In vitro maturation (IVM) of human oocytes is important in assisted reproductive technology (ART) laboratories. The present study aimed to investigate the effect of growth differentiation factor 9 (GDF-9) additions into IVM medium, and co-culture of cumulus cells (CCs) on IVM outcome. 385 immature oocytes in germinal vesicle (GV) stage were divided into four groups based on the IVM culture condition; 1) GV oocytes cultured in standard IVM medium (control), 2) GV oocytes co- cultured with CCs, 3) GV oocytes treated with GDF-9, and 4) GV oocytes co- cultured with CCs and treated with GDF-9, simultaneously. The presence of meiotic spindle (MS) and zona pellucida (ZP) birefringence were assessed in IVM oocytes, followed by intracytoplasmic sperm injection (ICSI). Finally, fertilization rate and embryo development in two-day-old embryos were evaluated. Maturation rates of GV oocytes were similar a whole groups following 24-48 h of incubation. A significant higher rate of oocytes matured in the medium treated with CCs reached 2 pronuclear (2PN) stage compared with those matured in the standard medium, respectively (76.5% vs. 51.6%, P= 0.01). The percentage of oocytes with visible MS was higher in all treated groups, but this difference was significant between the oocytes matured in medium supplemented with GDF-9 and control group (45% vs. 26.6% respectively, P= 0.05). Co-culturing of GV oocytes with CCs and/or supplementation of IVM medium with GDF-9 are potentially involved in improvement of fertilization and embryo development. Also, MS assessment provides an early predictor of embryo development.