Prenatal diagnosis of zellweger's syndrome by chorionic villus sampling-and a caveat

At 7·5 weeks gestation, two small chorionic villous biopsies were obtained from a woman at risk for Zellweger's cerebro-hepato-renal syndrome, and were separately established in culture. After 3 weeks, dihydroxyacetone phosphate acyltransferase (DHAP-AT) activity was measured in both cultures. The enzyme was markedly deficient in one cell strain and this was subsequently shown to have a male karyotype. However, the second culture had normal enzyme activity and a female karyotype. The pregnancy was terminated at 11·5 weeks gestation, and follow-up studies on fetal tissues confirmed a male fetus with markedly deficient DHAP-AT activity.     

Activities of alkaline phosphatase in first trimester chorion biopsy tissue

We have determined the activity of alkaline phosphatase in chorionic villous tissue obtained in the first trimester of pregnancy, in order to obtain the normal range of values as a prerequisite for application to the prenatal diagnosis of the rare bone disease hypophosphatasia. The activities found were a combination of intestinal and liver/bone/kidney types; traces of placental type were present in only one sample.     

Abnormal karyotype in the chorion,not confirmed in a subsequently aborted fetus

An abnormal fetal karyotype, containing a del 16(q21-qter) as an extra chromosome, was diagnosed in all 14 metaphases examined in a sample of chorionic villous biopsy material. After elective abortion a mosaicism for this cell-line together with a normal one was detected in the chorionic tissue. Fibroblast cultures from several fetal skin biopsies all revealed a normal karyotype.     

First-trimester prenatal diagnosis of osteogenesis imperfecta type II by DNA analysis and sonography

Osteogenesis imperfecta type II was diagnosed prenatally by analysis of DNA obtained from chorionic villus sampling (CVS) performed at 12 weeks of gestation in a woman who previously had had an affected child. The father had been shown to be mosaic for a mutation in the gene (COL1A2) which encodes the α2(I) chain of type I collagen. An affected fetus was predicted by detection of the mutation in amplified chorionic villus genomic DNA. Ultrasound examination at 13 weeks 4 days demonstrated femoral deformity and virtual absence of calvarial mineralization. In pregnancies at risk for osteogenesis imperfecta type II, sonographic evidence of skeletal abnormalities may be evident by 13 weeks' gestation.     

Human chorionic gonadotropin levels in pregnancies with aneuploid fetuses

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 secondtrimester 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 ≥ 18 weeks gestation and demonstrates that hCG evaluation is not useful at 9–11 weeks gestation for selecting pregnancies at risk for fetal aneuploidy.     

Chorionic villus sampling—short-term versus long-term culture in a subtle 2; 18 translocation

Prenatal diagnosis on chorionic villous tissue was performed for a woman with the karyotype 46,XX,t(2;18)(q32;q12)—a subtle ‘difficult’ translocation. The case illustrates the necessity of good quality cytogenetics for accurate prenatal diagnosis. For chorionic villi this can be obtained only with long-term culture.     

The importance of chorionic villus sampling after first trimester diagnosis of cystic hygroma

The prenatal diagnosis of The Turner Syndrome is described at a menstrual age of 12 weeks. Detection of cystic hygroma was followed by vaginal chorionic villous sampling (CVS) which revealed a 45,X karyotype. Early documentation of fetal karyotype in the presence of a cystic hygroma is essential for accurate diagnosis and genetic counselling.     

Microvillar enzyme analysis in amniotic fluid and the prenatal diagnosis of cystic fibrosis

The activities of two microvillar enzymes, gamma-glutamyltranspeptidase (GGTP) and alkaline phosphatase (ALP) have been determined in amniotic fluid (AF) samples from 39 pregnancies with a 1-in-4 risk of cystic fibrosis. Seventeen of these were investigated prospectively. A reduced proportion of the fetal specific intestinal ALP isoenzyme was found in 7 of a total of 13 pregnancies with cystic fibrosis and in one pregnancy of confirmed normal outcome. Eight of the affected pregnancies were tested for AF GGTP activity and depressed levels were found in 15. None of the 3 liveborn cystic fibrosis cases in the prospective series was identified by the ALP assay although 2 had significantly reduced GGTP activity. There were several amniotic fluid samples from cases of cystic fibrosis, trisomy 18 and normal outcome which had discordant GGTP and ALP results. Four of the 6 cases of cystic fibrosis misclassified by the ALP assay had amniocentesis at 15 or 16 weeks gestation. Evidence is presented which confirms a previous suggestion that amniocentesis after 17 weeks gestation improves the predictability of the ALP isoenzyme assay for the prenatal diagnosis of cystic fibrosis.     

Genetic diagnosis by chorionic villus sampling before 8 gestational weeks: Efficiency,reliability, and risks on 317 completed pregnancies

Transabdominal chorionic villus sampling (TA-CVS) was attempted in 328 high-risk pregnancies at 6–7 weeks of gestation. Sampling was feasible in 97.7 per cent of cases; chorionic tissue specimens of more than 10 mg were obtained in 94.4 per cent ofcases at the first needle insertion and in 100 per cent after a second attempt. Fetal karyotyping succeeded in 99.4 per cent of cases, while no diagnostic failures were reported in enzymatic and DNA analyses. Fetal loss rate in the first 4 weeks after CVS was significantly higher than in the later CVS series (7.2 vs. 2.5 per cent), but 50 per cent of losses were observed within 2 weeks in cases of inviable aneuploidies. A high incidence of severe limb abnormalities (1.6 per cent) was detected in pregnancies intended to continue, confirming the aetiological role of early CVS. Unclear visualization of the placental limits and poor control of the needle path are thought to be the main reasons for the vascular disruption of the chorionic plate, and thereby hypoxic embryo tissue damage. A better selection of cases, together with high-resolution vaginal ultrasound visualization, and analytical techniques requiring a minimal amount of tissue should avoid any teratogenic effect of early CVS.     

Alkaline phosphatase activity in amniotic fluid in pregnancies with fetal disorders

Alkaline phosphatase (ALP) activity was determined in 255 amniotic fluid samples collected by amniocentesis between 15 and 39 weeks of gestation. The samples were originally used for chromosomal analysis and/or alpha-fetoprotein measurements. The mean ALP activity in early amniotic fluid from pregnancies with fetal trisomy 18 and 21 syndromes was half of that found in the controls. Highly elevated ALP activity (over 10 times the median level) was found in 14 samples. Two of these pregnancies had normal outcome. Three samples were from pregnancies with intrauterine fetal death. Fetal disorders, including abdominal wall defect (four cases), Meckel's syndrome (two), hydrops fetalis syndrome (two) and genital anomaly (one), were observed in nine cases. Moderately elevated ALP activity (over three times the median) was found in 10 cases, including five pregnancies with a preterm labour shortly after the sample collection. The results indicate that elevated ALP activity in the third trimester amniotic fluid is often associated with fetal disorders.     

Oligohydramnios sequence in a live-born infant following chorionic villus sampling

We describe an infant born at 29 weeks' gestation with oligohydramnios sequence due to amniotic fluid leakage following chorionic villus sampling at 12 weeks. To our knowledge, this is the first such report.     

Prenatal diagnosis of haemoglobin disorders by cordocentesis at 12 weeks' gestation

Prenatal diagnosis of haemoglobin disorders is accepted to be a useful procedure to avoid births of infants with homozygous diseases. Advances in sampling and molecular techniques, such as polymerase chain reaction (PCR) and chorionic villus sampling (CVS), have made earlier and safer first-trimester prenatal diagnosis possible. However, these procedures need previous studies of at-risk couples, which can be very time-consuming when a number of different β-thalassaemia mutations occur in the region. We describe the possibility of making a first-trimester prenatal diagnosis by cordocentesis and fetal blood analysis at the 12th week of gestation. We found no statistically significant difference (p>0.05) between β/γ values in fetuses at the 12th and 18th weeks of gestation. In seven affected fetuses aborted at the 12th week of gestation, the diagnosis was confirmed in all cases by PCR analysis. These findings suggest that early cordocentesis could be an alternative procedure to CVS and PCR analysis.     

β-glucuronidase deficiency: Identification of an affected fetus with simultaneous sampling of chorionic villus and amniotic fluid

Four pregnancies at risk for mucopolysaccharidosis VII were monitored by chorionic villus sampling obtained in the first or second trimester of gestation. One fetus showed reduced β-glucuronidase activity following simultaneous sampling of chorionic villus and amniotic fluid at 17 weeks of gestation. The pregnancy was terminated. Subsequent assay of β-glucuronidase activity in the fetal tissues was consistent with a diagnosis of mucopolysaccharidosis VII, thus confirming that chorionic villus samples provide useful information for diagnosis of this condition.     

Serum PAPP-A measurements in first-trimester screening for Down syndrome

Serum PAPP-A measurements taken from 254 women in the first trimester are reported. Eleven chromosomal abnormalities were detected. The mean serum PAPP-A levels in cases of Down syndrome were 0.44 MOM at 9 weeks gestation, 0.15 MOM at 10 weeks, and 0.29 MOM at 11 weeks. The PAPP-A level at 10 weeks was below those of pregnancies which aborted spontaneously. At 11 weeks, the pregnancies with Down syndrome recorded the lowest PAPP-A levels at that gestation. On this small sample, offering chorionic villus sampling to women with singleton pregnancies and a PAPP-A level below 0.3 MOM (approximately 6.5 per cent of this at-risk group) would have detected all the Down syndrome fetuses at 10 weeks and 50 per cent at 11 weeks without selecting those cases destined to abort. This suggests that serum PAPP-A should continue to be investigated as a potential first-trimester screening test for Down syndrome.     

Immunoblot detection of enzyme proteins of peroxisomal β-oxidation in fibroblasts,amniocytes, and chorionic villous cells. Possible marker for prenatal diagnosis of Zellweger's syndrome

The amounts of enzyme proteins of peroxisomal β-oxidation in fibroblasts and chorionic villous cells from infants with Zellweger syndrome and in fibroblasts, amniocytes, and chorionic villi from healthy controls were measured by immunoblot analysis. Immunoreactive proteins of peroxisomal acyl-CoA oxidase and 3-ketoacyl-CoA thiolase were absent in fibroblasts and chorionic villous cells from the patients, yet these enzyme proteins were present in fibroblasts, cultured amniocytes, and chorionic villi from the normal controls. These results show that immunoblot analysis of peroxisomal β-oxidation enzymes in amniocytes and chorionic villous cells is of potential value for the prenatal detection of Zellweger syndrome.     

Maternal cell contamination in cultured chorionic villi: Comparison of chromosome Q-polymorphisms derived from villi,fetal skin,and maternal lymphocytes

Maternal cell contamination of chorionic villi (CV) samples used for first trimester prenatal diagnosis can cause obvious and/or unrecognized diagnostic dilemmas. The purpose of this investigation is to assess the frequency of maternal cell contamination (MCC) in chorionic villus samples and to evaluate selected parameters which might predict where contamination is more likely to have occurred. Maternal lymphocytes, chorionic villi from ultrasonically directed transcervical catheter aspiration, and fetal tissue were obtained at 8–11 weeks gestation from 45 patients undergoing elective termination. Quinacrine (Q) banded metaphases were compared from duplicate direct preparations of chorionic villi; cultured chorionic villi, fetal fibroblast tissue cultures, and maternal lymphocyte cultures. Q-polymorphisms in metaphase chromosomes were 100 per cent concordant between fetal tissue and direct CV preparation. However, evidence for maternal cell contamination occurred in 13.1 per cent of cultured chorionic villi preparations where polymorphisms were found to be identical between maternal and cultured CV and both distinct from fetal tissue preparations. Where MCC was identified, it was noted that CV cell cultivation interval was prolonged (24.2±6.8 days) compared with non-contaminated cultures (14.1±4.4 days) (p <0.05). We conclude that maternal cell contamination is a significant problem with chorionic villus sampling. Where direct preparations are not employed or when cultures are ‘slow growing’, MCC may be a significant and unrecognized complication re: fetal diagnosis. Direct preparations, multiple cultures, quinacrine banding, and maternal Q-polymorphism comparisons can minimize diagnostic dilemmas secondary to maternal cell contamination. Q-polymorphism comparisons between maternal and fetal chromosomes should be included in all instances where cultured chorionic villi are utilized for fetal diagnosis and where direct preparations are not available.     

Evaluation of transcervical chorionic villus sampling with a completed follow-up of 1550 consecutive pregnancies

Data from 1550 consecutive pregnancies after first-trimester prenatal diagnosis by transcervical chorionic villus sampling (TC-CVS) are presented. The sampling efficacy was 97.8 per cent; the mean amount of collected villus tissue was 23 mg (range 5–100 mg). There were 97 affected fetuses, mainly (73.2 per cent) with a chromosomal abnormality or a male karyotype in carriers of X-linked disease. Pregnancy termination in these and four other women for social reasons resulted in 1449 continuing pregnancies. In these pregnancies, the fetal loss rate up to 28 weeks of gestation was 5.1 per cent with the highest loss rate (3.9 per cent) before 16 weeks. When relating this fetal loss rate to maternal age, this was 6.1 per cent in the advanced maternal age group (⩾36 years) against 3.1 per cent in the younger age group. In 1376 pregnancies continuing beyond 28 weeks, the perinatal mortality rate was 1.1 per cent; the percentage of non-genetic congenital anomalies was 0.9 per cent. The reproductive pattern of women at high genetic risk after CVS followed by pregnancy termination was evaluated. Within 12 months after the first CVS followed by pregnancy termination, 70 percent of women again requested CVS in a subsequent pregnancy.     

Direct preparations from chorionic villi–relationship between villous morphology and mitotic index

It has been postulated that chorionic villi with abundant sprouts have a higher mitotic index and are therefore preferable for obtaining direct chromosome preparations from chorionic villus samples. This theory was tested by correlating villous morphology with mitotic index. Surprisingly, no statistically significant relationship was found. Choice of culture medium, however, was found to be important, with serum-free RPMI yielding a higher mitotic index than 40 per cent FCS in MEM. We conclude that villous morphology, as assessed in this study, is not a major factor in determining the success of direct chromosome preparations.