Helen Brandenburg

Amniocentesis or chorionic villus sampling in multiple gestations? Experience with 500 cases

500 women with multiple pregnancies underwent amniocentesis or chorionic villus (CV) sampling at our department between January 1988 and July 1997. The aim of this retrospective study was to evaluate the laboratory aspects and the consequences of discordant results in these pregnancies in relation to the method of sampling. Uncertain results in one or both samples, requiring further investigation were more frequent in CV samples (eight times in 163 paired samples, 5 per cent) than in amniotic fluid (AF) samples (once in 298 paired samples, 0·3 per cent). Sampling one fetus twice (erroneous sampling) was seen only once among 163 pregnancies with two CV samples in our study. Cross contamination due to mixed sampling was discovered in two of seven pregnancies that underwent DNA diagnosis in CV and might be a rather regular occuring phenomenon. In none of the 500 pregnancies mixed sampling caused diagnostic dilemmas. A third sampling problem, maternal cell contamination caused a diagnostic problem once among the AF samples. Selective fetal reduction appeared safer after CV sampling than after amniocentesis. Subsequently, CV sampling instead of amniocentesis has become the method of choice for prenatal diagnosis in multiple pregnancies in our department. Copyright

Prospective prenatal investigations on potential uniparental disomy in cases of confined placental trisomy

In most reported cases of uniparental disomy (UPD) associated with confined placental mosaicism (CPM), a high level of mosaicism or a full trisomy was found in chorionic villi. At the time that we started our investigations, it was not quite clear whether fetal UPD also existed in the more frequently occurring low levels of mosaicism. During a 4‐year period, a follow‐up amniocentesis was performed in all cases of mosaic or non‐mosaic trisomy detected in chorionic villus (CV) semi‐direct preparations and suspected to be confined to the placenta. We performed fluorescent in situ hybridization (FISH) on uncultured amniotic fluid cells to differentiate between generalized mosaicism and CPM. We found 29 cases of CPM and we determined the incidence of UPD in 23 of these cases. Normal biparental chromosome contributions were found in 22 cases. In one case, we detected a maternal heterodisomy for chromosome 16. UPD appeared to be a rare phenomenon in the cases of CPM (type I and/or type III) that we encountered in 3958 consecutively investigated CV samples, and is not the cause of the pregnancy complications found in seven out of 23 cases with CPM.

Accuracy of abnormal karyotypes after the analysis of both short- and long-term culture of chorionic villi

We report in detail the cytogenetic results of 1838 consecutive chorionic villus samples with the availability of both short‐term culture (STC‐villi) and long‐term culture (LTC‐villi) preparations in 1561 cases (84.9%). A high degree of laboratory success (99.5%) and diagnostic accuracy (99.8%) was observed; in four cases of low mosaicism, all four associated with the final birth of a normal child, a small risk of uncertainty was accepted. The combined analysis of STC‐ and LTC‐villi reduced follow‐up amniocenteses by one‐third in comparison with the analysis of STC‐villi alone. We believe that the desired level of quality and accuracy of prenatal cytogenetics in chorionic villi can only be achieved when both STC‐ and LTC‐villi are available. We conclude that CVS might then be the mode of prenatal diagnosis of first choice in pregnancies with a high (cytogenetic) risk. Copyright

FULL MONOSOMY 21, PRENATALLY DIAGNOSED BY FLUORESCENT IN SITU HYBRIDIZATION

We describe a case of full monosomy 21 which was prenatally diagnosed in chorionic villi by fluorescent in situ hybridization (FISH). Because of intrauterine fetal death, a curettage was performed and cytogenetic analysis of skin fibroblasts confirmed the presence of monosomy 21 in fetal cells. DNA investigations showed a paternal origin of the single chromosome 21. Inspection and autopsy of the fetus revealed several congenital malformations. Some of them have been reported in earlier studies of monosomy 21; others concern new observations. Regarding the eye, the following abnormalities were microscopically observed: absence of the anterior and posterior eye chambers, aniridy, a hypoplastic ciliary body, Peters anomaly, and a double retina with secondary dysplasia. In addition, malformations of the extremities were seen: partial, proximal syndactyly of digits 3 and 4 of the right hand; pes varus position of the right foot; and transverse reduction defect at the tarsals of the left foot. To our knowledge, this is the first case in which full monosomy 21 has been proven.

How useful is the in vitro expansion of fetal CD34+ progenitor cells from maternal blood samples for diagnostic purposes?

Fetal cells present in the maternal circulation are a potential source of fetal DNA that can be used for the development of a prenatal diagnostic test. Since their numbers are very low, amplification of fetal cells has been discussed for a long time. So far, most studies have focused on culturing fetal erythroid cells. In this study, we evaluated whether limiting numbers of fetal haemopoietic progenitor cells present in an excess of maternal cells were able to overgrow the maternal component. Therefore, we used a model system in which limiting numbers of male CD34+ umbilical cord blood cells were diluted in 400 000 female CD34+ peripheral blood cells. The number of XY positive cells derived from umbilical cord blood was determined using two‐colour in situ hybridization with X and Y chromosomal probes. We demonstrated a 1500‐fold relative expansion of male umbilical cord blood cells over the peripheral blood component after three weeks of liquid culture, which also corresponded to the extent of expansion of CD34+ cells derived from 20‐week fetal blood. However, application of the same culture protocol to maternal blood samples obtained at 7–16 weeks of gestation showed no preferential growth of fetal haemopoietic progenitor cells. This study, therefore, suggests that fetal primitive haemopoietic progenitor cells do either not circulate in maternal blood before 16 weeks of gestation, or require different combinations/concentrations of cytokines for their in vitro expansion. Copyright

The use of in vitro expanded erythroid cells in a model system for the isolation of fetal cells from maternal blood.

The development of a non‐invasive prenatal diagnostic test using fetal nucleated red blood cells (NRBCs) isolated from the maternal circulation is hampered by the low frequency of these cells in maternal blood, requiring extensive enrichment procedures before any analytical procedure can be performed. In order to improve and simplify these procedures, we have used in vitro expanded erythroid cells derived from male umbilical cord blood in a model system for the isolation of fetal NRBCs from maternal blood. Erythroblast cells were expanded in vitro to high cell numbers and were immunophenotypically identical to fetal NRBCs isolated from maternal blood. Magnetic activated cell sorting (MACS) isolation procedures were optimized using in vitro expanded male NRBCs diluted up to 1 in 400 000 with female peripheral blood mononucleated cells. The number of recovered male cells was determined using two‐colour fluorescence in situ hybridization with X and Y chromosomal probes. Using this model system, an NRBC isolation technique is described. It is based on a one‐step MACS enrichment protocol for CD71 positive cells, which showed a significant (Wilcoxon signed ranks test, p<0·05) two‐fold higher yield of male NRBCs than previously described MACS methodologies, in which CD71 positive cells were enriched after depletion of other cell types. Application of these isolation strategies to maternal blood samples resulted in a similar improved enrichment of male fetal cells after the direct enrichment of CD71 positive cells. Copyright

The effect of chorionic villus sampling on the number of fetal cells isolated from maternal blood and on maternal serum alpha-fetoprotein levels

Fetal cells are present in the circulation of pregnant women and can be isolated using density gradient centrifugation and magnetic cell sorting. In the present study, maternal cell preparations were depleted for CD45‐ and CD14‐positive cells and enriched for CD71‐positive cells. The number of fetal nucleated cells was determined using fluorescence in situ hybridization for X and Y chromosomes. Analysis of maternal blood samples taken before and after transabdominal chorionic villus sampling (TA‐CVS) showed an increase in the number of fetal cells in 10 out of 19 male pregnancies after the invasive procedure. This cellular transfusion was found to correlate with elevated maternal serum alpha‐fetoprotein levels. TA‐CVS‐induced cellular transfusion may form a good in vivo system to optimize fetal cell isolation procedures and to study fetal cell dynamics and characteristics.

Prenatal diagnosis of trisomy 9: cytogenetic, FISH, and DNA studies

A cytogenetic survey and follow‐up studies were performed in eight cases of full, mosaic, and pseudomosaic trisomy 9 prenatally diagnosed among 36 213 prenatal samples in our department between August 1970 and July 1996. Besides conventional chromosome analysis, interphase fluorescent in situ hybridization (FISH) was employed. FISH turned out to be a rapid and accurate method for verification of trisomy cell lines and could provide additional information to the prenatal cytogenetic results. FISH also enables the study of uncultured specimens of amniotic fluid, not accessible for traditional cytogenetic analysis. In three cases, retrospective DNA analysis showed the supernumerary chromosome 9 to be of maternal origin. The disomic cell lines in both mosaic trisomy 9 cases showed maternal uniparental disomy.

A case of early intrauterine parvovirus B19 infection

We present a case of parvovirus B19 infection in the first trimester, confirmed by polymerase chain reaction (PCR) in amniotic fluid and cord blood, that caused myocarditis, severe intrauterine growth retardation, and probably glomerulonephritis. Eventually a small‐for‐dates neonate was born, without any signs of the infection.

Abnormal karyotypes in semi-direct chorionic villus preparations of women with different cytogenetic risks

Among 3499 cytogenetically investigated semi‐direct chorionic villus samples, 219 (6·3 per cent) abnormal karyotypes were encountered. The karyotypes were considered certainly abnormal (generalized abnormal with high probability) in 109 cases (3·1 per cent), and in 110 cases (3·1 per cent) uncertainly abnormal (potentially confined to the placenta), requiring further investigation. Of these 110 uncertain abnormalities, the cytogenetic result turned out to be finally abnormal representing generalized abnormality in 36 cases (32·7 per cent), finally normal representing confined placental mosaicism (CPM) in 69 cases (62·7 per cent), and remained undetermined in 5 instances (4·5 per cent). The rate of the numbers of certainly abnormal and all (certainly+uncertainly) abnormal results, the certainty rate, and that of generalized abnormalities and all abnormalities (generalized abnormalities+CPM cases), the predictive value, are strongly correlated with the cytogenetic risk. Therefore, we advise chorionic villus sampling for cytogenetic investigation only in women with a cytogenetic risk equal to or exceeding that of a 40‐year‐old pregnant woman. Because of the high rate of prenatal follow‐up investigations after the finding of uncertain results in semi‐direct villi, semi‐direct and cultured villi should be karyotyped simultaneously. Copyright