Lysosomal enzyme levels in human amniotic fluid cells in tissue culture. I. α-glucosidase and β-glucosidase

A number of factors which may correlate with the levels of α-glucosidase and β-glucosidase in cultured amniotic fluid cells have been investigated. Fluctuations in enzyme activity occurred as passage numbers increased. Whereas α-glucosidase showed a consistently lower activity in the earlier passages compared to the later ones, the results for β-glucosidase were equivocal. Both enzymes showed an increase in activity correlated with the time taken by the cells to reach confluency in the third passage. When replicate cultures were assayed daily after subculture, neither enzyme showed any change correlated with time. When cultures were grown in parallel in Ham's F10 and Eagle's M.E.M. tissue culture media, the activity of both enzymes was unaffected. Cell strains cultured from serial samples of amniotic fluid from the same woman had differing enzyme levels unrelated to gestational age.     

Stem cells in human amniotic fluid

Amniotic fluid (AF) contains a heterogeneous population of cells of fetal origin in which stem cells are present. These cells are characterized by the expression of mesenchymal (CD73, CD90, CD105) and neural (Nestin, β3-tubulin, NEFH) markers, and also some markers of pluripotency (Oct4, Nanog), and they are capable of differentiating into diverse derivatives in vitro. We have shown that epithelial markers (Keratin 19, Keratin 18, and p63) are expressed in AF stem cells simultaneously with mesenchymal ones. During cloning, colonies of cells with fibroblastoid and epithelioid cells are formed. The status and differentiation potential of stem cells from AF have been discussed.     

Chromosome lesions in amniotic fluid cell cultures

Summary The frequencies of chromosome lesions were determined on 3537 mitoses in samples of varying sizes from cultures of 25 amniotic fluid specimens taken from patients at cytogenetic risk. The average percentage values of aberrant cells, including and excluding gaps, were 12.5 and 4.9, respectively. The corresponding values for fibroblasts and peripheral blood lymphocytes from normal adult donors, calculated under the same laboratory conditions, were 5.0 (including gaps) and 2.4 (excluding gaps) and 2.4 (including gaps) and 1.0 (excluding gaps), respectively. The hypothesis of a correlation between the increased incidence of chromosome lesions and the occurrence of abnormal karyotypes in amniotic fluid cell cultures is discussed.     

Chemical dilution and clearance studies to estimate amniotic fluid volume and amniotic fluid ingestion in normal baboon pregnancies

Amniotic fluid volume (AFV) and amniotic fluid ingestion rate (or fetal swallowing rate, FSR) were estimated by inulin and para-aminohippurate (PAH) dilution in 14 normal baboon pregnancies. Mean (± SE) AFV was significantly lower at 137–140 days of pregnancy (preterm) than at 173–178 days (term) (inulin: 326 ± 22.9 ml vs 483 ± 55.9 ml, P = 0.014; PHA:269 ± 39.4 ml vs 471 ± 39.4 ml, P = 0.002). In proportion to fetal weight, however, mean AFV was similar throughout the third trimester of pregnancy (inulin: 582 ± 40.9 ml/kg; PAH: 541 ± 39.8 ml/kg). Mean FSR was lower in preterm than in term animals when estimated by inulin dilution (587 ± 55.5 ml/day vs 784 ± 55.0 ml/day, P = 0.030) but not when estimated by PAH dilution (753 ± 65.7 ml/day vs 625 ± 50.6 ml/day). In proportion to their weights, however, preterm fetuses swallowed amniotic fluid more rapidly than term fetuses (inulin: 1,216 ± 117.6 ml/kg/day vs 840 ± 67.5 ml/kg/day, P = 0.025; PAH: 1,561 ± 142.9 ml/kg/day vs 682 ± 62.7 ml/kg/day, P < 0.001). Furthermore, our data suggest that the commonly accepted technique for estimating AFV may be based on inaccurate premises, that insulin may be a better marker than PAH to estimate AFV and FSR, and that needle aspiration of amniotic fluid does not appear to be an adequate technique to validate chemical dilution methods. Our data, however, provide estimates which indicate that the baboon is an appropriate animal model in which to seek refinements and validation of our techniques.     

Glycosaminoglycan composition of human amniotic fluid

The glycosaminoglycan composition of human amniotic fluid between 12–21 weeks gestation has been studied by Dowex column chromatography coupled with enzymatic analyses of the specific glycosaminoglycan in each column fraction. The total uronic acid recovered from the columns consisted of “glycopeptides” (7%), hyaluronic acid (34%), nonsulfated chondroitin (14%), chondroitin-4-sulfate (13%), chondroitin-6-sulfate (20%), dermatan sulfate (5%), and heparan sulfate (6%). Based on these studies a simple screening procedure was devised to detect increased quantities of heparan sulfate and dermatan sulfate in 5–10-ml samples of amniotic fluid and tested in the antenatal diagnosis of Hurler and Hunter's syndrome. A false negative result was recorded in a Hunter fluid obtained early gestation and a false positive result recorded in a normal fluid obtained at weeks. These data suggest that the time in gestation when amniotic fluid is sampled for chemical analysis is an important variable affecting glycosaminoglycan composition in both normal and pathological pregnancies.     

Collagenous constituents of amniotic fluid.

The amniotic fluid (AF) was fractionated by dialysis, gel filtration and SDS/PAGE, and submitted to the assay of collagenous constituents. The collagenous character of peptides and proteins of amniotic fluid was confirmed by hydroxyproline (Hyp) assay and treatment with bacterial collagenase followed by electrophoresis and gel filtration of the digestion products. It was found that AF contains collagen degradation products but the classical method of Hyp determination described by Woessner (Arch. Biochem. Biophys., 1961, 93, 440-447) gives overestimated values due to the interference with other AF components. Fractionation of AF on Sephadex G-100 column allowed to remove the interfering material and to estimate the actual Hyp content which equals to approx. 6.2 microg/ml. About 70% of Hyp was found in low molecular dialyzable products and the rest (about 30%) appears to be a constituent of nondialyzable collagenous polypeptides of the molecular mass of about 7.9-26.3 kDa. It is suggested that such collagenous polypeptides may be the products of proteolytic conversion of collagen precursor (procollagen) into the monomeric form of this protein. No high molecular forms of collagen, corresponding to alpha-subunits, were found.     

Proteomics analysis of human amniotic fluid

Amniotic fluid is a dynamic and complex mixture that reflects the physiological status of the developing fetus. In this study, the human amniotic fluid (AF) proteome of a 16-18-week normal pregnancy was profiled and analyzed to investigate the composition and functions of this fluid. Due to the complexity of AF, we utilized three different fractionation strategies to provide greater coverage. Two types of two-dimensional LC/MS/MS as well as an LC-SDS-PAGE-LC-MS/MS platform were used. A total of 16 AF samples between gestational ages of 16 and 18 weeks from women carrying chromosomally normal fetuses were analyzed by one of the three fractionation methods followed by a common reverse phase LC-MS/MS step. Mascot and The Global Proteome Machine engines were used to search the International Protein Index human database for peptide sequence identification. The list of proteins was generated by combining the results of both engines through the PeptideProphet of Scaffold software. All identified proteins were combined to generate the AF proteome comprising 1,026 unique gene matches or 842 non-redundant proteins. This list includes most of the currently used biomarkers for pregnancy-associated pathologic conditions such as preterm delivery, intra-amniotic infection, and chromosomal anomalies of the fetus. The subcellular localization, tissue expression, functions, and networks of the AF proteome were analyzed by various bioinformatic tools. These data will contribute to the better understanding of amniotic fluid function and to the discovery of novel biomarkers for prenatal diagnosis of fetal abnormalities.