with PCOS and in healthy controls using an -
enzyme-linked immunosorbent assay (ELISA) that detected free
-subunit, inhibin A, and, to a lesser extent, inhibin B (100% and
16.7% cross-reactivities, respectively). This assay has good
correlation with the results of the Monash RIA for the determination of
inhibin in human follicular fluid (29). We demonstrate that PCOS women
have higher serum -inhibin levels than controls. Furthermore, we
provide for the first time in vivo evidence that serum
-inhibin, in conjunction with and independently of LH, is linked to
the hyperandrogenism of PCOS.
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Subjects and Methods |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Controls. The control population is comprised of 61 healthy women [age, 18.1 ± 2.7 yr (mean ± SD); range, 15–27 yr] recruited on the occasion of an annual physical examination carried out at the Centre de Bilan de Santé, Institut Pasteur (Lille, France). The exclusion criteria were a history of menstrual disturbances (i.e. cycle length either <25 days or >35 days), hirsutism, or hormonal treatment during the 3 months before the study. Their body mass indexes (BMIs) ranged from 17–37 kg/m2 (mean ± SD, 22.7 ± 5 kg/m2).
Women with PCOS.
Seventy-two women with PCOS were recruited
into the study. They were referred to our department for
hyperandrogenism and/or ovulation disorder. Patients ranged in age from
14–37 yr (mean ± SD, 23.6 ± 4.8 yr), and BMI
ranged from 17–37 kg/m2 (mean ± SD,
25.6 ± 4.9 kg/m2). The diagnosis of PCOS was based on
the presence of at least one clinical criterion (hirsutism determined
by a modified Ferryman and Galway score >8, menstrual and/or ovulatory
disturbances, or minor signs such as acne or seborrhea) associated with
either one ultrasonographic criterion and/or one biological criterion.
Biological criteria were basal serum LH levels greater than 6 IU/L,
testosterone greater than 0.7 ng/mL, and/or androstenedione (A) greater
than 2.2 ng/mL, as previously reported (30). The ultrasonographic
criterion was an ovarian area more than 5.5 cm2 uni- or
bilaterally (31). Pelvic ultrasonographic examination was performed by
the abdominal route in all patients and by the vaginal route in
sexually active women. Any patient with one or more follicles with a
diameter greater than 10 mm was excluded from the study so as not to
confound the data with a dominant follicle. No patient had received
long term hormonal treatment or oral contraceptives for at least 3
months before the study. One patient with insulin-dependent diabetes
mellitus was excluded. Massively obese patients (i.e. BMI
>37 kg/m2) were also excluded from this study, as a
preliminary report by us (32) showed that they tended to have very low
levels of -inhibin as well as other endocrine abnormalities
independent of PCOS.
Sampling procedure
All controls and PCOS subjects underwent blood sampling in the early follicular phase (i.e. between days 3 and 5 after the last menstrual period). The last mestrual period occurred either spontaneously or after withdrawal of didrogesterone (Duphaston, Roussel Laboratories, Paris, France; 10 mg/day for 7 days) in amenorrheic or oligomenorrheic PCOS women. A total of 30 mL blood were drawn in each fasting subject at 0800 h. Five 5-mL aliquots were collected in tubes for assays of LH, FSH, inhibin, estradiol, and insulin, and one 5-mL aliquot was collected in a tube containing ethylenediamine tetraacetate for the assay of A.
Hormonal immunoassays
-Inhibin was measured by a two-site immunoenzymometric assay
(Inhibin - Easia, Medgenix, Fleurus, Belgium), as previously
described (33). Both antibodies used in this assay recognized distinct
epitopes on the -subunit of human inhibin (34). Human inhibin used
as a standard was extracted from the follicular fluid of women
undergoing hyperstimulation in an in vitro fertilization
program. This standard was calibrated against a reference pool of sera
from 30 healthy men (33), which was arbitrarily assigned a value of 1
U/mL (1 U of this standard corresponds to 400 pg recombinant 32-kDa
inhibin A). The minimal detectable amount of inhibin was 0.1 U/mL. The
intra- and interassays coefficients of variation were 6.5% and 8.3%,
respectively (33). No cross-reactions were observed with
inhibin-related proteins such as activins, follistatin, or TGFß.
Inhibin A (WHO reference preparation) and recombinant inhibin B
exhibited 100% and 16.7% cross-reactivities in this assay,
respectively (29).
Serum insulin levels were measured in duplicate by a RIA (Bi-insulin RIA, Diagnostics Pasteur, Marnes de Coquette, France) using an antipig insulin polyclonal antibody and human [125I]insulin as a tracer. Results are expressed as microinternational units per mL in terms of the WHO 66/304 reference preparation. This RIA displayed a 40% cross-reactivity with human proinsulin. Intra- and interassay coefficients of variation were less than 6% and 9%, respectively.
Serum gonadotropin levels were evaluated by two immunoassays provided by Cis Bio International (France). These immunometric assays used monoclonal antibodies raised against LH and FSH. Results are expressed as milliinternational units per mL in terms of the First International Reference Preparation 68/40 (LH) and the Second International Reference Preparation 78/549 (FSH). Intra- and interassay coefficients of variation were less than 5% for the two immunoassays.
Estradiol concentrations were measured in duplicate in unextracted serum by RIA using kits provided by Biomérieux (France). The detection limit was 12 pg/mL. The intra- and interassays coefficients of variation were 9.8% and 8.7%, respectively.
A was measured by RIA after extraction with ethyl ether using a kit provided by Immunotech (France). The limit of detection was 0.04 ng/mL. The intra- and interassay coefficients of variation were 8.1% and 11.9%, respectively.
Statistical methods
Data are expressed as the mean ± SD.
Statistical analysis for differences between the means of two
independent groups was performed by Student’s t test.
P < 0.05 was considered significant. Significant
relations between -inhibin and particular variables were evaluated
by the Pearson correlation coefficient. Comparisons within different
variables were investigated by multiple regression analysis, which
included partial correlation coefficients. Data were analyzed with SAS
statistical software (SAS Institute, Cary, NC).
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Results |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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The principal clinical and biochemical features of the control and
PCOS groups are presented in Table 1
. Higher serum
levels of -inhibin (Fig. 1), LH, and A were observed
in women with PCOS than in controls. Age and BMI were also greater in
the PCOS group, whereas estradiol and fasting insulin serum levels did
not differ. Finally, serum FSH was significantly lower in the PCOS
women than in the controls.
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-inhibin
Using simple regression analysis (Table 2), we
noted that A was the only factor correlated to circulating -inhibin
levels (r = 0.32; P < 0.01) in PCOS group (Fig. 2). As expected, LH and A were also highly correlated
among PCOS women (r = 0.48; P < 0.001; Fig. 2),
but not controls. -Inhibin remained correlated to A after
controlling for LH by partial regression analysis (r = 0.27;
P < 0.05). Likewise, the correlation between A and LH
was still significant after controlling for -inhibin levels (r
= 0.45; P < 0.001). Thus, LH and -inhibin appeared
to be two independent variables determining the variability of A levels
in PCOS women. In a theoretical model in which A would depend
exclusively on these two variables, multiple regression analysis
(incremental partial sum of squares) indicated that LH and -inhibin
accounted for 80% and 20% of the variability of A in PCOS,
respectively. The results of the correlation matrix were not
significantly modified when BMI and insulin were introduced into the
model. Likewise, no significant relationship was found between A and
fasting insulin in either control or PCOS women. Finally, -inhibin
did not show a significant correlation with age in either of the two
groups.
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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-subunits (1, 8). Recently,
two-site immunoassays have been developed that can specifically measure
serum levels of dimeric inhibin A or B with some sensitivity (11, 12).
Initial results obtained with these assays indicated that inhibin A and
inhibin B displayed different secretory patterns during the menstrual
cycle; inhibin A is mainly secreted in the luteal phase and in
pregnancy, whereas inhibin B rises in the early follicular phase
(11, 12, 13, 35). In men, inhibin B seems to be the form of inhibin that is
most important for the physiological control of FSH release (36, 37).
The antibodies in our inhibin ELISA recognize both bioactive
dimeric inhibins and free -subunit, which are both produced and
released by GC under the control of FSH (38, 39, 40). However, we estimate
that this lack of assay specificity does not hinder our results, since
we used this assay as an index of GC function and responsiveness to
FSH. With our assay, we were able to demonstrate that serum -inhibin
levels are increased in PCOS women, whereas most of the previous
studies have failed to demonstrate such a relationship, regardless of
the type of inhibin assay (25, 26, 27). Only Mizunuma et al.
reported a significantly higher -inhibin level in a series of 23
women with PCOS (28). There may be various explanations for the
discrepancies between our data and those of others. Firstly, patient
populations differed from one study to another because of different
selection criteria. Secondly, the small size of the populations could
have masked a difference. Hence, the large size of our patient and
control populations allowed us to use more powerful statistical tests.
Finally, inhibin immunoassays differ in their specificity (1, 29),
making comparisons between the results of different studies difficult.
The increase in -inhibin levels that we observed suggested that the
GC of polycystic ovaries are functionally active (41). This agrees with
the findings of other studies that the follicles in polycystic ovaries
are not simply atretic, but contain steroidogenically active GC when
tested in vitro (42). It is also in agreement with the
hypothesis that the follicles of anovulatory polycystic ovaries exhibit
premature maturational arrest (42, 43), a developmental stage at which
inhibin synthesis is increased (39).
This is the first report demonstrating in vivo that
-inhibin is positively related to A levels in PCOS women. This
raises the possibility that part of the circulating inhibin in PCOS
patients originates from the TC, which in PCOS patients are
hyperplastic and secrete increased amounts of A in response to the high
levels of LH. Jaatinen et al. (44) and Roberts et
al. (45) effectively demonstrated in vitro, using
in situ hybridization, that both GC and TC of normal and
polycystic ovaries express inhibin subunit messenger ribonucleic acid.
Moreover, -subunit messenger ribonucleic acid levels were higher in
TC compared to GC of polycystic ovaries, in contrast to the normal
ovary (45, 46). Accordingly, the serum levels of inhibin B in PCOS
patients were significantly and positively correlated to the LH level
in a recent study (47). However, the paradoxical decrease in this
peptide 24 h after hCG injection (47) brings into question whether
circulating inhibin in some way reflects TC secretory activity, in
parallel with A.
In our study, the relationship between -inhibin and A was noted to
be independent of LH, whereas no clear association was found between
-inhibin and LH. Consequently, we would favor the hypothesis that
-inhibin exerts a positive effect on A secretion through either a
paracrine (from GC) or autocrine (from TC) action independent of and in
addition to stimulation by LH (Fig. 3). In our series,
the influences of -inhibin and LH were estimated to be 20% and
80%, respectively, as determined by multiple regression analysis. Such
a conclusion was not unexpected in view of in vitro studies
showing that inhibin enhanced both insulin- and LH-stimulated A
production in cultured human TC (22, 23). It has been previously
proposed that abnormalities of the activin/inhibin paracrine or
autocrine system may be involved in the hyperandrogenism of PCOS (45, 48).
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In conclusion, our in vivo data agree with the in vitro models noting that LH is the major factor involved in the control of ovarian androgen production by the TC of polycystic ovaries (48). However, inhibin seems to play a small, but significant, regulatory role in the TC synthesis of A, acting in either a paracrine and/or autocrine fashion. Further studies are needed to determine whether this effect is exerted by inhibin A, inhibin B, or both.
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Acknowledgments |
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Footnotes |
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Received November 5, 1996.
Revised February 13, 1997.
Accepted March 6, 1997.
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References |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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C-containing forms in human serum by a
new ultrasensitive two site ELISA. J Clin Endocrinol Metab. 80:2926–2932.-inhibin and progesterone. Fertil Steril. 61:663–668.[Medline]
-subunit
immunoreactive material in maternal serum during spontaneous and
in vitro fertilization pregnancies. J Clin Endocrinol
Metab. 81:985–989.[Abstract]
, ßA and ßB mRNAs
in the normal human ovary and in polycystic ovarian syndrome. J
Endocrinol. 143:127–137.This article has been cited by other articles:
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  S. Jonard, Y. Robert, and D. Dewailly Revisiting the ovarian volume as a diagnostic criterion for polycystic ovaries Hum. Reprod., October 1, 2005; 20(10): 2893 - 2898. [Abstract] [Full Text] [PDF]
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 V. Mitchell, K. Steger, C. Marchetti, J.-C. Herbaut, P. Devos, and J.-M. Rigot Cellular expression of protamine 1 and 2 transcripts in testicular spermatids from azoospermic men submitted to TESE-ICSI Mol. Hum. Reprod., May 1, 2005; 11(5): 373 - 379. [Abstract] [Full Text] [PDF]
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 S. Luisi, P. Florio, F. M. Reis, and F. Petraglia Inhibins in female and male reproductive physiology: role in gametogenesis, conception, implantation and early pregnancy Hum. Reprod. Update, March 1, 2005; 11(2): 123 - 135. [Abstract] [Full Text] [PDF]
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S. Jonard and D. Dewailly The follicular excess in polycystic ovaries, due to intra-ovarian hyperandrogenism, may be the main culprit for the follicular arrest Hum. Reprod. Update, March 1, 2004; 10(2): 107 - 117. [Abstract] [Full Text] [PDF]
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S. Jonard, Y. Robert, C. Cortet-Rudelli, P. Pigny, C. Decanter, and D. Dewailly Ultrasound examination of polycystic ovaries: is it worth counting the follicles? Hum. Reprod., March 1, 2003; 18(3): 598 - 603. [Abstract] [Full Text] [PDF]
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 C. K. Welt, A. E. Taylor, K. A. Martin, and J. E. Hall Serum Inhibin B in Polycystic Ovary Syndrome: Regulation by Insulin and Luteinizing Hormone J. Clin. Endocrinol. Metab., December 1, 2002; 87(12): 5559 - 5565. [Abstract] [Full Text] [PDF]
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 C. Welt, Y. Sidis, H. Keutmann, and A. Schneyer Activins, Inhibins, and Follistatins: From Endocrinology to Signaling. A Paradigm for the New Millennium Experimental Biology and Medicine, October 1, 2002; 227(9): 724 - 752. [Abstract] [Full Text] [PDF]
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J. S.E. Laven, B. Imani, M. J.C. Eijkemans, F. H. de Jong, and B. C.J.M. Fauser Absent biologically relevant associations between serum inhibin B concentrations and characteristics of polycystic ovary syndrome in normogonadotrophic anovulatory infertility Hum. Reprod., July 1, 2001; 16(7): 1359 - 1364. [Abstract] [Full Text] [PDF]
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