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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.

Background Evidently, when undergoing GnRH-antagonist protocols, dual trigger has proven to produce not just better quality and quantity of oocytes but also pregnancy outcome. However, not much comparative studies have been published when PPOS protocol is used for ovarian stimulation. Can the same positive outcomes be expected after the patients have been exposed to the high level of progesterone required for PPOS protocols? Methods In this retrospective cohort study, patients undergoing PPOS protocols were separated into three groups based on the method employed for triggering final follicular maturation, which included: (a) human chorionic gonadotropin (hCG); (b) Gonadotropin-releasing hormone-agonist (GnRH-agonist); or (c)dual trigger (GnRH-agonist + hCG). Either in vitro fertilization or intracytoplasmic sperm injection (IVF/ICSI) was utilized for fertilization. Assessment comprised of their dynamic hormone profiles, embryonic analysis, and clinical outcomes. Results Of the 344 recruited patients, those fulfilling the Bologna criteria as poor ovarian responders and showing Estradiol (E2) 6500 pg/ml on the day of triggering, none of the three triggering methods demonstrated a significant advantage regarding the number of oocytes, percentage of matured oocytes, and rate of oocytes at fertilization or cleavage stages. Conclusions Implementing dual trigger for stimulating final follicular maturation in patients undergoing PPOS protocols is debatable. For poor ovarian response (POR) patients, dual trigger appeared to yield higher percentage of matured oocytes. In contrast, for hyper-responders, methods of triggering oocyte maturation did not affect the percentage of matured oocytes or the qualities of the embryos. For this group of patients, therefore, the agent used should be one that would reduce the risks of ovarian hyper-stimulation syndrome (OHSS).

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

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. Keywords: HEPES, protein transfection, intracellularly targeting

// Angel Chao 1, 2, * , Chiao-Yun Lin 1, 2, * , An-Ning Chao 3, * , Chia-Lung Tsai 4 , Ming-Yu Chen 1 , Li-Yu Lee 5 , Ting-Chang Chang 1, 2 , Tzu-Hao Wang 1, 2 , Chyong-Huey Lai 1, 2 and Hsin-Shih Wang 1 1 Department of Obstetrics and Gynecology, Chang Gung Memorial Hospital and Chang Gung University, Taoyuan, Taiwan 2 Gynecologic Cancer Research Center, Chang Gung Memorial Hospital, Kaohsiung, Taiwan 3 Department of Ophthalmology, Chang Gung Memorial Hospital and Chang Gung University, Taoyuan, Taiwan 4 Genomic Medicine Research Core Laboratory, Chang Gung Memorial Hospital, Taoyuan, Taiwan 5 Department of Pathology, Chang Gung Memorial Hospital and Chang Gung University, Taoyuan, Taiwan * These authors have contributed equally to this work Correspondence to: Tzu-Hao Wang, email: knoxtn@cgmh.org.tw Chyong-Huey Lai, email: sh46erry@ms6.hinet.net Keywords: gynecologic malignancies, LSD1, autophagy, p62 Received: October 25, 2016 Accepted: June 19, 2017 Published: August 10, 2017 ABSTRACT Lysine-specific demethylase 1 (LSD1) – also known as KDM1A – is the first identified histone demethylase. LSD1 is highly expressed in numerous human malignancies and has recently emerged as a target for anticancer drugs. Owing to the presence of several functional domains, we speculated that LSD1 could have additional functions other than histone demethylation. P62 – also termed sequestasome 1 (SQSTM1) – plays a key role in malignant transformation, apoptosis, and autophagy. Here, we show that a high LSD1 expression promotes tumorigenesis in gynecologic malignancies. Notably, LSD1 inhibition with either siRNA or pharmacological agents activates autophagy. Mechanistically, LSD1 decreases p62 protein stability in a demethylation-independent manner. Inhibition of LSD1 reduces both tumor growth and p62 protein degradation in vivo . The combination of LSD1 inhibition and p62 knockdown exerts additive anticancer effects. We conclude that LSD1 destabilizes p62 and inhibits autophagy in gynecologic cancers. LSD1 inhibition reduces malignant cell growth and activates autophagy. The combinations of LSD1 inhibition and autophagy blockade display additive inhibitory effect on cancer cell viability. A better understanding of the role played by p62 will shed more light on the anticancer effects of LSD1 inhibitors.

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.

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

// 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.

Introduction The impact of maternal age on euploidy rate and the outcome of PGT-Aisdemonstrated by many studies. But less understanding of the probability of finding at least one euploidy embryoaccordingto maternal age. In this study, weassess patients of different maternal age group in PGT-A cyclesusing aCGH and NGS: 1. Number of biopsy blastocyst per cycle; 2. Numbers of 2PN needed for aeuploidy embryo; 3. Euploidy rate; 4. The proportion of cycles having at least aeuploidy embryo; 5. Livebirth rate through frozen embryo transfer (FET). Materialand Methods We retrospectively analyzed the PGT-A cycles enrolled from 2012 to 2016. To compare the clinical outcomes, the included data were separated into groups based on maternal age at IVF stimulation start. TE biopsy was performed on Day5 to 7 post-retrieval. All biopsied blastocysts were subjected to vitrification immediately. Biopsied TE cells were analyzed using array-CGH and NGS for all 23 chromosomes. Patients transferred euploidy embryo on progesterone Day6 in FET cycles. Results The age groups for analysis were: 42. The cycles having biopsy were88.0%(22/25), 100%(18/18), 88.5%(23/26), 60%(9/15) and 82.3%(14/17). The number of biopsy blastocyst per cycle were 4.9(108/22), 5.8(105/18), 3.9(90/23), 3.2(29/9) and 1.6(23/14) and the number of 2PN needed for aeuploidy embryo were 6.8(218/32), 4.1(158/39), 7(189/27), 7.8(8/62) and 7.1(50/7). Euploidy rates were 29.6% (32/108), 37.1% (39/105), 30.0% (27/90), 27.6% (8/29), and 30.4% (7/23). The proportion of cycles having at least a euploidy embryo were 68.2%(15/22), 83.3%(15/18), 65.2%(15/23), 66.7%(6/9) and 35.7%(5/14). Pregnancy rate per FET were63.6%(7/11), 77.8%(7/9), 53.3%(8/15), 100%(1/1) and 50%(2/4). Live birth rates per FET were 63.6% (7/11), 77.8% (7/9), 46.7% (7/15), 100%(1/1) and 25% (1/4). Conclusions In the course of PGT-A, the clinical outcomes and live birth rate can be predicted according to maternal age. Maternal age increase does not affect the number of 2PN needed for aeuploidy embryo, but it seems that women aged between 35 and 37are more likelyto benefit from PGT- A (the highestopportunity of having at least a euploidy embryo). The results of this research can assist doctors in patient consultations and help patients set realistic expectations.

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 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

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.

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.

Introduction: More than 25% of patients diagnosed with endometrial carcinoma have an invasive primary cancer accompanied by metastasis. The kisspeptin has an important role in reproduction. In mammals, kisspeptin may regulate tumor progression of endometrial cancer. FAK and Src signaling have been considered important components of tumor progression. MMPs are largely implicated in promoting angiogenesis and tumor metastasis. In the present study, we examined the action of kisspeptin-promoted motility of endometrial cancer cells and the mechanisms of the action in endometrial cancer. Materials and methods: Endometrial cancer cell line Ishikawa and ECC-1 were derived from an endometrial adenocarcinoma. Kisspeptin agonist and antagonist were synthetic peptides. Cell motility was estimated by invasion and migration assay. The activities of MMP-2 was assessed by gelatin zymography. Immunoblot analysis was done to study the expression of kisspeptin receptor and the effects of kisspeptin in the activation of FAK,Src and MMP-2. Human FAK siRNA and Src siRNA were used to knock down the expression of FAK and Src to evaluate the effects of FAK and Src. MMP-2 inhibitor (OA-Hy) was pretreated for 30 min to evaluate the effects of MMP-2 in cell motility. Results: The kisspeptin regulated cell motility in a dose-dependent manner. Kisspeptin activated the phosphorylation of FAK and Src signaling and the phosphorylation was abolished by FAK siRNA and Src siRNA. Kisspeptin-regulated cell motility was suppressed in cells pretreated with FAK siRNA and Src siRNA. Moreover, FAK siRNA and Src siRNA abolished kisspeptin-induced activation of MMP-2. Inhibition of MMP-2 with 10μM OA-Hy suppressed cell motility in response to kisspeptin. Conclusion: Our study shows that the kisspeptin regulated the cell motility of endometrial cancer cells through the kisspeptin receptor, and the phosphorylation of FAK and Src-dependent activation of MMP-2. Our findings represent a new concept regarding the mechanisms of kisspeptin-regulated cell motility in endometrial cancer cells, suggesting the possibility of kisspeptin analogues as a potential therapeutic intervention for the treatment of human endometrial cancer. Citation Format: Hsien-Ming Wu, Yung-Yu Liang, Wei-Jung Chiu, Hsin-Shih Wang, Hong-Yuan Huang, Chyong-Huey Lai, Yung-Kuei Soong. Kisspeptin regulates the invasiveness of endometrial cancer cells through FAK/ Src signaling-dependent activation of matrix metalloproteinase (MMP)-2. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3481.

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.

Objective To investigate the efficacy of the PolScope on imaging the spindle morphology in oocytes at the metaphase II stage before vitrification and after thawing. Design In vitro study. Setting University infertility clinic and academic research laboratory. Intervention(s) Oocytes at the metaphase II stage that were obtained from superovulating mice were vitrified and then thawed. Main outcome measure(s) Morphological features of the spindle in oocytes were evaluated by both the PolScope and immunofluorescent staining. Result(s) Using the PolScope, the morphological features of the spindle of intact thawed oocytes were undetected by 3 hours of thawing in only 25% of cases. Most of the spindle images were recognized during the first hour of observation. Additionally, the statistical analysis of agreement of spindle morphology by both the PolScope and fluorescent staining showed a weighted Kappa value of 0.70, indicating good agreement. Oocytes with good spindle morphology verified by the PolScope before vitrification had a higher survival rate of intact oocytes after thawing compared with those with poor or undetected spindle images. Conclusion(s) The morphological features of the spindle in oocytes evaluated by the PolScope before freezing and after thawing are significantly correlated with those assessed by immunofluorescent staining after fixation. With the assistance of the PolScope, thawed oocytes with good spindle morphology can be verified and selected for further manipulation without fixation and staining.

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.