High resolution proton NMR spectroscopy of human amniotic fluid

Human amniotic fluid (HAF) is a dynamic system whose characteristics depend on continuous interchanges between fetal and maternal circulations. HAF reflects not only the environment of the fetus but may also provide information about fetal development or pathology. The concentration of HAF constituents varies with gestational age and pathological states. The number of the compounds currently implicated in fetal developmental pathology are relatively few. Currently used assay methods are not adequate to totally explain or predict the complex biochemistry of the fetus. The purpose of this work was to investigate HAF with NMR spectroscopy. In the present study HAF was obtained from 47 women undergoing routine amniocentesis. Cells were separated for karyological analysis and the supernatant was acid-extracted, lyophilized and re-suspended in D₂0 resulting in a concentration increase over native fluid. ¹H NMR spectra were obtained at 360 MHz and 60 MHz. Eighteen compounds including several amino acids, were identified using parallel reference and standard addition protocols. NMR spectroscopy detected compounds of known clinical importance including glucose, leucine, isoleucine, lactate and creatinine. In conclusion, we have demonstrated that a number of physiologically relevant compounds are readily observable in HAF using ¹H NMR spectroscopy. This technique can currently provide valuable information regarding HAF composition and has the potential of being used in vivo in the future.     

Preparation of high resolution chromosomes from amniotic fluid cells

A relatively simple method of obtaining high resolution chromosomes from amniotic fluid cells is described. The elongated chromosomes are achieved by adding ethidium bromide (5μg/ml) to the culture 4 1/2 hours before harvesting and the high resolution banding can be produced by usual banding procedures.     

Synchronization of amniotic fluid cells for high resolution cytogenetics

A high resolution technique was applied to amniotic fluid cells by synchronization. After inoculation, the cells were incubated for 30 h in the presence of either thymidine or 5-bromodeoxyuridine (BrdU). After removal of the blocking agent and addition of a low concentration of thymidine, the cells were incubated for another 6 1/2–7 h, then harvested in prometaphase without colcemid. This technique gives a mitotic index of 3·7 per cent after thymidine synchronization and of 3·2 per cent after BrdU synchronization, and more than half of the mitoses were in the earlier phases with the chromosomes showing more than 550 bands per haploid set. GBG, GTG, and RHG prometaphases are presented. Precise high-resolution banding of chromosomes of amniotic fluid cells can increase diagnostic accuracy.     

Prenatal detection of the congenital nephrotic syndrome (Finnish type) by trehalase assay in amniotic fluid

We report relatively high trehalase activity in the amniotic fluid of two fetuses affected with the congenital nephrotic syndrome of the Finnish type. The results suggest that prenatal detection of this condition can be done on this basis in conjunction with the α-fetoprotein and acetylcholinesterase tests in amniotic fluid.     

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.     

Prenatal diagnosis of mucopolysaccharidosis by two-dimensional electrophoresis of amniotic fluid glycosaminoglycans

Amniotic fluid from 72 pregnancies at risk for mucopolysaccharidosis have been analysed for glycosaminoglycans (GAG) by means of two-dimensional electrophoresis. Definite results were obtained in all cases and required only 3 days to report. Of the 66 cases for which GAG analysis was accompanied by enzyme assays on cultured amniotic fluid cells, there was agreement of results in 65. In the one case of disagreement the result of GAG analysis proved to be correct. Fourteen pregnancies were predicted to be affected and the predictions were confirmed by studies on fetal tissues and/or cultured cells, or in one case the birth of an affected infant. Of the 58 pregnancies predicted to be unaffected, 48 have so far gone to term and produced healthy infants. There have been no false negative predictions. The analytical method is simple, rapid, and, in this study, has been found completely reliable for prenatal diagnosis.     

Variables influencing growth and morphology of colonies of cells from human amniotic fluid

Growth of cells from amniotic fluid was studied with respect to cell concentration in the inoculum, blood contamination of the fluid, fluid colour, fluid clarity, gestational age of the pregnancy, and growth factors. Dependent variables measured were colony formation, colony size, and colony morphology after 7, 11, and 14 days of culture. The following conclusions were established from these studies: small sample volumes are the most efficient for producing colonies; cells from very bloody or dark brown fluids have a slower rate of growth; growth of cells from cloudy (noncontaminated) fluids is better than growth of cells from clear fluids; the proportion of colonies that are epithelioid varies with gestational age; the stimulating effect of 100 ng/ml fibroblast growth factor on cells from amniotic fluid was confirmed.     

Isoelectric focusing pattern of human amniotic fluid α-fetoprotein

Isoelectric focusing (IEF) of amniotic fluid α-fetoprotein (AFP) in thin-layer polyacrylamide gels containing 8 M urea followed by immunoblotting reveals at least nine bands, band I lying next to the cathode. Compared with 298 amniotic fluid samples from normal pregnancies, we found that the density of band V was increased in seven cases of fetal death. In 16 amniotic fluid samples from pregnancies with open neural tube defects (ONTO), band V disappeared or was markedly decreased. In seven cases with elevated AFP and positive acetylcholines-terase (AChE) due to contamination with fetal blood, no difference in pattern was observed compared with samples from normal pregnancies. It is suggested that IEF of AFP and subsequent immunoblotting are an apparently diagnostic test for ONTD and intrauterine fetal death (IUFD).     

Short communications prenatal detection of the autosomal recessive type of polycystic kidney disease by trehalase assay in amniotic fluid

We report relatively high trehalase activity in amniotic fluid of a fetus affected with the autosomal recessive type of polycystic kidney disease (type II), suggesting that prenatal detection of this condition could be done on this basis in conjunction with ultrasonography.     

Identification of acetylcholinesterase and cholinesterase in amniotic fluid by immune absorption technique

A method for immunological detection of acetylcholinesterase (AChE) and cholinesterase (ChE) in amniotic fluid is described. By addition of a small amount of antihuman-erythrocyte membrane antibody or anti-pseudocholinesterase antibody to the sample before electrophoresis the two esterase bands on polyacrylamide gel (PAG) can be absorbed away. Similar staining results can also be obtained by specific inhibition of the two esterases with either BW 284C51 (AChE inhibitor) or Lysivane (ChE inhibitor). In cases with a faint AChE band and in cases with blood contamination the immune absorption technique makes interpretation easier. Nearly identical staining results have been obtained by the immune absorption technique and the inhibition technique in the following samples with an AChE band: 34 samples from pregnancies with severe fetal malformation or intrauterine death (2 cases), 4 fetal serum samples, 4 samples of cerebrospinal fluid, 4 samples of fetal erythrolysate and 4 samples of adult erythrolysate. It can be concluded that an antibody prepared against erythrocyte AChE cross-reacts with AChE in cerebrospinal fluid, and that this antibody can be used for demonstration of AChE in amniotic fluid.     

Stimulation of human amniotic fluid cell proliferation and colony formation by cell plating on a naturally produced extracellular matrix

Human amniotic fluid cells exhibit a higher cloning efficiency and rate of cell proliferation when maintained on dishes coated with a naturally produced extracellular matrix (ECM) in comparison with the regular tissue culture plastic. In 22 out of 31 amniotic fluid samples there was by plating the cells on ECM a 2–6 fold increase in number and size of colonies and in the cell density per colony as detected by actual staining and viewing of each colony. These effects yielded, in 21 of 41 additional samples, a reduction ranging from 2–8 days, in the culture time elapsing between amniocentesis and the first harvesting of cells for chromosomal analysis. An even greater effect was obtained with primary cells that failed to attach to plastic surfaces and stayed floating in the medium but did attach and proliferate when seeded on ECM. Cells that were left firmly attached to ECM after the first trypsinization and harvesting of cells for chromosomal analysis yielded colonies ready for second karyotyping in less than half the time required for cells maintained on plastic. Studies with secondary cultures of human amniotic fluid cells have demonstrated a 5–10 fold decrease in serum requirement of cells cultured on ECM as compared with plastic. Addition of fibroblast growth factor (FGF) to the cultures further potentiated the effects of ECM. The ECM induced stimulation of cell attachment and proliferation was not associated with any chromosomal anomalies, nor did it interfere with the handling procedure. ECM coated dishes may be useful to reduce the time interval between amniocentesis and diagnosis, in particular when the amniotic cells exhibit an exceedingly slow rate of proliferation on plastic or when large quantities of cells are required for enzymatic studies.     

Magnetic resonance spectroscopy of the fetal brain

Fetal magnetic resonance spectroscopy (MRS) is technically feasible in utero and demonstrates similar findings to those observed in neonatal populations. MRS can provide additional information to conventional T1- and T2-weighted imaging of the fetal brain. It is of particular use when subtle changes are present on conventional fetal MRI sequences, and when imaging fetuses at risk of brain injury and metabolic abnormalities. Copyright © 2009 John Wiley & Sons, Ltd.     

Separation of amniotic fluid cell types in primary culture by percoll density gradient centrifugation

Amniotic fluid cells obtained by transabdominal amniocentesis at 15–17 weeks of gestation and cultured for 15 and 21 days were separated into three fractions by density gradient centrifugation in Percoll. Each fraction (or peak) corresponded to the following densities: peak A, 1·02–1·03 g/ml; peak B, 1·04–1·05; peak C, 1·05–1·06. Peak A was composed of both non-viable and viable cells; the latter adopted the morphology of epithelial cells in culture. Peak B contained a mixture of fibroblasts and epithelioid cells and peak C had only epithelioid cells. The variability of N-acetyl-β-hexosaminidase and alkaline phosphatase activities was reduced in peaks B and C as compared to that of peak A and of unseparated cells suggesting that more defined and homogeneous cell types for enzymatic determinations can be obtained by centrifugation in Percoll density gradient. In prenatal diagnosis of biochemical defects, the separation of cells would permit a more precise diagnosis by eliminating enzyme variability due to the presence of different cell types or non-viable cells.     

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.     

The oxidation of octanoic acid by cultured amniotic fluid cells: The effect of cell type and passage number

We have measured the rate of oxidation of [1⁻¹⁴C]octanoate in cultured amniotic fluid (AF) cells at various passages and in AF cell lines with different clonal morphology. It is possible that both the passage number and the cell type may influence the outcome of prenatal diagnosis of fatty acid oxidation defects using this technique. We found that there was no significant difference between the three major AF cell types (epithelial, large epithelial, and fibroblast) when analysed at identical passage number but there was a significant reduction in octanoate oxidation in all cell types with increasing passage. For reliable prenatal diagnosis, cell lines of similar low passage number should be used.     

Prenatal diagnosis of inherited metabolic disorders by quantitation of characteristic metabolites in amniotic fluid: Facts and future

An attempt is made to summarize as completely as possible what is known about the prenatal diagnosis of amino– and organic acidurias by direct measurement of characteristic metabolites in amniotic fluid, and to indicate which disorders can potentially be diagnosed prenatally by direct quantitation of metabolites. Furthermore, the disorders are mentioned in which the prenatal diagnosis was proven to be unsuccessful by this approach. The prenatal diagnoses of a case of propionic acidemia and a case of tyrosinemia type I in the 11th and 12th week of gestational age, respectively, are reported and the prospects of performing amniocentesis in the first trimester for prenatal diagnosis are discussed.     

46,XX/46,XY chromosome complement in amniotic fluid cell culture followed by the birth of a normal female child

Experience indicates that the most likely explanation for a mixture of 46,XX/46,XY cells in an amniotic fluid sample is that of maternal cell contamination and that a normal male child is to be expected at birth. We report the birth of a normal female child following prenatal diagnosis of such a mixture. Extensive postnatal studies failed to reveal an XY cell line. The possible sources of the XY cell line are discussed, as are the various techniques that were applied in an effort to discover it's origin. Cross-contamination of samples could be ruled out and there was no evidence of an unsuspected twin pregnancy. It is clear from this case that not all 46,XX/46,XY results obtained in amniotic fluid can be assumed to represent maternal cell contamination and some effort should be made to eliminate other potential sources for such a mixture.     

Prenatal diagnosis of congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency by steroid analysis in the amniotic fluid of mid-pregnancy: Comparison with HLA typing in 17 pregnancies at risk for CAH

Amniotic fluid (AF) levels of 17-hydroxyprogesterone (17OHP) and testosterone (T) were determined at 16–17 weeks in 17 pregnancies at risk for CAH and results compared to 75 normal controls. The fetus was predicted to be unaffected in 12 cases on the findings of normal AF levels of both 17OHP and T and the latter allowed a correct prediction of fetal sex in all instances. HLA typing confirmed normality in 12 cases revealing 5 carriers, 5 homozygous normal and 2 indeterminate. Steroid levels of the 2 groups were similar. Three fetuses were predicted to be CAH affected on unambiguously high levels of 17OHP and T (in female only). HLA typing was in agreement, and the diagnosis was confirmed in 2 abortuses and a female newborn by physical and hormonal studies. In the last 2 cases AF levels of OHP and T were normal but HLA (A/B/C) genotypes were identical to the CAH affected siblings. Normal physical and hormonal findings in the 2 aborted fetuses would exclude the possibility of an in utero virilizing form of CAH. The discrepancy could be explained on the basis that the fetuses had an allelic form of 21-hydroxylase deficiency or on the basis of recombination (not fully tested). It is concluded that a fully informative prenatal diagnosis of CAH should not rely entirely on HLA typing but on hormonal studies.