Relationship between menopausal hormone therapy and incidence of fractures in postmenopausal women.

OBJECTIVE: Long-term protective effects of menopausal hormone therapy (MHT) at fractures with different doses and components are controversial. We analyzed the effect of MHT on the incidence of spine and femur fractures according to MHT type, age at commencement, duration and dose of hormones in Korean women. METHOD: This retrospective study evaluated propensity score-matched patients with MHT from the Korean National Health Insurance Service database. Among women aged ≥50 years with menopause between 2004 and 2007, spine and femur fracture incidence until 2017 was analyzed in 36,446 women who had received MHT for >1 year. Estrogen-progesterone therapy (EPT), estrogen-only therapy (ET) or tibolone therapy was conducted. RESULTS: EPT significantly lowered the incidence of spine and femur fractures with a conventional dose, but not with a low dose. Tibolone significantly decreased the incidence of spine fractures in women aged 50-59 years when used for >5 years, and the incidence of femur fractures in women older than 60 years when used for >3 years. ET significantly lowered the risk of femur fractures when estradiol was used for >5 years. CONCLUSION: In menopausal women, all MHT including conventional-dose EPT, ET and tibolone tended to lower the incidence of fractures. The effects, however, varied with the type of fracture and type of MHT.

Folpet induces mitochondrial dysfunction and ROS-mediated apoptosis in mouse Sertoli cells.

Folpet is a phthalimide type of fungicide and has been used to control several crop diseases. Although it has adverse effects on the gastrointestinal tract, its mechanism and toxic effects on testis have not been demonstrated. In the present study, we elucidated the cytotoxic effect of folpet on the mouse Sertoli cell line, TM4. Our results revealed that folpet suppressed viability and proliferative capacity of TM4 cells and further inhibited 3D spheroid formation. Moreover, folpet impeded appropriate cell cycle progression and induced apoptotic cell death in TM4 cells. It disrupted the electrochemical gradient of mitochondria and calcium homeostasis in TM4 cells. Furthermore, endoplasmic reticulum stress-related proteins were activated in folpet-treated TM4 cells, and relative reactive oxygen species (ROS) production was also increased. N-acetylcysteine (NAC) treatment reinstated the folpet-induced ROS generation in TM4 cells. Additionally, NAC restored the proliferative capacity and reduced the apoptotic cells in folpet-treated TM4 cells. Collectively, we demonstrated that folpet causes ROS-mediated apoptotic cell death with mitochondrial dysfunction and calcium dysregulation in TM4 cells.

Reproductive toxicity of folpet through deregulation of calcium homeostasis in porcine trophectoderm and luminal epithelial cells during early pregnancy.

Folpet, a fungicide, is utilized even in cosmetics and pharmaceuticals. The LD50 of folpet in mammals, birds, and fish is relatively high. Recently, several negative effects of folpet on the respiratory system and cornea have been reported. However, there is no study on the negative effects of folpet on maternal-fetus interactions. In the present study, we used porcine trophectoderm (pTr) cells and porcine luminal epithelial (pLE) cells to investigate the toxic effects of folpet during implantation. Folpet treatment decreased cell proliferation and promoted apoptosis with cell cycle arrest. In addition, the ERK, JNK, and AKT signal pathways were activated by folpet treatment. Folpet treatment induced calcium overload in pTr and pLE cells mediating antimigratory and antiadhesive effects in both cell lines. Co-treatment with calcium chelates decreased the anti-implantation effect of folpet. Overall, our results demonstrated potential reproductive toxicity of folpet in pig.

Potential roles of aquaporin 9 in the pathogenesis of endometriosis.

Aquaporins (AQPs) are involved in cell migration, proliferation and carcinogenesis in tumor development and physiologic inflammatory processes, but their associations with endometriosis have not been fully evaluated. In this study, tissue samples were obtained from women undergoing laparoscopic surgery for endometriosis and other benign conditions. Analysis of expressions of AQP subtypes in eutopic and ectopic endometrium of patients with endometriosis (Eu-EMS and Ect-EMS, respectively) and eutopic endometrium of control patients without endometriosis (Eu-CTL) were performed using the NanoString nCounter System and western blotting. Human endometrial stromal cells (HESCs) were cultured and transfected with the siRNA of the AQP of interest. Among the AQP1-9 subtypes, endometrial expression of AQP2 and AQP8 was significantly increased, whereas AQP9 expression was significantly decreased in the Eu-EMS group compared to the Eu-CTL group. Comparison of expression of AQP2, AQP8 and AQP9 among Eu-EMS, Ect-EMS and Eu-CTL groups revealed significant differences for only AQP9. Expression of AQP9 in the Eu-EMS group was decreased compared with that in Eu-CTL. After transfection of AQP9 siRNA in HESCs, expressions of MMP2 and MMP9 were significantly elevated. Increased expression of phosphorylated ERK 1/2 and phosphorylated p38 MAPK proteins after transfection was also confirmed using western blot analysis. Increased migration and invasion potentials of HESCs after transfection were determined by migration and wound healing assays. These findings suggest that AQP9 may be involved in the pathogenesis of endometriosis and warrant further investigation as a potential therapeutic target for treating endometriosis.

Use of the Endometriosis Fertility Index to Predict Natural Pregnancy after Endometriosis Surgery: A Single-Center Study.

AIMS: Our objective was to predict natural pregnancy after endometriosis surgery using the endometriosis fertility index (EFI). METHODS: A retrospective medical records review was conducted, examining patients surgically treated for endometriosis at a single center in Korea between January 2009 and February 2015. In total, 68 women attempting natural conception were analyzed by assessing age, preoperative serum CA-125, body mass index, revised American Fertility Society (rAFS) stage, EFI, and pregnancy outcome. Kaplan-Meier estimates and log-rank tests were used to generate cumulative natural pregnancy rate curves based on an EFI cut-point. A receiver-operating characteristic (ROC) curve was plotted for EFI. RESULTS: Seventy-seven patients attempted conceptions, resulting in 33 natural and 9 assisted conceptions. Excluding assisted conceptions, the mean EFI scores of 68 women who were not pregnant and pregnant were 5.43 ± 0.36 and 6.88 ± 0.28 respectively. The relation between EFI and natural pregnancy was significant (cumulative overall pregnancy rate, p = 0.006), whereas rAFS stage was not (univariate logistics, p = 0.853). The cut-point for maximum natural pregnancy outcomes was 6 (area under ROC curve = 0.710, 95% CI 0.586-0.835). CONCLUSION: The EFI is a reliable staging system for predicting natural pregnancy after endometriosis surgery. EFI scores can be used to guide postoperative treatment of women with endometriosis.

Dinitramine induces cardiotoxicity and morphological alterations on zebrafish embryo development.

Dinitramine (DN), an herbicide in the dinitroaniline family, is used in agricultural areas to prevent unwanted plant growth. Dinitroaniline herbicides inhibit cell division by preventing microtubulin synthesis. They are strongly absorbed by the soil and can contaminate groundwater; however, the mode of action of these herbicides in non-target organisms remains unclear. In this study, we examined the developmental toxicity of DN in zebrafish embryos exposed to 1.6, 3.2, and 6.4 mg/L DN, compared to embryos exposed to DMSO (control) for 96 h. Visual assessments using transgenic zebrafish (fli1:eGFP) indicated abnormal cardiac development with enlarged ventricles and atria, decreased heartbeats, and impaired cardiac function. Along with cardiac development, vessel formation and angiogenesis were suppressed through activation of the inflammatory response. In addition, exposure to 6.4 mg/L DN for 96 h induced cell death, with upregulation of genes related to apoptosis. Our results showed that DN induced morphological changes and triggered an inflammatory response and apoptotic cell death that can impair embryonic growth and survival, providing an important mechanism of DN in aquatic organisms.

Saponin Extracts Induced Apoptosis of Endometrial Cells From Women With Endometriosis Through Modulation of miR-21-5p.

Among the several components in Korean red ginseng, the saponin components are known to have various pharmacologic activities. The objective of this study was to evaluate therapeutic effects of saponin extracts from Korean red ginseng on endometriosis and to identify microRNAs (miRNAs) associated with saponin treatment. This is an in vitro study which used human endometrial stromal cells (HESCs) obtained from patients who underwent laparoscopic surgery for endometriosis and other benign conditions. Human endometrial stromal cells were treated with saponin extracts, and microarray profiling was performed. Human endometrial stromal cells were then transfected with miRNAs identified in the profiling. After the saponin extract treatment, the expression of caspase 3 was significantly increased in HESCs. Microarray profiling revealed several miRNAs that were differentially expressed, and miR-21-5p was further validated. Expression of miR-21-5p was significantly upregulated in the endometrium of patients with endometriosis, compared with controls. Transfection of a miR-21-5p inhibitor significantly increased caspase 3 expression in HESCs. The apoptotic potential of saponin extracts and the miR-21-5p inhibitor were further validated in HESCs using flow cytometry analysis. In conclusions, treatment with saponin extracts significantly decreased the expression of miR-21-5p in HESCs from patients with endometriosis. Inhibition of miR-21-5p effectively increased the apoptotic potential of HESCs. These findings suggest that saponin extract treatment may have therapeutic potential for endometriosis via modulation of specific miRNAs.

Molecular cloning and characterization of the soybean DEAD-box RNA helicase gene induced by low temperature and high salinity stress.

A novel gene encoding a DEAD-box RNA helicase designated as GmRH was isolated from soybean. Amino acid sequence alignment and phylogenetic tree analysis revealed a close relationship between GmRH and other orthologous DEAD-box RNA helicases from other plant species. Structural motif analysis revealed that the bipartite lysine rich nuclear localization signal (NLS) is present in the N-terminal variable region of GmRH and that there are ten conserved motifs found in DEAD-box RNA helicase proteins. Southern blot analysis revealed the presence of 2 copies of GmRH in the soybean genome. Northern blot analysis demonstrated that the RNA expression of the GmRH was induced during low temperature or high salinity stress, but not by the exogenous application of abscisic acid or drought stress. Subcellular localization studies showed that GmRH((1-355))-GFP is localized in the nucleus, whereas GmRH((130-355))-GFP is localized both in the cytoplasm and in the nucleus. This provides the evidence that the N-terminal region predicted as NLS is essential for nuclear targeting of the GmRH protein in the plant cell. Purified GST-GmRH recombinant protein was shown to unwind dsRNA independent of ATP in vitro. Here, we propose that GmRH plays an important role in RNA processing during low temperature and high salinity stresses in plants.