Experimental design
This study was attained under the approval of the state committee on animal ethics, Shiraz University, Shiraz, Iran. Also, the testimonial of the European Council Directive (86/ 609/ EC) of November 24, 1980, regarding the standards in the protection of animals for experimental goals were followed.
Twenty-four adult female Sprague Dawley rats (200 ± 20 g) purchased from Comparative and Experimental Center of Medical Sciences Department of Shiraz Medical University. Animals with regular reproductive cycle (following 3 cycles checking), were selected and randomly dispensed into four groups as control (C) (received distill water), Treatment- Control (TC) (received 40 mg/kg hydroalcoholic extract of spearmint + 200 mg/kg flaxseed extract for 30 days by gavage). PCOS was induced in the next two groups, PCOS group, and Treatment group (T) by a single intramuscular injection of estradiol valerate (4 mg/rat). The treatment group received 40 mg/kg spearmint extract + 200 mg/kg flaxseed extract for 30 days by gavage, 7 weeks after injection of estradiol valerate, while PCOS group received distilled water during the same period. Animals were kept in the standard polypropylene cages at 20 -22 ˚C, 38% humidity and 12/12 hour light/dark cycle, fed with a standard pellet diet and had free access to tap water.
Preparation of hydroalcoholic extract of spearmint and flaxseed:
Fresh spearmint and flaxseed were purchased from a local market source in Shiraz. The plants' qualities were confirmed by a botanist in the biology department. After clearing and drying, the spearmint was completely ground; the resulting powder was placed in 70% alcohol for 72 hours. After straightening with filter paper, the rotary machine was used to concentrate extract under reduced pressure. The resultant semi-solid extract put into a lyophilizer machine for 24 hours in order to make a powder. The same procedure was used for flaxseed after grinding of the seeds.
Monitoring of reproductive cycle
In rats, the reproductive cycle lasts approximately 4-5 days. The characteristic of each cycle was based on the ratio of the three types of cells which are including; cornified, leukocytes and epithelial [25]. The reproductive cycle of rats was monitored through vaginal smear according to the method described by Caligioni (2009) for two weeks before the selection of rats for the experiment (only rats with regular cycles were selected) and also was checked during the last 8 days of the experiment (from day 85 of the experiment) (Fig.1).
Induction of PCOS and treatment
PCOS was induced in two groups (treatment and PCOS groups) of rats with regular estrous cycles 7 weeks after a single intramuscular injection of 4 mg of estradiol valerate/rat dissolved in 0.2 ml of sesame oil [26, 27].
Blood sampling and histological study
During the last week of treatment, again the reproductive cycle of all rats assessed. On day 93 (the last day of the experiment) rats were anesthetized by 2 % diethyl ether-saturated cotton ball in a chamber for 3–5 min., and euthanized by whole blood collection through heart puncture, and sera were used for measuring hormones including estrogen, progesterone, testosterone, and dehydroepiandrosterone (DHEA).
Ovaries were dissected out and subjected to histomorphometric study after fixation and tissue processing. Serial sections of ovaries were prepared at a thickness of 7 µm and stained with hematoxylin and eosin and the same section of all ovaries was selected for histological study. Different types of follicles including; primary, preantral, antral and cystic were evaluated under a light microscope (CX21 OLYMPUS, Japan) and were photographed by an adjusted digital camera (AM423U Eyepiece camera, Dino- Eye Taiwan). Granulosa and theca layer in different follicles were measured also follicle diameter was evaluated. Furthermore, the number of primary, preantral, antral and cystic follicles was measured by Dino Capture 2.0.
Hormone assay
The hormones including testosterone, estrogen, progesterone, and DHEA in serum were measured by the ELISA method using ELISA reader (Monobind, Inc. lake Forest, CA (92630) USA.
Statistical analysis
All results are presented as Mean ± standard error of the mean (Mean ± SEM). Statistical analysis was performed by (SPSS-24.0). Statistical differences between groups were compared with (one-way ANOVA). Pair group comparison was measured by the LSD test. Statistical significance was set at P<0.05.