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PubMed2006

Noninvasive prenatal detection of fetal trisomy 18 by epigenetic allelic ratio analysis in maternal plasma: Theoretical and empirical considerations.

BACKGROUND: The discovery of cell-free fetal DNA in maternal plasma has opened up new possibilities for noninvasive prenatal diagnosis. However, the use of maternal plasma fetal DNA for the direct detection of fetal chromosomal aneuploidies has not been reported. We postulate that the aneuploidy status of a fetus could be revealed by an epigenetic allelic ratio approach, i.e., by analyzing the allelic ratio of a single-base variation present within DNA molecules exhibiting a placental-specific epigenetic signature in maternal plasma. METHODS: Placental-derived fetal-specific unmethylated maspin (SERPINB5) promoter sequences on human chromosome 18 were detectable in placental-maternal DNA mixtures and in maternal plasma by bisulfite modification followed by methylation-specific PCR (MSP) and primer extension. The ratios between the extension products of the 2 alleles were calculated for heterozygous placentas, placental-maternal blood cell DNA mixtures, and maternal plasma samples. The allelic ratios were compared between pregnancies carrying trisomy 18 and euploid fetuses. RESULTS: The epigenetic allelic ratios of all tested trisomy 18 samples deviated from the reference range obtained from euploid samples (placental DNA, 1.135 to 2.052; placental-maternal DNA mixtures, 1.170 to 1.985; maternal plasma, 0.330 to 3.044; without skew correction on the raw mass spectrometric data). A theoretical model was established and validated that predicted that a minimum of 200 copies of genomic DNA after bisulfite conversion were required for distinguishing euploid and aneuploid fetuses with confidence. CONCLUSION: Epigenetic allelic ratio analysis of maternal plasma DNA represents a promising approach for noninvasive prenatal diagnosis of fetal chromosomal aneuploidies.

Chromosomes, Human, Pair 18↗
PubMed2005

The utility of an erythroblast scoring system and gender-independent short tandem repeat (STR) analysis for the detection of aneuploid fetal cells in maternal blood.

OBJECTIVE: The aim of this study was to determine whether fetal nucleated red blood cells (NRBCs) could be distinguished from maternal cells in peripheral blood using an erythroblast scoring system based on the unique morphological and hemoglobin staining characteristics of this cell type. Presumptive fetal NRBCs were further analyzed for the presence of paternally inherited DNA polymorphisms to prove fetal origin. METHODS: NRBCs were isolated by density gradient separation, CD15/45 depletion, and gamma hemoglobin positive selection from peripheral blood of nine women following termination of pregnancy for trisomy 21 (n=4), 18 (n=1), 13 (n=2), and other genetic abnormalities (n=2). Candidate fetal NRBCs, based on four discrete morphological and hemoglobin staining criteria, were then subjected to fluorescent PCR (polymerase chain reaction) amplification of chromosome 21 (D21S1411, D21S11) and chromosome 18 (D18S535) short tandem repeat (STR) DNA polymorphisms. RESULTS: In all cases, candidate fetal NRBCs were accurately identified on the basis of morphologic and hemoglobin staining characteristics and confirmed to be fetal in origin based on the presence of shared and nonshared polymorphic DNA alleles when compared to DNA isolated from maternal cells. CONCLUSIONS: Using the erythroblast scoring system and subsequent analysis of inherited DNA polymorphisms, we were able to distinguish fetal NRBCs from maternal cells and prove fetal origin independent of gender. These results suggest that this novel combined approach to fetal cell isolation and genetic analysis is a promising method for noninvasive prenatal diagnostic applications.

Aneuploidy↗
PubMed2000

Circulating DNA in plasma or serum.

Small amounts of DNA circulate in both healthy and diseased human plasma/serum, and increased concentrations of DNA are present in the plasma of cancer patients. Characteristics of tumor DNA have been found in genetic material extracted from the plasma of cancer patients. These features include decreased strand stability, the presence of specific oncogene or tumor suppressor gene mutations, microsatellite alterations, Ig rearrangements and hypermethylation of several genes. The results obtained in many different cancers have opened a new research area indicating that plasma DNA might eventually be a suitable target for the development of noninvasive diagnostic, prognostic and follow-up tests for cancer. Following the discovery of tumor derived DNA in plasma or serum, cell-free fetal DNA has also been found in maternal plasma and serum. This discovery provides an easily accessible source of fetal genetic material for prenatal diagnosis.

DNA, Neoplasm↗
PubMed1993

Observations of intrapartum fetal activities.

OBJECTIVE: The objective of this observational study was to document the frequency of biophysical activities such as breathing and body movements during spontaneous labor in the low-risk, term fetus. STUDY DESIGN: The fetuses of truly low-risk patients without medication during labor were observed serially with real-time ultrasonography for fetal breathing or body movements for a maximum scanning time of 1 hour. Fetal breathing movements, hiccups, and fetal body movements were recorded, as were cervical dilatation, status of the fetal membranes, and Apgar scores. RESULTS: Fetal breathing activity was recorded in 76% of fetuses in early labor and 90% in advanced labor. Fetal body movements were present during 85% of the observations. All babies had 5-minute Apgar scores > 7. CONCLUSION: Ultrasonographic evaluation of fetal breathing and body movements during spontaneous labor was associated with normal birth outcome in this low-risk population. The clinical utility of this noninvasive method in determining intrapartum fetal health remains to be tested in an unselected population.

Apgar Score↗
PubMed1993

Predicting hemolytic disease of the newborn: a comparison of the monocyte monolayer assay and the chemiluminescence test.

The ability of the monocyte monolayer assay (MMA) and the chemiluminescence test (CLT) to predict the clinical significance of alloantibodies associated with hemolytic disease of the newborn (HDN) was assessed by the use of 22 well-characterized antisera--predominantly anti-D--from alloimmunized pregnant women. Seventeen sera were obtained before delivery from women whose infants were antigen positive for the antibody specificities identified in the maternal serum. With testing of these 17 sera by MMA, 10 results were in agreement with the presence or absence of HDN, but there were 5 false-positive and 2 false-negative results. With the CLT, 16 results were in agreement with the presence or absence of HDN, and there was 1 false-negative result. Five sera were obtained from women whose infants were antigen negative for the antibody specificities identified in the maternal serum. The CLT and the MMA were both subject to false-positive results with these sera. These results suggest that the CLT may be more valuable than the MMA as a noninvasive test for predicting the clinical significance of alloantibodies in HDN.

Erythroblastosis, Fetal↗
PubMed2006

Molecular testing for transfusion medicine.

PURPOSE OF REVIEW: Molecular testing methods were introduced to the blood bank and transfusion medicine community more than a decade ago after cloning of the genes made genetic testing for blood groups, that is genotyping, possible. This review summarizes the progress made in the last decade in applying genotyping to prenatal practice and clinical transfusion medicine. RECENT FINDINGS: Assays that target allelic polymorphisms prevalent in all populations are reproducible and highly correlated with red blood cell phenotype. For some blood groups, assays that detect silencing mutations are also required for accurate typing, and for ABO and Rh, multiple regions of the genes must be sampled. Genotyping is a powerful adjunct to serologic testing and is superior for typing transfused patients, for D-zygosity determination, for noninvasive fetal typing, and for antigen-matching in sickle cell patients. SUMMARY: Implementation of molecular testing for transfusion medicine has been a conservative process and limited primarily to reference laboratory environments. With the development of high-throughput platforms, genotyping is poised to move into the mainstream, revolutionizing the provision of antigen-negative donor units. This will enable electronic selection of units antigen matched to recipients at multiple blood group loci, potentially eliminating alloimmunization and significantly improving transfusion outcomes.

Blood Group Incompatibility↗
PubMed2005

Diagnosis and management of human parvovirus B19 infection.

Parvovirus B19 is a common infection in children occurring in winter and spring months that can affect pregnant women and their unborn fetuses. Adverse fetal sequelae may include red blood cell aplasia due to bone marrow suppression, anemia, myocarditis, nonimmune hydrops, and fetal death. Fortunately, several noninvasive testing schemes are available to assess the fetus, and invasive testing is reserved for fetuses with marked anemia.

Antibodies, Viral↗
PubMed1997

PCR-based screening for cystic fibrosis carrier mutations in an ethnically diverse pregnant population.

As the most common lethal autosomal recessive disorder in North America, cystic fibrosis (CF) is an obvious candidate for general population carrier screening. Although the identification of the causative gene has made detection of asymptomatic carriers possible, the extreme heterogeneity of its mutations has limited the sensitivity of the available DNA screening tests and has called into question their utility when they are applied to patients with no family history of the disease. The purpose of this study was to determine the technical feasibility, patient acceptance and understanding, and psychosocial impact of large-scale CF carrier screening in an ethnically diverse pregnant population. A total of 4,739 pregnant women attending prenatal clinics located in both an academic medical center and a large HMO were invited in person to participate. Of this group, 3,543 received CF instruction and assessments of knowledge and mood, and 3,192 underwent DNA testing for the six most common CF mutations, by means of a noninvasive PCR-based reverse-dot-blot method. Overall participation rates (ranging from 53% at the HMO to 77% at the academic center) and consent rates for DNA testing after CF instruction (>98%) exceeded those of most other American studies. The PCR-based screening method worked efficiently on large numbers of samples, and 55 carriers and one at-risk couple were identified. Understanding of residual risk, anxiety levels, and overall satisfaction with the program were acceptable across all ethnic groups. Our strategy of approaching a motivated pregnant population in person with a rapid and noninvasive testing method may provide a practical model for developing a larger CF screening program targeting appropriate high-risk groups at the national level, and may also serve as a paradigm for population-based screening of other genetically heterogeneous disorders in the future.

Academic Medical Centers↗
PubMedDate not supplied

Favorable attitudes toward testing for chromosomal abnormalities via analysis of fetal cells in maternal blood.

PURPOSE: The NICHD Fetal Cell Isolation Study (NIFTY) was a multicentered project to isolate fetal cells from maternal blood to detect fetal chromosomal abnormalities. The project included a psychosocial component, which is the basis of this article. We examined the attitudes of high-risk pregnant women toward the availability of a maternal blood test to identify fetal chromosomal abnormalities, how women would respond to hypothetical normal and abnormal maternal blood testing results, and the factors associated with a woman's preference to have an invasive procedure in response to a normal maternal blood test. METHODS: High-risk pregnant women (N = 854) planning to have prenatal diagnostic invasive testing (amniocentesis or chorionic villus sampling) completed a survey. RESULTS: The women highly favored maternal blood testing. Almost all women would seek invasive testing after an abnormal blood test. Only half of the women would seek invasive testing after a normal blood test; these women were older, more willing to terminate their pregnancy, and valued the increased accuracy of invasive testing more highly than women who would not have invasive testing after a normal maternal blood test. CONCLUSIONS: Women having invasive diagnostic testing welcome a noninvasive procedure that uses fetal cells in maternal blood, and its availability would decrease invasive testing by approximately 50%. Research needs to examine the attitudes and anticipated responses of other risk groups as well as the effects of information about maternal blood test sensitivity and specificity on attitudes and responses.

Abortion, Eugenic↗
PubMed2003

Fetal nucleated red blood cells in peripheral blood of pregnant women: detection and determination of location on a slide using laser-scanning cytometry.

OBJECTIVE: The purpose of the study was to assess the feasibility of analysis of fetal nucleated red blood cells (NRBC) present in the maternal circulation by laser-scanning cytometry. METHODS: CD71-positive cells were obtained by magnetic cell sorting of peripheral blood of pregnant women after density centrifugation. Immunofluorescence for the Hbgamma-chain was combined with fluorescent staining of DNA (TO-PRO-3) and fluorescence in situ hybridization (FISH) with a Y-chromosome specific probe. The cells were scanned on a slide using a laser-scanning cytometer (LSC). Events double positive for Hbgamma and TO-PRO-3 were relocated and their morphology and FISH reactivity were visually assessed. Determination of male fetal sex with LSC was compared with findings from amniocentesis. RESULTS: In 8/15 pregnancies with male fetuses and in 0/9 with females (apart from one case with a male/female twin pregnancy), we detected Y-chromosome-positive NRBC. In pregnancies with female fetuses, Y-chromosome-positive cells other than NRBC were found in all women who had previously given birth to male babies, whereas women with no abortion and no male babies in their history did not present with Y-chromosome-positive non-NRBC. CONCLUSION: On the basis of automatic relocation of once-defined cells of fetal origin from the current pregnancy, laser-scanning cytometry is likely to facilitate repeated (poly-)FISH analysis and single-cell PCR for noninvasive prenatal diagnosis.

Adult↗
PubMed1998

Animal behavior models. Increased sensitivity to stressors and other environmental experiences after prenatal cocaine exposure.

Neural compensations occurring after prenatal cocaine exposure may often permit some functional recovery, although the cost of this reorganization may be a decrease in adaptability. As we have seen in our rodent model of prenatal cocaine exposure, latent deficits may become unmasked when offspring are exposed to cognitive and environmental demands and stressors. In adolescence and adulthood, offspring exposed gestationally to cocaine exhibit characteristic decreases in stress-induced immobility along with increases in aggression under the demands of social competition. Recently, we observed that cocaine-exposed offspring are also unusually sensitive to the long-term effects of early manipulation (noninvasive heart rate testing at 16 days of age). When tested in adulthood, cocaine-exposed offspring not receiving this early experience exhibited less immobility when tested in the presence of intermittent footshock or when subsequently examined in an open field as well as more locomotion in the open field than control offspring, findings reminiscent of previous work. By contrast, these effects were normalized (shock-induced immobility) or reversed (open field immobility and locomotion) in cocaine-exposed animals given the early experience. This marked susceptibility to the effects of early manipulation was less evident in control offspring and even in a group of stunted nutritional controls. Thus, cocaine-exposed offspring may exhibit increased sensitivity not only to environmental demands and stressors, but also to the potential moderating or beneficial effects of early experiences.

Aging↗
PubMed1998

Analysis of HLA-DQ alpha sequences for prenatal diagnosis in single fetal cells from maternal blood.

We have extended a previously developed method that allows prenatal DNA diagnosis of female fetuses through the isolation of single nucleated erythrocytes from maternal blood by developing a method that can distinguish between maternal and fetal nucleated erythrocytes. Nucleated erythrocytes were separated by a density-gradient method and then collected by micromanipulation. Sex was determined after primer extension preamplification (PEP) of the entire genome of a single cell, and human leukocyte antigen (HLA)-DQ alpha type was determined after further amplification of this gene. The HLA-DQ alpha genotype of fetal erythrocytes in maternal blood samples and their corresponding paternal and maternal lymphocytes were successfully determined in all cases. The accuracy of the method was determined by using single nucleated erythrocytes from umbilical cord blood from five normal deliveries. This is the first demonstration that the fetal HLA-DQ alpha gene sequences can be identified in a small aliquot of a single nucleated erythrocyte in maternal blood. We believe that this method ushers in a new era in which the reliability and accuracy of noninvasive prenatal DNA diagnosis from maternal blood is markedly improved.

Alleles↗
PubMedDate not supplied

Is routine amniocentesis for advanced maternal age still indicated?

OBJECTIVE: To evaluate a policy of selective rather than routine use of amniocentesis for advanced maternal age. METHOD: A consecutive series of 359 pregnant women aged 38-47 underwent nuchal translucency measurement (NTM) at 10-14 weeks, maternal serum screening (MSS) by alpha-fetoprotein (AFP) and human chorionic gonadotropin (hCG) at 15-17 weeks, and second trimester ultrasound at 21-23 weeks. Women with NTM <3 mm, MSS-derived risk <1/250, and a normal second trimester sonography were considered at low risk and were suggested not to have an amniocentesis. RESULTS: Either the NTM or MSS test was positive in 130 women; 105 (81%) of them elected to have an amniocentesis, versus 122 (53%) of 229 in whom both tests were negative (p < 0.001). Nineteen (5%) of 359 patients had NTM > or =3 mm; all 7 cases of Down's syndrome were in this group; 122 (34%) of 359 patients had a MSS-derived risk > or =1/250; 6 of the 7 cases of Down's syndrome were in this group: Ten patients had an abnormal second trimester ultrasound, 1 of which had trisomy 18. Of the 219 patients with MSS-derived risk <1/250, a NTM <3 mm, and a normal second trimester ultrasound, none had a baby with a chromosomal abnormality (95% confidence interval: 0-1.4%). CONCLUSION: Amniocentesis may be offered on a selective rather than routine basis in women over 38, based upon the results of noninvasive screening tests.

Adult↗
PubMed2002

Enrichment, immunomorphological, and genetic characterization of fetal cells circulating in maternal blood.

Fetal cells circulating in the peripheral blood of pregnant women are a potential target for noninvasive genetic analyses. They include epithelial (trophoblastic) cells, which are larger than peripheral blood leukocytes. We enriched circulating trophoblastic cells using the isolation by size of epithelial tumor cells (ISET) method. Peripheral blood was obtained at 11 to 12 weeks of pregnancy. Cells isolated by ISET were stained by hematoxylin and eosin or by immunohistochemistry. Large epithelial cells were microdissected and fetal cell identification was obtained by polymerase chain reaction with short tandem repeats and/or Y-specific primers. By analyzing only 2 ml of blood, we found a variable number (n = 1 to 7) of Y-positive cells (overall 15 of 23) in all of the six mothers carrying a male fetus. In contrast, none of the 26 cells isolated from seven mothers carrying a female fetus scored positive. Eleven cells were analyzed by using short tandem repeat-specific markers: six of them showed a fetal profile and five showed a maternal profile consistently with Y-specific results. Only one-fifth of the single cell DNA was used for fetal cell assessment, leaving enough material for further genetic tests. We also show that the ISET approach allows the performance of fluorescence in situ hybridization analyses and the detection of DNA point mutations in single microdissected cells. We conclude that this is a powerful approach to enrich circulating fetal cells and prove their fetal origin, and that it may have implications for noninvasive prenatal diagnosis of genetic disorders.

Cell Separation↗
PubMed2005

Noninvasive prenatal RHD genotyping by real-time polymerase chain reaction using plasma from D-negative pregnant women.

OBJECTIVE: Prenatal noninvasive determination of fetal Rh status is an important aid to the management of hemolytic disease of the fetus and newborn. We performed real-time polymerase chain reaction on fetal DNA derived from maternal plasma to determine fetal Rh status. STUDY DESIGN: Cell-free plasma DNA from 98 D-negative pregnant women was tested for the presence of exons 4, 5, and 10 of RHD. The presence of fetal DNA was confirmed by detection of SRY or biallelic insertion/deletion polymorphisms in the maternal plasma and buffy coat. RESULTS: Seventy-two D-positive infants and 26 D-negative infants were determined by serologic studies. All 3 RHD exon sequences were detected in 68 of 72 mothers of D-positive infants. The presence of fetal DNA in mothers of D-negative infants was confirmed in all 10 boys and in 14 of 16 girls. CONCLUSION: Fetal RHD genotyping in this study correctly predicted fetal Rh status in 92 of 98 (94%) cases.

Algorithms↗
PubMed1992

[Noninvasive screening for chromosome abnormalities in young pregnant patients].

Two thirds of the patients with Down's syndrome are born by women below 35 years of age. An extension of invasive prenatal diagnosis to these women is problematical due to the operative risks and the relatively high costs. Therefore, non-invasive screening methods are sought, apt to identify groups at high risk. Biochemical screening methods applied at 16 weeks of pregnancy (triple test) and the new first trimester tests are discussed. Ultrasound screening at 10 to 12 weeks is dealt with in more detail. A large fluid cushion over most of the back was documented not only in most cases of trisomy 21 but in trisomies 18 and 13 and in Turner's syndrome as well. Only few chromosomally normal fetuses with the same peculiarity were observed. Systematic first trimester screening for nuchal fluid accumulation seems to be a recommendable method for the detection of chromosome anomalies. It compares favourably with current methods of maternal serum screening performed at 16-18 weeks which require a manyfold higher number of invasive procedures.

Amniocentesis↗
PubMed1997

Influence of gestational age on fetal deoxyribonucleic acid retrieval in maternal peripheral blood.

OBJECTIVE: We wanted to verify whether gestational age influences the retrieval of fetal deoxyribonucleic acid in maternal blood to identify the best period for maternal blood sampling for a future noninvasive prenatal diagnoses. STUDY DESIGN: We amplified 81 deoxyribonucleic acid samples extracted from the peripheral blood of 27 pregnant women (18 bearing male fetuses and 9 bearing females) by nested polymerase chain reaction of the Y-specific sequence DYS14. We obtained three blood samples (one per gestational trimester) from each woman. Statistical evaluation was assessed by the McNemar test of symmetry. RESULTS: Polymerase chain reaction results in male-bearing pregnancies differed significantly between the first and second trimesters and between the second and third trimesters (p < 0.025) in parallel with a decrease in sensitivity in the second trimester (67%) compared with the first (94%) and third trimesters (100%). CONCLUSIONS: The drop in sensitivity from the first to the second trimester witnesses a variable concentration of fetal cells in maternal blood, with a negative balance in the second trimester. Therefore, to achieve an adequate polymerase chain reaction accuracy, the choice of gestational age is relevant and the first trimester seems to be more suitable than the second trimester.

DNA↗
PubMed2004

Prospective study of parental choice for aneuploidy screening in assisted conception versus spontaneously conceived twins.

The aim of this study was to evaluate and compare parental choice towards the use of aneuploidy screening and diagnostic techniques after assisted conception versus spontaneously conceived pregnancy. A total of 193 patients with consecutive twin pregnancies participated in the study, including 143 who spontaneously conceived and 50 who conceived following assisted reproduction treatment. Patients were investigated prospectively for their choice in the prenatal screening/diagnosis of aneuploidy. In the group of patients who conceived spontaneously, 111 (77.6%) opted initially for the non-invasive screening based on nuchal translucency measurements, whereas in the assisted conception group, 44 (88%) opted for the non-invasive screening. In the spontaneous conception group, 32 (22.4%) presented because they had a positive serum screening test or because they had opted for a chorionic villus sampling (CVS) or an amniocentesis for advanced maternal age. In the assisted conception group only six patients (12%) opted for amniocentesis. The difference in choice of noninvasive versus invasive screening between those with spontaneous and those with assisted conception was not significant, but those who had conceived spontaneously after the age of 37 years were significantly more likely (P < 0.05) to request an invasive diagnostic procedure through all maternal age groups. When needed, an amniocentesis was preferred to CVS. These data also indicate that the antenatal counselling needs to be adapted to couples presenting with an assisted conception twin pregnancy.

Adult↗