Imprinted genes are differentially methylated during gametogenesis to allow parental-specific monoallelic expression of genes. During mouse oogenesis, DNA methylation at imprinted genes is established during the transition from primordial to antral follicle stages. Studies in human and mouse suggest aberrant imprinting in oocytes following in vitro maturation and after superovulation with high doses of gonadotrophines. The exact mechanisms leading to aberrant imprinting are unknown. We examined the methylation status of differentially methylated regions of key imprinted genes (by bisulphite sequencing) in mouse metaphase II oocytes, grown in a long-term pre-antral follicle culture system and matured in vitro, in the presence of a physiological (10 IU/L) and a high (100 IU/L) recombinant FSH dose. Our results showed a normal DNA methylation at the studied regulatory sequences of Snrpn, Igf2r and H19, demonstrating that 1) prolonged culture and in vitro maturation do not per se modify the establishment of imprinting in oocytes and 2) supraphysiological FSH doses do not induce aberrant DNA methylation at the studied regulatory sequences in this system. © 2009 UBC Press.