DOI: https://doi.org/10.21203/rs.3.rs-2120435/v1
Background: This research aimed to appraise the 2100 MHz frequency of the new generation of mobile phones on the male rat reproductive system. The genital organ is one of the important systems for sustaining reproduction and generation. The consequences of mobile phone radiation exposure have been a growing general health worry in recent years.
Study design: 35 Wister albino male rats were randomly divided into five groups for this study. The groups were exposed to a 2100 MHz frequency electromagnetic field for 0, 15, 60,120, 180 min/day, 70 consecutive days. At the end of the experiment, serum testosterone levels were measured, and each group was evaluated for epididymal sperm parameters, including mobility, morphology, and viability. Then, immunohistochemistry staining was performed by Cleaved-Caspase 3 antibody to show apoptosis in the testicular tissue.
Results: Our results revealed that serum testosterone levels were significantly reduced in experimental groups (60 and 120 min). In addition, sperm motility was significantly reduced in experimental groups (60, 120, and 180 min), and a significant decline of sperm viability and apoptosis (P=0.001) was observed in all groups.
Conclusion: This study found a significant increase in the number of dead sperm and apoptosis and a significant reduction in motility, which could impair the spermatogenesis process, reduce sexual characteristics, and eventually lower the rate of fertility.
Today, High Frequency (HF) electromagnetic fields are found in devices such as mobile phones as one of the most common devices emitting electromagnetic waves, which are readily accessible for nearly half the world's crowd across disparate age groups. High Frequency (HF) is the idiom used to describe the section of the electromagnetic spectrum comprising the frequency range from 100 KHz to 300 GHz. The electric and magnetic fields, which make up the electromagnetic field, are interrelated and considered jointly for measurements at a high frequency. HF fields are applied in different technologies, particularly for communication, medicine, and heating purposes (e.g.microwave, ovens). Also, a remarkable body of work has been conducted on the relationship between HF fields and health outcomes such as headaches, concentration difficulty, sleep quality, cognitive function, cardiovascular effects, etc. [1]. The results of Kundi suggested that alternating electromagnetic fields have positive and negative effects on biological systems [2]. Accumulating results have indicated that RF-EMR potentially negatively affects human health [3]. Berg, in 1999 found that the intensity, duration of exposure, and frequency of waves were three important factors for the effects of electromagnetic waves on the cell body function [4]. Elsewhere, Kafaee Razavi et al. observed that exposure to electromagnetic fields with a 940 MHz frequency increased blood-brain barrier permeability and apoptosis across all exposed groups [5]. On the other hand, more research has shown that the radiation severity and duration of RF-EMR will influence the parameters of male reproduction valence; these parameters include the concentration [6], mobility [7, 8], morphology [9–13], the viability of sperm cells [6, 14, 15] and sperm apoptosis [16, 17].
Further, compared to controls, a significantly reduced infertility was seen in exposed rats (200 MHz frequency magnetic field). Ultrastructural variations in seminiferous tubules, Leydig cells, and spermatids were the main reasons for reduced fertility [18]. Thus, the present study evaluated whether 2100 MHz can cause sperm cell apoptosis and affect sperm morphology, percentage of motility, and the number of dead sperms. In addition, exposure to EMF adversely affects testicles and might influence spermatogenesis by impairing the functions of Sertoli and Leydig cells in experimental animals [19]. The purpose of our work is to investigate the possible changes in spermatogenesis and testosterone levels in association with the effects of mobile radiation (2100 MHz) on the reproduction system of male rats.
An antenna (output power: 2000 MW, power source lithium-ion battery 127, 2600 MAh) was used with a variable frequency band (1900 to 2600 MHz) to generate high-frequency electromagnetic waves.
Thirty-five adult male Wistar rats (weighing 300 to 400 g) were used in this experiment. After seven days of adaptation to the laboratory environment and stress control with 12 h light and 12 h dark situation.
The rats were randomly divided into five groups, including seven animals. After exposure to 2100 MHz (70 consecutive days) electromagnetic waves, the rats in control (0 min/day) and all experimental (15, 60,120,180 min/day) groups were euthanized by CO2 gas. Blood specimens were accumulated from the abdominal aorta. The serum was obtained by centrifugation at 3000 rpm for 10 minutes. Finally, the rats were killed with pity (Code of Medical Ethics: IR.UM.REC.1400.338).
the sperm samples were collected from the cauda epididymis to analyze sperm motility, morphology, and viability.
The cell culture environment provided for evaluating the sperm motility was composed of PBS (9ml) and FBS (1ml).
About 200 sperms were counted by a light microscope and ultimately calculated as percentages for each sample.
After preparing the cell culture environment, it was placed in an incubator at 37 ° C /0.5 h. The sperm suspension was placed on a slide and was counted with a light microscope.
For evaluation of sperm morphology, the sample of sperm was stained with Diff – Quick (White, red and blue 30 seconds, and washed with water) and was dried in the air.
From each sample (the sperm suspension and eosin – necro since), a drop with a micropipette (1000 µl) was placed on a slide whereby the smear was prepared, which was dried in the air.
Finally, the Right and left testes were weighed separately, and then the left testis was fixed in formalin solution for immunohistochemistry studies.
cleaved caspase-3 (cell signaling, primary antibody: 9661S) and Goat Anti-Rabbit IgG H&L (HRP) (Abcam, Secondary Antibody: AB6721) were prepared to investigate testis tissue apoptosis.
The kidney tissue was considered positive control, while the negative control was performed by removing the primary antibody from the testicular tissue. Finally, the level of apoptosis was measured at the same level.
To designation significant differences between the groups, a one-way analysis of variance (ANOVA) test was accomplished, and all differences were considered significant at the following: P ≤ 0.001, 0.005, and 0.05.
Statistical Analysis of Serum Testosterone
Statistical studies of the data obtained from the serum testosterone level showed that the serum testosterone levels of all experimental groups were lower than in the control group (o min). Specifically, there was a significant reduction in experimental groups of 60 and 120 min in comparison with the control group (P≤0.005) (Figure 1).
Sperm parameters
The results showed that the effects of the electromagnetic field (2100 MHz) caused a significant rise in the number of dead sperm cells across all experimental groups compared with the control group (P≤0.05) (Figure 2). On the other hand, the sperm motility had a significant reduction (P≤0.05) in the experimental groups (60, 120, and 180 min) (Figure 3). Finally, the results showed no change in the number of abnormal forms caused by exposure.
Immunohistochemistry results
Immunohistochemistry investigation showed that the percentage of apoptotic cells in all experimental groups significantly increased compared to the control group (P=0.001) (Figure 4).
In the present study, it was found that prolonged time (2-3 over per day) of exposure to 2100 MHz electromagnetic waves could cause changes in the level of testosterone hormone, increase the number of dead sperms and promote apoptosis in the testes. In the same way, Berg in 1999 reported that the intensity, duration of exposure, and frequency of waves are three important factors for the effects of electromagnetic waves on cellular function [4, 20], which is compatible with the present study.
Various and sometimes contradictory results have been reported on the effects of electromagnetic fields on testosterone levels. The present study showed that exposure to the electromagnetic field could significantly decrease (P≤0.004) the testosterone levels of the experimental groups (60 and 120 min). Interestingly, a remarkable increase was observed in testosterone level in the 180 min group compared to 60- and 120-min groups of exposure, although it was still lower than the control group value. It is possible that after 120 minutes, due to the severe reduction in testosterone, a rise in the LH production by the pituitary gland caused by the effect of electromagnetic waves on the CNS occurs alongside a negative feedback mechanism, which stimulates the Leydig cell to increase the testosterone hormone [21] further.
Al-Akhras et al. reported a significant increase in the serum levels of luteinizing hormone (LH) only after 18 weeks of exposure (P <0.005) [22].
Given that a series of testicular Leydig cells died by 2100 MHz electromagnetic waves, the serum level of testosterone in the 180 min group was lower than in the control group. Also, prolongation of the duration of exposure of rats affected by 2100 MHz electromagnetic waves increased the percentage of dead sperm cells, according to a report by Desai and Al-Akhras et al. [22, 23]. The reason behind the increased percentage of dead sperm cells could be mitochondrial, DNA, and nuclear destruction of sperm cells [23].
Ozguner et al. in 2005 reported that serum total testosterone level decreased significantly in the EMF group (p<0.05) [24], and Al-Akhras et al. reported testosterone levels were significantly decreased after 6 and 12 weeks of the exposure period [22] which was similar to 60 min and 120 min groups in our study. However, Al-Akhras et al. reported that no significant changes in testosterone levels were observed after 18 weeks in adult rats exposed to an electromagnetic field (50 Hz) [22] consistent with this finding, we observed that the testosterone level rose at 180 min in comparison with the 60 min and 120 min groups but in comparison with the control group decrease but the decrease was not significant.
Our results indicated that a high-frequency electromagnetic field (2100 MHz) negatively affects sperm parameters such as motility and viability characteristics (Figure 2 and Figure 3). An increase in the duration of exposure to electromagnetic waves (2100 MHz) by rats may ultimately lead to the destruction of the mitochondria and the epidermis's sperm nucleus. There may be a reason for the increase in the percentage of dead sperm cells. Figure 3 indicated that sperm motility significantly decreased in the experimental groups (60, 120, and 180 min). In addition, Figure 2 revealed that sperm viability diminished significantly across all experimental groups after 70 days of exposure (P≤0.05). Also, our result is in line with Baharara et al. [25] findings obtained in 2015 (50 Hz), Hamdi [26] (50 Hz), De Iuliis et al. [27] (Mobile phone radiation), Deepinder et al. [1] (radio-frequency), and Erogul et al. [28] (900 MHz) who reported diminished sperm motility.
In another study, Yan et al.'s results suggested that rats exposed to 6 hours of daily cellular phone propagation for 18 weeks exhibited a significantly higher incidence of sperm cell death than the control group rats did through chi-squared analysis. In addition, abnormal clumping of sperm cells was present in the rats exposed to cellular phone emissions while absent in control group rats [6]. The report of Yan results in increasing the meaning of dead sperm cells is consistent with the present study.
Apoptosis is a physiological process of selected cell elimination. As an antagonist of cell proliferation, apoptosis contributes to keeping the cell number in testicular tissue and helps to delete superfluous and damaged cells [29].
The studies of Kesari et al. [30] (Testes of rat) and Kafaee Razavi et al. [5] (rat brain
tissue) showed a significant rise in apoptosis across all experimental groups exposed to electromagnetic waves at a high frequency. Although the duration and the intensity of the electromagnetic field in the experimental groups were different, their results have been similar to ours.
The present study showed that an electromagnetic field of a new generation of mobile phone (2100MHz) increased apoptosis in spermatogenesis as well as the activity of caspase 3, which is congruent with Kesari et al. (2.45 GHz), who found Using flow cytometry a significant decrease in sperm percentage and an increase in apoptotic cells after cell phone exposure (2 hours a day for 35 days) [30].
The number of apoptotic cells was evaluated across all groups in the present study. It was observed that the groups affected by 2100 MHz electromagnetic waves had a significant rise in apoptosis. This result was in line with Kesari et al. [30] findings, though our frequency used here (2100MHz) was different from Kesari et al. (2.45 GHz) [30].
The present research outcomes suggested that using a new generation of mobile phone at 2100 MHz frequency could induce detrimental effects on reproductive parameters such as viability, motility, and testosterone levels, which lead to increased apoptosis and reduced apoptosis sexual characteristics, and ultimately diminished fertility. Therefore, to mitigate the harmful effects of these waves on the male genital system, it is recommended to avoid the unnecessary and prolonged use of mobile phones and other electromagnetic radiation emitters, especially in high-risk groups (children, adolescents, and pregnant women).
Acknowledgment
This research resulted from a Ph.D. thesis project, and we highly appreciate Mr. Poradibi, Histology laboratory technician and Navid medical laboratory, for their valuable assistance.
Ethical Approval
After the review conducted by the Ethical Approval Committee meeting at Ferdowsi University of Mashhad, it was accepted to work with laboratory animals with the Code of Medical Ethics: IR.UM.REC.1400.338.
Competing interests
The authors declare that they have no conflicts of interest in the research.
Authors' contributions
Fariba Ghasemiannejadjahromi: Writing manuscripts and doing practical work
Ahmadreza Raji: Guidance for the practical part of research and text rewriting
Mohsen Maleki:Guidance in the field of practical research pathology
Pezhman Mirshokraei:Guidance in the field of practical sperm research
Morteza Kafaeerazavi:Making an electromagnetic wave generating device
Funding
This work was supported by the National Institutes of Health [grant numbers 41494, [Ferdowsi University of Mashhad].
Availability of data and materials
The project data will be accessible in a file at any time you need. Also, I am doing stereology of the testicle tissue, and if necessary, the stereology data will also be available to you.