Embryo-derive TNF promotes decidualization via fibroblast activation.

Decidualization is a process in which endometrial stromal fibroblasts differentiate into specialized secretory decidual cells and essential for the successful establishment of pregnancy. The underlying mechanism during decidualization still remains poorly defined. Because decidualization and fibroblast activation share similar characteristics, this study was to examine whether fibroblast activation is involved in decidualization. In our study, fibroblast activation-related markers are obviously detected in pregnant decidua and under in vitro decidualization. ACTIVIN A secreted under fibroblast activation promotes in vitro decidualization. We showed that arachidonic acid released from uterine luminal epithelium can induce fibroblast activation and decidualization through PGI2 and its nuclear receptor PPARδ. Based on the significant difference of fibroblast activation-related markers between pregnant and pseudopregnant mice, we found that embryo-derived TNF promotes CPLA2α phosphorylation and arachidonic acid release from luminal epithelium. Fibroblast activation is also detected under human in vitro decidualization. Similar arachidonic acid-PGI2-PPARδ-ACTIVIN A pathway is conserved in human endometrium. Collectively, our data indicate that embryo-derived TNF promotes CPLA2α phosphorylation and arachidonic acid release from luminal epithelium to induce fibroblast activation and decidualization.

Primary Cilia Restrain PI3K-AKT Signaling to Orchestrate Human Decidualization.

Endometrial decidualization plays a pivotal role during early pregnancy. Compromised decidualization has been tightly associated with recurrent implantation failure (RIF). Primary cilium is an antenna-like sensory organelle and acts as a signaling nexus to mediate Hh, Wnt, TGFß, BMP, FGF, and Notch signaling. However, whether primary cilium is involved in human decidualization is still unknown. In this study, we found that primary cilia are present in human endometrial stromal cells. The ciliogenesis and cilia length are increased by progesterone during in vitro and in vivo decidualization. Primary cilia are abnormal in the endometrium of RIF patients. Based on data from both assembly and disassembly of primary cilia, it has been determined that primary cilium is essential to human decidualization. Trichoplein (TCHP)-Aurora A signaling mediates cilia disassembly during human in vitro decidualization. Mechanistically, primary cilium modulates human decidualization through PTEN-PI3K-AKT-FOXO1 signaling. Our study highlights primary cilium as a novel decidualization-related signaling pathway.

Circ-CCNB1 Modulates Trophoblast Proliferation and Invasion in Spontaneous Abortion by Regulating miR-223/SIAH1 axis.

CONTEXT: Spontaneous abortion (SA) is a common disorder in early pregnancy. Circular RNAs (circRNAs) have been reported to exert important regulatory effects on trophoblast function and embryo development. OBJECTIVE: The aim of this study was to explore whether and how circRNAs regulate trophoblast function in SA during early pregnancy. METHODS: Cell proliferation, 5-bromo-2-deoxyuridine (BrdU) staining, Transwell, immunofluorescence, Western blot, RNA pull-down, and dual luciferase reporter assays were performed to investigate the effect of circRNA cyclin B1 (circ-CCNB1) on trophoblast function in HTR-8/SVneo and JEG-3 cells. RESULTS: An in vitro study demonstrated that upregulation of circ-CCNB1 significantly inhibited trophoblast proliferation and invasion compared with the controls using HTR-8/SVneo and JEG-3 cells, respectively. Moreover, miR-223 was downregulated in the villous tissues of patients with SA and was further predicted and shown to negatively interact with circ-CCNB1, which is involved in trophoblast proliferation and invasion. Using bioinformatics tools and subsequent RNA pull-down and dual luciferase assays, we found that miR-223 directly targets seven in absentia homolog-1 (SIAH1) and that upregulation of miR-223 decreased circ-CCNB1-induced SIAH1 expression levels in HTR-8/SVneo cells. Interestingly, upregulation of circ-CCNB1 suppressed trophoblast proliferation and invasion through inhibition of CCNB1 nuclear translocation induced by SIAH1. Downregulation of SIAH1 enhanced circ-CCNB1-suppressed CCNB1 nuclear protein expression in trophoblast cells. CONCLUSION: Circ-CCNB1 served as a modulator of trophoblast proliferation and invasion by sponging miR-223, thus forming a regulatory network of circ-CCNB1/miR-223/SIAH1 in modulating CCNB1 nuclear translocation, which enabled us to elucidate the molecular mechanisms involved in normal embryo implantation or in SA.

A diarylheptanoid compound from Alpinia officinarum Hance ameliorates high glucose-induced insulin resistance by regulating PI3K/AKT-Nrf2-GSK3ß signaling pathways in HepG2 cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Alpinia officinarum Hance, a perennial natural medicine-food herb, has been traditionally used to treat colds, stomachache, and diabetes for thousands of years. 1,7-Diphenyl-4E-en-3-heptanone (DPH5), a diarylheptanoid isolated from the rhizome of A. officinarum has been reported to be safe and to have antioxidant and hypoglycemic effects, suggesting its potential in the treatment of insulin resistance (IR). AIM OF THE STUDY: Aim of to investigate the protective effect of DPH5 on IR and elucidate its underlying mechanism of action. MATERIALS AND METHODS: HepG2 cells were used as the research objects. Glucose uptake and reactive oxygen species (ROS) levels in high glucose-induced insulin-resistant HepG2 cells were assessed using flow cytometry. Glucose consumption and the levels of malondialdehyde (MDA) and superoxide dismutase (SOD) were analyzed using the corresponding assay kits. The expression of mRNA and proteins related to insulin signaling, glucose metabolism, and antioxidant factor, including insulin receptor substrate-1 (IRS1), phosphatidylinositol 3-kinase (PI3K), protein kinase B (AKT), translocation of glucose transporter-4, glycogen synthase kinase-3ß (GSK3ß), glucokinase (GCK), pyruvate kinase (PK), phosphoenolpyruvate carboxykinase (PEPCK), glucose-6-phosphatase (G6Pase), nuclear factor-erythroid 2 related factor 2 (Nrf2), heme oxygenase-1 (HO-1), NADPH quinoneoxidoreductase (NQO1), and glutathione peroxidase (GSH-Px) was determined using real-time quantitative polymerase chain reaction and western blotting. Furthermore, molecular docking was performed to determine the spatial mechanism of DPH5 on the key targets PI3K, AKT, Nrf2, and GSK3ß. RESULTS: DPH5 could improve IR that manifested as increased glucose uptake and glucose consumption in insulin-resistant HepG2 cells. Moreover, DPH5 could enhance antioxidant capacity by activating Nrf2/HO-1 elements, including increasing Nrf2, HO-1, SOD, NQO1, and GSH-Px expression and reducing MDA, ROS, and JNK levels, thereby improving oxidative stress and ultimately alleviating IR. Additionally, DPH5 could promote the expression of IRS1, PI3K, AKT, GSK3ß, GCK, and PK, and downregulate the expression of PEPCK and G6pase, thereby accelerating glucose utilization and enhancing insulin sensitivity. The mechanism underlying the effect of DPH5 in alleviating IR was related to the PI3K/AKT- and Nrf2/HO-1-mediated regulation of the GSK3ß signaling pathway, and the results were further confirmed using the specific inhibitors LY294002 and ML385. Results from molecular docking indicated that there were different regulatory sites and interacting forces between DPH5 and PI3K, AKT, Nrf2, and GSK3ß; however, the binding force was relatively strong. CONCLUSIONS: DPH5 improved oxidative stress and glucose metabolism via modulating the PI3K/AKT-Nrf2-GSK3ß pathway, thereby ameliorating IR. Overall, our findings suggest the potential of DPH5 as a natural medicine to treat type-2 diabetes mellitus.

High Maternal Serum Estradiol in First Trimester of Multiple Pregnancy Contributes to Small for Gestational Age via DNMT1-Mediated CDKN1C Upregulation.

High maternal serum estradiol (E2) levels in the first trimester of pregnancy are associated with a high incidence of low birth weight (LBW) and small for gestational age (SGA). This study aimed to investigate the effect of first-trimester high maternal serum E2 levels on fetal growth and the underlying mechanisms in multiple pregnancies. Maternal serum E2 levels of women at 8 weeks of gestation were measured. The expression levels of imprinted genes and DNMT1 were determined by RT-qPCR, and KvDMR1 methylation in embryo tissue, placenta, and newborn cord blood samples was examined by bisulfite sequencing PCR. The effect of E2 on CDKN1C expression was investigated in HTR8 cells. The incidence of SGA was significantly higher in multiple pregnancies reduced to singleton than that in primary singleton pregnancies (11.4% vs. 2.9%) (P < 0.01) and multiple pregnancies reduced to twins than primary twins (38.5% vs. 27.3%) (P < 0.01). The maternal serum E2 level at 8 weeks of gestation increased with the number of fetuses and was negatively correlated with offspring birth weight. CDKN1C and DNMT1 expression was significantly upregulated in embryo tissue, placenta, and cord blood from multiple pregnancies. Furthermore, there was a positive correlation between CDKN1C mRNA expression and KvDMR1 methylation levels. In HTR8 cells, DNMT1 mediated the estrogen-induced upregulation of CDKN1C, which might contribute to SGA. To minimize the risks of LBW and SGA, our findings suggest that abnormally high maternal serum E2 levels should be avoided during the first trimester of multiple pregnancies from assisted reproductive technology (ART).

In situ Synthesized Monosodium Urate Crystal Enhances Endometrium Decidualization via Sterile Inflammation During Pregnancy.

High level of uric acid (UA) is the major origin of gout, and is highly associated with various pregnant complications, such as preeclampsia and gestational diabetes. However, UA's level and role in the very early stage of pregnancy has not been uncovered. This study aims to investigate the relevance of serum UA and decidualization, an essential process for the establishment and maintenance of pregnancy in women and mice during the early stage of pregnancy. In this study, we first proved that expression level of UA synthase xanthine dehydrogenase (XDH) is highly increased along with decidualization of endometrial stromal cells in both in vitro and in vivo models. Furthermore, serum and endometrial levels of UA are higher in mice with decidualized uterin horn and in vitro decidualized stromal cells. The existence of monosodium urate (MSU) crystal was also confirmed by immunostaining. Next, the roles of MSU on decidualization were explored by both in vitro and in vivo models. Our data shows MSU crystal but not UA enhances the decidualization response of endometrial stromal cells, via the upregulation of inflammatory genes such Ptgs2 and Il11. inhibiting of Cox-2 activity abolishes MSU crystal induced higher expression of decidualization marker Prl8a2. At last, in women, we observed enriched expression of XDH in decidua compare to non-decidualized endometrium, the serum level of UA is significantly increased in women in very early stage of pregnancy, and drop down after elective abortion. In summary, we observed an increased serum UA level in the early stage of women's pregnancy, and proved that the increased level of UA results from the expressed XDH in decidualizing endometrium of both human and mouse, leading to the formation of MSU crystal. MSU crystal can enhance the decidualization response via inflammatory pathways. Our study has uncovered the association between UA, MSU, and decidualization during the early stage of pregnancy.

Regulation and Function of Laminin A5 during Mouse and Human Decidualization.

Decidualization is essential to the establishment of pregnancy in rodents and primates. Laminin A5 (encoding by Laminin α5) is a member of the laminin family, which is mainly expressed in the basement membranes. Although laminins regulate cellular phenotype maintenance, adhesion, migration, growth, and differentiation, the expression, function, and regulation of laminin A5 during early pregnancy are still unknown. Therefore, we investigated the expression and role of laminin A5 during mouse and human decidualization. Laminin A5 is highly expressed in mouse decidua and artificially induced deciduoma. Laminin A5 is significantly increased under in vitro decidualization. Laminin A5 knockdown significantly inhibits the expression of Prl8a2, a marker for mouse decidualization. Progesterone stimulates the expression of laminin A5 in ovariectomized mouse uterus and cultured mouse stromal cells. We also show that progesterone regulates laminin A5 through the PKA-CREB-C/EBPß pathway. Laminin A5 is also highly expressed in human pregnant decidua and cultured human endometrial stromal cells during in vitro decidualization. Laminin A5 knockdown by siRNA inhibits human in vitro decidualization. Collectively, our study reveals that laminin A5 may play a pivotal role during mouse and human decidualization via the PKA-CREB-C/EBPß pathway.

Aldosterone from endometrial glands is benefit for human decidualization.

Local renin-angiotensin system (RAS) in female reproductive system is involved in many physiological and pathological processes, such as follicular development, ovarian angiogenesis, ovarian, and endometrial cancer progress. However, studies on the functional relevance of RAS in human endometrium are limited, especially for renin-angiotensin-aldosterone system (RAAS). In this study, we defined the location of RAS components in human endometrium. We found that angiotensin II type-1 receptor (AT1R) and aldosterone synthase (CYP11B2), major components of RAAS, are specifically expressed in endometrial gland during mid-secretory phase. Aldosterone receptor, mineralocorticoid receptor (MR), is elevated in stroma in mid-secretory endometrium. In vitro, MR is also activated by aldosterone during decidualization. Activated MR initiates LKB1 expression, followed by phosphorylating of AMPK that stimulates PDK4 expression. The impact of PDK4 on decidualization is independent on PDHE1α inactivation. Based on co-immunoprecipitation, PDK4 interacts with p-CREB to prevent its ubiquitination for facilitating decidualization via FOXO1. Restrain of MR activation interrupts LKB1/p-AMPK/PDK4/p-CREB/FOXO1 pathway induced by aldosterone, indicating that aldosterone action on decidualization is mainly dependent on MR stimulation. Aldosterone biosynthesized in endometrial gland during mid-secretory phase promotes decidualization via activating MR/LKB1/p-AMPK/PDK4/p-CREB/FOXO1 signaling pathway. This study provides the valuable information for understanding the underlying mechanism during decidualization.

Effectiveness of acupuncture combined mecobalamin in the treatment of elderly diabetic peripheral neuropathy: A protocol of systematic review and meta-analysis.

BACKGROUND: Although previous studies have reported the effectiveness of acupuncture combined mecobalamin (AM) in the treatment of elderly diabetic peripheral neuropathy (EDPN), no systematic study has assessed its effectiveness and safety. Thus, this study will evaluate the effectiveness and safety of AM for the treatment of patients with EDPN. METHODS: Bibliographic electronic databases will be searched as follows: Cochrane Library, PUBMED, EMBASE, CINAHL, PsycINFO, WANGFANG, and China National Knowledge Infrastructure. All of them will be searched from each database initial to March 1, 2020 without language restrictions. All study selection, information extracted, and study quality evaluation will be performed by 2 independent authors. Any disagreements between 2 authors will be resolved by a third author via discussion. RevMan 5.3 software will be used for data pooling and meta-analysis performance if it is possible. RESULTS: This study will provide synthesis of current evidence of AM for patients with EDPN through primary outcome of glycemic profile, and secondary of neuropathic pain intensity, plantar tactile sensitivity, sensory nerve conduction velocity and motor nerve conduction velocity, health-related quality of life, and adverse events. CONCLUSION: This study will provide helpful reference for the efficacy and safety of AM for the treatment of patients with EDPN to the clinicians and further studies.Study registration number: INPLASY202040094.

Estradiol promotes trophoblast viability and invasion by activating SGK1.

BACKGROUND: 17ß-Estradiol (E2) is a critical regulator of trophoblast function during pregnancy. Serum- and glucocorticoid-inducible kinase (SGK1) has been shown to regulate specific cellular targets downstream of E2. However, whether and how SGK1 directly mediates the regulatory effects of E2 on trophoblasts functions remain unknown. METHODS: SGK1 expression in human villous samples and serum E2 levels were measured in women with early pregnancy loss (EPL) and healthy pregnant women. The effect of E2 on SGK1 regulation was assessed using luciferase reporter gene assay and Chromatin Immunoprecipitation assay. The mediation of regulatory effects of E2 by SGK1 on trophoblast functions including cell viability, invasion and related signaling molecules such as B cell leukemia/lymphoma 6, E-cadherin, matrix metalloproteinase 2, α-ENaC, vascular endothelial growth factor, and the phosphorylation status of FOXO1 and AKT were evaluated in HTR8/SVneo cells transfected with SGK1 knockdown plasmid with/without E2 treatment. RESULTS: SGK1 protein levels in human villous samples and serum E2 levels were decreased in patients with EPL compared to controls. E2 (10 nM) increased SGK1 promoter activity directly through estrogen receptor. E2-activated SGK1 enhanced cell viability, invasion and downstream targets in trophoblast cells. SGK1 knockdown abrogated the above responses to E2 treatment. CONCLUSIONS: SGK1 mediates the effects of E2 on trophoblast viability and invasion, suggesting that SGK1 acts as a key node in regulating the cross-talk at the feto-maternal interface during the development of placenta and might be a potential therapeutic target for EPL.

G Protein α Subunit 14 Mediates Fibroblast Growth Factor 2-Induced Cellular Responses in Human Endothelial Cells.

During pregnancy, a tremendous increase in fetoplacental angiogenesis is associated with elevated blood flow. Aberrant fetoplacental vascular function may lead to pregnancy complications including pre-eclampsia. Fibroblast growth factor 2 (FGF2) and vascular endothelial growth factor A (VEGFA) are crucial regulators of fetoplacental endothelial function. G protein α subunit 14 (GNA14), a member of Gαq/11 subfamily is involved in mediating hypertensive diseases and tumor vascularization. However, little is known about roles of GNA14 in mediating the FGF2- and VEGFA-induced fetoplacental endothelial function. Using human umbilical vein endothelial cells (HUVECs) cultured under physiological chronic low oxygen (3% O2 ) as a cell model, we show that transfecting cells with adenovirus carrying GNA14 complementary DNA (cDNA; Ad-GNA14) increases (p < 0.05) protein expression of GNA14. GNA14 overexpression blocks (p < 0.05) FGF2-stimulated endothelial migration, whereas it enhances (p < 0.05) endothelial monolayer integrity (maximum increase of ~35% over the control at 24 hr) in response to FGF2. In contrast, GNA14 overexpression does not significantly alter VEGFA-stimulated cell migration, VEGFA-weakened cell monolayer integrity, and intracellular Ca++ mobilization in response to adenosine triphosphate (ATP), FGF2, and VEGFA. GNA14 overexpression does not alter either FGF2- or VEGFA-induced phosphorylation of ERK1/2. However, GNA14 overexpression time-dependently elevates (p < 0.05) phosphorylation of phospholipase C-ß3 (PLCß3) at S1105 in response to FGF2, but not VEGFA. These data suggest that GNA14 distinctively mediates fetoplacental endothelial cell migration and permeability in response to FGF2 and VEGFA, possibly in part by altering activation of PLCß3 under physiological chronic low oxygen.

GNA11 differentially mediates fibroblast growth factor 2- and vascular endothelial growth factor A-induced cellular responses in human fetoplacental endothelial cells.

KEY POINTS: Fetoplacental vascular growth is critical to fetal growth. Fibroblast growth factor 2 (FGF2) and vascular endothelial growth factor A (VEGFA) are two major regulators of fetoplacental vascular growth. G protein α subunit 11 (GNA11) transmits signals from many external stimuli to the cellular interior and may mediate endothelial function. It is not known whether GNA11 mediates FGF2- and VEGFA-induced endothelial cell responses under physiological chronic low O2 . In the present study, we show that knockdown of GNA11 significantly decreases FGF2- and VEGFA-induced fetoplacental endothelial cell migration but not proliferation and permeability. Such decreases in endothelial migration are associated with increased phosphorylation of phospholipase C-ß3. The results of the present study suggest differential roles of GNA11 with respect to mediating FGF2- and VEGFA-induced fetoplacental endothelial function. ABSTRACT: During pregnancy, fetoplacental angiogenesis is dramatically increased in association with rapidly elevated blood flow. Any disruption of fetoplacental angiogenesis may lead to pregnancy complications such as intrauterine growth restriction. Fibroblast growth factor 2 (FGF2) and vascular endothelial growth factor A (VEGFA) are crucial regulators of fetoplacental angiogenesis. G protein α subunits q (GNAq) and 11 (GNA11) are two members of the Gαq/11 subfamily involved in mediating vascular growth and basal blood pressure. However, little is known about the roles of GNA11 alone with respect to mediating the FGF2- and VEGFA-induced fetoplacental endothelial function. Using a cell model of human umbilical cord vein endothelial cells cultured under physiological chronic low O2 (3% O2 ), we showed that GNA11 small interfering RNA (siRNA) dramatically inhibited (P < 0.05) FGF2- and VEGFA-stimulated fetoplacental endothelial migration (by ∼36% and ∼50%, respectively) but not proliferation and permeability. GNA11 siRNA also elevated (P < 0.05) FGF2- and VEGFA-induced phosphorylation of phospholipase C-ß3 (PLCß3) at S537 in a time-dependent fashion but not mitogen-activated protein kinase 3/1 (ERK1/2) and v-akt murine thymoma viral oncogene homologue 1 (AKT1). These data suggest that GNA11 mediates FGF2- and VEGFA-stimulated fetoplacental endothelial cell migration partially via altering the activation of PLCß3.

A potential role of galectin-1 in promoting mouse trophoblast stem cell differentiation.

Galectin-1 is highly expressed in blastocysts and trophoblast giant cells during implantation, and dysregulated galectin-1 is associated with many pregnancy-related abnormalities. Elevated galectin-1 contributes to cancer cells invasion. Here, we found that galectin-1 is expressed in mouse oocytes, preimplantation embryos (all stages), and trophoblast stem (TS) cells. Peak levels of galectin-1 mRNA and protein were detected on day 4 and day 5 after the induction of TS cells differentiation. Overexpression of galectin-1 increased TS cells migration and invasion, whereas knockdown of galectin-1 attenuated these effects. Additionally, knockdown of galectin-1 in TS cells decreased the expression of matrix metalloproteinase (MMP) 2/9, ZEB-1, Snail, N-cadherin, TGF-ß, Nodal, and phospho-Smad2/3, whereas the expression of E-cadherin was increased. In contrast, overexpression of galectin-1 in TS cells increased the expression of MMP2/9, ZEB-1, and N-cadherin, whereas the expression of E-cadherin was decreased. These findings suggest a potential role of galectin-1 in the differentiation of mouse TS cells.

Preeclampsia Downregulates MicroRNAs in Fetal Endothelial Cells: Roles of miR-29a/c-3p in Endothelial Function.

Context: Preeclampsia is a leading cause of fetal and maternal morbidity and mortality during pregnancy. Although the etiology of preeclampsia is unknown, preeclampsia offspring have increased risks of developing cardiovascular disorders in adulthood, implicating that preeclampsia programs fetal vasculature in utero. Objective: We hypothesize that preeclampsia alters expression profiles of endothelial microRNAs (miRNAs) in fetal endothelial cells and disturbs the vascular endothelial growth factor A (VEGFA)- and fibroblast growth factor 2 (FGF2)-induced endothelial function. Design and Setting: Unpassaged (P0) human umbilical vein endothelial cells (HUVECs) were isolated immediately after cesarean-section delivery from normotensive (NT) and preeclamptic (PE) pregnancies. Differentially expressed miRNAs between P0-HUVECs from NT and PE pregnancies were identified using a miRNA polymerase chain reaction (PCR) array and confirmed using reverse transcription quantitative PCR. To determine the function of these differentially expressed miRNAs, miRNAs of interest were knocked down in NT-HUVECs following by cell functional assays. Results: Sixteen miRNAs, including miR-29a/c-3p, were downregulated in P0-HUVECs from the PE group compared with the NT group. Bioinformatics analysis predicted the PI3K-v-akt murine thymoma viral oncogene homolog 1 (AKT) signaling pathway was dysregulated in P0-HUVECs from the PE group, which was associated with the miR-29a/c-3p downregulation. We further demonstrated that miR-29a/c-3p knockdown inhibited the VEGFA- and FGF2-induced endothelial migration as well as FGF2-induced AKT1 phosphorylation in HUVECs. However, miR-29a/c-3p knockdown did not alter the extracellular signal-regulated kinase 1/2 phosphorylation, cell proliferation, and endothelial monolayer integrity in response to VEGFA and FGF2 in HUVECs. Conclusions: Preeclampsia-downregulated miR-29a/c-3p may impair fetal endothelial function by disturbing the FGF2-activated PI3K-AKT signaling pathway, hence inhibiting endothelial cell migration.

Autoantibodies in Chinese patients with chronic hepatitis B: prevalence and clinical associations.

AIM: To investigate the prevalence of autoantibodies and their associations with clinical features in Chinese patients with chronic hepatitis B (CHB). METHODS: A total of 325 Chinese patients with CHB were enrolled in this retrospective, hospital-based study. Patients with chronic hepatitis C (CHC), autoimmune hepatitis (AIH), or primary biliary cirrhosis (PBC) were included, with healthy donors acting as controls. A panel of autoantibodies that serologically define AIH and PBC was tested by indirect immunofluorescence assay and line immunoassay. The AIH-related autoantibody profile included homogeneous anti-nuclear antibodies (ANA-H), smooth-muscle antibodies, anti-liver kidney microsome type 1, anti-liver cytosolic antigen type 1, and anti-soluble liver antigen/liver pancreas; the PBC-related antibodies were characterized by ANA-nuclear dots/membranous rim-like, anti-mitochondrial antibodies-M2 (AMA-M2), anti-BPO (recombinant antigen targeted by AMA-M2), anti-Sp100, anti-promyelocytic leukemia protein (anti-PML), and anti-gp210. The dichotomization of clustering was used to unequivocally designate the AIH or PBC profiles for each case. Anti-Ro52 antibodies were also tested. RESULTS: The prevalence of any autoantibody in CHB amounted to 58.2%, which was similar to the 66.2% prevalence in CHC, significantly higher than the 6.7% in the healthy controls (P < 0.001), and lower than the 100% found in AIH and PBC (P = 0.004 and P < 0.001, respectively). There were more anti-PML and anti-gp210 antibodies among the CHB patients than the CHC patients (11.1% vs 0%, P = 0.003; 12.6% vs 0%, P < 0.001, respectively). The prevalence and titer of AMA, anti-BPO, anti-PML, and anti-gp210 were higher in PBC than in those with CHB. Among the CHB patients, the prevalence of ANA, especially ANA-H, was significantly lower in patients with compensated and decompensated cirrhosis compared with patients without cirrhosis. Thirty-eight cases of hepatocellular carcinoma (HCC) in CHB showed a significant difference compared with non-HCC patients in the prevalence of anti-PML (0% vs 12.5%, P = 0.013). Dichotomization of the autoantibodies revealed that the PBC profile was more prevalent in patients with CHB than in those with CHC, and that it was strongly correlated with both compensated and decompensated cirrhosis. In contrast, the prevalence of the AIH profile was significantly higher in non-cirrhosis patients with CHB than in those with compensated cirrhosis (18.5% vs 8.2%, P = 0.039). Moreover, the AIH profile was also closely associated with hepatitis B e-antigen positivity. CONCLUSION: ANA-H could be an indicator of early-stage CHB. Dichotomizing the autoantibody profiles revealed that the PBC profile is strongly associated with cirrhosis in CHB.

Preliminary proteomic analysis on the alterations in follicular fluid proteins from women undergoing natural cycles or controlled ovarian hyperstimulation.

PURPOSE: To study the differences in protein expression profiles of follicular fluid (FF) between controlled ovarian hyperstimulation (COH) and natural ovulatory cycles. METHODS: Twelve infertile women undergoing in vitro fertilization and embryo transfer (IVF-ET), with matched clinical information, were retrospectively recruited in the IVF center of our university hospital, including six undergoing COH and another six with natural cycles. FF was sampled from dominant follicles with mature oocytes. Protein expression profiles in each FF sample were analyzed respectively using two-dimensional gel electrophoresis. Differentially expressed proteins were identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and validated by western blotting. Differentially expressed proteins were further analyzed using Ingenuity Pathway Analysis (IPA) software. RESULTS: Two proteins were downregulated and 11 proteins were upregulated (change ≥1.5-fold, P < 0.05) in the COH group. We identified one down-egulated and seven upregulated proteins using MALDI-TOF MS. Four differentially expressed proteins, including transferrin, complement component C3 (C3), haptoglobin and alpha-1-antitrypsin (AAT), were further validated by rate nephelometry and western blotting analyses. The IPA analysis revealed a significant network involved in the humoral immune and inflammatory responses. CONCLUSIONS: The eight differentially expressed proteins were related to immune and inflammatory responses in the ovary. Our results provide new insights into the influence of COH on follicular (spp) development and IVF outcomes.

Increased risk of gonadal malignancy and prophylactic gonadectomy: a study of 102 phenotypic female patients with Y chromosome or Y-derived sequences.

STUDY QUESTION: What is the optimal protocol of management for phenotypic female patients with Y chromosome or Y-derived sequences, in particular for adult patients? SUMMARY ANSWER: Immediate gonadectomy, long-term hormone therapy and psychological care are suggested to be the optimal management for older phenotypic female patients with Y chromosome or Y-derived sequences. WHAT IS KNOWN ALREADY: Phenotypic female patients with Y chromosome or Y-derived sequences are at increasing risk of developing gonadal tumors with age. Early diagnosis and safe guidelines of management for these patients are needed. STUDY DESIGN, SIZE, DURATION: One hundred and two phenotypic women with Y chromosome or Y-derived sequences were included in a straightforward, retrospective-observational study conducted over a period of 26 years from January 1985 to November 2010. PARTICIPANTS/MATERIALS, SETTING AND METHODS: Patients aged 16-34 years presenting to our Academic Department of Gynecology with symptoms of disorders of sex development were subjected to history taking, hormonal evaluation, conventional cytogenetic analysis, PCR, histopathology and immunohistochemistry. Features of the gonads were examined and the outcome of prophylactic gonadectomy evaluated. MAIN RESULTS AND THE ROLE OF CHANCE: Among the patients recruited in our study, 48 patients (47.1%) were diagnosed with complete/partial androgen insensitivity syndrome (CAIS/PAIS) (46XY), 33 cases (32.4%) with gonadal dysgenesis (46XY) and the remaining subjects (20.1%) with mixed gonadal dysgenesis (with sex chromosome structural abnormalities). The total incidence of malignancy was 17.6%. Seventeen patients (16.7%) had gonadoblastoma, while one patient (1.0%) with gonadal dysgenesis had dysgerminoma. Gonadoblastoma were observed in 2/21 patients with sex chromosome structural abnormalities (9.5%), 3/33 patients with gonadal dysgenesis (9.1%), 9/30 patients with CAIS (30.0%) and 3/18 patients with PAIS (16.7%). LIMITATIONS, REASONS FOR CAUTION: Selection bias in this cohort study may affect data interpretation due to the low incidence of disorders of sex development in the general population. WIDER IMPLICATIONS OF THE FINDINGS: The risk for malignant transformation may occur in early life and highly increase with age in patients with Y chromosome or Y-derived sequences. Optimal timing of gonadectomy should be decided by multiple factors including the subgroup of disorder, age and degree of patient's maturity. In addition, gonadal biopsy is suggested when the disease is diagnosed and any evidence of premalignancy warranties gonadectomy. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the National Key Scientific Research Project (2013CB967404), Natural Science Funds of Zhejiang Province (Y13H04005), Zhejiang Qianjiang talent plan (2013R10027), the Fundamental Research Funds for the Central Universities and Key Projects in the National Science & Technology Pillar Program during the Eleventh Five-Year Plan Period (2012BAI32B04). The authors have no conflicts of interest to declare. TRIAL REGISTRATION NUMBER None.

[Cloning and expressing of tissue inhibitor of metalloproteinases I gene fragment and preparation of monoclonal antibodies against the recombinant protein].

OBJECTIVE: To prepare the monoclonal antibody (mAb) against tissue inhibitor of metalloproteinases I (TIMP-I) fusion protein. METHODS: TIMP-I gene was amplified from fibrotic human liver tissue by RT-PCR, then ligated with pQE31 to form recombinant plasmid pQE-TIMP-I and transformed into E. coli BL21. The protein induced by IPTG was purified by 6 x His-tag and used to immunize the BALB/c mice. The specific monoclonal antibodies (mAbs) were prepared by the cell fusion technique. Western Blot were used to detect specificity of mAbs. RESULTS: The prokaryotic plasmid expressing the recombinant protein was constructed, and the TIMP-I recombinant protein was expressed and purified. Four hybridoma cell lines that secreted anti-TIMP-I mAbs were obtained. 3 of 4 mAbs were the IgG1 subtype. Western Blot indicated the mAbs showed specific combination with TIMP-I protein. CONCLUSION: The TIMP-I recombinant protein is highly purified and has strong antigenicity. The anti- TIMP-I mAbs were prepared successfully.

[Establishment of a purificatory method for alpha-fetoprotein variant by affinity adsorption].

OBJECTIVE: To establish a purificatory method of alpha-fetoprotein variant (AFP-L3) based on microspincolumn with lens culinaris agglutinin (LCA). METHODS: LCA was isolated by ammonium sulfate precipitation method from lens culinaris. AFP-L3 affinity adsorption microspincolumns which were made from LCA coupled with activated Sepharose 4B were prepared. By adding into the centrifuge column, serum was absorbed and eluted to purify AFP-L3. The results of purified AFP-L3 detection of 10 cases AFP positive sera by electro-chemiluminescence immunoassay were compared with traditional crossed affinity immunoelectrophoresis. RESULTS: 8 of 10 cases AFP-L3 concentration were greater than 5 ng/ml in purified sera. Six cases show positive reaction in affinity immune cross electrophoresis experiment. CONCLUSION: Successfully established purification method of AFP-L3 by affinity absorption based on microspincolumn. The method was more conducive to clinical laboratory applications due to its high sensitive and easy operation.

[The investigantion of comparative experiments for different clinical laboratory instruments].

OBJECTIVE: To estimate the consistency of two VITROS 3600 chemiluminescent analyzers according to the requirement of ISO15189. METHODS: Verification tests were made for precision and accuracy of anti-HCV in two instruments. While 40 serum samples including Anti-HCV negative (10 cases) , positive (10 cases) , and weakly positive (20 cases). and the test results were statistical analised. RESULTS: Two instruments negative and positive control samples intra-batch precision and coefficients of variation were 5% , 4% and 7. 14% , 7. 23% , inter-batch precision and coefficients of variation were 9. 47% , 7. 7% and 8.04%, 7. 6%, are less than requirement CV (15%) by ISO15189. The accuracy of two instrument were 100% , The test results of the control samples showed no significant difference (P < 0. 05). The correlation analysis of the test results of clinical samples R2 =0. 9984, with good consistency. CONCLUSION: Test results of two Vitros 3600 has good consistency and comparability.