Search

Purpose: Methods to isolate cell-free fetal DNA from maternal plasma are critical in developing noninvasive fetal DNA testing strategies. Given that plasma consists of heterogeneous DNA-size fragments in a complex mix of proteins, recovery and analysis of this DNA are understandably inefficient. To facilitate recovery, we performed qualitative and quantitative analysis of DNA isolated from maternal plasma., Methods: DNA isolated from maternal blood (n = 15) was compared using five different DNA isolation protocols: two conventional, two column-based, and one magnetic-bead based. Purity and concentration of DNA recovered were determined with a NanoDrop spectrophotometer. Real-time polymerase chain reaction quantification of the beta-globin and DYS1 loci was performed to determine total and fetal-specific genome equivalents, respectively., Results: DNA quality and quantity were different among the five methods tested. Although purity and concentration of total DNA were greatest with the conventional boiling-lysis approach, correct detection of a male fetus was achieved in only 62.5% of cases. DNA isolation using the magnetic beads yielded the highest quantity of total DNA (2018.83 +/- 4.09 GEq/mL), with 100% fetal DNA detection., Conclusions: Optimal plasma DNA recovery protocols must take into account DNA purity and concentration. We confirm that the magnetic-beads method provides a fast, simple, sensitive, and specific approach to purify plasma DNA. The resulting high-quality DNA facilitates efficient examination of fetal DNA sequences.

Background: Analysis of fetal DNA from maternal plasma by PCR offers great potential for noninvasive prenatal genetic diagnosis. To further evaluate this potential, we developed and validated a standard protocol to determine whether fetal DNA sequences could be reproducibly amplified and measured across multiple laboratories in a common set of specimens., Methods: Each of five participating centers in a National Institute of Child Health and Human Development consortium collected 20 mL of peripheral blood from 20 pregnant women between 10 and 20 weeks of gestation. The plasma fraction was separated according to a common protocol, divided, and frozen in five aliquots. One aliquot was shipped to each participating laboratory, where DNA was extracted according to a standard protocol. All plasma samples (n = 100) were then analyzed blindly for the presence and quantity of total DNA (GAPDH) and male fetal DNA (SRY) by real-time PCR. Genomic DNA was isolated from female and male cells at one center, quantified, and shipped to the others to serve as calibrators for GAPDH and SRY, respectively., Results: The amplification of known quantities of DNA was consistent among all centers. The mean quantity of male DNA amplified from maternal plasma when the fetus was male ranged from 51 to 228 genome equivalents (GE)/mL. Qualitative concordance was found overall among centers. The sensitivity of the assay for detection of male DNA when the fetus was male varied from 31% to 97% among centers. Specificity was more consistent (93-100%) with only four false-positive results obtained across the entire study., Conclusions: All centers were able to consistently amplify frozen and shipped DNA. The PCR procedure used here is reliable and reproducible. Centers that extracted and amplified more DNA per milliliter of maternal plasma had superior sensitivities of Y chromosome sequence detection. The specificity of the assay was more consistent among centers. A robust and thoroughly optimized protocol for the extraction of DNA from maternal plasma is needed to make testing of fetal DNA in maternal plasma a clinically relevant analytical tool.

Methods utilizing circulating cell-free RNA in plasma have clinical applications for cancer and prenatal genetic analysis. Given these potential roles, the feasibility of detecting placental specific RNA in dried maternal blood spots after storage at room temperature for varying lengths of time was investigated. Using real-time polymerase chain reaction (PCR), positive amplification of placental-specific β-human chorionic gonadotrophin transcripts was demonstrated in nine of 11 dried blood samples from first and second trimester pregnancies stored at room temperature for up to 4 weeks. This work demonstrates feasibility in isolation and amplification of placental mRNA using dried maternal blood spots. With the development of fetal and placental RNA markers, this approach would allow simplified collection, transport, and storage of samples for prenatal genetic diagnosis and pregnancy related complications. [ABSTRACT FROM AUTHOR]

Fetal DNA is found in the plasma of pregnant women that appears to be stable for PCR amplification. Although the underlying mechanism giving rise to this DNA in plasma remains unclear, the source of these fragments may be from apoptotic bodies (Apo-Bodies) created from dying cells. Trophoblast apoptosis is essential for normal placental development, given the enormous amount of proliferation, differentiation, and migration during pregnancy. Through flow cytometric analysis coupled with real-time PCR, our lab has shown that aggregates of acridine orange (AO)-stained material (apoptotic particles) are resistant to DNase treatment, disrupted by sodium dodecyl sulfate (SDS), and contain fetal DNA. Because the placenta continuously remodels in an hypoxic environment, our hypothesis is that fetal DNA in maternal plasma comes from hypoxia-induced dying trophoblasts and that this DNA circulates predominately in the form of Apo-Bodies. We have developed a model culture system for analysis of Apo-Bodies derived from JEG-3 cells, an extravillous trophoblastic cell line, undergoing various methods of cell death: hypoxia-induced, etoposide-induced, and heat stress (necrosis like)–induced cell death. Under conditions of similar propidium iodide (PI) uptake, suggesting comparable levels of death, both hypoxia- and etoposide-induced Apo-Bodies increase in concentration over time, whereas heat-induced levels of particles remain fairly constant, indicating that production of DNA-associated Apo-Bodies is a continuous process. Hypoxia, which is likely to be responsible for trophoblast cell death in vivo, produced membrane-bound Apo-Bodies containing DNA. Our results are consistent with the characteristics of membrane-bound particles containing fetal DNA found in maternal plasma. [ABSTRACT FROM AUTHOR]

Objective: The purpose of this study was to determine the association between first-trimester trisomy 21 screening markers (free human chorionic gonadotropin-β [hCG], pregnancyassociated plasma protein A [PAPP-A], and nuchal translucency) and adverse pregnancy outcome. Study design: This was a cohort study of 8012 patients enrolled in a National Institute of Child Health and Human Development-sponsored study of first-trimester trisomy 21 and 18 screening. Trisomy 21 and 18 risk results and individual marker levels in unaffected pregnancies and pregnancies with adverse outcomes were evaluated. Results: PAPP-A < 1st percentile (OR 5.4, 95% CI 2.8-10.3) and PAPP-A <5th percentile (OR 2.7, 95% CI 1.9-3.9) and free β-hCG < 1st percentile (OR 2.7, 95% CI 1.3-5.9) were associated with increased risk of intrauterine growth restriction (IUGR) with positive predictive values of 24.1%, 14.1%, and 14.3%, respectively. PAPP-A <5th percentile (OR 2.3 95% CI 1.1-4.7) and nuchal translucency >99th percentile (OR 3.5, 95% CI 1.1-11.3) were associated with increased risk of preterm delivery before 34 weeks. Increased risk at screening for trisomy 21 and 18 identified 16 of the 29 other chromosomal abnormalities (55%). Low free β-hCG, Iow PAPP-A, and increased nuchal translucency were all associated with an increased rate of fetal abnormality. Conclusion: Extreme values of first-trimester free β-hCG, PAPP-A, and nuchal translucency are all associated with adverse outcomes. The especially high predictive value for IUGR of PAPP-A levels below the 1st percentile suggests that patients within this group may benefit from increased surveillance for this condition. [ABSTRACT FROM AUTHOR]

The author offers insights on the potential of analyzing cell-free fetal DNA for a definitive diagnosis of chromosomal abnormalities. Traditionally, the invasive diagnostic procedures for chromosomal abnormalities include chorionic villus sampling and amniocentesis. One technique used for determining trisomic pregnancies is the massive parallel genomic sequencing. Based on several studies conducted, it was revealed that cell-free fetal DNA is valid for detecting trisomy 21.

Background: Screening for aneuploid pregnancies is routinely performed after 15 weeks of gestation and has a sensitivity of approximately 65 percent, with a false positive rate of 5 percent. First-trimester markers of aneuploidy have been developed, but their use in combination has not been adequately evaluated in clinical practice. Methods: We conducted a multicenter study of screening for trisomies 21 and 18 among patients with pregnancies between 74 and 97 days of gestation, based on maternal age, maternal levels of free β human chorionic gonadotropin and pregnancy-associated plasma protein A, and ultrasonographic measurement of fetal nuchal translucency. A screening result was considered to be positive for trisomy 21 if the calculated risk was at least 1 in 270 pregnancies and positive for trisomy 18 if the risk was at least 1 in 150. Results: Screening was completed in 8514 patients with singleton pregnancies. This approach to screening identified 85.2 percent of the 61 cases of Down's syndrome (95 percent confidence interval, 73.8 to 93.0), with a false positive rate of 9.4 percent (95 percent confidence interval, 8.8 to 10.1). At a false positive rate of 5 percent, the detection rate was 78.7 percent (95 percent confidence interval, 66.3 to 88.1). Screening identified 90.9 percent of the 11 cases of trisomy 18 (95 percent confidence interval, 58.7 to 99.8), with a 2 percent false positive rate. Among women 35 years of age or older, screening identified 89.8 percent of fetuses with trisomy 21, with a false positive rate of 15.2 percent, and 100 percent of fetuses with trisomy 18. Conclusions: First-trimester screening for trisomies 21 and 18 on the basis of maternal age, maternal levels of free β human chorionic gonadotropin and pregnancy-associated plasma protein A, and measurement of fetal nuchal translucency has good sensitivity at an acceptable false positive rate. N Engl J Med 2003;349:1405-13. [ABSTRACT FROM AUTHOR]

Objective: To determine whether preimplantation genetic diagnosis (PGD) and transfer of euploid embryos would decrease spontaneous abortion rates in recurrent miscarriage (RM) patients. Design: Controlled clinical study. Setting: In vitro fertilization centers and PGD reference laboratory. Patient(s): Recurrent-miscarriage patients with three or more prior lost pregnancies with no known etiology. Intervention(s): Biopsy of a single blastomere from each day 3 embryo, followed by fluorescence in situ hybridization analysis. Main Outcome Measure(s): The rate of spontaneous abortions in RM subjects undergoing PGD were compared with [1] their own a priori expectations and [2] a comparison group of women undergoing PGD for advanced maternal age (?35 years). Result(s): Before PGD, RM patients had lost 87% (262/301) of their pregnancies, with an expected loss rate of 36.5%. After, they only lost 16.7% pregnancies. This difference was mostly due to reduction in pregnancy loss in the ?35-years age subgroup, to 12% from an expected 44.5%. Conclusion(s): Preimplantation genetic diagnosis aneuploidy screening has a beneficial effect on pregnancy outcome in RM couples, especially those in which the woman is aged ?35 years. Our data indicate that PGD reduces the risk of miscarriage in RM patients to baseline levels. [Copyright &y& Elsevier]

Several letters to the editor are presented in response to the article "Preventing Preterm Births: Analysis of Trends and Potential Reductions With Interventions in 39 Countries With Very High Human Development Index," by Hannah H. Chang and colleagues in a 2013 issue.

We screened growth differentiation factor 9 coding regions for mutations in a Chinese sample of 100 women with premature ovarian failure and discovered four novel single-nucleotide polymorphisms: c.436C>T (p.Arg146Cys), c.588A>C (silent), c.712A>G (p.Thr238Ala), and c.1283G>C (p.Ser428Thr). Nonsynonymous single-nucleotide polymorphisms c.436C>T and c.1283G>C were also detected in the control population. The c.712A>G perturbation results in a missense mutation (p.Thr238Ala) and was not present in any of 96 controls. Substitution of the hydrophobic amino acid residue alanine for hydrophilic threonine may disrupt growth differentiation factor 9 function. [Copyright &y& Elsevier]

Objective: To review a 12-year experience of the world''s three largest preimplantation genetic diagnosis (PGD) centers.Design: Multicenter analysis of the clinical outcome of PGD.Setting: In vitro fertilization programs at the Reproductive Genetics Institute, Chicago, Illinois; Saint Barnabas Medical Center, West Orange, New Jersey; and SISMER, Bologna, Italy.Patient(s): Poor-prognosis IVF patients, patients carrying balanced chromosomal translocations, and couples at risk for producing children with Mendelian disorders.Intervention(s): In vitro fertilization, intracytoplasmic sperm injection, polar body removal, blastomere biopsy, and ET.Main outcome measure(s): DNA or chromosomal analysis of biopsied polar bodies or blastomeres, implantation and clinical pregnancy rates, and live-born pregnancy outcome.Result(s): A total of 754 babies have been born as a result of 4,748 PGD attempts, which shows the expanded application and the practical relevance of PGD for single-gene disorders, chromosomal aneuploidies and translocations, late-onset diseases with genetic predisposition, and nondisease testing in couples at need for human leukocyte antigens-matched offspring for treatment of affected siblings.Conclusion(s): Preimplantation genetic diagnosis is evolving to become a clinical option for couples at risk for producing offspring with Mendelian diseases, has a positive numerical impact in standard assisted reproduction practices through aneuploidy testing, and reduces by at least fourfold the spontaneous abortion rate in couples carrying translocations. [Copyright &y& Elsevier]