Immunocytochemical assessment of mitotic activity with an antibody to phosphorylated histone H3 in the macaque and human endometrium

Background: Determination of the mitotic index in sections of endometrium stained with haematoxylin and eosin (H&E) is difficult and time-consuming. We assessed the value of two mitotic markers as immunocytochemical reagents for measuring mitotic rates in endometrium. Methods: Mitotic protein monoclonal antibody 2 (MPM-2) and anti-phosphorylated histone H3 (Phospho H3) were applied to paraffin sections of rhesus macaque and human endometrium. Results: In estrogen-treated macaque endometrium the mean ± SEM mitotic indices were: H&E 1.5 ± 0.25%, Phospho H3 antibody 1.02 ± 0.23% and MPM-2 antibody 0.69 ± 0.17%; these were not statistically significantly different, but the Phospho H3 antibody gave a stronger and cleaner signal than the MPM-2 antibody. Comparisons were made between a computer-determined Phospho H3 index, the H&E-determined mitotic index and the Ki-67 index in samples of human endometrium across the cycle. All revealed that the highest proliferative rate occurred during the follicular phase, but the Phospho H3 and the mitotic indices were more highly correlated (R² = 0.89, P < 0.001) than the Ki-67 and mitotic indices (R² = 0.74, P < 0.05). Conclusions: The exceptionally high contrast staining and the excellent correlation between the Phospho H3 and mitotic indices validates the specificity of the Phospho H3 antibody as a new tool for the assessment of endometrial mitotic activity.