Differential Effects of the Hormonal and Copper Intrauterine Device on the Endometrial Transcriptome.

The contraceptive effectiveness of intrauterine devices (IUDs) has been attributed in part to a foreign body reaction in the endometrium. We performed this study to better understand mechanisms of action of contraceptives of by studying their effects on endometrial and cervical transcriptomes. We collected endometrial and cervical biopsies from women using the levonorgestrel-releasing intrauterine system (LNG-IUS, n = 11), copper intrauterine device (cu-IUD, n = 13) or levonorgestrel-containing combined oral contraceptives (COC, n = 12), and from women not using contraceptives (control group, n = 11). Transcriptional profiling was performed with Affymetrix arrays, Principal Component Analysis and the bioconductor package limma. In endometrial samples from cu-IUD users, there were no genes with statistically significant differential expression compared to controls. In LNG-IUS users, 2509 genes were differentially expressed and mapped predominantly onto immune and inflammatory pathways. The cervical samples showed no statistically significant differential gene expression compared to controls. Hormonal and copper IUDs have significantly different effects on the endometrial transcriptome, with the LNG-IUS transcriptome showing pronounced inflammation and immune activation compared to controls whereas the cu-IUD transcriptome was indistinguishable from luteal phase endometrium. These findings argue against a foreign body reaction as a common mechanism of action of IUDs

Progestins Related to Progesterone and Testosterone Elicit Divergent Human Endometrial Transcriptomes and Biofunctions.

Progestins are widely used for the treatment of gynecologic disorders and alone, or combined with an estrogen, are used as contraceptives. While their potencies, efficacies and side effects vary due to differences in structures, doses and routes of administration, little is known about their effects on the endometrial transcriptome in the presence or absence of estrogen. Herein, we assessed the transcriptome and pathways induced by progesterone (P4) and the three most commonly used synthetic progestins, medroxyprogesterone acetate (MPA), levonorgestrel (LNG), and norethindrone acetate (NETA), on human endometrial stromal fibroblasts (eSF), key players in endometrial physiology and reproductive success. While there were similar transcriptional responses, each progestin induced unique genes and biofunctions, consistent with their structural similarities to progesterone (P4 and MPA) or testosterone (LNG and NETA), involving cellular proliferation, migration and invasion. Addition of estradiol (E2) to each progestin influenced the number of differentially expressed genes and biofunctions in P4 and MPA, while LNG and NETA signatures were more independent of E2. Together, these data suggest different mechanisms of action for different progestins, with progestin-specific altered signatures when combined with E2. Further investigation is warranted for a personalized approach in different gynecologic disorders, for contraception, and minimizing side effects associated with their use

Global endometrial DNA methylation analysis reveals insights into mQTL regulation and associated endometriosis disease risk and endometrial function

Epigenomics; Genetic predisposition to disease; Urogenital reproductive disordersEpigenómica; Predisposición genética a la enfermedad; Trastornos reproductivos urogenitalesEpigenòmica; Predisposició genètica a la malaltia; Trastorns reproductius urogenitalsEndometriosis is a leading cause of pain and infertility affecting millions of women globally. Herein, we characterize variation in DNA methylation (DNAm) and its association with menstrual cycle phase, endometriosis, and genetic variants through analysis of genotype data and methylation in endometrial samples from 984 deeply-phenotyped participants. We estimate that 15.4% of the variation in endometriosis is captured by DNAm and identify significant differences in DNAm profiles associated with stage III/IV endometriosis, endometriosis sub-phenotypes and menstrual cycle phase, including opening of the window for embryo implantation. Menstrual cycle phase was a major source of DNAm variation suggesting cellular and hormonally-driven changes across the cycle can regulate genes and pathways responsible for endometrial physiology and function. DNAm quantitative trait locus (mQTL) analysis identified 118,185 independent cis-mQTLs including 51 associated with risk of endometriosis, highlighting candidate genes contributing to disease risk. Our work provides functional evidence for epigenetic targets contributing to endometriosis risk and pathogenesis. Data generated serve as a valuable resource for understanding tissue-specific effects of methylation on endometrial biology in health and disease.This work has been supported by the National Institutes of Health (NIH) NICHD R01 HD089511. It was also supported, in part, by funding from Wellbeing of Women (through sponsorship from PwC) (R42533) and the Medical Research Council (MR/N024524/1 and MR/N022556/1) and NIH HD094842 (Harvard/MSU). K.K. was supported by NIH NCI R37 CA233774. A.F.M. was supported by an Australian Research Council Future Fellowship (FT200100837). G.W.M. was supported by NHMRC Fellowship (GNT1177194)

DNA Methylation Profiles of Ovarian Epithelial Carcinoma Tumors and Cell Lines

BACKGROUND:Epithelial ovarian carcinoma is a significant cause of cancer mortality in women worldwide and in the United States. Epithelial ovarian cancer comprises several histological subtypes, each with distinct clinical and molecular characteristics. The natural history of this heterogeneous disease, including the cell types of origin, is poorly understood. This study applied recently developed methods for high-throughput DNA methylation profiling to characterize ovarian cancer cell lines and tumors, including representatives of three major histologies. METHODOLOGY/PRINCIPAL FINDINGS:We obtained DNA methylation profiles of 1,505 CpG sites (808 genes) in 27 primary epithelial ovarian tumors and 15 ovarian cancer cell lines. We found that the DNA methylation profiles of ovarian cancer cell lines were markedly different from those of primary ovarian tumors. Aggregate DNA methylation levels of the assayed CpG sites tended to be higher in ovarian cancer cell lines relative to ovarian tumors. Within the primary tumors, those of the same histological type were more alike in their methylation profiles than those of different subtypes. Supervised analyses identified 90 CpG sites (68 genes) that exhibited 'subtype-specific' DNA methylation patterns (FDR<1%) among the tumors. In ovarian cancer cell lines, we estimated that for at least 27% of analyzed autosomal CpG sites, increases in methylation were accompanied by decreases in transcription of the associated gene. SIGNIFICANCE:The significant difference in DNA methylation profiles between ovarian cancer cell lines and tumors underscores the need to be cautious in using cell lines as tumor models for molecular studies of ovarian cancer and other cancers. Similarly, the distinct methylation profiles of the different histological types of ovarian tumors reinforces the need to treat the different histologies of ovarian cancer as different diseases, both clinically and in biomarker studies. These data provide a useful resource for future studies, including those of potential tumor progenitor cells, which may help illuminate the etiology and natural history of these cancers

Identification of preferential target sites for human DNA methyltransferases

DNA methyltransferases (DNMTs) play an important role in establishing and maintaining DNA methylation. Aberrant expression of DNMTs and their isoforms has been found in many types of cancer, and their contribution to aberrant DNA methylation has been proposed. Here, we generated HEK 293T cells stably transfected with each of 13 different DNMTs (DNMT1, two DNMT3A isoforms, nine DNMT3B isoforms and DNMT3L) and assessed the DNA methylation changes induced by each DNMT. We obtained DNA methylation profiles of DNA repetitive elements and 1505 CpG sites from 808 cancer-related genes. We found that DNMTs have specific and overlapping target sites and their DNA methylation target profiles are a reflection of the DNMT domains. By examining H3K4me3 and H3K27me3 modifications in the 808 gene promoter regions using promoter ChIP-on-chip analysis, we found that specific de novo DNA methylation target sites of DNMT3A1 are associated with H3K4me3 modification that are transcriptionally active, whereas the specific target sites of DNMT3B1 are associated with H3K27me3 modification that are transcriptionally inactive. Our data suggest that different DNMT domains are responsible for targeting DNA methylation to specific regions of the genome, and this targeting might be associated with histone modifications

Genome-scale screen for DNA methylation-based detection markers for ovarian cancer.

The identification of sensitive biomarkers for the detection of ovarian cancer is of high clinical relevance for early detection and/or monitoring of disease recurrence. We developed a systematic multi-step biomarker discovery and verification strategy to identify candidate DNA methylation markers for the blood-based detection of ovarian cancer

Idiopathic Male Infertility Is Strongly Associated with Aberrant Promoter Methylation of Methylenetetrahydrofolate Reductase (MTHFR)

Abnormal germline DNA methylation in males has been proposed as a possible mechanism compromising spermatogenesis of some men currently diagnosed with idiopathic infertility. Previous studies have been focused on imprinted genes with DNA methylation in poor quality human sperms. However, recent but limited data have revealed that sperm methylation abnormalities may involve large numbers of genes or shown that genes that are not imprinted are also affected.Using the methylation-specific polymerase chain reaction and bisulfite sequencing method, we examined methylation patterns of the promoter of methylenetetrahydrofolate reductase (MTHFR) gene (NG_013351: 1538-1719) in sperm DNA obtained from 94 idiopathic infertile men and 54 normal fertile controls. Subjects with idiopathic infertility were further divided into groups of normozoospermia and oligozoospermia. Overall, 45% (41/94) of idiopathic infertile males had MTHFR hypermethylation (both hemimethylation and full methylation), compared with 15% of fertile controls (P<0.05). Subjects with higher methylation level of MTHFR were more likely to have idiopathic male infertility (P-value for trend  = 0.0007). Comparing the two groups of idiopathic infertile subjects with different sperm concentrations, a higher methylation pattern was found in the group with oligozoospermia.Hypermethylation of the promoter of MTHFR gene in sperms is associated with idiopathic male infertility. The functional relevance of hypermathylation of MTHFR to male fertility warrants further investigation

Promoter Methylation in Head and Neck Squamous Cell Carcinoma Cell Lines Is Significantly Different than Methylation in Primary Tumors and Xenografts

Studies designed to identify novel methylation events related to cancer often employ cancer cell lines in the discovery phase of the experiments and have a relatively low rate of discovery of cancer-related methylation events. An alternative algorithm for discovery of novel methylation in cancer uses primary tumor-derived xenografts instead of cell lines as the primary source of nucleic acid for evaluation. We evaluated DNA extracted from primary head and neck squamous cell carcinomas (HNSCC), xenografts grown from these primary tumors in nude mice, HNSCC-derived cell lines, normal oral mucosal samples, and minimally transformed oral keratinocyte-derived cell lines using Illumina Infinum Humanmethylation 27 genome-wide methylation microarrays. We found >2,200 statistically significant methylation differences between cancer cell lines and primary tumors and when comparing normal oral mucosa to keratinocyte cell lines. We found no statistically significant promoter methylation differences between primary tumor xenografts and primary tumors. This study demonstrates that tumor-derived xenografts are highly accurate representations of promoter methylation in primary tumors and that cancer derived cell lines have significant drawbacks for discovery of promoter methylation alterations in primary tumors. These findings also support use of primary tumor xenografts for the study of methylation in cancer, drug discovery, and the development of personalized cancer treatments

A genome-wide DNA methylation study in colorectal carcinoma

<p>Abstract</p> <p>Background</p> <p>We performed a genome-wide scan of 27,578 CpG loci covering 14,475 genes to identify differentially methylated loci (DML) in colorectal carcinoma (CRC).</p> <p>Methods</p> <p>We used Illumina's Infinium methylation assay in paired DNA samples extracted from 24 fresh frozen CRC tissues and their corresponding normal colon tissues from 24 consecutive diagnosed patients at a tertiary medical center.</p> <p>Results</p> <p>We found a total of 627 DML in CRC covering 513 genes, of which 535 are novel DML covering 465 genes. We also validated the Illumina Infinium methylation data for top-ranking genes by non-bisulfite conversion q-PCR-based methyl profiler assay in a subset of the same samples. We also carried out integration of genome-wide copy number and expression microarray along with methylation profiling to see the functional effect of methylation. Gene Set Enrichment Analysis (GSEA) showed that among the major "gene sets" that are hypermethylated in CRC are the sets: "inhibition of adenylate cyclase activity by G-protein signaling", "Rac guanyl-nucleotide exchange factor activity", "regulation of retinoic acid receptor signaling pathway" and "estrogen receptor activity". Two-level nested cross validation showed that DML-based predictive models may offer reasonable sensitivity (around 89%), specificity (around 95%), positive predictive value (around 95%) and negative predictive value (around 89%), suggesting that these markers may have potential clinical application.</p> <p>Conclusion</p> <p>Our genome-wide methylation study in CRC clearly supports most of the previous findings; additionally we found a large number of novel DML in CRC tissue. If confirmed in future studies, these findings may lead to identification of genomic markers for potential clinical application.</p

Differential Analysis of Ovarian and Endometrial Cancers Identifies a Methylator Phenotype

Despite improved outcomes in the past 30 years, less than half of all women diagnosed with epithelial ovarian cancer live five years beyond their diagnosis. Although typically treated as a single disease, epithelial ovarian cancer includes several distinct histological subtypes, such as papillary serous and endometrioid carcinomas. To address whether the morphological differences seen in these carcinomas represent distinct characteristics at the molecular level we analyzed DNA methylation patterns in 11 papillary serous tumors, 9 endometrioid ovarian tumors, 4 normal fallopian tube samples and 6 normal endometrial tissues, plus 8 normal fallopian tube and 4 serous samples from TCGA. For comparison within the endometrioid subtype we added 6 primary uterine endometrioid tumors and 5 endometrioid metastases from uterus to ovary. Data was obtained from 27,578 CpG dinucleotides occurring in or near promoter regions of 14,495 genes. We identified 36 locations with significant increases or decreases in methylation in comparisons of serous tumors and normal fallopian tube samples. Moreover, unsupervised clustering techniques applied to all samples showed three major profiles comprising mostly normal samples, serous tumors, and endometrioid tumors including ovarian, uterine and metastatic origins. The clustering analysis identified 60 differentially methylated sites between the serous group and the normal group. An unrelated set of 25 serous tumors validated the reproducibility of the methylation patterns. In contrast, >1,000 genes were differentially methylated between endometrioid tumors and normal samples. This finding is consistent with a generalized regulatory disruption caused by a methylator phenotype. Through DNA methylation analyses we have identified genes with known roles in ovarian carcinoma etiology, whereas pathway analyses provided biological insight to the role of novel genes. Our finding of differences between serous and endometrioid ovarian tumors indicates that intervention strategies could be developed to specifically address subtypes of epithelial ovarian cancer