Evaluation of Protamine Level in Human Sperm Samples Using Chromomycin A3 and Aniline Blue Staining

Background: Current microscopic experimental methods cannot diagnose DNA damages present in spermatozoa .Therefore, some methods are needed to address the abnormality of the genetic material status on the sperm samples. As reported by many investigators aniline blue staining technique has been used for identifying sperm chromatin condensation. Also, chromomycin A3 is used for evaluation of the degree of protamination of spermatozoa. This study aimed at evaluating these two different staining techniques on human sperm protamine status. Materials and Methods: Sperm samples were collected from 72 males [including 37 infertile men: (seven asetenotratospermic, two trato-espermic, and one azo-spermic) and 35 healthy fertile men]   attending the research and clinical center for infertility affiliated with Babol University of Medical Sciences. Measurement of sperm motility, volume and density of semen samples were carried out in andrology laboratory. In estimation with light microscopy aniline blue tool, in each slide, blue stained were assumed as normal spermatozoa, but dark blue stained were regarded as abnormal spermatozoa. Bright yellow stained chromomycin-reacted spermatozoa (CMA3+) were observed under fluorescent microscope with 460 nm filter considered as normal and yellowish green were assumed as abnormal. Statistical analysis results were expressed as mean ± SD. Results: The rate of reacted spermatozoa to aniline blue in the infertile group was higher than that of the healthy control group 42.8% ±8.7 vs. 17.9% ±6.4. Also, the rate of reacted spermatozoa to CMA3 in infertile and normal group was [53.6 ± 8.7 and 24.7% ±5.1], respectively. Conclusion: Infertility status could be assessed by staining the spermatozoa via aniline blue and CMA3 techniques. Combination of these two staining methods had the best predictive values for semen analysis compared to using just one method. Our results showed that both CMA3 and AB staining methods were successful in detecting sperm chromatin defects. .