Introduction
Polycystic ovary syndrome (PCOS) is a complex metabolic disease characterized by chronic anovulation and hyperandrogenism, affecting 5-10% of women of reproductive age (1). It is a health problem with many clinical findings such as anovulation, amenorrhea, oligomenorrhea, menstrual irregularities, dysfunctional uterine bleeding, and hirsutism. More serious long-term risks are developing infertility, endometrial hyperplasia, endometrial cancer, dyslipidemia, coronary artery disease, and possible breast cancer (2). The chance of implantation of the embryo in the uterus of PCOS patients decreases (3). Abnormal endocrine and paracrine factors, metabolic dysfunctions, and changes in the microenvironment during folliculogenesis cause failure in the oocyte maturation and embryonic development in women with PCOS (4,5). Impaired oocyte sufficiency in PCOS is inevitably associated with abnormal follicle development. In PCOS patients, insulin resistance and paracrine disorder of growth factors, including transforming growth factor-b disrupt the follicular environment. This changes the relationship between granulosa cells and oocytes and damages the cytoplasm of the oocyte and the nucleus maturation (6,7,8,9,10).
PCOS is characterized by abnormal secretion of gonadotropins (11). PCOS also is an increased synthesis of steroid hormones by the ovaries (11). In particular, the secretion of LH and T is increased in women with PCOS. The ratio of luteinizing hormone/follicle-stimulating hormone (LH/FSH) is elevated with PCOS, it results in the ovaries producing more androgens. Furthermore, in PCOS patients, the levels of insulin and insulin-like growth factors are elevated. This situation eventually increases the production of androgens by follicular theca cells and enhances LH function (12). Apoptosis and the cellular proliferation ratio are essential for the normal physiological function of the body. However, this balance is disturbed in PCOS patients. Some factors are related to this syndrome, including the tumor regulatory gene, p53 (13,14).
The most common secondary metabolites in plants are polyphenols and tannins are among the most widely studied phenolic compounds (15). Tannins are classified into hydrolyzed tannins and condensed tannins. Tannic acid (TA) is a kind of hydrolyzed tannin (16). Tannins have been reported to promote mammalian ovulation rate and embryo development (17). Also, TA prevents damage to biological molecules in the body by inhibiting hydroxyl radicals (18). TA has been accepted by the Food and Drug Administration as a safe chemical (19). TA can interact directly with biomacromolecules such as collagen, gelatin, and albumin due to its ability to form electrostatic interactions, hydrogen bonds, and hydrophobic interactions (20). Recently, it has been shown that TA can be used as an anti-cancer chemical in various animal cells (21). It has been shown that TA increases plasma androgen and estrogen in both pikas and root voles (22). It has been reported that low concentrations of tannins promote the in vitro embryo development of the mouse oocyte (23). However, there are very limited studies related to explaining why tannins could affect the reproduction of animals.
In the literature, there is no study demonstrating the effect of TA on experimental PCOS models in rats. The present study was carried out in an attempt to elucidate the effects of TA on letrozole-induced PCOS modelled rats with three main objectives. First, to determine whether TA administration in the PCOS model is correlated with circulating LH, FSH, and T concentrations, and to determine whether TA can regulate the p53 expressions, and third, to determine whether TA can improve ovarian architecture and folliculogenesis in PCOS modeled rats.
Material and Methods
The experimental study was performed in the laboratory of the Hamidiye Experimental Animals Production and Research Laboratory, University of Health Sciences Türkiye. Ethical approval of the project was obtained from the Hamidiye Animal Experiments Local Ethics Committee, University of Health Sciences Türkiye (no: 2019-03/01). All experimental animal procedures were performed following standard ethical guidelines. Scientific Research Projects Unit, University of Health Sciences Türkiye (no: 2019/071) supported this study.
PCOS Model and Experimental Groups
A total of 18 female adult Wistar Albino rats weighing 170-250 g and 7-9 weeks old were used. The rats were drunk into city water and fed with standard pellet feed in a room with a 12-h light 12 hours dark photoperiod. The temperature was 21-23 °C. Rats in the estrus phase were determined by vaginal cytology follow-up. From the 10th day until the last day of the experiment, vaginal smear analysis was performed every day. The estrus cycle was followed by methylene blue staining. While examining the preparations, the result was reached by taking the cell structures as a reference (24). Weight control of experimental animals was done every day.
Letrozole was dissolved in 1 mL 0.5% carboxymethyl cellulose (CMC). All agents were given the gavage method. The rats were randomly divided into 3 prospective groups as follows:
The control group (n=6): The group in which 0.5% CMC was given every day during the experimental period.
PCOS group (n=6): The group in which 1 mg/kg/day letrozole® (FL24873, Cymıt Químıca S.L., Spain) was given for 21 days then 1 mL of distilled water was given for 10 days.
PCOS+TA group (n=6): The group in which 1 mg/kg/day letrozole® was given for 21 days and then 40 mg/kg/day TA was given for 10 days.
Histopathological Assessment
At the end of the experiment, 75 mg/kg ketasol and 10 mg/kg xylazine anesthesia were applied intraperitoneally to all rats. All rats were sacrificed by exsanguination and blood samples were collected via intracardiac way. The left ovaries of all the groups were isolated for histopathological assessment. The ovaries were fixed with 10% formaldehyde for pathological examination and embedded in paraffin blocks after standard tissue processing. The blocks were sectioned with a thickness of 5 µm. Preparations were stained with HE and evaluated under light microscopy in terms of ovarian follicle number.
To assess ovarian architecture and follicle number, three sections of ovarian tissue were selected. Blinded observers evaluated the sections independently. Primordial, primary, secondary, graafian, and cystic follicles were counted as suggested by Souza et al. (25). Follicles were counted: As primordial follicle; surrounded by thin single layers of follicular epithelial cells surrounding the oocyte, primary follicle; the single or multilayered prismatic epithelium surrounding the oocyte, secondary follicle; follicle in which the oocyte is lined with more than two granulosa cells with a small antrum, graafian follicle; follicle containing a single large antrum, in which the oocyte is surrounded by some cumulus cells, cystic follicles; follicles with degrading granulosa cells in the thin layer of granulosa cells (26). The stained tissue samples were observed by a light microscope (Zeiss, Scope.A1, Germany).
Immunohistochemistry (IHC) Staining of p53
Apoptosis of the cells was measured by IHC staining of p53 [Novocastra™ Liquid Mouse Monoclonal Antibody, p53 protein (DO-7), United Kingdom]. Deparaffinized ovary slides were rehydrated and incubated in citrate buffer for antibody retrieval. The lides were incubated in 3% of H2O2 to quench the endogenous peroxidase activity. After blocking, slides were incubated with a p53 antibody at 4 °C overnight. Then, slides are incubated with biotinylated secondary antibodies, streptavidin-horseradish peroxidase, and DAB (3,3¢-diaminobenzidine) on the next day. Counterstaining was performed with a hematoxylin solution for 5 min, and then slides were mounted after dehydration. The stained tissue samples were observed by a light microscope (Zeiss, Scope.A1, Germany).
Biochemical Assessment
The blood was centrifuged at 12000 rpm for 10 minutes, serum samples were stored at -80 °C. Serum T (E0259Ra, BT Lab, Korea), FSH (EA0015Ra, BT Lab., Korea), and LH (E0179Ra, BT Lab., Korea) concentrations were measured by ELISA kits. The standard procedures were followed during ELISA measurements.
Statistical Analysis
Statistical analysis of the data was performed with SPSS 25.0 (Statistical Package for the Social Sciences) program. The distribution of variables was analyzed using the Shapiro-Wilk test and normally distributed variables were reported as mean ± standard deviation and non-normally distributed variables were reported as median (minimum-maximum). Normally distributed variable groups were examined by the One-Way ANOVA test and non-normally distributed variable groups were examined with the Kruskal-Wallis H test. Within-group differences were examined using the Bonferroni correction. Different time measures of dependent variables were examined using the Repeated Measure ANOVA test. P<0.05 was considered statistically significant.
Results
Evaluation of the Estrous Cycle
The experiment was successful in all rats. Estrus cycles were determined by methylene blue staining (Figure 1A). Regular estrus cycles were observed in the vaginal smear preparations of rats belonging to the control (G1) group. Vaginal smear preparations from rats belonging to the PCOS group (G2) exhibited an irregular estrous cycle; mostly in the estrus phase. It was observed that the estrous cycle was regular in the PCOS+TA group.
Evaluation of Body Weights
In all groups, the weight gain increased as the rat age increased weekly (Figure 1B). Accelerated weight gain was observed in the PCOS model group compared to the control and PCOS+TA group.
Light Microscopy
Normal ovarian morphology was exhibited in the control group in the histopathological analysis. Many cystic follicles were present in the PCOS group. The ovarian tissue of letrozole-induced PCOS rats depicted cystic expansion, enlarged cystic sinus follicles, and decreased granulosa cell layers (Figure 2). The improved ovarian architecture was detected in the PCOS+TA group as indicated by the decreasing number of cystic follicles and an increasing number of graafian follicles in comparison to the PCOS group. It has been observed that treatment with TA reduced the theca cell layer, increased granulosa cell layers, and improved mature follicles in ovaries.
Table 1 shows the data of the primordial follicle, secondary follicle, Graaf follicle, and cystic follicle numbers. The number of cystic follicles was significantly increased in the PCOS group compared to the TA treatment group (p<0.05). The number of primary follicles was significantly increased in the TA treatment group compared to the PCOS group (p<0.001). No significant change was observed in the number of primordial, secondary, and Graaf follicles between the experimental groups. Light microscopic results supported the analysis of follicular count (Figure 2).
IHC Staining of p53
Apoptosis was determined by assessing the expression of p53 (Figure 3). The p53 immunoreactivity in the ovaries of all animals belonging to the control and experimental groups was examined.
Immunoreactivity was not observed in any of the control and experimental groups. In the PCOS group, p53 staining in the nuclear areas was determined below 1%.
Biochemical Assessment
Table 2 shows the results of the biochemical parameters. In terms of hormone LH and T levels, the PCOS group had a significant increase compared to the control group (p<0.05). The increase in LH was significantly decreased in the PCOS+TA group (p<0.05). Although it was not significant, serum FSH level was increased in the PCOS+TA group.
Discussion
PCOS is a disease characterized by an androgen increase in the ovaries, ovulation disorder, and cystic structures in the ovaries (27). This endocrine disease affects 4-8% of women of reproductive age. 40% of women with PCOS are infertile due to anovulation disorder (28). The diagnosis of PCOS is based on many criteria. PCOS treatment, causes, genetic transmission, roles of related genes, and gene regulation have not been fully elucidated (29). TA is an antioxidant compound, contains polyphenolic compounds, so it can mitigate various conditions of oxidative stress (30). In this study, we hypothesize that the administration of TA might have a potential curative effect against PCOS-modelled rat ovaries. To test this hypothesis, serum LH, FSH, and T levels, the number of follicles, and p53 expression were examined. Our study showed that 40 mg/kg TA treatment significantly decreased the number of cystic follicles. Also, although TA treatment cannot fully improve the p53 expression, it can control the hormone profile, the ovarian follicular cell architecture, and folliculogenesis in PCOS.
Since anovulation is one of the symptoms of PCOS, the estrous cycle of rats was followed by a vaginal smear examination. It has been previously reported that letrozole-treated rats lost their regular estrus period (31). In our study, it was determined that the letrozole-administered rats exhibited an irregular estrous cycle, which depicts successful PCOS modeling. It was determined that letrozole caused prolongation of the estrus cycle in rats, apoptosis in granulosa cells, thinning of the membranes, and an increase in the number of antral follicles. In our study, it was determined that rats administered letrozole showed irregular and prolonged cycles and TA administration regulated the estrus cycle. These findings depicted that TA administration may control and regulate the estrus cyclicity in PCOS ovaries. Our finding are in accord with the literature (32).
Although obesity is not the only cause of PCOS, it plays a role in the pathophysiology of this disease. Glucose intolerance and insulin resistance have been found to be other causes of obesity in women with PCOS (33). In this study, the body weights of the rats were evaluated weekly, it was determined that the weights of the PCOS group increased in comparison to the control group. It was statistically determined that body weight gain decreased in the TA treatment group. These findings are consistent with previous studies in the literature (34).
To our knowledge, this study is the first report demonstrating a significant depletion in cystic follicles in the PCOS model by TA treatment. 40 mg/kg TA treatment decreased the elevated number of cystic follicles significantly in comparison to PCOS (p<0.05). These indicate the potential therapeutic effects of the TA treatment on the maintenance of folliculogenesis in the PCOS model (Table 1). Although no statistically significant difference was found, it was determined that the number of primordial follicles in the ovary was decreased in rats in the PCOS group. It was observed that the follicle reserve could not be preserved in the PCOS group due to the decrease in primordial follicular tissue, but it was preserved in the TA group. Furthermore, the number of cystic follicles was increased significantly in PCOS rats (p<0.05). The elevated number of cystic follicles is caused by hyperandrogenism (13). Since granulosa cells cannot transform into luteinized granulosa cells due to low FSH in PCOS, progesterone production and ovulation do not occur. A slowdown or hesitation at any stage of this mechanism causes the follicle to remain in the form of millimetric cysts in the ovaries. Therefore, with the monthly recurrence of these events, the number of millimetric cysts in the ovary increase (35). The effects of TA might be due to its potent antioxidant and anti-inflammatory properties. An increase in the number of primary follicle counts was significant in TA administered group compared to the PCOS group (p<0.001) (Table 1). In the PCOS group, the primary follicles were decreased; pointed out that some of these follicles in the folliculogenesis process have undergone atresia or formed secondary follicles. This idea is consistent with the increased secondary follicle counts, especially in the PCOS group. In histopathological evaluation, letrozole-induced PCOS rats depicted cystic expansion, enlarged cystic sinus follicles, and decreased granular cell layers. These features are consistent with the pathological changes observed in human PCOS ovaries (Figure 2).
In PCOS patients, the secretion of LH and T is increased on the other hand FSH is decreased. When the LH/FSH ratio is elevated, the ovaries preferentially synthesize androgens. Therefore, PCOS-diagnosed women have higher levels of LH and T (36). In PCOS, ovulation does not occur due to low FSH and there is no progesterone production since granulosa cells cannot transform into luteinized granulosa cells (37). In this study, a significant increase in LH and T was observed in the PCOS group (p<0.05) (Table 2). The increase in LH significantly decreased TA administered rats (p<0.05) (Table 2). Serum T level was also decreased in TA administered rats. This reveals that TA can control the serum LH and T levels in PCOS modelled rats. No significant difference was observed in serum FSH levels. In humans, a decrease in serum FSH levels is seen in PCOS patients, but no statistical difference was observed in rats (38). Serum FSH level was increased in the PCOS+TA group. This suggests that TA can control FSH release in PCOS. Although it was not significant, a slight increase in serum FSH levels can be explained by an increase in FSH level at the very beginning of the prolonged estrus phase in PCOS rats (38).
The largest receptor group of apoptotic signals are molecules that have important roles in the cell cycle. One of the proteins that is best understood to control apoptosis is the p53 protein (39). The balanced ratio of cellular apoptosis and proliferation is disturbed in patients with PCOS. There is evidence that the cell’s cycle-related tumor regulatory gene, p53, is responsible for the etiology of PCOS (13). The p53 has been demonstrated to play a role in granulosa cell apoptosis. Altered expression of p53 may contribute to the possible enhanced follicular resistance to apoptotic signals in PCOS (40). Cui et al. (41) investigated whether follicular dysplasia is associated with the regulation of apoptosis in ovarian granulosa cells. They showed that while the S phase was longer in the PCOS group in comparison to the control group, the G2/M phase was shorter in length and the cells in the PCOS group were subjected to apoptosis (41). In our study, an IHC study was applied to show the apoptotic cells in granulosa cells and to determine the effect of the p53 expression pattern on the pathogenesis of PCOS at the protein level. However, a significant immunopositivity could not be determined in immunohistochemical light microscopy examinations of p53 (Figure 3). This indicated that PCOS follicles do not go atrophy. Also indicated that TA treatment cannot induce the apoptotic p53 mechanism.
Study Limitations
The present study has certain limitations. The circulating LH, FSH, and T concentrations were measured as the final outcome, but it is not clear whether TA treatment concentration would successfully regulate pregnancy. Furthermore, the present study failed to show whether low or high TA treatment is more important in controlling hormonal disturbances. Also, as this was an animal study, the results could not be considered linear for humans.
Conclusion
We observed that TA can alleviate histological disturbances in the PCOS model of rats. To the best of our knowledge, we reported for the first time that TA may be an effective and good alternative therapeutic agent in ameliorating PCOS histological disturbances and hormonal regulation. However, further studies are needed to confirm the safety of TA and its proper dose for PCOS treatment.
Ethics
Ethics Committee Approval: Research Laboratory, University of Health Sciences Türkiye. Ethical approval of the project was obtained from the Hamidiye Animal Experiments Local Ethics Committee, University of Health Sciences Türkiye (no: 2019-03/01).
Informed Consent: All experimental animal procedures were performed following standard ethical guidelines.
Peer-review: Externally peer-reviewed.
Authorship Contributions
Surgical and Medical Practices: F.G., M.T., S.A., Concept: F.G., T.S., H.T.C., S.T., K.S., O.I., Design: F.G., T.S., H.T.C., S.T., K.S., O.I., Data Collection or Processing: F.G., K.S., M.T., M.E.P., Analysis or Interpretation: F.G., K.S., S.A., Literature Search: F.G., T.S., H.T.C., S.T., K.S., O.I., Writing: T.S., H.T.C., S.T., K.S., O.I.
Conflict of Interest: No conflict of interest was declared by the authors.
Financial Disclosure: The authors declared that this study received no financial support.