Promoter targeted bisulfite sequencing reveals DNA methylation profiles associated with low sperm motility in asthenozoospermia

Human Reproduction, Vol.31, No.1 pp. 24– 33, 2016 Advanced Access publication on November 30, 2015 doi:10.1093/humrep/dev283 ORIGINAL ARTICLE Andrology Promoter targeted bisulfite sequencing reveals DNA methylation profiles associated with low sperm motility in asthenozoospermia Ye Du1,†, Meiyan Li2,†, Jing Chen1, Yonggang Duan 1, Xuebin Wang 2, Yong Qiu 2, Zhiming Cai 1, Yaoting Gui 1,*, and Hui Jiang 2,* 1 Guangdong Key Laboratory of Male Reproductive Medicine and Genetics, Peking University Shenzhen Hospital, FuTian District, Shenzhen 518036, China 2BGI-Shenzhen, Shenzhen 518083, China *Correspondence address. E-mail: (Y.T.G.)/ (H.J.) Submitted on June 4, 2015; resubmitted on September 28, 2015; accepted on October 20, 2015 study question: Is there an association between sperm DNA methylation profiles and asthenozoospermia? summary answer: DNA methylation, at specific CpGs but not at the global level, was significantly different between low motile sperm cells of asthenozoospermic individuals and high motile sperm cells of normozoospermic controls. what is known already: Aberrant DNA methylation, both globally and restricted to a specific gene locus, has been associated with male infertility and abnormal semen parameters. study design, size, duration: This was a case–control study investigating the differences in DNA methylation at CpGs in promoter regions between high and low motile sperm cells from eight normozoospermic controls and seven asthenozoospermic patients. participants/materials, setting, methods: The liquid hybridization capture-based bisulfite sequencing method was used to determine DNA methylation at CpGs in promoter regions. The global inter-individual and intra-individual methylation variability were estimated by evaluating the methylation variance between and within different motile sperm fractions from the same or different individuals. Asthenozoospermia-associated differentially methylated or variable CpGs and differentially methylated regions were identified by comparing the DNA methylation of high motile sperm cells from normozoospermic controls with that of low motile sperm cells from asthenozoospermic patients. main results and the role of chance: In this study, we determined the global DNA methylation level (24.7%), inter-individual variance (14.4%) and intra-individual differences between high and low motile sperm fractions (3.9%). We demonstrated that there were no statistically significant differences in either the global DNA methylation level or global methylation variability between sperm from men with normozoospermia or asthenozoospermia. Between high motile sperm from men with normozoospermia and low motile sperm from men with asthenozoospermia, we identified 134 differentially methylated CpGs, 41 differentially methylated regions and 134 differentially variable CpGs. The genomic distribution patterns of the differential methylation spectrum suggested that gene expression may be affected in low motile sperm cells of asthenozoospermic patients. Finally, through a functional analysis, we detected 16 differentially methylated or variable genes that are required for spermatogenesis and sperm motility or dominantly expressed in testis. limitations, reasons for caution: The sample size in this study was limited, although the participants in the two groups were carefully selected and well matched. Our results must be verified in larger cohorts with the use of different techniques. Furthermore, our results were descriptive, and follow-up studies will be needed to elucidate the effect of differential methylation profiles on asthenozoospermia. wider implications of the findings: Our study identified asthenozoospermia-associated DNA methylation profiles and proposed a list of genes, which were suggested to be involved in the regulation of sperm motility through an alteration of DNA methylation. These results will provide promising clues for understanding the effect of DNA methylation on sperm motility and asthenozoospermia. † The authors consider that the first two authors should be regarded as joint First Authors. & The Author 2015. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: 25 DNA methylation profiles in asthenozoospermia study funding/competing interest(s): This study was funded primarily by the National Natural Science Foundation of China, Shenzhen Project of Science and Technology and the National Basic Research Program of China. The authors have no competing interests. Key words: asthenozoospermia / DNA methylation / low motile sperm / promoter targeted bisulfite sequencing / potential clinical markers Introduction Asthenozoospermia, mainly characterized by low sperm motility, is detected in .40% of infertile men (Jungwirth et al., 2012). The etiology of asthenozoospermia is multifactorial and closely related to endocrine disturbances, environmental factors, life experience and even aging, but the exact causal factors remain largely unknown (Luconi et al., 2006; Homan et al., 2007; Guo et al., 2014; Eslamian et al., 2015). In mouse studies, based on knock out and mutagenesis methods, mutations in some genes have been associated with altered sperm motility, but none have been confirmed in humans (Luconi et al., 2006). Although nearly 50% of infertility cases are estimated to be due to genetic defects (Ferlin et al., 2006; Zorrilla and Yatsenko, 2013), for some cases of male infertility, genetic factors do not appear to be the major cause (Aston and Carrell, 2009). An increasing body of evidence supports the idea that abnormal epigenetic changes also contribute to male infertility (Boissonnas et al., 2013). DNA methylation, one of the best studied epigenetic mechanisms, is an essential epigenetic modification typically associated with gene silencing (Lande-Diner et al., 2007). During the past decade, aberrant DNA methylation of both imprinted and non-imprinted genes have been found to be associated with abnormal semen parameters (e.g. sperm concentration and sperm motility) in infertile patients (Houshdaran et al., 2007; Kobayashi et al., 2007; Boissonnas et al., 2010; Hammoud et al., 2010; Marques et al., 2010; Poplinski et al., 2010; Pacheco et al., 2011; Sato et al., 2011). The development of large-scale DNA methylation detection technologies makes the study of DNA methylation of larger regions or even the whole genome feasible. Based on these technologies, aberrant sperm DNA methylation spectrums have been demonstrated to be associated with some types of male infertility (Houshdaran et al., 2007; Navarro-Costa et al., 2010; Wu et al., 2010; Pacheco et al., 2011; Aston et al., 2012; Ferfouri et al., 2013; Urdinguio et al., 2015). However, the majority of the previous studies have used predesigned DNA methylation microarrays, which are only able to interrogate a fixed and small fraction of the entire DNA methylome. This inc (...truncated)