EGF-Enhanced GnRH-II Regulation in Decidual Stromal Cell Motility through Twist and N-Cadherin Signaling.

Crucial roles in embryo implantation and placentation in humans include the invasion of the maternal decidua by extravillous trophoblasts and the motile behavior of decidual endometrial stromal cells. The effects of the epidermal growth factor (EGF) and GnRH-II in the endometrium take part in early pregnancy. In the present study, we demonstrated the coaction of EGF- and GnRH-II-promoted motility of human decidual endometrial stromal cells, indicating the possible roles of EGF and GnRH-II in embryo implantation and early pregnancy. After obtaining informed consent, we obtained human decidual endometrial stromal cells from decidual tissues from normal pregnancies at 6 to 12 weeks of gestation in healthy women undergoing suction dilation and curettage. Cell motility was evaluated with invasion and migration assays. The mechanisms of EGF and GnRH-II were performed using real-time PCR and immunoblot analysis. The results showed that human decidual tissue and stromal cells expressed the EGF and GnRH-I receptors. GnRH-II-mediated cell motility was enhanced by EGF and was suppressed by the knockdown of the endogenous GnRH-I receptor and EGF receptor with siRNA, revealing that GnRH-II promoted the cell motility of human decidual endometrial stromal cells through the GnRH-I receptor and the activation of Twist and N-cadherin signaling. This new concept regarding the coaction of EGF- and GnRH-promoted cell motility suggests that EGF and GnRH-II potentially affect embryo implantation and the decidual programming of human pregnancy.

Impact of growth hormone-releasing hormone antagonist on decidual stromal cell growth and apoptosis in vitro†.

Endometrial stromal cells remodeling is critical during human pregnancy. Growth hormone-releasing hormone and its functional receptor have been shown to be expressed in gynecological cancer cells and eutopic endometrial stromal cells. Recent studies have demonstrated the potential clinical uses of antagonists of growth hormone-releasing hormone as effective antitumor agents because of its directly antagonistic effect on the locally produced growth hormone-releasing hormone in gynecological tumors. However, the impact of growth hormone-releasing hormone antagonists on normal endometrial stromal cell growth remained to be elucidated. The aim of this study was to investigate the effect of a growth hormone-releasing hormone antagonist (JMR-132) on cell proliferation and apoptosis of human decidual stromal cells and the underlying molecular mechanisms. Our results showed that growth hormone-releasing hormone and the splice variant 1 of growth hormone-releasing hormone receptor are expressed in human decidual stromal cells isolated from the decidual tissues of early pregnant women receiving surgical abortion. In addition, treatment of stroma cells with JMR-132 induced cell apoptosis with increasing cleaved caspase-3 and caspase-9 activities and decrease cell viability in a time- and dose-dependent manner. Using a dual inhibition approach (pharmacological inhibitors and siRNA-mediated knockdown), we showed that JMR-132-induced activation of apoptotic signals are mediated by the activation of ERK1/2 and JNK signaling pathways and the subsequent upregulation of GADD45alpha. Taken together, JMR-132 suppresses cell survival of decidual stromal cells by inducing apoptosis through the activation of ERK1/2- and JNK-mediated upregulation of GADD45alpha in human endometrial stromal cells. Our findings provide new insights into the potential impact of growth hormone-releasing hormone antagonist on the decidual programming in humans.

Gonadotropin-releasing hormone type II (GnRH-II) agonist regulates the motility of human decidual endometrial stromal cells: possible effect on embryo implantation and pregnancy.

Invasion of the maternal decidua by extravillous trophoblast is an important process for embryo implantation and placentation in humans. Motile behavior of decidual endometrial stromal cells has been considered of critical importance for embryo implantation and programming of human pregnancy. The gonadotropin-releasing hormone (GnRH) effects in endometrium have raised concerns in reproduction. In the present study, we examined the action of GnRH-II agonist-promoted motility of human decidual endometrial stromal cells and the mechanisms of the action, indicating the role of GnRH-II agonist in embryo implantation and early pregnancy. Human decidual endometrial stromal cells were isolated from the decidual tissue from healthy women undergoing elective pregnancy termination of a normal pregnancy at 6- to 12-wk gestation, after informed consent. Cell motility was estimated by invasion and migration assay. Zymography and immunoblot analysis were performed to investigate the mechanisms of the GnRH-II action. The GnRH-I receptor (GnRH-IR) was expressed in human decidual tissue and endometrial stromal cells. The GnRH-II agonist promoted cell motility. Mitogen-activated protein kinase inhibitors abolished GnRH-II agonist-induced cell motility and activation of MMP-2 and MMP-9. GnRH-II agonist-mediated cell motility was suppressed by knockdown of endogenous GnRH-IR, MMP (matrix metalloproteinase)-2, and MMP-9 with small interfering RNA and MMP inhibitors. Our study demonstrates that the GnRH-II agonist promoted the cell motility of human decidual endometrial stromal cells through the GnRH-IR and the phosphorylation of extracellular signal-regulated protein kinase 1/2 and JNK-dependent activation of MMP-2 and MMP-9. Our findings represent a new concept regarding the mechanisms of GnRH-II-promoted cell motility, suggesting that GnRH-II agonist has strong effects on embryo implantation and decidual programming of human pregnancy.

Gonadotropin-releasing hormone type II (GnRH-II) agonist regulates the invasiveness of endometrial cancer cells through the GnRH-I receptor and mitogen-activated protein kinase (MAPK)-dependent activation of matrix metalloproteinase (MMP)-2.

BACKGROUND: More than 25% of patients diagnosed with endometrial carcinoma have an invasive primary cancer accompanied by metastases. Gonadotropin-releasing hormone (GnRH) plays an important role in reproduction. In mammals, expression of GnRH-II is higher than GnRH-I in reproductive tissues. Here, we examined the effect of a GnRH-II agonist on the motility of endometrial cancer cells and its mechanism of action in endometrial cancer therapy. METHODS: Immunoblotting and immunohistochemistry (IHC) were used to determine the expression of the GnRH-I receptor protein in human endometrial cancer. The activity of MMP-2 in the conditioned medium was determined by gelatin zymography. Cell motility was assessed by invasion and migration assay. GnRH-I receptor si-RNA was applied to knockdown GnRH-I receptor. RESULTS: The GnRH-I receptor was expressed in the endometrial cancer cells. The GnRH-II agonist promoted cell motility in a dose-dependent manner. The GnRH-II agonist induced the phosphorylation of ERK1/2 and JNK, and the phosphorylation was abolished by ERK1/2 inhibitor (U0126) and the JNK inhibitor (SP600125). Cell motility promoted by GnRH-II agonist was suppressed in cells that were pretreated with U0126 and SP600125. Moreover, U0126 and SP600125 abolished the GnRH-II agonist-induced activation of MMP-2. The inhibition of MMP-2 with MMP-2 inhibitor (OA-Hy) suppressed the increase in cell motility in response to the GnRH-II agonist. Enhanced cell motility mediated by GnRH-II agonist was also suppressed by the knockdown of the endogenous GnRH-I receptor using siRNA. CONCLUSION: Our study indicates that GnRH-II agonist promoted cell motility of endometrial cancer cells through the GnRH-I receptor via the phosphorylation of ERK1/2 and JNK, and the subsequent, MAPK-dependent activation of MMP-2. Our findings represent a new concept regarding the mechanism of GnRH-II-induced cell motility in endometrial cancer cells and suggest the possibility of exploring GnRH-II as a potential therapeutic target for the treatment of human endometrial cancer.

Regulation of oocyte and cumulus cell interactions by intermedin/adrenomedullin 2.

Ovarian folliculogenesis has been studied as a model of hormonal regulation of development and differentiation, cell death, and cell-cell communication. In addition to gonadotropins from the pituitary and follicular paracrine factors, oocyte secreted factors have been shown to play critical roles in the regulation of follicular cell functions. Except for the well characterized BMP family proteins, including GDF9 and BMP15, oocytes are known to secrete oocyte secreted factors that are important for the regulation of cumulus cell survival and the maintenance of tertiary structure of cumulus cell-enclosed oocyte complexes (COCs). Based on genomic screening and studies of COCs cultured in vitro, we showed that intermedin (IMD)/adrenomedullin 2 (ADM2) is a novel oocyte-derived ligand important for the regulation of cell interactions in COCs that functions, in part, by suppressing cumulus cell apoptosis. Consistently, we showed that suppression of IMD/ADM2 signaling in growing rat ovaries in vivo leads to oocyte atresia and aberrant cell cycle progression in follicular cells. Together, our studies indicated that mammalian oocytes deploy a G protein-coupled receptor ligand to coordinate normal interactions of oocytes and cumulus cells and provided a better understanding of how the tertiary structure of a COC is maintained as follicles undergo exponential growth during the late stages of folliculogenesis.

Gonadotrophin-releasing hormone antagonist induces apoptosis in human decidual stromal cells: effect on GADD45α and MAPK signaling.

BACKGROUND: The impact of gonadotrophin-releasing hormone (GnRH) antagonists used in IVF protocols on endometrial tissue remodeling, embryo implantation and the programming of early pregnancy is still unclear. Pregnancy and infant outcomes after treatment with GnRH antagonist for IVF are particular causes of concern. The purpose of this study was to investigate the mechanisms of GnRH antagonist-induced apoptosis of human decidual stromal cells and the effects of GnRH antagonist on the activation of ERK1/2, JNK and GADD45α signaling. METHODS: Human decidual stromal cells were isolated from decidual tissue. The expression of GnRH-I receptor (GnRH-IR) was examined by immunoblot analysis and immunohistochemistry. The cells were treated with the GnRH antagonist, Cetrorelix. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide assay was used to examine cell viability. Cleaved caspase-3 and terminal deoxynucleotidyl transferase-mediated dUDP nick-end labeling assay were used as indicators for cell apoptosis. Mitogen-activated protein kinase function was tested for the elucidation of intracellular signalings through the pre-treatment of stromal cells with ERK1/2 (U0126) and JNK (SP600125) inhibitors prior to the Cetrorelix treatment. To characterize the signaling pathway of GnRH antagonist, the endogenous GnRH-IR and GADD45α were knocked down by specific small-interfering RNA (siRNA). RESULTS: The GnRH-IR is expressed in human decidual stromal cells. Treatment with GnRH antagonist decreased cell viability, induced apoptosis and increased the phosphorylation of ERK1/2 and JNK. Cells pre-treated with U0126 and SP600125 were rescued from the GnRH antagonist-mediated inhibition of cell growth and did not exhibit GnRH antagonist-induced apoptosis and downstream GADD45α signaling. GnRH antagonist-mediated cell growth inhibition and apoptosis were also abolished by the knockdown of the endogenous GnRH-IR or GADD45α with siRNAs. CONCLUSIONS: The GnRH antagonist suppresses the growth of decidual stromal cells by inducing apoptosis through the GnRH-IR and through the ERK1/2 and JNK phosphorylation-dependent induction of GADD45α signaling. These results indicate that ERK1/2, JNK and GADD45α are coordinately regulated by the GnRH antagonist through the GnRH-IR to induce apoptosis in human decidual stromal cells, suggesting that the GnRH antagonist may play a role in decidual programming in human pregnancy.

Lead level in seminal plasma may affect semen quality for men without occupational exposure to lead.

BACKGROUND: Infertility affects approximately 10-15% of reproductive-age couples. Poor semen quality contributes to about 25% of infertile cases. Resulting from the direct effect on testicular function or hormonal alterations, heavy metals exposure has been related to impaired semen quality. The objective of this study was to assess the level of lead in the seminal plasma in men without occupational exposure to lead, and to determine the relationship between semen quality and lead concentration in the semen. METHODS: This is a prospective and nonrandomized clinical study conducted in University infertility clinic and academic research laboratory. Three hundred and forty-one male partners of infertile couples undergoing infertility evaluation and management were recruited to the study. Semen samples collected for the analyses of semen quality were also used for the measurement of lead concentrations. Semen samples were evaluated according to the WHO standards. RESULTS: All subjects were married and from infertile couples without occupational exposure to lead. There is a significant inverse correlation between the lead concentration in seminal plasma and sperm count. A higher semen lead concentration was correlated with lower sperm count, but not with semen volume, sperm motility or sperm morphology as assessed by simple linear regression. CONCLUSIONS: We found that semen lead concentration was significantly higher among the patients with lower sperm count. To our knowledge, this is the first study to demonstrate that a high level of lead accumulation in semen may reduce the sperm count contributing to infertility of men without occupational exposure to lead.

Stress-induced phosphoprotein 1 as a secreted biomarker for human ovarian cancer promotes cancer cell proliferation.

Ovarian cancers are frequently not diagnosed until advanced stages, resulting in a high case fatality rate. Because of this, more tumor markers, in addition to CA125, for detecting and monitoring ovarian cancer are needed. During a systematic search for potential biomarkers of ovarian cancer, we compared the protein profiles between tumor interstitial fluid and normal interstitial fluid of ovaries, rationalizing that abnormal levels of proteins in tumor interstitial fluid may be detected in peripheral blood and thus serve as easily accessible tumor markers. Here, we show that stress-induced phosphoprotein 1 (STIP1) was secreted by ovarian cancer tissues into the peripheral blood of patients, resulting in a significant increase of serum levels of STIP1 in cancer patients compared with those in age-matched normal controls. Our results further indicated that combined use of CA125 and STIP1 may increase early detection of ovarian cancer. Functionally, recombinant STIP1 significantly induced ERK phosphorylation, promoted DNA synthesis, and increased Ki-67 immunoreactivity in ovarian cancer cells, suggesting that STIP1 in vitro promotes cell proliferation. Colocalization of STIP1 and phospho-ERK in human ovarian cancer tissues also supports an in vivo activation of ERK by STIP1. Further understanding of molecular roles of STIP1 in human ovarian cancer may shed light on its pathophysiology and development of novel therapeutic strategies.

The role of diagnostic hysteroscopy before the first in vitro fertilization/intracytoplasmic sperm injection cycle.

OBJECTIVE: To assess the role of diagnostic hysteroscopy in pregnancy outcome in patients starting the first in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI). MATERIALS AND METHODS: A total of 567 infertile women who underwent first IVF treatment were reviewed retrospectively. Two hundred and fifteen (37.9 %) women underwent diagnostic hysteroscopy before the scheduled controlled ovarian hyperstimulation (COH). Two hundred and eighty-four (50.1 %) women only accepted transvaginal ultrasonography (TVU), and 68 (12.0 %) woman did not receive hysteroscopy and TVU before COH. Primary outcome measure was the live birth rate. Secondary outcome measures were the implantation rate, clinical pregnancy rate, and miscarriage rate. RESULTS: There was no difference among three groups with regard to number of oocytes retrieved, fertilization rate, implantation rate, clinical pregnancy rate, miscarriage rate, and live birth rate per cycle. The implantation rate and clinical pregnancy rate were significantly lower in the cycles with endometrial thickness <8 mm on human chorionic gonadotropin day (P = 0.001 and 0.018, respectively). Women with greater body mass index (>22) were associated with higher incidence of intrauterine lesions (22 of 36, 61.1 %). CONCLUSION: Diagnostic hysteroscopy prior to COH may not increase the implantation rate and live birth rate for the first IVF/ICSI programs. The efficacy of routinely performing diagnostic hysteroscopy before the first IVF program is needed to re-evaluate.

Extracellular Vesicle-Associated MicroRNA-138-5p Regulates Embryo Implantation and Early Pregnancy by Adjusting GPR124.

Functional embryo-maternal interactions occur during the embryo implantation and placentation. Extracellular vesicles with microRNA (miR) between cells have been considered of critical importance for embryo implantation and the programming of human pregnancy. MiR-138-5p functions as the transcriptional regulator of G protein-coupled receptor 124 (GPR124). However, the signaling pathway of miR138-5p- and GPR124-adjusted NLRP3 inflammasome activation remains unclear. In this study, we examine the roles of the miR138-5p and GPR124-regulated inflammasome in embryo implantation and early pregnancy. Human decidual stromal cells were isolated from the abortus tissue and collected by curettage from missed abortion patients and normal pregnant women at 6- to 12-week gestation, after informed consent. Isolated extracellular vesicles from decidua and decidual stromal cells were confirmed by transmission electron microscopy (TEM). Next-Generation Sequencing (NGS) and microarray were performed for miR analysis. The predicated target genes of the differentially expressed miR were analyzed to identify the target genes and their pathway. We demonstrated the down-regulation of miR-138-5p and the overexpression of GPR124 in spontaneous miscarriage compared to normal pregnancy. We also showed the excessive activation of the NLRP3 inflammasome in spontaneous miscarriage compared to normal pregnancy. Here, we newly demonstrate that the miR-138-5p and GPR124-adjusted NLRP3 inflammasome were expressed in extracellular vesicles derived from decidua and decidual stromal cells, indicating that the miR-138-5p, GPR124 and NLRP3 (NACHT, LRR, and PYD domains-containing protein 3) inflammasome have a potential modulatory role on the decidual programming and placentation of human pregnancy. Our findings represent a new concept regarding the role of extracellular vesicles, miR-138-5p, GPR124, and the NLRP3 inflammasome in normal early pregnancy and spontaneous miscarriage.

Site-specific endometrial injury improves implantation and pregnancy in patients with repeated implantation failures.

BACKGROUND: To test whether a site-specific hysteroscopic biopsy-induced injury in the endometrium during the controlled ovarian hyperstimulation cycle improves subsequent embryo implantation in patients with repeated implantation failure, a total of 30 patients who have had good responses to controlled ovulation stimulation but have failed to achieve pregnancy after two or more transfers of good-quality embryos were recruited in this prospective study. METHODS: A single, site-specific hysteroscopic biopsy-induced injury was generated on the posterior endometrium at midline 10-15 mm from the fundus during the D4-D7 period of the ongoing controlled ovarian hyperstimulation cycle in six patients. RESULTS: Patients received endometrial biopsy protocol achieved a pregnancy rate of 100%. By contrast, only 46% of patients with similar clinical characteristics (N = 24) achieved pregnancy without the hysteroscopic biopsy-induced endometrium injury (p < 0.05). CONCLUSIONS: Our proof-of-concept study demonstrates that a site-specific hysteroscopic endometrium injury performed during the ongoing in vitro fertilization (IVF) cycle, instead of injuries received during prior cycles, significantly improves clinical outcomes in patients with repeated implantation failure.

Supplementation with human menopausal gonadotropin in the gonadotropin-releasing hormone antagonist cycles of women with high AMH: Pregnancy outcomes and serial hormone levels.

OBJECTIVE: To evaluate the value of using both HMG and recombinant FSH (r-FSH) in the GnRH antagonist protocol for women with high AMH. MATERIALS AND METHODS: This retrospective, single-center cohort study was conducted from January 2013 to December 2018. Of 277 GnRH antagonist IVF/ICSI cycles in women with anti-Mullerian hormone (AMH) ≥5 µg/L, 170 cycles receiving the combination of r-FSH and HMG (77 with HMG added at the beginning of the GnRH antagonist cycle and 93 with HMG added after GnRH antagonist administration) and 107 cycles receiving r-FSH alone were analyzed. The dynamic hormone profiles and embryonic and clinical outcomes of the patients were evaluated. RESULTS: We observed significantly lower serum LH levels in the r-FSH + HMG groups during ovarian stimulation. The serum estradiol and progesterone levels were lower in the r-FSH + HMG groups on the trigger day. Nevertheless, there were no significant differences with respect to the number of oocytes retrieved, maturation, fertilization, blastocyst formation rate or ovarian hyperstimulation syndrome (OHSS). The implantation and live birth rates were increased in the r-FSH + HMG groups compared with the r-FSH alone group, with no statistical significance. CONCLUSIONS: HMG for LH supplementation in the GnRH antagonist protocol for patients with high AMH is not significantly superior to r-FSH alone in terms of ovarian response and pregnancy outcome. Nevertheless, HMG supplementation might be appropriate for women with an initially inadequate response to r-FSH or intracycle LH deficiency.

Genetic analysis of a Taiwanese family identifies a DMRT3-OAS3 interaction that is involved in human sexual differentiation through the regulation of ESR1 expression.

OBJECTIVE: To identify the genetic etiology of recurrent disorders of sex development (DSDs) in a Taiwanese family with 46,XY sex reversal and hypospadias. DESIGN: Genetic and functional studies. SETTING: Academic hospital. PATIENT(S): A three-generation family consisting of 22 members, with eight cases of 46,XY DSD, of whom four have 46,XY male-to-female sex reversal and four are 46,XY males with hypospadias. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Results of exome sequencing and in vitro protein and RNA analyses. RESULT(S): All patients with DSDs were found to carry heterozygous missense mutations in the doublesex and mab-3-related transcription factor 3 (DMRT3; rs187176004, c.A815C, p.K272T) and 2',5'-oligoadenylate synthetase 3 (OAS3; rs16942374, c.G2606A, p.R869H) genes. The DMRT3 mutation increased estrogen receptor 1 (ESR1) expression. Upon binding with the OAS3-RNase L complex, wild-type DMRT3 promoted degradation of ESR1 mRNA. However, the DMRT3A815C-OAS3G2606A complex interacted less strongly with ESR1 mRNA and RNase L, ultimately preventing ESR1 mRNA degradation. The interactions between DMRT3, OAS3, and RNase L were confirmed in the patients' testis. CONCLUSION(S): Our results indicate that DMRT3 and OAS3 are involved in human DSDs by controlling ESR1 expression.

Synergistic effects of pazopanib and hyperthermia against uterine leiomyosarcoma growth mediated by downregulation of histone acetyltransferase 1.

Pazopanib-a multitargeted tyrosine kinase inhibitor with prominent antiangiogenic effects-has shown promise in the treatment of soft-tissue sarcomas. Hyperthermia has been also applied as an adjunctive treatment to chemotherapy for these malignancies. Here, we show that pazopanib and hyperthermia act synergistically in inhibiting uterine leiomyosarcoma (LMS) cell growth. Compared with either treatment alone, the combination of pazopanib and hyperthermia exerted the highest antitumor activity in a xenograft model. Mechanistically, we found that combined treatment with pazopanib and hyperthermia inhibited histone acetyltransferase 1 (HAT1) expression in LMS cells. The Clock element on the HAT1 promoter was critical for pazopanib- and hyperthermia-induced HAT1 downregulation. Inhibition of HAT1-either by pazopanib and hyperthermia or through HAT1 silencing-was mediated by suppression of Clock. Accordingly, Clock protein reconstitution rescued both HAT1 levels and HAT1-mediated histone acetylation. Immunohistochemistry revealed a higher expression of HAT1 in uterine LMS than in leiomyomas (p = 0.007), with high HAT1 expression levels being associated with poor clinical outcomes (p = 0.007). We conclude that pazopanib and hyperthermia exert synergistic effects against LMS growth by inhibiting HAT1. Further preclinical studies on HAT1 as a potential drug target in uterine LMS are warranted, especially in combination with hyperthermia. KEY MESSAGES: Pazopanib and hyperthermia inhibit the growth of leiomyosarcoma. Their combined use inhibits HAT1 expression in leiomyosarcoma cells. The promoter Clock element is required for HAT1 downregulation. HAT1 expression is higher in leiomyosarcoma than in leiomyomas. An increased HAT1 expression is associated with poor clinical outcomes.

Gonadotropin-stimulated epidermal growth factor receptor expression in human ovarian surface epithelial cells: involvement of cyclic AMP-dependent exchange protein activated by cAMP pathway.

In addition to their critical roles in folliculogenesis and ovarian granulosa cell steroidogenesis, gonadotropins have been implicated as potential risk factors in ovarian epithelial carcinomas, most of which are derived from ovarian surface epithelium (OSE). However, the molecular mechanism underlying the effects of FSH and LH in OSE and its neoplastic counterpart is not well understood. We previously demonstrated that gonadotropins promote the growth of OSE cells by regulating the levels of epidermal growth factor receptor (EGFR) via the activation of ERK1/2 and PI3K pathways in immortalized human OSE (IOSE) cells. In this study, we investigated whether cAMP and its novel binding target, named exchange protein activated by cAMP (Epac), are involved in the gonadotropin-induced EGFR expression in OSE cells. Gonadotropins elevated intracellular cAMP levels in both IOSE and granulosa cells, and this increase was attenuated by SQ22536, an inhibitor of adenylyl cyclase (AC). The activation of the ERK1/2 and Akt pathways as well as the expression of EGFR was stimulated by reagents that elevate intracellular cAMP levels, via cAMP analog 8-bromo-cAMP and AC activator forskolin. A similar increase was observed when the cells were treated with a novel cAMP analog, 8-(4-chlorophenylthio)-2'-O-methyl adenosine-3',5'-cyclic monophosphate (8-CPT-2ME-cAMP), which activates Epac specifically but not PKA. Moreover, the gonadotropin-induced EGFR expression and ERK1/2 and Akt activation were abolished by overexpression of dominant negative Epac. Taken together, these results indicate that the AC/cAMP/Epac signaling pathway may mediate the up-regulation of EGFR by gonadotropins via ERK1/2 and Akt activation.

GnRH signaling in intrauterine tissues.

Type I GnRH (GnRH-I, GNRH1) and type II GnRH (GnRH-II, GNRH2), each encoded by separate genes, have been identified in humans. The tissue distribution and functional regulation of GnRH-I and GnRH-II clearly differ despite their comparable cDNA and genomic structures. These hormones exert their effects by binding to cell surface transmembrane G protein coupled receptors and stimulating the Gq/11 subfamily of G proteins. The hypothalamus and pituitary are the main origin and target sites of GnRH, but numerous studies have demonstrated that extra-hypothalamic GnRH and extra-pituitary GnRH receptors exist in different reproductive tissues such as the ovary, endometrium, placenta, and endometrial cancer cells. In addition to endocrine regulation, GnRH is also known to act in an autocrine and paracrine manner to suppress cell proliferation and activate apoptosis in the endometrium and endometrial cancer cells through several mechanisms. Both GnRH-I and GnRH-II exhibit regulatory roles in tissue remodelling during embryo implantation and placentation, which suggests that these hormones may have important roles in embryo implantation and early pregnancy. The presence of varied GnRH and GnRH receptor systems demonstrate their different roles in distinct tissues using dissimilar mechanisms. These may result in the generation of new GnRH analogues used for several hormone-related diseases.

Obstetric and perinatal outcomes of pregnancy in patients with repeated implantation failure.

OBJECTIVE: Despite the great advance of assisted reproductive technology (ART) in recent decades, many IVF patients failed to achieve a pregnancy even after multiple IVF-ET attempts. These patients are considered to have repeated implantation failure (RIF). While exhausting efforts have been devoted to the improvement of pregnancy rate in RIF patients, it is not clear whether RIF patients have aberrant obstetric or perinatal outcomes after they eventually achieved a pregnancy. MATERIALS AND METHODS: Taking advantage of a relatively large database of IVF-ET cycles at the Chang Gung Memorial Hospital, we compared obstetric and perinatal outcomes of RIF patients who have a successful pregnancy after IVF-ET treatment(s) to those of control IVF-ET patients. RESULTS: Because multiple pregnancies are associated with a high risk of obstetric complications, we restricted the analysis to patients who had singleton pregnancies. Analysis of a total of 596 control and 46 RIF cases showed the rates of almost all obstetric and perinatal outcomes investigated are not different between the two groups. However, the rate of placental abruption in the RIF group (4.35%) appeared to be significantly higher than that of controls (0.50%; OR = 8.99). This difference is still statistically significant after adjustment with the age (adjusted OR = 8.2). CONCLUSION: While the rates of a spectrum of obstetric and perinatal outcomes are normal in RIF patients, these patients could have an enhanced risk of placental abruption. However, investigations with a large sample size are needed to substantiate this inference.

Application of non-invasive detection of peripheral vascular dysfunction in ovarian hyperstimulation syndrome (OHSS): A pilot study of clinical relevance.

OBJECTIVE: The current study tested the hypothesis that vascular endothelial function, as reflected by the reactive hyperemia index (RHI), and biochemical factors, including VEGF, TNFα, CRP, inhibin A, and inhibin B, were involved in the pathogenesis of ovarian hyperstimulation syndrome (OHSS). MATERIALS AND METHODS: This study was conducted between June 2010 and June 2012, enrolling 15 patients with OHSS and 6 healthy control subjects <45 years of age. Detailed clinical parameters were reviewed, including serum VEGF, TNFα, CRP, inhibin A, inhibin B, and hematocrit. RHI assessed by novel automatic peripheral arterial tonography was used to evaluate the vascular endothelial function. RESULTS: Twenty-one subjects were evaluated. There was no significant difference between patients with OHSS and control subjects with respect to VEGF, TNFα, CRP, inhibin A and inhibin B. The RHI was not significantly different between patients with OHSS and control subjects (mean, 1.8 ± 0.4 vs. 1.7 ± 0.2). The hematocrit was significantly different between patients with OHSS and control subjects. CONCLUSIONS: Our preliminary data did not reveal direct evidence of vascular endothelial dysfunction in patients with OHSS. To identify whether RHI could reflect vascular endothelial dysfunction in patients with OHSS, more cases with different severities of OHSS should be recruited in the future study.

Utilization of HEPES for Enhancing Protein Transfection into Mammalian Cells.

The delivery of active proteins into cells (protein transfection) for biological purposes offers considerable potential for clinical applications. Herein we demonstrate that, with a readily available, inexpensive organic agent, the 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) method can be used for simple and efficient protein transfection. By mixing proteins with a pure HEPES solution before they are applied to live cells, proteins with various molecular weights (including antibodies, recombinant proteins, and peptides) were successfully delivered into the cytoplasm of different cell types. The protein transfection efficiency of the HEPES method was not inferior to that of commercially available systems that are both more expensive and time consuming. Studies using endocytotic inhibitors and endosomal markers have revealed that cells internalize HEPES-protein mixtures through endocytosis. Results that HEPES-protein mixtures exhibited a low diffusion coefficient suggest that HEPES might neutralize the charges of proteins and, thus, facilitate their cellular internalization. Upon internalization, the cytosolic antibodies caused the degradation of targeted proteins in TRIM21-expressing cells. In summary, the HEPES method is efficient for protein transfection and has potential for myriad clinical applications.