Studies on EMR and Male Infertility

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Within the last decade, there have been several well-designed and executed studies on the effects of mobile phone radiation on spermatozoa, covering three main areas, clinical/epidemiological, human (in vitro) and in vivo animal models. The association between RF-EMR and male infertility was initially suggested by an epidemio-logical study in 2001 where it was speculated that differences in fertility parameters from Chinese males in various professions may be due to EMR exposures [52] . More pertinent epidemiological data from a study in 2005 found negative correlations between mobile phone usage and various attributes of semen quality, particularly motility [53] . Studies on male reproduction in mouse or rat models showing that mobile phone radiation affected testicular histology, including decreased seminiferous tubule diameter, appeared in 1999 [ 54]. but wasn't investigated further until 2003 [55] and 2004 [56] with the effects of low frequency EMR on murine models. There were some conflicting data within these two latter studies; however, both indicated that testis histology was altered in the exposed group. The former study also showed a decrease in testis size, while the latter did not. The latter study did, however, show one of the first instances of DNA damage (fragmentation within spermatogonia only) in the male germ line after mobile phone exposure.

This work was immediately followed by an experimental study involving exposure of male mice to RF-EMR via a wave guide. Exposures were at a frequency of 900 MHz at 90 mW kg-1 for 12 h day-1 for 7 days. This study revealed a significant impact on the integrity of the sperm mitochondrial genome, but no effect on the nuclear DNA or microscopic parameters [57], somewhat confirming the detrimental effects of RF-EMR on DNA integrity. Then further negative impacts of mobile phone usage on semen quality in human males were observed in a study that found significant reductions in sperm motility after exposure to a mobile phone after only 5 min exposure (talk mode) at a 10 cm range in vitro [58]; shortly after, a study also reported losses of motility and vitality after mobile phone exposure for 6 h day-1 for 18 weeks on rats held 1 cm from mobile phones measured at an SAR ranging from 0.9 to 1.8 W kg-1 in standby and talk modes, respectively. The spermatozoa of animals exposed to mobile phones also exhibited an up-regulation of CAD-1 and ICAM-1 RNA levels [59] (proteins associated with cell adhesion).

In three elegant studies, some concerning links with mobile phone use and male infertility were presented. Fejes et al. [53] found over 371 men that the duration of possession and use of mobile phones negatively correlated with semen quality. Wdowiak et al. [60] similarly showed that lower motility, vitality and poor morphology correlated with the frequency of mobile phone use over the 304 men studied. Agarwal et al. [61] confirmed these findings showing that sperm cell motility, vitality, normal morphology as well as sperm counts were defective in men who used mobile phones more frequently [61]. Also within this earlier time frame of research, various researchers proposed that the RF-EMR also exerts a range of negative genotoxic effects on different cell types including mature sperm cells [57, 62, 63] . These effects include chromatid exchange, aneuploidy and defective chromosome recombination. The "real world" clinical significance of this body of work was not confirmed; however, these studies were greatly important in providing a platform for continuing high-quality research and focusing effort into the potential harmful effects of mobile phone use. The work in this field up until 2007 is reviewed by Deepinder et al. [64].

From 2008 to the present (2011), several additional studies have shown adverse effects; in a clinical setting, in model systems and studies on human spermatozoa in vitro. Importantly, some studies have begun to shed light on how RF-EMR may drive the adverse effects observed by some researchers in the past. Nevertheless, this latest period of research has also generated several studies showing no detrimental effects of RF-EMR or mobile phone use.

Despite several groups reporting no effects of RF-EMR on male reproduction, other groups with very similar experimental design have reported a common finding of lower motility [65]. Some groups also showed that signs of oxidative stress in the exposed cohort, where increases in markers such as 8-OH-dG [31] and lipid peroxidation and the reduction of antioxidant levels [65], were present in human sperm in vitro, as well as in murine models. One study reported an increase in testicular sperm count in the rat after 1.95 GHz cellular phone radiation with a SAR or 0.080.4 W kg-1 after 5 h day-1 for 5 weeks [66]. A further study in the rat using exposures of 90 min day-1, 5 days week-1 for 12 weeks and using 848.5 MHz frequency at an SAR of 2.0 W kg-1 found no changes compared to controls in testis histology or several other markers including lipid peroxidation, expression of p53, bcl2 or cas-pase [67], again exemplifying the range of conflicting data.

A detailed human (in vitro) study was completed by our research group in 2009 [31]. Our aims were to uncover a chain of cause and effect from RF-EMR radiation to the resulting motility and vitality loss and increased levels of DNA damage. We completed this by exposing purified human spermatozoa in a waveguide in a power-dependant fashion (0.4-27.5 W kg- 1 at 1.8 GHz). In step with increasing SAR, motility and vitality were significantly reduced, while the mitochondrial generation of reactive oxygen species and DNA fragmentation were significantly elevated (P < 0.001). Furthermore, we also observed highly significant relationships between SAR, mitochondrial ROS levels, the oxidative DNA damage bio-marker, 8-OH-dG, and DNA fragmentation after RF-EMR exposure. This study has identified that RF-EMR may interact with the mitochondria in the sperm mid-piece, which then

Fig. 1.2 Proposed oxidative stress model of the effects of mobile phone frequency radiation on the human spermatozoon. Evidence suggests that, like several other factors, radio frequency electromagnetic radiation (RF-EMR) can induce a non-thermal oxidative stress response in the gamete, possibly through interactions with NAD(P)H oxidases in the plasma membrane or by perturbation of mitochondria. This stress then leads to the range of adverse effects commonly observed under experimental conditions

Fig. 1.2 Proposed oxidative stress model of the effects of mobile phone frequency radiation on the human spermatozoon. Evidence suggests that, like several other factors, radio frequency electromagnetic radiation (RF-EMR) can induce a non-thermal oxidative stress response in the gamete, possibly through interactions with NAD(P)H oxidases in the plasma membrane or by perturbation of mitochondria. This stress then leads to the range of adverse effects commonly observed under experimental conditions leads to ROS generation and a state of oxidative stress. This stress manifests in a loss of motility and vitality (through lipid peroxidation) and the presence of oxidative DNA damage and DNA strand breaks in the nucleus. Our conclusion from this study was that RF-EMR in both the power density and frequency range of mobile phones enhances mitochondrial reactive oxygen species generation by human spermatozoa, decreasing the motility and vitality of these cells while stimulating DNA base adduct formation and, ultimately DNA fragmentation. This study shed light on a potential mechanism by which "real-life" mobile phone radiation may affect biology. These findings confirm other published data that RF-EMR can indeed impact the male germ line and further that extensive mobile phone use may have negative impacts on males of reproductive age, potentially affecting both their fertility and the health and wellbeing of their offspring. This work was then supported by Agarwal et al. [68], showing that human sperm in vitro suffered the same oxidative stress by and increase in ROS levels and a decrease in the total antioxidant capacity of the cells after exposure to a mobile phone for only 1 h. Whereas we hypothesis that the source of ROS is the sperm mitochondria, Agarwal et al. suggest that NADH oxidase on the plasma membrane is responsible. Nonetheless, there is growing confidence that ROS has a key role to play in the potential mechanism of adverse effects of RF-EMR on the male germ line (Fig. 1.2). The relationship of oxidative stress to the detrimental effects of mobile phone use is also reviewed in this recent article [69].

Certainly in the last 6 years, several papers have implicated ROS as the mediator for RF-EMR-based cellular damage [65] . This hypothesis is not confined to the germ line, where similar studies in other cell types also seem to conform to this proposed pathway [ 70, 71] . Several studies have demonstrated that antioxidant molecules such as melatonin, caffeic acid, phenyl ester, vitamin C and E have some protective or preventive effect against oxidative stress caused by RF-EMR [49, 50, 72]. More recent reports have also found similar results with other types of antioxidants including catechins (from green tea), ^-acetylcysteine [73], again strengthening the hypothesised role of ROS in the effects of RF-EMR on the body.

Besides some conformity and progress of our knowledge in this field, we cannot ignore the controversy in many findings. The roots of this disparity may arise from two main factors, firstly, the intention of the study, and secondly, the experimental setup. There are a fraction of studies within the field which attempt to obtain mechanistic information and the molecular consequences of RF-EMR on spermatozoa and male reproduction; this focus, in part, removes itself from clinical relevance, however is key to establishing a causative effect and may therefore resolve some of these inconsistencies. Understanding its aetiology and resolving any potential adverse health effects of this radiation is a major priority. The second aspect, the experimental design, is particularly difficult to address at this point in time as the interactions between RF-EMR and biology are complex and generally not well understood. Further to this, as with various other human studies, clinical investigation into RF-EMR is extremely difficult to control.

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