Non-Invasive Prenatal Screening for Chromosomal Abnormalities. What’s New?

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OBSTETRICS

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NON--INVASIVE PRENATAL SCREENING FOR CHROMOSOMAL ABNORMALITIES. WHAT'S NEW? J.M. Johnson, MD, FRCSC,l Doug Wilson, MD, FRCSC,2 1Associate

Professor, 2Professor, Head, Division of Maternal Fetal Medicine, 1,2Department of Obstetrics and Gynaecology, 1University of Toronto, 2University of British Columbia

ABSTRACT

Prenawl screening fOT chromosomal almarmalities is a rapidly changing field. Increasing emphasis is being plnced on rne development of noninvasit'e screening methods based on matemal biochemical markers and ultTasound findings. The purpose of this artic/e is to provide rne clinician with an update on rnese newer methods, and to discuss rneir advantages and disadvantages when compared with our current standard of care (rnatemal age-based screening). In addition, the clinical implications of intToducing such screening on a large-scale basis are discussed. RESUME

Le domaine du dipistage prenawl des anomalies chromosomiques est en p/eine evolution. Oe plus en plus, on met I' accent sur /e diveloppement de methodes non effractives de dipistage qui ont recours ades marqueurs biologiques marernels et aux ultTasons. Cet artic/e vise a presenter aux cliniciens une mise ajour sur /es plus nouvel/es methodes et aen soupeser /es avantages er /es inconvenients par comparaison ala pratique normale actueUe (/e dipistage selon I' age de la mere). On examine aussi /es implications cliniques de I' utibsation agrande echeUe de ces methodes de dipistage.

J SOC OBSTET GYNAECOL CAN KEY WORDS Prenawl screening, chromosomal almarmalities, biochemical markers, ultrasound. Received on June 24th, 1998. Revised and accepted on September 15th, 1998.

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1999;21(2):12-15

, , , INTRODUCTION

TABLE 2 DETECTION RATE (DR) AND FALSE POSITIVE RATE (FPR) FüR DIFFERENT SCREENING PARAMETERS FOR DOWN'S SYNDROME SCREENING

In recent years, the scope of prenatal genetic evaluation has expanded greatly with the development of a number of non-invasive screening methods (Table 1). As a consequence, many health care providers involved in obstetric care are counselling patients prior to and following such prenatal genetic screening. This article will review the current status of screening for chromosomal disorders in pregnancy and provide an update for clinicians on some of the newer screening methods.

MA

+~

DR(%)

FPR(%)

33

10

35 Yrs

MA + AFP

33

5

MA + AFP + hCG + uE3**

75

7

MA+ NT

80

5

MA + NT + free ß-hCGo (1 st trimester)

85

5

+ maternal age AFP=alpha-fetoprotein hCG=human chorionic gonadotrophin uE3=unconjugated estriol NT=nuchal translucency

PRENATAL SCREENING FOR CHROMOSOMAL ABNORMALITIES

•• Based on a cut-off 01 1:385 risk 01 D5 at livebirth. Data courtesy 01 the Maternal Serum Screening Program, Province of Ontario. • Under investigation at present in Canada.

SCREENING BA5ED ON MATERNAL AGE

of delivery. If all these women underwent amnioeentesis, about 33 percent of DS would be identified (DR 33%, FPR 10%) (Table 2). In practiee, less than 15 percent of DS cases are actually detected because fewer than half of these older women have diagnostic testing. Thus, with age-based screening, the vast majority of DS infants (approximately 70-80%, depending on the age-distribution of the population) will not be detected prenatally.

Approximately one in 160 live born infants will have a demonstrable chromosomal abnormality, at least half of which will lead to conditions requiring medical intervention. The most common liveborn chromosomal abnormality and the most common genetic cause of mental retardation is trisomy 21 or Down's syndrome (DS), occurring in approximately one in 700 livebirths. 1 The prevalence of DS and other chromosomal abnormalities increases with maternal age. 2 Reeognition of this fact has prompted the recommendation that all women who will be 35 years of age or over at the time of delivery (late maternal age) oe offered invasive prenatal testing with either amnioeentesis or chorionic villus sampling (CVS). Maternal age is a relatively poor basis for screening primarily because of the low detection rate (DR) and relatively high false-positive rate (FPR). In Ontario for example, approximately 10 percent of women are 35 years or older at the time

WHAT 15 THE MATERNAL SERUM SCREEN?

In the late 1980s, a new method of screening for fetal DS was introduced that takes into accaunt not only maternal age but also the coneentrations of various feto-placental produets in the maternal eireulation (alpha-fetoprotein IAFP], human ehorionic gonadotrophin [hCGj and uneonjugated estriol [uE3]). The method relies on the fact that the median maternal serum coneentrations of AFP and uE3 are

TABLE3

TABLE 1 INVASIVE AND NON-INVASIVE PRENATAL TESTING

LEVELS OF VARIOUS ANAL YTES IN OPEN NEURAL TUBE DEFECTS, DOWN'S SYNDROME AND TRISOMY 18

Non·invasive methods

Open Neural Tube Defect

Down's Syndrome

Trisomy 18

Increased

Decreased

Decreased

Human chorionic gonadotrophin (hCG)

N/A

Increased

Decreased

Unconjugated estriol (nE3)

N/A

Decreased

Decreased

Analyte

o Maternal age, genetic history o Maternal serum screening (MSS) o Ultrasound "The Genetic Sonogram"

Alpha-fetoprotein (AFP)

Under investigation at present o First trimester screening - Biochemical screening - Ultrasound screening (nuchal translucency) - Fetal cells in maternal circulation

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, , , a predetermined cut-off (e.g. 1:385 risk of DS at livebirth), the gestational age is first verified by ultrasound biometry (if not already done). If, on the basis of accurate fetal dating, the risks still exceed the cut-off, the woman is offered genetic counselling and amniocentesis. Ir is important to remember that MSS is a screening test. This means that most patients who are found to be screen positive for DS, trisomy 18 or oNTD will go on to have a normal baby, but such patients are eligible for genetic counselling and invasive diagnostic testing.

about 25 percent lower, and hCG ab out two times higher in DS pregnancies than in normal pregnancies (Table 3).3.4 These values are sufficiently different in DS pregnancies to allow for the identification of a 'high-risk' group who are then offered further testing. Using this approach, appraximately 70 to 80 percent of DS fetuses can be detected at a false positive rate (FPR) of seven to eight percent, depending on the risk cut-off chosen. This detection rate (DR) is much higher than that achieved with screening based on matemal age or using maternal serum alpha-fetoprotein alone (Table 2). lt also results in a lower false-positive rate and has the potential to result in fewer amniocenteses while a110wing screening to be available to a11 pregnant women in the population. In addition to screening for DS, the maternal serum alpha-fetoprotein (MSAFP) component alone screens for open neural tube defects (oNTDs).5 The detection rate and FPR for oNTDs depend upon the MSAFP cutoff level chosen. For example, at a cut-off of 2.2 multiples of the median (MOM), most programmes will achieve a DR greater than 80 percent at an FPR of less than five percent. The sensitivity approaches 100 percent when the AFP measurement is combined with a detailed ultrasound examination and the selective use of amniocentesis. The maternal serum screen (MSS) can also be used to screen for trisomy 18. Levels ofhCG, AFP and uE3 are a11 depressed in the presence of trisomy 18, and using these markers, approximately 60 to 80 percent of trisomy 18 cases can be detected at an FPR of less than one percent. 6 How

ROLE OF ULTRASOUND IN MATERNAL SERUM SCREENING

As the concentrations of the three markers are highly dependent upon gestational age, and an errar in gestational dating of greater than nine days can result in a large discrepancy in the risk calculation, many experts recommend a routine dating ultrasound examination prior to screening. Studies have shown that the use of a dating ultrasound to establish gestational age prior to MSS decreases the FPR by as much as 50 percent, leading to a major reduction in the incidence of screen positive individuals and, therefore, the rate of amniocentesis and maternal anxietyY The optimal time to perform this dating ultrasound is controversial. Many practitioners offer a single scan at 16 weeks, combining the dating scan with the complete (anatomical survey) scan. We favour offering the dating sc an between 10 and 14 weeks (optimally 12 weeks), with a complete scan between 18 and 20 weeks gestation. This is because we believe that 18 weeks is the optimal time to perform the anatomical survey. Performing the dating scan in the first trimester has several additional benefits: accurate dating, detection of multiple gestation, nuchal evaluation.

DOES THE MATERNAL SERUM SCREEN

WORK?

With MSS, all pregnant women are offered screening in the second trimester (15-22 weeks, optimally 16 weeks), and are provided with an estimate of the risk of their fetus being affected with DS, oNTD or trisomy 18. The risk estimate is derived from the individual tests of the three markers which are combined with maternal age, maternal weight, race and insulin-dependent diabetes (IDDM) status to produ ce a summary probability that the fetus has DS or trisomy 18. In women with a calculated prabability exceeding

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AVAILABILITY OF BIOCHEMICAL SCREENING

At present, biochemical screening using MSS is not uniformly available across Canada. In those centres where it is unavailable, the standard of care for prenatal genetic screening is to offer counselling and invasive testing to all women who will be 35 years or older at the time of delivery. A complete ultrasound scan at 16 to 20 weeks gestation is currently recommended by the SOGe. In centres where MSS is available, such screen-

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, , , ing should be performed as d ose to 16 weeks gestation as possible. Any wo man undergoing MSS should understand that while anormal screen may have decreased her risk, it does not rule out DS or other chromosomal abnormalities in her fetus.

TABLE4 SONOGRAPHIC MARKERS FOR DOWN'S SYNDROME (14-22 WEEKS GESTATION) • Struetural defeet • Mild ventrieulomegaly (2: 10 mm) • Nuehal thiekening (2: 6 mm)

USE OF UNCONJUGATED ESTRIOL

• Echogenie bowel (eehogenieity=bone)

Because uE3 adds relatively little sensitivity and specificity to OS screening, some centres have chosen to use a 'double screen,' using only AFP ,md hCG. We advocate the tripIe screen because of the influence of uE3 on the FPR. It has been shown, particularly in ultrasound-dated pregnancies, that uE3 decreases the FPR by approximately 25 percent while keeping the OR approximately the same (60%).9 Furthermore, the high sensitivity and specificity of screening for trisomy 18 appear to be dependent upon measuring the values of all three analytes. l l'

• Short humerus/femur Eehogenie intraeardiae foeus (ElF)

•

Renal pyeleetasis (2: 3 mm)

Further prospective studies are needed to verify this approach. SCREENING FOR CHROMOSOMAL ÄBNORMALITIES WITH ULTRASOUND

In the past 10 years, our ability to detect fetal malformations by ultrasound has increased dramaticalIy. In addition, a number of subtle abnormalities, tenned 'sonographic markers' have been shown to facHitate the prenatal diagnosis of chromosomally abnormal fetuses, especially those with Down's syndrome (T able 4). These 'markers' largely represent extrapolations from the phenotypic knowledge of infants born with chromosomal abnormalities. Ir is important to note that the phenotypic expression of chromosomal abnormalities in utero and at birth differs somewhat, with certain abnormalities being detected more

FIRST TRIMESTER BIOCHEMICAL SCREENING

The main disadvantage of MSS is that it is not performed until16 weeks gestation. By the time resulrs are available and invasive testing performed, if indicated, the woman is often at 18 to 20 weeks gestation. This prolonged waiting period is a time of major psychological distress for most couples. Moving the window of MSS into the first trimester would be a great advantage to both dinician and patient. Although most se co nd trimester markers in current use appear to be ineffective in the first trimester, matemal serum free-beta-hCG is an important exception, with several re ports showing that levels in OS are approximately twice the normal level." ·l l A second marker which has been found to be effective in first trimester screening is maternal serum pregnancy-associated plasma protein (PAPP-A). Recent retrospective reports reveal first-trimester maternal serum median PAPP-A levels to be markedly reduced at less than 0,4 MOM in over 100 cases of OS." In addition, there is a low correlation between the two markers, indicating that they can be combined to achieve a higher detection rate than either alone provides. In a study by Orlandi er al., screening for fetal OS using free-beta-hCG and PAPP-A between 10 to 14 weeks was associated with a detection rate for OS and trisomy 18 of 61 percent and 63 percent, respectively, at an FPR of five percent. 14

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FIGUREI MILD CEREBRAL VENTRICULAR DILATATION WITH DOWN'S SYNDROME

Transverse view of the cranium, through the lateral ventricles, demonstrates mild dilatation of the lateral ventricles (L) at 18 weeks gestation.

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, , , frequently prenatally (e.g. nuchal abnormalities). The 'markers' considered most predictive of fetal DS are described below.

FIGURE 2a CYSTIC HYGROMA

SONOGRAPHIC MARKERS FOR FETAL DOWN'S SYNDROME (14 TO

22

WEEKS

GESTATION) MILD OR BORDERLINE VENTRICULOMEGALY

Evaluation of the fetal cerebral ventricles is a standard part of the 18-week anatomic survey. The ventrides are measured in the axial plane of the fetal head along the posterior portion of the lateral ventricle. Their size remains relatively constant throughout gestation, with a mean diameter of 6.1 +/- 1.3 mm, with slightly larger ventricles in males than females (6.4 mm versus 5.8 mm).15,16 Ventriculomegaly is suspected when the lateral atrial ventricular diameter reaches 10 mm (Figure 1). The detection of mild ventricular dilatation may be an important clue to the presence of fetal abnormalities, induding chromosomal abnormalities. In aseries by Bromley et al. , five of 43 fetuses (12%) with mild ventriculomegaly (ventricular diameter 10-12 mm) had abnormal karyotypes (3 trisomy 21,2 trisomy 18); all of these fetuses had other abnormal findings. 17 Even in the absence of additional abnormalities (e.g. isolated ventriculomegaly), the risk of a chromosomal abnormality has been shown to be increased to approximately two to three percent. 16,17 While the outcome of mild, isolated ventriculomegaly with anormal karyotype is gene rally favourable, such children may be at an increased risk of mild to moderate developmental or motor handicaps in childhood. 15 ,17

Cystic hygroma with trisomy 18. Axial view through the head at 14 weeks shows large nuchal cystic hygroma (CH) with midline septation.

earliest abnormalities detectable by ultrasound. Although cystic hygromas are usually picked up in the first trimester, they mayaiso be detected in the second trimester if spontaneous demise has not occurred. The cystic hygroma is usually seen on ultrasound as sonolucent fluid spaces in the fetal neck, se para ted into compartments by membranes coursing in an anterior-posterior plane (Figures 2a and b). The prognosis is very poor for fetuses with large cystic hygromas associated with generalized lymphoedema and non-immune hydrops. Nuchal cystic hygroma carries a high risk of chro-

FIGURE 2b CYSTIC HYGROMA

NUCHAL ABNORMALITIES

Abnormal fluid collections or thickening in the posterior fetal neck carry a high risk for aneuploidy, even in the absence of other ultrasonographic markers. These findings may be categorized as a spectrum of abnormalities, ranging from cystic hygroma to nuchal thickening or increased nuchal fold to nuchal translucency (also known as nuchal oedema). NUCHAL CYSTIC HYGROMA

Nuchal cystic hygroma (eH) deserves special mention because it is one of the most common and

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, , , mosomal abnormality, usually Turner's syndrome (45XO); trisomies 21 and 18 comprise the other abnormal karyotypes. Cystic hygroma can be associated with normal chromosomes when Noonan's syndrome should be considered, particularly if rhe fetus is seen to be male. 18•19

ECHOGENIC BOWEL

The assoeiation between echogenic bowel and trisomy 21 was first established by Nyberg et al. , who no ted echogenic bowel in seven of 68 (10%) trisomy 21 fetuses scanned between 14 and 24 weeks gesration. 26 In addition, echogenie bowel has been shown to be a non-specific finding seen in so me fetuses wirh bowel atresia, volvulus, eongenital infeetion and me conium ileus secondary to eystic fibrosis Y Increased risks of intra-uterine growth restriction (IUGR) and fetal demise have also been described with echogenie abdominal masses. Reeognition of eehogenic bowel is subjective, although it is generally considered echogenic if its echogenicity on ultrasound is similar to that of surrounding bone. In our centre, the approach to echogenie bowel is as folIows: 1, Echogenic bowel in rhe mid-trimester fetus should prompt a seareh for additional abnormalities . Even if isolated, the risk for chromosomal abnormality is estimated as high as 5.5 times the baseline risk. Z6 2. Amniocentesis should be offered to identify chromosomal abnormality. In addition, testing for congenital infeetion and screening for cystic fibrosis should also be eonsidered. 3. If these tests are normal, serial ultrasonographie evaluation of fetal growth and antenatal biophysical profile testing should be considered.

NUCHAL FOlD

As many as 80 percent of infants with trisomy 21 will have redundant skin in the posterior part of the neck. I Y,W Benacerraf et al" in 1985, were the first to suggest the usefulness of measuring this feature to deteet fetuses with trisomy 21. 21 In aseries of 1,704 patients undergoing amnioeentesis between 15 and 20 weeks gestation for late maternal age, 11 fetuses had trisomy 21. Forty-five percent of these had nuchal thiekness of six mm or more compared with only 0.6 percent of normal fetuses. Subsequent prospectivestudies have reported variable results, with sensitivities ranging from 16 to 75 pereent, and false-positive rates from zero to eight pereent. ll- 14 The nu eh al fold measurement should be made from the outer skull table to the skin surface, with the transducer in an axial plane through the thalami, cerebellum and oeeipital bone (Figure 3). Improper angling ean produce an incorreetly wide value and false -positive results. 2S Beeause nuehal thickness inereases with gestational age, a threshold of five mm has been suggested between 14 and 18 weeks.

SHORT HUMERUS/FEMUR

FIGURE 3

A well-known eharacteristic of children with trisomy 21 is short stature associated with disproportionately short proximal long bones (femur and humerus). This charaeteristic has been suggested as a potentially useful method for screening fetuses for triso my 21 in the second trimester. There is a large overlap in bone measurement between the group with and the group without trisomy 21. The sensitivities and speeifici ries for deteeting trisomy 21 using femur length vary considerably among studies, Sensitivities as high as 69 pereent with false-positive rates as low as two to seven percent have been reported,2Z,28 but others have shown sensitivities of less than 20 percent for the detection of trisomy 21 using femur length alone. The humerus length appears to be a slightly more spe-

NUCHAL THICKENING AND TRISOMY 21

Axial view at 17 weeks shows nuchal thickening of 8 mm.

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, , , multiple. The most eommon finding is a single foeus in the left ventricle (Figure 4). The prevalence of these foci in routine second trimester sc ans is approximateIy one to three pereent, and histologie studies suggest that these foci are due to mineralization within a papillary muscle.: 9 Follow-up studies of fetuses with ElF have demonstrated normal cardiac function, suggesting that they are usually a benign variant with no cll111ca1 signifieance. However, an association with chromosomal abnormalities, particularly in trisomy 21, has been shown. As an isolated finding, it is estimated that ElF may increase the risk of trisomy 21 two-fold. 1"

FIGURE4 ECHOGENIC CHORDAE TENDINAE (INTRACARDIAC ECHOGENIC FOCUS) AND TRISOMY 13

ISOLATED MILD HYDRONEPHROSIS AND DOWN'S SYNDROME

Mild hydronephrosis or pyelectasis is a common finding dunng routine obstetric ultrasound evaluation. lf defined as an anteroposterior renal pelvic diameter of > 3.5 mm, mild pyelectasis has been found in two to 2.8 percent of the normal population, and in as many as 25 percent of fettIses with trisomy 21 (Figure 5).11 The prevalenee of chromosomal abnormalities has been estimated as 1.1 percent when isolated compared to 5.4 percent, 22.9 percent and 63.3 percent with one, two or three additional abnormalities, respective!y.J2 Snijders et al. estimate that iso la ted mild pyelectasis increases the risk of

Transverse view of the heart shows echogenie focus {arrow} in the left ventricle of the heart. The left ventricle and left atrium also appear disproportionately sma ll.

cifie indieator, but the differences among raees and populations make the use of this marker impraetical in general population screening fm trisomy 21." ECHOGENIC CARDIAC FOCI (ECHOGENIC

CHORDA TENDINAE)

Echogenic intracardiae foei (ElF), also referred tn as echogenic chorda tendinae, may be found in either or both ventricles of (he fetal heart, and may be single or

FIGURE 6

FIGURE 5

CHOROID PLEXUS CYSTS AND TRISOMY 18

RENAL PYELECTASIS AND TRISOMY 21

Transverse view of the kidney shows mild bi lateral renal pyelectasis with the renal pelvis measuring 7 mm in AP dimension.

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Transverse view at the level of the lateral ventricle shows large bilateral choroid plexus cysts (Cl.

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, , , should be the main factor in deciding whether or not fetal karyotyping should be performed. 2. If one additional abnormality is found, the baseline risk is increased about 20-fold. 3. If two or more additional abnormalities are found, the risk is increased by almost a thousand times and karyotyping should be offered, irrespective of matemal age.

trisomy 21 by 1.6-fold over the baseline risk. 33 OTHER SONOGRAPHIC MARKERS CHOROID PLEXUS CYSTS

Choroid plexus cysts (CPCs) are fluid-filled structures of varying size, surrounded by normal tissue in the choroid plexus of the lateral cerebral ventricles. These cysts are found in approximately one to three percent of fetuses at 16 to 24 weeks of gestation. 34 ,3S A higher frequency of CPCs has been observed in fetuses with chromosomal abnormalities, particularly trisomy 18. The risk for trisomy 18 increases with additional anomalies and with unusually large cysts (Figure 6). Among 38 fetuses with trisomy 18 and CPCs reported by Snijders et al. / 697 percent had other detectable anomalies, including 5.2 percent with one additional abnormality and 92.1 percent with two or more abnormalities. On the other hand, in a meta-analysis by Gross et al. ,37 only two cases of trisomy 18 were identified among 748 fetuses with apparently iso la ted CPCs (1:374). These data do not support offering karyotyping in fetuses with isolated CPCs. We support the following recommendations: 1. The detection of fetal CPCs should stimulate the sonographer to search for additional features of trisomy 18 (e.g. abnormal heart, clenched fists) . Jf the cysts are apparently isolated, the risk of trisomy 18 is only marginally increased, and maternal age

FETAL NUCHAL TRANSLUCENCY

(10-14

Nuchal translucency (NT) is a term used to describe a sonolucent area in the nuchal region of the neck, typically observed between 10 and 14 weeks (Figure 7).38.39 Nuchal translucency normally increases with gestational age, and thresholds for abnormal NT have been described in relation to crown rump length. In general, a measurement of 3.5 mm or more is considered abnormal. Increased NT has been associated with a variety of chromosomal abnormalities including trisomies 21, 18 and 13 and triploidy and Turner's syndrome. Among karyotypically normal fetuses with an abnormal NT measurement, an increased incidence of cardiovascular and pulmonary defects, skeletal dysplasias, congenital infection and metabolic and haematologic disorders have been noted. 39 It is feasible to combine matemal age with gestational age and NT thickness to derive an 'NT-adjusted risk' for aneuploidy. Using a computer programme to calculate the adjusted risk, Snijders et al. showed, in aseries of 100,000 patients, that using a cut-off of one in 300 estimated risk for trisomy 21, a detection rate of 82 percent for tri so my 21 and 78 percent for other chromosomal abnormalities could be achieved at an FPR of eight percent. 40 First-trimester scanning with measurement of NT thickness has the obvious advantage of offering early invasive testing (e.g. chorionic villus sampling) to women determined to be at risk. In addition, screening with NT thickness is potentially useful in multiple pregnancies because alternative methods of screening (e.g. maternal biochemistry).are not applicable in such cases. Selective termination of the affected fetus, if chosen, carries a lower risk of miscarriage of the normal fetus if performed before 16 weeks gestation than after 16 weeks gestation (5.4 versus 14.4%, respec-

FIGURE 7 NUCHAL TRANSLUCENCY AND TRISOMY 21 AT 11 WEEKS GESTATION

Megacystistis was also noted.

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, , , tively).41 In addition, NT screening may be FIGURE8 useful in monochorionic twin pregnancies in APPROACH TO THE PATIENT WITH AN ULTRASOUND-IDENTIFIED predicting the subsequent development of FETAL ABNORMALITY severe twin-to-twin transfusion syndrome FETAL ABNORMALITY (TTS). It has been suggested by Sebire et al. \ DETAILED SONOGRAPHIC EVALUATION that the underlying haemodynamic changes \ associated with TIS may manifest as increased Isolated abnormality* More than 1 abnormality* fetal NT thickness between 10 and 14 weeks \ \ gestationY Consider geneticlobstetric Refer for genetic counselling counselling and fetal karyotyping T 0 summarize, the following conclusions \ Risk of karyotypic abnormality depends on: Risk of karyotypic abnormality high about NT may be made: • Type of abnormality 1. Increased NT is a useful marker for • Maternal age (MA) (risk increases with chromosomal abnormalities including increasing MA) • Gestational age (GA) (risk increases with trisomies 21,18,13 and Turner's syndrome increasing GA) and triploidy in first trimester (10-14 *Consider adjunct screening methods (e.g. biochemical markers). **See Table 4. weeks). 2. An NT measurement of;::: 3.5 mm is considered abnormal and an indication for genetic counuploidy in dichorionic ge stations and TTS in selling and consideration of fetal monochorionic gestations. karyotyping. CURRENT STATUS OF NUCHAL 3. The 'NT-adjusted risk' for trisomies can be derived TRANSLUCENCY SCREENING IN by multiplying the background risk by a likelihood CANADA ratio which depends on the degree of deviation in NT from the normal median for CRL. This Whether NT screening should be performed in the approach is currently limited in Canada to centres general population or the high risk population or both with sonographers, genetic counsellors and physiremains a subject of extensive investigation. At precians 'certified' in nuchal scanning by the Fetal sent, offering NT screening is not within the standard of Medicine Foundation (FMF), United Kingdom, care in Canada. Efforts are under way to establish properforming NT screening under protocols for tocols for investigation so that further information can investigation. be obtained to determine whether NT screening should 4. The risk of fetal anomalies and the mortality be performed and, if so, how to introduce it most effirate increase with the degree of NT thickness. 5. Abnormal NT thickness commonly is resolved TABLE5 during the second trimester whether or not the chromosomes are abnormal. ABNORMALITIES ASSOCIATED WITH INCREASED PROBABILITY OF FETAL CHROMOSOME ABNORMALlTY, EVEN IF ISOLATED 6. Patients with an NT measurement of;::: 3.5 mm • Borderline ventriculomegaly with anormal karyotype are at increased risk of • Posterior fossa abnormalities birth defects including cardiac septal defects. • Cystic hygroma All should be followed up with a targeted obstetric • Nuchal fold sonogram, including detailed cardiac evaluation at • Nuchal translucency 18 to 22 weeks gestation. If a11 of these examina• Atrioventricular septal defect tions are normal, the prognosis is good and the • Double outlet right ventricle majority of such children can be expected to be • Omphalocoele normal at birth. Further long-term follow-up is • Duodenal atresia needed. • Echogenie bowel 7. Nuchal translucency screening is useful in multi• Genito-urinaryabnormalities • Non-immune hydrops ple gestation for predicting increased risk for ane-

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C9

steoporosis affects one in four \\omen OYer the ag-e or i)() in Canada. T\\·eln, to 2W'f of Canadian \H>IlH:' n who sustain a fracture of the hip will die \\"ithin six months. · That's where Premarin can .help. \\"hen taken long--term, Premarin has been shown to reduce >steoporotic fractures lJ\ .){)(1r .'" Premarin can also offer relief from hot flushes , night s\\·cats. Yaginal drYness, anclmam other symptoms of menopause. :\s \H~ ll , hormone replacement therapy otTers direct benefits on qua lity of life. ' So when your patients ask about osteoporosis and menopause , tell them you han· some \cry comforting nc\\·s.

, , , ciently. In order to ensure quality assurance with respect to NT measurements, it is anticipated that the 10 to 14-week certification course currently offered by The Fetal Medicine Foundation (FMF) in the UK will be adopted as a standard in Canada. At present, using a biometric cut-off (:2: 3.5 mm) is a satisfactory approach. If a measurement of 3.5 mm or greater is reported by a reliable sonograph er, the patient should be offered prompt genetic counselling and the option of invasive testing with appropriate follow-up if the karyotype is normal.

4. The maternal age. The risk of a chromosomal abnormality increases with increasing maternal age. 5. Past history of pregnancy. A historyof a previous fetus/baby with trisomy increases the age-related risk by 0.5 percent. These factors determine the background risk wh ich can then be multiplied by aseries of components which depend on the results of screening tests carried out during the pregnancy. Every time a new test is performed, the background risk is multiplied by the test factor to determine a new risk. This process is called sequential screening.

APPROACH TO THE PATIENT WITH ULTRASOUND FETAL ANOMALIES RISK ASSESSMENT

SONOGRAPHIC MARKERS AND RISK ASSESSMENT: 'THE GENETIC SONOGRAM'

The detection of a fetal abnormality by ultrasound, regardless of whether it appears 'major' or 'minor,' should prompt the sonographer to embark on a detailed search for associated malformations (Figure 8). In fetuses with multiple abnormalities, the frequency of chromosomal defects is high and fetal karyotyping should be offered, irrespective of maternal age. When apparently isolated or 'subtle' abnormalities (e.g. mild hydronephrosis) are present, there are large differences in the reported prevalence of associated chromosomal defects, and it is less clear whether karyotyping should be offered. This is especially true when the prevalence of the abnormality is high in the general population, and the prognosis, in the absence of a chromosomal defect, is good (e.g. mild pyelectasis) . Certain factors do influence the patient's risk and should be kept in mind. These include: 1. The type of defect. Certain abnormalities are associated with an increased probability of fetal chromosomal abnormality even if isolated. These are described in Table 5. 2. The number of abnormalities present. The risk of chromosomal abnormalities increases with the number of abnormalities present. 3. The gestational age of the fetus. The risk of a chromosomal abnormality decreases with increasing gestational age. This reflects the natural attrition of chromosomally abnormal fetuses in early pregnancy.

Methods for integrating sonographic markers into the patient's overall risk-assessment far aneuploidy have been proposed by a number of authors, and rely largely on the principle of sequential screening. In Nyberg's model, for example, each ultrasound marker is assigned a likelihood ratio (LR) which is then multiplied by the patient's age-related risk to give an 'age-adjusted ultrasound risk assessment' ('AAURA')." In this system, the sonographic marker of echogenic bowel is assigned an LR of 5.5. For a 34-year-old woman, the finding of echogenic bowel would increase her risk of fetal DS from a baseline of one in 342 (based on age alone) to one in 62 0/342 x 5.5}, which would result in her being offered amniocentesis. Conversely, anormal ultrasound examination could reduce her age-related risk by approximately 50 percent, but may miss up to one in every three DS fetuses (about 1/3 of DS fetuses will have anormal ultrasound examination). In another approach, described by Benacerraf et al. , 'points' are assigned based on specific ultrasound findings. For example, structural malformations are assigned two points and 'markers,' far example pyelectasis one point. Genetic counselling and amniocentesis are then offered to any patient with ascore of two or more. In arecent modification of this approach, points are also assigned for advanced maternal age, with one point for women aged 35 to 40 years inclusively, and two points for women over 40. 44

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, , , The advantages of ultrasound screening using such a system as 'AAURA' or the scoring index method are that it permits improved individual counselling about the risk of fetal OS following a second trimester sonogram. The sensitivity and specificity of the 'genetic sonogram,' as marker screening is now commonly referred to, in the detection of fetal OS have yet to be evaillated prospectively. This approach, while very promising, remains under investigation in Canada at present.

for fetal OS (approximately 87% at a 5% FPR). Further prospective trials are needed to verify this approach. DISADVANTAGES OF BIOCHEMICAL AND ULTRASOUND SCREENING

At present, the current standard of care is to offer all women biochemical screening, where available, at 16 weeks gestation, and an ultrasound examination between 16 and 20 weeks of gestation. Screening in the first trimester with either biochemistry or NT thickness is not available routinely in Canada. Many centres are evaluating the nuchal region in the first trimester and reporting abnormal measurements. It is, thus, possible that many women may be subjected to all three screening methods (i.e. a first trimester nllchal scan, and second trimester biochemical screen and sonogram). This could result in an increase in the rate of invasive testing, from a minimum of five percent for one method to a maximum of 18 percent if all three methods are used, without achieving an increase in the detection rate. If nuchal screening were performed, most chromosomal abnormalities would be detected in the first trimester, resulting in a decreased incidence of OS in ongoing pregnancies and, therefore, a reduction in the positive predictive value of se co nd trimester screening with either biochemistry or ultrasound. The only way such screening wOllld be effective would be if the apriori risk of each patient were modified after each screen to 'correct' for the change in the underlying prevalence (e.g. sequentia1 screening). While this approach appears feasible, flIrther prospective data are required before implementation can be considered. False-positive diagnoses remain a recognized disadvantage of both biochemical and 1Iitrasound screening, as they may markedly increase parental anxiety and lead to unnecessary invasive testing and obstetric intervention with its attendant risks. In addition, many of the newer ultrasound 'markers' in the presence of normal chromosomes, are associated with uncertain prognoses. While this also is an area of active research, more data are needed regarding the long-term implications of these findings.

BIOCHEMICAL MARKERS AND UL TRASOUND

Another important clinical issue is how to integrate ultrasound markers and biochemical results. A common clinical quest ion is whether a 'normal' ultrasound examination (18-20 weeks gestation) can be used to 'modify' the risk of fetal OS as determined by the patient's biochemical profile. In other words, are biochemical analytes and ultrasound markers independent variables with respect to risk for fetal OS? If so, a new risk could be generated based on the likelihood ratios derived from each screening method. For example, if a woman's risk of fetal OS based on her maternal serum screen is one in 150, and she has anormal 'genetic sonogram' at 18 weeks, her baseline risk may be reduced by as much as 50 percent, to one in 300. She may then decide not to undergo amniocentesis. At present, however, we believe that ultrasound is still not sensitive or specific enough to replace amniocentesis. Thus, the current standard of care would still be to offer amniocentesis to any woman with an abnormal biochemical screen, though she may wish to take her ultrasound information into consideration in making her decision. FIRST TRIMESTER BIOCHEMICAL AND ULTRASOUND MARKERS

In the first trimester, free-beta-hCG and PAPP-A appear to be effective markers for fetal OS.lllb In addition, both biochemical markers and fetal NT measurements have been shown to be independent variables when it comes to predicting fetal OS. Various studies have shown that combining the first trimester NT measurement with the biochemistry is associated with an increased detection efficiency

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, , , FETAL CELLS IN THE MATERNAL

4.

CIRCULATION

The transplacental passage of fetal cells into the matemal circlilation during pregnancy has been recognized for many years. Recently, considerable progress has been made in isolating these cells from the maternal circlilation and llsing them for prenatal diagnosis. Isolation of these cells is challenging due to their very small numbers and, at present, sllccessful prenatal diagnosis using these cells has been limited tu common aneuploidies and single gene disorders llsing such technologies as FISH and polymerase chain reaction (PCR). Clinicians need to be aware of advances in this area because the availability of such early noninvasive testing could greatly alter our entire approach to prenatal diagnosis!'

5.

6.

7.

8.

CONCLUSION

9.

In conclusion, tremendous advances have been made in recent years in developing non-invasive screening methods to identify women at increased risk of carrying fetuses with chromosomal abnormalities. Of these, the most promising new development is biochemical screening in the first trimester using free-ßhCG and PAPP-A in conjunction with nuchal translucency screening. While this approach is achieving increasing acceptance worldwide, the current standard of care for aneuploidy screening in Canada remains matemal-age-based screening. Materna1 serum screening in the seclmd trimester is not uniformly available here, and the 18-week screening ultrasound is currently recommended. The role of the 'genetic sonogram' remains to be cIarified. Further prospective evaluations of these screening tests willlikely be required before they are incorporated into clinical practice.

10. 11.

12.

13.

14.

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