Introduction
Polycystic ovary syndrome (PCOS) with a prevalence of 10-15%, is a complex endocrine disorder associated with dyslipidemia [1, 2]. For the treatment of PCOS, different methods such as lifestyle modification, medication, and surgery are used [3]. The most common method is the medication therapy but due to the many side effects of drugs, it is very important to find alternative methods.
Intestinal microbes (microbiome) protect against pathogens, regulate host metabolism and immunity, and modulate intestinal barrier integrity [4, 5]. Evidence suggests a possible role for the intestinal microbiome in the pathogenesis of PCOS [6]. Tremellen et al. proposed the theory of Dysbiosis of Gut Microbiota (DOGMA). According to this theory, a disrupted balance of intestinal microbes, due to the production of toxic products by these microbes, can lead to an inflammatory response by the host immune system and impair the function of insulin receptors (creating insulin resistance). The resulting hyperinsulinemia interferes with follicular growth and maturation and stimulates the production of androgens by ovarian monocytes. Thus, it causes all three classic features related to PCOS (irregular menstrual cycles, androgen excess, and polycystic ovaries) [7]. On the other hand, a high-sugar, high-fat, and low-fiber dietary regime as well as obesity increase the permeability of intestinal mucosa [8]. In case of intestinal damage which occurs in PCOS patients, Lipopolysaccharide (LPS) enters the bloodstream whose inflammatory effects play a key role in exacerbating the symptoms of PCOS [9]. 
Previous studies have shown the beneficial role of probiotics in various diseases such as gastrointestinal diseases and diabetes. Considering the effects of probiotic supplements on some metabolic disorders such as insulin resistance and inflammation, and the relationship of these disorders with PCOS, it seems that probiotics or synbiotics have favorable effects on PCOS [10, 11]. This study aims to investigate the effect of synbiotics on serum parameters including Follicle-Stimulating Hormone (FSH), Luteinizing Hormone (LH), triglyceride, cholesterol (CL), glucose, and insulin in LPS-treated rats with PCOS.
Methods
In this experimental study, 40 adult female Wistar rats weighing 180-200 g were prepared from the Pasteur Institute of Iran and were kept at a temperature of 22 ° C and in a 12-12 h light cycle. All had free access to water and food. For PCOS induction in animals, an aromatase inhibitor, namely letrozole (Iran Hormone Company, Iran) was used [12]. All rats in the estrous cycle were given 1 mg/kg of letrozole orally for 21 days. After daily administration, the vaginal smear test was conducted until the changes and irregularity happened in the estrous cycle and reached the stage of persistent vaginal cornification.
The rats were randomly divided into five groups of control (n=8, not received any treatment), PCOS (n=8, received 1 mg/kg of letrozole for 21 days by gavage), PCOS + LPS (n=8, received 3 mg/kg of LPS intraperitoneally prepared from SIGMA-Aldrich Company, Germany), PCOS + Synbiotic (n=8, received 20 CFU/ml of synbiotic supplement by gavage or by mixing in a drinking water prepared from Zist Takhmir Company, Iran), and PCOS+LPS+Synbiotic (n=8, received 3 mg/kg of LPS intraperitoneally plus synbiotic supplement). 
One day after receiving the last dose of the extracts (22nd day), the animals were anesthetized with 100/20 mg/kg of Ketamine/Xylazine [13]. After cutting their chest, 5 mL blood was collected from the ventricle with a 5-mm syringe. To show the effectiveness of the treatments, the serum levels of endocrine variables related to inflammation and PCOS were measured by ELISA kits and finally the data were evaluated using statistical tests (one-way ANOVA followed by Tukey’s post-hoc test) in SPSS v.22 software (IBM, USA). Kolmogorov-Smirnov test or Shapiro-Wilk test was used for evaluating the normality of data distribution. Data were described as Mean±SEM, and a P<0.05 was considered as the statistically significance level.
Results 
As shown in Table 1, letrozole increased serum levels of triglycerides (TG), cholesterol, insulin, FSH, LH, and glucose in PCOS rats compared to the control group (healthy rats) which was statistically significant (P<0.05).


Triglyceride, cholesterol, insulin, glucose, FSH, and LH levels in the PCOS and PCOS+LPS groups were increased significantly compared to the healthy group (P<0.05). Treatment with synbiotics in both intervention groups caused a significant reduction in triglyceride, cholesterol, insulin, glucose, FSH, and LH levels.
Discussion
 Analysis of serum markers in the present study showed a significant increase in triglyceride, cholesterol, FSH, LH, insulin, and glucose levels after PCOS induction by letrozole [14]. LPS, produced by the intestinal flora, are vital molecules in causing inflammatory and microbial diseases and have an endotoxin effect. LPS enters the bloodstream, binds to the LPS-binding protein (LBP), and CD14 toll-like receptor complex (TRL-4) on the surface of innate immune cells, activating the signaling pathway. This activity of the immune system interferes with the function of the insulin receptor, leading to increase in the serum level of insulin [15]. At the same time, the presence of a permeable gut increases TNF-α and IL-6, which is mediated by the activation of endotoxin-induced macrophages [16]. In PCOS patients, the expression of TNF-α and IL-6 is increased, which is related to insulin resistance [7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17]. Insulin resistance and chronic inflammation are closely related to each other; when the function of the intestinal barrier is disrupted, the endotoxin enters the bloodstream to cause chronic inflammation of the ovaries and insulin resistance, which enhances the occurrence and progression of PCOS [18].
Rashad et al. found that probiotic supplementation (L delbrueckii and L fermentum) for 12 weeks significantly reduced the fasting plasma glucose level and improved the lipid profile [19]. Ghanei et al. reported that probiotic supplementation for 12 weeks balanced inflammation in women with PCOS [20]. One study showed that consuming synbiotic pomegranate juice for 8 weeks in women with PCOS increased insulin sensitivity [21]. Nasri et al. and Samimi et al. showed that synbiotic supplementation (L acidophilus, L casei, B bifidum + inulin) for 12 weeks decreased serum insulin levels in women with PCOS [22, 23]. Synbiotic supplementation has beneficial effects in reducing the symptoms of PCOS including lipid profile, gonadotropins, insulin and glucose.


Ethical Considerations
Compliance with ethical guidelines
This study was approved by the Ethics Committee of Guilan University of Medical Sciences (Ethics Code: IR.GUMS.REC.1396.239).

Funding
This work was funded by Guilan University of Medical Sciences (Grant number: 96062508).

Authors' contributions
Conceptualization and design, data curation, data analysis, data interpretation, review, funding acquisition, and supervision: Mojtaba Hedayati Ch; Conceptualization and design, data curation, data analysis, data interpretation, review, funding acquisition, drafting, supervision, and statistical analysis: Mahmood Abedinzade; Conceptualization and design, data curation, data analysis, data interpretation, review, supervision, statistical analysis: Zahra Bostani Khalesi; Conceptualization and design, data curation, data analysis, data interpretation, review, and supervision: Behrooz Khakpour Taleghani; Investigation, drafting, and review: Seiyedeh Narjes Naseran, Tahere Pourmirzaee.

Conflicts of interest
The authors declared no conflict of interest.

Acknowledgements
The authors would like to thank the Vice-Chancellor for Research and Technology of Guilan University of Medical Sciences for financial support.


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