Jo-Ann Johnson, MD
Division of Maternal Fetal Medicine
Department of OB/GYN
University of Calgary
Calgary, AB

Learning Objectives:

1. To review the role of the 11-13⁺⁶ week scan in screening for chromosomal abnormalities and birth defects.
2. To provide an update on all aspects of nuchal translucency screening.
3. To review the current status of the "newer" first TM markers (nasal bone, ductus venosus, tricuspid regurgitation, facial angle)
4. To demonstrate that combining the 11-13⁺⁶ week scan with maternal biochemistry in the first TM not only predicts aneuploidy but also adverse pregnancy outcome.

Screening for chromosome disorders and neural tube defects is an established part of prenatal care that has been available to North American women for many years. Beginning with maternal age-based screening in the mid-1960’s, there are now a wide array of new and improved prenatal screening tests available, their development driven by advances in technology, concerns with safety and efficacy, and patient choice. These tests rely on various combinations of maternal serum biochemical and fetal ultrasound data obtained in the first and second trimesters, and are associated with significantly improved detection rates (DR) for chromosome disorders compared with maternal age or maternal serum screening (MSS). Offering invasive testing on the basis of maternal age alone is now considered obsolete in the context of these new screening tests and should be abandoned.

The majority of birth defects and chromosome abnormalities occur among otherwise low risk couples.

• Approximately 1:160 live born infants have a chromosome abnormality.
• Over 80% of Down syndrome infants are born to women under 35 years of age.
• All couples have a 2-3% risk of having a child with a birth defect. ¹

If prenatal diagnosis were restricted to high risk groups, the majority of abnormalities would go undetected. Thus, a major focus of prenatal diagnosis in recent years has been to develop effective non-invasive screening tools applicable to the entire pregnant population.

Every pregnant woman has a risk that her fetus will have a chromosomal abnormality. In order to determine a woman's individual risk, the background risk, comprised of 3 main factors (maternal age, gestational age, history of a previous fetus/baby with a chromosome defect) must be taken into consideration. ¹:

• The risk for fetal chromosomal abnormalities increases with increasing maternal age.
• The risk for fetal chromosomal abnormalities decreases with increasing gestational age.
• The history of a previous fetus or baby with a chromosome defect increases the risk for fetal chromosomal defect by about 0.75% over the age-related risk.

The background risk is then be adjusted by a factor, which depends on the results of a (or a series of) screening test(s). Theoretically, every time a test is carried out, the background risk is multiplied by the test factor to calculate the "adjusted risk" which then becomes the background risk for the next test.

It has been standard of care for many years to offer genetic counselling and invasive testing to all women ≥ 35 years of age at delivery (late maternal age or LMA). The recommendation based on maternal age was originally selected because the risk of a 35 year old mother having a child with a chromosomal abnormality was approximately equal to the risk of miscarriage due to the procedure, which in most centers is quoted to be 0.5% (1/200) ¹. This approach is now obsolete in the context of enhanced non-invasive screening modalities and the recently published guidelines by ACOG ² (http://www.acog.org/) and the SOGC ³ (http://sogc.medical.org/guidelines/documents/187E-CPG-February2007.pdf). The new SOGC guidelines recommends that it is no longer appropriate to offer women amniocentesis on the basis of maternal age alone; rather, all pregnant women should be offered multiple marker screening initially and only those with screening test results above a pre-determined cut-off should be offered invasive testing. In Canada, maternal age 40 and older is considered to have an a priori risk high enough to proceed directly to invasive diagnosis ³.

Screening using ultrasound is based on the fact that most fetuses with chromosomal abnormalities have either major structural malformations or minor abnormalities (commonly known as "markers") that can be detected by sonographic examination. The most effective sonographic marker of trisomy 21 and other chromosomal defects is increased nuchal translucency (NT) thickness at 11-13⁺⁶ weeks. Extensive studies over the last decade have examined the methodology of measuring NT, the development of the necessary algorithms for calculating the individual patient risk for trisomy 21 by NT in combination with maternal age and with various maternal serum biochemical markers, and the performance of this test ^{4, 5, 6, 7}. Another promising marker for trisomy 21, both in the first and second trimesters, is absence of the fetal nasal bone. There is also an extensive literature on the association between chromosomal abnormalities and a wide range of second trimester ultrasound findings (will not be discussed here).

Sagittal view 12 weeks showing NT Measurement.

Nuchal translucency (NT) refers to a sonolucent area in the fetal neck, typically observed in the first trimester (11-13⁺⁶ weeks). Increased NT is associated with chromosome abnormalities including trisomies 21, 18 and 13, triploidy and Turner’s syndrome ¹. In addition, increased NT thickness (≥ 3.0 mm) in the presence of normal chromosomes is associated with an increased incidence of certain birth defects including cardiac (septal defects), pulmonary (diaphragmatic hernia), renal and abdominal wall defects, as well as certain genetic syndromes, particularly hypokinesia disorders ⁸. An NT above the 99^th percentile has a sensitivity of 31% and specificity of 98.7% for major congenital heart defects when there is a normal fetal karyotype ⁹. One in 33 fetuses with NT above the 95^th percentile and 1 in 16 with NT above the 99^th percentile have a major cardiac defect detected ¹⁰. Finding an increased NT (≥ 3.5 mm) at 11-13⁺⁶ weeks gestation in association with a normal karyotype thus warrants offering a detailed ultrasound examination at 18-20 weeks, and a fetal echocardiogram ¹⁰.

Screening using NT measurements is also useful in multiple gestations where conventional methods (e.g. maternal serum screening (MSS)) are not applicable. In dichorionic diamniotic gestations (DCDA), discordancy for NT thickness is a useful marker for chromosome and other abnormalities; in monochorionic diamniotic (MCDA) gestations, it is also a useful marker for chromosome abnormalities but the false positive rate is higher because it is also a marker for twin-twin transfusion syndrome ¹¹.

In screening for chromosomal defects every NT measurement for a given crown–rump length represents a factor or "likelihood ratio" which is multiplied by the background risk to calculate a new risk. The likelihood ratio (LR) is defined as the sensitivity/false positive rate. A LR of greater than one suggests a positive association with a particular finding. In screening using NT, the larger the NT, the higher the LR becomes and therefore the higher the new risk. In contrast, the smaller the NT measurement, the smaller the LR becomes and therefore the lower the new risk ¹.

Example: In a woman aged 37 years at 12 weeks gestation, the age-related risk of trisomy 21 is 1/152. Ultrasound examination showed a fetal crown-rump length (CRL) of 56 mm and NT of 1.3 mm, which is below the normal median NT at this CRL. The risk for trisomy 21 was therefore reduced by a LR of 8 to 1 in 1051. If the NT were 3.5 mm, the risk would have increased by a LR of 18 and the final risk would have been 1 in 7.

There are several prospective studies examining the implementation of NT measurement in screening for trisomy 21 ⁴. In all studies, the sonographers had received appropriate training in the standard technique for the measurement of NT. Although different cut-offs were used for identifying the screen positive group, with consequent differences in the false positive and detection rates, they all reported high detection rates. The combined results on a total of 174,473 pregnancies, including 728 with trisomy 21, demonstrated a detection rate of 77% for a false positive rate of 4.7% ⁴.

Fetal NT at the 11-13⁺⁶ week scan, when measured with the appropriate technique, is currently the single most powerful marker for the detection of trisomy 21; for an invasive false positive testing rate (FPR) of 5%, about 75% of affected pregnancies can be identified.

Guidelines for measuring NT to maximize reproducibility and accuracy have been developed by the Fetal Medicine Foundation (FMF) UK (http://www.fetalmedicine.com/f-downs.htm). The Royal College of Obstetricians and Gynaecologists (UK) study group on first trimester assessment of trisomy 21 (DS) recommended that NT should be implemented only in centres with appropriately trained sonographers using high-quality equipment, and that the results should be subject to regular audit by an external agency (http://nscfa.web.its.manchester.ac.uk/nuchaltranslucency).
To achieve standardization and maintain quality, the use of NT in a clinical setting requires a program of quality control and maintenance of skills through an ongoing audit of NT measurements. Details of NT training and standards as per the FMF UK can be viewed at http://www.fetalmedicine.com/f-downs.htm, http://www.mfmedicine.com/phys_train2.aspx, http://fetalmedicine.com/usa/.

It is also possible to perform an anatomic survey at the 11-13⁺⁶ week scan, ranging from limited to detailed, and this, combined with the NT allows for triaging of pregnancies identified to be at risk into more appropriate levels of fetal surveillance while providing reassurance for "normal" pregnancies. In Europe, the United States and Canada, many centers have introduced NT screening as part of routine pregnancy screening.

In trisomy 21 pregnancies at 11-13⁺⁶ weeks, the maternal serum concentration of free β-hCG is higher (about 2 MoM) than in chromosomally normal fetuses whereas pregnancy associated plasma protein-A (PAPP-A) is lower (about 0.5 MoM). When combined with maternal age, the detection rate of trisomy 21 by first trimester biochemical screening is approximately 60% with a screen false positive rate of 5% ^12,13,14.

Ultrasound markers and biochemical markers appear to be independent predictors with respect to DS risk, hence it is possible to combine them to achieve a higher DR than with each test alone. Several studies have shown that screening for trisomy 21 using a combination of maternal age, fetal NT and maternal serum PAPP-A and free β-hCG is associated with a detection rate of about 85- 90% at an FPR of 5% ^15,16,17,18.

The combination of maternal age, NT thickness, free β-hCG and PAPP-A is known as combined first trimester combined screening or FTS. FTS is a major advance over mid-trimester maternal serum screening (triple test) as it is offered much earlier in pregnancy (11-13⁺⁶ weeks versus 15-20 weeks gestation) and is associated with a much better detection rate and lower false positive rate ¹⁸. It is important to note however, that FTS will alter the predictive value of biochemical screening in the second trimester and the significance of "markers" detected at the 18 week scan as well. Thus, if second trimester MSS (or ultrasound) is performed following first trimester NT screening, one should adjust the background risk accordingly ¹⁹.

Integrated prenatal screening (IPS) is based on the use of PAPP-A and NT in the first trimester and the Quad screen (AFP, estriol, hCG and inhibin A) in the second trimester with no results released until all the testing is completed. Wald proposed that integrating first and second trimester screening would result in a 83% DR for DS, with a 2.1% FPR at a term risk cut-off of 1:200²⁰. The major limitation of IPS is the fact it is a 2-step process not complete until 15-17 weeks of pregnancy, and requires that the results of the first trimester blood and NT scan are suppressed. Most believe that women have the right to know their results early and it is unethical to withhold the first trimester results.

Nasal Bone:
Ultrasound screening for delayed ossification of the fetal nasal bone can be done in the first or second trimester. First trimester assessment of the fetal nasal bone was first described by Cicero et al. and detected 77% of Down syndrome cases ²¹. Subsequent work has shown a DR of 68.8% and that the FPR depends upon maternal ethnicity (9% in Afro-Caribbeans, 5% in Asians and 2.2% in Caucasians) ²². A study of intra- and inter-operator variability in fetal nasal bone assessment during the first trimester showed good reproducibility ²³. It is a relatively difficult technique to master however, hence participation in a training and quality assurance program is considered essential before using this parameter in clinical assessment (http://www.fetalmedicine.com/f-downs.htm).

Tricuspid Regurgitation:
Recent studies suggest that examination of the fetal tricuspid flow at 11-13⁺⁶ weeks is associated with improved performance of screening for DS by maternal age and fetal nuchal translucency (NT). The finding of tricuspid regurgitation increases the risk for DS and this risk factor can be incorporated into the assessment with specialized software. Tricuspid regurgitation is also a risk factor for cardiac defects. Patients found to have TC regurgitation in the first trimester should be referred for a fetal echocardiogram. Of note, normal tricuspid flow is reassuring and will be particularly useful for patients with risks between 1 in 150 and 1 in 300 who are traditionally classified as screen positive, but with the presence of normal flow confirmed, may be sufficiently reassured. As with the NT and nasal bone scan, it is imperative that sonographers undertaking risk assessment by examination of the fetal tricuspid flow receive appropriate training and certification of their competence in performing the tricuspid flow scan (http://www.fetalmedicine.com/f-downs.htm).

Ductus Venosus:
The ductus venosus is a unique shunt that carries well-oxygenated blood from the umbilical vein through the inferior atrial inlet on its way across the foramen ovale. Blood flow in the ductus is characterized by high velocity during ventricular systole (S-wave) and diastole (D-wave) and by the presence of forward flow during atrial contraction (A-wave). This can be examined at 11-13⁺⁶ weeks of gestation by Doppler ultrasound. Studies have shown that the A-wave is absent or reversed in 60-90% chromosomally abnormal fetuses and in only about 5% chromosomally normal fetuses ²⁴. However, examination of ductal flow is time-consuming and requires skilled operators. It is therefore unlikely that this assessment will be incorporated into the routine first-trimester scan.
Nevertheless, assessment of ductal flow can potentially play a major role as a secondary method of screening in order to achieve a major reduction in the false-positive rate of primary screening for chromosomal abnormalities by a combination of maternal age, fetal NT and maternal serum free β-hCG and PAPP-A at 11-13⁺⁶ weeks.

Fronto maxillary facial angle:
Another feature typical of Down syndrome is a "flat face" ^25,26. Even though this can sometimes be subjectively appreciated on prenatal ultrasound, especially late in gestation, to date an objective estimation of facial flatness by prenatal ultrasound has been lacking. Recently, Sonek et al have developed a method to evaluate the position of the maxilla relative to the forehead utilizing fronto-maxillary facial (FMF) angle measurements ²⁷. A detailed review of the subject and description of the technique can be viewed at http://fetalmedicine.com/usa/pdf/Newsletter%20volume%203%20number%201.pdf (The FMF USA 11-13⁺⁶ Weeks Scan Project Newsletter Volume 3 Issue 1 Page 2) Since the FMF angle measurements does not change significantly with gestational age and is independent of nuchal translucency measurement, presence or absence of the nasal bone, and maternal serum biochemistries (free β-hCG and PAPP-A), it meets criteria for inclusion in the first trimester combined screening protocols and is likely to improve screening for trisomy 21 in the first trimester.
Mathematical modeling suggests that its inclusion would improve combined first trimester screening detection rate to 98% with a screen positive rate of below 5%. As is the case with all other first trimester markers, adherence to standardized techniques is crucial.

Pregnancy-associated plasma protein (PAPP-A) is a zinc-binding metalloproteinase responsible for the cleavage of the insulin growth factor binding protein-4 (IGFBP-4). PAPP-A plays an essential growth regulatory role in vivo by increasing the bioavailability and mitogenic effectiveness of the insulin growth factor (IGF) during early development ²⁸. Women who experienced spontaneous abortions had significantly low PAPP-A serum levels during the first trimester ²⁹. In addition, plasma levels of PAPP-A are also affected (low) in the event of IUGR or chromosomal abnormalities such as trisomy 21. A significant reduction in PAPP-A levels have also been observed between the 8^th and 14^th weeks of pregnancy among women who developed pre-eclampsia and also during the early second trimester (at the 17^th week) ^28-35.

More effective prenatal screening translates into fewer invasive tests (mainly amniocentesis) and a higher yield of abnormalities per amniocentesis performed. Earlier screening also affords women increased options with respect to pregnancy management, with the attendant reduction in costs, both medical and psychological.

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