Effect of Wi-fi Radiations on Sperms in Vitro

Effect of Wi-fi Radiations on Sperms in Vitro SPERM DNA FRAGMENTATION AND ROS. Omkar Pokharkar, Himanshu Patel, Vidisha Bhatt . ABSTRACT: All around the globe, studies are conducted to determine the effect of Wi-Fi on the quality of sperms both in vivo and in vitro. To determine the level of damage to the sperms in vitro, sperm chromatin dispersion test along with semen analysis was performed to map the motility, vitality, morphology and also the fragmentation in the sperms which are exposed and which are not exposed to radiations emitted by Wi-Fi. It was found after exposing sperm samples to Wi-Fi for long durations in a closed cabin placing sperm samples near Wi-Fi router can affect sperm quality to a great extent, reducing motility of sperms and causing DNA fragmentations in sperms. And unexposed samples were in better condition both in terms of motility and fragmentation. This study proved the ill effects of using Wi-Fi on laptops and mobiles on sperms in vitro. Keywords: Sperms, in vitro, fragmentation, DNA, motility, vitality, Wi-Fi radiations, ROS. INTRODUCTION: Wi-Fi radiations from laptops and mobiles can impair or damage sperms. Motility of the sperms are said to be reduced due to prolong exposure of sperms to radiations emitted by Wi-Fi (Wireless fidelity). This study was carried out to verify that radiations affect sperm motility and damages the DNA causing fragmentation. This experiment is to note the motility, vitality and levels of DNA damage after exposing sperm samples to Wi-Fi for certain period of time. DNA fragmentation is a way to accurately map the sperms with fragmented and non fragmented DNA. Sperms with fragmented DNA disperse no halos and sperms with non fragmented DNA disperse big halos and degrading sperms show small halos. If the radiation from Wi-Fi affects motility, vitality and DNA of spermatozoa it would be revealed after performing standard semen analysis according to the criteria set by WHO[1] and Sperm chromatin dispersion test (SCD). MATERIALS AND METHODS: For this type of study semen samples from 12 fertile men with no recent history of illness aged 22-29 were obtained in wide mouthed collecting jars during the period of sexual abstinence of 3 days. Each of the 12 Sperm samples were washed by swim up method which has high rate of success in obtaining viable sperms, for removal of debris and dead or immotile sperms and only motile sperms were used. This was to make sure that before exposing the samples to Wi-Fi there were all live sperms with good motility and no pre dead or immotile sperms were present to map accurate changes due to radiations. All 12 samples were subjected to semen analysis as per the criteria of world health organization. Motility, vitality, morphology, and ph was observed and noted down [1], this was done before exposing sperms to radiations. These results were compared to the results obtained after radiation exposure. Motility was calculated by using a formula: 100 X (number of motile spermatozoa)/ (total number o f spermatozoa counted). More than 400 spermatozoa per ejaculate were evaluated for estimation of motility. Each 12 Sperm samples were divided in 3 aliquots of 0.5 ml each and out of three aliquot, 2 aliquots were exposed to Wi-Fi radiations for different periods of time, 1st aliquot was exposed to radiation for 1 ½ hour and second aliquot was exposed for 3 hours. These 2 aliquots were labeled as test and one aliquot was considered as control sample and was kept in different room to avoid any radiations or other factors which would influence sperms. The 0.5ml aliquots of sperm samples were placed in tubes. After exposure of Wi-Fi radiations to sperms the motility, morphology, vitality and ph was observed again and results were recorded. The results from pre exposure and post exposure of sperms to radiations were compared later. The samples were exposed to radiations by keeping the samples in a closed cabin near the router of the Wi-Fi and several laptops and mobiles were kept in cl ose proximity with Wi-Fi enabled also the laptops were downloading and uploading data continuously during the time of exposure to max the radiations[2]. The samples were placed in close range of all laptops, router and mobiles; the distance between samples and Wi-Fi sources was about 1-2 inches. DNA FRAGMENTATION TEST (SCD): Then DNA fragmentation or Sperm chromatin dispersion test was carried out on all samples to check the percentage of DNA fragmentations in samples exposed for 90 minutes (1 ½ hour) and samples exposed for 180 minutes (3 hours) and these outcomes were compared with control samples (sample which was not subjected Wi-Fi or any other factor which will affect sperm). The solidified agarose gel tubes were boiled in water using the float at about 90 0C – 100 0C for 2 minutes so that the gel in the tube liquefies and then cool down the tubes at 37 0C for 5 minutes. Then 40  µ liter of semen sample from control samples was added and mixed with liquefied agarose gel tube, similarly 40  µ liter of semen sample from first test sample (Wi-Fi for 90 minutes) was obtained and was mixed with second agarose gel eppendroff. Again 40  µ liter of sample from second test sample (Wi-Fi for 180 minutes) was extracted and mixed with third agarose gel eppendroff. These 3 tubes represent the con trol and test sample suspensions respectively. Three pre coated slides were used to study and compare between 1 control sample (not exposed to Wi-Fi) and 2 test samples with varying periods of exposure to radiations. Then 150  µ liter of suspension from control tube was obtained with micropipette and placed on the coated slide and was covered with a cover slip. Similarly 150  µ liter of suspension from first test sample was obtained with micropipette and placed on second slide and was covered with cover slip. Again 150  µ liter of suspension from second test sample was obtained and placed on the third coated slide. These 3 slides were prepared simultaneously, air bubbles were avoided and the slides were transferred to a fridge to maintain the temperature around 40C–80C for 5 minutes. This step helps in solidification of gel on the slide. Then after 5 minutes, slides were obtained from fridge and the cover slips from the 3 slides were removed carefully such that gel inte grity is not affected. Then the slides were placed on even surface and was overlaid with 1 ml of acid denaturant each and was incubated at 22 0C for about 7 minutes and the solution was drained completely after 7 minutes. Then next step was to overlay 1 ml of lysis solution each on all 3 slides and was incubated for 20 minutes at room temperature. After 20 minutes the lysis solution was drained completely. Lysis solution possesses a pungent odor. Then all 3 slides were washed in slanting position with 20 ml distilled water with help of syringe or a dropper. In the next step all the 3 slides were sequentially dehydrated using dehydrating solution 1, 2, and 3 provided in the kit. Then the slides were allowed to air dry for few minutes. In this time period working stain was prepared using stain solution and stain dilution buffer. Working Stain was prepared by taking 400  µ liter of stain solution and mixing it with 100  µ liter of stain dilution buffer in a dilution tube. So for 3 slides the stain was prepared 3 times. This working stain must be used within 1 hour of preparation. After air drying all 3 slides, 200 -300  µ liter of working stain was overlaid each on all 3 slides representing control and test slides respectively. Then the slides were rocked by tilting in to and fro directions for 3 minutes to maintain even distribution of stain over the slide. After 3 minutes the slides were washed by dipping and moving in a couplin jar or a beaker filled with tap water. Then the slides were kept in slanting position to air dry. This marks the end to the procedure for making sperm DNA fragmentation slides of both control and test samples. Sperm DNA fragmentation was calculated by formula: 100 X (Number of spermatozoa with fragmented DNA)/ (Total number of spermatozoa counted) [3]. More than 500 spermatozoa per ejaculate were evaluated for estimation of sperm DNA fragmentation [3]. This study took around 25 days for completion (6th January – 31st Januar y, 2015). RESULTS: Normal Semen analysis of 12 samples before exposing it to Wi-Fi radiations showed motility percentage around (72  ± 4.18) and after exposure to radiations for 90 minutes the motility rate decreased to about (65  ± 3.2) and the samples exposed to 180 minutes showed further decrease in motility percent which ranged (56  ± 2.89). Similarly vitality percentage of all 33 sperm samples before exposure was about (71  ± 4.07) and after exposure for 90 minutes it shifted to (61  ± 5.78). And also samples exposed for 180 minutes indicated a decrease in vitality ranging from (48  ± 7.98). Morphology defects due to radiations were prominent when compared to the samples not exposed to Wi-Fi. Defective mid piece and several head defects were significant in test samples. On the other hand ph was not affected by radiations and was in the range of 7.2 -8.0 for both test and control samples before and after exposure. Table I. showing differences in sperm motility as the time duration of radiation exposure increased: I. A chart showing motility percentages Table II. Showing differences in sperm vitality as the time duration of radiation exposure increases: II. A chart showing vitality percentages. The principle of DNA fragmentation test lies in dispersion of a characteristic halos, which indicates the status of the spermatozoa. If a big halo is dispersed then the DNA of the sperm is not fragmented. On the other hand when small halo is dispersed by a sperm then it is on the verge of fragmentation/degradation and also when no halo is dispersed by sperm then it is a sign of fragmented DNA or the entire sperm is degraded [4]. DNA fragmentation test also known as sperm chromatin dispersion test was carried out for all 12 samples and the slides were observed under bright field microscope, it was revealed that the samples exposed to Wi-Fi radiations showed high percentages of DNA fragmentation. Whereas Sperm samples unexposed to Wi-Fi radiations showed fewer fragmentations. Fragmentation percentages of unexposed samples were in the range of (10  ± 5.11).Test samples exposed to Wi-Fi radiations for 90 minutes revealed (23  ± 6.1) of DNA fragmentations. On the other hand, samples e xposed to Wi-Fi radiations for 180 minutes showed (34  ± 7.04) of DNA fragmentation. Table III. Showing levels of DNA fragmentations caused due to Wi-Fi radiations: III. A chart showing percentage of DNA fragmentation in sperms. CONCLUSION: From the study conducted above it was discovered that Wi-Fi radiations had detrimental effects on sperm motility, vitality and morphology. Semen analysis of 12 samples proved that sperm motility was greatly reduced as the time of exposure to Wi-Fi radiations was increased. Morphological defects in head and mid piece were noticeable after comparing exposed and unexposed samples using bright field microscope and staining the slides with Giemsa stain. Analysis and calculations of DNA fragmentation caused by radiations revealed that the percentage of fragmentation also increased with increase in time of exposure to Wi-Fi. So from the findings mentioned above it was clear that Wi-Fi radiations from laptops and mobiles could cause damaging effects to sperms and could degrade their ability to stay motile and fertilize the egg. DISCUSSION: There is a rapid progress in fields such as electronics and telecommunication, computers etc making mankind smart and advance. First programmable computer was made by Charles Babbage in 1830’s. And later giant computer named â€Å"ENIAC† was built by John W. Mauchly and J. Presper Eckert at the University of Pennsylvania. Now in 21st century with advancement in technology, a new era of computers and laptops have emerged which are much smaller in size and are portable with wireless fidelity also known as Wi-Fi .It was long known that Wi-Fi radiations had the potential to impair sperm’s ability to function properly and could reduce the fertility of men habituated to use laptop and mobile Wi-Fi for long periods of time and almost every day. This experiment was a recreation of the situation in which sperms get exposed to radiations when an individual works on his laptop or mobile with Wi-Fi enabled. However, this was an artificial way to map the effects of radiations on sperms in vitro. In in vivo, effects of radiations must be much different than the results obtained when performed in vitro. The layers of testes acts as protective covering which absorbs much of the radiations and shields sperms from the devastating effects of radiations but only to some extent where as in in vitro there was not enough biological protection to sperm samples and were little more susceptible to the radiations. The samples placed in tubes were exposed to radiations by keeping the sample tubes in a closed cabin near the router of the Wi-Fi and several laptops (hp, dell and Acer) and mobiles (NOKIA, Samsung) were kept in close proximity (1-2 inches) with Wi-Fi enabled (3G) and also the laptops were downloading and uploading data continuously during the time of exposure to max out the radiations [2]. Samples were not placed beneath the laptop to avoid getting a combined effect of heat and Wi-Fi radiations on sperms because the study was purely about radiations and no t heat + radiations. However, keeping samples beneath laptop with Wi-Fi would mimic a person keeping laptop on his lap. But other factors such as heat would also contribute in DNA fragmentation which was undesirable in this study. It was possible to place the samples beneath the laptop with air conditioner turned on to maintain the temperature under the laptop as equal to room temperature (25-27 0C) even if the laptop generates heat [2]. But In this study, we completely eliminated the chance or risk of other factors which would influence the results. The study was focused only on effect of radiations. The results of this study were quite intimidating. From studies conducted around the world it is scientifically proven that laptops generating heat and Wi-Fi generating radiations for longer periods of time can affect fertility of men and mobiles or cell phones will do the same.[2][5][6][7][8][9][10]. So it is advisable to minimize the time span for usage of Wi-Fi and the connection mu st be disabled/turned off when not in use avoiding radiations and also laptops must be kept on working desks or tables during the use, avoiding the excess heat emitted by laptops which comes in close contact with testes or pelvis area. AKNOWLEDGEMENT: This material is based on the work supported by the Stem cure private ltd, centre for reproductive medicine and stem cell development, Ahmedabad, Gujarat, India; authors want to say thanks to, Mrunmayee Pande and Ramesh Parmar for assistance, Madhuri Patel for helpful input, Sejal Patel for technical support. Words are inadequate in offering thanks to the entire team of stem cure for encouragement and co-operation in carrying out the research and allowing the use of lab facilities. Authors would also like to thank D.Y. Patil University, school of biotechnology and bioinformatics, CBD Belapur, Navi Mumbai, India. REFERENCES: [1] WHO. (1999). Laboratory manual for examination of human semen and spermcervical mucus interactions (4 ed.). Cambridge, East Anglia, United kingdom: Canmbridge university press. [3] Akira Komiya, T. K. (2014, august 4). Clinical Factors Associated with Sperm DNA Fragmentation in Male Patients with Infertility. (A. E. Calogero, Ed.) The scientific world journal, 2014, 11.