Elisa Bevilacqua

Concerns with performance of screening for aneuploidy by cell-free DNA analysis of maternal blood in twin pregnancy

In singleton pregnancy, cell-free DNA (cfDNA) analysis of maternal blood has been reported to perform well in screening for trisomies 21, 18 and 13 in both highand low-risk patients1. There are also sufficient data to suggest that cfDNA testing is feasible in twin pregnancy, although it seems to be associated with a higher failure rate than in singleton pregnancy2. Apart from twin pregnancy, other potential causes of failure and discordant results of cfDNA analysis have been identified: fetoplacental mosaicism, maternal chromosomal abnormalities, vanishing twin, maternal weight, conception by in-vitro fertilization (IVF), low fetal DNA fraction and technical errors2,3. Recently, Takoudes and Hamar4 tested five laboratories offering cfDNA analysis by sending samples from two non-pregnant women, stating that they were carrying a singleton fetus. Three of the laboratories reported that the two non-pregnant women were carrying a genetically normal female fetus, raising concerns about the need for quality standards in non-invasive prenatal testing. In order to compare the performance, feasibility and reliability of cfDNA testing in twin pregnancy by various laboratories, we collected blood samples from a 32-year-old woman with a dichorionic twin pregnancy at 17 weeks of gestation (Patient 1) and from a 42-year-old non-pregnant woman (Patient 2) and sent them to two Belgian laboratories and two laboratories outside Belgium offering cfDNA analysis. Patient 1 had an IVF twin pregnancy with a high risk (1 in 4) of trisomy 21 at the first-trimester combined test for Fetus 1 (female) and a low risk (1 in 9761) for Fetus 2 (male). Amniocentesis at 16 weeks of gestation confirmed the presence of trisomy 21 in Fetus 1. We informed the laboratories that both patients were carrying twin pregnancies and that the gestational ages were 17 weeks and 12 weeks, respectively. Sending a sample from a non-pregnant woman to the various laboratories was justified on the basis of the lack of clarity regarding whether laboratories did or did not measure fetal fraction as a whole or for each individual fetus. The cfDNA test results reported by the four laboratories are presented in Table 1. Our results, like those of Takoudes and Hamar4, raise concerns about the quality of laboratory testing. Each laboratory gave a false-negative result for the pregnant woman carrying twins, one of whom presented fetal trisomy 21; thus, no laboratory outperformed the others in terms of test sensitivity. More concerns arise from these false-negatives being obtained despite the fact that fetal fraction values were far above the 4% limit that is often reported as being necessary for successful and reliable analysis. The fact that none of the laboratories detected the affected pregnancy also suggests that there may have been significant technical challenges with the woman’s blood

Comparison of conventional 2D ultrasound to magnetic resonance imaging for prenatal estimation of birthweight in twin pregnancy

BACKGROUND: During prenatal follow‐up of twin pregnancies, accurate identification of birthweight and birthweight discordance is important to identify the high‐risk group and plan perinatal care. Unfortunately, prenatal evaluation of birthweight discordance by 2‐dimensional ultrasound has been far from optimal. OBJECTIVE: The objective of the study was to prospectively compare estimates of fetal weight based on 2‐dimensional ultrasound (ultrasound–estimated fetal weight) and magnetic resonance imaging (magnetic resonance–estimated fetal weight) with actual birthweight in women carrying twin pregnancies. STUDY DESIGN: Written informed consent was obtained for this ethics committee–approved study. Between September 2011 and December 2015 and within 48 hours before delivery, ultrasound–estimated fetal weight and magnetic resonance–estimated fetal weight were conducted in 66 fetuses deriving from twin pregnancies at 34.3–39.0 weeks; gestation. Magnetic resonance–estimated fetal weight derived from manual measurement of fetal body volume. Comparison of magnetic resonance–estimated fetal weight and ultrasound–estimated fetal weight measurements vs birthweight was performed by calculating parameters as described by Bland and Altman. Receiver‐operating characteristic curves were constructed for the prediction of small‐for‐gestational‐age neonates using magnetic resonance–estimated fetal weight and ultrasound–estimated fetal weight. For twins 1 and 2 separately, the relative error or percentage error was calculated as follows: (birthweight – ultrasound–estimated fetal weight (or magnetic resonance–estimated fetal weight)/birthweight) × 100 (percentage). Furthermore, ultrasound–estimated fetal weight, magnetic resonance–estimated fetal weight, and birthweight discordance were calculated as 100 × (larger estimated fetal weight–smaller estimated fetal weight)/larger estimated fetal weight. The ultrasound–estimated fetal weight discordance and the birthweight discordance were correlated using linear regression analysis and Pearsons correlation coefficient. The same was done between the magnetic resonance–estimated fetal weight and birthweight discordance. To compare data, the χ2, McNemar test, Student t test, and Wilcoxon signed rank test were used as appropriate. We used the Fisher r‐to‐z transformation to compare correlation coefficients. RESULTS: The bias and the 95% limits of agreement of ultrasound–estimated fetal weight are 2.99 (–19.17% to 25.15%) and magnetic resonance–estimated fetal weight 0.63 (–9.41% to 10.67%). Limits of agreement were better between magnetic resonance–estimated fetal weight and actual birthweight as compared with the ultrasound–estimated fetal weight. Of the 66 newborns, 27 (40.9%) were of weight of the 10th centile or less and 21 (31.8%) of the fifth centile or less. The area under the receiver‐operating characteristic curve for prediction of birthweight the 10th centile or less by prenatal ultrasound was 0.895 (P < .001; SE, 0.049), and by magnetic resonance imaging it was 0.946 (P < .001; SE, 0.024). Pairwise comparison of receiver‐operating characteristic curves showed a significant difference between the areas under the receiver‐operating characteristic curves (difference, 0.087, P = .049; SE, 0.044). The relative error for ultrasound–estimated fetal weight was 6.8% and by magnetic resonance–estimated fetal weight, 3.2% (P < .001). When using ultrasound–estimated fetal weight, 37.9% of fetuses (25 of 66) were estimated outside the range of ±10% of the actual birthweight, whereas this dropped to 6.1% (4 of 66) with magnetic resonance–estimated fetal weight (P < .001). The ultrasound–estimated fetal weight discordance and the birthweight discordance correlated significantly following the linear equation: ultrasound–estimated fetal weight discordance = 0.03 + 0.91 × birthweight (r = 0.75; P < .001); however, the correlation was better with magnetic resonance imaging: magnetic resonance–estimated fetal weight discordance = 0.02 + 0.81 × birthweight (r = 0.87; P < .001). CONCLUSION: In twin pregnancies, magnetic resonance–estimated fetal weight performed immediately prior to delivery is more accurate and predicts small‐for‐gestational‐age neonates significantly better than ultrasound–estimated fetal weight. Prediction of birthweight discordance is better with magnetic resonance imaging as compared with ultrasound.

Screening for Sex Chromosome Aneuploidy by Cell-Free DNA Testing: Patient Choice and Performance

Objective: To study patient choice regarding testing for sex chromosome aneuploidy (SCA) and the performance of cell-free DNA (cfDNA) screening for SCA. Methods: Patient choice regarding screening for SCA and factors influencing this choice were evaluated in a single center. In a subsequent two-center study, cases that screened positive for SCA were analyzed to determine the positive predictive value (PPV) for each SCA. Results: In all, 1,957 (61.9%) of the 3,162 patients undergoing cfDNA testing opted for SCA screening. Regression analysis demonstrated that independent predictors of a patients decision for SCA were earlier gestational age, spontaneous conception, and cfDNA chosen as a primary method of screening. A total of 161 cases screened positive for SCA and follow-up data were available for 118 (73.3%). Forty-six of the 61 cases of 45,X were false-positive results and 15 were concordant with the fetal karyotype (PPV = 24.6%). Seventeen of the 22 cases of 47,XXX were false positive and 5 concordant (PPV = 22.7%). Eleven of the 30 cases of 47,XXY were false positive and 19 concordant (PPV = 63.3%). All 5 cases of 47,XYY were correctly identified, thus yielding a PPV of 100%. Conclusion: More than half of the patients undergoing cfDNA aneuploidy screening also opted for SCA testing, but they were less likely to do so in the presence of an increased risk of trisomy. SCAs involving the X chromosome had a lower PPV than those involving the Y chromosome.

A Longitudinal Study on Fetal Weight Estimation at Third Trimester of Pregnancy: Comparison of Magnetic Resonance Imaging and 2-D Ultrasound Predictions

Objective: To prospectively compare magnetic resonance (MR) estimation of fetal weight (MR-EFW) performed at third trimester with ultrasound (US) estimation of fetal weight (US-EFW) and actual birth weight, and to evaluate factors influencing fetal growth rate near term. Methods: US-EFW and MR-EFW were calculated at a median of 33.0 and 37.7 weeks of gestation in 37 fetuses and plotted on curve centiles to predict birth weights at 39.3 weeks of gestation. The median absolute relative errors for predicted US-EFW and MR-EFW were calculated. Regression analysis was used to investigate the effect of different variables on fetal growth rate at 35.2 weeks of gestation. Results: The relative error of actual birth weight as predicted by US at 33.0 weeks was significantly higher compared with MR (7.33 vs. 4.11%; p = 0.001). This was also the case for fetal weight predicted by US at 37.7 weeks as compared with MR (6.63 vs. 2.60%; p < 0.01). Fetal growth rate was significantly and independently positively associated with the mothers weight and with gestational age at estimation (p < 0.05 for both variables). Conclusion: Fetal weight estimates predicted using MR at third trimester are better than those given by prenatal US. Fetal growth rate depends on fetal and maternal characteristics.

Prenatal Screening for 22q11.2 Deletion Using a Targeted Microarray-Based Cell-Free DNA Test.

Objective: To determine the performance of a targeted microarray-based cell-free DNA (cfDNA) test (Harmony Prenatal Test®) for the identification of pregnancies at increased risk for 22q11.2 deletion. Methods: Test performance was determined in 2 steps including a total of 1,953 plasma samples. Analytical validation was performed in 1,736 plasma samples. Clinical verification of performance was performed in an additional 217 prospectively ascertained samples from pregnancies with fetal deletion status determined by diagnostic testing. Results: Analytical sensitivity was 75.4% (95% CI: 67.1–82.2%) based on 122 samples with deletions ranging from 1.96 to 3.25 Mb. In 1,614 presumed unaffected samples, specificity was determined to be at least 99.5% (95% CI: 99.0–99.7%). In the clinical cohort, 5 of 7 samples from pregnancies affected with 22q11.2 deletion were determined to have a high probability of deletion. There were no false positive results in the 210 unaffected samples in this cohort. These clinical data are consistent with the performance demonstrated in the analytical validation. Conclusions: cfDNA testing using a targeted microarray-based technology is able to identify pregnancies at increased risk for 22q11.2 deletions of 3.0 Mb and smaller while maintaining a low false positive rate.

Repeatability of estimated fetal weight: Comparison between MR imaging versus 2D ultrasound in at- and near-term patients

INTRODUCTION Our aim was to evaluate the intra- and inter-observer variability and the impact of operator experience on the estimation of fetal weight (EFW) as measured by 2-dimensional ultrasound (2D-US) and magnetic resonance (MR) imaging. MATERIAL AND METHODS We estimated fetal weight in 46 singleton pregnancies at 35.6-41.4 weeks gestation using 2D-US according to the Hadlock formula and using MR imaging according to the equation developed by Baker. Each examination was performed twice, once by an inexperienced operator and once by an experienced operator. The MR-EFW was derived from the planimetric measurement of fetal body volume (FBV) using an assisted semi-automated method. Intra- and inter-observer variability was evaluated by Bland-Altman analysis. Regression analysis was used to investigate the effect of maternal BMI, delivery weight, diabetes and fetal gender on the differences in US-EFW between the inexperienced and experienced operators. RESULTS US-EFW showed higher intra-observer variability than MR-EFW, irrespective of operator experience. The 95% limits of agreement of MR were narrower compared with those of the US measurements. Similarly, US-EFW showed higher inter-observer variability than MR-EFW. MR-EFW improvement over 2D-US for the limits of agreement was 77.9% for intra-observer variability and 74.5% for inter-observer variability. Regression analysis showed that the differences between US-EFW measurements were not related to any of the tested variables. CONCLUSIONS Operator experience has a marginal impact on the variability of US-EFW and no impact on MR-EFW variability. The variability in US-EFW measurements is unpredictable.

Cell-Free DNA Analysis in Maternal Blood: Differences in Estimates between Laboratories with Different Methodologies Using a Propensity Score Approach

Objectives: To evaluate the failure rate and performance of cell-free DNA (cfDNA) testing, mainly in terms of detection rates for trisomy 21, performed by 2 laboratories using different analytical methods. Methods: cfDNA testing was performed on 2,870 pregnancies with the HarmonyTM Prenatal Test using the targeted digital analysis of selected regions (DANSR) method, and on 2,635 pregnancies with the “Cerba test” using the genome-wide massively parallel sequencing (GW-MPS) method, with available outcomes. Propensity score analysis was used to match patients between the 2 groups. A comparison of the detection rates for trisomy 21 between the 2 laboratories was made. Results: In all, 2,811 patients in the Harmony group and 2,530 patients in the Cerba group had no trisomy 21, 18, or 13. Postmatched comparisons of the patient characteristics indicated a higher no-result rate in the Harmony group (1.30%) than in the Cerba group (0.75%; p = 0.039). All 41 cases of trisomy 21 in the Harmony group and 93 cases in the Cerba group were detected. Conclusions: Both methods of cfDNA testing showed low no-result rates and a comparable performance in detecting trisomy 21; yet GW-MPS had a slightly lower no-result rate than the DANSR method.

Postmortem examination of human fetuses: a comparison of 2-dimensional ultrasound with invasive autopsy

To assess the diagnostic accuracy of postmortem ultrasound performed by operators blinded to prenatal findings and to invasive autopsy results in fetuses at different gestational ages and to investigate the effect of various parameters on its diagnostic success.

P20.04: Fetal fraction following selective reduction in twin pregnancies: Poster discussion hub abstracts

Objectives: To examine the relationship of uterine artery pulsatility Index (PI) on the fetal fraction (FF) in maternal plasma cell-free (cf) DNA at 11+0-13+6 weeks of gestation and the possible association with other maternal and fetal characteristics. Methods: This is a cohort study of 1037 singleton pregnancies undergoing at 11–13 weeks of gestation screening by cf-DNA testing for fetal trisomies. CfDNA was extracted from maternal plasma and FF evaluated by the referral laboratory. Uterine artery velocity waveforms were evaluated at the time of maternal sampling by Doppler ultrasonography (WS80 Samsung Medical, Seoul, Republic of Korea) from both uterine arteries and the mean PI calculated. Since uterine artery mean PI changes with gestational age with gestational age, data are expressed as a multiple of the expected median (MoM). Maternal and fetal characteristics considered were maternal age, weight, body mass index (BMI), ethnicity, method of conception, cigarette smoking, fetal Crown–rump length (CRL). Multivariable regression analysis was used to determine significant predictors of the fetal fraction among uterine arteries mean PI MoM and maternal and fetal characteristics. Results: The median fetal fraction was 10.8% (interquantile range [IR] 6.2-15.9%). In 32 women (3.1%) FF resulted < 4%. In such pregnancies uterine artery PI Mom were significantly higher than in those with a FF > 4% (median 1.38; IR 1.05-1.89 vs. 0.98 IR 0.81-1.12 p<0.003). FF decreased with increased mean uterine artery PI MoM, maternal weight, BMI, CRL values and smoking. In multivariable model only maternal weight (p<0.001) and mean uterine PI MoM (p<0.04) resulted significant. Conclusions: FF in maternal plasma cfDNA is affected by maternal weight and uterine artery impedance to flow at 11+0-13+6 weeks of gestation. Uterine artery PI MoM values may have the potential to be incorporated in models to predict the amount of FF to be used in counselling parents concerning the likelihood of failure to obtain a result from cfDNA analysis.

Profile of women choosing the Harmony® Prenatal Test

ABSTRACT Introduction: The Harmony® Prenatal Test, a noninvasive cell-free DNA (cfDNA) method for major trisomies has been available since January 2013 at our unit, and tests were sent to the Ariosa Clinical Laboratory Improvement Amendments (CLIA) laboratory in California. From July 2017 onward, prenatal cfDNA has been reimbursed in Belgium for all pregnancies; however, since then samples are sent to a local laboratory. Little data are available on patient’s profile and choices toward cfDNA and on the performance of local technology transfer centers. Areas covered: The profiles and choices of women regarding this test were evaluated. Further, the performance of cfDNA at the local laboratory was compared to the one in California. Our results showed that women from the Netherlands, as compared to Belgium, were more likely to undergo cfDNA testing for maternal request and would be less likely to undergo karyotyping if cfDNA were unavailable, therefore are better candidates for cfDNA testing, when this is used as first-line screening. Expert commentary: Our findings highlight the importance of conducting these types of studies, before decisions about clinical implementation are made by national governments and ministries of health.