Non invasive prenatal diagnosis of fetal aneuploidy using cell free fetal DNA

Renga Barbara

CLINILAB, street Manzoni 418, Perugia, 06135 Italy

mail: barbararenga@yahoo.it

phone: +39 075393323


The discovery of cell free fetal DNA in maternal plasma has stimulated a rapid development of non-invasive prenatal testing. The recent advent of massively parallel sequencing has allowed the analysis of circulating cell-free fetal DNA to be performed with unprecedented sensitivity and precision. It is thus expected that plasma DNA-based non invasive prenatal testing will play an increasingly important role in the future of obstetric care. The present review summarizes recent advances in non invasive prenatal testing using cell free fetal DNA. The importance of genetic counseling, the current guidelines for the use of cffDNA screening in pregnancy,  as well as specific maternal conditions that can affect the performance of non invasive prenatal testing are also discussed in this review.


Approximately 3% to 5% of pregnancies are complicated by birth defects or genetic disorders. (1) Chromosomal abnormalities are present in approximately 1 in 150 live births, (2) and congenital malformations remain the leading cause of infant death and a leading cause of childhood death. (3) These chromosomal abnormalities include aneuploidy (defined as having one or more extra or missing chromosomes), translocations, duplications, and deletions. The most common chromosomal disorder is trisomy 21 (Down syndrome), with an incidence of 1 per 800 live births. (4) Trisomy 13 (Patau syndrome) and 18 (Edwards syndrome) can also result in live births, though with a significantly lower incidence (1 per 7500 live births for Trisomy 13 and 1 per 15000 live births for Trisomy 13 respectively). (2,4) Sex chromosome aneuploidies are less common than autosomal aneuploidies. (4) The only known viable monosomy is monosomy X (Turner syndrome), with an incidence of 1 per 5000 girls. Risk of aneuploidy increases with maternal (5); however other factors might also influence patients’ risk in any given pregnancy, including the presence of birth defects or soft markers on ultrasound and past obstetric history, particularly if it is notable for a prior pregnancy affected by aneuploidy or another genetic disorder. A past family history of aneuploidy increases current pregnancy risk of aneuploidy, especially if a parent is a balanced robertsonian translocation carrier, though most cases are sporadic and secondary to chromosomal nondisjunction. (2, 4)

Screening for chromosomal aneuploidy in pregnancy began in the 1960s with maternal age as the only available marker. Although antiquated, maternal age remains the most frequent method for identifying women at increased risk for fetal chromosome abnormalities and remains a determinant of who should be offered prenatal diagnostic testing (5).

Recent technological advances in methods to identify numerical and structural chromosome abnormalities and point mutations, such as array-based copy-number analysis, also known as chromosomal microarray analysis (CMA), and next-generation sequencing (NGS) has enabled scientists to use cell free fetal DNA (cffDNA) for a whole new generation of non invasive detection of fetal genetic abnormalities (6-15). The state of the art in the field of cffDNA based screening of aneuploidy, also called non invasive prenatal testing (NIPT), as well as the pivotal role of maternal health on NIPT have been discussed in this review.

Current guidelines for the use of cffDNA screening in pregnancy

Numerous professional societies, including the SMFM (the Society for Maternal Fetal Medicine), and ACOG (the American Congress of Obstetricians and Gynecologists) have published recommendations regarding the appropriate application of cffDNA screening in pregnancy (16-18). Initial guidelines (2011) from all major societies recommended limiting the use of cffDNA screening to only those pregnancies at increased risk of aneuploidy, where increased risk was typically defined as age 35 years or older at the time of delivery, ultrasound findings that suggest an increased risk of aneuploidy, positive first or second trimester screening tests for aneuploidy, a history of a previous pregnancy with trisomy, or a parental balanced Robertsonian translocation that increases the risk of trisomy 21 or 13. (16) In a 2015 committed opinion, ACOG and SMFM acknowledged that patients may choose cffDNA screening regardless of their risk status, and in those cases, the patients should receive pretest counseling to discuss the risks and benefits of screening (17). The SMFM also recommended that cffDNA microdeletion screening should not be routinely offered (17). Indeed, screening for conditions with such low prevalence will inevitably result in false positive results. Finally, both SMFM and ACOG do not recommended the use of cffDNA in multiple gestations as preliminary studies suggested that despite this form of screening is accurate results of larger prospective studies are mandatory before changing this recommendation (16-18).


Cell free fetal DNA for prenatal diagnosis

The majority of cffDNA in plasma derived from haematopoietic cells that release fragments of DNA during cell turnover, but a large variety of solid organs also contribute to the circulating plasma pool (19). In the non-pregnant state, the genomic profile in plasma cffDNA reflects the individual’s karyotype. However, during pregnancy, cffDNA from the placenta is also released into the maternal plasma and this can be used to detect fetal chromosomal or genetic abnormalities. The presence of fetal DNA in maternal plasma was first reported in 1997 using conventional polymerase chain reaction techniques to identify Y chromosome-specific DNA sequences (20). Fetal fraction (i.e. the average proportion of total cffDNA in maternal blood that derives from the placenta) increases with gestational age but is reliably greater than 10% as early as 10 weeks’ gestation. Notably, fetal fraction of greater than 4% is required for reliable analysis. Placenta-derived DNA fragments are shorter in length (143 base pairs) than maternally derived DNA fragments (166 base pairs). This difference in the fragment length profile is now being employed to improve the accuracy of prenatal aneuploidy screening (21).

In October 2011, the first commercial laboratory began offering NIPT using cell-free DNA for common aneuploidy conditions. Since that time, the utilization of this technique has markedly increased due to the very high reported detection rate and low false-positive rate, particularly for Down syndrome screening (99.7 %) (22). It is important to note that detection rates for trisomy 18, 13, and sex chromosome abnormalities (monosomy X) are significantly lower than for trisomy 21 (98.2, 99.0 and 95.8 % respectively) (22, 23).

At the present, cell-free DNA for aneuploidy screening is only recommended for women with high risk of aneuploidy. High risk is defined by American College of Obstetricians and Gynecologists (ACOG) and the Society for Maternal-Fetal Medicine (SMFM) as maternal age of 35 years or older at delivery, history of prior pregnancy with trisomy, ultrasonographic findings indicating an increased aneuploidy risk, high risk first-trimester or second trimester aneuploidy screening results, or parental balanced Robertsonian translocation with increased risk for trisomy 13 or 21 (24). Noteworthy, all patients that undergo a cffDNA test should understand the screening nature of the test as the test only detects common aneuploidies, the potential for unexpected results or failure to obtain test results, the possibility of obtain false positive results, and finally, the options for diagnostic testing. Patients should be counseled of these possibilities before proceeding with screening. A possible source of false positive results include: confined placental mosaicism, vanishing twin (In uterine demise of an aneuploid twin fetus), maternal aneuploidy (i.e. 47, XXX), maternal mosaicism, maternal copy number variations, maternal malignancy, prior organ transplant.

In this field of perinatal medicine, technology is rapidly expanding to offer more and more clinical data. For example, some companies offer screening for microdeletion conditions, such as 22q deletion and 1p36 deletion. Although most microdeletions are individually rare, the 22q microdeletion is reported to occur in about 1/3000 live births. The use of cell-free DNA as a screening tool for these rare microdeletion and microduplication conditions has not been validated by any well-designed study. A diagnosis should not be made or excluded by this testing and should be identified only by CVS or amniocentesis. The importance of pre-test counseling and discussing possible results and implications for prenatally ascertained information should be reviewed (25, 26).

Genomic approaches  for aneuploidy detection

There are a number of different NIPT techonolgies developed for aneuploidy detection, including Massive parallel sequencing (MPS), Chromosome-selective (or targeted) sequencing (CSS) and single nucleotide polymorphism (SNP) based sequencing.

            Massive parallel sequencing

The Massive parallel sequencing (MPS) process used in prenatal diagnosis is based on the random or shotgun sequencing of DNA molecules in maternal plasma (27-28).

With the advent of MPS, an even more powerful method for counting millions or even billions of DNA molecules has become available, as it  would allow virtually any DNA molecule contained within the plasma sample to be sequenced and to contribute towards the counting process (29-30).

MPS-based methods for the NIPT of fetal chromosomal aneuploidies have been available clinically since 2011 in a number of countries, including those in North America, Asia and Europe. Apart from abnormalities involving the entire chromosome, MPS-based analysis of maternal plasma DNA has also been shown to be useful for detecting Down syndrome caused by Robertsonian translocation as well as microdeletions and microduplications (30-32).

Statistically, MPS methods calculate the standard deviation of the expected count from each chromosome and allocate a “z-score” for each chromosome. If the number of DNA fragments from

chromosome 21 in the test sample is more than 3 standard deviations away from expected (i.e. z-score >3), this is considered a high risk result for trisomy 21 (33).

Chromosome-selective (or targeted) sequencing

In the CSS approach, the plasma DNA fragments undergo an enrichment process, so that only preselected fragments are read and sequenced, rather than the entire plasma mixture which contains DNA from all chromosomes (34, 35). The enrichment step involves a PCR-based reaction that amplifies segments of DNA that are unique to the targeted chromosome. For example, chromosome 21 represent only 1.3 % of the entire genome. A more efficiently strategy is to selectively sequence genomic regions of chromosome of interest so that sequencing power is focused on genomic regions of diagnostic interest. This would help to reduce the cost and to increase the throughput (34, 35).

The CSS approach has been stated to require approximately one million mappable reads per sample, which appears to be less than that required by the whole genome sequencing approach. One disadvantage of this approach is that off target chromosomal aneuploidies will not be detected .

The statistical method for CSS differs from that of MPS. Indeed, CSS uses the woman’s prior risk of aneuploidy (based on maternal and gestational age), the target chromosome counts, and the fetal fraction to calculate a final risk using an odds ratio approach. The result is provided as a risk result (rather than a categorical result), with a risk of 1 in 100 or greater being defined as high risk (34, 35).

SNP based approach

To date, two described SNP approaches incorporate genotype information:

Allele ratios. The first genotypic approach amplifies and sequences SNPs, counting the number of observed maternal and fetal alleles and generating an allele ratio between a chromosome-of-interest and reference chromosome to determine copy number (36). This method is similar to the counting methods in that it identifies samples as aneuploid when the allele ratio falls beyond an established threshold. The requirement for a reference chromosome also means this approach is incapable of detecting triploidy, and as this study only focused on chromosome 21, it is not clear whether this method will accurately detect copy number imbalances at other chromosomes. This method has not yet been developed commercially or validated in a clinical trial.

Genotype analysis with maximum likelihood estimation. The second approach uses targeted amplification of SNPs followed by NGS and sophisticated informatics analysis to identify fetal chromosomal copy number (37).This method differs from the targeted sequencing approach in that it specifically targets SNPs instead of non-polymorphic regions and uses a genotype-based analytic method rather than a counting approach to detect fetal aneuploidy. The method employs a massively multiplexed PCR amplification targeting 19,488 SNPs in a single reaction—at least two orders of magnitude greater than other reports of multiplexed PCRs (34).

While previous NIPT methods demonstrate high sensitivity and specificity when detecting autosomal trisomies, none accomplishes similar levels of accuracy with sex chromosome aneuploidy detection. This is partially attributed to variable amplification that results from different guanosine-cytosine (GC) levels in chromosomes 13 and X as compared to chromosomes 21 and 18, and is particularly problematic for methods that require a reference chromosome (38-40).

Since the SNP method analyzes the relative amount of alleles at polymorphic loci and does not utilize a reference chromosome, it is not subject to issues with amplification variation. Thus, it is expected to have consistent sensitivities across all regions interrogated. Indeed, clinical data indicates sensitivities of >99% for Trisomy 21, Trisomy 18, and Trisomy 13 (38, 41). It is also the only method that is capable of detecting, and has successfully reported detection of, triploidy (42).  The commercially available SNP NIPT test based on this methodology also routinely reports copy number for the X and Y chromosomes. Overall, this method reports an overall no-call rate of <6% for copy number calling at all five chromosomes implicated in at-birth abnormalities (13, 18, 21, X, and Y) (41, 42). However, the targeted approaches (CSS or SNP-based NIPT) will not detect off-target abnormalities, limiting the potential for incidental findings.

Influence of mathernal biology on NIPT performance

A small proportion of samples submitted to NIPT will not return an interpretable results. The most common reason for these “no call” results is a relatively low amount of placental cfDNA in maternal blood, or low fetal fraction. However, It is now apparent that some maternal influences can also interfere with NIPT performance. In particular, any condition which increases maternal cell turnover without increasing placental cell turnover could theoretically reduce the fetal fraction and then increase NIPT failure rates. For example, maternal obesity is a condition which associates with a two fold increase in plasma levels of cDNA of maternal origin without significant difference in the cfDNA of fetal origin (43, 47). Several studies demonstrated that obese women may have lower fetal fractions due to the increased apoptosis and necrosis in their adipose tissue (43, 47).

Autoimmune disease is also a known cause of increased cell turnover and non-pregnant patients with systemic lupus erythematosus (SLE) have elevated levels of circulating cfDNA as a recent study utilizing massively parallel sequencing has characterized in detail the abnormalities in plasma cfDNA in SLE patients (44, 47).

Several case reports showed a strong correlation between maternal malignancy with NIPT performance. A 2012 case report first described maternal malignancy as a cause of discordant NIPT results (45, 47). Subsequently, a case series obtained from a total cohort of 125,426 women provided details on 10 women that had discordant NIPT results due to an undiagnosed maternal cancer (prevalence 1 in 12,500) (46, 47). The cancer types included lymphoma, leukaemia, colorectal and anal cancers. Of the 39 cases in the total cohort that had an NIPT result indicating multiple aneuploidies, seven were due to asymptomatic maternal malignancies. However, not all neoplastic causes of abnormal NIPT results are malignant; some may be due to benign tumours such as uterine leiomyomata. In light of this accumulating data on maternal cancers, patients should be counseled of these possibilities before proceeding with screening.

Other statistically significant associations of maternal factors with fetal fraction have been reported, including maternal aneuplody (e.g. 47, XXX), maternal mosaicism (e.g. 45X/46XX), maternal copy number variations, prior organ transplant (may cause false positive result for male fetal sex), maternal medications, smoking and pre-existing hypertension) (46, 47).


Cell free fetal DNA based screening for the common autosomal aneuploidies is the most superior screening method for trisomy 21 to date, with unprecedented sensitivity and specificity. However, women who choose cell-free fetal DNA technology should be counseled that the test remains a screening test for aneuploidy at this time and that microdeletion testing continues to have poor positive predictive values due to the low prevalence of these disorders. Clinicians should be aware of specific maternal conditions that may affect the performance of NIPT, such as obesity, active autoimmune disease, neoplasms or mosaicism. In conclusion, the rapid development of NIPT using cell free fetal DNA in maternal plasma might impact clinical practice of prenatal diagnosis within a few years. This technology will be expected to make prenatal testing safer and more informative in the future.


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