Carolyn Troeger

Fetal DNA in maternal plasma is elevated in pregnancies with aneuploid fetuses

Current non‐invasive screening methods for the prenatal diagnosis of fetal aneuploidies are hampered by low sensitivities and high false positive rates. Attempts to redress this situation include the enrichment of fetal cells from maternal blood, or the use of fetal DNA in the plasma of pregnant women. By the use of real‐time quantitative polymerase chain reaction (PCR) it has recently been shown that circulatory male fetal DNA in maternal plasma is elevated in pregnancies with trisomy 21 fetuses. In this independent study we confirm and extend upon these results by showing that the levels of fetal DNA are also elevated in pregnancies with other chromosomal aneuploidies (mean=185.8 genome equivalents/ml; range=62.2–471.7) when compared to pregnancies with normal male fetuses (mean=81.9 genome equivalents/ml; range=28.8–328.9), p=0.005. This elevation was greatest for fetuses with trisomy 21, whereas it was not significant for fetuses with trisomy 18, p=0.356. These data suggest that a quantitative analysis of such fetal DNA levels may serve as an additional marker for certain fetal chromosomal abnormalities, in particular for trisomy 21. Copyright

Fluctuation of maternal and fetal free extracellular circulatory DNA in maternal plasma.

Objective To examine whether concentrations of free extra-cellular fetal circulatory DNA in maternal plasma are stable or fluctuate. Methods Consecutive blood samples were drawn from 13 healthy nonpregnant volunteers and from 16 healthy pregnant women over 3 days. DNA was isolated from the plasma fraction and quantified by real-time polymerase chain reaction (PCR). Results In nonpregnant controls the total amount of cell free DNA fluctuated by an average of 13.5-fold. In samples obtained from pregnant women the amount of maternal cell free DNA varied by an average of 21.5-fold. Because ten of those women were pregnant with male fetuses, the concentration of free fetal DNA in these cases was determined by a real-time PCR assay for the Y chromosome. The mean variation in free fetal DNA levels in male fetuses was 2.2-fold. Conclusion The degree of variation in free fetal DNA concentrations observed in this study was similar to published values, so these results imply that care should be exercised when considering quantitation of this fetal material for potential diagnostic or screening purposes.

Current applications of single-cell PCR

Abstract. The advent of the polymerase chain reaction (PCR) has revolutionised the way in which molecular biologists view their task at hand, for it is now possible to amplify and examine minute quantities of rare genetic material: the limit of this exploration being the single cell. It is especially in the field of prenatal diagnostics that this ability has been readily seized upon, as it has opened up the prospect of preimplantation genetic analysis and the use of fetal cells enriched from the blood of pregnant women for the assessment of single-gene Mendelian disorders. However, apart from diagnostic applications, single-cell PCR has proven to be of enormous use to basic scientists, addressing diverse immunological, neurological and developmental questions, where both the genome but also messenger RNA expression patterns were examined. Furthermore, recent advances, such as optimised whole genome amplification (WGA) procedures, single-cell complementary DNA arrays and perhaps even single-cell comparative genomic hybridisation will ensure that the genetic analysis of single cells will become common practice, thereby opening up new possibilities for diagnosis and research.

A comparison of different density gradients and antibodies for enrichment of fetal erythroblasts by MACS

The enrichment of fetal cells, in particular fetal erythroblasts from the blood of pregnant women offers a promising non‐invasive alternative for prenatal diagnosis. The purpose of this study was to compare the retrieval of erythroblasts by different density gradients and different antibodies against erythroid surface antigens, in both a model test system and in blood samples of pregnant women. We enriched erythroblasts from artificial mixtures of cord and adult blood (1:50) and from 16 ml of peripheral blood from pregnant women at a mean gestational age of 14+2 weeks. The yield of erythroblasts was calculated and compared using Wilcoxons matched‐pairs signed‐rank test.

Cell‐Free DNA in Urine

Abstract: Intrigued by the rapid clearance of free fetal DNA from the maternal circulation, we have investigated whether this fetal genetic material could be cleared via the kidney. For this purpose, we examined for the presence of Y chromosome‐specific DNA sequences in urine samples obtained from 8 women pregnant with male fetuses. No male‐specific sequences could be detected, despite the use of a very sensitive nested PCR assay nor a highly reproducible real‐time PCR assay. We did, however, detect maternal DNA sequences. To determine if this cell‐free DNA was derived from the kidney or another source, we next examined urine from female kidney transplant patients who had received male kidneys. Y chromosome‐specific sequences were indeed detectable by both nested and real‐time PCR in these samples, thereby confirming a recent report describing urinary DNA microchimerism. Quantitative analysis of serially obtained samples furthermore suggests that transplant‐derived sequences are elevated during periods of graft rejection. These results imply that the measurement of graft‐derived urinary DNA may serve as a new marker for kidney graft tolerance.

Multiplex and Real-Time Quantitative PCR on Fetal DNA in Maternal Plasma: A Comparison with Fetal Cells Isolated from Maternal Blood

Abstract: Fetal DNA has recently been detected in maternal plasma by PCR and has shown promise for the prenatal determination of fetal sex or rhesus D. In order to obtain the maximum amount of information from this fetal genetic material, we have devised a sensitive multiplex PCR method to permit simultaneous analysis for both the SRY locus and the rhesus D gene. Our studies show that this technique is very sensitive and specific. In the 22 cases from rhesus D negative women examined, we were able to determine both fetal genotypes correctly. In the parallel enrichment for fetal cells, fetal erythroblasts were only detected in 14 of the 19 cases. Our data also indicate that fetal DNA from rhesus D positive fetuses is present in maternal plasma even after prophylactic anti‐D treatment. Furthermore, since fetal cells have been reported to be elevated in pregnancies with aneuploid fetuses, we have quantified the amount of fetal DNA present in the maternal plasma of 10 such affected pregnancies by real‐time PCR. Our results indicate that fetal DNA is elevated under such circumstances when compared to gestationally matched normal pregnancies (mean of 7% in aneuploid samples versus 3.5% in normal pregnancies). These results indicate that the quantification of fetal DNA in maternal plasma may be an additional screening tool for pregnancies at risk of bearing an aneuploid fetus.

Disturbances in placental immunology: ready for therapeutic interventions?

Recent studies have provided new insight into aberrations in the immunological interplay between mother and fetus and their potential role in the development of recurrent fetal loss and preeclampsia. The action of anti-phospholipid antibodies in recurrent fetal loss is now proposed to involve the complement system, neutrophil activation and the production of TNFα by immune bystander cells. A clear involvement of the immune system is emerging in preeclampsia, involving mainly the innate arm, especially neutrophils. The activation of peripheral neutrophils by placentally released inflammatory debris triggers the induction of neutrophil extracellular traps (NETs), which may lead to an occlusion of the intervillous space, thereby further promoting a condition of placental hypoxia. It has, hence, been suggested that new therapeutic strategies be developed, including the possible use of TNFα antagonists in cases of recurrent miscarriage. These strategies need to be addressed with caution due to the possible induction of fetal congenital abnormalities.

Attitudes of Swiss mothers toward unrelated umbilical cord blood banking 6 months after donation

BACKGROUND :During the past decade, the use of umbilical cord blood (CB) as a source of transplantable hematopoietic stem cells has been increasing. Little is known about the psychosocial consequences that later affect parents after unrelated CB donation. The objective of this study was to evaluate the attitudes of mothers toward unrelated donation of umbilical CB for transplantation 6 months after giving birth.

Inability to Detect Cell Free Fetal DNA in the Urine of Normal Pregnant Women nor in Those Affected by Preeclampsia Associated HELLP Syndrome

Objective: Recent reports have indicated that cell-free fetal DNA can be detected in the urine of pregnant women. We attempted to reproduce those data. Methods: Urine samples were collected from 18 normal pregnant women (11 with a male fetus). Urinary DNA was examined by Y-chromosome-specific nested polymerase chain reaction (PCR) or real-time PCR. Samples were also examined from two pregnancies complicated by HELLP (hemolysis, elevated liver enzymes, and low platelets) syndrome, which had very high levels of cell-free fetal DNA in the maternal plasma. To validate our data, a quantitative comparison of different DNA extraction procedures used in the previous reports was performed. Results: In no instance were we able to detect any fetal DNA in maternal urine, although copious quantities of cell-free fetal DNA were present in the maternal plasma of those pregnancies affected by HELLP syndrome. Our quantitative analysis of the various extraction procedures used indicated that the commercial column elution method we used was comparable, if not superior, to the noncommercial methods used in previous reports. Conclusion: Our data strongly suggest that cell-free fetal DNA is not readily detectable in maternal urine, even under conditions known to increase kidney permeability.

Fetal cells in maternal blood: An update from Basel

OBJECTIVE The efficiency of two protocols for the enrichment of fetal cells from the blood of pregnant women was compared: a triple density gradient followed by twin magnetic separations (method A) versus a single density gradient and single magnetic separation (method B). STUDY DESIGN Blood samples were obtained from women prior to undergoing an invasive procedure. The processed samples, 87 by method A and 332 by method B, were examined for the presence of male cells by fluorescence in situ hybridisation. RESULTS The simpler protocol was found to be superior. The most critical component, however, is the ability of the reader to correctly evaluate the sample, where we observed large variations, with reader B attaining a sensitivity of 82.61% with a corresponding specificity of 86.96%. CONCLUSION A simpler enrichment protocol can be used from smaller blood samples to attain detection efficiencies which are similar to or superior to current noninvasive methods.