All you need to know about Non-invasive prenatal testing
Integration of NIPT in transforming prenatal care for women
Introduction
Being a mother is the most beautiful experience ever. The journey to motherhood is a mix of joy, excitement and some unprecedented changes. To make this journey comfortable and safe for both the mother and her baby, regular checkups and clinical screening tests are performed. These tests include ultrasound scans and biochemical blood work spanning different stages of pregnancy. Normal results from these scans and tests are indicative of a healthy, normal and a low-risk pregnancy. However, out of range results summon further investigations to detect any underlying complications.
For example, elevated alpha- fetoprotein levels indicate risk for neural tube defects (NTDs) in the foetus (unborn offspring). Similarly, increased nuchal translucency (NT) noticed during the ultrasound scans, is indicative of Down syndrome. Pregnancies with such complications are categorized as high-risk pregnancies. However, these markers have high false positive rates and are best used to identify pregnancies that may be affected as opposed to are affected. Other factors such as advancing maternal age, positive family history of birth defects, smoking, cancer and diabetes may also indicate a high risk pregnancy.
Women with a high-risk pregnancy have been offered prenatal genetic screening for common chromosomal abnormalities since 1960, via invasive methods such as Chorionic Villus Sampling (CVS) and Amniocentesis. Aneuploidy (presence of incorrect number of chromosomes) is the most common chromosomal abnormality responsible for one-third of all miscarriages and affects 1 in every 300 livebirths.
High prevalence of aneuploidy and the drawbacks of invasive procedures orchestrated the demand for a test that is non-invasive, detects aneuploidy accurately and is low cost to be offered to every high-risk pregnant woman. Non-invasive prenatal testing (NIPT) came as an advancement which revolutionized the traditional approach to prenatal screening and diagnosis. It fulfilled all the requirements of being minimally invasive as it requires just a maternal blood draw, it provided accurate detection of fetal aneuploidies, it is low-cost and fast enough to be used as a screening test, reducing the need for invasive prenatal procedures in many cases
NIPT and its origin
Clinical inception of NIPT dates back to October 2011, when it could only be performed for singleton pregnancies. NIPT is now routinely available as a standard of prenatal care to women with high-risk pregnancies and is a frequent choice by women with low risk pregnancies as well.
NIPT mainly screens for trisomy (presence of an additional chromosome) for chromosome 21, 18 and 13. In 1959, it was first established that Trisomy 21 causes Down syndrome. Soon after in 1960, trisomy 18 and 13 were found to cause Edwards and Patau syndromes respectively. Down syndrome is the most common chromosomal abnormality, affecting 1 in every 800 live births. It is also one of the leading causes for mental retardation in school-aged children. Edwards and Patau syndromes are the second and third most prevalent chromosomal disorders respectively. Neonates (new born child) born with Edwards or Patau syndromes have shortened life expectancy of about a year with several medical complications.
With technology advancement and more research, few NIPT screens may also offer to include trisomy 16, trisomy 22, sex-chromosome aneuploidy, triploidy and micro-deletion analysis. A normal euploid human cell has 46 chromosomes: 22 pairs of normal chromosomes and 2 sex- chromosomes (XX or XY). However, an aneuploid cell may have one chromosome less or more than the normal 46 chromosomes. In the rare case of triploidy, a cell can have a complete extra set of chromosomes (46+23=69). Micro deletions as the name suggests are the small sized deletions which can occur in genes across the human genome.
NIPT leverages the presence of Cell-free DNA (cfDNA) in maternal blood to use as a specimen type. It is called cell free because it circulates freely in maternal blood without being encapsulated in a cell. cfDNA is extracted from the maternal plasma. Plasma is yellow in color and forms the largest component of blood. It is separated from red cell components of the blood by centrifugation at a high speed. cfDNA originates from trophoblast cells and extrudes into maternal circulation via apoptosis (removal of cells by programmed cell death). Trophoblast cells are the first type of cells to differentiate from a fertilized egg to form placenta. Their main role is to provide nutrition to the growing embryo.
Maternal DNA contains about 10% of cffDNA (cell-free fetal DNA), also known as the fetal fraction. Percentage of fetal fraction increases with advancing gestational age. cffDNA contains the genetic imprint of the baby, which is analyzed for the presence of any chromosomal abnormalities. Results from NIPT are reported as low-risk/high-risk for every analyzed aneuploidy with respective risk calculations stating the probabilities of carrying an aneuploidy. As NIPT is only a screening test, the high- risk results are confirmed with Amniocentesis or CVS.
Significance of NIPT over invasive techniques
A major advantage of NIPT over invasive procedures is that it can be performed as early as at 9–10 weeks of gestation. This allows enough time for detailed investigations, if required; fostering informed choice and decision making.
NIPT is a highly sensitive and specific test which can identify trisomy 21, 18 and 13 more accurately than the traditional approaches. It has been observed that NIPT can identify trisomy 21 positive cases with 99% accuracy.
Another huge advantage of NIPT is that it can be performed on a simple maternal peripheral blood draw sample, making it a low-risk, non-invasive technique which can be referred to any pregnant woman without any risk for complications. While, the invasive prenatal tests like Amniocentesis and Chorionic Villus Sampling (CVS) involve detailed procedures which have been associated with increased chances of miscarriages.
Although NIPT can’t replace the diagnostic prenatal testing, these advantages have made it an acceptable technique which can be offered as a clinical primary screening test resulting in concurrent fewer invasive tests, preventing 0.5–1% miscarriages associated with Amniocentesis and CVS.
Technologies used in NIPT
Most of the NIPT tests are based on Next Generation Sequencing (NGS). The use of NGS to sequence cfDNA marked the new era for NIPT. It is a fast, robust, accurate and cost-effective technology to perform NIPT.
NGS uses massively parallel sequencing (MPS) to analyze millions to billions of cfDNA strands spanning across the foetal genome simultaneously. One sequencing pool accommodates more than one specimen. Usually 48- 96 specimens are sequenced at once.
There are two approaches to NGS testing as well. First approach is called the whole genome approach where the aneuploidies are detected by sequencing the whole cfDNA in maternal plasma. While, the second approach is called the targeted approach where some selective regions on the chromosome which are required to reliably detect fetal trisomy 21,18,13 and sex chromosome aneuploidies are sequenced in parallel. Much of the extra data which is generated with the whole genome approach remains unused. So the switch to targeted approach significantly reduced the test cost as well as the run time, generating the relevant information which is useful for downstream analysis. Both the approaches have their pros and cons and the debate is on-going.
Another approach called the rolling circle replication technology which involves direct capture and labelling of cfDNA fragments which can then be counted for presence of chromosome 21, 18 and, 13 aneuploidies. This technology is both PCR and NGS free. It doesn’t generate tons of data but rather it is a very precise and accurate approach to target just these commonly found aneuploidies.
Conclusion
NIPT has revolutionized healthcare offered to pregnant women by saving them from the anxiety and inconvenience of undesirable invasive procedures. With advancing technology and cutting edge analysis software, NIPT is striding towards fewer false positive results, accurate aneuploidy detection at low fetal fractions and reduced per specimen cost.
NIPT provides autonomy to parents for making informed choices and decisions. However, it also opens avenue for being misused as a sex determination test, which has led to government interventions to ban direct-to-consumer NIPT offerings and in some cases, disclosure of sex only when sex chromosomal abnormalities are found.
Another topic of ongoing debate is the use & disclosure of the extra genomic information generated during the genome-wide NIPT scans, incidental findings and data storage.
Adequate pre-test counselling and respecting the rights for coming generations can help to overcome some of controversies. However, applications and use will vary across countries and various healthcare systems. In spite of some challenges and differences, the future will see NIPT as a routine standard care practice for pregnant women.
