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We have developed a two-colour immunocytochemical staining method for the detection of fetal and embryonic haemoglobin in erythroid cells. The method was applied to study these haemoglobin types in fetal red cells. Specimens from fetal blood (10 weeks), cord blood and fetal liver (14 weeks) as well as chorionic villus samples (10-13 weeks) were stained for gamma and epsilon chains using CY3 and FITC labelled antibodies. Morphometric analysis was applied to determine cell size. Samples from organs involved in early embryonic development contained relatively large erythroblasts expressing the epsilon globin chain (megaloblasts); later in gestation the gamma chain was co-expressed by the same cells which ultimately became smaller and contained HbF (alpha 2 gamma 2) only. This phenomenon was confirmed in CVS samples in which all cell types were abundantly present. Since fetal erythroblasts are considered candidate cells for non-invasive prenatal diagnosis using FISH, we studied the phenotype of erythroblasts circulating in the maternal blood. The majority of erythroblasts in maternal blood appeared to be of the relatively small gamma globin-containing cell type. However, careful screening of the same maternal blood samples also revealed erythroblasts expressing epsilon or epsilon and gamma globins simultaneously, although at low frequency. Control specimens from non-pregnant women did not show nucleated red cells expressing either of the haemoglobin types. These observations may contribute to the better recognition of fetal cells in the maternal blood for prenatal diagnosis.

Prenatal testing of 12 pregnancies at risk for argininosuccinic aciduria due to argininosuccinate lyase (ASAL) deficiency and three pregnancies at risk for citrullinaemia due to argininosuccinate synthatase (ASAS) deficiency was performed by metabolite detection in amniotic fluid and measurement of enzyme activity in uncultured and cultured chorionic tissue and in cultured amniocytes. From our data and those of previous studies, amniotic fluid argininosuccinate measurement alone is clearly a reliable and rapid diagnostic test for both severe and mild ASAL deficiency if maternal ASAL deficiency can be excluded. For prenatal diagnosis of ASAS deficiency, however, both measurement of the amniotic fluid citrulline level and enzyme assay should be employed.

The finding of a 'faint-positive' acetylcholinesterase band in amniotic fluid samples of women at 15 weeks' gestation or above is associated with an increased risk of fetal abnormalities, most commonly gastroschisis. This finding warrants a targeted sonographic evaluation, in order to rule out significant fetal malformations.

We report the first prenatal diagnosis of an affected fetus with Chediak-Higashi syndrome (CHS). Diagnosis was accomplished via fetal blood sampling at 17 menstrual weeks and was confirmed after birth. Retrospective measurement of the largest acid phosphatase-positive lysosomes in cultured amniotic fluid cells and chorionic villus cells showed that in CHS these lysosomes are significantly larger than those in normal cells. This method may be used for prenatal diagnosis of CHS by amniocentesis and chorionic villus sampling (CVS).

Maternal serum CA 125 levels were determined at 9-11 menstrual weeks for 26 cases of trisomy 13 (n = 4), trisomy 18 (n = 7), trisomy 21 (n = 15), and appropriate controls. There were no statistically significant differences between groups.

Cytogenetic data are presented for 11,473 chorionic villus sampling (CVS) procedures from nine centres in the U.S. NICHD collaborative study. A successful cytogenetic diagnosis was obtained in 99.7 per cent of cases, with data obtained from the direct method only (26 per cent), culture method only (42 per cent), or a combination of both (32 per cent). A total of 1.1 per cent of patients had a second CVS or amniocentesis procedure for reasons related to the cytogenetic diagnostic procedure, including laboratory failures (27 cases), maternal cell contamination (4 cases), or mosaic or ambiguous cytogenetic results (98 cases). There were no diagnostic errors involving trisomies for chromosomes 21, 18, and 13. For sex chromosome aneuploidies, one patient terminated her pregnancy on the basis of non-mosaic 47,XXX in the direct method prior to the availability of results from cultured cells. Subsequent analysis of the CVS cultures and fetal tissues showed only normal female cells. Other false-positive predictions involving non-mosaic aneuploidies (n = 13) were observed in the direct or culture method, but these cases involved rare aneuploidies: four cases of tetraploidy, two cases of trisomy 7, and one case each of trisomies 3, 8, 11, 15, 16, 20, and 22. This indicates that rare aneuploidies observed in the direct or culture method should be subjected to follow-up by amniocentesis. Two cases of unbalanced structural abnormalities detected in the direct method were not confirmed in cultured CVS or amniotic fluid. In addition, one structural rearrangement was misinterpreted as unbalanced from the direct method, leading to pregnancy termination prior to results from cultured cells showing a balanced, inherited translocation. False-negative results (n = 8) were observed only in the direct method, including one non-mosaic fetal abnormality (trisomy 18) detected by the culture method and seven cases of fetal mosaicism (all detected by the culture method). Mosaicism was observed in 0.8 per cent of all cases, while pseudomosaicism (including single trisomic cells) was observed in 1.6 per cent of cases. Mosaicism was observed with equal frequency in the direct and culture methods, but was confirmed as fetal mosaicism more often in cases from the culture method (24 per cent) than in cases from the direct method (10 per cent). The overall rate of maternal cell contamination was 1.8 per cent for the culture method, but there was only one case of incorrect sex prediction due to complete maternal cell contamination which resulted in the birth of a normal male.(ABSTRACT TRUNCATED AT 400 WORDS)

The accuracy of biochemical and molecular prenatal diagnoses using chorionic villi as the fetal source was assessed by seven centres participating in the NICHD collaborative study on the safety and accuracy of chorionic villus sampling (CVS) and amniocentesis. Of 601 pregnancies studied, biochemical methods were used to determine the diagnosis in 283 fetuses at risk for 35 different metabolic disorders. Fifteen different lysosomal storage diseases accounted for 81 per cent of the biochemical prenatal diagnoses performed, with 57 per cent of these pregnancies at risk for Tay-Sachs disease. No errors were made in the biochemical diagnoses that predicted affected or unaffected fetuses. However, the diagnoses of certain disorders (e.g., mucopolysacchariodosis type IH, metachromatic leukodystrophy, and Krabbe disease) occasionally required confirmatory studies in cultured amniocytes because the enzyme results were inconclusive in direct and/or cultured villi or due to the presence of a pseudodeficiency allele. Of these, only the diagnosis of a fetus at risk for Krabbe disease remained inconclusive after special studies to discriminate between mutant and pseudo-deficiency alleles. Recombinant DNA techniques were used to predict the diagnosis of 318 fetuses at risk for 16 different disorders in which the defective disease gene could be detected either directly or by linkage analysis to a nearby polymorphic marker. Of these, 32 per cent were for haemoglobinopathies, 25 per cent for cystic fibrosis, 24 per cent for Duchenne or Becker muscular dystrophy, and 7 per cent for haemophilias. Pregnancies at risk for known disorders with specific molecular lesions (e.g., sickle cell disease) were accurately diagnosed in direct and/or cultured villi. Diagnoses requiring analyses with closely linked polymorphic markers were occasionally uniformative or inconclusive. Maternal contamination was not reported in any biochemical or molecular-based diagnosis. These studies document the high accuracy and rapidity of both biochemical and mutation-specific prenatal diagnoses with direct and cultured chorionic villi.

Factors found to be associated with pregnancy loss after transcervical CVS were race (higher for non-white), history of spontaneous abortion, unplanned pregnancy, history of spotting or bleeding during the pregnancy prior to CVS, and placental position (higher for fundal or lateral locations). Whether the increase in loss risk is due to the factor, per se, or the factor plus the CVS cannot be determined due to the lack of appropriate control data.

Cytogenetic data from the United States NICHD collaborative study of chorionic villus sampling (CVS) were used to evaluate the clinical significance of chorionic mosaicism. The 10,754 patients with normal cytogenetic results were compared with 108 patients (1.0 per cent) with placental mosaicism and 181 patients (1.6 per cent) with pseudo-mosaicism. Of the pregnancies intended to continue, the pregnancy loss rate was significantly greater in patients with placental mosaicism than in the cytogenetically normal cohort (8.6 vs. 3.4 per cent, p less than 0.05). However, there was no difference in the frequencies of abruptio placenta, preterm labour or delivery, small-for-gestational-age newborns, pregnancy-induced hypertension, or neonates with Apgar scores less than 7.

Immunochemical serum assays for human chorionic gonadotropin (hCG), the free ahCG subunit, and progesterone (P) were considered separately and in combination for their ability to screen for chromosomally abnormal pregnancies in the first trimester. Maternal serum was collected from 141 women undergoing chorionic villus sampling at 9-12 menstrual weeks. Trisomy 21 pregnancies had significantly higher hCG levels, while trisomy 18 and 13 pregnancies had markedly lower hCG and progesterone levels than those of chromosomally normal pregnancies. However, the discrimination of normal from aneuploid pregnancies was poor with either hCG alone, progesterone alone, or free ahCG alone. Much improved discrimination was obtained by combining hCG, free ahCG, and P into an aneuploidy index [(P/hCG)(free ahCG/hCG)]. This index distinguished 9 out of 17 (53 per cent) of the trisomy 21 pregnancies, while only misidentifying 5 out of 112 (4.5 per cent) of the normal pregnancies. The aneuploidy index thus appears promising as a first-trimester biochemical screen for aneuploid pregnancies.

This paper reports our experience with 55 fetuses identified in utero to have a cystic hygroma. The outcome of fetuses with an isolated cystic hygroma, cystic hygroma with non-immune hydrops, and cystic hygroma with multiple anomalies was evaluated. Approximately two-thirds of karyotypes were aneuploid, and a strong association of septation and aneuploidy existed. Only five cases, four of which had isolated hygromas, came to term and resulted in live births. Two of these involved small non-septated lesions which resolved in utero.

Maternal sera (MS) taken from 1396 women prior to chorionic villus sampling at 9-12 menstrual weeks were assayed for unconjugated oestriol (uE3) and alpha-fetoprotein (AFP). Median levels increased by 41 and 26 per cent per week respectively in normal pregnancies. There were 32 pregnancies with a chromosome abnormality. The median MS uE3 and AFP were 0.73 and 0.75 multiples of the median (MoM) respectively in the ten cases of Down's syndrome (DS) but not decreased in the other abnormalities. These results suggest that both uE3 and AFP may be useful in identifying DS in the first trimester. Additional prospective studies are needed to confirm these findings.

Three hundred and thirty-nine cases of multiple gestation underwent prenatal diagnosis by amniocentesis. The spontaneous abortion rate (to 28 weeks) in this group was 3.57 per cent compared with our singleton abortion rate of 0.60 per cent. The perinatal mortality rate (PMR) and prematurity rate were not different from the singletons, and compared favourably with the PMR reported in the literature for multiple gestations which did not undergo any intervention during pregnancy. This increased abortion rate following amniocentesis may only represent the increased natural loss rate in multiple gestations, and not indicate any increased risk added by the procedure.

Sera from women carrying either chromosomally normal or aneuploid fetuses in the first half of pregnancy were assayed for human chorionic gonadotropin (hCG) bioactivity in order to determine whether differences might provide the basis for a useful antenatal screen for aneuploidy. A mouse uterine weight assay was used to assess hCG bioactivity in sera from 35 patients undergoing chorionic villus sampling (12 normal pregnancies and 23 trisomic pregnancies) and in sera from 18 patients undergoing elective second-trimester abortion (12 presumed normal pregnancies, 3 trisomic pregnancies, and 3 pregnancies with neural tube defects). The hCG bioactivity to immunoactivity (B:I) ratio of normal pregnancies progressively decreased from 7.7 +/- 1.3 at 4-5 menstrual weeks, to 4.7 +/- 0.4 at 9-12 menstrual weeks, to 3.3 +/- 0.5 at 16-20 menstrual weeks. There were no significant differences in the B:I ratios between normal and aneuploid pregnancies in either the first-trimester (4.7 +/- 0.4 versus 5.2 +/- 0.3) or the second-trimester samples (3.3 +/- 0.5 versus 2.6 +/- 0.3), despite significantly greater hCG concentrations in the trisomic pregnancies. We conclude that while aneuploid pregnancies display dysfunctional regulation of hCG expression, the bioactivity of their hCG is normal and does not appear to form the basis for a useful screen for aneuploidy.

Four cases of multiple gestation with second trimester death of one fetus and subsequent damage to a survivor are reported. Monochorionic placentation was documented in three of the cases. Central nervous system lesions occurred in all cases, and bowel injury was noted in two of the damaged fetuses. Although rare maternal clotting problems have been reported in similar situations, none was noted in any of these four cases. Prior reports have indicated that losses in the first half of gestation were of no consequence to the surviving fetuses. These four cases contradict this suggestion, and indicate that close sonographic observation of the survivors is important in any multiple gestation where on fetus has died.

Chorionic villus sampling (CVS) has emerged as a first trimester alternative to amniocentesis for the prenatal detection of genetic disorders. We report our experience in 600 consecutive CVS procedures to better delineate the safety, efficacy and reliability of this new method of prenatal diagnosis. Adequate samples were obtained at the initial visit in 97 per cent of the cases, and successful cultures were established in 98.7 per cent of these patients. Chromosome abnormalities were detected in 5.9 percent of those pregnancies tested because of advanced maternal age (greater than or equal to 35 years). A discrepancy between the villus karyotype and that of the fetus was found in 2.0 per cent of cases, and most commonly consisted of mosaicism in the villus sample for a chromosomal abnormality that was not found in fetal samples. The risk of spontaneous abortion following the procedure was 6.3 per cent. We conclude that chorionic villus sampling is an acceptably safe and reliable procedure, but further investigation is needed before it can become an established technique in prenatal diagnosis.

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.

Maternal serum human chorionic gonadotropin (hCG) and the free alpha-hCG subunit were evaluated in 249 women from 9 to 11 weeks gestation who subsequently underwent chorionic villus sampling for determination of fetal karyotype and in 20 women of 18 or more weeks gestation who were ascertained to have an aneuploid fetus by genetic amniocentesis. Seven of the first-trimester pregnancies were determined to be aneuploid and six had hCG levels in the normal range (one triploid pregnancy had elevated hCG levels) whereas 12 of the 20 second-trimester cases had abnormal hCG levels and an additional three had elevated levels of alpha-hCG. This study confirms the previous report of abnormal maternal serum hCG levels in women with an aneuploid fetus at greater than or equal to 18 weeks gestation and demonstrates that hCG evaluation is not useful at 9-11 weeks gestation for selecting pregnancies at risk for fetal aneuploidy.

To examine the potential for prenatal diagnosis of genetic lipoprotein metabolic defects (e.g. abetalipoproteinemia. Tangier disease) we determined the normal concentrations of apolipoproteins (apo) A-I, A-II, B, and E in mid-trimester amniotic fluid and fetal plasma. The concentrations of apo A-I and apo A-II in amniotic fluid were 1--2 per cent of the respective levels in the mother's plasma, whereas apo B and apo E were undetectable in amniotic fluid. In contrast to amniotic fluid, all four apolipoproteins were detectable in fetal plasma, and the levels of apo A-I, apo B and apo E were in the range observed in the mothers: 160.2 +/- 103.1, 59.8 +/- 35.7 and 5.7 +/- 3.5 mg/dl respectively (mean +/- SD, n = 13). The fetal plasma level of apo A-II (28.3 +/- 12.4 mg/dl) was two-thirds that observed in the mother's plasma. The normal levels of these apolipoproteins in fetal plasma are well above the sensitivity of the methods, and their quantification requires only 10--20 microliters of fetal plasma. Determination of apolipoproteins in fetal blood obtained by fetoscopy thus may provide a method for the prenatal diagnosis of congenital apolipoprotein deficiencies.

The amount of fetal-maternal transfusion during invasive intrauterine diagnostic instrumentation was determined by measuring the increase in maternal serum alpha-fetoprotein (delta AFP) caused by the procedure. Fetal liver biopsy or fetoscopy for purposes other than blood sampling caused a mean delta AFP of 11.4 ng/ml and 34.2 ng/ml, respectively. Fetoscopy with fetal blood sampling produced a mean delta AFP of 211.8 ng/ml, while fetoscopy followed by placentesis caused a mean delta AFP of 462.8 ng/ml (representing a 1.07 ml fetal-maternal transfusion). Although this magnitude of fetal-maternal transfusion is an acceptable risk for the fetus, it is a sufficient transfusion to cause blood cell antigen sensitization.

A series of 8009 genetic amniocenteses were retrospectively examined to evaluate the relationship of the procedure to Rh isoimmunization. Of the 615 Rh negative women giving birth to Rh positive infants and estimated to be at risk, thirteen (2.1 per cent) were sensitized subsequently to the amniocentesis. Eleven of the sensitizations occurred early in the programs, and a combination of experience and ultrasound performed concurrently with the amniocentesis appear to have reduced the risk of isoimmunization to that of control data from the literature.

Two fetuses at risk for glucose-6-phosphatase deficiency had in utero liver biopsies. Analysis of each showed this enzyme activity to be in the normal range and the pregnancies continued. Neither child has any clinical or metabolic evidence of glucose-6-phosphatase deficiency.

A reliable and sensitive microassay for the measurement of liver glucose-6-phosphatase is described. Human fetal liver was assayed for glucose-6-phosphatase activity from 7.5 to 24 weeks of gestation and was found to have a mean activity of 2.11 nmol per min per mg of protein. This was approximately 30 per cent of the postnatal controls assayed by the same method, but there was no evidence of a change in activity during the gestational period examined. If fetal liver tissue can be reliably obtained, it may be possible to determine a deficiency of glucose-6-phosphatase in fetuses who are at risk.