OBJECTIVE: The purpose of this study was to determine the role of Ureaplasma urealyticum-derived lipid-associated membrane proteins (LAMPs) in the host innate immune system, specifically their effect on Toll-like receptors (TLRs). METHODS: LAMPs were derived from U. urealyticum strains, and human amniotic epithelial cells (HAECs) were isolated from healthy full-term placentas. Cytokine concentrations were determined by enzyme-linked immunosorbent assay (ELISA) and TLR2 mRNA by real-time PCR. Expression of TLR2 was confirmed by Western blotting and immunohistochemistry. RESULTS: LAMPs induced HAECs to produce inflammatory cytokines interleukin (IL)-6, IL-8, and tumor necrosis factor (TNF)-α. Cytokine production was reduced after blocking TLR2 using TLR2 inhibitor (anti-hTLR2-IgA). CONCLUSIONS LAMPs isolated from U. urealyticum induced TLR2-dependent up-regulation of inflammatory genes and cytokines in HAECs.
Amnion/cytology* ; Amniotic Fluid/cytology* ; Cytokines/metabolism* ; Dose-Response Relationship, Drug ; Epithelial Cells/metabolism* ; Female ; Humans ; Inflammation ; Interleukin-6/metabolism* ; Interleukin-8/metabolism* ; Lipids/chemistry* ; Lipopolysaccharides/metabolism* ; Membrane Proteins/metabolism* ; Placenta/metabolism* ; Pregnancy ; Toll-Like Receptor 2/metabolism* ; Tumor Necrosis Factor-alpha/metabolism* ; Up-Regulation ; Ureaplasma urealyticum/metabolism*

PURPOSE: To develop a model based on non-invasive clinical and ultrasonographic parameters for predicting the likelihood of subsequent histologic chorioamnionitis in women with preterm premature rupture of membranes (PPROM) and to determine whether the inclusion of invasive test results improves the predictive value of the model. MATERIALS AND METHODS: This retrospective cohort study included 146 consecutive women presenting with PPROM (20-33 weeks). Transvaginal ultrasonographic assessment of cervical length was performed. Maternal serum C-reactive protein (CRP) levels and white blood cell (WBC) counts were measured after amniocentesis. Amniotic fluid (AF) obtained by amniocentesis was cultured, and interleukin-6 (IL-6) levels and WBC counts were determined. The primary outcome measure was histologic chorioamnionitis. RESULTS: Risk scores based on serum CRP concentrations and gestational age (model 1) were calculated for each patient. The model was shown to have adequate goodness of fit and an area under the receiver operating characteristic curve (AUC) of 0.742. When including AF test results (e.g., AF IL-6 levels) in model 1, serum CRP concentrations were found to be insignificant, and thus, were excluded from model 2, comprising AF IL-6 levels and gestational age. No significant difference in AUC was found between models 1 and 2. CONCLUSION: For women with PPROM, the newly developed model incorporating non-invasive parameters (serum CRP and gestational age) was moderately predictive of histologic chorioamnionitis. The inclusion of invasive test results added no predictive information to the model in this setting.
Adult ; *Amniocentesis ; Amniotic Fluid/*cytology/microbiology ; C-Reactive Protein/*metabolism ; Chorioamnionitis/blood/*diagnosis/metabolism ; Cohort Studies ; Female ; Fetal Membranes, Premature Rupture/*blood ; *Gestational Age ; Humans ; Infant, Newborn ; Interleukin-6/blood ; Leukocyte Count ; Predictive Value of Tests ; Pregnancy ; ROC Curve ; Retrospective Studies ; Sensitivity and Specificity

OBJECTIVETo explore the genetic cause for a fetus with structural anomaly, and to correlate the phenotype with the genotype.

METHODSAmniotic fluid was obtained following the revelation of structural anomaly by ultrasonography. Cell culture and direct DNA extraction were performed in parallel. G-banded karyotyping analysis and chromosome microarray analysis (CMA) were subsequently carried out.

RESULTSG-banded karyotyping has suggested the fetus to be a normal male. However, CMA analysis has revealed the presence of a mosaic 3.24 Mb duplication of 1p36.33p36.32 (24%) and uniparental disomy (UPD) of chromosome 6. The genetic diagnosis for the fetus was therefore 46,XY, arr 1p36.33 p36.32(849,466-4,090,472)×2-3, (6)×2 hmz. The anomaly can probably explain the ultrasound findings in the fetus.

CONCLUSIONCompared with conventional cytogenetic methods, CMA has greater resolution and throughput, and can serve as a more efficient platform for the detection of chromosomal microdeletion, microduplication, loss of heterozygosity and UPD.

Adult ; Amniotic Fluid ; cytology ; metabolism ; Chromosome Aberrations ; Chromosome Duplication ; Chromosomes, Human, Pair 1 ; genetics ; Chromosomes, Human, Pair 6 ; genetics ; Female ; Fetal Diseases ; diagnosis ; genetics ; Humans ; Karyotyping ; Oligonucleotide Array Sequence Analysis ; methods ; Polymorphism, Single Nucleotide ; Pregnancy ; Prenatal Diagnosis ; methods ; Reproducibility of Results ; Sensitivity and Specificity ; Uniparental Disomy

OBJECTIVETo compare the results of fluorescence in situ hybridization (FISH) assay and conventional karyotyping analysis for the detection of chromosomal aneuploidies.

METHODSIn total 2607 amniotic fluid samples were subjected to an improved FISH technique. Meanwhile, karyotype analysis was also ordered for each sample.

RESULTSOf the 2607 samples, 62 abnormalities were identified by FISH, which included 62 cases of trisomy 21, 5 cases of 45,X, 12 cases of trisomy 18, 3 cases of trisomy 13, and 1 case of 47, XYY. Conventional karyotyping analysis has identified 63 cases of trisomy 21, 5 cases of 45,X, 12 cases of trisomy 18, 3 cases of trisomy 13, 1 case of 47, XYY, and 57 cases of balanced translocations. The success rate of FISH detection was 98.4% for trisomy 21, and 100% for 45,X, trisomy 18 and trisomy 13.

CONCLUSIONFor the detection of chromosomal aneuploidies, FISH assay is quick, simple, accurate and can reduce workload when aminocyte culture has failed. As an auxiliary method for amniocytic analysis, it can provide reference for the consultation of those with advanced age and high pregnancy risk.

Adult ; Amniocentesis ; methods ; Amniotic Fluid ; cytology ; metabolism ; Chromosomes, Human, Pair 18 ; genetics ; Chromosomes, Human, Pair 3 ; genetics ; Chromosomes, Human, Y ; genetics ; Down Syndrome ; genetics ; Female ; Fetal Diseases ; diagnosis ; genetics ; Humans ; In Situ Hybridization, Fluorescence ; methods ; Karyotype ; Karyotyping ; methods ; Middle Aged ; Pregnancy ; Reproducibility of Results ; Sensitivity and Specificity ; Sex Chromosome Aberrations ; Trisomy ; genetics ; Trisomy 18 Syndrome ; Turner Syndrome ; genetics ; Young Adult

OBJECTIVETo use different technologies to distinguish true and pseudo mosaicisms among cultured amniocytes in order to attain more accurate diagnosis.

METHODSWith informed consent, 20 mL of amniotic fluid was obtained from pregnant women at between 18 to 24 gestational week. Each amniotic fluid sample was processed as two separate lines for the culturing, observation, harvesting and analysis. All procedures were conducted conforming to the Technology Standards of Cytogenetic Prenatal Diagnosis of Fetal Chromosome Abnormalities issued by the Ministry of Health in 2010. Umbilical cord blood, fluorescence in situ hybridization (FISH), single nucleotide polymorphism array (SNP-array) and flow cytometer were applied when necessary.

RESULTSAmong 3910 cases, 128(3.3%) were detected as mosaicisms. Further analysis with the above technologies has verified 6 cases as true mosaicisms and the remaining 120 as pseudomosaicisms. For one case detected by karyotype analysis as 47, XXY/46, XY, the ratio of different cell lines was confirmed by FISH as 1:2. Another case, detected by karyotype analysis as 47, XX,+mar/46, XX (1:1), was verified by SNP-array as 18p duplication. A suspected polyploidy mosaicism was rejected by flow cytometry and cord blood karyotyping.

CONCLUSIONTwo separate cell cultures are important for distinguishing true and pseudo mosaicisms. Combined FISH, SNP-array and flow cytometry can attain more reliable and accurate diagnosis for mosaicisms.

Adult ; Amniotic Fluid ; cytology ; metabolism ; Cells, Cultured ; Chromosome Disorders ; diagnosis ; embryology ; genetics ; Chromosomes, Human, Pair 18 ; genetics ; Cytogenetic Analysis ; methods ; Female ; Fetal Diseases ; diagnosis ; genetics ; Genetic Testing ; methods ; Gestational Age ; Humans ; In Situ Hybridization, Fluorescence ; Karyotype ; Karyotyping ; Microarray Analysis ; methods ; Mosaicism ; Polymorphism, Single Nucleotide ; Pregnancy ; Prenatal Diagnosis ; methods ; Trisomy ; diagnosis ; genetics ; Trisomy 18 Syndrome

We established methods to isolate human amniotic fluid-derived progenitor cells (hAFPCs), and analyze the ability of hAFPCs to secrete human coagulation factor IX (hFIX) after gene modification. The hAFPCs were manually isolated by selection for attachment to gelatin coated culture dish. hFIX cDNA was transfected into hAPFCs by using a lentiviral vector. The hFIX protein concentration and activity produced from hAFPCs were determined by enzyme-linked immunosorbent assay (ELISA) and clotting assay. The isolated spindle-shaped cells showed fibroblastoid morphology after three culture passages. The doubling time in culture was 39.05 hours. Immunocytochemistry staining of the fibroblast-like cells from amniotic fluid detected expression of stem cell markers such as SSEA4 and TRA1-60. Quantitative PCR analysis demonstrated the expression of NANOG, OCT4 and SOX2 mRNAs. Transfected hAFPCs could produce and secrete hFIX into the culture medium. The observed concentration of secreted hFIX was 20.37% +/- 2.77% two days after passage, with clotting activity of 16.42% +/- 1.78%. The amount of hFIX:Ag reached a plateau of 50.35% +/- 5.42%, with clotting activity 45.34% +/- 4.67%. In conclusion, this study established method to isolate and culture amniotic fluid progenitor cells. Transfected hAFPCs can produce hFIX at stable levels in vitro, and clotting activity increases with higher hFIX concentration. Genetically engineered hAFPC are a potential method for prenatal treatment of hemophilia B.
Amniotic Fluid ; cytology ; Blood Coagulation ; Cell Culture Techniques ; Cell Separation ; methods ; DNA, Complementary ; Factor IX ; biosynthesis ; Genetic Engineering ; Genetic Vectors ; Humans ; Stem Cells ; cytology ; metabolism ; Transfection

The aim of this study was to determine whether intra-amniotic infection/inflammation (IAI) was associated with subsequent ruptured membranes in women with preterm labor and intact membranes who had a clinically indicated amniocentesis. This retrospective cohort study included 237 consecutive women with preterm labor (20-34.6 weeks) who underwent amniocentesis. The clinical and laboratory parameters evaluated included demographic variables, gestational age, C-reactive protein (CRP) and amniotic fluid (AF) white blood cell, interleukin-6 (IL-6) and culture results. IAI was defined as a positive AF culture and/or an elevated AF IL-6 level (>2.6 ng/mL). The primary outcome was ruptured membranes in the absence of active labor occurring within 48 hours of amniocentesis. Preterm premature rupture of membranes subsequently developed in 10 (4.2%) women within 48 hr of amniocentesis. Multivariate analysis demonstrated that only IAI was independently associated with the ruptured membranes occurring within 48 hr of amniocentesis. In the predictive model based on variables assessed before amniocentesis, only CRP level was retained. IAI is an independent risk factor for subsequent ruptured membranes after clinically indicated amniocentesis in preterm labor. Prior to amniocentesis, measurement of serum CRP level can provide a risk assessment for the subsequent development of ruptured membranes after the procedure.
Adult ; Amniocentesis/*adverse effects ; Amnion/physiopathology ; Amniotic Fluid/cytology/metabolism/microbiology ; Bacterial Infections/*etiology/microbiology ; C-Reactive Protein/analysis ; Cohort Studies ; Demography ; Female ; Gestational Age ; Humans ; Inflammation/*etiology ; Interleukin-6/metabolism ; Leukocytes/cytology ; Multivariate Analysis ; Mycoplasma/isolation & purification ; Obstetric Labor, Premature/*etiology ; Pregnancy ; ROC Curve ; Retrospective Studies ; Risk Factors ; Ureaplasma urealyticum/isolation & purification

The most promising treatment for stress urinary incontinence can be a cell therapy. We suggest human amniotic fluid stem cells (hAFSCs) as an alternative cell source. We established the optimum in vitro protocol for the differentiation from hAFSCs into muscle progenitors. These progenitors were transplanted into the injured urethral sphincter and their therapeutic effect was analyzed. For the development of an efficient differentiation system in vitro, we examined a commercial medium, co-culture and conditioned medium (CM) systems. After being treated with CM, hAFSCs were effectively developed into a muscle lineage. The progenitors were integrated into the host urethral sphincter and the host cell differentiation was stimulated in vivo. Urodynamic analysis showed significant increase of leak point pressure and closing pressure. Immunohistochemistry revealed the regeneration of circular muscle mass with normal appearance. Molecular analysis observed the expression of a larger number of target markers. In the immunogenicity analysis, the progenitor group had a scant CD8 lymphocyte. In tumorigenicity, the progenitors showed no teratoma formation. These results suggest that hAFSCs can effectively be differentiated into muscle progenitors in CM and that the hAFSC-derived muscle progenitors are an accessible cell source for the regeneration of injured urethral sphincter.
Amniotic Fluid/*cytology ; Animals ; Biological Markers/metabolism ; Cell Differentiation ; Cell Lineage ; Cell Transformation, Neoplastic ; Cells, Cultured ; Coculture Techniques ; Female ; Gene Expression Regulation ; Humans ; Immunohistochemistry ; Mice ; Mice, Inbred ICR ; Regeneration ; *Stem Cell Transplantation ; Stem Cells/*cytology/metabolism ; Urethra/physiology ; Urinary Incontinence, Stress/pathology/*therapy ; Urodynamics

OBJECTIVEThis research intends to amplify the entire coding region sequences of SLC25A13 mRNA which encodes citrin, and to investigate sequence features of the transcripts for this gene in cultured human amniocytes. This study will provide laboratory evidence for prenatal diagnosis of neonatal intrahepatic cholestasis caused by citrin deficiency (NICCD) at mRNA level.

METHODSOne amniocyte sample was collected from a pregnant woman who underwent prenatal diagnosis of citrin deficiency and whose fetus has proven a carrier of 851del4 mutation by genomic DNA analysis. Another amniocyte sample, as a control, was from a fetus without family history of citrin deficiency. Total RNA was extracted from cultured amniocytes, cDNA was synthesized, and then nested-PCR was performed to amplify the entire coding region sequences of SLC25A13. The PCR products were cloned and analyzed by sequencing.

RESULTSThe entire coding region of SLC25A13 gene was successful amplified from two cultured human amniocytes. The splice variant of SLC25A13, SLCA (normal mRNA), was identified in the two samples. SLCB (CAG insertion between exon 9-10) was identified in the control. SLCC (exon 5-11 skipping), but not transcriptional product from the allele with 851del4 mutation, was identified in the 851del4 mutation carrier.

CONCLUSIONSThis study demonstrated that the entire coding region of SLC25A13 cDNA can be successfully amplified from two cultured human amniocytes, and revealed exon 5-11 skipping as a novel SLC25A13 transcript. Normal mRNA predominated in the transcripts in normal control and 851del4 mutation carrier, suggesting that the two fetuses were not at risk for NICCD. These SLC25A13 transcription features provided laboratory evidence for prenatal diagnosis of NICCD.

Amniotic Fluid ; cytology ; metabolism ; Calcium-Binding Proteins ; deficiency ; Cholestasis, Intrahepatic ; diagnosis ; Cloning, Molecular ; Female ; Humans ; Mitochondrial Membrane Transport Proteins ; genetics ; Organic Anion Transporters ; deficiency ; Polymerase Chain Reaction ; Pregnancy ; Prenatal Diagnosis ; methods ; RNA, Messenger ; analysis ; Sequence Analysis, DNA ; Transcription, Genetic

OBJECTIVETo reprogram amniotic fluid cells into pluripotent stem cells in order to create an optimal internal control model for directed cell differentiation.

METHODSHuman amniotic fluid-derived cells (hAFDCs) from heterozygotic twin fetuses were induced by retroviral vectors encoding Oct4, Sox2, c-Myc and Klf4. In vivo pluripotency, differentiation capacity and karyotype of hAFDCs induced pluripotent stem cells (hAFDCs-iPSCs) were determined.

RESULTShAFDC-iPSCs derived from heterozygotic twins have maintained self renewal, with expression of high pluripotency marker gene detected at both mRNA and protein levels. The cells have maintained their differentiation capacity both in vitro and vivo, and showed normal karyotypes after long-term culturing in vitro.

CONCLUSIONhAFDCs-iPSCs derived from heterozygotic twins have good consistency in terms of genetic background, and can provide a good internal control for directed differentiation of iPSCs, and may be used an ideal source for autologous cell replacement therapy in the later life of the fetus.

Amniotic Fluid ; cytology ; metabolism ; Cell Differentiation ; genetics ; Cell Line ; Female ; Fetus ; metabolism ; Heterozygote ; Humans ; Induced Pluripotent Stem Cells ; cytology ; metabolism ; Karyotype ; Pluripotent Stem Cells ; cytology ; metabolism ; Pregnancy ; Twins