Lactate Suppresses Retroviral Transduction in Cervical Epithelial Cells through DNA-PKcs Modulation

Recently, we have shown the molecular basis for lactate sensing by cervical epithelial cells resulting in enhanced DNA repair processes through DNA-PKcs regulation. Interestingly, DNA-PKcs is indispensable for proper retroviral DNA integration in the cell host genome. According to recent findings, the mucosal epithelium can be efficiently transduced by retroviruses and play a pivotal role in regulating viral release by cervical epithelial cells. This study examined the effects of lactate on lentiviral transduction in cervical cancer cells (HeLa, CaSki, and C33A) and model glioma cell lines (DNA-PKcs proficient and deficient). Our study showed that L- and D-lactate enhanced DNA-PKcs presence in nuclear compartments by between 38 and 63%, which corresponded with decreased lentiviral transduction rates by between 15 and 36%. Changes in DNA-PKcs expression or its inhibition with NU7441 also greatly affected lentiviral transduction efficacy. The stimulation of cells with either HCA1 agonist 3,5-DHBA or HDAC inhibitor sodium butyrate mimicked, in part, the effects of L-lactate. The inhibition of lactate flux by BAY-8002 enhanced DNA-PKcs nuclear localization which translated into diminished lentiviral transduction efficacy. Our study suggests that L- and D-lactate present in the uterine cervix may play a role in the mitigation of viral integration in cervical epithelium and, thus, restrict the viral oncogenic and/or cytopathic potential.

Single-cell analysis defines the lineage plasticity of stem cells in cervix epithelium

Information about the dynamic change and post-injury regeneration of cervical epithelium is relatively rare, even though it is tightly related to gynecologic malignancy. Here, using a feeder cell-based culturing system, we stably cloned mouse and human P63 and KRT5 expressing cells from the adult cervix as putative cervical stem/progenitor cells (CVSCs). When subjected to differentiation, the cultured cells gave rise to mature cervical epithelium by differentiating into squamous or glandular cells. The ability of endogenous mouse CVSCs to reconstitute cervical epithelium after injury was also evident from the genetic lineage tracing experiments. Single-cell transcriptomic analysis further classified the CVSCs into three subtypes and delineated their bi-lineage differentiation roadmap by pseudo-time analysis. We also tracked the real-time differentiation routes of two representing single CVSC lines in vitro and found that they recapitulated the predicted roadmap in pseudo-time analysis. Signaling pathways including Wnt, TGF-beta, Notch and EGFR were found to regulate the cervical epithelial hierarchy and implicated the different roles of distinct types of cells in tissue homeostasis and tumorigenesis. Collectively, the above data provide a cloning system to achieve stable in vitro culture of a bi-lineage stem/progenitor cell population in the cervix, which has profound implications for our understanding of the cervix stem/progenitor cell function in homeostasis, regeneration, and disease and could be helpful for developing stem cell-based therapies in future.

Neural network analysis of some molecular parameters of the cervical epithelium for cervical cancer diagnostics

Background. A personalized approach is the basis for the specialized care for cancer patients. The relevance of cervical cancer (CC) is still high. The searches for diagnostic criteria of cervical epithelium malignancy are continuing. The application ohm technologies has led to a big number results, the analysis of which is often difficult. The neural network data analysis allows to solve these problems.Objective: to create a technology for diagnosing cervical intraepithelial neoplasia (CIN) and CC, based on a neural network analysis of some molecular parameters.Materials and methods. The research carried out among patients with CIN III (n = 15), patients with CC stages I–IV (n = 49). The control group consisted of female volunteers without cervical pathology (n = 15). Studied molecular parameters: the spectrum of fatty acids was determined in cervical biopsies, proteins OPN, ICAM-1 were studied in blood serum, proteins of the immune cycle sCD25, sCD27 – in the cervical epithelium. Research methods: gas-liquid chromatography, flow cytometry.Results. Significant differences of fatty acids spectrum, local level sCD27 were revealed in among the studied groups. The multilayer perceptron included C18:2ω6, OPN, ICAM-1, sCD25, sCD27. The performed neural network analysis of the molecular data allows to diagnose CIN III (Se = 0.92; Sp = 0.87; AUC = 0.94; p˂0.001) and CC (Se = 1.00; Sp = 1.00; AUC = 1.00; p˂0.001).Conclusion. The created model makes it possible to diagnose CIN III and CC with high accuracy. The configuration of the multilayer perceptron allows confirming the pathophysiological relationships between the studied molecular parameters, to expand the understanding of the mechanisms of cervical carcinogenesis.

Comparative evaluation of the results of an immunocytochemical study of P16/Ki-67 coexpression in patients with cervical intraepithelial neoplasia associated with human papillomavirus

Aim. To evaluate the results of an immunocytochemical study of p16/Ki-67 double staining in the cervical epithelium of patients with cervical intraepithelial neoplasia (CIN) associated with high-risk human papillomavirus (HPVhr) in comparison with patients without cervical pathology. Materials and methods. The comparative study included the results of examination of 75 patients divided into 4 groups. Patients with CIN1 comprised the 1st main group (n=21), women with CIN2CIN3 the 2nd main group (n=26), the comparison group consisted of patients with latent HPV infection (n=15) and the control group (n=13). The average age of women with cervical HPV infection was 26.46.13 years. Methods of investigation: liquid cytology, colposcopic, histological examination; methods for determining HPVhr DNA; immunocytochemical examination for determining double staining of p16/Ki-67 markers, statistical analysis. Results. A positive p16/Ki-67 double staining test prevailed among patients with CIN (31.9%) compared to patients without cervical pathology (3.6%) (p=0.003) and correlated with the severity of colposcopic signs (rs=+0.397, p=0.0004). In the 1st main group of patients with verified CIN1 and in the comparison group of patients with latent infection, isolated cases of a positive test of double staining of p16/Ki-67 markers in the epithelium were recorded without significant differences between the groups (9.5 и 6.6%, p0.05). In the 2nd main group of patients with verified CIN2, CIN3, a positive test of double staining of p16 and Ki-67 was observed in every second case, dominating relative to the 1st group, the comparison group and the control group (p=0.003, p=0.005, р=0.001 respectively). In the control group, a negative double staining test was established in all cases. Conclusion. Every second patient with CIN2+ associated with HPVhr has a positive test of double staining of the cervical epithelium, with CIN1 it is observed in 9.5% of cases (p=0.003). Among patients with CIN1, there were no differences in the expression of p16/Ki-67 in epithelial cells compared to women without cervical pathology. The data of the immunocytochemical study of p16/Ki-67 in the cervical epithelium of HPVhr positive patients with CIN should be taken into account when choosing a differentiated management strategy.

Genotoxicity evaluation of levonorgestrel-releasing intrauterine system (LNG-IUS) in exfoliated cervical cells using the micronucleus (MN) test

Purpose: This study aimed to determine whether any relationships exist between the levonorgestrel-releasing intrauterine system (LNG-IUS) and micronuclei or other nuclear anomalies, including condensed chromatin, karyorrhexis, and karyolysis, on the cervical epithelium in young women. Methods: A prospective observational study was conducted. The study population comprised healthy women aged ≤40 years who were referred for birth control. Cervical smears that were obtained from 18 women before and three months after LNG-IUS insertion were tested for micronuclei and other nuclear anomaliesusing the micronucleus test. Results: The results revealed no statistically significant difference (P>0.05) in the frequency of micronucleated exfoliated cervical mucosa cells after LNG-IUS exposure. However, LNG-IUS was able to increase other nuclear alterations closely related to cytotoxicity. Conclusions: Data indicated that exposure to LNG-IUS may not be a factor in inducing chromosomal damage, but it can promote cytotoxicity.

Karnini Yonivyapad (Cervical Erosion): A Review

Health status of women is essential for healthy society. In today's fast and competitive world, there is change in the lifestyle of women which leads to their physical and mental stress, women mostly get affected by this environment and are prone to gynaecological problems. Common health problem or gynaecological problem in women is white discharge, foul smell discharge, itching at vulvar region, burning micturition. Cervical erosion is a condition seen in the age group of 20-40 years with more of Kapha dominance. Kandu, Yoni Srava and other features of Kapha Pittadushti are seen in cervical erosion. It is seen as red circular granular surface surrounding the cervical os bathed in discharges. On the study of etiological factors, sign and symptoms the disease is found to be caused by Kapha – Pittadosha Prokopa affecting Yoni and Garbhasayamukha leading to Samprapti which manifests as cervical erosion. Based on symptomatology, cervical erosion can be correlated with Karnini Yonivyapad. Karnini is an abnormal proliferation of cervical epithelium which may produce abnormal discharge, alter the vaginal pH and cervical mucus plug. Chikitsa of Karnini Yonivyapad is Yoni Prakshalana,Yoni Pichu Dharana, Yoni Varti Dharana, Agni Karma and Kshara Karma. Kaphahara line of treatment should be done. Diet also plays an important role in cervical erosion so proper Pathya Apathtya are to be followed.

Diagnostic informative value of liquid-based cytology optimized with genetic methods for the differential diagnosis of precancerous and malignant diseases of the cervix

Purpose of the study. Was to assess diagnostic informative value of liquid-based cytology optimized with genetic methods for the differential diagnosis of precancerous and malignant diseases of the cervix.Materials and methods. The study included 381 patients. Cervical pathologies were diagnosed with liquid-based cytology only and liquid-based cytology optimized with genetic methods of assessing the expression of miRNA‑20a, miRNA‑375, miRNA‑21 and –23b. Results of liquid-based cytology and genetic methods were verified by histological examination of the material. Statistical analysis was performed using descriptive statistics methods with the calculation of the mean and standard error of the mean. The mean values were compared with the help of the Mann-Whitney test.Results. Diagnostic results of liquid-based cytology were consistent with histological results in 107 (73.8 %) of 145 cervical cancer (CC) patients, in 52 (57.1 %) of 91 patients with high-grade squamous intraepithelial lesions (HSIL), and in 30 (65.2 %) of 46 patients with low-grade squamous intraepithelial lesions (LSIL). Optimization of liquid-based cytology by assessing the expression of miRNA‑21 and miRNA‑23b in the cervical epithelium improved the diagnostic sensitivity of the method from 73.8 % to 80 %, and its specificity from 94.1 % to 97.9 %. The diagnostic sensitivity and specificity of liquid-based cytology for differential diagnosis of CC and HSIL was 87 % and 78.8 %, respectively. Optimization of liquidbased cytology by assessing the expression of miRNA‑20a and miRNA‑375 in the cervical epithelium for the differential diagnosis of CC and HSIL improved the diagnostic sensitivity of the method from 87 % to 95.1 %, and its specificity from 78.8 % to 93.9 %.Conclusions. We revealed the most informative pairs of miRNAs in the cervical epithelium, as an analysis of their expression expanded the possibilities of liquid-based cytology both as a method for diagnosing CC and as a method for the differential diagnosis between CC and HSIL.

The cytological component of cervical cancer screening: causes of false negative and false positive results, and ways to avoid them

Cervical cancer (CC) screening is a major component of secondary prevention of CC and involves screening all women at risk of developing this disease, most of whom are asymptomatic. Cytology remains an important component of CC screening in the era of primary screening by genotyping the human papillomavirus. Papanikolaou staining is the method of choice for CC screening. This review highlights the causes of false negative results for various methods of Pap tests and how they can be prevented. A detailed analysis of conditions accompanied by a high probability of false positive abnormal results of the Pap test, an explanation of the pathophysiological basis of this phenomenon, clinical and cytological criteria for differential diagnosis is also presented.Pap test is a screening test. The aim of the cytological examination in CC screening is to assign the patient to a group with absent neoplastic changes in the cervical epithelium, a group with neoplastic changes in the cervical epithelium present, or a group when it is impossible to make an accurate differential diagnosis between benign reactive changes and neoplasia. The Bethesda Cytology Reporting System is used to unify and standardize these categories in most countries of the world. Benign conditions are a common cause of false positive reports of cellular atypia on cervical screening, as evidenced by a large number of studies. This fact should be taken into account both in the interpretation of the results and, if possible, in the planning of cervical screening. The most common conditions that are accompanied by reactive changes in the cervical epithelium, which can be incorrectly assessed as atypia, are: reactive and reparative inflammatory changes, atrophy, metaplasia, reactive changes caused by intrauterine devices. In this regard, when working with the cytological component of cervical screening, it is important for the clinician to understand the basic principles of assessing the cervical epithelium, which will allow using the descriptive part of the report to determine tactics if a false negative or false positive screening result is suspected.Thus, understanding the impact of common benign conditions on the cervical epithelium makes it possible to rationally plan cytological cervical screening and correctly interpret its results in order to achieve the best clinical results that are not limited to the detection of precancerous conditions.

Prognostic value of microRNA assessment in cervical epithelial cells of patients with mild dysplasia

Background. Currently, there are no reliable markers for the prognosis of the low-grade squamous intraepithelial lesion (LSIL) of the cervical epithelium. Scientific literature provides with inconsistent recommendations regarding the management of the young patients with a cytological diagnosis of LSIL. The progression of cervical dysplasia and the development of cervical cancer are associated with characteristic alterations of the microRNA expression profile.Objective: to assess the prognostic value of microRNA in LSIL. Materials and methods. Samples (cytological smears) obtained from patients diagnosed with LSIL (n = 36), but with a different course of the disease over the next 6–12 months. Analysis of miRNA expression was carried out by the method of “two-tailed” reverse transcription and subsequent PCR.Results. The expression level of miR-126, miR-21, miR-1246, miR-182 was statistically significantly different in the compared groups, but the predictive value of the analysis of individual molecules was low (AUC <0.65). Calculation of the concentration ratios of the “reciprocal” pairs of microRNAs made it possible to obtain a more effective prognostic marker. ROC analysis of such ratios (miR-126/miR-182, miR-21/miR-182, miR-1246/miR-182) yielded AUC values: 0.82–0.89, sensitivity: 0.71–0.92; specificity: 0.86.Conclusions. Analysis of a panel of microRNA marker molecules in the material of the cervical epithelium and calculation of the concentration ratios of “reciprocal” pairs is a promising method for prognosis of LSIL course.