Extended Abstract
Introduction
Polycystic Ovary Syndrome (PCOS) is an endocrine disorder [1]. The disease is heterogeneous and has various causes including immunological, hormonal and neurological factors [3]. Mild chronic inflammation is a new proposed mechanism in PCOS pathogenesis which indicates the role of the immune system. Given the side effects of classic medications, it is important to identify and introduce alternative therapies. Fennel, with its phytoestrogenic, antioxidant and anti-diabetic properties, is used in traditional Iranian medicine to treat PCOS. Due to the role of inflammation in PCOS immunopathogenesis and the anti-inflammatory effects of fennel [6], this study aimed to study the effect of fennel hydroalcoholic extract on serum antioxidant power, Nitric Oxide (NO) concentration and percentage of Treg cells in a rat model of PCOS.
Materials and Methods
In this experimental study, 18 immature 21-day-old BALB / C mice weighing about 14 grams were used. The mice were divided into three groups of 6, including control group, untreated PCOS-induced group, and fennel-treated group. The control group received 0.1 ml of sesame oil subcutaneously for 21 days. The untreated and fennel-treated groups received 60 mg /kg Dehydroepiandrosterone (DHEA) plus 0.1 ml sesame oil subcutaneously for 21 days to induce PCOS [16]. After 21 days, the fennel-treated group received 0.5 ml of fennel extract (500 mg/kg, i.p.) for 21 days. Mice were weighed every 3 to 4 days with a scales and estrous cycle for three periods during 42 days was determined by a vaginal smear. The serum sample was used to measure the NO concentration by Griess reaction method and the total antioxidant power was measured by the Ferric Reducing/Antioxidant Power (FRAP) assay. From the left ovary, 7 micron sections were prepared and stained with hematoxylin eosin. Splenocytes were separated from the spleen by perfusion and used to count Treg cells by flow cytometry analysis.
Results
The efficiency of the maceration method was 4.7%. The average weight in the study groups did not change significantly. At the start of DHEA injection in mice, estrous cycles were regular, but gradually the cycles became irregular. This irregularity is one of the signs of follicular cyst in the ovary. After treatment with fennel extract, the cycles became regular again. Fennel increased the number of antral follicles, but this increase was not significant. This plant significantly reduced ovarian cysts (P=0.005) (Figure 1). The number of corpus luteum in the PCOS group decreased significantly compared to the control group (P=0.033). Prescribing fennel increased the number of corpus luteum follicles, but this increase was not significant. The NO concentration in the untreated PCOS-induced group increased significantly compared to the control group (P=0.001). 

In the group treated with fennel extract, the NO concentration decreased significantly compared to the untreated group (P=0.002) (Figure 2). FRAP levels in the untreated PCOS-induced group decreased compared to the control group, but this decrease was not significant. FRAP levels in fennel-treated group increased compared to the untreated group, but this increase was not significant. The number of Treg cells in untreated PCOS mice decreased significantly compared to the control group (P=0.008). Treatment with fennel extract significantly increased the number of Treg cells (P=0.003) (Figure 3).

Discussion
Induction of PCOS in mice caused irregular estrous cycles, a significant increase in the number of cystic ovarian follicles, and a significant decrease in the number of corpus luteum follicles. These results indicate the successful induction of PCOS in the animal model of PCOS. Estrous cycles became normal after treatment with fennel extract, which indicates the effectiveness of fennel hydroalcoholic extract in reducing the symptoms of PCOS.
Fennel is a plant with anti-inflammatory, antioxidant and phytoestrogenic properties [10, 11]. Injecting fennel extract significantly reduced the number of cystic follicles. One of the important characteristics of this disease is a decrease in the rate of ovulation which causes a significant reduction in the number of corpus luteum follicles in polycystic ovaries [21]. Studies have shown that flavonoid compounds in plants with antioxidant activity can reduce oxidative stress in ovarian tissue and reduce the number of cystic follicles in this tissue [22].
The use of fennel extract significantly reduced NO level compared to the untreated group. Excessive NO production in the uterus can lead to inflammation and miscarriage [27]. Oxidative stress in patients with PCOS plays an important role in altering follicular structure and overgrowth of mesenchymal ovarian tissue [28].
The number of Treg cells in mice with PCOS decreased significantly compared to the control group, and the administration of fennel extract significantly increased the number of these cells. Some studies have shown that patients with PCOS have a lower Treg rate than healthy people that can lead to mild chronic inflammation, which is suggested as a new mechanism in PCOS pathogenesis and the role of immune system in this disease [29].
Ethical Considerations
Compliance with ethical guidelines
This study was approved by the Research Ethics Committee of Arak University of Medical Sciences (Code: IR.ARAKMU.REC.1397.312).
Funding
This study was extracted from a research proposal (Code: 3175). It was financially supported by the Deputy for Research and Technology of Arak University of Medical Sciences.
Authors' contributions
All authors met the writing standards based on the recommendations of the International Committee of Medical Journal Editors (ICMJE).
Conflicts of interest
The authors declare no conflict of interest.
Acknowledgements
The authors would like to thank the Deputy for Research of Arak University of Medical Sciences for their support.