Maternal alpha-fetoprotein screening: two years' experience in a low-risk district.

Over a two-year period, 3479 pregnant women in the Kings'' Lynn Health District were screened for neural tube defects by estimation of maternal serum alpha-fetoprotein. Most pregnancies were scanned by sonar for fetal maturity. Eight women had fetuses with open neural tube defects; four with anencephaly were associated with very high alpha-fetoprotein values. Of the four with open neural tube defects without anencephaly, only one was detected by screening and confirmed after amniocentesis. One other had a raised serum alpha-fetoprotein but a normal amniotic fluid value. The other two affected fetuses were missed. This disappointing outcome was attributed to the poor predictive value of alpha-fetoprotein in detecting open neural tube defects (anencephaly apart) rather than to errors in its estimation or in assessment of fetal maturity by sonar scan. We question the validity of screening, particularly in areas of intermediate or low incidence.     

Demonstration of astrocytes in cultured amniotic fluid cells of three cases with neural-tube defect

Summary We have investigated the origin of rapidly adhering (RA) cells in three cases of neural tube defects (two anencephali, one encephalocele). We were able to demonstrate the presence of glial fibrillary acidic (GFA) protein in variable percentages (4–80%) of RA cells cultured for 4–6 days by use of indirect immunofluorescence with GFA antiserum. Cells cultured from amniotic fluids of normal pregnancies and fetal fibroblasts were completely GFA protein negative. GFA protein is well established as a highly specific marker for astrocytes. Demonstration of astrocytes may prove to be a criterion of high diagnostic value for neural tube defects. The percentage of astrocytes decreased with increasing culture time, while the percentage of fibronectin positive cells increased both in amniotic fluid cell cultures from neural tube defects and normal pregnancies.     

Ring chromosome 13 in a polymalformed anencephalic.

In the 33rd week of pregnancy an amniocentesis was performed because of low estriol. X-ray indicated the presence of anencephaly and a premature delivery was induced. Necropsy, in addition to anencephaly, showed a wide variety of malformations. The fetal karyotype determined from cultured amniotic fluid cells revealed a ring chromosome 13.     

Amniotic fluid cell morphology in early antenatal prediction of abortion and low birth weight.

The morphology of rapidly adherent (RA) amniotic fluid cells was examined in 201 pregnant women referred for amniocentesis because of two sequential high serum alpha-fetoprotein (AFP) concentrations. Out of 43 amniotic fluid samples containing increased amounts of AFP, 42 had neural or peritoneal cells predominating among the RA cells, the outcome being an infant with a neural-tube defect or exomphalos. In the other case with a raised amniotic fluid AFP concentration but only anterior placental cells the infant was normal. In 25 amniotic fluid samples containing normal amounts of AFP distinctive new patterns of RA cells were observed, termed fetal distress cells. These pregnancies resulted in five spontaneous abortions and 20 infants with birth weights under 2500 g. Fetal distress cells were not detected in any of the remaining 133 samples. One pregnancy was terminated because of a chromosomal abnormality, and there were seven twin pairs not recognised on ultrasonography before amniocentesis. The remaining 125 pregnancies went to term, resulting in infants with birth weights exceeding 2500 g. The results suggest that RA-cell morphology will prove to be of value in the early antenatal prediction of spontaneous abortion and low birth weight.     

THE NATURE AND ORIGIN OF THE SOLUBLE PROTEIN IN HUMAN AMNIOTIC FLUID

1. Amniotic fluid surrounds the human fetus and is separated from the uterus by the amnion, chorion and placenta. The ability to obtain samples of amniotic fluid from women by a simple procedure has encouraged studies on the nature and origin of the fluid, and on its use for the diagnosis of a variety of clinical conditions. The fluid contains cells, which are of fetal origin, and can be grown in a tissue culture. Cyto-genetic and biochemical analyses can therefore be used to detect chromosomal aberrations and inborn errors of metabolism in the fetus. 2. The supernatant of amniotic fluid contains many of the solutes typical of extracellular fluid. In particular, it contains a wide range of proteins and those which are of fetal origin are likely to be of use in the prenatal diagnosis of fetal disease. This review examines the nature and origin of the soluble protein in amniotic fluid, and discusses the diagnostic uses of the proteins which are of fetal origin. 3. In other mammals, the arrangement of the fetal membranes is different from that in man, and these differences are reflected by changes in the nature of the amniotic fluid. Thus data from other animals have little applicability to man. 4. Electrophoresis and immunoelectrophoresis have established that the major proteins in amniotic fluid are also present in maternal and fetal sera. Their concentrations in the fluid are influenced by their molecular weight and proteins larger than about 2.5 times 10⁶ may be excluded. Towards term, phenotyping studies show that a number of serum proteins in amniotic fluid are of maternal origin. In the case of group-specific component (Gc) this has been shown to be so throughout pregnancy. Such proteins must enter the fluid by diffusing across either the chorion or the chorionic plate and then the amnion. 5. It has been previously claimed that various serum proteins in amniotic fluid are of fetal origin. For albumin and IgG there are data that strongly support a maternal origin. The evidence on the origin of insulin is inconclusive. The concentration of β₂-microglobulin in amniotic fluid exceeds that in maternal serum and is probably too high also for fetal serum to be its major source. It has a wide tissue distribution and probably enters the fluid from surrounding structures. 6. Alpha-fetoprotein in amniotic fluid is of fetal origin as it is present in maternal serum at far lower concentrations. It is found in fetal serum, urine and yolk sac, but it is not clear how it enters the amniotic fluid of normal fetuses. The concentrations of Gc and alpha-fetoprotein have been measured in amniotic fluid and in their sera of origin. The relative concentration of Gc in amniotic fluid was found to be much greater than that of alpha-fetoprotein and the concentration gradients of these marker proteins can be compared with data for other proteins. In this way further evidence has been obtained that the albumin, α₁,-antitrypsin and transferrin in amniotic fluid are mainly of maternal origin throughout pregnancy. 7. Immunological studies have shown that at least three proteins of non-serum origin are present in amniotic fluid and they have also been located in the amnion and uterine decidua. 8. The enzymes present in amniotic fluid are summarized. Many lysosomal enzymes are clearly of fetal origin since they show altered specific activities in the appropriate cases where the fetus is affected with an inborn error of metabolism. For other enzymes, analysis of specific activity gradients can help to decide the extent to which an enzyme is of serum origin, although this will not exclude the possibility of a maternal (uterine) contribution. The results of such analyses suggest that, relative to the serum protein in amniotic fluid, the greatest concentrations of the minor non-serum proteins in the fluid occurs between thirteen and eighteen weeks of pregnancy and also towards term. 9. Some inborn errors of metabolism may be diagnosed prenatally by measuring the specific activity of the respective enzyme in amniotic fluid. However, the presence of different enzymes with similar substrate specificities has prevented this in Pompe's disease. 10. In cases where the fetus is affected with anencephaly or spina bifida there is an increase in the concentration of alpha-fetoprotein in the amniotic fluid. This has provided a way of detecting these diseases early enough to allow termination of pregnancy. 11. The discovery of new proteins in fetal serum and in the tissues surrounding the amniotic cavity would seem to provide the best chance of extending the uses of amniotic fluid into the other areas of prenatal medicine.     

A counseling dilemma involving anencephaly, acrania and amniotic bands.

A suggested fetal anencephaly on routine office ultrasound examination resulted in a diagnosis of fetal acrania when targeted ultrasonography was performed by a consultant. Following pregnancy termination, examination of the abortus revealed partial cranial destruction secondary to an amniotic band. It is often difficult to distinguish between anencephaly, acrania, and amniotic band sequence prenatally, but postnatal differentiation is imperative for accurate risk assessment in genetic counseling.     

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.     

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.     

Assessment of Fetal Lung Maturity by Estimation of Amniotic Fluid Palmitic Acid

The measurement of palmitic acid in amniotic fluid has been shown to be a rapid means of assessing the lecithin concentration. The level of palmitic acid increases quickly when the fetal lung matures at about 35 weeks'' gestation, and the level in amniotic fluid obtained 24 hours or less before delivery clearly distinguishes which infants are likely to develop respiratory distress syndrome and which are mature.     

Free Amino-acid Concentrations in Fetal Fluids

The pattern of free amino-acid concentrations in maternal venous plasma, fetal umbilical arterial plasma, fetal urine, and amniotic fluid at 15 to 20 weeks'' gestation has been determined. Free amino-acid concentrations were greater in fetal plasma than in maternal plasma, amniotic fluid, or fetal urine.The ratios of amino-acid concentrations in fetal umbilical arterial plasma and urine indicate that the fetal kidney can effectively conserve amino-acids, possibly reaching an adult level of competence in this respect.There was little correlation between amino-acid concentrations in the fluids analysed with the exception of that between amniotic fluid and fetal urine.     

Estimation of gestational age from study of amniotic fluid and clinical assessment.

Study of 108 samples of amniotic fluid obtained between 28 and 42 weeks'' gestation from 101 patients revealed that in normal pregnancies the creatinine concentration, lecithin/sphingomyelin (L/S) ratio and percentage of fat cells correlated better with the gestational age of the newborn--assessed by clinical criteria--than did the bilirubin and sodium concentrations. A creatinine concentration of 1.75 mg/dL or more, an L/S ratio of 4 or more and a fat cell percentage of 10 or more correlated significantly with a gestational age of 37 weeks or more. In abnormal pregnancies (those with obstetric or medical complications, or both) the mean creatinine concentration in the amniotic fluid was significantly less than expected for gestational age in fetal dysmaturity and greater than expected when the mother had diabetes. The mean L/S ratio in the amniotic fluid was elevated when the mother had hypertension or smoked and in cases of fetal dysmaturity or long interval between rupture of the membranes and delivery, whereas it was significantly lower than normal when the mother had diabetes. The mean bilirubin concentration in the amniotic fluid was significantly lower than normal when the mother had hypertension. When the mother had diabetes, maturity of the fetal lung, liver, skin and brain appeared to be delayed, according to the values for the amniotic fluid constituents.     

Chromosome Analysis before Birth and its Value in Genetic Counselling

Chromosome analysis of amniotic cell cultures was achieved in 29 out of 30 consecutive patients who were referred for genetic counselling during pregnancy. Amniocentesis was performed without any apparent untoward maternal or fetal complication. The only pregnancy terminated was that of a carrier of X-linked granulomatous disease, in whom the amniotic cells showed that the fetus was male and also had Down''s syndrome (trisomy G). Chromosome analysis in the remaining 28 patients showed normal karyotypes. The interval between amniocentesis and a definitive karyotype varied from 7 to 31 (average 18·4) days.The reliability of chromosome analysis from amniotic cell culture and of fetal sex determination by means of the sex chromatin and Y-fluorescence techniques was studied further in amniotic fluid from cases of therapeutic abortion and of rhesus incompatibility. The fetal sex was correctly determined in all cases. It is concluded that antenatal diagnosis of genetic disease by amniocentesis now permits a more practical approach to genetic counselling.     

Cell morphology in long-term cultures of normal and abnormal amniotic fluids

Summary The cell morphology of long-term cultures of amniotic fluid cells from 10 fetuses with a neural tube defect (NTD) and three with omphalocele was examined and compared to 30 long-term cultures of normal amniotic fluids as well as a long-term culture of human fetal brain. Cultures from the amniotic fluids of the fetuses with NTD and omphalocele showed cells with the same general characteristics as normal amniotic fluid cells. However, the cultures of amniotic fluid cells from NTD pregnancies had an additional cell type also seen in fetal brain culture. This was a neuroblast-like cell, with small rounded refractile morphology and long branching processes forming clusters of varying sizes which lay on top of large flat cells. These neuroblast-like cells diminished in number with time in culture and were not present in subcultures. Their possible neuronal origin is discussed.     

The detection of neural tube closure defects by exfoliative cytology of amniotic fluid

Cytospin preparations of amniotic fluid samples from 200 pregnancies, taken between 16 and 20 weeks' gestation, were examined without knowledge of the fluid alpha-fetoprotein (AFP) level. The specimens were taken because of the possibility of neural tube closure defect. All but eight cases showed predominantly squamous cells, amnion cells, macrophages and blood cells. AFP levels in these fluids were within the normal range in 178 cases, unequivocal in 11, undetectable in 2 and raised in 1; none of the babies in these cases had a neural tube closure defect. In eight cases a large population of small cells with dark nuclei and a population of large, foamy macrophages were present in addition to the other cell types; all of these babies had a neural tube closure defect (five anencephaly and three anencephaly with spina bifida). This technique may provide a useful additional method of diagnosis of neural tube closure defects.     

Sampling pure fetal blood by fetoscopy in second trimester of pregnancy.

A technique for fetal blood-sampling in the second trimester of pregnancy (between 16 and 22 weeks'' gestation) combining fetoscopy with real-time ultrasound was used in 48 attempts at fetal blood-sampling. Specimens containing fetal red cells with or without amniotic fluid or maternal blood, and adequate for diagnosing haemoglobinopathies, were obtained in 45 of the 48 fetoscopies. Sampling was successful in all 18 patients with a posterior placenta, and in 27 of the 30 with an anterior placenta. In 22 of the last 27 consecutive fetoscopies pure fetal blood was taken; the placenta was anterior in 16 and posterior in six. Out of 17 cases sampled between 18 and 22 weeks'' gestation pure fetal blood was obtained in 16. The volume of the samples varied from 50 to 500 microliter. The ability to obtain pure fetal blood consistently even when the placenta is anterior will increase knowledge of fetal physiology and the scope of prenatal diagnosis.     

Bovine fetal fluid cells in vitro: fate and fetal sex prediction accuracy

The in vitro fate of bovine fetal fluid cells and the efficiency of fetal sex predication from cultures of these cells are studied using aspirates from live animals and pregnant uteri collected from the slaughterhouse. Over 70 percent of bovine amniotic fluid samples aspirated from pregnant uteri at the time of slaughter yielded cultures adequate for chromosome analysis whereas only 10 percent of allantoic fluid samples produced growth of cells satisfactory for chromosome analysis. Fetal sexing accuracy was 100 percent in all samples studied. Seven readily recognizable cell types were noted in cultures of fetal fluid cells obtained at various stages of gestation. In a majority of cases, the in vitro morphology of cells from both fetal cavities was similar to that observed in primary human amniotic fluid cell cultures.     

Bovine fetal fluid cells in vitro: Fate and fetal sex prediction accuracy

Summary The in vitro fate of bovine fetal fluid cells and the efficiency of fetal sex prediction from cultures of these cells are studied using aspirates from live animals and pregnant uteri collected from the slaughterhouse. Over 70% of bovine amniotic fluid samples aspirated from pregnant uteri at the time of slaughter yielded cultures adequate for chromosome analysis, whereas only 10% of allantoic fluid samples produced growth of cells satisfactory for chromosome analysis. Fetal sexing accuracy was 100% in all samples studied. Seven readily recognizable cell types were noted in cultures of fetal fluid cells obtained at various stages of gestation. In a majority of cases, the in vitro morphology of cells from both fetal cavities was similar to that observed in primary human amniotic fluid cell cultures.     

Citrullinemia: prenatal diagnosis of an affected fetus.

We monitored a pregnancy in a family at risk for citrullinemia due to argininosuccinic acid (ASA) synthetase deficiency. ASA synthetase activity in cultured epithelioid amniotic fluid cells from the fetus at risk was less than 2% of control epithelioid amniotic fluid cell activity. An increased concentration of citrulline was found in the at-risk amniotic fluid (0.14 mumol/ml) as compared with fluid from six controls and one at-risk but unaffected pregnancy (trace). The pregnancy was terminated, and the in utero diagnosis was confirmed by assay of ASA synthetase activity in cultured fetal skin fibroblasts (4.4% of control activity). In addition, all five fetal tissues studied had significant accumulation of citrulline, whereas control fetal tissues had none. These data provide evidence that, if precise control is maintained over tissue culture variables, citrullinemia can be diagnosed successfully in utero by microassay of ASA synthetase activity in cultured amniotic fluid cells. They also suggest that amniotic fluid citrulline concentrations provide strong adjunctive evidence for this prenatal diagnosis.     

Rat alpha-fetoprotein: isolation, radioimmunoassay and fetal-maternal distribution during pregnancy.

A method is described for the isolation of mg quantities of two forms of rat alpha-fetoprotein (AFP) from amniotic fluid by preparative disc-gel column electrophoresis using a continuous elution system. AFP isolated by this method is suitable for use as an antigen, can be labelled for use in a radioimmunoassay and serves as a reference standard. The characteristics of a new antiserum to AFP are also described. The protocol for a radioimmunoassay is outlined which permits the measurement of AFP in several fetal-maternal physiological compartments throughout gestation. Levels of AFP in fetal liver and fetal plasma suggest that secretion of AFP from liver occurs soon after synthesis with minimal hepatic storage. The pattern for AFP in maternal serum parallels that observed in amniotic fluid and fluctuations in maternal serum levels of AFP appear to be buffered by AFP accumulation in amniotic fluid. Fetal clearance of AFP under normal conditions may be relatively constant from Days 11-20 of gestation since an amniotic fluid: maternal serum AFP ratio of 30:1 is maintained during this period.     

Oxytocin and vasopressin in the rat do not readily pass from the mother to the amniotic fluid in late pregnancy

In order to see whether the mother contributes to the vasopressin or oxytocin levels of amniotic fluid, these peptides were measured under conditions (1) in which the fetus lacks vasopressin (Brattleboro strain) and (2) where high maternal oxytocin and vasopressin plasma levels were induced by means of a controlled-delivery Accurel-collodion device. No vasopressin could be demonstrated in amniotic fluid of vasopressin-deficient fetuses present in a heterozygous (i.e., vasopressin-synthetizing mother). High peptide levels on the maternal side of Wistar rats generally failed to affect the amniotic fluid levels. The increase that was occasionally seen in amniotic vasopressin was probably due to fetal release concomitant with growth retardation. Amniotic vasopressin is derived from the fetus. Since amniotic fluid oxytocin is neither derived from the mother nor from the fetal brain, other fetal sources should be considered.