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Benzo[a]pyrene (BaP) is a polycyclic aromatic hydrocarbon and a potent inducer of carcinogenesis. Many studies have reported that the carcinogenic effects of BaP might be due to its intermediate metabolites and to reactive oxygen species (ROS) that cause oxidative damage to the cells. However, the mechanisms of BaP-induced oxidative damage in cervical tissue are still not clear. We studied these mechanisms in female ICR mice treated with BaP either orally or intraperitoneally by measuring (1) several general biomarkers of oxidative stress in serum, (2) mitochondrial function in the cervix, and (3) the morphology of mitochondria in cervical tissue. BaP treatment (1) significantly lowered levels of vitamins A, C, and E and of glutathione; (2) reduced activities of superoxide dismutase, catalase, glutathione peroxidase, and glutathione S-transferases; and (3) significantly increased lipid peroxidation levels. In addition, significant increases in the levels of superoxide anion, hydrogen peroxide, and hydroxyl radical were observed. These results were confirmed by morphological changes in mitochondria and by decreases in membrane potential levels and in succinate dehydrogenase and malate dehydrogenase activities. The changes in these biomarkers and mitochondrial damage were BaP-dose-dependent and eventually induced both cell apoptosis and necrosis in cervical tissue. As mitochondria are the major sites of ROS generation, these findings show that mitochondrial decay greatly contributes to BaP-induced cervical damage., (Copyright © 2010 Elsevier Inc. All rights reserved.)

The initial host response in a primary chlamydial infection is the onset of acute inflammation. However, we still know very little about the early temporal events in the induction of the acute inflammatory response and how these events relate to the initial chlamydial developmental cycle in an actual genital infection. Because it was critical to initiate a synchronous infection in the endocervix in the first 24 h to evaluate the sequential expression of the host response, we developed the surgical methodology of depositing Chlamydia muridarum directly on the endocervix. Cervical tissue was collected at 3, 12, and 24 h after inoculation and the expression array of chemokines, cytokines, and receptors was assessed to characterize the response during the initial developmental cycle. Polymorphonuclear leukocyte (PMN) infiltration was first observed at 12 h after inoculation, and a few PMNs could be seen in the epithelium at 24 h. Electron microscopic analysis at 24 h showed that virtually all inclusions were at the same stage of development, indicating a synchronous infection. Several chemokine and cytokine genes were expressed as early as 3 h after infection, but by 12 h, 41 genes were expressed. Thus, activation of the host response occurs both with the introduction of elementary bodies into the host and early replication of reticulate bodies. No significant response was observed when UV-inactivated organisms were inoculated into the cervix at any time interval. This model provides an ideal opportunity to investigate the mechanisms by which the early inflammatory response is induced in vivo.

Aims: Gastric mucin expression has been demonstrated in a group of endocervical glandular lesions. The aim of this study was to gain further insight into endocervical lesions with a gastric phenotype., Methods and Results: Various types of tunnel clusters (TC) were examined for gastric mucin by alcian blue/periodic acid-Schiff staining and immunohistochemistry for HIK1083. Five of 34 cases of TC expressed gastric mucin defined by PAS dominant neutral mucin and immunopositivity for pyloric gland mucin. Histologically, TC expressing gastric mucin showed lobular arrangements of small to medium-sized glands composed of mucin-rich columnar cells and were classified as Flumann's type A TC. Neither type B TC nor normal endocervical glands expressed PAS dominant neutral mucin and none of them was immunopositive for pyloric gland mucin. Five patients with type A TC of gastric phenotype ranged in age from 33 to 79 years (mean 58 years) and were multiparous. Type A TC of gastric phenotype, ranging from 2 to 4 mm in maximum diameter, were incidental findings in hysterectomy specimens., Conclusion: Type A TC of gastric phenotype could be related to lobular endocervical glandular hyperplasia of gastric phenotype. The pathogenesis of gastric metaplasia in TC remains unclear.

As more attention is paid to cervical cancer screening in the postmenopausal population, increased numbers of atrophic specimens will be evaluated in the cytology laboratory. In addition, specimens that have cell patterns that mimic the nonestrogen or partially estrogen-stimulated state occur in a variety of situations, including pregnancy, the postpartum period, and in individuals who are treated with progesterone. A firm understanding of the cellular changes that are within the range of normal in such circumstances is critical to ensure the specificity of interpretation. This article has detailed the conditions under which nonestrogen stimulated patterns occur and addressed the cytologic changes that are noted. Hints to avoid pitfalls have been offered. There is no substitute for a thorough evaluation of each case, and with continued experience and understanding of these principles, the correct interpretations, and, ultimately, correct management of patients, can be optimized.

We tested the hypothesis that extensively keratinized squamous intraepithelial lesions (SILs) are difficult to grade precisely by identifying 100 Papanicolaou smears with a keratinizing SIL that had been originally judged difficult to grade. Of these, 65 were confirmed as low-grade SIL (LSIL) or high-grade SIL (HSIL) on subsequent biopsy. The 65 smears were reviewed independently by 3 cytopathologists who graded each case as LSIL or HSIL (by Bethesda System criteria). The accuracy of the grade was determined by the subsequent biopsy results; accuracy was compared with that of a historic control group of SILs with biopsy follow-up. In the study group, biopsies showed LSIL in 41 cases and HSIL in 24. The mean accuracy for a smear diagnosis of LSIL was 60% for the study group and 92% for the control group. For a smear diagnosis of HSIL, the accuracy was 60% for the study group and 95% for the control group. The overall kappa value for the study group confirmed poor interobserver agreement. Some keratinizing SILs are difficult if not impossible to grade precisely using standard criteria. For such lesions, the diagnosis "SIL, grade cannot be determined due to extensive keratinization" is justified.

Changes of condyloma are sometimes difficult to distinguish from changes seen with inflammation or with squamous metaplasia (SM) in the uterine cervix. Cervical condylomas show increased proliferative activity, so we sought to discover whether this increased activity is more than that seen in SM or with inflammation. Mitotic activity per 10 high power fields was measured in 22 cases with SM, 22 cases with exocervix only, 26 cases with inflammation, and 34 cases with condylomas. The percentage of basal cells staining for MIB-1 was compared between 27 cases with condyloma, 11 cases each with SM and inflammation, and 13 cases with exocervix. Mitotic activity was significantly increased in cervical condylomas (7.6 +/- 6.6, mean +/- standard deviation) compared with SM (1.4 +/- 1.4, P < .0001), inflamed epithelium (1.6 +/- 1.4, P < .0001), and exocervix (1.4 +/- 1.5, P < .0001). More than 15% of basal cells stained for MIB-1 in 18 of the 27 condylomas, 1 of 11 cases with SM, 1 of 11 cases with inflammation, and none of the 13 cases with exocervix (P = .003, .003, and .0006 respectively). Expression of MIB-1 in the surface half of the epithelium was seen in 18 (66%) of the 27 cases with condyloma but in none of the 24 controls (P < .00001). MIB-1 in the surface half of the epithelium and/or in more than 15% of the basal cells was seen in 24 (90%) of the 27 condyloma cases and 1 (4%) of the 24 control cases (P < .00001). A finding of 6 or more mitoses per 10 high power fields, expression of MIB-1 in the surface half of the epithelium, and more than 15% of basal cells staining for MIB-1 should support a diagnosis of condyloma over metaplastic or inflammatory changes.

Expression of syndecan-1, a cell surface proteoglycan that binds growth factors and extracellular matrix components, was studied in normal and pathological human uterine cervix using immunohistochemical methods. Normal cervical squamous epithelium showed positive staining for syndecan-1 in all cell layers, except the basal cell layer, whereas endocervical columnar epithelium stained weakly. In non-neoplastic reactive lesions, metaplastic squamous cells were positive for syndecan-1, whereas columnar cells showed weak or negative staining. In cervical condylomas, cells showing koilocytotic atypia were positive for syndecan-1. The progression of cervical intraepithelial neoplasia (CIN) grade I to grade III was associated with reduced syndecan-1 expression and localization of syndecan-1 to more superficial cell layers. In squamous cell carcinomas (SCCs), syndecan-1 expression correlated with histological differentiation, being absent from most poorly differentiated tumours. The results suggest that loss of syndecan-1 from atypical cells is an early event during cervical carcinogenesis and show a close association of syndecan-1 expression with preserved epithelial morphology and differentiation.

Aim: To investigate the distribution of cytokeratins 10, 13, 14 and 19 in biopsy specimens taken from acetowhite and non-acetowhite areas of the cervix., Method: Cervical biopsy specimens were taken from both acetowhite and non-acetowhite areas from 44 patients who presented with abnormal cervical cytology. The specimens were snap frozen in liquid nitrogen and multiple sections taken from each specimen. Staining was performed for cytokeratins 10, 13, 14, 19 and NADPH diaphorase enzyme. The areas of each section positive for the various markers were measured., Results: Cytokeratin 10 positive cells were greatly increased in number in acetowhite biopsy specimens compared with non-acetowhite samples (45.1% v 2.8%; p < 0.0001). Cytokeratin 19 was also increased, but to a lesser extent (17.8% v 5.5%; p < 0.0001). In contrast, the almost universal expression of cytokeratin 13 was reduced in acetowhite biopsy specimens (86.2% v 96.9%; p < 0.0001). Cytokeratin 14 was found diffusely in the basal region of the stratified squamous epithelium and was marginally more apparent in the acetowhite biopsy specimens (p = 0.04)., Conclusion: It is suggested that the presence of cytokeratin 10 may be an essential requirement for the formation of acetowhite change in association with the cellular swelling caused by acetic acid.

Epithelial distribution of filaggrin, a histidine-rich protein related to squamous terminal differentiation, was investigated in 87 cervical biopsies using an avidin-biotin-peroxidase technique with a monoclonal anti-human filaggrin antibody (AKH1). Normal squamous cervical epithelium exhibited a positive homogeneous immunoperoxidase stain in the upper parabasal, intermediate and superficial cell layers. Similar findings were obtained in cervical condylomas, although full-thickness staining was observed in 35.7% of the cases (P < 0.001). Filaggrin expression in CIN was inversely related to the severity of the lesion (P < 0.001). An irregular staining pattern was present in most high-grade CIN. Filaggrin expression was closely connected to the degree of tumour differentiation (P < 0.05) in squamous cell carcinomas of the cervix. Abnormal filaggrin stainings identified a premalignant/malignant cervical squamous lesion with a positive predictive value of 92.3%. Non-squamous epithelia showed lack of filaggrin expression. Filaggrin may therefore be considered a marker of squamous differentiation in both the normal and pathological human uterine cervix.

Four cases of unusual lesions of endocervix are presented. They were all incidental findings, showing no obvious infiltrative and metastatic properties to attest their malignant nature. Two lesions were entirely confined to the endocervical mucosa. The main characteristics of these proliferative processes thus were an abnormal architecture with branched or small glands, a hypermucinous benign-appearing epithelium of endocervical type, and stromal smooth-muscle. In contrast with normal endocervical mucosa, all lesions contained prominent and variegated endocrine cells. These 4 cases were quite comparable to 3 other observations previously reported. This homogeneous group of endocervical lesions does not correspond to a well-defined type of endocervical neoplasia. It shares morphological analogies with adenoma malignum. The relationship with adenoma malignum as well as with some other gynaecological neoplasms is discussed. The recognition of these small-sized and highly differentiated lesions is largely facilitated by the use of the Grimelius reaction to detect argyrophilic cells.

Keratin 19 (K-19) expression has been strongly correlated with dysplasia in oral epithelium. Expression of K-19 was evaluated by immunoperoxidase staining in formalin-fixed normal ectocervical tissue, normal endocervical tissue, cervical dysplasia, squamous metaplasia, atrophic epithelium, cervical condylomas, and invasive carcinoma to determine if a correlation of K-19 expression with dysplasia was present in the cervical epithelium. Uniform expression of K-19 was seen in endocervical epithelium and in the basal layer of normal ectocervical epithelium in all areas where these epithelia were present. Cervical dysplasia without associated condylomatous changes showed increased expression of K-19 in suprabasal epithelium, corresponding to the level of immature cells. Squamous metaplasia was characterized by scattered cells with increased staining (patch-quilt pattern). There was considerable overlap in the patterns of K-19 expression in dysplastic and metaplastic epithelium. Thus K-19 staining pattern could not be used as a distinctive marker for dysplasia in the cervical epithelium. Atrophic epithelium showed a characteristic uniform but low-level expression of K-19 in suprabasal areas. This pattern may be of diagnostic use in differentiating atrophic lesions from dysplasia. Condylomas showed focal loss of K-19 in the basal layer, suggesting induction of premature differentiation in the basal layer by human papillomavirus infection. Invasive carcinomas showed variable patterns. K-19 is a marker of immature cervical squamous epithelium, with generally distinctive but sometimes overlapping patterns of expression in various diagnostic categories.

Cystatin A was immunohistochemically demonstrated in the normal squamous epithelium of the uterine cervix, particularly in the parabasal and superficial cell layers whereas it was absent or scanty in the basal cells and in areas with parakeratosis. Cystatin A was also found in neoplastic lesions (dysplasia, carcinoma in situ and squamous cell carcinoma), but less abundant than in normal squamous epithelium. The immunoreaction in intraepithelial neoplasia was closely related to the degree of morphological maturation of the squamous cells with more abundant cystatin A in low grade dysplasia and less in high grade dysplasia and carcinoma in situ. In squamous cell carcinoma, cystatin A was often abundant in highly differentiated areas and almost absent in poorly differentiated ones. Cystatin A was found in the squamous epithelium in herpes and in condylomatous lesions. It was also found in the cytoplasm of neutrophils, but not in lymphocytes and plasma cells. In unspecific cervicitis, cystatin A was found extracytoplasmatically as small vesicles in the epithelial-stromal junction. The implications of cystatin A in neoplastic, virus, and inflammatory processes are discussed.

We investigated the use of three monoclonal antikeratin antibodies on routinely formalin-fixed and paraffin-embedded punch and cone biopsies of the normal human uterine cervix and its metaplastic and premalignant lesions. Monoclonal antibodies used were AE8, which is specific for keratin 13; 34BE12, which reacts with keratins of the stratified squamous epithelium; and CAM5.2, which is specific for keratin 8. All these antibodies performed well in routinely processed surgical pathology material. AE8 antibody stained the suprabasal layer of the normal squamous epithelium. Squamous metaplasia and dysplasia were stained in 50% of the cases. Normal suprabasal distribution of the keratin 13, however, was lost in all positive dysplasia cases. CAM5.2 reacted with normal columnar cells in all cases, and squamous metaplasia was focally positive in 20% of the cases. Dysplasia showed a positive reaction in 30% to 40% of the cases. The 34BE12 antibody was reacting with the full thickness of the squamous epithelium. Squamous metaplasia and dysplasia were positive in 80% of the cases. In addition, 34BE12 stained reserve cell hyperplasia, making it a useful marker for this condition. Our results demonstrate that keratin immunohistochemistry with the above-listed antibodies gives pathogenetically interesting information on cervical lesions.

A study was undertaken to determine the potential value of involucrin immunostaining, a protein synthesized by mature squamous epithelial cells, in distinguishing benign from neoplastic lesions in cervical pathology. A total of 146 cervical biopsies were analyzed using an indirect immunoperoxidase method and polyclonal antibody. A suprabasal homogeneous cytoplasmic staining pattern was consistently observed in normal squamous cervical epithelium. In contrast, 43.7% of cervical condylomas showed involucrin at all levels of the epithelium including the basal layer. Variable patterns were seen in cervical intraepithelial neoplasia (CIN), with 46% of full-thickness stainings, although no significant difference was obtained among the different grades of CIN lesions. Distribution of involucrin was correlated (P less than 0.05) with the degree of tumor differentiation in squamous cell carcinomas, being absent in 71.4% of poorly differentiated carcinomas and focally present in 75% of well-differentiated carcinoma. Lesions of endocervical origin, either benign or malignant, were entirely negative for involucrin. It is concluded that involucrin seems unable to establish a reliable differential diagnosis between benign and neoplastic conditions in cervical pathology, and should therefore be considered only a specific marker of squamous differentiation in both normal and pathological human uterine cervix.

Normal and pathologically changed structures of human cervix uteri were examined for estrogen receptor (ER) content using monoclonal antiestrophilin antibody (H 222 SP gamma) and the peroxidase-antiperoxidase method (PAP). The study was performed on fresh-frozen cervical specimens from 30 women; the staining was evaluated semiquantitatively. The ER expression in cervical mucosa was generally strong and comparable to the ER expression by proliferating endometria. The only exception was the endocervical stroma, which showed distinctly weaker specific staining than other cervical structures. This might account for ER concentration gradients observed in biochemical studies. The ER content in endocervical glands varied in different cases and in different areas of the same specimen, but no correlation to the functional activity of the genital tract or to the morphology of the glands could be found. The squamous epithelium of the portio vaginalis contained relatively large amounts of ER in the basal, parabasal, and intermediate layers. The superficial layer was ER-negative. The only factor we observed that strongly influenced the ER content in cervical mucosa was a local inflammatory process. Our results suggest that the synthesis of ER in cervical epithelium can be influenced by underlying stroma.

In this study using a panel of anticytokeratin antibodies and an indirect immunoperoxidase method, we examined cervical squamous epithelia including mature stratified epithelium, immature squamous metaplasia, CIN 1, 2 and 3, wart virus infection and squamous carcinoma. Changes from the normal patterns of staining were inconsistently seen in CIN 1 and 2, but in CIN 3 the changes were more marked, and consisted of a loss of stratification of the staining pattern and a patchy reduction in staining. Invasive carcinomas showed a similar staining pattern to CIN 3 lesions.

A selected group of 51 patients with histological features of cervical warty atypia was observed for 1 year. Initial features were compared with a cohort of 50 patients with CIN. Twenty-four patients showed resolution of the warty atypia, in 19 it remained unchanged, and in eight patients CIN or microinvasive disease was present on repeat biopsy. The DNA patterns obtained by tissue imprint of the initial biopsies and subsequent integrated densitometry, using a Vickers M85 scanning densitometer, showed that warty atypias could be objectively distinguished from CIN, but that the method could not predict the group of warty atypias that would resolve. There was no clinical feature or group of features which allowed a means of predicting the outcome of such lesions.