Sahar Houshdaran

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.

Genome-Scale Screen for DNA Methylation-Based Detection Markers for Ovarian Cancer

Background 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. Methodology/Principal Findings We used the Illumina Infinium platform to analyze the DNA methylation status of 27,578 CpG sites in 41 ovarian tumors. We employed a marker selection strategy that emphasized sensitivity by requiring consistency of methylation across tumors, while achieving specificity by excluding markers with methylation in control leukocyte or serum DNA. Our verification strategy involved testing the ability of identified markers to monitor disease burden in serially collected serum samples from ovarian cancer patients who had undergone surgical tumor resection compared to CA-125 levels. We identified one marker, IFFO1 promoter methylation (IFFO1-M), that is frequently methylated in ovarian tumors and that is rarely detected in the blood of normal controls. When tested in 127 serially collected sera from ovarian cancer patients, IFFO1-M showed post-resection kinetics significantly correlated with serum CA-125 measurements in six out of 16 patients. Conclusions/Significance We implemented an effective marker screening and verification strategy, leading to the identification of IFFO1-M as a blood-based candidate marker for sensitive detection of ovarian cancer. Serum levels of IFFO1-M displayed post-resection kinetics consistent with a reflection of disease burden. We anticipate that IFFO1-M and other candidate markers emerging from this marker development pipeline may provide disease detection capabilities that complement existing biomarkers.

Human Endometrial Fibroblasts Derived from Mesenchymal Progenitors Inherit Progesterone Resistance and Acquire an Inflammatory Phenotype in the Endometrial Niche in Endometriosis

ABSTRACT Human endometrium undergoes cyclic regeneration involving stem/progenitor cells, but the role of resident endometrial mesenchymal stem cells (eMSC) as progenitors of endometrial stromal fibroblasts (eSF) has not been definitively demonstrated. In endometriosis, eSF display progesterone (P4) resistance with impaired decidualization in vivo and in vitro. To investigate eMSC as precursors of eSF and whether endometriosis P4 resistance is inherited from eMSC, we analyzed transcriptomes of eutopic endometrium eMSC and eSF isolated by fluorescence-activated cell sorting (FACS) from endometriosis (eMSCendo, eSFendo) and controls (eMSCcontrol, eSFcontrol) and their derived primary cultures. Differentially expressed lineage-associated genes (LG) of FACS-isolated eMSC and eSF were largely conserved in endometriosis. In culture, eSFcontrol maintained in vitro expression of a subset of eSF LG and decidualized in vitro with P4. The eMSCcontrol cultures differentiated in vitro to eSF lineage, down-regulating eMSC LG and up-regulating eSF LG, showing minimal transcriptome differences versus eSFcontrol cultures and decidualizing in vitro. Cultured eSFendo displayed less in vitro LG stability and did not decidualize in vitro. In vitro, eMSCendo differentiated to eSF lineage but showed more differentially expressed genes versus eSFendo cultures, and did not decidualize in vitro, demonstrating P4 resistance inherited from eMSCendo. Compared to controls, cultures from tissue-derived eSFendo uniquely had a pro-inflammatory phenotype not present in eMSCendo differentiated to eSF in vitro, suggesting divergent niche effects for in vivo versus in vitro lineage differentiation. These findings substantiate eMSC as progenitors of eSF and reveal eSF in endometriosis as having P4 resistance inherited from eMSC and a pro-inflammatory phenotype acquired within the endometrial niche.

Human Endometrial DNA Methylome Is Cycle-Dependent and Is Associated With Gene Expression Regulation

Human endometrium undergoes major gene expression changes, resulting in altered cellular functions in response to cyclic variations in circulating estradiol and progesterone, largely mediated by transcription factors and nuclear receptors. In addition to classic modulators, epigenetic mechanisms regulate gene expression during development in response to environmental factors and in some diseases and have roles in steroid hormone action. Herein, we tested the hypothesis that DNA methylation plays a role in gene expression regulation in human endometrium in different hormonal milieux. High throughput, genome-wide DNA methylation profiling of endometrial samples in proliferative, early secretory, and midsecretory phases revealed dynamic DNA methylation patterns with segregation of proliferative from secretory phase samples by unsupervised cluster analysis of differentially methylated genes. Changes involved different frequencies of gain and loss of methylation within or outside CpG islands. Comparison of changes in transcriptomes and corresponding DNA methylomes from the same samples revealed association of DNA methylation and gene expression in a number of loci, some important in endometrial biology. Human endometrial stromal fibroblasts treated in vitro with estradiol and progesterone exhibited DNA methylation changes in several genes observed in proliferative and secretory phase tissues, respectively. Taken together, the data support the observation that epigenetic mechanisms are involved in gene expression regulation in human endometrium in different hormonal milieux, adding endometrium to a small number of normal adult tissues exhibiting dynamic DNA methylation. The data also raise the possibility that the interplay between steroid hormone and methylome dynamics regulates normal endometrial functions and, if abnormal, may result in endometrial dysfunction and associated disorders.

Repeated assessment by high-throughput assay demonstrates that sperm DNA methylation levels are highly reproducible

OBJECTIVE To assess reliability of a high-throughput assay of sperm DNA methylation. DESIGN Observational study comparing DNA methylation of sperm isolated from 3 divided and 12 longitudinally collected semen samples. SETTING Academic medical center. PATIENT(S) One man undergoing screening semen analysis during evaluation of an infertile couple and 2 healthy fertile male volunteers. INTERVENTION(S) Spermatozoa were separated from seminal plasma and somatic cells using gradient separation. DNA was extracted from spermatozoa, and DNA methylation was assessed at 1,505 DNA sequence-specific sites. MAIN OUTCOME MEASURE(S) Repeatability of sperm DNA methylation measures, estimated by correlation coefficients. RESULT(S) DNA methylation levels were highly correlated within matched sets of divided samples (all r ≥ 0.97) and longitudinal samples (average r = 0.97). CONCLUSION(S) The described methodology reliably assessed methylation of sperm DNA at large numbers of sites. Methylation profiles were consistent over time. High-throughput assessment of sperm DNA methylation is a promising tool for studying the role of epigenetic state in male fertility.

Alterations in Deoxyribonucleic Acid (DNA) Methylation Patterns of Calca, Timp3, Mmp2, and Igf2r Are Associated With Chronic Cystitis in a Cyclophosphamide-induced Mouse Model

OBJECTIVE To determine whether epigenetic changes occur during cyclophosphamide-induced chronic bladder inflammation in mice. MATERIALS AND METHODS Epigenetic changes play a role in the regulation of inflammatory genes in noncancer diseases such as asthma and chronic obstructive pulmonary disease. However, epigenetic (deoxyribonucleic acid [DNA] methylation) changes during chronic bladder inflammation have not been previously described. Chronic cystitis was induced in 3 groups of adult CD-1 male mice using multiple weight-based intraperitoneal cyclophosphamide injections during a 3-month period. Histopathologic and MethyLight assays were performed on specimens with chronic bladder inflammation at multiple points to monitor cystitis progression and DNA methylation changes compared with the control specimens. RESULTS Histopathologic analysis showed the most extensive edema and urothelial sloughing at the 1-month point. MethyLight analyses revealed statistically significant changes in DNA methylation associated with the Calca, Timp3, Mmp2, and Igf2r genes in the chronic bladder injury model. The changes in DNA methylation associated with chronic cystitis were DNA hypomethylation of the Calca gene in the control tissue and DNA hypermethylation for the Calca, Timp3, Mmp2, and Igf2r genes compared with that in the control tissue. CONCLUSION DNA methylation changes were noted in the Calca, Timp3, Mmp2, and Igf2r genes during chronic cystitis in a murine model. Epigenetic changes appear to play a role in the regulation of inflammatory bladder genes during chronic cystitis; however, additional studies are needed to elucidate the pathways associated with these genes.

In vitro evidence that platelet-rich plasma stimulates cellular processes involved in endometrial regeneration

PurposeThe study aims to test the hypothesis that platelet-rich plasma (PRP) stimulates cellular processes involved in endometrial regeneration relevant to clinical management of poor endometrial growth or intrauterine scarring.MethodsHuman endometrial stromal fibroblasts (eSF), endometrial mesenchymal stem cells (eMSC), bone marrow-derived mesenchymal stem cells (BM-MSC), and Ishikawa endometrial adenocarcinoma cells (IC) were cultured with/without 5% activated (a) PRP, non-activated (na) PRP, aPPP (platelet-poor-plasma), and naPPP. Treatment effects were evaluated with cell proliferation (WST-1), wound healing, and chemotaxis Transwell migration assays. Mesenchymal-to-epithelial transition (MET) was evaluated by cytokeratin and vimentin expression. Differential gene expression of various markers was analyzed by multiplex Q-PCR.ResultsActivated PRP enhanced migration of all cell types, compared to naPRP, aPPP, naPPP, and vehicle controls, in a time-dependent manner (p < 0.05). The WST-1 assay showed increased stromal and mesenchymal cell proliferation by aPRP vs. naPRP, aPPP, and naPPP (p < 0.05), while IC proliferation was enhanced by aPRP and aPPP (p < 0.05). There was no evidence of MET. Expressions of MMP1, MMP3, MMP7, and MMP26 were increased by aPRP (p < 0.05) in eMSC and eSF. Transcripts for inflammation markers/chemokines were upregulated by aPRP vs. aPPP (p < 0.05) in eMSC and eSF. No difference in estrogen or progesterone receptor mRNAs was observed.ConclusionsThis is the first study evaluating the effect of PRP on different human endometrial cells involved in tissue regeneration. These data provide an initial ex vivo proof of principle for autologous PRP to promote endometrial regeneration in clinical situations with compromised endometrial growth and scarring.

Effects of noncavity-distorting fibroids on endometrial gene expression and function

Abstract Uterine fibroids are a common finding in infertility patients. Impaired implantation and decidualization have been proposed to contribute to compromised fertility. Data are limited on the endometrial transcriptome from subjects with uterine fibroids, as well as endometrial receptivity and decidualization potential of endometrial stromal fibroblasts (eSF) from women with fibroids. Our objective was to investigate the endometrial transcriptome of women with noncavity-distorting intramural fibroids and compare them to control subjects with no uterine pathology throughout the menstrual cycle. We also evaluated endometrial receptivity gene expression and basic endometrial functions such as decidualization, proliferation, and apoptosis in women with fibroid uterus. Results showed that large numbers of transcripts were significantly dysregulated throughout the menstrual cycle in fibroid subjects compared to controls. However, there were essentially no differences in the expression of receptivity markers at the tissue level, as well as decidualization markers in tissue and eSF in subjects with fibroids compared to controls. However, eSF from women with a fibroid uterus exhibited decreased proliferation potential and increased apoptosis upon decidualization. These data indicate preserved implantation and decidualization potential despite observed gene expression changes in endometrium from women with noncavity-distorting fibroids compared to controls. How this phenomenon and altered proliferation/apoptosis may contribute to impairment of endometrial function in subfertile patients warrants further investigation. Summary Sentence Decidualization and implantation potentials are not affected in the endometrium of women with noncavity-distorting fibroids compared to controls, despite a large number of differentially regulated genes.