PCOS is an endocrine disorder seen in the majority women of reproductive age and in which cystic ovaries are observed in cases in which follicular development steps are not fully completed due to increased androgen levels. Various symptoms occur in patients with PCOS, but particularly obesity, anxiety, depression, and blood pressure problems. A high prevalence of obesity and depression is observed in patients with PCOS (Kerchner et al. 2009; Hung et al. 2014; Hart et al. 2015; Cooney and Dokras 2017).
Depression, one of the most frequently observed syndromes in patients with PCOS, is a mental illness that manifests with a decreased sensitivity to stimuli and reinforcement hopelessness and pessimism. The mechanism involved is a complex one. Recent studies have shown a very close relationship between PCOS and depression. Individuals with PCOS are eight times more likely to be depressed than healthy individuals (Kerchner et al. 2009; Hung et al. 2014; Hart et al. 2015; Cooney and Dokras 2017). The relationship between PCOS and depression has generally been examined in clinical studies, and analyses have been performed using anxiety and depression diagnostic questionnaires. The high prevalence of depression observed in patients with PCOS can be attributed to three factors - high androgen levels observed in PCOS, insulin resistance, and infertility (Kerchner et al. 2009; Hung et al. 2014; Hart et al. 2015; Cooney and Dokras 2017). The present study investigated the effects of AMI on ovarian tissues in a rat model of PCOS.
The animals were weighed at the beginning and end of the experiment. The weights of the animals in the PCOS group increased at the end of the study compared to the initial values. No significant difference was observed in the other groups. Considering the obesity rate of 40–70% in adolescents with PCOS, this finding in the present study is quite possible (Vatopoulou and Tziomalos 2020). Sam (2007) showed that exposure to high androgen levels in post-menopausal women causes an increase in visceral adipose tissue. The weight gain occurring in PCOS has been ascribed to insulin and glucose metabolism (Melekoglu et al. 2019; Zeng et al. 2019). No increase in weight between the beginning and end of the experiment in the PCOS + AMI group in this study, suggesting that AMI may have a positive effect on weight gain in patients with PCOS. This can be explained by the reduction of depression and the decrease in food intake that comes with depression. It is also possible that this pathway proceeds via neuropeptide Y (NPY), implicated in both PCOS and depression.
Corpus luteum volumes, follicle volumes and numbers, and cystic structure volumes were analyzed for the quantitative evaluation of morphological changes in the ovary in all groups. Stereological analysis showed that PCOS produced no change in primordial and antral follicle volumes, but increased preantral follicle volume. No statistically significant difference was also observed between the AMI and PCOS + AMI groups. Analysis of follicle numbers and volumes revealed no statistically significant difference between any of the study groups. Although an increase in volume was observed in the PCOS group, no statistically significant difference in follicle numbers was observed, although a decrease was observed in the PCOS group compared to the Cont group. A study involving subcutaneous injection of dehydroepiandrosterone in rats to induce a PCOS model observed large primary follicles at ovarian morphological examination (Misugi et al. 2017). Considering all this information and the data from the present study data, it may be concluded that PCOS increases preantral follicle volume without causing any increase in the number of preantral follicles. This situation results from hormonal imbalances and increased androgen levels under the effect of insulin, changing the FSH/LH balance and inducing follicle development. Similarly, although comparable results were observed between the PCOS + AMI and PCOS groups, the difference was not statistically significant. This shows that AMI treatment is insufficient to ameliorate the adverse effects of PCOS at the primordial and preantral follicle levels.
Analysis of antral follicle numbers in this study revealed a decrease in the PCOS group compared to the Cont group, but there was no significant difference between the PCOS + AMI group and the PCOS group. This situation can be regarded as a natural result of PCOS development. Follicles with a diameter of 2–5 mm in the developmental stage form cystic structures. The absence of a significant difference between the PCOS + AMI group and PCOS group shows that AMI treatment does not affect antral follicle formation.
In addition to follicular changes, the most important parameter in PCOS pathology is the cystic structures located in the ovarian cortex that have not yet completed their development. Many studies show that patients with PCOS have cystic follicles with a diameter of 2 mm (Alsamarai et al. 2009; Azziz 2018; Behmanesh et al. 2019). Although the histological structure of the cystic follicle is evaluated differently in many studies, the thin granulosa layer and the very thick and well-developed theca internal layer are characteristic features of cystic structures (Wang et al. 2017; Manneråset al. 2007; Yaba and Demir. 2012). In their study of 40 rats, Behmanesh (2019) induced a PCOS model with estradiol valerate and reported a cystic structure with impaired follicular maturation due to an altered FSH/LH ratio in the animals in the PCOS group (Behmanesh et al. 2019). Those authors observed increases in cystic structures as well as preantral follicles in rats exposed to the PCOS model. Although there was no statistically significant difference between the groups in the analyses performed in that study, small-volume cystic structures with the criteria described were observed in the PCOS and PCOS + AMI groups (Wu et al. 2014). Due to the changes in FSH and LH levels, the follicular transformation into a cystic structure without development explains the small volume of cystic structures observed in the PCOS group compared to other groups in that study. Takahashi et al. reported cystic structures with small diameters in ovaries with PCOS (Takahashi et al. 1994). The small volume cystic structures observed in the PCOS + AMI group in that study also showed that the application of AMI was insufficient. Other studies have reported the presence of a thickened tunica albuginea layer among the histopathological findings, together with a prominent theca follicle and hyperplasia, increase interstitial cells, and increases in the numbers of cells and volume of the corpus luteum are observed in groups with PCOS (Takahashi et al. 1994; Wang et al. 2012zükara 2013). In the present study, the presence of a thick tunica albuginea was particularly noteworthy at histopathological evaluation of the ovaries from the PCOS group. Dark-stained cells with an angular structure that had lost their spherical structure, most of which had unclear nuclear borders, were observed among the granulosa cells in the follicles. In addition, the outer borders of the oocyte structures in the follicles could not be distinguished. Fragmentation of the nuclei, loss of the nuclear membrane, and a difficult-to-select zona pellucida structure were observed in this group. Lipid vacuoles of theca cells increase in number in PCOS pathology (Gözükara 2013). Histopathological results similar to those in PCOS were also observed in the Ami group, which exhibited oocyte and follicular damage, a thick tunica albuginea layer, and thick and irregular granulosa cells. All these findings suggest that the effects of AMI on the ovary snow need to be investigated more extensively. At histopathological analysis of the PKOS + AMI group, although the healthy follicle numbers and thick corpus luteum were higher than in the PCOS group, some damaged follicular and cystic structures were also observed. The presence of degenerated cells and structures at ultrastructural examination showed the negative effect of AMI on PCOS. These results show that the effect of PCOS cannot be reduced by AMI treatment. In addition to all these results, intense hilus cells, which are involved in testosterone secretion, were found in the PCOS group. Although the number of hilus cells in a healthy ovarian tissue varies, they increase in number in the postmenopausal ovary (Gilks and Clement 2012). Considering the increase in testosterone levels, a characteristic of PCOS, a greater hilus cell density is a possible outcome. This result reveals the need for further research into hilus cell structures and testosterone levels in ovarian structures with PCOS.
The majority of studies of PCOS have observed marked increases in serum LH levels (Azziz 2018; Yıldırım and Memişoğulları 2011; Teede et al. 2010). A study of 20 individuals with PCOS showed that LH levels increased compared to the healthy group and that the ovarian morphology changed accordingly. Another study involving three-week-old Wistar albino rats, produced a PCOS model observed that LH levels increased significantly compared to the control group, although the change in FSH was not significant (Sun et al. 2016). Another study of D vitamin treatment in a PCOS model reported increases in both LH and FSH levels (Çelik 2016). In the present study, both LH and FSH levels decreased in the PCOS group compared to the Cont group. However, only the decrease in FSH levels was statistically significant. Recent studies suggest that the principle marker of androgen level increases in PCOS is a rise in free testosterone levels rather than an increase in LH. A previous study showed that LH levels decreased in PCOS (Tessaro et al. 2015). The FSH and LH results in the present study were compatible with the previous literature. However, the fact that testosterone levels were not determined in this study cannot fully explain this situation. No statistical difference in FSH levels was found between the PCOS and PCOS + AMI groups, although there was a significant difference between the Cont and PCOS + AMI groups. Although this was not statistically significant, the LH value in the PCOS + AMI group was closer to that in the Cont group. This suggests that AMI treatment may cause changes in FSH and LH levels.
The association between PCOS and oxidative stress has been demonstrated by numerous previous studies (Gonzalez et al. 2006; Kuşçu and Var 2009; Blair et al. 2013). Gonzalez et al. showed that the hyperglycemia observed in PCOS patients affects androgen levels by stimulating the secretion of ROS from mononuclear cells. The present study revealed an increased CAT level in the PCOS and AMI groups compared to the Cont group, but there was no significant difference in the PCOS + AMI group. No significant differences in SPD levels were found among any of the groups. The increase in CAT levels in the PCOS group is consistent with previous studies in the literature.
Although a statistically insignificant decrease in SOD is not expected, there are studies reporting decreased SOD levels in individuals with PCOS (Liu and Zhang 2012; Seleem et al. 2014). Several studies have reported that AMI exacerbates oxidative stress (Viola et al. 2000; Cordero et al. 2010; Mytych et al. 2019; Sehonova et al. 2019). The results in the PCOS + AMI group showed that the use of AMI together with PCOS causes a decrease in oxidative stress in rats, even though AMI and PCOS individually cause an increase in oxidative stress.
A previous study of ovarian morphology in rats with PCOS involving AMI showed that the numbers of follicles and the corpus luteum in the PCOS group decreased compared to the control group, while the number of atretic follicles and cystic follicles was significantly higher than the control group (Li et al. 2019). Li et al. stated that the total number of preantral and antral follicles, and the size of the corpus luteum increased significantly in the group treated with AMI, while the number of atretic follicles and cystic follicles decreased. However, these results are not consistent with our stereological and histopathological results. Considering all the data obtained from the PCOS + AMI group in this study, AMI treatment appears to have no positive effect on ovarian morphology.
In conclusion, AMI appears to exhibit no protective or curative effect against the deleterious effects of PCOS on ovarian follicles and hormonal levels, although it may exhibit antioxidant activity capable of reducing the oxidative stress that may occur with PCOS. In addition, the use of AMI alone caused morphological changes in the ovary due to oxidative stress.