International Journal of

Introduction
According to several studies, presence of oxidative stress could cause molecular and genetic defects leading to infertility (1). Oxidative stress is usually associated with aerobic metabolism that generate pro-oxidant molecules (free radicals) or reactive oxygen species (ROS) (including hydroxyl radicals, superoxide anion, hydrogen  peroxide,  and
nitric oxide). There are complex interactions between the prooxidants and antioxidant molecules resulting in maintenance of intracellular homeostasis. Whenever there is an imbalance between the pro-oxidants and antioxidants, a state of oxidative stress is initiated. Some cells possess specific mechanisms to produce ROS required for cellular functions in low concentrations. Depending on ROS tissue concentration, they can exert beneficial physiologic effects and play a role in fertilization processes. For example, free radicals can influence the oocytes, sperm, and embryos in their micro environments, including follicular fluid, hydrosalpingeal fluid, and peritoneal fluid. These micro environments have a direct effect on quality of oocytes, sperm oocyte interaction, implantation, and early embryo development. Free radicals can also have pathological damage to cellular components including lipids, proteins and nucleic acids. There is a complex interplay of cytokines, hormones, and other stressors (such as irradiation, e.g. UV sunlight) that affects cellular generation of free radicals; these molecules act further through the modification of many transcription factors and gene expression (2). The modification may result in structural and functional alterations and impair many cellular processes. Pathological mechanisms of cell injury include lipid peroxidation, DNA damage, and apoptosis. Organisms have developed efficient protective mechanisms against excessive accumulation of ROS. ROS could be neutralized by an elaborate antioxidant defense system consisting of enzymes such as catalase, superoxide dismutase and glutathione peroxidase/reductase, and numerous non-enzymatic antioxidants such as vitamin C, vitamin E, vitamin A, pyruvate, glutathione, taurine and hypotaurine.
Whenever ROS levels become pathologically elevated, antioxidants begin to work and help to minimize the oxidative damage, repair it or prevent it altogether. The male and female genital tracts are rich in both enzymatic and non-enzymatic antioxidants. Follicular fluid contains high levels of antioxidants, which protect oocytes from ROS-induced damage. Significantly lower selenium levels were detected in follicular fluid of patients with unexplained infertility compared with those with tubal infertility or couples with male factor infertility. Elevated levels of ROS in peritoneal fluid may be the cause of infertility in some women with no other obvious causes.
Elevated levels of ROS can damage the ovum (after its release from the ovary), the zygote/embryo and most importantly, spermatozoa. Spermatozoa are very sensitive to oxidative stress. Based on some studies, 30-80% of unselected infertile men have oxidative stress-related infertility. Oxidative stress appears to be due to increased generation of ROS rather than a depletion of antioxidants. Therefore it is important to identify the source of increased ROS generation.
Antioxidant supplementation may or may not be effective depending on the pathology of the infertility (3). A number of drugs with antioxidative properties have been postulated to have a possible role in the management of idiopathic problems. However, WHO recommends application of traditional drugs in medical health service system. So recently, there has been a significant interest in finding natural antioxidants from plant materials to replace synthetic medicines (4). New studies have indicated that oral administration of Satureja Khuzestanica essential oil (SKEO) to rats induces a significant antioxidative property without appearance of any toxicity or unwanted effects.
Satureja Khuzestanica is an endemic plant in the Southern part of Iran. Its fame is due to the medical uses as analgesic and antiseptic in folk medicine resulted from the essential oil existing in that. There are marked differences between and within the subspecies of Satureja essential oil composition in the world. Recent studies indicated significant antioxidtive, antidiabetic, antihyperlipidemic, and reproduction stimulatory effects for oral administration of SKEO to rats.
No toxicity or adverse effects were observed (5) and also a significant increase in the number of implantation and live fetuses in female rats receiving SKEO was investigated (4). The present work aimed to review the effect of antioxidative drugs in infertility and the effectiveness of Satureja Khuzestanica in treating the problem.
 
Materials and methods
To investigate the effect of antioxidants on infertility, published literature was obtained by searching MEDLINE on the Pubmed search system and databases on Scopus interface. Filters were applied to limit the retrieval to randomized controlled trials. Regular alerts were established on MEDLINE and information retrieved through alerts is current to August 2010.
Parallel searches were performed on Cochrane library with all record status and No filters were applied to limit the retrieval by study type. The key words, antioxidants and infertility were used and controlled vocabulary such as the National Library of Medicine’s MeSH (Medical Subject Headings) and keywords were applied. The search was restricted to English language articles on human population. The articles obtained were screened based on the title and abstract and then selected for inclusion in the report. The criteria for article inclusion were study design (mainly randomized controlled trials), population (patient of any age with any primary problem of infertility were included in the study), intervention (any antioxidant), comparator (mainly placebo) and outcome (any clinical outcome). Duplicate publications of identical trials were excluded. Full text articles were obtained for all selected articles if possible and abstract was used for the rest. The Cochrane Reviewers’ Handbook applied to evaluate the quality of the articles. To investigate the effectiveness of Satureja Khuzestanica, the key word field of Pubmed and Scopus were searched without any limitation to be implicated. The search term was “Satureja Khuzestanica” and all the relevant literature was included in the study.
 
Results
By initial searching, a collection of 83 articles were obtained of which 22 studies (5-26) were identified based on their titles and abstracts and all of them included in the review. 15/ 22 of the studies were about the effectiveness of antioxidants in infertility (6-20) (Table I) and 7/ 22 of them were concerning the effectiveness of SKEO (5, 21-26) (Table II). Only 4 of the studies were randomized controlled trials with allocation concealment (6-9) and 3 of them were performed in in Vitro environment (13, 15, 20).
Based on the study by Keskes-Ammar et al sperm motility and viability are inversely correlated with semen MDA (Malonedialdehyde is produced from oxidation of polyunsaturated fatty acids) levels (12). Spermatozoal MDA concentration is also higher in men with decrease sperm motility (19).  Also DNA methylation has a significant negative correlation with sperm DNA fragmentation and seminal reactive oxygen species (ROS) production (16). The first observation of a correlation between the anti-oxidant substances and ROS generation in human semen was reported in 1995 (18) which are supported by several of other studies (11, 12, 16, 19). According to references (6, 8, 11, 12) (Table I), antioxidative supplementation produces a significant improvement of sperm motility. Two of the included studies showed that the total and per cycle pregnancy rates among the cases treated with antioxidant is higher than the control group (11, 19). The percentage of DNA-fragmented spermatozoa is markedly reduced by antioxidative drug therapy (7, 14, 15, 20). Antioxidative therapy may also be effective on improving the sperm count at least in a subset of oligospermic males (9). Proven fertile men has higher blood and spermatozoa levels of omega-3 fatty acids compared with the infertile patients and the ratio of serum omega-6/omega-3 fatty acids is significantly higher in infertile patients in comparison to fertile controls (17). Among all the evidences supporting the usefulness of anti oxidants in infertility, in the study by Tarin et al no significant effect of Ascorbate on fertilization, number of cells and embryo grade or percentage of embryos was observed (13). The information obtained from the studies evaluating the effectiveness of SKEO, are summarized in table II.
In the study of Vosough- ghanbari et al in 2008, the patients treated with SK showed significant decrease in total cholesterol, LDLC and increase in HDL-C and total antioxidant power (TAP) compared to baseline while the patients in the placebo group showed no changes (21). In the study by Abdollahi et al (22), SKEO therapy decreased the normal lipid peroxidation level and increased significantly the antioxidant capacity. Blood glucose and triglycerides levels were also decreased significantly. In the study by Ghazanfari et al (23), elevated lipid peroxidation and myeloperoxidase activity was significantly restored by SKEO administration, also SKEO -treated groups showed significantly lower score values of macroscopic and microscopic characters when compared to the experimentally induced IBD group. Generally, the beneficial effect of SKEO was comparable to that of prednisolone. Saadat and colleagues (24) showed that SKEO therapy did not affect on the blood glucose levels but hepatic phosphoenolpyruvate carboxykinase (PEPCK) activity decreased by 26% and hepatic glycogen phosphorylase (GP) increased by 24% in comparison to control group. One of the studies included in our review showed that SKEO could protect rats treated with cyclophosphamide from hemorrhagic cystitis (25) and in another study; protection of the reproductive system in these animals was observed (26).
The effects of SKEO on infertility were investigated by Haery et al (5) in which the potency and fecundity of the male rats administered SKEO were significantly higher than those of the age-matched control group. Fertility index and litter size were also significantly improved as well as decreasing in post implantation loss in mated females of the treated groups. There was no effect on the serum LH by SKEO therapy but concentration of FSH increased significantly in the group treated with SKEO. SKEO increased significantly serum testosterone levels in all doses used but no change was observed in serum concentration of estradiol. In male rats treated with SKEO, the number of spermatogonium, spermatid cords, Leydig cells, and spermatozoids was increased according to histopathological analysis. The sertoli cells were also hypertrophic in this group.
In our searching strategy, no appropriate RCT on infertility in women was found, however we encountered with the study of Ghanem et al in which significantly higher pregnancy rate was found among the patients treated with combination of clomiphen and an antioxidative drug (10). Also Tamura and colleagues investigated the effect of melatonin, an antioxidant, on intra-follicular oxidative stress in vitro and realized their significant negative relationship (20).


Table I. Human studies evaluating antioxidants effectiveness in infertility.



Table II. The studies evaluating the anti oxidative effects of Satureja Khuzestanica essential oil.


Discussion
The results obtained from the trials included in our mini-review, confirm the protective and usefulness of antioxidants on infertility and reproduction system. For example, the higher blood and spermatozoa levels of omega-3 fatty acids in comparison to the infertile patients (17), is in agreement with the usefulness of antioxidants in infertility because Omega-3 fatty acids are known potentially important antioxidants. These substances are an element in all cell membranes and maintain the properties of the lipid bilayers. The lipid bilayers also exist in the spermatozoa membrane and are essential for the fluidity and flexibility of spermatozoa, and successful fertilization.
There is also a close relationship between oxidative stress and poor oocyte quality (20) but unfortunately in our searching strategy, no randomized clinical trial was found on women infertility treatment with antioxidants. Therefor appropriate controlled studies designing in this population is needed.
However, Ghanem et al observed significantly higher pregnancy rate among the patients treated with combination of clomiphen and an anti-oxidative drug (10). But there was no comparison between a group treated with antioxidant alone and a group as control. Therefore the effectiveness of the antioxidant used in the trial, could not be judged well.
Despite all the evidences represent the usefulness of anti oxidants in infertility, the study of Tarin et al showed no significant effectiveness of ascorbate on fertilization, number of cells and embryo grade or percentage of embryos. However, the positive effect of antioxidants on fertilization and embryo development in vitro cannot be totally ruled out until the effects of other, non-physiological concentrations of ascorbate and longer-term embryo cultures have been tested (13).
Given the effectiveness of antioxidants in management of infertility, Satureja Khuzestanica which has established antioxidative properties (22, 23) could be proposed to represent reproduction stimulatory effects.
Of the studies in which the antioxidative moiety of SKEO has been assessed, the trial of  Vosough-ghanbari et al could be pointed which was performed in 2008 and showed the patients treated with SK, represent significant decrease in total cholesterol, LDLC and increase in HDL-C and total antioxidant power (TAP) compared to baseline (21). Generally, it appears that plants particularly those with high levels and strong antioxidative compounds have an important role in improvement of disorders relating to oxidative stress such as diabetes mellitus (27).
SKEO which has antioxidative properties should be useful in diabetes and its complications. The results obtained from the study also confirmed the effectiveness of SK on improving patients’ total cholesterol, LDL-C, HDL-C and TAP (21).
Ghazanfari et al (23) showed significantly lower score values of macroscopic and microscopic characters were observed, compared to the experimentally induced IBD group. As elevated lipid peroxidation and myeloperoxidase activity was also significantly restored by SKEO administration, it seems that antioxidative property is one of the mechanisms by which this plant might have protective effects.
Saadat et al (24) realized that SKEO therapy did not affect the blood glucose levels but decreased hepatic phosphoenolpyruvate carboxykinase (PEPCK) activity by 26% and increased hepatic glycogen phosphorylase (GP) by 24% in comparison to control group. Disturbance of hepatic glucose metabolism has been proposed as a mechanism of anti-diabetic action of SKEO which could be in relation with antioxidative effect of this plant.
The PEPCK gene in liver is present in most models of diabetes, and is thought to contribute to the increased hepatic glucose output seen in diabetes. Thus any medicine to alter hepatic gluconeogenesis or glycogenolysis might have significant effect on glucose hemostasis.
According to existing reports, troglitazone also inhibits expression of the PEPCK gene in isolated hepatocytes by an antioxidative property which is due to continuation of the alpha-tocopherol (vitamin E) moiety in its chemical structure (28). Furthermore metformin which inhibits hepatic gluconeogenesis, produces concurrent antioxidative effects anbhave most benefits in treatment of diabetes (29) in view of the ability of SKEO to reduce hepatic PEPCK activity and considering above statements, the first mechanism that may come to mind is its antioxidative properties.
The human and animal studies of Satureja Khuzestanica show significant antioxidative potential of the plant. Injury of the plant cells is associated with the occurrence of oxidative mechanisms that may explain why an abundance of antioxidative compounds have been extracted from plant tissue (4) Various animal models including inflammatory bowel disease, diabetes, and hyperlipidema in rat have been investigated and most of them have been treated by SKEO according to its antioxidative properties. As a result, the effectiveness of SKEO in infertility could be assumed by considering its antioxidative properties. Of course this hypothesis has been established in rats by the study of Haeri et al in which histopathological analysis in male rats treated with SKEO demonstrated the increase of the number of spermatogonium, spermatid cords, Leydig cells, and spermatozoids and hypertrophic Sertoli cells (5). The result of the study also supports the previous discovery on the reproduction stimulation indicated by enhancing the number of live fetuses per litter in dams treated by SKEO (22). The results were approved by decreased blood lipid peroxidation and increased total antioxidant power (5).
Flavonoids, mainly p-cymene and carvacrol have been documented in the SKEO and have been well analyzed by GC-mass. Their positive effects are due to their ability to inhibit lipid peroxidation, chelate redox-active metals, and attenuating other processes involving reactive oxygen species. Also carvacrol represents significant antioxidant properties (30-32). The increase in the weight of the testis and weights of the epididymis, seminal vesicles and ventral prostate in SKEO-treated rats would be the result of decrease in lipid peroxidation. Therefore, improved fertility observed in the study of Haeri et al might be due to the antioxidant effect of SKEO.

Conclusion
 
Although the methods and clinical settings of the controlled trials included in our mini-systematic review were diverse, 14/ 15 (93.33%) of the trials proved that there is an improvement in either sperm quality or pregnancy rate after antioxidative therapy, however, randomized placebo clinical trials with adequate power are needed to guide clinical practice (33).
     As the antioxidative properties of SKEO have been established and its effectiveness also has been observed in vitro, its effectiveness in human infertility could be hypothesized. Further preclinical evaluations and clinical trials in humans are necessary to identify a possible place for SKEO in therapies of infertility (5).