Effects of radiofrequency electromagnetic field (RF-EMF) exposure on male fertility: A systematic review of experimental studies on non-human mammals and human sperm in vitro.

BACKGROUND: The World Health Organization is coordinating an international project aimed at systematically reviewing the evidence regarding the association between radiofrequency electromagnetic field (RF-EMF) exposure and adverse health effects. Reproductive health outcomes have been identified among the priority topics to be addressed. OBJECTIVES: To evaluate the effect of RF-EMF exposure on male fertility of experimental mammals and on human sperm exposed in vitro. METHODS: Three electronic databases (PubMed, Scopus and EMF Portal) were last searched on September 17, 2022. Two independent reviewers screened the studies, which were considered eligible if met the following criteria: 1) Peer-reviewed publications of sham controlled experimental studies, 2) Non-human male mammals exposed at any stage of development or human sperm exposed in vitro, 3) RF-EMF exposure within the frequency range of 100 kHz-300 GHz, including electromagnetic pulses (EMP), 4) one of the following indicators of reproductive system impairment:Two reviewers extracted study characteristics and outcome data. We assessed risk of bias (RoB) using the Office of Health Assessment and Translation (OHAT) guidelines. We categorized studies into 3 levels of overall RoB: low, some or high concern. We pooled study results in a random effects meta-analysis comparing average exposure to no-exposure and in a dose-response meta-analysis using all exposure doses. For experimental animal studies, we conducted subgroup analyses for species, Specific Absorption Rate (SAR) and temperature increase. We grouped studies on human sperm exposed in vitro by the fertility status of sample donors and SAR. We assessed the certainty of the evidence using the GRADE approach after excluding studies that were rated as "high concern" for RoB. RESULTS: One-hundred and seventeen papers on animal studies and 10 papers on human sperm exposed in vitro were included in this review. Only few studies were rated as "low concern" because most studies were at RoB for exposure and/or outcome assessment. Subgrouping the experimental animal studies by species, SAR, and temperature increase partly accounted for the heterogeneity of individual studies in about one third of the meta-analyses. In no case was it possible to conduct a subgroup analysis of the few human sperm in vitro studies because there were always 1 or more groups including less than 3 studies. Among all the considered endpoints, the meta-analyses of animal studies provided evidence of adverse effects of RF-EMF exposure in all cases but the rate of infertile males and the size of the sired litters. The assessment of certainty according to the GRADE methodology assigned a moderate certainty to the reduction of pregnancy rate and to the evidence of no-effect on litter size, a low certainty to the reduction of sperm count, and a very low certainty to all the other meta-analysis results. Studies on human sperm exposed in vitro indicated a small detrimental effect of RF-EMF exposure on vitality and no-effect on DNA/chromatin alterations. According to GRADE, a very low certainty was attributed to these results. The few studies that used EMP exposure did not show effects on the outcomes. A low to very low certainty was attributed to these results. DISCUSSION: Many of the studies examined suffered of severe limitations that led to the attribution of uncertainty to the results of the meta-analyses and did not allow to draw firm conclusions on most of the endpoints. Nevertheless, the associations between RF-EMF exposure and decrease of pregnancy rate and sperm count, to which moderate and low certainty were attributed, are not negligible, also in view of the indications that in Western countries human male fertility potential seems to be progressively declining. It was beyond the scope of our systematic review to determine the shape of the dose-response relationship or to identify a minimum effective exposure level. The subgroup and the dose-response fitting analyses did not show a consistent relationship between the exposure levels and the observed effects. Notably, most studies evaluated RF-EMF exposure levels that were higher than the levels to which human populations are typically exposed, and the limits set in international guidelines. For these reasons we cannot provide suggestions to confirm or reconsider current human exposure limits. Considering the outcomes of this systematic review and taking into account the limitations found in several of the studies, we suggest that further investigations with better characterization of exposure and dosimetry including several exposure levels and blinded outcome assessment were conducted. PROTOCOL REGISTRATION: Protocols for the systematic reviews of animal studies and of human sperm in vitro studies were published in Pacchierotti et al., 2021. The former was also registered in PROSPERO (CRD42021227729 https://www.crd.york.ac.uk/prospero/display_record.php?RecordID = 227729) and the latter in Open Science Framework (OSF Registration DOI https://doi.org/10.17605/OSF.IO/7MUS3).

Effects of Radiofrequency Electromagnetic Field (RF-EMF) exposure on pregnancy and birth outcomes: A systematic review of experimental studies on non-human mammals.

BACKGROUND: The World Health Organization is coordinating an international project aimed at systematically reviewing the evidence regarding the association between radiofrequency electromagnetic field (RF-EMF) exposure and adverse health effects. Within the project, 6 topics have been prioritized by an expert group, which include reproductive health outcomes. OBJECTIVES: According to the protocol published in 2021, a systematic review and meta-analyses on the adverse effects of RF-EMF exposure during pregnancy in offspring of experimental animals were conducted. METHODS: Three electronic databases (PubMed, Scopus and EMF Portal) were last searched on September 8 or 17, 2022. Based on predefined selection criteria, the obtained references were screened by two independent reviewers. Studies were included if they met the following criteria: 1) original, sham controlled experimental study on non-human mammals exposed in utero, published in peer-reviewed journals, 2) the experimental RF-EMF exposure was within the frequency range 100 kHz-300 GHz, 3) the effects of RF-EMF exposure on fecundity (litter size, embryonic/fetal losses), on the offspring health at birth (decrease of weight or length, congenital malformations, changes of sex ratio) or on delayed effects (neurocognitive alterations, female infertility or early-onset cancer) were studied. Study characteristics and outcome data were extracted by two reviewers. Risk of bias (RoB) was assessed using the Office of Health Assessment and Translation (OHAT) guidelines. Study results were pooled in a random effects meta-analysis comparing average exposure to no-exposure and in a dose-response meta-analysis using all exposure doses, after exclusion of studies that were rated at "high concern" for RoB. Subgroup analyses were conducted for species, Specific Absorption Rate (SAR) and temperature increase. The certainty of the evidence was assessed using the Grading of Recommendations, Assessment, Development and Evaluations (GRADE) approach. RESULTS: Eighty-eight papers could be included in this review. Effects on fecundity. The meta-analysis of studies on litter size, conducted at a whole-body average SAR of 4.92 W/kg, did not show an effect of RF-EMF exposure (MD 0.05; 95% CI -0.21 to 0.30). The meta-analysis of studies on resorbed and dead fetuses, conducted at a whole-body average SAR of 20.26 W/kg, showed a significant increase of the incidence in RF-EMF exposed animals (OR 1.84; 95% CI 1.27 to 2.66). The results were similar in the dose-response analysis. Effects on the offspring health at birth. The meta-analysis of studies on fetal weight, conducted at a whole-body average SAR of 9.83 W/kg, showed a small decrease in RF-EMF exposed animals (SMD 0.31; 95% CI 0.15 to 0.48). The meta-analysis of studies on fetal length, conducted at a whole-body average SAR of 4.55 W/kg, showed a moderate decrease in length at birth (SMD 0.45; 95% CI 0.07 to 0.83). The meta-analysis of studies on the percentage of fetuses with malformations, conducted at a whole-body average SAR of 6.75 W/kg, showed a moderate increase in RF-EMF exposed animals (SMD -0.45; 95% CI -0.68 to -0.23). The meta-analysis of studies on the incidence of litters with malformed fetuses, conducted at a whole-body average SAR of 16.63 W/kg, showed a statistically significant detrimental RF-EMF effect (OR 3.22; 95% CI 1.9 to 5.46). The results were similar in the dose-response analyses. Delayed effects on the offspring health. RF-EMF exposure was not associated with detrimental effects on brain weight (SMD 0.10; 95% CI -0.09 to 0.29) and on learning and memory functions (SMD -0.54; 95% CI -1.24 to 0.17). RF-EMF exposure was associated with a large detrimental effect on motor activity functions (SMD 0.79; 95% CI 0.21 to 1.38) and a moderate detrimental effect on motor and sensory functions (SMD -0.66; 95% CI -1.18 to -0.14). RF-EMF exposure was not associated with a decrease of the size of litters conceived by F2 female offspring (SMD 0.08; 95% CI -0.39 to 0.55). Notably, meta-analyses of neurobehavioural effects were based on few studies, which suffered of lack of independent replication deriving from only few laboratories. DISCUSSION: There was high certainty in the evidence for a lack of association of RF-EMF exposure with litter size. We attributed a moderate certainty to the evidence of a small detrimental effect on fetal weight. We also attributed a moderate certainty to the evidence of a lack of delayed effects on the offspring brain weight. For most of the other endpoints assessed by the meta-analyses, detrimental RF-EMF effects were shown, however the evidence was attributed a low or very low certainty. The body of evidence had limitations that did not allow an assessment of whether RF-EMF may affect pregnancy outcomes at exposure levels below those eliciting a well-known adverse heating impact. In conclusion, in utero RF-EMF exposure does not have a detrimental effect on fecundity and likely affects offspring health at birth, based on the meta-analysis of studies in experimental mammals on litter size and fetal weight, respectively. Regarding possible delayed effects of in utero exposure, RF-EMF probably does not affect offspring brain weight and may not decrease female offspring fertility; on the other hand, RF-EMF may have a detrimental impact on neurobehavioural functions, varying in magnitude for different endpoints, but these last findings are very uncertain. Further research is needed on the effects at birth and delayed effects with sample sizes adequate for detecting a small effect. Future studies should use standardized endpoints for testing prenatal developmental toxicity and developmental neurotoxicity (OECD TG 414 and 426), improve the description of the exposure system design and exposure conditions, conduct appropriate dosimetry characterization, blind endpoint analysis and include several exposure levels to better enable the assessment of a dose-response relationship. PROTOCOL REGISTRATION AND PUBLICATION: The protocol was published in Pacchierotti et al., 2021 and registered in PROSPERO CRD42021227746 (https://www.crd.york.ac.uk/prospero/display_record.php?RecordID=227746).

Pyrogenic synthetic amorphous silica (NM-203): Genotoxicity in rats following sub-chronic oral exposure.

The genotoxicity of nano-structured synthetic amorphous silica (SAS), a common food additive, was investigated in vivo in rats. A 90-day oral toxicity study was performed according to OECD test guideline 408 and the genotoxicity of pyrogenic SAS nanomaterial NM-203 was assessed in several organs, using complementary tests. Adult Sprague-Dawley rats of both sexes were treated orally for 90 days with 0, 2, 5, 10, 20, or 50 mg SAS/kg bw per day. Dose levels were selected to approximate expected human dietary exposures to SAS. DNA strand breaks were evaluated by the comet assay in blood, bone marrow, liver, and spleen according to OECD test guideline 489; mutations induced in bone marrow precursors of erythrocytes were assessed by the Pig-a assay and chromosome/ genome damage by the micronucleus assay in blood (OECD test guideline 474) and colon. No treatment-related increases of gene (Pig-a) or chromosome/genome (micronucleus) mutations were detected in the blood. The percentage of micronucleated cells was not increased in the colon of treated rats. Among the organs analyzed by the comet assay, the spleen was the only target showing a weak but biologically relevant genotoxic effect.

Effects of sub-chronic oral exposure to pyrogenic synthetic amorphous silica (NM-203) in male and female Sprague-Dawley rats: focus on reproductive systems.

Synthetic amorphous silica (SAS) consists of agglomerates and aggregates of primary particles in the nanorange (<100 nm) and it is the E551 authorized food additive. The potential risks for human health associated to dietary exposure to SAS are not completely assessed; in particular, data on male and female reproductive systems are lacking. A 90-day oral toxicity study with pyrogenic SAS nanomaterial NM-203 was carried out on the basis of the OECD test guideline 408 in the frame of the NANoREG project. Adult Sprague-Dawley rats of both sexes were orally treated for 90 days with 0, 2, 5, 10, 20 and 50 mg SAS/kg bw per day. Dose levels were selected to be as close as possible to the expected human exposure to food additive E551. The present paper provides specific information on potential effects on male and female reproductive systems, through the evaluation of serum biomarkers, sperm count, histopathological analysis of testis, epididymis, ovary and uterus and real-time PCR on uterus; potential genotoxic alterations were evaluated by comet assay on testis, sperm and ovary. NM-203 did not induce histophatological and genotoxic effects in male reproductive system. In female rats, ovary is not target of NM-203 and only tissue-specific effects on uterus were recorded up to 10 mg/kg bw per day. To our best knowledge, this is the first study providing data on male and female reproductive systems after long-term, repeated oral exposure at dose levels close to dietary human exposure, which identifies a limited concern only for female reproductive health.

Effects of Radiofrequency Electromagnetic Field (RF-EMF) exposure on male fertility and pregnancy and birth outcomes: Protocols for a systematic review of experimental studies in non-human mammals and in human sperm exposed in vitro.

BACKGROUND: Radiofrequency Electromagnetic Fields (RF-EMF) at environmental level have been reported to induce adverse effects on the male reproductive system and developing embryos. However, despite the number of experiments conducted since the 1970s, the diversity of testing approaches and exposure conditions, inconsistencies among results, and dosimetric flaws have not yet permitted a solid assessment of the relationship between RF-EMF exposure and such effects, warranting a more systematic and methodologically rigorous approach to the evaluation of available data. OBJECTIVES: This study aims at evaluating the effects of RF-EMF exposure on male fertility and pregnancy outcomes by a systematic review (SR) of experimental studies, conducted in compliance with international guidelines. The evidence will be organized into three streams: 1) Studies evaluating the impact of RF-EMF on the male reproductive system of experimental mammals; 2) studies evaluating the impact of RF-EMF on human sperm exposed in vitro; 3) studies evaluating the impact of RF-EMF on adverse pregnancy, birth outcomes and delayed effects in experimental mammals exposed in utero. STUDY ELIGIBILITY AND CRITERIA: Eligible studies will include peer-reviewed articles reporting of original results about effects of controlled exposures to RF-EMF in the frequency range 100 kHz-300 GHz on the selected outcomes without any language or year-of-publication restrictions. Eligible studies will be retrieved by calibrated search strings applied to three electronic databases, PubMed, Scopus and EMF Portal and by manual search of the list of references of included papers and published reviews. STUDY APPRAISAL AND SYNTHESIS METHOD: The internal validity of the studies will be evaluated using the Risk of Bias (RoB) Rating Tool developed by National Toxicology Program/Office of Health Assessment and Translation (NTP/OHAT) integrated with input from the SYRCLE RoB tool. Given sufficient commensurate data, meta-analyses will be performed, otherwise narrative syntheses will be produced. Finally, the certainty of the effects of RF-EMF exposure on male fertility and pregnancy and birth outcomes will be established following GRADE. FUNDING: The study is financially supported by the World Health Organization. REGISTRATION: OSF Registration DOI https://doi.org/10.17605/OSF.IO/7MUS3; PROSPERO CRD42021227729, CRD42021227746.

Hazard identification of pyrogenic synthetic amorphous silica (NM-203) after sub-chronic oral exposure in rat: A multitarget approach.

Food additive E551 consists of synthetic amorphous silica (SAS), comprising agglomerates and aggregates of primary particles in the nanorange (<100 nm), which potential nanospecific risks for humans associated to dietary exposure are not yet completely assessed. In NANoREG project, aim of the study was to identify potential hazards of pyrogenic SAS nanomaterial NM-203 by a 90-day oral toxicity study (OECD test guideline 408). Adult Sprague-Dawley rats of both sexes were orally treated with 0, 2, 5, 10, 20 and 50 mg SAS/kg bw per day; dose levels were selected to be as close as possible to E551 dietary exposure. Several endpoints were investigated, the whole integrative study is presented here along with the results of dispersion characterization, tissue distribution, general toxicity, blood/serum biomarkers, histopathological and immunotoxicity endpoints. No mortality, general toxicity and limited deposition in target tissues were observed. NM-203 affected liver and spleen in both sexes. Proposed NOAEL 5 mg/kg bw per day in male rats for enlarged sinusoids in liver. In female rats, TSH and creatinine levels were affected, proposed LOAEL 2 mg/kg bw per day. Overall, these data provide new insight for a comprehensive risk assessment of SAS exposure by the oral route.

Can sustained exposure to PFAS trigger a genotoxic response? A comprehensive genotoxicity assessment in mice after subacute oral administration of PFOA and PFBA.

PFAS (perfluoroalkyl substances) are considered non-genotoxic. However, PFAS exposure has been associated with the induction of oxidative stress in vitro and in vivo, and the possible induction of indirect genotoxic effects under sustained PFAS exposure has not been investigated. In order to shed light on this aspect, in this study a comprehensive assessment of genotoxicity was carried out in mice administered with perfluorooctanoic acid (PFOA, 0.1, 1 and 5 mg/kg body weight) and its C4 analogue perfluorobutyric acid (PFBA, 5 mg/kg body weight) for five weeks through drinking water. Markers of cell toxicity, oxidative stress and DNA strand breaks were measured in liver, the main target of toxicity of PFOA in rodents; systemic genotoxicity was also assessed by the analysis of micronuclei in reticulocytes and spleen lymphocytes, and germ cell effects by the Comet assay on testis cells. PFOA administration at the highest dose (5 mg/kg body weight) induced marked liver hypertrophy with signs of cell injury (elevated ALT and AST), with no concurrent evidence of lipid peroxidation and oxidative stress (decreased antioxidant capacity). Only mild liver hypertrophy, with no other signs of toxicity, was determined by PFBA administration. No evidence of treatment related genotoxicity was observed in any experimental group. Overall, data indicate that under the experimental conditions of this study, severe liver toxicity induced by PFOA administration is not associated with oxidative stress. Accordingly, no genotoxic effect is observed in liver and in the other tissues examined. Milder evidence of liver toxicity, with no genotoxicity, and a lower tendency to bioaccumulation were observed in PFBA treated mice.

Study of TiO2 P25 Nanoparticles Genotoxicity on Lung, Blood, and Liver Cells in Lung Overload and Non-Overload Conditions After Repeated Respiratory Exposure in Rats.

Inhaled titanium dioxide (TiO2) nanoparticles (NPs) can have negative health effects, and have been shown to cause respiratory tract cancer in rats. Inflammation has been linked to oxidative stress, and both have been described as possible mechanisms for genotoxicity of NPs, but rarely examined side-by-side in animal studies. In the present study, a wide range of complementary endpoints have been performed to study TiO2 P25 NP-induced genotoxicity in lung overload and non-overload conditions. Additionally, lung burden, inflammation, cytotoxicity and oxidative stress have also been evaluated in order to link genotoxicity with these responses. To assess quick and delayed responses after recovery, endpoints were evaluated at two time points: 2 h and 35 days after three repeated instillations. This study confirmed the previously described lung overload threshold at approximately 200-300 cm2 of lung burden for total particle surface area lung deposition or 4.2 µl/kg for volume-based cumulative lung exposure dose, above which lung clearance is impaired and inflammation is induced. Our results went on to show that these overload doses induced delayed genotoxicity in lung, associated with persistent inflammation only at the highest dose. The lowest tested doses had no toxicity or genotoxicity effects in the lung. In blood, no lymphocyte DNA damage, erythrocytes chromosomal damage or gene mutation could be detected. Our data also demonstrated that only overload doses induced liver DNA lesions irrespective of the recovery time. Tested doses of TiO2 P25 NPs did not induce glutathione changes in lung, blood or liver at both recovery times.

No genotoxicity in rat blood cells upon 3- or 6-month inhalation exposure to CeO2 or BaSO4 nanomaterials.

In the course of a 2-year combined chronic toxicity-carcinogenicity study performed according to Organisation for Economic Co-operation and Development (OECD) Test Guideline 453, systemic (blood cell) genotoxicity of two OECD representative nanomaterials, CeO2 NM-212 and BaSO4 upon 3- or 6-month inhalation exposure to rats was assessed. DNA effects were analysed in leukocytes using the alkaline Comet assay, gene mutations and chromosome aberrations were measured in erythrocytes using the flow cytometric Pig-a gene mutation assay and the micronucleus test (applying both microscopic and flow cytometric evaluation), respectively. Since nano-sized CeO2 elicited lung effects at concentrations of 5mg/m3 (burdens of 0.5mg/lung) in the preceding range-finding study, whereas nano-sized BaSO4 did not induce any effect, female rats were exposed to aerosol concentrations of 0.1 up to 3mg/m3 CeO2 or 50mg/m3 BaSO4 nanomaterials (6h/day; 5 days/week; whole-body exposure). The blood of animals treated with clean air served as negative control, whereas blood samples from rats treated orally with three doses of 20mg/kg body weight ethylnitrosourea at 24h intervals were used as positive controls. As expected, ethylnitrosourea elicited significant genotoxicity in the alkaline Comet and Pig-a gene mutation assays and in the micronucleus test. By contrast, 3- and 6-month CeO2 or BaSO4 nanomaterial inhalation exposure did not elicit significant findings in any of the genotoxicity tests. The results demonstrate that subchronic inhalation exposure to different low doses of CeO2 or to a high dose of BaSO4 nanomaterials does not induce genotoxicity on the rat hematopoietic system at the DNA, gene or chromosome levels.

Exposure to persistent organic pollutants and sperm DNA methylation changes in Arctic and European populations.

Persistent organic pollutants (POPs), such as PCBs (polychlorinated biphenyls) and DDT [1,1,1-trichloro-2,2-bis(4-chlorophenyl)ethane], are environmental contaminants with potential endocrine disrupting activity. DNA methylation levels in peripheral blood lymphocytes have been associated with serum concentrations of POPs in Greenland Inuit and Korean populations. Greenland Inuits are characterized by the highest worldwide POP levels. In this cross-sectional study we evaluated the relationship between serum POP concentrations and DNA methylation levels in sperm of non-occupationally exposed fertile men from Greenland, Warsaw (Poland), and Kharkiv (Ukraine). Serum levels of PCB-153 [1,2,4-trichloro-5-(2,4,5-trichlorophenyl)benzene], as a proxy of the total PCBs body burden, and of p,p'-DDE [1,1-dichloro-2,2-bis(4-chlorophenyl)ethylene], the main metabolite of DDT were measured. Sperm DNA methylation level was assessed globally by flow cytometric (FCM) immunodetection of 5-methyl-cytosines and at specific repetitive DNA sequences (Alu, LINE-1, Satα) by PCR-pyrosequencing after bisulfite conversion. Multivariate linear regression analysis was applied to investigate correlations between serum POP concentrations and DNA methylation. No consistent associations between exposure to POPs and sperm DNA methylation at repetitive DNA sequences were detected. A statistically significant global decrease in methylation was associated with exposure to either POP by FCM analysis. This is the first study to investigate environmental exposure to POPs and DNA methylation levels considering sperm as the target cells. Although POP exposure appears to have a limited negative impact on sperm DNA methylation levels in adult males, the global hypomethylation detected by one of the methods applied suggests that further investigation is warranted.

Genotoxicity Induced by Foetal and Infant Exposure to Magnetic Fields and Modulation of Ionising Radiation Effects.

BACKGROUND: Few studies have investigated the toxicity and genotoxicity of extremely low frequency magnetic fields (ELF-MF) during prenatal and neonatal development. These phases of life are characterized by cell proliferation and differentiation, which might make them sensitive to environmental stressors. Although in vitro evidences suggest that ELF-MF may modify the effects of ionizing radiation, no research has been conducted so far in vivo on the genotoxic effects of ELF-MF combined with X-rays. AIM AND METHODS: Aim of this study was to investigate in somatic and germ cells the effects of chronic ELF-MF exposure from mid gestation until weaning, and any possible modulation produced by ELF-MF exposure on ionizing radiation-induced damage. Mice were exposed to 50 Hz, 65 µT magnetic field, 24 hours/day, for a total of 30 days, starting from 12 days post-conception. Another group was irradiated with 1 Gy X-rays immediately before ELF-MF exposure, other groups were only X-irradiated or sham-exposed. Micronucleus test on blood erythrocytes was performed at multiple times from 1 to 140 days after birth. Additionally, 42 days after birth, genotoxic and cytotoxic effects on male germ cells were assessed by comet assay and flow cytometric analysis. RESULTS: ELF-MF exposure had no teratogenic effect and did not affect survival, growth and development. The micronucleus test indicated that ELF-MF induced a slight genotoxic damage only after the maximum exposure time and that this effect faded away in the months following the end of exposure. ELF-MF had no effects on ionizing radiation (IR)-induced genotoxicity in erythrocytes. Differently, ELF-MF appeared to modulate the response of male germ cells to X-rays with an impact on proliferation/differentiation processes. These results point to the importance of tissue specificity and development on the impact of ELF-MF on the early stages of life and indicate the need of further research on the molecular mechanisms underlying ELF-MF biological effects.

Exposure to perfluoroalkyl substances and sperm DNA global methylation in Arctic and European populations.

Perfluoroalkyl substances (PFASs) are widely used in a variety of industrial processes and products, and have been detected globally in humans and wildlife. PFASs are suspected to interfere with endocrine signaling and to adversely affect human reproductive health. The aim of the present study was to investigate the associations between exposure to PFASs and sperm global methylation levels in a population of non-occupationally exposed fertile men. Measurements of PFASs in serum from 262 partners of pregnant women from Greenland, Poland and Ukraine, were also carried out by liquid chromatography tandem mass spectrometry. Perfluorooctane sulfonate (PFOS), perfluorooctanoic acid (PFOA), perfluorohexane sulfonic acid (PFHxS), and perfluorononanoic acid (PFNA) were detected in 97% of the blood samples. Two surrogate markers were used to assess DNA global methylation levels in semen samples from the same men: (a) average DNA methylation level in repetitive DNA sequences (Alu, LINE-1, Satα) quantified by PCR-pyrosequencing after bisulfite conversion; (b) flow cytometric immunodetection of 5-methyl-cytosines. After multivariate linear regression analysis, no major consistent associations between PFASs exposure and sperm DNA global methylation endpoints could be detected. However, since weak but statistically significant associations of different PFASs with DNA hypo- and hyper-methylation were found in some of the studied populations, effects of PFASs on sperm epigenetic processes cannot be completely excluded, and this issue warrants further investigation.

X-ray induced DNA damage and repair in germ cells of PARP1(-/-) male mice.

Poly(ADP-ribose)polymerase-1 (PARP1) is a nuclear protein implicated in DNA repair, recombination, replication, and chromatin remodeling. The aim of this study was to evaluate possible differences between PARP1(-/-) and wild-type mice regarding induction and repair of DNA lesions in irradiated male germ cells. Comet assay was applied to detect DNA damage in testicular cells immediately, and two hours after 4 Gy X-ray irradiation. A similar level of spontaneous and radiation-induced DNA damage was observed in PARP1(-/-) and wild-type mice. Conversely, two hours after irradiation, a significant level of residual damage was observed in PARP1(-/-) cells only. This finding was particularly evident in round spermatids. To evaluate if PARP1 had also a role in the dynamics of H2AX phosphorylation in round spermatids, in which γ-H2AX foci had been shown to persist after completion of DNA repair, we carried out a parallel analysis of γ-H2AX foci at 0.5, 2, and 48 h after irradiation in wild-type and PARP1(-/-) mice. No evidence was obtained of an effect of PARP1 depletion on H2AX phosphorylation induction and removal. Our results suggest that, in round spermatids, under the tested experimental conditions, PARP1 has a role in radiation-induced DNA damage repair rather than in long-term chromatin modifications signaled by phosphorylated H2AX.

Direct and delayed X-ray-induced DNA damage in male mouse germ cells.

Sperm DNA integrity is essential for the accurate transmission of paternal genetic information. Various stages of spermatogenesis are characterized by large differences in radiosensitivity. Differentiating spermatogonia are susceptible to radiation-induced cell killing, but some of them can repair DNA damage and progress through differentiation. In this study, we applied the neutral comet assay, immunodetection of phosphorylated H2AX (γ-H2AX) and the Sperm Chromatin Structure Assay (SCSA) to detect DNA strand breaks in testicular cells and spermatozoa at different times following in vivo X-ray irradiation. Radiation produced DNA strand breaks in testicular cells that were repaired within the first few hours after exposure. Spermatozoa were resistant to the induction of DNA damage, but non-targeted DNA lesions were detected in spermatozoa derived from surviving irradiated spermatogonia. These lesions formed while round spermatids started to elongate within the testicular seminiferous tubules. The transcription of pro-apoptotic genes at this time was also enhanced, suggesting that an apoptotic-like process was involved in DNA break production. Our results suggest that proliferating spermatogonia retain a memory of the radiation insult that is recognized at a later developmental stage and activates a process leading to DNA fragmentation.

The usefulness of combining traditional sperm assessments with in vitro heterospermic insemination to identify bulls of low fertility as estimated in vivo.

To date, no single in vitro assessment can estimate bull fertility. This research was aimed at evaluating the ability of a series of laboratory assessments to assign 50 Holstein Friesian bulls grouped as low (ER-NRR<-1.5), medium (-0.5

Kinetics of gamma-H2AX induction and removal in bone marrow and testicular cells of mice after X-ray irradiation.

Male germ cells have been shown to differ in their DNA damage response (DDR) with respect to somatic cells. In addition, DDR pathways are modulated along spermatogenesis, accompanying profound chromatin modifications. Histone H2AX phosphorylation is a fundamental step of DDR. Few data are available on the long-term kinetics of phosphorylated H2AX (γ-H2AX) after in vivo irradiation. We have investigated, by microscopic and flow cytometric immunochemistry, γ-H2AX induction and removal in testicular cells of irradiated mice, in comparison with bone marrow cells. In unirradiated testicular cells, much higher levels of γ-H2AX were measured by flow cytometry with respect to bone marrow cells. Irradiation induced a redistribution of γ-H2AX into discrete foci detectable by microscopy. In irradiated bone marrow, the percentage of labelled cells peaked at 1 h and rapidly declined, in agreement with data on in vitro cell lines. In contrast, spermatocytes and round spermatids showed persistent labelling until 48 h. During this time, in spermatids, topological changes were observed in γ-H2AX foci from a pattern of many uncountable dots to a pattern of few large spots. Observations of testicular sections confirmed this trend in the reduction of foci number in spite of substantially invariable percentages of labelled cells in the analysed timeframe. To assess whether γ-H2AX persistence in testicular cells was due to unrepaired DNA breaks, we performed comet assay and immunofluorescence analysis of Mdc1, a marker of DDR different from γ-H2AX. Comet assay showed that most breaks were repaired within 2 h. Forty-eight hours after irradiation, contrary to γ-H2AX foci that remained detectable in 80% of initially labelled cells, Mdc1 foci were observed in only 20-30% of cells. These data suggest that, at long times after irradiation, mechanisms additional to impairment of DNA break repair may account for the long persistence of γ-H2AX foci in male germ cells.

Sperm DNA fragmentation induced by DNAse I and hydrogen peroxide: an in vitro comparative study among different mammalian species.

Sperm DNA damage may have adverse effects on reproductive outcome. Sperm DNA breaks can be detected by several tests, which evaluate DNA integrity from different and complementary perspectives and offer a new class of biomarkers of the male reproductive function and of its possible impairment after environmental exposure. The remodeling of sperm chromatin produces an extremely condensed nuclear structure protecting the nuclear genome from adverse environments. This nuclear remodeling is species specific, and differences in chromatin structure may lead to a dissimilar DNA susceptibility to mutagens among species. In this study, the capacity of the comet assay in its two variants (alkaline and neutral) to detect DNA/chromatin integrity has been evaluated in human, mouse, and bull sperm. The hypothesis that chromatin packaging might influence the amount of induced and detectable DNA damage was tested by treating sperm in vitro with DNAse I, whose activity is strictly dependent upon its DNA accessibility. Furthermore, hydrogen peroxide (H2O2) was used to assess whether spermatozoa of the three species showed a different sensitivity to oxidative stress. DNAse I-induced damage was also assessed by the sperm chromatin structure assay and the TUNEL assay, and the performances of these two assays were compared and correlated with the comet assay results. Results showed a different sensitivity to DNAse I treatment among the species with human sperm resulting the most susceptible. On the contrary, no major differences among species were observed after H2O2 treatment. Furthermore, the three tests show a good correlation in revealing sperm with DNA strand breaks.

Toxic and genotoxic effects of oral administration of furan in mouse liver.

In this study, the effects induced in mouse liver by repeated oral exposure to furan were investigated. To this aim, the compound was given for 28 days by daily gavage to male B6C3F1 mice at 2, 4, 8 and 15 mg/kg body weight (b.w.)/day. Twenty-four hours after last administration, animals were sacrificed, liver was excised and the following parameters were evaluated: histological alterations, apoptosis, cell proliferation, polyploidy, overall DNA methylation, gene expression and DNA damage by the immunofluorescence detection of foci of phosphorylated histone H2AX (gamma-H2AX) and by alkaline comet assays, using both standard and modified protocols for the detection of DNA cross links. Liver DNA damage by comet assays was also evaluated in mice receiving furan as a single acute oral dose (15, 100 or 250 mg/kg b.w.). Microscopic analysis of liver sections indicated that repeated oral administration of furan was moderately toxic, producing mild histological alterations with necrotic figures, apoptosis and limited regenerative cell proliferation. The flow cytometric analysis of DNA content in single-cell suspensions of liver cells showed a statistically significant increase in polyploid (8N) cells at the highest dose. No treatment-related changes in overall DNA methylation, gamma-H2AX foci, DNA strand breaks and cross links were observed at the end of the 4-week exposure period. However, several genes involved in DNA damage response, beyond stress and liver toxicity, were over-expressed in mice treated with the highest furan dose (15 mg/kg b.w./day). Acute administration of furan induced evident liver toxicity at the highest dose (250 mg/kg b.w.), which was associated with a significant increase of DNA damage in the alkaline comet assay and with a distinct decrease in gamma-ray-induced DNA migration. Overall, the results obtained suggest that the contribution of genotoxicity to the mechanism of furan carcinogenicity in mouse liver should not be dismissed.

Xenobiotic activity in serum and sperm chromatin integrity in European and inuit populations.

Lipophilic persistent organic pollutants (POPs) are ubiquitous in the environment and suspected to interfere with hormone activities and reproduction. In previous studies we demonstrated that POP exposure can affect sperm DNA integrity and differences between Inuits and Europeans in sperm DNA integrity and xenobiotic activity were observed. The aim of this study was to investigate possible relations between human sperm chromatin integrity and the xenobiotic serum activity of lipophilic POPs assessed as effects on the estrogen (ER), androgen (AR), and/or aryl hydrocarbon (AhR) receptors. Human sperm chromatin integrity was assessed as DNA fragmentation index (%DFI) and high DNA stainability (%HDS) using the flow cytometric sperm chromatin structure assay (SCSA). Xenobiotic receptor activities were determined using chemically activated luciferase gene expression (CALUX) assay. The study included 53 Greenlandic Inuits and 247 Europeans (Sweden, Warsaw (Poland) and Kharkiv (Ukraine)). A heterogeneous pattern of correlations was found. For Inuits, ER and AhR activities and %DFI were inversely correlated, whereas a positive correlation between AR activity and %DFI was found for Europeans. In contrast, no correlation between receptor activities and %HDS was observed for Inuits but for Europeans positive and negative correlations were observed between ER and AR activities and %HDS, respectively. We suggest that the different patterns of xenobiotic serum activities, in combination with diet associated factors and/or genetics, might be connected to the observed differences in sperm chromatin integrity between the Inuits and Europeans.

Sperm chromatin structure assay parameters measured after density gradient centrifugation are not predictive for the outcome of ART.

BACKGROUND: The sperm chromatin structure assay (SCSA) parameter DNA fragmentation index (DFI) has been shown to predict in vivo and in vitro fertility. So far most SCSA studies have been based on SCSA analysis performed on neat semen. The aim of this study is to assess whether SCSA analysis of sperm prepared by density gradient centrifugation (DGC) could add more information in regard to the prediction of treatment outcome. METHODS: The study included 510 assisted reproductive technique (ART) cycles. SCSA was performed in neat semen and post DGC. SCSA results were expressed in terms of DFI and high DNA stainability (HDS) cell fractions. The outcome parameter was clinical pregnancy (CP). RESULTS: Scatter-plot diagrams demonstrated that for DGC samples, no DFI cut-off values could be set for in vivo or in vitro fertility. In intrauterine insemination, IVF and ICSI groups the mean difference (95% CI) in DFI post DGC between those who achieved CP and those who did not was 0.2% (-1.7 to 2.0%), 0.4% (-1.9 to 2.8%) and 1.3% (-3.1 to 5.9%), respectively, none of these being statistically significant. The corresponding differences for HDS were 0.1% (-1.3 to 1.5%), 0.1% (-0.7 to 0.9%) and 0.6% (-1.6 to 2.7%), respectively (all P-values >0.6). CONCLUSIONS: SCSA performed in semen prepared by DGC cannot predict the outcome of ART.