Ovarian steroidogenic response to human chorionic gonadotrophin in obese women with polycystic ovary syndrome: effect of metformin

Riitta M. Koivunen1, Laure C. Morin-Papunen1, Aimo Ruokonen2, Juha S. Tapanainen1 and Hannu K. Martikainen1,3

1 Department of Obstetrics and Gynaecology and 2 Department of Clinical Chemistry, University Hospital of Oulu, Kajaanintie 52A, FIN-90220 Oulu, Finland


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
BACKGROUND: The aim of the present study was to investigate the steroidogenic response pattern to HCG in obese women with polycystic ovary syndrome (PCOS) and the possible effects of metformin treatment on it. METHODS: A single injection of human chorionic gonadotrophin (HCG, 5000 IU) was given to 12 obese [body mass index (BMI) 27 kg/m2] women with PCOS and to 27 control women. Blood samples for assays of 17{alpha}-hydroxyprogesterone (17-OHP), androstenedione, testosterone and oestradiol were collected at baseline and 1, 2 and 4 days after the injection. Responses to HCG were also assessed in the PCOS women after 2-month treatment with metformin (500 mgx3 daily). RESULTS: Serum 17-OHP and oestradiol concentrations peaked at 24 h in the PCOS women and preceded the maximum testosterone concentration, which was seen at 48 h. In the control women the maximum concentrations of all these steroids were reached 96 h after HCG. After metformin treatment, the basal serum testosterone concentration and the areas under the androstenedione (AUCA) and testosterone (AUCT) response curves after HCG decreased significantly. CONCLUSIONS: The results demonstrate that obese PCOS women have a male-type steroidogenic response pattern to a single injection of HCG and a higher androgen secretory capacity than control women, which may be explained by the increased thecal cell activity in the polycystic ovary. The slight alleviation of hyperandrogenism brought about by metformin therapy appears to be due to its effect on ovarian steroidogenesis possibly mediated by decreased insulin action.

Key words: human chorionic gonadotrophin/metformin/polycystic ovary syndrome/steroidogenesis


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Polycystic ovary syndrome (PCOS), characterized by anovulation and hyperandrogenism, is the most common endocrinopathy in women of reproductive age (Franks, 1995Go). Insulin resistance and hyperinsulinaemia have been shown to play a central role in the pathogenesis of PCOS by acting on ovarian and adrenal steroidogenesis (Burghen et al., 1980Go; Chang et al., 1983Go; Dunaif and Graf, 1989Go; Nestler et al., 1989Go; Mesiano et al., 1997Go), and on sex hormone-binding globulin (SHBG) synthesis (Plymate et al., 1988Go; Nestler et al., 1989Go).

The mechanisms leading to increased androgen production in PCOS are not completely understood. Luteinizing hormone (LH) is known to induce androgen production in theca cells, augmented by insulin and insulin-like growth factors (IGFs) (Barbieri et al., 1984Go; Poretsky and Kalin, 1987Go). Women with PCOS have been shown to exhibit 17{alpha}-hydroxyprogesterone (17-OHP) hyperresponsiveness to gonadotrophin-releasing hormone (GnRH) agonists and human chorionic gonadotrophin (HCG), which has been interpreted as an indication of cytochrome P450c17{alpha} overactivity (Barnes and Rosenfield, 1989Go; Ehrmann et al., 1992Go; Rosenfield et al., 1994Go; Ibanez et al., 1996Go; Gilling-Smith et al., 1997Go; Levrant et al., 1997Go). In addition, in theca cell culture studies, accumulation of androstenedione and 17-OHP has been shown to be higher in polycystic ovaries than in normal ovaries, suggesting increased activity of 17{alpha}-hydroxylase and 17,20-lyase as an intrinsic feature of PCOS theca cells (Gilling-Smith et al., 1994Go). Recently, it has been reported that basal and cAMP-dependent CYP17 gene transcription is increased in PCOS theca cells (Wickenheisser et al., 2000Go).

Metformin is a biguanide antihyperglycaemic drug used in the treatment of type 2 diabetes mellitus. We and other groups (Nestler and Jakubowicz, 1996Go; Velazquez et al., 1997Go; Morin-Papunen et al., 1998aGo), but not all (Crave et al., 1995Go; Ehrmann et al., 1997Go), have shown that insulin-sensitizing therapy with metformin results in a reduction of hyperinsulinaemia and hyperandrogenism in obese and non-obese women with PCOS.

The aim of the present study was to investigate the steroidogenic response pattern to HCG in obese PCOS women and the possible effects of metformin treatment on it. The testicular steroidogenic response to HCG in men is well characterized. In women, however, the follow-up time after HCG administration has been short (24–48 h) (Barnes and Rosenfield, 1989Go; Ehrmann et al., 1992Go; Rosenfield et al., 1994Go; Ibanez et al., 1996Go; Gilling-Smith et al., 1997Go; Levrant et al., 1997Go). In this study the steroidogenic response to a single injection of HCG was followed up for 96 h in control and PCOS women.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Subjects
27 control women volunteers [body mass index (BMI), 23.0 ± 3.0 kg/m2], contacted through an advertisement in Oulu University Hospital, and 12 obese women with PCOS (BMI, 33.1 ± 5.3 kg/m2, P = 0.01) underwent an HCG test. The mean (± SD) age of the control women was 31.5 ± 6.0 years (range 22–38) and that of the PCOS women was 29.2 ± 4.8 years (range 18–34, P = 0.05). All of the control women had regular cycles and normally appearing ovaries in transvaginal ultrasonography. All of the PCOS women had oligomenorrhoea (intermenstrual interval >36 days) or amenorrhoea (intermenstrual interval >6 months) and hyperandrogenism (serum testosterone >=2.7 nmol/l and/or hirsutism score >7) (Ferriman and Gallwey, 1961Go), and all of them had polycystic ovaries observed in transvaginal ultrasonography (at least 10 subcapsular follicles of 3–8 mm diameter in one plane in one ovary, and increased stroma) (Adams et al., 1986Go).

All of the PCOS women were recruited from our Reproductive Endocrinology Unit and in all cases serum concentrations of FSH (4.2 ± 2.0 IU/l) and LH (7.1 ± 2.1 IU/l) were assessed in the early follicular phase after spontaneous or progestin-induced bleeding, and an oral glucose tolerance test (OGTT) was performed 1–7 months before this study. All of these women had normal fasting glucose and two of the women had impaired glucose tolerance (IGT) (Anonymous, 1997Go). Diabetics, smokers, alcohol users and those using sex hormones or other medication known to affect lipoprotein metabolism during the 2 months preceding the study were excluded. Late onset adrenal hyperplasia in PCOS subjects was excluded on the basis of a normal serum 17-hydroxyprogesterone concentration (17-OHP <9 nmol/l).

HCG test
HCG stimulation was performed by giving a single i.m. injection of HCG (5000 IU Pregnyl®, Organon, Oss, The Netherlands) on cycle day 1–4 after spontaneous or, in the presence of amenorrhoea, progestin-induced menstruation. Blood samples from all subjects for assay of serum 17-OHP, androstenedione, testosterone and oestradiol were collected before, and 1, 2 and 4 days after the injection.

All blood samples were collected at 0700–1000 h after the subject had been at rest in a sitting position for 20 minutes.

Metformin treatment
All of the PCOS women (n = 12) were given metformin (metformin hydrochloride, Diformin®, Leiras, Finland, 500 mgx3 daily) for two months. Blood samples for serum testosterone, androstenedione, oestradiol and 17-OHP assays were collected at baseline and 1, 2 and 4 days after HCG injection, given before and after the treatment. Blood samples for insulin, leptin and sex hormone-binding globulin (SHBG) analyses were obtained at baseline, before and after metformin treatment.

Transvaginal ultrasonography (General Electric RT-x200, Milwaukee, Wisconsin, with a 6.5 MHz probe) was performed to measure ovarian volumes and the number of follicles. Volumes were determined by using the formula for the volume of an ellipsoid: 0.523xlengthxwidthxthickness (Robert et al., 1995Go).

The studies were approved by the Ethics Committee of the University of Oulu, Finland, and informed written consent was obtained from each subject.

Assays
Serum concentrations of SHBG were analysed by fluoroimmunoassay (Wallac Ltd, Turku, Finland), and commercial radioimmunoassays were used for the analysis of serum androstenedione, 17-OHP (Diagnostic Products Corporation, Los Angeles, CA, USA), oestradiol, insulin (Pharmacia Diagnostics, Uppsala, Sweden) and leptin (Linco Research, Inc., St Charles, MO, USA), following the instructions of the manufacturers. Serum testosterone concentrations were determined by using an automated chemiluminescence system (Ciba-Corning ACS-180, Medfield, MA, USA). Serum glucose was determined by the enzymatic reference method with hexokinase using Cobas Integra 700 automatic analyzer (Hoffmann-La Roche, Basel, Switzerland).

The free androgen index (FAI) was calculated according to the equation testosterone (nmol/l)x100/SHBG (nmol/l). Areas under the curve (AUCs) for 17-OHP, androstenedione, testosterone and oestradiol were calculated by the trapezoidal method. The fasting glucose to insulin ratio was calculated to assess insulin resistance. We used a threshold value <0.250 mmol/mU (<4.5 mg/µU), which has been shown to provide the best combination of sensitivity and specificity as well as the best positive and negative predictive values as a screening test for predicting insulin resistance in PCOS (Legro et al, 1998Go). We used also another formula to describe insulin resistance i.e. the homeostasis model assessment (HOMA): insulin resistance = (fasting glucosexfasting insulin)/22.5 (Matthews et al, 1985Go; Duncan et al, 1995Go).

Internal quality control of the hormone determinations was carried out in each analytical run by including three quality control samples representing low, medium and high serum concentrations of the hormones. External quality control of the hormone assays was organized by national (Labquality Ltd, Helsinki, Finland) and international (Murex Biotech Ltd, Dartford, UK) companies. The intra- and inter-assay coefficients of variation were 1.3 and 5.1% for SHBG respectively, 5.0 and 8.6% for andostenedione, 5.0 and 5.4% for 17-OHP, 4.0 and 5.6% for testosterone, 5.8 and 6.1% for oestradiol, 1.4 and 2.4% for glucose, 5.3 and 7.6% for insulin and 5.0 and 6.0% for leptin.

Statistical analysis
Student's t-test was used for differences between controls and PCOS women, if the variables were normally distributed. The Mann–Whitney U-test was used for variables showing a skewed distribution after logarithmic transformation. The limit of significance was set at P < 0.05.

In PCOS women paired t-tests were used for comparison of normally distributed variables, with or without log transformation, before and after metformin treatment. A paired Wilcoxon nonparametric rank sum test was used for variables with a persisting skewed distribution after log transformation. P-values were two-tailed, with 0.05 considered as the limit of statistical significance.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Controls and subjects with PCOS
The basal serum androstenedione (P = 0.01) and testosterone (P = 0.003) concentrations were higher in PCOS women than in the control women, while the serum 17-OHP and oestradiol concentrations did not differ significantly (Figure 1Go).




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Figure 1. (a) Serum 17-OHP, (b) androstenedione, (c) testosterone and (d) oestradiol responses (mean ± SEM) to a single injection of HCG (5000 IU) in control women (squares, n = 27) and in PCOS women (n = 12) before (circles) and after (triangles) 2 months of metformin treatment. Areas under the curve (AUC) for androstenedione (AUCA) and testosterone (AUCT) were compared in PCOS women before and after metformin treatment. *P < 0.05 compared with PCOS women after metformin treatment. **P <= 0.01 compared with PCOS women before metformin treatment.

 
A peak in the serum 17-OHP and oestradiol concentrations was seen in PCOS women at 24 h after HCG injection (Figure 1Go). Serum testosterone concentrations were constantly significantly higher in PCOS women than in controls and reached a maximum level at 48 h. In the control women the maximum concentrations of all steroids measured were reached at 96 h (Figure 1Go).

In the control group, obese control women (BMI >=27 kg/m2, n = 5) showed similar responses to HCG as did lean control women (data not shown).

Metformin study
Serum basal testosterone (P = 0.05), insulin (P = 0.04) and leptin (P = 0.03) levels were decreased significantly after 2 months of metformin treatment. There were no changes in the 17-OHP and oestradiol response profiles or in the androstenedione/17-OHP ratio (2.7 ± 0.9 [SD] versus 2.4 ± 1.0; NS) after the treatment (Table IGo, Figure 1Go).


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Table I. Clinical and hormonal parameters (mean, 95% confidence intervals in parentheses) in obese PCOS women (n = 12) before and after the 2 months treatment with metformin
 
Serum 48 h and 96 h testosterone, and 48 h androstenedione concentrations decreased significantly after 2 months of metformin treatment (P < 0.05). Similarly, the AUC for androstenedione (AUCA) (894.4 ± 350.6 [SD] versus 738.4 ± 276.4 nmol/lxh, P = 0.05) and the AUC for testosterone (AUCT) (152.5 ± 44.9 [SD] versus 114.1 ± 43.7 nmol/lxh, P = 0.04) decreased significantly after 2 months of treatment. No significant change was observed in the AUC's for 17-OHP (AUC17-OHP) or oestradiol (AUCE2) (Figure 1Go).

Before the treatment the fasting glucose to insulin ratio was under the threshold value of 0.250 mmol/mU (<4.5 mg/µU) indicating insulin resistance and it tended to improve during the treatment (NS, Table IGo). HOMA decreased significantly during the treatment indicating improved insulin sensitivity (P = 0.01, Table 1Go).


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Obese women with PCOS displayed a distinctly different steroidogenic response pattern to a single dose of HCG compared with the control women followed up for 4 days. Peak peripheral serum testosterone concentrations were reached in the PCOS women at 48 h after HCG injection, preceded by peak levels of 17-OHP and oestradiol at 24 h. In contrast, in the control women all steroids measured reached their maximum serum concentrations at 96 h.

The rapid 17-OHP and oestradiol responses to HCG in PCOS women are in accordance with the results of previous studies in which short-term responses (up to 24–48 h) to HCG (Ibanez et al., 1996Go; Gilling-Smith et al., 1997Go; Levrant et al., 1997Go) or to GnRH (Ehrmann et al., 1992Go; Rosenfield et al., 1994Go; Ibanez et al., 1996Go) have been studied. These findings have been interpreted to imply that `dysregulation' of the enzyme P450c17{alpha}, leading to enhanced activities of both 17{alpha}-hydroxylase and 17,20-lyase, plays a central role in the pathogenesis of ovarian hyperandrogenism associated with PCOS. The male-type steroidogenic response pattern to HCG seems to be associated with PCOS and possibly not with obesity, since in controls the response pattern was not related to BMI. Higher number of follicles and theca cells in PCOS women may have an effect on the 17-OHP and oestradiol responses to HCG. Whether the response to HCG is dependent on age, because the number of follicles is known to decline with age (Faddy et al., 1992Go), remains to be studied.

In this study the steroid responses to HCG were followed for a longer time than in the previous studies, and it was shown that the levels of 17-OHP and oestradiol began to decline at 48 h, in contrast to the control women, who showed peak 17-OHP and oestradiol values at 96 h after HCG. The steroid response patterns observed in the obese PCOS women were identical to those found in normal men (Smals et al., 1979Go; Martikainen et al., 1980Go). It has been suggested that oestradiol inhibits 17-lyase activity, leading to an accumulation of 17-OHP, possibly preventing its further conversion to testosterone in the human testis (Forest et al., 1979Go; Martikainen et al., 1980Go; Tapanainen et al., 1983Go). Although our observations on peripheral serum steroid concentrations do not necessarily reflect cellular events, it is tempting to speculate that a similar regulatory mechanism may be operative in the polycystic ovary, with increased theca cell activity. Furthermore, the time course of 17-OHP and oestradiol responses suggests that oestradiol, in contrast to a previous suggestion (Ibanez et al., 1996Go), may regulate 17,20-lyase activity in the human ovary.

The observed significant and rapid increase in oestradiol after HCG in obese PCOS women is not readily explained by the two-cell model of ovarian steroidogenesis. On the basis of the present data several mechanisms may be proposed. Firstly, there is a considerable amount of data indicating that human theca cells are capable of forming oestradiol throughout the life span of the antral follicle (McNatty et al., 1979Go; Gilling-Smith et al., 1994Go). Thus, the rapid release of oestradiol from polycystic ovaries after HCG-stimulation observed in this study may reflect the existence of a releasable pool of steroids in the theca cells. Secondly, androgens might exert a rapid paracrine effect on granulosa cells by up-regulating their aromatase activity (Haning et al., 1993Go). The response of oestradiol to HCG may also be due to direct activation of granulosa cells, since it has been shown that granulosa cells from small individual follicles obtained from anovulatory polycystic ovaries are prematurely responsive to LH (Willis et al., 1998Go). Endogenous FSH present in the serum may potentiate LH action by inducing LH-receptors (Knecht et al., 1986Go). The higher serum levels of 17-OHP and oestradiol at 4 days after HCG in the ovulatory control women compared with PCOS subjects may result from normal follicular development and increased steroid secretion by the dominant follicle.

The improvement of hyperandrogenism observed during metformin treatment has been considered to be the result of a decrease in serum insulin concentrations (Velazquez et al., 1994Go; Nestler and Jakubowicz, 1996Go; Nestler, 1997Go; Diamanti-Kandarakis et al., 1998Go; Morin-Papunen et al., 1998aGo), although not all studies support this concept (Crave et al., 1995Go; Acbay and Gundogdu, 1996Go; Ehrmann et al., 1997Go). In this study fasting serum insulin concentrations were decreased and insulin sensitivity improved during metformin treatment in obese and insulin resistant PCOS women suggesting that the slight alleviation of hyperandrogenism brought about by metformin may be mediated by decreased insulin action.

In the present study, we observed a significant decrease in basal serum testosterone and a slight but non-significant decrease in basal serum androstenedione concentrations during metformin treatment. In addition, we observed significant decreases in AUCT and AUCA after HCG during metformin treatment. Given that significant decreases in GnRH-agonist- and HCG-stimulated 17-OHP responses have been observed during metformin treatment, it has been suggested that improvement of hyperinsulinaemia by metformin treatment could reduce ovarian cytochrome P450c17{alpha} activity (Nestler and Jakubowicz, 1996Go, 1997Go; la Marca et al., 2000Go). In the present study the response of 17-OHP to the HCG challenge test was not significantly decreased by metformin, although insulin levels decreased. In contrast to the study of la Marca et al. the first sample after HCG in the present study was collected at 24 h, and therefore the possible differences before this could not be detected (la Marca et al.., 2000Go). However, in the study of la Marca et al. a significant decrease after metformin treatment was demonstrated also 24 h after the HCG, which is not in accordance with our results. Our data suggest that if the increased response of 17-OHP to HCG in women with PCOS is partly due to hyperactivity of P450c17{alpha} (Rosenfield et al., 1994Go; Levrant et al., 1997Go), short-term metformin treatment may not improve hyperandrogenism by affecting this step of androgen biosynthesis.

A significant decrease of serum leptin concentration, in parallel with serum testosterone changes, was observed during metformin treatment. This is in accordance with previous studies in which serum leptin decreased after 2–6 months of metformin treatment (Morin-Papunen et al., 1998bGo, 2000Go; Pasquali et al., 2000Go). Since mRNA for leptin receptors has been found in both ovarian granulosa and theca cells (Agarwal et al., 1997), leptin may directly regulate ovarian function. Insulin has been shown to stimulate leptin production in vitro (Kennedy et al., 1997Go) and in vivo (Kolaczynski et al., 1996Go). Thus, the improvement of hyperinsulinemia in our study may be related to the decrease of serum leptin concentrations.

The present results, as well as those of our previous studies (Morin-Papunen et al., 1998aGo, 2000Go), showed that the serum SHBG concentration and the FAI did not change significantly during 2–6 months of metformin treatment. This is in contrast to some previous studies where increased serum SHBG concentrations during metformin treatment have been observed (la Marca et al., 1999Go, 2000Go; Unluhizarci et al., 1999Go).

In conclusion, this study indicates that obese PCOS women have a male-type steroidogenic response pattern to a single dose of HCG. The differences observed in steroid responses between normal and PCOS women might be explained by higher theca cell activity or mass in polycystic ovaries. The present results suggest that the slight alleviation of hyperandrogenism brought about by metformin therapy may be explained by decreased ovarian steroidogenesis.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
This work was supported by grants from the Sigrid Jusenius Foundation and the Academy of Finland.


    Notes
 
3 To whom correspondence should be addressed. E-mail: hmartika{at}cc.oulu.fi Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
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Submitted on May 11, 2001; accepted on September 6, 2001.