Human Reproduction Vol.20, No.12 pp. 3333–3340, 2005 doi:10.

1093/humrep/dei258
Advance Access publication August 25, 2005.

The effects of rosiglitazone and metformin on oxidative stress

and homocysteine levels in lean patients with polycystic ovary
syndrome

Murat Yilmaz1,4, Neslihan Bukan2, Göksun Ayvaz3, Ayhan Karakoç3, Füsun Törüner3,
Nuri Çakir3 and Metin Arslan3
1
Department of Endocrinology and Metabolism, Faculty of Medicine, Kirikkale University, 71100 Kirikkale, 2Departments of
Biochemistry and 3Endocrinology and Metabolism, Faculty of Medicine, Gazi University, Ankara, Turkey
4
To whom correspondence should be addressed. E-mail: murartt@hotmail.com

BACKGOUND: Oxidative stress and hyperhomocysteinaemia are risk factors for cardiovascular diseases. The aim of

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this study was to assess the effects of rosiglitazone and metformin on cardiovascular disease risk factors such as insulin
resistance, oxidative stress and homocysteine levels in lean patients with polycystic ovary syndrome (PCOS). MEHODS:
Fifty lean patients (BMI <25 kg/m2) with PCOS and 35 healthy subjects were included this study. Serum homocysteine,
sex steroids, fasting insulin, fasting glucose and lipid levels were measured. Total antioxidant status (TAS; combines
concentrations of individual antioxidants) and malonyldialdehyde concentration (MDA) were determined. Insulin res-
istance was evaluated by using the homeostasis model insulin resistance index (HOMA-IR), quantitative insulin sensitiv-
ity check index (QUICKI), Area under the curve insulin (AUCI) and the insulin sensitivity index (ISI). Patients were
divided into two groups. One group was treated with metformin (n = 25) and the other received rosiglitazone (n = 25) for
12 weeks. All measurements were repeated at the end of 12 weeks. RESULTS: Compared with healthy women, those
with PCOS had significantly elevated serum MDA, homocysteine, HOMA-IR, AUCI and lipoprotein a levels, and signif-
icantly decreased serum TAS, QUICKI and ISI. Serum free testosterone levels showed a significant positive correlation
with MDA, AUCI and HOMA-IR, and a negative correlation with TAS, ISI and QUICKI in PCOS patients. HOMA-IR
and AUCI significantly decreased, while QUICKI and ISI significantly increased after treatment in both groups. Serum
TAS level increased and serum MDA level decreased after the rosiglitazone treatment, but these parameters did not
change after the metformin treatment. Serum homocysteine and lipid levels did not change in either group, while serum
androgen levels and LH/FSH ratio significantly decreased after the treatment period in only the rosiglitazone-treated
group. CONCLUSION: Elevated insulin resistance, oxidative stress and plasma homocysteine levels and changes in
serum lipid profile (risk factors for cardiovascular disease) were observed in lean PCOS patients. Rosiglitazone seemed
to decrease elevated oxidative stress when compared with metformin treatment in lean PCOS patients.

Key words: insulin resistance/metformin/oxidative stress/PCOS/rosiglitazone

Introduction mellitus (DM) (Ehrmann et al., 1999) and cardiovascular dis-

Polycystic ovary syndrome (PCOS) is one of the most common ease (CVD) (Guzick, 1996; Legro, 2003).
endocrinopathies in women, affecting 5–10% of the population Oxidative stress means an imbalance between the production
(Frank, 1995). PCOS is a heterogeneous disorder characterized of reactive oxygen species and the antioxidant defence system,
by menstrual irregularities, clinical and/or biochemical hyper- which buffers the oxidative damage. Oxidative stress is impli-
androgenism, and hyperinsulinaemia secondary to reduced cated in the pathogenesis of several diseases, such as atheroscle-
insulin sensitivity (Homburg, 2003). Approximately half of all rosis, DM and carcinogenesis (Betteridge et al., 2000). Oxidative
women with PCOS are overweight or obese. Independently of stress also impairs insulin action, as has been demonstrated in
the presence of obesity, these women are frequently insulin- type 2 diabetics, and this impairment might be due to several fac-
resistant and therefore they have hyperinsulinaemia, which may tors, such as membrane fluidity alterations, decreased availability
play a pathogenic role in the disease (Dunaif et al., 1989; of nitric oxide and increased intracellular calcium content (Caimi
Dunaif, 1997). Insulin resistance and the resulting hyperinsuli- et al., 2003). Serum total antioxidant status (TAS) combines the
naemia lead patients to a cluster of long-term metabolic disor- concentrations of individual antioxidants, such as vitamin C and E,
ders, including impaired glucose tolerance, type 2 diabetes β-carotene and thiol groups, and also their synergists. TAS is

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M.Yilmaz et al.

sensitive to the changes in plasma antioxidant levels and degrees was defined according to the 2003 Rotterdam consensus criterion
of oxidative stress (Garibaldi et al., 2001). Malonyldialdehyde (Rotterdam ESHRE/ASRM, 2004). The ethics committee of Gazi
(MDA) is a marker of lipid peroxidation and increases in oxida- University, Faculty of Medicine, Ankara, approved the study protocol.
tive stress states (Knight et al., 1987; Betteridge, 2000). Recent All patients gave their signed informed consent.
Patients who had DM, hyperprolactinaemia, congenital adrenal
studies have documented increased oxidative stress in patients
hyperplasia, thyroid disorders, Cushing’s syndrome (1 mg dexametha-
with PCOS (Sabuncu et al., 2001; Fenkçi et al., 2003), which
sone suppression test), hypertension, vitamin B12 and folate defi-
may increase the risk of CVD in such patients. ciency, hepatic or renal dysfunction were excluded from the study.
Homocysteine (Hcy) is an intermediate product formed during Confounding medications, including oral contraceptive agents, anti-
the breakdown of the amino acid methionine, and may undergo lipidaemic drugs, hypertensive medications, and insulin-sensitizing
remethylation to methionine or trans-sulphuration to cystathione drugs which may affect the metabolic criteria, were questioned.
and cysteine. Elevated serum levels of Hcy, called hyperhomo- Patients were also excluded if they had used any anti-androgen agent,
cysteinaemia, have adverse effects on the cardiovascular system combined estrogen–progestin or an oral hypoglycaemic agent within
(Fonseca et al., 1999). Elevated plasma Hcy levels are consid- 90 days prior to enrolment.
ered to be an independent risk factor for CVD (Clarke et al., 1991). BMI (kg/m2) and waist to hip (W/H) ratio were calculated. Weight
In recent studies, serum Hcy concentrations were found to be and height were measured in light clothing without shoes. Waist cir-
cumference was measured at the narrowest level between the costal
elevated in PCOS women, suggesting that an alteration in Hcy
margin and the iliac crest, and the hip circumference was measured at
metabolism may play a role in the increased cardiovascular
the widest level over the buttocks while the subject was standing and
risk associated with PCOS (Loverro et al., 2002; Randeva breathing normally. Degree of hirsutism was determined by Fer-
et al., 2002; Vrbikova et al., 2002; Schachter et al., 2003;

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riman–Gallwey scoring (Ferriman and Gallwey, 1961).
Wijeyaratne et al., 2004). Menstrual cyclicity was assessed by recording the menses in the
Experimental and clinical evidence suggest indirectly that last 6 months prior to the study. The menstrual patterns were defined
moderate hyperhomocysteinaemia may predispose affected indi- as follows. Cycles between 22 and 45 days were noted as regular,
viduals to endothelial dysfunction through a mechanism that those between 46 and 180 days as irregular, those lasting 21 days or
involves the generation of reactive oxygen species (Kanani less as polymenorrhoea, those between 46 and 180 days as oligomen-
et al., 1999). Hyperhomocysteinaemia-induced oxidative stress orrhoea, and those lasting 180 days or more as amenorrhoea.
may occur as a consequence of either decreased expression and Serum levels of FSH, LH, prolactin, dehydroepiandrosterone sul-
phate (DHEA-S), insulin, cortisol and thyroid-stimulating hormone
activity of key antioxidant enzymes or increased enzymatic gen-
(TSH) were measured with specific chemiluminescence assays from
eration of superoxide anions (Eberhardt et al., 2000; Lentz et al.,
the Abbott Architect system (Chicago, USA). Serum levels of 17 OH-
2000; Faraci, 2003; Loscalzo, 2003; Ungvari et al., 2003). progesterone, free testosterone, androstenedione were measured by
Use of oral anti-hyperglycaemic drugs, predominantly met- radioimmunoassay (RIA). Serum levels of total cholesterol, high-den-
formin and thiazolidinediones, have been shown to improve sity lipoprotein (HDL) cholesterol, low-density lipoprotein (LDL)
insulin sensitivity and ovarian function of women with PCOS. cholesterol and triglyseride were measured using an Abbott-Aeroset
Metformin inhibits hepatic glucose production and enhances (Chicago, IL, USA) autoanalyser with original kits. Lipoprotein a [Lp
peripheral tissue sensitivity to insulin, resulting in a decrease in (a)], apoprotein A (Apo A) and apoprotein B (Apo B) levels were
insulin secretion (Bailey and Turner, 1996). In women with determined by nephelometric assay using a Beckman 360 protein
PCOS, many studies have demonstrated the efficacy of met- array system. Serum vitamin B12 level was measured using an Immu-
formin in improving menstrual cycle pattern, ovulation and lyte 2000 (Los Angeles, CA, USA) analyser with the chemilumines-
cence method. Folate level was determined with a Tosoh (Tokyo,
pregnancy outcomes (Morin-Papunen et al., 1998; Unluhizarci
Japan) analyser. Plasma Hcy levels were measured by HPLC using
et al., 1999; La Marca et al., 2000; Moghetti et al., 2000;
Chromsystems kits with fluorescence detector (München, Germany).
Kocak et al., 2002; Baillargeon et al., 2004; Hoeger, et al., A standard 75 g oral glucose tolerance test (OGTT) and a test of the
2004). Rosiglitazone, an agonist of peroxisome proliferator- insulin response to oral glucose loading were performed after 10–12 h
activating receptor-γ (PPAR-γ), increases the uptake and utility of fasting between 8.30 and 10.30 a.m. Glucose tolerance state was
of glucose in the periphery and decreases hepatic gluconeogen- evaluated using the criteria of American Diabetes Association (Expert
esis (Goldstein, 1999). Most of the studies have shown that Committee on the Diagnosis and Classification Follow-up Report on
rosiglitazone reduces plasma androgen levels, with consequent the Diagnosis of Diabetes Mellitus, 2003). The responses of glucose
improvement in ovulatory reproductive function in PCOS and insulin to the OGTT were analysed by calculating the area under
patients (Cataldo et al., 2001; Ghazeeri et al., 2003; Shobokshi the curve insulin (AUCI) and area under the curve glucose (AUCG)
and Shaarawy, 2003; Baillargeon et al., 2004; Belli et al., 2004; using the trapezoidal method. The insulin sensitivity index (ISI) was
calculated by the Matsuda method (Matsuda and DeFronzo, 1999). The
Kilicdag et al., 2005a; Sepilian and Nagamani, 2005).
homeostasis model (HOMA) insulin resistance (HOMA-IR) index
The aim of this study was to evaluate the effects of rosiglita-
was calculated according to the following formula: fasting glucose
zone and metformin on cardiovascular risk factors, such as (mmol/l) × fasting insulin (μU/ml)/22.5 (Matthews et al., 1985). The
insulin resistance, homocysteine levels and oxidative stress in quantitative insulin sensitivity check index (QUICKI) was calculated
lean PCOS patients. according to the following formula: 1/[log fasting serum insulin (μU/ml)
+ log fasting plasma glucose (mg/dl)] (Katz et al., 2000).
Serum TAS was measured spectrophotometrically using ABTS
Materials and methods [2,2′-azino-di-(3- ethyl benzthiazoline sulphate)] method (Total Anti-
Fifty lean patients with PCOS (BMI <25 kg/m2) and 35 age- and oxidant Status kit; Randox, Antrim, UK). Serum MDA levels were
weight-matched healthy subjects were included in the study. PCOS determined by spectrophotometry at 532 nm after boiling the sample

3334
 Oxidative stress in lean PCOS patients

and condensing it with thiobarbituric acid. The results were expressed of PCOS patients and control subjects are shown in Table I.
as nmol/ml (Yoshioka et al., 1979). The clinical and endocrinological parameters of the metformin
After the screening period, patients were assigned to metformin and and rosiglitazone groups are shown in Table II. The clinical
rosiglitazone treatment groups consecutively. The study was designed and endocrinological parameters after the rosiglitazone and
as open-labelled. The first patient recruited into the study was treated
metformin treatments are shown in Table III.
with rosiglitazone, then metformin was given to the second patient.
Then, the patients were quasi-randomized, i.e. subjects with odd num-
bers received rosiglitazone (n = 25) 4 mg/day for 12 weeks, whilst those Insulin resistance, BMI and W/H ratio
with even numbers received metformin (n = 25) 850 mg twice daily.
Compared with healthy women, those with PCOS had signifi-
Effects of metformin and rosiglitazone on menstrual cyclicity were
evaluated by accessing the changes in frequency of cycles after treatment. cantly elevated HOMA-IR (P < 0.001) and AUCI (P < 0.001)
These assessments were performed by two independent obstetricians. levels, and significantly decreased QUICKI (P < 0.001) and
Liver enzymes were measured monthly during the study. At the end of 12 ISI (P < 0.001) levels. After treatment, the HOMA-IR and
weeks all laboratory and anthropometric measurements were repeated. AUCI levels declined significantly (P < 0.001), while QUICKI
and ISI levels significantly increased (P < 0.001). BMI
Statistical analysis increased in the rosiglitazone group (P < 0.05) but decreased in
For comparison of all the variables between the PCOS and control the metformin group (P < 0.05). W/H ratio decreased in the
groups, the unpaired t-test was used. For comparison of all quantita- metformin group (P < 0.05) but did not change in the rosiglita-
tive variables, clinical, biochemical and hormonal parameters between zone group (P > 0.05). With multiple regression analysis, the
rosiglitazone and metformin groups the unpaired t-test was used. reduction in insulin resistance was calculated to be independ-

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Menstrual cycle rates were compared by using the χ2test. The paired ent from the decrease in BMI in the metformin groups
t-test was performed to compare the basal and end values within the
(HOMA-IR, β = 0.123; AUCI, β = 0.136; QUICKI, β = 0.119;
same group. Multiple regression analysis was conducted with HOMA,
ISI, β = 0.127; P > 0.05).
AUCI, ISI, QUICKI, BMI, TAS, MDA, lipids, Hcy and androgens.
Pearson correlation analysis was used to explore correlations between
the variables. Results are shown as mean ±SD. Statistical significance Oxidative stress
was accepted at P < 0.05. Data analysis was performed using the stat-
istical software package SPSS for Windows (Version 10.0 for Windows; Women with PCOS had significantly higher serum MDA lev-
SPSS, Inc., Chicago, IL). els than healthy women (P < 0.001). However, TAS levels
were significantly lower in women with PCOS compared with
healthy women (P < 0.005). TAS levels (P < 0.005) increased
Results and MDA levels (P < 0.001) declined after the rosiglitazone
Mean age, BMI and smoking were similar between PCOS and treatment, but these parameters did not change after the met-
control groups. The clinical and endocrinological parameters formin treatment (P > 0.05).

Table I. Basal clinical and endocrinological parameters in patients with PCOS and control subjects

PCOS (mean ± SD) Controls (mean ± SD) P

(n = 50) (n = 35)

Age (years) 24.12 ± 4.89 24.35 ± 5.02 NS

BMI (kg/m2) 21.92 ± 3.21 22.45 ± 4.89 NS
W/H ratio 0.83 ± 0.08 0.83 ± 0.07 NS
Smoking 10/40 6/29 NS
LH (mIU/ml) 9.96 ± 4.76 5.89 ± 1.99 <0.001
FSH (mIU/ml) 6.04 ± 2.18 5.82 ± 1.88 NS
LH/FSH ratio 1.65 ± 0.18 1.01 ± 0.14 <0.001
Free testosterone (pg/ml) 2.99 ± 1.06 2.05 ± 0.55 <0.001
DHEA-S (μg/dl) 312.63 ± 108.56 182.73 ± 63 <0.001
Androstenedione (ng/ml) 2.96 ± 1.62 1.58 ± 0.51 <0.001
HOMA-IR 3.20 ± 1.52 2.19 ± 0.98 <0.001
QUICKI 0.315 ± 0.02 0.378 ± 0.03 <0.001
ISIOGTT 4.25 ± 2.64 6.39 ± 3.76 <0.001
TAS (mmol/l) 0.98 ± 0.08 1.25 ± 0.12 <0.005
MDA (nmol/ml) 7.31 ± 3.07 3.44 ± 1.43 <0.001
Hcy (μmol/l) 13.76 ± 6.28 9.32 ± 4.52 <0.005
Vitamin B12 (pg/ml) 312.87 ± 72.61 306.12 ± 68.35 NS
Folic acid (ng/ml) 11.52 ± 3.14 11.83 ± 3.39 NS
AUCI (μIU/ml/min) 7012.35 ± 812.98 4487.12 ± 619.4 <0.001
AUCG (mg/dl/min) 13 984.63 ± 1718.6 13 523.98 ± 1611.12 NS
Total cholesterol (mg/dl) 163.62 ± 59.35 166.71 ± 63.23 NS
LDL cholesterol (mg/dl) 100.63 ± 26.35 93.68 ± 23.11 NS
HDL cholesterol (mg/dl) 48.36 ± 11.56 59.87 ± 9.48 <0.05
Triglyceride (mg/dl) 78.01 ± 32.67 72.87 ± 45.59 NS
Apo A (mg/dl) 110.21 ± 27.81 136.15 ± 23.85 <0.05
Apo B (mg/dl) 85.19 ± 21.87 84.92 ± 21.66 NS
Lp(a) (mg/dl) 21.98 ± 12.76 17.38 ± 10.3 <0.05

3335
M.Yilmaz et al.

Table II. Basal clinical and endocrinological parameters of PCOS patients in rosiglitazone and metformin groups

Rosiglitazone group (mean ± SD) Metformin group (mean ± SD) P

(n = 25) (n = 25)

Age (years) 23.92 ± 4.64 24.31 ± 4.75 NS

BMI (kg/m2) 21.82 ± 3.44 22.16 ± 3.57 NS
W/H ratio 0.83 ± 0.08 0.83 ± 0.09 NS
LH (mIU/ml) 10.08 ± 7.81 9.89 ± 5.62 NS
FSH (mIU/ml) 6.11 ± 2.27 5.96 ± 2.47 NS
LH/FSH ratio 1.65 ± 0.18 1.65 ± 0.12 NS
Free Testosterone (pg/ml) 2.95 ± 1.06 3.01 ± 1.34 NS
DHEA-S (μg/dl) 308.11 ± 112.56 317.25 ± 131.38 NS
Androstenedione (ng/ml) 2.95 ± 1.58 2.96 ± 1.46 NS
Hcy (μmol/l) 13.94 ± 6.72 13.52 ± 5.98 NS
QUICKI 0.321 ± 0.023 0.313 ± 0.021 NS
ISIOGTT 4.26 ± 2.61 4.23 ± 2.58 NS
HOMA-IR 3.21 ± 1.45 3.18 ± 1.19 NS
TAS (mmol/l) 0.95 ± 0.07 1.00 ± 0.12 NS
MDA (nmol/ml) 7.46 ± 3.07 7.10 ± 3.72 NS
AUCI (μIU/ml/min) 6973.81 ± 856.12 7061.11 ± 912.87 NS
AUCG (mg/dl/min) 14 312.82 ± 1718.61 13 767.32 ± 1542.17 NS
Total C (mg/dl) 161.16 ± 31.85 166.68 ± 36.43 NS
LDL cholesterol (mg/dl) 101.83 ± 25.55 99.72 ± 26.34 NS

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HDL cholesterol (mg/dl) 48.22 ± 12.47 49.31 ± 16.01 NS
Triglyceride (mg/dl) 77.48 ± 34.43 78.47 ± 29.87 NS
Apo A (mg/dl) 110.27 ± 28.84 108.73 ± 25.67 NS
Apo B (mg/dl) 84.68 ± 21.73 86.18 ± 20.60 NS
Lp(a) (mg/dl) 21.54 ± 13.61 22.06 ± 14.87 NS

There was no correlation between oxidative stress and Hcy Homocysteine and lipid levels
levels, lipid levels and insulin resistance parameters (P > 0.05). Serum Hcy levels were significantly elevated in women with
Serum free testosterone showed a significant positive correla- PCOS compared with healthy women (P < 0.005). No significant
tion with MDA (P < 0.05, r = 0.41) and a negative correlation difference in serum fasting total cholesterol, LDL cholesterol,
with TAS (P < 0.05, r = –0.33) in PCOS patients. triglyseride and Apo B levels were observed between PCOS

Table III. Clinical and endocrinological parameters after the rosiglitazone and metformin treatments

Metformin Rosiglitazone

Before treatment After treatment P Before treatment After treatment P

(n = 25) (n = 25) (n = 25) (n = 25)
Age (years) 24.31 ± 4.75 23.92 ± 4.64
BMI (kg/m2) 22.16 ± 3.57 20.08 ± 3.14 <0.05 21.82 ± 3.44 23.13 ± 3.70 <0.05
W/H ratio 0.83 ± 0.09 0.82 ± 0.08 <0.05 0.83 ± 0.08 0.83 ± 0.08 NS
LH (mIU/ml) 9.89 ± 5.62 8.12 ± 4.62 NS 10.08 ± 7.81 7.47 ± 6.06 <0.005
FSH (mIU/ml) 5.96 ± 2.47 5.12 ± 2.38 NS 6.11 ± 2.27 5.75 ± 2.18 NS
LH/FSH ratio 1.65 ± 0.12 1.58 ± 0.11 NS 1.65 ± 0.18 1.28 ± 0.12 <0.005
Free testosterone (pg/ml) 3.01 ± 1.34 2.76 ± 1.27 NS 2.95 ± 1.06 2.04 ± 0.92 <0.005
DHEA-S (μg/dl) 317.25 ± 131.38 282.61 ± 121.56 NS 308.11 ± 112.56 218.73 ± 78.05 <0.005
Androstenedione (ng/ml) 2.96 ± 1.46 2.65 ± 1.41 NS 2.95 ± 1.58 2.17 ± 0.51 <0.005
Hcy (μmol/l) 13.52 ± 5.98 13.46 ± 6.01 NS 13.94 ± 6.72 13.02 ± 5.88 NS
HOMA-IR 3.18 ± 1.19 2.32 ± 0.87 <0.001 3.21 ± 1.45 2.02 ± 0.91 <0.001
AUCI (μIU/ml/min) 7061.1 ± 912.8 4412.6 ± 764.9 <0.001 6973.81 ± 856.12 4118.3 ± 652.95 <0.001
QUICKI 0.313 ± 0.021 0.382 ± 0.031 <0.001 0.321 ± 0.023 0.392 ± 0.031 <0.001
ISIOGTT 4.23 ± 2.58 6.19 ± 3.36 <0.001 4.26 ± 2.61 6.78 ± 3.75 <0.001
AUCG (mg/dl/min) 13767.3 ± 1542.1 13654.1 ± 1586.2 NS 14312.8 ± 1718.6 13832.1 ± 1705.4 NS
MDA (nmol/ml) 7.10 ± 3.72 6.35 ± 2.98 NS 7.46 ± 3.07 4.02 ± 2.34 <0.001
TAS (mmol/l) 1.00 ± 0.12 1.11 ± 0.13 NS 0.95 ± 0.07 1.21 ± 0.22 <0.005
Total cholesterol (mg/dl) 166.68 ± 36.43 156.72 ± 32.98 NS 161.16 ± 31.85 164.35 ± 32.09 NS
LDL cholesterol (mg/dl) 99.72 ± 26.34 95.36 ± 25.12 NS 101.83 ± 25.55 97.13 ± 27.42 NS
HDL cholesterol (mg/dl) 49.31 ± 16.01 51.54 ± 15.87 NS 48.22 ± 12.47 54.56 ± 16.01 NS
Triglyceride (mg/dl) 78.47 ± 29.87 73.71 ± 27.98 NS 77.48 ± 34.43 78.47 ± 29.87 NS
Apo A (mg/dl) 108.73 ± 25.67 110.86 ± 26.52 NS 110.27 ± 28.84 116.73 ± 22.85 NS
Apo B (mg/dl) 86.18 ± 20.60 78.94 ± 19.43 NS 84.68 ± 21.73 80.38 ± 20.60 NS
Lp(a) (mg/dl) 22.06 ± 14.87 21.19 ± 13.85 NS 21.54 ± 13.61 21.60 ± 19.44 NS
Vitamin B12 (pg/ml 311.19 ± 71.74 309.23 ± 71.12 NS 313.62 ± 72.38 314.48 ± 73.92 NS
Folic acid (ng/ml) 11.19 ± 3.09 11.58 ± 3.17 NS 11.57 ± 3.26 11.62 ± 3.29 NS

3336
 Oxidative stress in lean PCOS patients

and control subjects (P > 0.05). In PCOS patients, Lp(a) levels Talbott et al., 1998). The present study investigated both
were significantly elevated (P < 0.05) while HDL cholesterol classical CVD risk factors, such as insulin resistance and dysli-
and Apo A levels were lower when compared with the control pidaemia, and more recently emerging risk factors, such as
group (P < 0.05). Serum Hcy, vitamin B12, folic acid and lipid serum Hcy and oxidative stress.
levels did not change after treatment in either group (P > 0.05). Although the mechanisms leading to the development of
There was no correlation between Hcy and lipids, TAS, MDA, PCOS are still not completely understood, it has become
serum androgens and insulin resistance (P > 0.05). apparent that insulin resistance and hyperinsulinaemia may
play pivotal roles in the pathophysiology of PCOS (Dunaif
Menstrual disturbance and androgen levels et al., 1989; Dunaif, 1997). Insulin resistance is associated
Compared with controls, women with PCOS had significantly with obesity. However, hyperinsulinaemia and insulin resist-
elevated free testosterone, androstenedione, DHEA-S and LH ance are not considered to be related to obesity in patients with
levels (P < 0.001). Serum androgen levels and LH/FSH ratio PCOS (Dunaif et al., 1989) and insulin secretion is defective
decreased significantly in the rosiglitazone group (P < 0.005) also in lean patients with PCOS (Holte et al., 1994; Ehrmann
but did not change in the metformin group after treatment (P > et al., 1995; Holte et al., 1995; Cibula, 2004). In the present
0.05). The decrease in serum androgen levels in rosiglitazone- study, insulin resistance, as detected by the markers HOMA-IR,
treated patients was found to be independent of the decrease in AUCI, QUICKI and ISI, was significantly higher in lean
insulin resistance (β = 0.134, P > 0.05). patients with PCOS than in healthy controls.
Nineteen women had menstrual disturbance (13 oligomenor- A few previous studies have assessed the effects of
metformin and rosiglitazone in lean women with PCOS. In a

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rhoea, three secondary amenorrhoea, three polymenorrhoea) in
the metformin group before treatment. Nine (47.3%) of the 19 recent study, lean PCOS patients without insulin resistance
women with menstrual disturbance achieved regular cycles were given separate and combined treatments of metformin
after the therapy. Twenty women had menstrual disturbance and rosiglitazone. After the treatment, insulin resistance
(13 oligomenorrhoea, four secondary amenorrhoea, three improved only in the metformin group. Compared with the
polymenorrhoea) in the rosiglitazone group before treatment. placebo group, those patients who received metformin and
Eleven (55%) of the 20 women with menstrual disturbance rosiglitazone had a significantly higher rate of menstrual regu-
achieved regular cycles after rosiglitazone therapy. This differ- larization, increased ovulation and decreased androgen levels
ence did not reach significance (P > 0.05). (Baillargeon et al., 2004). Maciel et al. demonstrated that lean
women with PCOS respond better to treatment with met-
Comparison between post-treatment values of metformin and formin, compared with obese women (Maciel et al., 2004). In
rosiglitazone groups the present study, insulin resistance improved, although serum
androgen levels did not significantly change in the metformin-
After treatment, serum LH, free testosterone, DHEA-S,
treated subjects. However, both insulin resistance and hyperan-
androstenedione and MDA levels were significantly higher in
drogenism improved in the rosiglitazone-treated subjects. Both
the metformin group compared with the rosiglitazone group
groups displayed a similar increase in menstrual cycles.
(P < 0.05). HOMA-IR, AUCI, ISI, QUICKI, TAS, Hcy and
Oxidative stress is an accepted risk factor for the develop-
lipoprotein levels were similar in the two groups after treat-
ment of CVD (Betteridge, 2000). PCOS is also associated with
ment (P > 0.05). Although pre- and post-treatment TAS values
an increased risk of CVD. However, the oxidative stress state
in each group were statistically similar (P > 0.05), in the rosigl-
and the effects of current therapies on this state are not well
itazone group TAS was found to have increased following
established in PCOS patients. There are only two studies show-
treatment compared with the pretreatment levels (P < 0.005).
ing that oxidative stress is increased, and these studies
BMI was lower in the metformin group compared with the ros-
concerned obese PCOS patients (Sabuncu et al., 2001; Fenkçi
iglitazone group after treatment (P < 0.05), although W/H ratio
et al., 2003). In our study, serum TAS levels were significantly
was similar (P > 0.05).
lower in lean women with PCOS compared with healthy
controls. Higher serum MDA levels were found in patients than
Side-effects in controls. These findings implicate the presence of increased
All patients completed the study and were analysed for the oxidative stress in lean patients with PCOS. As the first
primary outcome after 12 weeks of follow-up. Ten patients derivative of thiazolidinedione used to treat DM, troglitazone is
(40%) in metformin group had gastrointestinal side-effects, chemically related to α-tocopherol, which is known as ananti-
such as nausea, vomiting, abdominal pain and diarrhoea. None oxidant (Garg et al., 2000). Hydroxylation of the phenyl and
of the subjects had elevated liver enzymes. pyridine rings in the chemical structure of rosiglitazone may
facilitate the scavenging of hydroxyl radicals. In animal
models, rosiglitazone has decreased oxidative stress, but this
Discussion issue has not been investigated in human studies (Sivarajah
Recent studies have shown that PCOS is not only a gynaeco- et al., 2003; Bagi et al., 2004). In some studies, the effects of
logical condition affecting women of reproductive age but also metformin on oxidative stress have been evaluated in diabetic
a comprehensive syndrome with a variety of associated patients, and conflicting data were obtained. Bonnefont-
metabolic disorders, such as insulin resistance and dyslipidae- Rousselot et al. demonstrated that metformin reduced
mia (Wild et al., 2000; Dunaif et al., 1989; Dunaif, 1997; oxidative stress (Bonnefont-Rousselot et al., 2003), while
3337
M.Yilmaz et al.

Pavloviç et al. showed that it increases oxidative stress (Boulman et al., 2004) did not detect a significant difference
(Pavloviç et al., 2000). We observed a significant increase in between patients with PCOS and healthy controls in terms of
the serum TAS level and a decrease in serum MDA level with Hcy levels. Increased serum Hcy level is an independent risk
rosiglitazone but not with metformin treatment. These results factor for CVD, which may put these subjects at a higher risk
suggest that rosiglitazone is capable of reducing oxidative of developing CVD. On the other hand, lowering plasma Hcy
stress in patients with PCOS. improves endothelial function in individuals with coronary
Oxidative stress is known to increase in parallel with factors artery disease and decreases the incidence of major cardiac
such as age, smoking, hyperhomocysteinaemia, hyperandroge- events (Schnyder et al., 2001). Four studies have elucidated the
naemia and insulin resistance. In one study, slightly higher impact of PCOS treatment on Hcy. Randeva et al. showed that
plasma MDA concentrations were reported in males than in regular exercise significantly lowers plasma Hcy in young,
females in a healthy population (Bolzan et al., 1997). Another overweight or obese women with PCOS (Randeva et al.,
study, also on antioxidant enzymes in healthy individuals, 2002). Vrbikova et al. reported that metformin treatment in
showed higher superoxide dismutase and lower glutathione women with PCOS may increase Hcy levels (Vrbikova et al.,
peroxidase activities in women than in men (Knight et al., 2002). Kilicdag et al., 2005a found the same results with
1987). It is not known whether hyperandrogenaemia has any metformin and rosiglitazone treatment in a similar study popu-
effect on oxidant and antioxidant status inwomen with PCOS. lation. They also showed that neither drug improved insulin
As for studies regarding PCOS, no correlation was found resistance and serum androgen levels (Kilicdag et al., 2005a).
between MDA, antioxidants and serum androgens (Sabuncu The same authors also reported that metformin treatment in
et al., 2001; Fenkçi et al., 2003). In contrast with previous studies, PCOS patients can lead to increase in Hcy levels and B-group

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we found a significant positive correlation between MDA and vitamins and folic acid administration decreased Hcy levels in
serum free testosterone and a significant negative correlation these patients (Kilicdag 2005b). In our study, there was no
between TAS and serum free testosterone in lean patients with change in Hcy levels in either treatment group.
PCOS. The present study has not shown a significant relation- Dyslipidaemia has been reported in women with PCOS. In
ship between oxidative stress and age, insulin resistance or accordance with previous studies, we noted an unfavourable
Hcy levels. The proportion of smokers was similar between lipid profile, including increased Lp(a) levels and decreased
PCOS and control subjects (20 and 17% respectively). HDL cholesterol and Apo A levels. These are considered to be
Metformin and glitazones may reduce androgen levels by risk factors for CVD (Wild et al., 2000; De Leo et al., 2003;
reducing pituitary gonadotrophin secretion, ovarian androgen Legro, 2003). No significant change in total cholesterol, LDL
secretion and adrenal androgen secretion, and by increasing the cholesterol, HDL cholesterol, triglyceride, Apo A, Apo B or
plasma levels of sex hormone binding globulin (SHBG) (De Leo Lp(a) was found after rosiglitazone and metformin therapies.
et al., 2003). The decrease in the circulating adrenal androgen Oxidative stress, hyperhomocysteinaemia, insulin resist-
levels might be secondary to the direct inhibitory effect of gli- ance and hyperlipidaemia are accepted risk factors for CVD.
tazones on the adrenal cortex (Azziz et al., 2003). Rosiglita- The present study confirmed that such risk factors are present
zone has also a direct but weaker inhibitory effect on both in patients with PCOS. These findings show that the risk of
P450c17 and 3β-Hydroxy steroid dehydrogenase II (3β-OH development of CVD is increased in PCOS. A decrease in
steroid DH II) than troglitazone (Arlt et al., 2001). Rosiglitazone insulin resistance could be reduced by both metformin and
inhibits two key enzymes involved in androgen biosynthesis; rosiglitazone. However, only rosiglitazone was able to lower
this effect is independent of its insulin-lowering effect. Met- oxidative stress in patients with PCOS. Studies performed on
formin has no direct effect on these enzymes, although it may PCOS have indicated that metformin could increase Hcy
decrease androgen levels by decreasing insulin levels (La Marca levels, although our study did not support this. These findings
et al., 2000). The present study concluded that a decrease in suggest that metformin may not facilitate the development of
serum androgen levels in patients treated with rosiglitazone is CVD by increasing Hcy levels and rosiglitazone may protect
independent of the decrease in insulin resistance. As already against CVD by lowering oxidative stress.
described, men appear to have higher oxidative stress than In the present study, we have evaluated the effects of these
women in healthy populations, and we found a positive corre- drugs on metabolic and hormonal parameters for 12 weeks,
lation between MDA levels and serum free testosterone levels although this was a short follow-up period for such a long-
and a significant negative correlation between TAS levels and lasting illness. Treatment for PCOS may continue for years
serum free testosterone levels. The reduction in oxidative stress because it is a chronic disorder, and these drugs are efficient as
in the rosiglitazone group may be explained by the decrease in long as they are administered. After re-evaluation of the
serum testosterone levels as a result of rosiglitazone therapy. patients at the end of 12 weeks, they were advised to continue
Recently, early atherosclerosis and increased risk of CVD their treatments in the same manner.
have been reported in patients with PCOS compared with In conclusion, in the present study elevated insulin resist-
healthy controls (Loverro et al., 2002; Randeva et al., 2002; ance, oxidative stress and plasma Hcy levels and changes in
Vrbikova et al., 2002; Schachter et al., 2003; Wijeyaratne serum lipid profile were found in our lean PCOS patients.
et al., 2004). Increased Hcy levels in patients with PCOS may These findings show that the risk of development of CVD is
partly explain these findings. In the present study we found increased in lean PCOS patients. Furthermore, we found that
higher Hcy levels in lean PCOS patients than control subjects. rosiglitazone seemed to decrease elevated oxidative stress
However, Orio et al. (Orio et al., 2003) and Boulman et al. when compared with metformin treatment. However, neither
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rosiglitazone nor metformin therapy for 12 weeks had any Fenkçi V, Fenkçi S, Yilmazer M and Serteser M (2003) Decreased total anti-
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