M J Xavier1, B Nixon1, S D Roman1,2, R J Scott3,4,5, J R Drevet6, R J Aitken1,3,4. 1. Priority Research Centre for Reproductive Science, Faculty of Science, The University of Newcastle, Callaghan, NSW, Australia. 2. Priority Research Centre for Drug Development, Faculty of Science, The University of Newcastle, Callaghan, NSW, Australia. 3. Faculty of Health and Medicine, The University of Newcastle, Callaghan, NSW, Australia. 4. Medical Genetics, Hunter Medical Research Institute, New Lambton Heights, NSW, Australia. 5. Division of Molecular Medicine, Pathology North, John Hunter Hospital, New Lambton Heights, NSW, Australia. 6. GReD Laboratory, CNRS UMR6293-INSERM U1103-Clermont Université, Clermont-Ferrand, France.
Abstract
STUDY QUESTION: Do all regions of the paternal genome within the gamete display equivalent vulnerability to oxidative DNA damage? SUMMARY ANSWER: Oxidative DNA damage is not randomly distributed in mature human spermatozoa but is instead targeted, with particular chromosomes being especially vulnerable to oxidative stress. WHAT IS KNOWN ALREADY: Oxidative DNA damage is frequently encountered in the spermatozoa of male infertility patients. Such lesions can influence the incidence of de novo mutations in children, yet it remains to be established whether all regions of the sperm genome display equivalent susceptibility to attack by reactive oxygen species. STUDY DESIGN, SIZE, DURATION: Human spermatozoa obtained from normozoospermic males (n = 8) were split into equivalent samples and subjected to either hydrogen peroxide (H2O2) treatment or vehicle controls before extraction of oxidized DNA using a modified DNA immunoprecipitation (MoDIP) protocol. Specific regions of the genome susceptible to oxidative damage were identified by next-generation sequencing and validated in the spermatozoa of normozoospermic males (n = 18) and in patients undergoing infertility evaluation (n = 14). PARTICIPANTS/MATERIALS, SETTING, METHODS: Human spermatozoa were obtained from normozoospermic males and divided into two identical samples prior to being incubated with either H2O2 (5 mm, 1 h) to elicit oxidative stress or an equal volume of vehicle (untreated controls). Alternatively, spermatozoa were obtained from fertility patients assessed as having high basal levels of oxidative stress within their spermatozoa. All semen samples were subjected to MoDIP to selectively isolate oxidized DNA, prior to sequencing of the resultant DNA fragments using a next-generation whole-genomic sequencing platform. Bioinformatic analysis was then employed to identify genomic regions vulnerable to oxidative damage, several of which were selected for real-time quantitative PCR (qPCR) validation. MAIN RESULTS AND THE ROLE OF CHANCE: Approximately 9000 genomic regions, 150-1000 bp in size, were identified as highly vulnerable to oxidative damage in human spermatozoa. Specific chromosomes showed differential susceptibility to damage, with chromosome 15 being particularly sensitive to oxidative attack while the sex chromosomes were protected. Susceptible regions generally lay outside protamine- and histone-packaged domains. Furthermore, we confirmed that these susceptible genomic sites experienced a dramatic (2-15-fold) increase in their burden of oxidative DNA damage in patients undergoing infertility evaluation compared to normal healthy donors. LIMITATIONS, REASONS FOR CAUTION: The limited number of samples analysed in this study warrants external validation, as do the implications of our findings. Selection of male fertility patients was based on high basal levels of oxidative stress within their spermatozoa as opposed to specific sub-classes of male factor infertility. WIDER IMPLICATIONS OF THE FINDINGS: The identification of genomic regions susceptible to oxidation in the male germ line will be of value in focusing future analyses into the mutational load carried by children in response to paternal factors such as age, the treatment of male infertility using ART and paternal exposure to environmental toxicants. STUDY FUNDING/COMPETING INTEREST(S): Project support was provided by the University of Newcastle's (UoN) Priority Research Centre for Reproductive Science. M.J.X. was a recipient of a UoN International Postgraduate Research Scholarship. B.N. is the recipient of a National Health and Medical Research Council of Australia Senior Research Fellowship. Authors declare no conflict of interest.
STUDY QUESTION: Do all regions of the paternal genome within the gamete display equivalent vulnerability to oxidative DNA damage? SUMMARY ANSWER: Oxidative DNA damage is not randomly distributed in mature human spermatozoa but is instead targeted, with particular chromosomes being especially vulnerable to oxidative stress. WHAT IS KNOWN ALREADY: Oxidative DNA damage is frequently encountered in the spermatozoa of male infertilitypatients. Such lesions can influence the incidence of de novo mutations in children, yet it remains to be established whether all regions of the sperm genome display equivalent susceptibility to attack by reactiveoxygen species. STUDY DESIGN, SIZE, DURATION: Human spermatozoa obtained from normozoospermic males (n = 8) were split into equivalent samples and subjected to either hydrogen peroxide (H2O2) treatment or vehicle controls before extraction of oxidized DNA using a modified DNA immunoprecipitation (MoDIP) protocol. Specific regions of the genome susceptible to oxidative damage were identified by next-generation sequencing and validated in the spermatozoa of normozoospermic males (n = 18) and in patients undergoing infertility evaluation (n = 14). PARTICIPANTS/MATERIALS, SETTING, METHODS:Human spermatozoa were obtained from normozoospermic males and divided into two identical samples prior to being incubated with either H2O2 (5 mm, 1 h) to elicit oxidative stress or an equal volume of vehicle (untreated controls). Alternatively, spermatozoa were obtained from fertility patients assessed as having high basal levels of oxidative stress within their spermatozoa. All semen samples were subjected to MoDIP to selectively isolate oxidized DNA, prior to sequencing of the resultant DNA fragments using a next-generation whole-genomic sequencing platform. Bioinformatic analysis was then employed to identify genomic regions vulnerable to oxidative damage, several of which were selected for real-time quantitative PCR (qPCR) validation. MAIN RESULTS AND THE ROLE OF CHANCE: Approximately 9000 genomic regions, 150-1000 bp in size, were identified as highly vulnerable to oxidative damage in human spermatozoa. Specific chromosomes showed differential susceptibility to damage, with chromosome 15 being particularly sensitive to oxidative attack while the sex chromosomes were protected. Susceptible regions generally lay outside protamine- and histone-packaged domains. Furthermore, we confirmed that these susceptible genomic sites experienced a dramatic (2-15-fold) increase in their burden of oxidative DNA damage in patients undergoing infertility evaluation compared to normal healthy donors. LIMITATIONS, REASONS FOR CAUTION: The limited number of samples analysed in this study warrants external validation, as do the implications of our findings. Selection of male fertility patients was based on high basal levels of oxidative stress within their spermatozoa as opposed to specific sub-classes of male factor infertility. WIDER IMPLICATIONS OF THE FINDINGS: The identification of genomic regions susceptible to oxidation in the male germ line will be of value in focusing future analyses into the mutational load carried by children in response to paternal factors such as age, the treatment of male infertility using ART and paternal exposure to environmental toxicants. STUDY FUNDING/COMPETING INTEREST(S): Project support was provided by the University of Newcastle's (UoN) Priority Research Centre for Reproductive Science. M.J.X. was a recipient of a UoN International Postgraduate Research Scholarship. B.N. is the recipient of a National Health and Medical Research Council of Australia Senior Research Fellowship. Authors declare no conflict of interest.