Introduction: Semen parameters and specially sperm morphology is used for the assessment of semen sample and for the prediction of the fertilization. However there is semen samples with normal sperm morphology but with hidden sperm chromatin anomalies. Since sperm morphology and chromatin anomalies reduce the fertilization rat independently, the purpose of this study is to consider the relation between the semen parameters with in vitro fertilization rate and the sperm chromatin status. Materials and Method: 101 infertile couples were randomly selected from the IVF candidate referred to Isfahan fertility and infertility canter. The semen samples were collected on the day of oocyte recovery major portions of the semen were prepared for routine IVF insemination and the rest was used for the following sperm nuclear maturity tests: chromomycin A3 (CMA3) fluorescence test, simple acridine orange test - with heat shock, aniline blue staining and SDS - test as well as semen parameters. Results: The highest significant correlation was obtained in CMA3 fluorescence test and aniline blue staining respectively. Sperm morphology and protamine dificiency can be measured by CMA3 which showed independent relationships to in vitro fertilization however among nuclear maturity tests, only CMA3 fluorescence showed an independent relationship to sperm morphology. Using ROC analysis, CMA3 fluorescence tests showed the highest sensitivity and pecifity with both in vitro fertilization rate and the morphology as compared to other nuclear maturing test. Since sperm morphology independent effects in vitro fertilization rate, on the other hand, CMA3 fluorescence test has also independent relation with sperm morphology and in vitro fertilization rate, therefore, we suggests that CMA3 fluorescence test can be used as a complementary sperm functional test along with the routine semen analysis.