Timeline of pre- and postovulatory aging in vivo (A, B) and in vitro (C, D).

A, B) For in vivo maturation of oocytes, follicle maturation was stimulated by PMSG on day 0. Ovulation was induced by hCG 48 h later. Control oocytes were collected from the ampullae the next morning. For preovulatory in vivo aging, ovulation was delayed by the GnRH antagonist cetrorelix for 3 d (A). Oocytes for postovulatory aging were collected at the same time as controls and cultured in M2 medium for further 12 or 24 h (B). C, D) For in vitro growth and maturation of oocytes, preantral follicles were cultured for 12 d in the presence of rLH and rFSH to the antral follicle stage. Ovulation was induced with rhCG/rEGF and control oocytes were collected after 18 h. To obtain preovulatory-aged oocytes, ovulation was induced with rhCG/rEGF on day 15 of follicle culture instead of day 12 (C). For postovulatory aging, ovulation was triggered with rhCG/rEGF and oocytes were incubated for additional 12 h before collection (D).