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 significance 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)
Rotterdam diagnostic criteria for PCOS
Rotterdam (2003) Diagnostic criteria for PCOS - two out of three of:
Clinical Hyperandrogenism (Ferriman-Gallwey Score > 8) or Biochemical Hyperandrogenism (Elevated Total/Free Testosterone)
Oligomenorrhea (Less Than 6–9 Menses per Year) or Oligo-Ovulation
Polycystic Ovaries on Ultrasound ( > = 12 Antral Follicles in One Ovary or Ovarian Volume > = 10 cm3)
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 identifier 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 paraffin.
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 each drug.
Preparation of Capsules: Root and Stem of Ferula assa foetida L. (gum) was purchased from a medicinal plant market (Adonis Gol Darou), a supplier of herbal medicines,Tehran, Iran. In Department of phytopharmaceuticals (Traditional Pharmacy), School of Pharmacy, Shiraz University of Medical Sciences with Herbarium Voucher Number 981, Ferula assa foetida essence was produced by distilling 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 paraffin. 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.
Testosterone, DEHAS Prolactin (reference number: 725090), TSH FSH and LH were measured by using the enzyme-linked immune sorbent assay method using the Eliza Kit (Monobind Inc., Germany).
In all groups, Participants underwent abdominal sonography for evaluating ovarian volumes, number of follicles of both ovaries, and endometrial thickness at 4.4-MHz (Alpinion, Seoul, South Korea) by a one sonographer in the follicular phase of menstrual cycle except in participants with oligo or amenorrhea
Ethics Cnsideration: The study was approved by the Ethics and Research Committee of Jahrom University of Medical Sciences (reference number: ums.REC.1393.023) and registered in the Iranian Randomized Clinical Trial (IRCT2016040427207N1).
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 efficacy of the Ferula with the placebo before and after 3 months interventions. P values less than 0.05 were considered significant.