OBJECTIVE: To investigate the role of miRNAs in amniotic fluid exosomes in growth and development of fetuses with Down syndrome (DS). METHODS: Amniotic fluid were collected from 20 fetuses with DS and 20 normal fetuses (control) to extract amniotic exosome miRNA. MicroRNA sequencing technique was used to identify the differentially expressed miRNAs between the two groups, for which gene ontology (GO) and pathway analysis was performed. Three differentially expressed miRNAs with the strongest correlation with DS phenotype were selected for qPCR verification. Dual luciferase reporter assay was used to verify the activity of let-7d-5p for targeted regulation of BACH1. RESULTS: We identified 15 differentially expressed miRNAs in DS as compared with the control group, among which 7 miRNAs were up-regulated and 8 were down-regulated. Target gene prediction results showed that the differentially expressed miRNAs targeted 17 DS-related genes. GO analysis revealed that the main functions of the target genes involved protein binding, protein transport, ATP binding, transferase activity and synapses. Pathway analysis revealed that the functional pathways were closely related with the development of the nervous system. qPCR results showed that the expression levels of miR-140-3p and let-7d-5p were significantly lower in DS group than in the control group (P < 0.05), as was consistent with miRNA sequencing results; the expression level of miR-4512 was significantly higher in DS group than in control group (P < 0.05), which was contrary to miRNA sequencing results. The results of double luciferase reporter gene assay confirmed that let-7d-5p was capable of targeted regulation of BACH1 expression. CONCLUSION Let-7d-5p in amniotic fluid exosomes may promote oxidative stress events in the brain of fetuses with DS by regulating BACH1 expression.
Amniotic Fluid/metabolism* ; Down Syndrome/genetics* ; Exosomes ; Female ; Humans ; MicroRNAs/metabolism* ; Pregnancy

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

Amniotic Fluid ; drug effects ; metabolism ; Female ; Fetus ; drug effects ; metabolism ; Gene Expression Regulation, Developmental ; drug effects ; genetics ; Humans ; MicroRNAs ; genetics ; pharmacology ; Placenta ; metabolism ; Pregnancy ; RNA, Plant ; genetics ; pharmacology ; Umbilical Cord ; drug effects ; metabolism

BACKGROUNDAmniotic fluid (AF) supernatant contains cell-free fetal DNA (cffDNA) fragments. This study attempted to take advantage of cffDNA as a new material for prenatal diagnosis, which could be combined with simple quantitative fluorescent polymerase chain reaction (QF-PCR) to provide an ancillary method for the prenatal diagnosis of trisomy 21 syndrome.

METHODSAF supernatant samples were obtained from 27 women carrying euploid fetuses and 28 women carrying aneuploid fetuses with known cytogenetic karyotypes. Peripheral blood samples of the parents were collected at the same time. Short tandem repeat (STR) fragments on chromosome 21 were amplified by QF-PCR. Fetal condition and the parental source of the extra chromosome could be determined by the STR peaks.

RESULTSThe sensitivity of the assay for the aneuploid was 93% (26/28; confidence interval, CI: 77%-98%) and the specificity was 100% (26/26; CI: 88%-100%). The determination rate of the origin of the extra chromosome was 69%. The sensitivity and the specificity of the assay in the euploid were 100% (27/27).

CONCLUSIONSTrisomy 21 can be prenatally diagnosed by the QF-PCR method in AF supernatant. This karyotype analysis method greatly reduces the requirement for the specimen size. It will be a benefit for early amniocentesis and could avoid pregnancy complications. The method may become an ancillary method for prenatal diagnosis of trisomy 21.

Amniotic Fluid ; metabolism ; Chromosomes, Human, Pair 21 ; genetics ; Down Syndrome ; diagnosis ; genetics ; Female ; Humans ; Microsatellite Repeats ; genetics ; Polymerase Chain Reaction ; methods ; Pregnancy ; Prenatal Diagnosis ; methods

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