Special Feature

Fetal DNA Testing for Aneuploidy Detection

By John C. Hobbins, MD, Professor of Obstetrics and Gynecology, University of Colorado School of Medicine, Aurora, Colorado, is Associate Editor for OB/GYN Clinical Alert.

Dr. Hobbins reports no financial relationships relevant to this field of study.

Synopsis: A new prenatal test that indirectly quantifies fetal DNA in maternal plasma has been shown to be highly sensitive in detecting trisomy 21 and has the potential to identify other forms of fetal aneuploidy.


Currently, screening for fetal aneuploidy involves a variety of first-line methods that put into play combinations of maternal serum analytes and ultrasound exams in the first and second trimester, with invasive procedures such as chorionic villus sampling (CVS) and amniocentesis being reserved for those at higher risk by history, age, or worrisome results from first-line screening. As will be discussed, each screening combination comes with its own predictive accuracy and now patients and providers can choose from a buffet of screening options, including going right to the main course (amniocentesis or CVS) if one wants 100% accuracy at a small risk of fetal loss (we quote a 1 in 400 risk with amniocentesis and about a 1 in 100 risk for CVS).

Development of a Fetal DNA Test for Aneuploidy Detection

It has been known for many years that fetal cells appear in small numbers in the maternal circulation, and for at least 2 decades investigators have been trying to separate these fetal cells from maternal blood for karyotyping. This has been a very difficult and expensive task since there is only about one fetal cell in each cc of maternal blood.

Fortunately, in 1997 the quest took a new direction when Dennis Lo's group in Hong Kong demonstrated the presence of DNA of male fetuses in the plasma of pregnant women,1 thereby making possible the use of cell free DNA (cfDNA) as a source of fetal chromosome information. Since then the following observations have been made regarding fetal cfDNA:

  1. The fractional percentage of fetal DNA in maternal plasma rises throughout pregnancy with a spike in the last few weeks.2
  2. cfDNA has a short life span in plasma of about 16 minutes in immediately postpartum women, suggesting a rapid turnover in pregnancy.3
  3. Its origin is most likely the placenta and not nucleated red blood cells or other sources.4
  4. It is found in maternal plasma as early as 22 days post-conception, raising the possibility of early diagnosis of fetal aneuploidy.5
  5. It has already been established as a source for fetal gender and Rh testing.6
  6. The total amount of fetal cfDNA is elevated in Down syndrome (1.7-fold) and preeclampsia (5-fold).7
  7. The concentration of fetal cfDNA is diluted by increased blood volume, making the detection of small differences in DNA concentrations more difficult in patients with increased body mass index.8

Current Investigation of Fetal cfDNA and Aneuploidy

Very recent modifications in methodologies have allowed easier sequencing of tiny fetal DNA fragments in maternal serum through a technique called massively parallel shotgun sequencing (MPSS). The method is based on the idea that Down syndrome fetuses, possessing another copy of chromosome 21, will contribute an extra amount of chromosome 21 cfDNA to the maternal serum pool, raising the level of total chromosome 21 cfDNA. However, since the mother already is contributing at least 80% of her DNA to her plasma pool and is adding her own two copies of chromosome 21 cfDNA to the mix, incremental differences are small — but, fortunately, still detectable. The amount of fetal chromosome 21 cfDNA is calculated by first establishing a normal range in euploid male fetuses from a reference ("training") population. Then "Z scores," or a similar index, "normalized chromosome values," are fashioned by comparing the amount of chromosome 21 cfDNA in the tested samples with what would be expected in the normal reference range.

In 2008, two groups reported an ability to detect trisomy 21 with this technique.9,10 Their work was expanded, allowing publication in 2011 of two clinical trials. The Hong Kong group screened 753 high-risk patients for trisomy 21.11 Eighty-six fetuses had trisomy 21 and all were detected using MPSS. This was accomplished at a 2.1% false-positive rate (100% sensitivity and 97.9% specificity). In 1.4%, an answer could not be obtained.

The second group, representing a California company,12 used another MPSS system while reporting similar results in a smaller patient sample consisting of 39 fetuses with trisomy 21, but later more clinical investigators (8) and high-risk patients (4664) were added. The expanded study yielded 100% sensitivity for detecting the 212 fetuses with trisomy 21, at a false-positive rate of 0.2%. In only 0.8 % of cases were they unable to obtain an answer from submitted samples.

The latest information comes from another corporate group, also from California. Initially the group was able to detect 13 of 13 fetuses with trisomy 21 in the 1,004 patients initially tested with their DNA sequencing product.13 Taking this one step further, an abstract was just presented at the recent annual Society of Maternal-Fetal Medicine meeting14 in which the same test was applied to 2882 high-risk patients in multiple sites around the United States. All 89 of the trisomy 21 fetuses were identified at a false-positive rate of zero (100% sensitivity and 100% specificity) — a feat that is rarely attainable with any test. Also, 35 of 36 fetuses with trisomy 18 and 11 of 14 fetuses with trisomy 13 were detected.

These tests could have huge clinical impact, as well as tremendous commercial potential. So, naturally, there has been a rush to market these products. Currently, the company responsible for the second study8,12 (Sequenom®) has launched its product. However, the company linked to the third study13,14 (Verinata Health™) is more than ready to go.

Where Will DNA Testing Fit into Our Already Complicated Screening Process?

This test has been neither labeled as a screening test nor a definitively diagnostic test. It is a "somewhere in between" test. Also, even though the possibility of 100% negative predictive value is very exciting, the cost of the procedure would have to be appropriate for this method to replace, or to be used in conjunction with, other screening tests. In addition, even if expanded data continue to show a 1% false-positive rate, it would not yet represent a replacement for invasive testing (although it certainly would narrow down the need for CVS and amniocentesis).

For comparison, here is a summary of the most commonly used screening options available today for fetal aneuploidy:

  1. First trimester combined screen. This puts into play serum hCG and PAPP-A, along with ultrasound nuchal translucency (NT) assessment. No second trimester aneuploidy testing is done and the answer is given as soon as it is available. According to FASTER trial data, the sensitivity for trisomy 21 is 86% at a screen positive rate of 5%.15
  2. Integrated screen. First trimester NT and serum PAPP-A + hCG and second trimester quad screen. Only one answer is given at the end of the full testing process.
  3. Stepwise sequential screen. First trimester NT and serum PAPP-A and hCG with the first trimester result given to the patient. If there is no invasive testing, a quad screen is added at 15-19 weeks and a final answer is then given.
  4. Contingent screen. Same first trimester testing, but if the risk is above 1 in 50 for trisomy 21, invasive testing is offered. If the risk for trisomy 21 is below 1 in 1,000, then no further testing is needed. If the risk is between these two levels, then a quad screen is obtained and a final answer is derived from all the data. This has obvious potential for savings in cost and angst.
  5. The genetic sonogram. This second trimester exam is almost always used in conjunction with, rather than instead of, the above screening strategies.

Below are accumulated data from the FASTER trial15 regarding the sensitivities for three of the above commonly used screening tests at a screen positive rate of 5%, depicted with or without the addition of a genetic sonogram.

  Standard After Sonogram
Integrated 93% 98%
Step-Wise 97% 98%
Contingent 95% 97%

What is the Present Status of Fetal DNA Testing?

At the moment, the Sequenom® test is being offered in some areas of the country to patients at high risk for aneuploidy — e.g., advanced maternal age, a history of trisomy 21, or concerning results from existing screening studies, including ultrasound. The test is offered any time after 10 weeks. Depending on the type of plan, some insurance companies are currently covering testing with the Sequenom® product, but Medicaid and others are not. The out-of-pocket price is very expensive, but may be negotiable. As far as we know, no insurance company is covering DNA testing if the patient is not in a high-risk category. Obviously, the cost status likely will change as the story unfolds and as other competitive products come onto the market. Also, the turnaround time presently is about 10 days, but it has been postulated that with increased demand and refinement of the methodology, the turnaround time could be the same as for current first and/or second trimester biochemistry screening.

Since the public is now aware of this test (through recent newspaper articles and a Time magazine piece on February 27, 2012), patients are requesting it. The companies are on a short leash, so they are only formally offering DNA testing to those in the high-risk categories above. It is unclear if they are interdicted from performing the test in low-risk patients who are demanding it and are prepared to pay. In the meantime, since a tsunami-like demand for this test could happen before we are fully prepared to handle it, here are some suggestions if the test is readily available in your area:

  1. In lieu of a formal counseling session, fold the new information into a handout for patients to read in the waiting room regarding the present status of DNA testing, including the fact that the accuracy of the test needs further study.
  2. Apprise those in the high-risk category of its availability as an adjunctive test, and not necessarily as a replacement first-line test.
  3. Offer it to those who have a desire for CVS or amniocentesis, but who have a higher risk of fetal loss (e.g., difficult access, recent vaginal bleeding, sub-chorionic hematoma, failed attempt at sampling, etc).
  4. Apprise low-risk patients demanding invasive testing that cfDNA may be an option, with the caveat that it might not be 100% accurate.
  5. It could be particularly useful as a second-line test for patients in otherwise low-risk categories who have isolated ultrasound findings (echogenic intracardiac focus, mild pyelectasis, short long bones, etc).

Some providers have decided to discuss the test as an option only if patients bring it up. While waiting for more data to surface, this may be an acceptable tactic regarding low-risk patients. But for how long? It is always a conundrum when a product, no matter how exciting the potential, is marketed before the full story has unfolded.

Frankly, if the cost can be whittled down appreciably and expanded data show similar predictive accuracies as reported above, our present screening strategies will be turned topsy-turvy by possibly negating the need for universal serum testing and even genetic sonograms. However, it will never eliminate the need for a detailed ultrasound examination to rule out structural abnormalities.

Only time will tell.


  1. Lo YM, et al. Presence of DNA in maternal plasma and serum. Lancet 1997;350:485-487.
  2. Lo YM, et al. Quantitative analysis of DNA in maternal plasma and serum: Implications for noninvasive prenatal diagnosis. Am J Hum Genet 1998;62:768-775.
  3. Lo YM, et al. Rapid clearance of fetal DNA from maternal plasma. Am J Hum Genet 1999;64:218-224.
  4. Bianchi DW. Circulating fetal DNA: Its origin and diagnostic potential. Placenta 2004;25(Suppl A):S93-S101.
  5. Guibert J, et al. Kinetics of SRY gene appearance in maternal serum: Detection by real time PCR in early pregnancy after assisted reproductive technique. Hum Reprod 2003;18:1733-1736.
  6. Lo YM, et al. Prenatal diagnosis of fetal RhD status by molecular analysis of maternal plasma. N Engl J Med 1998;339:1734-1738.
  7. Leung TN, et al. Increased maternal plasma DNA concentrations in women who eventually develop preeclampsia. Clin Chem 2001;47:137-139.
  8. Palomaki GE, et al. DNA sequencing of maternal plasma to detect Down syndrome: An international clinical validation study. Genet Med 2011;13:913-920.
  9. Chiu RW, et al. Noninvasive diagnosis of fetal chromosomal aneuploidy by massively parallel genomic sequencing of DNA in maternal plasma. Proc Natl Acad Sci U S A 2008;105:20458-20463.
  10. Fan HC, et al. Noninvasive diagnosis of fetal aneuploidy by shotgun sequencing DNA from maternal blood. Proc Nat Acad Sci U S A 2008;105:16266-16271.
  11. Chiu RW, et al. Non-invasive prenatal assessment of trisomy 21 by multiplexed maternal plasma DNA sequencing: Large scale validity study. BMJ 2011;342:c7401.
  12. Ehrich M, et al. Noninvasive detection of fetal trisomy 21 by sequencing of DNA in maternal blood: A study in a clinical setting. Am J Obstet Gynecol 2011;204:205.e1-11.
  13. Sehnert AJ, et al. Optimal detection of fetal chromosome abnormalities by massively parallel DNA sequencing of cell-free fetal DNA from maternal blood. Clin Chem 2011;57:1042-1049.
  14. Rava R, et al. Genome wide fetal aneuploidy detection by sequencing of maternal plasma DNA: Diagnostic accuracy in a prospective, blinded, multicenter study. Abstract #837. Am J Obstet Gynecol 2012;206:S367.
  15. Aagaard-Tillery KM, et al; First and Second Trimester Evaluation of Risk Research Consortium. Role of second-trimester genetic sonography after Down syndrome screening. Obstet Gynecol 2009; 114:1189-1196.