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To assess the influence of structural differences in sperm nuclei on embryo development in intracytoplasmic sperm injection (ICSI).Semen obtained from forty-four infertile patients who underwent ICSI was examined. In assessing blastocyst development, only those patients who had successfully obtained over five fertilized eggs were included to exclude any oocyte factors (n = 22). Spermatozoa were assessed using excitation fluorescence flow cytometry with acridine orange and the sperm chromatin dispersion (SCD) test.There was a significant positive correlation between the COMP values obtained from flow cytometry and blastocyst formation. (r = 0.477, p = 0.025). There was a significant negative correlation between the SCD values representing DNA fragmentation and blastocyst formation. (r = 0.796, p < 0.001). COMP values and SCD values were independent parameters to assess sperm nuclear quality regarding embryo development in vitro (r = 0.224, p = 0.080).Results suggest that injection of spermatozoa with fewer disulfide bonds and less nuclear DNA fragmentation could achieve better blastocyst formation in human ICSI. Assessment of sperm chromatin should help to predict embryo development after ICSI.