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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.

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 ERβ, 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.

Background: The molecular assays that test gene expression, transcriptional, and epigenetic regulation are increasingly diverse and numerous. The information generated by each type of assay individually gives an insight into the state of the cells tested. What should be possible is to add the information derived from separate, complementary assays to gain higher-confidence insights into cellular states. At present, the analysis of multi-dimensional, massive genome-wide data requires an initial pruning step to create manageable subsets of observations that are then used for integration, which decreases the sizes of the intersecting data sets and the potential for biological insights. Our Significance-based Modules Integrating the Transcriptome and Epigenome (SMITE) approach was developed to integrate transcriptional and epigenetic regulatory data without a loss of resolution., Results: SMITE combines p-values by accounting for the correlation between non-independent values within data sets, allowing genes and gene modules in an interaction network to be assigned significance values. The contribution of each type of genomic data can be weighted, permitting integration of individually under-powered data sets, increasing the overall ability to detect effects within modules of genes. We apply SMITE to a complex genomic data set including the epigenomic and transcriptomic effects of Toxoplasma gondii infection on human host cells and demonstrate that SMITE is able to identify novel subnetworks of dysregulated genes. Additionally, we show that SMITE outperforms Functional Epigenetic Modules (FEM), the current paradigm of using the spin-glass algorithm to integrate gene expression and epigenetic data., Conclusions: SMITE represents a flexible, scalable tool that allows integration of transcriptional and epigenetic regulatory data from genome-wide assays to boost confidence in finding gene modules reflecting altered cellular states.

Background: Preeclampsia, traditionally characterized by high blood pressure and proteinuria, is a common pregnancy complication, which affects 2-8 % of all pregnancies. Although children born to women with preeclampsia have a higher risk of hypertension in later life, the mechanism of this increased risk is unknown. DNA methylation is an epigenetic modification that has been studied as a mediator of cellular memory of adverse exposures in utero. Since each cell type in the body has a unique DNA profile, cell subtype composition is a major confounding factor in studies of tissues with heterogeneous cell types. The best way to avoid this confounding effect is by using purified cell types. However, using purified cell types in large cohort translational studies is difficult. The amnion, the inner layer of the fetal membranes of the placenta, is derived from the epiblast and consists of two cell types, which are easy to isolate from the delivered placenta. In this study, we demonstrate the value of using amnion samples for DNA methylation studies, revealing distinctive patterns between fetuses exposed to proteinuria or hypertension and fetuses from normal pregnancies., Results: We performed a genome-wide DNA methylation analysis, HpaII tiny fragment Enrichment by Ligation-mediated PCR (HELP)-tagging, on 62 amnion samples from the placentas of uncomplicated, normal pregnancies and from those with complications of preeclampsia or hypertension. Using a regression model approach, we found 123, 85, and 99 loci with high-confidence hypertension-associated, proteinuria-associated, and hypertension- and proteinuria-associated DNA methylation changes, respectively. A gene ontology analysis showed DNA methylation changes to be selecting genes with different biological processes in exposure status. We also found that these differentially methylated regions overlap loci previously reported as differentially methylated regions in preeclampsia., Conclusions: Our findings support prior observations that preeclampsia is associated with changes of DNA methylation near genes that have previously been found to be dysregulated in preeclampsia. We propose that amniotic membranes represent a valuable surrogate fetal tissue on which to perform epigenome-wide association studies of adverse intrauterine conditions.

Background: The ovulatory LH surge rapidly alters the expression of steroidogenesis-related genes such as steroidogenic acute regulatory protein (StAR) in granulosa cells (GCs) undergoing luteinization. We recently reported that histone modifications contribute to these changes. Histone modifications are regulated by a variety of histone modification enzymes. This study investigated the changes in gene expression of histone modification enzymes in rat GCs undergoing luteinization after the induction of ovulation. The extracellular regulated kinase (ERK)-1/2 is a mediator in the intracellular signaling pathway stimulated by the ovulatory LH surge and regulates the expression of a number of genes in GCs. We further investigated whether ERK-1/2 is involved in the regulation of the histone modification at the StAR promoter region in GCs undergoing luteinization., Results: GCs were obtained from rats treated with equine chorionic gonadotropin (CG) before (0 h) and after human (h) CG injection. The expressions of 84 genes regulating histone modifications or DNA methylation were measured using a PCR array. Five genes (HDAC4, HDAC10, EZH2, SETDB2, and CIITA) were identified as histone acetylation- or histone methylation-related genes, and were significantly altered after hCG injection. None of the genes were related to DNA methylation. mRNA levels of EZH2, SETDB2, HDAC4, and HDAC10 decreased and CIITA mRNA levels increased 4 or 12 h after hCG injection. GCs isolated after eCG injection were incubated with hCG for 4 h to induce luteinization. StAR mRNA levels were significantly increased by hCG accompanied by the increase in H3K4me3 of the StAR promoter region. StAR mRNA expression was inhibited by the ERK inhibitor with the significant decrease of H3K4me3. These results suggest that hCG increases StAR gene expression through the ERK-1/2-mediated signaling which is also associated with histone modification of the promoter region., Conclusions: Gene expressions of histone modification enzymes change in GCs undergoing luteinization after ovulation induction. This change may play important roles in regulating the expression of various genes during the early stage of luteinization, which may be critical for the subsequent corpus luteum formation.

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.

Background: Neovascularization is necessary for follicular growth. Vascularization is first observed in preantral follicles, and thereafter the vasculature markedly increases in follicles undergoing development. Neovascularization includes angiogenesis and vasculogenesis. Vasculogenesis is the formation of new blood vessels by bone marrow-derived endothelial progenitor cells. It is unclear whether vasculogenesis occurs during follicular growth. Blood vessels must be mature to be functional blood vessels. Mature blood vessels are characterized by the recruitment of pericytes. However, it is unclear where pericytes come from and whether they contribute to neovascularization in the follicle during follicular growth. In this study, we investigated whether bone marrow-derived progenitor cells that differentiate into vascular endothelial cells or pericytes contribute to neovascularization during follicular growth., Methods: A parabiosis model was used in this study. Six-week-old wild-type and transgenic female mice expressing green fluorescent protein (GFP) were conjoined between the lateral abdominal regions to create a shared circulatory system. After 6 weeks, the ovaries were obtained and immunostained for CD31/CD34 (a vascular endothelial cell marker), platelet-derived growth factor receptor-β (PDGFR-β) (a pericyte marker), and GFP (a bone marrow-derived cell marker)., Results: Cells that were positive for CD34 and PDGFR-β were observed in the stroma adjacent to the primary or early preantral follicles and in the theca cell layer of the follicles from the late preantral stage to the preovulatory stage. CD31/CD34 and GFP double-positive cells were observed in the theca cell layer of the follicle from the antral stage to the preovulatory stage while the number of double-positive cells in the preovulatory follicles did not increase. PDGFR-β and GFP double-positive cells were observed in the theca cell layer of the preovulatory follicle but not in the smaller follicle., Conclusions: Locally existing endothelial cells and pericytes in the stroma play a central role in the neovascularization during follicular growth, while bone marrow-derived endothelial cells and pericytes partially contribute to this process.

Melatonin (N-acetyl-5-methoxytryptamine) is secreted during the dark hours at night by pineal gland, and it regulates a variety of important central and peripheral actions related to circadian rhythms and reproduction. It has been believed that melatonin regulates ovarian function by the regulation of gonadotropin release in the hypothalamus-pituitary gland axis via its specific receptors. In addition to the receptor mediated action, the discovery of melatonin as a direct free radical scavenger has greatly broadened the understanding of melatonin's mechanisms which benefit reproductive physiology. Higher concentrations of melatonin have been found in human preovulatory follicular fluid compared to serum, and there is growing evidence of the direct effects of melatonin on ovarian function especially oocyte maturation and embryo development. Many scientists have focused on the direct role of melatonin on oocyte maturation and embryo development as an anti-oxidant to reduce oxidative stress induced by reactive oxygen species, which are produced during ovulation process. The beneficial effects of melatonin administration on oocyte maturation and embryo development have been confirmed by in vitro and in vivo experiments in animals. This review also discusses the first application of melatonin to the clinical treatment of infertile women and confirms that melatonin administration reduces intrafollicular oxidative damage and increase fertilization rates. This review summarizes our recent works and new findings related to the reported beneficial effects of melatonin on reproductive physiology in its role as a reducer of oxidative stress, especially on oocyte maturation and embryo development.

Background: Blood flow in the corpus luteum (CL) is closely related to luteal function. It is unclear how luteal blood flow is regulated. Standardized ovarian-stimulation protocol with a gonadotropin-releasing hormone agonist (GnRHa long protocol) causes luteal phase defect because it drastically suppresses serum LH levels. Examining luteal blood flow in the patient undergoing GnRHa long protocol may be useful to know whether luteal blood flow is regulated by LH., Methods: Twenty-four infertile women undergoing GnRHa long protocol were divided into 3 groups dependent on luteal supports; 9 women were given ethinylestradiol plus norgestrel (Planovar) orally throughout the luteal phase (control group); 8 women were given HCG 2,000 IU on days 2 and 4 day after ovulation induction in addition to Planovar (HCG group); 7 women were given vitamin E (600 mg/day) orally throughout the luteal phase in addition to Planovar (vitamin E group). Blood flow impedance was measured in each CL during the mid-luteal phase by transvaginal color-pulsed-Doppler-ultrasonography and was expressed as a CL-resistance index (CL-RI)., Results: Serum LH levels were remarkably suppressed in all the groups. CL-RI in the control group was more than the cutoff value (0.51), and only 2 out of 9 women had CL-RI values < 0.51. Treatments with HCG or vitamin E significantly improved the CL-RI to less than 0.51. Seven of the 8 women in the HCG group and all of the women in the vitamin E group had CL-RI < 0.51., Conclusion: Patients undergoing GnRHa long protocol had high luteal blood flow impedance with very low serum LH levels. HCG administration improved luteal blood flow impedance. This suggests that luteal blood flow is regulated by LH.

Physical activity in childhood is important as it may establish adult behavior. However, few studies on physical activity in children have been conducted, especially in Asian children. We performed anthropometric measurements of 159 school children in two grades (grade 5: 10-11 years old and grade 8: 13-14 years old) from urban areas of Korea (n=79) and China (n=80). The total daily energy expenditure (TEE) was estimated for 7 consecutive days using an accelerometer. The mean height, weight, and body mass index (BMI) for boys and girls in both countries exceeded the US national reference median (CDC, 2000). Physical activity levels (PALs) were significantly higher in the grade 5 group (10-11 years old) and in girls than in boys for both grades. No significant difference in PALs or daily step-counts (STPs) was observed between 'normal' and 'overweight' subgroups based on BMI, although negative correlations were found between weight, BMI, or %body fat vs. PAL or STP among Korean girls and Chinese boys (r=0.32-0.38, all p<0.05). Daily variation in physical activity was observed in Korean children. In the Koreans (boys and girls, both grades pooled), TEE and STP were significantly lower than the 7-day average on Sundays, whereas for the Chinese population, STP did not clearly differ between the weekends and the week averages. In summary, PALs were higher in the fifth grade boys and girls than in the eighth grade children; interestingly, girls tended to have higher PALs than boys. Daily variation in physical activity was observed in Korea; children were less active on Sundays.