Diagnostic application of first trimester trophoblast sampling in 100 pregnancies

Summary The results of the diagnostic application of first trimester trophoblast sampling in 100 pregnancies are reported in detail. Further improvement of the method for routine, direct chromosome analysis resulted in a technique which proved to be fast, simple, and efficient. We found that short-term incubation of villi permits the application of many experimental methods, such as visualization of sister chromatid exchanges and bromodeoxyuridine (BrdU) incorporation. Fetal karyotyping was successful in each of the 96 pregnancies in which fetal material was obtained from a total of 98 fetuses. There were 42 males and 56 females, and an abnormal chromosome constitution was found in 12 cases. Two trisomic fetuses were found among the eight pregnancies at risk for Duchenne muscular dystrophy, and this indicates that fetal sexing (which is achieved with our method in two hours) should not be performed without chromosome visualization. The results indicate a risk of 8% of an abnormal fetus for mothers aged 35 years or more, while the risk of failure of sampling and of spontaneous abortion after villi sampling were 4 and 6%, respectively. Enzyme determinations were performed in three pregnancies at risk for gangliosidosis GM₁, Niemann-Pick disease, and Hurler syndrome. In this last case inconsistency between the results of the assay of iduronidase on chorionic villi and amniotic fluid cells was found. This unexplained error indicates the need for extensive characterisation in chorionic villi of the series of enzymes involved in metabolic diseases.     

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.     

Improved technique for the expression of fragile-X in cultured amniotic fluid cells

Summary An improved technique for inducing fra(X) expression in cultured cells was obtained by using diazepam for mitotic arrest and 5-fluorodeoxyuridine (FUdR) for the induction of fra(X) expression. The method was developed using cultured fibroblast and urinary cells from fra(X) patients. Prenatal studies were performed on cultured amniotic fluid cells in five pregnancies at risk for fra(X). In two cases the cultured cells showed a 46,XY, fra(X) karyotype. One of the pregnancies was terminated and the diagnosis was confirmed by chromosome studies on several fetal tissues including chorionic villi and by histopathologic changes in the lymphatic vessels of the fetal testes. The fra(X) was also demonstrated in chorionic villi in a case in which amniotic fluid cells were not studied. Chorionic villi were isolated after a spontaneous abortion, the cultured cells had a 45,X karyotype and in addition 5% of the cells were fra(X) positive.     

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.     

Cytogenetic analysis of chorionic villi: a technical assessment

Summary Eighty-five samples of chorionic villi from women undergoing prenatal diagnosis at 8 to 12 weeks' gestation were subjected to cytogenetic analysis. Samples were prepared by a direct technique that permits limited analysis within two hours and by a short-term culture technique that permits detailed structural analysis within one week. An adequate number of cell divisions for cytogenetic analysis was obtained from 96% of living fetuses. Using both the direct technique and short-term culture, satisfactory banded chromosomal preparations were made in 93% of cases. Eleven of 12 pregnancies (92%) shown by ultrasound to be dead shortly before sampling, had cytogenetic abnormalities. Further studies are needed to develop banding definition equivalent to that available on cultured amniocytes.     

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.     

229例流产绒毛染色体核型分析及两种长期培养方法比较

目的:探讨早期自然流产绒毛染色体核型分析在自然流产病因检测中的应用价值,并比较两种长期培养方法的差别。方法:选择孕早期自然流产的孕妇229例,在无菌条件下,从宫腔内取出绒毛,同时或单独经胰酶消化法与切碎贴壁法进行细胞培养,传代之后常规进行G显带,在显微镜下做核型分析。结果:229例流产胎儿绒毛,培养成功206例,成功率为89.96%。异常核型105例,异常率为50.97%,数目异常者101例,占异常核型的96.19%,以16三体最为多见。胰酶消化法的培养成功率及收获时间都显著优于直接贴壁法,差异具有统计学意义(P0.05)。结论:自然流产绒毛染色体核型分析对流产查因具有实用价值。胰酶消化法较切碎贴壁法对流产绒毛长期培养及染色体核型分析更具实用性。     

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.     

Fetal mortality at the time of chorionic villi sampling

Summary To estimate the background fetal loss rates among women who might be candidates for chorionic villi sampling (CVS) for prenatal diagnosis, we examined the frequency of spontaneous abortion and of non-viable fetuses in two groups of women thought to be pregnant at 8–12 weeks' gestation. Among 1519 women over 35 years given an appointment for amniocentesis 1978–1981, 9.8% had a spontaneous abortion prior to 16 weeks' gestation. For those under observation before week 12, the loss rate by 16 weeks was 15.3%. Among all 190 candidates for elective termination of pregnancy between 6 and 12 weeks' gestation, 12.6% were found to have a non-viable fetus at the scheduled date of abortion. The frequency of non-viability was 14% among those seen before week 12. The data suggest that the background loss rate between the time of CVS and the time of amniocentesis is approximately 1–2% and is unlikely to be higher than 9%. Until randomized clinical trials of the procedure are completed we will not know how much, if at all, the loss rate associated with CVS is increased above this background. Nevertheless, knowledge of these background risk estimates may be useful in counseling women considering participating in trials of CVS.     

Method of sampling chorionic villi in first trimester of pregnancy under guidance of real time ultrasound.

Samples of chorionic villi were obtained in the first trimester by aspiration using a cannula passed transcervically under the guidance of real time ultrasound. In initial studies in 47 anaesthetised patients immediately before therapeutic abortion a method was developed giving a success rate of 89%. In 10 patients successful sampling was performed as an outpatient procedure without anaesthesia. In all, seven diagnostic procedures were undertaken and four of the five unaffected pregnancies continued. The technique of chorionic villous sampling using real time ultrasound is simple to learn and yields material for biochemical analysis and chromosomal study without the need for tissue culture. The exact obstetric risk, however, remains to be defined.     

Karyotyping results of the material of spontaneous abortions and miscarriages after using assisted reproductive technologies

The results of a molecular cytogenetic study of the material of spontaneous abortions and nonprogressive pregnancies in the first trimester in 43 couples who were treated with various ART methods are presented in this paper. Chromosomal pathologies (CPs) were present in 28 (65%) samples of chorionic villi. A comparative analysis of the frequency and types of CPs in groups composed according to the pathological states in the semen of males was performed. Recommendations to improve the efficiency of ART are developed based on the data.     

Investigations of chorionic villi after chorionic villus sampling (CVS)

Summary This report documents the first 262 cases of chorionic villus sampling (CVS) performed in parallel with cytogenetic and morphological investigations. Histomorphological examination of these CVS specimens gave suitable results in about 96% (251 cases). Of the latter, 201 samples (80.1%) exhibited villi and 176 (70.1%), maternal tissue. Viability and maturation of the chorionic villi were determined light microscopically even in cases with few villus trees. Smooth avascular villi with poorly defined margins observed under an inverted microscope, less than 10 mitoses after short-term incubation, and reduced growth of cell cultures were significantly correlated with sampling at the chorion laeve by means of histomorphologic criteria. Villi from cases exhibiting cytogenetically proved chromosomal abnormalities were characterized by molar degeneration or stromal fibrosis, or both, in 4 out of 9 cases, including 3 mosaics. In early abortions (within 3 weeks after CVS), an unexpectedly high rate of pathohistological changes within maternal tissue was evident. These results need further confirmation by investigation of a greater number of samples with immunohistochemical and morphometric methods.     

The natural history of cytogenetically abnormal fetuses detected at midtrimester amniocentesis which are not terminated electively: new data and estimates of the excess and relative risk of late fetal death associated with 47,+21 and some other abnormal karyotypes.

We report the results of an ongoing survey of rates of spontaneous death of fetuses with chromosome abnormalities detected at second-trimester amniocentesis in which the mother did not elect abortion. Estimated excess risks (and conservative 90% confidence intervals) of spontaneous fetal death for various cytogenetic abnormalities are as follows: 47,+21, 25.6% (18.0%-34.0%); 47,+18, 63.8% (49.3%-79.8%); 47,+13, 36.5% (11%-69.7%); 45,X, 65.3% (41.0%-84.2%); and mosaic 45,X/46,XX, 10.8% (1.0%-26.8%). There is little evidence for an excess risk of fetal death, at least following amniocentesis, for 47,XXX, 47,XXY, or 47,XYY. The excess risks of fetal death were adjusted for the likelihood that a fetus of normal karyotype would undergo spontaneous fetal death in a population of older maternal age similar to that in which prenatal cytogenetic diagnosis is undertaken. The absolute fetal death rates when this factor is ignored are about 3.5% higher (i.e., may be derived by adding 3.5% to the values given). The excess risks are those which are most appropriate for use in estimating the contribution of chromosome abnormalities to spontaneous fetal death.     

Familial translocation t(17;22), including the segregation in five consecutive abortuses

Summary A cytogenetic study of a family with t(17;22) is presented. The translocation has been detected in four generations. The proband was a female who has had ten pregnancies, seven of which had resulted in spontaneous abortion. The material of five aborted fetuses was available for cytogenetic examination. The karyotypes revealed four different forms of chromosomal imbalance, most of them due to 3:1 segregation. The only living proband's offspring was born after amniocentesis and had a balanced translocation t(17;22).     

Prenatal fetal karyotyping and maternal serum alpha-fetoprotein screening.

Prenatal karyotyping was undertaken in 569 consecutive amniotic fluid samples where the indication for amniocentesis was two sequential raised maternal serum alpha-fetoprotein concentrations. In 475 successful cultures five chromosome abnormalities were found--four constitutional anomalies (47,XXY; 47,XYY; an inherited inv(8) (p23q11); and a de-novo translocation t(6;7) (p11;p22) and a culture-derived anomaly (trisomy 2) found in amniotic fluid cells but not in the fetus aborted because it had spina bifida. Of the pregnancies complicated by constitutional abnormalities, only the pregnancy in which the de-novo translocation was detected was terminated. No chromosome abnormalities were detected in the 17 pregnancies which miscarried after amniocentesis. These results provide little justification for including fetal karyotyping as an essential part of maternal serum alpha-fetoprotein screening programmes.     

Does the karyotype of a spontaneous abortion predict the karyotype of a subsequent abortion? Evidence from 273 women with two karyotyped spontaneous abortions.

At least two spontaneous abortions were karyotyped in 273 women during cytogenetic surveys in New York City and Honolulu. These pairs were analyzed using maximum-likelihood logistic-regression analysis to adjust for maternal age and location. There was a significantly increased risk for a chromosomally normal spontaneous abortion after a previous abortion with a normal karyotype. There was no increased risk for trisomy in a second spontaneous abortion following either a previous trisomic abortion or an abortion with another abnormal karyotype. This is unexpected, given the increased risk for trisomy found among live births and at prenatal diagnosis in young women with a previous trisomic birth. The most likely explanation is that the increased recurrence risk for trisomy is restricted to trisomy for only one or a few chromosomes, for reasons such as parental trisomy mosaicism. These data predict no increased risk of chromosome abnormality in future pregnancies after either (1) spontaneous abortions with trisomies of a kind that are always lethal in utero or (2) multiple early abortions in the presence of normal parental karyotypes.     

Effect of chorionic villus sampling and early pregnancy counselling on uptake of prenatal diagnosis.

An early pregnancy counselling clinic was introduced to improve the uptake of prenatal diagnosis and to offer chorionic villus sampling to women aged 38 and over by their expected date of delivery. Ninety eight (62%) unselected older mothers were seen before 11 weeks'' gestation, and 23 (32%) of those with viable pregnancies elected to undergo chorionic villus sampling compared with 38 (52%) electing amniocentesis. A quarter of the patients booking before 11 weeks had a miscarriage. Because of the future potential demand for chorionic villus sampling counselling during pregnancy and referral of eligible patients should occur as early as possible.     

Placental mosaicism and intrauterine survival of trisomies 13 and 18.

Cytogenetic analysis of 14 placentas from live newborn infants or from terminated pregnancies with trisomies 13 and 18 revealed that all were mosaic. The mosaicism was confined to the cytotrophoblast and not detected in villous stroma, chorionic plate, or amnion. The percentage of cells with a normal karyotype varied from 12% to 100%, the average being 70%. No such confined mosaicism could be detected in 12 placentas of trisomy 21 fetuses. These findings suggest that a postzygotic loss of a trisomic chromosome in a progenitor cell of trophectoderm facilitates the intrauterine survival of trisomy-13 and -18 conceptuses. They also imply that it is placental function which determines the intrauterine survival and that the mother plays no active role in rejection of trisomic conceptions. The combination of both a pre- and post-zygotic cell division defect in viable trisomy-13 and -18 conceptions points to the possibility of a genetic predisposition to such events. The detection of only a diploid cell line in the cytotrophoblast of some pregnancies with trisomies 13 and 18 also suggests that direct preparation is unreliable for prenatal diagnosis of these trisomies on chorionic villi sampling and that long-term villous culture should be used.     

Distribution of mosaicism in human placentae

Traditional first trimester chorionic villus sampling (CVS) for prenatal diagnosis can be performed by cytogenetic analysis of cytotrophoblast or chorionic villous stroma. Approximately 2% of pregnancies studied by CVS show confined placental mosaicism (CPM) involving either cytotrophoblast, stroma or both. We present the results of a cytogenetic study of nine term placentae from pregnancies with prenatally diagnosed CPM. The aneuploid cell lines involved trisomies for chromosomes 7,9,16, and X. The cytotrophoblast and villous stroma from multiple biopsies of these placentae were examined using a combination of interphase and metaphase cytogenetic analysis. CPM was detected in all nine of the term placentae and both tissue-specific and site-specific patterns of mosaicism could be discerned. These results indicate that the analysis of villous stroma and cytotrophoblast from multiple placental biopsies is necessary to improve our understanding of the evolution of CPM during pregnancy and its effect on the fetus. Received: 1 May 1995 / Revised: 11 August 1995     

Ethical considerations regarding parental decisions for termination following prenatal diagnosis of sex chromosome abnormalities

Termination rates following prenatal diagnosis of sex chromosome abnormalities have been reported to be in a very wide spectrum (12.7-86.5%) in various studies. The different attitudes in management of prenatal diagnosed sex chromosome abnormalities may depend on several factors as the type of the abnormality, the indication for prenatal testing, the number of previous healthy children and whether the pregnancy was assisted or spontaneous. In the current study, we look at prenatal diagnostic procedures carried out in our department over a period of 5 years (2002-2007). We did not detect sex chromosome abnormalities in the 43 cordocenteses and the 26 chorionic villus samples. Among the 1130 amniocentesis patients, 12 cases (1.06%) were diagnosed as having sex chromosome abnormalities. Five (41.67%) of 12 pregnancies with sex chromosome abnormalities were terminated (one case with 47,XXY, one case with 46,X,del(X), and three cases with 45,X karyotype); whereas seven pregnancies (58.33%) continued. Among the factors influencing parents' decision-making, the attitude of the health-care professional giving the post-diagnosis counseling seems to be the most important, next to the socio-economic and educational status of the parents.