Special Feature: Non-Invasive Approach to Prenatal Diagnosis

By John C. Hobbins, MD

The american college of obstetrics and gynecology issued a statement many years ago that has been ingrained into the thinking of providers caring for patients of "advanced maternal age." The recommendation was that all pregnant women 35 years or older be offered an amniocentesis. What evolved almost immediately was that the phrase became "recommended that these individuals have amniocentesis." Many patients today are referred to us for an amniocentesis without mention by their providers of the availability of any other type of prenatal diagnostic testing. Often they expect that we will cover this prior to the procedure, but in some cases the message is "you are 38 years old. You need an amniocentesis." The reasons for the slow movement towards a more noninvasive approach are diverse, but often represent reluctance by a provider to settle for a diagnostic regimen that does not have a 100% accuracy. However, the ultimate decision about diagnostic testing should be in the hands of the patient.

In this special feature, up-to-date information will be provided regarding the risks and benefits of various diagnostic methods available today for patients of advanced maternal age (AMA).

Biochemical Testing

Maternal serum alpha-fetoprotein (MSAFP) testing was begun in the 1970s to screen for neural tube defects. Merkatz first noticed that MSAFP levels were low in many pregnancies complicated by Down syndrome (DS). Later, it was realized that when one used a 2-variable formula [maternal age and MSAFP in multiples of the median (MoM)], 30% of fetuses with DS would be screened in.

When it became clear that the addition of 2 other analytes, human chorionic gonadotropin (hCG) and estriol (E3) enhanced the predictive value of the test, these were added to bring the method up to a "triple screen." In an overall pregnant population this has a sensitivity for DS of about 60% at around a 5% screen positive rate. This sensitivity rose to about 80% in a population older than 35 years of age, using a higher preset screen positive rate of about 15%.

Now we have the quad screen. Interestingly, there is no uniformity among laboratories regarding what this fourth analyte should be. Some labs break down hCG into "total hCG" and the alpha or beta subunits, while most now add the analyte with the most available data regarding efficacy, inhibin-A. The incorporation of inhibin-A adds at least another 5% to the sensitivity without an increase in the screen positive rate.

The weakest performer is estriol, and in Europe many colleagues have found it to be cost-effective to drop this from the diagnostic package.

The beauty of the triple or quad screen is that it will often drop the risk for an AMA patient far below the risk of amniocentesis. The drawback is that the test will miss 15% of pregnancies complicated by DS. Another inherent downside is that many individuals will be unnecessarily concerned when notified that they have a "positive test." However, with proper counseling, this can be effectively neutralized. To many patients, a positive screen means that their fetus has DS, when, in fact, their risk may be, let’s say, 1:200, giving her a 99.5% chance that the fetus is normal. Also, when describing the test, the fact that providers use the word "false positive" is misleading, since this would mean the test indicated that the fetus actually had a problem for which it was being screened, and the test was wrong. The term "screen positive" has a "softer" connotation.

First-Trimester Biochemistry

The 2 best performers are beta subunit of HCG (beta HCG), which tends to be elevated in DS, and pregnancy-associated plasma protein-A (PAPP-A), which is depressed. The average beta HCG in DS is 1.9 MoM and the PAPP-A is 0.44 MoM.

The sensitivity when used together is about 62% for DS at a screen positive rate of 5%.


First trimester: In a beautifully designed study, Nicolaides and his team were the first to demonstrate how careful assessment of fetal nuchal translucency in the first trimester could be used to screen for DS and other aneuploidies. At first, they began using a measurement made from the inner portion of the nuchal membrane to the inner aspect of the fetal neck (NT), and, by establishing an arbitrary cutoff of 2.5 mm, they grossly adjusted the risk for a given patient. The London group then prospectively assessed the efficacy of an algorithm taking into account the patient’s age, the crown-rump length of the fetus, and the NT thickness. Their findings in more than 100,000 patients were suggestive of a 77% sensitivity for DS at a 5% screen-positive rate and 82% when a cutoff of 1:300 was used.

Unfortunately, the first American study using this same concept, yielded only a 33% sensitivity and about the same time it was postulated that the performance of the London group was not quite as good as was initially reported since the authors did not account for the nearly 40% spontaneous loss rate in DS fetuses. In other words, there was complete ascertainment in the screen-positive fetuses who had second trimester invasive testing and in those who delivered toward the end of pregnancy, but chromosomal analysis was not necessarily done on all those who had spontaneous abortions. Using this principle, Haddow pointed out that the sensitivity would be more towards 60%.

Now it is clear from well constructed studies in the literature, including 2 American studies with very large numbers (BUN and FASTER trials), that Nicolaides’ sensitivity figures stand up to the newer figures when quality control for NT assessment is adhered to.

For example, a collection of pooled data from 30 studies indicated a sensitivity for DS of 80% with a screen positive rate of 6%.

After the inception of the NT theme, another marker for DS also has come along. The fetal nasal bone, which, in a seminal study from London, was found to be absent in 73% of first trimester fetuses with DS. Recently, there has been a suggestion that assessment of the nasal bone is not as useful in the first trimester as in the second trimester, when the size of the nasal bone can be better quantified.

It is clear that the combination of NT testing and first trimester biochemistry is better than either alone, but, although the sensitivity is increased by only a few percentage points, from a public health standpoint this may well be worth the extra cost of the 2 since the screen-positive rate is not increased.

The data available from the combined test (biochemistry and NT) are limited, but from BUN study results, published in the New England Journal of Medicine in 2003, with a cut off of 1:270 and a screen positive rate of 9%, 85% of DS fetuses would be screened in with NT, PAPP-A, and beta HCG. The FASTER trial data, published in abstract forum in the American Journal of Obstetrics and Gynecology, shows a 90% sensitivity at a 14% screen positive rate with the NT, PAPP-A and free beta hCG combination.

The Genetic Sonogram

There are 3 major components of a genetic sonogram. The first one involves measurements of the humerus and femur, which tend to be shorter in DS. Interestingly, only about 20% of DS fetuses will have measurements that are > 2 standard deviations below the mean for gestation, but a much larger percentage will be about a week less than dates.

The second part involves an attempt to rule out fetal anomalies such as cardiac defects, CNS or renal abnormalities. About 20% of fetuses with DS will have a "major abnormality," which includes ventriculomegaly.

The third part, varying from center to center, consists of a search for markers for DS. The best performers, and therefore the most commonly evaluated, are the nuchal skin fold thickness (NSFT), the size of the nasal bone, the presence of echogenic bowel, and iliac angle assessment. Less commonly evaluated are the middle bone of the fifth digit, ear length, frontal lobe length, and sandal gap.

The echogenic intracardiac focus (EIF) has been the source of controversy and an angst producer for diagnosticians. It is more commonly found in DS but, as with most of the other markers, it has been noted in up to 5% of normal second trimester fetuses. There is a growing feeling that if it is isolated, it should not raise the risk for a given fetus, and if found in a low-risk patient (younger than 35 years of age and/or with normal biochemical testing), it could be classified as a normal variant.

Many individual studies and pooled data show the sensitivity of a genetic sonogram to be about 75%. Once the sensitivity and specificity of a given test are known, then a likelihood ratio (LR) can be calculated. If a test has a negative LR of 0.3, which a genetic sonogram has in some hands, one can drop the risk for a given patient from her pretest value by 70%.

We have taken the approach of adjusting DS risk downward following a careful genetic sonogram by 50% (adding a cushion). This would take a 35-year-old with a risk of 1:280 to a posttest risk of 1:560. If an isolated marker (with the exception of an NSFT) is found, then we will not change her risk, and if the fetus has a major anomaly or more than one marker, we will raise her risk.

Some patients will start with a quad or triple screen risk that is higher than her age-related risk. In these patients we have been cautious with our counseling even though there is growing evidence that the same principle can be applied to those with nonreassuring biochemistry.

Invasive Testing

CVS: We quote a risk of between 1 and 1.5% procedure related risk to CVS. These data are hard to come by because of the non-randomized nature of the experience in literature. An older Canadian randomized trial, comparing CVS and amniocentesis, suggests the risk of CVS to be about 0.7% higher than that of amniocentesis. Certainly, there is evidence that the risk is lowest in experienced hands.


Although individuals and centers have reported loss rates that are at great variance with each other, the only RCT in the literature shows a procedure related risk of 1% with a spontaneous loss rate in the nonamnio group of 0.7%. This leaves one to question any individual operator citing an amniocentesis loss rate of less than 7/1000.

Unpublished data from the FASTER trial suggest the difference in loss rate between screen positive patients having amniocentesis and those not having this procedure was surprisingly low and might have to do with our inherently high spontaneous loss rate in any screen positive patient.

The "ball park" loss rate that we quote is 1:200. This was the figure used long ago when 35 was arbitrarily chosen as an age at which the risk of DS was roughly equivalent to the risk of amniocentesis.

The Integrated Screen

This puts into play first trimester ultrasound, first trimester biochemistry, and second trimester biochemistry. The method has the ability to screen in more than 90% of DS fetuses with a false positive rate of 5.4%, as stated in the FASTER abstract.

Some AMA patients want 100% assurance that their fetus does not have DS, and these individuals will always choose to have an amniocentesis. However, there is a growing number of patients who want the best non-invasive information available with which to weigh their risk of fetal DS against the risk of amniocentesis. In our experience the majority of patients today will decline amniocentesis if there is a mis-match in these risks. Whatever the patient’s choice, it is the job of their provider to give them the most up to date information with which to base their decision.

Suggested Reading

  • Merkatz IR, et al. An association between low maternal serum alpha-fetoprotein and fetal chromosomal abnormalities. Am J Obstet Gynecol. 1984;148(7): 886-894.
  • Snijders RJ, et al. UK multicenter project on assessment of risk of trisomy 21 by maternal age and fetal nuchal-translucency thickness at 10-14 weeks of gestation. Lancet. 1998;351:343-346.
  • Haddow JE. Antenatal screening for Down’s syndrome: Where are we and where next? Lancet. 1998; 352:336-337.
  • Haddow JE, et al. Screening of maternal serum for fetal Down’s syndrome in the first trimester. N Engl J Med. 1998;338:955-961.
  • Malone FD, D’Alton ME. First-trimester sonographic screening for Down syndrome. Obstet Gynecol. 2003; 102:1066-1079.
  • Cicero S, et al. Absence of nasal bone in fetuses with trisomy 21 at 11-14 weeks of gestation: An observational study. Lancet. 2001;358:1665-1667.
  • Wapner R, et al. First-Trimester screening for trisomies 21 and 18. N Engl J Med. 2003;349(15): 1405-13.
  • Malone FD, et al. First- and second-trimester evaluation of risk (FASTER) trial: Principal results of the NICHD multicenter Down syndrome screening study. Abstract #1. Am J Obstet Gynecol. 2003;189(6):S56.
  • Tabor A, et al. Randomised controlled trial of genetic amniocentesis in 4606 low-risk women. Lancet. 1986; 1(8493):1287-1293.
  • Hobbins JC, et al. The genetic sonogram in screening for Down syndrome. J Ultrasound Med. 2001;20: 569-572.
  • Wald NJ, et al. Integrated screening for Down’s syndrome based on tests performed during the first and second trimesters. N Engl J Med. 1999;341:461-467.

John C. Hobbins, MD, Professor and Chief of Obstetrics, University of Colorado Health Sciences Center, Denver, is Associate Editor for OB/GYN Clinical Alert.