Amniotic fluid digestive enzymes: Diagnostic value in fetal gastrointestinal obstructions

The diagnostic value of amniotic fluid gamma-glutamyl-transpeptidase (GGTP) and intestinal alkaline phos-phatase (iALP) was evaluated in 55 patients who underwent amniocentesis for karyotyping because fetal gastric or small bowel dilatation had been detected by ultrasound. Gastrointestinal malformation was confirmed in 46 cases and there was no gastrointestinal anomaly in nine cases. Prenatal ultrasound was suggestive of gastroduodenal dilatation in 34 cases (group I) and small bowel dilatation in 21 cases (group II). In group I, amniotic fluid GGTP above the 99th percentile was 71 per cent sensitive and 100 per cent specific for a true anatomical defect of the digestive tract (mainly duodenal atresia). In group II, high levels of GGTP and/or iALP were 69 per cent sensitive and 83 per cent specific for a fetal digestive tract anomaly. In other words, when digestive tract dilatations were diagnosed by prenatal sonography, abnormal amniotic fluid enzyme activities were strongly suggestive of such an anomaly, the possibility of which was not precluded by normal amniotic fluid iALP and GGTP activities. But amniotic fluid digestive enzyme activities do not help in defining the prognosis.     

Microvillar enzyme assays in amniotic fluid and fetal tissues at different stages of development

A systematic study of microvillar enzyme activities in the amniotic fluid in correlation with their values in different fetal tissues during development has been undertaken. Microvillar enzymes appeared in the amniotic fluid at the time of disappearance of the anal membrane, 12–13 weeks, and declined from the 18th week until the 24th week. The study of fetal tissues and fluids has shown that gamma-glutamyltranspeptidase is mainly of liver origin. The significant decrease of the activities of these amniotic fluid enzymes has been the basis of prenatal diagnosis of cystic fibrosis. These assays may be useful for the diagnosis of certain digestive tract abnormalities at later stages of pregnancy.     

Maternal contamination of amniotic fluid demonstrated by DNA analysis

DNA from 16 sets of samples comprising DNA from uncultured amniotic fluid cells, cultured amniotic fluid cells, fetal tissue, and maternal blood was analysed by the polymerase chain reaction (PCR) with AC-repeat primers. The analysis was performed to investigate the presence of contaminating maternal cells in amniotic fluid which would affect the reliability of DNA studies for prenatal diagnosis. In three sets, maternal contamination of uncultured amniotic fluid cells was detected. In one of the three sets, maternal contamination was present in both uncultured and cultured amniotic fluid cells. The use of amniotic fluid cells as a source of DNA for prenatal diagnosis should be limited to cases where the purity of the DNA can be demonstrated prior to the diagnostic test being performed. This limitation in the use of amniotic fluid DNA also extends to other forms of diagnosis relying on the purity of amniotic fluid samples, particularly the new in situ hybridization methods currently being developed.     

Extraction of DNA from amniotic fluid cells for the early prenatal diagnosis of genetic disease

Ten-ml samples of amniotic fluid were taken from pregnancies being terminated at 8–14 weeks' gestation. DNA was extracted from the amniotic cells by sequential centrifugation and analysed using the polymerase chain reaction (PCR). Fifteen samples were analysed for evidence of maternal contamination using Mfd5 oligo-nucleotide primers for repeat polymorphisms. Ten amniotic fluid samples were tested for the Delta-F₅₀₈ deletion characteristic of cystic fibrosis to demonstrate a diagnostic application for the technique. In each case, DNA extracted from fetal tissue from the same pregnancy was included in the controls. In 14 of the 15 cases tested with the Mfd5 primers, both the amniotic fluid DNA and the fetal DNA showed no evidence of contaminating DNA. In one case, neither the amniotic fluid cells nor the fetal cells yielded results. In nine of the ten cases tested with the Delta-F₅₀₈ primers, the amniotic fluid cell DNA provided accurate information about the genetic status of the fetus; in the tenth, the fetal DNA failed to amplify. The results indicate that adequate DNA can be extracted from amniotic fluid from 8 weeks' gestation onward and these samples are suitable for prenatal diagnosis using PCR.     

Correct prenatal diagnosis of a hurler fetus where amniotic fluid cell cultures were of maternal origin

Contamination of amniotic fluid cell cultures by maternal cells can be expected to lead to misdiagnosis of fetal genotype in 0·1 to 0·5/100 cultures, when assays are carried out directly on cultured cells. Chemical analysis of the cell-free amniotic fluid supernatant may overcome this source of error and has the added advantages of speed and independence from amniotic cell culture failure. We describe a pregnancy at risk for Hurler's disease where amniotic cells cultured at amniocentesis had a female karyotype and an α-iduronidase activity towards both phenyl and 4-methylumbelliferyl substrates at the lower end of the normal range, suggesting a heterozygous fetus. An affected fetus was predicted, however, because of a high concentration of dermatan sulphate in the amniotic fluid. The discrepancy between these findings was shown to be due to maternal cell contamination of amniotic fluid cell cultures by the birth of a male infant with Hurler's disease.     

Influence of extracellular matrix on the proliferation of human amniotic fluid cells in vitro

Factors which influence the proliferation of human amniotic fluid cells in vitro have potential importance in reducing the time for prenatal diagnosis. Fibroblast growth factor (FGF) has been shown to be mitogenic for human amniotic fluid cells. The observation that cells which respond to FGF in vitro produce their own extracellular matrix (ECM), led to the use of an ECM as a substrate to assess proliferation. Pooled amniotic fluid cells maintained on an ECM prepared from bovine corneal endothelial cells demonstrated a significant increase in proliferation when compared with cells maintained on plastic substrate in the presence or absence of FGF. If FGF was added to cultures of amniotic fluid cells maintained on ECM, further increases in proliferation were noted compared with cells maintained on ECM in the absence of FGF. These results indicate that the substrate upon which amniotic fluid cells are maintained can have a profound influence on their proliferation.     

Detection and enumeration of colonic mucosal cells in amniotic fluid using a colon epithelial-specific monoclonal antibody

Since its introduction, prenatal diagnosis of chromosomal and metabolic disorder by mid-trimester amniocentesis has relied upon the use of a mixture of fetal cells obtained from amniotic fluid. Little knowledge has been gained in the sorting of these cells for diagnosis of tissue-specific disorders. In an attempt to determine the contribution of fetal colonic mucosal cells to the overall amniocyte population, we used the colonic epithelial-specific monoclonal antibody (MC-Ab) 7E₁₂H₁₂, IgM isotype. Specimens of the small intestine, colon, buccal mucosa, kidney, urinary bladder, and umbilical cord were obtained from electively aborted normal fetuses of 12–28 weeks' gestation. All of these specimens were examined with 7E₁₂H₁₂ by the immunoperoxidase technique. The MC-Ab reacted with the colonic epithelial cells but not with any of the other tissues. In addition, 40 amniotic fluid samples obtained from women between 16 and 18 weeks of gestation, who underwent amniocentesis because of advanced maternal age, were tested using a fluorescent activated cell sorter. Among the amniotic fluid specimens examined, 18·4 ± 10·3 percent cells reacted with 7E₁₂H₁₂. Double immunofluorescence studies revealed that all Mc-Ab-stained cells contained secretory component, confirming that they were epithelial in origin. All fetuses whose amniotic fluid was analysed had normal karyotypes and amniotic fluid alpha-fetoprctein levels that were also normal. This study demonstrates that cell-specific Mc-Ab can be used to detect colon cells in the amniotic fluid and that colon cells contribute significant numbers in the mixture of amniotic fluid cells. This technique could be helpful in the prenatal diagnosis of disorders in which the flow of amniotic fluid through the fetal intestine is impaired, such as cystic fibrosis, imperforate anus, Hirschsprung aganglionic megacolon, and intestinal atresia.     

Association of generalized dystrophic epidermolysis bullosa with positive acetylcholinesterase and markedly elevated maternal serum and amniotic fluid alpha-fetoprotein

A case of fatal generalized dystrophic epidermolysis bullosa is described in a prematurely born female whose mother had strikingly elevated mid-trimester serum and amniotic fluid alpha-fetoprotein concentrations, a positive amniotic fluid acetylcholinesterase band, and negative serial ultrasound studies. This case lends further support to an association between autosomal recessive generalized dystrophic epidermolysis bullosa and increased levels of alpha-fetoprotein, positive amniotic fluid acetylcholines'terase, and normal ultrasound findings.     

Prenatal diagnosis of congenital adrenal hyperplasia due to 21-hydroxylase deficiency by amniotic fluid steroid analysis

The concentration of 17OH-progesterone was measured in second trimester amniotic fluid samples from 12 mothers who previously had had an infant with congenital adrenal hyper-plasia due to 21-hydroxylase deficiency. In 4 affected pregnancies, the concentrations were more than 2 S.D. higher than those determined in 44 samples from normal pregnancies (mean ± S.D., 8·1 ± 2·4 nmol/1). The remaining 8 pregnancies were predicted to be unaffected based on the results of amniotic fluid concentrations within the normal range. In each instance, the infant was normal. The results indicate that measurement of amniotic fluid 17OH-progester-one concentrations during the second trimester is an accurate prenatal test for 21-hydroxylase deficiency. The results should be supplemented with determination of fetal sex by karyotype analysis on the amniotic fluid cells.     

Prenatal diagnosis of herlitz junctional epidermolysis bullosa by amniocentesis

Herlitz junctional epidermolysis bullosa (HJEB) is a severe blistering disorder which usually results in death during infancy. We have previously shown that the anchoring filament protein laminin-5 (kalinin/nicein), which mediates keratinocyte attachment and dermal–epidermal cohesion, is abnormally expressed in individuals with HJEB. Laminin-5 was detected by Western blot analysis in amniotic fluid from 44 consecutive normal secondtrimester control pregnancies, but was undetectable in second-trimester amniotic fluid from four pregnancies with fetuses affected by HJEB. In one case of severe non-Herlitz JEB, laminin-5 was detected in both amniotic fluid and skin. In human amniotic fluid, the laminin-5 a3 subunit was processed to a major 165 kD species and a minor 145 kD species and the β2 subunit was partially processed to 105 kD. Although laminin-5 was covalently associated with laminin-6 (K-laminin) in amniotic membrane, no covalent interaction was detected in amniotic fluid. Laminin-5 from amniotic fluid strongly supported keratinocyte attachment. These results suggest that Western blot analysis of second-trimester amniotic fluid is useful in determining the prenatal diagnosis of HJEB and that laminin-5 may serve a physiologically important function in amniotic fluid.     

Prenatal diagnosis of citrullinemia: Elevated levels of citrulline in the amniotic fluid in the three affected pregnancies

In three pregnancies at risk for citrullinemia affected fetuses were predicted both by strongly increased levels of citrulline in the amniotic fluid and by the reduced incorporation of ¹⁴C-citrulline into TCA-precipitable material in cultured amniotic fluid cells. The prenatal diagnoses of affected fetuses were confirmed after termination of the pregnancies by direct and indirect assays of argininosuccinate synthetase in the fetal livers and fibroblasts respectively. Measurement of the citrulline concentration in amniotic fluid appears to be a valuable adjunct in the prenatal diagnosis of citrullinemia.     

S-100 protein and neuron-specific enolase in amniotic fluid as markers of abdominal wall and neural tube defects in the fetus

The aim of this study was to determine whether there is increased leakage of neuron-specific enolase (NSE) and S-100 protein into amniotic fluid in pregnancies with neural tube defects, since both these proteins are produced by neural tissue, and to compare the value of these substances for detecting such defects with that of the more conventional techniques of alpha-fetoprotein (AFP) and acetylcholinesterase (AChE) gel electrophoresis. Amniotic samples from 25 mid-pregnancies (15–17 weeks' gestation) with neural tube defects (14 with open spina bifida and 11 with anencephaly) and from seven mid-pregnancies with abdominal wall defects were compared with a control material consisting of 80 amniotic fluid samples from 80 consecutive mid-pregnancy amniocenteses, with normal karyotypes and AFP concentrations. All of the above cases of abnormalities were primarily detected through increased AFP levels in the amniotic fluid. Amniotic fluid samples from 13 pregnancies with fetuses with autosomal chromosomal abnormalities and seven amniotic fluid samples contaminated with blood were also included in the investigation. It is concluded from the results that the conventional AFP assay combined with AChE gel electrophoresis is the best method for screening amniotic fluid for neural tube defects and defects of the abdominal wall. Neither NSE nor S-100 assay alone proved to be superior for the detection of these cases in mid-trimester amniotic fluid. The S-100 assay, however, could give additional information in cases where AChE gel electrophoresis is not decisive; for example, in samples contaminated with blood.     

Prenatal diagnosis of hunter syndrome

Sixteen pregnancies at risk for Hunter syndrome have been monitored by amniocentesis. Iduronate 2-sulphate sulphatase levels were measured in amniotic fluid, cultured amniotic fluid cells and cord blood. Thirteen of the pregnancies resulted in normal livebirths, two are continuing and one affected pregnancy was terminated. Reduced enzyme levels were observed in either amniotic fluid, cells or cord blood for four female fetuses. Such fetuses are likely to be carriers expressing reduced enzyme levels. The affected male fetus had reduced enzyme activity in amniotic fluid; insufficient cells were cultured for enzyme estimation, however no enzyme activity was detected in fetal liver after termination. Eight cord blood enzyme estimations have been performed, five confirming normal male infants.     

Glial fibrillary acidic protein in amniotic fluids from pregnancies with fetal neural tube defects

The glial fibrillary acidic protein (GFAP) is the subunit protein of intermediate filaments in astrocytes and closely related cell types. By means of an enzyme immunoassay we have determined the concentration of GFAP in amniotic fluids from normal pregnancies and from pregnancies complicated by various fetal malformations. The group of 20 cases of fetal anencephaly had a significantly higher mean amniotic fluid GFAP concentration (115 μg/1±133.6 (S.D.), range 6–378 μg/1) than the control group of 117 normal pregnancies (13 μg/1k±5.5 (S.D.), range 0–31 μg/1), (P<0.001). Two cases of fetal encephalocele likewise had very high amniotic fluid GFAP concentrations. None of the other cases of fetal malformations investigated, including 12 cases of spina bifida, had increased amniotic fluid GFAP concentrations. We conclude that determination of the amniotic fluid GFAP concentration may give additional information in the prenatal diagnosis of fetal nervous system malformations.     

HUMAN CHORIONIC GONADOTROPHIN AND ALPHA-FETOPROTEIN LEVELS IN MATCHED SAMPLES OF AMNIOTIC FLUID,EXTRAEMBRYONIC COELOMIC FLUID,AND MATERNAL SERUM IN THE FIRST TRIMESTER OF PREGNANCY

Separately identified samples of amniotic fluid and extraembryonic coelomic fluid obtained by high resolution transvaginal ultrasound-guided amniocentesis from 32 women between 7 and 12 weeks of pregnancy were analysed for human chorionic gonadotrophin (hCG) and alpha-fetoprotein (AFP). There was a highly significant difference between the hCG levels in amniotic fluid (median level 6.3 U/ml; range 1.6–310.0 U/ml) and those in extraembryonic coelomic fluid (median level 400.0U/ml; range 135.0–2250.0U/ml) (p<0.001; Mann-Whitney U/–test). The levels of AFP were very similar in amniotic fluid (median 26.0 kU/ml; range 10.0–116.5 kU/ml) and extraembryonic coelomic fluid (median level 24.1 kU/ml; range 12.4–94.4 kU/ml).     

Reduction of sera requirements in amniotic fluid cell culture

Reduction in serum requirement for culture of primary human amniotic fluid cells can be achieved by the addition of 10 growth-promoting factors to the nutrient medium. This supplemented medium preserves cell types normally found in amniotic fluid cell cultures supplemented with 20–30 per cent fetal bovine serum. The volume of amniotic fluid required to initiate culture can be as little as 1 ml. Amniotic fluid samples contaminated with red blood cells with no visible clot also grow well in the low serum medium. Cell-free amniotic fluid combined with equal parts of supplemented medium is useful in initiating cell culture.     

Prenatal diagnosis of Fabry's disease by direct analysis of chorionic villi

Six pregnancies of three carriers for X-linked Fabry's disease, were monitored by chromosome and enzyme analysis. Two affected male fetuses were detected by the demonstration of α-galactosidase deficiency in amniotic fluid cells and chorionic villi respectively. The use of chorionic villi enabled a diagnosis within a few hours after sampling in the ninth week of pregnancy whereas the use of amniotic fluid cells in the earlier case required two weeks of culturing after amniocentesis in the 16th week. Four female fetuses were found; heterozygosity was demonstrated in one by analysis of clones in the primary amniotic fluid cell culture.     

Amniotic fluid analysis in a fetus with laryngeal atresia

Complete laryngeal atresia is a rare congenital malformation that is known to cause hypertrophy of the fetal lung in utero. A fetus with laryngeal atresia was found to have markedly immature amniotic fluid lung maturity studies at term. Inappropriately low amniotic fluid lung maturity studies may be an important clue to the diagnosis of this condition.     

Enhancement of amniocyte growth on a precoated surface

This study investigates whether cell-free amniotic fluid facilitates cell attachment to the surface of culture plates and thereby promotes rapid amniocyte growth. Isolated or pooled cell-free amniotic fluid samples at different volumes were added to culture plates. Trypsinized subcultures, grown in Eagle's minimum essential alpha medium supplemented with fetal bovine serum (4–20 per cent), were monitored by cell counts. The results demonstrated growth stimulation on culture plates precoated with amniotic fluid. The minimal time for coating the culture plates was 6h. Maximal coating was observed after an overnight incubation with 2–3 ml of the fluid per culture vessel. No synergistic effect from addition of fetal bovine serum to amniotic fluid was observed. A freshly coated surface provided the best amniocyte growth. When primary cultures are grown on a precoated surface, there is an increase in colony counts in 80 per cent of the samples tested. This method may be used to improve amniocyte growth, especially in samples with relatively small numbers of cells.     

Determination of maternal serum acetylcholinesterase in pregnancies with fetal neural tube defects

We have investigated the occurrence of acetylcholinesterase (AChE) (E.C.3.1.1.7) in fetal serum, amniotic fluid and maternal serum using an immuno-chemical assay-technique employing both polyclonal and monoclonal antibodies. Fetal serum had increased amounts of AChE, which is due to an increase in the 10.5S form of the enzyme. This form was also found in amniotic fluids of pregnancies with a fetal neural tube defect (NTD), but not in normal amniotic fluid. The increase in amniotic fluid AChE was however, not reflected in the maternal serum.