P-6: Flow Cytometry: Assessment of Protamine Deficiency by CMA3 Staining and Apoptotic Bodies

Background: Chromomycin A3 (CMA3) staining, with the slide method or fluorescence microscopy, is widely used for indirect assessment of protamine deficiency in a semen sample. Flow cytometry is the most suitable tool which to improve assessment accuracy, both in terms of statistical analysis and to prevent observer variation. This study provides a simple procedure to account for merocyanine 540 (M540) or apoptotic bodies which results in underestimation of the percentage of CMA3 positivity using propidium iodide (PI) staining. Therefore, the aim of this study was to evaluate the percentage of CMA3 by using PI staining to exclude M540 bodies that prevent underestimation of CMA3 staining. Materials and Methods: Semen samples were collected from 104 infertile men who referred to the Andrology Unit of the Isfahan Fertility and Infertility Center. Semen samples were initially assessed according to WHO and, washed twice with Ham’s. Each sample was divided into two portions. One portion was used as a control while the other was processed for density gradient centrifugation. Each portion was assessed for CMA3 staining by both the slide and flow cytometry methods. Results: Detection of CMA3 staining is more appropriate with fluorescence detector 3 (FL3) rather than fluorescence detector 2 (FL2). Conclusion: This study for the first time provides the basis for assessment of CMA3 staining for flow cytometry. However, since the maximum excitation for CMA3 is not covered by the 488 nm laser therefore, we recommend further experimentation using a flow cytometer with optimal excitation.