Polycystic ovarian syndrome (PCOS) is an ovarian dysfunction syndrome with the combination of heterogeneous symptoms and signs; it is considered the most prevalent endocrinopathy resulting in anovulation up to 10% of women at reproductive age (
N-acetyl cysteine (NAC) is an acetylated variant of L-cysteine containing sulfhydryl groups which acts as a powerful antioxidant, antiapoptotic and free-radical scavenger; it also stimulates production of glutathione (
Since the effects of co-administration of MTF and NAC compared to MTF and NAC alone in reducing metabolic and hormonal factors such as insulin, leptin and MDA in patients with PCOS has not been evaluated yet, we have evaluated and compared the efficacy of co-treatment of MTF and NAC on clinical, metabolic and hormonal aspects during course of ovulation induction in PCOS individual undergoing ICSI cycle.
Through a prospective randomized clinical trial, placebo controlled pilot study, in the interval between July 2012 and February 2013, 80 infertile PCOS individuals at the age of 25-35 years, candidate of ICSI, were enrolled. This study was conducted in the
Patients needed to fulfill PCOS diagnostic criteria which was based on the Rotterdam consensus workshop in 2003 (
An approval from the Research Ethics Committee of Royan Institute, Iran, and an informed consent from participants were obtained. All patients were asked to avoid any changes in their normal physical activity and diet and also not to take any new pharmacotherapy during the study.
The patients were examined by the gynecologists. The subjects were randomly selected to receive either MTF and NAC or placebo. Eighty patients were divided into four groups (N=20): i. NAC (NAC group) which received NAC (Holzkrichen, Germany, batch no. 6N5483; 600 mg three times daily), ii. MTF group which received MTF (Glucophage, Merck, West Drayton, UK; 500 mg three times daily), iii. MTF+NAC group which were co-treated with MTF and NAC with the offered doses three times daily, and iv. placebo (PLA group) which received oral rehydration salts (ORS, Poursina, Tehran, Iran; batch no.30) three times daily, for a period of six weeks. Before beginning treatment, patients were also asked to report any possible adverse effects during the treatment, and they were evaluated for any presenting main complaints at the end of the treatment period.
A total number of 20 patients (6 from MTF group, 4 from NAC group, 6 from MTF+NAC group and 4 from PLA group) were dropped out due to intolerance of medication, monofollicular development, and failure of ovulation induction during ICSI cycle, and ultimately, 60 patients remained in the study.
Patients with PCOS undergoing ICSI treatment using a long GnRH agonist protocol received MTF, NAC, or MTF+NAC tablets, randomly, from third day of menses of previous cycle until the day of oocyte aspiration.
All patients received oral contraceptive pills (OCPs) for 21 days starting simultaneously with MTF, NAC or MTF+NAC on day 3 of menses of the cycle prior to the treatment cycle. Ovarian down-regulation was initiated with daily buserelin acetate 1 mg (Suprefact, Aventis, Germany), beginning on day 19 of the preceding menstruation (Luteal phase) and after ovarian down-regulation was achieved day 2 of last menstrual period (LMP) and the dose was then reduced to 0.5 mg when the thickness of the endometrium was <4 mm. Ovarian stimulation began with daily injections of average 2 ampoule of recombinant follicle stimulating hormone (rFSH; Gonal-f, Merck Serono S.A., Geneva, Switzerland). Following cycle monitoring, using vaginal sonography (HS 4000, Honda Electronics Co., Japan), ovulation was induced by the administration of average 10,000 IU human chorionic gonadotropin (HCG; Pregnyl, Organon, Netherlands). When at least three follicles had reached diameters of 16-18 mm, transvaginal oocyte aspiration was performed with ultrasound guidance under general anesthesia 36 hours after injection of HCG.
The body mass index (BMI), the waist/hip ratio (WHR) and the blood pressures were recorded for each patient once before the treatment on the third day of menstruation and once in the day of ovum pick up (OPU) of ICSI cycle. Fasting blood samples were collected from each individual once before the treatment on day 3 of menses of previous cycle before ICSI and once during oocyte aspiration. The peripheral blood sample taken from each patient were immediately centrifuged for 10 minutes at 3000 rpm (EBA20, Hettich, UK), and serum samples were stored at -70˚C for future evaluadtion and analysis.
The serum levels of follicle stimulating hormone (FSH, mIU/ml, Cat.N.DE1288), luteinizing hormone (LH, mIU/ml, Cat.N.DE1289), prolactin (PRL, ng/ml, Cat.N.DE1291), total testosterone (TT, ng/ml, Cat.N.DE1559), fasting insulin (mIU/L, Cat.N.DE2935), estradiol (E2, pg/ml, Cat.N.DE2693) and dehydroepiandrosterone sulfate (DHEA-S, ng/ml, Cat.N.DE1562) in all samples were measured using ELISA enzyme immunoassay (Demeditec Diagnostics GmbH, Germany) for hormonal profile. The serum levels of fasting blood sugar (FBS, mg/dl), cholesterol (Chol, mg/dl), high density lipoprotein cholesterol (HDL, mg/dl), low density lipoprotein cholesterol (LDL, mg/dl), triglyceride (TG, mg/dl) and very low density lipoprotein cholesterol (VLDL, mg/dl) were measured using electro-chemical luminescent technique kits (E-411, Roche Company Germany). Serum levels of anti-Mullerian hormone (AMH, ng/ml) were assessed using a second generation enzyme immunoassay (AMH-EIA kit, Cat.N.A92269C, Immunotech Beckman Coulter Company, USA) according to the supplier’s instructions. Serum leptin (ng/ml) level was also measured with the LEPTIN (Human) ELISA Kit (Cat.N.KA0025, Abnova Corporation, Taiwan) using sandwich enzyme immunoassay technique. The level of serum malondialdehyde (MDA, μM), a naturally occurring product of lipid peroxidation, was measured using the thiobarbituric acid (TBA) colorimetric method by TBARS Assay Kit (Cat.N.KA1381, Abnova Corporation, Taiwan).
The normality of continuous variables were confirmed using the Kolmogrov-Smirnov test and data were reported as means ± SD. Data were analyzed with one-way ANOVA and the Tukey’s test for post-hoc analysis. Chi-squared test was used for the statistical analysis where appropriate. Means were considered significantly different at p<0.05. Pearson’s correlation test was performed to define the correlation between variables. Multivariate linear regression analysis was done to test the effect of leptin on hyperinsulinemia and on increased stress oxidative. All data were analyzed with a Statistical Package for the Social Sciences (SPSS Inc., Chicago, IL, USA) version 16.
There were no significant differences in the age, duration of marriage, duration of infertility, the occurrence of oligomenorrhoea, amenorrhoea and hirsutism between the four groups are presented in table 1.
Also, there were no significant differences in weight, height, BMI, waist size, hip size and WHR in the treatment groups are presented in table 2.
Clinical characteristics of the four groups of PCOS patients
|Parameters||Treatment groups with|
Data are shown as mean ± SD. Analysis was performed by ANOVA followed by the Tukey’s test for multiple comparisons.
* ; Analysis was performed by Pearson Chi-squared test for comparisons, NS; No differences were observed between the mean of variables in the experimental groups compared with placebo, NAC; N-acetyl cysteine, MTF; Metaformin and PLA; Placebo.
Clinical features before and after treatment in PCOS patients
|Parameters||Treatment groups with|
Serum levels of LH, total testosterone, cholesterol and triglyceride, insulin and leptin significantly reduced in the MTF and NAC groups compared to the placebo (p<0.05). But levels of LH, total testosterone, cholesterol and triglyceride had no significant reduction in the MTF+NAC groups compared to the placebo (p>0.05). The serum levels of insulin and leptin reduced significantly after treatment in the MTF+NAC group compared to the placebo (p<0.01). Unlike the MTF+NAC and NAC groups, the level of MDA in the MTF group was not significantly decreased compared to placebo group (p>0.05). Levels of FBS, FSH, PRL, E2, DHEA-S, LDL, HDL, VLDL and AMH ratio were not significantly different in treatment groups and to those of the placebo group, data are presented in table 3.
Biochemical and hormonal parameters before and after treatment in serum of PCOS patients
|Parameters||Treatment groups with|
Data are shown as mean ± SD. Analysis was performed by ANOVA followed by the Tukey’s test for multiple comparisons. Significant differences for the comparison between treatments are in bold type.
a; Differences were observed between in the experimental groups compared with placebo,b; MET group compared with placebo 6 weeks after treatment.(p < 0.05) , c; NAC group compared with placebo 6 weeks after treatment.(p < 0.05) and d;. MTF+NAC group compared with placebo 6 weeks after treatment (p< 0.05). NS; No differences were observed between the mean values of variables in the experimental groups compared with placebo (p > 0.05).
Our results showed a significant positive correlation between insulin with BMI (r=0.553, p=0.0001), LH (r=0.371, p=0.004), E2 (r=0.699, p=0.0001) and TT (r=0.544, p=0.0001) before drug administration. Although the serum levels of leptin showed a significant positive correlation with BMI (r =0.379, p=0.003), insulin (r=0.592, p=0.0001), E2 (r=0.547, p=0.0001) and TT (r =0.549, p=0.0001), but no significant correlation was found between leptin concentrations with LH (r=0.168, p=0.201) and FSH (r=-0.143, p=0.276) before drug treatment. However, there was only significant positive correlation between leptin and insulin (r=0.562, p=0.0001) after drug administration. Our results also revealed that MDA levels showed a significant positive correlation with insulin (r=0.307, p=0.017) and TT (r=0.332, p=0.009) before drug administration, data are demonstrated in table 4. Moreover, there was only significant positive correlation between MDA and insulin (r=0.591, p=0.0001) after drug administration (
Upon performing multivariate analyses after adjusting for BMI, LH, FSH, E2, TT, insulin and MDA on leptin there was a strong statistically significant relationship between leptin and both insulin (p<0.01) and MDA (p<0.05,
Considering the amount of data and length of the manuscript, the data regarding clinical outcomes of ICSI will be presented in a different manuscript.
Correlations between blood serum variables in the four groups of PCOS patients before and after treatment
|Parameters||Insulin||P value||leptin||P value||MDA||P value|
Significant differences for the comparison between treatments are in bold type. MDA; Malondialdehyde, E2; Estradiol, LH; Luteinizing hormone, FHS; Follicle stimulating hormone and TT; Total testostrone.
Multiple linear regression analysis of the leptin level on insulin and MDA before and after treatment
|Before treatment||After treatment|
|Coefficient||Standard error||P value||95% Coefficient interval||Coefficient||Standard error||P value||95% Coefficient interval|
Significant differences for the comparison between treatments are in bold type. MDA; Malondialdehyde.
PCOS has been a research subject over the past six decades, while it is associated with insulin resistance, hyperandrogenism, obesity, dyslipidemia and infertility (
Considering the benefits of MTF and NAC consumption in patients with PCOS, we decided to investigate whether a combination therapy of MTF and NAC during the course of ovarian stimulation during ICSI cycle in patients with PCOS will introduce a better effect in reducing hyperinsulinism and hyperandrogenism and in normalizing other biochemical and endocrine parameters rather than using MTF or NAC alone. We should stress that our study is a pilot study, and there is a lack of literature on combined administration of these drug, especially during course of ovarian stimulation.
Our results showed that 6 weeks of co-treatment with NAC and MTF is significantly effective in reducing the levels of insulin compared with the control. Although this result supports previous studies which have reported a significant change in the fasting insulin levels after the consumption of MTF and NAC compared with the placebo group(
Concomitant with reduced hyperinsulinaemia, a significant decline in the serum levels of leptin upon treatment with MTF, NAC and MTF+NAC groups was observed compared to the control group. This observation was consistent with background literature on MTF and NAC (
Our results revealed a significant reduction in the levels of TT after 6 weeks of treatment with NAC and MTF alone, which is in consistent with previous findings (
Several studies have pointed out an increase in oxidative stress in PCOS patients which is influenced by the following factors: age, hyperhomocysteinemia, hyperandrogenemia and insulin resistance (
There was no significant reduction in the FBS, PRL, E2, DHEA-S, LDL, HDL and VLDL levels after 6 weeks of treatment with NAC and MTF group, which is in agreement with previous findings (
Despite the fact that MTF and NAC have different influence of action (i.e. one as insulin sensitizers and other as an antioxidant), we observed no beneficial effect of these drugs which administered together during course of ovulation induction for ICSI. In contrast to our expectation, the beneficial effect of each drug on reducing LH, TT, cholesterol and TG which was observed when MTF or NAC was administered alone rather than combination therapy. Therefore, we conclude that co-administration of these drugs might not be useful for PCOS patient during course of ovulation stimulation. However, the beneficial effects of co-administration of the two drugs for routine treatment of PCOS patient remain to be evaluated.