Effects of Ferula Assa-Foetida on Clinical, Hormonal and Sonography Parameters in Young Girls with Polycystic Ovary Syndrome: Pilot Randomized, Placebo Controlled, Triple-Blinded

Background: Polycystic ovarian syndrome is the most common endocrine disorder in reproductive aged women. As a result of side effect of pharmaceutical medications women are interested in using alternative medicines to treat. To determine the comparative effects of Ferula assa-foetida on androgenic hormone levels and ovarian features in patient with polycystic ovarian syndrome (PCOS). Methods: In this triple-blinded controlled clinical trial, 34 student participants were randomly divided in two groups. Intervention group received 100 mg of oleo-gum resin of Ferula assa- foetida, control group received oral paran (Placebo) twice daily for 3 months. The ecacy of this herbal medicines was measured after the 3-month intervention. Hormonal assay for evaluating Testosterone, DEHAS, Prolactin, TSH, FSH, LH levels and also abdominal sonography for evaluating ovarian volumes, number of follicles of both ovaries, and endometrial thickness. were measured before and after the study. Results: In this study, no signicant hormonal changes were observed (p value>0.5). Although the greatest reduction in the number of ovarian follicles and ovarian volume was reported in the Ferula assa-foetide group (p value <0.01). Conclusion: Use of Ferula assa-foetida can be effected in decrease of ovarian volume and ovarian follicles number in young girls with PCOS. Trial registration: the Iranian Randomized Clinical Trial (IRCT2016040427207N1).


Background
Polycystic ovarian syndrome (PCOS) is a heterogeneous endocrine disorder affecting 6%-20% of reproductive-aged women according to various diagnostic criteria (1), AYOUBI, ARSHAMI (2). The 2003 European Society of Human Reproduction and Embryology/American Society for Reproductive Medicine Rotterdam, describe PCOS as ful lling two of the following three criteria; anovulation or oligo-ovulation, clinical and/or biochemical hyperandrogenism, and polycystic ovaries on ultrasound (Rotterdam consensus) (3). Androgen disturbances increase the prevalence of several comorbidities, including insulin resistance, diabetes mellitus, hyperlipidemia, and cardiovascular disease (4). In addition, these features are accompanied by hirsutism, acne, and an ovulatory cycles leading to menstrual irregularities and infertility (5).
Several treatments are recommended for PCOS such as life style modi cation (6), drug therapy(7) and herbal medicine (8).
In Iran, some women with gynecological disorder such as dysmenorrhea (9) and oligomenoreha (10) use herbal medicines such as Ferula assa-foetida,that is locally known as Anghuzeh. Owing toits anti hirsutism, antibacterial, anthelmintic, and anticancer properties (11), this herbal medicine has been traditionally used for different purposes: abdominal pain, cancer and colic in children (12) and also for treatment of vaginal infection (13). This medicinal plant belong to the Umbelliferae (Apiaceae) family. The biochemical activities of Umbelliferae which are attributed to terpenoids such as ferutinin, may modulate estrogen signaling in the same way as phytoestrogens. Due to the potential selectivity of ferutinin for Estrogen receptor Alph (ERα) and Estrogen Receptor Beta (ERβ), they may be useful as elective Estrogen Receptor Modulators (SERMs (14). Some study evaluated the effects of Ferula assa-foetida in histopathology of testis in male wistar rat and showed a signi cant decreasing trend in terms of testosterone hormones (2,15,16).
Since Polycystic ovary syndrome is a disorder primarily characterized by signs and symptoms of androgen excess (17), and based on some study results that Ferula can decrease testosterone in gonad and blood (2,16), then use of Ferula is one way for PCOS improving in patients, the present study aimed to investigate the effect of Ferrula assa-foetida on PCOS symptoms.

Methods
Participants: This study was conducted on students who were studying in Jahrom University of Medical Sciences.
Sample size estimation: There was no previous comparable study on which to base data for a Sample size calculation, then to estimate an acceptable sample size, the Cohen ' s table was used (17). For sample size Calculation, a signi cance level of 0.05, a power level of 0.80, an effect size of 1.05 based on blood Testosterone changes and SD = 2, were determined. The minimum sample size was determined 17.
Inclusion criteria: In this study, the participants were young girls aged 18-30 years that diagnosed PCOS based on Rotterdam criteria (Table 1)  The exclusion criteria were medical conditions such as androgen-secreting tumors, hyper prolactinemia, thyroid disorders and Cushing's syndrome (determined by suitable laboratory assay), pregnancy and lactation.
Randomization: After being informed about the research methods and objectives, each participant gave written informed consent prior to data collection. Each participant was given a unique identi er and assigned randomly to one of two groups. Each Participant in intervention group received active compounds in capsular form (each capsule contained 100 mg of oleo-gum resin of Ferula assa-foetida) twice daily for 3 months. In the same way, participants in placebo group received capsules of 100 mg prepared from oral para n.
Blinding: The manufacturer produced 17 asafetida boxes that contain 180 Capsules (100 mg) and 15 placebo boxes. The subjects were requested to use that drug for 12 weeks, twice a day (each 12 hours) after meals. Each box was assigned a number from 1 to 30 and each participant received one box according to table randomization. Participant groups received the capsule from the container that had the same label. Therefore, the participants, and investigator, were all unaware of the capsule contents. After 3 months interventions, the contents of the capsules given to each group were revealed by the pharmacist. After study was completed ,the researcher received information about the numbers and the nature of the Root and Stem with steam and then for this study they were formed into pearl-shaped pills (100 mg). The major components of Ferula assa foetida included 65% oleo g um resin, 20% essence, 25% stem (18).Ferula assa foetida drug toxicity was assessed in a past study and its safety had been reported (12). The placebo contained 100 mg para n. In order to isotropy, each pearl was placed inside a capsule and was coded.
Data Collection: The primary outcome was clinical parameters (BMI, FG score, Menstruation periods), that data were obtained at the beginning of the study and at the end of the study period. Secondary outcome were hormonal (Dehydroepiandrosterone sulfate (DHEAS), Free testosterone (FT), Follicle-stimulating hormone (FSH), Luteinizing hormone (LH) and Prolactin(PRL)) and sonography parameters (Endometrial thickness, ovarian volume and Number of follicle in both Ovary). A venous blood sample was obtained from the studied population to evaluate the changes in the level of biochemical factors. Plasma was separated and kept at -20ºC until it was assayed for DHEAS, FT, FSH, and LH using immunoassay kits (Monobind Inc, CA, US) according to the manufacturer's instructions. Statistical Analysis: All statistical analysis was performed using SPSS statistical software version 14 and Graph pad Prism (version 6). Data are presented as mean and standard deviation. The normal distribution of the data was tested by Kolmogorov-Smirnov test and a non-parametric test was conducted to evaluate the objectives. clinical, hormonal and sonography parameters were analyzed by using Kruskal Wallis tests to compare the e cacy of the Ferula with the placebo before and after 3 months interventions. P values less than 0.05 were considered signi cant.

Results
Off the 34 participants recruited into this study, six subjects did not complete the trial and were excluded from the analysis. Finally 28 students completed 3 months of the intervention (Fig. 1).
The mean ages in this study were 22.93 ± 1.5 years in the Ferula group and 23.14 ± 2.7 years in the placebo groups, respectively.
No signi cant differences were shown between the two groups before and after intervention in Clinical parameters (BMI, FG score and menstruation) ( Table 2). All of hormonal parameters (TSH, FSH, LH, DHEAS, PROLACTIN, and free testosterone) before and after intervention were no signi cantly different between the two groups (Table 3). All of sonography parameters in two groups before intervention were not signi cantly different but 3 months after intervention. There were remarkable reductions in the ovarian volume and number of follicles in both ovaries in the Ferula group vs placebo group endometrial thickness was not signi cant after intervention between two groups (Table 4).

Discussion
This randomized, placebo-controlled clinical trial was conducted to evaluate the e cacy of Ferula assafoetida in improving the features of PCOS.
In our study, BMI in both groups was not signi cantly different before and after the intervention, and this might be related to the duration of intervention, which was three months. Based on recent PCOS international guidelines, a six month intervention duration is optimal to reduce the BMI in PCOS women (19) some studied reported that Soy protein diet can decrease weight in obese women (20,21). Also, the meta-analysis reported by Zhang et al. (2013) concluded that Soy Iso avone supplementation could improve glucose metabolism and insulin control in non-Asian postmenopausal women (22).
Result showed that F-G score for the hirsutism severity was not signi cant after intervention, same as result of oral fennel used in PCOS women (23) and in consist with Javidnia et al. result that idiopathic hirsutism patients were treated with creams containing 1% and 2% fennel extract over 12 weeks (24).
In this study, assa-foetida was not effective on the length of the menstruation cycle in PCOS women, and this could be related to the dosage and duration of treatment which is comparable to the results of treatment with fennel in PCOS. However, the use of another herbals such as Vitex (25), Cinnamon (26), Mentha longifolia (27) Trigonella foenum-graecum (28) have been effective for oligomenorrhea treatment.
The ndings of this study showed that Ferula assa-foetida exhibited effects on ovarian volumes and the number of ovarian follicles in women with PCOS. To the authors' knowledge there has been no study that has evaluated the effects of assa-foetida in patients with PCOS.
Hormonal level in this study did not signi cantly change after intervention and this was in accordance with following studies. One study investigated the e cacy of fenugreek seed extract on PCOS that decrease in the LH to FSH ratio was reported (29). In another study, use of fennel in women with PCOS was not signi cantly change blood hormonal level (23). Use of Urtica dioica in hyper-androgenic women was effective in testosterone level decrease after treatment (30). Also, serum levels of luteinizing hormone (LH), triglyceride (TG), low density lipoprotein cholesterol (LDL), dehydroepiandrostrone sulfate (DHEAS) and testosterone were signi cantly decreased after 3 months therapy in Genistein group (31).
Ovarian characteristics such as ovarian volume and ovarian follicular numbers in this study were signi cantly improved after intervention such as metformin therapy in PCOS women that decrease both ovarian volume (32,33). Also, use of fennel in PCOS women could decrease follicular number of both ovarian (23). Trigonella foenum-graecum decreased signi cantly polycystic-appearing ovaries in ultrasound scans (28)and decreased both ovarian volume (29).
Some of the limitations of this study were small sample size, poor generalizability and the Duration of treatment. Thus, we suggest a greater sample size with a longer treatment time for future studies to evaluate this herbal drug. According to budget limitations the effect size which The authors used where determined to high, so the study was unable to detect small differences. The informed consents were obtained from all of university students above 18 years.

Funding
No funding was received.

Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Competing interest
We wish to con rm that there are no known con icts of interest associated with this publication and there has been no signi cant nancial support for this work that could have in uenced its outcome.

Authors Contributions
S.A and F.GH were involved in study design, preparation of intervention.
F.SH and M.T contributed to critical discussion and manuscript drafting. All authors read and approved the nal manuscript. CONSORTextensionforPilotandFeasibilityTrialsChecklist.doc