New promising methods of prenatal diagnostics

Non-invasive prenatal testing (NIPT) provides a new reliable method for early detection of the most common aneuploidies. The sensitivity and specificity of NIPT for the detection of fetal trisomy 21 are >99% with a false positive rate of 0.1-0.4%. Especially in women with increased risk for aneuploidies, e.g. advanced maternal age or increased risk in the first trimester test (ETT), NIPT can be useful. Qualified counseling of pregnant women as a basis for decision-making for any form of prenatal diagnostics is of central importance. In a very high-risk population and especially in cases of abnormal ultrasound findings, NIPT is not primarily indicated because approximately 30% of chromosomal defects are not detected by NIPT.

In Switzerland, the majority of pregnant women between the 11th and 14th week of pregnancy (SSW) are offered the so-called first trimester test (ETT), which allows a risk calculation for trisomy 21, 13 and 18 based on maternal age and two maternal serum parameters (beta-HCG and PAPP-A) in combination with sonographic nuchal fold measurement. Measurement of the nuchal fold, which may be widened in trisomies but also in other chromosomal aberrations, fetal syndromes, or malformations, is feasible when the fetal vertex length is 45-84 mm. With a sensitivity of about 90% and a false-positive rate of 3-5%, the most common numerical chromosomal abnormalities can be detected [1]. The costs of sonography as well as ETT are covered by health insurance in Switzerland. Most pregnant women take the option of sonography in the 11th-14th week of pregnancy. SSW and the ETT true (Fig. 1).

The maternity guidelines also specify a second fetal organ screening at 20-22 weeks gestation. SSW provided. In addition to malformations of the organs and fetal growth, the focus is also on so-called soft markers as indirect indicators of aneuploidies. Soft markers include hypoplastic nasal bone, aberrant right subclavian artery (ARSA), choroid plexus cysts, or “white spot” in the heart. Disadvantages of soft markers are their low sensitivity and high false positive rate [2]. A third ultrasound examination in the 30th-32nd week. This is common practice, since numerous fetal diseases, especially late growth retardation, amniotic fluid anomalies, but also malformations of the brain, urogenital and gastrointestinal tracts, can often only be diagnosed in the third trimester.

If the ETT results in a risk assessment that is greater than the average risk of a 35-year-old pregnant woman (1:300), or if sonographic abnormalities are found, the patient is usually recommended further diagnostic workup. For the detection of numerical and structural chromosomal abnormalities, invasive diagnosis by chorionic villus sampling or amniocentesis from the 11th or 16th week of pregnancy remains the gold standard.

The risk of miscarriage associated with invasive exploration is 0.5-1%, so the indication should be very strict and only after detailed patient education [3].

Non-invasive prenatal testing (NIPT) method

Today’s NIPTs are based on Dennis Lo’s discovery that cell-free fetal DNA can be detected in maternal plasma and serum [4,5]. Since 2012, NIPT have been available in Switzerland for testing the most common fetal aneuploidies. The costs of about 900-1300 CHF have to be paid by the patients themselves at the moment.

Approximately 10-13% of the cell-free DNA circulating in the maternal circulation consists of fetal (placental) DNA, which is released from the placenta into maternal blood throughout pregnancy and is undetectable within a few hours after birth. Using Next Generation Sequencing, also known as Massive Parallel Genomic Sequencing, these fragments of cell-free DNA can be sequenced within a few days.

Four different NIPTs are currently available in Switzerland (Tab. 1) . These offer testing for fetal trisomy 21, 13, 18 and sex determination. Whole Genome Sequencing (^PraenaTest®, ^Prendia®) amplifies and sequences the entire cell-free DNA (maternal and fetal cell-free DNA). After assigning the DNA fragments to the corresponding chromosome, the amount of DNA fragments is compared to a reference genome. In the case of fetal trisomy 21, a small difference in quantity is found in comparison to the reference, corresponding to the small fetal portion of the total cell-free DNA.

So-called Targeted Sequencing (^Harmony®), in which only fragments of specific target chromosomes (e.g. chromosome 21, 13 and 18) are amplified and sequenced, is a somewhat more cost-effective analysis.

Another method that can distinguish maternal from fetal DNA by sequencing single nucleotide polymorphisms (SNP) is based on the Panorama Test®. The SNP technology has the advantage that triploidies are detected, since it is not a pure comparison of the quantity of DNA. On the other hand, it is not suitable in situations where the fetus is genetically unrelated to the maternal leukocytes, e.g. after maternal bone marrow transplantation or after egg donation.

For twin pregnancies and sex chromosome disorders, overall experience with NIPT is still very limited, yet most companies offer them.

A minimum percentage of cell-free fetal DNA in maternal blood of 4-8% is required to obtain a meaningful result. Up to 5% of the analyses cannot be evaluated due to an insufficient amount of fetal DNA.

Role of NIPT in prenatal diagnostics.

An advantage of NIPT compared to ETT for the detection of aneuploidies is the significantly lower rate of false-positive results (0.1-0.4% vs. 3-5%). Also, the high sensitivity and specificity allow reliable detection of the three most common chromosomal disorders without risking invasive diagnostics. Sensitivity to detect trisomy 21 is 99% at >, 89% for trisomy 13, and 98% for trisomy 18 [6].

The positive predictive value (PPV) and thus the probability that a fetus with a positive test result actually has the disease depend crucially on the prevalence of the disease in the collective. The PPV is as high as 91% in high-risk collectives [7] and is much lower at 45% in the low-risk collective [8].

However, the negative predictive value is 100% even in the low-risk population, so that a fetal trisomy 21 can be largely excluded in the case of a negative result [8].

It should be noted, however, that NIPT cannot be classified as diagnostic tests. In practice, this means that an abnormal result must be confirmed by means of invasive diagnostics, e.g. before an abortion is performed.

Time window and analysis duration

Depending on the provider, a NIPT is possible from the ninth or tenth SSW until birth. The processing of the sample usually takes about ten working days, so that the tested person can expect a result after two weeks at the latest. Express processing within one week is also offered in some cases.

Who should get tested?

NIPTs are not recommended as general screening tests at this time. However, caring gynecologists and primary care physicians should now educate all pregnant women about the possibility of NIPT. The medical consultation is of central importance in this context [9]. In particular, the respective possibilities and limitations of the different methods and ethical considerations should play a role. Key points in counseling for NIPT are summarized in Table 2 and 3 and Figure 2.

The question of general NIPT screening as part of statutory prenatal care is being discussed nationally and internationally. Because general screening would result in substantial costs, two-stage screening based on the results of the ETT appears to be more cost-effective. However, in Switzerland, it is mainly the patients with a low or intermediate risk of fetal aneuploidies who undergo NIPT [6]. Thus, the need for increased certainty in the diagnosis of trisomy appears to be the most common reason for performing NIPT, regardless of risk calculation.

According to the ACOG, ISUOG, and SGGG guidelines, NIPT may be considered primarily in the following situations [10]:

Maternal age >35 years
Abnormal ETT with unremarkable sonography
Trisomy in a previous pregnancy
Balanced Robertsonian translocation of parents.

NIPT is not recommended for abnormal ultrasound findings such as markedly increased nuchal translucency or fetal anomaly because approximately 30% of chromosomal defects are not detected by NIPT [11,12]. In this case, invasive diagnostics are recommended. In this high-risk population, the delay of a definitive diagnosis by upstream NIPT should also be considered.

Counseling is different for parents who wish to continue the pregnancy in any case in the presence of a fetal anomaly, especially trisomy 21. Here, a NIPT can be a tool to prepare for a possible disease of the child without endangering the pregnancy. A decision tree, such as the one shown in Figure 2, can be helpful in counseling a pregnant patient.

Future of prenatal diagnostics

An expansion of the spectrum of chromosomal disorders that can be diagnosed by NIPT is necessary due to the rapid development of these technology to be expected. Already, structural chromosomal abnormalities, especially microdeletions, are being added to the wide range of analyses. Validation for the respective diseases is still pending.

It is conceivable that as NIPT analyses become faster and thus less expensive, serum biochemistry (beta-HCG and PAPP-A) will become obsolete, replacing ETT as a broad screening method for aneuploidies. Sonography in the 11th-14th decades. However, the first antenatal check-up with nuchal fold measurement and early organ diagnostics will continue to be an essential part of prenatal care.

The rapid medical progress in the field of prenatal diagnostics requires differentiated counseling of pregnant women. Referral to a university-based perinatal center for consultation on the current state of options can promote decision-making in terms of patient empowerment.

Literature:

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