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The histologic features of cervical intraepithelial neoplasia (CIN 1), caused by infection by the human papillomavirus (HPV), can overlap with those of its mimics that can lead to an over diagnosis of this sexually transmitted disease. In this study, 67 consecutive cervical biopsies that were diagnosed as CIN 1 from the surgical files of Ohio State University Medical Center were analyzed. Twenty controls (10 CIN 1 cervical biopsies and 10 normal cervical tissues) were also studied. The 87 biopsies were reevaluated blinded to the original diagnosis and the results were correlated with detection of HPV DNA by in situ hybridization and glycogen by the periodic acid solution (PAS)/PAS-D stain, respectively. HPV was detected by in situ hybridization in 55/67 cases (82%); no virus was evident in the negative controls whereas each of the 10 CIN 1 controls was virus positive. A PAS test demonstrated in the mature squamous component of the negative controls a strong signal in cells with prominent and uniform halos, which was lost with diastase treatment, indicative of abundant glycogen. The PAS/PAS-D tests in the CIN 1 lesions showed rare variable sized glycogen deposits in the dysplastic cells. Nine (15%) cases initially diagnosed as CIN 1 were HPV negative by in situ hybridization and had halolike cells that were strongly and uniformly positive for glycogen. This data underscores the value of glycogen and HPV analyses in improving the specificity of the diagnosis of CIN 1.