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

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

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.

Most human pre-implantation embryos are mosaics of euploid and aneuploid cells. To determine the fate of aneuploid cells and the developmental potential of mosaic embryos, here we generate a mouse model of chromosome mosaicism. By treating embryos with a spindle assembly checkpoint inhibitor during the four- to eight-cell division, we efficiently generate aneuploid cells, resulting in embryo death during peri-implantation development. Live-embryo imaging and single-cell tracking in chimeric embryos, containing aneuploid and euploid cells, reveal that the fate of aneuploid cells depends on lineage: aneuploid cells in the fetal lineage are eliminated by apoptosis, whereas those in the placental lineage show severe proliferative defects. Overall, the proportion of aneuploid cells is progressively depleted from the blastocyst stage onwards. Finally, we show that mosaic embryos have full developmental potential, provided they contain sufficient euploid cells, a finding of significance for the assessment of embryo vitality in the clinic.

/pdf/mouse-model-of-chromosome-mosaicism-reveals-lineage-specific-35jexl5y6n.pdf

Mouse model of chromosome mosaicism reveals lineage-specific depletion of aneuploid cells and normal developmental potential

Aneuploidy has been well-documented in blastocyst embryos, but prior studies have been limited in scale and/or lack mechanistic data. We previously reported preclinical validation of microarray 24-chromosome preimplantation genetic screening in a 24-h protocol. The method diagnoses chromosome copy number, structural chromosome aberrations, parental source of aneuploidy and distinguishes certain meiotic from mitotic errors. In this study, our objective was to examine aneuploidy in human blastocysts and determine correspondence of karyotypes between trophectoderm (TE) and inner cell mass (ICM). We disaggregated 51 blastocysts from 17 couples into ICM and one or two TE fractions. The average maternal age was 31. Next, we ran 24-chromosome microarray molecular karyotyping on all of the samples, and then performed a retrospective analysis of the data. The average per-chromosome confidence was 99.95%. Approximately 80% of blastocysts were euploid. The majority of aneuploid embryos were simple aneuploid, i.e. one or two whole-chromosome imbalances. Structural chromosome aberrations, which are common in cleavage stage embryos, occurred in only three blastocysts (5.8%). All TE biopsies derived from the same embryos were concordant. Forty-nine of 51 (96.1%) ICM samples were concordant with TE biopsies derived from the same embryos. Discordance between TE and ICM occurred only in the two embryos with structural chromosome aberration. We conclude that TE karyotype is an excellent predictor of ICM karyotype. Discordance between TE and ICM occurred only in embryos with structural chromosome aberrations.

Comprehensive analysis of karyotypic mosaicism between trophectoderm and inner cell mass

Disclosed herein are methods for determining the copy number of a chromosome in a fetus in the context of non-invasive prenatal diagnosis. In an embodiment, the measured genetic data from a sample of genetic material that contains both fetal DNA and maternal DNA is analyzed, along with the genetic data from the biological parents of the fetus, and the copy number of the chromosome of interest is determined. In an embodiment, the maternal serum is measured using a single-nucleotide polymorphism (SNP) microarray, along with parental genomic data, and the determination of the chromosome copy number is used to make clinical decisions pertaining to the fetus.

Methods for non-invasive prenatal ploidy calling

Origins and rates of aneuploidy in human blastomeres.

Purpose 
The metaphase karyotype is often used as a diagnostic tool in the setting of early miscarriage; however this technique has several limitations. We evaluate a new technique for karyotyping that uses single nucleotide polymorphism microarrays (SNP). This technique was compared in a blinded, prospective fashion, to the traditional metaphase karyotype.


Methods 
Patients undergoing dilation and curettage for first trimester miscarriage between February and August 2010 were enrolled. Samples of chorionic villi were equally divided and sent for microarray testing in parallel with routine cytogenetic testing.


Results 
Thirty samples were analyzed, with only four discordant results. Discordant results occurred when the entire genome was duplicated or when a balanced rearrangement was present. Cytogenetic karyotyping took an average of 29 days while microarray-based karytoyping took an average of 12 days.


Conclusions 
Molecular karyotyping of POC after missed abortion using SNP microarray analysis allows for the ability to detect maternal cell contamination and provides rapid results with good concordance to standard cytogenetic analysis.

/pdf/informatics-enhanced-snp-microarray-analysis-of-30-1t22s6s3ky.pdf

George Gemelos

Papers

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

Comprehensive analysis of karyotypic mosaicism between trophectoderm and inner cell mass

Methods for non-invasive prenatal ploidy calling

Origins and rates of aneuploidy in human blastomeres.

Informatics enhanced SNP microarray analysis of 30 miscarriage samples compared to routine cytogenetics.