Circulating follistatin concentrations are higher and activin concentrations are lower in polycystic ovarian syndrome

Robert J. Norman1,4, Clyde R. Milner1, Nigel P. Groome2 and David M. Robertson3

1 Reproductive Medicine Unit, the Queen Elizabeth Hospital, University of Adelaide, 28 Woodville Road, Woodville, South Australia 5011, Australia, 2 Oxford School of Biological and Molecular Sciences, Oxford Brookes University, Oxford, UK and 3 Prince Henry's Institute of Medical Research, PO Box 5152, Clayton, Victoria 3168, Australia


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Familial polycystic ovarian syndrome (PCOS) has been proposed to be linked to a site near the follistatin gene. We studied the concentrations of circulating follistatin, activin A and inhibin B in well-characterized subjects with PCOS (n = 108) and controls without PCOS (n = 20). Mean (± SEM) concentrations of follistatin were higher (P < 0.05) in PCOS (0.27 ± 0.03 ng/ml) than controls (0.15 ± 0.02 ng/ml) and activin A were lower (P < 0.05) in PCOS (0.20 ± 0.01ng/ml) than controls (0.24 ± 0.02 ng/ml). Inhibin B concentrations were not different between the two groups: PCOS (0.06 ± 0.01ng/ml), and controls (0.06 ± 0.01ng/ml). It is proposed that higher concentrations of follistatin with lower concentrations of activin A may relate to follicular development not proceeding beyond 8–10 mm and may be partly responsible for the lack of pre-ovular follicle development in PCOS.

Key words: activin/follistatin/inhibin/PCOS


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Polycystic ovarian syndrome (PCOS) is a condition found in 5–10% of women and may lead to a spectrum of diseases ranging from menstrual abnormalities through to diabetes and cancer (Dunaif, 1994Go; Franks, 1997Go). As a heterogeneous condition with an ill-defined set of diagnostic criteria due to a lack of understanding in the aetiology of its cause (Ben-Rafael and Orvieto, 2000Go), there have been many suggestions as to its origin. While there is a distinct familial aetiology for PCOS (Franks, 1997Go), it is uncertain whether PCOS is caused by genetic or environmental factors.

In a recent publication following extensive recruitment of siblings with hyperandrogenaemia compatible with PCOS, a proposal was made that there is a linkage of PCOS to the follistatin gene (Urbanek et al., 1999Go). Follistatin is a well-known intra-ovarian regulator which binds activin, a protein also found in the ovary and partly responsible for follicular growth (Findlay, 1993Go; Schneyer et al., 1996Go; Hillier, 1999Go). Follistatin also binds other members of the transforming growth factor ß (TGFß) family, including bone morphogenetic proteins (Iemura et al., 1998Go). Mice with gene-targeted hyperexpression of the follistatin gene have impaired follicular development (Guo et al., 1998Go). Overexpression of follistatin has been associated with the arrest of follicular development, an increase in ovarian androgen development and a decrease in serum FSH concentrations—all cardinal features of PCOS. Follistatin concentrations increase in the early follicular phase, then fall and then have a short surge mid-cycle. It has been noted that in patients with PCOS there is an absence of this mid-cycle surge (Lockwood et al., 1998Go).

The human ovary expresses mRNA for all three subunits of activin and inhibin ({alpha}, ßA, ßB) and follistatin (Burns et al., 1990Go; Schwall et al., 1990Go; Eramaa et al., 1993Go; Roberts et al., 1993Go) as well as the proteins for activin and inhibin subunits and follistatin. These proteins are detectable in the circulation with cyclic, specific variations for inhibin and activin A and can be measured in follicular fluid from small and large follicles for all three molecules (Schneyer et al., 1996Go; Lambert-Messerlian et al., 1997Go; Magoffin and Jakimiuk, 1997Go, 1998Go; Lau et al., 1999Go). Activin A is produced by follicles in culture (Smitz et al., 1998Go) and has been proposed to promote proliferation of granulosa cells, enhance FSH receptor expression, decrease LH-induced androgen production, increase pituitary FSH secretion and generally promote follicle growth (Li et al., 1995Go; Silva and Knight, 1998Go; Smitz et al., 1998Go). Follistatin is a binding protein for activin and inhibits most of these actions of activin (Hillier and Miro, 1993Go; de Winter et al., 1996Go; Hillier, 1999Go). Both proteins have effects on oocyte maturity and development, with activin promoting post-fertilization development and follistatin inhibiting this process. In mice where activin receptors have been inactivated by recombinant technology, FSH concentrations are low and animals are infertile. Follistatin knockout animals die early in life (Matzuk et al., 1995Go) while overexpressing animals are infertile, have small ovaries and exhibit a block in folliculogenesis between primary and secondary follicles (Guo et al., 1998Go).

Data relating to follistatin, activin A and inhibin B concentrations in PCOS are scanty. In this study we show that circulating follistatin concentrations are increased and activin A concentrations decreased in subjects with PCOS while inhibin B values did not alter.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
We have previously described the group of patients with PCOS in whom we have measured their metabolic status. Patients were included in the PCOS group if they exhibited a raised concentration of serum testosterone or androstenedione together with a low concentration of sex hormone binding globulin. In addition, ultrasound demonstrated the presence of >=8 peripheral cysts <10 mm in diameter with increased stroma in at least one ovary (Norman et al., 1995bGo). Subjects with PCOS (n = 108) were identified by the presence of hyperandrogenaemia, polycystic ovaries on ultrasound and menstrual abnormalities with no recent ovulation as evidenced by progesterone concentrations. Controls (n = 20) were included if they had a regular cycle (>=22 and <33 days), did not have high serum androgens and had normal ovarian ultrasound morphology. Blood was obtained from women with PCOS who were at least 3 weeks from their last period and did not have an LH surge in the following 2 weeks. Blood was taken from controls between day 3 and day 5 of the cycle. Sera was stored at –70°C prior to assay. Follistatin (FS-288), activin A and inhibin B were measured by two-site enzyme-linked immunosorbent assay by D.M.R. in Clayton using reagents supplied by N.P.G. (Groome et al., 1996Go; Knight et al., 1996Go; Evans et al., 1998Go). Personal experience has shown these peptides to be stable for many years in the purified form. Repeated determinations of inhibin B in frozen serum samples shows no evidence of loss with time, and is in accordance with a previous study (Blaakaer et al., 1996Go). The detection limit for follistatin was 19 pg/ml and for activin A and inhibin B 10 pg/ml). Inter- and intra-assay coefficients of variation were <7%. All other immunoassays were performed as previously described (Norman et al., 1995aGo,bGo).

Statistical analysis was performed after assessing the distribution of the data. Parametric comparisons were performed using Student's t-test; non-parametric comparisons utilized the Mann-Whitney test and Spearman's test of correlation. P < 0.05 was considered significant.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Subjects with PCOS had slightly higher body mass index (BMI) values and lower age than controls (Table IGo). Fasting glucose and insulin concentrations were decreased in control women. As expected, concentrations of testosterone (PCOS 5.2 ± 0.3, controls 2.1 ± 0.2 nmol/l), LH (PCOS 11.9 ± 1.1, controls 9.2 ± 2.9 IU/l) and insulin (PCOS 15.8 ± 1.2, controls 10.4 ± 2.2 mIU/l) were higher in PCOS subjects. Results for follistatin, activin A and inhibin B are shown in Table IIGo: the concentrations of follistatin were statistically significantly higher (P < 0.05) in subjects with PCOS. Activin A concentrations were significantly lower (P < 0.05) in PCOS subjects and hence the ratio of activin A to follistatin was lower (P < 0.01) in PCOS. Inhibin B concentrations were not significantly different between groups. Correlations between activin A, follistatin, inhibin B and some of the steroids and glucose concentrations are shown in Table IIIGo. Inhibin B showed significant associations with LH, fasting insulin, fasting glucose, oestradiol and FSH in the PCOS group. There were no significant associations between follistatin, activin A and inhibin B in any group (Table IVGo). However, there was a significant association between activin A and BMI for both groups, even though the correlation between activin A and BMI for controls was negative (r = –0.51), whereas for PCOS subjects the correlation was positive (r = 0.26). For inhibin B there was a strong association with BMI for the PCOS group.


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Table I. Data associated with the biochemical and clinical presentation for the polycystic ovarian sydnrome (PCOS) and control subjects
 

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Table II. Mean, SEM and significance (Mann-Whitney test) of activin A (ng/ml), follistatin (ng/ml), inhibin B (ng/ml) and the activin A:follistatin ratio for the control (n = 20) and polycystic ovarian sydnrome (PCOS) (n = 108) subjects
 

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Table III. Spearman correlation coefficients between each of the proteins (follistatin activin A and inhibin B) and LH, FSH, oestradiol, progesterone, testosterone, free androgen index (FAI), fasting insulin and fasting glucose in controls and polycystic ovarian syndrome (PCOS) subjects
 

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Table IV. Significance (Spearman P value) of the association between activin A, follistatin and inhibin B with each other, and with age and body mass index (BMI), for the control and polycystic ovarian sydnrome (PCOS) subjects
 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
This investigation has shown that circulating concentrations of follistatin are higher, and of activin A are lower, in PCOS subjects compared to those with no evidence of this syndrome. While the exact contribution of the ovary to circulating concentrations of these proteins is not known, these data raise the possibility that alterations in secretion of follistatin and activin from the ovary or other organs may explain the change in circulating concentrations of the proteins. This follows the initial observation that PCOS is most closely linked to areas near the follistatin gene in genetic studies in a large cohort of PCOS siblings (Urbanek et al., 1999Go) although more recent data have shown the association to be much weaker than previously thought (Liao et al., 2000Go).

Several studies have examined the patterns of inhibin and activin in follicular fluid from subjects with PCOS. Erickson et al. were the first to report that there was no difference in the follicular fluid concentrations of follistatin between normal and PCOS follicles (Erickson et al., 1995Go). They also observed no difference between atretic and healthy follicles. Examination of follicular fluid from small ovaries (Magoffin and Jakimiuk, 1998Go) found no differences in the activin A and follistatin concentrations between controls and PCOS in size-matched follicles, whereas inhibin B concentrations were higher in dominant follicles from control ovaries. The advantage of both these studies was the use of unstimulated ovaries. Another study indicated that follicular aspirates from women undergoing IVF treatment had normal concentrations of follistatin although the rate of increase in follistatin concentrations from small to large follicles was less than expected in PCOS (Lambert-Messerlian et al., 1997Go). The implications of this observation are clouded by the fact that aspirates were obtained after ovarian stimulation and administration of human chorionic gonadotrophin. The origins of circulating follistatin and activin are unclear, especially as follistatin has been shown to remain constant across the menstrual cycle and is not altered by administration of GnRH antagonist which acutely lowers FSH and LH concentrations (Kettel et al., 1996Go).

Liao et al. recently produced preliminary results in follistatin gene mutations in PCOS (Liao et al., 2000Go). Although they did not find a mutation in the coding region of the Chinese population, there remains the possibly that there may be changes in follistatin in other ethnic populations or indeed changes in the regulation of the follistatin gene.

Inhibin B has been measured in follicular fluid and blood from women with PCOS. Two studies have shown higher concentrations in the circulation (Anderson et al., 1998Go; Lockwood et al., 1998Go), although their populations of no more than 10 subjects makes a comparison difficult. This higher concentration has not been confirmed in follicular fluid (Lambert-Messerlian et al., 1997Go; Magoffin and Jakimiuk, 1998Go). In our study using a much larger population size, we were not able to confirm any changes in the peripheral blood using the identical assays in the studies of Anderson or Lockwood (Anderson et al., 1998Go; Lockwood et al., 1998Go).

The current study implicates follistatin and activin in the pathophysiology of PCOS in that follistatin is higher and activin is lower in the circulation of women with this condition. The study has a large number of well-characterized patients with PCOS clearly distinguished from the control group. All control subjects had blood taken in the early follicular phase at a time that follicles are similar in size to those that characterize the PCO ovary. However, the PCOS subjects were heavier yet slightly younger than control women, raising the issue that comparisons may be affected by these parameters. It has been found that there was no relationship of activin A and age in women even though there was a transient rise at about the time of menopause (Loria et al., 1998Go). Inhibin B probably does not change with age until menopause is imminent (Burger et al., 1999Go) and changes cannot be explained on the basis of age. Multivariate analysis showed no relationship between age and any of the analytes measured. There is no significant association between follistatin and BMI; however, activin A shows a significant association with BMI which is negatively correlated in the control group and positively correlated in the PCOS women. It is well established that PCOS women are often overweight with an increase in their BMI. We have shown that the larger PCOS women have a positive correlation with activin A concentrations. This is in contrast to the control group whose activin A concentrations were negatively correlated with increasing BMI. The follistatin concentrations in the PCOS group were nearly twice as high as for the control group. It is postulated that the higher follistatin concentrations have a much greater effect on reducing the activity of activin A, thus reducing the FSH effect from the pituitary with a consequential effect on ovulation rate. This is the opposite effect to that of age, where activin A increases as women become older and the FSH concentration in blood increases (Reame et al., 1998Go).

Follicles in PCOS are arrested at 8–10 mm in diameter and a variety of intrafollicular regulators have been implicated, including epidermal growth factor, insulin-like growth factor (IGF) and TGFß (Hillier and Miro, 1993Go; Findlay, 1994Go; Hillier, 1999Go). Overexpression of follistatin could be one mechanism whereby follicular growth is impeded. The previous studies that failed to show any difference in follicular fluid concentrations of follistatin could be explained by thecal production of follistatin and the rich blood supply in this area of the ovary leading to rapid clearance into the blood. The lack of difference in inhibin B does not accord with two previous studies in this area (Lambert-Messerlian et al., 1997Go; Magoffin and Jakimiuk, 1998Go). More investigations are needed before the true situation with inhibin B is resolved.

The current study raises the possibility that follistatin from the ovary or other tissues could inhibit follicle development in women prone to PCOS. Given the information relating abnormalities in other growth factors, including IGF and insulin, follistatin and activin regulation may be areas for diagnosis and pharmacological intervention in this condition.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The studies were funded in part from the NHMRC of Australia (Program Grant 983212 DMR) and the Reproductive Medicine Unit, The University of Adelaide. The technical assistance of Enid Pruysers is gratefully acknowledged.


    Notes
 
4 To whom correspondence should be addressed at: Reproductive Medicine Unit, the Queen Elizabeth Hospital, University of Adelaide, 28 Woodville Road, Woodville, South Australia 5011, Australia. E-mail: robert.norman{at}adelaide.edu.au Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
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Submitted on September 13, 2000; accepted on January 4, 2001.