prenatal screening for down syndrome - sm journals · [1]. down syndrome is the leading cause of...

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Gr upSMPrenatal Screening for Down Syndrome

INTRODUCTIONDown syndrome ([DS] trisomy 21) is the most common cause of intellectual disability

worldwide, affects approximately 1:500 pregnancies and is seen in 1:800 to 1:1,000 live births [1]. Down syndrome is the leading cause of prenatal chromosome abnormalities, accounting for 53% of all reported chromosome conditions. Genetic prenatal diagnosis (PD) for DS, since its introduction in the late 1960s, has evolved significantly. In order to limit the number of invasive procedures and improved detection rate. The majority of public current screening programs [2] for DS generally combine initial non-invasive risk screening strategies. Conventionally, prenatal testing (screening or invasive) for Down syndrome has been offered to women aged 35 or above because they are at a higher risk than younger women. However, the detection rate is as low as 30% while the rate of invasive testing can be as high as 30%. The American College of Obstetrics and Gynecology (ACOG) recommends that all pregnant women [3], regardless of their age, be offered screening for DS. Consequently, the routine offer of medical tests to pregnant women is usually a two-tier procedure, in many Western countries with a public health setting. This approach can detect 85% of Down syndrome at an invasive testing rate of 5%. The introduction

Shell Fean Wong1*1Department of Maternal Fetal Medicine, University of University of Hong Kong, Hong Kong

*Corresponding author: Shell Fean Wong, Department of Maternal Fetal Medicine, University of Hong Kong, Pok Fu Lam Road, Hong Kong, Email: [email protected]

Published Date: May 25, 2016


of new genomics based Non-Invasive Prenatal Testing (NIPT) using cff DNA (cell free fetal DNA) screening which is currently not publicly funded in most jurisdictions [4]. These tests have the potential to offer earlier results during pregnancy and to substantially reduce the number of invasive procedures [5-7]. In this review, we look at the historical evolvement of PD for DS, from the past to future.

SCREENING: PAST TO PRESENT Maternal Age & Risk Factors

In the early 1980s, maternal age was effectively the only screening tool available for detection of DS and invasive diagnostic tests were offered to all women aged 35 years and above. These tests were only offered to women younger than 35 years if there was known family history of the disorder [8]. However this approach was inappropriate and unsustainable for numerous reasons. Firstly, maternal age alone is not an effective screening test as it has a DR of less than 35%, meaning that most fetuses with DS were undetected and many women with unaffected fetuses were subjected to unnecessary invasive testing [9]. Secondly, as the average maternal age was beginning to rise, resources to perform invasive testing for all these women were unavailable [9,10]. In the early 1970s, about 5% of pregnant women were aged 35 years or more, and this group contained about 30% of the total number of fetuses with trisomy 21. Therefore, screening on the basis of maternal age, with a cut-off of 35 years to define the high-risk group, was associated with a 5% screen-positive rate (also referred to as false-positive rate, because the vast majority of fetuses in this group are normal) and a detection rate of 30%. In the subsequent years, in developed countries there was an overall tendency for women to get pregnant at an older age, so that now about 20% of pregnant women are 35 years or older and this group contains about 50% of the total number of fetuses with trisomy 21.

If a woman has a previously affected child, or either one of the couple carries balanced structural rearrangements of chromosome 21, she is at high risk of carrying a fetus with Down syndrome. A direct invasive test is recommended for these women. NIPT may also be an option. Other family history of Down syndrome, unless associated with a translocation involving chromosome 21, is usually not associated with a significantly increased risk to warrant direct invasive tests.

Maternal Serum Screening

The initial opportunity to improve screening arose in 1984, when several studies identified an association between low Alpha-Fetoprotein (AFP) levels (around a 25% reduction) in maternal serum and DS [11-13]. AFP is a large serum glycoprotein produced by both the yolk sac and the fetal liver, and is considered to function in a similar way as albumin in adults [14]. DiMaio, et al. identified that using a cut-off for risk at which 5% of women under 35 are offered invasive testing, around 25-30% of pregnancies in which the fetus has DS will be detected using AFP serum biomarker alone [12]. The identification of this marker for DS detection was a serendipitous


scientific discovery, initially raised AFP levels were used to identify pregnancies that were potentially affected by fetal neural tube defects, particularly anencephaly, and it was only during this cohort that the link between low AFP levels and an increased incidence of DS was identified. Now AFP is used clinically worldwide for screening of DS after the first trimester as one of the biochemical serum markers used in the quadruple test.

Since then, various pregnancy associated maternal serum markers for DS have been evaluated. Pregnancies with DS are associated with altered maternal serum concentrations of various fetoplacental products, including AFP, free β-hCG, inhibin A and unconjugated Estriol (uE3) and PAPP-A [13-19].

In screening using maternal serum biochemical markers, the measured concentration of the markers is converted into a Multiple of the Median (MoM) of unaffected pregnancies at the same gestation. The Gaussian distributions of log10 (MoM) in trisomy 21 and unaffected pregnancies are then derived, and the ratio of the heights of the distributions at a particular MoM, which is the likelihood ratio for trisomy 21, is used to modify the a priori maternal age-related risk to derive the patient-specific risk.

Second trimester

Early attempts at incorporating maternal serum markers into screening for aneuploidies focused on the second trimester of pregnancy and demonstrated a substantial improvement in detection rates of DS, compared with screening by maternal age. At a false-positive rate of 5%, the detection rate improves from 30% in screening by maternal age alone to 60 to 65% by combining maternal age with serum AFP and free β-hCG (double test), 65 to 70% with the addition of uE3 (triple test) and 70 to 75% with the addition of inhibin A (quadruple test) [20-22]. If intact hCG rather than free β-hCG is used, the detection rates are reduced by about 5%.

First trimester

In the last decade biochemical testing has moved to the first trimester because when this is combined with the ultrasound marker of fetal Nuchal Translucency (NT) thickness, the performance of screening is superior to second-trimester screening. In trisomy 21 pregnancies, the maternal serum concentration of free β-hCG is about twice as high and PAPP-A is reduced to half compared with euploid pregnancies. The measured serum concentrations of these placental products are affected by maternal characteristics, including racial origin, weight, smoking and method of conception (artificial reproductive technology) as well as the machine and reagents used for the analysis. Consequently, in the calculation of risk for aneuploidies using these products it is necessary to take into account the effects of these maternal variables in defining MoMs before comparing affected and unaffected pregnancies [23]. In euploid pregnancies, the average adjusted value for both free β-hCG and PAPP-A is 1.0 MoM at all gestations, whereas in trisomy 21 the average free β-hCG is 2.0 MoM and the average PAPP-A is 0.5 MoM and they both increase with gestation.


In screening for DS by maternal age and serum free β-hCG and PAPP-A, the detection rate is about 65% for a false-positive rate of 5%. The performance is better at 9 to 10 weeks than at 13 weeks because the difference in PAPP-A between trisomic and euploid pregnancies is greater in earlier gestations [23-26]. Although the difference in free β-hCG between trisomic and euploid pregnancies increases with gestation, the magnitude of the difference is smaller than that of the opposite relation of PAPP-A.

Screening by Ultrasound

Second trimester genetic sonogram

The genetic sonogram is a systematic algorithm combining multiple individual ultrasound markers during the second trimester to improve DS risk assessment. In the second trimester scan, each chromosomal defect has its own syndromal pattern of detectable abnormalities [27-28]. For example, trisomy 21 is associated with cardiac defects, duodenal atresia, nasal hypoplasia, increased nuchal fold and prenasal thickness, intracardiac echogenic foci, and echogenic bowel, mild hydronephrosis, shortening of the femur and more so of the humerus, sandal gap and mid-phalanx hypoplasia of the fifth finger and widened iliac angle [29]. The absence of any marker on a second trimester scan conveys a 60-80% reduction in prior risk of DS based on advanced maternal age or serum screen risk. On the other hand, risk is adjusted in the presence of multiple sonographic markers by multiplying age-related risk by the product of the respective ultrasound markers’ likelihood ratios.

Although this concept was applied in high risk referral populations [30], a large meta-analysis concluded that sonographic markers are not of practical value in the low-risk population probably due to the variability in obtaining and interpreting these makers, operator experience, sonographic equipment and quality control [31]. Therefore, a genetic sonogram is not recommended as the primary screening method for fetal Down syndrome. Its use is mainly for women who want to reduce the need of invasive tests following a positive integrated test or second trimester serum test. Furthermore, most of these studies were performed on Caucasian subjects. Ethnic variations in these markers, such as the humerus length and prevalence of echogenic intracardiac focus in normal fetuses must be taken into consideration. Caution is needed when applying these markers to the other ethnicities.

First Trimester

Screening by fetal nuchal translucency thickness: Extensive research in the last 20 years has established that the measurement of fetal NT thickness provides effective and early screening for trisomy 21 and other major aneuploidies [32-34]. The optimal gestational age for measurement of fetal NT is 11 to 13 weeks and 6 days. The minimum fetal Crown–Rump Length (CRL) should be 45 mm and the maximum 84 mm. The lower limit is selected to allow the sonographic diagnosis of many major fetal abnormalities, which would have otherwise been missed, and the upper limit is such to provide women with affected fetuses the option of an earlier and safer form of


termination. Fetal NT can be measured either by transabdominal or transvaginal sonography and the results are similar. The ability to achieve a reliable measurement of NT is dependent on appropriate training of sonographers, adherence to a standard ultrasound technique in order to achieve uniformity of results among different operators.

The main advantage of first trimester over second trimester screening is that it allows earlier prenatal diagnosis (provided that chorionic villus sampling is available) and thus earlier pregnancy termination if aneuploidy is detected. However, approximately 9% of all DS fetuses viable in the first trimester are lost spontaneously before the second trimester, [35] and thus early detection may lead to some unnecessary invasive diagnostic and pregnancy termination procedures.

Fetal NT increases with CRL and therefore it is essential to take gestation into account when determining whether a given NT thickness is increased. There are essentially two approaches to quantifying the deviation of NT from the normal median. One approach is to subtract the normal median from the NT measurement and to produce a deviation in millimeters referred to as delta NT [36-37]. The other approach is to divide NT by the normal median to produce a MoM value [38]. In the calculation of patient-specific risks for trisomy 21, the a priori maternal age-related risk is multiplied by the likelihood ratio for a measured NT, which is the ratio of the heights of distributions of measurements in trisomy 21 and unaffected pregnancies. Recently, a new approach has been proposed for quantifying the deviation in the measured NT from the normal. This is based on the observation that in both aneuploid and euploid pregnancies, fetal NT follows two distributions, one which is CRL dependent and another which is CRL independent [39]. In this mixture model, the distribution in which NT increases with CRL is observed in about 95% of euploid fetuses and 5% with DS. The median CRL-independent NT was 2.0 mm for the euploid group and 3.4 for DS.

Several prospective interventional studies in hundreds of thousands of pregnancies have demonstrated that firstly, fetal NT is successfully measured in more than 99% of cases, secondly, the risk of chromosomal abnormalities increases with both maternal age and fetal NT thickness and thirdly, in pregnancies with low fetal NT the maternal age-related risk is decreased. For a 5% false-positive rate, fetal NT screening identifies 75 to 80% of fetuses with DS and other major aneuploidies [40].

Screening by fetal NT thickness and serum biochemistry: There is no significant association between fetal NT and maternal serum free β-hCG or PAPP-A in either trisomy 21 or euploid pregnancies, and therefore the ultrasononographic and biochemical markers can be combined to provide more effective screening than either method individually (41-43). Several prospective interventional studies in many thousands of pregnancies have demonstrated that for a 5% false-positive rate, the first-trimester combined test identifies about 90% of trisomy 21 pregnancies [44- 49].


There are 2 available options; either combined the ultrasound & blood test at the same setting or in 2 separate occasions. One option in first-trimester combined screening for trisomy 21 is to perform bio-chemical and ultrasonographic testing as well as to counsel women in One-Stop Clinics for Assessment of Risk (OSCAR) [45,50]. The ideal gestation for OSCAR is 12 weeks because the aim of the first-trimester scan is not just to screen for trisomy 21 but also to diagnose an increasing number of fetal malformations, and in this respect the ability to visualize fetal anatomy is best at 12 weeks [51]. The detection rate of trisomy 21 with OSCAR at 12 weeks is about 90% at a false-positive rate of 5%.

An alternative strategy for first-trimester combined screening is for biochemical testing and ultrasound scanning to be carried out in two separate visits, with the first done at 9 to 10 weeks and the second at 12 weeks [52-54]. It has been estimated that this approach would improve the detection rate from 90% to 93 to 94%. A third option would be to perform the scan at 12 weeks and optimize the performance of biochemical testing by measuring PAPP-A at 9 weeks and free β-hCG at the time of the scan at 12 weeks or even later with an estimated detection rate of 95%. The cost and patient acceptability of the alternative policies of first trimester testing will depend on the existing infrastructure of antenatal care. The potential advantage of two- or three-stage screening in terms of detection rate may be eroded by the likely increased non-compliance with the additional steps.

Additional first-trimester sonographic markers: In addition to NT, other highly sensitive and specific first-trimester sonographic markers of DS are absence of the nasal bone, increased impedance to flow in the ductus venosus and tricuspid regurgitation. Absence of the nasal bone, reversed a wave in the ductus venosus and tricuspid regurgitation are observed in about 60, 66 and 55% of fetuses with DS and in 2.5, 3.0 and 1.0%, respectively, of euploid fetuses [55-61]. Assessment of each of these ultrasound markers can be incorporated into first-trimester combined screening by maternal age, fetal NT and serum free β-hCG and PAPP-A resulting in improvement of the performance of screening with an increase in detection rate to 93 to 96% and a decrease in false-positive rate to 2.5% [61-63]. A similar performance of screening can be achieved by a contingent policy in which first-stage screening by maternal age, fetal NT and serum free β -hCG and PAPPA is offered to all cases. Patients with a risk of 1 in 50 or more are considered to be screen positive and those with a risk of less than 1 in 1000 are screen negative.

Combination & Two Stage Tests

Integrated first and second trimester test

For women who wants to choose a more sensitive screening or to reduce the false positive rate (thus reducing the risk of procedure related fetal loss), a two staged integrated first and second trimester test, combining NT and all the first and second trimester serum markers, can theoretically achieve a detection rate of around 90% with a false positive rate of around 2% [21]. However, the risk estimate will only be available after the second trimester test is completed. Furthermore, some women may miss the second part of the screening test.


Step-wise sequential screening: This means women undergo a first trimester screening first. Based on this result, they are classified into three risk categories: high, intermediate and low risk. Only those at high risk will be offered definitive diagnosis by CVS; those determined to be at low risk will have no further screening and those at intermediate risk will go on to the second trimester screening test [34,64]. It was calculated that for an overall false positive rate of 5%, 94% Down syndrome detection rates can be achieved, with 70% of the cases detected in the first trimester, and only 15% of women requiring a second trimester test [64].

The principles of sequential contingent screening can be applied to a two-stage test, all completed in the first trimester. Instead of waiting for a second trimester serum test, women with intermediate risk can be offered further first-trimester ultrasound assessment for presence or absence of the fetal nasal bone, increased resistance to flow in the ductus venosus and for triscupid regurgitation [65]. A recently described first-trimester sonographic marker of trisomy 21 is increased flow in the fetal hepatic artery [66,67]. This marker is also likely to find an application in the assessment of the intermediate risk group after first-stage combined screening. Patients with a positive secondary ultrasound marker will be offered CVS, whereas those with absence of these markers will be considered screened negative. This approach can still identify more than 90% of affected fetuses while reducing the false positive rate to 2-3% [65]. However, ultrasound assessment of these markers is technically demanding. This approach cannot be widely applied.

Patients with the intermediate risk of 1 in 51 to 1 in 1000, which constitutes about 15% of the total population, have second-stage screening with nasal bone, ductus venosus or tricuspid blood flow which modifies their first-stage risk. If the adjusted risk is 1 in 100 or more, the patients are considered to be screen positive and those with a risk of less than 1 in 100 are screen negative [65].

An alternative first-trimester contingent screening policy consists of maternal serum biochemistry in all pregnancies followed by fetal NT only in those with an intermediate risk after biochemical testing. Studies examining the potential performance of such policy have estimated that the detection rates and false-positive rates would be 80 to 90% and 4 to 6%, respectively, and measurement of fetal NT would be necessary in only 20 to 40% of cases [68-71]. The advantage of biochemical testing as a first-stage policy relies on its apparent simplicity.

Fetal DNA: Non-invasive Prenatal Testing (NIPT)

The presence of cffDNA (cell free fetal DNA) released by the fetus into the circulation of its mother was reported in 1997 [72]. By analyzing this source of fetal genetic material, obtainable through a blood sample from a pregnant woman, cffDNA screening has been developed [73] and proposed as potentially changing the approach to PD for DS and other conditions such as other significant trisomies for chromosomes 13 and 18 [73-75]. Cell free fetal DNA is thought to be derived from the placenta, which undergoes continual remodelling throughout pregnancy [76]. Once a mother delivers, cffDNA is rapidly cleared. This means that any fetal DNA present originates from the current rather than previous pregnancies [77].


‘Next generation’ sequencing generates masses of DNA sequence data at relatively low cost. It is the most common method used to identify numeric chromosomal abnormalities. This relatively new technology is used to define the relative proportion of DNA fragments originating from different chromosomes. If a fetus is trisomic, then the proportion of DNA fragments related to that specific chromosome will be increased relative to other chromosomes. A positive result is reported when the number of fragments of an individual chromosome is more than three standard deviations from the mean of reference chromosomes. The absolute difference in the proportion of fragments is very small as the abnormal fetal genome is diluted by normal maternal genome. However, the advantage of sequencing technology is that millions of fragments are counted, allowing these small differences to be resolved. After sequencing, bioinformatic analysis determines whether there is evidence of a numeric chromosomal abnormality [73-75].

NIPT does not necessarily differentiate between fragments of maternal and fetal DNA, although at least 4% of cffDNA needs to be fetal in origin to be able to resolve differences between euploid and trisomic samples [75]. Approximately 2-5% of samples will have lower levels of cffDNA and in these circumstances it is not possible to report a result.

It is important to recognize that NIPT is not a diagnostic test, but a very effective screening test. Recent meta-analysis showed that the pooled sensitivity was 99.3% (95% CI 98.9% to 99.6%) for DS. The pooled specificity was 99.9% (99.9% to 100%). In 100 000 pregnancies in the general obstetric population we would expect 417 cases of DS to be detected by NIPT, with 94 false positive results. Sensitivity was lower in twin than singleton pregnancies, reduced by 9% for DS. Pooled sensitivity was also lower in the first trimester of pregnancy, in studies in the general obstetric population, and in cohort studies with consecutive enrolment [78].

Most studies were performed in ‘high-risk’ populations (advanced maternal age, previous history, abnormal ultrasound, increased risk after routine screening) but there are also data that support testing in an unselected population [79,80]. Based on sensitivity and specificity results, likelihood ratios can be calculated - a positive result effectively increases a patient’s a priori risk of having an affected pregnancy almost 1000-fold and a negative test reduces a patient’s risk 250-fold.

Professional groups have stated that cffDNA screening could be an option for prenatal fetal aneuploidy detection. Professional groups [4,5,81-83] have published clinical recommendations regarding the use of cfDNA screening for fetal aneuploidy detection. Together, the American Congress of Obstetricians and Gynecologists, the National Coalition for Health Professional Education in Genetics and the National Society of Genetic Counselors in the USA, the International Society for Prenatal Diagnosis, the Society of Obstetricians and Gynecologists of Canada, and the California Technology Assessment Forum stated that cfDNA screening could be an option for fetal aneuploidy detection in high-risk pregnancies after non-directive counseling by qualified personnel. It is obvious that a shift has already started in the routine care of pregnancies [4,84].


The American College of Medical Genetics and Genomics has not limited their recommendation to women at high risk for fetal chromosome abnormality in accordance with the health insurer Blue Cross [85,86]. New clinical advances in cfDNA screening may necessitate clinical policy modifications for considering new potential uses and applications for other conditions [87-90].

Test failures

The rate of analytic failure (failure of the cffDNA testing) ranged from 0% to 12.7% [91] and among 5789 pregnancies with resampling, 803 (13.9%) also failed the repeat cffDNA testing. There were five papers in this review that reported indeterminate results (results in a range defined as neither positive nor negative) for DS [92-95] ranging from 0% (0/2042) to 11.1% (5/45). In the study with no indeterminate results they used eight-plex testing, and where the initial score was indeterminate they repeated using one-plex which corrected any indeterminate results. There is some evidence that the rate of test failure is higher when gestational age is lower, and in trisomic pregnancies. Pergament, et al [96] found that failure rate at <9 weeks was 26/95 (27.4%), between 9.0 and 9.9 weeks was 6/50 (12.0%), and more than 10 weeks was 53/900 (5.9%). The same study found aneuploidy incidence was increased (20/86 (23.3%)) in samples that did not return a result when compared with the aneuploidy incidence in samples with a cffDNA testing result (105/966 (10.9%), p=0.004). Norton, et al [97] did not find an association between test failure and gestational age in 18 510 women between 10 and 14 weeks gestation, but found that the prevalence of aneuploidy in the group with test failure (1 in 38 (2.7%)) was higher than the prevalence of 1 in 236 (0.4%) in the overall cohort (p<0.001) [78].

Including test failures in an intention to diagnose analysis in the meta-analysis decreased sensitivity estimates by 1.7% for DS and decreased specificity estimates by nearly 2%. Excluding test failures from the calculations of test accuracy may have caused overestimation of accuracy. Similarly in the subgroup analysis sensitivity estimates were lower by 6.1% for DS for cohort studies with consecutive sampling in comparison to all other studies. Test accuracy did not appear to differ systematically between different technology, or by publication year. Estimates of test sensitivity were higher in high-risk populations, in studies including pregnancies in the second and third trimester, and in singleton pregnancies. In high-risk populations, defined in a variety of ways, pooled sensitivity estimates were 1.4% higher than in the general obstetric population for DS. Twin pregnancies had 8.3% lower sensitivity estimates than singletons for DS.

Application of NIPT in current practice

When the new test is compared to combined first trimester screening purely on sensitivity and specificity results, NIPT appears to be better. Combined first trimester screening does, however, provide other information. Ultrasound screening allows accurate dating of the pregnancy, recognition of structural (rather than chromosomal) anomalies and identification of multiple pregnancies. It may also identify pregnancies at risk of other adverse obstetric outcomes such as pre-eclampsia and fetal growth restriction. NIPT can be offered to pregnant women together with


an ultrasound and NT assessment. This will provide early detection of major fetal anomalies and identified fetus with increased NT.

At present, most national and international guidelines suggest that non-invasive prenatal testing should be restricted to women with a high risk of an affected pregnancy [81-84]. NIPT has only been validated in women with an increased risk of fetal aneuploidies; according to the American Congress of Obstetricians and Gynecologists (ACOG), risk factors include: 1) maternal age 35 years or older at delivery; 2) fetal ultrasonographic findings indicating an increased risk of aneuploidy; 3) history of a prior pregnancy with a trisomy; 4) positive test result for aneuploidy, including first trimester, sequential, or integrated screen, or a quadruple screen; or 5) a parental balanced Robertsonian translocation with increased risk of fetal trisomy 13 or trisomy 21 [4]. Although it is highly specific, the prevalence of a Down syndrome pregnancy is low in women who have not had previous screening or who are considered to have a low risk after prenatal screening. The positive predictive value (proportion of positive results that are true positives) in an unselected population is at best 50%. In other words, one in two positive test results in low-risk women are likely to be false positives – and test results need to be confirmed by amniocentesis before any intervention.

If NIPT is restricted to patients who have previously been screened for Down syndrome and found to have a high risk, then a positive result will imply that the fetus is indeed affected, and a negative result will imply the fetus is unlikely to be affected. False positive results have been reported and all positive results should be confirmed by amniocentesis. Using quantitative fluorescent polymerase chain reaction, the result can be confirmed within 24 hours. If women have not had any previous screening or are considered to be low risk after prenatal screening, confirmation of a positive result will be more important.

Options for screening strategies

As non-invasive prenatal testing is so sensitive, one option is to offer this test to women who have had a high-risk result from combined first trimester screening. It has been suggested that this may lead to 80% reduction in the current invasive testing rate. While this will improve the overall specificity of the screening strategy, it does not take advantage of the high sensitivity of non-invasive prenatal testing for the population as a whole.

An alternative strategy is to offer all women noninvasive prenatal testing and an ultrasound scan. However, this will increase the cost of the screening program quite significantly.

A third strategy would be to change the reporting strategy of combined first trimester screening to identify three groups; high intermediate and low risk groups. For high-risk group (>1 in 50) these women can be offered invasive testing. For an intermediate-risk group (1 in 50 to 1 in 1000), these women can be advised about the availability of, and offered, NIPT. Those in the low-risk group (<1 in 1000) reassured. These pregnant women may choose to have NIPT at their own expense. Such strategy may increase detection rate with a lower false positive rate.


In the presence of NIPT, it is not known whether the counsellor or obstetrician should inform all pregnant women about this test. It is uncertain whether the counsellor may be liable if DS was not detected and the pregnant woman was not previously informed of this test.

OTHER MATTERSMultiple Pregnancy

NT measurement which is fetus-specific, seems to be a promising method of screening in these women [98]. The addition of maternal serum analytes may improve the sensitivity of first trimester screening. A 80% detection rate of Down syndrome for a 5% false positive rate using NT and first trimester serum makers has been reported [99]. However, a large discordance between the NT in a pair of monochorionic twins is more likely to be an early sign of twin-twin transfusion, rather than a risk of chromosomal aneuploidy.

The data regarding the performance of NIPT in twin pregnancy are limited [100,101]. Although small studies show that this screening is accurate, the ACOG proposes that larger prospective studies are needed before this test can be offered to women with twin pregnancy. Thus, ACOG concludes that NIPT is not recommended for twin pregnancy. There are no available data on higher order multiples. On meta-analysis on sensitivity, the sensitivity of NIPT was also lower in twin than singleton pregnancies, reduced by 9% for DS (91%) [78].

Ethics

The main justification for offering prenatal genetic diagnosis is the promotion of reproductive autonomy and informed decision-making by pregnant women. Reproductive autonomy is of utmost importance in PD [102,103]. Since there is no cure for DS, efficient prenatal screening and diagnosis enable couples to make informed reproductive choices [104,105]. PD differs from other diagnostic procedures in medicine insofar as most conditions tested cannot be cured or substantially alleviated and the only option following an undesired result is therefore to decide whether to accept the child’s condition and prepare for his or her birth or to terminate the pregnancy. Consequently, the main reason for offering prenatal genetic testing is to enhance the reproductive autonomy of the pregnant woman and/or the couple [102,104] Invasive genetic diagnosis, second-trimester ultrasound screening [106], and first-trimester risk assessment [107], were considered controversial at the time of their introduction but have since become important autonomy-enhancing strategies in obstetric practice [108,109]. In a practice bulletin published in 2007, the American College of Obstetrics and Gynecology recommended that prenatal screening for aneuploidy should be offered to all women, regardless of age [3]. Nevertheless, choices may be more limited in the context of a publicly funded screening program where costs are a constraining factor than in settings where women have to fully pay themselves [110,111].


DISCUSSION Genetic PD for DS, since its introduction in the late 1960s, has evolved significantly. In order

to limit the number of invasive procedures and improved detection rate. The majority of public current screening programs [2] for DS generally combine initial non-invasive risk screening strategies. Conventionally, prenatal testing (screening or invasive) for Down syndrome has been offered to women aged 35 or above because they are at a higher risk than younger women. However, the detection rate is as low as 30% while the rate of invasive testing can be as high as 30%. Currently, most screening programs offer a two-tier procedure, combination of maternal age, ultrasound scan and biochemical analytes, in many Western countries with a public health setting. This approach can detect 85% of Down syndrome at an invasive testing rate of 5%. The introduction of new genomics-based NIPT using cffDNA screening which is currently not publicly funded in most jurisdictions (Table 1) [4]. These tests have the potential to offer earlier results during pregnancy and to substantially reduce the number of invasive procedures [5-7].

Table 1: Performance of different methods of screening for DS.

Screening Method Detection rate (%) FPR (%)

Maternal Age 30 5

First trimester

MA + fetal NT 75 5

MA+ serum free beta-hCG and PAPP-A 60-70 5

MA + NT + free beta-hCG and PAPP-A (combined test) 85-90 5

Combined test + nasal bone or tricuspid flow or ductus venosus flow 93-96 2.5

Second Trimester

MA + serum AFP, hCG (double test) 55–60 5

MA + serum AFP, free β-hCG (double test) 60–65 5

MA + serum AFP, hCG, uE3 (triple test) 60–65 5

MA + serum AFP, free β-hCG, uE3 (triple test) 65–70 5

MA + serum AFP, hCG, uE3, inhibin A (quadruple test) 65–70 5

MA + serum AFP, free β-hCG, uE3, inhibin A (quadruple test) 70–75 5

MA + NT + PAPP-A (11-13 weeks) + quadruple test 90–94 5

NIPT 99 1-2

The implications for policymakers and clinicians are that NIPT using cffDNA has very high sensitivity and specificity, and can contribute to screening programs for DS. NIPT is highly accurate but not perfect. This is particularly true when considering populations in terms of risk and gestational age. Test performance is better in high-risk populations as well as in studies including pregnancies in the second and third trimester [78]. Consideration of NIPT as a screening test for the general obstetric population primarily tested in the first trimester of pregnancy has to take into account the lower sensitivity of NIPT in this population. There is also some indication that higher maternal weight, and conception by In Vitro Fertilisation (IVF) are potential predictors of


NIPT test failure [112] suggesting that NIPT may not work equally well in all subpopulations. We consider that for this reason NIPT should not be regarded as a diagnostic test and that confirmation of a positive NIPT result by amniocentesis or CVS is necessary to make a diagnosis of trisomy. This is essential if parents are considering termination of pregnancy on the basis of trisomy, because in the general obstetric population as many as 20% of positive NIPT results for DS may be false positive. Because the source of cffDNA is the placenta, confined placental mosaicism may explain a proportion of discordant NIPT results [113]. Furthermore, early fetal demise of an affected fetus [112,113] and unknown chromosomal abnormality in the mother [113,114] can lead to false positive results. Finally, in some cases discordance between NIPT and fetal karyotype results might be due to lab error. The role of low fetal fraction as contributor to false positive or false negative results is unclear: Zhang, et al [114] reported no major influence, whereas Quezada, et al [115] found lower fetal fractions in discordant than in those with concordant results.

Communicating to clinicians and patients that this genetic test is not perfect will be key for safe implementation, and pretest and post-test information provision and counselling for positive and negative NIPT results should be given careful consideration. The NIPT test may be particularly attractive to parents who are not considering termination of pregnancy, but who would like to know in advance if their pregnancy is affected by a trisomy, since NIPT gives broadly accurate results, without the slightly increased risk of miscarriage associated with invasive procedures such as amniocentesis and CVS. The final consideration for implementation is the range of test failure rates from <1% to >12%, with some evidence that presence of trisomy may be a predictor of test failure. Quality assurance to minimize test failures would minimize delays due to repeated testing, which may be a priority for pregnant women. However, if the test failure is due to insufficient fetal fraction a retest is also likely to fail.

Currently, this test is used worldwide, mostly provided directly by private providers rather than national health systems. Further research into how the test is being interpreted and understood by clinicians and pregnant women will be key to understanding the balance of benefits and harms from the provision of the test. In particular, how this understanding leads to decisions about whether to continue the pregnancy, and whether this may be influenced by how the test is presented to parents both by companies, and by clinicians. Finally if it is implemented into national screening programs, keeping accurate records of outcomes and test failures would enable the test performance to be evaluated in practice.

In future when the cost of NIPT is lower and more researches are available on the general population. NIPT can be offered to more pregnant women. This will reduce the false positive and unnecessary invasive procedures. NIPT should not be used as a diagnostic test, as there is a discrepancy between the test positive result and the final diagnostic result.


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