What is the PrenatalSAFE® test?

PrenatalSAFE® is a non-invasive prenatal test (NIPT) which evaluates if a foetus has a higher or lower chance of having certain chromosomal abnormality, such as three copies of chromosome 21 instead of the normal two that causes Down’s Syndrome.

Who can have the PrenatalSAFE® test?

Pregnant women with a gestational age of at least 10 weeks. The test can be offered to both single and twin pregnancies, obtained either by natural conception or by assisted reproductive techniques, such as In vitro fertilization (IVF). For non-identical twins or a vanishing twin pregnancy, test sensitivity is reduced to 95%.

The PrenatalSAFE® cannot be used:

• For pregnancies greater than twins (e.g., Triplets)

• If the mother has the chromosomal abnormality being investigated

How does the PrenatalSAFE® test work?

During pregnancy, the placenta sheds the baby’s (foetal) DNA into the mother’s blood. A blood sample is taken from the expectant mother. The circulating foetal DNA is isolated from the plasma component of the maternal blood and is then evaluated to determine the risk of having a baby with a chromosomal abnormality (e.g., three copies or trisomy for chromosome 21 that causes Down’s syndrome). It is important to note, that the PrenatalSAFE® test is a screening test and is not a diagnostic test. If a highrisk (detected) result is obtained, follow-up diagnostic testing is required.

What chromosomal disorders does the PrenatalSAFE® test investigate?

• The PrenatalSAFE® 3 test investigates the presence of Trisomy 21 (Down’s Syndrome), Trisomy 18 (Edward’s Syndrome) and Trisomy 13 (Patau’s syndrome)

• The PrenatalSAFE® 5 test investigate the presence of Trisomy 21, Trisomy 18, Trisomy 13 and aneuploidies (abnormal numbers) of the sex chromosomes e.g., Turner’s Syndrome,

• Both tests have the option to report foetal sex

Does Foetal sex have to be reported with the PrenatalSAFE® test and how is it reported?

If a sex chromosome aneuploidy is tested and detected, the foetal sex will be disclosed when the abnormality is reported. If no sex chromosome aneuploidy is detected, foetal sex will not be reported unless requested. In twin pregnancies, one result will be reported for both foetuses. In all other cases, the foetal sex is stated as male, or female based on the presence or absence of the Y chromosome.

What is Trisomy 21 (Down’s Syndrome)

Trisomy 21 is the presence of an extra copy of chromosome 21 and is also known as Down’s syndrome. It is the most common genetic cause of learning disability and is estimated to be present in 1/700 births. However, the chance that a foetus has Down’s Syndrome increases as maternal age increases.

What is Trisomy 18 (Edward’s Syndrome)

Trisomy 18 is caused by the presence of an extra copy of chromosome 18 and is also known as Edward’s Syndrome. It is linked to a high risk of miscarriage and causes severe mental disability. Newborns with trisomy 18 often have congenital heart defects, as well as other pathological conditions that reduce their life expectancy. It is estimated that trisomy 18 is present in 1/5,000 births. However, the chance increases as maternal age increases.

What is Trisomy 13 (Patau’s Syndrome)

Trisomy 13 is caused by the presence of an extra copy of chromosome 13 and is also known as Patau’s syndrome. It is linked to a high risk of miscarriage. Newborns with trisomy 13 may have heart defects and other pathological conditions, which means that survival beyond the age of one year is rare. It is estimated that trisomy 13 is present in 1/16,000 births. However, the chance increases as maternal age increases.

What are Sex Chromosome Aneuploidies?

Usually, females have two copies of the X Chromosome (XX) and males have one X and one Y chromosome (XY). A sex chromosome aneuploidy is an abnormal number of sex chromosomes. The most frequent sex Chromosome aneuploidy is Monosomy X, which is clinically known as Turner Syndrome (XO). Females with Turner Syndrome have a short stature and reproductive difficulties in association with other possible clinical manifestations such as congenital heart disease, neurosensory hypoacusis, renal abnormalities such as horseshoe kidney and predisposition to some autoimmune diseases (thyroiditis, diabetes, coeliac diseases, etc.). Other sex chromosome aneuploidies that can be found with the test are Klinefelter syndrome (XXY) and Jacobs syndrome (XYY)

What type of results are expected from the PrenatalSAFE® test?

DETECTED

Aneuploidy detected: indicates that the test has produced a result consistent with an aneuploidy of one (or more) of the chromosomes investigated. This result. however, is not diagnostic and therefore does not guarantee that the foetus has such a condition. The recommended follow-up is an invasive prenatal diagnosis test, such as chorionic villus sampling (CVS) or amniocentesis.

NOT - DETECTED

Aneuploidy not detected: indicates that the test did not detect any aneuploidies for the chromosomes investigated. This result, however, does not guarantee that the foetus does not carry these abnormalities. See test limitations.

Inconclusive result (approximately 1%)

In such cases, the expectant mother will be asked for a new blood sample in order to repeat the test. The re-test might still produce an “Inconclusive result” and if that happens invasive prenatal diagnosis is recommended because there have been reports in the scientific literature suggesting an increase in the incidence of foetal aneuploidies in samples with inconclusive results, for example due to a low foetal fraction.

How reliable is the PrenatalSAFE® test?

Sensitivity, specificity, and limitations: Pertile et al., 2021. Concordance of the test for trisomy 13, trisomy 18 and trisomy 21 is 99.9% for singleton pregnancies (Pertile et al., 2021). For foetal sex, concordance was 100% for both ‘female’ and ‘male’ (based on newborn physical exam) and for XX and XY (based on cytogenetic results). For sex chromosome aneuploidies, concordance was 90.5% for monosomy X (19/21), 100% for XXY (23/23), and 91.7% for XYY (11/12) (Pertile et al. 2021 – DOI: 10.1093/clinchem/hvab067)

What are the limitations of the PrenatalSAFE® test?

Non-invasive prenatal testing of circulating cell-free foetal DNA isolated from a sample of maternal blood is a screening test and although this test is very accurate, the results are not diagnostic and need to be evaluated in the context of the patient’s clinical and family history. In addition, the test is not a substitute for invasive prenatal diagnosis (CVS or amniocentesis). The test has been validated on single and twin pregnancies, monozygotic or dizygotic, starting from week 10 of gestation. The test cannot rule out the presence of all foetal chromosomal abnormalities. PrenatalSafe® 3 evaluates only the aneuploidies of chromosomes 13, 18, 21, PrenatalSafe® 5 also evaluates the aneuploidies of the sex chromosomes (X and Y). However, PrenatalSafe® 5 cannot be performed on dichorionic twin pregnancies. PrenatalSafe® cannot detect balanced chromosomal rearrangements, foetal and/or placental chromosomal mosaicism (i.e., the presence of two cell lines with a different chromosomal arrangement) or any malformations/defects outside the scope of the test. In dichorionic twin pregnancies it is not possible to distinguish the condition of the individual foetuses or to report on aneuploidies of the sex chromosomes. However, the presence/absence of the Y chromosome can be detected. If the Y chromosome is detected, it is not possible to discern whether only one or both foetuses are male. In pregnancies that have started as twins or multiples, followed by the loss of one or more foetuses with reabsorption of the gestational chamber (vanishing twin), cell-free foetal DNA of the lost foetus may also be present in the maternal blood. This could interfere with the quality of the results, with consequent false positive results if the cause of the abortion was due to the presence of chromosomal aneuploidies affecting one of the chromosomes investigated in the above-mentioned foetus. Similarly, there may be a mismatch in sex results (e.g., male sex diagnosis, where the presence of the Y chromosome originates from the DNA of the lost foetus). Malignancy/tumour in the expectant mother could produce false positive test results. The test is based on the quantification of cell-free foetal DNA fragments circulating in maternal blood, which are placental in origin. Placental chromosomal mosaicism (frequency: 1-2%), a condition where the chromosomal composition of the placenta differs from that of the actual foetus, may lead to false positive or false negative results, hence sensitivity and specificity of <100%. A false positive result occurs when the test detects a chromosomal aneuploidy (confined to the placenta) but the invasive diagnostic test does not. A false negative result occurs when the test does not detect a chromosomal aneuploidy but the invasive diagnostic test (e.g., performed after an abnormality is detected in a late-gestation ultrasound scan) does or a child is born with the chromosomal disorder. If the expectant mother has a chromosomal abnormality in a homogeneous or mosaic form, the test might lead to a false positive result and cannot give any information about the presence of the abnormality in the foetus. A "NOT-DETECTED” result significantly reduces the chances that the foetus has an aneuploidy at the level of the examined chromosomes, but it cannot guarantee that the chromosomes are actually normal or that the foetus is healthy. Due to the above limitations, in the event of a positive result, it is recommended to have a consultation with a geneticist and confirm the result by invasive testing. Pregnancies with abnormal ultrasound findings suggestive of foetal disease should undergo invasive prenatal diagnosis, such as molecular foetal karyotype on chorionic villi or amniotic fluid.