Background
Polycystic ovary syndrome is one of the major causes of anovulatory infertility in women. The etiology and pathology of PCOS are still questioned, but imbalances of testosterone, estrogen, progesterone, LH and FSH, obesity, insulin resistance, and hyperinsulinemia are crucial factors for ovarian hyperandrogenism and chronic anovulation [
1]. In addition to infertility, patients with PCOS are at a risk of miscarriage [
2], which has been reported during the first trimester of pregnancy in approximately 30 to 35 % of patients [
3].
The protocols proposed for the treatment of PCOS include a diet plan, exercise and physical activity, drug treatments, and surgical procedures [
4]. Drug treatments include the administration of metformin, glitazones, spironolactone, estrogen, and clomiphene citrate [
5]. In recent years, metformin alone or in combination with other drugs is used to treat PCOS. Metformin, a biguanide antihyperglycemic drug, is utilized to treat type 2 diabetes. In women with PCOS associated with anovulation and resistance to clomiphene citrate, the administration of clomiphene citrate in combination with metformin satisfactorily increases the ovulation and pregnancy rate [
6].
Many studies have reported that metformin can induce oocyte maturation and improve oocyte quality [
7]. However, the role of metformin in promoting
in vitro oocyte maturation remains unknown. The controversial effects of metformin on
in vitro maturation (IVM) of oocytes have been demonstrated. Lee et al. demonstrated the effects of metformin on the
in vitro developmental potential of porcine oocytes and embryos; further, they clearly asserted that metformin augments the actions of insulin as an insulin-sensitizing agent on the cytoplasmic aspect of oocyte maturation and the preimplantation embryonic development during
in vitro production [
8]. In addition, the administration of metformin in combination with oral contraceptives to women with PCOS can increase the probability of
in vitro oocyte maturation in relation with miscarriage rate [
9]. In contrast, Tosca et al. demonstrated that metformin activates adenosine monophosphate-activated protein kinase (AMPK), which inhibits the germinal vesicle breakdown (GVBD) in cumulus oocyte complexes (COCs) and most COCs arrested at the germinal vesicle (GV) stage, but not denuded bovine oocytes, [
10]. Bilodeau-Goeseels et al. also reported that metformin, as an activator of AMPK, inhibited GVBD in bovine cumulus-enclosed oocytes and denuded oocytes and that AMPK might play contradictory roles in the regulation of bovine and murine oocyte maturation, [
11].
Despite the ethical and technical restrictions on human experimentations, so far, the animal models that can display all features of human PCOS has not been well introduced. On the other hand, the effects of supplements on the
in vitro oocyte maturation to be clearly determined, when all the main characteristics of PCOS are observed. Sabatini et al. revealed that metformin reduced the IVM of oocytes of the wild type and leptin deficient transgenic (ob/ob) mice models, but not in leptin receptor mutant mice (db/db) models, [
12]. The
in vitro maturation of oocytes consists of two principle features: nuclear and cytoplasmic maturation [
8]. Thus far, the precise role of metformin in promoting all the aspects of oocyte maturation in animal models of PCOS has not been identified. The purpose of this study was that, despite the fact that EV PCOS-induced rat model lack some endocrine and metabolic features, whether metformin contributes to the quality of the
in vitro maturation of oocytes? Therefore, the present study was designed to investigate the effects of metformin as an additive supplement on nuclear and cytoplasmic maturation as well as the expansion of cumulus cells as the third criterion of oocyte maturation in in EV PCOS-induced rats.
Discussion
Because of ethical and technical restrictions on human experimentation, researchers have used animal PCOS models. The animal models of PCOS have proven useful in determining the causes of PCOS, to perform preclinical trials, and to examine ovarian morphology, hormonal disorders, and the pathogenesis of anovulation in PCOS. Thus far, a persuasive animal model that can replicate all features of human PCOS has not been established. Among mammals, despite the fact that rhesus monkeys and sheep show major ovarian morphological changes [
16], rodents are versatile and a more suitable and unique animal for PCOS models [
17,
18]. Compare to rhesus monkeys and sheep, rats and mice are inexpensive, readily available, and easy to handle and maintain [
16], they have a short reproductive cycle, short estrous cycles, and a short gestational period [
17,
19]. Rodent models of PCOS have shown hyperandrogenism and hormonal alteration; ovarian morphological changes, including the presence of multi-cystic follicles; and metabolic disorders [
17]. Rodent models of PCOS can be attained by a variety of methods, including constant exposure to light [
20], genetic manipulation [
17], and administration of hormones, such as testosterone (T) [
17], dihydrotestosterone (DHT) [
21], EV [
22,
23], and letrozole (a non-steroidal aromatase inhibitor) [
21,
24]. Among these hormones, T, DHT, and letrozole show many characteristics of human PCOS, including acyclicity, anovulation, polycystic ovaries, hyperandrogenism, and insulin resistance [
17]. These features are dependent on the dose of hormones, menstrual cycle phase and duration of treatment, and the waiting time for induction of PCOS [
25]. Several injections and/or gavages of T, DHT, and letrozole are likely to increase the possibility of stress and high mortality rates during the experiment and it is also a time-consuming method. However, after a single injection of the EV, it will take about 8–12 weeks to induce PCOS, therefor the stress of injection is reduced, it is well known that stress causes irregularities in the menstrual cycle. The EV-induced PCOS model displays characteristic morphological alterations in the ovary [
26], particularly the multi-cystic follicle; as previously shown [
27,
28], and mention shortly in results section, which is typically observed in human PCOS. In addition, the EV-induced PCOS model is used in many laboratories, develops hypertension and increases sympathetic activity [
29]; these effects lead to increased blood glucose as in this study and it is utilized for autoimmune disorder responses in PCOS [
30]. However, the major restrictions of the EV-induced PCOS model are a lack of exactly endocrine and metabolic features associated with human PCOS [
17]. It seems that EV can creates a reversible PCO adult rat model, but not PCOS [
16,
31,
32], Gonzalo Cruz et al. established an irreversible PCO model when neonatal rat exposure to EV [
33]. Recently, Caldwell et al. have demonstrated that long-term DHT administration in mice imitates an extensive of PCOS features [
34].
The results of this present study show that a single intramuscular injection of EV induces experimental PCOS in rats, which can be identified by observing the cystic and atretic follicles as previously indicated in our laboratory [
27,
28] and alterations in the serum levels of gonadotropins and increases blood glucose level. In our study, as in the study of Stener-Victorin et al. [
29], EV may affect the hypothalamic–pituitary–adrenal axis and the ovary, following a hormonal disorder. Various PCOS animal models have shown dissimilar hormonal changes, but our findings confirm the results of studies by Singh [
19], and Stener-Victorin et al. [
29], who have reported alterations in the serum levels of gonadotropins.
Obesity is a subordinate symptom of PCOS in women [
1]. In this study, weight gain in the PCOS-induced rats was lower than that in the control rats, which is comparable with the results of Stener-Victorin et al. [
29]. The administration of EV increases adrenal glucocorticoid production, which enhances lipid metabolism, and leads to a decrease in the body weight [
29]. However, the obesity is not always observed in women with PCOS [
35,
36]. In addition, the amplification of sympathetic activities in women with PCOS was found to increase lipid metabolism and body activity, and consequently, decreases the body weight [
31].
Metformin is an insulin-sensitizing drug [
8,
37], usually prescribed in patients with PCOS, to induce ovulation, reduce the symptoms of hyperinsulinemia [
8], and improve insulin sensitivity to decrease the serum levels of androgen [
37]. It has been reported that metformin contributes to
in vitro maturation of oocytes, which are collected from patients with PCOS [
38], and associated with insulin, but not alone, have beneficial effects on oocyte maturation, oocyte quality and production of embryo [
8]. Metformin improves the action of insulin on oocyte glutathione (GSH) content, which knocks out the free radicals in the oocytes, resulting in enhanced oocyte competence [
37]. These results suggest that metformin accompany with insulin may increase the cytoplasmic maturity of oocytes during IVM. Mansfield et al. demonstrated that metformin in the culture medium has a direct effect on cumulus and theca cells and mediates enzyme activities for the synthesis of steroid hormones. In addition, metformin has inhibitory effects on progesterone and estradiol production on the
in vitro culture of granulosa cells; progesterone is the secreted end point in the steroid synthesis pathway in these cells [
39]. Progesterone plays a role in bovine oocyte maturation, particularly in cytoplasmic maturation, but represents a different role and is dependent on cells (oocyte and/or cumulus cells) and the cell progesterone receptors [
40]. In addition, progesterone induces meiosis resumption in cultured bovine COCs in a concentration-dependent manner [
41]. Our data show that metformin does not affect all features of nuclear and/or cytoplasmic maturation. These controversial effects of metformin on nuclear and cytoplasmic maturation lead us to believe that the EV-induced PCOS animal model does not clearly present all the features of hyperandrogenism and hyperglycemia. It has been reported that metformin is more effective in a batch of transgenic PCOS-induced mice that categorized by hyperleptinemia and hyperinsulinemia [
12]. The lacks of our study are that we have not measured leptin and insulin levels, but hyperglycemia was observed in the EV-induced PCOS animal model, which it may be due to the increase in sympathetic activity [
29]. In the other hand it is believed that patients with the most obvious hyperandrogenism have most benefited from metformin treatment [
42], therefore, in our study the metformin inability to show all aspects of
in vitro oocyte maturation and may be resulted from deficiency of EV to induce PCOS.
It is clearly known that the expansion of cumulus cells of COCs is a criterion for oocyte maturation. Nagyova reviewed the mechanisms involved in ovarian follicular processes, including the expansion of cumulus cells, the hyaluronan synthesis and progesterone production in COCs. The expansion of cumulus cells in mouse, porcine, bovine, and rat depends on a specific factor, “cumulus expansion enabling factor”, which secreted by the oocytes and/or in some mammals, by cumulus cells [
43]. It was concluded that optimal cumuli expansion promotes embryonic development in bovine oocytes. It is suggested that glutathione is needed for the expansion of cumulus cells and that hyaluronan accumulates in the expanded cumulus cells. Hyaluronan, which builds up within cumulus cells in porcine COCs during cumuli expansion, disrupts cell junctions of COCs and promotes meiotic resumption in oocytes [
44]. Our findings concur with those of previous studies and show that in the presence of metformin in PCOS-induced rats, a higher number of cumulus cells of COCs (53.03 %) are completely expanded as compared to those in the absence of metformin (32.83 %). However, this contradicts the findings of Tosca et al. who concluded that metformin inhibits cumuli expansion and oocyte meiotic resumption in bovine COCs (not denuded oocytes), [
45]. These results indicate that the presence of metformin in the culture medium may enhance the expansion of cumulus cells, but not in PCOS-induced rats, because metformin acts first on cumulus cell to dissociate around the oocyte [
45]. Despite of metformin has a role on the resumption of meiotic division, a small percentage of oocytes reaches to MII stage. Bilodeau-Goeseels et al. reported that both metformin and aminoimidazole- carboxamide ribofuranoside (AICAR) (activators of AMPK) have inhibitory effects on cumulus cells expansion and nuclear maturation in bovine, but not mouse and are greater in COCs than in denuded oocytes due to the presence of cumulus-oocyte projections [
11]. Also Nicolas reported that AMPK has same effects on cumulus cells expansion and nuclear maturation in porcine [
46]. Metformin decreased progesterone and estradiol productions
in vitro in human, rat and bovine granulosa cells [
10,
39]. In relevant to the species, different mechanisms involved on estradiol production in granulosa cells and metformin may also reduce steroid levels in granulosa cells from follicular cysts. [
10]. Increased estradiol and progesterone concentrations have been reported in the ovarian cyst [
47].
Resumption of meiotic division is the other principle of oocyte maturation and takes place largely in IVM. In our study, resumption of meiotic division was slightly increased in the presence of metformin, particularly in the PCOS-induced rats. Although the resumption of oocyte meiotic division is the first step of oocyte maturation, completion of oocyte maturation occurs when the oocyte reaches to the MII stage. In contrast to bovine [
45] and porcine [
48], which metformin arrested COCs at the GV stage, in our study 65.11 % of COCs were initiated meiotic resumption at the first 6 h of IVM in metformin supplemented medium. While, in PCOS-induced rats a fairly significant increase from 57.12 % in metformin-untreated oocytes to 64.28 % in metformin-treated oocytes. Our data show that, no differences were observed in the percentage of the MII stage between metformin-treated and –untreated oocytes, as reported in bovine [
45], these data clearly show that low percentage of oocytes reached to the MII of maturation. Thus, the nuclear maturation of oocytes in the PCOS-induced rats may be not affected by metformin. Tosca et al. reported that metformin decreases the number of cumulus and theca cells to generate steroid hormone genes, thus indirectly promoting the nuclear maturation of oocytes [
10]. In contrast to our findings, Bilodeau-Goeseels et al. concluded that metformin activates AMPK, which inhibits GVBD in bovine COCs and denuded oocytes, but enhances oocyte meiotic resumption in mice, which is similar to that observed in the PCOS-induced rats in our study. Several studies also reported that AMPK may have contradictory roles in the management of bovine and murine oocyte maturation; it seems that a different mechanism is used to stimulate AMPK in rodents [
11]. The difference between the rate of cumulus cell expansion and nuclear maturation in PCOS-induced rat in presence and absence of metformin, may be as a result in a lack of orchestration between these phenomena.
Cytoplasmic maturation is a criterion for deducing the developmental competence of oocyte maturation
in vitro and includes numerous morphological and biochemical features. The morphological changes include the reduction of the Golgi complex volume, increase in the number of lipid droplets and cortical granules, and enlargement of PVS [
49]. The results of this study at the level of light and electron microscopy show that metformin influences few features of oocyte maturation. Cumulus cells around the oocytes in the metformin-treated group were dark, due to nuclear heterochromatin [
50], and had lower cell density than the other groups. However, Nottola et al. [
51] demonstrated that the presence of dark cumulus cells is related to the accumulation of lipid droplets in the ooplasm, which shows active steroidogenesis in healthy and mature granulosa cells. Our data show that the number of cell junctions between the other cumulus cells and oocytes were decreased, and these tended to expand. According to the findings of Tosca et al. [
10], metformin reduces the production of steroids and enzymes in cumulus cells and consequently, causes a decrease in the number of connections between cumulus cells and oocytes; thus, cumulus cells initiate the expansion. Our findings on the effects of metformin, such as a declining trend of cellular links and redistribution of some cytoplasmic organelles, corroborate the findings of Suzuki et al. [
52] with human oocytes and granulosa cells cultured in Ham’s F-10. An increase in the number of apoptotic cells was observed in cumulus cell in the metformin-treated group. Note that a non-uniform zona pellucida, non-obvious oolemma, and narrow PVS were observed in the absence of metformin. It is well known that the enlargement of PVS is a morphological change in matured oocytes [
49,
53]. As we have shown, the presence of cortical granules close to the inside of oolemma is a symptom of oocyte maturation, which is required to prevent polyspermy [
54].