Flow Cytometry: A Novel Approach for Indirect Assessment of Protamine Deficiency by CMA3 Staining, Taking into Account the Presence of M540 or Apoptotic Bodies

Abstract

Background: Chromomycin A3 (CMA3) staining, either by 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 to improve assessment accuracy, both in terms of statistical analysis and for prevention of observer variation. This study provides a simple procedure to account for merocyanine 540 (M540) or apoptotic bodies, which result in underestimation of the percentage of CMA3 positivity, by using propidium iodide (PI) staining. Therefore, this study aims to evaluate the percentage of CMA3 by PI staining to exclude M540 bodies that prevent underestimation of CMA3 staining.

Materials and methods: This study is an experimental study. Semen samples collected from 104 infertile men who referred to the Andrology Unit of the Isfahan Fertility and Infertility Center were initially assessed according to World Health Organization (WHO) criteria. Samples were washed twice with Ham's. Each sample was divided into two portions, a control and the other processed for density gradient centrifugation (DGC). Each portion was assessed for CMA3 staining by both the slide and flow cytometry methods. Coefficients of correlation and student t-test were carried out using the Statistical Package for the Social Studies (SPSS 11.5).

Results: Detection of CMA3 staining was more appropriate with fluorescence detector 3 (FL-3) rather than fluorescence detector 2 (FL-2) in the evaluation of protamine deficiency to exclude M540 bodies.

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, we recommend further experimentation using a flow cytometer with optimal excitation.

Keywords: Flow Cytometry; Merocyanine; Protamine; Sperm.

Fig 1

(A) Dot plot of M540 versus Yo-Pro-1 staining and (B) PI histogram in a semen sample. (C). Correlation between the percentage of PI negative cells and M540 positive/ Yo-Pro-1 negative. (D) Dot plot of M540 versus Yo-Pro-1 staining and (E) PI histogram in a processed sample by DGC. (F) Comparison of the percentage of PI negative cells and M540 positive/ Yo-Pro-1 negative before and after processing. Common letters are significantly different at p<0.05.

Fig 2

Comparison of the percentage of sperm with CMA3 positivity by fluorescence microscopy and flow cytometry using fluorescence detectors 2 or 3 (FL-2 and FL-3) in semen and DGC. Common letters are significantly different at p<0.05.

Fig 3

Correlations of the percentage of CMA3 positivity assessed by fluorescence microscopy and flow cytometry using fluorescence detector 2 or 3 (FL-2 and FL-3).

Fig 4

Comparison of the percentage of sperm with CMA3 positivity between the slide method and flow cytometry using fluorescence detectors 2 or 3 (FL-2 and FL-3). Common letters are significantly different at p<0.05.