C Consales1, G Leter1, J P E Bonde2, G Toft3, P Eleuteri1, T Moccia4, A Budillon4, B A G Jönsson5, A Giwercman6, H S Pedersen7, J K Ludwicki8, V Zviezdai9, D Heederik10, M Spanò11. 1. Laboratory of Toxicology, Unit of Radiation Biology and Human Health, ENEA Casaccia Research Center, Via Anguillarese 301, 00123 Rome, Italy. 2. Department of Occupational and Environmental Medicine, Bispebjerg University Hospital, DK-2400 Copenhagen NV, Denmark. 3. Department of Occupational Medicine, Aarhus University Hospital, DK-8000 Aarhus C, Denmark. 4. Experimental Pharmacology Unit, Department of Experimental Oncology, National Cancer Institute - G. Pascale, 80131 Naples, Italy. 5. Division of Occupational and Environmental Medicine, Department of Laboratory Medicine, Lund University, SE-22185 Lund, Sweden. 6. Reproductive Medicine Centre, Skåne University Hospital Malmö, Lund University, Malmö SE-20502, Sweden. 7. Centre for Arctic Environmental Medicine, Greenland Institute of Natural Resources, 3900 Nuuk, Greenland. 8. Department of Toxicology and Risk Assessment, National Institute of Public Health - National Institute of Hygiene, 00971 Warsaw, Poland. 9. Department of Social Medicine and Organization of Public Health, Kharkiv National Medical University, 61022 Kharkiv, Ukraine. 10. Institute for Risk Assessment Sciences, Utrecht University, Utrecht, The Netherlands. 11. Laboratory of Toxicology, Unit of Radiation Biology and Human Health, ENEA Casaccia Research Center, Via Anguillarese 301, 00123 Rome, Italy marcello.spano@enea.it.
Abstract
STUDY QUESTION: Which are the main determinants, if any, of sperm DNA methylation levels? SUMMARY ANSWER: Geographical region resulted associated with the sperm methylation status assessed on genome-wide repetitive sequences. WHAT IS KNOWN ALREADY: DNA methylation level, assessed on repetitive sequences from peripheral blood lymphocyte, can vary with age, gender, alcohol consumption and white blood cell counts. STUDY DESIGN, SIZE, DURATION: A cross-sectional study. Individual data were collected from 269 young healthy men of proven fertility living in three geographical regions: Inuits from Greenland, Caucasians from Warsaw (Poland) and Kharkiv (Ukraine). Semen samples were collected between May 2002 and February 2004 and aliquots were immediately frozen. PARTICIPANTS/MATERIALS, SETTING, METHODS: We estimated sperm DNA global methylation level (DGML) in two ways. First DNA methylation in repetitive DNA sequences (LINE-1, Satα and Alu) was quantified by PCR pyrosequencing after bisulfite conversion and second by flow cytometry (FCM) using fluorescently labeled monoclonal antibodies anti-5-methylcytosine. We analyzed whether personal characteristics and habits, body mass index, semen quality parameters, sperm chromatin integrity, biomarkers of accessory gland function and the plasma concentration of reproductive hormones were associated with sperm DNA methylation levels in men. Associations were evaluated by analysis of variance and linear regression analyses. MAIN RESULTS AND THE ROLE OF CHANCE: The geographical location emerged as the main determinant when using the methylation level in repetitive sequences. FCM DGML results were not associated with those from repetitive sequence analysis. No other consistent associations between methylation markers and the assessed variables were identified across countries. LIMITATIONS, REASONS FOR CAUTION: The methods used are only surrogates of the actual sperm methylome and the methylation levels at individual specific loci were not explored. WIDER IMPLICATIONS OF THE FINDINGS: Sperm DGML is relatively independent from semen quality parameters and is a new candidate biomarker for epidemiological studies of the impact of environmental contaminants on male fertility. STUDY FUNDING/COMPETING INTERESTS: The study is part of the project CLEAR (Climate change, Environmental contaminants and Reproductive health) supported by the European Commission 7th framework program, contract no: FP7-ENV-2008-1-226217. No competing interest is declared.
STUDY QUESTION: Which are the main determinants, if any, of sperm DNA methylation levels? SUMMARY ANSWER: Geographical region resulted associated with the sperm methylation status assessed on genome-wide repetitive sequences. WHAT IS KNOWN ALREADY: DNA methylation level, assessed on repetitive sequences from peripheral blood lymphocyte, can vary with age, gender, alcohol consumption and white blood cell counts. STUDY DESIGN, SIZE, DURATION: A cross-sectional study. Individual data were collected from 269 young healthy men of proven fertility living in three geographical regions: Inuits from Greenland, Caucasians from Warsaw (Poland) and Kharkiv (Ukraine). Semen samples were collected between May 2002 and February 2004 and aliquots were immediately frozen. PARTICIPANTS/MATERIALS, SETTING, METHODS: We estimated sperm DNA global methylation level (DGML) in two ways. First DNA methylation in repetitive DNA sequences (LINE-1, Satα and Alu) was quantified by PCR pyrosequencing after bisulfite conversion and second by flow cytometry (FCM) using fluorescently labeled monoclonal antibodies anti-5-methylcytosine. We analyzed whether personal characteristics and habits, body mass index, semen quality parameters, sperm chromatin integrity, biomarkers of accessory gland function and the plasma concentration of reproductive hormones were associated with sperm DNA methylation levels in men. Associations were evaluated by analysis of variance and linear regression analyses. MAIN RESULTS AND THE ROLE OF CHANCE: The geographical location emerged as the main determinant when using the methylation level in repetitive sequences. FCM DGML results were not associated with those from repetitive sequence analysis. No other consistent associations between methylation markers and the assessed variables were identified across countries. LIMITATIONS, REASONS FOR CAUTION: The methods used are only surrogates of the actual sperm methylome and the methylation levels at individual specific loci were not explored. WIDER IMPLICATIONS OF THE FINDINGS: Sperm DGML is relatively independent from semen quality parameters and is a new candidate biomarker for epidemiological studies of the impact of environmental contaminants on male fertility. STUDY FUNDING/COMPETING INTERESTS: The study is part of the project CLEAR (Climate change, Environmental contaminants and Reproductive health) supported by the European Commission 7th framework program, contract no: FP7-ENV-2008-1-226217. No competing interest is declared.
Authors: Abdolrahman Khezri; Birgitte Narud; Else-Berit Stenseth; Anders Johannisson; Frøydis Deinboll Myromslien; Ann Helen Gaustad; Robert C Wilson; Robert Lyle; Jane M Morrell; Elisabeth Kommisrud; Rafi Ahmad Journal: BMC Genomics Date: 2019-11-27 Impact factor: 3.969