Villus Tissue Research Articles

Compensatory role of wild-type p53-induced phosphatase in trophoblastic apoptosis in response to hypoxia

Objective To investigate the mechanism of wild-type p53-induced phosphatase (Wip1) in regulating p53-dependent apoptosis of trophoblasts for further understanding the etiology of preeclampsia (PE). Methods Placenta tissues were collected from normal (n=15) and PE (n=13) gravidas who underwent caesarean section in the First Affiliated Hospital of Chongqing Medical University from June 2017 to December 2018. Chorionic villus and decidua tissues were collected from another 10 women who aborted in early pregnancy. Two in vitro trophoblastic hypoxia cultures were established by subjecting human chorionic trophoblast cells (HTR8/SVneo) to either hypoxia intervention in incubator (HII) or simulated ischemic buffer (SIB). Wip1 expressions at the transcriptional and protein levels were determined by real-time quantitative polymerase chain reaction and Western blotting, respectively. The localization of Wip1 in placental tissues and HTR8/SVneo cells was determined by immunohistochemistry and immunofluorescence. Cell apoptosis was assessed by flow cytometry after viral infection and hypoxia. And the changes of pathway-related molecules including p53, phospho-p53 (p-p53), mouse double minute 2 homolog (Mdm2) and cleaved caspase3 (cl-cas3) were measured by Western blotting. The impact of Wip1 on Mdm2-p53 interaction was examined by co-immunoprecipitation. NVP-CGM097, an Mdm2-p53 specific inhibitor, was administered in PE cell models to verify the regulation of Wip1 on trophoblastic apoptosis through Mdm2-p53 pathway. Independent student's t-test, Welch's t-test and one-way analysis of variance were used as statistical methods. Results (1) Wip1 expression, which was mainly in trophoblast cells, was significantly elevated in human PE placentas (mRNA: 1.711±0.141 vs 0.860±0.126, t=4.496; protein: 0.449±0.027 vs 0.192±0.019, t=7.902) and in both in vitro trophoblastic PE models (protein in HII: 1.376±0.086 vs 0.977±0.114, t=2.792; SIB: 1.243±0.057 vs 0.381±0.045, t=11.910) compared with the corresponding control groups (all P<0.05). (2) Compared with corresponding control groups, overexpression of Wip1 suppressed the hypoxia-induced upregulation of p53 (HII: 0.185±0.024 vs 0.572±0.072; SIB: 0.400±0.067 vs 0.803±0.064), cl-cas3 (HII: 0.243±0.034 vs 0.529±0.072; SIB: 0.179±0.011 vs 0.368±0.025) and p-p53/p53 protein expression (HII: 1.326±0.129 vs 2.100±0.187; SIB: 0.473±0.028 vs 0.925±0.036) and also reduced the apoptosis rate [HII: (8.925±1.092)% vs (17.610±1.980)%; SIB: (13.910±1.886)% vs (24.650±1.622)%], which in turn promoted Mdm2-p53 binding (all P<0.05). However, knockdown of Wip1 gene expression in HTR8/SVneo cells brought about opposite effects (all P<0.05). (3) Neither overexpression nor knockdown of Wip1 influenced p53 or cl-cas3 expression when Mdm2-p53 interaction was blocked by NVP-CGM097. Conclusions Mdm2-p53 interaction promoted by Wip1 upregulation could compensate for the trophoblastic p53 accumulation in response to hypoxia, while exogenous upregulation of Wip1 in trophoblasts may reverse hypoxia-induced apoptosis. Therefore, this might provide a new therapeutic target for PE. Key words: Pre-eclampsia; Cell hypoxia; Trophoblasts; Protein phosphatase 2C; Apoptosis

Evaluation of serum biomarkers and efficacy of MTX in women with ectopic pregnancy.

Ectopic pregnancy occurs when a fertilized ovum attaches outside the uterus. As a complication in approximately 1‑2% of all pregnancies, ectopic pregnancies may cause catastrophic hemorrhage as a result of invading maternal blood vessels. Therefore, early diagnosis and timely treatment are crucial for women with ectopic pregnancy. In this study, we aimed to identify and determine the efficacy of serum biomarkers for the prompt diagnosis of ectopic pregnancy. For this purpose, the serum concentrations of progesterone, β human chorionic gonadotropin (β‑hCG) and cancer antigen‑125 (CA125) were detected by solid‑phase, competitive binding chemiluminescent enzyme immunoassays. Flow cytometry was used to analyze the percentage of CD3+ Tcells in women with ectopic pregnancy. Pathological analysis of tubal and villus tissues was performed by hematoxylin and eosin (H&E) staining. After receiving an injection of methotrexate (MTX), patients were examined by transvaginal ultrasound to detect the size of the echogenic mass. The results revealed that the serum levels of progesterone, β‑HCG and CA125 were significantly decreased in women with ectopic pregnancy, whereas the percentage of CD3+ T cells was increased in women with ectopic pregnancy. Histopathological examination revealed blood clots with small tissue fragments of a tubal‑type epithelium and incomplete pile structures. Five days after the MTX injection, an echogenic mass was found with a size of 1.7x1.2x1.6cm that contained a gestational sac‑like structure and a yolk sac. On the whole, the findings of this study indicate the at the joint detection of progesterone, β‑HCG, CA125 serum levels and the CD3+ T cell percentage could be applied as a reliable indicator for the early diagnosis of ectopic pregnancy. MTX administration was determined to be an efficacious approach for the treatment of ectopic pregnancy.

Increased miRNA-518b inhibits trophoblast migration and angiogenesis by targeting EGR1 in early embryonic arrest†.

Evidence indicates that microRNAs (miRNAs) play essential roles in early embryonic development. The miRNA-518 family is a special biomarker of the placenta, and miRNA-518b is abnormally expressed in placental tissue in preeclampsia. Early growth response protein 1 (EGR1), a zinc finger transcriptional factor, plays an essential role in regulating cell differentiation, angiogenesis, and migration. Moreover, earlier studies have shown that EGR1 protein plays a key role in implantation. However, little is known about the role of miR-518b and EGR1 on early embryonic arrest (EEA) in humans. In our study, increased miR-518b along with decreased EGR1 was found in human villus tissues with EEA. Furthermore, we demonstrated by luciferase assay that miR-518b is a direct regulator of EGR1. After comparing the effect of silencing EGR1, vascular endothelial growth factor (VEGF) individually, and EGR1/VEGF in combination, we found that EGR1 can inhibit migration and angiogenesis of HTR-8 SVneo cells by decreasing the VEGF expression. Hypoxia plays an initial role in early embryonic development, and we found that hypoxia reduces the expression of miR-518b and increases the expression of EGR1 and VEGF to facilitate migration and angiogenesis in a hypoxic model of HTR-8/SVneo cell line. Our findings provide new insights into the role of miR-518b in EEA and implicate the potential application of miR-518b in the diagnosis and development of intervention for EEA.

Trophoblast-Derived CXCL16 Decreased Granzyme B Production of Decidual γδ T Cells and Promoted Bcl-xL Expression of Trophoblasts.

Decidual γδ T cells are known to regulate the function of trophoblasts at the maternal-fetal interface; however, little is known about the molecular mechanisms of cross talk between trophoblast cells and decidual γδ T cells. Expression of chemokine C-X-C motif ligand 6 (CXCL16) and its receptor CXCR6 was evaluated in first-trimester human villus and decidual tissues by immunohistochemistry. γδ T cells were isolated from first-trimester human deciduae and cocultured with JEG3 trophoblast cells. Cell proliferation and apoptosis-related molecules, together with cytotoxicity factor and cytokine production, were measured by flow cytometry analysis. Expression of CXCL16 and CXCR6 was reduced at the maternal-fetal interface in patients who experienced unexplained recurrent spontaneous abortion as compared to healthy pregnancy women. With the administration of pregnancy-related hormones or coculture with JEG3 cells, CXCR6 expression was upregulated on decidual γδ T cells. CXCL16 derived from JEG3 cells caused a decrease in granzyme B production of decidual γδ T cells. In addition, decidual γδ T cells educated by JEG3-derived CXCL16 upregulated the expression of Bcl-xL in JEG3 cells. This study suggested that the CXCL16/CXCR6 axis may contribute to maintaining normal pregnancy by reducing the secretion of cytotoxic factor granzyme B of decidual γδ T cells and promoting the expression of antiapoptotic marker Bcl-xL of trophoblasts.

Evaluation of chromosomal constitution in choronic villi samples of aborted fetuses after embryo transfers of euploid blastocysts

Introduction Preimplantation genetic testing of aneuploidies (PGT-A) using Next Generation Sequencing (NGS) platforms decreases significantly the risk of abortion from aneuploidy reason, nevertheless some fetuses are unfortunately aborted despite PGT-A from another sakes. NGS platforms provide high dynamic range and are able to detect copy number variations starting at 20% mosaicism. Material of trophectoderm biopsy includes several trophectoderm cells and it is quite often discussed whether such sample is representative enough to reflect the inner cell mass chromosomal status. The aim of this retrospective study is to reveal a representativeness and a significance of a trophectoderm biopsy sample evaluation with respect to the chromosomal constitution during the further embryonic development and assignments of concordance between trophectoderm biopsy day 5-6 and the finding in chorionic villi of aborted fetuses. Material and methods 59 chorionic villi samples were obtained from aborted fetuses during the surgical curettage for incomplete spontaneous or missed early abortions during a period January 2016 till March 2018. All samples were cleaned, washed and stored in a phosphate buffer and after DNA isolation the chromosome copy number was evaluated using 24sure array (Illumina). 42 samples were obtained from fetuses conceived after embryo transfers, 10 embryos were transferred without PGT-A and 32 were analyzed using PGT-A (VeriSeq™ PGS Kit by Illumina) before an embryo transfer. In all study cases a single embryo transfer was performed. All 32 PGT-A embryos were originally diagnosed as euploid. 17 samples were derived from spontaneously conceived embryos. The samples were divided into 3 groups. Group A consists of 10 non-PGT-A samples, group B of 17 samples from spontaneously conceived fetuses and group C of 32 samples from fetuses after PGT-A. Results In total, 23,7 % (14/59) chorionic villi samples were diagnosed as aneuploid. In the group A (non-PGT-A fetuses) 50% (5/10) were aneuploid, in the group B (spontaneous conceptions) 47% (8/17) samples were aneuploid while in the group C (PGT-A fetuses) only 3% (1/32) were aneuploid. Euploid status was concordant in 31 out of 32 PGT-A analyzed embryos (97%). Conclusions Our retrospective study showed that 50% of aborted fetuses after IVF with no PGT-A were aneuploid. This corresponds very well with the aneuploidy rate observed for products of spontaneous conception in infertile couples (47%). Chorionic villi samples from aborted fetuses derived from previously negatively tested blastocysts were aneuploid in 3% of cases (1/32). The concordance between an euploid status of trophectoderm samples and chorionic villi tissue from aborted fetuses is 97%. This confirms the figures referred previously in another studies comparing evaluation of trophectoderm and inner cell mass of the embryo and fully supports routine implementation of PGT-A into assisted reproductive techniques. The slight discordance is possible to be explained by unequal distribution of aneuploid/mosaic cell lines within the trophectoderm tissue at the blastocyst stage when the biopsy is performed.

An Enhanced Approach for Automation the Diagnosis of Iron Deficiency Anemia Based on Quantitative Analysis of Red Blood Cells in Intestine Villi Tissue

Recent years have witnessed a huge revolution in developing automated diagnosis for different diseases such as cancer using medical image processing. Many researchers have been conducted in this field. Analyzing medical microscopic images provide pathology medical track with large information about the status of the patients and the progress of the diseases and help in detecting any pathological changes in tissues. Automation of the diagnosis of these images will lead to a better, faster and enhanced diagnosis for different hematological and histological images. This paper proposes an automated approach for analyzing blood smear microscopic images to help in diagnosing anemia using quantitative analysis of red blood cells in intestine villi tissue. The diagnoses depends on counting the number of blue and red stained blood cells that contain iron in each villi separately, then, it calculates the percentage of blue cells and red cells in the experimented image. The experimental results have shown that using digital image processing techniques through processing the image into different stages as including noise removal, image sharpening, enhancing contrast, find region of interest, isolating color, removing edges, and counting cells leads to a successful outcome and the diagnose of anemia.

The Enhancer RNA lnc-SLC4A1-1 Epigenetically Regulates Unexplained Recurrent Pregnancy Loss (URPL) by Activating CXCL8 and NF-kB Pathway

Backgrounds: Enhancer RNAs (eRNAs) are subsets of lncRNAs and transcribed from enhancers, whose regulatory effects on gene expressions are an emerging area of interest. However, the roles of eRNAs in regulating the trophoblates and unexplained recurrent pregnancy loss (URPL), still remain elusive. Methods: We profiled eRNAs in the villus tissues from URPL patients and matched normal controls by RNA-seq and VISTA Enhancer Browser. Results: We identified lnc-SLC4A1-1, which was transcribed from an active enhancer marked with H3K27ac and H3K4me1 and so-called eRNA, highly expressed in villus of URPL patients. Gain-of-function experiments indicated that lnc-SLC4A1-1 facilitated the trophoblast cells migration and apoptosis. Mechanistically, as eRNAs, lnc-SLC4A1-1 retained in the nuclei and recruited transcription factor NF-κB to bind to CXCL8, resulting in increased H3K27ac in CXCL8 promoter and subsequent elevation of CXCL8 expression. Activation of CXCL8 exacerbated inflammatory reactions in trophoblast cells by inducing TNF-α and IL-1β, whose effects could be blocked by antagonist of lnc-SLC4A1-1. Interpretation: Therefore, these findings indicate that as an eRNA, lncSLC4A1-1 alters trophoblast functions through activation of immune responses and regulating NF-κB/CXCL8 axis. Our study provides new insights in understanding the lncRNA/eRNA's functions in pathological pregnancy, potentially informing on therapeutic strategies for URPL. Funding Statement: This work was supported by the National Natural Science Foundation of China (81671461, 81402706 and 81630085), National Key Research and Development Program (2016YFC1000207) and the Priority Academic Program for the Development of Jiangsu Higher Education Institutions (Public Health and Preventive Medicine). Declaration of Interests: The authors state: None declared. Ethics Approval Statement: This study was approved by the Institutional Ethics Committee of Nanjing Medical University. All activities involved in this study were done under full compliance with government policies and the Helsinki Declaration. All experiments protocol was approved by the Institutional Review Board of Nanjing Medical University prior to the study.

Fetal aneuploidy diagnosed at celocentesis for early prenatal diagnosis of congenital hemoglobinopathies.

Currently, prenatal diagnosis of genetic disorders requires chorionic villus sampling or amniocentesis carried out after 11 and 16weeks of gestation, respectively. Celocentesis is a procedure for prenatal diagnosis that could be used from as early as 7weeks. The present investigation evaluated the feasibility of performing diagnosis for monogenic diseases using celomic fluid containing cells of fetal origin. Analysis consisted of 489 singleton pregnancies undergoing celocentesis for the prenatal diagnosis of hemoglobinopathies (n=367) or before surgical termination of pregnancy for social indications (n=122). Embryo-fetal cells were isolated from celomic fluid using CD71 antibodies or by micromanipulation. Quantitative fluorescent polymerase chain reaction of short tandem repeat sequences of chromosomes 13, 18, 21, X and Y were used to determine the presence of maternal DNA. 357/489 (73%) of celomic fluid samples were contaminated with maternal cells. In two cases, diagnosis was not possible due to the high contamination of celomic fluid. Eighty-seven (23.8%) fetuses were affected by hemoglobinopathies and, in five cases, chromosomal aneuploidies were found, including three cases of trisomy 21, one of trisomy 13 and one of triploidy. In all cases, the diagnosis of hemoglobinopathies and chromosomal abnormalities was confirmed by molecular and traditional cytogenetic analysis after amniocentesis, chorionic villus or placental tissue collection following pregnancy termination. The findings of this study demonstrate that embryo-fetal cell selection from celomic fluid allows reliable and early prenatal diagnosis of hemoglobinopathies and can give more information on any fetal aneuploidy following the control of maternal contamination by quantitative fluorescent-PCR.

Interaction between Kynurenine and Aryl Hydrocarbon Receptor in Regulating the Balance of T helper 17 Cells and Regulatory T-cells in Decidua during Early Gestation

Objective: To investigate whether kynurenine/aryl hydrocarbon receptor (AHR) affects the maternal-fetal tolerance by involving the differentiation of T helper 17 (Th17)/ regulatory T (Treg) cells, and to provide theoretical basic for new treatment of unexplained abortion. Methods: Flow cytometry (FCM) was used to detect the expression of AHR in peripheral/decidual CD4+ T, Treg, and Th17 cells. The effect of Kyn on the differentiation of peripheral/decidual naive T-cells under Treg-/Th17-polarizing condition was detected by FCM; enzyme-linked immunosorbent assay was performed to examine the level of Kyn in villus and decidual tissues from normal pregnancy (NP) and unexplained abortion (UA). Student's t-test in the case of two groups or one-way ANOVA in multiple groups was used. Results: AHR expression in CD4+ T-cells was decreased in decidua versus blood in early pregnancy (P Conclusions: Kyn/AHR promotes Th17 and restricts Treg cells' differentiation, which is involved in maintaining the balance of Treg/Th17 cells at the maternal–fetal interface.

In Utero Transplantation of Placenta-Derived Mesenchymal Stromal Cells for Potential Fetal Treatment of Hemophilia A

Hemophilia A (HA) is an X-linked recessive disorder caused by mutations in the factor VIII (FVIII) gene leading to deficient blood coagulation. The current standard of care is frequent infusions of plasma-derived FVIII or recombinant B-domain-deleted FVIII (BDD-FVIII). While this treatment is effective, many patients eventually develop FVIII inhibitors that limit the effectiveness of the infused FVIII. As a monogenic disorder, HA is an ideal target for gene or cell-based therapy. Several studies have investigated allogeneic stem cell therapy targeting in utero or postnatal treatment of HA but have not been successful in completely correcting HA. Autologous in utero transplantation of mesenchymal stem cells is promising for treatment of HA due to the naive immune status of the fetal environment as well as its potential to prevent transplant rejection and long-term FVIII inhibitor formation. HA can be diagnosed by chorionic villus sampling performed during the first trimester (10 to 13 wk) of gestation. In this study, we used an established protocol and isolated placenta-derived mesenchymal stromal cells (PMSCs) from first trimester chorionic villus tissue and transduced them with lentiviral vector encoding the BDD-FVIII gene. We show that gene-modified PMSCs maintain their immunophenotype and multipotency, express, and secrete high levels of active FVIII. PMSCs were then transplanted at embryonic day 14.5 (E14.5) into wild-type fetuses from time-mated pregnant mice. Four days after birth, pups were checked for engraftment, and varying levels of expression of human green fluorescent protein were found in the organs tested. This study shows feasibility of the approach to obtain PMSCs from first trimester chorionic villus tissue, genetically modify them with the FVIII gene, and transplant them in utero for cell-mediated gene therapy of HA. Future studies will involve evaluation of long-term engraftment, phenotypic correction in HA mice, and prevention of FVIII inhibitor development by this approach.

Chromosomal karyotype in chorionic villi of recurrent spontaneous abortion patients.

Recurrent spontaneous abortion (RSA) is a multifactorial disease of which the exact causes are still unknown. In the current study, we aimed to analyze the distribution of abnormal embryonic karyotypes in RSA. 781 RSA patients of 17 hospitals in Shanghai from January 2014 to September 2016 were enrolled. Fetal villus tissues were collected during uterine curettage and then cultured in situ for karyotyping. All of the 781 cases were successfully cultured. There were 393 cases of abnormal karyotypes, accounting for 50.3% of the total cases. Women with abnormal embryonic karyotype were significantly older compared to those with normal karyotype (P < 0.001). The majority of patients with abnormal karyotype fell among age groups of 25-29 and 30-34. There were 247 cases of aneuploidy, accounting for 62.8% of the total abnormal karyotype cases. Autosomal trisomy was the primary form of aneuploidy (189/247, 76.5%), and the most common types were trisomy-16 (n = 69), trisomy-22 (n = 28), trisomy-21 (n = 21), trisomy-15 (n = 15), and trisomy-13 (n = 10). Abnormal karyotype is a major factor related to RSA. Further studies are needed to elucidate the etiology of RSA in order to achieve more effective prevention and treatment.

CCL2 Enhances the Viability of Human Chorionic Trophoblast Cell Line HTR-8/SVneo Cells by Inhibiting Interleukin-24 Expression

Background: To investigate the regulatory effect of interleukin-24 (IL-24) on cell viability of human chorionic trophoblast cell line (HTR-8/SVneo cells). Methods: Immunohistochemical staining was used to detect the expression of IL-24 and its receptors IL-20R1, IL-20R2, and IL-22R1 in villus tissue at early normal pregnancy. The effect of thymic stromal lymphopoietin (TSLP) and chemokine CCL2 on the expression of IL-24 in human chorionic trophoblast cell line HTR-8/SVneo cells was analyzed by In-cell Western. In addition, the effect of recombinant human IL-24 (rhIL-24) and CCL2 on the viability of HTR-8/SVneo cells was analyzed by MTT assay. Results: IL-24 and its receptors showed a strong positive staining in trophoblasts at early normal pregnancy. Compared with control group, expression of IL-24 in HTR-8/SVneo cells was significantly inhibited after in vitro stimulation of recombinant human CCL2 protein (rhCCL2) (P Conclusions: CCL2 enhances the viability of human trophoblast cell line HTR-8/SVneo cells in vitro by inhibiting the secretion of IL-24, which may be beneficial to blastocyst implantation and placental development.

Abnormal expression of microRNA‑575 leads to missed abortion through regulating apoptosis and angiogenesis

Numerous microRNA (miR) are important for placental development and function. miR-575 has been demonstrated to be upregulated in maternal placenta in patients who have experienced a miscarriage. The present study aimed to explore the role of abnormal expression of miR-575 in missed abortion (MA) and to further analyze the potential molecular mechanisms. Embryo villus tissue samples were extracted from 10 childless women with MA and 10 fertile women without a history of MA. Additionally, human choriocarcinoma cells, JEG-3, were used in the present study, which were transfected with miR-575 mimic, inhibitor and scramble. The expression of miR-575 in embryo villus tissues and in JEG-3 cells was detected by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Apoptosis in villus tissues of patients with MA and in JEG-3 cells of miR-575 mimic, inhibitor and scramble groups were detected by flow cytometry. Furthermore, the expression levels of apoptosis-related proteins, including B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X protein (Bax) and phosphorylated (p)-p53, and angiogenesis-related proteins, including vascular endothelial growth factor (VEGF) and angiopoietin 2 (Ang-2), were measured by RT-qPCR and western blotting. Additionally, the target of miR-575 was predicted and clarified by luciferase reporter assay. miR-575 was significantly overexpressed in MA villus tissue compared with normal tissue (P<0.05). The percentage of apoptotic cells in MA embryo villus tissue was significantly higher than that in normal tissue (P<0.05). After JEG-3 cells were transfected with miR-575 inhibitor, the expression of miR-575 and the percentage of apoptotic cells decreased significantly compared with the control (P<0.05). MiR-575 suppression significantly increased the expression of Bcl-2 (P<0.05), and decreased the expressions of Bax (P<0.05) and p-p53 (P<0.01) compared with the control. Furthermore, miR-575 suppression significantly increased the expressions of angiogenesis-related proteins, Ang-2 and VEGF (P<0.01). Superoxide dismutase 2 was identified as the target of miR-575. Therefore, abnormal expression of miR-575 may lead to MA through regulating apoptosis and angiogenesis. Inhibition of miR-575 may inhibit apoptosis and promote angiogenesis in MA.

Effect of the p53-tristetraprolin-stathmin-1 pathway on trophoblasts at maternal-fetal interface.

ProblemTo reveal the effect of p53–tristetraprolin–stathmin-1 signaling on trophoblasts and recurrent spontaneous abortion (RSA).Method of studyStathmin-1 (STMN1), p53, and tristetraprolin (TTP) expression in paraffin-embedded villus tissue was determined using immunohistochemistry. HTR-8/SVneo cells were treated with doxorubicin to activate p53; STMN1 and TTP levels were detected by quantitative reverse transcription–PCR and western blotting. Western blotting and immunofluorescence were used to investigate STMN1 expression after TTP overexpression or knockdown in HTR-8 cells.ResultsSTMN1 was downregulated and p53 was upregulated in the villus tissue from patients with RSA. Doxorubicin decreased STMN1 expression but enhanced TTP expression in HTR-8 cells. In vitro, TTP overexpression inhibited STMN1 production; TTP knockdown promoted it. TTP downregulated STMN1 expression in trophoblasts by directly binding its 3ʹ untranslated region.ConclusionsTTP modulates trophoblast function and interacts with STMN1 and p53, and is related to pregnancy outcomes.

Synthetic Receptors Induce Anti Angiogenic and Stress Signaling on Human First Trimester Cytotrophoblast Cells.

The cytotrophoblast (CTB) cells of the human placenta have membrane receptors that bind certain cardiotonic steroids (CTS) found in blood plasma. One of these, marinobufagenin, is a key factor in the etiology of preeclampsia. Herein, we used synthetic receptors (SR) to study their effectiveness on the angiogenic profile of human first trimester CTB cells. The humanextravillous CTB cells (Sw.71) used in this study were derived from first trimester chorionic villus tissue. Culture media of CTB cells treated with ≥1 nM SR level revealed sFlt-1 (Soluble fms-like tyrosine kinase-1) was significantly increased while VEGF (vascular endothelial growth factor) was significantly decreased in the culture media (* p < 0.05 for each) The AT2 receptor (Angiotensin II receptor type 2) expression was significantly upregulated in ≥1 nM SR-treated CTB cells as compared to basal; however, the AT1 (Angiotensin II receptor, type 1) and VEGFR-1 (vascular endothelial growth factor receptor 1) receptor expression was significantly downregulated (* p < 0.05 for each). Our results show that the anti-proliferative and anti-angiogenic effects of SR on CTB cells are similar to the effects of CTS. The observed anti angiogenic activity of SR on CTB cells demonstrates that the functionalized-urea/thiourea molecules may be useful as potent inhibitors to prevent CTS-induced impairment of CTB cells.

Manufacture and preparation of human placenta-derived mesenchymal stromal cells for local tissue delivery

BackgroundIn this study we describe the development of a Current Good Manufacturing Practice (CGMP)-compliant process to isolate, expand and bank placenta-derived mesenchymal stromal cells (PMSCs) for use as stem cell therapy. We characterize the viability, proliferation and neuroprotective secretory profile of PMSCs seeded on clinical-grade porcine small intestine submucosa extracellular matrix (SIS-ECM; Cook Biotech). MethodsPMSCs were isolated from early gestation placenta chorionic villus tissue via explant culture. Cells were expanded, banked and screened. Purity and expression of markers of pluripotency were determined using flow cytometry. Optimal loading density and viability of PMSCs on SIS-ECM were determined using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) cell proliferation and fluorescent live/dead assays, respectively. Growth factors secretion was analyzed using enzyme-linked immunosorbent assays (ELISA). ResultsPMSCs were rapidly expanded and banked. Viable Master and Working Cell Banks were stable with minimal decrease in viability at 6 months. All PMSCs were sterile, free from Mycoplasma species, karyotypically normal and had low endotoxin levels. PMSCs were homogeneous by immunophenotyping and expressed little to no pluripotency markers. Optimal loading density on SIS-ECM was 3–5 × 105 cells/cm2, and seeded cells were >95% viable. Neurotrophic factor secretion was detectable from PMSCs seeded on plastic and SIS-ECM with variability between donor lots. DiscussionPMSCs from early gestation placental tissues can be rapidly expanded and banked in stable, viable cell banks that are free from contaminating agents, genetically normal and pure. PMSC delivery can be accomplished by using SIS-ECM, which maintains cell viability and protein secretion. Future work in vivo is necessary to optimize cell seeding and transplantation to maximize therapeutic capabilities.

Effect of 2-octylcyanoacrylate on placenta derived mesenchymal stromal cells on extracellular matrix

PurposeDetermine the effect of 2-octylcyanoacrylate on placenta derived mesenchymal stromal cells (PMSCs) seeded onto extracellular matrix (ECM) in order to assess its biocompatibility as a potential adhesive for in-vivo fetal cell delivery. MethodsPMSCs isolated from chorionic villus tissue were seeded onto ECM. A MTS proliferation assay assessed cellular metabolic activity at various time points in PMSC-ECM with direct, indirect, and no glue contact. Conditioned media collected prior to and 24 hours after glue exposure was analyzed for secretion of human brain-derived neurotrophic factor, hepatocyte growth factor, and vascular endothelial growth factor. ResultsDirect and indirect contact with 2-octylcyanoacrylate results in progressively decreased cellular metabolic activity over 24 hours compared to no glue controls. Cells with direct contact are less metabolically active than cells with indirect contact. 24 hours of glue exposure resulted in suppression of growth factor secretion that is near complete with direct contact. DiscussionExposure to 2-octylcyanoacrylate results in decreased metabolic activity and decreased measurable secretion of growth factors by PMSCs seeded onto ECM. Thus, the application of 2-octylcyanoacrylate glue should be limited when working with cell-engineered scaffolds as its inhibitory effects on cell growth and secretory function can limit the therapeutic potential of cell-based interventions.

Uterine Expression of NDRG4 Is Induced by Estrogen and Up-Regulated during Embryo Implantation Process in Mice

Embryo implantation is an essential step for the establishment of pregnancy and dynamically regulated by estrogen and progesterone. NDRG4 (N-myc down-regulated gene 4) is a tumor suppressor that participates in cell survival, tumor invasion and angiogenesis. The objective of this study was to preliminarily explore the role of NDRG4 in embryo implantation. By immunohistochemistry (IHC) and quantitive RT-PCR (qRT-PCR), we found that uterine expression of NDRG4 was increased along with puberal development, and its expression in adult females reached the peak at the estrus stage during the estrus cycle. Furthermore, uterine NDRG4 expression was significantly induced by the treatment of estradiol (E2) both in pre-puberty females and ovariectomized adult females. Uterine expression pattern of NDRG4 during the peri-implantation period in mice was determined by IHC, qRT-PCR and Western blot. It was observed that NDRG4 expression was up-regulated during the implantation process, and its expression level at the implantation sites was significantly higher than that at the inter-implantation sites. Meanwhile, an increased expression in NDRG4 was associated with artificial decidualization as well as the activation of delayed implantation. By qRT-PCR and Western blot, we found that the in vitro decidualization of endometrial stromal cells (ESCs) was accompanied by up-regulation of NDRG4 expression, whereas knockdown of its expression in these cells by siRNA inhibited the decidualization process. In addition, Western blot analysis showed that NDRG4 protein expression was decreased in human villus tissues of recurrent miscarriage (RM) patients compared to normal pregnant women. Collectively, these data suggested that uterine NDRG4 expression could be induced by estrogen, and NDRG4 might play an important role during early pregnancy.

Expression of centromere protein-C (CENP-C) in villus tissue of the first-trimester spontaneous abortion and its correlation with chromosome segregation

Objective To investigate the expression of centromere protein-C (CENP-C) in villus tissue of the first-trimester spontaneous abortion (SA) and the correlation study of CENP-C expression with chromosome segregation. Methods Fluorescence in situ hybridization (FISH) and G-banded karyotype analysis were used to detect the numerical chromosome abnormality in 94 villus tissues of women with SA. The participants were separated into case group (n=30) and control group (n=30) according to the results with FISH. The qRT-PCR and Western blotting analysis were used to assess the expression level of CENP-C. Results Forty-eight (51.06%) cases had observed the numerical chromosome abnormality, including 30 positive cases and the positive rate was 31.91%. The main types of variation included trisomy 16, 21, 22, X monosomy and triploid. The expression levels of CENP-C mRNA and protein in case group were statistically higher than that in control group (P Conclusion Expression of CENP-C in the villus tissues of women might be related to SA induced by chromosomal aneuploid.

Deep-sequencing identification of differentially expressed miRNAs in decidua and villus of recurrent miscarriage patients.

PurposeMicroRNAs (miRNAs) are small non-coding RNA molecules that play critical roles in post-transcriptional gene expression regulation. The aim of this study was to identify differentially expressed miRNAs in decidua and villus of recurrent miscarriage (RM) patients.MethodsParticipants were recruited at the outpatient Department of Gynecology and Obstetrics, The Second Hospital of Tianjin Medical University, China. Decidua and villus tissues were collected by curettage from recruited RM patients and normal pregnant women with their informed consent. MiRNAs expression profiles in decidua or villus were respectively determined by the deep-sequencing analysis. The predicated target genes of these differentially expressed miRNAs were analyzed by miRWalk. The differential expressions of four miRNAs in decidua and four miRNAs in villus between the six pairs of RM patients and normal pregnant women were confirmed by qRT-PCR analysis. The expression patterns of two predicated target genes, Bcl-2 and Pten, in the same six pairs of decidual or villus tissues were detected by Western blotting analysis, respectively.ResultsTotally 18 RM patients and 15 normal pregnant women were recruited. Thirty-two miRNAs in decidua and four miRNAs in villus of RM patients were screened out to be significantly up-regulated compared to that of normal pregnant women, and five miRNAs in villus of RM patients were screened out to be remarkably down-regulated compared to that of normal pregnant women (P value < 0.05 and Fold change >2). These differentially expressed miRNAs were predicted to target a large number of genes that involved in cell apoptosis, p53 signaling pathway, cell cycle and other cellular bio-functions. Differential expressions of hsa-miR-516a-5p, -517a-3p, -519a-3p and -519d in decidua, as well as hsa-miR-1, -372, -100-5p and -146a-5p in villus, were validated by qRT-PCR analysis. In the decidual of RM patients, expression of hsa-miR-516a-5p, -517a-3p, -519a-3p and -519d were significantly up-regulated compared to normal pregnancy. In the villi of RM patients, expression of hsa-miR-100 and -146a-5p were significantly higher, while hsa-miR-1 and -372 were significantly lower compared to normal pregnancy. Furthermore, the expression of Bcl-2 and Pten, a predicated target gene of hsa-miR-1 or hsa-miR-372 respectively, was significantly up-regulated in the villi of RM patients.ConclusionsThese data suggested that the pathogenic process of RM might be associated with the alteration of miRNAs expression profiles in decidua and villus. Especially, the aberrant placental expression of hsa-miR-1 and -372 might be involved in the progression of RM, but need to be further investigated by larger studies in the future.