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Variations in DNA methylation levels in the placenta are thought to influence gene expression and are associated with complications of pregnancy, like fetal growth restriction (FGR). The most important cause for FGR is placental dysfunction. Here, we examined whether changes in DNA methylation, followed by gene expression changes, are mechanistically involved in the etiology of FGR. In this retrospective case-control study, we examined the association between small-for-gestational-age (SGA) children and both DNA methylation and gene expression levels of the genes WNT2, IGF2/H19, SERPINA3, HERVWE1, and PPARG in first-trimester placental tissue. We also examined the repetitive element LINE-1. These candidate genes have been reported in the literature to be associated with SGA. We used first-trimester placental tissue from chorionic villus biopsies. A total of 35 SGA children (with a birth weight below the 10th percentile) were matched to 70 controls based on their gestational age. DNA methylation levels were analyzed by pyrosequencing and mRNA levels were analyzed by real-time PCR. None of the average DNA methylation levels, measured for each gene, showed a significant difference between SGA placental tissue compared to control tissue. However, hypermethylation of WNT2 was detected on two CpG positions in SGA. This was not associated with changes in gene expression. Apart from two CpG positions of the WNT2 gene, in early placenta samples, no evident changes in DNA methylation or expression were found. This indicates that the already reported changes in term placenta are not present in the early placenta, and therefore must arise after the first trimester., (© 2016 by the Society for the Study of Reproduction, Inc.)

Epigenetic modifications, such as aberrant DNA promoter methylation, are frequently observed in cervical cancer. Identification of hypermethylated regions allowing discrimination between normal cervical epithelium and high-grade cervical intraepithelial neoplasia (CIN2/3), or worse, may improve current cervical cancer population-based screening programs. In this study, the DNA methylome of high-grade CIN lesions was studied using genome-wide DNA methylation screening to identify potential biomarkers for early diagnosis of cervical neoplasia. Methylated DNA Immunoprecipitation (MeDIP) combined with DNA microarray was used to compare DNA methylation profiles of epithelial cells derived from high-grade CIN lesions with normal cervical epithelium. Hypermethylated differentially methylated regions (DMRs) were identified. Validation of nine selected DMRs using BSP and MSP in cervical tissue revealed methylation in 63.2-94.7% high-grade CIN and in 59.3-100% cervical carcinomas. QMSP for the two most significant high-grade CIN-specific methylation markers was conducted exploring test performance in a large series of cervical scrapings. Frequency and relative level of methylation were significantly different between normal and cancer samples. Clinical validation of both markers in cervical scrapings from patients with an abnormal cervical smear confirmed that frequency and relative level of methylation were related with increasing severity of the underlying CIN lesion and that ROC analysis was discriminative. These markers represent the COL25A1 and KATNAL2 and their observed increased methylation upon progression could intimate the regulatory role in carcinogenesis. In conclusion, our newly identified hypermethylated DMRs represent specific DNA methylation patterns in high-grade CIN lesions and are candidate biomarkers for early detection.

Objective: Lymph node status in early-stage vulvar cancer can be accurately assessed by the sentinel-node (SN) procedure. Molecular techniques, such as DNA-methylation assay, might improve SN assessment. In this study, we selected methylation markers for vulvar cancer and determined if these methylation markers were suitable for lymph node assessment., Methods: We performed methylation specific PCR on DNA isolated from primary tumors, metastatic lymph nodes, and negative lymph nodes from twenty vulvar cancer patients using the following genes: P16INK4a, MGMT, TWIST1, CADM1, TERT, and TFPI2. For P16INK4a and MGMT immunohistochemistry was performed on primary tumors and metastatic lymph nodes in order to explore intratumor heterogeneity in gene expression patterns., Results: TERT was methylated in all vulvar cancers, P16INK4a in 13/20, TFPI2 in 12/20, CADM1 in 11/20, MGMT in 9/20, and TWIST1 in 7/20. A panel of three methylation markers (P16INK4a, TERT and TFPI2) reached a sensitivity of 67% and specificity of 100% for detection of metastatic lymph nodes. Immunohistochemistry showed intratumor heterogeneity for expression of P16INK4a and MGMT in respectively 55% and 45% of primary tumors., Conclusions: Our study shows methylation for one or more methylation markers in all vulvar cancers. Despite a specificity of 100% our panel of three methylation markers had only moderate sensitivity for metastatic lymph node detection, thereby limiting its applicability for lymph node assessment. Intratumor heterogeneity for expression of P16INK4a and MGMT may reflect intratumor heterogeneity for methylation patterns and thereby in general explain the moderate sensitivity of our marker panel for detection of metastases., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Cervical neoplasia-specific biomarkers, e.g. DNA methylation markers, with high sensitivity and specificity are urgently needed to improve current population-based screening on (pre)malignant cervical neoplasia. We aimed to identify new cervical neoplasia-specific DNA methylation markers and to design and validate a methylation marker panel for triage of high-risk human papillomavirus (hr-HPV) positive patients. First, high-throughput quantitative methylation-specific PCRs (QMSP) on a novel OpenArray™ platform, representing 424 primers of 213 cancer specific methylated genes, were performed on frozen tissue samples from 84 cervical cancer patients and 106 normal cervices. Second, the top 20 discriminating methylation markers were validated by LightCycler® MSP on frozen tissue from 27 cervical cancer patients and 20 normal cervices and ROCs and test characteristics were assessed. Three new methylation markers were identified (JAM3, EPB41L3 and TERT), which were subsequently combined with C13ORF18 in our four-gene methylation panel. In a third step, our methylation panel detected in cervical scrapings 94% (70/74) of cervical cancers, while in a fourth step 82% (32/39) cervical intraepithelial neoplasia grade 3 or higher (CIN3+) and 65% (44/68) CIN2+ were detected, with 21% positive cases for ≤CIN1 (16/75). Finally, hypothetical scenario analysis showed that primary hr-HPV testing combined with our four-gene methylation panel as a triage test resulted in a higher identification of CIN3 and cervical cancers and a higher percentage of correct referrals compared to hr-HPV testing in combination with conventional cytology. In conclusion, our four-gene methylation panel might provide an alternative triage test after primary hr-HPV testing., (Copyright © 2011 UICC.)

Objective: To explore the feasibility of DNA methylation analysis for the detection of cervical neoplasia in self-obtained cervico-vaginal lavages., Methods: Lavages collected by a self-sampling device and paired cervical scrapings were obtained from 20 cervical cancer patients and 23 patients referred with an abnormal cervical smear (15 with high-grade cervical intraepithelial neoplasia (CIN2+) and 8 without CIN). All lavages and scrapings were analyzed by liquid based cytology (LBC), Hybrid Capture II (HC-II) for hr-HPV DNA detection and by DNA methylation analysis (JAM3, TERT, EPB41L3 and C13ORF18). Concordance between lavages and scrapings was measured by Cohen's Kappa (k)., Results: In lavages and scrapings from cervical cancer patients (n=20), methylation analysis was positive in 19 (95%) and 19 (95%), HC-II in 16 (80%) and 15 (75%) and LBC in 15 (75%) and 19 (95%), respectively. In lavages and scrapings from CIN2+ patients (n=15), methylation analysis was positive in 10 (67%) and 12 (80%), HC-II in 15 (100%) and 15 (100%) and LBC in 11 (73%) and 12 (80%), respectively. Concordance between cervical scrapings and lavages (n=43) was for LBC k=0.522 (p<0.001), hr-HPV testing k=0.551 (p<0.001) and DNA methylation analysis k=0.653 (p<0.001)., Conclusions: DNA methylation analysis in cervico-vaginal lavages obtained by a self-sampling device is feasible and its diagnostic performance appears to be at least comparable to the detection of cervical neoplasia by cytomorphology and hr-HPV. Our pilot study suggests that detection of cervical neoplasia by DNA methylation analysis in cervico-vaginal lavages warrants exploration of its use in large prospective studies., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Objectives: To determine methylation status of nine genes, previously described to be frequently methylated in cervical cancer, in squamous intraepithelial lesions (SIL)., Methods: QMSP was performed in normal cervix, low-grade (L)SIL, high-grade (H)SIL, adenocarcinomas and squamous cell cervical cancers, and in corresponding cervical scrapings., Results: Only CCNA1 was never methylated in normal cervices and rarely in LSILs. All other genes showed methylation in normal cervices, with CALCA, SPARC and RAR-beta(2) at high levels. Methylation frequency of 6 genes (DAPK, APC, TFPI2, SPARC, CCNA1 and CADM1) increased with severity of the underlying cervical lesion. DAPK showed the highest increase in methylation frequency between LSIL and HSIL (10% vs. 40%, p<0.05), while CCNA1 and TFPI2 were most prominently methylated in cervical cancers compared to HSILs (25% vs. 52%, p<0.05, 30% vs. 58%, p<0.05). CADM1 methylation in cervical cancers was related to depth of invasion (p<0.05) and lymph vascular space involvement (p<0.01), suggesting a role in invasive potential of cervical cancers. Methylation ratios in scrapings reflected methylation status of the underlying lesions (p<0.05)., Conclusion: Methylation of previously reported cervical cancer specific genes frequently occurs in normal epithelium. However, frequency of methylation increases during cervical carcinogenesis, with CCNA1 and DAPK as the best markers to distinguish normal/LSIL from HSIL/cancer lesions.

Purpose: Recently, we reported 13 possible cervical cancer-specific methylated biomarkers identified by pharmacologic unmasking microarray in combination with large-genome computational screening. The aim of the present study was to perform an in-depth analysis of the methylation patterns of these 13 candidate genes in cervical neoplasia and to determine their diagnostic relevance., Experimental Design and Results: Five of the 13 gene promoters (C13ORF18, CCNA1, TFPI2, C1ORF166, and NPTX1) were found to be more frequently methylated in frozen cervical cancer compared with normal cervix specimens. Quantitative methylation analysis for these five markers revealed that both CCNA1 and C13ORF18 were methylated in 68 of 97 cervical scrapings from cervical cancer patients and in only 5 and 3 scrapings, respectively, from 103 healthy controls (P < 0.0005). In cervical scrapings from patients referred with an abnormal Pap smear, CCNA1 and C13ORF18 were methylated in 2 of 43 and 0 of 43 CIN 0 (no cervical intraepithelial neoplasia) and in 1 of 41 and 0 of 41 CIN I, respectively. Furthermore, 8 of 43 CIN II, 22 of 43 CIN III, and 3 of 3 microinvasive cancer patients were positive for both markers. Although sensitivity for CIN II or higher (for both markers 37%) was low, specificity (96% and 100%, respectively) and positive predictive value (92% and 100%, respectively) were high., Conclusion: Methylation of CCNA1 and C13ORF18 in cervical scrapings is strongly associated with CIN II or higher-grade lesions. Therefore, these markers might be used for direct referral to gynecologists for patients with a methylation-positive scraping.