We developed a new method for an objective assessment of the meconium content in amniotic fluid. By establishing a standard scale through a serial dilution of a known amount of meconium into the amniotic fluid, we developed a new method 'mecometer 'that can objectively measure the meconium content in meconium-stained amniotic fluid samples. The objectivity and reliability of this mecometer were verified by 300 student volunteers. At least 70% of the volunteers could objectively measure and digitally describe the meconium content in meconium-stained amniotic fluid samples. We believe our newly developed mecometer is a very simple, reliable, and portable method, not requiring any instruments.
Amniotic Fluid/*metabolism ; Densitometry/methods ; Humans ; Infant, Newborn ; Meconium/*metabolism

Amniotic Fluid ; metabolism ; Biomarkers ; metabolism ; Diagnosis, Differential ; Embolism, Amniotic Fluid ; metabolism ; pathology ; Female ; Humans ; Infant, Newborn ; Keratin-13 ; metabolism ; Pregnancy ; Respiratory Aspiration ; metabolism ; pathology

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

OBJECTIVETo optimize the method for preparing samples for amniotic fluid proteomics study.

METHODSPregnant rats were sacrificed with an overdose of Chloral hydrate at E17. The fetuses and amniotic fluid were harvested. The samples were processed by three different methods including trichloroacetic acid (TCA)-acetone precipitation (protocol 1), TCA-acetone precipitation combined with an Albumin and IgG Removal Kit (protocol 2), and a Centrifugal Filter concentrating combined with an Albumin and IgG Removal Kit (protocol 3). The samples were run through a two-dimensional electrophoresis gel, stained and analyzed with a Image Master 6.0 software. Protein spots were identified with a LCQ Deca XP mass spectrometer.

RESULTSThe total numbers of protein spots for samples processed by protocol 1, 2 and 3 were 253 ± 28, 749 ± 32 and 782 ± 27, respectively. And there was a significant difference between protocol 1 has and other two methods. Those with MW > 50 kDa were 57± 14, 45 ± 13 and 41 ± 14, respectively. Protocol 2 differed significantly from protocol 3. Protein number of samples with MW < 50 kDa was 196± 29, 702± 35 and 735 ± 29, respectively. Again, protocol 1 has differed significantly from other two methods.

CONCLUSIONBy removing albumin and IgG from the serum, low abundance proteins can be enriched with little loss of high abundance proteins. Centrifugal Filter concentrating combined with Albumin and IgG Removal Kit can be effectively applied for amniotic fluid proteomics study.

Amniotic Fluid ; metabolism ; Animals ; Electrophoresis, Gel, Two-Dimensional ; Female ; Pregnancy ; Proteome ; Proteomics ; methods ; Rats

OBJECTIVETo detect the change of nerve growth factor (NGF) level in human amniotic fluid during gestation, and to explore the relationship between this change and fetal ventriculomegaly (VM).

METHODSThe studied subjects (collected from 2004 to 2007) were divided into four groups, including the second-trimester pregnancy group (n=113), third-trimester pregnancy group (n=110), fetal cerebral VM group (n=12), and healthy control group (n=12) which matched with the VM group in gestational weeks. The amniotic fluid specimens were obtained during amniocentesis or cesarean section. The NGF levels in amniotic fluid were detected with enzyme-linked immunosorbent assay.

RESULTSA significantly negative correlation was found between gestational age and the NGF level in amniotic fluid (r=−0.6149, P<0.0001). The NGF level in patients with fetal VM was significantly lower than that in healthy controls (33.95±29.24 pg/mL vs. 64.73±16.21 pg/mL, P=0.024).

CONCLUSIONNGF levels in amniotic fluid may be a sensitive marker for fetal VM.

Adult ; Amniotic Fluid ; chemistry ; Female ; Humans ; Hydrocephalus ; metabolism ; Nerve Growth Factor ; analysis ; Pregnancy

To assess the influence of different sampling methods on Human Amniotic Fluid (HAF) during metabonomics analysis, and to establish a metabolite profile database for normal human amniotic fluid, four experimental groups (the group of freeze-drying, of freeze-thawing, of storage at -20 degrees C, and of keeping in room temperature) and a control group were investigated by use of 1H-NMR spectroscopy, respectively; the data of H-NMR spectroscopy was treated by principal components analysis (PCA). The results showed that, by comparison with the control, there were distinct differences in the experimental groups except the group of storage at -20 degrees C. Therefore, It is possible to use 1H-NMR-based metabonomics technique for analysis of HAF; moreover, during the tests, careful treatments of HAF should be institued to minimize the influence on the samples.
Amniotic Fluid ; metabolism ; Female ; Humans ; Magnetic Resonance Spectroscopy ; methods ; Metabolome ; Metabolomics ; methods ; Pregnancy ; Principal Component Analysis ; Specimen Handling ; methods ; standards

In the rat, the neural plate appears at day 9 and the neural tube closes at day 10.3. During neurulation, the neuroepithelium is exposed directly to amniotic fluid and blood circulation begins at day 10.5. Accordingly, amniotic fluid may be an important source of nutrition for normal development of the nervous system. Among many different components of amniotic fluid, glucose is known as common currency of metabolism and the developing embryo is more dependent on this. The purpose of this study is to provide basic data on the capacity of amniotic fluid as a source of glucose for neurulating rat embryos. In the first part of this paper, isolated days 10, 11 and 16 rat embryos with intact amnion were used to pursue the change of the glucose concentratons in the amniotic fluid. The day 10 embryo amniotic fluid glucose disappeared after 20 minutes, and the day 11 amniotic fluid glucose disappeared after 33 minutes. The day 16 amniotic fluid glucose showed no significant changes during 40 minutes. In the second part of this paper, the author determined the time required for glucose concentraton in the day 10 amniotic fluid to be 0 mg% at glucose free Hanks` solution. The day 10 amniotic fluid glucose disappeared afttar 10 minutes. Another embryos were exposed to glucose free Hank`s solution for 10 minutes, and switched immediately to regular Hank`s for measuring the changes of amniotic fluid glucose, that is `charging Phenomena`. During the first 15 minutes amniotic glucose was charged to nearly normal level, and after that it decreased. These changes were similar to the results from the first experiment. These results indicate that neurulating embryo has a potential for restoring its amniotic glucose concentration to the normal level rapidly. So harmful effects of hypoglycemic states may be compensated by this `charging phenomena` of amniotic fluid during neurulation.
Amnion ; Amniotic Fluid* ; Animals ; Blood Circulation ; Embryonic Structures* ; Female ; Glucose* ; Metabolism ; Nervous System ; Neural Plate ; Neural Tube ; Neurulation ; Rats*

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

BACKGROUNDBioactive proteins, such as cytokines and chemokines, have not been systematically evaluated in healthy and preeclamptic pregnancies. We aimed to investigate the difference of these proteins between healthy and preeclamptic pregnancies in order to help clarify their potential roles in the pathogenesis of hypertension and proteinuria in preeclampsia.

METHODSSamples of amniotic fluid and maternal/umbilical cord blood were collected from normal pregnancies and women with preeclampsia for examination of bioactive proteins. Fifty-three pregnant women were enrolled in this study. Of them, 30 pregnant women were recruited as healthy controls, and 23 pregnant women were diagnosed with preeclampsia. An antibody array was used to screen for higher levels of cytokines and related proteins in amniotic fluid than in the blood samples, and these proteins were then selected for quantification by immunoassay.

RESULTSInterleukin-1 receptor 4, hepatocyte growth factor, and urokinase plasminogen activator receptor were significantly elevated in the blood of preeclampsia patients. In particular, interleukin-1 receptor 4 was 8-fold higher in preeclampsia patients than in the healthy pregnancies. Moreover, in cord blood samples hepatocyte growth factor and interleukin-8 were significantly higher in preeclampsia patients.

CONCLUSIONSBecause of the biologic activities, Interleukin-1 receptor 4, hepatocyte growth factor, urokinase plasminogen activator receptor and interleukin-8 in maternal and/or cord blood could play a role in the pathogenesis of hypertension and proteinuria in preeclampsia.

Adult ; Amniotic Fluid ; metabolism ; Chemokines ; analysis ; physiology ; Cytokines ; analysis ; physiology ; Female ; Humans ; Hypertension ; etiology ; L-Lactate Dehydrogenase ; blood ; Pre-Eclampsia ; metabolism ; Pregnancy ; Proteinuria ; etiology

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