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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.

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.

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. Keywords: RIF, Repeated implantation failure, Placental abruption, IVF, Embryo transfer

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

Objective: We have recently reported that stress-induced phosphoprotein 1 (STIP1) is over-expressed in endometriosis/adenomyosis tissues. STIP1 may also be involved in immune regulation, thus we attempted to study the association between STIP1 single nucleotide polymorphisms (SNPs) and endometriosis/adenomyosis. Materials and methods: Five STIP1 SNPs (rs7941773, rs2845597, rs4980524, rs2282490, and rs2236647) were selected for genotyping with matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) in 286 patients with endometriosis/adenomyosis and 288 healthy postmenopausal controls. In vitro studies included luciferase promoter reporter assays and western blot analysis for STIP1 and MMP9 proteins. Results: The frequency of the G allele at rs4980524 was significantly higher in patients with endometriosis/adenomyosis than in control women. The promoter reporter with rs4980524 GG genotype significantly increased luciferase activity than that with TT genotype in endometrial cancer RL95-2 cells, and the primary endometrial stromal cells carrying rs4980524 GG genotype expressed higher protein levels of STIP1 and MMP9 than those carrying the TT one. Conclusion: The G/G allele of STIP1 SNP rs4980524 is associated with the increased expression of STIP1 and MMP9 in endometriosis. Further validation in independent cohorts of endometriosis patients may prove its usefulness as a genetic risk maker for endometriosis/adenomyosis. Keywords: Endometriosis, SNP, STIP1

Objective Although major advances have greatly improved the outcomes of assisted reproductive technology in the last two cascades, there remains significant difficulty in achieving pregnancy for many patients even after repeated attempts of IVF. Interestingly, recent studies have shown that transcutaneous electrical acupoint stimulation (TEAS) can improve the reproductive outcomes of select IVF patients. To determine the utility of TEAS in improving IVF outcomes in patients with a history of implantation failure, we conducted a retrospective study of clinical outcomes of women, who had a prior history of unsuccessful pregnancy outcome after IVF-embryo transfer (IVF-ET), following TEAS treatment. Materials and methods A total of 25 patients, who had failed to conceive after multiple IVF cycles in which good embryos were transferred, received noninvasive low frequency TEAS treatment prior to and during an IVF-ET cycle. The clinical outcomes, including biochemical pregnancy rate, clinical pregnancy rate and implantation rate, were compared to those of prior cycles which received only standard IVF treatment. Results Analysis of reproductive outcomes showed that implantation rate and clinical pregnancy rate increased significantly in IVF cycles that included the TEAS treatment when compared to prior cycles that received only the standard IVF treatment in this cohort of patients. Conclusions This surprising finding indicated that TEAS treatment is a promising technique to improve reproductive outcomes in difficult cases of IVF-ET. Because TEAS treatment is noninvasive and has high reproducibility, and can be applied with limited training, further refinement of this procedure would not only substantiate the beneficial effects of TEAS, but also allow the technique to be more effective and reproducible.

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 2ʹ,5ʹ-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.

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.

Endometrial cancers expressing estrogen and progesterone receptors respond to hormonal therapy. The disappearance of steroid hormone receptor expression is common in patients with recurrent disease, ultimately hampering the clinical utility of hormonal therapy. Here, we demonstrate for the first time that nucleophosmin (NPM1/B23) suppression can restore the expression of estrogen receptor α (ESR1/ERα) in endometrial cancer cells. Mechanistically, B23 and activator protein-2γ (TFAP2C/AP2γ) form a complex that acts as a transcriptional repressor of ERα. Our results indicate that B23 or AP2γ knockdown can restore ERα levels and activate ERα-regulated genes (e.g., cathepsin D, EBAG9, and TFF1/pS2). Moreover, AP2γ knockdown in a xenograft model sensitizes endometrial cancer cells to megesterol acetate through the upregulation of ERα expression. An increased immunohistochemical expression of AP2γ is an adverse prognostic factor in endometrial cancer. In summary, B23 and AP2γ may act in combination to suppress ERα expression in endometrial cancer cells. The inhibition of B23 or AP2γ can restore ERα expression and can serve as a potential strategy for sensitizing hormone-refractory endometrial cancers to endocrine therapy.

Stress-induced phosphoprotein-1 (STIP1), an adaptor protein that coordinates the functions of HSP70 and HSP90 in protein folding, has been implicated in the development of human gynecologic malignancies. This case-control study investigates STIP1 serum levels and tissue expression in relation to endometriosis/adenomyosis in Taiwanese population. Female patients with surgically confirmed endometriosis/adenomyosis were compared with women free of endometriosis/adenomyosis. Serum STIP1 levels were measured using an enzyme-linked immunosorbent assay and surgical tissues were analyzed by immunohistochemistry. Both epithelial and stromal cells in surgical tissues of endometriosis and adenomyosis expressed STIP1 and MMP-9. Notably, MMP-9 expression was significantly decreased when STIP1 expression was knocked-down. In vitro experiments revealed that STIP1 was capable of binding to the MMP-9 promoter and enhanced its transcriptional expression. The preoperative serum STIP1 levels of patients with endometriosis/adenomyosis were significantly higher than those of the controls. In brief, our data suggest an association between STIP1 levels and endometriosis/adenomyosis.

Objective Benign mature teratoma during pregnancy is common, mostly discovered incidentally by antenatal sonography. However, repeated pregnancy coincident with ovarian mature teratoma is rarely reported. The cases of teratoma with rapid growing characteristics are even more unique. Case report A 17-year-old woman was pregnant at 6 weeks of gestation with a left ovarian teratoma. She underwent artificial abortion followed by surgical removal of the teratoma. However, eleven years after the surgery, a right ovarian teratoma was found incidentally by antepartum sonography at 21 weeks of gestation. The right ovarian teratoma developed uneventfully, with rapid growth during pregnancy. Abdominal delivery at term was accomplished without any complication. Conclusion Younger patients and patients with bilateral or large size dermoid cysts should be followed up closely. Further studies are needed for better understanding of its natural clinical course and the mechanism of progression. The treatment options should be made individually, weighing the risks of torsion, rupture, or obstruction of labor versus the potential for unnecessary surgical risk to mother and fetus.

Objectives: To analyze the relationship between in vitro fertilization (IVF) pregnancy rate and basal serum hormone levels before patients begin an IVF course. Materials and methods: In this retrospective study, we analyzed patients with anti-Müllerian hormone (AMH) data and IVF data from January 2009 to October 2012. Pregnancy rates were calculated by AMH and follicle stimulating hormone quartiles and analyzed using the independent samples t test. Furthermore, patients were divided into three groups by age. The Chi-square test was used to assess the association between the parameters and IVF pregnancy rates. Results: From the 910 IVF treatment courses, 377 (41.4%) clinical pregnancies resulted. The pregnant and nonpregnant groups differed significantly in age and FSH and AMH levels. The pregnancy rate was 53.3% for patients aged 38 years. The pregnancy rate was 53.4% for patients with FSH levels 8.9 mIU/mL. The pregnancy rate was 56.8% for patients with AMH levels >4.0 ng/mL and 20.0% for patients with AMH levels

Adenomyosis was found to have negative impacts on embryo implantation. Leukemia inhibitory factor (LIF), proposed to be a molecular marker for endometrial receptivity, works through the LIF receptor (LIFR) on both the embryo and the endometrium. We aimed to evaluate the endometrial expression of LIF and LIFR and its subsequent signaling in patients with adenomyosis during the window of implantation (WOI). Endometrium was obtained during the WOI from patients with adenomyosis (age

Objective To study the impact of integrin-linked kinase (ILK) in endometrial stromal cells (ESCs) during decidualization. Design Laboratory study with the use of human endometrium. Setting University hospital. Patient(s) Fertile reproductive-age women who had not received hormonal treatment for 3 months before tissue collection. Intervention(s) Endometrium tissue collection, in vitro decidualization of isolated ESCs, and small interfering (si) RNA transfection. Main Outcome Measure(s) Immunohistochemistry, ELISA, Western blot analysis, methylthiazolyl tetrazolium assay, and immunofluorescence staining. Result(s) In vivo expression of ILK is significantly increased in distended-fusiform stromal cells of late secretory endometrium and in cobblestone-shaped decidual cells of early pregnancy. During in vitro decidualization for up to 8 days, confluent cultures of isolated ESCs consistently displayed increased ILK expression and morphologic transformation from fibroblast-like to polygonal cells. Subsequent ILK knockdown by siRNA transfection reversed this transformation, accompanied by decreased phosphorylation of glycogen synthase kinase (GSK) 3β and decreased viable cell numbers. Immunofluorescence staining of the decidualized ESCs demonstrated linkage of increased levels of ILK at the tips of the fan-shaped organization of actin stress fibers located in the submembranous area, which expanded the decidual cells into a typical polygonal appearance. Knock-down of ILK abrogated the polymerization and organization of actin fibers, which reverted the cells to their undecidualized morphology. Conclusion(s) During human endometrial decidualization, ILK is essential for morphologic transformation of ESCs through organization of the actin cytoskeleton; it may also function through subsequent GSK3β signaling, which requires further studies.

Unopposed estrogen exposure is an important factor in the tumorigenesis of endometrial cancer. Nucleophosmin/B23 (NPM/B23), a phosphoprotein that has pleiotropic functions in cells, plays an important role in various cancers. However, the regulatory role of NPM/B23 in estrogen signaling in endometrial cancer has not been explored. Here, we report that NPM/B23 was required for estrogen-induced endometrial proliferation, and the increase in NPM/B23 was estrogen receptor α-dependent. Furthermore, estrogen increased NPM/B23 protein levels by repressing its ubiquitination and subsequently stabilizing the protein. The overexpression of the alternate reading frame (ARF) suppressed the estrogen-induced increase in the NPM/B23 protein levels, indicating that ARF inhibited the observed estrogen-mediated NPM/B23 stabilization. Our results suggest that one of the effects of estrogen on endometrial proliferation is the suppression of the NPM/B23-ARF interaction and the subsequent increase in NPM/B23 protein levels. This novel characterization of NPM/B23 in estrogen-mediated cell proliferation may extend our understanding of the tumorigenesis of steroid hormone-related cancers.

To assess the role of diagnostic hysteroscopy in pregnancy outcome in patients starting the first in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI).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.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 thickness8 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 %).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.

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 GADD45a 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 GADD45a 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 GADD45a signaling. GnRH antagonist-mediated cell growth inhibition and apoptosis were also abolished by the knockdown of the endogenous GnRH-IR or GADD45a 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 GADD45a signaling. These results indicate that ERK1/2, JNK and GADD45a 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.

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.

Objective: To evaluate the pregnancy courses and obstetric outcomes in patients conceived after conservative treatment of endometrial cancer. Materials and Methods: Case series and systemic review of pregnancy women after fertility-sparing treatment of endometrial cancer. Patients with early stage endometrial cancer were identified through Tumor Registry in Chang Gung Memorial Hospital between 1990 and 2005 and MEDLINE search. Diagnosed cases were managed by fertility-sparing therapies. Pregnancies followed by assisted reproductive technology and spontaneous or ovulation with intrauterine insemination were designated as Group 1 and Group 2, respectively. Results: Five livebirths in three patients with two sets of twin pregnancy were delivered. Adding 47 women in the MEDLINE search literature, there were 65 deliveries with 77 livebirths. Groups 1 and 2 had 15 and 50 deliveries, respectively. Group 1 had 23 livebirths including four sets of twins and two sets of triplets, whereas 54 livebirths consisted of two sets of twins and one set of triplets were noted in Group 2 (p = 0.003). Seven preterm deliveries were noted in Group 1 and three in Group 2 (p = 0.001). Cesarean rate was 93.3% versus 22.0% (p

Objective To investigate the eutopic endometrial interleukin-18 (IL-18) system including interleukin-18 (IL-18), IL-18 receptor (IL-18R), and IL-18 binding protein (IL-18BP), mRNA, and protein expression in patients with adenomyosis. Design A clinical and molecular study. Setting Clinical and academic research setting in a university medical center. Patient(s) Twenty-eight samples of human eutopic endometria were obtained from surgical specimens of normal cycling women undergoing hysterectomy for uterine adenomyosis (n = 19); the control group (n = 9) was women undergoing hysterectomy for benign reason including uterine fibroids. Intervention(s) Quantitative competitive polymerase chain reaction (QC PCR) and immunohistochemistry studies were performed. Main Outcome Measurement(s) The differences of the IL-18 system mRNA and the ratio of IL-18BP to IL-18 in the eutopic endometrium of uterine adenomyosis and control group were analyzed. Result(s) IL-18 system mRNA and protein expression was demonstrated in the eutopic endometrium of both adenomyosis and control women. Quantitative competitive PCR demonstrated that endometrial IL-18R mRNA and the ratio of IL-18BP to IL-18 were significantly increased in adenomyosis patients in comparison to the control group. Pearson's correlation showed a significant correlation between IL-18 and IL-18R in the eutopic endometrium of women with uterine adenomyosis, but not the control group. Conclusion(s) The expression of the eutopic endometrial IL-18 system and the ratio of antagonist to agonist at the level of the endometrial-myometrial interface (EMI) may possibly be responsible for the pathologic process of adenomyosis.

Objective to understand postpartum weight retention among women of different body sizes at pre-pregnancy, and to explore factors influencing this weight retention. Design a comparative, descriptive study. Pre-pregnancy body mass indices (BMIs) were determined from hospital records. Dependent variables were measured by self-report questionnaires. Setting a 3,900-bed medical centre in northern Taiwan that delivers approximately 4,000 children each year. Participants a systematic sample of 810 women who had delivered children at a medical centre. In total, 602 valid questionnaires were collected. Findings the average pre-pregnancy and six month postpartum BMIs were 21.5 [standard deviation (S.D.)=3.32] and 22.48 (S.D.=3.39), respectively. Overweight (OW) and obesity (OB) increased from 18.27% before pregnancy to 27.57% at six months postpartum. Gestational weight gain (GWG) was 14.36, 14.37, 13.07 and 11.15kg among women in the underweight (UW), normal weight (NW), OW and OB groups, respectively; and six month postpartum weight retention was 3.32, 2.57, 1.67 and −0.29kg. GWG was the most significant predictor of postpartum weight change across groups. Key conclusions women with different body sizes at pre-pregnancy have common and specific risks for postpartum weight retention. Implications for practice individualised interventions should be developed to manage postpartum weight for women of different body sizes. Interventions should establish criteria for GWG and provide strategies to improve body image and modify lifestyle. The overall goal should be to manage postpartum weight gain and to promote women's health.

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.

This study evaluated the relationship between pregnancy rate and semen cadmium concentration. This prospective and nonrandomized clinical study analyzed 341 male partners of infertile couples undergoing infertility evaluation and management. Semen samples were collected to analyze semen quality and cadmium concentrations. The main outcome was pregnancy during 60-day infertility treatment. Simple linear regression analysis revealed an association between semen cadmium concentration NS sperm count (r = −0.150, P = 0.0416) in nonsmoking subjects (n = 184). In both smokers and nonsmokers, semen cadmium concentrations were significantly higher in non-pregnant patients than in pregnant patients. In nonsmokers, Cox multi-variable fertility ratio analysis demonstrated an association between semen cadmium concentration and fertility (fertility ratio of log semen cadmium = 0.24; 95% confidence intervals (CI) = 0.12–0.47, P

Summary: Objective: Whole genome amplification (WGA) is a crucial procedure for genomic DNA analysis from limited sources, such as in forensic analysis, embryo biopsy for preimplantation genetic diagnosis, or needle aspiration biopsies. Several strategies for WGA have been developed for either genotyping or microarray-based comparative genome hybridization (array-CGH) during the past decade. Nevertheless, there were few studies in which various WGA methods had been performed side-by-side and results evaluated with multiple methods. Materials and Methods: Ease of performance, qualitative accuracy, and quantitative fidelity of different WGA methods, such as degenerate oligonucleotide-primed polymerase chain reaction (DOP-PCR), ligation-mediated PCR (LM-PCR) and strand displacement amplification (SDA), were compared in amplifyinggenomic DNA derived from karyotype-confirmed amniocytes and the cancer cell line SAOS2. Results: Using analysis with microsatellite markers, single nucleotide polymorphism markers, and array-CGH, our results suggested that: (1) genomic DNA amplified from DOP-PCR resulted in false positive and negative results by analysis with array-CGH; (2) SDA is the easiest performance method among the three WGA methods; and (3) amplified DNA products generated by LM-PCR best reflect the original genomic DNA. Conclusion: The amplified DNA products generated by LM-PCR best reflect the original genomic DNA. Key Words: array-CGH, DOP-PCR, LM-PCR, SDA, whole genome amplification

// Chia-Lung Tsai 1, * , Angel Chao 1, 2, * , Shih-Ming Jung 3 , Chi-Neu Tsai 4 , Chiao-Yun Lin 2 , Shun-Hua Chen 2, 5 , Shih-Che Sue 6 , Tzu-Hao Wang 1, 2, 7 , Hsin-Shih Wang 2 , Chyong-Huey Lai 2 1 Genomic Medicine Research Core Laboratory, Chang Gung Memorial Hospital, Taoyuan, Taiwan 2 Department of Obstetrics and Gynecology, Chang Gung Memorial Hospital and Chang Gung University, Taoyuan, Taiwan 3 Department of Pathology, Chang Gung Memorial Hospital and Chang Gung University, Taoyuan, Taiwan 4 Graduate Institute of Clinical Medical Sciences, Chang Gung University, Taoyuan, Taiwan 5 Graduate Institute of Biomedical Science, School of Medicine, Chang Gung University, Taoyuan, Taiwan 6 Department of Life Sciences, Institute of Bioinformatics and Structural Biology, National Tsing Hua University, Taiwan 7 School of Traditional Chinese Medicine, College of Medicine, Chang Gung University, Taoyuan, Taiwan * These authors have contributed equally to this work Correspondence to: Tzu-Hao Wang, email: knoxtn@cgmh.org.tw Keywords: JAK2, STIP1, cancer Received: March 08, 2016 Accepted: June 17, 2016 Published: July 8, 2016 ABSTRACT Overexpression of stress-induced phosphoprotein 1 (STIP1) − a co-chaperone of heat shock protein (HSP) 70/HSP90 – and activation of the JAK2-STAT3 pathway occur in several tumors. Combined treatment with a HSP90 inhibitor and a JAK2 inhibitor exert synergistic anti-cancer effects. Here, we show that STIP1 stabilizes JAK2 protein in ovarian and endometrial cancer cells. Knock-down of endogenous STIP1 decreased JAK2 and phospho-STAT3 protein levels. The N-terminal fragment of STIP1 interacts with the N-terminus of JAK2, whereas the C-terminal DP2 domain of STIP1 mediates the interaction with HSP90 and STAT3. A peptide fragment in the DP2 domain of STIP1 (peptide 520) disrupted the interaction between STIP1 and HSP90 and induced cell death through JAK2 suppression. In an animal model, treatment with peptide 520 inhibited tumor growth. In summary, STIP1 modulates the function of the HSP90-JAK2-STAT3 complex. Peptide 520 may have therapeutic potential in the treatment of JAK2-overexpressing tumors.

Reduced scleral collagen accumulation has been found in the development of myopia. Single nucleotide polymorphisms (SNPs) at the type I collagen alpha-1 gene (COL1A1) may cause different susceptibilities to myopia. We conducted a case-control study to systematically examine COL1A1 as a candidate gene for high myopia. A case was defined as spherical refractionor=-6 D and controlor=-1.5 D. The study comprised 471 cases and 623 controls, and ten tagging SNPs were genotyped. None of the SNPs reached the significant level of 0.05. Subset analysis on cases with a strong family history did not demonstrate significant results. We could not find an interaction between gene and near work. Exploratory analyses by changing the cutoff values to re-define cases and controls did not improve the results. Haplotype analysis did not yield significant association with myopia. This study failed to demonstrate COL1A1 as a significant risk factor for high myopia.

Salmonella enterica serovar Choleraesuis (S. Choleraesuis), a highly invasive serovar among non-typhoidal Salmonella, usually causes sepsis or extra-intestinal focal infections in humans. S. Choleraesuis infections have now become particularly difficult to treat because of the emergence of resistance to multiple antimicrobial agents. The 4.7 Mb genome sequence of a multidrug-resistant S. Choleraesuis strain SC-B67 was determined. Genome wide comparison of three sequenced Salmonella genomes revealed that more deletion events occurred in S. Choleraesuis SC-B67 and S.Typhi CT18 relative to S. Typhimurium LT2. S. Choleraesuis has 151 pseudogenes, which, among the three Salmonella genomes, include the highest percentage of pseudogenes arising from the genes involved in bacterial chemotaxis signal-transduction pathways. Mutations in these genes may increase smooth swimming of the bacteria, potentially allowing more effective interactions with and invasion of host cells to occur. A key regulatory gene of TetR/AcrR family, acrR, was inactivated through the introduction of an internal stop codon resulting in overexpression of AcrAB that appears to be associated with ciprofloxacin resistance. While lateral gene transfer providing basic functions to allow niche expansion in the host and environment is maintained during the evolution of different serovars of Salmonella, genes providing little overall selective benefit may be lost rapidly. Our findings suggest that the formation of pseudogenes may provide a simple evolutionary pathway that complements gene acquisition to enhance virulence and antimicrobial resistance in S. Choleraesuis.

Objective Conventional chromosomal studies or fluorescent in situ hybridization takes days to diagnose fetal aneuploidies during amniocentesis. Here, we evaluated the value of mass spectrometry-based clinical proteomics analysis on amniotic fluid supernatant (AFS) as a rapid detection of fetal aneuploidies. Methods Proteomics profiles generated by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) after fractionating samples with functionalized magnetic beads were used for differentiating 60 normal karyotypic from 20 aneuploid AFS. After the discriminating models were generated using genetic algorithm, we evaluated the clinical efficacy of the models in detecting aneuploidies in two batches (each n = 30) of AFS prior to the release of chromosomal diagnoses. Results Within hours, the two-step proteomics analysis of AFS with the C18 model, followed by the weak cation exchange model, was able to detect aneuploid AFS at 3.3% disease prevalence rate with 100% sensitivity, 72 to 96% specificity, 11 to 50% positive predictive value, and 100% negative predictive value. Conclusion Clinical proteomics analysis of AFS using magnetic beads-based sample preparation and MALDI-TOF-MS can be used as a rapid detection for fetal aneuploidies. With perfect sensitivity and negative predictive value of the two-step proteomics method, it may be used for rapid detection of aneuploid AFS immediately after amniocentesis. Further large-scale examinations are apparently needed to verify the clinical value of this rapid detection. Copyright © 2005 John Wiley & Sons, Ltd.

The study was undertaken to test whether human telomerase reverse transcriptase (hTERT) transcripts in an individual blastomere could be used as an indicator for embryo development.Group A consisted of day 3 normal cleaving embryos at 4- to 8-cell stage, which were surplus and not allocated for uterine transfer. Group B consisted of arrested or fragmented embryos at the same stage, which were considered to be compromised. After blastomere dissociation, RNA purification, reverse transcription, and hTERT PCR amplification, the amplified product was separated by 3% gel electrophoresis.Eighty-six (90.5%) of the 95 intact blastomeres in group A and 78 (70.9%) of the 110 blastomeres in group B demonstrated hTERT mRNA expression. The difference was statistically significant (P0.05, chi-square). Eight (38.1%) of the 21 embryos in group A and 22 (62.9%) of the 35 embryos in group B had at least one blastomere that did not express hTERT mRNA under this procedure. The difference was not significant (P0.05, chi-square).General hTERT mRNA transcripts can be detected in most of the individual blastomeres but cannot be used as an indicator for early embryo development. Further investigations are necessary to elucidate its clinical application.

Brain-specific angiogenesis inhibitor 1 (BAI1)-associated protein 2-like 1 (BAIAP2L1), also known as insulin receptor tyrosine kinase substrate (IRTKS), is involved in plasma membrane protrusion and actin formation during cell morphogenesis and migration. BAIAP2L1 is recently reported to promote cell proliferation through activation of the EGFR-ERK pathway in hepatocellular carcinoma. In this study, we report the first comprehensive study of BAIAP2L1 upregulation in human ovarian cancer. Upregulation of BAIAP2L1 in ovarian tumors was first found during RNA screening and confirmed by immunohistochemical studies on ovarian cancers and other cancer types. Significant upregulation of BAIAP2L1 in ovarian cancer was validated by analyzing multiple independent cohorts in publicly available data sets. Furthermore, BAIAP2L1 protein expression in metastatic lesions was higher than the corresponding primary tumors. Functional assays in ovarian cancer cells revealed that BAIAP2L1 is involved in promoting cell proliferation and avoiding apoptosis. In conclusion, results of this study not only indicate that BAIAP2L1 can be used as a biomarker for human ovarian cancer but also reveal its role in cancer biology. Further elucidation of the role of BAIAP2L1 in context of the insulin receptor signaling pathways of cancer cells is warranted for developing cancer therapeutics by targeting cancer-specific metabolism.

Six hundred thirty-seven Taiwanese Chinese women including 300 patients with endometriosis and 337 controls without endometriosis were enrolled to investigate the association between nonsynonymous single nucleotide polymorphism of the FSH receptor gene and the risk of endometriosis. For the A/G polymorphism of FSH receptor gene (Asn680Ser), a univariate analysis for women with endometriosis demonstrated that both the GG genotype (680Ser/Ser) and GA genotype (680Ser/Asn) were associated with a significantly lower risk of endometriosis.

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.

Preclinical studies of amniotic fluid-derived cell therapy have been successful in the research of neurodegenerative diseases, peripheral nerve injury, spinal cord injury, and brain ischemia. Transplantation of human amniotic fluid stem cells (AFSCs) into rat brain ventricles has shown improvement in symptoms of Parkinson's disease and also highlighted the minimal immune rejection risk of AFSCs, even between species. Although AFSCs appeared to be a promising resource for cell-based regenerative therapy, AFSCs contain a heterogeneous pool of distinct cell types, rendering each preparation of AFSCs unique. Identification of predictive markers for neuron-prone AFSCs is necessary before such stem cell-based therapeutics can become a reality. In an attempt to identify markers of AFSCs to predict their ability for neurogenesis, we performed a two-phase study. In the discovery phase of 23 AFSCs, we tested ZNF521/Zfp521, OCT6, SOX1, SOX2, SOX3, and SOX9 as predictive markers of AFSCs for neural differentiation. In the validation phase, the efficacy of these predictive markers was tested in independent sets of 18 AFSCs and 14 dental pulp stem cells (DPSCs). We found that high expression of SOX9 in AFSCs is associated with good neurogenetic ability, and these positive correlations were confirmed in independent sets of AFSCs and DPSCs. Furthermore, knockdown of SOX9 in AFSCs inhibited their neuronal differentiation. In conclusion, the discovery of SOX9 as a predictive marker for neuron-prone AFSCs could expedite the selection of useful clones for regenerative medicine, in particular, in neurological diseases and injuries.

The purpose of this study was to investigate the possible regulatory roles of insulin-like growth factors (IGF) and IGF binding protein-1 (IGFBP-1) on postmenopausal endometrium in women undergoing hormone replacement therapy (HRT). Endometrial tissues and blood samples were collected from 25 postmenopausal women with abnormal uterine bleeding before and after HRT. As a control, blood and endometrial samples were also obtained at mid-luteal phase (days 18-23 of the menstrual cycle) from 10 women with benign uterine leiomyoma during surgical intervention. Expression of mRNA for IGF-I, IGF-II, type 1-IGF receptor, IGFBP-1, oestrogen receptor (ER) and progesterone receptor (PR) in the endometrium was explored by a semiquantitative reverse transcription-polymerase chain reaction (RT-PCR). Serum concentrations of IGFBP-1, oestradiol, progesterone, follicle stimulating hormone (FSH) and sex hormone-binding globulin (SHBG) were also determined. In the endometrium obtained from postmenopausal women with abnormal uterine bleeding (before HRT), expression of IGFBP-1 was undetectable, whereas PR expression was abundant. HRT significantly up-regulated expression of IGFBP-1 but down-regulated PR. Moderate expression of IGF-I, IGF-II, type 1-IGF receptor and ER was identified in the postmenopausal endometrium before HRT. HRT significantly down-regulated IGF-I, up-regulated IGF-II, but had no effects on expression of type 1-IGF receptor and ER. In conclusion, up-regulation of IGFBP-1 collaboratively with down-regulation of IGF-I may account for the protective effect of progesterone in HRT on the endometrium.

Granulosa-lutein cell growth differentiation factor-9 (GDF-9), a member of the transforming growth factor beta (TGF-beta) superfamily, is an oocyte-derived factor expressed throughout the development of the maturing follicle and is essential in early follicular development and fertility. Both GDF-9 messenger RNA and protein are detected in granulosa-lutein cells. A statistically significant correlation between GDF-9 expression and the number of dominant follicles on the day human chorionic gonadotropin is administered also is demonstrated. Human granulosa-lutein cell GDF-9 expression may play a role in folliculogenesis dependent on follicle-stimulating hormone in the in vitro fertilization cycle.

The antineoplastic agent paclitaxel (TaxolTM), a microtubule stabilizing agent, is known to arrest cells at the G2/M phase of the cell cycle and induce apoptosis. We and others have recently demonstrated that paclitaxel also activates the c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) signal transduction pathway in various human cell types, however, no clear role has been established for JNK/SAPK in paclitaxel-induced apoptosis. To further examine the role of JNK/SAPK signaling cascades in apoptosis resulting from microtubular dysfunction induced by paclitaxel, we have coexpressed dominant negative (dn) mutants of signaling proteins of the JNK/SAPK pathway (Ras, ASK1, Rac, JNKK, and JNK) in human ovarian cancer cells with a selectable marker to analyze the apoptotic characteristics of cells expressing dn vectors following exposure to paclitaxel. Expression of these dn signaling proteins had no effect on Bcl-2 phosphorylation, yet inhibited apoptotic changes induced by paclitaxel up to 16 h after treatment. Coexpression of these dn signaling proteins had no protective effect after 48 h of paclitaxel treatment. Our data indicate that: (i) activated JNK/SAPK acts upstream of membrane changes and caspase-3 activation in paclitaxel-initiated apoptotic pathways, independently of cell cycle stage, (ii) activated JNK/SAPK is not responsible for paclitaxel-induced phosphorylation of Bcl-2, and (iii) apoptosis resulting from microtubule damage may comprise multiple mechanisms, including a JNK/SAPK-dependent early phase and a JNK/SAPK-independent late phase.

The essential cellular functions associated with microtubules have led to a wide use of microtubule-interfering agents in cancer chemotherapy with promising results. Although the most well studied action of microtubule-interfering agents is an arrest of cells at the G2/M phase of the cell cycle, other effects may also exist. We have observed that paclitaxel (Taxol), docetaxel (Taxotere), vinblastine, vincristine, nocodazole, and colchicine activate the c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) signaling pathway in a variety of human cells. Activation of JNK/SAPK by microtubule-interfering agents is dose-dependent and time-dependent and requires interactions with microtubules. Functional activation of the JNKK/SEK1-JNK/SAPK-c-Jun cascade (where JNKK/SEK1 is JNK kinase/SAPK kinase) was demonstrated by activation of a 12-O-tetradecanoylphorbol-13-acetate response element (TRE) reporter construct in a c-Jun dependent fashion. Microtubule-interfering agents also activated both Ras and apoptosis signal-regulating kinase (ASK1) and coexpression of dominant negative Ras and dominant negative apoptosis signal-regulating kinase exerted individual and additive inhibition of JNK/SAPK activation by microtubule-interfering agents. These findings suggest that multiple signal transduction pathways are involved with cellular detection of microtubular disarray and subsequent activation of JNK/SAPK.

Purpose We studied the relationship between high myopia and the MMP3 and TIMP1 genes. Design Case-control study. Methods We enrolled 150 high myopic individuals (≤ −6 diopters) and 262 controls (≥ −1.5 diopters) initially, and another 216 cases and 474 controls were enrolled for genotyping promising polymorphisms for replication. Thirteen polymorphisms were genotyped in the initial data. Logistic regression was used to test for genetic effects. Subset analyses were performed according to the education level and family history. Results There was no significant association between any polymorphism and high myopia in the initial data. Among the highly educated subjects, the 5A/6A polymorphism at the MMP3 gene suggested a potential relationship with high myopia, and it was genotyped in the follow-up samples. However, this polymorphism failed to show any significant results in the overall subjects. Conclusions The two genes may not play a crucial role for high myopia in young Taiwanese men.

Objective To investigate circulating levels of insulin-like growth factor-binding protein 1 (IGFBP-1) and IGFBP-3 in the mother and the fetus in pregnancies complicated by pre-eclampsia, and the relationship between serum levels of IGFBPs and fetal birthweight. Design A prospective study over an 18 month period. Setting A tertiary care academic medical centre. Participants Sixty-six pregnant women with pre-eclampsia (35 cases of mild/moderate pre-eclampsia and 31 cases of severe pre-eclampsia) and 78 nonpra-eclamptic pregnant women of matched gestational weeks and maternal ages. Main outcome measures Serum concentrations of IGFBP-1 and IGFBP-3 at the time of delivery. Results In pre-eclampsia associated with intrauterine growth retardation (IUGR), maternal and cord serum IGFBP-1 levels at the time of delivery were elevated. By contrast, circulating IGFBP-3 levels in both the mother and the fetus were lower in pre-eclampsia with IUGR than in nonpre-eclamptic pregnancy. However, there was no difference in serum IGFBP-1 and IGFBP-3 levels between pre-eclampsia without IUGR and nonpre-eclamptic pregnancy in both the mother and the fetus. Conclusions In pre-eclampsia, elevated concentrations of circulating IGFBP-1 and decreased serum IGFBP-3 levels were observed in both the mother and the fetus. However, these changes may simply reflect low birthweight in pre-eclampsia.

Co-culturing embryos on helper cells can mimic the in-vivo environment, thereby enhancing embryo development in vitro. Insulin-like growth factors (IGF) and their binding proteins (IGFBP) also enhance embryo development. To investigate the kinds of IGFBP produced by various cell monolayers and the effects of IGFBP-3 on mouse embryo co-culture systems, 2-cell ICR mouse embryos were cultured in either human tubal fluid medium alone or in the presence of Vero cells, human oviductal cells or endometrial cells. The helper cells were analysed immunohistochemically to investigate the types of IGFBP produced by various cell monolayers. The concentrations of IGF-I and IGFBP-3 in media obtained from the culture of embryos alone, cells alone or cells plus embryos were determined by radioimmunoassays. On day 7, more blastocysts hatched in the co-culture groups (73% in the Vero cell group, 76% in the endometrial cell group and 74% in the oviductal cell group) than in the control group (43%) (P < 0.0001). The results of immunohistochemistry revealed that (i) all three cell groups produced a lot of IGFBP-1, -2 and -3, but only a little of IGFBP-4 and -5; and (ii) IGFBP-1, -2, and -3 were present in blastocysts in either the presence or absence of helper cells. The IGF-I secreted by cell monolayers or embryos was undetectable (detection limit 0.83 microg/l). The IGFBP-3 concentrations in media obtained from co-cultured embryos and cells were significantly higher than in media without embryos (median values in oviductal cell culture medium, 165 versus 127 microg/l, P = 0.04; median values in endometrial cell culture medium, 277.5 versus 183.5 microg/1, P = 0. 0002; median values in Vero cell culture medium, 219 versus 120 microg/l, P = 0.011). Although IGFBP-3 concentration in the medium that contained embryos alone was undetectable by radioimmunoassay (detection limit 1.1 microg/l), immunohistochemistry demonstrated the presence of IGFBP-3 in the embryos. Co-culture in systems in which there was an increased production of IGFBP-3 led to an improved development of mouse embryos. IGFBP can improve the binding of IGF to cell surface receptors of target tissue, and thus enhance the effect of limited IGF concentrations in promoting embryo development in a co-culture system. We conclude that Vero cells, human endometrial cells and oviductal cells produce IGFBP-1, -2, -3, -4 and -5. IGFBP-3 may play a role in embryotrophic potential by either regulating the action of IGF or directly enhancing embryo development.

Estrogens and tamoxifen (an antiestrogen) exert their actions by activation of estrogen receptor (ER) through genomic and non-genomic mechanisms and are implicated in the development of endometrial cancer. Previous reports have demonstrated that estradiol and tamoxifen induce proliferation of human endometrial cancer cells through GPR30 (non-genomic ER) signaling pathway. Herein, we demonstrate that phosphorylation of focal adhesion kinase (FAK) is involved in cell migration induced by estradiol, tamoxifen and G1 (a GPR30 agonist) through the transmembrane ER (GPR30) in endometrial cancer cell lines with or without ERα (Ishikawa and RL95-2). Additionally, the GPR30-mediated cell migration was further abolished by administration of either specific RNA interference targeting GPR30 or an FAK inhibitor. Moreover, we have validated that the signaling between GPR30 and phosphorylated FAK is indeed mediated by the EGFR/PI3K/ERK pathway. Clinically, a significant correlation between levels of GPR30 and phophorylated FAK (pFAK) observed in human endometrial cancer tissues with low or without ERα further suggested that estrogen-induced phosphorylation of FAK and cell migration were most likely triggered by GPR30 activation. These results provided new insights for understanding the pathophysiological functions of GPR30 in human endometrial cancers.

The aim of this study was to investigate circulating levels of insulin-like growth factor-binding protein-3 (IGFBP-3) in women with normal pregnancy. A total of 468 pregnant women at various gestational ages and 30 non-pregnant females were recruited in this study. Serum concentration of IGFBP-3 was determined by radioimmunoassay. Maternal serum levels of IGFBP-3 increased as pregnancy progressed. The concentration of IGFBP-3 in pregnancy serum was positively associated with the gestational age. The mean values of circulating IGFBP-3 in the second and third trimesters of pregnancy were higher than those in the first trimester of pregnancy and in non-pregnant females. In addition, there was a highly significant correlation between IGFBP-3 and IGF-I in pregnancy serum. We conclude that, during pregnancy, IGFBP-3 appears to be the principal binding protein for circulating IGF-I and may be involved in the regulation of fetal growth.

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.

Endometriosis is determined by genetic factors, and the prevalence of genetic polymorphisms varies greatly depending on the ethnic group studied. The objective of this study was to investigate the relationship between single nucleotide polymorphisms (SNPs) of 9 genes involved in estrogen biosynthesis and metabolism and the risks of endometriosis. Three hundred patients with endometriosis and 337 non-endometriotic controls were recruited. Thirty four non-synonymous SNPs, which change amino acid residues, were analyzed using matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF MS). The functions of SNP-resulted amino acid changes were analyzed using multiple web-accessible databases and phosphorylation predicting algorithms. Among the 34 NCBI-listed SNPs, 22 did not exhibit polymorphism in this study of more than 600 Taiwanese Chinese women. However, homozygous and heterozygous mutants of 4 SNPs - rs6165 (genotype GG+GA, 307(Ala/Ala)+307(Ala/Thr)) of FSHR, rs 6166 (genotype GG+GA, 680(Ser/Asn)+680(Ser/Ser)) of FSHR, rs2066479 (genotype AA+AG, 289(Ser/Ser)+289(Ser/Gly)) of HSD17B3 and rs700519 (genotype TT+TC, 264(Cys/Cys)+264(Cys/Arg)) of CYP19, alone or in combination, were significantly associated with decreased risks of endometriosis. Bioinformatics results identified 307(Thr) of FSHR to be a site for O-linked glycosylation, 680(Ser) of FSHR a phosphorylated site by protein kinase B, and 289(Ser) of HSD17B3 a phosphorylated site by protein kinase B or ribosomal protein S6 kinase 1. Results of this study suggest that non-synonymous polymorphisms of FSHR, HSD17B3 and CYP19 genes may modulate the risk of endometriosis in Taiwanese Chinese women. Identification of the endometrosis-preferential non-synonymous SNPs and the conformational changes in those proteins may pave the way for the development of more disease-specific drugs.

Objective To evaluate interrelationships between levels of insulin-like growth factor I (IGF-I), insulin-like growth factor binding protein (IGFBP-I), and ovarian steroids in late preovulatory follicular fluid (FF) after pituitary desensitization with GnRH agonist and controlled ovarian hyperstimulation. Design Follicular fluid and matched serum were collected during the ovum pick-up. Insulin-like growth factor binding protein I, E 2 , P, and androstenedione (A) were measured by means of RIA, whereas T was measured by means of fluoroimmunoassay and IGF-I by means of immunoradiometric assay. Setting University teaching hospital. Participants Fifty-nine patients underwent 62 ovum pick-ups for IVF. Main Outcome Measures Levels of IGF-I, IGFBP-I, E 2 , P 4 (representative of follicular maturity), T, and A in FF and the volume of this fluid were determined. Levels of E 2 to A (representative of aromatase activity) in FF were examined in relation to the volume of FF. Results Levels of FF-IGF-I decreased with increasing volume of FF. This observation is unprecedented. In contrast, FF-P increased with increasing volume of FF. The reverse relationships with FF volume between FF-IGF-I and FF-P may not be causal because no correlation was found between concentrations of FF-IGF-I and FF-P. Our findings are consistent with and complementary to those in previous reports. The follicular fluid E 2 :A ratio, FF-E 2 , FF-A, FF-T, and FF-IGFBP-I were not found to be correlated with volume of FF. Conclusion Decreased levels of FF-IGF-I with unaltered levels of FF-IGFBP-I in larger follicles may favor a shift toward diminished action of IGF-I at the time of immediate preovulation. The physiological significance of this shift remains uncertain as it is not reflected in the levels of steroids in FF.

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.

Context: The cause of preeclampsia remains unknown. Excessive antiangiogenic proteins have been proposed to play a pathogenic role in preeclampsia.Objective: Our objective was to determine the differences in soluble endoglin (sEndoglin), soluble fms-like tyrosine kinase receptor-1 (sFLT1), leptin, adiponectin, and endothelin 1 concentrations between normal and preeclampsia amniotic fluid (AF). Such results may help us understand the pathophysiology of preeclampsia.Methods: We performed a nested case-control study. Seventy-one women with preeclampsia were matched to 71 normotensive controls. The preeclamptic women were broken into two subgroups according to the association with fetal intrauterine growth restriction (IUGR). AF concentrations of sEndoglin, sFLT1, leptin, adiponectin, and endothelin 1 were measured by ELISA. Receiver-operating characteristics curve analysis was used to compare the discriminative values of these potential biomarkers. Functional network analysis was performed using MetaCore to reveal the common functions of the interacting proteins.Results: Increased AF concentrations of sFLT1, sEndoglin, endothelin 1, and leptin were found in women who later developed preeclampsia. sFLT1, sEndoglin, leptin, and adiponectin were significantly higher in the preeclampsia with IUGR than those without IUGR. Leptin has the largest area under the curve (0.753). Network analysis revealed that elevated amniotic proteins are involved in the inflammatory process of the human placenta.Conclusions: Significant elevation of leptin can be detected in AF 2 months earlier than the appearance of symptoms; thus, it may be used as a predictive marker for preeclampsia. The increase of these antiangiogenic proteins supports the roles of inflammation and oxidative stress in pathogenesis of preeclampsia.

SummaryGenome-wide association studies (GWAS) use high-throughput genotyping technology to relate hundreds of thousands of genetic markers (genotypes) to clinical conditions and measurable traits (phenotypes). This review is intended to serve as an introduction to GWAS for clinicians, to allow them to better appreciate the value and limitations of GWAS for genotype-disease association studies. The input of clinicians is vital for GWAS, since disease heterogeneity is frequently a confounding factor that can only really be solved by clinicians. For diseases that are difficult to diagnose, clinicians should ensure that the cases do indeed have the disease; for common diseases, clinicians should ensure that the controls are truly disease-free.

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.

To investigate intrinsic alterations in ovarian steroidogenesis in women with clomiphene citrate (CC)-resistant polycystic ovary syndrome (PCOS) who ovulated after metformin treatment.Fifty-six women of reproductive age (18-40 years) diagnosed with CC-resistant PCOS received metformin for 12 weeks. If ovulation was successfully induced by CC after metformin treatment, the women were classified as responders. Circulating levels of follicle-stimulating hormone (FSH), luteinizing hormone (LH), estradiol (E2), testosterone (T), free testosterone (freeT), fasting insulin and sex hormone-binding globulin (SHBG) were determined at weeks 0, 4 and 12 of metformin treatment.Thirty-seven women with CC-resistant PCOS finished the treatment course. There were no significant differences in circulating levels of FSH, E2, T, freeT and fasting insulin at weeks 0, 4, and 12 between responders and nonresponders. At week 4, responders showed significantly higher LH levels and higher LH/FSH ratios than non-responders (p0.005 and p0.05, respectively). After the 12-week treatment, responders demonstrated higher serum E2/T ratios and lower freeT levels than non-responders (p0.05 and p0.05, respectively).The results suggest that in women with CC-resistant PCOS, elevated E2/T is associated with a better response to metformin.