Construction of ceRNA network and key gene screening in cervical squamous intraepithelial lesions.

BACKGROUND: This study aimed to construct an endogenous competition network for cervical squamous intraepithelial lesions using differential gene screening. METHODS: GSE149763 was used to screen differentially expressed long non-coding RNAs (lncRNAs) and mRNAs to predict correlated microRNAs (miRNAs). The correlated miRNAs and GSE105409 were used to screen differentially expressed miRNAs for differential co-expression analysis, and the co-expressed differentially expressed miRNAs were used to predict correlated mRNAs. Differentially expressed mRNAs, miRNAs, and lncRNAs were visualized, and differential gene screening, enrichment, and pathway analysis were performed. RESULTS: The ceRNA network of cervical squamous intraepithelial was successfully established and a potential differentially expressed network was identified. The key genes were VEGFA and FOS, and the key pathway was the MAPK signaling pathway. CONCLUSIONS: The differential expression and potential effects of the lncRNA BACH1-IT1/miR-140-5p/VEGFA axis, key genes, VEGFA and FOS, and MAPK signaling in CIN were clarified, and the occurrence and potential effects of CIN were further clarified. The underlying molecular mechanism provides a certain degree of reference for subsequent treatments and experimental research.

Correlation between Lactobacillus and expression of E-cadherin, ß-catenin, N-cadherin, and Vimentin in postmenopausal cervical lesions.

BACKGROUND: To detect the correlation between Lactobacillus vaginalis and the expression of epithelial-mesenchymal transition (EMT)-related factors, E-cadherin, ß-catenin, N-cadherin, and Vimentin, in postmenopausal cervical squamous intraepithelial lesions (SILs) and cervical squamous cell carcinoma (SCC), and to explore the possible mechanism. METHODS: From January 2016 to January 2020, 30 postmenopausal patients with low-grade SIL (LSIL), 18 patients with high-grade SIL (HSIL), and 30 patients with SCC who underwent colposcopy biopsy in the Outpatient Department of the First Affiliated Hospital of Inner Mongolia Medical University were selected as the experimental group, and 30 postmenopausal normal women were selected as the control group. The expression of 16SrRNA of Lactobacillus vaginalis in each group was determined by the 16S third-generation full-length amplification sequencing technique. The mRNA expression levels of E-cadherin, ß-catenin, N-cadherin, and Vimentin were detected by quantitative real-time polymerase chain reaction (qPCR). The correlation between the 16SrRNA expression level of Lactobacillus vaginalis and the mRNA expression level of the EMT-related proteins was compared among all groups. RESULTS: (I) The progression of postmenopausal cervical SILs to cervical SCC was significantly positively correlated with age, number of pregnancies, smoking, pH value, positive rate of HPV16, and negatively correlated with total Lactobacillus 16SrRNA expression (P<0.0001). (II) The level of vaginal microbiota in postmenopausal women showed that Lactobacillus iners was dominant. With the progression of the disease, the expression levels of 16SrRNA in Lactobacillus iners and Lactobacillus total vagina decreased gradually, and the differences were statistically significant (P<0.05). (III) With the disease progresses. The expression of total Lactobacillus 16SrRNA was positively correlated with the mRNA expression of ß-catenin and E-cadherin (r>0; P<0.05), and negatively correlated with the mRNA expression of Vimentin and N-cadherin (r<0; P<0.05). CONCLUSIONS: In postmenopausal women, Lactobacillus vaginalis interacts with HPV and is associated with the occurrence of EMT, promoting the development of cervical lesions.

Transformed low-rank ANOVA models for high-dimensional variable selection.

High-dimensional data are often encountered in biomedical, environmental, and other studies. For example, in biomedical studies that involve high-throughput omic data, an important problem is to search for genetic variables that are predictive of a particular phenotype. A conventional solution is to characterize such relationships through regression models in which a phenotype is treated as the response variable and the variables are treated as covariates; this approach becomes particularly challenging when the number of variables exceeds the number of samples. We propose a general framework for expressing the transformed mean of high-dimensional variables in an exponential distribution family via ANOVA models in which a low-rank interaction space captures the association between the phenotype and the variables. This alternative method transforms the variable selection problem into a well-posed problem with the number of observations larger than the number of variables. In addition, we propose a model selection criterion for the new model framework with a diverging number of parameters, and establish the consistency of the selection criterion. We demonstrate the appealing performance of the proposed method in terms of prediction and detection accuracy through simulations and real data analyses.

CCR2 and CCR5 genes polymorphisms in women with cervical lesions from Pernambuco, Northeast Region of Brazil: a case-control study.

Polymorphisms in chemokine receptors play an important role in the progression of cervical intraepithelial neoplasia (CIN) to cervical cancer (CC). Our study examined the association of CCR2-64I (rs1799864) andCCR5-Δ32 (rs333) polymorphisms with susceptibility to develop cervical lesion (CIN and CC) in a Brazilian population. The genotyping of 139 women with cervical lesions and 151 women without cervical lesions for the CCR2-64I and CCR5-Δ32 polymorphisms were performed using polymerase chain reaction-restriction fragment length polymorphism. The individuals carrying heterozygous or homozygous genotypes (GA+AA) for CCR2-64I polymorphisms seem to be at lower risk for cervical lesion [odds ratio (OR) = 0.37, p = 0.0008)]. The same was observed for the A allele (OR = 0.39, p = 0.0002), while no association was detected (p > 0.05) with CCR5-Δ32 polymorphism. Regarding the human papillomavirus (HPV) type, patients carrying the CCR2-64Ipolymorphism were protected against infection by HPV type 16 (OR = 0.35, p = 0.0184). In summary, our study showed a protective effect ofCCR2-64I rs1799864 polymorphism against the development of cervical lesions (CIN and CC) and in the susceptibility of HPV 16 infection.

CCR2 and CCR5 genes polymorphisms in women with cervical lesions from Pernambuco, Northeast Region of Brazil: a case-control study

Polymorphisms in chemokine receptors play an important role in the progression of cervical intraepithelial neoplasia (CIN) to cervical cancer (CC). Our study examined the association of CCR2-64I (rs1799864) andCCR5-Δ32 (rs333) polymorphisms with susceptibility to develop cervical lesion (CIN and CC) in a Brazilian population. The genotyping of 139 women with cervical lesions and 151 women without cervical lesions for the CCR2-64I and CCR5-Δ32 polymorphisms were performed using polymerase chain reaction-restriction fragment length polymorphism. The individuals carrying heterozygous or homozygous genotypes (GA+AA) for CCR2-64I polymorphisms seem to be at lower risk for cervical lesion [odds ratio (OR) = 0.37, p = 0.0008)]. The same was observed for the A allele (OR = 0.39, p = 0.0002), while no association was detected (p > 0.05) with CCR5-Δ32 polymorphism. Regarding the human papillomavirus (HPV) type, patients carrying the CCR2-64Ipolymorphism were protected against infection by HPV type 16 (OR = 0.35, p = 0.0184). In summary, our study showed a protective effect ofCCR2-64I rs1799864 polymorphism against the development of cervical lesions (CIN and CC) and in the susceptibility of HPV 16 infection.

Correlation of plasma nitrite/nitrate levels and inducible nitric oxide gene expression among women with cervical abnormalities and cancer.

Cervical cancer is caused by infection with high risk human papillomavirus (HR-HPV). Inducible nitric oxide synthase (iNOS), a soluble factor involved in chronic inflammation, may modulate cervical cancer risk among HPV infected women. The aim of the study was to measure and correlate plasma nitrite/nitrate levels with tissue specific expression of iNOS mRNA among women with different grades of cervical lesions and cervical cancer. Tissue biopsy and plasma specimens were collected from 120 women with cervical neoplasia or cancer (ASCUS, LSIL, HSIL and invasive cancer) and 35 women without cervical abnormalities. Inducible nitric oxide synthase (iNOS) mRNA from biopsy and plasma nitrite/nitrate levels of the same study subjects were measured. Single nucleotide polymorphism (SNP) analysis was performed on the promoter region and Ser608Leu (rs2297518) in exon 16 of the iNOS gene. Differences in iNOS gene expression and plasma nitrite/nitrate levels were compared across disease stage using linear and logistic regression analysis. Compared to normal controls, women diagnosed with HSIL or invasive cancer had a significantly higher concentration of plasma nitrite/nitrate and a higher median fold-change in iNOS mRNA gene expression. Genotyping of the promoter region showed three different variations: A pentanucleotide repeat (CCTTT) n, -1026T > G (rs2779249) and a novel variant -1153T > A. These variants were associated with increased levels of plasma nitrite/nitrate across all disease stages. The higher expression of iNOS mRNA and plasma nitrite/nitrate among women with pre-cancerous lesions suggests a role for nitric oxide in the natural history of cervical cancer.

Rotura de cuerno uterino rudimentario en gestación de 22 semanas. A propósito de un caso y revisión de la literatura / Rupture of a rudimentary uterine horn at 22 weeks of pregnancy: A propos of a case and review of the literature

El útero unicorne con cuerno rudimentario es una anomalía mulleriana rara con una alta incidencia de complicaciones obstétricas que afecta al 4,5% de las mujeres. La gestación albergada en él ocurre en uno de cada 76.000 embarazos con un riesgo de rotura uterina de un 50-80% y ocurre normalmente a final del segundo trimestre del embarazo. El diagnóstico precoz reduce la morbimortalidad, pero la sensibilidad diagnóstica por ecografía es solo del 30%, dada la baja prevalencia de la enfermedad. Presentamos el caso de una gestante de 22 semanas, con cesárea previa, con abdomen agudo y shock hipovolémico por rotura de un cuerno rudimentario uterino (AU)

Unicornuate uterus with rudimentary horn is a rare Müllerian anomaly with a high incidence of obstetric complications, affecting 4.5% of women. Pregnancy located in the rudimentary horn occurs in 1 in 76,000 pregnancies with a risk of uterine rupture of 50-80%. Rupture usually occurs at the end of the second trimester of pregnancy. Early diagnosis reduces morbidity and mortality, but ultrasound diagnosis has a sensitivity of only 30%, due to the low prevalence of this entity. We report the case of a woman at 22 weeks of pregnancy with a previous cesarean delivery, who presented with acute abdomen and hypovolemic shock due to a ruptured rudimentary horn (AU)

[Association of interferon-γ gene polymorphism and risk of cervical HPV infection].

OBJECTIVE: To investigate the association of interferon (IFN) γ gene polymorphisms and risk and prognosis of HPV cervical infection. METHODS: PCR-ASP was used for detecting IFN-γ rs2430561 polymorphism in 179 HPV positive patients and 328 HPV negative normal controls. RESULTS: The frequency of A allele of 63.7% (228/358) was significantly higher than the frequency of T allele of 36.3% (130/358) in HPV positive group (P = 0.045). The frequencies were 41.3% (74/179) in AA genotype and 14.0% (25/179) in TT genotype, women carrying AA genotype increased the risk of HPV infection compare with those with TT genotype (OR = 1.784, 95% CI: 1.031 - 3.088, P = 0.039). During follow-up, the rate of HPV positive again in AA genotype was 83.8% (62/74), while TT genotype was 20.0% (5/25). In the analysis of Kaplan-Meier, the cumulative HPV negative rates of AA, TA and TT genotype exhibited significantly different (P = 0.008). The cumulative HPV negative rate of AA genotype was the lowest (1.1% - 5.9%). CONCLUSIONS: IFN-γ rs2430561 polymorphisms confer the susceptibility to HPV infection. Women with AA genotype exhibited higher risk of infection and inclined to be continuous status and recurrence after HPV infection.

Florid reactive lymphoid hyperplasia (lymphoma-like lesion) of the uterine cervix.

Lymphoma-like lesion (LLL) of the female genital tract is an older term in the literature that describes a florid reactive lymphoid proliferation that can be misinterpreted as lymphoma. Multiple causes of LLL have been suggested but most cases remain unexplained. We describe the clinicopathologic features of 6 patients with LLL involving the uterine cervix. Five patients presented with abnormal Papanicolaou test (Pap smear), and 3 patients had a biopsy procedure performed prior to detection of LLL in a loop electrosurgical excision procedure (LEEP). In each specimen, surface epithelial erosion was associated with a superficial, polymorphous lymphoid infiltrate with numerous scattered large cells, without cellular necrosis or sclerosis. Squamous dysplasia was present in 4 patients. Immunohistochemical studies revealed a mixed population of B- and T-lymphoid cells. T-cells were more numerous but B-cells and formed aggregates or sheets in areas. The large cells were predominantly B-cells positive for CD20 and negative for CD3 in all cases. CD30 was positive 3 cases, and Epstein-Barr virus-encoded RNA was positive in 3 cases. Assessment for clonality in 1 patient using polymerase chain reaction (PCR) methods revealed monoclonal immunoglobulin heavy chain (IgH) gene rearrangements. At last clinical follow-up there was no evidence of progressive or systemic disease. We conclude that LLL of the cervix has a number of etiologies and that a prior surgical procedure, present in 3 patients in this study, is another possible etiology. As has been reported by others, monoclonal IgH gene rearrangements can be detected in this entity which has a benign clinical course.

Prevalence and type distribution of human papillomavirus infection in women from North Sardinia, Italy.

BACKGROUND: Human papillomavirus (HPV) has been associated with several disorders of the genital tract, skin and oropharynx. The aims of our study were to evaluate the prevalence of HPV infection in women between 15 and 54 years of age in North Sardinia, Italy, to identify the prevalence of High Risk - Human papillomaviruses (HR-HPV) genotypes and to establish a correlation between molecular and cytological results. METHODS: From 2007 to 2009 we consecutively enrolled women aged 15-54 years admitted to public and private outpatient settings. All the participants filled in a questionnaire about the socio-cultural state, sexual activity and awareness about HPV. 323 cervical specimens were tested for HPV-DNA and HPV genotypes with INNO-LiPA HPV Genotyping CE Amp kit. Samples showing positivity to some HPV genotypes were re-tested using "in house" quantitative Real-Time PCR assays. RESULTS: Overall HPV-DNA positivity was detected in 35.9% of the women. The prevalence of HR-HPV infection among HPV positive samples was 93.1% with a specific prevalence of HPV 16, 51, 31, 53 and 18 of 54.3%, 37.9%, 10.3%, 6.9% and 5.2%, respectively. Co-infection with any HPV, HR-HPV, LR-HPV and HR/LR-HPV type was 18.3%, 14.9%, 0.9% and 2.5%, respectively; HPV 16/51 co-infection was detected in 64.6% of the HR-HPV co-infection group. The most frequent HPV-genotypes detected were 16 (32.5%) and 51 (22.7%). Among the 57 patients harboring mono-infection the most prevalent HPV genotypes were 16 (38.6%) and 31(10.5%). A multivariate analysis identified a statistical significant association between HPV infection and age and between HPV infection and previous sexual transmitted diseases. A statistically significant association between cytological cervical lesions and generic HPV exposure was identified. CONCLUSIONS: To our knowledge, this is the first survey evaluating the prevalence of HPV infection in Northern Sardinia and drawing attention to the unusual high proportion of genotype HPV 51. Given the recent implementation of a widespread immunization program with vaccines not containing HPV 51, it has been relevant to prove the high prevalence of this HPV genotype from the start of the vaccination campaign, in order to avoid in the future attributing to the vaccination program a possible selection effect (HPV replacement).

Clinical significance of human telomerase RNA gene (hTERC) amplification in cervical squamous cell lesions detected by fluorescence in situ hybridization.

BACKGROUND: Genomic amplification of the human telomerase RNA gene (hTERC), located in the chromosome 3q26 region, has been documented in tumorigenesis. The present study was designed to detect hTERC amplification in cervical lesions and evaluate whether this might serve as a supportive biomarker to cytopathology or histopathology in the diagnosis of cervical lesions. METHODS: Liquid-based thin-layer cytopathologic examination and detection of amplification by fluorescence in situ hybridization (FISH) was conducted in 130 women, along with assessment of human papillomavirus DNA, colposcopy with biopsy, and histopathologic examination. RESULTS: In cytopathologic examinations, hTERC amplification rates for negative for intraepithelial lesion or malignancy (NILM),atypical squamous cells of undetermined significance (ASCUS), low-grade squamous intraepithelial lesion (LSIL), high-grade squamous intraepithelial lesion (HSIL) and squamous cell carcinoma (SCC) cases were 0% (0/10), 4% (1/25), 20% (6/30), 77% (27/35), and 100% (10/10), respectively. The difference among abnormal cellular change groups was statistically significant (P< 0.05). In histopathologic examinations, hTERC amplification rates in normal squamous cell with or without inflammatory, cervical intraepithelial neoplasia 1 (CIN 1), CIN 2, CIN 3 and SCC cases were 3.8% (2/52), 18.2% (6/33), 66.7% (6/9), 84.6% (22/26), 100% (10/10), respectively. There were significant differences among CIN1, CIN2, CIN3 and SCC cases (P< 0.05). The hTERC amplification was more specific than HPV positivity in differentiating lowgrade from high-grade cervical disorders (specificity: 88.5% vs. 70.8%, P< 0.05). CONCLUSIONS: FISH detection of hTERC amplification could be an effective adjunct to cytopathologic or histopathologic examination for differential diagnosis of low- and high-grade cervical squamous cell disorders.

Genetic diversity of HPV-16 E6, E7, and L1 genes in women with cervical lesions in Liaoning Province, China.

INTRODUCTION: High-risk human papillomaviruses (HPVs) play a cardinal role in the etiology of cervical cancer. The most prevalent type, HPV-16, shows intratypic sequence variants that are known to differ in oncogenic potential and geographic distribution. Intratype variations in oncogenic E6/E7 and capsid L1 proteins of HPV-16 are associated with risk of viral persistence and progression. METHODS: This study was designed to analyze sequence variations in E6, E7, and L1 genes of HPV-16 in patients with cervical lesion to identify the most prevalent and novel HPV-16 variants in northern China. RESULTS: Our results showed that HPV-16 variants with respect to E6 and E7 were high prevalence of the Asian lineage: 48.3% and 51.4%, respectively. Sequences of the E6 gene revealed 4 amino acid changes of variants D25E and L83V, with 48.3% (69/143) and 11.2% (16/143), respectively, and variants H78Y and E113D in this study. The results also showed the prevalence of 4 hot spots of E7 nucleotide variations leading to N29H, N29S, and 2 silent variations, nucleotide G666A and nucleotide T846C, with 4.2% (6/142), 43% (61/142), 32.4% (46/142), and 43% (61/142), respectively. The following L1 variations were found in this study: L103F, P104K, P104Y, P104S, D105G, P106S, N108P, F109V, C172S, H228D, and T292A. It was also found that 448S was inserted and 465D was deleted in the L1 amino acid sequences of all the samples. No significant relationship between HPV-16 variants and high-grade lesions was found. CONCLUSIONS: The study provides some new data on the genetic diversity of HPV-16, which may help to understand the oncogenic potential of the virus and design the diagnosis reagents and vaccine of HPV in China. Furthermore, in-depth studies are needed to determine the clinical and biological effects of these variants.

Lack of HPV 16 and 18 detection in serum of colposcopy clinic patients.

BACKGROUND: Persistent infection with high-risk human papillomavirus (HPV) types is necessary for the development of high-grade cervical dysplasia and cervical carcinoma. The presence of HPV DNA in the blood of cervical cancer patients has been reported; however, whether HPV DNA is detectable in the blood of patients with pre-invasive cervical disease is unclear. OBJECTIVES: The objectives of this study were to determine if HPV 16 and HPV 18 DNA could be detected in the serum of colposcopy clinic patients, and if serum HPV detection was associated with grade of cervical disease and HPV cofactors. STUDY DESIGN: Samples were selected from a biorepository collected from non-pregnant, HIV-negative women ages 18-69 attending colposcopy clinics at two urban public hospitals. Cervical disease status was based on review of colposcopy, biopsy and cytology findings. Serum HPV DNA detection was conducted using a novel PCR and mass spectroscopy-based assay. RESULTS: Of the 116 adequate serum samples, all (100%) were negative for HPV 16 and HPV 18. Over half (51.7%) of participants had cervical HPV 16 and/or HPV 18 infection. Nearly one-third (31.1%) had high grade, 10.3% had low grade, and 50.9% had no cervical disease. Nearly one-third (28.5%) had ever regularly smoked cigarettes, 70.7% had early onset of sexual intercourse, and 75% had ever used oral contraceptives. CONCLUSIONS: In this colposcopy clinic population with a range of clinical characteristics and established HPV cofactors, HPV DNA was undetectable in their serum. Our findings suggest that serum HPV DNA detection is not a cervical cancer screening tool.

[p16INK4A overexpression is a useful marker for uterine cervix lesions]. / La surexpression de p16 INK4A est un marqueur utile des lésions du col utérin.

The histological criteria of uterine cervix lesions are well known. However, there is a poor diagnostic reproducibility especially concerning low-grade precancerous lesions. Therefore, the aim of our study was to evaluate the utility of p16INK4A overexpression as a surrogate biomarker of precancerous lesions of the uterine cervix. A retrospective study was carried out by the International Center for Research on Cancer, Lyon, on 79 uterine cervix lesions. Specimens included 4 normal tissue samples, 24 benign lesions, 9 low-grade precancerous lesions (CIN1), 40 high-grade precancerous lesions (CIN2-3) and 2 squamous cell carcinomas. Immunohistochemistry was used to find p16INK4A expression. HPV infection was detected by HPV testing. No p16INK4A expression was detected in normal tissues and benign lesions of the uterine cervix. p16INK4A immunolabeling was weak in CIN1 cases (77.8%). Strong and diffuse p16INK4A expression was detected among all precancerous lesions (CIN2-3) and squamous cell carcinomas. p16INK4A overexpression was associated to the CIN grade (p<0.0001) and high-risk HPV infection (p<0.0001). In conclusion, p16INK4A overexpression should be regarded as a surrogate biomarker of precancerous lesions of the uterine cervix. p16INK4A overexpression is useful in reducing the variability during evaluation of suspicious biopsies of the uterine cervix.

Florid reactive lymphoid hyperplasia of the lower female genital tract (lymphoma-like lesion): a benign condition that frequently harbors clonal immunoglobulin heavy chain gene rearrangements.

Lymphoma-like lesions (LLL) of the lower female genital tract are florid reactive inflammatory processes that mainly occur in women in their reproductive years. Histologically, they are characterized by a dense lymphoid infiltrate with admixed large cells that is often suspicious for lymphoma. In contrast to lymphoma, however, they are superficial lesions that typically show surface erosion and a mixed lymphoid infiltrate and do not have evidence of a mass, deep invasion, or prominent sclerosis. With the advent of widespread molecular genetic testing, it would seem that LLLs should be polyclonal helping make the correct diagnosis. However, we have found cases with morphologic and immunophenotypic features of LLLs and evidence of clonal rearrangement of the immunoglobulin heavy chain (IGH) gene, potentially leading to misdiagnosis. We examined the clinicopathologic features and outcome of 12 patients with LLL (9 in the cervix and 3 in the endometrium). The patients ranged in age from 18 to 54 (median 37) years and came to medical attention because of squamous dysplasia (8 patients), vaginal bleeding (3), or adnexal mass (1). One patient had an endocervical polyp, but otherwise none had a discrete mass. The specimens contained a dense, polymorphous inflammatory infiltrate, commonly associated with mucosal erosion. Immunohistochemical studies showed a mixture of B and T cells without immunoglobulin light chain restriction. Four cases (all cervical) had a clonal IGH gene rearrangement by PCR. There was no evidence of lymphoma on staging or on follow-up in any patient, including the 4 patients with clonal IGH rearrangement, after a mean follow-up of 3.5 years (range: 4 mo to 13 y). In summary, we describe 12 patients with LLL of the lower female genital tract. Four of the 9 cases (44%) analyzed by PCR had a clonal IGH gene rearrangement. The clinical and pathologic features of these cases suggest that a clonal IGH rearrangement in this setting does not warrant a diagnosis of lymphoma. Careful correlation of clinical, histologic, immunophenotypic, and genetic features is required to avoid misdiagnosis and inappropriate treatment. Routine microscopic findings and detailed clinical information remain paramount in establishing the correct diagnosis.

Biomarkers in cervical screening: quantitative reverse transcriptase PCR analysis of P16INK4a expression.

Molecular insights into the human papillomavirus (HPV)-induced cervical carcinogenesis led to the discovery of biomarkers for cervical disease. The detection of cellular proteins that are overexpressed by HPV-infected cells, such as tumor suppressor protein p16(INK4a), might play an important role in future cervical cancer screening strategies. P16(INK4a) immunostaining correlates with the severity of cytological and histological abnormalities, but shows some methodological shortcomings such as the lack of standardized methodology and interobserver variability. This study evaluated quantitative reverse transcriptase PCR (RT-PCR) as an alternative tool to analyze p16(INK4a) overexpression as a biomarker for transforming HPV-infections in a liquid-based cervical cytology (LBC) setting. Sixty LBC samples, divided in three groups based on their cytological diagnosis, were subjected to HPV typing and analysis of p16 expression by immunocytochemistry and RT-PCR. The analytical sensitivity of the RT-PCR was determined by spiking HeLa and HaCaT cells. P16(INK4a) expression measured by RT-PCR did not correlate with the cytological diagnosis or HPV status (HPV-positivity, infection type and HPV16-positivity). The spiking experiment proved that, to detect increased biomarker expression by RT-PCR, about 1.0% dysplastic cells is required within a pool of normal keratinocytes. In conclusion, RT-PCR analysis of biomarker expression is not appropriate for cervical screening purposes. In typical LBC samples, the biomarker transcripts of the dysplastic cells are diluted by the RNA of the normal cells in such a manner that their overexpression cannot be detected by RT-PCR.

Clinical significance of hTERC gene amplification detection by FISH in the screening of cervical lesions.

This study evaluated the clinical significance of hTERC gene amplification detection by fluorescence in situ hybridization (FISH) in the screening of cervical lesions. Cervical specimens of 50 high risk patients were detected by thin liquid-based cytology. The patients whose cytological results were classified as ASCUS or above were subjected to the subsequent colposcopic biopsies. Slides prepared from these 50 cervical specimens were analyzed for hTERC gene amplification using interphase FISH with the two-color hTERC probe. The results of the cytological analysis and those of subsequent biopsies, when available, were compared with the FISH-detected hTERC abnormalities. It was found that the positive rates of hTERC gene amplification in NILM, ASCUS, LSIL, HSIL, and SCC groups were 0.00, 28.57%, 57.14%, 100%, and 100%, respectively. The positive rates of hTERC gene amplification in HSIL and SCC groups were significantly higher than those in NILM, ASCUS and LSIL groups (all P<0.05). The mean percentages of cells with hTERC gene amplification in NILM, ASCUS, LSIL, HSIL, and SCC groups were 0.00, 10.50%, 36.00%, 79.00%, and 96.50%, respectively. Patients with HSIL or SCC cytological diagnoses had significantly higher mean percentages of cells with hTERC gene amplification than did patients with NILM, ASCUS or LSIL cytological diagnoses (all P<0.05). It was concluded that two-color interphase FISH could detect hTERC gene amplification to accurately distinguish HSIL and ISIL of cervical cells. It may be an adjunct to cytology screening, especially high-risk patients.

Genomic amplification of the human telomerase RNA gene for differential diagnosis of cervical disorders.

To evaluate genomic amplification of the human telomerase RNA gene (TERC) as a supportive approach to cytopathology or histopathology in diagnosis of low-grade and high-grade uterine cervical lesions, 1,033 Chinese women at three medical centers had liquid-based thin-layer cytopathologic examination and TERC detection by fluorescence in situ hybridization (FISH). Human papillomavirus DNA testing, colposcopy with or without biopsy, and histopathologic examination were conducted as needed. In cytopathologic examination, genomic amplification of TERC was found in 30 of 659 (4.6%) normal or benign cellular changes; in 23 of 170 (13.5%) atypical squamous cells of undetermined significance (ASCUS); in 8 of 28 (28.6%) atypical squamous cells with high-grade squamous intraepithelial lesion possible (ASC-H); and in 26 of 103 (25.2%) low-grade (LSIL) and 64 of 73 (87.7%) high-grade (HSIL) squamous intraepithelial lesions; with pairwise significant difference (P< 0.05) in each, except ASC-H and LSIL (chi(2) = 0.127, P = 0.72). In histopathologic examination, TERC was amplified in 28 of 671 (4.2%) normal, inflammatory, or wart cases; in 17 of 233 (7.3%) cervical intraepithelial neoplasia grade 1 cases (CIN 1); in 27 of 39 (69.2%) CIN 2 cases; in 57 of 67 (85.1%) CIN 3 cases; and in 22 of 23 (95.7%) cervical cancer cases; with pairwise significant difference in each (P < 0.05). The number of cells with abnormal signals increased and the abnormal signal patterns were diversified with increasing severity of cervical dysplasia. FISH detection of TERC amplification may provide an effective, noninvasive approach in conjunction with cytopathologic or histopathologic evaluation for differential diagnosis of low- and high-grade cervical disorders.

[Evaluation of genomic amplification of the human telomerase RNA component gene in the screening of cervical lesions].

OBJECTIVE: To investigate the genomic amplification of the human telomerase RNA component (hTERC) gene in cervical cytology and evaluate its role in screening of cervical lesions. METHODS: A total of 301 cases were recruited, with liquid-based cytology diagnoses as normal (n = 203), atypical squamous cells (ASC, n = 66), low-grade squamous intraepithelial lesions (LSIL, n = 18), and high-grade squamous intraepithelial lesions (HSIL, n = 14). Following cytological examination, the slides were analyzed using a two-color fluorescence in situ hybridization (FISH) probe targeted to chromosome 3q26 containing hTERC. The hTERC findings were compared to the cytologic and histologic results, as well as high-risk human papilloma viruses (HPV) results. RESULTS: Genomic amplification of hTERC was found in 3.0% (6/203) of normal specimens, 21.2% (14/66) of ASC, 44.4% (8/18) of LSIL and 92.9% (13/14) of HSIL, with a significant difference in each pair wise (all P < 0.05). Significantly more cells with 3q26 gain were found in cervical intraepithelial lesion (CIN)II than in CINI (75.0% vs. 20.0%), as well as in CINIII (86.7% vs. 20.0%) and squamous cervical cancer (SCC) than in CINI (100.0% vs. 20.0%)(all P < 0.01). The sensitivity of hTERC amplification was significantly higher than cytological screening (82.6% vs. 17.4%, P < 0.01), and its specificity was higher than high-risk HPV test (67.8% - 73.5% vs. 25.6% - 27.7%, P < 0.01) in the diagnosis of HSIL (CINII - III). The abnormal hTERC signal type mostly was 2:3 in CINI (84.9%); whereas in CINII - III, 2:3, 2:4 and 4:4 accounted for 44.6%, 24.8% and 17.8%, respectively. CONCLUSION: Testing the gain of chromosome 3q26 in cytological specimens using specific probe for hTERC is powerful in screening of HSIL, and the amplification patterns of 2:4 and 4:4 may serve as potential prognosis markers.

Infertility and abnormal cervical mucus in two sisters who are compound heterozygotes for the cystic fibrosis (CF) DeltaF508 and R117H/7T mutations.

OBJECTIVE: To describe two cases of infertile sisters who are compound heterozygote carriers of the cystic fibrosis (CF) DeltaF508 and R117H/7T mutations and who were found to have significantly abnormal cervical mucus. DESIGN: Case reports and review of literature. SETTING: Infertility practice based in an academic medical center. PATIENT(S): Two sisters (ages 34 and 42), compound heterozygote carriers of CF mutations, who presented with involuntary infertility. INTERVENTION(S): The partners of both patients tested negative for CF. The evaluation of both sisters did not indicate other causes of infertility aside from advanced maternal age in the 42-year-old patient. Both sisters underwent natural-cycle intrauterine insemination. MAIN OUTCOME MEASURE(S): Pregnancy conception. RESULT(S): The 34-year-old patient has subsequently conceived twice through natural-cycle inseminations. CONCLUSION(S): This is the first reported case of infertility due to a cervical mucus factor in a patient who is a compound heterozygote of the DeltaF508 and R117H/7T mutations. This case is important not only because of the distinct phenotypic abnormality seen with specific CF mutations but also because of the associated genotype.