Hiromi Asada

Oxidative stress impairs oocyte quality and melatonin protects oocytes from free radical damage and improves fertilization rate

Abstract:  We investigated the relationship between oxidative stress and poor oocyte quality and whether the antioxidant melatonin improves oocyte quality. Follicular fluid was sampled at oocyte retrieval during in vitro fertilization and embryo transfer (IVF‐ET). Intrafollicular concentrations of 8‐hydroxy‐2′‐deoxyguanosine (8‐OHdG) in women with high rates of degenerate oocytes were significantly higher than those with low rates of degenerate oocytes. As there was a negative correlation between intrafollicular concentrations of 8‐OHdG and melatonin, 18 patients undergoing IVF‐ET were given melatonin (3 mg/day), vitamin E (600 mg/day) or both melatonin and vitamin E. Intrafollicular concentrations of 8‐OHdG and hexanoyl‐lysine adduct were significantly reduced by these antioxidant treatments. One hundred and fifteen patients who failed to become pregnant with a low fertilization rate (50%) in the previous IVF‐ET cycle were divided into two groups during the next IVF‐ET procedure; 56 patients with melatonin treatment (3 mg/day) and 59 patients without melatonin treatment. The fertilization rate was improved by melatonin treatment compared to the previous IVF‐ET cycle. However, the fertilization rate was not significantly changed without melatonin treatment. Oocytes recovered from preovulatory follicles in mice were incubated with H2O2 for 12 hr. The percentage of mature oocytes with a first polar body was significantly reduced by addition of H2O2 (300 μm). The inhibitory effect of H2O2 was significantly blocked by simultaneous addition of melatonin. In conclusion, oxidative stress causes toxic effects on oocyte maturation and melatonin protects oocytes from oxidative stress. Melatonin is likely to improve oocyte quality and fertilization rates.

DNA methyltransferase expression in the human endometrium: down-regulation by progesterone and estrogen

BACKGROUND Epigenetic regulation may be involved in modulation of gene expression during the normal cyclic changes of the human endometrium. We investigated expression of DNA methyltransferases (DNMTs) in endometrium during the menstrual cycle and the influence of sex steroid hormones on DNMT in endometrial stromal cells (ESC) in culture. METHODS Expression of DNMT1, DNMT3a and DNMT3b was assessed by immunohistochemistry and real-time RT-PCR in endometrial tissue (n = 42 women). ESC (n = 3 women) were cultured with estradiol and medroxyprogesterone acetate (E + MPA) for 17 days, and DNMT mRNA levels were measured by real-time RT-PCR. RESULTS Nuclei of both epithelial and stromal cells immunostained for DNMT1, DNMT3a and DNMT3b during each phase of the menstrual cycle. Tissue levels of DNMT1 and DNMT3a mRNA were significantly lower in the mid-secretory phase than in the proliferative phase (P < 0.01). For DNMT3b, the change in mRNA levels showed a similar trend to that for DNMT3a. In ESC culture, DNMT3a and DNMT3b mRNA levels were significantly decreased by E + MPA treatment (P < 0.01 and P < 0.05, respectively) at Day 8 and Day 17. CONCLUSIONS DNMT mRNAs declined in the human endometrium during the secretory phase, and E + MPA down-regulated DNMT3a and DNMT3b mRNAs in ESC in culture. These results suggest that DNMTs have regulatory functions in gene expression that is associated with decidualization.

Potential link between estrogen receptor-α gene hypomethylation and uterine fibroid formation

Uterine leiomyomas are the most common uterine tumors in women. Estrogen receptor-alpha (ER-alpha) is more highly expressed in uterine leiomyomas than in normal myometrium, suggesting a link between uterine leiomyomas and ER-alpha expression. DNA methylation is an epigenetic mechanism of gene regulation and plays important roles in normal embryonic development and in disease progression including cancers. Here, we investigated the DNA methylation status of the ER-alpha promoter region (-1188 to +229 bp) in myometrium and leiomyoma. By sodium bisulfite sequencing, 49 CpG sites in the proximal promoter region of ER-alpha gene were shown to be unmethylated in both leiomyoma and normal myometrium. At seven CpG sites in the distal promoter region of the ER-alpha gene, there was a variation in DNA methylation status in myometrium and leiomyoma. Further analysis of the DNA methylation status by bisulfite restriction mapping among 11 paired samples of myometrium and leiomyoma indicated that CpG sites in the distal region of ER-alpha promoter are hypomethylated in leiomyomas of nine patients. In those patients, ER-alpha mRNA levels tended to be higher in the leiomyoma than in the myometrium. In conclusion, there was an aberrant DNA methylation status in the promoter region of ER-alpha gene in uterine leiomyoma, which may be associated with high ER-alpha mRNA expression.

Protective role of melatonin in progesterone production by human luteal cells.

Abstract:  This study investigated whether melatonin protects luteinized granulosa cells from reactive oxygen species (ROS) as an antioxidant to enhance progesterone production in the follicle during ovulation. Follicular fluid was sampled at the time of oocyte retrieval in women undergoing in vitro fertilization and embryo transfer (IVF‐ET). Melatonin concentrations in the follicular fluid were positively correlated with progesterone concentrations (r = 0.342, P < 0.05) and negatively correlated with the concentration of 8‐hydroxy‐2′‐deoxyguanosine (8‐OHdG), an oxidative stress marker (r = −0.342, P < 0.05). The progesterone and 8‐OHdG concentrations were negatively correlated (r = −0.246, P < 0.05). Luteinized granulosa cells were obtained at the time of oocyte retrieval in women undergoing IVF‐ET. Cells were incubated with H2O2 (30, 50, 100 μm) in the presence or absence of melatonin (1, 10, 100 μg/mL). Progesterone production by luteinized granulosa cells was significantly inhibited by H2O2. Melatonin treatment overcame the inhibitory effect of H2O2. Twenty‐five patients who had luteal phase defect (serum progesterone concentrations <10 ng/mL during the mid‐luteal phase) were divided into two groups during the next treatment cycle: 14 women were given melatonin (3 mg/day at 22:00 hr) throughout the luteal phase and 11 women were given no medication as a control. Melatonin treatment improved serum progesterone concentrations (>10 ng/mL during the mid‐luteal phase) in nine of 14 women (64.3%), whereas only two of 11 women (18.1%) showed normal serum progesterone levels in the control group. In conclusion, melatonin protects granulosa cells undergoing luteinization from ROS in the follicle and contributes to luteinization for progesterone production during ovulation.

Aberrant DNA methylation status in human uterine leiomyoma

Aberrant DNA methylation has been implicated in tumorigenesis. This study was undertaken to establish the genome-wide DNA methylation profile in uterine leiomyomas and to investigate whether DNA methylation status is altered in uterine leiomyomas. For this purpose, restriction landmark genomic scanning (RLGS) was performed on a paired sample of leiomyoma and adjacent normal myometrium. The RLGS profile revealed 29 aberrant methylation spots (10 methylated and 19 demethylated) in leiomyoma in comparison with myometrium. One of the differently methylated genomic loci was newly identified as GS20656 from the human genome sequence database. In 9 of the 10 paired samples, the DNA methylation levels of the first exon of GS20656 were significantly lower in leiomyoma than in myometrium, suggesting the existence of a genomic locus under epigenetic regulation in uterine leiomyomas. Unexpectedly, DNA methyltransferase 1 (DNMT1) and DNMT3a mRNA expression levels were higher in leiomyoma than in myometrium. These facts suggest that other epigenetic factors besides DNMT are involved in local changes of DNA methylation at genome loci. The present study indicates not only aberrant genome-wide DNA methylation status in uterine leiomyomas but also the existence of a genomic locus that is differently methylated between normal myometrium and uterine leiomyoma.

Genome-Wide DNA Methylation Analysis Reveals a Potential Mechanism for the Pathogenesis and Development of Uterine Leiomyomas

Background The pathogenesis of uterine leiomyomas, the most common benign tumor in women, remains unclear. Since acquired factors such as obesity, hypertension and early menarche place women at greater risk for uterine leiomyomas, uterine leiomyomas may be associated with epigenetic abnormalities that are caused by unfavorable environmental exposures. Principal Findings Profiles of genome-wide DNA methylation and mRNA expression were investigated in leiomyomas and in myometrium with and without leiomyomas. Profiles of DNA methylation and mRNA expression in the myometrium with and without leiomyomas were quite similar while those in leiomyomas were distinct. We identified 120 genes whose DNA methylation and mRNA expression patterns differed between leiomyomas and the adjacent myometrium. The biological relevance of the aberrantly methylated and expressed genes was cancer process, including IRS1 that is related to transformation, and collagen-related genes such as COL4A1, COL4A2 and COL6A3. We also detected 22 target genes of estrogen receptor (ER) alpha, including apoptosis-related genes, that have aberrant DNA methylation in the promoter, suggesting that the aberrant epigenetic regulation of ER alpha-target genes contributes to the aberrant response to estrogen. Conclusions Aberrant DNA methylation and its related transcriptional aberration were associated with cancer processes, which may represent a critical initial mechanism that triggers transformation of a single tumor stem cell that will eventually develop into a monoclonal leiomyoma tumor. The aberrant epigenetic regulation of ER alpha-target genes also may contribute to the aberrant response to estrogen, which is involved in the development of uterine leiomyomas after menarche.

Progesterone Increases Manganese Superoxide Dismutase Expression via a cAMP-Dependent Signaling Mediated by Noncanonical Wnt5a Pathway in Human Endometrial Stromal Cells

CONTEXT Manganese superoxide dismutase (Mn-SOD), an antioxidant enzyme in the mitochondria, protects cells by scavenging superoxide radicals in human endometrial stromal cells (ESCs). Mn-SOD increases in ESCs during decidualization induced by progesterone. OBJECTIVE The present study investigated the molecular mechanism for Mn-SOD expression induced by progesterone in human ESCs. METHODS ESCs were incubated with medroxyprogesterone acetate (MPA; 10(-6) m) or dibutyryl-cAMP (0.5 mm) for 17 d. To determine whether a cAMP-dependent signaling pathway is involved in the MPA-induced Mn-SOD expression, ESCs were treated with H89, an inhibitor of cAMP-dependent protein kinase A. A chromatin immunoprecipitation assay was performed to examine the binding of cAMP-binding protein to the cAMP-response element on the Mn-SOD gene promoter. To examine the involvement of Wnt5a signaling, anti-Wnt5a antibodies were used to neutralize the Wnt5a activities. RESULTS Mn-SOD and Wnt5a mRNA levels and intracellular cAMP concentrations were significantly increased by MPA. These increases were accompanied by an increase in the mRNA expression of IGF-binding protein-1, a marker of decidualization. The increase in Mn-SOD mRNA levels by MPA or dibutyryl-cAMP was completely inhibited by H89. The chromatin immunoprecipitation assay revealed that MPA induced cAMP-binding protein binding with cAMP-response element on the Mn-SOD gene promoter. The increase in intracellular cAMP concentrations by MPA was completely inhibited by treatment with anti-Wnt5a antibodies. MPA treatment had no effects on β-catenin expression. CONCLUSIONS Progesterone increased Mn-SOD expression via a cAMP-dependent pathway in ESCs during decidualization. cAMP-dependent signaling stimulated by progesterone is mediated by noncanonical Wnt5a pathways that signal independently of β-catenin.

Changes in Histone Modification and DNA Methylation of the StAR and Cyp19a1 Promoter Regions in Granulosa Cells Undergoing Luteinization during Ovulation In Rats

The ovulatory LH surge induces rapid up-regulation of steroidogenic acute regulatory (StAR) protein and rapid down-regulation of aromatase (Cyp19a1) in granulosa cells (GCs) undergoing luteinization during ovulation. This study investigated in vivo whether epigenetic mechanisms including histone modifications are involved in the rapid changes of StAR and Cyp19a1 gene expression. GCs were obtained from rats treated with equine chorionic gonadotropin (CG) before (0 h) and after human (h)CG injection. StAR mRNA levels rapidly increased after hCG injection, reached a peak at 4 h, and then remained higher compared with 0 h until 12 h. Cyp19a1 mRNA levels gradually decreased after hCG injection and reached their lowest level at 12 h. A chromatin immunoprecipitation assay revealed that levels of histone-H4 acetylation (Ac-H4) and trimethylation of histone-H3 lysine-4 (H3K4me3) increased whereas H3K9me3 and H3K27me3 decreased in the StAR promoter after hCG injection. On the other hand, the levels of Ac-H3 and -H4 and H3K4me3 decreased, and H3K27me3 increased in the Cyp19a1 promoter after hCG injection. Chromatin condensation, which was analyzed using deoxyribonuclease I, decreased in the StAR promoter and increased in the Cyp19a1 promoter after hCG injection. A chromatin immunoprecipitation assay also showed that binding activities of CAATT/enhancer-binding protein β to the StAR promoter increased and binding activities of phosphorylated-cAMP response element binding protein to the Cyp19a1 promoter decreased after hCG injection. These results provide in vivo evidence that histone modifications are involved in the rapid changes of StAR and Cyp19a1 gene expression by altering chromatin structure of the promoters in GCs undergoing luteinization during ovulation.

Luteal blood flow and luteal function

BackgroundBlood flow in the corpus luteum (CL) is associated with luteal function. The present study was undertaken to investigate whether luteal function can be improved by increasing CL blood flow in women with luteal phase defect (LFD).MethodsBlood flow impedance in the CL was measured by transvaginal color-pulsed-Doppler-ultrasonography and was expressed as a resistance index (RI). The patients with both LFD [serum progesterone (P) concentrations < 10 ng/ml during mid-luteal phase] and high CL-RI (≥ 0.51) were given vitamin-E (600 mg/day, n = 18), L-arginine (6 g/day, n = 14) as a potential nitric oxide donor, melatonin (3 mg/day, n = 13) as an antioxidant, or HCG (2,000 IU/day, n = 10) during the subsequent menstrual cycle.ResultsIn the control group (n = 11), who received no medication to increase CL blood flow, only one patient (9%) improved in CL-RI and 2 patients (18%) improved in serum P. Vitamin-E improved CL-RI in 15 patients (83%) and improved serum P in 12 patients (67%). L-arginine improved CL-RI in all the patients (100%) and improved serum P in 10 patients (71%). HCG improved CL-RI in all the patients (100%) and improved serum P in 9 patients (90%). Melatonin had no significant effect.ConclusionVitamin-E or L-arginine treatment improved luteal function by decreasing CL blood flow impedance. CL blood flow is a critical factor for luteal function.

Importance of C/EBPβ binding and histone acetylation status in the promoter regions for induction of IGFBP-1, PRL, and Mn-SOD by cAMP in human endometrial stromal cells.

Dynamic changes of gene expressions occur in human endometrial stromal cells (ESCs) during decidualization. CCAAT/enhancer-binding proteinβ (C/EBPβ) regulates the expression of a number of decidualization-related genes. In addition to transcription factors, it is important to know the role of epigenetic mechanisms, such as histone modifications in the regulation of decidualization-related genes. This study investigated the molecular and epigenetic mechanisms by which cAMP up-regulates the expression of IGF-binding protein-1 (IGFBP-1), prolactin (PRL), and manganese superoxide dismutase (Mn-SOD) in ESC. ESCs isolated from proliferative phase endometrium were incubated with cAMP to induce decidualization. IGFBP-1, PRL, and Mn-SOD mRNA expressions were determined by real-time RT-PCR. The C/EBPβ binding and histone modification status (acetylation of histone-H3 lysine-27 [H3K27ac]) in the promoter were examined by chromatin immunoprecipitation assay. Knockdowns of C/EBPβ were performed using the small interfering RNA method. cAMP induced mRNA expressions of IGFBP-1 and PRL accompanied by the increases in both C/EBPβ binding activities and H3K27ac levels in the promoters. The stimulatory effects of cAMP on mRNA levels and H3K27ac levels were completely abolished by C/EBPβ knockdown. cAMP increased Mn-SOD mRNA levels and C/EBPβ binding activities in the enhancer region. C/EBPβ knockdown inhibited Mn-SOD mRNA levels. The H3K27ac levels in the enhancer were high before cAMP stimulus but were not further increased by cAMP and were not inhibited by C/EBPβ knockdown. These results show that C/EBPβ regulates the expression of IGFBP-1 and PRL by altering the histone acetylation status of their promoters but differently regulates Mn-SOD gene expression in human ESC during decidualization.