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

与众多关于母性因素对表观遗传编程的研究相比，关于父性因素对下一代的影响知之甚少。这一建议的目的是确定进化上保守的精子甲基化模式，这些模式受到父亲年龄的影响，并找出这些表观遗传变化在多大程度上传递给下一代。为了检测人类精子甲基组中精液质量、父亲年龄和辅助生殖技术(ART)结局的表观遗传学特征，我们将对四组24个精液样本进行简化代表性亚硫酸盐测序(RRBS)，每个样本具有正常和异常的精液参数，分别导致或不导致怀孕/活产。对于每组12个样品的子集，将通过氧化(OX)亚硫酸氢盐测序来研究羟甲基化。为了确定在非人类灵长类动物和牛模型中类似的与年龄相关的甲基化变化，我们将分别对16只不同年龄段的普通绒猴的精子样本和从同一12头公牛身上收集的年轻和老年牛精子样本进行RRBS和RRoxBS。在三个物种中，精子甲基化和父亲年龄之间的相同相关性将指向功能相关的基因/路径，容易受到父亲年龄的影响。预测父亲年龄和/或ART结局的10个最有希望的候选基因将通过在多达1000个人类精子样本的独立复制队列中进行亚硫酸氢盐焦磷酸测序来验证。为了研究人类年龄相关的精子甲基化变化传递给后代的情况，将通过IVF/ICSI受孕的脐带血样本与分析后的精子样本进行鉴定。深度亚硫酸氢盐测序与SNP分型相结合，可以区分父亲和母亲的等位基因甲基化，并将精子甲基化与父亲的等位基因甲基化联系起来(在二倍体体细胞中)。在胚胎发育和妊娠等许多方面，牛模型比啮齿动物更接近人类。用3头公牛的精液分别在幼年和老年对屠宰场的卵母细胞进行体外成熟和受精，得到牛胚泡胚胎。亚硫酸氢盐焦解测序和Taqman分析将揭示父亲年龄对胚胎rDNA甲基化和表达的可能影响。单个胚泡的RNA测序将用于比较年轻公牛和老年公牛精子受孕的胚胎的转录。总而言之，我们的研究将提供关于精子甲基组与父亲年龄、ART结局、胚胎发生和传递给后代表观基因组的关联的全面信息。为了更好地评估ART的表观遗传安全性，迫切需要这样的数据。

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.