Immunophenotyping of mitotic cells from long-term cultures of chorionic villi

Chromosomal mosaicism in chorionic villus samples (CVS) may arise from different sources, such as clonal diversity within the chorionic tissue or contamination with maternal cells. To determine the origin of karyotyped cells, we compared the immunocytochemical features of mitotic cells in CVS long-term cultures with histological sections of their tissue of origin, i.e. chorionic villi. Immunolabelling of intermediate filaments specific for epithelial cells (cytokeratin) and mesenchymal cells (vimentin) established that mitoses yielded from CVS long-term cultures indeed stem from independently growing clones derived from both the epithelial and mesenchymal parts of the chorionic villi. Thus, mosaicism in CVS cultures may reflect true genetic heterogeneity within the biopsy. However, epithelial chorionic cells undergo in vitro metaplasia leading to co-expression of cytokeratins and vimentin. Fetal-specific immune markers (-HCG and SP1-glycoprotein) are not reliably expressed in CVS cell culture.     

Chromosome analysis in chorionic villi samples from the first trimester elective terminations

Chorionic villi sampling (CVS) has become a first trimester alternative to amniocentesis for prenatal diagnosis. The cytogenetic findings in 150 experimental samples are presented. The ages of the mothers ranged from 12 to 35 years, but the majority of them were 18 and 19 years of age. Various parameters of culturing and processing the samples in order to improve the method, were investigated. Short term incubation for 48 h was the method routinely employed in processing the biopsies for cytogenetic analysis. In the first series of 100 cases one mosaic case (46,XX/45,X), one Robertsonian translocation (13;14), one marker chromosome and one fragment were found. The foetal tissues were not analysed for chromosomes. In the second series of 50 samples, one case of mosaicism was found in the chorionic villi (46,XX/47,XX, 18q-), but this abnormality was absent in the foetal tissue. One variant inv(9) was observed in the foetal tissue as well as in the chorionic villi. In all other cases the karyotypes from the chorionic villi samples matched those of the corresponding foetal samples. There was no maternal contamination in this series of 50 samples. The discrepancies in the cytogenetic results from other investigators are discussed.     

Confined chorionic mosaicism in prenatal diagnosis

Summary Confined chorionic mosaicism, detected commonly on chorionic villus sampling (CVS) and occasionally in cultured amniotic fluid cells, is described in five pregnancies that showed confined chorionic mosaicism for trisomies 12, 13, 14, 17 and a marker chromosome. Cytogenetic findings in these pregnancies support the conclusion that within chorion some chromosomal mosaicism are confined to the trophectoderm derivatives while others to the extra-embryonic mesoderm. The etiology of confined chorionic mosaicism is discussed in relation to a significant role of multiple cell lineages contributing to the early development of placenta. The need is indicated for the use of both direct and long-term cultures in CVS prenatal diagnosis, and for the confirmatory testing of fetal blood or amniotic fluid in cases where mosaicism is detected in chorionic villi.     

Prenatal diagnosis of a true fetal tetraploidy in direct and cultured chorionic villi

Prenatal diagnosis of a true fetal tetraploidy in direct and cultured chorionic villi: Tetraploidy is characterized by four complete sets of chromosomes (4n= 92). Although it has been frequently reported in spontaneous abortions, tetraploidy is extremely rare in term pregnancy. Most of late surviving patients are diploid/tetraploid mosaics and present severe mental and physical impairment. Up to date, only five tetraploidies were ascertained in the prenatal stage in amniocytes and/or fetal blood lymphocytes. No one has been reported in chorionic villi probably because tetraploidy is generally considered in this tissue as a false positive result due to confined placental mosaicism (CPM) or placental culture artefacts. We report here on a case of tetraploidy detected in chorionic villi because of fetal cystic hygroma. We discuss the reliability of this diagnosis and propose guidelines in the follow-up of tetraploidies detected after chorionic villus sampling (CVS). Thus a misdiagnosis of this poor condition will be avoided at best and an appropriate genetic counseling will be given to the parents.     

Variation in spontaneous chromosomal damage as a function of biologic rhythms in women

Summary The alphafetoprotein (AFP) concentration in maternal serum was determined before and after chorionic villus sampling (CVS). A significant increase of 20% or more in the pre-CVS level was noted immediately after sampling in 59% of 837 pregnancies indicating some degree of feto-maternal haemorrhage. The increase in the AFP concentration in maternal serum was correlated with the weight of the tissue sample but not with the number of sampling attempts. A correlation of AFP increase and frequency of spontaneous abortions following CVS was suggested only in the group with an AFP increase of more than 100% or with a continuing rise in the first hour following CVS. CVS in early pregnancy obviously did not interfere with maternal serum AFP screening for neural tube defects in the second trimester. Although AFP measurement before and after CVS seems to have no immediate diagnostic application, in the research phase of CVS it may help to identify those procedures that are the least traumatic.     

Trisomy 16 confined to chorionic villi and unfavourable outcome of pregnancy.

Two cases of trisomy 16 confined to placental tissue associated with an unfavourable outcome of the pregnancy are reported. In the first case, after a diagnosis of an apparent non-mosaic trisomy 16 at chorionic villi sample (CVS), an intrauterine fetal death occurred at the 22nd week. In the second case a mosaic with trisomy 16 was found in chorionic villi and the fetus was still-born at 38 weeks. From a comparison of their cases with those of the literature, the authors conclude that a trisomy 16 confined to placental tissue has a negative effect on fetal growth and pregnancy outcome.     

No effect of maternal smoking in early pregnancy observed on chromosome aberrations in chorionic villus samples

The effect of maternal smoking on first trimester chorionic villus samples (CVS) was studied by analysing the frequency of chromosome aberrations (CAs) among 20 non-smoking and 20 smoking mothers. The aberrations were classified as chromosome- and chromatid-type breaks and gaps. No statistically significant differences were found in the frequencies of CAs between non-smoking mothers (5.4% or 2.0% gaps excluded) and smoking mothers (3.5% or 1.0% gaps excluded).     

Prenatal enzymatic diagnosis of Krabbe disease (globoid-cell leukodystrophy) using chorionic villi

Summary Sixteen pregnancies in families with children enzymatically diagnosed as having Krabbe disease (KD) were monitored for prenatal KD using the assay of galactosyl ceramide -galactosidase (GCG) in uncultured chorionic villi (CV), cultured CV, or cultured amniotic fluid cells (AFC). Prenatal KD diagnoses were made for 5 pregnancies on the basis of lower than 10% normal GCG activity in cultured CV or AFC. Uncultured CV were studied in 3 out of the 5 KD embryos, although the GCG activities of 14%–23% as compared with control villi were diagnostically inconclusive; the relatively high activities were considered to be caused by maternal GCG contamination of these very small villus samples. Although the villi from 6 of the other pregnancies yielded more conclusive results, the use of uncultured CV alone is not recommended for prenatal KD diagnosis, this material being subject to possible uncontrolled contamination with maternal enzyme.     

Efficient direct chromosome analyses and enzyme determinations from chorionic villi samples in the first trimester of pregnancy

Chorionic villi were obtained by an aspiration technique which proved to be the best of four alternative procedures. We report in detail the series of experiments which led to (1) successful, rapidly growing cell cultures practically free of maternal cell contamination (the use of hormone-supplemented Chang medium greatly increased the growth rate); (2) an efficient direct method to obtain high quality metaphases from the Langhans cells of the cytotrophoblast tissue and with which the fetal karyotype is defined within a few hours of chorionic villi sampling; and (3) successful testing for the activity of eight enzymes directly from the villi samples, thus showing that this material is suitable for a rapid, direct diagnosis of the related metabolic diseases.     

Cytogenetic analysis of 750 spontaneous abortions with the direct-preparation method of chorionic villi and its implications for studying genetic causes of pregnancy wastage

Altogether, 750 cases of spontaneous abortion between the fifth and 25th week of gestation were analyzed cytogenetically by the direct-preparation method using chorionic villi. The majority of cases (68%) were derived from early abortions before the 12th week of gestation. The frequency of abnormal karyotypes was 50.1%; trisomy was predominant (62.1%), followed by triploidy (12.4%), monosomy X (10.5%), tetraploidy (9.2%), and structural chromosome anomalies (4.7%). Among trisomies, chromosomes 16 (21.8%), 22 (17.9%), and 21 (10.0%) were prevalent. The frequency of chromosomally abnormal abortions increased with maternal age but only because of an increase of trisomy. Polyploidy and monosomy X, however, decreased. Mean maternal age was significantly increased for trisomies 16, 21, and 22 and was highest for trisomies 18 and 20. The results obtained are within the range of variability reported earlier from tissue culture-type studies. A consistent feature during our study is the excess of females in chromosomally normal abortions (male:female sex ratio 0.71). According to the methodology applied, maternal cell contamination and undetected 46,XX molar samples cannot have influenced the sex ratio. However, a bias introduced by social status or maternal age cannot be excluded. With the more rapid and convenient direct preparation of chorionic villi, reliable cytogenetic data on causes of spontaneous abortions can be obtained.     

Level of alpha-fetoprotein in maternal serum before and after chorionic villus sampling

To evaluate incidence and severity of feto-maternal transfusion post-chorionic villus sampling (CVS), maternal serum AFP (MSAFP) were determined for 88 patients before and 15 minutes after CVS. To know whether MSAFP elevation could have clinical implications, a questionnaire was sent to the patients researching if they have had haemorrhages, temperature, spontaneous abortion or premature delivery in the period post CVS. Results of the present study indicate that 44.7% of patients present MSAFP significative elevation (greater than or equal to 8 micrograms/l), a variable elevation (from 8 to 423 micrograms/l). There is no relation between MSAFP elevation and unfavourable events post CVS.     

First-trimester diagnosis on chorionic villi obtained by direct vision technique

Summary An improved technique for direct vision chrionic biopsy that gives a clear view of the amniotic sac was developed. With this technique, used in 48 women prior to vacuum aspiration and in six cases for diagnosis (karyotyping or enzyme analysis), it was possible to obtain chorionic villi free from contamination by maternal tissue. It was also possible to pick out villi (rich in blood vessels and with abundant buds on their surface) found to be most capable of growing in vitro. In the diagnostic cases, the pregnancies have continued uneventfully since the sampling; one pregnancy is now in the 32nd week.     

Chorionic villi sampling: cytogenetic and clinical findings in 500 pregnancies.

The cytogenetic findings were analysed in a series of 500 pregnancies in which chorionic villi sampling was performed. In all cases a direct method was used, karyotyping being successful in 481 cases (96.2%). The main indication for sampling was maternal age over 36 (412 cases; 82.4%). Abnormal laboratory findings resulted in 24 terminations of pregnancy (4.8%); in addition five unexpected balanced chromosome rearrangements were detected. Twelve of 15 cytogenetic discrepancies were detected at amniocentesis, two after termination, and one at spontaneous abortion. Complete follow up data were available for the first 250 patients, among whom nine pregnancies (3.6%) ended in spontaneous abortion before the 20th week. There were no false negative findings. Seventy additional chromosome studies were performed because of failure of chorionic villi sampling or equivocal results, or for confirmation. Counselling before chorionic villi sampling should include the possibility that subsequent amniocentesis may be needed should mosaicism or other unexpected abnormalities be found. The success rate and accuracy of karyotyping chorionic villi samples by the direct method are acceptable but distinctly less than those of karyotyping cultured amniotic fluid cells.     

Somatic stability in chorionic villi samples and other Huntington fetal tissues

We have studied different tissues from two affected fetuses with Huntington's disease (HD). In the first case the analysis was performed at 11 weeks of pregnancy; CAG repeats from seven different tissues were compared with the results obtained in the chorionic villi sample (CVS). We found 42 CAG repeats in all samples. In the second case the study was done at 12 weeks; eight tissues (including brain) were studied and compared with the CVS; in all of them, 44 CAG repeats were obtained. Our results show a somatic stability in the different analyzed tissues and suggest that mitotic instability can be a secondary consequence of neuronal degeneration and gliosis. Likewise, our data show great viability in the prenatal diagnosis (PD) of Huntington's disease using samples from any tissue.     

Maternal age-specific rates of 47,+21 and other cytogenetic abnormalities diagnosed in the first trimester of pregnancy in chorionic villus biopsy specimens: comparison with rates expected from observations at amniocentesis.

Results are presented on chromosome analyses made on 4,481 embryos or fetuses studied through chorionic villus sampling (CVS) in whom there was no known bias to presence of a chromosome abnormality except advanced parental age. We excluded from the analysis most cases in which mosaicism was diagnosed or in which there were cytogenetic discrepancies among samples obtained from the conceptus. There remain 48 cases of 47,+21, 39 cases of other nonlethal abnormalities, and 12 lethal abnormalities diagnosed in 4,481 studied. A regression analysis (restricted to the 3,848 cases diagnosed in the 35-49-year maternal age interval) was done on rates of (1) 47,+21, (2) other abnormalities excluding lethals or (3) including them, and (4) all abnormalities excluding lethals or (5) including them. The model used was y = exp(bx + c), where y is the rate of abnormality, x is maternal age at time of CVS (the modal age of the procedure was 10 gestational weeks from the last menstrual period), and b and c were, respectively, (1) 0.288 and -15.527; (2) 0.272 and -15.173; (3) 0.253 and -14.141; (4) 0.282 and -14.753; and (5) 0.271 and -14.195. We also derived rates of abnormalities at the time of CVS that would be predicted from rates (of nonmosaics) at amniocentesis after adjustment for the difference in gestational age between the usual times that these two procedures are done. The difference between the numbers of abnormalities predicted on the basis of these adjusted amniocentesis rates and the numbers observed at CVS provides an estimate of the spontaneous loss of embryos and fetuses between the usual gestational ages of these procedures. In these data, for 47,+21 the estimated proportion lost is 21% but the result is not significant at the .05 level. For other abnormalities excluding lethals the estimated spontaneous loss is 29% (P approximately .05); including lethals it is 44%. For all abnormalities, excluding lethals, pooled together, the estimate is 24%; including lethals it is 33%. The last three values are all significant at the .05 level or lower. The observed rates of abnormalities at CVS would be approximately 10% to 15% higher if one pooled diagnosed mosaics with the nonmosaics, but the estimated proportion of spontaneous fetal loss would be lower.     

Cell cycle studies in chorionic villi

Summary We have studied the cell cycle of cells obtained from chorionic villi in direct and culture preparations by incorporation of the thymidine analogue BrdU to produce latelabelling or sister chromatid differentiation patterns. We have, therefore, been able to estimate the duration of the cell cycle and, more specifically, the length of some of its phases. While results for chorionic villus sample cells in culture resembled those obtained for fibroblasts, data for the spontaneously dividing trophoblastic cells in direct preparations were different. Villi exposed to BrdU immediately after sampling showed a slight delay in the incorporation of the analogue and a lower percentage of labelled cells compared to villi treated after an overnight incubation, probably due to a temporary effect of the sampling technique. Results from semi-direct protocols suggest that cells have a G2 of no more than 4h, and a mid-S phase of 10–16h. The G1 period is very variable. After 48 h incubation with BrdU, only 4% of cells reach their second generation, whereas this percentage increases up to 70% after 72h, indicating that under these experimental conditions most cells have a cell cycle of approximately 36 h. The average number of sister chromatid exchanges was similar in both direct preparations and cultures: 5.2±2.1 SCE per cell.     

DNA analysis of first-trimester chorionic villous biopsies: test for maternal contamination.

We investigated the reliability of chorionic villous biopsy as a method to obtain tissues reflecting the genetic constitution of the embryo. In 12 pregnancies before elective termination, we searched for detectable maternal tissue after careful dissection of villi from small 2-5-mg specimens that yielded 7 micrograms of DNA per mg tissue. In Southern blotting experiments (1-2 micrograms DNA per lane), restriction fragment length polymorphisms (RFLPs) at an autosomal (D14S1) and a sex chromosomal (DXYS1) locus allowed recognition of maternally and embryonically derived alleles. Pure villi were obtained in six of the seven informative cases. One biopsy was not dissected satisfactorily; a mixture of embryonic and maternal DNA was found. Nonvillous tissues were mostly maternally derived in eight informative cases. Sex determination by molecular analysis (alleles at the DXYS1 locus) agreed with the karyotypes of uncultured or cultured villi. In one continuing pregnancy, distinct RFLPs indicated maternal inheritance of the alpha-thalassemia 1 trait in a female embryo without detectable maternal contamination. Reliable prenatal diagnosis depends on the specimen's purity. Maternal contamination can be evaluated by DNA analyses.     

Pyruvate carboxylase activity in chorionic villi: possibility of application to prenatal diagnosis

Pyruvate carboxylase (PC) activity was assayed in 27 chorionic villi samples (CVS) obtained at 9-12 weeks of gestation. The kinetic properties of the CVS enzyme were similar to those of liver PC; more than 75% of PC activity was recovered in the mitochondrial fraction of CVS. Apparent Km for pyruvate, ATP, acetyl CoA and HCO3- in the presence of saturation concentrations of the other reactants, were 0.3, 0.44, 0.015 and 6.0 mmol/l, respectively. The optimum pH was 7.5-8.0. The activity of PC in CVS was 3.2 +/- 0.3 nmol/min/mg protein, which is severalfold higher than that of amniotic fluid fibroblasts.     

Angiogenic factors measured in aspirated placental tissue between the 10 + 6 and 18 + 3 weeks of gestation

This study was designed to determine the level of vascular endothelial growth factor-A (VEGF-A), basic fibroblast growth factor (bFGF) and endothelial nitric oxide synthase (eNOS) in chorionic villi during in first and second trimester, and their association with nuchal translucency (NT) measured by ultrasound. Seventy-five singleton healthy pregnancies with no detected congenital malformation were collected for NT measurements and chorionic villus sampling (CVS). Concentrations of angiogenic factors were assayed in chorionic villi sampled between 10 + 6 and 18 + 3 weeks of gestation. ENOS level was steady during this gestational period, while the concentrations of VEGF-A and bFGF significantly decreased. Placental concentrations of VEGF-A and bFGF correlated positively with each other (semi-partial correlation in multivariable linear regression (r): 0.90) and both correlated negatively with the eNOS level (r: -0.64 and r: -0.83, respectively). NT was positively correlated with eNOS concentration and negatively correlated with bFGF levels (r: 0.85 and r: -0.78, respectively). Inverse correlation was found between gestational age and VEGF-A and bFGF concentrations (r: -0.57 and r: 0.73, respectively). Alterations of angiogenic factors in chorionic villi might be an adjunct modality to NT and foetal growth as sonographic markers.     

Use of prenatal diagnostic procedures in pregnancies affected with birth defects, Hawaii, 1986-2002

BACKGROUND: Information on the utilization of prenatal ultrasound (US), amniocentesis (AC), and chorionic villus sampling (CVS) in pregnancies affected by birth defects in the United States is limited. The intent of this study was to report on the utilization of these procedures in Hawaii. METHODS: Cases were all infants and fetuses of any pregnancy outcome with birth defects, included in a Hawaii birth defects registry, and delivered during 1986-2002. The rates of prenatal US, AC/CVS, and prenatal diagnosis were calculated. RESULTS: Prenatal US was performed in 76% of the cases and AC/CVS in 14% of the cases. Prenatal diagnosis of a birth defect was made in 16% of the cases. The prenatal US, AC/CVS, and prenatal diagnosis rates in 1998-2002 were 1.5, 1.5, and 1.7 times the rates in 1986-1991, respectively. Among all birth defects, the AC/CVS rate for women aged <35 years was 7% and for women aged > or =35 years was 48%. Among chromosomal abnormalities, the AC/CVS rate for women aged <35 years was 36% and for women aged > or =35 years was 66%. CONCLUSIONS: Only a fraction of the Hawaii birth defects cases was prenatally diagnosed. The rates for prenatal US, AC/CVS, and prenatal diagnosis among pregnancies affected by birth defects were higher in 1998-2002 than in 1986-1991. AC/CVS rates were lower for maternal age <35 years.