Effects of coenzyme Q10 on ovarian surface epithelium-derived ovarian stem cells and ovarian function in a 4-vinylcyclohexene diepoxide-induced murine model of ovarian failure.

BACKGROUND: Several studies have shown that coenzyme Q10 (CoQ10) can rescue ovarian aging and that ovarian surface epithelium (OSE)-derived ovarian stem cells (OSCs) are useful for treating infertility due to ovarian aging. However, few studies have examined the effect of CoQ10 on OSCs. This study was aimed to investigate whether CoQ10 activates OSCs and recovers ovarian function in a 4-vinylcyclohexene diepoxide (VCD)-induced mouse model of ovarian failure. METHODS: Forty female C57BL/6 mice aged 6 weeks were randomly divided into four groups (n = 10/group): a control group administered saline orally, a CoQ10 group administered 150 mg/kg/day of CoQ10 orally in 1 mL of saline daily for 14 days, a VCD group administered 160 mg/kg/day of VCD i.p. in 2.5 mL of saline/kg for 5 days, and a VCD + CoQ10 group administered VCD i.p. for 5 days injection and CoQ10 (150 mg/kg/day) orally for 14 days. After treatment, follicle counts were evaluated by hematoxylin and eosin (H&E) staining, and ovarian mRNA expressions of Bmp-15, Gdf-9, and c-Kit were examined by quantitative real-time PCR. Serum FSH, AMH, and ROS levels were also measured. Oocyte-like structure counts and the expressions of Oct-4 and MVH were also evaluated after culturing OSE for 3 weeks. In a second experiment, 32 female mice were administered CoQ10 as described above, induced to superovulate using PMSG and hCG, and mated. Numbers of zygotes and embryo development rate were examined. RESULTS: Postcultured OSE showed significant increases in the numbers of oocyte-like structure and that the expression of Oct-4 and MVH were higher in the VCD + CoQ10 group than in the VCD group (p < 0.05). Numbers of surviving follicles from primordial to antral follicles, numbers of zygotes retrieved and embryo development rate to blastocyst were significantly greater in the VCD + CoQ10 group than in the VCD group (p < 0.01). Serum AMH level and ovarian expressions of Bmp-15, Gdf-9 and c-Kit were also significantly greater in the VCD + CoQ10 group than in the VCD group (p < 0.05). In contrast, serum ROS level was significantly lower in the VCD + CoQ10 group than in the VCD group (p < 0.05). CONCLUSION: This study shows that CoQ10 stimulates the differentiation of OSE-derived OSCs and confirms that CoQ10 can reduce ROS levels and improve ovarian function and oocyte quality in mice with VCD-induced ovarian failure.

Dipeptide YA is Responsible for the Positive Effect of Oyster Hydrolysates on Alcohol Metabolism in Single Ethanol Binge Rodent Models.

Accumulative alcohol hangovers cause liver damage through oxidative and inflammatory stress. Numerous antioxidant and anti-inflammatory reagents have been developed to reduce alcohol hangovers, but these reagents are still insignificant and have limitations in that they can cause liver toxicity. Oyster hydrolysate (OH), another reagent that has antioxidant and anti-inflammatory activity, is a product extracted through an enzymatic hydrolysis process from oysters (Crassostrea gigas), which can be easily eaten in meals. This study was aimed at determining the effects of OH on alcohol metabolism, using a single high dose of ethanol (EtOH) administered to rodents, by monitoring alcohol metabolic enzymes, oxidative stress signals, and inflammatory mediators. The effect of tyrosine-alanine (YA) peptide, a main component of OH, on EtOH metabolism was also identified. In vitro experiments showed that OH pretreatment inhibited EtOH-induced cell death, oxidative stress, and inflammation in liver cells and macrophages. In vivo experiments showed that OH and YA pre-administration increased alcohol dehydrogenase, aldehyde dehydrogenase, and catalase activity in EtOH binge treatment. In addition, OH pre-administration alleviated CYP2E1 activity, ROS production, apoptotic signals, and inflammatory mediators in liver tissues. These results showed that OH and YA enhanced EtOH metabolism and had a protective effect against acute alcohol liver damage. Our findings offer new insights into a single high dose of EtOH drinking and suggest that OH and YA could be used as potential marine functional foods to prevent acute alcohol-induced liver damage.

Differential expression of visfatin, leptin, stromal cell derived factor-1α, endothelial nitric oxide synthase, and vascular endothelial growth factor in human leiomyomas.

AIM: This study was aimed to understand expressions of the visfatin, leptin, stromal cell derived factor (SDF)-1α, endothelial nitric oxide synthase (eNOS), and vascular endothelial growth factor (VEGF) in human uterine leiomyomas (UL) and normal myometrium. METHOD: This study investigated expression of visfatin, leptin, SDF-1α, eNOS and VEGF in 23 uterine leiomyoma patients and 10 normal myometrium by RT-PCR and western blot. Messenger RNA transcripts of SDF-1α, eNOS, VEGF and hypoxia inducible factor-1α (HIF-1α) were analyzed according to the size of UL by real-time PCR. RESULTS: There were no significant differences in expressions of visfatin and leptin between UL compared with normal myometrium. However, expressions of eNOS, SDF-1α and VEGF were significantly higher in both intramural and subserosal UL compared with normal myometrium. The expression of SDF1-α was significantly increased in small UL (<5 cm) compared to the large UL (≥5 cm), whereas the expressions of eNOS, VEGF and HIF-1α were higher in large UL than small UL. CONCLUSIONS: This study shows that expression of SDF-1α, eNOS and VEGF were significantly higher in UL than myometrium with a different expression pattern according to the size of UL. However, expressions of visfatin and leptin had no significant differences between the two groups.

Uterine artery embolization using progressively larger calibrated gelatin sponge particles.

PURPOSE: To assess the effectiveness and safety of uterine artery embolization (UAE) using progressively larger calibrated gelatin sponge particles for symptomatic uterine fibroids. MATERIAL AND METHODS: Thirty patients with symptomatic uterine fibroids underwent UAE. Calibrated gelatin sponge particles were used in all patients, beginning with 355-500 µm particles, progressively increasing to 500-710 µm and finally to 710-1000 µm particles. Changes in tumor, uterine volume, and tumor infarction rate were assessed using pelvic magnetic resonance imaging (MRI). The level of complication, improvement of clinical symptoms, and Uterine Fibroid Symptom and Quality of Life (UFS-QOL) score were assessed. RESULTS: MR imaging revealed the mean largest tumor volume reduction was 56.23 ± 16.25% at three months and 72.61 ± 14.47% at 12 months after the procedure. 100% infarction of the dominant fibroids was 91.27 ± 5.02% at three months and 96 ± 5.20% at 12 months after the procedure. Menorrhagia improved markedly in all 23 patients. Bulk-related symptoms improved in 12 (92.30%) of 13 patients. The baseline UFS-QOL score was 43.13 and improved to 11.88 (p < 0.001). No major complications were observed. CONCLUSION: UAE using progressively larger calibrated gelatin sponge particles is an effective and safe treatment for symptomatic uterine fibroids.

Evaluation of YD MolecuTech Real HPV Assay in Comparison with Roche Cobas HPV Assay and BD Onclarity HPV Assay for Detection of HPV Infection.

OBJECTIVE: Human papillomavirus (HPV) is a double-stranded DNA virus that belongs to the papillomavirus family. High-risk (HR) genotypes of HPV are associated with cervical cancer. The combination of molecular HPV testing and cytology results in an increased detection of high-grade cervical lesions. This study compares the performance of a newly developed MolecuTech Real HPV 16/18/HR assay to that of the cobas HPV assay and Onclarity HPV Assay in Korea. METHODS: A SurePath liquid-based cytology device (BD diagnostics, NC, USA) was used to prospectively collect cervical swab specimens. Onclarity HPV Assay (Onclarity; BD diagnostics), Cobas 4800 HPV Test (Cobas; Roche, Rotkreuz, Switzerland), and MolecuTech Real HPV 16/18/HR (MolecuTech; YD, Yongin, Korea) were performed to detect HPV genotypes. RESULTS: Of the 438 cervical specimens, 13.7% showed the HR-HPV genotype. The concordance rates between Onclarity and MolecuTech, cobas and MolecuTech, and Onclarity and Cobas were 94.9% (kappa=0.754), 95.7% (kappa=0.768), and 95.5% (kappa=0.791), respectively. Moreover, no statistically significant differences in HPV genotyping results were observed in the cytology-positive specimens. CONCLUSIONS: The MolecuTech Real HPV 16/18/HR assay showed good agreement in the detection of HR HPV genotypes, and similar analytical performance for the detection of HR HPV genotypes in samples with abnormal cytological findings.

Endobronchial Ultrasound Using Guide Sheath-Guided Transbronchial Lung Biopsy in Ground-Glass Opacity Pulmonary Lesions without Fluoroscopic Guidance.

Diagnosing ground-glass opacity (GGO) pulmonary lesions poses challenges. This study evaluates the utility of radial probe endobronchial ultrasound-guided transbronchial lung biopsy (RP-EBUS-TBLB) in diagnosing GGO pulmonary lesions. A total of 1651 RP-EBUS procedures were performed during the study period. This study analyzed 115 GGO lesions. The EBUS visualization yield was 80.1%. Of 115 lesions, 69 (60%) were successfully diagnosed. The average size of diagnosed lesions was significantly larger than that of undiagnosed lesions (21.9 ± 7.3 vs. 17.1 ± 6.6 mm, p < 0.001). Diagnostic yield varied by lesion size: 50.0% for lesions <20 mm, 65.1% for 20-30 mm lesions, and 85.7% for lesions >30 mm. The mixed blizzard sign on EBUS appeared in 60.6% of mixed GGO lesions, with no cases in pure GGO lesions. Multivariable analyses showed that lesion size (odds ratio [OR], 1.10; 95% confidence interval [CI], 1.00-1.16; p < 0.001) and mixed blizzard sign on EBUS (OR, 20.92; CI, 7.50-58.31; p < 0.001) were significantly associated with diagnostic success. Pneumothorax and hemoptysis occurred in 1.7% and 2.6% of patients, respectively. RP-EBUS-TBLB without fluoroscopic guidance is a viable diagnostic approach for GGO pulmonary lesions with acceptable complications.

Non spherical polyvinyl alcohol versus gelatin sponge particles for uterine artery embolization for symptomatic fibroids.

PURPOSE: To compare clinical and magnetic resonance imaging (MRI) outcomes after uterine artery embolization (UAE) with non spherical polyvinyl alcohol (nPVA) versus gelatin sponge particles. MATERIAL AND METHODS: During ten months, from Jan 2011 to Oct 2011, 60 patients underwent UAE in a standardized manner. nPVA (n = 30) or gelatin sponge particles (n = 30) were used in all patients. Pelvic MRI and clinical symptoms were reviewed before and after the procedure. Changes in tumor, uterine volume and infarction rate of dominant tumor were assessed using MRI. RESULT: At three months after embolization, the outcome data were collected. The improvement of the mean bleeding score was more pronounced with gelatin sponge particles than with nPVA particles (p = 0.035). The mean volume reduction rate and infarction rate of dominant fibroid were 47.9 ± 20.8%, 97.7 ± 5.2% and 60.2 ± 18.1, 96 ± 7.0% after UAE with nPVA compared to gelatin sponge particles, respectively. CONCLUSION: This study showed the superiority of gelatin sponge particles over nPVA in terms of improvement of mean bleeding score and volume reduction rate of dominant fibroid at three months follow-up after UAE, although the infarction rate of dominant fibroid was similar across groups at three months.

Dual effects of fluoxetine on mouse early embryonic development.

Fluoxetine, a selective serotonin reuptake inhibitor, regulates a variety of physiological processes, such as cell proliferation and apoptosis, in mammalian cells. Little is known about the role of fluoxetine in early embryonic development. This study was undertaken to investigate the effect of fluoxetine during mouse early embryonic development. Late two-cell stage embryos (2-cells) were cultured in the presence of various concentrations of fluoxetine (1 to 50µM) for different durations. When late 2-cells were incubated with 5µM fluoxetine for 6h, the percentage that developed into blastocysts increased compared to the control value. However, late 2-cells exposed to fluoxetine (5µM) over 24h showed a reduction in blastocyst formation. The addition of fluoxetine (5µM) together with KN93 or KN62 (calcium/calmodulin-dependent protein kinase II (CaMKII) inhibitors) failed to increase blastocyst formation. Fluoxetine treatment inhibited TREK-1 and TREK-2, members of the two-pore domain K(+) channel family expressed in mouse embryos, activities, indicating that fluoxetine-induced membrane depolarization in late 2-cells might have resulted from TREK inhibition. In addition, long-term exposure to fluoxetine altered the TREK mRNA expression levels. Furthermore, injection of siRNA targeting TREKs significantly decreased blastocyst formation by ~30% compared to injection of scrambled siRNA. Long-term exposure of fluoxetine had no effect on blastocyst formation of TREK deficient embryos. These results indicate that low-dose and short-term exposures of late 2-cells to fluoxetine probably increase blastocyst formation through activation of CaMKII-dependent signal transduction pathways, whereas long-term exposure decreases mouse early embryonic development through inhibition of TREK channel gating.

K(+) efflux through two-pore domain K(+) channels is required for mouse embryonic development.

Numerous studies have suggested that K(+) channels regulate a wide range of physiological processes in mammalian cells. However, little is known about the specific function of K(+) channels in germ cells. In this study, mouse zygotes were cultured in a medium containing K(+) channel blockers to identify the functional role of K(+) channels in mouse embryonic development. Voltage-dependent K(+) channel blockers, such as tetraethylammonium and BaCl(2), had no effect on embryonic development to the blastocyst stage, whereas K(2P) channel blockers, such as quinine, selective serotonin reuptake inhibitors (fluoxetine, paroxetine, and citalopram), gadolinium trichloride, anandamide, ruthenium red, and zinc chloride, significantly decreased blastocyst formation (P<0.05). RT-PCR data showed that members of the K(2P) channel family, specifically KCNK2, KCNK10, KCNK4, KCNK3, and KCNK9, were expressed in mouse oocytes and embryos. In addition, their mRNA expression levels, except Kcnk3, were up-regulated by above ninefold in morula-stage embryos compared with 2-cell stage embryos (2-cells). Immunocytochemical data showed that KCNK2, KCNK10, KCNK4, KCNK3, and KCNK9 channel proteins were expressed in the membrane of oocytes, 2-cells, and blastocysts. Each siRNA injection targeted at Kcnk2, Kcnk10, Kcnk4, Kcnk3, and Kcnk9 significantly decreased blastocyst formation by ~38% compared with scrambled siRNA injection (P<0.05). The blockade of K(2P) channels acidified the intracellular pH and depolarized the membrane potential. These results suggest that K(2P) channels could improve mouse embryonic development through the modulation of gating by activators.

Acetylcholine controls mouse oocyte maturation via downregulation of cAMP.

1. In mice, acetylcholine (ACh) plays an important role in oocyte activation and embryonic development. However, the role of ACh in mouse oocyte maturation has not been investigated. 2. In the present study, the effects of 100 µmol/L and 1 mmol/L ACh on maturation processes of murine germinal vesicle (GV) intact oocytes (GV oocytes) exposed to 10 and 100 µmol/L 3-isobutyl-1-methylxanthine (IBMX), an inhibitor of cyclic nucleotide phosphodiesterase, were evaluated morphologically and immunologically. It has been shown that IBMX inhibits the resumption of meiosis by preventing cAMP breakdown. 3. In the present study, at the start of in vitro culture 100% of oocytes were at the GV stage. After 18 h culture, 95 ± 3, 0 and 85.8 ± 10.2% of oocytes had passed the GV stage in the control, IBMX and IBMX + ACh groups, respectively. The IBMX-induced inhibition of the maturation process was significantly attenuated by approximately 90% by ACh in groups treated with 10 µmol/L IBMX + 100 µmol/L ACh and 100 µmol/L IBMX + 1 mmol/L ACh. Although cAMP levels were high in oocytes treated with 100 µmol/L IBMX, levels were reduced in groups treated simultaneously with 100 µmol/L ACh. Furthermore, compared with mature oocytes, ACh-treated GV oocytes exhibited significantly lower (by approximately 2.3-fold) or absent Ca(2+) peaks. 4. The results of the present study indicate that maturation of GV oocytes, arrested by IBMX treatment, is resumed following ACh treatment and that this effect is due to downregulation of cAMP rather than changes in intracellular Ca(2+) levels.

Role of Visfatin in Restoration of Ovarian Aging and Fertility in the Mouse Aged 18 Months.

The activation of dormant primordial follicles and ovarian angiogenesis has been attempted as a new treatment strategy for age-related ovarian aging. This study examined whether visfatin rescues age-related fertility decline in female mice aged 18 months, and whether this effect relates to the mTOR/PI3K signaling pathways for activation of primordial follicles and ovarian angiogenesis. Female mice were intraperitoneally injected with 0.1 ml of 500 ng/ml or 1000 ng/ml of visfatin three times at intervals of 2 days, and both ovaries were provided for H&E staining. In another experiment, the mice were superovulated with pregnant mare's serum gonadotropin and human chorionic gonadotropin, and were mated with males. After 18 h, zygotes were collected and cultured for 4 days, and numbers and embryo developmental competency of zygotes retrieved were evaluated. The expression of mTOR/PI3K signaling pathway regulated genes (4EBP1, S6K1, and RPS6) and angiogenic factors (VEGF, visfatin, and SDF-1α) in the ovary were examined. As well, visfatin-treated mice were mated with male mice for 2 weeks, and the pregnancy outcome was monitored up to 3 weeks. Visfatin significantly increased the total numbers of follicles compared with control. Numbers of zygotes retrieved, blastocyst formation rate, and pregnancy rate were significantly increased at 500 ng/ml of visfatin (2.83%, 40.0%, and 80%, respectively) compared with control (0, 0, and no pregnancy). Ovarian expressions of S6K1, RPS6, VEGF, visfatin, and SDF-1α were significantly stimulated at 500 ng/ml of visfatin. These results show that visfatin treatment of an optimal dose rescues age-related decline in fertility, possibly by stimulating mTOR/PI3K signaling.

Prediction of ovarian aging using ovarian expression of BMP15, GDF9, and C-KIT.

IMPACT STATEMENT: Ovarian aging is becoming a more important issue in terms of fertility preservation and infertility treatment. Serum anti-Mullerian hormone (AMH) level and antral follicle count (AFC) are being practically used as markers of ovarian aging as well as ovarian reserve in human. However, these factors have some drawbacks in assessing ovarian aging and reserve. Therefore, the identification of ovarian expressions of BMP15, GDF9, and C-KIT according to female could be applied as a potent predictor of ovarian aging. This work provides new information on the development of diagnosis and treatment strategy of age-related fertility decline and premature ovarian insufficiency.

A rare case of squamous cell carcinoma in situ arising in mature cystic teratoma.

Mature cystic teratoma (MCT) is the most common ovarian tumor. Secondary malignant tumors rarely arise in MCTs, and squamous cell carcinoma (SCC) is the most common form of such tumors. MCT-derived SCC in situ (CIS) is mostly found together with invasive SCC; it is seldom detected alone. A 44-year-old woman with breast cancer was found to have a left ovarian cyst (size >8 cm) before treatment. She underwent bilateral salpingo-oophorectomy, and frozen biopsy showed MCT with focal proliferation of squamous epithelium and mild atypism. However, definitive pathologic diagnosis confirmed CIS arising in MCT. In addition, germline BRCA 1/2 test and human papillomavirus test of tumor tissue yielded negative results. This report is the first case of its kind in Korea. Our report can aid in clinical decision making and serve as a basis for follow-up studies on this rare type of CIS arising in MCT.

Effects of three-area laser-assisted zona thinning in 8-cell human embryos on pregnancy outcomes in vitro fertilization.

OBJECTIVE: This study conducted a preliminary examination of the effects of three-area laser-assisted zona thinning (LAZT) during the cleavage stage of embryo development on the hatching process in human in vitro fertilization-embryo transfer (IVF-ET) with subjects of advanced female age or frozen-thawed (FT) embryos. METHODS: Eight-cell stage embryos were treated with LAZT in three areas of the zona pellucida at 120° intervals. The control group was embryos without LAZT. Of the 72 consecutive fresh cycles and the 28 FT embryo transfer cycles, the patients in 55 fresh cycles and 17 FT cycles declined LAZT, and those cycles were defined as the control group. RESULTS: In the fresh cycles, the pregnancy rates were similar in the LAZT and control groups. However, in the FT cycles, the pregnancy rate was significantly higher in the LAZT group than in the control group (45.5% in the LAZT group vs. 23.5% in the control group, p<0.05). CONCLUSION: These results show that multi-area LAZT resulted in significantly improved pregnancy outcomes in human 8-cell embryos compared to controls.

The effects of uterine artery embolization on ovarian reserve.

OBJECTIVE: To evaluate the effects of UAE for symptomatic uterine fibroids on ovarian reserve based on AMH. STUDY DESIGN: This was a retrospective study conducted between March 2011 and October 2014. All women underwent UAE. At baseline and at the 3-month and 12-month follow-up visits, serum anti-Müllerian hormone (AMH), follicle stimulating hormone (FSH), luteinizing hormone (LH), and estradiol (E2) levels were assessed, and ovarian volume and antral follicle count (AFC) were evaluated in each patient. RESULTS: There were no statistically significant differences in serum E2, LH, or FSH levels or in ovarian volume 3 or 12 months after UAE (P=0.8194, P=0.3976, P=0.4766, and P=0.6822, respectively). However, AMH and AFC were significantly different 3 and 12 months after the procedure (P=0.00, P=0.029 and P=0.00, P=0.00, respectively). AMH levels remained low after 12 months of follow-up compared to the expected AMH levels. A statistically significant recovery of serum AMH at 12 months compared to at 3 months in those <40 years of age (P=0.00), but not in those ≥40 years (P=0.837). CONCLUSIONS: Ovarian reserve appears to be affected by UAE in premenopausal women. However, younger ovaries (according to biological ovarian age) exhibit a greater capacity for recovery after ovarian damage. Therefore, larger studies are needed for more conclusive results.

Identification of differentially expressed genes in bovine follicular cystic ovaries.

Follicular cystic ovary (FCO) is one of the most frequently diagnosed ovarian diseases and is a major cause of reproductive failure in mammalian species. However, the mechanism by which FCO is induced remains unclear. Genetic alterations which affect the functioning of many kinds of cells and/or tissues could be present in cystic ovaries. In this study, we performed a comparison analysis of gene expression in order to identify new molecules useful in discrimination of bovine FCO with follicular cystic follicles (FCFs). Normal follicles and FCFs were classified based on their sizes (5 to 10 mm and ≥25 mm). These follicles had granulosa cell layer and theca interna and the hormone 17ß-estradiol (E(2))/ progesterone (P(4)) ratio in follicles was greater than one. Perifollicular regions including follicles were used for the preparation of RNA or protein. Differentially expressed genes (DEG) that showed greater than a 2-fold change in expression were screened by the annealing control primer (ACP)-based PCR method using GeneFishing™ DEG kits in bovine normal follicles and FCFs. We identified two DEGs in the FCFs: ribosomal protein L15 (RPL15) and microtubule-associated protein 1B (MAP1B) based on BLAST searches of the NCBI GenBank. Consistent with the ACP analysis, semi-quantitative PCR data and Western blot analyses revealed an up-regulation of RPL15 and a down-regulation of MAP1B in FCFs. These results suggest that RPL15 and MAP1B may be involved in the regulation of pathological processes in bovine FCOs and may help to establish a bovine gene data-base for the discrimination of FCOs from normal ovaries.