Therapeutic Effects of Vitex (Vitagnus castus) Extract on In vitro Maturation and In vitro Fertilization Oocytes of Mice as Model of Polycystic Ovary Syndrome

Nowadays, to get rid of the side effects of chemical drugs, the use of medicinal plants is taken into consideration. Polycystic Ovary Syndrome (PCOs) is an important reproductive and endocrine disorder in women in the reproductive age. Also, Similar to PCOs, some disorders have been identified in other animals such as sheep, rat and mouse. PCOs is known as one of the most important endocrine disorders and a common cause of anovulation and infertility. There are some studies about the effects of vitex (Vitagnus castus) on different aspects of PCOs animals such as endocrinology (hormonal profiles), ovarian histology and changes in estrus cycle, but there is no study about the effects of vitex on embryo qualities or efficiency of embryo production. The aim of this study was evaluation of vitex extract on in vitro maturation (IVM) and in vitro fertilization (IVF) of oocytes in mice exposured to polycystic ovary syndrome.

A total of 32 adult virgin NMRI female mice were kept in a temperature-controlled room under a 12 hours on and 12 hours off light cycle. Animals were randomly and equally allocated into four experimental groups included: 1) Control group (con) which did not receive any injection or other manipulations, 2) polycystic ovary syndrome group (PCO) received an IM injection of 4 mg/kg estradiol valerate dissolvedin 0.4 ml of sesame oil) (Aburaihan Co., Iran), to induce PCOs, and groups 3 and 4 were induced PCO that orally received vitex extract at 365 mg/kg/day and 730 mg/kg/day dosage, respectively for 30 days. Each eight of them were housed in to a cage with unlimited food and water access. Thirty days after the estradiol valerate injection, animals were IM administered by 7.5 IU of PMSG. Subsequent of 48 hours, the mice in experimental groups were anesthetized with ketamine and xylazine and then sacrificed, after that their ovaries were removed and separated from other tissues. In order to provide oocytes, GV oocytes were isolated from ovaries and cultured in TEM199. After insemination of MII oocytes resulted from incubation of GV oocytes, the investigation of fertilized oocytes (zygotes), blastocysts and hatched embryos were carried out by inverted microspore. Then the ovaries were fixed at least for a day in formaldehyde 10%. Prepared tissues according to standard protocols were used for counting of different type of ovarian follicles. Tissue staining was carried out with hematoxylin and eosin. All types of follicles were counted in each ovary.

Numerous experimental models for PCOs have been developed using estradiol valerate. Estradiol valerate can produce a PCOs model in mouse with interrupting in metabolic and physiologic processes. In this study, morphological examination of the ovaries showed that exposure of young adult female mice to a single dose of exogenous estradiol valerate can inhibit ovulation and consequently induces PCOs. The amounts of serum testosterone and estradiol substantially decreased in treated groups in comparison with PCO mice (p < 0.05). Increase of testosterone is one of the most important symptoms in PCOs, but use of vitex extract decrease testosterone concentration whether in groups consuming 365 or 730 mg/kg/day that is because of less number of follicular cysts in these groups versus PCO group (p < 0.05). In this regard it is observed total number of follicles with less than 70 µm and more than 200 µm significantly was higher than PCO group (p < 0.05). These results are expected because polycystic ovary is a disorder of follicle development. In PCOs, follicles arrest in immature stages and therefore dominant follicle selection will not occur. This may be due to lack of positive estrogen feedback to the hypothalamus and pituitary axis. As the result of this lacking, LH surge will not occur and therefore ovulation will be disrupted and finally follicular cysts remain on the ovaries. The number of matured oocytes (MII) in PCO group + 730 mg/kg/day was significantly higher than PCO group (p < 0.05). Also there were no significant differences in percentage of fertilized oocytes (zygotes) and Two-cell embryos between experimental groups (p>0.05). On the other hand, the percentage of produced blastocyst and hatched embryos in group PCO + 365 mg/kg/day was higher than PCO group (p < 0.05). With administration of vitex extract more ovarian follicles ovulated and their oocytes were higher in quality (matured oocytes) that finally, produced more blastocysts and hatched embryos.

The result of present study showed that PCOs can be induced in adult mice with a single dose injection of Estradiol valerate. Also it seems vitex extract consumption can induce more oocytes for fertilization and consequently, production of embryos in animals with PCO syndrome.Conflict of interest: None declared.Keywords: PCOS, Vitex, In Vitro Maturation, In Vitro Fertilization.