Bruno Brambati

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.

Low maternal serum levels of pregnancy associated plasma protein A (PAPP-A) in the first trimester in association with abnormal fetal karyotype.

Objective To assess the relation between maternal serum pregnancy associated plasma protein A (PAPP‐A) in the first trimester and the outcome of pregnancy by karyotype.

Selective termination for structural, chromosomal, and mendelian anomalies: International experience

OBJECTIVE Our purpose was to evaluate the outcomes of selective termination for fetal anomalies at 8 centers with the largest known experiences worldwide. STUDY DESIGN Outcomes in 402 cases of selective termination in pregnancies with dizygotic twins from 8 centers in 4 countries were analyzed by year, gestational age at procedure, and indication. Reductions of fetuses were as follows: 2 to 1, n = 345; 3 to 2, 39; >/=4 to 2 or 3, n = 18. Potassium chloride was used in all procedures. RESULTS Selective termination resulted in delivery of a viable infant or infants in >90% of cases. Loss up to 24 weeks occurred in 7.1% of cases in which the final result was a singleton fetus and in 13.0% of cases in which the final result was twins. Loss was 6.6% as a result of structural abnormalities, 7.0% for chromosomal abnormalities, and 10% for mendelian abnormalities (difference not statistically significant). Loss rates for procedures were as follows: 9-12 weeks, 5.4%; 13-18 weeks, 8.7%; 19-24 weeks, 6.8%; and >/=25 weeks, 9.1% (difference not statistically significant). Mean gestational age at delivery was 35.7 weeks. No differences were seen in outcomes by maternal age. The rate of very early premature deliveries has fallen in recent years. There were no known cases of disseminated intravascular coagulation or serious maternal complications. CONCLUSION (1) Selective termination, in the most experienced hands, can be technically performed in all 3 trimesters with good outcomes in >90% of cases. (2) The previously observed increase in second- versus first-trimester losses has diminished. (3) Third-trimester procedures, where legal, can be performed with a good outcome for the surviving fetus.

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.

Rapid detection of chromosome aneuploidies by quantitative fluorescence PCR: first application on 247 chorionic villus samples

We report the results of the first major study of applying quantitative fluorescence polymerase chain reaction (QF-PCR) assays for the detection of major chromosome numerical disorders. The QF-PCR tests were performed on a total of 247 chorionic villus samples, which were analysed blind, without any knowledge of the results obtained using conventional cytogenetic analysis. The aims of this investigation were to evaluate the detection power and accuracy of this approach by testing a large number of fetal samples and to assess the diagnostic value of each of the chromosome specific small tandem repeat (STR) markers used. In addition, we introduced three more markers specific for chromosomes 13, 18, and X to allow an accurate analysis of samples homozygous for a particular STR. Fluorescent labelled primers were used to amplify 12 STRs specific for chromosomes 21, 18, 13, X, and the amylogenin-like DNA sequence AMXY, expressed on the X and Y chromosomes. In this blind study of 247 fetal samples, 222 were correctly diagnosed by QF-PCR as normal for each of the five chromosomes investigated; 20 were diagnosed by QF-PCR as trisomic for chromosomes 21, 18, or 13, in agreement with the cytogenetic tests. Only one false negative result was observed, probably owing to the mishandling of the sample, which had been transferred through three laboratories before being analysed by QF-PCR. The 247 samples also included four cases of mosaicism or translocation; one case of mosaic trisomy 21 was detected by QF-PCR and the other cases were not identified by QF-PCR. The results of this investigation provide clear evidence that the QF-PCR assays are powerful adjuncts to conventional cytogenetic techniques and can be applied for the rapid and accurate prenatal diagnosis of the most frequent aneuploidies.

International, collaborative experience of 1789 patients having multifetal pregnancy reduction: a plateauing of risks and outcomes.

Objective: To develop the most up-to-date, complete data base of multifetal pregnancy reduction (MFPR) from cases, and to provide the best counseling for couples with multifetal pregnancies. Methods: From nine centers in five countries, 1789 completed MFPR cases were collected and outcomes evaluated. Pregnancy losses were defined as through 24 weeks and deliveries categorized in groups of 25-28, 29-32, 33-36, and 37 or more weeks. Results: Overall, the pregnancy loss rate was 11.7% but varied from a low of 7.6% for triplets to twins and increased with each additional starting number to 22.9% for sextuplets or higher. Early premature deliveries (25-28 weeks) were 4.5% and varied with starting number. Loss rates by finishing number were highest for triplets and lowest for twins, but gestational age at delivery was highest for singletons. Conclusions: Multifetal pregnancy reduction has been shown to be a safe and effective method to improve outcome in multifetal pregnancies. Outcomes are worse with higher-order gestations and support the need for continued vigilance of fertility therapy.

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.

CONCENTRATION OF CHORIONIC GONADOTROPHIN AND CHORIONIC SOMATOMAMMOTROPHIN IN MATERNAL SERUM, AMNIOTIC FLUID AND CORD BLOOD SERUM AT TERM

The mean concentration of chorionic gonadorophin in maternal serum at term was 9.45±0.93 LU. per ml. and that of chorionic somatomammotrophin was 6.34±0.39μg. per ml. In the amniotic fluid the levels were respectively 0.380±0.048 I.U. per ml. and 0.547±0.067 μg. per ml. In the cord blood serum mean values of 0.027±0.003 I.U. per ml. of HCG and 0.027±0.003 μg. per ml. of HCS were found.

First trimester fetal diagnosis of genetic disorders: clinical evaluation of 250 cases.

Chromosome and enzyme determinations were performed in 250 pregnancies between the 7th and the 12th week of gestation. The majority of the tests were performed for risk of chromosomal abnormalities and 75% of the women were 35 years old or more. We describe a chorionic villi sampling (CVS) technique which proved to be highly efficient, with a diagnostic success rate of 97.7%. In the light of our experience we suggest that CVS is best performed between the 9th and 10th weeks of pregnancy. The average weight of the aspirated specimen was 20 mg with a lower limit of 5 mg which proved sufficient for diagnostic purposes. No major maternal complications were encountered and the slight bleeding observed in 14% of the cases during the days following the CVS should be considered a harmless effect of the aspiration technique. The proportion of fetal losses may lie between 4 and 7%. Paediatric monitoring of the 93 infants born so far and ultrasound examination of the pregnancies still in progress at the time of writing did not reveal any negative effect of CVS. Fetal-maternal transfusion and intrauterine infection are problems which need further basic investigations.

Chorionic villus sampling and amniocentesis.

Purpose of review The advantages and disadvantages of common invasive methods for prenatal diagnosis are presented in light of new investigations. Recent findings Several aspects of first-trimester chorionic villus sampling and mid-trimester amniocentesis remain controversial, especially fetal loss rate, feto-maternal complications, and the extension of both sampling methods to less traditional gestational ages (early amniocentesis, late chorionic villus sampling), all of which complicate genetic counseling. A recent randomized trial involving early amniocentesis and late chorionic villus sampling has confirmed previous studies, leading to the unquestionable conclusion that transabdominal chorionic villus sampling is safer. The old dispute over whether limb reduction defects are caused by chorionic villus sampling gains new vigor, with a paper suggesting that this technique has distinctive teratogenic effects. The large experience involving maternal and fetal complications following mid-trimester amniocentesis allows a better estimate of risk for comparison with chorionic villus sampling. Summary Transabdominal chorionic villus sampling, which appears to be the gold standard sampling method for genetic investigations between 10 and 15 completed weeks, permits rapid diagnosis in high-risk cases detected by first-trimester screening of aneuploidies. Sampling efficiency and karyotyping reliability are as high as in mid-trimester amniocentesis with fewer complications, provided the operator has the required training, skill and experience.