Combined histological and DNA methylome profiling approaches may provide insights into the pathophysiology of ovarian endometriomas.

Purpose: To test the theory that invaginated ovarian surface epithelium and endometrial implants on the ovary form ovarian endometriomas. Methods: Adhesion sites of ovarian endometrioma on the peritoneum and consecutive ovarian endometrioma cyst wall, called non-adhesion sites, were histologically examined. DNA methylomes of the adhesion sites, non-adhesion sites, and blueberry spots were compared with those of ovary, endometrium, and peritoneum. Results: The non-adhesion sites showed an ovarian surface epithelium-like structure near the adhesion site, which continued to a columnar epithelium-like structure. Calretinin staining was strong in the ovarian surface epithelium-like structure but weak in the columnar epithelium-like structure. Estrogen receptors were absent in the ovarian surface epithelium-like structure, but present in the columnar epithelium-like structure. The adhesion sites had endometrial gland-like structures that expressed estrogen receptors. Analyses of DNA methylomes classified the non-adhesion sites and ovaries into the same group, suggesting that ovarian endometriomas originate from the ovarian surface epithelium. The adhesion sites, blueberry spots and peritoneum were classified in the same group, suggesting that the adhesion sites and blueberry spots originate from the peritoneum. Conclusions: The present results support the invagination theory. Ovarian endometriomas consist of invaginated ovarian surface epithelium with celomic metaplasia and endometrium implants on the peritoneum.

Wilms tumor 1 regulates lipid accumulation in human endometrial stromal cells during decidualization.

We previously reported that the transcription factor Wilms tumor 1 (WT1) regulates the expression of insulin-like growth factor-binding protein-1 (IGFBP-1) and prolactin (PRL) during decidualization of human endometrial stromal cells (ESCs). However, other roles of WT1 in decidualization remain to be fully clarified. Here, we investigated how WT1 regulates the physiological functions of human ESCs during decidualization. We incubated ESCs isolated from proliferative-phase endometrium with cAMP to induce decidualization, knocked down WT1 with siRNA, and generated three types of treatments (nontreated cells, cAMP-treated cells, and cAMP-treated + WT1-knockdown cells). To identify WT1-regulated genes, we used gene microarrays and compared the transcriptome data obtained among these three treatments. We observed that WT1 up-regulates 121 genes during decidualization, including several genes involved in lipid transport. The WT1 knockdown inhibited lipid accumulation (LA) in the cAMP-induced ESCs. To examine the mechanisms by which WT1 regulates LA, we focused on very low-density lipoprotein receptor (VLDLR), which is involved in lipoprotein uptake. We found that cAMP up-regulates VLDLR and that the WT1 knockdown inhibits it. Results of ChIP assays revealed that cAMP increases the recruitment of WT1 to the promoter region of the VLDLR gene, indicating that WT1 regulates VLDLR expression. Moreover, VLDLR knockdown inhibited cAMP-induced LA, and VLDLR overexpression reverted the suppression of LA caused by the WT1 knockdown. Taken together, our results indicate that WT1 enhances lipid storage by up-regulating VLDLR expression in human ESCs during decidualization.

The clinical outcome of Dienogest treatment followed by in vitro fertilization and embryo transfer in infertile women with endometriosis.

BACKGROUND: Endometriosis is considered to be the most intractable cause of female infertility. Administering any type of treatment for endometriosis before in vitro fertilization and embryo transfer (IVF-ET) is an important strategy for improving the IVF-ET outcomes for infertile women with endometriosis. In fact, treatment with a gonadotropin-releasing hormone (GnRH) agonist just before IVF-ET has been reported to improve the clinical outcome in endometriosis patients. However, the benefit of Dienogest (DNG), a synthetic progestin, treatment just before IVF-ET remains unclear. METHODS: Sixty-eight infertile women with Stage III or IV endometriosis (ovarian endometrial cyst < 4 cm) were recruited for this study. The subjects were divided into 2 groups: a DNG group (n = 33) and a control group (n = 35). DNG was administered orally every day for 12 weeks prior to the conventional IVF-ET cycle in the DNG group. Standard controlled ovarian hyperstimulation with the GnRH agonist long protocol was performed in the control group. The numbers of mature follicles and retrieved oocytes, fertilization rates, implantation rates, and clinical pregnancy rate were compared between the two groups. In addition, the concentrations of inflammatory cytokines, oxidative stress markers, and antioxidants in follicular fluids were also measured. RESULTS: The numbers of growing follicles, retrieved oocytes, fertilized oocytes, and blastocysts were significantly lower in the DNG group than in the control group. The fertilization and blastocyst rates were also lower in the DNG group than in the control group. Although there was no significant difference in the implantation rate between the groups, the cumulative pregnancy rate and live birth rate were lower in the DNG group than in the control group. There was no significant difference in the abortion rate. Our results failed to show that DNG reduces the inflammatory cytokine levels and oxidative stress in follicular fluids. CONCLUSIONS: Administering DNG treatment just before IVF-ET did not provide any benefits to improve the clinical outcomes for infertile women with endometriosis.

SATB2 and NGR1: potential upstream regulatory factors in uterine leiomyomas.

PURPOSE: We attempted to identify the genes involved in the pathogenesis of uterine leiomyomas, under a hypothesis that the aberrant expression of upstream regulatory genes caused by aberrant DNA methylation is involved in the onset and development of uterine leiomyomas. METHODS: To find such genes, we compared genome-wide mRNA expression and DNA methylation in uterine leiomyomas and adjacent normal myometrium. Analysis of the data by Ingenuity Pathway Analysis software identified SATB2 which is known to be an epigenetic regulator, and NRG1 as candidate upstream regulatory genes. To infer the functions of these genes, human uterine smooth muscle cell lines overexpressing SATB2 or NRG1 genes were established (SATB2 or NRG1 lines), and their transcriptomes and pathways were analyzed. RESULTS: SATB2 and NRG1 were confirmed to be hypermethylated and upregulated in most uterine leiomyoma specimens (nine to 11 of the 11 cases). Among the established cell lines, morphological changes from spindle-like forms to fibroblast-like forms with elongated protrusions were observed in only the SATB2 line. Pathway analysis revealed that WNT/ß-catenin and TGF-ß signaling pathways which are related to the pathogenesis of uterine leiomyomas were activated in both SATB2 and NRG1 lines. In addition, signaling of growth factors including VEGF, PDGF, and IGF1, and retinoic acid signaling were activated in the SATB2 and NRG1 lines, respectively. CONCLUSIONS: These results indicate that SATB2 and NRG1 overexpression induced many of the signaling pathways that are considered to be involved in the pathogenesis of uterine leiomyomas, suggesting that these genes have roles as upstream regulatory factors.

Genome-wide DNA methylation analysis revealed stable DNA methylation status during decidualization in human endometrial stromal cells.

BACKGROUND: During decidualization in endometrial stromal cells (ESCs), expressions of a number of genes and epigenetic modifications of histones are altered. However, there is little information about whether DNA methylation, which is another epigenetic mechanism, also changes during decidualization. Here, we examined the genome-wide DNA methylation profiles in ESCs during decidualization and their associations with the changes of gene expressions and histone modifications. RESULTS: ESCs were incubated with estradiol and medroxyprogesterone acetate for 14 days to induce decidualization. The genome-wide DNA methylation profiles were compared between the non-decidualized ESCs and the decidualized ESCs. Of 482,005 CpGs, only 23 CpGs (0.0048%) showed different DNA methylation statuses. The DNA methylation statuses of the differentially expressed genes and the regions with different histone modifications (H3K4 tri-methylation and H3K27 acetylation) were also compared between the ESCs. In the upregulated and downregulated genes in decidualized ESCs, DNA methylation statuses around the promoter region of the genes did not significantly differ between the ESCs. In the regions with different histone modification, DNA methylation statuses did not differ between the ESCs. The differentially expressed genes and the differential histone modification regions were hypomethylated. CONCLUSIONS: Culturing ESCs with estrogen/progesterone did not distort the physiological pattern of DNA methylation, although mRNA expression and histone modifications were dynamically altered. A genome-wide DNA methylation analysis revealed stable DNA methylation statuses during decidualization in human endometrial stromal cells. DNA hypomethylation is maintained for the variable changes of histone modifications and gene expression during decidualization.

Glucose regulates the histone acetylation of gene promoters in decidualizing stromal cells.

Decidualization stimuli activate the insulin signaling pathway and increase the glucose uptake in human endometrial stromal cells (ESCs). The inductions of prolactin (PRL) and IGF-binding protein-1 (IGFBP1), specific markers of decidualization, were inhibited by incubating ESCs under low glucose concentrations. These results suggested that decidualization stimuli activate the insulin signaling pathway, which contributes to decidualization through the increase of glucose uptake. Here, we investigated the mechanisms by which glucose regulates decidualization. ESCs were incubated with cAMP to induce decidualization. We examined whether low glucose affects the expression levels of transcription factors that induce decidualization. Forkhead box O1 (FOXO1) expression was significantly suppressed under low glucose conditions. Knockdown of FOXO1 by siRNA inhibited the expression levels of PRL and IGFBP1 during decidualization. Taken together, our results showed that low glucose inhibits decidualization by decreasing FOXO1 expression. We also examined the levels of histone H3K27 acetylation (H3K27ac), which is related to active transcription, of the promoter regions of FOXO1, PRL and IGFBP1 by ChIP assay. The H3K27ac levels of these promoter regions were increased by decidualization under normal glucose conditions, but not under low glucose conditions. Thus, our results show that glucose is indispensable for decidualization by activating the histone modification status of the promoters of PRL, IGFBP1 and FOXO1.

Aberrant DNA methylation suppresses expression of estrogen receptor 1 (ESR1) in ovarian endometrioma.

BACKGROUND: In ovarian endometriomas (OE), the expression statuses of various steroid hormone receptors are altered compared with their expression statuses in eutopic endometrium (EE). For example, in OE, the expressions of estrogen receptor 1 (ESR1), which encodes ERα, and progesterone receptor (PGR) are downregulated, while the expression of ESR2, which encodes ERß, is upregulated. The causes of these changes are unclear. DNA methylation of a specific region of a gene can result in tissue-specific gene expression. Such regions are called tissue-dependent and differentially methylated regions (T-DMRs). We previously reported that the tissue-specific expression of ESR1 is regulated by DNA methylation of a T-DMR in normal tissues. In the present study, we examined whether aberrant DNA methylation of the T-DMR is associated with the altered expressions of ESR1, ESR2 and PGR in OE. RESULTS: Gene expression levels of ESR1, ESR2 and PGR were measured by quantitative RT-PCR. The expression levels of ESR1 and PGR were significantly lower and the expression level of ESR2 was significantly higher in OE than in EE. DNA methylation statuses were examined with an Infinium HumanMethylation450K BeadChip and sodium bisulfite sequencing. DNA methylation at the T-DMRs of ESR1 were significantly higher in OE than in EE, but no significant differences were observed in the DNA methylation statuses of ESR2 and PGR. CONCLUSIONS: Aberrant DNA methylation of the T-DMR was associated with the impaired expression of ESR1, but not the altered expressions of ESR2 and PGR, in OE.

C/EBPß regulates Vegf gene expression in granulosa cells undergoing luteinization during ovulation in female rats.

The ovulatory LH-surge increases Vegf gene expression in granulosa cells (GCs) undergoing luteinization during ovulation. To understand the factors involved in this increase, we examined the roles of two transcription factors and epigenetic mechanisms in rat GCs. GCs were obtained from rats treated with eCG before, 4 h, 8 h, 12 h and 24 h after hCG injection. Vegf mRNA levels gradually increased after hCG injection and reached a peak at 12 h. To investigate the mechanism by which Vegf is up-regulated after hCG injection, we focused on C/EBPß and HIF1α. Their protein expression levels were increased at 12 h. The binding activity of C/EBPß to the Vegf promoter region increased after hCG injection whereas that of HIF1α did not at this time point. The C/EBPß binding site had transcriptional activities whereas the HIF1α binding sites did not have transcriptional activities under cAMP stimulation. The levels of H3K9me3 and H3K27me3, which are transcriptional repression markers, decreased in the C/EBPß binding region after hCG injection. The chromatin structure of this region becomes looser after hCG injection. These results show that C/EBPß regulates Vegf gene expression with changes in histone modifications and chromatin structure of the promoter region in GCs undergoing luteinization during ovulation.

The distal upstream region of insulin-like growth factor-binding protein-1 enhances its expression in endometrial stromal cells during decidualization.

We have previously shown that decidualization of human endometrial stromal cells (ESCs) causes a genome-wide increase in the levels of acetylation of histone-H3 Lys-27 (H3K27ac). We also reported that the distal gene regions, more than 3 kb up- or downstream of gene transcription start sites have increased H3K27ac levels. Insulin-like growth factor-binding protein-1 (IGFBP-1) is a specific decidualization marker and has increased H3K27ac levels in its distal upstream region (-4701 to -7501 bp). Here, using a luciferase reporter gene construct containing this IGFBP-1 upstream region, we tested the hypothesis that it is an IGFBP-1 enhancer. To induce decidualization, we incubated ESCs with cAMP and found that cAMP increased luciferase expression, indicating that decidualization increased the transcriptional activity from the IGFBP-1 upstream region. Furthermore, CRISPR/Cas9-mediated deletion of this region in HepG2 cells significantly reduced IGFBP-1 expression, confirming its role as an IGFBP-1 enhancer. A ChIP assay revealed that cAMP increased the recruitment of the transcriptional regulators CCAAT enhancer-binding protein ß (C/EBPß), forkhead box O1 (FOXO1), and p300 to the IGFBP-1 enhancer in ESCs. Of note, C/EBPß knockdown inhibited the stimulatory effects of cAMP on the levels of H3K27ac, chromatin opening, and p300 recruitment at the IGFBP-1 enhancer. These results indicate that the region -4701 to -7501 bp upstream of IGFBP-1 functions as an enhancer for IGFBP-1 expression in ESCs undergoing decidualization, that C/EBPß and FOXO1 bind to the enhancer region to up-regulate IGFBP-1 expression, and that C/EBPß induces H3K27ac by recruiting p300 to the IGFBP-1 enhancer.

Novel Function of a Transcription Factor WT1 in Regulating Decidualization in Human Endometrial Stromal Cells and Its Molecular Mechanism.

The Wilms tumor suppressor gene (WT1) encodes an essential transcription factor regulating mammalian urogenital development. However, the function of WT1 in human endometrium is still unclear. The current study examined the involvement of WT1 in the regulation of IGF-binding protein-1 (IGFBP-1) and prolactin (PRL), which are specific markers of decidualization, in human endometrial stromal cells (ESCs) undergoing decidualization. ESCs isolated from proliferative-phase endometrium were incubated with cyclic adenosine monophosphate (cAMP) to induce decidualization. cAMP increased WT1 expression with the induction of IGFBP-1 and PRL. Knockdown of WT1 by small interfering RNA inhibited cAMP-induced expression of IGFBP-1 and PRL. cAMP also induced the recruitment of WT1 to the IGFBP-1 and PRL promoters. To investigate the mechanism by which WT1 is upregulated by cAMP, we focused on C/EBPß, a gene that regulates the expression of many genes during decidualization. Knockdown of C/EBPß decreased cAMP-increased WT1 expression. cAMP increased the recruitment of C/EBPß to the WT1 enhancer that is located approximately 14,000 bp downstream from the transcription start site. To test the endogenous function of the WT1 enhancer region on WT1 expression, the endogenous WT1 enhancer region was deleted by CRISPR/Cas9 system in HEK293 cells. The increase of WT1 expression by cAMP was not observed in the enhancer-deleted clones. Chromatin immunoprecipitation assay revealed that this enhancer region has high levels of H3K27ac and H3K4me1, which are active enhancer marks. These results show the role of WT1 in regulating decidualization in human ESCs. C/EBPß is an upstream gene that regulates WT1 expression by binding to the novel enhancer region.

Retinoic acid has the potential to suppress endometriosis development.

BACKGROUND: Despite endometriosis is common estrogen dependent disease afflicting women in reproductive age, the pathogenesis has not been fully elucidated. Retinoic acid has various functions in cells as biologic modulator, and aberrant retinoid metabolism seems to be involved in the lesions of endometriosis. In order to evaluate the potential of all-trans retinoic acid (ATRA) for therapeutic treatment, a transcriptome analysis and estradiol measurements in cultured endometriotic cells and tissues were conducted. METHODS: The mRNA expression levels in ATRA-treated endometriotic stromal cells (ESC) isolated from ovarian endometrial cysts (OEC) were investigated. Estradiol production in OEC tissues was also investigated. RESULTS: In the isolated ESC culture supplemented with ATRA for four days, total RNA was extracted followed by a transcriptome analysis using GeneChip. Forty-nine genes were upregulated and four genes were down-regulated by the ATRA treatment. Many upregulated genes were associated with the negative regulation of cellular proliferation. In addition, ATRA treatment decreased the mRNA expression of 17-beta-dehydrogenase 2 (HSD17B2) which converts estradiol into estrone in a dose-dependent manner, and the ELISA measurements indicated that estradiol production in the OEC tissue was inhibited by ATRA treatment. CONCLUSIONS: Retinoic acid has the potential to suppress endometriosis development.

Case of pure ovarian squamous cell carcinoma resistant to combination chemotherapy with paclitaxel and carboplatin but responsive to monotherapy with weekly irinotecan.

Primary ovarian squamous cell carcinoma is uncommon, and the optimal treatment strategy for this disease has not yet been established. A 71-year-old woman diagnosed with FIGO stage IIb pure ovarian squamous cell carcinoma underwent cytoreductive surgery followed by combination chemotherapy with paclitaxel and carboplatin. After the second treatment course, a recurrent mass grew rapidly, and serum tumor maker levels increased. Monotherapy with weekly irinotecan was then instituted. This second-line chemotherapy was remarkably effective, and the patient subsequently underwent complete interval debulking surgery with a pathological complete response after the third treatment course. Weekly irinotecan is an effective choice for primary ovarian squamous cell carcinoma resistant to combination chemotherapy with paclitaxel and carboplatin.