Cell-free DNA screening and sex chromosome aneuploidies

Cell-free DNA (cfDNA) testing is increasingly being used to screen pregnant women for fetal aneuploidies. This technology may also identify fetal sex and can be used to screen for sex chromosome aneuploidies (SCAs). Physicians offering this screening will need to be prepared to offer comprehensive prenatal counseling about these disorders to an increasing number of patients. The purpose of this article is to consider the source of information to use for counseling, factors in parental decision-making, and the performance characteristics of cfDNA testing in screening for SCAs. Discordance between ultrasound examination and cfDNA results regarding fetal sex is also discussed. © 2015 John Wiley & Sons, Ltd.     

Commentary: The federal ‘Prenatally and Postnatally Diagnosed Conditions Awareness Act’

The recently enacted federal law, the ‘Prenatally and Postnatally Diagnosed Conditions Awareness Act’ (United States Public Law 110–374) seeks to improve opportunities for parents and pregnant women to anticipate and understand the likely life course of children born with Down syndrome and other (unspecified) conditions. The law is in part a response to the continued growth of prenatal screening and testing. For example, the American College of Obstetricians and Gynecologists' Practice Bulletin 77 recommends that ‘Screening and invasive diagnostic testing for aneuploidies be available to all women who present for prenatal care before 20 weeks of gestation regardless of maternal age.’ Emerging technologies anticipate an era in which the scope of prenatal screening and testing will be much larger than it is today. Inevitably, more women will find themselves facing the hard question of whether to continue or end a pregnancy in which a fetus has been found to have a significant abnormality. While the new federal law is not likely to have a major impact on obstetric practice, it may be a harbinger of renewed wide-scale public debate concerning the ethics of prenatal screening. Copyright © 2009 John Wiley & Sons, Ltd.     

Amniotic fluid-AFP in Down syndrome and other chromosome abnormalities

80·2 Per cent of 111 Down syndrome pregnancies had amniotic fluid (AF) alpha fetoprotein (AFP) levels on or below the median and 10·8 per cent at or below 0·5 MoM compared with 41·9 and 1·4 per cent of controls. These differences were even more striking when the gestational age was < 18 weeks compared with ⩾ 18 weeks. No such association was seen for other chromosome abnormalities including trisomy 18,45,X and mosaics, 47,XXY, 47,XXX, and other structural abnormalities and triploidy, even when high levels due to defects such as omphalocele and cystic hygroma were excluded. All cases of trisomy 13 and 80 per cent with 47,XYY had AF-AFP levels above the median. Selection of cases for karyotyping by a low level of AF-AFP would clearly fail to detect aneuploidies other than Down syndrome and is not recommended. A possible weak association between low maternal serum (MS) and AF-AFPs in Down syndrome was most evident at < 18 weeks, suggesting that MS screening between 16 and 18 weeks may be the most informative time.     

Compromise ultrasound dating policy in maternal serum screening for Down syndrome

Routine ultrasound biometry is the method of choice for gestational dating when screening for Down syndrome. However, it is costly and an alternative policy is to restrict ultrasound to women most likely to have menstrual dating errors. This was evaluated by statistical modelling with parameters from 14 274 women screened between January 1997 and July 2001 using free beta-human chorionic gonadotrophin (free β-hCG), α-fetoprotein (AFP) and unconjugated estriol (uE₃). A total of 12 711 (89%) women had both ultrasound and menstrual gestations, but in 4101 (29%) women either the last menstrual period (LMP) was uncertain or a pill-withdrawal period, or there were irregular or abnormal length cycles. The LMP was not entered in the test request form for a further 1404 (9.8%) women. Routine ultrasound dating yielded a predicted detection rate higher than for menstrual dating by 3.9–7.1%, depending on the marker combination and cut-off. The false-positive rate was reduced by 0.2–1.1%. Selectively scanning the 39% with unreliable dates increased detection by 2.6–4.6%, and reduced the false-positive rate by 0.04–0.6%. Some centres only use the ultrasound estimate of gestation when it differs from the menstrual estimate by more than 7 days. Such a rule reduces the gain in detection rate to 2.5–4.6% for routine ultrasound and 1.7–3.1% with the compromise policy; the false-positive rate reductions are 0.06–0.6% and 0.0–0.3%, respectively. We conclude that if routine ultrasound is not financially and practically feasible, the compromise policy yields a clinically important improvement in screening performance compared to menstrual dating. Copyright © 2002 John Wiley & Sons, Ltd.     

In situ hybridization studies for the detection of common aneuploidies in CVS

We have attempted to evaluate the efficiency of interphase cytogenetics in the detection of specific aneuploidies in chorionic villus samples. For this purpose, we used alphoid repetitive sequences specific for the chromosomes involved in the common aneuploidies, namely probes for chromosomes 13, 18, 21, X, and Y. These probes were applied to normal and abnormal CVS cases, as well as to a few mosaic cases. Results from these preliminary studies indicate that the technique can be very efficient for the detection of specific aneuploidies and can be particularly useful in the analysis of mosaic cases, which usually requires the screening of high number of metaphases.     

Non-invasive prenatal testing in the management of twin pregnancies

Twin pregnancies are common and associated with pregnancy complications and adverse outcomes. Prenatal clinical management is intensive and has been hampered by inferior screening and less acceptable invasive testing. For aneuploidy screening, meta-analyses show that non-invasive prenatal testing (NIPT) through analysis of cell-free DNA (cf-DNA) is superior to serum and ultrasound-based tests. The positive predictive value for NIPT is driven strongly by the discriminatory power of the assay and only secondarily by the prior risk. Uncertainties in a priori risks for aneuploidies in twin pregnancies are therefore of lesser importance with NIPT. Additional information on zygosity can be obtained using NIPT. Establishing zygosity can be helpful when chorionicity was not reliably established early in pregnancy or where the there is a concern for one versus two affected fetuses. In dizygotic twin pregnancies, individual fetal fractions can be measured to ensure that both values are satisfactory. Vanishing twins can be identified by NIPT. Although clinical utility of routinely detecting vanishing twins has not yet been demonstrated, there are individual cases where cf-DNA analysis could be helpful in explaining unusual clinical or laboratory observations. We conclude that cf-DNA analysis and ultrasound have synergistic roles in the management of multiple gestational pregnancies.     

Second-trimester levels of maternal urinary gonadotropin peptide in down syndrome pregnancy

Urinary gonadotropin peptide (UGP; β-core fragment), a major metabolite of human chorionic gonadotropin (hCG), was shown recently to be markedly elevated in Down syndrome pregnancy between 19 and 22 weeks of gestation. To confirm and extend this finding, we obtained maternal urine and matching maternal serum samples from 14 cases of Down syndrome and six other aneuploidies between 17 and 21 weeks of gestation. UGP was measured in all these samples and in 91 singleton control urines. Results were corrected for urinary creatinine level and expressed as multiples of the control median (MOM). hCG levels were assayed in all serum samples from the cases and compared with previously established reference values. The median UGP level in Down syndrome cases was 5.34 MOM (range 2.71–12.57); 88 per cent of the values were above the 95th centile of control levels after modelling. The median maternal serum hCG level for the same cases was 2.20 MOM (range 0.84–3.40); 36 per cent of the values were above the 95th centile. The level of UGP in every case including all other aneuploidies was higher than the comparable maternal serum hCG level. Elevated UGP measurements are strongly associated with fetal Down syndrome during the second trimester and could contribute to improved Down syndrome screening protocols that are more accessible and less expensive than are currently available.     

First-trimester alpha-fetoprotein screening for Down syndrome

Screening for Down syndrome and other chromosomal aneuploidies by biochemical parameters in maternal serum is well established for the second trimester. With screening as late as 16 weeks of gestation, the option of chorionic villus sampling (CVS) unfortunately is lost. In our study population, the maternal serum alpha-fetoprotein (MSAFP) concentration was determined in 2471 women in the first trimester immediately prior to CVS. Although in this sample MSAFP tended to be lower in Down syndrome (DS) pregnancies than in pregnancies with a chromosomally normal fetus, at this early gestational age neither a fixed cut-off level of 0·5 multiples of the normal median (MOM) nor one of 0·6 MOM was suitable for identifying pregnancies at higher risk for DS. This also applied to trisomy 18, although on average MSAFP in trisomy 18 pregnancies was lower than in normal and DS pregnancies.     

Copy number analysis of meiotic and postzygotic mitotic aneuploidies in trophectoderm cells biopsied at the blastocyst stage and arrested embryos

Preimplantation genetic testing for aneuploidy (PGT-A) by copy number analysis is now widely used to select euploid embryos for transfer. Whole or partial chromosome aneuploidy can arise in meiosis, predominantly female meiosis, or in the postzygotic, mitotic divisions during cleavage and blastocyst formation, resulting in chromosome mosaicism. Meiotic aneuploidies are almost always lethal, however, the clinical significance of mitotic aneuploidies detected by PGT-A is not fully understood and healthy live births have been reported following transfer of mosaic embryos. Here, we used single nucleotide polymorphism genotyping of both polar bodies and embryo samples to identify meiotic aneuploidies and compared copy number changes for meiotic and presumed mitotic aneuploidies in trophectoderm cells biopsied at the blastocyst stage and arrested embryos. PGT-A detected corresponding full copy number changes (≥70%) for 36/37 (97%) maternal meiotic aneuploidies. The number of presumed mitotic copy number changes detected exceeded those of meiotic origin. Although mainly in the mosaic range, some of these mitotic aneuploidies had copy number changes ≥70% and would have been identified as full aneuploidies. Interestingly, many arrested embryos had multiple mitotic aneuploidies across a broad range of copy number changes, which may have arisen through tripolar spindle and other mitotic abnormalities.     

Outcome of fetal echogenic bowel: A systematic review and meta-analysis

The main aim of this systematic review was to explore the outcome of fetuses with isolated echogenic bowel (EB) on antenatal ultrasound. Inclusion criteria were singleton pregnancies with isolated EB no associated major structural anomalies at the time of diagnosis. The outcomes observed were: chromosomal anomalies, cystic fibrosis (CF), associated structural anomalies detected only at follow-up scans and at birth, regression during pregnancy, congenital infections, intra-uterine (IUD), neonatal (NND) and perinatal (PND) death. Twenty-five studies (12 971 fetuses) were included. Chromosomal anomalies occurred in 3.3% of the fetuses, mainly Trisomy 21 and aneuploidies involving the sex chromosomes. Cystic fibrosis occurred in 2.2%. Congenital infections affected 2.2%, mainly congenital Cytomegalovirus (CMV) infection. The majority of fetuses with EB experienced regression or disappearance of the EB at follow-up scans. Associated anomalies were detected at a follow-up scan in 1.8%. Associated anomalies were detected at birth and missed at ultrasound in 2.1% of cases. IUD occurred in 3.2% of cases while the corresponding figures for NND and PND were 0.4% and 3.1%. Fetuses with EB are at increased risk of adverse perinatal outcome, highlighting the need for a thorough antenatal management and postnatal follow-up. Assessment during pregnancy and after birth should be performed in order to look for signs of fetal aneuploidy, congenital infections and associated structural anomalies.     

First trimester serum screening for down's syndrome

Screening for Down's Syndrome has been shown to be effective at 10 weeks of pregnancy. A multicentre study (the First trimester serum screening study) has shown that there are two biochemical markers of choice at this time in pregnancy, namely pregnancy associated placental protein A (PAPP-A) and the free β-sub-unit of human chorionic gonadotrophin (free β-hCG). When used together with maternal age these two biochemical markers have an estimated detection rate of 62% and a 5% false-positive rate. The results are consistent with those obtained from a systematic review of the world literature. Other markers are less predictive of Down's syndrome though there is still some uncertainty over the value of dimeric inhibin-A at 10 weeks of pregnancy. Nuchal translucency measurement, from an ultrasound examination performed at about 10 weeks of pregnancy, is associated with Down's syndrome and is emerging as an important potential screening marker. At present there is uncertainty over its quantitative performance and performance when combined with biochemical markers. The resolution of these issues is currently the subject of active research. Ten week screening for Down's syndrome is an advance that is now technically possible though there is still insufficient information to justify its use in routine screening practice.     

Down syndrome maternal serum marker screening after 18 weeks' gestation

Women having access to prenatal care late in pregnancy may still wish to benefit from maternal serum screening for Down syndrome. Therefore, we established reference values for α-feto protein (AFP) and free β-human chorionic gonadotrophin (β-hCG), and assessed the diagnostic value of maternal serum marker screening at 18–35 weeks' gestation based upon a series of 4072 sera from unaffected pregnancies and 118 sera from pregnant women with fetuses affected by Down syndrome. Using a 1/250 risk cut-off, a detection rate of 72.9% (95% CI = 71.5–74.3%) was achieved with a false-positive rate of 7.51% (95% CI = 6.71–8.3%). This was not significantly different from the percentages observed in our 14–17 weeks routine screening (50 596 patients): 71.9% (95% CI = 71.5–72.3%) and 6.48% (95% CI = 6.28–6.68%), respectively. Detection and screen-positive rates were, respectively, 51.3% (95% CI = 35.6–67.0%) and 5.95% (95% CI = 5.12–6.68%) in women aunder 35 years of age, and 84.8% (95% CI = 76.9–92.7%) and 24% (95% CI = 20.7–27.3%) in women aged 35 years and over. In conclusion, maternal serum marker screening is feasible at 18 weeks' gestation and later, which may be of interest in selected cases. Copyright © 2002 John Wiley & Sons, Ltd.     

First-trimester assessment of placenta function and the prediction of preeclampsia and intrauterine growth restriction

Preeclampsia and intrauterine growth restriction (IUGR) are major contributors to perinatal mortality and morbidity worldwide. Both are characterized by impaired trophoblastic invasion of the maternal spiral arteries and their conversion from narrow muscular vessels to wide non-muscular channels. Despite improvement in the understanding of the pathophysiology of these conditions, ability to accurately identify pregnant woman who will develop them is limited. This greatly impairs the development and testing of preventive interventions. While different measures of placental dysfunction have been associated with increased risk for adverse pregnancy outcomes, the ability of any single one to accurately predict these outcomes is poor. Developing predictive tests is further challenged by difficulty in the timing of the measurements, as both the structural and biochemical characteristics of the placenta change with increasing gestational age. The ideal screening test would accurately predict the development of adverse pregnancy outcomes early enough to provide a window for preventive interventions. Improvement in ultrasound technology provides potentially useful novel tools for evaluating placental structure, but measuresments need to be standardized in order to be useful. Maternal serum analyte screening is a noninvasive test of placental biochemical function, but present serum marker alone is not sufficiently accurate to suggest its routine use in clinical practice. The use of first trimester biochemical markers in combination with uterine artery Doppler screening is promising as a potential screening tool. Prospective longitudinal studies using standardized methodology are necessary to further evaluate the choice of parameters and strategies of combination to achieve the best predictive models. Copyright © 2010 John Wiley & Sons, Ltd.     

Maternal serum free α-human chorionic gonadotrophin levels in twin pregnancies: Implications for screening for Down's syndrome

Maternal serum free α-human chorionic gonadotrophin (free α-hCG) levels were determined in twin and singleton pregnancies at 15–22 weeks of gestation using a set of stored serum samples relating to 200 twin pregnancies and 600 singleton control pregnancies matched for gestational age and duration of storage. Free α-hCG values are, on average, 1·66 times greater in twin pregnancies than in singleton pregnancies (95 per cent confidence interval 1·56–1·76). If maternal serum free α-hCG is used in screening for Down's syndrome, values in twin pregnancies can be adjusted using this result so that screening can be performed in twin pregnancies as well as in singleton pregnancies.     

Is maternal alpha-fetoprotein screening still of value in a low-risk area for neural tube defects?

Estimation of maternal serum alpha-fetoprotein (AFP) was used as a screening method for the detection of neural tube defects (NTDs) in 6344 women over three years. Of 88 (1.4 per cent) who had one or more serum AFP levels equal to, or greater than, 2.5 multiples of the median (MoM) for the relevant gestational age, 43 (0.68 per cent) underwent amniocentesis. There were eight NTDs. Four of these were screened by serum AFP, and all cases of spina bifida had serum AFP levels greater than 3.0 MoM, including one small open defect which was not seen on ultrasound. The other four cases of NTD, which were not screened, were identified by ultrasound. Of 64 singleton pregnancies 32 (50 per cent) had serum AFP levels between 2.5 and 3.0 MoM, and low birthweight (⪕2500 g) occurred in 29 per cent. Because of improvements in ultrasound techniques and the apparent falling incidence of NTD, the role of serum AFP as the primary screening procedure should be regularly reviewed. Effective screening is dependent on mothers booking early.     

Genetic diagnosis by chorionic villus sampling before 8 gestational weeks: Efficiency,reliability, and risks on 317 completed pregnancies

Transabdominal chorionic villus sampling (TA-CVS) was attempted in 328 high-risk pregnancies at 6–7 weeks of gestation. Sampling was feasible in 97.7 per cent of cases; chorionic tissue specimens of more than 10 mg were obtained in 94.4 per cent ofcases at the first needle insertion and in 100 per cent after a second attempt. Fetal karyotyping succeeded in 99.4 per cent of cases, while no diagnostic failures were reported in enzymatic and DNA analyses. Fetal loss rate in the first 4 weeks after CVS was significantly higher than in the later CVS series (7.2 vs. 2.5 per cent), but 50 per cent of losses were observed within 2 weeks in cases of inviable aneuploidies. A high incidence of severe limb abnormalities (1.6 per cent) was detected in pregnancies intended to continue, confirming the aetiological role of early CVS. Unclear visualization of the placental limits and poor control of the needle path are thought to be the main reasons for the vascular disruption of the chorionic plate, and thereby hypoxic embryo tissue damage. A better selection of cases, together with high-resolution vaginal ultrasound visualization, and analytical techniques requiring a minimal amount of tissue should avoid any teratogenic effect of early CVS.     

The clinical implementation of non-invasive prenatal diagnosis for single-gene disorders: challenges and progress made

Recently, we have witnessed the rapid translation into clinical practice of non-invasive prenatal testing for the common aneuploidies, most notably within the United States and China. This represents a lucrative market with testing being driven by companies developing and offering their services. These tests are currently aimed at women with high/medium-risk pregnancies identified by serum screening and/or ultrasound scanning. Uptake has been impressive, albeit limited to the commercial sector. However, non-invasive prenatal diagnosis (NIPD) for single-gene disorders has attracted less interest, no doubt because this represents a much smaller market opportunity and in the majority of cases has to be provided on a bespoke, patient or disease-specific basis. The methods and workflows are labour-intensive and not readily scalable. Nonetheless, there exists a significant need for NIPD of single-gene disorders, and the continuing advances in technology and data analysis should facilitate the expansion of the NIPD test repertoire. Here, we review the progress that has been made to date, the different methods and platform technologies, the technical challenges, and assess how new developments may be applied to extend testing to a wider range of genetic disorders. © 2013 John Wiley & Sons, Ltd.     

Population screening for gestational hypertensive disorders using maternal,fetal and placental characteristics: A population-based prospective cohort study

Objective

To determine screening performance of maternal, fetal and placental characteristics for selecting pregnancies at risk of gestational hypertension and preeclampsia in a low-risk multi-ethnic population.

Method

In a prospective population-based cohort among 7124 pregnant women, we collected maternal characteristics including body mass index, ethnicity, parity, smoking and blood pressure in early-pregnancy. Fetal characteristics included second and third trimester estimated fetal weight and sex determined by ultrasound. Placental characteristics included first and second trimester placental growth factor concentrations and second and third trimester uterine artery resistance indices.

Results

Maternal characteristics provided the best screening result for gestational hypertension (area-under-the-curve [AUC] 0.79 [95% Confidence interval {CI} 0.76-0.81]) with 40% sensitivity at 90% specificity. For preeclampsia, the maternal characteristics model led to a screening performance of AUC 0.74 (95% CI 0.70-0.78) with 33% sensitivity at 90% specificity. Addition of second and third trimester placental ultrasound characteristics only improved screening performance for preeclampsia (AUC 0.78 [95% CI 0.75-0.82], with 48% sensitivity at 90% specificity).

Conclusion

Routinely measured maternal characteristics, known at the start of pregnancy, can be used in screening for pregnancies at risk of gestational hypertension or preeclampsia within a low-risk multi-ethnic population. Addition of combined second and third trimester placental ultrasound characteristics only improved screening for preeclampsia.

     

Four-marker serum screening for Down's syndrome

The value of measuring the separate sub-units of human chorionic gonadotrophin (free α-hCG and free β-hCG) instead of total hCG together with alpha-fetoprotein (AFP) and unconjugated oestriol (uE₃) was examined to determine the effect on the performance of serum screening for Down's syndrome between 15 and 22 weeks of pregnancy. The study was based on stored serum samples relating to 75 singleton pregnancies with fetal Down's syndrome and 367 unaffected singleton pregnancies, matched for maternal age, gestational age, and duration of storage of the serum sample, supplemented by data from 970 white women with unaffected pregnancies. Using the four serum markers AFP, uE₃, free β-hCG, and free α-hCG, in addition to maternal age, 65 per cent of Down's syndrome pregnancies were detected for a 5 per cent false-positive rate compared with 59 per cent with the conventional triple test (AFP, uE₃, total hCG with maternal age). If gestation was based on an ultrasound scan examination, the detection rate was 72 per cent using the four serum markers compared with 67 per cent with the triple test. As an alternative illustration, if the detection rate was kept at 60 per cent and gestation was estimated by an ultrasound scan examination the four-marker test reduced the false-positive rate by one-third from 3 per cent using the triple test to 2 per cent with the four-marker test. Screening performance was hardly affected by adjusting marker levels for maternal weight. The four-marker test is, both from a medical and from a financial perspective, the most effective method of prenatal screening for Down's syndrome suitable for routine use.     

Fetal curarization for prenatal magnetic resonance imaging

Fetal magnetic resonance (MR) imaging was performed at 33 weeks of gestation for investigation of a posterior fossa abnormality found at ultrasound screening. Fetal movements were abolished by vecuronium injected under ultrasound guidance into the umbilical vein. MR images showed atrophy of the left cerebellar lobe with cisternal dilatation. These were confirmed postnatally by CT scan.