The prenatal determination of glucose-6-phosphatase activity by fetal liver biopsy

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.     

Fetal intestinal microvilli in human amniotic fluid

The intestinal microvilli of fetal origin in human amniotic fluid were purified by Ca²⁺ precipitation of contaminating organelles followed by differential centrifugation of microvillar membranes. In the purified preparation, the specific activity of the microvillar marker-enzymes maltase and sucrase increased about 77-fold over that in cell-free amniotic fluid. Significant contamination of the purified preparation by endoplasmic reticulum (microsomes) and lysosomes was ruled out on the basis of a low content of the marker enzymes glucose-6-phosphatase (microsomes) and acid phosphatase (lysosomes). Amniotic fluid microvilli contain typical enzymes of the fetal intestine including maltase, sucrase, trehalase, alkaline phosphatase and γ-glutamyltransferase, and their morphology by electron microscopy resembles that of vesiculated intestinal microvilli. Prenatal detection of genetic diseases due to a deficiency of a protein expressed in these membranes or associated to abnormal microvilli seems feasible.     

Possibility of prenatal diagnosis of hereditary triose phosphate isomerase deficiency

Prenatal diagnosis has been performed on umbilical cord blood of an 18 weeks fetus of heterozygous triosephosphate isomerase (TPI) deficient parents. After excluding maternal blood contamination, TPI activity was measured and found to be 60 per cent of the normal mean whereas the value of glucose-6-phosphate dehydrogenase activity was in the normal range of fetal blood. In addition, the analysis of the characteristics of fetal TPI, i.e. K_m measurements for glyceraldehyde-3-phosphate, heat stability tests and electrophoretic studies, did not show any evidence of a special form of TPI in fetal blood. These results were consistent with the heterozygous state and were confirmed at birth.     

Serum hCG assay: A method for detection of contamination of fetal blood samples

Serum human chorionic gonadotrophin (hCG) can be assayed in specimens obtained by percutaneous fetal blood sampling to check for the absence of maternal blood or amniotic fluid contamination. In order to assess the accuracy of this approach, we measured serum hCG in 44 pure fetal blood samples obtained by intracardiac puncture. The mean fetal serum hCG concentration was 52 IU/l, and the ratio of maternal to fetal serum hCG concentration never exceeded 1 · 1 per cent, which represents the smallest contamination rate detectable by this method.     

Immunocytochemical localization of peroxisomal β-oxidation enzymes in human fetal liver

In the majority of congenital peroxisomal disorders, β-oxidation of very long chain fatty acids is deficient. We have investigated the appearance and localization of the three peroxisomal β-oxidation enzymes in normal fetal liver (fertilization age between 5 and 18 weeks) with protein A- gold immunocytochemistry and silver enhancement for light microscopic visualization. With specificity-tested polyclonal antibodies, acyl-CoA-oxidase, bifunctiooal enzyme, and 3-oxoacyl-CoA thiolase were localized in the peroxisomes of the parenchymal cells, which appear as brown or black granules. In the youngest specimen, no immunopositive reaction was obtained. A weak reaction with anti-thiolase was obtained at the age of 6–7 weeks. At a fertilization age of 8 weeks, peroxisomes could be distinctly visualized after immunostaining for all three enzymes. From a staining series with anti-thiolase on simultaneously treated slides, it appears that the amount of antigen per peroxisome and the organelle size increase between the seventh and eighteenth weeks. These data should enable a more specific diagnosis in fetal liver biopsies from pregnancies at risk and after termination of pregnancy.     

Fetal liver alanine: Glyoxylate aminotransferase and the prenatal diagnosis of primary hyperoxaluria type 1

Primary hyperoxaluria type 1 (PH1) is caused by a deficiency of the hepatic peroxisomal enzyme alanine: glyoxylate aminotransferase (AGT, EC 2.6.1.44) (Danpure and Jennings, FEBS Lett., 201 , 20–24, 1986). The activity of AGT has been measured in fetal livers of gestational age 14–21 weeks. Activity increases up to 17 weeks and then levels off between 17 and 21 weeks. At this time, the mean AGT activity is about 30 per cent of the mean normal postnatal level. As in adult liver, the AGT enzyme activity and the AGT immunoreactive protein are peroxisomal. Prenatal diagnosis has been performed by measuring AGT enzyme activity and immunoreactive AGT protein on liver biopsies from two fetuses at risk for primary hyperoxaluria type 1. One was unaffected and one was affected.     

Risk assessment of amniocentesis between 11 and 15 weeks: Comparison to later amniocentesis controls

We studied 693 consecutive early amniocenteses (prior to 15 weeks) and found a spontaneous abortion rate to 28 weeks' gestation of 1·5 per cent. A control group of women having standard amniocentesis (15–20 weeks) experienced a 0·6 per cent fetal loss in the same period. There were no other apparent differences between the two groups. Early amniocentesis results are generally available 4–6 weeks before standard amniocentesis and 1–3 weeks after chorionic villus sampling (CVS). Alpha-fetoprotein (AFP) can be accurately assayed in 11- to 15-week amniotic fluid samples but additional studies are necessary to determine the accuracy of neural tube defect (NTD) detection. Including the present study, over 5800 early amniocenteses have been reported and the results suggest that this is a relatively safe prenatal diagnostic test and an alternative to CVS and later amniocentesis.     

Prenatal diagnosis of carbamoyl-phosphate synthetase deficiency by fetal liver biopsy

In a pregnancy at risk for carbamoyl-phosphate synthetase (CPS) deficiency, prenatal diagnosis was attempted by fetal liver biopsy, performed at 18 weeks of gestation. CPS activity was absent and the diagnosis was confirmed after termination of the pregnancy. The technique employed for fetal liver biopsy is described together with an evaluation of its possible role in prenatal diagnosis.     

Transabdominal villus sampling in early second trimester: A safe sampling method for women of advanced age

Transabdominal chorionic villus sampling (TA-CVS) was performed in 707 viable singleton pregnancies to exclude chromosomal abnormalities. Maternal age ranged between 36 and 49 years (mean 37·9 years); gestational age varied between 10·2 and 18·3 weeks (mean 13·3 weeks). In 639 women (90·4 per cent), a sufficient amount of chorionic tissue (⩾ 10 mg) was obtained after one needle insertion; in 66 women (9·3 per cent) two insertions were needed. An abnormal chromosome pattern was established in 19 cases (2·9 per cent). Vaginal bleeding or spotting within 28 days after TA-CVS occurred in 11 cases (1·5 per cent). The completed follow-up of 678 chromosomally normal pregnancies showed an overall fetal loss rate of 2·6 per cent before 28 weeks. The overall perinatal mortality was 0·9 per cent. When relating fetal loss to gestational age at TA-CVS, this was 6·6 per cent in women sampled before 12 weeks against only 1·8 per cent after 12 weeks. At the same time, the percentage of fetal loss occurring within 2 weeks following the procedure was 75 and 30 per cent, respectively. It is suggested that these data reflect the decline in spontaneous abortion rate during this particular period of pregnancy. It is concluded that TA-CVS is an effective procedure which, when performed after the natural decrease of fetal loss, appears to be a safe option for women of advanced maternal age.     

Early amniocentesis and fetal nuchal translucency in women requesting karyotyping for advanced maternal age

Fetal nuchal translucency was measured at 11–14 weeks' gestation in 97 pregnancies referred for early amniocentesis for advanced maternal age. The nuchal translucency was abnormal in 11 fetuses and the fetal karyotype was abnormal in five of these 11 cases. The karyotype was normal in 86 cases with normal nuchal translucency. The culture failure and miscarriage rates associated with early amniocentesis were 3·3 per cent and 2·2 per cent respectively. Amniotic fluid leakage occurred in 6 per cent of cases. In women requesting fetal karyotyping for advanced maternal age without additional biochemical screening, fetal nuchal translucency should be measured at 11–14 weeks. If the nuchal thickness is ≥ 3 mm, a first-trimester diagnostic procedure is indicated; however, if it is <3 mm, amniocentesis should be delayed until 16 weeks' gestation.     

Risks of transabdominal chorionic villus sampling before the 12th week of amenorrhea

The authors report on a series of 210 chorion villus sampling diagnoses made with a needle by the transabdominal route. The rate of fetal loss was 4·2 per cent. Placental localization was important: fetal losses were 8 per cent when the placenta was strictly posterior (transamniotic route), whereas it was only 1·6 per cent when it was not posterior. Moreover, all fetal losses occurred (apart from one at 12·5 weeks of amenorrhea) before the 12th week of amenorrhea. The authors suggest that choriocentesis by the transabdominal route should not be performed before the 12th week of amenorrhea, and that the amniotic membrane should not be disturbed before the 13th week of amenorrhea.     

Amylo-1,6-glucosidase activity in cultured cells: A deficiency in type III glycogenosis with prenatal studies

Deficiency of amylo-1,6–glucosidase activity was expressed in parallel in liver and skin fibroblasts from a patient with type III glycogenosis. In crude extracts of control liver and muscle, amylo-1, 6–glucosidase (M.W. 164000) was identified by immunoprecipitation; no cross-reacting material was found in the patient's liver. Assay of amylo-1,6–glucosidase activity in cultured skin fibroblasts from the affected family revealed less than 10 per cent of control value in mutant homozygous cells whereas in cells from the parents, activity was reduced to 40–60 per cent of the control value. Activity in cultured amniotic fluid cells was similar to that of control fibroblasts. In cultured amniotic fluid cells obtained during the mother's subsequent pregnancy, the normal amylo–1,6–glucosidase activity measured, predicted correctly the outcome of this pregnancy prior to the 20th week of gestation.     

‘Late CVS’ international registry compilation of data from 24 centres

Data on 2058 late CVS cases, i.e., placental biopsies after 12 completed weeks of pregnancy, were collected from 24 centres. Two major groups of indications with or without ultrasound findings suspicious of fetal chromosomal abnormalities can be differentiated. In the first group, the rates of cytogenetic anomalies (21 per cent) and fetal losses (10 per cent) are high. The respective figures for the low-risk group are 6 per cent for chromosome anomalies and 2 per cent for total fetal losses. To evaluate this rapidly spreading new method further, more data are required and will be collected by the CVS registry based in Philadelphia, U.S.A.     

First trimester diagnosis of Wolman's disease

Wolman's disease was diagnosed in the first trimester of pregnancy by the direct demonstration of acid lipase deficiency in chorionic villi. The diagnosis was confirmed by studies on cultured chorionic villus cells and fetal skin fibroblasts. Acid lipase activity was assayed with both 4-methylumbelliferyl-palmitate and radiolabelled cholesterol oleate as substrates. The higher specificity of the enzyme for the latter, natural, substrate makes it superior in prenatal diagnosis.     

The impact of supportive intervention after second trimester termination of pregnancy for fetal abnormality

The reactions of women who had had a termination of pregnancy for fetal abnormality in the second trimester have been studied retrospectively using a semi-structured questionnaire. The severity of the grief reaction was measured and the outcome at 6months was compared with the findings from a previous study in South Wales which had led to the introduction of skilled support from genetic fieldworkers and formal genetic counselling after the termination. Of the 69 women interviewed, 55 (80 per cent) experienced an acute grief reaction and 17 (25 per cent) had not resolved their grief 6 months after the termination, compared with 37 (77 per cent) and 22 (46 per cent) out of 48 respectively in the previous study. Fifty-seven (83 percent) women had found the fieldworker's intervention useful or very useful, some describing her support as essential. An association between poor resolution of the grief reaction with increasing maternal age and with poor perceived support from partners was noted. Improved follow-up support and counselling have lessened the adverse emotional consequences and support should therefore be offered to all women undergoing termination for fetal malformation.     

Transabdominal chorionic villus sampling for fetal genetic diagnosis. Technical and obstetrical evaluation of 100 cases

First trimester fetal diagnosis was established in 100 pregnancies at risk by transabdominal chorionic villus sampling (TA-CVS). Forty-eight per cent of the women were 35 years or more at the time of sampling. Using the double needle technique, both the aspiration and the diagnostic success rate were 100 per cent. The mean amount of villi aspirated was 28·2 mg (10–50 mg). The mean needle time was 3 min. Vaginal spotting appeared in 2 per cent of the women. Four women had therapeutic abortion due to abnormal findings and one for social reasons. Three fetuses with normal karyotypes were lost. Excluding the therapeutic abortions, the fetal loss rate was 3±2 per cent. The fetal loss rate in the amniocentesis control group (n = 200) was 3±6 per cent. The cytogenetic diagnosis was carried out by the direct preparation technique as well as by chorion villus cultivation. All karyotypes were confirmed by lymphocyte cultures from umbilical cord blood or heel blood from the newborn or from aborted fetal tissue. Transabdominal CVS is deemed a safe and easy tool for achieving chorionic villi in the first trimester.     

Prenatal diagnosis of chromosomal abnormalities from chorionic biopsy samples: Improved success rate using a modified direct method

Several methods for fetal chromosome analysis using chorionic biopsy samples were compared. A modified direct method for culturing villi was considered to be the method of choice and details are presented of 186 pregnancies tested prenatally. The success rate in obtaining a fetal karyotype with the direct method was 93 per cent. The fetal loss rate in the prenatal series was 4.3 per cent and congenital abnormalities in the babies already born did not differ from the expected incidence.     

Detection of fetal HLA-DQα sequences in maternal blood: A gender-independent technique of fetal cell identification

The objective of this study was to detect fetal HLA-DQα gene sequences in maternal blood. HLA-DQα genotypes of 70 pregnant women and their partners were determined for type A1. We specifically sought couples where the father, but not the mother, had genotype A1. In 12 women, maternal blood samples were flow-sorted. Candidate fetal cells were isolated and amplified by using PCR primers specific for a paternal HLA-DQα A1 allele. Fetal HLA-DQα A1 genotype was predicted from sorted cells; amniocytes or cheek swabs were used for confirmation. Six of twelve sorted samples had amplification products indicating the presence of the HLA-DQα A1 allele; 6/12 did not. Prediction of the fetal genotype was 100 per cent correct, as determined by subsequent amplification of amniocytes or cheek swabs. We conclude that paternally inherited uniquely fetal HLA-DQα gene sequences can be identified in maternal blood. This system permits the identification of fetal cells independent of fetal gender, and has the potential for non-invasive prenatal diagnosis of paternally inherited conditions.     

Chromosomal prenatal diagnosis: Study of 936 cases of intrauterine abnormalities after ultrasound assessment

Nine hundred and thirty-six prenatal chromosomal analyses were performed by four cytogenetic centres after ultrasound diagnosis of fetal abnormalities, amniotic fluid disorders, fetal growth retardation, and fetal or placental abnormalities. During the same period, 6515 fetal karyotypes were analysed because of maternal age. Frequencies of chromosomal aberrations in each case were respectively 4·4, 6·7 and 15·8 per cent, compared with 3·18 per cent when the fetal karyotype was performed because of maternal age. High rates of chromosomal aberrations are observed in cases of cervical hygroma, limb abnormalities, omphaloceles, duodenal stenosis, hydrocephalus, and facial abnormalities. In the case of polymalformations, this rate was 29·2 per cent. When malformations were seen together with an amniotic fluid disorder or growth retardation, 21·5 per cent chromosomal aberrations were observed. This frequency was 10·4 per cent when growth retardation was associated with an amniotic fluid disorder. Trisomy 13, 18, 21 and monosomy X accounted for 4/5 of all abnormalities in which we observed a high rate of triploidies (4·9 per cent) and balanced (3·3 per cent) or unbalanced (9·8 per cent) non-Robertsonian structural abnormalities. Sonographic ascertainment of these aberrations and prenatal characteristics of major anomalies are discussed.     

Discrepant karyotypes after second- and third-trimester combined placentacentesis/amniocentesis

Cytogenetic data are presented from a total of 1306 consecutive pregnancies with successful diagnosis obtained from both chorionic villi after short-time culture (CVS-SC) and amniotic fluid cell cultures (AC); samples had been taken simultaneously at combined placentacentesis (placental biopsy) and amniocentesis during the second (92·8 per cent) and third (7·2 per cent) trimesters. Concordant results were obtained in 1218 pregnancies with a normal karyotype and in 62 pregnancies with an aberrant fetal karyotype. Discrepant, i. e. false-positive and false-negative, results were found in 26 cases (2 per cent). From these data the accuracy of CVS-SC, defined as the proportion of all correct diagnoses, is calculated to be 98 per cent. Three non-mosaic and 14 mosaic false-positive results obtained after CVS-SC could not be confirmed by AC. Related to 1235 true normal fetal karyotypes, the specificity of CVS-SC, i.e. the proportion of normal karyotypes correctly diagnosed, amounts to 98·6 per cent. In nine pregnancies, an aberrant fetal karyotype detected after AC was missed by CVS-SC. The sensitivity of CVS-SC, i.e. the proportion of abnormal fetuses correctly diagnosed (62 out of 71), amounts to 87·3 per cent in our study group.