Follicular fluid progesterone downregulated HPGD and COX2 in granulosa cells via suppressing NF-ĸB in endometriosis†.

Increasing evidences showed that ovulatory dysfunction, possibly caused by luteinized unruptured follicular follicle syndrome (LUFS), is one of the reasons for endometriosis-related infertility. The present study was conducted to explore the potential effect of elevated progesterone in follicular fluid (FF) on ovulation in endometriosis. A prospective study including 50 ovarian endometriosis patients and 50 control patients with matched pairs design was conducted with alterations in FF and peritoneal fluid (PF) components identified by metabolomics analyses and differentially expressed genes in granulosa cells (GCs) identified by transcriptome analysis. Patients with endometriosis exhibited a significantly higher progesterone level in serum, FF, and PF. Granulosa cells from endometriosis patients revealed decreased expression of HPGD, COX-2, and suppressed NF-ĸB signaling. Similarly, progesterone treatment in vitro downregulated HPGD and COX2 expression and suppressed NF-ĸB signaling in granulosa tumor-like cell line KGN (Bena Culture Collection, China) and primarily cultured GCs, as manifested by decreased expressions of IL1R1, IRAK3, reduced pIĸBα/IĸBα ratio, and nucleus translocation of p65. On the contrary, TNF-α treatment increased expression of IL1R1, IRAK3, pIĸBα, p65, and HPGD in GCs. One potential p65 binding site was identified in the promoter region of HPGD by chromatin immunoprecipitation. In conclusion, we found that intrafollicular progesterone might downregulate HPGD and COX-2 in GCs via suppressing the NF-ĸB signaling pathway, shedding light on the mechanism underlying the endometriosis-related ovulatory dysfunction.

Curative effect of kangfuyan capsule combined with antibiotic treatment on pelvic inflammatory disease.

This study aims to investigate the curative effect of Kangfuyan capsule in the treatment of damp-heat and blood stasis type of pelvic inflammatory disease (PID), and its influence on serum inflammatory factors IL-6, CRP and TNF-α. A total of 83 patients with PID were randomly divided into two groups: Western medicine group (control group, n=41) received oral antibiotics (azithromycin + metronidazole) alone and the traditional Chinese medicine combined with Western medicine group (experimental group, n=42) received Kangfuyan capsule based on Western medicine therapy. Clinical efficacy between these two groups and the influence of drugs in serum inflammatory factors (IL-6, CRP and TNF-α) were compared. The total effective rate was 78.05% in the control group and 97.62% in the experimental group and difference between these two groups was statistically significant (P<0.01). The symptoms and signs in the two groups significantly improved after treatment (P<0.05) and improvement rate was significantly better in the experimental group than in the control group (P<0.05). After treatment, serum inflammatory factor levels in the two groups were significantly lower than levels before treatment (P<0.05) and improvement rate was significantly better in the experimental group than in the control group (P<0.05). Kangfuyan capsule combined with antibiotics can effectively relieve the symptoms and signs of patients, improve the efficiency of treatment, provide high safety, and does not increase adverse reactions. The possible mechanism may be that this therapy suppresses chronic PID by reducing serum inflammatory factor (IL-6, CRP and TNF-α) levels.

Phoenixin-14 concentrations are increased in association with luteinizing hormone and nesfatin-1 concentrations in women with polycystic ovary syndrome.

BACKGROUND: Polycystic ovary syndrome (PCOS) is commonly characterized by obesity, insulin resistance (IR), hyperandrogenemia and hirsutism. Following the reported relationship between phoenixin-14 and gonadotropin production in rat hypothalamic-pituitary-gonadal axis, the present study was designed to investigate the circulating concentrations of phoenixin-14 and their associations with the concentrations of sex hormones including luteinizing hormone (LH), follicular stimulating hormone (FSH), estradiol (E2), progesterone (P4) and total testosterone (TT) in PCOS patients. METHODS: A total of 41 women with diagnosed PCOS using Rotterdam criteria and 37 healthy individuals were enrolled in the study. RESULTS: Serum phoenixin-14 concentration in PCOS patients (n=41) was 0.515±0.044ng/ml, significantly higher than that in healthy controls (0.289±0.046ng/ml, n=37). PCOS patients had higher serum LH, dehydroepiandrosterone and fasting blood glucose concentrations, and higher index of homeostasis model of assessment-IR than those in healthy women. Correlation analysis showed significantly positive correlations of phoenixin-14 with LH, FSH, TT, P4, BMI and nesfatin-1 concentrations, and significantly negative correlations with E2 and serum insulin (FSI) concentrations, respectively. CONCLUSIONS: Compared to control women, PCOS patients had significantly increased serum phoenixin-14, LH and androgen concentrations. The positive correlations of phoenixin-14 concentrations with LH and TT concentrations suggest a possible role of phoenixin-14 in the development of PCOS.

XCI-escaping gene KDM5C contributes to ovarian development via downregulating miR-320a.

Mechanisms underlying female gonadal dysgenesis remain unclarified and relatively unstudied. Whether X-chromosome inactivation (XCI)-escaping genes and microRNAs (miRNAs) contribute to this condition is currently unknown. We compared 45,X Turner Syndrome women with 46,XX normal women, and investigated differentially expressed miRNAs in Turner Syndrome through plasma miRNA sequencing. We found that miR-320a was consistently upregulated not only in 45,X plasma and peripheral blood mononuclear cells (PBMCs), but also in 45,X fetal gonadal tissues. The levels of miR-320a in PBMCs from 45,X, 46,XX, 46,XY, and 47,XXY human subjects were inversely related to the expression levels of XCI-escaping gene KDM5C in PBMCs. In vitro models indicated that KDM5C suppressed miR-320a transcription by directly binding to the promoter of miR-320a to prevent histone methylation. In addition, we demonstrated that KITLG, an essential gene for ovarian development and primordial germ cell survival, was a direct target of miR-320a and that it was downregulated in 45,X fetal gonadal tissues. In conclusion, we demonstrated that downregulation of miR-320a by the XCI-escaping gene KDM5C contributed to ovarian development by targeting KITLG.

Avian influenza virus in pregnancy.

The unprecedented epizootic of avian influenza viruses, such as H5N1, H5N6, H7N1 and H10N8, has continued to cause disease in humans in recent years. In 2013, another novel influenza A (H7N9) virus emerged in China, and 30% of those patients died. Pregnant women are particularly susceptible to avian influenza and are more likely to develop severe complications and to die, especially when infection occurs in the middle and late trimesters. Viremia is believed to occur infrequently, and thus vertical transmission induced by avian influenza appears to be rare. However, avian influenza increases the risk of adverse pregnancy outcomes, including spontaneous abortion, preterm birth and fatal distress. This review summarises 39 cases of pregnant women and their fetuses from different countries dating back to 1997, including 11, 15 and 13 infections with H7N9, H5N1 and the 2009 pandemic influenza (H1N1), respectively. We analysed the epidemic features, following the geographical, population and pregnancy trimester distributions; underlying diseases; exposure history; medical timelines; human-to-human transmission; pathogenicity and vertical transmission; antivirus treatments; maternal severity and mortality and pregnancy outcome. The common experiences reported in different countries and areas suggest that early identification and treatment are imperative. In the future, vigilant virologic and epidemiologic surveillance systems should be developed to monitor avian influenza viruses during pregnancy. Furthermore, extensive study on the immune mechanisms should be conducted, as this will guide safe, rational immunomodulatory treatment among this high-risk population. Most importantly, we should develop a universal avian influenza virus vaccine to prevent outbreaks of the different subtypes. Copyright © 2016 John Wiley & Sons, Ltd.

Hyaluronic acid reagent functional chitosan-PEI conjugate with AQP2-siRNA suppressed endometriotic lesion formation.

To identify a new drug candidate for treating endometriosis which has fewer side effects, a new polymeric nanoparticle gene delivery system consisting of polyethylenimine-grafted chitosan oligosaccharide (CSO-PEI) with hyaluronic acid (HA) and small interfering RNA (siRNA) was designed. There was no obvious difference in sizes observed between (CSO-PEI/siRNA)HA and CSO-PEI/siRNA, but the fluorescence accumulation in the endometriotic lesion was more significant for (CSO-PEI/siRNA)HA compared with CSO-PEI/siRNA due to the specific binding of HA to CD44. In addition, the (CSO-PEI/siRNA)HA nanoparticle gene therapy significantly decreased the endometriotic lesion sizes with atrophy and degeneration of the ectopic endometrium. The epithelial cells of ectopic endometrium from rat models of endometriosis showed a significantly lower CD44 expression than control after treatment with (CSO-PEI/siRNA)HA. Furthermore, observation under an electron microscope showed no obvious toxic effect on the reproductive organs. Therefore, (CSO-PEI/siRNA)HA gene delivery system can be used as an effective method for the treatment of endometriosis.

Binding of FGF2 to FGFR2 in an autocrine mode in trophectoderm cells is indispensable for mouse blastocyst formation through PKC-p38 pathway.

Fibroblast growth factors (FGF1, FGF2 and FGF4) and fibroblast growth factor receptors (FGFR1, FGFR2, FGFR3 and FGFR4) have been reported to be expressed in preimplantation embryos and be required for their development. However, the functions of these molecules in trophectoderm cells (TEs) that lead to the formation of the blastocyst as well as the underlying mechanism have not been elucidated. The present study has demonstrated for the first time that endogenous FGF2 secreted by TEs can regulate protein expression and distribution in TEs via the FGFR2-mediated activation of PKC and p38, which are important for the development of expanded blastocysts. This finding provides the first explanation for the long-observed phenomenon that only high concentrations of exogenous FGFs have effects on embryonic development, but in vivo the amount of endogenous FGFs are trace. Besides, the present results suggest that FGF2/FGFR2 may act in an autocrine fashion and activate the downstream PKC/p38 pathway in TEs during expanded blastocyst formation.

Auricular acupressure reduces anxiety levels and improves outcomes of in vitro fertilization: a prospective, randomized and controlled study.

The study was to explore whether auricular acupressure (AA) can relieve anxiety during the period from trans-vaginal oocyte retrieval to the embryo transfer in IVF treatment and whether AA can improve the outcomes of IVF. 305 infertile patients with tubal blockage who were referred for IVF were included. The women were randomized into a control group with 102 cases, a Sham-AA group with 102 cases and an AA group with 101 cases. The anxiety levels were rated with Spielberger's State Trait Anxiety Inventory and the Amsterdam Preoperative Anxiety and Information Scale. Data of clinical pregnancy rate (CPR), implantation rate (IR) and live birth rate (LBR) were obtained. The levels of neuropeptide Y (NPY) and transforming growth factor alpha (TGF-alpha) in the follicular fluids were detected with ELISA. After treatment, in AA group, the levels of state anxiety, preoperative anxiety and need-for-information were significantly lower, whereas CPR, IR, LBR and NPY levels in the follicular fluids were markedly higher than Sham-AA group and control group. We concluded that AA could help to reduce anxiety levels associated with IVF and improves the outcomes of IVF partly through increasing the levels of NPY in the follicular fluids.

Identification of a mutation in GDF9 as a novel cause of diminished ovarian reserve in young women.

STUDY QUESTION: Do any mutations in growth differentiation factor 9 (GDF9) have a role in diminished ovarian reserve (DOR) in young women? SUMMARY ANSWER: The GDF9 p.R146C mutation may be a source of DOR in some young women. WHAT IS KNOWN ALREADY: DOR affects 10% of women under 37 years of age and is associated with accelerated expenditure of follicles. GDF9 is an oocyte-secreted factor that plays a critical role in follicular development and female fertility. Several GDF9 variants have been linked to ovarian dysfunction. STUDY DESIGN, SIZE, DURATION: This case-control study included 139 women with DOR and 152 controls aged under 37 years. PARTICIPANTS/MATERIALS, SETTING, METHODS: All women were recruited in a Chinese tertiary center and underwent DNA sequencing of GDF9 gene. We then determined the molecular and biological properties of mutant GDF9 proteins using protein expression, structural prediction and functional analyses. MAIN RESULTS AND THE ROLE OF CHANCE: We identified two mutations in the proregion of GDF9 gene: c.169T > G (p.D57Y) and c.436T > C (p.R146C). The p.R146C mutation was found in three women with DOR but was absent in the control population. This mutation was also associated with significant reductions in GDF9 mature protein secretion in cultured cells. Functional studies with human granulosa cells (GCs) showed that the p.R146C mutation reduced the abilities of GDF9 to stimulate GC proliferation and to activate the Smad2 pathway. Protein structure modeling predicted that p.R146C disrupted an α-helix in GDF9 protein. In contrast with p.R146C, the p.D57Y mutation, found in both the DOR and control groups (6 versus 2), had no obvious deleterious effects. LIMITATIONS, REASONS FOR CAUTION: Larger studies in varying populations may validate the role of GDF9 mutation in young women with DOR. WIDER IMPLICATIONS OF THE FINDINGS: These results may provide new insights into the pathophysiological mechanisms of early-onset DOR.

Aquaporin-1 plays a crucial role in estrogen-induced tubulogenesis of vascular endothelial cells.

CONTEXT: Aquaporin-1 (AQP1) has been proposed as a mediator of estrogen-induced angiogenesis in human breast cancer and endometrial cancer. Elucidation of the molecular mechanisms governing AQP1-mediated, estrogen-induced angiogenesis may contribute to an improved understanding of tumor development. OBJECTIVE: Our objective was to identify the estrogen-response element (ERE) in the promoter of the Aqp1 gene and investigate the effects and mechanisms of AQP1 on estrogen-induced tubulogenesis of vascular endothelial cells. SETTING: The study was conducted in a university hospital in eastern China. MAIN OUTCOME MEASURES: Immunohistological, real-time PCR and Western blot analyses were used to determine the expression AQP1 mRNA and protein in vascular endothelial cells. Chromatin immunoprecipitation analyses and luciferase reporter assays identified ERE-like motif in the promoter of the Aqp1 gene. RESULTS: Expression of AQP1 in blood vessels of human breast and endometrial carcinoma tissues were significantly higher than controls. Estradiol (E2) dose-dependently increased the expression levels of AQP1 mRNA and protein in human umbilical vein endothelial cells (HUVECs). A functional ERE-like motif was identified in the promoter of the Aqp1 gene. AQP1 colocalized with ezrin, a component of the ezrin/radixin/moesin protein complex, and, ezrin colocalized with filamentous actin in HUVECs. Knockdown of AQP1 or ezrin with specific small interfering RNA significantly attenuated the formation of transcytoplasmic filamentous actin stress fibers induced by E2 and inhibited E2-enhanced cell proliferation, migration, invasion, and tubule formation of HUVECs. CONCLUSIONS: Estrogen induces AQP1 expression by activating ERE in the promoter of the Aqp1 gene, resulting in tubulogenesis of vascular endothelial cells. These results provide new insights into the molecular mechanisms underpinning the angiogenic effects of estrogen.

Down-regulation of S100A11, a calcium-binding protein, in human endometrium may cause reproductive failure.

BACKGROUND: Low expression levels of S100A11 proteins were demonstrated in the placental villous tissue of patients with early pregnancy loss, and S100A11 is a Ca2+-binding protein that interprets the calcium fluctuations and elicits various cellular responses. OBJECTIVES: The objective of the study was to determine S100A11 expression in human endometrium and its roles in endometrial receptivity and embryo implantation. METHODS: S100A11 expression in human endometrium was analyzed using quantitative RT-PCR, Western blot, and immunohistochemical techniques. The effects of S100A11 on embryo implantation were examined using in vivo mouse model, and JAr (a human choriocarcinoma cell line) spheroid attachment assays. The effects of endometrial S100A11 on factors related to endometrial receptivity and immune responses were examined. Using a fluorescence method, we examined the changes in cytosolic Ca2+ and Ca2+ release from intracellular stores in epidermal growth factor (EGF)-treated endometrial cells transfected with or without S100A11 small interfering RNA. RESULTS: S100A11 was expressed in human endometrium. S100A11 protein levels were significantly lower in endometrium of women with failed pregnancy than that in women with successful pregnancy outcomes. The knockdown of endometrial S100A11 not only reduced embryo implantation rate in mouse but also had adverse effects on the expression of factors related to endometrial receptivity and immune responses in human endometrial cells. Immunofluorescence analysis showed that S100A11 proteins were mainly localized in endoplasmic reticulum. The EGF up-regulated endometrial S100A11 expression and promoted the Ca2+ uptake and release from Ca2+ stores, which was inhibited by the knockdown of S100A11. CONCLUSIONS: Endometrial S100A11 is a crucial intermediator in EGF-stimulated embryo adhesion, endometrium receptivity, and immunotolerance via affecting Ca2+ uptake and release from intracellular Ca2+ stores. Down-regulation of S100A11 may cause reproductive failure.

Small-conductance calcium-activated K(+) channels 3 (SK3) regulate blastocyst hatching by control of intracellular calcium concentration.

BACKGROUND: The present study was designed to investigate the expression of small-conductance calcium-activated K(+) channels 3 (SK3) in preimplantation embryos and to explore their role in the underlying mechanism of blastocyst hatching. METHODS: Human preimplantation embryos were donated by patients who achieved successful pregnancy with in vitro fertilization. Mouse preimplantation embryos in different stages were collected and cultured with or without siRNA cell injection. The expression of SK3 was examined by RT-PCR, quantitative real-time PCR, western blot and immunofluorescence. Functional expression of SK3 was investigated using the patch-clamp technique. [Ca(2+)]i was measured by fluorescent imaging. Embryos were cultured in vitro to investigate the effect of SK3 knockdown or apamin, an SK3 inhibitor, on blastocyst hatching and F-actin formation. RESULTS: In human blastocysts, the level of SK3 expression was significantly lower in blastocysts that failed to hatch than in blastocysts that hatched successfully. In mouse embryos, SK3 mRNA and protein were not found in zygotes, but were detected from the 2-cell stage onward, with the highest levels observed in blastocysts. SK3 was predominately located in the trophectoderm cell membrane of expanded blastocysts. SK3 knockdown in trophectoderm cells not only suppressed the SK3 current, but also reduced [Ca(2+)]i elevation and membrane potential hyperpolarization induced by thapsigargin. Although the formation of expanded blastocysts was not affected, blastocyst hatching and F-actin formation were significantly inhibited after SK3 knockdown in trophectoderm cells. CONCLUSIONS: SK3-mediated [Ca(2+)]i elevation and membrane potential hyperpolarization in trophectoderm cells are important for blastocyst hatching, and defects in SK3 expression may contribute to infertility.

Functional expression of large-conductance calcium-activated potassium channels in human endometrium: a novel mechanism involved in endometrial receptivity and embryo implantation.

BACKGROUND: Large-conductance calcium-activated potassium channels (BK(Ca) channels) mediate physiological processes in nonexcitable cells. OBJECTIVE: The aim of the study was to determine BK(Ca) channel expression in human endometrium and its role in endometrial receptivity and embryo implantation. METHODS: BK(Ca) channel expression in human endometrium is described at different phases of the menstrual cycle using quantitative real time-PCR and Western blot techniques. Their effects on embryo implantation were examined using JAr spheroid attachment assays and in vivo mouse model. We examined their effects on endometrial receptivity factors, nuclear factor-κB (NF-κB) activity using quantitative real time-PCR, Western blot, and EMSA analyses. Changes in electrophysiological properties and cytosolic free Ca(2+) were measured in endometrial cells with or without specific BK(Ca) blocker or transfected with BK(Ca) small interfering RNA using patch-clamp and fluorescence analyses, respectively. RESULTS: BK(Ca) channels are expressed in human endometrial cells in a phase-related fashion during the menstrual cycle (proliferative, 0.20 ± 0.02, vs. mid-secretory, 0.72 ± 0.07; P < 0.01). Blocking BK(Ca) channel function or knockdown of endogenous BK(Ca) channel expression not only decreased JAr spheroid attachment rate and embryo implantation rate in mice but also significantly reduced the expression levels of endometrial receptive factors, including leukemia inhibitory factor, integrin ß3, claudin-4, and DKK-1, in human endometrial cells. Blocking BK(Ca) channels also reduced BK(Ca)-regulated NF-κB activity, cytosolic Ca(2+) concentrations, and membrane potentials in human endometrial cells. CONCLUSIONS: These observations demonstrate that BK(Ca) channels: 1) are expressed in endometrial cells; 2) affect embryo implantation by mediating endometrial receptive factors; and 3) alter the activity of NF-κB and homeostasis of Ca(2+) in the human endometrial cells.

Identification of estrogen response element in the aquaporin-2 gene that mediates estrogen-induced cell migration and invasion in human endometrial carcinoma.

BACKGROUND: Accumulating evidence suggests that aquaporins (AQP) can facilitate cell migration, invasion, and proliferation in tumor development in addition to water transport. OBJECTIVE: The aim of this study was to examine AQP2 expression in the endometrial tissues from patients with endometrial carcinoma (EC) and determine the roles and mechanisms of AQP2 in estrogen-related cell migration, invasion, adhesion, and proliferation of Ishikawa (IK) cells. APPROACH: AQP2 expression levels were measured in human endometrial cells and estradiol (E(2))-treated IK cells, and the estrogen-response element was identified. After blocking down and up-regulating the endogenous expression of AQP2 in IK cells, cell morphology, capacity for invasion, migration and adhesion, and expression markers of membrane/cytoskeleton were analyzed. RESULTS: AQP2 was expressed in endometrial tissues from patients with EC and endometriosis, both of which are estrogen-dependent diseases. In IK cells, E(2) dose-dependently increased AQP2 expression, which was blocked by the estrogen receptor inhibitor ICI182780. An estrogen-response element was identified in the AQP2 promoter. E(2) significantly increased the migration, invasion, adhesion, and proliferation of IK cells. AQP2 knockdown attenuated E(2)-enhanced migration, invasion, and adhesion. AQP2 knockdown reduced not only the E(2)-enhanced expression of F-actin and annexin-2 but also the E(2)-induced alteration of cell morphology. Moreover, higher expression levels of F-actin and annexin-2 were detected in the endometrial tissues from patients with EC. CONCLUSIONS: AQP2 mediates E(2)-enhanced migration, invasion, and adhesion through alteration of F-actin and annexin-2 expression and reorganization of F-actin, and inhibition of AQP may be a potential method for antitumor therapy.