Factors affecting levels of circulating cell-free fetal DNA in maternal plasma and their implications for noninvasive prenatal testing.

OBJECTIVE: Sufficient fetal DNA in a maternal plasma sample is required for accurate aneuploidy detection via noninvasive prenatal testing, thus highlighting a need to understand the factors affecting fetal fraction. METHOD: The MaterniT21™ PLUS test uses massively parallel sequencing to analyze cell-free fetal DNA in maternal plasma and detect chromosomal abnormalities. We assess the impact of a variety of factors, both maternal and fetal, on the fetal fraction across a large number of samples processed by Sequenom Laboratories. RESULTS: The rate of increase in fetal fraction with increasing gestational age varies across the duration of the testing period and is also influenced by fetal aneuploidy status. Maternal weight trends inversely with fetal fraction, and we find no added benefit from analyzing body mass index or blood volume instead of weight. Strong correlations exist between fetal fractions from aliquots taken from the same patient at the same blood draw and also at different blood draws. CONCLUSION: While a number of factors trend with fetal fraction across the cohort as a whole, they are not the sole determinants of fetal fraction. In this study, the variability for any one patient does not appear large enough to justify postponing testing to a later gestational age.

Maternal plasma DNA testing for aneuploidy in pregnancies achieved by assisted reproductive technologies.

PURPOSE: We sought to compare measurements of circulating cell-free DNA as well as Down syndrome test results in women with naturally conceived pregnancies with those conceived using assisted reproductive technologies. METHODS: Data regarding assisted reproductive technologies were readily available from seven enrollment sites participating in an external clinical validation trial of nested case/control design. Measurements of circulating cell-free fetal and total DNA, fetal fraction (ratio of fetal to total DNA), chromosome-specific z-scores, and karyotype results were available for analysis. RESULTS: Analyses were restricted to 632 euploid (5.2% assisted reproductive technologies) and 73 Down syndrome (13.7% assisted reproductive technologies), including 16 twin pregnancies. No differences were found for fetal or total circulating cell-free DNA, or for the fetal fraction in euploid (P = 0.70) or Down syndrome (P = 0.58) pregnancies by method of conception. There appeared to be systematic z-score reductions for chromosomes 21, 18, and 13 in assisted reproductive technologies versus natural euploid pregnancies (P = 0.048, 0.0032, and 0.36, respectively). CONCLUSION: Assisted reproductive technologies and naturally conceived pregnancies contribute similar levels of circulating cell-free DNA into maternal circulation. Small differences in the z-scores of pregnancies achieved by assisted reproductive technologies were observed and do not appear to be test-related artifacts. However, the findings need confirmation before any consideration of changes to testing and reporting protocols.

Noninvasive prenatal detection of aneuploidy.

Noninvasive prenatal testing (NIPT) uses cell-free fetal DNA from the plasma of pregnant women to provide valuable information about the potential risks for fetal aneuploidy. This article provides a historical overview of both invasive diagnostic testing and serum screening approaches, both biochemical and the newer molecular noninvasive prenatal testing assays, used to identify patients who would be best served by invasive testing.

Noninvasive prenatal detection of sex chromosomal aneuploidies by sequencing circulating cell-free DNA from maternal plasma.

OBJECTIVE: Whole-genome sequencing of circulating cell free (ccf) DNA from maternal plasma has enabled noninvasive prenatal testing for common autosomal aneuploidies. The purpose of this study was to extend the detection to include common sex chromosome aneuploidies (SCAs): [47,XXX], [45,X], [47,XXY], and [47,XYY] syndromes. METHOD: Massively parallel sequencing was performed on ccf DNA isolated from the plasma of 1564 pregnant women with known fetal karyotype. A classification algorithm for SCA detection was constructed and trained on this cohort. Another study of 411 maternal samples from women with blinded-to-laboratory fetal karyotypes was then performed to determine the accuracy of the classification algorithm. RESULTS: In the training cohort, the new algorithm had a detection rate (DR) of 100% (95%CI: 82.3%, 100%), a false positive rate (FPR) of 0.1% (95%CI: 0%, 0.3%), and nonreportable rate of 6% (95%CI: 4.9%, 7.4%) for SCA determination. The blinded validation yielded similar results: DR of 96.2% (95%CI: 78.4%, 99.8%), FPR of 0.3% (95%CI: 0%, 1.8%), and nonreportable rate of 5% (95%CI: 3.2%, 7.7%) for SCA determination CONCLUSION: Noninvasive prenatal identification of the most common sex chromosome aneuploidies is possible using ccf DNA and massively parallel sequencing with a high DR and a low FPR.

DNA sequencing of maternal plasma reliably identifies trisomy 18 and trisomy 13 as well as Down syndrome: an international collaborative study.

PURPOSE: To determine whether maternal plasma cell-free DNA sequencing can effectively identify trisomy 18 and 13. METHODS: Sixty-two pregnancies with trisomy 18 and 12 with trisomy 13 were selected from a cohort of 4,664 pregnancies along with matched euploid controls (including 212 additional Down syndrome and matched controls already reported), and their samples tested using a laboratory-developed, next-generation sequencing test. Interpretation of the results for chromosome 18 and 13 included adjustment for CG content bias. RESULTS: Among the 99.1% of samples interpreted (1,971/1,988), observed trisomy 18 and 13 detection rates were 100% (59/59) and 91.7% (11/12) at false-positive rates of 0.28% and 0.97%, respectively. Among the 17 samples without an interpretation, three were trisomy 18. If z-score cutoffs for trisomy 18 and 13 were raised slightly, the overall false-positive rates for the three aneuploidies could be as low as 0.1% (2/1,688) at an overall detection rate of 98.9% (280/283) for common aneuploidies. An independent academic laboratory confirmed performance in a subset. CONCLUSION: Among high-risk pregnancies, sequencing circulating cell-free DNA detects nearly all cases of Down syndrome, trisomy 18, and trisomy 13, at a low false-positive rate. This can potentially reduce invasive diagnostic procedures and related fetal losses by 95%. Evidence supports clinical testing for these aneuploidies.

DNA sequencing of maternal plasma to identify Down syndrome and other trisomies in multiple gestations.

OBJECTIVE: Studies on prenatal testing for Down syndrome (trisomy 21), trisomy 18, and trisomy 13 by massively parallel shotgun sequencing (MPSS) of circulating cell free DNA have been, for the most part, limited to singleton pregnancies. If MPSS testing is offered clinically, it is important to know if these trisomies will also be identified in multiple pregnancies. METHOD: Among a cohort of 4664 high-risk pregnancies, maternal plasma samples were tested from 25 twin pregnancies (17 euploid, five discordant and two concordant for Down syndrome; one discordant for trisomy 13) and two euploid triplet pregnancies [Correction made here after initial online publication.]. Results were corrected for GC content bias. For each target chromosome (21, 18, and 13), z-scores of 3 or higher were considered consistent with trisomy. RESULTS: Seven twin pregnancies with Down syndrome, one with trisomy 13, and all 17 twin euploid pregnancies were correctly classified [detection rate 100%, 95% confidence interval (CI) 59%-100%, false positive rate 0%, 95% CI 0%-19.5%], as were the two triplet euploid pregnancies. CONCLUSION: Although study size is limited, the underlying biology combined with the present data provide evidence that MPSS testing can be reliably used as a secondary screening test for Down syndrome in women with high-risk twin gestations.

Performance of a Multianalyte 'Rule-Out' Assay in Pregnant Individuals With Suspected Preeclampsia.

BACKGROUND: The ability to diagnose preeclampsia clinically is suboptimal. Our objective was to validate a novel multianalyte assay and characterize its performance, when intended for use as an aid to rule-out preeclampsia. METHODS: Prospective, multicenter cohort study of pregnant individuals presenting between 280/7 and 366/7 weeks' with preeclampsia-associated signs and symptoms. Individuals not diagnosed with preeclampsia after baseline evaluation were enrolled in the study cohort, with those who later developed preeclampsia, classified as cases and compared with a negative control group who did not develop preeclampsia. Individuals with assay values at time of enrollment ≥0.0325, determined using a previously developed algorithm, considered at risk. The primary analysis was the time to develop preeclampsia assessed using a multivariate Cox regression model. RESULTS: One thousand thirty-six pregnant individuals were enrolled in the study cohort with an incidence of preeclampsia of 30.3% (27.6%-33.2%). The time to develop preeclampsia was shorter for those with an at-risk compared with negative assay result (log-rank P<0.0001; adjusted hazard ratio of 4.81 [3.69-6.27, P<0.0001]). The performance metrics for the assay to rule-out preeclampsia within 7 days of enrollment showed a sensitivity 76.4% (67.5%-83.5%), negative predictive value 95.0% (92.8%-96.6%), and negative likelihood ratio 0.46 (0.32-0.65). Assay performance improved if delivery occurred <37 weeks and for individuals enrolled between 28 and 35 weeks. CONCLUSIONS: We confirmed that a novel multianalyte assay was associated with the time to develop preeclampsia and has a moderate sensitivity and negative likelihood ratio but high negative predictive value when assessed as an aid to rule out preeclampsia within 7 days of enrollment. REGISTRATION: The study was registered on Clinicaltrials.gov (Identifier NCT02780414).

DNA sequencing of maternal plasma to detect Down syndrome: an international clinical validation study.

PURPOSE: Prenatal screening for Down syndrome has improved, but the number of resulting invasive diagnostic procedures remains problematic. Measurement of circulating cell-free DNA in maternal plasma might offer improvement. METHODS: A blinded, nested case-control study was designed within a cohort of 4664 pregnancies at high risk for Down syndrome. Fetal karyotyping was compared with an internally validated, laboratory-developed test based on next-generation sequencing in 212 Down syndrome and 1484 matched euploid pregnancies. None had been previously tested. Primary testing occurred at a CLIA-certified commercial laboratory, with cross validation by a CLIA-certified university laboratory. RESULTS: Down syndrome detection rate was 98.6% (209/212), the false-positive rate was 0.20% (3/1471), and the testing failed in 13 pregnancies (0.8%); all were euploid. Before unblinding, the primary testing laboratory also reported multiple alternative interpretations. Adjusting chromosome 21 counts for guanine cytosine base content had the largest impact on improving performance. CONCLUSION: When applied to high-risk pregnancies, measuring maternal plasma DNA detects nearly all cases of Down syndrome at a very low false-positive rate. This method can substantially reduce the need for invasive diagnostic procedures and attendant procedure-related fetal losses. Although implementation issues need to be addressed, the evidence supports introducing this testing on a clinical basis.

Fetal RHD genotype detection from circulating cell-free fetal DNA in maternal plasma in non-sensitized RhD negative women.

OBJECTIVE: To examine the performance of the SensiGene Fetal RHD Genotyping Laboratory Developed Test (RHD Genotyping LDT) using circulating cell-free fetal DNA (ccff DNA) extracted from maternal plasma. METHODS: ccff DNA was extracted from maternal blood from non-sensitized women with singleton pregnancies in two cohorts, one with a serotype reference (11-13 weeks' gestation) and one with the reference source (6-30 weeks' gestation). The presence of three RHD exon sequences (exons 4, 5, 7), the psi-pseudogene, three Y-chromosome sequences (SRY, DBY and TTTY2), and the X/Y-chromosome TGIF gene control were determined using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry-the RHD Genotyping LDT. RESULTS: The cohort with a serotype RhD reference showed correct classification in 201 of 207 patients, a test accuracy of 97.1%, with a sensitivity and specificity for prediction of RhD serotype of 97.2 and 96.8%, respectively. The cohort with a genotype RHD reference showed correct classification in 198 of 199 patients, indicating a test accuracy of 99.5% with a sensitivity and specificity for prediction of RHD genotype of 100.0 and 98.3%, respectively. CONCLUSION: Fetal RHD genotyping can accurately be determined using ccff DNA in the first and second trimesters of pregnancy.

Fetal sex determination using circulating cell-free fetal DNA (ccffDNA) at 11 to 13 weeks of gestation.

OBJECTIVE: To examine the performance of a mass spectrometry-based detection platform using three Y-chromosome sequences for fetal sex determination from circulating cell-free fetal DNA (ccffDNA) in maternal blood in the first trimester of pregnancy. METHODS: We extracted ccffDNA for the determination of fetal sex from stored maternal plasma obtained at 11 to 13 weeks' gestation from singleton pregnancies with documented fetal gender. Mass spectrometry was used to examine 236 specimens for the presence of three Y-chromosome sequences (SRY, DBY and TTTY2). The sample was classified as male, female or inconclusive depending on the detection of three, one/none and two sequences, respectively. RESULTS: Three (1.3%) of the 236 cases were classified as invalid due to the absence of a well-defined spectral peak for TGIF and 22 (9.3%) were reported as inconclusive. In the 211 cases with a valid result, the fetal sex was correctly identified in 90 of 91 male babies and 119 of 120 female babies giving an accuracy of 99.1% and sensitivity and specificity for prediction of male fetuses of 98.9 and 99.2%, respectively. CONCLUSION: Fetal sex determination can be accurately determined from maternal ccffDNA in the first trimester of pregnancy using mass spectrometry analysis.

A randomized clinical trial comparing the effect of maternal intravenous hydration and placebo on the amniotic fluid index in oligohydramnios.

OBJECTIVE: To compare the treatment of acute intravenous hydration with placebo in term pregnant women manifesting oligohydramnios. METHODS: All patients with oligohydramnios who met the inclusion criteria were offered participation in this randomized, double-blind, placebo-controlled study. After ruling out rupture of membranes and maternal and fetal complications, we recruited 44 women with third trimester singleton pregnancies having an amniotic fluid index (AFI) of less than 6. Patients were randomized into treatment or control groups. Patients in the treatment group received a continuous intravenous infusion of (1/2) normal saline (NS) at a rate of 1000 mL/h for two hours. Patients in the placebo group received an intravenous infusion of (1/2) NS at 10 mL/h for two hours. The AFI was re-assessed by the same sonographer one hour after the hydration was completed. Both the patient and the examiner were blinded to the study groups. RESULTS: Maternal age, parity, gestational age, and birth weight were not significantly different between the two groups. The AFI increased significantly in both treatment (3.8 +/- 1.2 vs. 5.3 +/- 2.5, p < 0.05) and placebo (4 +/- 1.3 vs. 5.5 +/- 2.4, p < 0.05) groups. Moreover, the changes in AFI did not significantly differ between the treatment and the placebo groups (1.2 +/- 2.1 vs. 1.5 +/- 2.1, respectively; p > 0.05). CONCLUSIONS: There are statistically significant short-term increases in the AFI in patients with oligohydramnios whether the patients are treated with intravenous fluids or not. The short-term increase in AFI may reflect physiologic diurnal variations in the amniotic fluid.

Prenatal screening for open neural tube defects.

The era of prenatal screening for serious birth defects began in the 1970s with the discovery that amniotic fluid and maternal serum levels of alpha-fetoprotein (AFP) were increased in pregnancies affected by fetal open neural tube defects. Since then, prenatal screening has become a part of routine obstetric care. In this article, the use of AFP in prenatal screening for open neural tube defects is discussed in the context of the laboratory and the laboratory's interactions with the practicing obstetrician.

Hereditary breast and ovarian cancer: what the primary care physician should know.

In recent years, testing for cancer susceptibility genes has entered the clinical setting. The practicing physician needs to be familiar with this evolving area of medicine to be able to counsel and/or refer high-risk patients such as those with a strong personal or family history of cancer. The following is a review of the clinically pertinent information regarding hereditary breast and ovarian cancers resulting from mutations in BRCA genes. A special emphasis is placed on the different options available for BRCA mutation carriers, because many interventions have already proven to be highly efficacious. The increased risk of cancer seen in hereditary nonpolyposis colorectal cancer (HNPCC) is not part of this review but is mentioned briefly.

Non-invasive prenatal chromosomal aneuploidy testing--clinical experience: 100,000 clinical samples.

OBJECTIVE: As the first laboratory to offer massively parallel sequencing-based noninvasive prenatal testing (NIPT) for fetal aneuploidies, Sequenom Laboratories has been able to collect the largest clinical population experience data to date, including >100,000 clinical samples from all 50 U.S. states and 13 other countries. The objective of this study is to give a robust clinical picture of the current laboratory performance of the MaterniT21 PLUS LDT. STUDY DESIGN: The study includes plasma samples collected from patients with high-risk pregnancies in our CLIA-licensed, CAP-accredited laboratory between August 2012 to June 2013. Samples were assessed for trisomies 13, 18, 21 and for the presence of chromosome Y-specific DNA. Sample data and ad hoc outcome information provided by the clinician was compiled and reviewed to determine the characteristics of this patient population, as well as estimate the assay performance in a clinical setting. RESULTS: NIPT patients most commonly undergo testing at an average of 15 weeks, 3 days gestation; and average 35.1 years of age. The average turnaround time is 4.54 business days and an overall 1.3% not reportable rate. The positivity rate for Trisomy 21 was 1.51%, followed by 0.45% and 0.21% rate for Trisomies 18 and 13, respectively. NIPT positivity rates are similar to previous large clinical studies of aneuploidy in women of maternal age ≥ 35 undergoing amniocentesis. In this population 3519 patients had multifetal gestations (3.5%) with 2.61% yielding a positive NIPT result. CONCLUSION: NIPT has been commercially offered for just over 2 years and the clinical use by patients and clinicians has increased significantly. The risks associated with invasive testing have been substantially reduced by providing another assessment of aneuploidy status in high-risk patients. The accuracy and NIPT assay positivity rate are as predicted by clinical validations and the test demonstrates improvement in the current standard of care.

Prenatal diagnosis of 47,XXX.

We report 2 cases of 47,XXX that were diagnosed prenatally and were screened positive for trisomy 21 by biochemical and ultrasound markers. These cases underline the importance of discussing the sex chromosome abnormalities during the genetic counseling after an abnormal triple screen test or ultrasound examination.