Impact of erythropoietin and myoinositol versus metformin on insulin resistance in a rat model of polycystic ovary syndrome

Abstract This study aimed to evaluate the therapeutic role of erythropoietin (EPO) or myoinositol versus metformin (MET) in improving the reproductive functions and glucose tolerance in a rat model of polycystic ovary (PCOS). Oral letrozole (LTZ) was used for induction of PCOS in wester rats for 21 days, after that, MET, EPO and myoinositol were administered for the following 21 days. The LTZ-induced PCOS rats have lost their oestrous cyclicity and become fixed at the diestrus phase, developed insulin resistance, abnormal sex and gonadotrophin hormone serum levels, increased cystic follicles, decreased number of the growing follicles and very little or no corpora lutea on microscopic examination, which were reversed by the three drugs, MET, EPO and myoinositol. MET and myoinositol were mostly equally effective in improving the reproductive manifestations of the disease. However, EPO was most effective in decreasing the insulin level observed in this LTZ-induced model of PCOS.


Introduction
Polycystic ovary syndrome (PCOS) is the most prevalent endocrine disorder and the leading cause of infertility among women in their reproductive age, affecting approximately 15% of them (Conde et al. 2019, Heshmati et al. 2021).This disease is considered to be multifactorial involving multisystem dysfunction, mainly reproduction, endocrine and metabolic (Maqbool et al. 2019).The disease is manifested clinically by menstrual abnormalities, acne, hirsutism, frequent anovulation and recurrent miscarriages (Maliqueo et al. 2014).The endocrine abnormalities include increased androgens, luteinizing hormone and decreased progesterone levels (Hachey et al. 2020).However, the metabolic disturbances of PCO include insulin resistance (IR), obesity, lipid abnormalities and type 2 diabetes mellitus (Rababa'h et al. 2020).
Obesity is a common feature in PCO patients.It augments the adverse metabolic and reproductive outcomes of PCOS (Jeanes and Reeves 2017).As it increases IR with compensatory hyperinsulinemia, which in turn increases adipogenesis and decreases lipolysis (Tosi et al. 2015).Also, inflammatory adipokines produced by excessive adipose tissue, in turn, promote hyperinsulinemia, which increases obesity, providing a vicious feedback cycle (Glueck and Goldenberg 2019).
Obesity is likely not a cause of PCOS, as PCOS relatively also common among thin individuals.However, obesity does exacerbate many aspects of the phenotype, especially cardiovascular risk factors such as glucose intolerance and dyslipidemia.
Women with PCOS have intrinsic IR independent of the extent of obesity and magnitude of androgen concentrations.Even lean women with PCOS manifest IR however obesity exacerbates it (Witchel et al. 2019).
The relationship between hyperandrogenemia and hyperinsulinemia has long been studied.Several studies have demonstrated a positive correlation between fasting insulin levels and androgen levels (Lobo et al. 1983).Furthermore, the severity of hyperinsulinaemia correlates with the degree of clinical expression of the syndrome (Robinson et al. 1993).
Letrozole (LTZ) is a non-steroidal aromatase inhibitor that was used recently for the induction of PCO in rats.In female rats, continuous prepubertal LTZ treatment causes disrupted oestrous cyclicity, larger ovarian weight, ovarian cysts, atretic follicles and lack of corpora lutea in adulthood (Maliqueo et al. 2013).Besides, rats treated with LTZ develop increased levels of LH and Testosterone level as occurs in PCOS while Progesterone level is reduced indicating anovulation.Besides those reproductive abnormalities of PCOS, LTZ treatment produces many metabolic features of PCOS such as increased body weight and IR (Kauffman et al. 2015).
Metformin (MET) is widely introduced to treat IR and hyperglycaemia in PCOS patients (Ayaz et al. 2013).Several studies have shown that MET could improve ovulation, pregnancy rates and live birth rates in PCOS patients (Di Pietro et al. 2015).
Erythropoietin (EPO) is a haematopoietic growth factor, produced mainly by the kidney in response to hypoxia and stimulates red blood cell formation in the bone marrow (Katz et al. 2010, Jelkmann 2013, Jelkmann 2016).Another attractive effect was reported for EPO, was its effects on glucose metabolism; its ability to reduce blood sugar, improve insulin sensitivity (Tuzcu et al. 2004, Scully et al. 2011, Meng et al. 2013) via increasing the expression of glucose transporter 4 (GLUT4), 5' AMP-activated protein kinase (AMPK) and glucose 6 phosphatase which are important enzymes for glucose transport and utilisation by the cells (Kuo et al. 2018).
EPO and its analogues are currently used and FDA approved for the treatment of anaemia secondary to either chronic renal failure or chemotherapy-induced anaemia in patients with cancer (Noxon et al. 2017).
Myoinositol (MYO) is a cyclitol naturally present in animal and plant cells, either in its free form or as a compound structure of phospholipids or inositol phosphate derivatives (Celentano et al. 2016).These inositol phosphate derivatives act as secondary messengers for many hormones (including insulin) and mediators inside the eukaryotic cells, particularly the inositol triphosphates (IP3), phosphatidylinositol phosphate lipids (PIP2/PIP3) and possibly inositol glycans (Chau et al. 2005).
MYO can increase glucose utilisation by tissues and increase insulin sensitivity.MYO is a precursor for inositol phosphoglycans (IPGs) which provoke phosphorylation of insulin receptor substrates (IRS), besides, phosphoinositide 3 kinase (PI3K) and protein kinase B/Akt (PKB/Akt) that account for most, but not all, intracellular actions of insulin (Gateva et al. 2018).For these reasons, Myo is important for the regulation of a wide range of cell functions, including cell growth and survival, development and function of peripheral nerves, osteogenesis and reproduction; It was found that MYO could restore normal ovulatory activity and Increase oocyte and egg quality causing improvement in fertilisation rates (Ciotta et al. 2011).Also, in the PCOS-endometrium, MYO could activate the AMPK pathway, increase GLUT-4 levels and, subsequently, increase glucose uptake by human endometrial cells.Therefore, MYO may be used as an effective treatment option in insulin-resistant PCOS women (Cabrera-Cruz et al. 2020).This study aimed to investigate the potential therapeutic role of EPO and MYO versus the widely used MET drug in treating the metabolic and reproductive parameters of a rat model of PCOS.

Animals
50 female wester rats, 4 weeks old of about 90: 100-g body weight, were purchased from the Faculty of Science, Sohag University and housed in the Medical Animal Laboratory in Sohag faculty of medicine.Animals gained free access to food and tap water.Rats were housed in Polycarbonate cages (20 Â 40 Â 20 cm, 5 rats/cage) at normal light/dark cycle and room temperature.The study was approved by The Medical Research Ethics Committee of Faculty of Medicine, Sohag University, registration number: Soh-Med-21-02-48, considering the care and use of laboratory animals.
The rats were divided into five groups (10 rats each); the control group received 2 ml carboxymethylcellulose (CMC) 0.5% by gastric gavage for 21 days then water by gavage for the next 21 days over the experimental period, groups 2-5 are PCO model groups that received LTZ (0.5 mg/kg dissolved in 0.5% CMC for 21 days) for induction of PCOS.After 21th day, rats in group 2 served as PCO model group and received water for the end of the study, group3 (MET T PCO G) received MET in a dose of 150 mg/kg orally for 3 weeks (Patel and Shah 2018) Group 4 (EPO T PCO G): received intraperitoneal injection of EPO in a dose of 100 IU per kg 3 days per week for 3 weeks (G€ unal et al. 2019).Group 5 (MYO T PCO G) received MYO in a dose with 420 mg/kg in 2 ml H 2 O for 3 weeks (Bevilacqua et al. 2019).LTZ, MYO, MET and EPO were purchased from Sigma-Aldrich company.Figure 1 explains the experimental timeline and animal groups.Because EPO is administered intraperitoneally in this study another control group that received IP saline injection for the last 21 days was included in the study.The data of this group is present in Supplementary file 1.

Bodyweight, glucose tolerance and insulin assessment
Bodyweight was weekly measured for assessment of obesity.Glucose tolerance test (GTT) was done twice during the study period, The first one on the 21st day of the study, and the second time on the 42nd day of the study period.Rats have fasted for 8 h with free access to water.Rats were tested for GTT independent of the cycle stage.Basal blood glucose was then measured using a handheld glucometer at (time zero) then a single intraperitoneal injection of glucose (2 g/kg body weight) was done, then blood glucose measured subsequently at 15 min, 30, 60, 90 and 120 min postadministration.Measurement of blood glucose was carried out using a glucometer (Bionime corporation, Right test GM300) and glucose-test strips (code 556) (Kauffman et al. 2015).

Measurement of serum insulin level
Serum insulin levels were measured using enzyme-linked immunosorbent assay (ELISA) kits CSB-E05070r: With detection range 15.6-1000 nIU/mL.According to manufacturer instruction.

Vaginal smear examination for the assessment oestrous cycle
Starting from the 2nd week of the study, a vaginal smear for each animal was done daily to determine the stage of the oestrus cycle using the light microscopic analysis of the predominant cell type in the vaginal epithelial smears (Grzesiak et al., 2021).To collect cells from the vaginal canal, approximately 0.2 ml of saline is drawn into the pipette or dropper.The tip of the dropper is gently inserted into the vaginal orifice at a depth of approximately 5-10 mm in rats and then the saline flushed into the vagina and back out 2 or 3 times.If after the first flush the fluid is cloudy, subsequent flushings may not be necessary.When inserting and flushing into the vaginal orifice, care must be taken not to insert the tip too deep to avoid cervical stimulation.After the lavage, a small drop of the sample is then placed on the slide in a thin layer (smear) and allowed to dry.Toluidine blue stain is used for the identification of the different cell types and is recommended for staining vaginal smears (Cora et al., 2015).

Collection of samples
At the end of the experiment, all rats were anaesthetized and blood samples were withdrawn from the heart of each animal then centrifuged at 3000 rpm, serum was preserved at À20 C till the time of the biochemical assay.After the rats were sacrificed, the abdominal cavities of the rats were opened and the ovaries were dissected.The right ovaries were taken for histological assessment, while the left ovaries were placed in liquid nitrogen and stored at À80 C for realtime PCR assay of the genetic expression of GLUT4 and AMPK genes.For a better assessment of hormones and assessment of follicular growth, rats were sacrificed when they were in the dioestrous phase of the cycle.

Quantitative real-time PCR (qRT-PCR) analysis of GLUT4and AMPK
Tissue samples were put immediately in liquid nitrogen and stored at À80 C to preserve their mRNA until they were extracted and then used to measure Glucose transporter 4 (GLUT4), 5 0 AMP-activated protein kinase (AMPK) in ovarian tissue by real-time polymerase chain reaction (real-time PCR).

Reverse transcription
The extracted RNA was transcribed into cDNA using RNA reverse transcriptase kits (RevertAid First Strand cDNA Synthesis kits (#K1622), Thermo Scientific).The thermal cycler was programmed at 25 C for 10 min, 37 C for 120 min, 85 C for 5 min and 4 C for 20 h.

Real-time PCR
The prepared cDNA was used in the q PCR analyzer (Step One, Applied Biosystems, Singapore) and the specific primers using the MAXIMA SYBR Green qPCR Master Mix (#K0251, Thermo scientific), The forward and reverse primer sequences used in this assay are illustrated in Table 1, with the following program: 1 cycle at 95 C for 10 min; 40 cycles of 95 C for 15 s, 60 C for 30 s and 72 C for 30 s. Fold expression (2 ÀDDct ) was calculated according to the relative expression of housekeeping gene b-actin

Histopathological examinations
The right ovaries were dissected and fixed in 10% buffered formalin for 24 h at room temperature and washed in water.
For light microscopy, fixed tissues were dehydrated in ascending grades of ethanol alcohol, cleared in xylene and embedded in paraffin.5um thick sections were mounted in slides stained with hematoxylin and eosin to be examined (Ibrahim et al. 2018).

Morphometric study of the ovarian follicles
The digital photos were taken in a microscopic photography unit, Leica ICC50 Wetzlar (Germany) at the Histology Department, Faculty of Medicine, Sohag University.Ovarian follicles count was determined on H&E stained sections.The mean count was measured in 10 high-power fields (Â100) for each specimen using Image J 1.51n software (National institutes of health USA Java 1.8.0_66).

Statistical analysis
Data were analyzed by using GraphPad Prism 9 (GraphPad, Inc., San Diego, CA).One-way (ANOVA) was used to compare more than two groups followed by the Bonferroni test for multiple comparisons to detect differences between groups.All data were expressed as mean ± standard deviation of the mean (SD).Statistical significance was considered at a level of p < .05.

Bodyweight
It was measured every week all over the experiment, during the first 21 days of the study, all rats receiving LTZ (PCO model) showed significantly increasing body weight in comparison to the control G.However, during the next 21 days, all groups received treatment (MET, EPO and MYO), showed significantly decreasing body weight when compared to the PCO model (Figure 2(A)).

Glucose tolerance test (GTT)
It was measured twice; at the 21st, and 42nd of the study.Glucose homeostasis is typically impaired in LTZ-treated rats.We found that fasting glucose levels were significantly elevated, in addition to significantly delayed glucose clearance after intraperitoneal injection on day 21 st of the study in comparison to the control G (Figure 2(B)).When GTT was repeated at the 42nd of the experiment, it revealed that glucose tolerance was highly improved in MET, EPO and MYOtreated groups in comparison with the PCOS group (Figure 2(C)).

Insulin hormone level (ng/ml)
The mean level of the three treated groups; MET (2.41 ± 0.39 mg/ ml), EPO (1.65 ± 0.43 mg/ml) and MYO-treated groups (2.6 ± 0.36 mg/ml) showed a significant decrease in Insulin level when compared to PCOS group (3.82 ± 0.84).It was interesting to find that EPO had significantly decreased insulin level more than MYO (p ¼ .0008)and MET (p ¼ .0106)(Figure 2(D)).MET, EPO and MYO increased estradiol hormone (E 2 ), progesterone and FSH while decreased LH and testosterone serum levels in the rat model of PCOS The three treated groups; MET, EPO and MYO showed a significant increase in E 2 level when compared to PCOS G.The MYO-treated group showed a significant increase in E 2 level when compared to the EPO group (p ¼ .0105).But there was no significant difference between the MET and the MYO groups nor in-between the MET and EPO group (Figure 3(A)).
As regard progesterone level: The three treated groups: MET, EPO and MYO showed a significant increase in progesterone level when compared to the PCOS group.No significant difference between the three treatment groups (Figure 3(B)).
The three drugs also significantly increased The FSH level when compared to the PCOS group.MET significantly increased the FSH level more than MYO (p ¼ .0004).However, no significant difference between either EPO or the other groups (Figure 3(C)).In the MET, EPO and MYO-treated groups (Table 2), the examination of vaginal smears showed 20% of rats of the MYO-treated group started to return to their cyclicity in comparison with 10% of MET and 5% in EPO-treated groups after the first week of treatment.However, 80% of rats of the MYO-treated group returned to their cyclicity in comparison with 70% in the MET group and 60% in EPO-treated groups after the 2nd week of treatment.All rats in all treated groups returned to their cyclicity while the PCOS group was still captured in the dioestrus phase after 3 weeks of treatment.

Ovarian tissue examination
Light microscopic examination of the ovarian tissue of the control group revealed that the ovary was covered with thin tunica albuginea.There were numerous follicles at different stages occupying the cortex (primary, growing and mature Graafian follicles).Numerous corpus luteum were frequently observed.Medullary tissue contained blood vessels, septa and connective tissue (Figure 6(A1,A2)), While PCOS group: the cortical tissue revealed multiple follicular cysts with an irregular thickness of granulosa cells and hypertrophied thickened theca cells.Most of the granulosa cells have degenerated with pyknotic nuclei.Few primary follicles could also be seen.There were atretic haemorrhagic follicles.Corpora Lutea were absent indicating anovulation (Figure 6(B1,B2)).
The MET-treated PCO group showed signs of improvement in the ovarian structure as decreased number of the cystic follicles, numerous corpora lutea indicating ovulation, numerous follicles at different stages of development as  The percentage % of animals with regular cycle in different groups.primary follicles and mature Graafian follicles (Figure 6(C)).As regards the EPO treated PCO group, the ovarian cortex also contained multiple corpora lutea, however, they were apparently less frequent than those observed in the MET group.Follicles of different developmental stages: primary follicles, numerous mature follicles could be observed in addition to numerous atretic follicles (Figure 6(D)).The MYO group showed the same picture of improvement, however with more corpora lutea and fewer or no cystic follicles (Figure 6(E)).
LTZ impaired the process of folliculogenesis while MET, EPO and MYO increased follicular growth and ovulation in PCO rats Table 3 shows the morphometric analysis of ovarian follicles which reveals that: the number of the primordial and cystic follicles increased, while the antral follicles and corpora lutea decreased significantly in the LTZ group in comparison with the control group, this is because the increased primordial follicles undergo cystic degeneration and do not grow into antral follicle or ovulation.On the contrary, the MET, EPO and MYO-treated groups showed a decreased number of primordial follicles and cystic follicles while significantly increased antral follicles and corpora lutea when compared to the LTZ PCO rats.The number of the antral follicles and corpora lutea in the MET and MYO groups were insignificantly different from each other nor the control group.However, the EPO-treated group significantly increased cystic follicles when compared to the MET group (p ¼ .017)and the MYO group (p ¼ .023),which indicate better follicular development in both MET and MYO groups.

Discussion
PCOS is a common disease among females in their reproductive period, characterized by hyperandrogenemia, frequent anovulatory cycles and infertility.Impaired glucose tolerance and elevated plasma insulin concentrations are present in about 70% of lean and 95% of overweight women diagnosed with PCOS (Kupreeva et al. 2019).Hyperinsulinemia may stimulate ovarian testosterone production, decrease serum sex hormone-binding globulin concentrations and impair ovulation (Maqbool et al. 2019).
In this study, PCOS was induced by oral administration of LTZ (0.5 mg/kg for 21 days) in prepubertal female wester rats aged 4 weeks.Development of the disease was confirmed by daily examination of the vaginal smears which revealed the arrest of the cyclicity in the dioestrus phase in all LTZ-treated rats.Besides, the LTZ-treated rats showed a significant decrease in the levels of E 2 , progesterone and FSH, while a significant increase in LH and testosterone levels compared to the control group, those results were in line with (Akintayo et al., 2021) who used LTZ with the same dose.
Also, LTZ induced IR as indicated by a significant increase in fasting glucose and insulin levels with impaired glucose clearance in GTT.Besides, a decrease in GLUT4 and AMPK mRNA expression levels when compared to the control group, this was following Zhang et al. (2020) who observed a significant reduction in GLUT4 mRNA levels in the PCOS model animals relative to controls.
Histological examination of the ovaries of the LTZ-treated group showed multiple cysts with thinning of granulosa cells and corpora Lutea were absent, indicating anovulation.This is in agreement with (Atef et al. 2019) who found that the PCOS rats showed multiple cystic follicles with attenuation of granulosa cell layer and absence of corpora lutea.
In this study, MET was used at a dose of 150 mg/kg orally from the 4th week to the 6th week to ensure the effectiveness of MET in the management of PCO and to compare its effect with that of EPO and MYO as they can also improve IR.
MET-treated PCOS group showed significant improvement in ovulation, increase in the levels of E 2 , progesterone and FSH while, a decrease of testosterone, LH level and insulin level when compared to LTZ-treated rats.This group also showed a significant improvement in glucose tolerance and increased GLUT4 and AMPK mRNA levels compared to the PCOS model.Histopathological examination of this group shows numerous corpora lutea and very few cystic follicles.
These results are in line with Rababa'h et al. (2020) and Kupreeva et al. (2019) who concluded that MET can increase glucose uptake, IR and endocrine aberrations in the PCOS rodent model.MET triggers the activation of AMP-activated protein kinase (AMPK), leading to downregulation of the expression of the gluconeogenic genes.Also, the increase in AMP concentration may inhibit the activity of adenylate cyclase, an important mediator of glucagon action, thus furtherly, inhibit gluconeogenesis (Miller et al. 2013).
EPO was used at a dose of 100 IU/kg day after day for 21 days (intraperitoneal injection) for treatment of PCO in group 4. EPO showed a better effect than MET in decreasing fasting insulin and glucose levels and increasing the glucose clearance from the circulation.These results are following Meng et al., who found that EPO had decreased blood glucose level and inhibited gluconeogenesis (Meng et al., 2013) in high fat diet-fed mouse.The increased expression of GLUT4 and AMPK noticed in this study was also previously reported by Kudo et al., and Kuo et al., in their studies on diet-induced obesity in mice and streptozotocin-induced diabetic rats successively.Increase expression of these genes is important for glucose transport and utilization by the cells (Kodo et al., 2017, Kuo et al., 2018).
In addition, the EPO-treated PCO group showed normalization of all reproductive parameters including oestrus cyclicity, sex and gonadotrophin hormones, increased number of the corpora lutea and growing follicles and decreased cystic and atretic follicles.
No previous studies in the literature used EPO for the treatment of PCO, but EPO was found to be produced by the human ovarian follicles during their maturation; granulosa, theca interna cells and lutein cells of the ovary in an estrogen-dependent manner (Yasuda et al., 2001).Besides, EPO plays an important role in angiogenesis of the uterine blood vessels during the cyclical changes in the proliferative and secretory phases of the endometrium (Yasuda et al., 1998).Recently, Conde et al. (2019), showed that an in-vitro treatment of bovine oocyte with EPO may have an antiapoptotic effect on the cumulus cells and a positive effect on the oocyte cytoplasmic maturation, moreover the number of blastomeres in the cleaved embryos have increased, with a better outcome of the produced embryos (Conde et al., 2019).
EPO is involved in angiogenesis in many tissues either physiological as in uterine blood vessels, hypoxia-induced as in the brain or myocardial ischaemia (Lu et al. 2012, Im et al. 2020), or tumour angiogenesis (Bhat et al. 2019, Liu et al. 2020).So adjustment of the minimal effective dose and duration for treatment of PCO should be taken into consideration in the next studies.
Myo-inositol (MYO) belongs to the vitamin B family.Animal and plant food is rich in MYO.Both MYO and Dchiro-inositol are insulin second messengers, besides, MYO is involved in follicular gonadotropin pathways which orchestrate ovulation (Lagan a et al. 2018).
In this study, MYO (420 mg\kg) was given orally, after induction of the PCO syndrome for 3 weeks.Vaginal smear and ovarian tissue examination showed the best result in comparison with the LTZ or with the MET and EPO-treated groups.Serum analysis of this group showed improvement of the condition as there was a significant decrease in the levels of testosterone and LH, in addition to a significant increase in E2 and progesterone levels, compared to LTZtreated group.There was also a significant increase in GLUT4 and AMPK mRNA.These results are in line with Zhang et al.
(2020) who used MYO in treating animal model of PCO with IR.They found a significant decrease in the serum levels of LH, LH/FSH ratio and testosterone, while an increased serum level of E2.Our results are supported by Merviel et al., andNas andTUU (Nas andT} u} u 2017, Merviel et al. 2021) who found that MYO could represent a possible alternative in the treatment of IR patients without diabetes or pre-diabetes in all those cases where MET cannot be used.Indeed, in their clinical studies, MYO and MET showed the same efficacy in the treatment of menstrual cycle disorders related to IR. Anyway, while MET side effects are widely reported in the literature, no side effects of MYO have been reported so far.

Conclusion
This study aimed to compare the therapeutic effects of EPO and MYO to the widely spread MET drug.This study showed that MET and MYO-treated PCO groups are nearly equally effective in restoring the normal hormonal levels, restoration of folliculogenesis and ovulation as proved by histologic examination of the ovaries and vaginal epithelial cells.
EPO also had beneficial effects on the reproductive parameters of PCO but its effect was less than those of MET and MYO according to the level histologic examination, however, EPO was the best one in improving IR as indicated by the decreased serum insulin level and better glucose tolerance.These results suggest that IR plays an important role in the pathogenesis of the PCO but it is not the only factor that controls the outcome results of treatment.Tosi, F., et al., 2015. Total

Figure 2 .
Figure 2. Effect of MET, EPO and MYO on (A) body weight, (B) GTT at the 21st day, (C) GTT at the 42nd day of the study, (D) blood insulin level in LTZ-treated rats (PCO G). p Values was considered significant when p < .05. a p < .05 vs. control G; b p < .05vs the PCOs model.c p < .05vs MET-treated PCO G.

Figure 5 .
Figure 5. Phases of the vaginal smear (A) pro-oestrus phase; characterised by the predominance of nucleated epithelial cells with clear margins.(B) Oestrus phase characterised by the predominance of cornified cells (large anucleated cells) with irregular margins, (C) Metoestrus phase shows many cornified cells plus infiltration of leucocytes.(D) Dioestrus phase, characterised by the predominance of leucocytes with the absence of cornified cells.

Table 1 .
Primer sequences used for gene expression analysis by quantitative PCR.

Table 2 .
Result of vaginal smear examination in different groups.

Table 3 .
Mean number of primordial follicles, antral follicles, cystic follicles and corpus lutea in different groups.Data represented as mean ± SD.Data analysed by ANOVA test, Post hoc, Boneferoni test.(N ¼ 10 rats per group).p Values were considered significant when p < .05. a p < .05 vs. control G.; b p < .05vs PCOS G. c p < .05vs MET-treated group.