Interleukin-2 gene methylation levels and interleukin-2 levels associated with environmental exposure as risk biomarkers for preterm birth.

AIM: To compare interleukin-2 levels (IL-2) and IL-2 gene site 1 methylation levels between preterm newborns (PN) and full-term newborns (FN) and investigate their association with the environmental exposure of their mothers during pregnancy. METHODS: IL-2 and IL-2 gene site 1 methylation levels were assessed in 50 PN and 56 FN. Newborns' mothers filled in questionnaires about their living and occupational environments, habits, diets, and hobbies. RESULTS: The mothers of PN were significantly more frequently agrarian/rural residents than the mothers of FN. PN had significantly higher IL-2 levels, and significantly lower methylation of IL-2 gene site 1 levels than FN. CONCLUSION: IL-2 levels, hypomethylation of the IL-2 gene site 1, and the mother's rural residence (probably due to pesticide exposure) were predictive biomarkers for preterm birth. For the first time, we present the reference values for the methylation of IL-2 gene site 1 in PN and FN, which can be used in the clinical setting and biomonitoring.

The HBM4EU chromates study - Outcomes and impacts on EU policies and occupational health practices.

Within the EU human biomonitoring initiative (HBM4EU), a targeted, multi-national study on occupational exposure to hexavalent chromium (Cr(VI)) was performed. Cr(VI) is currently regulated in EU under REACH (Registration, Evaluation, Authorisation and Restriction of Chemicals) and under occupational safety and health (OSH) legislation. It has recently been subject to regulatory actions to improve its risk management in European workplaces. Analysis of the data obtained within the HBM4EU chromates study provides support both for the implementation of these regulatory actions and for national enforcement programs and may also contribute to the updating of occupational limit values (OELs) and biological limit values for Cr(VI). It also provides useful insights on the contribution of different risk management measures (RMMs) to further reduce the exposure to Cr(VI) and may support the evaluation of applications for authorisation under REACH. Findings on chrome platers' additional per- and polyfluoroalkyl substances (PFAS) exposure highlight the need to also pay attention to this substance group in the metals sector. A survey performed to evaluate the policy relevance of the HBM4EU chromates study findings supports the usefulness of the study results. According to the responses received from the survey, the HBM4EU chromates study was able to demonstrate the added value of the human biomonitoring (HBM) approach in assessment and management of occupational exposure to Cr(VI). For future occupational studies, we emphasise the need for engagement of policy makers and regulators throughout the whole research process to ensure awareness, relevance and uptake of the results in future policies.

HBM4EU Chromates Study-Genotoxicity and Oxidative Stress Biomarkers in Workers Exposed to Hexavalent Chromium.

A study was conducted within the European Human Biomonitoring Initiative (HBM4EU) to characterize occupational exposure to Cr(VI). Herein we present the results of biomarkers of genotoxicity and oxidative stress, including micronucleus analysis in lymphocytes and reticulocytes, the comet assay in whole blood, and malondialdehyde and 8-oxo-2'-deoxyguanosine in urine. Workers from several Cr(VI)-related industrial activities and controls from industrial (within company) and non-industrial (outwith company) environments were included. The significantly increased genotoxicity (p = 0.03 for MN in lymphocytes and reticulocytes; p < 0.001 for comet assay data) and oxidative stress levels (p = 0.007 and p < 0.001 for MDA and 8-OHdG levels in pre-shift urine samples, respectively) that were detected in the exposed workers over the outwith company controls suggest that Cr(VI) exposure might still represent a health risk, particularly, for chrome painters and electrolytic bath platers, despite the low Cr exposure. The within-company controls displayed DNA and chromosomal damage levels that were comparable to those of the exposed group, highlighting the relevance of considering all industry workers as potentially exposed. The use of effect biomarkers proved their capacity to detect the early biological effects from low Cr(VI) exposure, and to contribute to identifying subgroups that are at higher risk. Overall, this study reinforces the need for further re-evaluation of the occupational exposure limit and better application of protection measures. However, it also raised some additional questions and unexplained inconsistencies that need follow-up studies to be clarified.

Polymorphisms in DNA Repair and Xenobiotic Biotransformation Enzyme Genes and Lung Cancer Risk in Coal Mine Workers.

BACKGROUND: Currently coal mining employs over 7 million miners globally. This occupational setting is associated with exposure to dust particles, heavy metals, polycyclic aromatic hydrocarbons and radioactive radon, significantly increasing the risk of lung cancer (LC). The susceptibility for LC is modified by genetic variations in xenobiotic detoxification and DNA repair capacity. The aim of this study was to investigate the association between GSTM1 (deletion), APEX1 (rs1130409), XPD (rs13181) and NBS1 (rs1805794) gene polymorphisms and LC risk in patients who worked in coal mines. METHODS: The study included 639 residents of the coal region of Western Siberia (Kemerovo region, Russia): 395 underground miners and 244 healthy men who do not work in industrial enterprises. Genotyping was performed using real-time and allele-specific PCR. RESULTS: The results show that polymorphisms of APEX1 (recessive model: ORadj = 1.87; CI 95%: 1.01-3.48) and XPD (log additive model: ORadj = 2.25; CI 95%: 1.59-3.19) genes were associated with increased LC risk. GSTM1 large deletion l was linked with decreased risk of LC formation (ORadj = 0.59, CI 95%: 0.36-0.98). The multifactor dimensionality reduction method for 3-loci model of gene-gene interactions showed that the GSTM1 (large deletion)-APEX1 (rs1130409)-XPD (rs13181) model was related with a risk of LC development. CONCLUSIONS: The results of this study highlight an association between gene polymorphism combinations and LC risks in coal mine workers.

Seminal Plasma Protein N-Glycan Peaks Are Potential Predictors of Semen Pathology and Sperm Chromatin Maturity in Men.

BACKGROUND: Male infertility is increasingly becoming a health and demographic problem. While it may originate from congenital or acquired diseases, it can also result from environmental exposure. Hence, the complexity of involved molecular mechanisms often requires a multiparametric approach. This study aimed to associate semen parameters with sperm DNA fragmentation, chromatin maturity and seminal plasma protein N-glycosylation. METHODS: The study was conducted with 166 participants, 20-55 y old, 82 normozoospermic and 84 with pathological diagnosis. Sperm was analyzed by Halosperm assay and aniline blue staining, while seminal plasma total protein N-glycans were analyzed by ultra-high-performance liquid chromatography. RESULTS: Sperm DNA fragmentation was significantly increased in the pathological group and was inversely correlated with sperm motility and viability. Seminal plasma total protein N-glycans were chromatographically separated in 37 individual peaks. The pattern of seminal plasma N-glycan peaks (SPGP) showed that SPGP14 significantly differs between men with normal and pathological semen parameters (p < 0.001). The multivariate analysis showed that when sperm chromatin maturity increases by 10%, SPGP17 decreases by 14% while SPGP25 increases by 25%. CONCLUSION: DNA integrity and seminal plasma N-glycans are associated with pathological sperm parameters. Specific N-glycans are also associated with sperm chromatin maturity and have a potential in future fertility research and clinical diagnostics.

Comparison of Estradiol, Testostosterone, and CYP19 Methylation Levels between Full-Term and Preterm Newborns.

Estradiol (E), testosterone (T), and their ratio are crucial axis in life. Especially during intrauterine growth, they orchestrate the complex development of organs and their interaction, which have lifelong impact on health and an organism's capacity to respond to environmental stressors. The aim of this study was to compare for the first time E, T, and their ratio levels with aromatase (CYP19) gene methylation levels between preterm newborns (PN) and full-term newborns (FN) with respect to their mother's environmental exposure and diet. In this study, 56 FN of 37-42 weeks of gestation age (GA) and 46 PN at GA 27-36 weeks were analysed for E and T levels and CYP19A1 gene pI.3/II promoter region methylation. Results showed there was no difference in E levels between PN and FN, but there were significantly lower levels of T in PN than in FN (2.81 nmol vs. 3.76 nmol, respectively) and consequently a significantly higher E/T ratio in PN than in FN (5278.04 vs. 2891.23, respectively). CYP19A1 methylation was significantly lower in PN than in FN (86.04% vs. 90.04%, respectively). CYP19A1 methylation was significantly reduced in newborns whose mothers reported daily milk consumption. Our study is the first to provide referent values for CYP19A1 methylation levels in FN and PN and shows that PN and FN significantly differ in CYP19A1 methylation levels, T levels, and E/T ratio. Future research should further investigate the mechanisms involved in GA-dependent CYP19A1 methylation levels and mechanisms of sex hormone disturbances which may contribute to preterm birth.

Reproductive Health Risks Associated with Occupational and Environmental Exposure to Pesticides.

A marked reduction in fertility and an increase in adverse reproductive outcomes during the last few decades have been associated with occupational and environmental chemical exposures. Exposure to different types of pesticides may increase the risks of chronic diseases, such as diabetes, cancer, and neurodegenerative disease, but also of reduced fertility and birth defects. Both occupational and environmental exposures to pesticides are important, as many are endocrine disruptors, which means that even very low-dose exposure levels may have measurable biological effects. The aim of this review was to summarize the knowledge collected between 2000 and 2020, to highlight new findings, and to further interpret the mechanisms that may associate pesticides with infertility, abnormal sexual maturation, and pregnancy complications associated with occupational, environmental and transplacental exposures. A summary of current pesticide production and usage legislation is also included in order to elucidate the potential impact on exposure profile differences between countries, which may inform prevention measures. Recommendations for the medical surveillance of occupationally exposed populations, which should be facilitated by the biomonitoring of reduced fertility, is also discussed.

Environmental and occupational exposures associated with male infertility.

The upsurge in male infertility over the last two decades, possibly due to environmental exposure, has raised significant interest, particularly boosted by reports from fertility clinics, which showed that chronic diseases and hereditary or other medical conditions might only partially explain current incidence of male infertility. Both environmental and occupational settings may have a significant role in exposure to complex mixtures of endocrine disruptors (ED), which play a major role in fertility disorders. The aim of this review is to give an insight into the current knowledge on exposure settings which may be associated with male infertility. Our study relied on a systematic search of PubMed, Scopus, and Web of Science for articles published between January 2000 and September 2020. It showed that some well documented factors associated with male infertility include smoking, and physiological disturbances or chronic diseases such as obesity and diabetes, which in turn, may also reflect lifestyle choices and environmental exposures, especially to EDs such as phthalates, bisphenols, pesticides, and flame retardants. However, the number of studies on the aetiology of male infertility is still too low in comparison with the size of affected population. Occupational health follow-ups and medical surveillance do not collect any data on male infertility, even though ED chemicals are part of many technological processes.

Comparison of Androgen Receptor, VEGF, HIF-1, Ki67 and MMP9 Expression between Non-Metastatic and Metastatic Stages in Stromal and Tumor Cells of Oral Squamous Cell Carcinoma.

OBJECTIVES: Oral squamous cell carcinoma (OSCC) is the most common oral malignancy with low survival as it is very often diagnosed at an advanced stage, which is why the accurate profiling of the tumor is essential. The aim of this study was to, for the first time, compare in OSCC the frequency of AR, VEGF, MMP9, HiF1beta and Ki67 between the non-metastatic and metastatic disease. MATERIALS AND METHODS: In the study, 96 non-metastatic and 91 metastatic OSCC patients were analysed for AR, VEGF, MMP9, HiF1beta and Ki67 levels by immunohistochemistry. RESULTS: All of the tested biomarkers significantly differed between non-metastatic and metastatic disease. A significant association was found between >/=20% AR positive epithelium cells in cytoplasm, Ki67 and VEGF in cancer stroma. Ki67, HiF1beta, VEGF and MMP9 were significantly associated with TNM stages. CONCLUSION: Our results show for the first time an interplay between AR, VEGF, MMP9, HiF1beta and Ki67 in OSCC which may contribute to better diagnostics and therapy selection.

In vivo acridine orange human spermatozoa staining-A new perspective for RNA detection and spermatozoa morphology evaluation.

Significant increases in male infertility and the still unresolved questions on the compatibility and interpretation of current methods in infertility diagnostics call for new protocols. Morphology, genome damage, RNA content and quantity are currently in practice as the major parameters in evaluation of sperm quality. However, results of various methods are not always in mutual concordance. In this study, in vivo acridine orange (AO) staining, which is presently in application in the estimation of genome damage in reticulocytes, was adjusted for spermatozoa staining. Ten men suffering from oligoasthenoteratozoospermia (OAT) and 10 healthy fertile men were analysed using in vivo AO staining. Microscopic analysis was performed by fluorescent and confocal fluorescent microscopy. Our results show that this method preserves spermatozoa membranes, which enables new insight into spermatozoa genome damage, RNA content in residual cytoplasm, damage of neck area with mitochondrion and tail pathology. The introduced method explains the difference between results of sperm DNA fragmentation assay and the globally used AO staining and opens new options for the development of automated systems. In conclusion, the results of our study offer (a) an innovative approach to the analysis of spermatozoa pathology, (b) enable localization and quantification of RNA in residual cytoplasm, (c) a significant contribution to research of aetiology of infertility in men, (d) open new perspectives for the automatization of sperm quality estimation and (e) improve the personalized approach in the selection of in vitro fertilization protocols.

Biomonitoring of occupational exposure to phthalates: A systematic review.

INTRODUCTION: Phthalates, a group of ubiquitous industrial chemicals, have been widely used in occupational settings, mainly as plasticizers in a variety of applications. Occupational exposure to different phthalates has been studied in several occupational settings using human biomonitoring (HBM). AIM: To provide a comprehensive review of the available literature on occupational exposure to phthalates assessed using HBM and to determine future data needs on the topic as part of the HBM4EU project. METHODS: A systematic search was carried out in the databases of Pubmed, Scopus, and Web of Science for articles published between 2000 and September 4, 2019 using the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. A total of 22 studies on the occupational HBM of phthalates was considered suitable for review. RESULTS AND DISCUSSION: Among the reviewed studies, 19 (86%) focused on DEHP, an old phthalate that is now subject to authorization and planned to be restricted in the EU. Concentrations of MEHHP, one of its metabolites, varied up to 13-fold between studies and across sectors when comparing extreme geometric means, ranging from 11.6 (similar to the general populations) to 151 µg/g creatinine. Only 2 studies focused on newer phthalates such as DiNP and DPHP. Concerning the geographical distribution, 10 studies were performed in Europe (including 6 in Slovakia), 8 in Asia, and 4 in North America, but this distribution is not a good reflection of phthalate production and usage levels worldwide. Most HBM studies were performed in the context of PVC product manufacturing. Future studies should focus on: i) a more uniform approach to sampling timing to facilitate comparisons between studies; ii) newer phthalates; and iii) old phthalates in waste management or recycling. CONCLUSION: Our findings highlight the lack of recent occupational HBM studies on both old and new phthalate exposure in European countries and the need for a harmonized approach. Considering the important policy actions taken in Europe regarding phthalates, it seems relevant to evaluate the impact of these actions on exposure levels and health risks for workers.

Rogue versus chromothriptic cell as biomarker of cancer.

New molecular cytogenetic biomarkers may significantly contribute to biodosimetry, whose application is still globally diverse and not fully standardized. In 2011, a new term, chromothripsis, was introduced raising great interest among researchers and soon motivating further investigations of the phenomenon. Chromothripsis is described as a single event in which one or more chromosomes go through severe DNA damage very much resembling rogue cells (RC) described more than 50 years ago. In this review, we for the first time compare these two multi-aberrant cells types, RC versus chromothriptic cells, giving insight into the similarities of the mechanisms involved in their etiology. In order to make a better comparison, data on RC in 3366 subjects from studies on cancer patients, Chernobyl liquidators, child victims of the Chernobyl nuclear plant accident, residentially and occupationally exposed population have been summarized for the first time. Results of experimental and epidemiological analysis show that chromothriptic cells and RC may be caused by exposure to high LET ionizing radiation. Experience and knowledge collected on RC may be used in future for further investigations of chromothripsis, introducing a new class of cells which include both chromothriptic and RC, and better insight into the frequency of chromothriptic cell per subject, which is currently absent. Both cell types are relevant in investigations of cancer etiology, biomonitoring of accidentally exposed population to ionizing radiation and biomonitoring of astronauts due to their exposure to high LET ionizing radiation during interplanetary voyages.

The Impact of Mother's Living Environment Exposure on Genome Damage, Immunological Status, and Sex Hormone Levels in Newborns.

Background: The aim of this study was to compare for the first time IL-6 (Interleukin 6), testosterone (T) and estradiol (E) levels, their ratio (E/T), micronucleus (MN), and nuclear bridge (NB) frequency between newborns with regard to their mother's residency and diet. Our results should enable an assessment of the possible environmental endocrine effects and interaction between biomarkers, pointing to possible associated health risks. Methods: Fifty full-term newborns of both sexes, whose mothers were healthy and not occupationally exposed to any known carcinogen, were analyzed. All of the mothers filled in a detailed questionnaire. Results: The results showed significantly higher levels of E in newborns of mothers with agricultural residency than those born by mothers with urban residency. Significantly, lower levels of E were measured in newborns of mothers who drank milk and carbonated beverages more frequently. Testosterone was significantly higher in boys of mothers with agricultural residency than from mothers with urban residency. Residence and other parameters had no impact on the difference in MN frequency. IL-6 levels were higher in newborns of mothers with agricultural residency. NB levels were significantly associated with E. A significant association between E levels and IL-6 was found. Conclusion: Our results were the first to show a significant impact of the mother's agricultural residency and diet on their newborns' sex hormone and IL-6 levels and their association.

Androgen Receptor as a Biomarker of Oral Squamous Cell Carcinoma Progression Risk.

BACKGROUND/AIM: Oral squamous cell carcinoma (OSCC) is a cancer with poor prognosis due to therapy resistance, locoregional recurrences, and distant metastases. There is on increased interest in profiling the androgen receptor (AR) in cancer biology. The aim of this study was to compare AR and Ki-67 levels in the neoplastic epithelium and stroma between non-metastatic and metastatic stages of OSCC. PATIENTS AND METHODS: Tissue specimens of 101 non-metastatic and 95 metastatic OSCC patients were analyzed by immunohistochemistry. RESULTS: More than 20% of AR-positive cytoplasmic staining of OSCC epithelium was significantly associated with nuclear AR levels in the epithelium and increased AR levels in the stroma. In metastatic OSCC patients, Ki-67 was significantly higher than in non-metastatic OSCC patients. CONCLUSION: More than 20% of AR-positive cytoplasmic staining in neoplastic OSSC epithelium is a significant predictor of OSCC progression risk.

Environmental and behavioural head and neck cancer risk factors.

OBJECTIVES: The high incidence of head and neck cancer (HNC), significantly associated with living environment and behaviour, can be prevented more efficiently. The aim of this study was to evaluate the environmental and behavioural risk factors for HNC. METHODS: Using a detailed questionnaire on social status, education, living and occupational environment exposures, family cancer and lifestyle, HNC patients (103 cases, 76.7% of men) were compared with control subjects (244 subjects, 73% of men) balanced by age: mean (standard deviation) 63.8 (9.3) and 63.8 (9.0) for cases and controls, respectively. RESULTS: The results of this study showed that smoking and low education were significant risk factors for HNC regardless of sex. Family HNC and breast cancer were significant predictors of HNC risk. CONCLUSION: The study confirmed previous results that smoking and low education are significantly associated with HNC. Additionally, results pointed to significant HNC and breast cancer risk in HNC patient's families that may have originated from passive smoking or a smoking habit stemming from social environments that support it. Better dissemination programmes regarding smoking risks for children and adults are needed, targeting not only individuals but also families.

Genome damage in children with classical Ehlers-Danlos syndrome - An in vivo and in vitro study.

Ehlers-Danlos syndrome (EDS) is a heritable connective tissue disorder characterized by skin hyperextensibility, abnormal wound healing, and joint hypermobility with prevalence 1:20 000. Its incidence is probably underestimated due to unknown number of subjects having mild symptoms who may have never been diagnosed through entire life time. Classical EDS is characterized by pathogenic variants of genes encoding type V collagen. The biological effects and health risks of patients with EDS exposure to low doses of ionizing radiation is poorly understood. The aim of this study was to investigate biological effect of low doses of ionizing radiation in children with EDS. Background values of chromosome aberrations in children suffering from classical EDS were determined and compared with control subjects. The in vitro experiment was performed by γ-irradiation of blood lymphocytes from EDS patients and healthy subjects at low doses (0.1, 0.2 and 0.3 Gy). Results show a significant increase level of spontaneous and radiation-induced chromosomal aberrations in children suffering from EDS in comparison with the control subjects (p < 0.05). In conclusion, children with EDS express higher background chromosome aberration frequency and increased radiosensitivity. These findings suggest specific susceptibility of EDS patients and importance of future investigation on risks of diagnostics and therapy which include radiation and genotoxic agents.

Potential Health Risk of Endocrine Disruptors in Construction Sector and Plastics Industry: A New Paradigm in Occupational Health.

Endocrine disruptors (EDs) belong to large and diverse groups of agents that may cause multiple biological effects associated with, for example, hormone imbalance and infertility, chronic diseases such as diabetes, genome damage and cancer. The health risks related with the exposure to EDs are typically underestimated, less well characterized, and not regulated to the same extent as, for example, carcinogens. The increased production and utilization of identified or suspected EDs in many different technological processes raises new challenges with respect to occupational exposure settings and associated health risks. Due to the specific profile of health risk, occupational exposure to EDs demands a new paradigm in health risk assessment, redefinition of exposure assessment, new effects biomarkers for occupational health surveillance and definition of limit values. The construction and plastics industries are among the strongest economic sectors, employing millions of workers globally. They also use large quantities of chemicals that are known or suspected EDs. Focusing on these two industries, this short communication discusses: (a) why occupational exposure to EDs needs a more specific approach to occupational health risk assessments, (b) identifies the current knowledge gaps, and

Chromosome aberrations in peripheral blood lymphocytes of lung cancer patients exposed to radon and air pollution.

Lung cancer is one of the most common forms of cancer. The aim of this study was to validate chromosome aberrations in peripheral blood lymphocytes of lung cancer patients living in a region with high air pollution and increased background radon levels as a biomarker of cancer risk. A total of 417 lung cancer patients and 468 control participants were analysed using a chromosome aberration assay in peripheral blood lymphocytes. The results showed that chromatid-type aberrations (2.26±1.58 vs. 1.60±1.58) and chromosome-type aberrations (CSAs) (0.96±1.36 vs. 0.42±0.70) in lung cancer patients were increased significantly in comparison with the controls. The most significant two-fold increase was detected for CSAs (nonsmoking patients: 0.84±1.54 vs. 0.41±0.73%, smoking patients: 0.99±1.31 vs. 0.44±0.67%). The frequency of dicentric and ring chromosomes, double minutes and rogue cells was significantly higher (P=0.002, 0.00002, 0.01, 0.0007) in the lung cancer patients. As both analysed groups lived in the same environment, our results show that increased radon levels were not the only source for the detected genome damage. Using binomial logistic regression, the estimated odds ratios and 95% confidence intervals adjusted for the main confounders (smoking, occupational exposure, age) were 1.31 (1.20-1.40) for chromatid-type aberrations, 1.28 (1.17-1.33), and 1.68 (1.49-1.88) for CSAs. It may be suggested that lung cancer patients show a significant increase in genome damage that may be caused by an interplay between exposure and individual low capacity of DNA repair, leading to genome instability.

Significance of stroma in biology of oral squamous cell carcinoma.

The worldwide annual incidence of oral squamous cell carcinoma (OSCC) is over 300,000 cases with a mortality rate of 48%. This cancer type accounts for 90% of all oral cancers, with the highest incidence in men over 50 years of age. A significantly increased risk of developing OSCC exists among smokers and people who consume alcohol daily. OSCC is an aggressive cancer that metastasizes rapidly. Despite the development of new therapies in the treatment of OSCC, no significant increase in 5-year survival has been recorded in the past decades. The latest research suggests focus should be put on examining tumor stroma activation within OSCC, as the stroma may contain cells that can produce signal molecules and a microenvironment crucial for the development of metastases. The aim of this review is to provide an insight into the factors that activate OSCC stroma and hence faciliate neoplastic progression. It is based on the currently available data on the role and interaction between metalloproteinases, cytokines, growth factors, hypoxia factor and extracellular adhesion proteins in the stroma of OSCC and neoplastic cells. Their interplay is additionally presented using the Systems Biology Graphical Notation in order to sublimate the collected knowledge and enable the more efficient recognition of possible new biomarkers in the diagnostics and follow-up of OSCC or in finding new therapeutic targets.

Association Between RASSF1A Promoter Methylation and Testicular Germ Cell Tumor: A Meta-analysis and a Cohort Study.

BACKGROUND: The RAS association domain family protein 1a (RASSF1A) is a prominent tumor suppressor gene showing altered promoter methylation in testicular germ cell tumors (TGCT). RASSF1A promoter hypermethylation might represent an early event in TGCT tumorigenesis. We investigated whether the RASSF1A promoter methylation in peripheral blood of TGCT patients can be associated with testicular cancer risk. MATERIALS AND METHODS: Following a meta-analysis, we performed a cohort study including 32 testicular cancer patients and 32 healthy controls. Promoter methylation of the RASSF1A and O6-methylguanine-DNA-methyltransferase (MGMT) genes was analyzed using bisulfite pyrosequencing of DNA from peripheral blood. RESULTS: Meta-analysis showed an odds ratio (OR) of 7.69 for RASSF1A promoter methylation as a risk factor for TGCT. Cohort study found altered methylation of the RASSF1A promoter in blood of TGCT patients. Methylation was higher in TGCT patients before BEP chemotherapy. CONCLUSION: The meta-analysis indicates a role of the RASSF1A promoter hypermethylation from peripheral blood in TCGT. We confirmed that finding in our cohort study, which represents the first report of changed RASSF1A promoter methylation in peripheral blood TGCT.