Co-Administration of Metformin and N-Acetyl Cysteine Fails to Improve Clinical Manifestations in PCOS Individual Undergoing ICSI

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 (1). A rise in the serum levels of androgen, insulin and luteinizing hormone (LH), menstrual dysfunction, hirsutism, infertility and obesity are some of the complications which are associated with PCOS (2). In addition, endometrial hyperplasia, cancer, type II diabetes, hypertension and dyslipidemia are considered as long term consequences of this syndrome (3). Studies have concluded that treatment with insulin-sensitizing agents such as metformin (MTF) results in a decrease of the levels of serum lipids, androgen and insulin; regulation of menstrual cycles; and promotion of both spontaneous and induced ovulation which leads to an increase in pregnancy rate (4). MTF has been used in the treatment of PCOS for a long time; however, gastrointestinal side effects, hypoglycemia and an increase in the serum homocysteine levels in some patients are associated with consuming MTF (5, 6). Hyperhomocysteinaemia is regarded as risk factors for cardiovascular diseases, thrombophilia, pre-eclampsia and recurrent abortion (7, 8).

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 (9, 10), lowers the serum homocysteine levels and promotes detoxification (11). Since clinical evidence suggests that hyperhomocysteinaemia leads to endothelial dysfunction through generation of reactive oxygen species (12), NAC can preserve vascular integrity and protect against ischemic injuries (13).

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.

PCOS has been a research subject over the past six decades, while it is associated with insulin resistance, hyperandrogenism, obesity, dyslipidemia and infertility (16). The beneficial effects of MTF, an insulin sensitizer (5-7), and NAC (9, 11, 17) on biochemical and clinical aspects of PCOS have been demonstrated by many studies. MTF appears to reduce the output of hepatic glucose, declining insulin concentrations and reducing the androgen production by theca cells (18). On the other hand, NAC is commonly used as a safe drug which increases the cellular levels of reduced glutathione. NAC also influences insulin receptor activity and significantly decreases T levels and free androgen index values (9, 17).

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(17), and this is the first report of the reduced level of insulin following combination therapy. There is also some contrary reports (9, 19, 20) which could probably be due to the differences in patient inclusion criteria, dosing and duration of therapy, and genetic background of individuals.

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 (21). These observations are in line with previous report on leptin which is considered to play an important role in the pathogenesis of PCOS that influences the LH production (22). Furthermore, in our study, a significant positive correlation was observed between leptin and insulin concentrations before and after treatment, indicating that insulin reduction was responsible for lowering leptin secretion. Moreover, it should be emphasized that when multiple regression analysis was performed, such correlation was found. Other study (23) have also shown that leptin concentrations correlated significantly and positively with insulin in women with PCOS. On the other hand, in a study of Yarali et al. (19), six weeks of treatment with 850 mg MTF twice daily made no significant decrease in serum leptin levels compared to the placebo, which is probably due to the differences in drug doses and protocols of ovulation induction.

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 (17). Insulin directly stimulates androgen production from theca-cells by increasing the activity of cytochrome P450-C17a, and it also induces adrenal esteroidogenesis (16). Therefore, MTF lowers the level of serum androgens through direct inhibition of androgen production in human ovarian theca cells and also by improving insulin sensitivity, indirectly (24). On the other hand, NAC decreases the expression of several key enzymes involved in T production such as 17β-hydroxysteroid dehydrogenase (17β-HSD) and 3β-hydroxysteroid dehydrogenase (3β-HSD)(25), leading to a decrease in the levels of T production. However, in contrary to our expectations, MTF and NAC combination did not cause a significant decrease in testosterone levels, probably due to the effects of drug interference which need future investigation. In our study, as well as previous study (22), leptin concentrations correlated significantly and positively with TT in PCOS patients, which likely suggests that a decrease in leptin levels can reduce esteroidogenesis. An abnormal lipid profile is a common finding in PCOS which elevates levels of total cholesterol, triglycerides, and LDL, while decreases levels of HDL (26). In our study, similar to previous study (27), an improved lipid profile was obtained following NAC and MTF administration by means of reduced TG and total cholesterol. These effects were not observed in combination therapy, and we have no suggestion for these observations, but drug co-treatment remains to be explored and needs future validation.

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 (28, 29). Leptin has peripheral actions to stimulate vascular inflammation, oxidative stress and cardiovascular disease (30). Leptin via Janus kinases(JNK) pathway [one of the intracellular signaling pathways; mitogen-activated protein (MAP)-kinase] increases the transcription factors activity of NF-kB (nuclear factor-kB) and AP-1(activator protein-1), thereby increases reactive oxygen species (ROS) through induction of cellular oxidation (31). A significant positive relationship between increased levels of MDA, as a marker of lipid-peroxidation and of increased oxidative stress, and the total testosterone and insulin levels in PCOS patients was observed in this study, confirming the existence of increased oxidative stress in patients with PCOS (32). In our study, following treatment, the level of MDA in the MTF+NAC group was not significantly different from the NAC group, but has a significant reduction compared to MTF group. This could be due to the fact that MTF may raise oxidative stress through increased homocysteine levels which is associated with increased generation of superoxide anions and decreased activity of antioxidant enzymes (32). Since NAC, as an antioxidant, inhibits the transcription factors activity of NF-kB and AP-1, it influences the activity of the signaling pathway of MAP-kinase and results in a reduction in the oxidative stress (11-13); therefore, it is possible that NAC can decrease elevated oxidative stress in PCOS patients taking MTF.

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 (19) about MTF+NAC group as compared with the control. However, the reduction of AMH level in the treatment groups, which was close to be significant (p=0.051), is consistent with literature, so it has been suggested that long-term administration is required for a reduction in the raised AMH level observed in PCOS individuals (33), indicating that these drugs can reduced the number of AMH producing follicle which is appropriate for PCOS patient.

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.