An investigation of the potential effects of amitriptyline on polycystic ovary syndrome induced by estradiol valerate

Polycystic ovarian syndrome (PCOS) is frequently observed in adolescent women and usually progresses with depression. The aim of this study was to examine the effects of amitriptyline (Ami), a drug used in the treatment of depression, in individuals with PCOS. Forty 12-week-old female Wistar albino rats were randomly divided into five groups: control, sham, PCOS, Ami, and PCOS + Ami. To induce the syndrome in the PCOS groups, a single dose of 4 mg/kg estradiol valerate was administered by intraperitoneal injection; 10 mg/kg Ami was administered by intraperitoneal injection for 30 days in the Ami groups. After 30 days, all the animals were sacrificed and blood, ovary, and brain tissues were collected and subjected to routine tissue processing. Stereological, histopathological analyses were performed on the ovarian sections, while luteinizing hormone (LH), follicle-stimulating hormone (FSH), catalase (CAT), and superoxide dismutase (SOD) levels were investigated in blood samples. The volume of the corpus luteum and preantral follicles increased in the PCOS group, while a decrease was determined in the number of antral follicles using stereological methods. Biochemical analysis revealed that FSH levels increased and CAT enzyme levels decreased in the PCOS group. Significant morphological changes were observed in ovaries from the PCOS group. The volume of the corpus luteum in the PCOS + Ami group decreased compared to the PCOS group. Serum FSH levels decreased in the PCOS + Ami group, while CAT enzyme levels increased compared to the PCOS group. Degenerative areas were also seen in the PCOS + Ami group ovaries. Ami administration was unable to sufficiently ameliorate the morphological and biochemical changes caused in the ovarian tissues by PCOS. In addition, this study is one of the few studies examining the effects of amitriptyline, an antidepressant frequently used in depression treatment of individuals with PCOS. We also observed firstly that use of amitriptyline caused PCOS-like ovarian morphology in healthy rat ovaries, while it had a healing effect by volume decreasing of cystic structures in the ovary with PCOS.


Introduction
Polycystic ovary syndrome (PCOS), one of the most prevalent diseases among women of reproductive age, is an endocrine disorder characterized by anovulation, hyperandrogenism, and polycystic ovary (Witchel 2006). Diagnosis of PCOS in a patient with the diagnostic criteria defined by the National Institute of Health (NIH), and revised at the Rotterdam Conference in 2003, was based on three criteria: chronic anovulation, hyperandrogenism, and polycystic ovary. At least two of these criteria must be present for a patient to be diagnosed with PCOS (Witchel 2006;Norman et al. 2007).
Chronic anovulation, the first criterion, involves longterm menstruation (oligomenorrhea) or amenorrhea. Oligomenorrhea is observed in 60% of cases, and these patients' menstrual cycles are completed in 35-day periods. Hypothalamic dysfunction is present in patients with amenorrhea, which is observed in 40% of cases (Teede et al. 2010). Hyperandrogenism, another PCOS diagnostic criteria, is characterized by increased androgen levels. Increased androgen release causes both clinical and biochemical symptoms. Hirsutism and acne are the most frequent clinical symptoms, but these are not used alone to define hyperandrogenism.

3
Circulating free androgens are measured as a biochemical marker of hyperandrogenism. Increased luteinizing hormone (LH) and free testosterone levels are other common symptoms of PCOS (Rotterdam 2004). Polycystic ovary is a term used for ovaries containing 10-15 follicles 2-9 mm in diameter and thickened tunica albuginea in the cortex. The volume of the ovarian stroma usually increases in PCOS, and can exceed 10 mL. A manifestation in only one ovary is sufficient for diagnosing polycystic ovary (Rotterdam 2004). Although polycystic ovary and PCOS are frequently confused, the two are quite distinct entities. Patients with polycystic ovaries should meet at least two of the diagnostic criteria to be considered to have this syndrome (Norman et al. 2007).
The pathophysiology of PCOS has yet to be fully explained. However, hypothalamic-pituitary dysfunction, exaggerated adrenarche, intraovarian factors, insulin resistance, hyperinsulinemia, obesity, genetic factors, abnormal granulosa cells, and enzymatic disorders are among the causes of the syndrome (Ehrmann 2005). Disruption of the hypothalamic-pituitary axis causes high amounts of gonadotropin to be secreted from the hypothalamus, thus altering the levels of LH secreted from the pituitary gland. The cells most affected by changes in LH levels are theca follicle cells in ovarian follicles and androgen synthesis increases, in line with the rise in LH levels. Increasing androgen levels play a role in developing numerous symptoms seen in patients with PCOS. Increasing estrogen levels in peripheral tissues and rising free androgen levels cause an increase in LH release and inhibit follicle-stimulating hormone (FSH) release with negative feedback. Anovulation develops as a result of these changes. Since FSH cannot be completely inhibited, the stimulation of the follicles continues. However, oocyte development does not occur during the development of the stimulating follicles, and ovulation cannot occur. Instead, these follicles appear as cystic structures in the ovary for 5-6 months (Ehrmann 2005). Many recent studies have shown that insulin resistance also plays an important role in PCOS. Insulin affects the theca cells and raises androgen levels, thus reducing the release of sex hormone-binding globin in the liver, and the level of free testosterone gradually increases as a result (Dumesic et al. 2008;McNeilly and Duncan 2013).
Depression is one of the most common PCOS symptoms (Cooney and Dokras 2017;Hung et al. 2014). However, whether depression is the cause or symptom of the disease is still debatable. However, the treatment of depression ameliorates the symptoms of PCOS. For this reason, antidepressant therapy is frequently employed in patients with both diseases. The most commonly used antidepressants in the treatment of depression are tricyclic antidepressants. Amitriptyline (Ami) is one such tricyclic antidepressant. Its mechanism of action has not been fully explained, although some drugs, such as serotonin and norepinephrine, are thought to suppress the reuptake of neurotransmitters from the membrane (Bryson and Wilde 1996).
In the literature, the number of studies examining the effects of amitriptyline, especially on the ovary, is very limited, and there is only one study examining the changes caused by amitriptyline in ovarian morphology. The purpose of this study was to investigate the microscopic and biochemical changes occurring in the PCOS rat model following the administration of Ami, a tricyclic antidepressant, in the treatment of PCOS symptoms.

Experimental animals and procedure
This study was supported by the Ondokuz Mayıs University Project Management Office (PYO. TIP.1904.19.002) and was approved by the Experimental Animal Studies Ethics Committee of Ondokuz Mayıs University (HADYEK 2017(HADYEK /54, 02.03.2018. All animal experiments were completed with the ARRIVE guidelines and were carried out in accordance with the U.K. Animals (Scientific Procedures) Act, 1986 and associated guidelines, and in accordance with the EU Directive 2010/63/EU for animal experiments. This experiment was strictly done according to these guidelines. A total of 40 female Wistar albino 12-week-old 200-250 g rats were used and divided into five equal groups; control (Cont), sham (Sham), Ami, polycystic ovary syndrome (PCOS), and PCOS plus Ami treatment (PCOS + Ami). The sham group received a single injection of 0.2 mL sesame oil. For the induction of PCOS, the PCOS and PCOS + Ami groups received a single injection of 4 mg/kg estradiol valerate dissolved in 0.2 mL sesame oil (Alivandi Farkhad and Khazali 2019; Li et al. 2019). The Ami groups received 10 mg/kg Ami injections for 30 days. The rats were housed in standard plastic cages at 50% ± humidity at room temperature in a 12-h light/12-h dark cycle. The body weight of the rats at the beginning and end of the experiment was measured.

Tissue procedures and analysis
At the end of experimental procedures, the rats were anesthetized with intramuscular ketamine (10 mg/100 g body weight; Sigma Chemical Comp. St. Louis, MO, USA) and prilocaine hydrochloride (0.25 mg/100 g body weight; Sigma Chemical Comp., St. Louis, MO, USA). The ovarian tissues were removed, and 1 cc of blood was collected from the heart for biochemical analysis.

The light microscopic procedures
For the light microscopic and stereological analysis, tissue samples were washed for twelve hours and were processed with graded alcohol (70%, 80%, 96%, and 100%) for dehydration and treated with xylene for clearing (Sigma Chemical Comp, St. Louis, MO, USA). After that, the processed tissue samples embedded in paraffin (Merck, Darmstadt, Germany) for the infiltration step. Sections were cut to a thickness of 5 or 25 μm using a rotary microtome and taken on the slides (Leica RM 2135, Leica Instruments, Nussloch, Germany). The slides were stained with hematoxylin and eosin for light microscopic and stereological analysis. For the histopathological analysis, sections were examined with a light microscope (Leica DM 7000, Leica Microsystems GmbH, Germany).
For histopathological and stereological analyses, structures with a thin layer of granulosa, a thickened theca layer, and a degenerated nuclear structure were defined as cysts (Wu et al. 2014;Wang et al. 2012;Lim et al. 2011).

The electron microscopic procedures
For transmission electron microscopic analysis, ovarian tissue samples were processed with osmium tetroxide for twelve hours, and passed through graded acetone (50%, 70%, 95%, and 100%) and propylene oxide (Sigma Chemical Comp., St. Louis, MO, USA), and araldite, and embedded in resin blocks. Semi-thin sections of 0.5 μm and thin sections of 70 nm were taken from these blocks with a ultramicrotome microtome (Leica RM 2135, Leica Instruments, Nussloch, Germany). Semi-thin sections were stained with toluidine blue, while thin sections were made visible using uranyl acetate and lead citrate solutions. Semi-thin sections were examined with a light microscope (Olympus BX43, Center Valley, PA) and images were taken with a digital camera (Olympus SC50, Center Valley, PA) connected to the microscope with lenses of 20×, 40×, and 100× magnification (numerical aperture 0.40; 0.65; 1.25 oil). Thin sections were examined with an electron microscope (JEOL JSM-7001F, JEOL Ltd., Tokyo, Japan).
For scanning electron microscopic analysis, one ovary from each group was passed through acetone series (25%, 50%, 75%, and 100%) and processed via critical point drying, and then plated with gold-palladium. After the tissue processing, the sections were examined with an scanning electron microscope (JEOL JSM-7001F, JEOL Ltd., Tokyo, Japan).

Biochemical analysis
For the biochemical analysis, 1 cc of blood samples collected from the animals' hearts was centrifuged at 2000 rpm for 15 min. Levels of LH, FSH, superoxide dismutase (SOD) (Sunred Biological Technology Co., Ltd, Shanghai, China), and catalase (CAT) (Cayman Chemical Company, Germany) in the serum samples were analyzed using the procedures described by the relevant kits.

Volume analysis
The Cavalieri method was used for volume analysis. Images of sections of ovarian tissue 5 μm in thickness were taken with a microscope with a camera attachment (Leica DM 7000, Leica Microsystems GmbH, Germany) and opened in ImageJ software. A 9000 μm 2 point counting grid was used to estimate cyst, corpus luteum, and follicle volumes.
The grid was randomly superimposed onto the image, and those points intersecting with the area of interest were counted. Next, the volume was calculated using the formula V = t × a(p) × ∑p, where V is volume, t is section thickness, a/p is area represented by a point in the area measurement scale, and ∑p is the total number of points falling on the surfaces of the slices.

Estimation of follicle number
Twenty-five micrometer-thick sections were used for follicle number estimation. Follicle numbers were calculated using the optical fractionator method in the Stereoinvestigator system (StereoInvestigator, MicroBrightField Bioscience Williston, VT, USA). The optical fractionator involves counting the particles formed by the optical disector volume. Estimation of primordial, preantral, and antral follicle number was performed using this method, which allowed particles within a given counting frame to be counted. In the analysis, the counting frame (x, y) was 120 × 120 μm in size, the step interval (x, y) was 200 × 200 μm, the disector height was 10 μm, and the upper and lower safety intervals were 3 μm.

Statistical analysis
The numerical data obtained were analyzed on SPSS software (SPSS version 21.0; SPSS Inc., Chicago, IL, USA) and expressed as mean ± standard error (SE). The Shapiro-Wilk test was used for normal distribution assumptions. Normally distributed data were compared between the groups using one-way analysis of variance (ANOVA) and the Tukey test. The Kruskal-Wallis and Tamhane tests were used to compare multiple groups without normal distribution. p values < 0.05 were considered statistically significant, and p values < 0.01 were considered highly statistically significant.

Ethical approval
This animal study was approved by the Experimental Animal Studies Ethics Committee of Ondokuz Mayıs University (HADYEK 2017/54, 02.03.2018).

Body weight
The animals' weights were recorded at the beginning and end of the experiment. Weights increased at the end of the experiment in the PCOS group (p = 0.048). No significant differences were determined in the other groups (p > 0.05) (Fig. 1).

Volume of cyst, corpus luteum, and follicles
In terms of the volume analysis of the corpus luteum located in the ovarian cortex; a statistically significant volume increase was observed between the Cont and PCOS groups (p = 0.047, one-way ANOVA). There are a statistically significant volume decrease between the PCOS and Ami groups (p = 0.034, one-way ANOVA), and a highly significant decrease in the volume of PCOS + Ami group in comparison with PCOS group was observed (p = 0.004; oneway ANOVA) (Fig. 2). The volume of the corpus luteum was higher in the PCOS group than in the Cont, Ami, and PCOS + Ami groups.
No statistically significant difference was observed between the groups in terms of primordial and antral follicle volumes (p > 0.05, one-way ANOVA). The preantral follicle volume results from the PCOS group were significantly higher than those in the Cont group (p = 0.021, oneway ANOVA) (Fig. 3).
The thin granulosa layer, thickened theca layer, and degenerative nuclear structures were determined for cyst volume analysis. No statistically significant difference of cyst volume was observed among the groups (p > 0.05, oneway ANOVA) (Fig. 4).

Follicle numbers
The number of follicles in the ovarian tissue was estimated using the optical fractionator method. No significant Fig. 1 The animals average weight values at the beginning and end of the experiment. Cont control group, Sham sham group, Ami amitriptyline group, PCOS polycystic ovary syndrome group, PCOS + Ami polycystic ovary syndrome and amitriptyline treatment group. *Statistical significance p < 0.05

Fig. 2
Volume results (± SE) for the corpus luteum in ovarian samples from all groups. A significant difference (*) was observed between the Cont, PCOS, and PCOS + Ami groups (p < 0.05). A highly significant difference (**) was observed between the PCOS and PCOS + Ami groups (p < 0.01) Fig. 3 The volume of primordial, preantral, and antral follicles (± SE) in all groups. There was a significant difference between the Cont and PCOS group for volume of the preantral follicle (p < 0.05, one-way ANOVA). For the volume of the primordial and antral follicles, no significant difference was observed between the groups (p > 0.05, one-way ANOVA) difference was determined between any of the groups in terms of numbers of primordial and preantral follicles (p > 0.05, one-way ANOVA). A significant decrease was found in the PCOS group in comparison with the Cont group (p = 0.039, one-way ANOVA) in terms of antral follicle number. The same decrease of antral follicle numbers was observed in the PCOS group when compared with the Ami group (p = 0.017, one-way ANOVA). Likewise, there was a significant difference between the Ami and PCOS + Ami groups (p = 0.035, one-way ANOVA) in terms of antral follicle numbers. The number of follicles in the Ami group was higher than in the PCOS + Ami group. A decreased number of antral follicles in PCOS + Ami group were found in comparison with the Sham group (p = 0.002, one-way ANOVA) (Fig. 5). Figure 6 shows the levels of FSH (a), LH (b), CAT (c), and SOD (d) in the blood sera (IU/L). There were no significant differences between any of the groups in terms of LH levels (p > 0.05, one-way ANOVA) (Fig. 6b). However, serum FSH levels differed significantly between the Cont and PCOS + Ami groups (p = 0.004, one-way ANOVA) and highly significantly between the Cont and PCOS groups (p = 0.001, one-way ANOVA) (Fig. 6a). In the Cont group, FSH levels were higher than the PCOS and PCOS + Ami groups. Highly significant differences in CAT levels were observed between the Cont and Sham (p = 0.001, one-way ANOVA), Cont and PCOS (p = 0.001, one-way ANOVA), and Cont and Ami (p = 001, one-way ANOVA), PCOS + Ami, and PCOS (p = 0,015, one-way ANOVA), (Fig. 6c) groups. The CAT level in the Cont group was lower than in the Sham, PCOS, and Ami groups. In addition, the level of CAT in PCOS + Ami was lower than the level of CAT in the PCOS group. The only significant difference in serum SOD levels was observed between the Cont and PCOS + Ami groups (p = 0.031, one-way ANOVA) (Fig. 6d). There was a significant decrease in the level of CAT in the PCOS + Ami group compared with the Cont group.

Microscopic characteristics of ovarian tissue under the light microscope
While the epithelium surrounding the ovary appeared in the form of a healthy, cuboidal epithelium in the Cont and Sham groups, it appeared as a cubic and prismatic structure in the PCOS and PCOS + Ami groups. A simple squamous epithelium was observed in the Ami group. The tunica albuginea layer in the PCOS, PCOS + Ami, and Ami groups was thicker than in the Cont and Sham groups (Fig. 7). When the follicle structures were examined, the oocyte borders were clear; the zona pellucida, the first-row granulosa cells surrounding the oocyte, and the theca layers were healthy, and the glassy membrane could be easily distinguished in the Cont and Sham groups (Figs. 7, 8). In the PCOS and PCOS + Ami groups, deformed granulosa cells were observed, in which the theca layers of the follicles were more prominent and thought to have entered apoptosis within the follicles. The borders of the oocytes in the follicles could not be distinguished, and the zona pellucida layer was indistinct. In the Ami group, the follicles were degenerated, similarly to the PCOS group. The density of hilus cells, characterized by spherical nuclei and lipid droplets in their cytoplasm, in the PCOS and PCOS + Ami groups was particularly remarkable. In the PCOS and PCOS + Ami groups, the density of cystic structures characterized by a thin layer of granulosa in the Fig. 4 Volume results (± SE) for cyst structures in all the study groups. No significant differences were observed between the groups

Fig. 5
Follicle numbers (± SE) in all the study groups. Significant differences (*) were observed between the Cont and PCOS, PCOS and Ami, and Ami and PCOS + Ami groups (p < 0.05). Highly significant difference (**) was determined between the Sham and PCOS + Ami, and Sham and PCOS groups (p < 0.01) cortex and a thick theca follicle was also striking. The presence of cystic structures similar to those in the PCOS group was also noteworthy in the Ami group (Fig. 8).

Microscopic characteristics of ovarian tissue under the scanning and transmission electron microscopes
Examination of the surface properties of the ovary revealed that the epithelium around the ovary was a simple cuboidal epithelium with a healthy appearance in the Cont and Sham groups. However, in the PCOS group, the outer surface was severely damaged, and the simple cuboidal epithelium had assumed other forms in some places. In the Ami and PCOS + Ami groups, the epithelium was mostly healthy. In addition, the epithelium in the PCOS + Ami group had indistinct borders, and the morphology of the connective tissue elements was severely impaired (Fig. 9, left column).
Examination of the corpus luteum structures in the ovarian cortex revealed that the luteal cells in the Cont and Sham groups exhibited a healthy structure with the typical endoplasmic reticulum, secretory vesicles, and mitochondria. In the PCOS group, the cytoplasm and mitochondria of the luteal cells were darkly stained, and the agranular endoplasmic reticulum cisterns were enlarged. The corpus luteum was healthy in the Ami and PCOS + Ami groups (Fig. 9, right column).

Discussion
PCOS is an endocrine disorder seen in the majority of women during their reproductive age, and in which cystic ovaries are observed in cases where follicular development steps are not fully completed due to increased androgen levels. Various symptoms occur in patients with PCOS, 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 and Doherty 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 of 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 and Doherty 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: Fig. 6 Biochemical analysis results from all the study groups (IU/L). a Serum FSH concentration results (± SE) for all groups. A significant difference (*) was observed between the Cont and PCOS + Ami groups (p < 0.05), and a highly significant difference was observed (**) between the Cont and PCOS + Ami groups (p < 0.01). b Serum LH (± SE) levels in all the study groups. No significant difference was observed between any of the groups. c Serum CAT concentra-tion results (± SE) from all the study groups. A highly significant difference (**) was observed between the Cont and Sham, Cont and PCOS, Cont and Ami, and PCOS and PCOS + Ami groups (p < 0.01). d Serum SOD concentration results (± SE) from all the study groups. A significant difference (*) was observed between the Cont and PCOS + Ami groups (p < 0.05) 1 3 high androgen levels observed in PCOS, insulin resistance, and infertility (Kerchner et al. 2009;Hung et al. 2014;Hart and Doherty 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 was observed 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 (Bidzińska-Speichert et al. 2012).
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 volumes. No statistically significant difference was 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 found in the PCOS group compared with 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. 2006). 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 with 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 the 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. 2012;Manneråset al. 2007;Yaba and Demir 2012). In their study of 40 rats, Behmanesh and coworkers (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 with 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 Fig. 9 Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) images from all the study groups. The first row shows the outer surface features of the ovaries, while changes are observed at the organelle level in the lower row. Lc lutein cell, N nucleus. Asterisk indicates lipid granule, arrow indicates mitochondria, arrowhead indicates endoplasmic reticulum. Bars: a, i 3 μm, b, j 1 μm c, d, f, h 2 μm, e 4 μm, g 5 μm 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, increased interstitial cells, and increased number of cells and volume of the corpus luteum observed in groups with PCOS (Takahashi et al. 1994;Wang et al. 2012;Gozukara et al. 2016). In the present study, the presence of a thick tunica albuginea was particularly noteworthy in the histopathological evaluation of the ovaries from the PCOS group. Dark-stained cells, with an angular structure and loss of 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 (Gozukara et al. 2016). 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 now need to be investigated more extensively. In the histopathological analysis of the PCOS + 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 may 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 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 on 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 with the healthy group and that the ovarian morphology changed accordingly. Another study involving 3-week-old Wistar albino rats produced a PCOS model where LH levels were increased significantly compared with the control group; although, the change in FSH was not significant (Sun et al. 2016). Another study of vitamin D treatment in a PCOS model reported increases in both LH and FSH levels (Çelik et al. 2018). In the present study, both LH and FSH levels decreased in the PCOS group compared with 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 patients with PCOS affects androgen levels by stimulating the secretion of reactive oxygen species (ROS) from mononuclear cells. The present study revealed an increased CAT level in the PCOS and Ami groups compared with the Cont group, but there was no significant difference in the PCOS + Ami group. No significant differences in SOD 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 with the control group, while the number of atretic follicles and cystic follicles was significantly higher than in the Cont 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.