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RESEARCH ARTICLEOpen AccessGenome-wide promoter methylation profile ofhuman testis and epididymis: identified fromcell-free seminal DNAChunlin Wu1, Xiaofang Ding2, Honggang Li1,3*, Changhong Zhu1,3and Chengliang Xiong1,3AbstractBackground: DNA methylation analysis is useful for investigation of male fertility in mammals, whereas the relianceon tissues limits the research on human. We have previously found the presence of high concentration of cell-freeseminal DNA (cfsDNA) in human semen. We proposed that some testis and epididymis-specific methylated promoterscould be detected in human cfsDNA, and thus hold promise as noninvasive epigenetic biomarkers for male infertility,of which most cases are caused by defects in testicular sperm production or epididymal sperm maturation.Results: The ejaculate of successfully vasectomized men does not contain any secretion from testis and epididymis.Here we compared genome-wide promoter methylation profiles in cfsDNA between health donors and post-vasectomy men. Promoters of 367 testis and epididymis-specific hypomethylated genes and 134 hypermethylatedgenes were identified. Subsequent validation by Methyl-DNA immunoprecipitation and MethyLight analysis confirmedthe result of promoter microarray. Gene Ontology analysis revealed many genes involved in male reproduction.Conclusion: We detected the testis and epididymis-specific methylated promoters in human cfsDNA, which may beused for noninvasive epigenetic biomarkers for the study and diagnosis of male infertility.Keywords: DNA methylation, Cell-free seminal DNA, Testis, Epididymis, Vasectomy, Noninvasive diagnosisBackgroundCell-free nucleic acids, including DNA and RNA, existubiquitously as cell-free or being absorbed at the cell sur-face of living organisms [1]. They are found to be releasedvia apoptotic or necrotic cells, and be actively secreted byliving cells [1,2]. Cell-free DNA, as a type of cell-free nu-cleic acids, can be isolated from various human body fluidsincluding blood plasma [3,4], urine [5], cerebrospinal fluid[6], bronchoalveolar lavage fluid [7], amniotic fluid [8],and seminal plasma [9,10]. The isolated cell-free DNA canbe used to examine DNA integrity, microsatellite instabil-ity, loss of heterozygosity, mutations, polymorphisms, andDNA methylation [2]. In recent years, considerable atten-tion has been paid to taking advantage of tissue or cell-specific DNA methylation markers in cell-free DNA,which represents one of the most promising approachesfor detection and prognosis of cancers, and prenatal diag-nosis [2,4,8].Cell-free seminal DNA (cfsDNA) has been detected inhuman semen [10]. Our previous work has isolatedcfsDNA and described its characteristics [9]. Given thathuman ejaculate is a mixture secreted from bilateral testes,epididymides, seminal vesicles, bulbourethral glands, andthe prostate [11], cfsDNA should contain DNA epigeneticinformation of these organs. Moreover, these DNA epi-genetic information should be undetectable in the blood,because DNA should not pass the physical barrier be-tween the blood and the male reproduction system includ-ing blood-testis barrier and blood-epididymis barrier.DNA epigenetic modifications are essential for sperm-atogenesis. Remethylation occurs from the prosperma-togonia stage onwards and results in mature spermatozoawith an adequate DNA methylation pattern [12]. Themethylation status of some genes is changed in some con-ditions that cause male infertility [13]. We presumed thatthe promoters of testis and epididymis-specific methylated* Correspondence: lhgyx@hotmail.com1Family Planning Research Institute/Center of Reproductive Medicine, TongjiMedical College, Huazhong University of Science and Technology, Wuhan430030, China3Wuhan Tongji Reproductive Medicine Hospital, Wuhan 430013, ChinaFull list of author information is available at the end of the article© 2013 Wu et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the CreativeCommons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, andreproduction in any medium, provided the original work is properly cited.Wu et al. BMC Genomics 2013, 14:288http://www.biomedcentral.com/1471-2164/14/288