The study aimed to evaluate the role of chromatin packaging (CMA3 staining), sperm morphology during sperm-zona binding, sperm decondensation and the presence of polar bodies in oocytes that failed in vitro fertilization (IVF). The percentage CMA3 staining categorized the data into three groups, < 44%, n = 10; > or = 44-59%, n = 10; and > or = 60%, n = 29. Morphology groups were < or = 4% (n = 11); > 4-14% (n = 19); and > 4% (n = 19). One hundred and seventy-two oocytes that failed IVF were evaluated for sperm-zona binding, ooplasma penetration and sperm decondensation. Odds ratio analyses indicated that being in the > or = 60% CMA3 staining group resulted in a 15.6 fold increase in the risk of decondensation failure, relative to CMA3, staining of < 44%. For morphology, there was a 2.17 fold decrease in the risk of fertilization failure in the morphology group with > 4-14% normal cells, while it increased 2.45 fold for the morphology group with < or = 4% normal cells. Using CMA3 fluorescence to discriminate, 51% of the oocytes in the group with elevated CMA3 fluorescence had no sperm in the ooplasma compared to 32% and 16% penetration failure in the CMA3 staining groups > or = 44-59% and < 44%, respectively. Sperm chromatin packaging quality and sperm morphology assessments are useful clinical indicators of human fertilization failure. Immunofluorescence techniques could be used to provide a clear diagnosis of failed fertilization.