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Abstract. cervical cancer is a leading cause of mortality in women. Molecular and epidemiological data have unequivocally confirmed that high-risk human papillomaviruses (HPVs) are a major etiological agent of this malignancy, as host epigenetic alterations are induced in response to viral infection. The present study evaluated the methylation status of cpG islands surrounding miR-124a, miR-34b and miR-203 in 29 cervical cancer precursor lesions, 31 cervical tumors and 30 normal control samples, with the aim of identifying potential markers of cervical cancer. Direct quantitative methylation-specific PCR (qMSP) was used to evaluate the degree of methylation in the samples. HPV DNA was detected and genotyped using the Linear Array HPV Genotyping Test. Data were statistically analyzed using the Kruskal-Wallis test. Differences in miRNA hypermethylation between the tumor and control samples were highly significant for all the genes tested (p<0.0001). Significant results were also obtained regarding the hypermethylation of miR-124a and miR-203 in the precursor lesions compared to the control samples. among the 29 patients with precursor lesions, 68.97% (20/29) presented high risk (hr)-HPV genotypes and 31.03% (9/29) were diagnosed with low risk (lr)-HPV. Significant results (p=0.0266) were obtained for miR-124a (hr-HPV group, mean 41.32; lr-HPV group, mean 6.74), revealing a strong association between the methylation process and the hr-HPV genotype. Borderline results (p=0.058) were obtained for miR-203 (hr-HPV group, mean 44.05; lr-HPV group, mean 3.33). These results confirm the involvement of epigenetic alterations in cervical oncogenesis. The lr-HPV precursor lesions had a methylation percent pattern similar to that of the normal samples, while the results for the hr-HPV precursor lesions and tumors indicate a possible involvement of the hr-HPV genotype in the miRNA methylation process.

Alterations in DNA methylation patterns in several genes may lead to abnormal male sexual development and infertility. This study investigated the promoter methylation status of MTHFR and SNRPN in infertile men from Romania by quantitative methylation-specific PCR in order to investigate possible correlations with sperm abnormalities. The study groups included patients (n=27) with a median age of 31 years (range 26-41 years) as well as controls (n=11) with a median age of 30 years (range 24-37 years) recruited from couples seeking advice for infertility. DNA was isolated from sperm samples and promoter methylation was assessed using direct. Significant trends were detected for both genes that indicate a tendency towards promoter hypermethylation in spermatozoa with low motility (MTHFR P=0.0032, r=0.23; SNRPN P=0.0003, r=0.32) and poor morphology (MTHFR P=0.0012, r=0.27; SNRPN P=0.0003, r=0.33) but no trend was found in cases of low sperm count (MTHFR r=0.007; SNRPN r=0.06). The data indicate that the methylation patterns of the promoters of MTHFR and SNRPN are associated with changes in sperm motility and morphology, which could lead to male infertility. A large number of studies are now focused on the causes of male infertility. Among these are epigenetic modifications, which are important contributors to reproductive pathology in the male by providing dynamic changes of the phenotype according to the environmental and metabolic factors. The most known epigenetic modification is DNA methylation and alterations in this pattern in several genes could induce male infertility. The present study aims to investigate the promoter methylation status of the genes for methylene tetrahydrofolate reductase (MTHFR) and small nuclear ribonucleoprotein polypeptide N (SNRPN) in infertile males from Romania, in order to establish a correlation with sperm parameters. MTHFR is an enzyme involved in the folate pathway and in de novo nucleotide biosynthesis but also a good example for gene-environment interaction in phenotype development. SNRPN is involved in both somatic cell expression and inheritance of the imprint and the methylation pattern of its gene seems to correlate not only with imprinted disorders but also with infertility. Our study includes patients (n=27, median age 31 years, range 26-41 years) recruited from men seeking advice for couple infertility and control group (n=11, median age 30.5 years, range 24-37 years). The data we obtained indicated significant correlations between hypermethylation of the investigated genes and sperm motility and morphology. No significant correlation between DNA methylation and sperm number was found. Our data suggest that methylation pattern of MTHFR and SNRPN is linked with sperm anomalies of motility and morphology and therefore male infertility.

Recently long non‐coding RNAs were identified as new factors involved in gene expression regulation. To gain insight into expression pattern of these factors related to E7 HPV18 oncogene, this study uses HeLa cell culture transfected with E7‐siRNA. Gene expression profile was investigated using microarray analysis. After analysing the microarray results, we identified 15,387 RNA species differentially expressed in E7‐siRNA‐transfected cells compared with controls (fold change >2). The expression profiles of lncRNA species highlighted 731 lncRNAs and 203 lincRNAs. We selected two lincRNAs (LINC01101 and LINC00277) and we evaluated the expression profile in HPV‐induced neoplasia. Both lincRNAs investigated display a significantly reduced pattern of expression in cervical lesions and cancer, associated with clinical parameters. A connection between HPV presence and lincRNAs was noted. hrHPV‐positive samples exhibit significantly reduced LINC01101 and LINC00277 expression level (P < 0.05). These results provide new insights into involvement of lncRNA in HPV‐induced cervical cancer, enriching our understanding of their potential role in this pathology.

The aim of this study was to investigate the role of methylenetetrahydrofolate reductase (MTHFR) polymorphisms and MTHFR methylation pattern in cervical lesions development among women from Romania, a country with high prevalence of human papillomavirus (HPV) cervical infections. To achieve this goal, blood samples and cervical cytology specimens (n = 77)/tumour tissue specimens (n = 23) were investigated. As control, blood and negative cytological smears (n = 50) were used. A statistically significant association was found between T allele of C677T polymorphism and cervical lesions, heterozygote women presenting a threefold increased risk (normal/cervical lesions and tumours: wild homozygote 34/41 (0.68/0.41), heterozygote 14/51 (0.28/0.51), mutant homozygote 2/8 (0.04/0.08); OR = 3.081, P = 0.0035). Using χ square test for the control group, the HPV-negative and HPV-positive patients with cervix lesions, a significant correlation between viral infection and T allele of C677T polymorphism (P = 0.0287) was found. The MTHFR promoter was methylated in all HGSIL and tumour samples, significant differences being noted between HPV-positive samples, control group and cases of cervical dysplastic lesions without HPV DNA (P < 0. 0001) and between samples from patients with high-risk (hr)HPV versus low-risk (lr)HPV (P = 0.0026). No correlations between polymorphisms and methylation were observed. In Romania, individuals carrying T allele are susceptible for cervical lesions. MTHFR promoter methylation is associated with cervical severity lesions and with hrHPV.