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*

OBJECTIVETo diagnose chromosomal abnormalities in amniotic fluid cells by combining karyotyping and single nucleotide polymorphism array (SNP-array) analysis, and to explore the application of SNP-array in routine clinical practice.

METHODSConventional G banding was used to karyotype a fetal amniotic fluid sample and the corresponding peripheral blood samples from the parents, followed by SNP-array analysis of the fetal genomic DNA from the amniotic fluid.

RESULTSThe karyotype of the amniocytes was 47, XX, +mar. The marker chromosome was further identified as psu idic (22) (q11.2) by SNP-array analysis, revealing tetraploidy of a 1.7 Mb fragment in 22q11.1-q11.2 interval that involves the critical region for Cat eye syndrome.

CONCLUSIONA rare chromosomal abnormality was identified by combining conventional G banding and SNP-array. The high resolution SNP-array could provide more detailed information for determining the origin of chromosomal abnormalities.

Adult ; Amniotic Fluid ; cytology ; Aneuploidy ; Chromosome Disorders ; genetics ; Chromosomes, Human, Pair 22 ; genetics ; Eye Abnormalities ; genetics ; Female ; Humans ; Isochromosomes ; Karyotyping ; Polymorphism, Single Nucleotide ; Pregnancy ; Tetraploidy

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

PURPOSE: Stem cell-based therapies represent new promises for the treatment of urinary incontinence. This study was performed to assess optimized cell passage number, cell dose, therapeutic efficacy, feasibility, toxicity, and cell trafficking for the first step of the pre-clinical evaluation of human amniotic fluid stem cell (hAFSC) therapy in a urinary incontinence animal model. MATERIALS AND METHODS: The proper cell passage number was analyzed with hAFSCs at passages 4, 6, and 8 at week 2. The cell dose optimization included 1x10(4), 1x10(5), and 1x10(6) cells at week 2. The in vivo cell toxicity was performed with 0.25x10(6), 0.5x10(6), and 1x10(6) cells at weeks 2 and 4. Cell tracking was performed with 1x10(6) cells at weeks 2 and 4. RESULTS: The selected optimal cell passage number was smaller than 6, and the optimal cell dose was 1x10(6) for the mouse model. In our pre-clinical study, hAFSC-injected animals showed normal values for several parameters. Moreover, the injected cells were found to be non-toxic and non-tumorigenic. Furthermore, the injected hAFSCs were rarely identified by in vivo cell trafficking in the target organs at week 2. CONCLUSION: This study demonstrates for the first time the pre-clinical efficacy and safety of hAFSC injection in the urinary incontinence animal model and provides a basis for future clinical applications.
Amniotic Fluid/*cytology ; Animals ; Cell Movement ; Disease Models, Animal ; Humans ; Injections ; Mice ; Stem Cell Transplantation/*methods ; Stem Cells/*cytology ; Treatment Outcome ; Urinary Incontinence/*therapy

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

Oocyte-like cells (OLC) can be generated by stem cells after the induction and differentiation in vitro, and maturated when transplanted in vivo to improve the development potential. Human amniotic fluid stem cells (hAFSC) were cultured for 10 days in porcine follicle fluid (pFF) that was extracted from the medium follicle with high levels of hormones and Bmp 15 protein. After the induction, the cell aggregates showed the germ cell-like cells and produced the germ cell marker oct4, and triggered epigenetic changes with high expression of methylation transferase gene dnmt3b. The cell aggregates were packaged into porcine theca folliculi to form grafts, which were then transplanted into mouse renal capsule. After one month of transplantation, the morphology of OLC from a graft was not only similar to oocytes, but also expressed the germ cells markers (oct4, nanog, stella, ifitm3, dazl, nanos3, bmp15, and gd9). The results demonstrate that the in vivo differentiation model was useful for OLC development.
Amniotic Fluid ; cytology ; Animals ; Biomarkers ; Cell Differentiation ; Female ; Humans ; Mice ; Oocytes ; cytology ; Ovarian Follicle ; Stem Cells ; cytology ; Swine

OBJECTIVETo reprogram the 1q21.1 microdeletion pluripotent stem cells in order to establish an ideal model for further studying its pathogenesis.

METHODSHuman amniotic fluid-derived cells induced pluripotent stem cells (hAF-iPSCs) were induced from amniotic fluid cells harboring the 1q21.1 microdeletion by retroviral vectors encoding Oct4, Sox2, c-Myc and Klf4. Characteristics of the 1q21.1 microdeletion hAF-iPSCs were determined, which included in vitro pluripotency, karyotype, microarray analysis, the capacity of differentiation in vivo and in vitro, etc.

RESULTShAF-iPSCs derived from amniotic fluid cells harboring the 1q21.1 microdeletion have maintained self renewal, with expression of pluripotency marker genes detectable at mRNA level. Stem cell surface antigens were tested by immunocytochemistry. The 1q21.1 microdeletion hAF-iPSCs showed a normal karyotype after long-term culturing in vitro, and harbored the same microdeletion as confirmed by microarray analysis. The cells have maintained their differentiation capacity both in vivo and in vitro.

CONCLUSIONThe hAF-iPSCs harboring the 1q21.1 microdeletion have all the characteristics of normal pluripotent stem cells, and can be used for directed differentiation into specific cells, which may provide an ideal model for studying the pathogenesis of 1q21.1 microdeletion in vitro.

Abnormalities, Multiple ; embryology ; genetics ; physiopathology ; Adult ; Amniotic Fluid ; cytology ; Animals ; Cell Differentiation ; Chromosome Deletion ; Chromosomes, Human ; genetics ; Chromosomes, Human, Pair 1 ; genetics ; Female ; Fetal Diseases ; genetics ; physiopathology ; Gene Deletion ; Humans ; Induced Pluripotent Stem Cells ; cytology ; Male ; Megalencephaly ; embryology ; genetics ; physiopathology ; Mice ; Mice, SCID ; Models, Biological ; Pregnancy ; 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