Chorionic villus culture for first trimester diagnosis of chromosome defects: Evaluation by two london centres

The maceration technique for the culture of chorionic villi has been applied by St Mary's Hospital and King's College Hospital, to a total of 225 villus aspirates of which 100 were from diagnostic cases. Two hundred and eighteen (96·8 per cent) of these were successfully karyotyped, and chromosome abnormalities were detected in 15 cases. Of the cases with a normal karyotype, 113 were male and 90 were female. Sixty-nine (77 per cent) of the female cultures were demonstrated to be fetal and a further two of these may be verified at delivery. The use of Chang medium was found to accelerate cell growth thereby reducing the interval between sampling and reporting to less than 2 weeks. An unlimited quantity of metaphase spreads was obtained suitable for the application of routine banding techniques, and comparable to those obtained from amniotic fluid culture. Experience with diagnostic cases reinforced our view that culture should always back up direct analysis and this is discussed with particular reference to the occurrence of mosaicism in the trophoblast.     

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.     

Prenatal diagnosis by chorionic villus sampling: Lessons of the first 600 cases

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 per cent of those pregnancies tested because of advanced maternal age (≥ 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.     

Practical experience using transabdominal chorionic villus biopsies taken after 16 weeks' gestation for rapid prenatal diagnosis of chromosomal abnormalities

Placental biopsy was performed on 81 patients at greater than 16 weeks' gestation. The major indication for such biopsies was an increased risk of chromosomal abnormality because of either abnormal ultrasound findings or late presentations for advanced maternal age. Six abnormal karyotypes resulting in elective termination were found. The use of rapid karyotyping by this procedure as an alternative to amniocentesis or fetal blood sampling is discussed.     

Estimation of the weight of chorionic villus samples obtained from first trimester pregnancies by transcervical aspiration

Eight obstetricians experienced in chorionic villus sampling were asked to estimate the weight of samples of villi with and without a reference photograph. Referral to the photograph significantly improved the accuracy of their estimations.     

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.     

Prenatal cytogenetic diagnosis after transabdominal chorionic villus sampling in the first trimester

First trimester prenatal cytogenetic diagnosis was attempted in 350 pregnancies after trans-abdominal chorionic villus sampling. The cytogenetic investigation was performed using both a short-term method (24 h incubation) and cell culture. Adequate samples were obtained in 99·1 per cent and in all these cases the fetal karyotype was established. A chromosome abnormality was found in 2·0 per cent of cases. A discrepancy between the karyotype obtained after 24 h incubation and the karyotype in cell culture was observed in 2·3 per cent. Maternal cell contamination in the cultures was confirmed in 13 of 181 cases where the 24 h incubation revealed a male karyotype. Studies of culture morphology showed that colonies of convoluted cells may serve as a marker for contamination with maternal cells in culture. For the present, we recommend using a short-term method as well as cell culture for cytogenetic investigation until the problems with karyotype discrepancy and maternal cell contamination have been further clarified.     

Prenatal diagnosis of β-thalassaemia by second-trimester chorionic villus sampling

In this study we evaluated the feasibility of second-trimester transabdominal chorionic villus sampling for prenatal diagnosis of β-thalassaemia in 80 pregnancies at risk presenting in the second trimester at the Antenatal Service. Sampling was carried out from 13 to 20 weeks and was successful in all cases. The amount of chorionic villi obtained varied from 10 to 40 mg, which was sufficient to make fetal diagnosis by oligonucleotide analysis within 10 days from sampling in all cases. No fetal losses occurred. From these results we conclude that transabdominal chorionic villus sampling is a useful procedure for prenatal diagnosis of β-thalassaemia in those couples presenting after the first trimester.     

Ultrastructure of first trimester chorionic villi with regard to the prenatal diagnosis of genodermatoses

Hopes are held out for chorion villus sampling, a technique which is gaining more and more importance for the first trimester prenatal diagnosis of chromosomal aberrations and metabolic abnormalities. A variety of inherited skin diseases can be diagnosed postnatally and prenatally (in the second trimester) by ultrastructural diagnostic markers. For evaluation of prenatal diagnosis in the first trimester, we investigated chorionic villi derived from the trophoblast layer of the early pregnancy by light microscopy and conventional electron microscopy. The ultrastructure of the cellular layers covering the villi, i.e., the inner cytotrophoblast and the outer syncytiotrophoblast, as well as that of the connective tissue of the inner extraembryonic mesoderm, are thoroughly described in relation to the ultra-structural changes in certain genodermatoses including epidermolyses and keratinization disorders. We found that chorionic villi have only a few of the characteristics differentiated in skin, and none of the structures which are relevant to the diagnosis of genodermatoses. In our view, the ultrastructural approach is not suitable for first trimester prenatal diagnosis of genodermatoses in chorionic villi.     

Transcervical chorionic villus biopsy with a brush

A brush delivered to the biopsy site by a metal introducer and cannula set was tested as an alternative implement for transcervical collection of chorionic villi. This implement was easy to use and readily identified by ultrasound. With only one attempt at sampling, the overall collection rate for 83 patients was 65 per cent. With practice 84 per cent successful collection was achieved. Gestations between 8 and 11 weeks was the best time for collection of chorionic villi. A single sampling can produce adequate material (15 mg wet weight) suitable for diagnostic purposes. No gestation sac was perforated but some slight bleeding followed the procedure. Appreciable success following a single sampling attempt coupled with the low complication rate suggests that this technique may have clinical application and deserves further investigation.     

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.     

First trimester diagnosis of cystinosis using intact chorionic villi

Cystinosis was diagnosed in the first trimester of pregnancy by studies of uptake and retention of [³⁵S]-cystine by intact biopsy samples of chorionic villi. The diagnosis was confirmed by similar studies on cell cultures of villi and fetal skin fibroblasts.     

Transabdominal chorionic villi sampling for first trimester fetal diagnosis. First 26 pregnancies followed to term

A consecutive series of 26 women followed to term after first trimester transabdominal chorionic villi sampling is presented. The clinical application of transabdominal chorionic villi sampling (TA-CVS) seems to have certain advantages, especially from the patients' point of view, but also in regard to successful sampling and to the complication ratio. The results in this clinical trial revealed no cases of abortions, no signs of placental damage and no cases of vaginal bleeding or infections.     

Exclusion of citrullinaemia in the first trimester of pregnancy by direct assay of argininosuccinate synthetase in chorionic villi

Citrullinaemia was presumed to be excluded in a fetus at risk by the direct assay of argininosuccinate synthetase in chorionic villi. The diagnosis was confirmed after amniocentesis by normal argininosuccinate synthetase activity in the cultured amniotic fluid cells and by a normal citrulline concentration in the amniotic fluid. The prediction of a normal fetus was confirmed at term by the birth of a non-citrullinaemic boy.     

A simple technique for obtaining high quality chromosome preparations from chorionic villus samples using FdU synchronization

A fluorordeoxyuridine (FdU) synchronization technique was applied to 30 chorionic villus samples (CVS) from patients undergoing first trimester fetal diagnosis. The villi were incubated for 15 h in the presence of FdU. The block in DNA synthesis was subsequently released using thymidine and after an additional 5 h of incubation the mitotic cells were arrested in metaphases using a high concentration of colcemid. This method results in improved morphology of the chromosomes and a high mitotic index. A diagnostic chromosome analysis could be obtained in each clinical case using at least 15 well-spread metaphases. G-banded karyotypes were prepared of four metaphases in each case. The diagnostic procedure was completed within 48 h from the time of CVS. Use of this technique significantly improves the success rate of ‘direct’ chromosome analyses from CVS in a busy cytogenetic laboratory.     

Experience with first trimester prenatal diagnosis of Menkes disease

We have performed 28 first trimester diagnoses for Menkes disease in 27 high risk pregnancies by direct copper measurement on chorionic villi (c.v.) Two male fetuses were found to be affected because of significantly increased copper content. In one male fetus a slightly increased copper content was observed indicating an exogenous copper contamination of the sample. This view was supported by normal results observed after abortion. Three out of 15 diagnostic c.v. samples with a female karyotype showed increased copper levels. In two of these cases, part of the copper content might have been released from the cannulae used for these particular biopsies. Histochemical visualization of copper accumulation in fixed chorionic villi of two affected fetuses and one female fetus was observed. [⁶⁴Cu]-uptake studies have been performed on 11 diagnostic and 10 control c.v. samples. As the control samples in some cases were found to incorporate more [⁶⁴Cu] than the corresponding diagnostic sample, this method cannot at present be used for diagnosis. Compiled results on newborn females gave evidence that two carriers expressed the paternal X-chromosome, and two carriers expressed the maternal X-chromosome in chorionic villi.     

Randomized clinical trial of transabdominal versus transcervical chorionic villus sampling methods

The relative advantages and disadvantages of transabdominal (TA) and transcervical (TC) chorionic villus sampling (CVS) in terms of fetal risks and efficacy were evaluated in a clinical trial conducted on 1194 women randomized at 7–12 weeks' gestation. The results of the study indicate that, if any, the relative risk of fetal loss following either procedure is less than double that of the alternative technique when performed by a skilled operator. Overall, the fetal loss rate (spontaneous abortions following randomization, terminations of pregnancy, and perinatal deaths) is 16.5 and 15.5 per cent, respectively, among women allocated to TA- and TC-CVS. The two procedures are equally effective, although TA-CVS is associated with a significantly lower rate of repeat device insertions; on the other hand, a higher weight of chorionic tissue is obtained, on average, with TC-CVS. Bleeding is more common following TC-CVS, while peritoneal reaction developed only after TA-CVS. No diagnostic problems specifically related to one sampling technique were identified.     

First trimester chorionic villus sampling versus mid-trimester genetic amniocentesis-preliminary results of a controlled prospective trial

This controlled prospective study assesses the relative risks of first trimester chorionic villus sampling (CVS) versus mid-trimester gentic amniocentesis (GA). CVS subjects and amnio-centesis controls were comparable with regard to several confounding variables which might influence the risk of pregnancy loss including maternal age, smoking, alcohol consumption, gestational age at study entry, and history of vaginal bleeding or poor prior reproductive outcome. The most common indication for prenatal diagnosis was advanced maternal age (n = 511). In this subgroup, spontaneous abortion (<24 weeks) occurred in 2·9 per cent of CVS subjects versus 4−3 per cent of amniocentesis controls. The sum of spontaneous and therapeutic abortions (<24 weeks) was identical (5·3 per cent) in both groups. Therefore, intervention in the CVS group (i.e., therapeutic abortion for cytogenetic abnormalities) did not influence the observed risk of pregnancy loss. Overall perinatal mortality rates were also similar in both groups. No significant differences were identified for a number of pregnancy outcome parameters including 5 min Apgar score, birth weight, body length, head circumference, gestational age at delivery, preterm delivery, fetal growth retardation, congenital malformations, and neonatal complications. Preliminary results of this controlled prospective study suggest that chorionic villus sampling carries a low and acceptable risk.     

High resolution chromosomes from first trimester trophoblast cultures

A simple method for obtaining prometaphase chromosomes from cultured first trimester cells involves the addition of BrdU and FdU 11 h before harvest and ethidium bromide 1.5 h before harvest. High resolution R-banding is obtained by acridine orange staining.     

An improved method of direct chromosome preparation from chorionic villus and high resolution banding technique

The trophoblast was dissociated from the underlying mesenchymal layer either with acetic acid after short-term prefixation or with mechanical power after fixation twice. The colcemid treatment time was shorted to 16 min and trypsin solution of low pH (6.2) was used for banding. By these steps, the quality of chromosome banding was greatly improved and complete standard chromosome diagnoses were made in 24 of 24 cases. With the modified technique, high resolution banding chromosomes were consistently obtained after short-term incubation.