Paternal age effects on the sperm epigenome and its impact on the next generation

• Applicant: Professor Dr. Thomas Haaf
• Subject Area: Reproductive Medicine, Urology ; Human Genetics
• Term: from 2019 to 2023
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 422212342

Compared to numerous studies on epigenetic programming by maternal factors, little is known about the influence of paternal factors on the next generation. Aim of this proposal is to identify evolutionarily conserved sperm methylation patterns which are influenced by paternal age and to find out to which extent these epigenetic changes are transmitted into the next generation. To detect epigenetic signatures for semen quality, paternal age and outcome of assisted reproductive technologies (ART) in the human sperm methylome, we will perform reduced representation bisulfite sequencing (RRBS) on four groups of 24 carefully matched (for known confounding factors) sperm samples each, with normal and abnormal semen parameters, respectively, either leading or not leading to a pregnancy/live birth. For a subset of 12 samples per group, hydroxymethylation will be studied by oxidative (ox) bisulfite sequencing. To identify similar age-related methylation changes in non-human primates and the bovine model, RRBS and RRoxBS will be performed on sperm samples from 16 common marmosets of different age classes and bovine sperm samples collected at young and old ages, respectively from the same 12 bulls. Identical correlations between sperm methylation and paternal age in three species will point to functionally relevant genes/pathways susceptible to paternal ageing. The 10 most promising candidate genes each for predicting paternal age and/or ART outcome will be validated by bisulfite pyrosequencing in an independent replication cohort of up to 1000 human sperm samples. To study transmission of human age-related sperm methylation changes to the offspring, informative cord blood samples conceived by IVF/ICSI with the analysed sperm samples will be identified. Deep bisulfite sequencing combined with SNP typing allows one to distinguish between paternal and maternal allele methylation and to correlate sperm methylation with paternal allele-methylation (in diploid somatic cells). In many aspects including embryo development and gestation, the bovine model is more similar to the human than rodents. Bovine blastocyst embryos will be produced by in vitro maturation and fertilization of slaughterhouse oocytes with sperm samples from three bulls at young and old age, respectively. Bisulfite pyrosequencing and Taqman assays will reveal possible paternal age effects on embryonal rDNA methylation and expression. RNA sequencing of individual blastocysts will be used to compare the transcriptomes of embryos conceived by sperm from young vs. old bulls. Collectively, our study will provide comprehensive information on the association of the sperm methylome with paternal age, ART outcome, embryogenesis and transmission to the offspring epigenome. Such data are urgently needed to better assess the epigenetic safety of ART.

• DFG Programme: Research Grants