1 Departments of Obstetrics and Gynecology and 3 Clinical Chemistry, Oulu University Hospital, Oulu and 2 Departments of Obstetrics and Gynecology and Physiology, Turku University Hospital, Turku, Finland
4 To whom correspondence should be addressed at: Department of Obstetrics and Gynecology, Oulu University Hospital, PO Box 5000, FIN-90014 Oulu, Finland. Email: juha.tapanainen{at}oulu.fi
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Abstract |
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Key words: anti-Müllerian hormone/follicle pool/metformin/ovarian ageing/PCOS
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Introduction |
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Polycystic ovary syndrome (PCOS) is the most common endocrinological disorder in women of fertile age. It is characterized by menstrual irregularity, androgen excess, polycystic ovaries (PCO) and disturbances in glucose metabolism. Since women with PCOS are known to have an excessive amount of small antral follicles in the ovaries and at the same time increased serum AMH levels (Pigny et al., 2003; Laven et al., 2004
; Mulders et al., 2004
), it is possible that AMH may indeed play a role in PCOS, being one of the factors that cause/reflect functional or morphological features typical of the syndrome.
Insulin-lowering agents, such as metformin, have been shown to improve hyperinsulinaemia, hyperandrogenism, menstrual pattern and ovulatory function in women with PCOS (Velazquez et al., 1994; Nestler and Jakubowicz, 1997
; Morin-Papunen et al., 1998
; Elter et al., 2002
), although the exact mechanism of action of this drug remains controversial. Up to now, there are only few data on the possible effects of metformin on ovarian morphology (ovarian follicle number and volume) (De Leo et al., 1999
; Stadtmauer et al., 2001
; Elter et al., 2002
) and no data on its potential effect on serum AMH levels in women with PCOS. Thus, it was of interest to study whether the improvement of hyperandrogenism induced by metformin treatment (Velazquez et al., 1994
; Morin-Papunen et al., 2003
) would be associated with changes in serum AMH levels and ovarian morphology in women with PCOS. Furthermore, our recent studies have shown that ovarian steroid production capacity differs significantly between healthy subjects and women with PCOS (Piltonen et al., 2003
, 2004
). The pronounced androgen production in PCOS lasts the whole of a woman's reproductive life, while in healthy women ovarian androgen secretion capacity decreases after the age of 30 years. This difference may partly be a result of a higher number of antral follicles in women with PCOS. Since serum AMH concentrations have been shown to correlate with the number of remaining follicles, it was also of interest to analyse serum AMH levels in relation to ovarian ageing and endocrine function in women with PCOS.
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Patients and methods |
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Fasting blood samples for basal serum AMH, LH, FSH, inhibin B, androstenedione, testosterone and estradiol (E2) assays were taken on days 15 of the menstrual cycle or on days 15 of progestin-induced menstrual bleeding. In healthy women, serum progesterone levels were 7.1 nmol/l, confirming the follicular phase of the menstrual cycle.
Twenty-six subjects with PCOS (aged 2041 years, BMI 1841 kg/m2) were treated with metformin. Fourteen women were treated with metformin (metformin hydrochloride, Diformin; Bristol-Myers Squibb Co., Leiras, Finland) at 1500 mg/day for 6 months (Morin-Papunen et al., 1998). The other 12 women were treated with metformin at 1000 mg/day for 3 months and thereafter the dose was doubled to 2000 mg/day for the next 3 months (Morin-Papunen et al., 2000
, 2003
).
Serum AMH concentrations were determined by enzyme immunoassay (Immunotech-Beckman Coulter, Marseille, France). Serum concentrations of testosterone and progesterone were analysed by using an automated chemiluminescence system (Advia Centaur, Bayer Corporation, New York). Inhibin B concentrations were analysed by enzyme-linked immuosorbent assay (ELISA) (Serotec Ltd., Oxford, UK). Serum concentrations of FSH and LH were analysed by fluoroimmunoassays (Perkin Elmer, Wallac Ltd., Turku, Finland) and radioimmunoassays were used for androstenedione (Diagnostic Products Corporation, Los Angeles, CA) and E2 (Orion Diagnostica, Oulunsalo, Finland), following the instructions of the manufacturers. The intra- and interassay coefficients of variation were 5.1 and 6.6%, respectively, for AMH, 3.8 and 4.3% for FSH, 4.9 and 6.5% for LH, 5.2 and 6.4% for inhibin B, 5.0 and 8.6% for androstenedione, 4.0 and 5.6% for testosterone, 5.7 and 6.4% for E2 and 3.7 and 5.4% for progesterone.
Data were analysed by means of the Statistical Program for Social Science (SPSS Inc., Chicago, IL). To compare basal serum hormone levels between control women and women with PCOS, and inside the PCOS group when setting the age divisions at 25, 30 and 35 years, the independent samples t-test was used for normally distributed variables and the MannWhitney test for variables with skewed distribution. Pearson's (normal distributions) and Spearman's (skewed distributions) correlation coefficients (r) were calculated to correlate age, follicle number, BMI, testosterone, androstenedione, E2, inhibin B, LH and FSH data with serum AMH levels. Multiple linear regression analysis and analysis of variance (ANOVA) were used to adjust for the impact of BMI. Paired samples t-test (normally distributed variables) and Wilcoxon test (variables with skewed distribution) were used to compare the changes in serum AMH levels, ovarian follicle count and ovarian volume before and after 6 months of metformin treatment. The limit of statistical significance was set at P0.05.
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Results |
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Discussion |
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There are several factors that have been reported to be associated with AMH secretion. For example, FSH has been shown to decrease the expression of AMH and its type II receptors in granulosa cells (Baarends et al., 1995), and a negative correlation between FSH and AMH levels has been observed in several studies (Seifer et al., 2002
; van Rooij et al., 2002
; Fanchin et al., 2003
; Pigny et al., 2003
). A negative association between serum AMH and FSH was also observed in the present study in healthy women but not in women with PCOS. The difference between healthy women and those with PCOS can be explained by unchanged serum FSH levels until late reproductive age (Piltonen et al., 2004
) and high although variable AMH levels in women with PCOS. In contrast to the study by Laven et al. (2004)
, no correlation was observed between serum LH and AMH levels. This difference could be a result of different age distributions in the studies. In our study, the age range was wider, and especially in women close to 40 years of age the individual variation of both LH and AMH levels was larger. Furthermore, it is also possible that the induction of menstrual bleeding by progestin in some subjects could have affected the results since it has been shown to decrease follicular phase LH concentrations (Anttila et al., 1992
), which may have weakened the correlation of LH and AMH.
Since pronounced androgen secretion is one of the typical features of PCOS, it was interesting to study whether AMH levels, in addition to their correlation to follicular count, also correlate to serum androgen levels, thus reflecting the degree of hyperandrogenism. Serum AMH concentration correlated significantly with serum androstenedione and testosterone levels in women with PCOS, and a similar correlation was observed between AMH and androstenedione concentrations in control subjects. These findings strengthen the results of previous studies (Pigny et al., 2003; Laven et al., 2004
) and confirm the importance of small ovarian follicles in the production of both AMH and androgens.
Normal (Pigny et al., 2003) or decreased (Cook et al., 2002
) serum E2 levels and a negative correlation between AMH and E2 concentrations have been shown in women with PCOS. The relationship between E2 and increased AMH levels could be explained by an inhibitory effect of AMH on aromatase activity (Vigier et al., 1989
; Rouiller-Fabre et al., 1998
) that could cause the well-known increase in androgen levels and unchanged/decreased levels of E2 in PCOS subjects, as suggested earlier (Laven et al., 2004
). The present and previous (Pigny et al., 2003
) results do not, however, support this concept since no correlation between AMH and E2 concentrations was observed. It is possible, however, that aromatase activity is not maximal at early follicular phase when the measurements were performed, and this could have affected the results.
There was a significant age-related decrease in AMH levels in both control subjects and those with PCOS, although serum AMH levels were always higher in women with PCOS. In fact, serum AMH levels became undetectable in the majority of control women after the age of 38 years, whereas in women with PCOS markedly increased serum AMH levels could still be observed. After dividing the healthy women into two groups, setting the age limit at 25 years, serum AMH levels were significantly lower in the older women, indicating that the decline in AMH secretion begins early. The age-related decrease in AMH levels in control women is also supported by the results of previous studies in which a negative correlation between age and serum AMH levels has been reported (de Vet et al., 2002; Fanchin et al., 2003
; Mulders et al., 2004
), although this has not been the case in all studies (Pigny et al., 2003
). In a previous study, we reported an age-related rise in FSH concentrations in control women which was already evident after 25 years (Piltonen et al., 2003
), as was the decline in AMH levels in the present study. It is possible that AMH as a direct marker of ovarian ageing will turn out to be a better marker than FSH, which reflects the changes in the ovarian environment via feedback systems. Inhibin B, another direct marker of ovarian function, has also been used to predict menopausal transition. In our previous studies, no significant correlation between age and inhibin B levels was observed in either healthy women or those with PCOS (Piltonen et al., 2003
, 2004
), and nor did inhibin B concentrations correlate with those of AMH in the present study.
Earlier studies have shown that the prevalence of PCO decreases with age (Koivunen et al., 1999; Bili et al., 2001
). Furthermore, the women with PCOS have been shown to gain regular menstrual cycles when ageing, probably due to age-related follicle loss in the ovaries (Elting et al., 2000
). In accordance with this, we observed a decreasing tendency in follicle number with age in PCOS subjects which became significant after adjustment for BMI. The relatively high serum levels of AMH in women with PCOS in the oldest age-group (>35 years), however, indicate that the follicles and thereby fertility may be preserved for longer in women with PCOS. Whether this affects menopausal transition and whether these women enter the menopause later remains open.
Although the positive effect of metformin on menstrual pattern, ovalutory function and hyperandrogenism in women with PCOS is widely documented (Velazquez et al., 1994; Nestler and Jakubowicz, 1997
; Morin-Papunen et al., 1998
; Elter et al., 2002
), studies concerning its effect on ovarian morphology/ultrasonographic appearance are few (Elter et al., 2002
), and no data on serum AMH levels during metformin treatment have existed. The present results show for the first time that serum AMH levels decrease during metformin treatment. It is most likely that the decrease is simply related to the decrease of follicle number, but the contribution of the improvement of hyperandrogenism, insulin action or menstrual pattern cannot be excluded. Whether subjects showing lowered serum AMH levels during metformin treatment have special features remains to be studied in a larger number of subjects. Since the ovarian appearance, i.e. the number of ovarian follicles/volume, is not likely to change in a short period of time, a longer follow-up may be needed to observe even more profound changes in serum AMH levels than in the present study.
In conclusion, serum levels of AMH decreased with age both in women with PCOS and in control subjects, and became undetectable in most of the control women before the age of 38 years. In women with PCOS, AMH levels remained 2- to 3-fold higher throughout the reproductive years but decreased during metformin treatment, as did the follicle number and ovarian volume. This may indicate that metformin has beneficial effects on follicle growth in women with PCOS. The measurement of serum AMH could be a useful tool for estimating follicle number as well as for diagnosing PCO/PCOS and for assessing ovarian ageing.
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Acknowledgements |
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Submitted on November 2, 2004; resubmitted on January 10, 2005; accepted on February 18, 2005.