Morphological and Biochemical Changes in the Rat Ovaries Following Electromagnetic Field Exposure

Purpose This study aims to investigate the effect of electromagnetic eld (EMF) exposure which is environmental toxic agent on rat ovary. Methods A total of 30 female Wistar albino rats are randomly divided into three groups (n=10) as Control, Sham, and EMF groups. The rats are not exposed any processes in Control group. The rats are kept in special cage for the same duration as in the EMF group without any EMF exposure in Sham group while the rats are exposed to 900 MHz EMF with a special mechanism in EMF group. At the end of day 28, the ovarian tissues are collected. Stereological and biochemical examinations are performed. Results and homogenized inappropriate buffer solution (4800 ml). Catalase (CAT), superoxide dismutase (SOD), lipid peroxidation (LPO) and glutathione (GSH) levels were assessed in homogenates. CAT test was performed using H 2 O 2 at 240nm whereas SOD measurement was performed using slightly modied version of Sun et al. LPO level was examined using thiobarbituric acid while GSH activities was measured according to slightly modied version of Sedlak and Lindsay.


Introduction
Exposure to electromagnetic eld (EMF) causes negative effects on human health such as fatigue, stress, pain and ache in muscles. The World Health Organization (WHO) has called these effects "idiopathic environmental intolerance attributed to electromagnetic elds" (IEI-EMF) [1]. In addition to these effects, EMF may be reason of some illness such as Alzheimer's disease (AD), Amyotrophic Lateral Sclerosis (ALS), childhood asthma and cancer [2]. Widely used cell phones, Wi-Fi, and various kinds of connected devices generate high frequency electromagnetic elds (HF-EMF) and exposure to EMF becomes unavoidable [3].
EMF has negative consequences on male productivity. It can cause reduced sperm motility, elevated abnormal sperm percentage, loss of spermatocytes and spermatids, slightly elevated testes caspase-3 activity [4]. Also, it can cause morphological changes in sperm such as signi cant reduction in sperm head area and acrosome percentage of the head area, signi cant decrease in sperm binding to the hemizona. Sperm fertilization potential is affected negatively because of these changes [5]. The ovaries may be further affected by environmental stress factors such as radiation, which may result in germ cell apoptosis. EMF exposure caused by infertility, separation of thecal layer of primary follicles, irregular thickness of the zona layer, and reduction number of ovarian follicles in rats [6,7]. When the effects of EMF on ovarian tissue are examined, the zona granulosa and thecal layers become thin, granulosa cells shrink, mitotic activity and leukocyte in ltration reduce [8]. Also, ovarian follicles are affected negatively such as isolation with dilated vessels showing in ltration in ovarian stroma is seen [7]. In another research, especially in prenatal period, exposure to 2450 MHz EMF cause postnatal growth restriction and delay in puberty in female rat are found [9]. In this experimental research, we aimed to investigate the effects of EMF which is one of the very important environmental toxic agents on ovarian tissues using unbiased stereological methods and biochemical approaches.

Methods
This experimental study was performed following Animal Experiments Local Ethics Committee of the Ondokuz Mayis University approval with the number of 2018-07. A total of 30 female rats of Wistar Albino (12 weeks old) were randomly divided into three groups (10 animals each) as Control, Sham, and EMF. During the experiment, subjects were kept under a constant dark light cycle of 12 hours; drinking water and standard rat feed ad libitum were given. The weight of all animals was measured, and data were recorded before starting the experiment. Groups and transactions are as follows: Control group: This group will not be processed.
Sham group: This group was subjected to stress conditions in a special cage for the same duration as the electromagnetic eld group. However, this group was never exposed to electromagnetic eld. EMF group: This group was exposed to 900 MHz electromagnetic eld for 28 days (60 min / day) [10] with a special mechanism [11].
On the 28th day of the experiment, vaginal smear method was used to check the estrus cycle of the animals in all groups and the data were recorded. At the end of day 28, the weight of all animals was measured, and the data were compared with the initial results. After the experimental procedures, the subjects were perfused transcardially under ketamine (0.1) and xylazine (0.5) anesthesia and ovarian tissues were removed. Stereological and biochemical analyzes were performed on the obtained tissues samples.
Applied methods:

Gonadal Somatic Index
Gonadal somatic index (GSI) is an indicator for the cyclic status of the animals. It basically manifests maturation of Graa an follicles and decreases in the acyclic animals compared with cyclic counterparts.

Stereological Method
Ovarian tissues from rats in all groups were xed in 10% formalin for 24-48 hours before embedded in para n for light microscopic examination. After depara nization and dehydration procedures, 20 µm thick sections (tissues were subjected to sectioning based on our previous studies for stereological methods) and stained with hematoxylin-eosin for histological examination and stereological analysis.
The sections prepared for examination were examined under Olympus BH 40 camera attachment light microscope and stereological and histopathological evaluations were made by taking photographs of all groups. Ovarian volume, cortex / medulla ratio, follicle volumes were calculated by using stereological method which is called Cavalieri method.

Cavalieri method
Cavalieri method was used to calculate ovarian volume, cortex/medulla ratio and follicle volume. This method is a branch of stereology which is one of the unbiased approach [13] and mainly is based on the calculation of the volume of an equally spaced and parallel sliced structure in sections taken by systematic random sampling. By calculating the surface area of the related structures on the surfaces facing the same direction, volumetric data are obtained by multiplying the total surface area and average cross-sectional thickness. For the area calculation, a point counting grid which consists of systematic (+) signs representing points separated at equal intervals from each other is used. The area occupied by a point is known in the point counting grid and is called the area associated with the point and shown as [a (p)]. In this context, the points falling on the area of interest were counted and the total number (Σp) was obtained. The obtained numbers were multiplied by a (p) to achieve surface area (A) measurements for ovarian volume, cortex/medulla ratio and follicle volume [14].
The following formula was used during the calculations; The volumes of the respective parameters were calculated using histological images taken on x10 magni cation.

Biochemical procedures
Regarding the biochemical parameters, ovarian tissues were harvested (200 mg) from each animal and homogenized inappropriate buffer solution (4800 ml). Catalase (CAT), superoxide dismutase (SOD), lipid peroxidation (LPO) and glutathione (GSH) levels were assessed in homogenates. CAT test was performed using H 2 O 2 at 240nm whereas SOD measurement was performed using slightly modi ed version of Sun et al. LPO level was examined using thiobarbituric acid while GSH activities was measured according to slightly modi ed version of Sedlak and Lindsay.

Statistical Analysis
The data analyzed using SPSS 21.0 for Mac (IBM Corporation) software and the comparisons between the groups are performed by One Way ANOVA (Tukey-Post-Hoc) test. p<0.05 value is considered as statistically signi cant.

Gonadal Somatic Index
Concerning the GSI values, there are statistical differences between the Control and EMF (p=0.001), and Sham and EMF (p=0.001) groups, respectively whereas no statistical difference is found between the Control and Sham groups (p>0.05) ( Table 1).

Stereological Results
Regarding the stereological evaluation; statistical differences were observed between the Control and EMF (p=0.001), Sham and EMF (p=0.001) groups respectively in terms of ovarian volumes. There were statistical differences between the Control and EMF (p=0.001), Sham and EMF (p=0.001) groups respectively in terms of medullary volumes. Also, there were statistical differences between the Control and EMF (p=0.001), Sham and EMF (p=0.001) groups respectively in terms of primary follicle volumes. Similarly, statistical differences were observed between the Control and EMF (p=0.005), Sham and EMF (p=0.005) groups respectively in terms of secondary follicle volumes. In addition, there were statistical differences between the Control and EMF (p=0.001), Sham and EMF (p=0.001) groups respectively in terms of both secondary follicle antrum and Graa an follicle antrum volumes. No statistical differences were found between the Control and Sham groups (p>0.05) in points of above parameters, stereologically. Finally, there were also no statistical differences between the Control and EMF (p>0.05), Sham and EMF (p>0.05), Control and Sham (p>0.05) groups, respectively in terms of Graa an follicle volumes ( Table 2).

Biochemical Analyses
Biochemical analyses show that CAT levels signi cantly decreased between the Control and EMF (p=0.001), and Sham and EMF (p=0.000) groups, respectively. Regarding the SOD levels, there are statistically signi cant differences between the Control and EMF (p=0.000), and Sham and EMF (p=0.000) groups, respectively. Similarly, there are signi cant statistical differences are found between the Control and EMF (p=0.000), and Sham and EMF (p=0.000) groups, respectively in points of LPO levels. There are no statistical differences between the Control and Sham groups in terms of the above parameters (p>0.05). Finally, no statistical differences are found between the all groups in point of GSH levels (p=0.05) ( Table 3).
The obtained results are given in Figure 1 visually (Figure 1).

Discussion
Previous studies regarding the effect of magnetic elds on serum gonadotropin levels revealed con icting results [15,16], this con icting results may originate from cyclic changes of these hormones, for this reason in our study, we aimed to assess gonadal stromal index to gure out this effect, our data showed that exposure to EMF resulted in signi cantly lower GSI. This result was consistent with the previous study by Roshangaret al. [17] which showed exposure to EMF during the developmental period which interfered with both oocyte differentiation, folliculogenesis and reduced fertility, this result was claimed to be secondary to decreasing ovarian reservoir. Technological devices such as electrical appliances, communication devices, radio and television transmitters and sub-stations and mobile phone base stations emit electromagnetic waves and generate electromagnetic eld. It causes negative health consequences and damage various tissues in living organism [18].Various studies have been done about the damage to tissues in various systems such as nervous system and endocrine system [19,20,21]. The effect of EMF exposure on the reproductive system was also investigated. Since there is no standard methodology for the evaluation of male and female reproductive systems, some contradictory results have been obtained in these studies. Several studies have reported that EMF reduces fertility potential [21], sperm concentration, motility, and seminiferous tubule diameter [22,23] and increases abnormal sperm morphology [8]. However, there are also experimental studies reporting that EMF does not affect sperm count in testes or epididymis and does not alter sperm motility or morphology. It has been reported in previous animal experiments that EMF increases oocyte DNA damage, apoptosis and oxidative stress in the endometrium and ovary and reduces the number of follicles in the female reproductive system [24,25]. Other studies have emphasized that EMF does not have a negative effect on the male and female reproductive system [6,26-28]. In our research, we found that there are signi cant statistical changes in volumes of medulla, primary follicle, secondary follicle, secondary antrum, graa an antrum.
In their work with RE-DNA, which can play an important role in cell functionality that can be replicated and  2019) found that EMF might cause to an impairment of mitochondrial function [30]. In their work with cancer cell, Storch et al. (2016) found that EMF causes to increase in reactive oxygen species (ROS) [31]. It is known that ROS and oxidative stress can cause DNA damage, general and speci c gene expressions and cell apoptosis [18]. Because of these changes, volume changes may have been in the research.
Few studies have examined histopathologically the effects of EMF on the female reproductive system. In their studies on the effect of EMF to ovarian follicle reservoir in prenatal exposure, Türedi et al. (2016) found that EMF can cause increased apoptosis in the rat ovarium, impaired the follicular development process and as a result of these conditions, a decrease in follicular reservoirs in the ovarium can be seen [23]. In another study, Okatan et al. (2018) found that EMF cause a decreases in mitotic activity in follicle cells, follicle cell dimensions, thickness in the zona granulosa layer, thickness in the thecal layer and secondary follicle numbers whereas an increase in leukocyte in ltration, hyperchromasia in granulosa cells, and biochemical parameters [8]. Our ndings suggest that, there is an increase in the volume of secondary follicles. In the literature review on role of mitochondria in the oxidative stress induced by EMF on reproductive systems, Santini et al. (2018) found that EMF can cause morphological, histological and biochemical changes. These include delayed puberty, accelerated loss of primordial follicles in the adult, no differences in ovarian follicle count, changes in the volume and the primordial follicle content of newborn ovaries, reduction of primordial ovarian follicles. Our results are consistent with these results. There are also other studies in the literature that are manifesting increased apoptosis in ovaries, reduced primordial and tertiary follicles, increased atretic follicle, vasocongestion, stromal brosis, lost oocytes in primary follicles, extended degeneration, vacuolization, and loss of connection with cumulus cells in granulosa cells, and increased Total antioxidant status (TAS), total oxidant status (TOS), and oxidative stress index (OSI) [26]. Increased apoptosis, TAS, TOS and OSI may be the cause of these volume changes. Based on our study results, it may be concluded that EMF is one of the most important environmental toxic facts and exposure to 900-MHz EMF may negatively affect the ovarian tissues.

Conclusions
The vast majority of the electromagnetic eld studies in the literature indicated that it has harmful effects of on human health. Especially, electromagnetic eld emitted from cell phones, are thought to have many harmful effects including. However, despite molecular and epidemiologic studies on both experimental animals and humans, the effects of electromagnetic eld on ovarian tissues which related to female reproductive system is still a topic for discussion. Based on our study results, it may be concluded that EMF is one of the most important environmental toxic facts and exposure to 900-MHz EMF may negatively affect the ovarian tissues.   Figure 1