Effects of six kinds of sperm staining methods on human sperm size and evaluation of their staining effects

BACKGROUND: Large‐ and small‐headed sperm are common morphological abnormalities. If different sperm staining methods affect sperm size, they will make a difference in the accuracy of sperm morphological analysis results. In this case, the normal reference values of sperm head parameters for different staining methods should be established. METHODS: Six sperm staining methods, including Papanicolaou, Diff‐Quik, Shorr, Hematoxylin–eosin (HE), Wright, and Wright‐Giemsa staining, were used to stain the sperm smears of 25 semen samples, respectively. Sperm head parameter's length (L), width (W), area (A), perimeter, acrosomal area (Ac), and the derived values L/W and Ac/A of 2500 sperm (100 for each specimen) per staining method were measured by a computer‐aided sperm morphological analysis system. RESULTS: The highest sperm head length and width were observed with the Wright‐Giemsa and Wright staining, followed by the Diff‐Quik. The lowest sperm head length and width were observed with the Papanicolaou staining, and the sperm head length and width of HE and Shorr staining were between those of Papanicolaou and Diff‐Quik staining. There was the same trend in changes in sperm head area and perimeter. Diff‐Quik and Shorr staining could clearly distinguish acrosome and nucleus, followed by HE staining, whereas the boundary between acrosome and nucleus was not evident in Papanicolaou, Wright, and Wright‐Giemsa staining. CONCLUSION: Different staining methods influence sperm size, and the normal reference values of sperm head parameters of each staining method should be established. Diff‐Quik and Shorr staining may be suitable methods for routine sperm morphological analysis.