Aki Matsuoka

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.

Changes in blood-flow impedance of the human corpus luteum throughout the luteal phase and during early pregnancy

OBJECTIVE To examine changes in blood flow in the corpus luteum throughout the luteal phase and during early pregnancy. DESIGN Longitudinal and cross-sectional prospective studies. SETTING University hospital and city general hospital. PATIENT(S) Sixty-one women with normal menstrual cycles and normal luteal function, 13 women with hCG-induced ovulatory cycle, 10 women with luteal phase defect, six women with luteinized unruptured follicle (LUF), and 17 pregnant women (4-10 weeks of gestation). INTERVENTION(S) Blood-flow impedance in the corpus luteum was assessed by transvaginal color-pulsed Doppler ultrasound. MAIN OUTCOME MEASURES Resistance index (RI) in the corpus luteum. RESULT(S) In the normal menstrual cycle, the RI of the preovulatory follicle was high and significantly decreased after ovulation. Luteal-RI further decreased during the early to midluteal phase but significantly increased during the late luteal phase. Those changes in luteal-RI were similar to those of the hCG-induced ovulatory cycle. Luteal-RI during the midluteal phase was significantly higher in the patients with luteal phase defect than in women with normal luteal function. Luteal-RI of the LUF patients remained high throughout the luteal phase. In pregnant women, luteal-RI remained at the midluteal phase level until 7 weeks of gestation and significantly increased thereafter. CONCLUSION(S) The change in luteal-RI was associated with corpus luteum development and corpus luteum regression. Luteal-RI was closely associated with luteal function.

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.

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.

Prostaglandin F2α (PGF2α) stimulates PTGS2 expression and PGF2α synthesis through NFKB activation via reactive oxygen species in the corpus luteum of pseudopregnant rats

This study was undertaken to investigate how prostaglandin F(2α) (PGF(2α)) increases PGF(2α) synthesis and PTGS2 expression in the corpus luteum of pseudopregnant rats. We further investigated the molecular mechanism by which PGF(2α) stimulates PTGS2 expression. PGF(2α) (3 mg/kg) or phosphate buffer as a control was injected s.c. on day 7 of pseudopregnancy. Ptgs2 mRNA expression and PGF(2α) concentrations in the corpus luteum were measured at 2, 6, and 24 h after PGF(2α) injection. PGF(2α) significantly increased Ptgs2 mRNA expression at 2 h and luteal PGF(2α) concentrations at 24 h. PGF(2α) significantly decreased serum progesterone levels at all of the times studied. Simultaneous administration of a selective PTGS2 inhibitor (NS-398, 10 mg/kg) completely abolished the increase in luteal PGF(2α) concentrations induced by PGF(2α). PGF(2α) increased NFKB p65 protein expression in the nucleus of luteal cells 30 min after PGF(2α) injection, and electrophoretic mobility shift assay revealed that PGF(2α) increased binding activities of NFKB to the NFKB consensus sequence of the Ptgs2 gene promoter. Simultaneous administration of both superoxide dismutase and catalase to scavenge reactive oxygen species (ROS) inhibited the increases of nuclear NFKB p65 protein expression, lipid peroxide levels, and Ptgs2 mRNA expression induced by PGF(2α). In conclusion, PGF(2α) stimulates Ptgs2 mRNA expression and PGF(2α) synthesis through NFKB activation via ROS in the corpus luteum of pseudopregnant rats.