Comparison of three staining methods for the morphological evaluation of human spermatozoa  Ralf Henkel, Ph.D., Gerhard Schreiber, M.D., Anne Sturmhoefel, M.D., Uta-Christina Hipler, Ph.D., Dirk Henrik Zermann, M.D., Roelof Menkveld, Ph.D.  Fertility and Sterility  Volume 89, Issue 2, Pages 449-455 (February 2008) DOI: 10.1016/j.fertnstert.2007.03.027 Copyright © 2008 American Society for Reproductive Medicine Terms and Conditions

Figure 1 Human sperm morphology after staining smears according to (A) Papanicolaou and (B) Shorr and (C) with Testsimplets®. Increased background staining on the Testsimplets® slide is clearly visible. Fertility and Sterility 2008 89, 449-455DOI: (10.1016/j.fertnstert.2007.03.027) Copyright © 2008 American Society for Reproductive Medicine Terms and Conditions

Figure 2 Bland-Altman plots for the comparison of the three techniques regarding normal sperm morphology (A–C) and flagellar defects (D–F). The deviation for the Testsimplets® method is obvious, especially for flagellar defects. In addition, B, C, E, and F for the comparison with the Testsimplets® show that the variation strongly depends on the magnitude of the measurement. N = 94. Fertility and Sterility 2008 89, 449-455DOI: (10.1016/j.fertnstert.2007.03.027) Copyright © 2008 American Society for Reproductive Medicine Terms and Conditions

Figure 3 Mountain plots for the comparison of the Shorr and Testsimplets® methods with the Papanicolaou stain. (A) Normal sperm morphology; (B) flagellar defects. Whereas the Shorr and Papanicolaou methods correspond quite well with each other, the deviation from zero for the Testsimplets® stain results compared with the Papanicolaou stain results as reference shows that the two methods are not unbiased. Moreover, the long tail to the right-hand side of the Testsimplets® plot in B is indicative of large differences between the two methods, underlining the underscoring of flagellar defects with the Testsimplets® method. Fertility and Sterility 2008 89, 449-455DOI: (10.1016/j.fertnstert.2007.03.027) Copyright © 2008 American Society for Reproductive Medicine Terms and Conditions