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This prospective study was designed to evaluate and clarify further whether the position of the polar body (PB) in relation to injection site during intracytoplasmatic sperm injection (ICSI) has an impact on fertilization and developmental rates and consequently clinical pregnancy outcome. The study included 264 patients undergoing 306 ICSI cycles from September 2007 to January 2009 performed by the same practitioner. Of all oocytes retrieved, 1736 were in metaphase II (MII). From every woman reaching ovum pick up, all MII-collected oocytes were allocated to 1 of the 4 groups according to PB orientation. In group A, MII oocytes were injected with the PB at 6 o’clock, group B with the PB at 7 o’clock, group C with the PB at 11 o’clock, and a group D with the PB at 12 o’clock. A significantly higher proportion of fertilized oocytes were produced from oocytes that had been injected with the PB at 11 o’clock (79.2%) as compared to those at 6 o’clock (70.5%), 7 o’clock (64.4%), and 12 o’clock (68.8%). Furthermore, embryos derived from oocytes that were injected with the PB at 11 o’clock appeared to be of higher quality score than those of the other groups of oocytes. A higher clinical pregnancy rate (28.7%) was obtained after the transfer of embryos from oocytes that had been injected with the PB at 11 o’clock. Given the higher fertilization, developmental, and pregnancy rate in the 11 o’clock group, it is suggested that this may be the preferred position of the PB at ICSI.