Measurement of epigenetic alterations from patient’s tissues in myoma, adenomyoma and endometriosis

Background: Myoma, adenomyoma, and endometriosis are estrogen-dependent gynecologic diseases and result in reproductive dysfunction and pelvic pain in women. However, these gynecologic diseases have a complex and poorly understood etiology, involving both genetic and environmental factors. Epigenetic alterations, heritable changes that can modify gene expression without affecting genetic sequence, are associated with the development and progression of numerous pathological states and diseases. Therefore, there is great potential for the use of epigenetics as biomarkers to better understand the early-stage biological responses and molecular mechanisms of gynecologic diesases. We aimed to examine levels of global DNA and gene-specific methylation, which are epigenetic alterations that could be associated with development of gynecologic diseases, including myoma, adenomyoma, and endometriosis. Methods: We measured global DNA methylation (LINE-1) as well as disease relevant gene-specific methylation (i.e. ER, PR, and aromatase) using pyrosequencing assay. For this measurement, gene-specific primers for the selected genes were designed using the Pyro-Mark assay design software. Genomic DNAs from each tissue were extracted, and underwent bisulfite modification to convert unmethylated cytosine residues to uracil. A Pyromark Q96 MD was used for all subsequent pyrosequencing. Samples were processed in duplicates on plates with water controls. Percent methylation of a sample was calculated by averaging all of the interrogated CpG sites. Results: Different methylation levels of selected genes were measured from myoma, adenomyoma, and endometriosis tissues. Our obtained results suggest that epigenetic changes are involved in development of different types of gynecologic diseases.