Abstract:
Objective: To examine an IVF cohort for imprinted and genome-wide DNA methylation abnormalities. Design: Retrospective study. Setting: Research laboratory. Patient(s): DNA samples from a previously described IVF cohort that comprised 66 IVF-conceived prepubertal children (IVF, n ¼ 34; intracytoplasmic sperm injection, n ¼ 32) and 69 matched naturally conceived controls. Intervention(s): DNA methylation was examined at four imprinted gene loci (H19, SNRPN, KCNQ1OT1, and IGF2) and satellite 2 using methylationsensitive quantitative polymerase chain reaction (MSQ-PCR) followed by bisulfite sequencing at H19, SNRPN, and KCNQ1OT1. Methylated DNA immunoprecipitation (MeDIP) microarray with validation using the Sequenom MassARRAY EpiTYPER platform was also used. Main Outcome Measure(s): Percentage of DNA methylation by MSQ-PCR, differential methylation based on microarray signal intensity, and percentage DNA methylation as determined by Sequenom MassARRAY EpiTYPER were compared. Result(s): No differences in percentage of methylation between the IVF and control group were observed at H19, KCNQ1OT1, SNRPN, or IGF2. Absence of aberrant imprinting was confirmed using bisulfite sequencing. Methylation of satellite 2 repeats (a surrogate for global methylation) showed no difference between the IVF and control groups. MeDIP was used to screen for differences in promoter methylation. Subsequent quantification of methylation of eight candidate genes using the Sequenom MassARRAY EpiTYPER system did not reveal any differential methylation. Conclusion(s): Low-level imprinting errors are not common in the IVF population.
