cEcsr

d

Infertility

An Evaluation of the Effects ofLong-term Cell Phone Use on the TestesVia Light and Electron Microscope AnalysisSerkan Çelik, I. Atilla Aridogan, Volkan Izol, Seyda Erdogan, Sait Polat, andSaban Doran

OBJECTIVE To investigate whether the low-intensity electromagnetic waves transmitted by cell phones causehistopathological or ultrastructural changes in the testes of rats.

MATERIALS ANDMETHODS

Wistar-Kyoto male rats were placed into either a control group or a group that was exposed toan electromagnetic field (EMF). Two cell phones with Specific Absorbation Rate values of 1.58were placed and left off in cages that housed 15 rats included in the control group, and four cellphones were placed and left on in cages that housed 30 rats included in the experimental group.After 3 months, weights, seminiferous tubule diameters, and spermatogenic cell conditions of alltestes of the rats were evaluated. One half of each testis was examined also under an electronmicroscope.

RESULTS No significant differences were observed between the testis weights, seminiferous tubule diam-eters, and histopathological evaluations between rats that had and had not been exposed to EMF.Electron microscope analysis revealed that the membrana propria thickness and the collagenfiber contents were increased and the capillary veins extended in the experimental group.Common vacuolization in the cytoplasm of the Sertoli cells, growth of electron-dense structures,and existence of large lipid droplets were noted as the remarkable findings of this study.

CONCLUSION Although the cells that had been exposed to long-term, low-dose EMF did not present anyfindings that were contrary to the control conditions, the changes observed during ultrastructuralexamination gave the impression that significant changes may occur if the study period were tobe extended. Longer studies are needed to better understand the effects of EMFs on testis

tissue. UROLOGY 79: 346–350, 2012. © 2012 Elsevier Inc.

asWrEatbreac

Cell phones are increasingly being used in modernsociety. According to data obtained by the In-ternational Telecommunication Union (ITU) of

the United Nations, the number of cell phone users willexceed 5.5 billion by the end of 2011. This growth in cellphone use has prompted interest in understanding therelationship that electromagnetic fields (EMFs) havewith human health.1,2 Experimental studies have beenonducted to investigate this issue and have reported thatMFs inhibit the signal transmission between cells andould potentially cause genetic mutation and immuneuppression, in addition to adverse effects on the neu-oendocrine and reproductive systems.

The effects of cell phone-induced EMFs on the repro-uctive system and fetal development have attracted the

Funding Support: This study is supported by the Academic Research Projects Unit ofthe University of Çukurova (grant number TF2009LTP42).

From the Department of Urology, Department of Pathology, and Department ofHistology, University of Cukurova, Faculty of Medicine, Adana, Turkey

Reprint requests: Volkan Izol, M.D., University of Cukurova, Faculty of Medicine,

Department of Urology, 01330, Adana, Turkey; E-mail:volkanizol@yahoo.com

Submitted: July 20, 2011, accepted (with revisions): October 25, 2011

346 © 2012 Elsevier Inc.All Rights Reserved

ttention of researchers, and accordingly, the number oftudies investigating this issue has gradually increased.

hereas it has been asserted in some studies that theeproductive system of males is negatively affected byMFs, others have reported that EMFs have no effect orpartial effect on testicular tissues and function. Consis-

ent results have not been obtained in these studiesecause the frequency and amplitude of the applied cur-ent, the intensity of the induced magnetic field, and thexposure time have been different in each study, and thessociated effects of cell phones on the reproductive systemannot be accurately defined as a result.3-6

The fact that male individuals generally carry their cellphones in their pockets close to their testes in standbymode increases the importance of this study regarding theeffects of EMFs on the male reproductive system. Al-though laboratory studies have been conducted usinghigh-dose electromagnetic waves for short periods, it israre in daily life for a person to be exposed to high dosesof EMFs.7 Therefore, in this study, we focused on exam-ining the long-term effects of low-intensity EMF, and we

analyzed the functional, histopathological, and ultra-

0090-4295/12/$36.00doi:10.1016/j.urology.2011.10.054

structural changes caused by the electromagnetic wavesof cell phones on the testes of rats. In contrast to otherstudies in the literature, we examined testicular tissuesusing an electron microscope.

MATERIAL AND METHODS

A total of 45 Wistar Kyoto male rats, 4 to 5 months of age withbody weights between 300 and 400 g, were used for this study.The subjects were placed in standard rat cages in a controlledenvironment at Çukurova University, Medical Sciences Exper-imental Research and Implementation Centre. Each cage ac-commodated a maximum of 7 or 8 rats. All activities within thescope of the study were performed with the approval of theethics committee, under the supervision of a veterinarian and incompliance with the provisions of the Strasbourg UniversalDeclaration of Animal Rights of 1986. The rats were fed waterand pellet feeds that were specifically prepared for rodents.Veterinary and experienced personnel of the center removedany associated animal waste and supplied water and feed to theanimals.

ExposureThe rats were separated into 2 groups. Fifteen of the rats werekept as a control group in 2 cages, whereas the remaining 30 ratsconstituted the experimental group of the study and were keptin 4 cages. Two cell phones with SAR values of 1.58 wereplaced in cages that housed the 15 rats included in the controlgroup, and another four cell phones with SAR values of 1.58were placed in cages that housed the 30 rats that constitutedthe experimental group. In each cage 7 or 8 rats were accom-modated and the cell-phone exposure was equal for each ofthem. During the study, the cell phones that were placed in thecontrol group cages were left off, and the cell phones that wereplaced in the experimental group cages were kept on continu-ously.

At the end of the third month, the rats were intraperitoneallyadministered a combination of 6 mg/kg of 2% xylazine hydro-chloride (Rompun) and 75 mg/kg ketamine hydrochloride(Ketalar) for anesthesia. The skin, subcutaneous tissue, fascia,and peritoneum of the rats were opened with 3-cm incisions onthe lower abdomen. Afterward, the testes of each rat werelocated and carefully dissected from the surrounding tissues, andthe rats were killed by cervical dislocation.

Histopathological and Ultrastructural EvaluationDepending on the group and the order of excision, the prepa-rations were placed in vials that contained formaldehyde forhistopathological evaluation or vials that contained Bouin’ssolution for ultrastructural evaluation with an electron micro-scope. All vials were numbered.

Light Microscopy StudyFor the histopathological evaluation, 0.3-cm samples weretaken from the testis tissues, fixed in 10% formol, and placed incassettes. Bracketing was performed after the cassettes werepassed through the formol–alcohol–xylol–paraffin set. Histo-pathological samples (5 �m thick), which were obtained fromparaffin sections, were evaluated under a light microscope. Foreach sample, 50 randomly selected seminiferous tubule diame-

ters were measured via an Olympus Bx 51 light microscope at

UROLOGY 79 (2), 2012

10� magnification in combination with the Leica ApplicationSuite (image aquisition) software. In addition, for each section,50 randomly selected seminiferous tubules were evaluated usingthe Johnsen classification.8

Electron Microscopic StudyThe testis tissue sections for electron microscopy analysis wereconditioned in a 5% glutaraldehyde solution, which was pre-pared with Millonig’s phosphate buffer. Approximately 500–Å-thick incisions were examined under a Jeol JEM (1400) trans-mission electron microscope, and micrographs were obtained asa result. The propria, Sertoli cells, and cells included in thespermatogenic series and interstitium were evaluated.

Statistical AnalysisSPSS 18 statistical software was used to evaluate the dataobtained in this study. The distributions of the continuousvariables, testis weights and seminiferous tubule diameters inthe control and study groups were analyzed via Mann WhitneyU test. The distributions of the Johnsen tissue scorings in the 2groups were evaluated via the chi-square method. Differenceswere considered statistically significant when calculated P val-ues were less than 0.05.

RESULTSFor the purposes of this study, the excised testes wereweighed on an assay balance that had an 0.001-g accu-racy. The testis weights taken from the control rats werenot significantly different from the rats in the experimen-tal group (P � .94).

Similarly, there were no significant differences be-tween the seminiferous tubule diameters (P � .96) andJohnsen tissue scores (� � 0.05) of the testes obtainedfrom the 2 groups. With regard to spermatogenesis, theseminiferous tubules were observed to be nearly full inthe histopathological analysis of the preparations.

The electron microscopic changes were consistentamong all the exposed rats and no changes were detectedin the control group of rats. In the electron microscopeexamination performed on the control group, the semi-niferous tubule was observed to be complex and coveredwith a multilayer normal epithelium and included Sertolicells and spermatogenic cells (spermatogoniums, sper-matocytes, spermatids, and spermatozoons) (Fig. 1).

In the experimental group, clearly discernible increasesin the membrane propria thickness and collagen fiberamount were observed. The thickness and the collagenfiber amount of the membrane propria were evaluatedqualitatively. The nucleus had an irregular contour, withdeep invaginations. It was remarkable that the basallamina, which became thicker in some sections and dis-played an irregular and bilaminar structure, extendedprotrusions towards the seminiferous tubule (Fig. 2). Inaddition, electron-dense structures, mitochondria, andmicropinocytotic vesicles were observed to be present inthe cytoplasm of myoid cells, which became denser (Fig.2). Widespread vacuolization was associated with agranu-

lar endoplasmic reticulum cysterna widening in the cy-

347

Scs

d

toplasm of Sertoli cells, and an increased the number ofelectron-dense and cup-shaped mitochondria, an in-crease in the number of electron-dense structures andlarge lipid droplets were noted as other remarkable ob-servations (Fig. 3). The presence of tight junctions be-tween adjacent Sertoli cells was apparent. It was observedthat because of the contractions of spermatogonia on thebasal lamina, the distances between cells increased, andthe nuclei had irregular contours. Synaptonemal com-plexes were identified in the nuclei of primary spermato-cytes that had relatively normal appearances, whereas thedeveloped Golgi complexes and grouped mitochondriawith electron-dense appearances in the center of sper-matocytes were observed in the cytoplasm. Cytoplasmic

Figure 1. Control group. Normal membrane propria (MP),ertoli cells (S), spermatocytes (Spt), and synaptonemalomplexes (white arrow) are seen in the thin incision of theeminiferous tubule. Nucleus (N), mitochondrion (M). Bar �

0.5 �m.

Figure 2. Experimental group. Electron microscopic view ofthe seminiferous tubule. Thickness of the membrana pro-pria (MP) is observed to increase. Irregularity of the basallamina (BL) and its protrusion towards the tubule (arrow) areseen. Myoid cells (MH) are seen in addition to electron-dense coloring. The collagen fiber (COL) amount increases.The Sertoli cell nucleus has an irregular contour, with deep-ened invaginations (white arrow). Tight junctions (*) be-tween Sertoli cells (S) are normally observed. M � mitochon-rion; N � nucleus; Spg � spermatogonium. Bar � 1 �m.

bridges were also apparent for this group (Fig. 4). Fibro-

348

blasts, macrophages, plasma cells and mast cells withnormal structures and distributions were also found to bepresent in the interstitial tissues.

COMMENTIn addition to the environmental and noise pollutionproblems in contemporary society, electromagnetic pol-lution is increasingly becoming an issue. Especially inrecent years, the gradually increasing use of cell phoneshas increased human exposure to EMF levels that aremuch higher than those observed in nature.

Although this study was conducted for a longer periodcompared with previous studies in the literature, ourstudy revealed results that were similar to those of pre-

Figure 3. Experimental group. Electron microscopic view ofthe seminiferous tubule. Widespread vacuolization due tothe widening of the SER cysternas (black arrow), an in-creased incidence in electron-dense structures (white ar-row), and huge lipid droplets (*) and cup-shaped mitochon-dria (M) are observed in the cytoplasm of Sertoli cells (S). Aspermatocyte (Spt) in lysis and a cell in a mitotic state(arrowhead) are seen. Bar � 1 �m.

Figure 4. Experimental group. Electron microscopic view ofthe seminiferous tubule and membrana propria (MP). Theempty spaces around the cell periphery are observed inspermatogonia (Spg) because of contraction (black arrow).Well-developed Golgi complexes (white arrow) and groupedmitochondria (M). N � nucleus. Bar � 2 �m.

vious studies in terms of histopathological data. In many

UROLOGY 79 (2), 2012

e1tsmttsnfuaMi(a

lhrMisdeedtwsn

sdtpp

ew

fwsoo

r9at

Up

studies in the literature, testicular tissues were exposed tohigher intensity electromagnetic waves relative to whatan individual would encounter during his/her daily life.Thus, the ability of the results from those studies toaccurately depict the situation for the entire population iscontroversial. However, in our study, we conducted ul-trastructural analyses on testicular tissues that were ex-posed to EMFs using an electron microscope, and weidentified certain changes in the experimental groupcompared to the control group.

Electromagnetic waves are transmitted by devices thatproduce radio frequency waves, such as cell phones, andmay cause thermal and nonthermal effects in living or-ganisms. Thermal effects are defined as the conversion ofthe electromagnetic energy absorbed by the body intoheat and the resulting increase in body temperature(0.1°C in average); most likely, these effects can be easilycompensated for by normal mechanisms of the body.9

The studies conducted by Saunders and Kowalczuk10

and by Kowalczuk et al11 for determining the thermalffects of EMFs on murine tissues demonstrated that.7-GHz (50 mW/cm2) and 2.45-GHz waves may affecthe seminiferous epithelium, primary spermatocytes,perm count, and morphology in mice; however, electro-agnetic waves transmitted by cell phones cause non-

hermal effects in living organisms when the inducedemperature increase does not exceed 0.1°C.12 In thistudy, we examined the biological effects of electromag-etic fields. EMFs are known to cause hyperthermic ef-

ects when the amount of energy absorbed in 1 second pernit mass (kg) of the body exceeds 4 W in 1 hour. Inddition, EMFs in the frequency range of 300 GHz to 10Hz are known to have nonthermal effects on organ-

sms. Thus, we preferred to apply an EMF of 1.8 GHz1.58 W/kg SAR) because there should not be a notice-ble temperature rise during exposure.

Although Hecht and Balzer13 have stated that theong-term effects of EMF has been reported from 200ours up to 20 years, the exact duration of EMF exposureequired to induce long-term effects is still not known.

ost of the activities that are related to reproduction andmplantation, including meiosis and impregnation, areensitive to toxic effects. High rates of cell division andifferentiation in developing fetuses and seminiferouspithelia make them more sensitive to such effects; how-ver, in our study, we did not observe any apparentegeneration in the seminiferous epithelium in the his-opathological examination. Although spermiogenesisas observed to be normal, we found some abnormal

perm morphologies in the electron microscope exami-ations of the group that was subjected to EMF.Salama et al7 evaluated changes in rectal temperature,

perm count, sperm mobility and seminiferous tubuleiameter in rabbits that had been exposed to cell phoneshat were in the standby position for 8 hours a day for aeriod of 12 weeks, and they observed decreases in all the

arameters. Similarly, after subjecting rats to 900-MHz

UROLOGY 79 (2), 2012

lectromagnetic waves for 30 minutes per day, 5 days aeek, for a period of 4 weeks, Özgüner et al14 did not

observe any significant changes in the testis weights,germinal epithelium thicknesses, Johnsen biopsy scores,and FSH and LH levels of the subjects, whereas theyreported a decrease in the seminiferous tubule diameterand an increase in the amount of serum testosterone.Although they used a very short study period, they iden-tified changes in the seminiferous tubule diameters,which differ from the findings reported in this study.Despite the fact that we used a study period that wassimilar to that of Salama et al,7 we did not observe asignificant change in seminiferous tubule diameter.

Ji Yoon et al15 subjected male rats to 2.45-GHz EMFsor 8 weeks and reported no significant changes in testiseights, seminiferous tubule diameters, sperm counts,

perm morphologies, and LH-FSH values; however, theybserved increases in Leydig cell number and testoster-ne amounts.The apoptosis effect of EMFs on the testicular tissues of

ats was also evaluated. The rats were exposed to a00-MHz EMF for 2 hours a day for 10 months, andpoptosis was determined not to be induced by thisreatment.16

Because of their study on rats subjected to 1.3-GHz(6.3 W/kg SAR) and 1.3-GHz (9 W/kg SAR) micro-waves, Lebovitz and Johnson17 showed that there was nochange in daily sperm production, the number of epidid-ymal sperm, sperm morphology, and testis weight andfunction. Other similar studies did not report significantchanges in these parameters.18,19 All of the previousworks support the results obtained in our study.

CONLUSIONSIn this study, we did not obtain any data providingevidence that the cells exposed to low-dose EMF for along period display extraordinary characteristics; how-ever, the changes observed in the ultrastructural exami-nation gave us the impression that significant changesmay take place during a longer study period. This periodshould be long enough for the rats to breed, as this mayshow us the fertility or infertility of the rats after they areexposed to EMF. Additional studies using longer periodswith quantitative measurements of the electron micro-scopic changes, the lack of which was a limitation of thisstudy, are needed to clearly identify any clinical changesthat are caused by the effects of EMFs on testicular tissue.

Acknowledgments. The authors thank the staff of Çukurovaniversity, Medical Sciences Experimental Research and Im-

lementation Centre.

References1. Sage S. An overview of radiofrequency/microwave radiation studies

relevant to wireless communication and data. In: Proceedings ofthe International Conference on Cell Tower Sitting. Linking Sci-

ence & Public Health, Salzburg, 2000, 73-90.

349

2. Fejes I, Závaczki Z, Koloszár S, et al. Hypothesis: Safety of usingmobile phones on male fertility. Arch J Androl. 2007;53:105-106.

3. Dasdag S, Akdag MZ, Aksen F, et al. Whole body exposure of ratsto microwaves emitted from a cell phone does not affect the testes.Bioelectromagnetics. 2003;24:182-188.

4. Desai NR, Kesari KK, Agarwal A. Pathophysiology of cell phoneradiation: oxidative stress and carcinogenesis with focus on malereproductive system. Reprod Biol Endocrinol. 2009; 7:114-123.

5. Yan JG, Agresti M, Bruce T, et al. Effects of cellular phoneemissions on sperm motility in rats. Fertil Steril. 2007;88:957-964.

6. Schoenwolf G, Bleyl S, Brauer P, et al. Urogenital system. In:Churchill L, ed. Larsen’s Human Embryology. New York: Elsevier;2009:479-542.

7. Salama N, Kishimato T, Kanayama HO. Effect of exposure to amobile phone on testicular function and structure in adult rabbit.Int J Androl. 2010;33:88-94.

8. Johnsen SG. Testicular biopsy score count-a method for registra-tion of spermatogenesis in human testes: normal values and resultsin 335 hypogonadal males. Hormones. 1970;1:2-25.

9. Van Leeuwev GM, Lagendijk JJ, Van Leersum BJ, et al. Calcula-tion of chance in brain temperatures due to exposure to a mobilephone. Phys Med Biol. 1999;44:2367-2379.

10. Saunders RD, Kowalczuk CI. Effects of 2.45 GHz microwave radi-ation and heat on mouse spermatogenic epithelium. Int J Radiat Biol

Relat Stud Phys Chem Med. 1981;40:623-632.

350

11. Kowalczuk CI, Saunders RD, Stapleton HR. Sperm count andsperm abnormality in male mice after exposure to 2.45 GHz mi-crowave radiation. Mutat Res. 1983;122:155-161.

12. Dewhirst MW, Lora-Michiels M, Viglianti BL, et al. Carcinogeniceffects of hyperthermia. Int J Hyperthermia. 2003;19:236-251.

13. Hecht K, Balzer HU. Biological Effects of Electromagnetic Fields onHumans in the Frequency Range of 0 to 3. Berlin: Institut fürStressforschung; 1997:1-4.

14. Özgüner M, Koyu A, Cesur G, et al. Biological and morphologicaleffects on the reproductive organ of rats after exposure to electro-magnetic field. Saudi Med J. 2005;26:405-410.

15. Ji Yoon K, Hyun Tae K, Ki Hak M, et al. Long-term Exposure ofRats to a 2.45 GHz electromagnetic field: effects on reproductivefunction. Korean J Urol. 2007;48:1308-1314.

16. Dasdag S, Akdag MZ, Ulukaya E, et al. Mobile phone exposuredoes not induce apoptosis on spermatogenesis in rats. Arch Med Res.2008;39:40-44.

17. Lebovitz RM, Johnson L. Acute, whole body microwave exposureand testicular function of rats. Bioelectromagnetics. 1987;8:37-43.

18. Cairnie AB, Harding RK. Cytological studies in mouse testis irra-diated with 2.45 GHz continuous wave microwaves. Radiat Res.1981;87:100-108.

19. Gurisik E, Warton K, Martin DK, et al. An in vitro study of theeffects of exposure to a GSM signal in two human cell lines:monocytic U937 and neuroblastoma SK–N–SH. Cell Biol Int. 2006;

30:793-799.

UROLOGY 79 (2), 2012