1. What is Non-invasive Prenatal Testing/ Screening (NIPT/ NIPS)?
- A screening test during pregnancy to look for certain fetal chromosomal and genetic abnormalities;
- Involves taking blood from the pregnant women for analysis; the test itself does not post risks of miscarriage to the fetus and hence it is regarded as ‘non-invasive’;
- Currently the most sensitive and specific screening test for fetal aneuploidies
2. How can NIPT achieve its goal?
- DNA fragments (around 150-200 base pairs in size) are found in the pregnant women’s plasma. These cell-free DNA consist of those released from the mother’s cells (~80-90%) and those released from the placenta (10-20%). In majority (> 99%) of cases, the DNA in the placenta is identical to that of the fetus;
- Sequencing techniques are used to analyse the cell-free DNA levels;
- If the levels of the cell-free DNA for the specific chromosome or genetic condition are increased or decreased, there is a chance that the fetus is affected with that condition
3. What is fetal fraction in the report?
- ‘Fetal fraction’ refers to the percentage of placenta DNA among the total DNA levels in the plasma sample;
- Enough placental DNA must be present in the mother’s blood to allow reliable results. Most laboratories use a fetal fraction of 4% as the minimal requirement for the test to be valid;
- The fetal fraction usually increases with gestation. Hence, NIPT is usually arranged after 10 weeks of gestation;
- In case the fetal fraction is < 4%, no result can be generated and a further sampling at later gestation would be needed
4. What conditions can be tested in NIPT?
- Trisomy 21 (Downs syndrome), trisomy 18 (Edwards syndrome) and trisomy 13 (Patau syndrome) which are the 3 commonest chromosomal abnormalities;
- Commonly tested also for sex chromosome aneuploidies, such as XO (Turners syndrome), XXX (triple X syndrome), XXY (klinefelter syndrome) and XYY (Jacob syndrome); the fetal gender will then be known but the couple have the option of not displacing the gender in the report if they do not want to know;
- Also commonly tested for micro-deletion disorders including the 22q11.2 Deletion syndrome (De George syndrome) which carries a reported incidence of 1 in 2,000-3,000 livebirths. Other micro-deletion disorders commonly tested include Angelman syndrome, Prader-Willi syndrome, Cri-du-chat Syndrome, Langer-Giedion Syndrome, 1p36 Deletion syndrome and 2q33.1 Deletion syndrome. The later micro-deletion disorders are rare, with an incidence between 1 in 5,000 to 1 in 15,000 livebirths;
- With the advance of technology, screening for all 23 pairs of chromosomes and additional micro-deletions/ microduplications with lesion size > 3 MB is increasingly performed by various laboratories;
- Performance of NIPT for the common disorders (Based on safeT21 test)
Disorders | Incidence | Detection rate | False +ve rate |
T21 Downs syndrome | 1 in 900 | 99.65% | 0.03% |
T18 Edwards syndrome | 1 in 4,000 | >99.9% | 0.01% |
T13 Patau syndrome | 1 in 5,000 | >99.9% | 0.02% |
Sex chromosome aneuploidies | 1 in 1,500 | 99.82% | 0.16% |
De George syndrome | 1 in 2,000 | 94.1% | <0.01% |
5. What are the indications for NIPT?
- As secondary screening, i.e. after a positive Downs screening, either a first trimester combined NT and biochemistry screening (OSCAR) or a second trimester biochemical screening (Triple test)- the latter 2 tests have higher false positive rates compared with NIPT
- As primary screening- common choice in the private sector in Hong Kong
6. What are the limitations of NIPT?
6.1 ‘Fetal’ DNA are of placental in origin
- The so-called ‘fetal’ DNA in the maternal plasma are of placental in origin;
- In majority (> 99%) of cases, the DNA in the placenta is identical to that of the fetus;
- In a small percentage of pregnancies (~1-2%), the placenta showed a condition called ‘confined placental mosacism’ (CPM). It means that in the placenta, there are a combination of cells with chromosomal abnormality and other cells with normal karyotyping. This finding is only confined within the placenta while the fetus has normal karyotyping;
- CPM is believed to be one of the causes for false positive NIPT results
6.2 The issue of vanished twin
- Sometimes a pregnancy may begin as a twin pregnancy but then one of the twins has spontaneously reduced in the first trimester, leaving behind an ongoing singleton pregnancy;
- If this vanished twin has chromosomal abnormalities, the ‘fetal’ DNA released by this twin to the maternal circulation can cause false positive NIPT results;
- The presence of ‘fetal’ DNA from the vanished twin in the maternal circulation can be as long as 8-9 weeks. There was one report of prolonged persistence of DNA from the vanished twin for 15 weeks
6.3 The issue of maternal DNA
- In the pool of DNA in the mother’s plasma, 80-90% belong to the mother; the maternal DNA might also affect the NIPT results;
- If the mother has chromosomal or genetic abnormalities, it could affect the NIPT results, especially if the mother has the condition of mosacism, i.e. she has a combination of cells with chromosomal abnormality and normal karyotyping;
- Certain maternal autoimmune or haematological conditions may lead to release of background maternal DNA into the circulation, causing difficult interpretation of the data;
- Maternal uterine fibroids and occult malignancies have also been reported to lead to non-reportable NIPT results
6.4 Non-reportable results
- Overall, ~0.5-1% of NIPT cannot provide a result at the initial blood sampling;
- Causes include
- (i) low fetal fraction,
- (ii) borderline results,
- (iii) maternal factors including haematological diseases, autoimmune conditions, fibroids, malignancy, obesity,
- (iv) medication such as low molecular weight heparin and intravenous immunoglobulin. Sometimes no cause can be found;
- Depending on the situation, a second sample is usually drawn at a later gestation. However, some may not get a result in the end still. In that case, it is important to discuss with a specialist doctor on the alternate options of screening including detailed fetal morphology scan, alternate Downs screening or amniocentesis
6.5 False positive and false negative
- While NIPT is the most sensitive and specific screening method at present, it still carries false positive (abnormal results but normal fetus) and false negative (normal result but abnormal fetus) rates;
- NIPT is NOT a diagnostic test and cannot exclude all chromosomal or genetic abnormalities
7. How is invasive prenatal diagnosis (chorionic villous sampling or amniocentesis) different from NIPT?
- Invasive prenatal diagnosis involves obtaining fetal or placental cells by invasive procedure for prenatal diagnosis. Commonly performed invasive procedures include chorionic villous sampling (CVS) or amniocentesis;
- Invasive prenatal diagnosis aims to confirm or refute the diagnosis of the suspected fetal chromosomal or genetic abnormalities;
- Currently, the conditions screened by NIPT have remained more limited compared with those that can be examined from the placental or fetal cells obtained from CVS or amniocentesis;
- The invasive prenatal diagnosis procedures, however, carries a small risk of miscarriage. This risk has to be weighed balance against the chance of the fetal abnormalities tested
8. What is the difference between NIPT and OSCAR tests?
- OSCAR refers to the first trimester combined nuchal translucency (NT) and biochemical screening. It involves ultrasound to determine the NT of the fetus and taking the pregnant woman’s blood for assay of two hormones. A risk for having Downs syndrome, Edwards syndrome and Patau syndrome will be generated based on factors of the pregnant woman’s age, the NT thickness and the levels of the two hormones;
- NIPT has a higher detection rates and much lower false positive rate compared with OSCAR. NIPT can also test for more abnormalities
9. Should nuchal translucency (NT) still have to be checked if NIPT is opted for instead of OSCAR?
- NT is an important screening test not just for fetal chromosomal abnormalities, but also for fetal structural defects in particular congenital heart diseases. NT is also a good screening test for some genetic conditions;
- It has been shown that among fetuses with an increase in NT > 99th centile in the first trimester, use of NIPT alone will miss up to 19 % of genetic abnormalities;
- Hence, a NT scan is still recommended even if NIPT is chosen
10. What does it mean if NIPT shows an abnormal result?
- An abnormal NIPT report suggests that further investigation(s) would be needed to diagnose or refute the suspected abnormality. It is important to realise that among those with abnormal results, not all fetuses are affected; the implications for the different categories of abnormalities might be different;
- For trisomy 21, the chance of the fetus being affected is high with an abnormal result. This is particularly the case if abnormal ultrasound features such as increase in NT is noted on a first trimester ultrasound. However, ultrasound features are only found in ~60% of Downs fetuses in the first trimester and hence absence of these ultrasound markers cannot exclude the diagnosis. An early diagnosis by CVS between 11-14 weeks is possible and is particularly recommended if ultrasound markers are present. However, it is subjected to a rare but potential error due to CPM. An amniocentesis after 16 weeks will avoid this potential problem but apart from the anxiety of waiting, there is an implication on the method of termination of pregnancy if the abnormality is confirmed;
- For trisomies 13 and 18, over 95% of affected fetuses will have ultrasound features. Hence, it is recommended to have an early fetal morphology scan. If abnormal features are identified, a CVS immediately is recommended for rapid diagnosis and an early termination of pregnancy can be offered if the diagnosis is confirmed. However, if no abnormal ultrasound features can be seen, an amniocentesis should be performed after 16 weeks. An amniocentesis helps to avoid error due to CPM as discussed above;
- For sex chromosome aneuploidies (SCA), there are several possibilities including
- (i) full fetal SCA,
- (ii)mosaic SCA,
- (iii)confined placenta mosacism (CPM) and
- (iv)maternal mosaic SCA
Some fetuses with XO (Turner syndrome) can have increase in NT (cystic hygroma) but most other SCA do not have ultrasound features. Hence, unless cystic hygroma is noted on early ultrasound when a CVS should be arranged, an amniocentesis is recommended for a clear-cut diagnosis;
- For other chromosomal defects and deletion disorders, the situation is different for each specific condition. Hence, the decision should be made case by case;
- The implications for each abnormality is different. Therefore, it is important to see a specialist doctor in this field for better counselling and diagnosis
11. The positive predictive values (PPV) of NIPT for different abnormalities
- Most laboratories quoted data on sensitivity (detection rate) and false positive rate. These two are related to the test itself for a condition or disease. However, for the couple given an abnormal report, the positive predictive value (PPV) is a more relevant information.
- PPV refers the chance (%) of truly abnormal when the test result is abnormal. Interestingly, the PPV is related to the prevalence (i.e. how common) of the disease tested. For more common conditions, the PPV will be higher and for rare conditions, the PPV will be much lower.
- In the literature, the PPV of NIPT for the different categories of abnormalities have been reported. They are presented in the table below. The data serve to provide ballpark figures to assist in counselling
Disorders | PPV (overall) |
T21 Downs syndrome | 80-95% |
T18 Edwards syndrome | 60-80% |
T13 Patau syndrome | 15-65% |
Sex chromosome aneuploidies | 35-60% |
Microdeletion disorders | 30-75% |
Other chromosomal anomalies | 20-30% |
12. Conclusions
NIPT is a rapid developing screening in the last 10 years. With the advance of technology, expanded screening for more and more chromosomal or genetic disorders is made possible. As a result, increasingly more chromosomal or genetic disorders missed in the past will be picked up through this non-invasive screening method. However, it is important to realise that this test has remained a screening tool and has its limitation. Prenatal counselling before and after the test is therefore very important, in particular when faced with an abnormal result.
