DNA methylation patterns are altered in numerous diseases and often correlate with clinically relevant information such as disease subtypes, prognosis and drug response. With suitable assays and after validation in large cohorts, such associations can be exploited for clinical diagnostics and personalized treatment decisions. Here we describe the results of a community-wide benchmarking study comparing the performance of all widely used methods for DNA methylation analysis that are compatible with routine clinical use. We shipped 32 reference samples to 18 laboratories in seven different countries. Researchers in those laboratories collectively contributed 21 locus-specific assays for an average of 27 predefined genomic regions, as well as six global assays. We evaluated assay sensitivity on low-input samples and assessed the assays' ability to discriminate between cell types. Good agreement was observed across all tested methods, with amplicon bisulfite sequencing and bisulfite pyrosequencing showing the best all-round performance. Our technology comparison can inform the selection, optimization and use of DNA methylation assays in large-scale validation studies, biomarker development and clinical diagnostics.

Cortactin is required for endothelial barrier function and leukocyte recruitment in vivo.

The maintenance of vascular barrier integrity is crucial during many pathophysiological processes and requires coordinated molecular events including signaling and cytoskeletal remodeling. Cortactin is an actin‐binding protein that regulates actin dynamics and related signaling events. To analyze the role of cortactin in vascular barrier functions, we generated cortactin‐deficient mice. Classical Miles assays revealed that vascular permeability is increased in these mice. Despite this leakiness, leukocyte extravasation in the TNF‐α‐stimulated cremaster was inhibited. Mechanistically, the permeability defect in the absence of cortactin was caused by reduced levels of active Rap1 in endothelial cells and could be rescued by activating Rap1 via the GTPase exchange factor EPAC. The defect in leukocyte extravasation was due to impaired interactions between leukocytes and endothelial cells, as documented by an increased rolling velocity and decreased numbers of adhering leukocytes in postcapillary venules. Impaired rolling interactions were linked to contributions of β2‐integrin‐ligands to this process and firm adhesion was compromised by the loss of ICAM‐1‐containing leukocyte docking structures. These results are the first physiological evidence that cortactin is crucial for orchestrating molecular events leading to proper endothelial barrier function and leukocyte recruitment in vivo.

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.

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.