Silvia Bosi

Methylenetetrahydrofolate reductase gene promoter hypermethylation in semen samples of infertile couples correlates with recurrent spontaneous abortion

STUDY QUESTIONnIs the methylation status of the methylenetetrahydrofolate reductase (MTHFR) promoter region in semen samples associated with recurrent spontaneous abortion (RSA)?nnnSUMMARY ANSWERnMTHFR promoter hypermethylation is more frequent in semen samples from RSA couples than in semen samples from infertile couples with no history of RSA (NRSA) and affects the whole sperm population significantly more often.nnnWHAT IS KNOWN ALREADYnModifications to the MTHFR gene such as polymorphisms and promoter methylations are associated with male infertility.nnnSTUDY DESIGN, SIZE AND DURATIONnRetrospective cohort study of semen samples from 20 RSA couples, 147 NRSA couples and 20 fertile men between 2011 and 2012.nnnMATERIALS, SETTING AND METHODSnDNA from the semen samples of RSA, NRSA and fertile men were analyzed by methylation-specific PCR amplification using primers which anneal to the methylated or unmethylated cytosine-phosphodiester bond guanine (CpG) islands within the promoter region of MTHFR. The specificity of the PCR products was assessed by DNA sequencing.nnnMAIN RESULTS AND THE ROLE OF CHANCEnThe methylated MTHFR epigenotype (including samples where it co-existed with unmethylated MTHFR epigenotypes) was detected in 75% of RSA men, 54% of NRSA men and 15% of fertile men. MTHFR methylation was observed in the whole sperm population in semen samples from 55% of RSA men compared with 8% in NRSA men (P < 0.05) and 0% in fertile men (P < 0.05). DNA sequencing analysis was fully concordant with the PCR results and revealed that when MTHFR methylation occurred, CpG islands within the promoter region were 100% methylated (hypermethylation of MTHFR promoter).nnnLIMITATIONS, REASONS FOR CAUTIONnThe relatively small sample size of RSA infertile couples.nnnWIDER IMPLICATIONS OF THE FINDINGSnThe hypermethylation of the MTHFR gene promoter should be taken into consideration as a novel putative risk factor in RSA etiology.nnnSTUDY FUNDING/COMPETING INTEREST(S)nOur institution has received an FAR research grant from the University of Ferrara, Ferrara, Italy. No competing interests declared.

Gene Expression Changes in Progression of Cervical Neoplasia Revealed by Microarray Analysis of Cervical Neoplastic Keratinocytes

To evaluate the gene expression changes involved in neoplastic progression of cervical intraepithelial neoplasia. Using microarray analysis, large‐scale gene expression profile was carried out on HPV16‐CIN2, HPV16‐CIN3, and normal cervical keratinocytes derived from two HPV16‐CIN2, two HPV‐CIN3 lesions, and two corresponding normal cervical tissues, respectively. Differentially expressed genes were analyzed in normal cervical keratinocytes compared with HPV16‐CIN2 keratinocytes and in HPV16‐CIN2 keratinocytes compared with HPV16‐CIN3 keratinocytes; 37 candidate genes with continuously increasing or decreasing expression during CIN progression were identified. One of these genes, phosphoglycerate dehydrogenase, was chosen for further characterization. Quantitative reverse transcription‐polymerase chain reaction and immunohistochemical analysis confirmed that expression of phosphoglycerate dehydrogenase consistently increases during progression of CIN toward cancer. Gene expression changes occurring during CIN progression were investigated using microarray analysis, for the first time, in CIN2 and CIN3 keratinocytes naturally infected with HPV16. Phosphoglycerate dehydrogenase is likely to be associated with tumorigenesis and may be a potential prognostic marker for CIN progression. J. Cell. Physiol. 230: 806–812, 2015.

Antibodies reacting with Simian Virus 40 capsid protein mimotopes in serum samples from patients affected by uveal melanoma

The uveal melanoma (UM) is the most common human intraocular tumour. Simian Virus 40 (SV-40) is a small DNA tumor virus detected in some malignancies, including the cutaneous melanoma. In this study an indirect ELISA using synthetic peptides that mimic SV-40 antigens, was employed to detect antibodies against SV-40 in serum samples from UM patients. Our report indicates a significant higher prevalence of antibodies against SV-40 capsid protein antigens in serum samples from UM patients compared to controls. Our data suggest an association between UM and SV-40, indicating that patients affected by uveal melanoma tested SV-40-positive could be treated by innovative therapies.

Establishment of keratinocyte colonies from small-sized cervical intraepithelial neoplasia specimens

The size of human cervical intraepithelial neoplasia (CIN) biopsies is usually very small and standard methods do not allow an adequate number of keratinocytes to be isolated for culturing purposes. In this study, a new approach to establish keratinocyte cultures from small CIN a tissue fragments was developed. Neoplastic specimens and corresponding normal tissues, which were used as controls, were digested with collagenase. Tissue‐derived fibroblasts and keratinocytes were co‐cultured in calcium and serum medium. Single keratinocyte colonies from primary cultures were expanded using a culture medium optimized in our laboratory. Primary keratinocyte colonies, as well as expanded colonies, were tested for epithelial and cervical markers such as 5, 14, 17, and 19 keratins, and p63 by immunofluorescence. Our results indicate that a variable number of primary keratinocyte colonies could be detected in neoplastic cultures, depending on the grade of cervical lesions from which the colonies originated. Single colonies, when cultured with our new medium, grew at a high rate with uniform size and morphology for some passages. Epithelial and p63 markers were expressed in keratinocyte colonies, as well as in expanded colonies. In conclusion, our study reports a rapid and easy culturing system which enables keratinocyte colonies from minute cervical tumor tissues to be obtained. Moreover, using the new culture medium, keratinocyte colonies can be expanded at a high proliferative rate. J. Cell. Physiol. 227: 3787–3795, 2012.