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

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

OBJECTIVETo evaluate the feasibility of rapid prenatal diagnosis of chromosome aneuploidies by interphase fluorescence in situ hybridization (FISH) using uncultured amniotic fluid.

METHODSBacterial artificial chromosome (BAC) DNA probes were prepared and validated by using cultured peripheral blood. Interphase FISH for chromosomes 13, 18, 21, X and Y was performed in 60 amniotic fluid samples for the rapid prenatal diagnosis of chromosome aneuploidies, and the results were compared with the karyotypes from conventional cytogenetic analysis.

RESULTSOf all 60 cases, 58 were concordant with their karyotypes, and 1 case of inv(9) and another case of t(2,12) were identified by karyotyping. Two cases of trisomy 21 and 1 case of trisomy 18 were detected by FISH and confirmed with conventional cytogenetics (sensitivity=100%). There were no false-positive or false-negative results.

CONCLUSIONThis evaluation demonstrated that FISH employing BAC DNA probes could accurately and rapidly detect aneuploidies involving the above 5 chromosomes. However, as it does not identify structural chromosome aberrations and aneuploidies involving other chromosomes, it is not a substitute for conventional chromosome analysis, and the negative FISH result should be carefully interpreted.

Adult ; Amniotic Fluid ; cytology ; metabolism ; Aneuploidy ; Culture Techniques ; Female ; Humans ; In Situ Hybridization, Fluorescence ; methods ; Karyotyping ; Male ; Pregnancy ; Prenatal Diagnosis ; methods ; Time Factors

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*

Rapid prenatal diagnosis of common chromosome aneuploidies have been successful through quantitative fluoresent PCR (QF-PCR) assays and small tandem repeat (STR) markers. The purpose of our study was to investigate the clinical feasibility for rapid prenatal detection of Down syndrome using the quantitative fluorescent PCR in uncultured amniocytes. DNA was extracted from uncultured amniotic fluid of normal karyotype (n=200) and of Down syndrome (n=21). It was amplified using QF-PCR with four STR markers located on chromosome 21. Among normal samples, the ranges of diallelic peaks for at least one STR marker were 1.0-1.3 for D21S11, 1.0-1.4 for D21S1411 and 1.0-1.5 for D21S1270. Down syndrome samples showed trisomic triallelic patterns or trisomic diallelic patterns. The sensitivity, specificity, and efficiency of the assay for detecting Down syndrome were 95.4%, 100%, and 99.5%, respectively. Rapid prenatal diagnosis of Down syndrome using QF-PCR is a reliable technique that aids clinical management of pregnancy.
Alleles ; Amniotic Fluid/*cytology ; Chromosomes, Human, Pair 21 ; DNA/metabolism ; Down Syndrome/*diagnosis ; Female ; Human ; Korea ; Microscopy, Fluorescence/*methods ; Polymerase Chain Reaction/*methods ; Polymorphism (Genetics) ; Pregnancy ; Prenatal Diagnosis/*methods ; Sensitivity and Specificity ; Support, Non-U.S. Gov't ; Tandem Repeat Sequences ; Time Factors

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

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

Complete or partial triplication of human chromosome 21 results in Down syndrome (DS). To analyze differential gene expressions in amniotic fluid (AF) cells of DS, we used a DNA microarray system to analyze 102 genes, which included 24 genes on chromosome 21, 28 genes related to the function of brain and muscle, 36 genes related to apoptosis, 4 genes related to extracellular matrix, 8 genes related to other molecular function and 2 house-keeping genes. AF cells were collected from 12 pregnancies at 16-18 weeks of gestation in DS (n=6) and normal (n=6) subjects. Our DNA microarray experiments showed that the expressions of 11 genes were altered by at least 2-folds in DS, as follows. Ten genes, COL6A1, CASP5, AKT2, JUN, PYGM, BNIP1, OSF-2, PRSS7, COL3A1, and MBLL were down-regulated and GSTT1 was only up-regulated. The differential expressions of GSTT1 and COL3A1 were further confirmed by semi-quantitative RT-PCR for each sample. The gene dosage hypothesis on chromosome 21 may explain the neurological and other symptoms of DS. However, our results showed that only two genes (COL6A1 and PRSS7), among 24 genes on chromosome 21, were down-regulated in the AF cells of DS. Our data may provide the basis for a more systematic identification of biological markers of fetal DS, thus leading to an improved understanding of pathogenesis for fetal DS.
Amniocentesis ; Amniotic Fluid/*cytology/*metabolism ; Apoptosis ; Cells, Cultured ; Chromosomes, Human, Pair 21 ; Collagen Type III/biosynthesis ; DNA, Complementary/metabolism ; Down Syndrome/*genetics/metabolism ; Down-Regulation ; Gene Dosage ; Gene Expression ; *Gene Expression Regulation ; Glutathione Transferase/biosynthesis ; Humans ; Models, Genetic ; *Oligonucleotide Array Sequence Analysis ; Research Support, Non-U.S. Gov't ; Reverse Transcriptase Polymerase Chain Reaction ; Time Factors ; Up-Regulation

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