Therapeutic Effects of Cynodon dactylon (C. dactylon) against Polycystic Ovary Syndrome Induced by Letrozole in Adult Rats: Ovarian and Uterine Aspects.

Polycystic ovary syndrome (PCOS) is the most common endocrine and metabolic disorder in women of childbearing age 15 to 49 years and affects about PCOS is a female reproductive disorder with a prevalence rate of 2.2 to 26% in the world and 19.5% in Iranian women ( 1 ). PCOS is a complex polygenic disorder influenced by environmental factors, such as those contributing to obesity and inherent abnormalities in ovarian steroidogenesis and follicular development ( 2 ). The syndrome is characterized by persistently rapid gonadotropin-releasing hormone (GnRH) pulses, an excess of luteinizing hormone (LH), and insufficient follicle-stimulating hormone (FSH) secretion, all of which contribute to different PCOS-derived disorders such as excessive ovarian androgen production and ovulatory dysfunction ( 3 ). Additionally, many women with PCOS exhibit insulin resistance, and compensatory hyperinsulinemia enhances ovarian and adrenal androgen production while increasing androgen bioavailability through reduced levels of sex hormone-binding globulin (SHBG). Genome-wide association studies have implicated numerous genes, including those for gonadotropin receptors, the beta subunit of FSH, insulin receptor, differentially expressed in normal and neoplastic cells domain-containing protein 1A (DENND1A), and thyroid adenoma-associated protein (THADA) ( 2 ). Furthermore, "programming" through environmental or hormonal imprinting may also contribute to the development of PCOS. In other words, it is a different syndrome with completely heterogeneous features including, hormonal imbalance in the ratio of LH to FSH, increasing the concentration of androgens and steroids, decreasing progesterone concentration, reproductive disorder, impaired glucose concentration, insulin resistance, menstrual irregularities, obesity, depression, and type II diabetes ( 4 ). Despite the prevalence of PCOS, the underlying cause has not yet been identified. Causes factors of PCOS include hormone imbalances, epigenetic changes in the fetus, genetic abnormalities, lifestyle, and environmental factors ( 5 ). Various pathophysiological factors may play different roles in individual patients with PCOS ( 3 ). Ovarian steroidogenesis, which requires gonadotropin stimulation, is influenced significantly by LH, a key factor in PCOSrelated hyperandrogenemia ( 3 ). Progesterone primarily regulates GnRH pulse frequency; however, in PCOS, the GnRH pulse generator exhibits relative resistance to progesterone's negative feedback effects ( 6 ). This resistance, mediated by androgen excess, can be reversed by the androgen-receptor blocker flutamide ( 7 ). Consequently, high GnRH pulse frequencies favor LH production and limit FSH production, promoting androgen production and disrupting normal follicular development. PCOS is associated with inherent abnormalities in ovarian and adrenal steroidogenesis. Cultured ovarian theca cells from women with PCOS secrete excess androgens and precursors, and these women exhibit exaggerated ovarian steroidogenic responses to gonadotropin stimulation ( 8 ). Hyperinsulinemia contributes to hyperandrogenemia by augmenting LH-stimulated androgen production by ovarian theca cells, potentiating corticotropin-mediated adrenal androgen production, and inhibiting hepatic synthesis of SHBG, thereby increasing free testosterone levels. The precise effects of hyperinsulinemia on gonadotropin secretion remain unclear ( 3 ). On the other side, PCOS can also lead to several serious pathological conditions, including type 2 diabetes mellitus, dyslipidemia, subclinical vascular disease (e.g., elevated coronary artery calcium scores, impaired endothelial function, and increased carotid-artery intimamedia thickness) ( 9 ). The risk of endometrial cancer is estimated to be 2.7 times higher among women with PCOS compared to those without the syndrome ( 10 ). Women with PCOS also face increased risks of pregnancy complications, such as preeclampsia and gestational diabetes, obstructive sleep apnea, and emotional distress, including depression and anxiety. The most important hormonal disease in PCOS is hyperandrogenism, which causes an increasing concentration of testosterone in the blood ( 11 ). Androgens also increase ovarian stimulation with LH, abnormal steroid production, hyperinsulinemia, and increased insulin increasing the response of androgens to LH ( 12 ). PCOS also causes various uterine disorders like endometrial hyperplasia and cancer and affects women’s fertility ( 13 ). Thus, the treatment of PCOS seems necessary and crucial. The goal of PCOS treatment is to relieve symptoms, correct sexual and ovulatory cycles, restore fertility, and prevent long-term complications ( 14 ). Metformin, a commonly prescribed medication for the management of PCOS in humans ( 15 ). Metformin is known for its insulin-sensitizing effects and has been shown to reduce hyperandrogenism and improve menstrual regularity in women with PCOS ( 16 ). However, all of implemented medications that have been used to cure PCOS (including metformin) have side effects such as hot flashes, arthritis, muscle aches, and psychological side effects such as irritability, mood swings, and depression ( 17 ). Therefore, scientists have been searching to discover brand-new anti-PCOS compounds. One of the most valuable sources of therapeutic compounds is plants. Medicinal plants have been used because of their natural and effective compounds for the treatment of PCOS ( 18 ). Due to the very long history of using medicinal plants in the treatment of diseases, human knowledge in identifying, mechanism of action, separation of effective compounds in them and industrialization of herbal medicine is increasing every day. Bermuda grass, Cynodon dactylon (C. dactylon) , is a weed of the Poaceae family, which is a weed with various medicinal properties, including treatment of urinary tract infections, cardiovascular disease, antimicrobial properties, antiviral activity, and antihypertensive impacts ( 19 ). Notably, Known chemical compounds in C. dactylon plant extract include flavonoids, alkaloids, glycosides, terpenoids, saponins, tannins, resins, phytosterol, carbohydrates, and proteins ( 20 ). Moreover, C. dactylon is effective in causing hypolipidemic, anti-hysteria, and also the treatment of thirst, anorexia, burning sensation in the body, itching, reverting fertility and hair loss ( 19 ). Leaves of C. dactylon is used orally to treat stomach aches and entire plant extract and to treat menstrual problems ( 21 ). Rhizome extracts are also anti-inflammatory, diuretic, and antiseptic ( 21 ). Besides, administration of aqueous extract of entire plant of C. dactylon to female eats leads to some fascinating effects on the reproductive system including a significant increase in the serum estradiol concentration, reduction in the level of FSH and LH in a significant manner, increase in the weight of the uterus and significant decrease in the weight of the ovaries, and disturbing the estrous cycle ( 22 ). Interestingly, toxicity with the extracts of this plant creates some adverse conditions such as increase in uterine weight and uterine proliferation in histopathology and reduced deciduoma formation ( 23 ). These findings demonstrate remarkable potential of the extracts of C. dactylon on the reproductive system of rat, especially rats with PCOS. All in all, by implementing Metformin, a commonly prescribed medication for the management of PCOS in humans, and also by including a metformin-treated group, we aimed to benchmark the therapeutic potential of C. dactylon against a clinically established treatment. Notably, Metformin was included in this study to provide a standard comparison for evaluating the efficacy of the C. dactylon extract. To be more specific, the aim of the present study was to investigate the effect of hydro alcoholic extract of C. dactylon on induced PCOS by letrozole in adult rats.

The assessment of the estrous cycle involved examining vaginal smears collected from rats over the final 14 days of both PCOS induction and subsequent treatment with metformin and C. dactylon. Analysis revealed that rats in the PCOS group exhibited irregular estrous cycles compared to the other experimental groups. Specifically, the estrous cycle in the PCOS group predominantly showed diestrus phases without a discernible regular pattern ( Fig .1A ). Interestingly, rats treated with metformin and C. dactylon initially displayed irregular estrous cycles which gradually normalized with prolonged treatment. The number of complete cycles observed in the control group exceeded those in the metformin and C. dactylon -treated groups. Statistical analysis indicated a significant difference in estrous cycles between the control and PCOS groups (P<0.001, Fig .1B ). Estrous cycles in the control group were notably more regular compared to the PCOS group, which exhibited irregular cycles and a prevalence of diestrus phases (P<0.001). Furthermore, the estrous cycles in the metformin and C. dactylon treatment groups showed a more regular pattern compared to the PCOS group (P<0.001). Additionally, rat body weight was measured at three distinct time points: initial weight, weight post-PCOS induction, and weight after treatment with C. dactylon and metformin. This assessment aimed to evaluate the impact of letrozole-induced syndrome, temporal effects, and their interactions across all groups. Significant differences were observed, with an increase in weight noted among rats in the PCOS-induced group. Conversely, rats treated with metformin and C. dactylon extract exhibited a significant weight reduction. Moreover, a notable difference was evident between the control group and the PCOS group (P<0.05, Fig .1C ). Therapeutic effects of Cynodon dactylon (C. dactylon) on stages of estrus cycle and weights after induction of PCOS in letrozole-induced adult rats. A. Diagrams illustrating various stages of the estrus cycle in the control group, PCOS group, metformin-treated PCOS rats, and C. dactylon extract-treated PCOS rats. B. Proportion of stages of the estrus cycle during three study periods: before induction (evaluation), during induction (induction), and after induction (treatment) in the four study groups. Lines above the columns indicate significant differences between the columns (P<0.05). C. Mean and standard error of body weight in rats during different study periods in the four groups. Stars of the same color as the lines denote statistical differences on each day. PCOS; Polycystic ovary syndrome, *, P<0.001, **; P<0.01, and ***; P<0.05. The study results revealed significant variations in the mean serum concentration of LH across different treatment groups. Specifically, the metformin-treated group exhibited a significantly higher LH concentration compared to the control, PCOS, and C. dactylon-treated groups (P0.05, Fig .2A ). Furthermore, there was no significant difference in serum FSH levels between the metformin-treated group and the PCOS group (P>0.05, Fig .2B ). However, a significant difference in the mean serum concentrations of LH to FSH ratio was noted in the metformin and C. dactylon -treated group (P=0.04, Fig .2C ). Regarding testosterone levels, the mean serum concentration in the extract-treated group showed a significant difference compared to the control, PCOS, and metformin-treated groups (P<0.05, Fig .2D ). Both the metformin and extract-treated groups exhibited a significant decrease compared to the PCOS group (P<0.05, Fig .2D ). Additionally, a significant difference in the mean serum concentration of estrogen was observed in the extract-treated group compared to the PCOS group (P=0.03, Fig .2E ). For progesterone, while the control group exhibited a significant difference from the PCOS group (P=0.008), no significant difference was found between the groups treated with metformin and extract (P>0.05, Fig .2F ). In summary, the findings indicate that induction of PCOS significantly increased mean serum concentrations of testosterone, estrogen, FSH, and LH (P<0.05), while significantly decreasing mean serum concentrations of progesterone. These results suggest that PCOS induction altered mean serum concentrations of estradiol, testosterone, and LH, resulting in a significant increase in androgens and estrogen. Furthermore, C. dactylon treatment led to a significant decrease in mean serum concentrations of estrogen and testosterone, and an increase in mean serum concentrations of progesterone. Serum hormone concentrations (means and standard errors) to evaluate the therapeutic effects of Cynodon dactylon against polycystic ovary syndrome induced by letrozole in adult rats. A. LH, B. FSH, C. LH/FSH, D. Testestrone, E. Estrogen, and F. Progestrone. PCOS; Polycystic ovary syndrome, FSH; Follicle-stimulating hormone, LH; Luteinizing hormone, *, **, and ***; Denote significance at the 1, 2, and 5% probability levels, respectively. The assessment included the observation and quantification of primary follicles, secondary follicles, tertiary follicles, morphologically atretic follicles, and corpus luteum across different experimental groups ( Fig .3AJ ). Generally, the control group demonstrated a higher count of secondary follicles, tertiary follicles, morphologically atretic follicles, and corpus luteum compared to the PCOS group. Although the number of tertiary follicles in PCOS rats was lower compared to the control group, this difference did not reach statistical significance. However, a significant difference was observed in the number of morphologically atretic follicles among the groups (P<0.05). Further histological examination of ovarian cortex follicles revealed a numerically higher mean number of secondary follicles in the control group compared to the PCOS group, although this difference was not statistically significant (P>0.05). The PCOS group exhibited a reduced presence of corpus luteum (P=0.030). Moreover, while the mean number of tertiary follicles was lower in the PCOS group compared to the control group, this difference was not statistically significant (P>0.05). The results indicated a significant decrease in the number of primary follicles and morphologically atretic follicles in the PCOS group, accompanied by a significant increase in cystic follicles (P<0.05). However, no significant differences were observed for secondary follicles, tertiary follicles, and corpus luteum.Comparison with the metformin-treated group at a dose of 250 mg/kg within the PCOS group revealed significant alterations in the number of atretic follicles, cysts, and corpus luteum (P<0.05). In contrast, the group treated with C. dactylon extract, compared to the PCOS group, exhibited a significant increase in the number of secondary follicles and corpus luteum, along with a significant decrease in the number of cystic follicles (P<0.05). Notably, this comparison did not reveal significant differences for primary and tertiary follicles. Ovarian follicle counts (means and standard errors) to evaluate the therapeutic effects of Cynodon dactylon against polycystic ovary syndrome induced by letrozole in adult rats. A. Number of primary follicle/slice, B. Number of secondary follicle/slice, C. Number of tertiary follicle/slice, D. Number of cystic follicle/slice, E. Number of atretic follicle/slice, F. Number of corpus luteum/slice. No statistically significant difference in the means was found. *, **, and *** indicate significance at the 1, 2, and 5% probability levels, respectively. Histological changes in ovaries of rats with letrozole-induced polycystic ovary syndrome (PCOS). Identification of corpus luteum (black stars) and normal tertiary follicles (arrows). PCOS-induced cystic follicles marked with black stars [hematoxylin and eosin staining (H&E)]. G. Control group, H. PCOS group, I. Metformin-treated group, and J. C. dactylon treatment group. Upon comparing the mean averages of antral structures to the total tertiary follicles, the antral area in the control group exhibited a significant difference compared to the PCOS group treated with C. dactylon (P=0.003, Fig .4AD ). Furthermore, the granulosa area in relation to the total tertiary follicles displayed a significant difference, with the control group differing significantly from the PCOS groups treated with metformin and C. dactylon extract (P<0.05); however, the theca area did not exhibit a significant difference across all groups. The measured antral region area significantly increased in the control group compared to the group treated with C. dactylon extract (P=0.02), but no significant difference was observed in the area of granulosa between the groups. The diagram illustrating the theca area in tertiary follicles revealed a significant increase in the control group compared to the PCOS group and the treatment group with C. dactylon extract. Additionally, the metformin-treated group displayed a significant decreasing trend compared to the PCOS and extract-treated groups (P<0.05). However, no significant difference was found in the total area of tertiary follicles between the groups. Comparison of the area of different layers of tertiary follicles across groups: absolute and relative measurements. A-D. Means and standard errors of the area of different layers of tertiary follicles were observed in different groups. There is no statistically significant difference in the mean. E-G. Means and standard errors of the area of different layers of tertiary follicles relative to total follicles were observed in different groups. No statistically significant difference in the mean was found. *, **; Denote significance at the 1, and 5% probability levels, respectively, and PCOS; Polycystic ovary syndrome. In morphometric studies focusing on the theca cell layer in different groups ( Fig .4E-G ), the results indicated a significant increase in the thickness of the theca layer in the PCOS group, indicative of PCOS induction. The theca layer was then measured in the group treated with C. dactylon extract, and the results were compared with the PCOS and control groups. This comparison revealed that C. dactylon reduced the thickness of this layer in rat ovaries, leading to the release of the oocyte-cumulus complex in antral follicles. This effect is likely attributed to the anti-inflammatory properties of C. dactylon , which reduces inflammation and collagen in the follicular sheath, ultimately decreasing the thickness of this layer. The results demonstrated significant findings regarding the uterine morphology across different treatment groups. Specifically, the mean luminal area of uterine tissue in the C. dactylon extract-treated group showed a significant reduction compared to the PCOS group (P=0.038, Fig .5A ). Additionally, the mean area of the endometrial layer in the metformin-treated group significantly decreased compared to the control, PCOS, and extract-treated groups (P<0.05, Fig .5B ). Moreover, the myometrial layer area in the control group was significantly reduced compared to both the metformin and extract-treated groups (P<0.05, Fig .5C ), and the myometrial layer in the metformin-treated group also significantly decreased compared to the PCOS group (P=0.002, Fig .5C ). In terms of the perimetrial layer, both the metformin and extract-treated groups exhibited a significant decrease compared to the control and PCOS groups (P<0.05, Fig .5D ). Additionally, a significant difference was observed in the comparison of the total uterine area among the metformin and extract treatment groups compared to the PCOS and control groups (P<0.05, Fig .5E ). Regarding glandular structure, the mean gland counts per area significantly increased in the PCOS group compared to the control, metformin, and extract-treated groups (P<0.05, Fig .5F ). Furthermore, the mean gland counts per area in the metformin and extract treatment groups significantly differed compared to the control group (P<0.05, Fig .5F ). Ovary and uterine histomorphometry. A-F. Means and standard errors of the area of different areas of uterine tissue were observed in different groups. There is no statistically significant difference in the mean. G-J. Mean and standard errors of different areas of uterine tissue to the whole uterus were observed in different groups. There is no statistically significant difference in the mean. *, **, and ***; Significant at 1, 2, and 5% probability levels, respectively. Histological changes in uterus of rats with letrozole-induced polycystic ovary syndrome (PCOS) are stained with hematoxylin and eosin stain- ing (H&E): K. Control group, L. PCOS group, M. Metformin-treated group, and N. C. dactylon treatment group. When examining the relative mean area of different uterine layers to the whole uterus ( Fig .5G-N ), the endometrial layer in the extract-treated group exhibited a significant difference compared to the control and metformin-treated groups (P<0.05), though not compared to the PCOS group. Moreover, a significant increase in the mean area of the perimetrial layer relative to the whole uterus was noted in the metformin-treated group compared to the PCOS and extract-treated groups (P<0.05, Fig .5J ). However, no significant differences were observed in the mean luminal area and myometrial layer diagrams between the groups. Overall, the findings suggest that C. dactylon extracts have a significant reducing effect on the luminal area of the perimetrial layer, endometrial glands, and total uterine area compared to the PCOS group, indicating a potential therapeutic benefit in this syndrome. Based on the molecular docking analysis, Ar-tumerone displayed the most binding affinity to anti-Müllerian hormone receptor type-2 (AMHR2), chemokine receptor type 7 (CXCR7), Estrogen receptor beta (ERβ), FSH receptor (FSHR), LH receptor (LHR), and tyrosine kinase type B (TrkB) ( Table 1 ). Besides, Tumerone had the most tendency to angiotensin II type 2 receptor (AT2R), β-adrenoreceptor, and chemokine receptor type 4 (CXCR4) ( Table 1 ). Moreover, Tricyclo[6.3.0.0( 1 , 5 )] undec-2-en-4-one, 2,3,5,9- tetramethyl demonstrated the most in-silico binding affinity to activin receptor (actR) and androgen receptor (AR). Curlone displayed the most affinity to Toll-like receptor 4 (TLR-4) and thyroid hormone receptor alpha (TRα). Ultimately, 3-Tert-butyl-4-hydroxyanisole showed the most binding affinity to Estrogen receptor alpha (ERα) ( Table 1 ). The detailed information about the amount of binding affinity and amino acids participating in the molecular interaction between compounds originating from the hydro alcoholic extract of C. dactylon and receptors involved in PCOS is demonstrated in Table 1 and Fig.S1 (See Supplementary Online Information at www.ijfs.ir), respectively. The values of binding affinity (Kcal/mol) between compounds originating from the hydro alcoholic extract of C. dactylon and receptors involved in polycystic ovary syndrome (PCOS) actR; activin receptor, AMH; Anti-müllerian hormone, AMHR2; AMH receptor type-2, AR; Androgen receptor, AT2R; Angiotensin II type 2 receptor, CXCR4; C-X-C chemokine receptor type 4, CXCR7; C-X-C chemokine receptor type 7, ER; Estrogen receptor, FSHR; follicle stimulating hormone receptor, LHR; LH receptor, NEFA; Nonesterified fatty acids, TLR-4; Toll-like receptor 4, TRα; Thyroid hormone receptor alpha, and TrkB; Tyrosine kinase type B.

The present survey demonstrated that C. dactylon extract exhibited a significant increase in the number of secondary follicles and corpus luteum, along with a significant decrease in the number of cystic follicles. However, the mentioned extract did not reveal significant differences for primary and tertiary follicles. In fact, the extract of C. dactylon could improve the structure of ovaries in the Letrozole-induced PCOS rats. To the best of our knowledge, this finding about the effects of C. dactylon extract on the number of various types of follicles in PCOS rats has been reported for the first time. Notably, Prior research examining the impact of C. dactylon extract on the ovaries of rats with PCOS has primarily focused on parameters such as ovarian weight and tissue mass size ( 24 ). The other remarkable result of the present study is that the estrous cycles in the C. dactylon treatment groups exhibited a more regular pattern compared to the PCOS group. Interestingly, previous studies have also demonstrated that the administration of C. dactylon extracts to the Letrozole-induced PCOS rats can lead to the improvement of estrous cycles ( 24 ). Thus, early studies confirm the mentioned findings of our work. Besides, the current study demonstrates that administering C. dactylon extract to rats with PCOS results in a significant weight reduction compared to the control group. Previous research has similarly indicated that treatment with C. dactylon extract leads to weight and body mass reduction in rats with Letrozole-induced PCOS ( 24 ). Consequently, the findings of this study are consistent with those of prior investigations. The current study revealed that C. dactylon extract does not significantly affect the levels of LH and FSH in rats with PCOS, a finding that contradicts previous research. Prior studies have indicated that C. dactylon extract can significantly reduce LH and FSH levels ( 25 ). However, this study found that the extract decreased testosterone and estrogen levels while increasing progesterone levels in the serum of rats with PCOS. Previous research has shown that C. dactylon extract can increase estrogen levels and decrease testosterone levels in PCOS-affected rats ( 25 ). Additionally, another study demonstrated that this extract could elevate progesterone levels in rats with chronic menopausal syndrome ( 26 ), aligning with our findings. Our study has demonstrated that C. dactylon extract exerts various effects on the uterine tissue of rats with PCOS. These effects include a reduction in the mean luminal area, maintenance of myometrium thickness, reduction of the perimetrium layer, a decrease in the number of glands in the uterine endometrium, and a reduction in the luminal area. Notably, previous studies have reported that this extract decreases the weight of the uterus and ovaries, as well as the uterine tissue mass, in rats with PCOS ( 24 ). Therefore, our findings are consistent with those of earlier research. Our docking analysis revealed that Artumerone exhibited the highest affinity for the AMHR2 receptor. Previous studies have established that AMH is secreted by granulosa cells (GCs) derived from pre-antral and small antral ovarian follicles and exerts its effects via the AMHR2 receptor. Furthermore, it has been shown that serum levels of AMH and AMHR2 are elevated in individuals with PCOS, and the severity of PCOS correlates directly with the expression of the gene encoding AMHR2 ( 27 ). This indicates that higher levels of the AMHR2 receptor in women with PCOS are associated with greater disease severity. Although the precise pathophysiological role of AMHR2 in PCOS remains unclear, existing studies suggest that this receptor is involved in the condition's pathophysiology ( 28 ). In conclusion, the data obtained from our molecular docking analysis, combined with findings from previous studies on the role of AMHR2 in PCOS, suggest that one possible mechanism underlying the therapeutic effect of the C. dactylon extract is the influence of Ar-tumerone on the AMHR2 receptor. Additionally, past studies have investigated the role of the CXCR7 receptor in the pathophysiology of PCOS. These studies have shown that the expression of CXCR7 is lower in rats with PCOS compared to healthy rats ( 29 ). Moreover, activation of CXCR7 has been found to suppress apoptosis in ovarian GCs, suggesting that reduced expression of this receptor in the reproductive system of rats with PCOS increases apoptosis in these cells ( 29 ). Consequently, activation of the CXCR7 receptor in rats with PCOS may ameliorate the condition. Coupled with our current study’s finding that Ar-tumerone has the highest affinity for this receptor, it can be inferred that one of the molecular mechanisms through which C. dactylon extract improves PCOS in rats is via the activation of the CXCR7 receptor by Ar-tumerone. Besides, regarding the role of ERβ in PCOS, previous studies have indicated that ERβ has two distinct roles in individuals with PCOS. Activation of ERβ in the uterus increases the risk of endometriosis. Conversely, the inactivation of this receptor in the ovaries of individuals with PCOS increases the possibility of abnormal follicular development, poor fertility, and fewer ovulation numbers ( 30 ). Thus, ERβ has a dual role in the reproductive system of individuals with PCOS. On the other side, our study demonstrated that in an in-silico environment, ERβ exhibited the highest affinity for Ar-tumerone in the hydro alcoholic extract of C. dactylon . Given that our study also showed the extract’s therapeutic effect on PCOS, it is plausible that another molecular mechanism involved in the improvement of PCOS by C. dactylon extract is the interaction of Ar-tumerone with the ERα receptor. Moreover, our molecular docking analysis revealed that among all compounds in the hydro alcoholic extract of C. dactylon , Ar-tumerone exhibited the highest affinity for the FSHR. Previous research has highlighted the role of FSHR in the pathophysiology of PCOS, demonstrating that FSHR expression is decreased in rats with PCOS. This decrease in FSHR levels has been suggested to play a significant role in abnormal folliculogenesis and ovulation disorders ( 31 ). Therefore, it can be inferred that another molecular mechanism by which the hydro alcoholic extract of C. dactylon . In addition, previous studies have demonstrated a decrease in LHR levels in individuals with PCOS. This reduction in LHR levels results in persistently elevated serum LH levels, leading to anovulation in affected individuals. Moreover, increasing LHR levels through various interventions can alleviate PCOS symptoms and enhance ovarian function in these patients ( 32 ). On the other hand, the current study identified Ar-turmerone as the compound with the highest affinity for LHR among the constituents of the hydro alcoholic extract of C. dactylon. These findings suggest that one potential mechanism by which the extract of C. dactylon ameliorates PCOS may be through the beneficial effects of Ar-turmerone on LHR levels and function in PCOS-affected rats. Previous studies have demonstrated that activation of the AT2R receptor by its agonists in rats with PCOS can lead to an improvement in the syndrome through ovarian non-stratified fatty acid (NEFA) uptake and a reduction in serum testosterone levels ( 33 ). Furthermore, our research has indicated that tumerone, present in the hydro-alcoholic extract of C. dactylon , exhibits the highest affinity for this receptor in an in-silico environment. Thus, it can be inferred that one of the potential molecular mechanisms through which the hydro alcoholic extract of this plant ameliorates PCOS is the activation of this receptor by tumerone. Additionally, the molecular docking analysis conducted in the current study has revealed that tumerone exhibits the highest affinity for binding to the β-adrenoreceptor. It is noteworthy that previous research on this receptor has demonstrated that blocking it by administering propranolol in rats with PCOS can enhance ovulation by reducing serum testosterone levels and decreasing the number of ovarian cysts ( 34 ). Therefore, it can be hypothesized that another potential mechanism by which the hydro-alcoholic extract of C. dactylon ameliorates PCOS in affected rats is the activation of the β-adrenoreceptor by the tumerone molecule present in the extract. Furthermore, previous research has examined the role of the CXCR4 receptor in the pathophysiology of PCOS. These studies have demonstrated that CXCR4 expression is lower in rats with PCOS compared to healthy controls. Additionally, the activation of CXCR4 has been shown to suppress apoptosis in ovarian GCs, indicating that reduced expression of this receptor in the reproductive system of PCOS-affected rats leads to increased apoptosis in these cells ( 29 ). Thus, activation of the CXCR4 receptor in PCOS-affected rats may alleviate the condition. Considering our study’s finding that tumerone exhibits the highest affinity for this receptor, it can be inferred that one of the molecular mechanisms by which C. dactylon extract ameliorates PCOS in rats is through the activation of the CXCR4 receptor by tumerone. Prior surveys have displayed that the ActR gene in ovarian GCs may play a key role in mediating PCOSrelated disorders, particularly abnormal ovarian folliculogenesis and ovulation dysfunction ( 31 ). Besides, Tricyclo[6.3.0.0( 1 , 5 )]undec-2-en-4-one, 2,3,5,9- tetramethyl showed the most in-silico tendency to this receptor. Therefore, increasing the expression of ActR or activation of this receptor may be one of the possible mechanism by which the extract of C.dactylon treated PCOS in the present study. Moreover, the other receptor that Tricyclo[6.3.0.0( 1 , 5 )] undec-2-en-4-one, 2,3,5,9- tetramethyl had the highest amount of binding affinity is AR. Interestingly, based on the early studies, the activation of AR by continuous administration of nonaromatizable androgen dihydrotestosterone (DHT) and the aromatase inhibitor letrozole could crate PCOS and PCOS-derived disorders including anovulation, increased body weight, increased body fat, and enlarged mesenteric adipocytes, as well as elevated leptin levels and insulin resistance ( 35 ). Therefore, this can be hypothesized that the inhibition of AR by Tricyclo[6.3.0.0( 1 , 5 )]undec-2-en-4-one, 2,3,5,9- tetramethyl may have a pivotal role in the treatment of PCOS through administration of C.dactylon extract. Based on previous studies, inhibition of TLR-4 by Quercetin, a compound derived from Chinese medicinal herbs, leads to the inflammatory microenvironment in ovarian tissue and decreasing insulin resistance ( 36 ). Therefore, inhibition of this receptor can cause treatment of PCOS in rats. Notably, Curlone demonstrated the most affinity to TLR-4 and it has the potential to inhibit this receptor and cause positive effects on PCOS condition. Furthermore, the treatment of PCOS rats with Melatonin can treat PCOS by creating a cross-talk between its two receptors, ERα, TRα, and Dio2in thyroid and ovarian tissue. This cross-talk resulted in decrease in the circulating level of gonadotropins (LH and FSH), and testosterone, as well as cure PCOS ( 37 ). On the other side, Curlone may affect TRα and lead PCOS to be treated in rats because of its high binding affinity to TRα. Regarding the role of ERα in PCOS, previous studies have highlighted that this receptor has two distinct roles in individuals with PCOS. Activation of ERα in the uterus of PCOS patients increases the rate of endometrial hyperplasia, the risk of endometrial cancer, and the likelihood of miscarriage and pregnancy loss. Conversely, inactivation of ERα in PCOS patients leads to elevated serum LH levels, resulting in increased infertility, formation of ovarian cysts, and a decrease in the number of ovulating follicles ( 30 ). Therefore, ERα exhibits a dual role in the reproductive system of individuals with PCOS. Moreover, our study has demonstrated that, in an in-silico environment, 3-Tert-butyl-4-hydroxyanisole, a compound found in the hydro alcoholic extract of C. dactylon , shows the highest affinity for Erα. Given our findings that the extract of this plant has a therapeutic effect on PCOS, it is plausible to suggest that another molecular mechanism involved in the amelioration of PCOS by the C. dactylon extract is the interaction of the 3-Tert-butyl4-hydroxyanisole molecule with ERα. The current study demonstrates that metformin can regularize estrous cycles in rats with PCOS. Previous studies have similarly indicated that metformin exerts significant positive effects in regularizing estrous cycles by reducing the number of small follicles, decreasing the number of ovarian cysts, increasing the number of antral follicles, and improving ovarian blood supply ( 38 ). Additionally, our study has shown that metformin can reduce the weight of uterine and ovarian tissues in rats with polycystic ovaries ( 24 ), corroborating findings from previous research on the impact of metformin on uterine and ovarian tissue weight in these animals. Moreover, the current study indicates that metformin increases levels of LH and FSH in rats with PCOS. This contrasts with previous studies, which reported no significant effect of metformin on FSH and LH levels in rats with chronic menstrual syndrome. Additionally, our study found that metformin can increase progesterone levels while decreasing estrogen and testosterone levels in rats with PCOS. Previous research, however, has shown that metformin does not significantly affect serum estrogen levels and has no notable impact on serum testosterone and progesterone levels in rats with PCOS ( 39 ). Additionally, the current study demonstrated that metformin reduces several key indicators in the uterus of rats with PCOS, including luminal area, endometrial area, myometrial area, perimetrial area, total area, and glandular count. To our knowledge, early surveys have also demonstrated that Metformin can stop the luminal area from becoming greater and also it can suppress the number of glands in the uterine tissue of rats with chronic menstrual syndrome ( 39 ). Our study demonstrates that the extract of, C. dactylon exhibits therapeutic effects on PCOS in rats, corroborating findings from previous research on this condition ( 24 ). Beyond its established anti-inflammatory, antibacterial, and anti-ulcer properties ( 40 ), C. dactylon also shows significant anti-PCOS activity. These results suggest that, C. dactylon extract could be a promising candidate for treating PCOS in humans. This finding highlights the necessity for further research, particularly clinical trials, to explore the potential of, C. dactylon extract in human therapeutics. The current study primarily investigated the effects of C. dactylon hydro-alcoholic extract on select aspects of letrozole-induced PCOS. However, the comprehensive nature of PCOS encompasses diverse hormonal, metabolic, and reproductive changes. The investigation did not extensively explore all dimensions of PCOS, including long-term impacts, genetic influences, and broader metabolic implications. Furthermore, while the study highlighted potential hormonal and histological impacts of C. dactylon , it did not provide in-depth mechanistic insights at the molecular level. Future research should aim to elucidate the specific pathways and molecular interactions through which C. dactylon exerts its effects on PCOS, thus enhancing understanding of its therapeutic mechanisms. Moreover, the study focused on the positive impacts of C. dactylon on PCOS symptoms, but did not comprehensively examine potential secondary effects or adverse reactions. Subsequent research efforts should address the safety profile of C. dactylon to ensure its suitability as a therapeutic intervention for PCOS.

Our findings indicate that C. dactylon significantly improves both the condition and function of ovaries and ovarian follicles in letrozole-induced PCOS rats, while also alleviating symptoms associated with PCOS. Additionally, the C. dactylon extract positively influences critical components of uterine tissue in letrozole-induced PCOS rats, including the luminal and perimetrial areas, endometrial glands, and total uterine area, enhancing overall uterine function. Molecular docking analysis has elucidated aspects of the molecular mechanisms through which C. dactylon exerts its therapeutic effects on PCOS. However, the limitations of this study should be acknowledged, and further research is necessary to comprehensively explore the full spectrum of C. dactylon 's effects on PCOS.

This experimental study was conducted in compliance with the relevant guidelines and regulations of the Animal Care Committee of Shiraz University. All experimental protocols received approval from the Research Committee’s Ethical Committee at Shiraz University (IR.SU.REC.1394.27418), with the approval number of 98gcb1m148075 and an approval date of June 18, 2020. The preparation of the C. dactylon extract was performed. The entire plant, excluding the rhizome, was collected, cleaned, and dried. The dried plant material was then powdered using a homogenizer. This powder was subjected to extraction using 70% ethanol as the solvent. The solvent was subsequently removed using a rotary evaporator at 70°C with medium speed, and the extract was further concentrated at 50°C in the rotary evaporator to obtain a pure semi-solid mass. In this study, sixteen female Sprague-Dawley rats weighing on average 200 ± 20 g were procured from the Center of Comparative and Experimental Medicine, Shiraz University of Medical Sciences. The rats were housed in the animal facility at Aliabad Agricultural Training Center under controlled conditions of 22 ± 2°C temperature, 38% humidity, and a 12-hour light/dark cycle. They were provided with ad libitum access to standard rodent food supplemented with carrot and parsley. The animals were acclimatized to these conditions for a minimum of seven days before commencing the therapeutic interventions. The reproductive cycle of the rats was determined. Briefly, the cycle was assessed through vaginal smears collected over 14 days and examined with a light microscope (CX21, Olympus, Japan). Each rat was gently restrained by holding its tail, and a wet cotton swab was inserted into the vagina. The swab was carefully rotated and then withdrawn, and the sample was smeared onto a clean, grease-free microscope slide. The slides were airdried and stained with methylene blue or crystal violet. The different stages of the estrous cycle were identified under a binocular microscope. Sixteen rats with a regular reproductive cycle were selected, and 12 of them received 1 mg/kg body mass of letrozole by gavage to induce PCOS over 21 days. At the end of the PCOS induction period, the rats were randomly divided into four groups: a control group, a PCOS group, a C. dactylon -treated group (500 mg/kg body weight), and a Metformin-treated group (150 mg/kg body weight). These treatments were administered for four weeks following the determination of the reproductive cycle. Proestrus: Characterized by the presence of numerous small, round, nucleated epithelial cells with similar morphology. These cells exhibit basophilic nuclei and are often seen in clusters. Both nucleated and some cornified epithelial cells are present. Estrus: Defined by the abundance of cornified epithelial cells without nuclei, alongside some well-developed nucleated epithelial cells. Metestrus: Identified by a mixture of predominantly cornified epithelial cells, both with and without nuclei, as well as a few neutrophils. The smear shows mostly cornified epithelial cells, neutrophils, and some nucleated epithelial cells. Diestrus: Characterized by a higher number of neutrophils and a lower number of cornified epithelial cells. Rats were anesthetized with ether for sampling, and blood was collected via cardiac puncture. The blood samples were transferred to tubes without anticoagulants, allowed to clot, and then centrifuged at 2000 rpm for 15 minutes. The resulting serum was stored at -20°C until analysis. Serum concentrations of testosterone (sensitivity: 0.2 ng/mL, Catalog # RK-61M, Institut des Isotopes Ltd, Budapest, Hungary), estradiol (sensitivity: 0.2 pg/ mL, Catalog # KIP0629, DIAsource Immunoassays, SA, Louvain-la-Neuve, Belgium), and progesterone (sensitivity: 0.05 ng/mL, Catalog # KIP1458, DIAsource Immunoassays SA, Louvain-la-Neuve, Belgium) were measured by radioimmunoassay. Serum FSH (sensitivity: 0.09 mIU/mL, Catalog # RF01N, Gyeonggi-do, South Korea) and LH (sensitivity: 0.22 mIU/mL, Catalog # RF03N, Gyeonggi-do, South Korea) concentrations were measured by radioimmunometry. Histomorphometric evaluation was conducted on the entire ovarian and uterine slides, encompassing all follicles and the various layers of the uterus. The histological distribution of the ovarian and uterine slides was examined. In brief, ovaries and uteri were sampled and preserved in a 10% formalin buffer solution for 48 hours, with the fixation solution changed after 24 hours. Subsequently, the samples were immersed in paraffin twice, for 75 and 45 minutes respectively. Serial sections of ovarian tissue (6 μm thickness) and uterine tissue (5 μm thickness) were prepared using a Leica RM 2025 rotary microtome (Leica Microsystems, Wetzlar, Germany). These sections were deparaffinized in xylene, rehydrated, and stained with hematoxylin and eosin (H&E) for histological examination. To conduct the H&E staining process, slides of both ovarian and uterine tissues were placed in xylene twice, followed by immersion in 100, 96, and 76% ethanol twice for each concentration. The slides were then stained with hematoxylin and subsequently placed in an acid fuchsin solution. After staining with eosin dye and rinsing with distilled water, the slides underwent further immersion in 76, 96, and 100% ethanol, and xylene twice for each step. Between each stage, the slides were rinsed in distilled water to enhance the contrast between structures. Finally, the prepared slides were observed and imaged using a light microscope (OPTIKA, Italy, Ponteranica) and a microscopic digital camera (TCH-1.4CICE, Canada). The slides were preserved for further analysis. For counting primary, secondary, and tertiary follicles, morphologically atretic follicles, corpora lutea, and cystic follicles, the entire ovarian serial sections were imaged using a microscopic digital camera (TCH-1.4CICE, Canada). Uterine transverse sections were similarly imaged and analyzed. The analysis of ovarian structures and uterine layers was conducted using ImageJ software (Fiji-ImageJ x64, US National Institutes of Health). Specifically, imported images were converted to 8-bit format using the "Image type" function in the "Image" menu. Subsequently, the scale for all images of ovarian and uterine sections was established using the "Set scale" option in the "Analyze" menu. To analyze the areas, the borders of ovarian structures or uterine layers were manually delineated and duplicated. The area measurements were then obtained using the "Measure" function in the "ROI manager" panel and saved in Excel (.csv) format. Counting of ovarian structures was performed manually following verification of identification criteria. For the molecular docking analysis, receptors involved in the pathophysiology of PCOS were initially identified from previous studies (Table S1, See Supplementary Online Information at www.ijfs.ir). The amino acid sequences (ASTA sequences) of these receptors were then retrieved from the UniProt online database. Subsequently, the three-dimensional structures of these receptors were modeled using the SWISS-MODEL online tool, and these modeled structures were utilized in the molecular docking analysis. In the next step, the three-dimensional structures of all compounds identified in the hydro alcoholic extract of C. dactylon from previous studies were obtained from the PubChem online database for the molecular docking process (Table S2, See Supplementary Online Information at www.ijfs.ir). AutoDock Vina software was employed to perform the molecular docking, with the conformation exhibiting the highest binding energy (Kcal/mole) selected as the optimal conformation. Finally, the details of the interactions between ligands and receptors were identified and visualized using PyMOL (Schrödinger, LLC. (2021). PyMOL [Software]. Version 2.5. Retrieved from https://pymol.org/2/) and Discovery Studio Visualizer software. This research was conducted in a completely randomized design. Data of histomorphometry and serum hormone concentrations were tested by Kolmogorov-Smirnov test to ensure normal distribution. Then, logarithmic normalized data were analyzed by one-way ANOVA test using by IBM SPSS Statistics 26 (SPSS for Windows, version 26, SPSS Inc, Chicago, Illinois, USA) and GraphPad Prism software version 5.01 for Windows (GraphPad Inc., San Diego, CA, USA) and the mean fewer squares of the groups were compared at the statistical level of 0.05. Besides, Graphs were created with GraphPad Prism software (version 5.01 for Windows, GraphPad Inc., San Diego, CA, USA). In addition, estrus cycle data were analyzed.