Ranjit Akolekar

Non-invasive prenatal assessment of Trisomy 21 by multiplexed maternal plasma DNA sequencing: large scale validity study

Objectives To validate the clinical efficacy and practical feasibility of massively parallel maternal plasma DNA sequencing to screen for fetal trisomy 21 among high risk pregnancies clinically indicated for amniocentesis or chorionic villus sampling. Design Diagnostic accuracy validated against full karyotyping, using prospectively collected or archived maternal plasma samples. Setting Prenatal diagnostic units in Hong Kong, United Kingdom, and the Netherlands. Participants 753 pregnant women at high risk for fetal trisomy 21 who underwent definitive diagnosis by full karyotyping, of whom 86 had a fetus with trisomy 21. Intervention Multiplexed massively parallel sequencing of DNA molecules in maternal plasma according to two protocols with different levels of sample throughput: 2-plex and 8-plex sequencing. Main outcome measures Proportion of DNA molecules that originated from chromosome 21. A trisomy 21 fetus was diagnosed when the z score for the proportion of chromosome 21 DNA molecules was >3. Diagnostic sensitivity, specificity, positive predictive value, and negative predictive value were calculated for trisomy 21 detection. Results Results were available from 753 pregnancies with the 8-plex sequencing protocol and from 314 pregnancies with the 2-plex protocol. The performance of the 2-plex protocol was superior to that of the 8-plex protocol. With the 2-plex protocol, trisomy 21 fetuses were detected at 100% sensitivity and 97.9% specificity, which resulted in a positive predictive value of 96.6% and negative predictive value of 100%. The 8-plex protocol detected 79.1% of the trisomy 21 fetuses and 98.9% specificity, giving a positive predictive value of 91.9% and negative predictive value of 96.9%. Conclusion Multiplexed maternal plasma DNA sequencing analysis could be used to rule out fetal trisomy 21 among high risk pregnancies. If referrals for amniocentesis or chorionic villus sampling were based on the sequencing test results, about 98% of the invasive diagnostic procedures could be avoided.

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

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.

First-Trimester Prediction of Hypertensive Disorders in Pregnancy

This study aimed to establish a method of screening for pregnancy hypertension by a combination of maternal variables, including mean arterial pressure, uterine artery pulsatility index, pregnancy-associated plasma protein-A, and placental growth factor in early pregnancy. The base-cohort population constituted of 7797 singleton pregnancies, including 34 case subjects who developed preeclampsia (PE) requiring delivery before 34 weeks (early PE) and 123 with late PE, 136 with gestational hypertension, and 7504 cases subjects (96.3%) who were unaffected by PE or gestational hypertension. Maternal history, uterine artery pulsatility index, mean arterial pressure, and pregnancy-associated plasma protein-A were recorded in all of the cases in the base cohort, but placental growth factor was measured only in the case-control population of 209 cases who developed hypertensive disorders and 418 controls. In each case the measured mean arterial pressure, uterine artery pulsatility index, pregnancy-associated plasma protein-A, and placental growth factor were converted to a multiple of the expected median (MoM) after correction for maternal characteristics found to affect the measurements in the unaffected group. Early PE and late PE were associated with increased mean arterial pressure (1.15 MoM and 1.08 MoM) and uterine artery pulsatility index (1.53 MoM and 1.23 MoM) and decreased pregnancy-associated plasma protein-A (0.53 MoM and 0.93 MoM) and placental growth factor (0.61 MoM and 0.83 MoM). Logistic regression analysis was used to derive algorithms for the prediction of hypertensive disorders. It was estimated that, with the algorithm for early PE, 93.1%, 35.7%, and 18.3% of early PE, late PE, and gestational hypertension, respectively, could be detected with a 5% false-positive rate and that 1 in 5 pregnancies classified as being screen positive would develop pregnancy hypertension. This method of screening is far superior to the traditional approach, which relies entirely on maternal history.

Competing risks model in early screening for preeclampsia by biophysical and biochemical markers.

Objective: To develop models for prediction of preeclampsia (PE) based on maternal characteristics, biophysical and biochemical markers at 11–13 weeks’ gestation in which the gestation at the time of delivery for PE is treated as a continuous variable. Methods: This was a screening study of singleton pregnancies at 11–13 weeks including 1,426 (2.4%) that subsequently developed PE and 57,458 that were unaffected by PE. We developed a survival time model for the time of delivery for PE in which Bayes’ theorem was used to combine the prior information from maternal characteristics with uterine artery pulsatility index (PI), mean arterial pressure (MAP), serum pregnancy-associated plasma protein-A (PAPP-A) and placental growth factor (PLGF) multiple of the median (MoM) values. Results: In pregnancies with PE, there was a linear correlation between MoM values of uterine artery PI, MAP, PAPP-A and PLGF with gestational age at delivery and therefore the deviation from normal was greater for early than late PE for all four biomarkers. Screening by maternal characteristics, biophysical and biochemical markers detected 96% of cases of PE requiring delivery before 34 weeks and 54% of all cases of PE at a fixed false-positive rate of 10%. Conclusions: A new model has been developed for effective first-trimester screening for PE.

Noninvasive prenatal diagnosis of fetal trisomy 18 and trisomy 13 by maternal plasma DNA sequencing.

Massively parallel sequencing of DNA molecules in the plasma of pregnant women has been shown to allow accurate and noninvasive prenatal detection of fetal trisomy 21. However, whether the sequencing approach is as accurate for the noninvasive prenatal diagnosis of trisomy 13 and 18 is unclear due to the lack of data from a large sample set. We studied 392 pregnancies, among which 25 involved a trisomy 13 fetus and 37 involved a trisomy 18 fetus, by massively parallel sequencing. By using our previously reported standard z-score approach, we demonstrated that this approach could identify 36.0% and 73.0% of trisomy 13 and 18 at specificities of 92.4% and 97.2%, respectively. We aimed to improve the detection of trisomy 13 and 18 by using a non-repeat-masked reference human genome instead of a repeat-masked one to increase the number of aligned sequence reads for each sample. We then applied a bioinformatics approach to correct GC content bias in the sequencing data. With these measures, we detected all (25 out of 25) trisomy 13 fetuses at a specificity of 98.9% (261 out of 264 non-trisomy 13 cases), and 91.9% (34 out of 37) of the trisomy 18 fetuses at 98.0% specificity (247 out of 252 non-trisomy 18 cases). These data indicate that with appropriate bioinformatics analysis, noninvasive prenatal diagnosis of trisomy 13 and trisomy 18 by maternal plasma DNA sequencing is achievable.

Procedure‐related risk of miscarriage following amniocentesis and chorionic villus sampling: a systematic review and meta‐analysis

To estimate procedure‐related risks of miscarriage following amniocentesis and chorionic villus sampling (CVS) based on a systematic review of the literature and a meta‐analysis.

Maternal serum placental growth factor at 11 + 0 to 13 + 6 weeks of gestation in the prediction of pre‐eclampsia

To investigate the potential value of maternal serum placental growth factor (PlGF) in first‐trimester screening for pre‐eclampsia (PE).

Aspirin versus Placebo in Pregnancies at High Risk for Preterm Preeclampsia

Background Preterm preeclampsia is an important cause of maternal and perinatal death and complications. It is uncertain whether the intake of low‐dose aspirin during pregnancy reduces the risk of preterm preeclampsia. Methods In this multicenter, double‐blind, placebo‐controlled trial, we randomly assigned 1776 women with singleton pregnancies who were at high risk for preterm preeclampsia to receive aspirin, at a dose of 150 mg per day, or placebo from 11 to 14 weeks of gestation until 36 weeks of gestation. The primary outcome was delivery with preeclampsia before 37 weeks of gestation. The analysis was performed according to the intention‐to‐treat principle. Results A total of 152 women withdrew consent during the trial, and 4 were lost to follow up, which left 798 participants in the aspirin group and 822 in the placebo group. Preterm preeclampsia occurred in 13 participants (1.6%) in the aspirin group, as compared with 35 (4.3%) in the placebo group (odds ratio in the aspirin group, 0.38; 95% confidence interval, 0.20 to 0.74; P=0.004). Results were materially unchanged in a sensitivity analysis that took into account participants who had withdrawn or were lost to follow‐up. Adherence was good, with a reported intake of 85% or more of the required number of tablets in 79.9% of the participants. There were no significant between‐group differences in the incidence of neonatal adverse outcomes or other adverse events. Conclusions Treatment with low‐dose aspirin in women at high risk for preterm preeclampsia resulted in a lower incidence of this diagnosis than placebo. (Funded by the European Union Seventh Framework Program and the Fetal Medicine Foundation; EudraCT number, 2013‐003778‐29; Current Controlled Trials number, ISRCTN13633058.)

Maternal Plasma DNA Analysis with Massively Parallel Sequencing by Ligation for Noninvasive Prenatal Diagnosis of Trisomy 21

BACKGROUND Noninvasive prenatal diagnosis of trisomy 21 (T21) has recently been shown to be achievable by massively parallel sequencing of maternal plasma on a sequencing-by-synthesis platform. The quantification of several other human chromosomes, including chromosomes 18 and 13, has been shown to be less precise, however, with quantitative biases related to the chromosomal GC content. METHODS Maternal plasma DNA from 10 euploid and 5 T21 pregnancies was sequenced with a sequencing-by-ligation approach. We calculated the genomic representations (GRs) of sequenced reads from each chromosome and their associated measurement CVs and compared the GRs of chromosome 21 (chr21) for the euploid and T21 pregnancies. RESULTS We obtained a median of 12 x 10(6) unique reads (21% of the total reads) per sample. The GRs deviated from those expected for some chromosomes but in a manner different from that previously reported for the sequencing-by-synthesis approach. Measurements of the GRs for chromosomes 18 and 13 were less precise than for chr21. z Scores of the GR of chr21 were increased in the T21 pregnancies, compared with the euploid pregnancies. CONCLUSIONS Massively parallel sequencing-by-ligation of maternal plasma DNA was effective in identifying T21 fetuses noninvasively. The quantitative biases observed among the GRs of certain chromosomes were more likely based on analytical factors than biological factors. Further research is needed to enhance the precision for measuring for the representations of chromosomes 18 and 13.

Prediction of small-for-gestation neonates from biophysical and biochemical markers at 11-13 weeks.

Objective: To develop a model for prediction of small-for-gestational age (SGA) neonates in the absence of preeclampsia (PE) based on maternal factors and biophysical and biochemical markers at 11–13 weeks’ gestation. Methods: Screening study in 1,536 SGA and 31,314 non-SGA pregnancies based on maternal characteristics, fetal nuchal translucency (NT) thickness, serum pregnancy-associated plasma protein-A (PAPP-A) and free β-human chorionic gonadotrophin (β-hCG). We also measured mean arterial pressure (MAP), uterine artery pulsatility index (PI) and performed case-control studies for measurement of maternal serum concentration of placental growth factor (PLGF), placental protein 13 (PP13) and A Disintegrin And Metalloprotease (ADAM12). Regression analysis was used to develop a model for the prediction of SGA. Results: In the SGA group, uterine artery PI and MAP were increased and serum PAPP-A, free β-hCG, PLGF, PP13, and ADAM12 and fetal NT were decreased. At a false positive rate of 10%, the estimated detection rate by a combination of maternal factors and biophysical and biochemical markers at 11–13 weeks was 73% for SGA requiring delivery before 37 weeks and 46% for those delivering at term. Conclusions: Half of pregnancies with SGA neonates in the absence of PE could potentially be identified at 11–13 weeks.