Clinics of M€ unster, M€ unster, Germany SUMMARYRecent studies have shown associations of aberrant DNA methylation in spermatozoa with idiopathic infertility. The analysis of DNA methylation of specific genes could therefore serve as a valuable diagnostic marker in clinical andrology. For this purpose, rapid and reliable detection methods, reference values and the temporal stability of spermatozoal DNA methylation need to be established and demonstrated. In this prospective study, swim-up purified semen samples from 212 consecutive patients (single samples), 31 normozoospermic volunteers (single samples) and 10 normozoospermic volunteers (four samples at days 1, 3, 42 and 45 plus a fifth sample after 180-951 days) were collected. Spermatozoal DNA was isolated, bisulphite converted and DNA methylation was analysed by pyrosequencing. DNA methylation of the maternally imprinted gene MEST was measured in samples of 212 patients and 31 normozoospermic volunteers and the temporal stability of eight different genes and two repetitive elements was examined in consecutive samples of 10 normozoospermic volunteers. MEST DNA methylation was significantly associated with oligozoospermia, decreased bi-testicular volume and increased FSH levels. A reference range for spermatozoal MEST DNA methylation (0-15%) was established using the 95th percentile of DNA methylation in normozoospermic volunteers. Using this reference range, around 23% of our patient cohort displayed an aberrant MEST DNA methylation. This epigenetic aberration was found to be significantly associated with bi-testicular volume, sperm concentration and total sperm count. DNA methylation in normozoospermic volunteers was stable over a time period of up to 951 days in contrast to classical semen parameters. Our data show that MEST DNA methylation fulfils the prerequisites to be used as routine parameter and support its use during andrological workup if a prognostic value can be shown in future.

The X-linked RHOX cluster encodes a set of homeobox genes that are selectively expressed in the reproductive tract. Members of the RHOX cluster regulate target genes important for spermatogenesis promote male fertility in mice. Studies show that demethylating agents strongly upregulate the expression of mouse Rhox genes, suggesting that they are regulated by DNA methylation. However, whether this extends to human RHOX genes, whether DNA methylation directly regulates RHOX gene transcription and how this relates to human male infertility are unknown. To address these issues, we first defined the promoter regions of human RHOX genes and performed gain- and loss-of-function experiments to determine whether human RHOX gene transcription is regulated by DNA methylation. Our results indicated that DNA methylation is necessary and sufficient to silence human RHOX gene expression. To determine whether RHOX cluster methylation associates with male infertility, we evaluated the methylation status of RHOX genes in sperm from a large cohort of infertility patients. Linear regression analysis revealed a strong association between RHOX gene cluster hypermethylation and three independent types of semen abnormalities. Hypermethylation was restricted specifically to the RHOX cluster; we did not observe it in genes immediately adjacent to it on the X chromosome. Our results strongly suggest that human RHOX homeobox genes are under an epigenetic control mechanism that is aberrantly regulated in infertility patients. We propose that hypermethylation of the RHOX gene cluster serves as a marker for idiopathic infertility and that it is a candidate to exert a causal role in male infertility.

Purpose Assess short-and mid-term impact of cryopreservation on DNA methylation status of different genes in spermatozoa. Methods Semen samples from 10 healthy normozoospermic men were collected at the Department of Clinical Andrology of the Centre of Reproductive Medicine and Andrology (Muenster, Germany). Each was divided into four equal aliquots: 1) untreated, 2) diluted in cryoprotectant, 3) short term (2 days) cryopreserved and 4) mid term (4 weeks) cryopreserved. Samples were "swim-up" purified prior to analysis. DNA fragmentation was measured using comet assay and Flow cytometric evaluation with Acridine Orange (FCEAO). The degree of methylation of nine genes was determined by bisulfite pyrosequencing of genomic DNA. Result(s) Analysis of three maternally imprinted genes (LIT1, SNRPN, MEST), two paternally imprinted genes (MEG3, H19), two repetitive elements (ALU, LINE1), one spermatogenesis-specific gene (VASA) and one gene associated with male infertility (MTHFR) in semen samples demonstrated no alteration in methylation pattern regardless of duration of cryopreservation. Conclusion(s) The lack of any changes in the sub-fraction of the genome examined in our study, implies that sperm DNA methylation is unaffected by cryopreservation and suggests that this daily clinical routine is safe in terms of DNA methylation.