Immediate and long-term applications of technology.

PIP: The study of human genetics and developments therein have truly made great strides in recent years. The tendency to immediately introduce new technology into medical practice, however, suggests the urgent need to consider the ethics of our recently developed and developing technological prowess, as well as the long- and short-term impacts of these changes. The authors therefore review and consider published field experiences specifically related to prenatal diagnosis. Noninvasive screening tests during pregnancy, the isolation of fetal cells from maternal circulation for prenatal diagnosis, amniocentesis, and molecular genetic techniques which analyze DNA are considered. In response to the increasing number of prospective parents who want to have a prenatal assessment of normal fetal well-being, voluntary ultrasound screening programs have developed. New available techniques make the diagnosis of fetal maldevelopments more frequent and more precise. The practice seems safe within the limits of current practice. As for the isolation of fetal cells from maternal circulation for prenatal diagnosis, large clinical pilot trials are needed to especially determine the method's sensitivity and specificity for use in either screening or diagnosis. This approach should be under tight control to avoid potential misuse.^ieng

Detection of fetal trisomies 21 and 18 from maternal blood using triple gradient and magnetic cell sorting.

PROBLEM: The need for an inexpensive and reproducible technique for noninvasive prenatal diagnosis by fetal cell isolation from maternal blood. METHOD: For enrichment of fetal cells we used a combination of triple density gradient and magnetic sorting (MACS) of (anti-CD71) transferrin receptor antibody labeled cells followed by fluorescence in situ hybridization (FISH) with chromosome-specific DNA probes for detection of fetal aneuploidies. We identified 15 cases of fetal trisomy (five cases with a trisomy 18 and ten cases with a trisomy 21) with subsequent chromosome-specific FISH. RESULTS: We found in all of the aneuploid pregnancies that the percentage of cells with three hybridization signals did not overlap with those of normal controls independent from gestational ages and previous invasive procedures. CONCLUSIONS: Our new approach for noninvasive prenatal diagnosis has proven to be reliable in this first series.

Fetal DNA in uterine vein blood.

OBJECTIVE: To investigate whether the ratio of fetal cells in the maternal circulation differs before and after the blood passes through the maternal lung. METHODS: We performed polymerase chain reaction-based Y-sequence analysis of DNA derived from antecubital vein blood obtained before and 2 hours after cesarean delivery, and from uterine vein blood obtained during cesarean of 14 women carrying male fetuses. RESULTS: Fetal DNA was detected in 17 tested specimens and, as estimated by comparison with parallel dilution series, the fetal-maternal DNA ratio was 1:10(5) to 1:10(6). However, there was no significant difference in the amount of Y-chromosomal DNA detectable between uterine vein blood and peripheral blood after polymerase chain reaction and Southern hybridization. In DNA derived from peripheral blood after delivery, the intensity of Y-specific fetal DNA sequences was also not significantly increased. CONCLUSION: Our results argue against the often-stated hypothesis of different ratios of fetal to maternal DNA in the uterine vein versus peripheral blood, and indicate that even delivery does not seem to increase the flow of fetal cells into the maternal circulation.

Magnetic cell sorting and the transferrin receptor as potential means of prenatal diagnosis from maternal blood.

OBJECTIVE: We wanted to test whether the recently described method of using the transferrin receptor system for fluorescence-activated cell-sorter enrichment of nucleated red blood cells can be used for prenatal diagnosis from maternal blood. STUDY DESIGN: Instead of the laborious, expensive fluorescence-activated cell-sorter system, we used the newly described magnetic-activated cell sorter. RESULTS: An effective enrichment could be achieved with separation of lymphocyte subsets. With the transferrin receptor, however, the enrichment was very inefficient because of the poor specificity of the antibody itself. Even in umbilical cord blood only 25% of nucleated red blood cells were labeled as demonstrated by immunogold silver enhancement of transferrin receptor-labeled cells. CONCLUSION: In spite of the availability of a fast and effective separation method (magnetic-activated cell sorter) the use of the transferrin receptor antigen alone is not likely to enable a reliable identification of fetal cells in maternal circulation.

Fetal cells in the maternal circulation.

The analysis of fetal cells from the maternal circulation would be the least invasive method of prenatal diagnosis. Potential fetal cell types to enter the maternal circulation are lymphocytes, trophoblast cells and nucleated erythrocytes. With conventional methods, such as cytology and interphase or metaphase cytogenetics, the ratio of fetal to maternal cells was overestimated in the past. Currently most groups use polymerase chain reaction-based Y-sequence analysis for the detection of fetal cells in pregnancies with male fetuses, either with or without prior enrichment of fetal cells. For fetal cell separation, fluorescence-activated cell sorting and immunomagnetic beads have been applied, and recently our group has used discontinuous density gradient centrifugation for this purpose. We have shown that the transferrin receptor antigen alone is not sufficient for enrichment of fetal nucleated erythrocytes. Despite some initial promising results with fluorescence in situ hybridization, the reproducibility and reliability of the techniques are still limited, mainly due to the lack of very specific cell markers and the very low and variable concentrations of fetal cells among numerous maternal cells.

Ratio of fetal to maternal DNA is less than 1 in 5000 at different gestational ages in maternal blood.

Using Southern hybridization with the DNA probe pY3.4, we were not able to detect fetal DNA in blood of 36 pregnant women carrying male fetuses. Gestational ages ranged from 8-40 weeks of pregnancy. Using the same DNA probe, we were able to detect the male-specific signal in experimental dilution series down to 1/5000 on autoradiograms. We conclude that the ratio of fetal DNA in maternal circulation, in contrast to previous estimations, must be lower than 1/5000.

Transabdominal placental biopsy in the second and third trimesters of pregnancy: what is the risk of maternal contamination in DNA diagnosis?

We investigated the risk of maternal contamination in antenatal DNA diagnosis after second- and third-trimester transabdominal placental biopsy. For this purpose, we compared the restriction fragment length polymorphism (RFLP) patterns of 11 chorionic villus DNA samples after late chorionic villus sampling (CVS) with those of the corresponding maternal DNA. Ten of 11 aspirated tissue samples were not separated from maternal contamination before DNA extraction. All 11 mother-embryo pairs were informative for analysis of maternal contamination, ie, the mother showed one RFLP allele not present in the embryo. In none of the 11 cases did the fetal DNA show maternal contamination after molecular hybridization, although ten samples were contaminated with maternal tissue macroscopically and microscopically. Despite some maternal tissue admixture, the risk of contamination seems to be lower in the second- and third-trimester CVS than in first-trimester CVS, based on previous reports and our own experiences. This is most likely due to the anatomically closer contact of villi and decidua in the first trimester of pregnancy.

Three cases of 45,X/46,XYnf mosaicism. Molecular analysis revealed heterogeneity of the nonfluorescent Y chromosome.

Three patients with 45,X/46,XYnf mosaicism were investigated by Southern hybridization using both X- and Y-specific DNA probes. Our patients seem to be hemizygous for the X chromosomal loci tested. Single-copy and low-copy repeated Y chromosomal sequences assigned to the short arm, centromere, and euchromatin of the long arm have been detected in our patients, suggesting the Y chromosomal origin of the marker chromosome both in male and female cases studied. Densitometry of autoradiographs revealed a double dose of Yp-specific fragments of the DXYS1 locus. None of the patients tested showed either the 3.4- or the 2.1-kb Hae III male-specific repeated DNA sequences. It seems likely that the Ynf is a pseudodicentric chromosome with duplication of Yp and euchromatic Yq sequences, the Yq heterochromatin being lost. Our findings indicate structural heterogeneity of the marker chromosome and in addition provide further information on the relative position of DNA sequences detected by DNA probes 50f2, M1A, and pDP105.