1. Impact of 1800 MHz GSM-like frequency associated with oxidative stress in micronuclei formation in rats’ ovarian tissue
Dr. ALI SAEED AL-CHALABI
Dr. Mohd Azam Khan B. Goriman Khan
Dr. Abdul Rahman Bin Aziz
Dr. Erkihun Aklilu
Faculty of Veterinary Medicine, Universiti Malaysia Kelantan (UMK)
Dr. Fareq Malek
Dr. Susanna. H. Ronald
School of Electrical System Engineering, Universiti Malaysia Perlis (UniMAP).

2. Introduction
Exposure to electromagnetic fields from mobile communications has been increasing during the past 10 years. A lot of concerns have been raised due to the introduction of the new technologies without the provisions of adequate public information about the potential health consequences.
Damages to the reproductive organs can cause subfertility and abnormality in embryonic development. , Several non-infective factors, including exposures to non-ionizing EMF, especially RF fields used in mobile communications have been proposed as possible risk factors contributing to infertility (Poulletier de Gannes et al., 2013).
Oxidative stress is a status result due to imbalance between oxidant agents and antioxidant system, contributing to the production of free radicals which are responsible for oxidative damage to proteins, lipids and DNA in the body (Halliwell 2007).
Recent works have performed to explain the role of oxidative stress as harmful effect on living tissues induced by GSM signals (Avci et al. 2012; Dasdag et al. 2012; Gharib et al. 2012; Kerman and Senol 2012; Kismali et al. 2012; Ozgur et al. 2013; Tök et al. 2014; Mattsson and Simkà 2014; Yakymenko et al. 2014).
Most of the fields on the impacts of EMW on the potency of the reproductive tract and its direct or indirect effect on female fertility have reported contradictory results.

3. This is perhaps due to variations in the experimental design in terms of a source of EMWs, frequency, exposure duration and the specific absorption rate (SAR) used.
In the experiment ran out by researchers on the impact of EMW on young rats aged 21 days from mothers exposed during pregnancy to a mobile phone for 11 hours and 45 minutes in standby and 15 minutes in speech mode to evaluate the ovarian follicle activity, the number of follicles and growth were significantly affected due to apoptosis among follicles, hyperplasia in ovarian stroma and elongation cell mitosis time (Gul, Celebi, and Uğraş 2009).
Vahid et al Suggested that mobile phone wave can increase ovarian and atresia follicles as well as variations in sex hormone levels in Wister rats, which were exposed to mobile phone 12 times for 10 min. (Calling mode) for 1 month and 2 weeks.
In contrast, other studies reported no adverse effects on reproduction activity and fertility in rats and mice exposed to EMWs (GSM /Wi-Fi signals) (Akdag et al. 2013; Bayat et al. 2012; Gathiram, Kistnasamy, and Lalloo 2009; Poulletier de Gannes et al. 2013).
Our work points to assess the correlation between chronic exposure to 1800 MHz GSM signals, “heavy use of mobile phone” induced oxidative stress and histopathological alterations in ovarian tissue.

4. Methodology
Thirty female Sprague-Dawley type rats at an average body weight of 200 g. and three months old were used throughout the study.
Especially designed exposure Plexiglas box (60cm x 40cm x 20 cm) was used during the RF-EMR exposure time. Whole body irradiation for 30 and 60 days respectively at an average 2h/day exposure at specific absorption rate level of W/Kg by using a GSM signal generator was done.
Figure 1. It shows the EMF exposure setup using GSM like radiofrequency generator system.

5. Experimental design

6. Results 1. Effect of 1800 MHz EMF on body and sex organ weights
Table 1: Effect of 1800 MHz GSM electromagnetic field on body, ovarian and uterine weight
Target
Groups
Body weight (g)
Ovarian weight(g)
Uterine weight (g)
Unexposed animals
± 14.3
0.094 ± 0.01
0.694 ± 0.09
EMF Exposure for 30 days
291.6 ± 11.8
0.106 ± 0.01
0.700 ± 0.1
EMF Exposure for 60 days
297.5 ± 10.04
0089 a± 0.01
0.721 ± 0.1
P value
0.588
0.024
0.773
Values are Mean ± S.D. a p< 0.05 statistically significant
2. Effect of 1800 MHz EMF on oxidative stress parameters
Table 2: Effect of 1800 MHz GSM electromagnetic field on oxidative stress parameters
Parameters
Groups
Ovary
Uterus
Serum
GSH-PX (mIU/mg)
MDA (nmol/mg)
MT (pg/ml)
Unexposed animals
19.74 ± 1.06
0.92 ± 0.12
9.88 ± 1.2
0.69 ± 0.15
41.32 ±7.06
EMF Exposure for 30 days
18.43 c ± 1.28
1.16 c ± 0.23
9.15 ± 0.89
0.9 ± 0.14
34.99 b ± 2.52
EMF Exposure for 60 days
18.35 c ± 1.33
1.12 b ± 0.26
8.46 c ± 1.08
1.48 c ± 0.67
32.24 b ± 3.91
P value
0.004
0.002
0.005
0.006
Values are Mean ± S.D. b p< 0.01, c p< statistically significant

7. 3. Effect of 1800 MHz EMF on histopathological changes in ovarian tissues
Figure 2: Ovarian sections from a female control group. Normal well follicular development of the Graffian follicle with mature oocyte surrounded by granulosa cells with normal zona pellucida normal ooplasm with normal chromatin condensation in nucleus 100 X. H & E stain.

8. Ovarian sections of EMF exposure group for 30 days1 2 3
Ovarian sections of EMF exposure group for 30 days
Figure 3: Graffian follicles show oocyte with micronucleus formation indicated by (2) enclosed by thin, irregular zona pellucida indicated by (1), granulosa cells were separated from each other and vacuolation in ooplasm and granulosa cell layer, autophagy granulosa cells are strong contact with the oocyte indicated by (3) 100 X. H & E stain. 1 *
Figure 4: Graffian follicle shows thin and degenerated zona pellucida indicated by (1), sever ooplasm vacuolation with micronuclei formation indicated by (2). 40 X, H & E stain. 2

9. Ovarian sections of EMF exposure group for 60 days.
Figure 5: (A) Ovary showing a decrease in ovarian follicle numbers with congestion in the blood vessels. (C.L) corpus luteum, (3) secondary follicle, (4) atretic follicle, (M) matrix. 4 X. (B) Graffian follicles (a) macrophage strong contact with the oocyte nucleus (N), (T.I) theca interna, and degenerative oocytes with vacuolization in ooplasm and granulosa cell layer enclosed by thin, irregular zona pellucida indicated by (z.p), 100 X. (C) Graffian follicle with micronucleus formation (a) and vacuolation of granulosa cell layer and ooplasm indicated by arrows 100 X. (D) Graffian follicle showing vacuolation in ooplasm and granulosa cell layer surrounded by disruptions and thinning of the zona pellucida 100 X. H & E stain.

10. DISCUSSION & CONCLUSION
Imbalance in the oxidant and antioxidant system leads to enhanced production of free radicals who have the ability to attack large molecules such as proteins and lipids and nucleic acids (DNA), micronuclei appearance is one the DNA damages shapes, leading to occur apoptosis, which results in a defect in the function of cells.
Theca interstitial cells are sensitive to free radicals' level and reduction in antioxidant triggers apoptosis and antioxidants with precisely different mechanisms of action stimulate a course of actions consistent with the apoptosis mechanism in an ovarian mesenchyme.
Our data suggest that the potential alteration of antioxidant capacity associated with excessive production of free radicals may contribute to ovarian oxidative damage, which could be related to the pathogenesis and progression of ovarian disorders leading to infertility under effect of radio-frequency electromagnetic radiation emitted from mobile phone.

11. References
Avci, Bahattin, Ayşegül Akar, Birşen Bilgici, and Özgür Korhan Tunçel “Oxidative Stress Induced by 1.8 GHz Radio Frequency Electromagnetic Radiation and Effects of Garlic Extract in Rats.” International journal of radiation biology 88(11): 799–805.
Dasdag, Suleyman et al “Effect of 900 MHz Radio Frequency Radiation on Beta Amyloid Protein, Protein Carbonyl, and Malondialdehyde in the Brain.” Electromagnetic Biology and Medicine 31(1): 67–74.
Gharib, Ola Ali, Usama Abdelnaby Abd Ellatif, Nadia Mohammad Abdellah, and Marwa Abdelhameed Mohammad “Radio-Protective Response on the Environmental Pollutant Induced Oxidative Stress.” Advances in Bioscience and Biotechnology 03(07): 989–96.
Halliwell1, B “Biochemistry of Oxidative Stress.” inflammation 35(5): 1147–50.
Kerman, Memduh, and Nilgun Senol “Oxidative Stress in Hippocampus Induced by 900 MHz Electromagnetic Field Emitting Mobile Phone : Protection by Melatonin.” Biomedical Research 23(1): 147–51.
Kismali, Gorkem et al “The Influence of 1800 MHz GSM-like Signals on Blood Chemistry and Oxidative Stress in Non-Pregnant and Pregnant Rabbits.” International journal of radiation biology 88(5): 414–19.
Mattsson, Mats-Olof, and Myrtill Simkà “Grouping of Experimental Conditions as an Approach to Evaluate Effects of Extremely Low-Frequency Magnetic Fields on Oxidative Response in in Vitro Studies.” Frontiers in Public Health 2(September): 1–11.
Ozgur, Elcin et al “Effects of Prenatal and Postnatal Exposure to GSM-like Radiofrequency on Blood Chemistry and Oxidative Stress in Infant Rabbits, an Experimental Study.” Cell biochemistry and biophysics 67(2): 743–51.
Poulletier de Gannes, Florence et al “Rat Fertility and Embryo Fetal Development: Influence of Exposure to the Wi-Fi Signal.” Reproductive toxicology 36: 1–5.
Tök, Levent et al “Effects of Melatonin on Wi-Fi-Induced Oxidative Stress in Lens of Rats.” Indian journal of ophthalmology 62(1): 12–15.
Yakymenko, Igor, Evgeniy Sidorik, Diane Henshel, and Sergiy Kyrylenko “Low Intensity Radiofrequency Radiation: A New Oxidant for Living Cells.” Oxid Antioxid Med Sci 3(1): 1–3.