The effects of melatonin and metformin on histological characteristics of the ovary and uterus in letrozole-induced polycystic ovarian syndrome mice: A stereological study

Abstract Background Polycystic ovarian syndrome (PCOS) with anovulation, hyperandrogenism, ovarian and uterine histological changes, menstrual irregularities, etc. signs is an infertility type. It seems that melatonin and metformin can improve these abnormalities. Objective To evaluate the effects of melatonin and metformin on the ovary and uterus in PCOS-induced mice using stereological methods. Materials and Methods Seventy-two adult female BALB/c mice (8-wk-old, 20-30 gr) were randomly divided into control (distilled water, gavage), PCOS (90 µg/kg letrozole, gavage), PCOS+metformin (500 mg/kg, gavage), PCOS+melatonin (10 mg/kg, intraperitoneal injection), and PCOS+melatonin control (0.5% ethanol saline) groups (n = 12/each). Another PCOS group was kept for a month to ensure that PCOS features remained. Finally, a stereological evaluation of the uterus and ovary was carried out, and vaginal cytology and serum testosterone levels were assessed. Results PCOS mice treated with metformin and melatonin had lower testosterone levels, body weight, and more regular estrus cycles than the PCOS group (p ≤ 0.001). A significant decrease in conglomerate and daughter gland numbers, and primary, secondary, atretic, and cystic follicles numbers with a significant increase in primordial and Graafian follicles, and corpus luteum numbers (p ≤ 0.001) was seen in these treated mice. Also, endometrial vessels' volume and length significantly increased, but ovarian, endometrial, myometrial, stromal, and glands volume, and endometrial and myometrial thickness dramatically declined (p ≤ 0.001). Conclusion It appears that metformin and melatonin could restore the PCOS phenotype including estrus cycle irregularity, high testosterone level, and ovarian and uterine micromorphology to the control levels. However, the 2 treatments had similar effects on the examined parameters.


Introduction
Endocrine problems can leads to the polycystic ovarian syndrome (PCOS), affecting 4-20% of women of reproductive age. PCOS is characterized by at least 2 of 3 criteria: low ovulation or anovulation, hyperandrogenism (HA), and polycystic ovary and/or ovarian volume increase (1).
Animal models of PCOS revealed histopathological lesions in the uterus, such as necrosis of stromal mesenchymal cells and lumen epithelial cell hyperplasia (2). In addition, ovaries of PCOS rats showed atretic follicles or degenerating granulosa cells, a double or triple increase of primary, preantral, and cystic follicles number, and a decrease in the antral follicles and corpus luteum using the stereology method, which in turn leads to an increase in ovarian size (3,4).
PCOS has no definitive treatment, but medications such as metformin (N, N-dimethyl biguanide) reduce insulin resistance and androgen levels, improve ovarian morphology and increase mature oocytes in PCOS patients (5). Furthermore, metformin can prevent abnormal endometrial structure development and apoptosis (6). Due to the direct relationship between reducing oxidative stress and increasing oocyte maturation, the use of alternative approaches such as antioxidants has attracted a huge deal of attention today (7).
Melatonin (N-acetyl-5-methoxy tryptamine) is a neurohormone synthesized and secreted by the pineal gland, which has antioxidant effects (8). In addition, melatonin has been found in the fluid of the ovarian follicles, which can reduce the damage prompted by oxidative stress in the follicle and thus may increase the fertilization and pregnancy chance in PCOS cases (9), but it needs more exploration. Also, a histological study showed melatonin's ameliorate effect on PCOS mice' ovarian morphology by morphometric method.
This led to an increase in the thickness of the granulosa layer and a decrease in the thickness of the theca layer, as well as a decrease in the number of cystic follicles and an increase in the number of corpus luteum (10).
Although 2-dimensional data can be obtained by morphometric methods, stereology-based techniques are preferred. Stereology is a 3dimensional interpretation of 2-dimensional sections of a substance or tissue that uses random and systematic sampling rather than random selection of specimens; therefore, each part of the area to be studied has the same probability of selection (11).
Given the above issues and using the morphometric method to assay structural changes in most research, this study aims to investigate the effects of metformin (as a common PCOS treatment) and melatonin (as a new treatment) on the ovary and uterus in the PCOS mouse model by stereological techniques, sexual cycle, and serum testosterone levels.

Materials and Methods
A summary of the work is shown in figure 1.

Animals
The

Weekly weight of mice
The animals were weighed once a week from purchase until the end of the study.

Determining the sexual cycle
A daily vaginal smear was taken (between 9:00-

Hormonal assay
The mice were sacrificed, blood was collected and serum isolation was done, and the samples were frozen at -80°C. Then, the serum nucleator (Figure 2a). For this purpose, the block of the ovary was randomly put on the φ-clock, and a random number was selected. Then, the block was located on the θ-clock along its cutting surface on the 0-0 axis. This was done randomly for the other block pieces (13). Successive 4 and 25 μm thick sections were obtained and stained with hematoxylin and eosin (H&E) (Figure 2b).

Estimation of the volume of the ovary
The total volumes of the ovary were estimated based on the "Cavalieri method" at the final

Estimation of the volumes of the cortex and medulla
Estimation of the cortex and medulla total volume (4 µm sections) was performed using "point-counting method" (Figure 1) at the final ×52 magnification by the following equations: ∑P (structure) indicates the total points hitting the selected structure.

Estimation of the number of the different follicles and corpus luteum
The "optical disector method" was utilized to calculate the number of primordial, primary, secondary, Graafian, atretic, and cystic follicles,

Preparation of the uterus tissue sections
The right and left uterine horns were separated, and after measuring their weight the primary volume was measured by the immersion method.
Then, based on the length of the uterine horn,

Estimation of the volumes of the uterine structures
The volumes of the lumen, perimetrium, myometrium, and endometrium (epithelium, gland, stroma, and blood vessels) layers from micrometer sections of the right uterine horn were assessed by the "point-counting method" as described before (13). The volume density and total volume of uterine structures was measured by the following formula, respectively:

Estimation of the length of the blood vessels
The length density of the uterine blood vessels from 4 µm sections was evaluated using an unbiased counting frame previously described and by the following formula: ∑Q is the total number of blood vessels whose diameter was more than 10 µm, ∑P also stands for the number of frame points hitting the uterine tissue, while a/f is the counting frame area at the last ×20 magnification. The total length of blood vessels was estimated by:

Estimation of the number of uterine glands
The number of uterine glands was estimated using the optical disector method described before (14). The following formula assessed the numerical density and total number of the gland, respectively:

Morphology of the endometrial gland
The morphological types of endometrial glands were recognized and categorized to assess hyperplastic or neoplastic changes in the endometrium. The normal endometrial gland is a simple tubular gland with a thin lumen that may be oval, circular, or elongated. The cystic endometrial glands are big and round. A gland endometrial with the daughters refers to glands with irregular shapes and sizes that make up the daughter glands within the epithelium or at the level of the lumen of the mother's glands

Estimation of the endometrial and myometrial thickness
To assess the endometrial and myometrial thickness, the harmonic mean thickness of the 4 µm sections of mentioned layers was evaluated based on the "orthogonal intercepts method" (17).
The probe of stereology (isotropic lines) at the final

Ethical considerations
All animal procedures were approved

Statistical analysis
The obtained data are presented as mean ± S.D.
The results were statistically analyzed using GraphPad Prism 9 (GraphPad Software Inc., San Diego, CA) by ANOVA, followed by the Tukey post hoc test to evaluate the difference between the studied parameters in the 3 groups (control, PCOS, and PCOS + metformin).
In addition, an independent t test was used to determine the differences between PCOS + melatonin and its corresponding control groups, and also PCOS + melatonin and PCOS groups. The first type of error (p-value) was 5%, and the confidence interval (CI) was 95%.
Significant differences were considered at p < 0.05.

Results
Metformin and melatonin as treatment groups

Body weight
Metformin and melatonin reduced the elevated body weight of PCOS mice after the 3 rd wk and brought it to the control groups' levels.

Determining the estrus cycle
Metformin and melatonin regulated the sexual cycle and significantly increased proestrus, and

Serum testosterone levels
Metformin and melatonin significantly reduced elevated serum testosterone levels in the PCOS mice (p = 0.001, CI = 95%).

Volumes of the ovary, cortex, and medulla
Metformin and melatonin significantly reduced the increased volume of the ovary, cortex, and medulla compared to the PCOS group (p ≤ 0.001, p ≤ 0.001, and p = 0.03, respectively, CI = 95%) ( Figure 3).

Numbers of the ovarian follicles and corpus luteum
Metformin and melatonin significantly increased the declined number of corpus luteum and primordial and Graafian follicles in the PCOS mice, and conversely decreased the elevated number of primary, secondary, atretic, and cystic follicles (Table I, Figure 4).

Volume of the uterus
Metformin and melatonin significantly reduced the uterine volume of the PCOS mice (p ≤ 0.001, CI = 95%) (Figure 5a).

Volumes of the endometrium, myometrium, and perimetrium
The volume of myometrium and endometrium in the PCOS mice reduced after treatment with metformin (p = 0.01 and p = 0.02, respectively, CI = 95%), and melatonin (p = 0.01, CI = 95%) (Figure 5b, c). Perimetrium volume did not exhibit a significant difference in any of the study groups.

Volumes of the endometrial structures
Metformin and melatonin decreased the volume of the endometrial glands, stroma, and epithelium of PCOS mice (p = 0.03, and p ≤ 0.001, respectively, CI = 95%), and increased their endometrial vessel's volume (p ≤ 0.001, CI = 95%). The lumen volume showed no significant difference in the studied groups (Figure 5d, e, f, and g).

Numbers and volumes of the endometrial gland types
Metformin and melatonin decreased the numbers and volumes of the endometrial total, daughter, and conglomerate glands and increased the normal glands' number and volume in PCOS mice. Cystic glands did not show significant differences in different groups (Table II, Figure 4).

Thickness of the endometrium and myometrium
Increased endometrial and myometrial thickness in PCOS mice reduced after treatment with metformin and melatonin (p ≤ 0.001, CI = 95%) ( Figure 4 and Figure 5h, i).

Length of the endometrial vessels
Metformin and melatonin increased the endometrial vessels' length of PCOS mice and brought it to their control groups' levels (p ≤ 0.001, CI = 95%) (Figure 5j).
There was no significant difference between the PCOS+melatonin and its corresponding control groups in any study parameters. Also, metformin and melatonin were not significantly different in their effectiveness. Besides, after 1 month, body weight, serum testosterone levels, and continuous diestrus stage in the PCOS support group were the same as the second wk after the beginning of letrozole gavage (p = 0.3, CI = 95%) and so were considered as PCOS range.

Discussion
In this study, induction of PCOS with our new dose of letrozole in mice led to diestrus stage continuity, weight gain, and elevated serum testosterone levels. The volume of the ovaries, cortex, and medulla, the number of primary, secondary, atretic, and cystic follicles increased.
Conversely, the number of primordial, and Graafian follicles, and corpus luteum decreased. In PCOS, the number of atretic follicles increased due to follicular melatonin concentration reduction and oxidative stress amplification, and the resulting follicular damage (9). Consistent with our results, a study found that melatonin increased corpus luteum and antral follicles and decreased ovarian cysts in PCOS rats (35). Many studies have assigned these effects to the role of melatonin in the regulation of VEGF and its receptors, but its exact mechanisms are still unknown (38). According to the evidence of decreased uterine blood flow and VEGF expression in PCOS (28), it is assumed that increased VEGF expression under the influence of melatonin was effective in increasing the volume and length of endometrial vessels, which of course, requires more investigations.

Limitations
At the time of the study, there was a time limitation for performing some other tests, including metabolic tests.

Conclusion
This study demonstrated the potential of Further investigations are required to confirm these findings through clinical studies.