Effects of Kelulut Honey on Oestrus Cycle Regulation and Histomorphological Changes in Letrozole-Induced Polycystic Ovary Syndrome Rats: A Preliminary Study

Polycystic ovary syndrome (PCOS) is a complex reproductive, metabolic, and endocrine disorder that affects women of reproductive age. Kelulut honey is stingless bee honey that possesses anti-inflammatory, anti-cancer, anti-diabetic, and potent antioxidative activities in most conditions. However, its value in improving PCOS remains to be elucidated. Thus, this preliminary study aimed to determine the effective dose of Kelulut honey in oestrus cycle regulation and ovarian histomorphological changes in letrozole-induced PCOS rats. PCOS was induced in all-female Sprague Dawley (SD) rats with 1 mg/kg/day of letrozole except for the control group for 21 days. Kelulut honey was then orally administered to the PCOS rats at the dose of 0.5, 1, or 2 g/kg/day, respectively, for 35 days. The oestrous cycle was determined through vaginal smears, while ovarian histomorphological changes were observed by haematoxylin and eosin (H&E) staining. The untreated PCOS rats were characterised by irregular oestrous cyclicity, hyperglycaemia, and aberrant ovarian histology. In this study, Kelulut honey (1 g/kg/day) increased the number of corpus luteum and antral follicles (p < 0.05), improved the cystic follicle, and normalised the oestrus cycle (p < 0.05). This preliminary study demonstrated that Kelulut honey, particularly at a dose of 1 g/kg/day, has the potential to alleviate oestrus cycle dysregulation and ovarian histomorphological changes occurring in PCOS.


Introduction
Polycystic ovary syndrome (PCOS) is a complex disorder of reproductive, endocrine, metabolic, and psychological health issues [1]. Its prevalence may reach up to 20% in women of reproductive age, depending on the diagnostic criteria [2]. Women with PCOS may experience hyperandrogenism, irregular menstrual cycles, dyslipidaemia, excessive body weight, hirsutism, and infertility [3]. Infertility has been reported in 40% of women with PCOS [1]. In addition, an estimated 80% of women with anovulatory infertility have PCOS, making PCOS the most common cause of anovulatory infertility [3,4].
Despite its high prevalence and health implications, PCOS is still underdiagnosed, with its underlying aetiology and pathophysiology still not fully clarified [5,6]. The most suggested aetiology of PCOS is abnormalities of ovarian steroidogenesis, which lead to functional ovarian hyperandrogenism [3,7]. Other pathophysiologic abnormalities include insulin-resistant, hyperinsulinism, gonadotrophin secretion abnormalities, and follicular arrest [8]. Hyperandrogenism in PCOS disrupts the physiological rhythm of the normal hormonal cycle in women, hence affecting the menstrual cycle. Hyperandrogenism also affects the ovary in PCOS, manifested mainly by the increased number of subcapsular follicle cysts, follicular arrest, decreased thickness of the granulosa layer, and absence

Animal Preparation
This study was approved by the National University of Malaysia Animal Ethics Committee (Ethical Approval Code FISIO/FP/2020/MOHD HELMY/14-MAY/1104-JUNE-2020-MAY-2023). Female Sprague-Dawley (SD) rats weighing 120-150 g that showed at least two consecutive regular oestrus cycles were used in this study. The rats were obtained from the Laboratory Animal Resources Unit, Faculty of Medicine, National University of Malaysia. All rats were individually caged, acclimatised for one week, maintained under a 12 h light and 12 h dark cycle in an air-conditioned room at 24 ± 2 • C, and given standard food pellets and water ad libitum. Throughout the treatment period, animals were weighed twice a week, and vaginal smear was observed daily under a light microscope to identify the oestrus stage.

Animal Treatment
Female rats with the above-mentioned regular oestrus cycles were chosen and divided into two groups. The first group (control group, n = 4) received vehicles (distilled water) throughout the study (56 days). The second group (n = 20) was given 1 mg/kg/day of letrozole orally, once daily for 21 days, to induce PCOS according to the method described by Kafali et al. [27]. Daily vaginal smears were obtained and examined under Olympus BX40 light microscope (Olympus Corporation, Tokyo, Japan) to determine the oestrus stage. In addition, the rats' body weight was recorded every week. The rats were confirmed to develop PCOS by oestrus cycle irregularities. Besides, PCOS rats showed the critical characteristics of PCOS, including hyperglycaemia, hyperandrogenism, and numerous cysts, as previously reported [28]. Four PCOS rats (letrozole-induced PCOS group) were sacrificed at the end of the induction period (21 days) to confirm successful PCOS induction. PCOS was successfully induced in all rats treated with 1 mg/kg/day of letrozole for 21 days, as evidenced by the changes in the percentage of dioestrous days, blood glucose levels, and ovarian histomorphological parameters ( Figures A1 and A2).
The other PCOS rats (n = 16) were then randomly divided into four experimental groups (n = 4 per group): Untreated PCOS rats that received distilled water (PCOS), PCOS rats treated with 0.5 g/kg/day of Kelulut honey (KH0.5), PCOS rats treated with 1 g/kg/day of Kelulut honey (KH1), and PCOS rats treated with 2 g/kg/day of Kelulut honey (KH2) orally for 35 days, respectively. Treatment duration and doses of Kelulut honey given were based on the study by Sahlan et al. [29]. At the end of the 35-day period, all the animals were sacrificed by ketamine-xylazine overdose (0.3 mL/100 g body weight) [30]. The ovaries were preserved in 10% buffered formalin and processed for histomorphological analysis.

Determination of Fasting Blood Glucose
This test was carried out prior to sacrificing the rats. Rats were fasted for eight hours, and blood glucose was determined using a handheld glucometer in a tail blood sample (Accucheck performa, Roche Diagnostics, Basel, Switzerland).

Determination of Oestrous Cycle
Vaginal smears were carried out on all the rats at 9:00 am daily using cotton bud immersed in 0.9% saline. A cotton bud was then rolled to the glass slide to collect the vaginal secretion. Oestrous cycles were tracked until the end of the study. Methylene blue was used to stain the cells, and a light microscope was used to examine them. As previously reported, the smears were classified as one of the four oestrous cycle stages [31]. The proestrous phase is a smear with a high percentage of rounded and nucleated epithelial cells. In contrast, the smears with predominantly irregular cornified cells without a nucleus are considered the oestrous phase. Leukocytes were small, round cells with no nucleus, primarily in the dioestrous phase. Finally, the metoestrous phase is defined as a smear with the same proportion of leukocyte, cornified, and nucleated epithelial cells [31].

Histomorphological Analysis of the Ovaries
The ovarian tissues were kept in 10% buffered formalin for 48 h prior to paraffin embedding. Next, paraffin-embedded tissues were sectioned at 5 µm thickness using Leica RM2245 microtome (Leica Biosystems, Wetzlar, Germany), air-dried in a vertical position, dewaxed, rehydrated, and stained with haematoxylin and eosin. The slides were then visualised using an Olympus BX40 light microscope (Olympus Corporation, Tokyo, Japan). The captured image was transferred and analysed with the ImageJ software.
The technique for counting ovarian follicles was adopted from another study [32]. The number of cysts, corpus luteum, and antral and atretic follicles were counted throughout each ovary serial segment. The presence of an oocyte with a nucleus was used to identify healthy follicles. Antral follicles are defined as having two or more layers of cuboidal granulosa cells, whether the cavity is visible or not. The follicles are considered atretic follicles with ovum degeneration or pyknotic granulosa cells. The cystic follicle is a large fluid-filled structure with a thickened theca interna cell layer and an attenuated granulosa cell layer [32].

Statistical Analysis
The data were presented as a mean ± SEM. T-test, one-way ANOVA, or Tukey's multiple comparison tests were used to determine the differences between the groups using GraphPad Software (GraphPad Inc., San Diego, CA, USA). Statistical significance was defined as p < 0.05.

Effects of Kelulut Honey on the Blood Glucose Levels and Body Weight Gain
Letrozole induction caused the blood glucose levels to be increased compared with normal control (8.87 ± 0.25 vs. 6.825 ± 0.57, p < 0.05). Treatment with all three different doses of Kelulut honey did not cause a significant reduction in the blood glucose levels, as shown in Figure 1a. Meanwhile, Figure 1b illustrates the effects of Kelulut honey on body weight gain. The percentage of body weight gain was significantly increased in PCOS rats (91.69 ± 5.48 vs. 44.68 ± 10.05, p < 0.05). In contrast, no difference in body weight was observed with the Kelulut honey treatment.

Effects of Kelulut Honey on the Oestrus Cycle
In the present study, rats in the normal control group showed a regular oestrus cyclicity ( Figure 2). However, untreated PCOS rats exhibited a significantly higher percentage of dioestrous days than control rats (81.10 ± 3.65 vs. 44.64 ± 3.86, p < 0.05). Meanwhile, treatment with 1 g/kg per day of Kelulut honey significantly lowered the percentage of the dioestrous day compared with the PCOS control group (63.68 ± 5.27 vs. 81.10 ± 3.65, p < 0.05). In addition, the rat vaginal epithelium staining used to recognise different stages

Effects of Kelulut Honey on the Oestrus Cycle
In the present study, rats in the normal control group showed a regular oestrus cyclicity ( Figure 2). However, untreated PCOS rats exhibited a significantly higher percentage of dioestrous days than control rats (81.10 ± 3.65 vs. 44.64 ± 3.86, p < 0.05). Meanwhile, treatment with 1 g/kg per day of Kelulut honey significantly lowered the percentage of the dioestrous day compared with the PCOS control group (63.68 ± 5.27 vs. 81.10 ± 3.65, p < 0.05). In addition, the rat vaginal epithelium staining used to recognise different stages in the oestrus cycle is shown in Appendix B.

Effects of Kelulut Honey on the Ovarian Histomorphological Changes
Transverse sections of ovaries from the normal control group showed normal histological morphology with appearances of abundant corpus luteum and healthy follicles with a fewer number of atretic and cystic follicles ( Figure 3. In contrast, fewer numbers of corpus luteum and antral follicles with numerous follicular ovarian cysts were observed in the untreated PCOS group. Treatment with 1 g/kg/day of Kelulut honey significantly increased the corpus luteum (10.5 ± 1.29 vs. 6 ± 0.82, p < 0.05) and antral follicle counts (3.75 ± 0.96 vs. 1.5 ± 0.58, p < 0.05) while reducing the cystic follicles (6 ± 0.82 vs. 10.5 ± 1.29, p < 0.05) compared with the untreated PCOS group (Figure 4). In addition, cystic follicles were reduced in the other two doses of Kelulut honey (0.5 and 2 g/kg/day) compared with the untreated PCOS group (6.75 ± 0.5/6.75 ± 1.7 vs. 10.5 ± 1.29, p < 0.05). Additionally, all groups showed an increased (p < 0.05) count of atretic follicles compared with the normal control group.

Effects of Kelulut Honey on the Ovarian Histomorphological Changes
Transverse sections of ovaries from the normal control group showed normal histological morphology with appearances of abundant corpus luteum and healthy follicles with a fewer number of atretic and cystic follicles (Figure 3. In contrast, fewer numbers of corpus luteum and antral follicles with numerous follicular ovarian cysts were observed in the untreated PCOS group. Treatment with 1 g/kg/day of Kelulut honey significantly increased the corpus luteum (10.5 ± 1.29 vs. 6 ± 0.82, p < 0.05) and antral follicle counts (3.75 ± 0.96 vs. 1.5 ± 0.58, p < 0.05) while reducing the cystic follicles (6 ± 0.82 vs. 10.5 ± 1.29, p < 0.05) compared with the untreated PCOS group (Figure 4). In addition, cystic follicles were reduced in the other two doses of Kelulut honey (0.5 and 2 g/kg/day) compared with the untreated PCOS group (6.75 ± 0.5/6.75 ± 1.7 vs. 10.5 ± 1.29, p < 0.05). Additionally, all groups showed an increased (p < 0.05) count of atretic follicles compared with the normal control group.

Discussion
Letrozole, an aromatase inhibitor, effectively induces PCOS in rats [33,34]. It resulted in excess androgens accumulating in the ovary, affecting the follicle development and leading to anovulation and insulin resistance which are the hallmark of PCOS [35,36]. Letrozole-induced PCOS rats also developed hyperandrogenism, ovarian cyst formation, abnormal follicles, hyperglycaemia, and oxidative stress similar to human PCOS [28]. Our study recorded similar findings in PCOS rats, including ovarian cyst formation, atretic follicle, altered oestrus cycle, and hyperglycaemia. Impaired glucose metabolism is highly prevalent in PCOS women [37,38]. Our current results showed that fasting blood glucose was highest in PCOS rats, which was expected from PCOS induction. Kelulut honey treatment did not reduce the fasting blood glucose in PCOS rats or increase it. A similar finding was reported in a clinical trial, whereby Kelulut honey daily intake for 30 days did not cause any changes in fasting blood glucose in patients with impaired fasting glucose [39].
This study showed that Kelulut honey supplementation did not affect body weight. Honey supplementation has caused a variety of results in body weight. A study originated in Nigeria found that Obudu honey supplementation for 29 weeks causes body weight to increase [40]. A previous study showed that Kelulut honey supplementation decreases

Discussion
Letrozole, an aromatase inhibitor, effectively induces PCOS in rats [33,34]. It resulted in excess androgens accumulating in the ovary, affecting the follicle development and leading to anovulation and insulin resistance which are the hallmark of PCOS [35,36]. Letrozole-induced PCOS rats also developed hyperandrogenism, ovarian cyst formation, abnormal follicles, hyperglycaemia, and oxidative stress similar to human PCOS [28]. Our study recorded similar findings in PCOS rats, including ovarian cyst formation, atretic follicle, altered oestrus cycle, and hyperglycaemia. Impaired glucose metabolism is highly prevalent in PCOS women [37,38]. Our current results showed that fasting blood glucose was highest in PCOS rats, which was expected from PCOS induction. Kelulut honey treatment did not reduce the fasting blood glucose in PCOS rats or increase it. A similar finding was reported in a clinical trial, whereby Kelulut honey daily intake for 30 days did not cause any changes in fasting blood glucose in patients with impaired fasting glucose [39].
This study showed that Kelulut honey supplementation did not affect body weight. Honey supplementation has caused a variety of results in body weight. A study originated in Nigeria found that Obudu honey supplementation for 29 weeks causes body weight to increase [40]. A previous study showed that Kelulut honey supplementation decreases the percentage of body weight gain and BMI in a high-fat diet-induced obese rat model more efficiently than orlistat, an anti-obesity drug [41]. Meanwhile, another study found that Kelulut honey might protect rats against metabolic syndrome-induced changes such as increased omental fat mass, triglyceride levels, adipocyte area, and adipocyte perimeter [42]. Obesity has been identified as a risk factor for PCOS [3]. Women with PCOS are overweight or obese in 38-88% of cases [43]. Clinically significant improvements in PCOS characteristics can be achieved with a moderate weight loss of 5-10% [44]. Letrozole treatment significantly increases body weight in treated rats. However, Kelulut honey does not alter the rat weight might be due to each stingless bee honey's unique botanical origin, determining its nutritional value. In addition, the duration of honey treatment also plays a vital role in inducing body weight alteration [40].
PCOS is mainly associated with ovarian physiological disturbance [45,46]. The letrozole-treated rat's ovarian histology was comparable to that of human PCOS patients [27]. In both cases, it is feasible to see the follicular arrest, increased number of subcapsular follicle cysts, atretic and decreased thickness of granulosa layer, and absence of corpus luteum [9,27]. In this study, PCOS rats' ovarian sections exhibited a high number of cystic and atretic follicles and a low count of corpus luteum. These findings were in agreement with several other studies in which PCOS has a detrimental effect on corpus luteum, cystic and atretic follicles [34,47,48]. We showed that 1 g/kg/day of Kelulut honey supplementation significantly increased the corpus luteum count compared to untreated PCOS rats, indicating restoration of ovulation and ovarian function [49].
The presence of cystic follicles was the main finding in PCOS animals [27,28,34]. In this study, cystic follicles were reduced in all Kelulut honey-treated groups. Similarly, in another study, cystic follicles were also decreased with quercetin treatment in PCOS rats [47]. Besides, defective folliculogenesis is one of the PCOS features that is mainly caused by androgen excess [50]. We found that 1 g/kg/day of Kelulut honey treatment significantly reduced antral follicle counts in PCOS rats. This indicates that Kelulut honey brings a closer recovery into the ovary's physiological state. In another study, antral follicles developed well in PCOS rats upon soy isoflavone treatment [48]. The same trend was recorded in atretic follicles. Although no statistically significant value was recorded, treatment with Kelulut honey reduced the number of the atretic follicle.
The histology of vaginal smears is an important marker of ovarian physiology [51]. In this study, cessation of the oestrus cycle in PCOS rats began on day eight and above, indicating ovarian dysfunction such as cyst formation. Restoration of oestrus cycle was seen in 1 g/kg/day Kelulut honey treatment on PCOS rat, which was in line with its restoration of corpus luteum count. This indicates normalisation toward a physiological state [52]. A similar finding has been reported by Rajan et al. in which the percentage of the diestrous day was reduced with soy isoflavones treatment in PCOS rats [48].
We demonstrated for the first time that Kelulut honey possesses beneficial effects on reproductive disorders in PCOS rats by regaining the oestrus cycle regularities, improving antral follicles and corpus luteum as well, and reducing ovarian cysts. Kelulut honey has been known for its high antioxidative properties [19,20]. Previously, Arentz et al. concluded that various herbal plants might have beneficial effects on PCOS [53,54], which may be due to the antioxidant content possessed by the herbal plants. However, the studies suggest further investigations to support the effectiveness of nutritional supplements and herbal medicine for women with PCOS [53,54]. Kelulut honey's antioxidative value is contributed by the nectar gathered from various plants collected by the bees [18]. Particularly in this study, Kelulut honey was harvested by a local beekeeper who planted different herbal plants as the source for the stingless bee nectar. Furthermore, honey has benefited female reproductive organs, mainly due to its antioxidant content [17,55]. Hence, the beneficial effect recorded in this study may be attributed to the high antioxidant content of Kelulut honey.

Conclusions
This preliminary study demonstrates that Kelulut honey, especially at the dose of 1 g/kg/day, can alleviate the reproductive disturbance in PCOS by regaining the oestrus cycle regularities, improving antral follicles and corpus luteum as well, and reducing ovarian cysts. In light of this, Kelulut honey could be further investigated as a complementary treatment for women with PCOS to improve their reproductive disease potentially. However, this present study has its limitations of not determining sex steroid hormone levels, insulin resistance status, and oxidative stress status, as well as elucidating the bioactive compounds of Kelulut honey. Therefore, further studies are needed to explore Kelulut honey's potential to improve PCOS conditions.

Conflicts of Interest:
The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.