Objective To review clinical validation or implementation studies of maternal blood cell-free (cf) DNA analysis and define the performance of screening for fetal trisomies 21, 18 and 13 and sex chromosome aneuploidies. Methods Searches of PubMed, EMBASE and The Cochrane Library were performed to identify all peer-reviewed articles on cfDNA testing in screening for aneuploidies between January 2011, when the first such study was published, and 4 January 2015. Results In total, 37 relevant studies were identified and these were used for the meta-analysis on the performance of cfDNA testing in screening for aneuploidies. These studies reported cfDNA results in relation to fetal karyotype from invasive testing or clinical outcome. Weighted pooled detection rates (DR) and false-positive rates (FPR) in singleton pregnancies were 99.2% (95% CI, 98.5–99.6%) and 0.09% (95% CI, 0.05–0.14%), respectively, for trisomy 21, 96.3% (95% CI, 94.3–97.9%) and 0.13% (95% CI, 0.07–0.20) for trisomy 18, 91.0% (95% CI, 85.0–95.6%) and 0.13% (95% CI, 0.05–0.26%) for trisomy 13, 90.3% (95% CI, 85.7–94.2%) and 0.23% (95% CI, 0.14–0.34%) for monosomy X and 93.0% (95% CI, 85.8–97.8%) and 0.14% (95% CI, 0.06–0.24%) for sex chromosome aneuploidies other than monosomy X. For twin pregnancies, the DR for trisomy 21 was 93.7% (95% CI, 83.6–99.2%) and the FPR was 0.23% (95% CI, 0.00–0.92%). Conclusion Screening for trisomy 21 by analysis of cfDNA in maternal blood is superior to that of all other traditional methods of screening, with higher DR and lower FPR. The performance of screening for trisomies 18 and 13 and sex chromosome aneuploidies is considerably worse than that for trisomy 21. Copyright © 2015 ISUOG. Published by John Wiley & Sons Ltd.

https://obgyn.onlinelibrary.wiley.com/doi/pdf/10.1002/uog.17484

Analysis of cell‐free DNA in maternal blood in screening for fetal aneuploidies: updated meta‐analysis

In this prospective, multicenter, blinded study conducted at 35 international centers, we assigned pregnant women presenting for aneuploidy screening at 10 to 14 weeks of gestation to undergo both standard screening (with measurement of nuchal translucency and biochemical analytes) and cfDNA testing. Participants received the results of standard screening; the results of cfDNA testing were blinded. Determination of the birth outcome was based on diagnostic genetic testing or newborn examination. The primary outcome was the area under the receiver-operatingcharacteristic curve (AUC) for trisomy 21 (Down’s syndrome) with cfDNA testing versus standard screening. We also evaluated cfDNA testing and standard screening to assess the risk of trisomies 18 and 13. Results Of 18,955 women who were enrolled, results from 15,841 were available for analysis. The mean maternal age was 30.7 years, and the mean gestational age at testing was 12.5 weeks. The AUC for trisomy 21 was 0.999 for cfDNA testing and 0.958 for standard screening (P = 0.001). Trisomy 21 was detected in 38 of 38 women (100%; 95% confidence interval [CI], 90.7 to 100) in the cfDNA-testing group, as compared with 30 of 38 women (78.9%; 95% CI, 62.7 to 90.4) in the standard-screening group (P = 0.008). False positive rates were 0.06% (95% CI, 0.03 to 0.11) in the cfDNA group and 5.4% (95% CI, 5.1 to 5.8) in the standard-screening group (P<0.001). The positive predictive value for cfDNA testing was 80.9% (95% CI, 66.7 to 90.9), as compared with 3.4% (95% CI, 2.3 to 4.8) for standard screening (P<0.001). Conclusions In this large, routine prenatal-screening population, cfDNA testing for trisomy 21 had higher sensitivity, a lower false positive rate, and higher positive predictive value than did standard screening with the measurement of nuchal translucency and biochemical analytes. (Funded by Ariosa Diagnostics and Perinatal Quality Foundation; NEXT ClinicalTrials.gov number, NCT01511458.)

/pdf/cell-free-dna-analysis-for-noninvasive-examination-of-1hjukc4445.pdf

Cell-free DNA Analysis for Noninvasive Examination of Trisomy

Noninvasive prenatal screening for fetal aneuploidy, 2016 update: a position statement of the American College of Medical Genetics and Genomics.

To review clinical validation or implementation studies of maternal blood cell‐free (cf) DNA analysis and define the performance of screening for fetal trisomies 21, 18 and 13 and sex chromosome aneuploidies (SCA).

Analysis of cell-free DNA in maternal blood in screening for aneuploidies: updated meta-analysis: Cell-free DNA in screening for aneuploidies

Objective
To determine the effects of gestational age and maternal weight on percent fetal cell-free DNA (cfDNA) in maternal plasma and the change in fetal cfDNA amounts within the same patient over time

Methods
The cfDNA was extracted from maternal plasma from 22 384 singleton pregnancies of at least 10 weeks gestation undergoing the HarmonyTM Prenatal Test The Harmony Prenatal Test determined fetal percentage via directed analysis of cfDNA

Results
At 10 weeks 0 days to 10 weeks 6 days gestation, the median percent fetal cfDNA was 102% Between 10 and 21 weeks gestation, percent fetal increased 01% per week (p < 00001), and 2% of pregnancies were below 4% fetal cfDNA Beyond 21 weeks gestation, fetal cfDNA increased 1% per week (p < 00001) Fetal cfDNA percentage was proportional to gestational age and inversely proportional to maternal weight (p = 00016) Of 135 samples that were redrawn because of insufficient fetal cfDNA of the initial sample, 76 (56%) had greater than 4% fetal cfDNA in the sample from the second draw

Conclusion
Fetal cfDNA increases with gestation, decreases with increasing maternal weight, and generally improves upon a blood redraw when the first attempt has insufficient fetal cfDNA © 2013 John Wiley & Sons, Ltd

/pdf/gestational-age-and-maternal-weight-effects-on-fetal-cell-2gz6gb59aj.pdf

Gestational age and maternal weight effects on fetal cell‐free DNA in maternal plasma

Objective This study aims to develop a noninvasive prenatal test on the basis of the analysis of cell-free DNA in maternal blood to detect fetal aneuploidy at chromosomes 13, 18, 21, X, and Y. Methods A total of 166 samples from pregnant women, including 11 trisomy 21, three trisomy 18, two trisomy 13, two 45, X, and two 47,XXY samples, were analyzed using an informatics-based method. Cell-free DNA from maternal blood was isolated, amplified using a multiplex polymerase chain reaction (PCR) assay targeting 11000 single nucleotide polymorphisms on chromosomes 13, 18, 21, X, and Y in a single reaction, and sequenced. A Bayesian-based maximum likelihood statistical method was applied to determine the chromosomal count of the five chromosomes interrogated in each sample, along with a sample-specific calculated accuracy for each test result. Results The algorithm correctly reported the chromosome copy number at all five chromosomes in 145 samples that passed a DNA quality test, for a total of 725/725 correct calls. The average calculated accuracy for these samples was 99.92%. Twenty-one samples did not pass the DNA quality test. Conclusions This informatics-based method noninvasively detected fetuses with trisomy 13, 18, and 21, 45,X, and 47, XXY with high sample-specific calculated accuracies for each individual chromosome and across all five chromosomes. © 2012 John Wiley & Sons, Ltd.

http://www.prenataltest.net/images2/ASHG2012.pdf

Noninvasive prenatal aneuploidy testing of chromosomes 13, 18, 21, X, and Y, using targeted sequencing of polymorphic loci.

http://journals.lww.com/greenjournal/Documents/Aug2014_JournalClub_Pergament.pdf

Single-nucleotide polymorphism-based noninvasive prenatal screening in a high-risk and low-risk cohort.

The invention provides methods for simultaneously amplifying multiple nucleic acid regions of interest in one reaction volume as well as methods for selecting a library of primers for use in such amplification methods. The invention also provides library of primers with desirable characteristics, such as minimal formation of amplified primer dimers or other non-target amplicons.

Highly multiplex pcr methods and compositions

Methods for simultaneous amplification of target loci

/pdf/session-16-innovations-in-reproductive-genetics-32iwjdto7e.pdf

Michael Dodd

Papers

Noninvasive prenatal aneuploidy testing of chromosomes 13, 18, 21, X, and Y, using targeted sequencing of polymorphic loci.

Single-nucleotide polymorphism-based noninvasive prenatal screening in a high-risk and low-risk cohort.

Highly multiplex pcr methods and compositions

Methods for simultaneous amplification of target loci

Session 16: Innovations in reproductive genetics