G. L. Terzoli

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

SummaryChorionic 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.

Diagnostic application of first trimester trophoblast sampling in 100 pregnancies

SummaryThe 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.

Seizure and EEG Patterns in Angelman's Syndrome

We studied the seizure and polygraphic patterns of 18 patients with Angelmans syndrome. All patients showed movement problems. Eleven patients were also reported to have long-lasting periods of jerky movements. The polygraphic recording showed a myoclonic status epilepticus in nine of them. Seven patients had partial seizures with eye deviation and vomiting, similar to those of childhood occipital epilepsies. These seizures and electroencephalographic patterns suggest that Angelmans syndrome occurs in most of the patients as a nonprogressive, age-dependent myoclonic encephalopathy with a prominent occipital involvement. These findings indicate that, whereas ataxia is a constant symptom in Angelmans syndrome, the occurrence of a transient myoclonic status epilepticus may account for the recurrence of different abnormal movements, namely the jerky ones. (J Child Neurol 1995;10:467-471).

First trimester fetal karyotyping: one thousand diagnoses

SummaryCytogenetic investigations for diagnostic purposes were performed on 1000 first trimester samples of chorionic villi (CVS) in two laboratories using similar techniques. Fetal karyotyping was the primary indication for CVS in 912 and maternal age was the major indication in 758 of them. The risk category “previous child/fetus with chromosome abnormality” included 74 diagnoses, while the category “chromosome abnormality in one of the parents” included 38 diagnoses. Sex determination was the primary indication for CVS in 53 pregnancies. The overall incidence of chromosomal abnormalities was 70, of which 47 were balanced and 23 unbalanced. The results are detailed for each of the risk categories and the incidence of abnormal karyotypes is given for each year of maternal age. In the maternal age of 35–37 years the incidence of unbalanced karyotypes was 2.9% and in the years 38 onwards it was 6.6%. The incidence of unbalanced karyotypes was about 4% when the sampling was made in the weeks 9 to 12 but six abnormal karyotypes were found among 39 CVS performed at the eight week of gestation. The 11 trisomies of the type not found at birth were clustered between the 8th and the 10th week of pregnancy. The technical problems encountered in this experience and the preliminary estimates of fetal loss are discussed.

Discordance Between Prenatal Cytogenetic Diagnosis after Chorionic Villi Sampling and Chromosomal Constitution of the Fetus

Discordance between the prenatal cytogenetic diagnosis at amniocentesis and the outcome of pregnancy was found in nine of 5580 amniocenteses by Loft and Tabar (1984), who estimated that the cytogenetic accuracy rate ranges from 99.2% to 99.9%. These estimates are based on the discordances and errors recorded in the literature, including contamination by maternal cells and mosaicism.

Cytogenetics of Chorionic Villi Sampling: Technical Developments and Diagnostic Applications

Trophoblast sampling during the first trimester of pregnancy has provided a new kind of fetal material for the prenatal diagnosis of genetic diseases. Studies on chorionic villi sampling (CVS) and trophoblastic cell culture were reported in the early 1970s by Kullander and Sandhal (1973) and by Hahnemann (1974) (Table 1), but while chorionic villi proved to be obtainable, throphoblastic cells showed low growth potential under culture conditions, with the result that cultures of nearly half the samples failed. It followed that cytogenetic analysis was limited to fetal sex prediction by X and Y chromatin assays. In addition, the identification and selection of pure fetal material was not easy, and it was soon realized that maternal cell contamination of trophoblast cultures was a serious problem that occurs much more frequently than with amniotic fluid cell cultures. Because of these technical difficulties, investigations were confined to experimental materials, and CVS was not used for the diagnosis of chromosomal abnormalities until 1983 (Brambati and Simoni 1983)

The offspring of marriage between two first cousins with the same reciprocal translocation t(2;7)(p11;q31)

SummaryA marriage between two first cousins who have the same 2/7 balanced translocation is reported. The chromosome rearrangement was primarily detected in amniotic fluid cells cultured for prenatal chromosome analysis because of advanced maternal age. The translocation was also found in the couples two normal children and in three other members of the family. The possible zygotic chromosome constitutions following 2:2 meiotic segregation in consanguineous parents with the same translocation are discussed.

Effect of Incubation Time and Serum Concentration on the Number of Mitoses in Aspirated Villi Samples

Chromosome preparations by direct method may be performed either immediately after chorionic villi sampling or after incubation at 37 °C in a CO2 incubator with complete medium (Simoni et al. 1983,1984). In order to define the optimal conditions for fetal chromosome study, we investigated the effects of different incubation times and serum concentrations on the number of mitoses. We found that 2 days’ incubation before processing aspirated villi specimens and a low concentration of serum result in a significant increase in the number of dividing cells in the Langhans’ layer. The introduction of both of these modifications in the preparation of specimens by the direct method may be advantageous in the diagnostic use of first trimester chorionic villi samples.