Gynecological Endocrinology, August 2011; 27(8): 587–592
The impact of oral contraceptives and metformin on anti-Mu¨llerian hormone serum levels in women with polycystic ovary syndrome and biochemical hyperandrogenemia DIMITRIOS PANIDIS1, NEOKLIS A. GEORGOPOULOS2,3, ATHANASIA PIOUKA1, ILIAS KATSIKIS1, ALEXANDROS D. SALTAMAVROS2,3, GEORGE DECAVALAS3, & EVANTHIA DIAMANTI-KANDARAKIS4
Gynecol Endocrinol Downloaded from informahealthcare.com by Hacettepe Univ. on 01/27/12 For personal use only.
Division of Endocrinology and Human Reproduction, Second Department of Obstetrics and Gynaecology, Aristotle University of Thessaloniki, Thessaloniki, Greece, 2Division of Reproductive Endocrinology, 3Department of Obstetrics and Gynaecology, University of Patras Medical School, Patras, Greece, and 4Division of Endocrinology, First Department of Medicine, Laiko Hospital, Medical School, University of Athens, Athens, Greece (Received 17 February 2010; revised 25 June 2010; accepted 5 July 2010) Abstract Objective. To assess the impact of metformin and of two different oral contraceptives (OCs) containing cyproterone acetate and drospirenone, on serum anti-Mu¨llerian hormone (AMH) levels, in a cohort of women with polycystic ovary syndrome (PCOS) with hyperandrogenism. Design. Prospective randomised study. Setting. Division of Endocrinology and Human Reproduction, Aristotle University of Thessaloniki. Patients. Forty-five (45) women with PCOS diagnosed according to the criteria proposed in 1990 by the NIH. Interventions. Women with PCOS were randomised into three groups, all treated for 6 months: Group A received an OC containing 35 mg ethinylestradiol plus 2 mg cyproterone acetate, Group B received an OC containing 30 mg ethinylestradiol plus 3 mg drospirenone and Group C received metformin 850 mg 6 2. Main outcome measure(s). Anti-Mu¨llerian hormone levels were measured by a specific ELISA. Results. AMH was significantly decreased under treatment with 35 mg ethinylestradiol plus 2 mg cyproterone acetate (p ¼ 0.002 at 3 months and p 5 0.001 at 6 months). Treatment with 30 mg ethinylestradiol plus 3 mg drospirenone, and treatment with metformin 850 mg 6 2 did not significantly affect serum AMH levels. AMH was significantly decreased under OCs treatment compared to metformin 850 mg 6 2 (p ¼ 0.005). Conclusion(s). AMH serum levels were significantly decreased under treatment with 35 mg ethinylestradiol plus 2 mg cyproterone acetate, due to decrease in androgens and suppression of gonadotropins. Keywords: Polycystic ovary syndrome, hyperandrogenism, anti-Mu¨llerian Hormone, oral contraceptives, metformin
Introduction Polycystic ovary syndrome (PCOS) is the most common endocrinopathy of reproductive age women  and is the leading cause of anovulatory infertility in women . PCOS is characterized by hyperandrogenism (hirsutism and/or biochemical hyperandrogenemia) and oligo/anovulation, and is also highly associated with obesity and insulin resistance (IR) . Oligo/anovulation in PCOS is, apparently, due to an excessive early follicular growth and a subsequent follicular arrest as the selection of a follicle from the increased pool of growing/selectable follicles and its further maturation to a dominant follicle does not occur . Anti-Mu¨llerian hormone (AMH) is a member of the transforming growth factor-b (TGF-b) superfamily of glycoproteins that has been found to play an important role in chronic anovulation by inhibiting the initial recruitment of primordial follicles  and by promoting follicular arrest . Indeed, we  and others [8–10] have found increased
AMH levels in both serum [7–10] and follicular fluid  of women with PCOS. Women with PCOS are usually treated with an oral contraceptive (OC), while both lean and obese PCOS with IR might benefit from treatment with metformin. Treatment with OCs is known to normalise menstrual function and to ameliorate hirsutism and acne, while the effects on IR are controversial . On the opposite, treatment with metformin is beneficial for weight and IR reduction, still the effect on menstrual cycle and hyperandrogenism is rather weak. Few data are available on the impact of these treatment modalities on serum AMH levels in women with PCOS. The use of OCs was shown not to affect serum AMH levels both in normal women , and in women with PCOS , while metformin treatment was shown to decrease AMH serum levels . The aim of the present study was to assess the impact of metformin and of two different regimens of OCs containing different progestins, namely cyproterone acetate and drospirenone, on serum AMH levels in a well characterised cohort PCOS women with hyperandrogenenemia recruited
Correspondence: Dimitrios Panidis, 119, Mitropoleos Str., 54622, Thessaloniki, Greece. E-mail: email@example.com ISSN 0951-3590 print/ISSN 1473-0766 online ª 2011 Informa UK, Ltd. DOI: 10.3109/09513590.2010.507283
D. Panidis et al.
on the basis of the stricter criteria proposed in 1990 by the National Institute of Child Health and Human Development Conference on PCOS .
Materials and methods
Gynecol Endocrinol Downloaded from informahealthcare.com by Hacettepe Univ. on 01/27/12 For personal use only.
Subjects The study included forty-five (45) women with PCOS, mean age 21.07 + 3.21 years and mean BMI 21.52 + 1.01 kg/m2, which were recruited from the outpatient endocrine clinic. Only normal weight (BMI 5 25 kg/m2) were included in the study. Diagnosis of PCOS was based on the presence of: chronic anovulation (fewer than six spontaneous bleeding episodes per year), and biochemical hyperandrogenemia in accordance with the criteria proposed in 1990 by the National Institute of Child Health and Human Development Conference on PCOS . All women presented polycystic ovarian morphology on ultrasound examination. Hyperandrogenemia was defined as testosterone levels 460 ng/ml. This value was derived from the mean value + 2SD of 100 control women. Other common causes of hyperandrogenism such as prolactinoma, congenital adrenal hyperplasia, Cushing syndrome and virilizing ovarian or adrenal tumours were excluded. The study was prospective and randomised. Randomisation was non-blind and was based on patients’ chronological presence at the outpatient endocrine infirmary, namely the first one in Group A the second in Group B, the third in Group C, etc. For each patient a prescription of the recommended medication was given in order of appearance for treatment, indifferent of BMI and IR. Forty-five (45) women with PCOS were randomly divided into three groups: Group A (n ¼ 15, age ¼ 20.67 + 4.13 years, BMI ¼ 21.04 + 1.97 kg/m2), which comprised women who were treated for 6 months with an OC containing 35 mg ethinylestradiol plus 2 mg cyproterone acetate, Group B (n ¼ 15, age ¼ 22.00 + 2.07 years, BMI ¼ 21.69 + 2.33 kg/m2), which comprised women who were treated for 6 months with an OC containing 30 mg ethinylestradiol plus 3 mg drospirenone. Both OCs were given in a sequential way. Group C (n ¼ 15, age ¼ 20.53 + 3.09 years, BMI ¼ 21.83 + 1.73 kg/m2), which comprised women who were treated for 6 months with metformin 850 6 2 mg. All the above mentioned Groups of PCOS women did not differ statistically for age (p ¼ 0.454) and BMI (p ¼ 0.351). Furthermore, no woman reported use of any medication that could interfere with the normal function of the hypothalamic–pituitary–gonadal axis, during the last 6 months. A written informed consent was obtained from all women and the study was approved by the Ethical Committee of the Institution. None of the PCOS women included in the study was lost to follow-up during the 6 months of treatment.
Methods At baseline, blood samples were collected between the third and seventh day after the beginning of a spontaneous bleeding episode, at 09:00, after an overnight fast. Under treatment, blood samples were collected during OC treatment between the fifth and the seventh day of the fourth and seventh cycle (reflecting 3 and 6 months of treatment), while patients were on OCs or metformin. All assays of hormonal levels and plasma glucose were carried out at the Department of Biochemistry of the Aristotle University of Thessaloniki School of Medicine.
Plasma glucose concentrations were measured using a glucose oxidase technique with an auto analyser (Roche/ Hitachi 902; Roche Diagnostics GmbH, Manheim, Germany). Luteinizing Hormone (LH), Follicle Stimulating Hormone (FSH) and prolactin levels were measured with an enzyme-linked immunoassay (EIA), using commercial kits (Nichols Institute Diagnostics, CA). Testosterone was measured with a Direct RIA kit (Sorin, Biomedica); D4androstenedione with a Gamma Coat [125I] RIA kit (Incstar Corp.); Dihydroepiandrosterone Sulfate (DHEAS) with direct RIA solid-phase coated tubes (Zer Science Based Industries Ltd); 17a-OH-progesterone with an ImmuChem Double Antibody [125I] RIA kit (ICN Pharmaceuticals, Inc.); insulin with a Coat-A-count Insulin kit (Diagnostic Products Corp.); and sex-hormone binding globulin (SHBG) with an immunoradiometric assay (IRMA) kit (SHBG: [125I] IRMA Kit, Orion Diagnostica). The intra-assay coefficients of variation (CV) were 1.5% for FSH, 0.7% for LH, 2.7% for prolactin, 3.8% for insulin, 4.1% for 17a-OH-progesterone, 1.3% for testosterone, 5.9% for androstendione, 9.4% for DHEA-S and 5.8% for SHBG. The average inter-assay CV were 3.2% for FSH, 1.7% for LH, 3.4% for prolactin, 4.4% for insulin, 6.3% for 17a-OH-progesterone, 2.2% for testosterone, 9.2% for androstenedione, 12.1% for DHEA-S, 7.8% and for SHBG. AMH concentrations were measured with an enzymatically amplified two-side immunoassay [DSL-10-14400 Active Mu¨llerian Inhibiting Substance/AMH enzymelinked immunosorbent (ELISA) kit, DSL laboratories, Webster, TX]. The theoretical sensitivity of the method is 0.006 ng/ml, the intra-assay coefficient of variation for high values 3.3% and the inter-assay coefficient of variation for high values 6.7%. Free androgen index (FAI) was calculated according to the equation: testosterone (nmol/l) 6 0.0347 6 100/SHBG (nmol/l). HOMA-IR was derived from the equation: [glucose (mmol/l) 6 insulin (mIU/ml)]/22.5. QUICKI was derived from the equation: 1/log (fasting insulin) þ log (fasting glucose).
Statistics All analyses were performed using the statistical package SPSS, v.16.0 (SPSS Inc., Chicago, IL). We performed a sample size calculation for the difference in mean values for serum AMH under different treatment regimens. A total of 15 patients will enter this crossover study. By including a minimum of 15 patients in each study group, the probability is 90% that the study will detect a treatment difference at a two sided 5.0% significance level. We prefer to use non-parametric methods as fewer assumptions have to be made and especially because of the small number of patients that consists the three different study Groups. Analysis of data was done by using Kruskal– Wallis one way analysis of variance. The Mann–Whitney test was used to evaluate differences between groups. All parameters are shown as the mean + SD. Correlations were assessed by using Spearman’s rank correlation. Correlations with a critical value of p 5 0.05 were considered significant.
Results The basal hormonal features for all women with PCOS as well as for the three different groups of treatment are summarised in Table I. Concerning all these parameters,
p ¼ 0.073 p ¼ 0.953 p ¼ 0.869 p ¼ 0.249 p ¼ 0.650 p ¼ 0.442 p ¼ 0.616 p ¼ 0.896 p ¼ 0.403 p ¼ 0.005 p ¼ 0.741 p ¼ 0.655 p ¼ 0.332 p ¼ 0.123 p ¼ 0.715 p ¼ 0.967 p ¼ 0.001 p ¼ 0.012 p ¼ 0.006 p ¼ 0.232 p ¼ 0.233 p ¼ 0.185 p 5 0.001 p 5 0.001 p ¼ 0.002 p ¼ 0.164 p ¼ 0.128 p ¼ 0.342 p ¼ 0.754 p ¼ 0.232 Values are mean + SD.
21.04 + 1.97 5.40 + 1.56 10.18 + 8.64 81.79 + 32.22 2.73 + 0.98 2707 + 1209 1.02 + 0.63 47.06 + 17.34 6.00 + 1.53 94.00 + 11.00 8.09 + 6.31 16.39 + 7.40 1.95 + 1.72 0.363 + 0.036 9.10 + 2.95 BMI FSH (mIU/ml) LH (mIU/ml) Testosterone (ng/dl) Androstenedione (ng/ml) DHEAS (ng/ml) 17-OH-Progesterone (ng/ml) SHBG (nmol/l) FAI Glucose (mg/dl) Insulin (mIU/ml) Glucose/Insulin ratio HOMA-IR QUICKI AMH (ng/ml)
21.56 + 2.02 6.00 + 1.63 11.25 + 7.66 83.03 + 24.58 3.07 + 1.00 3052 + 1025 1.38 + 0.74 47.46 + 18.62 6.63 + 2.52 98.47 + 13.10 9.30 + 5.93 13.80 + 6.26 2.34 + 1.73 0.349 + 0.031 9.08 + 3.32
21.69 + 2.34 6.04 + 1.75 10.76 + 6.86 83.14 + 22.12 3.39 + 1.09 2947 + 946 1.75 + 0.88 55.16 + 19.78 5.88 + 2.66 99.00 + 4.81 8.64 + 4.80 13.93 + 5.63 2.11 + 1.18 0.348 + 0.023 8.90 + 3.49
21.97 + 1.69 6.56 + 1.46 12.81 + 7.65 84.15 + 19.20 3.10 + 0.88 3503 + 768 1.36 + 0.53 40.16 + 16.64 8.01 + 2.72 102.40 + 18.90 11.17 + 6.46 11.07 + 4.65 2.95 + 2.11 0.336 + 0.028 9.24 + 3.70
21.05 + 1.99 1.02 + 1.05 1.83 + 2.35 50.78 + 14.86 2.04 + 0.61 2036 + 860 0.57 + 0.48 213.00 + 61.18 0.87 + 0.27 89.07 + 6.37 12.94 + 7.25 9.33 + 5.48 2.90 + 1.76 0.337 + 0.031 5.28 + 0.84
21.67 + 2.30 2.47 + 3.06 4.14 + 6.32 55.59 + 27.33 2.86 + 1.21 2486 + 1083 1.33 + 0.77 251.79 + 93.57 0.89 + 0.60 88.14 + 11.25 11.54 + 6.08 10.40 + 6.46 2.60 + 1.53 0.344 + 0.038 8.42 + 3.57
20.75 + 1.32 6.49 + 3.76 13.60 + 16.69 76.04 + 18.48 2.96 + 0.83 3808 + 1307 1.51 + 1.05 40.89 + 13.76 7.15 + 2.90 85.93 + 6.41 10.24 + 8.76 11.97 + 6.12 2.21 + 2.03 0.354 + 0.03 7.77 + 2.82
p ¼ 0.967 p 5 0.001 p ¼ 0.002 p ¼ 0.003 p ¼ 0.033 p ¼ 0.099 p ¼ 0.038 p 5 0.001 p 5 0.001 p ¼ 0.155 p ¼ 0.065 p ¼ 0.007 p ¼ 0.152 p ¼ 0.049 p 5 0.001
0–6 months, Group C 0–6 months, Group B 0–6 months, Group A 6 months – Group C 6 months – Group B 6 months – Group A Baseline – Group C Baseline – Group B Baseline – Group A Baseline – all groups
Table I. General characteristics of women with PCOS as a whole as well as at baseline and after 6 months treatment with 35 mg ethinylestradiol þ 2 mg cyproterone acetate (Group A), with 30 mg ethinylestradiol þ 3 mg drospirenone (Group B) and with Metformin 850 mg 6 2.
Gynecol Endocrinol Downloaded from informahealthcare.com by Hacettepe Univ. on 01/27/12 For personal use only.
AMH in PCOS under treatment
no statistical significant difference was detected between all PCOS groups at baseline. No statistical significant difference in Weight and BMI was noted for all treatment groups after 6 months of treatment (Table I). A tendency for a slight decrease in weight was noted for women under metformin treatment (60.52 + 5.53 versus 57.77 + 4.16, p ¼ 0.168). Menstrual function was normalised in all patients under treatment with an OC (Groups A and B) (mean cycle length 28 + 2). Under metformin treatment in 10 out of 15 patients (66.66%) menstrual function was normalised with a menstrual cycle length between 27 and 32 days. Concerning the rest five patients under metformin treatment three experienced successively ovulatory versus anovulatory cycles, while for the rest two menstrual cycles remained anovulatory during the whole period of treatment. Six months of treatment with 35 mg ethinylestradiol plus 2 mg cyproterone acetate (Group A) led to a significant decrease in serum LH (p ¼ 0.002), FSH (p 5 0.001), testosterone (p ¼ 0.003), androstenedione (p ¼ 0.033), 17OH-progesterone (p ¼ 0.038), FAI (p 5 0.001), glucose/ insulin ratio (p ¼ 0.007) and QUICKI (p ¼ 0.049), as well as to a significant increase in SHBG (p 5 0.001). Six months of treatment with 30 mg ethinylestadiol plus 3 mg drospirenone acetate (Group B) led to a significant decrease in serum LH (p ¼ 0.001), FSH (p ¼ 0.012), testosterone (p ¼ 0.006), FAI (p 5 0.001), SHBG (p 5 0.001) as well as to serum Glucose (p ¼ 0.002) (Table I). Six months of treatment with metformin 850 mg 6 2 (Group C) led to a statistically significant decrease only in fasting glucose (p ¼ 0.005) (Table I). Under treatment with an OC containing 35 mg ethinylestradiol plus 2 mg cyproterone acetate (Group A), AMH serum levels were significantly decreased from 9.10 + 2.95 ng/ml at baseline to 7.45 + 2.36 ng/ml after 3 months and to 5.28 + 0.84 ng/ml after 6 months of treatment ( p 5 0.001 and p ¼ 0.002, respectively) (Figure 1). Under treatment with an OC containing 30 mg ethinylestradiol plus 3 mg drospirenone (Group B), AMH serum levels ranged from 8.90 + 3.49 ng/ml at baseline to 7.06 + 3.12 ng/ml after 3 months and to 8.42 + 3.57 ngl/ml after 6 months of treatment (p ¼ ns) (Figure 1). Under treatment with metformin 850 mg 6 2 (Group C), AMH serum levels ranged from 9.24 + 3.70 ng/ml at baseline to 9.92 + 3.80 ng/ml after 3 months and to 7.77 + 2.82 ng/ml after 6 months of treatment (p ¼ ns) (Figure 1). Serum AMH levels were significantly decreased under treatment with an OC containing 35 mg ethinylestradiol plus 2 mg cyproterone acetate (Group A) compared to treatment with metformin 850 mg 6 2 (Group C) both at 3 and at 6 months (p ¼ 0.041 and p ¼ 0.005, respectively) (Figure 1). Serum AMH levels were also significantly decreased under treatment with an OC containing 30 mg ethinylestradiol plus 3 mg drospirenone (Group B) compared to treatment with metformin 850 mg 6 2 (Group C) both at 3 and at 6 months (p ¼ 0.032 and p ¼ 0.005, respectively) (Figure 1). Concerning serum AMH levels at baseline and their change under the influence of different treatment regimens the following correlations were detected: at baseline, in Group A, serum AMH correlated only with serum 17-OHprogesterone (Spearman’s r ¼ 0.560, p ¼ 0.030) and the observed change of serum AMH levels after 6 months did not correlate with the observed change of serum 17-OHProgesterone (Spearman’s r ¼ 0.493, p ¼ 0.062). In Group B, serum AMH levels at baseline did not correlate significantly with any parameter. In Group C, serum
D. Panidis et al.
Figure 1. Serum AMH levels at baseline and after 3 and 6 months treatment with 35 mg ethinylestradiol þ 2 mg cyproterone acetate, with 30 mg ethinylestradiol þ 3 mg drospirenone and with metformin 850 mg 6 2. Values are mean + SD.
AMH levels correlated at baseline with serum testosterone (Spearman’s r ¼ 0.533, p ¼ 0.041), fasting glucose (Spearman’s r ¼ 0.587, p ¼ 0.021), HOMA-IR (Spearman’s r ¼ 0.568, p ¼ 0.027), and QUICKI (Spearman’s r ¼ 70.568, p ¼ 0.027). After 6 months of treatment with metformin, the observed change in serum AMH levels correlated only with the observed change in fasting insulin (Spearman’s r ¼ 0.762, p ¼ 0.001). Finally, after 6 months of treatment, serum AMH levels were positively correlated to testosterone (p ¼ 0.021), 17-OH-progesterone (p ¼ 0.032) and FAI (p ¼ 0.046) in the group of women with PCOS under 35 mg ethinylestradiol plus 2 mg cyproterone acetate (Group A).
Discussion AMH is produced by the granulosa cells of early developing follicles  and reflects the continuous, non-cycling growth of small follicles in the ovary . AMH has been found to be increased in the serum of women with PCOS [6–10]. AMH levels are not influenced by hormonal fluctuations and remain constant throughout the menstrual cycle, making it a promising diagnostic marker for patients with PCOS . Women with PCOS are usually treated with an OC, while obese patients with PCOS, especially those with IR might benefit from treatment with metformin. Several studies have suggested that the use of OCs aggravates IR and worsens glucose tolerance in women with PCOS. On the contrary, metformin improves insulin sensitivity and, in addition, may decrease circulating androgen levels and may improve menstrual cyclicity, thus addressing the traditional goals of long-term treatment . The aim of the present study was to assess the impact of metformin and of two different regimens of OCs containing different progestins, namely cyproterone acetate and drospirenone, on serum AMH levels in women with PCOS. The data of the present study clearly demonstrate a significant decrease of serum AMH levels under treatment with an OC containing 35 mg ethinylestradiol plus 2 mg cyproterone acetate, while no statistically significant change was noted under treatment with an OC containing 30 mg ethinylestradiol plus 3 mg drospirenone or under treatment with metformin 850 mg 6 2. Our data are clearly contra-
dictory to the data previously presented by Somunkiran et al. , who reported no change in serum AMH levels after 6 months of treatment with 35 mg ethinylestradiol plus 2 mg cyproterone acetate. This discrepancy might be attributed to the different selection of patients with PCOS between the two studies. Somunkiran et al. recruited their PCOS patients according to the criteria of the Rotterdam PCOS  consensus workshop group, namely the presence of two of the following three criteria: oligomenorrhea or amenorrhea, clinical or biochemical signs of hyperandrogenism, and ultrasonographic polycystic ovarian morphology , while patients with PCOS in our study were recruited to fulfil (and) the stricter criteria proposed in 1990 by the National Institute of Child Health and Human Development Conference on PCOS . As all women with PCOS included in this study had also ultrasonographic polycystic ovarian morphology they belonged to the 1A category according to the Rotterdam criteria which is the most severe form of the syndrome. As a consequence, all patients in the present study presented hyperandrogenemia, while in the study of Somunkiran et al. only 56.6% had clinical or biochemical signs of hyperandrogenism. Nevertheless, the decrease in serum testosterone levels achieved was not as significant as in the present study (p ¼ 0.05 versus p ¼ 0.003), although in both studies the same regimen was used. The decrease in serum AMH levels in this study was mostly attributed to the additional influence of cyproterone acetate, the antiandrogenic progestin component of the OC used. Indeed, with the use of cyproterone acetate serum androgen levels were significantly reduced compared to treatment with both an OC containing drospirenone and metformin. The decrease in serum androgen levels with the use of an OC containing drospirenone, although significant, was not at the magnitude achieved with the use of cyproterone acetate and only concerned testosterone and SHBG and not androstenedione and 17-OH-progesterone. Nevertheless, in the present study, serum AMH levels after 6 months of treatment with 35 mg ethinylestradiol plus 2 mg cyproterone acetate were significantly related to serum testosterone and 17-OH-Progesterone levels, as well as to FAI. In a previous study, we have shown that AMH levels were higher in anovulatory and hyperandrogenemic women with NIH-defined ‘classical’ PCOS, compared to both ovulatory women with PCOS morphology on ultrasound and hyperandrogenemia and to anovulatory women with PCOS morphology on ultrasound but normal androgen
AMH in PCOS under treatment levels . In this study, the strong positive association between LH and AMH levels, the significantly higher LH concentrations in women with ‘severe’ PCOS along with the highest levels of serum AMH, could not possibly be accounted for by any other PCOS-associated hormonal or metabolic defect other than hyperandrogenemia and chronic anovulation . Therefore, the findings of the present study add additional evidence that AMH levels reflect the severity of PCOS, traditionally defined by its two cardinal elements, i.e. oligo-anovulation and hyperandrogenemia . Somunkiran et al. , presented data concerning women with PCOS from whom 80% presented menstrual dysfunction, while our cohort of women with PCOS were all anovulatory raising the possibility that the discrepancy in the results recorded could be partly attributed, besides the differences in hyperandrogenemia and to differences in menstrual function. Ovulatory disorders in women with PCOS are caused by an increased early follicular growth, resulting in a larger reserve of follicles and/or a defective follicular selection, leading to follicular arrest . Since intra-ovarian androgens are also responsible for defective follicular selection and follicular arrest, it has been proposed that the intraovarian hyperandrogenism by increasing the AMH intraovarian level could exert an inhibiting effect on the selection process . The excess in AMH production by polycystic ovaries might be the result of the increased number of follicles 2–9 mm in diameter caused by the intra-ovarian excess of androgens , still it might not be the sole determinant of serum AMH, as we have previously shown that the total number of 2–9 mm-sized follicles, although an independent determinant, contributed only an additional 5.3% to the variance of AMH levels, as opposed to 18% by LH levels alone and an additional 9.5% by serum testosterone . It should be noted that serum testosterone might not accurately reflect intra-ovarian androgen levels, still we might speculate that the observed correlation between serum AMH and androgens levels might be even more pronounced in the intra-ovarian level. An additional effect of OC administration on serum AMH levels could be attributed to the suppression of pituitary gonadotropins. In particular, the administration of 35 mg ethinylestradiol plus 2 mg cyproterone acetate led to the most significant suppression of both LH and FSH, while the administration of metformin had no effect at all on pituitary gonadotropins. We have previously shown that increased serum LH levels was the most significant independent link between PCOS-associated disorders of ovulation and the observed increase in serum AMH as it contributed as much as 18% in the variance of circulating AMH . It is postulated that premature LH action on the granulosa cells of anovulatory women with PCOS contribute to the follicular arrest, being the link between PCOS-associated disorders of ovulation and the observed increase in ovarian production of AMH . It should be noted that, compared to treatment with metformin, treatment with an OC containing drospirenone led also to a statistical significant decrease in AMH serum levels after 6 months of treatment. This is also due to the combined effect of decreased serum androgen levels and suppressed pituitary gonadotropins, achieved under OC containing drospirenone, still to a lesser extent compared to an OC containing cyproterone acetate. Finally, a third major effect of treatment with metformin or an OC containing cyproterone acetate or drospirenone is their influence on IR. It is well known that, IR nonspecifically contributes to PCOS-associated follicular arrest [22,23]. In the present study which concerned normal
weight hyperandrogenic women with PCOS with no differences in surrogate markers of IR between the three treatment groups, the administration of an OC containing cyproterone acetate led to a borderline significant deterioration of IR, by decreasing both the glucose/insulin ratio and the QUICKI index of IR, still all these differences remained within normal range. It should be emphasised that both Metrformin and OC containing drospirenone had their major effect on fasting glucose levels and not on insulin. It has been previously shown that in women with PCOS metformin significantly decreased body weight and increased insulin sensitivity, while OC containing cyproterone acetate had no effect on IR [24,25]. In a previous study, we have shown that serum AMH levels were significantly lower in overweight or obese compared to normal weight women with PCOS, while these patients did not differ in their indices of IR, suggesting that obesity per se influence serum AMH levels . In the present study BMI was not modified in all groups after 6 months of treatment, therefore the major effect of metformin was not achieved and thus additional time should be given in order to observe the net effect of metformin administration on body weight reduction, IR amelioration and eventually its effect on serum AMH levels. Therefore, the findings of this study suggest that although obesity and, to a lesser extent IR, play a role in modulating serum AMH levels, the decrease in serum AMH levels under treatment could not be attributed to these factors and are mainly due to hyperandrogenism and chronic anovulation. Indeed, Carlsen et al. demonstrated that the administration of metformin in women with PCOS, although led to a significant reduction in body weight and an improvement of IR, had no effect on serum AMH levels . Finally, the results of the present study should be interpreted with caution taking into account the limitations of this study. The number of women with PCOS included in each study group is relatively small and also the serum levels of AMH do not necessarily reflect the intra-follicular content of AMH. In conclusion, AMH serum levels reflect the severity of PCOS and are significantly increased in its ‘classical’ phenotypic forms, based on the 1990 criteria, as opposed to its recently introduced subtypes. Moreover, AMH levels are significantly decreased under treatment with OCs containing antiandrogenic progestins such as the association of 35 mg ethinylestradiol plus 2 mg cyproterone acetate, through decrease in serum androgens and the suppression of pituitary gonadotropins. Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper. References 1. Carmina E, Lobo RA. Polycystic ovary syndrome (PCOS): arguably the most common endocrinopathy is associated with significant morbidity in women. J Clin Endocrinol Metab 1999;84:1897–1899. 2. Urbanek M, Du Y, Silander K, Collins FS, Steppan CM, Strauss JF 3rd, Dunaif A, Spielman RS, Legro RS. Variation in resistin gene promoter not associated with polycystic ovary syndrome. Diabetes 2003;52:214–217. 3. Norman RJ, Dewailly D, Legro RS, Hickey TE. Polycystic ovary syndrome. Lancet 2007;370:685–697. 4. Jonard S, DeWailly D. The follicular excess in polycystic ovaries, due to intra-ovarian hyperandrogenism, may be the main culprit for the follicular arrest. Hum Reprod Update 2004;10:107–117.
D. Panidis et al.
5. Durlinger AL, Gruijters MJ, Kramer P, Karels B, Ingraham HA, Nachtigal MW, Uilenbroek JT, Grootegoed JA, Themmen AP. Anti-Mu¨llerian hormone inhibits initiation of primordial follicle growth in the mouse ovary. Endocrinology 2002;143:1076–1084. 6. Durlinger AL, Gruijters MJ, Kramer P, Karels B, Kumar TR, Matzuk MM, Rose UM, de Jong FH, Uilenbroek JT, Grootegoed JA, Themmen AP. Anti-Mu¨llerian hormone attenuates the effects of FSH on follicle development in the mouse ovary. Endocrinology 2001;142:4891–4899. 7. Piouka A, Farmakiotis D, Katsikis I, Macut D, Gerou S, Panidis D. Anti-Mu¨llerian hormone levels reflect the severity of PCOS, but are negatively influenced by obesity: relationship with increased Luteinizing hormone levels. Am J Physiol Endocrinol Metab 2009;296:238–243. 8. Pigny P, Robert Y, Cortet-Rudelli C, Decanter C, Jonard S, DeWailly D. Elevated serum level of antimu¨llerian hormone in patients with polycystic ovary syndrome: Relationship to the ovarian follicle excess and the follicular arrest. J Clin Endocrinol Metab 2003;88:5957–5962. 9. Pigny P, Jonard S, Robert Y, DeWailly D. Serum antimu¨llerian hormone as a surrogate for antral follicle count for definition of the polycystic ovary syndrome. J Clin Endocrinol Metab 2006;91:941–945. 10. Siow Y, Kives S, Hertweck P, Perlman S, Fallat ME. Serum mu¨llerian inhibiting substance levels in adolescent girls with normal menstrual cycles or with polycystic ovary syndrome. Fertil Steril 2005;84:938–944. 11. Pellatt L, Hanna L, Brincat M, Galea R, Brain H, Whitehead S, Mason H. Granulosa cell production of antimu¨llerian hormone is increased in polycystic ovaries. J Clin Endocrinol Metab 2007;92:240–245. 12. Nader S, Diamanti-Kandarakis E. Polycystic ovary syndrome, oral contraceptives and metabolic issues: new perspectives and a unifying hypothesis. Hum Reprod 2007;22:317–322. 13. Streuli I, Fraisse T, Pillet C, Ibecheole V, Bischof P, de Ziegler D. Serum antimu¨llerian hormone levels remain stable throughout the menstrual cycle and after oral or vaginal administration of synthetic sex steroids. Fertil Steril 2008; 90:395–400. 14. Somunkiran A, Yavuz T, Yucel O, Ozdemir I. Anti-Mu¨llerian hormone levels during hormonal contraception in women with polycystic ovary syndrome. Eur J Obstet Gynecol Reprod Biol 2007;134:196–201. 15. Piltonen T, Morin-Papunen L, Koivunen R, Perheentupa A, Ruokonen A, Tapanainen JS. Serum anti-Mu¨llerian hormone
levels remain high until late reproductive age and decrease during metformin therapy in women with polycystic ovary syndrome. Hum Reprod 2005;20:1820–1826. Zawadski JK, Dunaif A. Diagnostic criteria for polycystic ovary syndrome. In: Dunaif A, Givens JR, Haseltine FP, Merriam GE, editors. Polycystic ovary syndrome, Mass. USA: Blackwell Scientific Boston; 1992. pp 377–384. Baarends WM, Uilenbroek JT, Kramer P, Hoogerbrugge JW, van Leeuwen EC, Themmen AP, Grootegoed JA. AntiMu¨llerian hormone and anti-Mu¨llerian hormone type II receptor messenger ribonucleic acid expression in rat ovaries during postnatal development, the estrous cycle, and gonadotropin-induced follicle growth. Endocrinology 1995;136:4951– 4962. Visser JA, de Jong FH, Laven JS, Themmen AP. AntiMu¨llerian hormone: a new marker for ovarian function. Reproduction 2006;131:1–9. La Marca A, Stabile G, Artenisio AC, Volpe A. Serum antiMu¨llerian hormone throughout the human menstrual cycle. Hum Reprod 2006;21:3103–3107. Nestler JE. Metformin for the treatment of the polycystic ovary syndrome. N Engl J Med 2008;358:47–54. Rotterdam ESHRE/ASRM-sponsored PCOS Consensus Workshop Group Revised 2003 consensus on diagnostic criteria and long-term health risks related to polycystic ovary syndrome (PCOS). Hum Reprod 2004;19:41–47. Dunaif A, Futterweit W, Segal KR, Dobrjansky A. Profound peripheral insulin resistance, independent of obesity, in the polycystic ovary syndrome. Diabetes 1989; 38:1165–1174. Dunaif A. Insulin resistance and the polycystic ovary syndrome: mechanism and implication for pathogenesis. Endocr Rev 1997;18:774–800. Wu J, Zhu Y, Jiang Y, Cao Y. Effects of metformin and ethinyl estradiol-cyproterone acetate on clinical, endocrine and metabolic factors in women with polycystic ovary syndrome. Gynecol Endocrinol 2008;24:392–398. Luque-Ramı´rez M, Alvarez-Blasco F, Botella-Carretero JI, Martı´nez-Bermejo E, Lasuncio´n MA, Escobar-Morreale HF. Comparison of ethinyl-estradiol plus cyproterone acetate versus metformin effects on classic metabolic cardiovascular risk factors in women with the polycystic ovary syndrome. J Clin Endocrinol Metab 2007;92:2453–2456. Carlsen SM, Vanky E, Fleming R. Anti-Mu¨llerian hormone concentrations in androgen-suppressed women with polycystic ovary syndrome. Hum Reprod 2009;24:1732–1738.