Flow Cytometry: A New Approach for Indirect Assessment of Sperm Protamine Deficiency

Flow cytometry (FCM) has been extensively used to study mammalian sperm in the areas of clinical andrology and reproductive toxicology. FCM provides a powerful advantage over microscopy technique in terms of rapid, accurate and reproducible technology for the quantification of various cell characteristics, including chromatin status. During spermiogenesis, histones are replaced by protamines resulting in a very condensed structure of sperm chromatin. Infertile men have an increased sperm histone: protamine ratio than fertile counterparts. Chromomycin A3 (CMA3) staining represents a useful tool for assessing the packaging quality of sperm chromatin and allows indirect visualization of protamine deficiency. Routinely, fluorescence microscope is used for evaluation of protamine deficiency by CMA3. Considering the advantages of FCM and increasing use of CMA3 in assessment of protamine deficiency in the literature and its possible use as a diagnostic test, the aim of this study is to standardize this procedure for routine laboratory analysis. Semen samples were collected from 85 infertile men who referred to Isfahan Fertility and Infertility Center. A portion of semen sample was used for routine semen analysis according to WHO criteria and the remainder were evaluated to standardize CMA3 staining procedure for fixation, the number of sperm and duration of exposure to CMA3. The results were compared with standard fluorescent microscopic procedure. Percentage CMA3 positive sperm were compared between flow cytometry and standard fluorescent microscopic procedure. Our results show that fixation, the number of sperm and duration of exposure to CMA3 can affect on FCM outcomes. In addition we show that the samples can be fixed, stained with CMA3, stores and then assessed for FCM. The optimal conditions for FCM assessment of CMA3 are: fixation, concentration of 0.25 mg/ml, sperm density of 2 million/ml and exposure for 60 minutes.