Presence of the major progesterone-induced proteins of the sheep endometrium in fetal fluids

Allantoic and amniotic fluids were collected on Days 60 (n = 3), 100 (n = 4), and 140 (n = 3) of pregnancy. The presence of uterine milk proteins (UTM-proteins) in these samples was evaluated by Ouchterlony immunodiffusion and enzyme-linked immunoabsorbant assay (ELISA). Eight of ten samples of allantoic fluid and three of ten samples of amniotic fluid produced one or two immunoprecipitin bands against antiserum to UTM-proteins. Each band fused with immunoprecipitin bands from UTM-proteins purified from uterine fluid. Data from a semi-quantitative ELISA indicated that allantoic fluid from all ewes and amniotic fluid from six of ten ewes contained immunoreactive UTM-proteins. Concentrations of UTM-proteins in these fluids were not statistically affected by day of gestation (p greater than 0.10), but tended to decline as gestation advanced. Greater concentrations of UTM-proteins were detected in allantoic fluid than in amniotic fluid (p less than 0.05). The physical characteristics of the immunoreactive material in allantoic and amniotic fluids were examined by polyacrylamide gel electrophoresis and Western blotting. The immunoreactive material was found to possess pIs and molecular weights identical to UTM-proteins. These results indicate that fetal fluids contain material that reacts with antiserum to UTM-proteins and has physical properties similar to UTM-proteins. It is likely, therefore, that the UTM-proteins are transported across the placenta during gestation, perhaps to serve some function in the fetal compartment.     

Appearance of paoproteins of pulmonary surfactant in human amniotic fluid.

We have isolated and partially characterized the protein found in surface-active material from adult human lungs, and have determined the time of appearance of this protein in amniotic fluid. Fluids were drawn at gestational ages from 12 to 44 wk, and were assayed for their concentration of apoprotein by an agglutination immunoassay. Surfactant apoprotein ((defined as that protein which is reproducibly found in purified preparations of surface active material) was usually first detected from 30 to 32 wk gestation, and its concentration increased almost fivefold to a maximum at 37 wk. The change in apoprotein concentration was approximately paralleled by the change in phospholipid concentration. At all gestational ages there was wide variability in both phospholipid and apoprotein concentrations, and the time of appearance of the apoprotein in amniotic fluid differed among fetuses. The results suggest that the presence of surfactant apoprotein in amniotic fluid is coincident with the biochemical and morphological maturation of the fetal lung, and are additional evidence that this apoprotein is cosecreted with the lipids of surface-active material.     

Excretory secretory antigens of filarial parasite Setaria digitata

Excretory Secretory (ES) material isolated from the culture fluid of S. digitata was highly antigenic. Neither oesophagus nor excretory cells and excretory pore of the parasite showed reasonable fluorescence with ES antisera. However, the uterine tissue and the egg showed strong fluorescence. The egg showed fluorescence mainly in the space between embryo and egg membrane (amniotic fluid). The amniotic fluid was highly antigenic and appears to be the most important source of ES material released by the filarial parasites.     

Pulmonary amniotic fluid embolism in a rhesus macaque (Macaca mulatta)

A pregnant female rhesus macaque died spontaneously during stage two labor. Gross and histopathologic findings included severe pulmonary edema, with low numbers of blood vessels containing pale basophilic mucinous material (Alcian Blue positive and PTAH negative), consistent with intravascular amniotic fluid-derived mucin resulting in pulmonary amniotic fluid embolism.     

Metabolic activation of benzo(a)pyrene by human amniotic fluid

The in vitro activation of benzo(a)pyrene was studied in amniotic fluid from ten 4-month pregnant women. Benzo(a)pyrene monooxygenase and epoxide hydrolase activities were in the same range in amniotic fluid as in human liver. Glutathione epoxide transferase activity was markedly lower than in hepatocytes. Human amniotic fluid also catalyzed the formation of hydrocarbon metabolites mutagenic to Salmonella typhimurium TA98 (Ames system). Profiles of amniotic fluid aromatic hydrocarbons from non smokers exhibited low benzo(a)pyrene concentration (less than 0.1 ng/ml).     

Maternal cell contamination in amniotic fluid samples as a consequence of the sampling technique

Maternal cell contamination in amniotic fluid samples is easily detected by in situ hybridization if the karyotype of the fetus differs from the karyotype of the mother. One out of two amniotic fluid samples appears to contain more than 20% maternal cells. Bloody samples often contain even more than 50% maternal cells. Maternal cells can also be identified on the basis of their nuclear morphology. Maternal cell contamination is regularly observed in pregnancies with an anterior placenta, whereas it is rare in posterior placenta pregnancies. The maternal cells are therefore thought to be artificially introduced into the amniotic fluid sample, as a result of placental bleeding during amniocentesis. The implications of maternal cell contamination for prenatal diagnosis using uncultured amniotic fluid samples are discussed.     

Argininosuccinic aciduria: prenatal studies in a family at risk.

We have monitored two successive pregnancies in a family which we found to be at risk for argininosuccinic aciduria. We measured argininosuccinic acid (ASA) concentrations in amniotic fluid and utilized an indirect assay of ASA lyase activity in cultured amniotic fluid cells. The assay procedure is based on the uptake of 14C from [14C]citrulline and of [3H]leucine into protein. ASA was easily measured in amniotic fluid from the first fetus at risk, whereas none was detectable in control fluids. Amniotic fluid cells cultured from this fetus had only 5.5% of control ASA lyase activity. The pregnancy was terminated, and hepatic ASA lyase activity in the fetus was shown to be about 1.3% of control values. In addition, eight fetal tissues were analyzed for ASA, and all had significant accumulation. ASA was not detected in amniotic fluid from the second fetus at risk, and ASA lyase activity in cultured cells was 80% of control activity. Enzymatic analysis of erythrocyte lysate confirmed the diagnosis of an unaffected child (ASA lyase = 46% of control) and indicated heterozygosity. Thus, we provide further evidence that argininosuccinic aciduria can be diagnosed successfully in utero by indirect assay of ASA lyase activity in cultured amniotic fluid cells. In addition, high amniotic fluid ASA concentrations provide strong adjunctive evidence for such a prenatal determination, and may prove to be sufficient for diagnosis.     

Isolation and characterization of a pulmonary glycoprotein from human amniotic fluid.

A glycoprotein of the molecular weight of 36 000 has been isolated from human amniotic fluid. The glycoprotein was found to contain sialic acid, galactose, mannose, fucose, glucosamine, hydroxyproline and relatively high amounts of glycine. End-group analyses resulted in a single NH2-terminal residue indicating that the glycoprotein was homogeneous. The data indicate that this unique collagen-like glycoprotein, which is immunologically identical to a major alveolar glycoprotein found in alveoli of patients with alveolar proteinosis, is also a major protein in the human amniotic fluid. The idea that the pulmonary constituents enter the amniotic fluid cavity during fetal lung development is also confirmed by this report.     

Soluble Leukocyte-Associated Ig-Like Receptor-1 in Amniotic Fluid Is of Fetal Origin and Positively Associates with Lung Compliance

The soluble form of the inhibitory immune receptor leukocyte-Associated Ig-like Receptor-1 (sLAIR-1) is present in plasma, urine and synovial fluid and correlates to inflammation. We and others previously showed inflammatory protein expression in normal amniotic fluid at term. We hypothesized that sLAIR-1 is present in amniotic fluid during term parturition and is related to fetal lung function development. sLAIR-1 was detectable in all amniotic fluid samples (n=355) collected during term spontaneous deliveries. First, potential intra-uterine origins of amniotic fluid sLAIR-1 were explored. Although LAIR-1 was expressed on the surface of amniotic fluid neutrophils, LAIR-1 was not secreted upon ex vivo neutrophil stimulation with LPS, or PMA/ionomycin. Cord blood concentrations of sLAIR-1 were fourfold lower than and not related to amniotic fluid concentrations and placentas showed no or only sporadic LAIR-1 positive cells. Similarly, in post-mortem lung tissue of term neonates that died of non-pulmonary disorders LAIR-1 positive cells were absent or only sporadically present. In fetal urine samples, however, sLAIR-1 levels were even higher than in amniotic fluid and correlated with amniotic fluid sLAIR-1 concentrations. Second, the potential relevance of amniotic fluid sLAIR-1 was studied. sLAIR-1 concentrations had low correlation to amniotic fluid cytokines. We measured neonatal lung function in a convenient subset of 152 infants, using the single occlusion technique, at a median age of 34 days (IQR 30-39). The amniotic fluid concentration of sLAIR-1 was independently correlated to airway compliance (ρ=0.29, P=.001). Taken together, we show the consistent presence of sLAIR-1 in amniotic fluid, which originates from fetal urine. Concentrations of sLAIR-1 in amniotic fluid during term deliveries are independent from levels of other soluble immune mediators. The positive association between concentrations of amniotic fluid sLAIR-1 and neonatal lung compliance suggests that amniotic fluid sLAIR-1 may be useful as a novel independent marker of neonatal lung maturation.     

Intestinal and renal origin of trehalase activity in rabbit amniotic fluid

To utilize specific fetal markers in amniotic fluid for prenatal detection of fetal anomalies, it is necessary to determine the precise tissue origin of these markers. In rabbit fetuses, we distinguished between intestinal and renal forms of trehalase (alpha,alpha'-trehalose-1-D-glucohydrolase, EC 3.2.1.28) in amniotic fluid on the basis of differences in net electric charges. Trehalase was solubilized from purified brush-border membranes of fetal rabbit kidney and intestine by Triton X-100 treatment, whereas the trehalase activity in amniotic fluid was soluble. The kinetic properties of trehalase from intestine, kidney and amniotic fluid were very similar. The Mr of the soluble amniotic fluid trehalase was between 72,600 and 66,300 from hydrodynamic parameters, depending on the amount of sugar bound to the enzyme, and 48,500 by radiation inactivation, a method which detects only the protein part of the enzyme. For membrane-bound trehalase from kidney and intestine in situ the radiation inactivation method also gave a molecular size of around 49,000. Isoelectric focusing of freshly solubilized membranes allowed us to distinguish between renal and intestinal forms of trehalase in rabbit fetuses on the basis of different isoelectric points. Each trehalase form was also present in the amniotic fluid but in varying proportions depending on the gestational age at which the amniotic fluid was collected. The results suggest that early in gestation amniotic fluid trehalase activity originates exclusively from the fetal kidney but that more and more intestinal enzyme is released into the amniotic cavity as the fetus develops. Similar results were also obtained when ion-exchange chromatography was used to separate the various trehalase forms. The development of trehalase activity in rabbit fetal kidney and intestine correlates well with its occurrence in the amniotic fluid; trehalase activity in the kidney develops early in gestation whereas the intestinal trehalase activity develops just before term.     

Fetal surfactant as a source of arachidonate in human amniotic fluid

The factors responsible for the onset of labor in women are not well understood but it is clear that parturition is associated with increased production of prostanoids and release of arachidonic acid by intrauterine tissues. Pulmonary surfactant is secreted from the fetal lung into the amniotic fluid where its concentration increases toward term. In this paper we have shown that the ability of fetal surfactant to stimulate prostaglandin production by amnion cells is greatly enhanced by pre-incubating surfactant with amniotic fluid. This is due to the release of fatty acids, including arachidonate, from the lipids of fetal surfactant by the sequential action of phospholipase C and diglyceride lipase. Thus, in addition to providing the amnion with a source of arachidonate derived from the intracellular transfer of arachidonate from surfactant phosphatidylcholine to phosphatidylethanolamine and phosphatidylinositol in amnion cells, fetal surfactant also contributes to the pool of free arachidonate in amniotic fluid.     

Stimulation of myometrial and decidual prostaglandin production by amniotic fluid from term,but not midtrimester pregnancies

The effect of amniotic fluid obtained from second trimester (16–20 wks) and term pregnancies (38–41 wks) on the production of PGE and F by human amnion, decidua and myometrium at term was determined using tissue slices incubated in vitro. Midpregnancy amniotic fluid neither inhibited nor stimulated the prostanoid production by any of the tissues. In contrast, term amniotic fluid obtained before as well as after the onset of labor markedly increased the production of both PGE and PGF in decidua and myometrium from levels in Krebs solution. The prostanoid production (PGE + PGF) in amnoin was not significantly increased but the proportion of PGF was raised during incubations in term amniotic fluid. In decidua and myometrium the increase in PGE and PGF production in term amniotic fluid was approximately 200 and 400 percent respectively, from control values in Krebs solution. We propose that the stimulatory activity in term amniotic fluid in responsible for the accelerated synthesis of prostaglandins after of membranes, which is reflected in raised PGF metabolite levels in maternal circulation. It may also be the reason for the rise in amniotic fluid prostaglandin levels around the 35th week of gestation, and perhaps for the onset of labor.     

Estimation of the Stokes radii of serum proteins for a study of protein movement from blood to amniotic fluid.

The effective hydrodynamic (Stokes) radii of fifteen serum proteins were estimated by Sephadex G 200 gel filtration and immunochemical methods. The Waldmann-Meyer and Birch equation (Protides Biol. Fluids, Proc. 21st Colloq. (1974) (Peeters, H., ed.), Vol. 21, pp. 653-656, Pergamon Press, Oxford) was used for maximum accuracy. Three replicate column runs were made to assess the precision for the size determinations (coefficients of variation 0.6-2.7(). Quantive two-dimensional immunoelectrophoresis was used to measure eleven proteins in specimens of serum and amniotic fluid collected from twelve normal pregnancies. There was a close inverse linear relationship between the amniotic fluid/serum ratios of the proteins and their Stokes radii. This indicates that the movement of a protein from the blood to amniotic fluid is determined by the size of the protein. The linear correlation of protein amniotic fluid/serum ratio with Stokes radius was better than that with molecular weight. This demonstrates that, as an expression of protein size, Stokes radius should be used in preference to molecular weight when protein filtration systems are being investigated.     

Evaluation of arachidonic acid in amniotic fluid before and during labor

Arachidonic acid is considered to be one of the precursors in prostaglandin synthesis. For this reason, arachidonic acid was measured in a series of amniotic fluid. Samples in order to rest the hypothesis that the beginning of uterine activity is accompanied by a rise of its concentration. In fact, it could be shown that in amniotic fluid from patients in labor arachidonic acid levels are much higher than in amniotic fluid from patients without uterine activity.     

A simple procedure for the purification of rat alpha-fetoprotein

A simple purification procedure for obtaining a high yield of electrophoretically and immunologically pure rat α-fetoprotein from amniotic fluid is described. Rat amniotic fluid is passed through an anti-rat albumin immunoabsorbent column to remove albumin. The albumin-free eluate is then chromatographed on DEAE-Sephacel to separate α-fetoprotein from transferrin and other minor protein contaminants. This two-step purification procedure results in a recovery of approximately 70% of the rat α-fetoprotein originally present in the amniotic fluid.     

Effect of centrifugation on amniotic fluid phospholipid composition.

The first step in the determination of phospholipid in amniotic fluid is generally the removal of cells and debris from the fluid by centrifugation. Low-speed centrifugation of the supernatant is reported to have the same phospholipidic profile and L/S, PG/S and PI/S ratios similar to those of the uncentrifuged amniotic fluid sample. With high-speed centrifugation almost all the pulmonary surfactant seems to be recovered in pellet with the same characteristics as the uncentrifuged amniotic fluid.     

Pemphigoid gestationis autoantigen, transmembrane collagen XVII, promotes the migration of cytotrophoblastic cells of placenta and is a structural component of fetal membranes.

In pemphigoid gestationis (PG), autoantibodies target collagen XVII, a hemidesmosomal transmembrane protein, which is an important element in cutaneous epithelial adhesion and signalling. We report that collagen XVII is expressed in the first trimester and term syncytial and cytotrophoblastic cells of normal placenta and in epithelial cells of amniotic membrane. Immunoelectron microscopy confirmed the localization of collagen XVII to the hemidesmosomes of amniotic epithelium. Examination of three PG placentas showed mild villitis, but there were no differences between collagen XVII expression levels or immunostaining signals as compared to normal placenta. Collagen XVII expression was also detected in cultured extravillous trophoblast HTR-8/SVneo cells, where collagen XVII expression was upregulated by PMA and TGF-beta1. Interestingly, the presence of Col15, the cell migration domain of collagen XVII, induced the migration of HTR-8/SVneo cells in transmigration assay. Analysis of amniotic fluid samples at different gestational weeks revealed that a large quantity of collagen XVII ectodomain was shed into amniotic fluid throughout pregnancy. Biochemical and immunoblotting analysis indicated that the ectodomain in amniotic fluid is structurally very similar to the ectodomain produced by cultured keratinocytes. Cultured cells from amniotic fluid samples also expressed collagen XVII. Our results suggest that collagen XVII may contribute to the invasion of extravillous trophoblasts during placental development and is also required for the integrity of amniotic basement membrane. Although the exact pathomechanism of PG is still largely unknown, the clinical symptoms of PG are initiated after the expression of collagen XVII in placenta during the first trimester of pregnancy.     

Cryopreservation does not alter karyotype, multipotency, or NANOG/SOX2 gene expression of amniotic fluid mesenchymal stem cells

Cryopreservation of mesenchymal stem cells from amniotic fluid is of clinical importance, as these cells can be harvested during the prenatal period and stored for use in treatments. We examined the behavior of mesenchymal stem cells from human amniotic fluid in culture that had been subjected to cryopreservation. We assessed chromosomal stability through karyotype analysis, determined whether multipotent capacity (differentiation into adipogenic, chondrogenic, and osteogenic cells) is maintained, and analyzed SOX2 and NANOG expression after thawing. Five amniotic fluid samples were cryopreserved for 150 days. No chromosomal aberrations were observed. The expression levels of NANOG and SOX2 also were quite similar before and after cryopreservation. Capacity for differentiation into adipogenic, chondrogenic, and osteogenic tissues also remained the same. We conclude that cryopreservation of amniotic fluid does not alter karyotype, NANOG/SOX2 gene expression, or multipotent capacity of stem cells that have been collected from amniotic fluid during pregnancy.     

Prenatal diagnosis of propionic acidemia by measuring methylcitric acid in dried amniotic fluid on filter paper using GC/MS

Propionic acidemia is a frequent inborn error of metabolism. Methylcitric acid, a key indicator of propionic acidemia, increases in the amniotic fluid of affected fetuses. For prenatal diagnosis, the methylcitric acid in amniotic fluid can be measured by stable-isotope dilution GC/MS. Here, we quantified this indicator in samples of amniotic fluid that had been dried on filter paper and transported at ambient temperatures, and compared the results with data obtained from the original amniotic fluid. We then used the filter-paper method to screen at-risk fetuses and obtained a clear-cut diagnosis in each case.     

Isolation of osteogenic progenitors from human amniotic fluid using a single step culture protocol

Background

Stem cells isolated from amniotic fluid are known to be able to differentiate into different cells types, being thus considered as a potential tool for cellular therapy of different human diseases. In the present study, we report a novel single step protocol for the osteoblastic differentiation of human amniotic fluid cells.

Results

The described protocol is able to provide osteoblastic cells producing nodules of calcium mineralization within 18 days from withdrawal of amniotic fluid samples. These cells display a complete expression of osteogenic markers (COL1, ONC, OPN, OCN, OPG, BSP, Runx2) within 30 days from withdrawal. In order to test the ability of these cells to proliferate on surfaces commonly used in oral osteointegrated implantology, we carried out cultures onto different test disks, namely smooth copper, machined titanium and Sandblasted and Acid Etching titanium (SLA titanium). Electron microscopy analysis evidenced the best cell growth on this latter surface.

Conclusion

The described protocol provides an efficient and time-saving tool for the production of osteogenic cells from amniotic fluid that in the future could be used in oral osteointegrated implantology.