Chromatin compaction and methylation status are biomarkers to detect the pattern and quality of sperm DNA prior to ART. The purpose of this study is to compare two criteria of sperm chromatin compaction and global methylation status in relation to the functional quality of human spemratozoa. The confocal microscopy and flowcytometry showed the Immunocytofluorescent pattern of ChromomycinA3 (CMA3) staining and the 5-methyl cytosine, sequencely. The CMA3positivity level showed a quality relation dependency (p<0.0001), also significant correlation (P<0.05) with the flowcytometry level of global methylation. Moreover confocal microscopy from CMA3 stained head of sperm demonstrated the spatial pattern of chromatids. Overall the results of this study support the concept that perfect spermatozoa collected from the high density Percoll fraction possesses higher compaction related to hypomethylated nuclear DNA. Interestingly the results of ChromomycinA3 assay demonstrated spatial geometry of chromatids in the sperm head. Also direct significant correlation with methylation status suggesting that during the development of spermatozoa, failure to chromatin compaction is associated with more extensive methylation of sperm DNA in poor quality of spermatozoa.