John M. G. van Vugt

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.

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.

Plasma placenta growth factor levels in midtrimester pregnancies.

OBJECTIVE Previous studies have shown decreased levels of placenta growth factor in serum of pregnant women with preeclampsia. The aim of this study was to investigate whether levels of placenta growth factor are decreased before the clinical onset of preeclampsia, and whether placenta growth factor levels are decreased in pregnancies complicated by intrauterine growth restriction. METHODS From an ongoing longitudinal study, 101 plasma samples were collected from 72 pregnant women at weeks 11–21 of gestation. Placenta growth factor levels were determined retrospectively in plasma using an enzyme‐linked immunosorbent assay. Correlations between plasma concentrations of placenta growth factor and pregnancy outcome were evaluated. RESULTS Plasma samples of 72 patients were analyzed. Forty‐four patients had no pregnancy complications, 18 developed preeclampsia, and 10 women had pregnancies complicated by intrauterine growth restriction. Between week 17 and week 21 of pregnancy, a significantly lower level of placenta growth factor was found in plasma of patients who later developed preeclampsia (n = 10), compared with control pregnancies (n = 25, P = .004). In women with a growth‐restricted baby at birth (n = 5), levels of placenta growth factor were also low. CONCLUSIONS Our results show that plasma placenta growth factor levels are decreased before preeclampsia is clinically evident. The data suggest that placenta growth factor may be useful to determine the relative risk of developing preeclampsia and intrauterine growth restriction.

Size-based molecular diagnostics using plasma DNA for noninvasive prenatal testing

Significance Noninvasive prenatal testing (NIPT) using fetal DNA in maternal plasma has been rapidly adopted worldwide. Current NIPT for fetal chromosomal disorders are based on the counting of DNA molecules in maternal plasma. Here, we show that plasma DNA-based molecular diagnostics can also be built around DNA fragment size, instead of count. First, we demonstrate that the fetal DNA fraction in maternal plasma can be rapidly measured by size analysis, even simply using microchip-based capillary electrophoresis. Second, we show that plasma DNA size analysis can be used for the detection of multiple types of fetal chromosomal aneuploidies with high accuracy. This strategy has many potential diagnostic applications, e.g., in oncology and transplantation monitoring. Noninvasive prenatal testing using fetal DNA in maternal plasma is an actively researched area. The current generation of tests using massively parallel sequencing is based on counting plasma DNA sequences originating from different genomic regions. In this study, we explored a different approach that is based on the use of DNA fragment size as a diagnostic parameter. This approach is dependent on the fact that circulating fetal DNA molecules are generally shorter than the corresponding maternal DNA molecules. First, we performed plasma DNA size analysis using paired-end massively parallel sequencing and microchip-based capillary electrophoresis. We demonstrated that the fetal DNA fraction in maternal plasma could be deduced from the overall size distribution of maternal plasma DNA. The fetal DNA fraction is a critical parameter affecting the accuracy of noninvasive prenatal testing using maternal plasma DNA. Second, we showed that fetal chromosomal aneuploidy could be detected by observing an aberrant proportion of short fragments from an aneuploid chromosome in the paired-end sequencing data. Using this approach, we detected fetal trisomy 21 and trisomy 18 with 100% sensitivity (T21: 36/36; T18: 27/27) and 100% specificity (non-T21: 88/88; non-T18: 97/97). For trisomy 13, the sensitivity and specificity were 95.2% (20/21) and 99% (102/103), respectively. For monosomy X, the sensitivity and specificity were both 100% (10/10 and 8/8). Thus, this study establishes the principle of size-based molecular diagnostics using plasma DNA. This approach has potential applications beyond noninvasive prenatal testing to areas such as oncology and transplantation monitoring.

'Identical' twins with discordant karyotypes

A chromosomal abnormality in one of the fetuses of a monozygotic twin pregnancy is a rare phenomenon. In the prenatal unit of our cytogenetics laboratory we have recently come across two such heterokaryotypic twin pregnancies. In both cases ultrasound abnormalities were detected in one fetus of each twin pair. Chromosomal analysis showed that one twin pregnancy was discordant for trisomy 21 and the other for 45,X. Ultrasonographic examination suggested a monochorionic twin pregnancy in each case and DNA studies confirmed that both sets of twins were monozygotic. Both pregnancies were terminated. Biopsies taken from different sites of the placentas showed chromosomal mosaicism in both cases. There was no clear correlation between the karyotype found close to the site of the umbilical cord insertion in the placenta and the karyotype of the fetus. Sampling of amniotic fluid from both sacs is recommended in diamniotic twin pregnancies if one (or both) of the fetuses has ultrasound abnormalities, even if the twins are apparently monochorionic. Copyright

Markers for Presymptomatic Prediction of Preeclampsia and Intrauterine Growth Restriction

Preeclampsia and intrauterine growth restriction are both characterized by placental malfunction. The pathological processes of abnormal trophoblast invasion, partial absence of maternal spiral artery modification, increased apoptosis of trophoblast cells, and placental ischemia are all associated with the release of specific molecules. These proteins, as well as cell‐free fetal DNA and RNA might be detected in the maternal peripheral circulation, quantified, and used for early identification and prediction of preeclampsia and intrauterine growth restriction, prior to the appearance of the clinical symptoms. As preeclampsia and intrauterine growth restriction are associated with increased maternal, perinatal, and neonatal morbidity and mortality, early identification of these pregnancy associated complications will permit the design of appropriate preventive measures. In this review a variety of factors reported to be useful as potential markers for early detection of pregnancies at increased risk will be discussed. Molecules associated with the establishment of the placenta and essential in fetal–maternal interactions, like interleukin 2‐receptor, insulinlike growth factor‐1, and insulinlike growth factor binding protein‐1, placenta growth factor, hepatocyte growth factor, inhibin A, activin A, and human chorionic gonadotrophin seem to be the most likely candidates for presymptomatic markers for preeclampsia and/or intrauterine growth restriction. Detection and discrimination of these molecules through the placental RNA in maternal plasma based strategy has become a realistic option.

The consequences of implementing non-invasive prenatal testing in Dutch national health care: a cost-effectiveness analysis

OBJECTIVE Non-invasive prenatal testing (NIPT) using cell-free fetal DNA in maternal plasma has been developed for the detection of fetal aneuploidy. Clinical trials have shown high sensitivity and specificity for trisomy 21 (T21) in both high-risk and average-risk populations. Although its great potential for prenatal medicine is evident, more information regarding the consequences of implementing NIPT in a national programme for prenatal screening is required. STUDY DESIGN A decision-analytic model was developed to compare costs and outcomes of current clinical practice in The Netherlands using conventional screening only, with two alternatives: implementing NIPT as an optional secondary screening test for those pregnancies complicated by a high risk for T21, and implementing NIPT as primary screening test, replacing conventional screening. Probability estimates were derived from a systematic review of international literature. Costs were determined from a health-care perspective. Data were analysed to obtain outcomes, total costs, relative costs and incremental cost-effectiveness ratios (ICERs) for the different strategies. Sensitivity analysis was used to assess the impact of assumptions on model results. RESULTS Implementing NIPT as an optional secondary, or as primary screening test will increase T21 detection rate by 36% (from 46.8% to 63.5%) and 54% (from 46.8% to 72.0%), simultaneously decreasing the average risk of procedure-related miscarriage by 44% (from 0.0168% to 0.0094% per pregnant woman) and 62% (from 0.0168% to 0.0064% per pregnant woman), respectively. None of the strategies clearly dominated: current clinical practice is the least costly, whereas implementing NIPT will cause total costs of the programme to increase by 21% (from €257.09 to €311.74 per pregnant woman), leading to an ICER of k€94 per detected case of T21, when utilised as an optional secondary screening test and by 157% (from €257.09 to €660.94 per pregnant woman), leading to an ICER of k€460 per detected case of T21, when utilised as primary screening test. However, implementing NIPT as triage test did result in the lowest expected relative costs per case of T21 diagnosed (k€141). CONCLUSION NIPT should be implemented in national health care as an optional secondary screening test for those pregnancies complicated by a high risk for T21.

Decreased plasma levels of metastin in early pregnancy are associated with small for gestational age neonates.

To investigate whether pregnancies with small for gestational age (SGA) neonates, defined as customized birth weight below the 10th centile, are associated with altered levels of metastin in maternal plasma in the first trimester.

The role of magnetic resonance imaging in prenatal diagnosis of fetal anomalies

Magnetic resonance imaging (MR) has become a useful adjuvant in evaluating fetal structural anomalies when ultrasound (US) is equivocal. It has a significant promise in confirming a US suspected abnormality and providing new information that was previously not available. The first studies on prenatal MR were hindered by fetal motion and long acquisition times. This degraded imaging and, therefore, maternal or fetal sedation was needed. Since fast and ultrafast MR with scan times of <1 s have become available, the amount of motion artifacts is decreased and sedation is no longer needed.MR has proved to be especially beneficial in detecting CNS anomalies. Agenesis of the corpus callosum, migration abnormalities and abnormalities of the posterior fossa are better seen on MR. Masses in the fetal neck and thorax can be identified on MR, as some abdominal anomalies. However, the fetal skeletal is difficult to visualize with MR. In the future, it is most likely that real time MR will become clinically available which would improve MR imaging even more.

Sequentially combined hormone replacement therapy reduces impedance to flow within the uterine and central retinal arteries in healthy postmenopausal women.

OBJECTIVE The purpose of this study was to investigate the long-term effects of combined hormone replacement therapy on the impedances of the uterine, central retinal, and ophthalmic arteries in healthy postmenopausal women. STUDY DESIGN In a prospective controlled study we randomly assigned 30 healthy postmenopausal women (mean age, 52 +/- 3 years) to 2 groups. Women in the hormone replacement therapy group (n = 15) received 1 mg micronized 17beta-estradiol daily sequentially combined with 5 or 10 mg dydrogesterone for 14 days of each 28-day cycle during 12 months and 2 mg 17beta-estradiol combined with 10 mg dydrogesterone thereafter for a period of 3 months. The control group (n = 15) received no treatment. Color Doppler ultrasonography was used to measure the impedance to flow (pulsatility index) within the uterine, central retinal, and ophthalmic arteries in the 17beta-estradiol phase at baseline and after 3, 12, and 15 months. RESULTS With respect to values in the control group, 12 months of hormone replacement therapy was associated with a significantly lower (by 39%) mean pulsatility index of the uterine artery (decrease from baseline of 25% in hormone replacement therapy group and increase of 14% in control group) and a significantly lower (by 29%) mean pulsatility index of the central retinal artery (decrease of 9% in hormone replacement therapy group and increase of 20% in control group). After 3 months this effect was already evident. During hormone replacement therapy the reductions in mean pulsatility index values of the uterine and central retinal arteries with respect to baseline were larger (both P =.002) in the women with high pretreatment pulsatility index values than in those with low pretreatment values. The baseline pulsatility index of the uterine artery correlated positively with age and with duration of amenorrhea (r = 0.42, P =. 01; r = 0.48, P =.008; respectively). CONCLUSION These results suggest that 12 months of sequentially combined hormone replacement therapy with a low dose of estradiol (1 mg) lowers arterial impedance in specific vascular territories. These data may help in understanding the effects of hormone replacement therapy on the cerebral circulation.