KH Nicolaides, NJ Sebire, RJM Snijders

‘The hair is not black, as in the real Mongol, but of a brownish colour, straight and scanty. The face is flat and broad, and destitute of prominence. The cheeks are roundish, and extended laterally. The eyes are obliquely placed, and the internal canthi more than normally distant from one another. The palpebral fissure is very narrow. The forehead is wrinkled transversely from the constant assistance which the levatores palpebrarum derive from the occipito-frontalis muscle in opening of the eyes. The lips are large and thick with transverse fissures. The tongue is long, thick, and is much roughened. The nose is small. The skin has a slight dirty yellowish tinge, and is deficient in elasticity, giving the appearance of being too large for the body.’

The above is an extract from the paper ‘Observations on an ethnic classification of idiots’ by Langdon Down, published in 1866¹. Down, who was a physician at the London Hospital, coined the phrase Mongolian idiots because he felt that a subgroup of his patients had a resemblance to the Mongolian peoples and this fitted in with his theory of ‘retrogression’ of ethnic type. Down’s theory of ethnic regression was in keeping with Darwin’s contemporary scientific reasoning for evolution. In 1924, Crookshank suggested that the regression was not merely to a primitive Oriental human type but also to the orangutan². Even though the theory of ethnic regression was proven to be inaccurate, Down’s description of the appearance of the skin was the basis for the observation, made more than one century later, that affected individuals during the 3rd month of intrauterine life, have a subcutaneous collection of fluid behind the neck (Figure 1), which can be visualized by ultrasound as nuchal translucency (Figure 2).

Figure 1 - Fetus with subcutaneous collection of fluid at the back of the neck. Image kindly provided by Dr Eva Pajkrt, University of Amsterdam.	Figure 2 - Ultrasound picture of a 12-week fetus with trisomy 21, demonstrating increased nuchal translucency thickness

Langdon Down in 1866 and Fraser and Mitchell in 1876 recognized that the condition was congenital, dating from intrauterine life, and in 1914 Goddard found that there was no increased incidence within families^1,3,4. A number of conditions were advocated as potential causes of Down’s syndrome, including syphilis, tuberculosis, parental alcoholism, epilepsy, insanity, nervous instability and mental retardation in a close relative, thyroid deficiency, hypoplasia of the fetal adrenal glands, dysfunction of the fetal pituitary and abnormality of the fetal thymus^1,6–13.

The association between Down’s syndrome and increased maternal age was noted in 1909 by Shuttleworth⁶, who examined 350 cases and reported that:

‘It would seem fair inference... that more than half of the Mongolian imbeciles in institutions are last-born children, mostly of long families, and that in a considerable proportion – from one-half to one-third – the mothers were at the time of gestation approaching the climacteric period, and that in consequence the reproductive powers were at a low ebb. Which of the two factors – the advanced age of the mother or her exhaustion by a long series of previous pregnancies – is the more potent factor is open to doubt.’⁶

As a result of the above observation, hypotheses were based upon theoretical degeneration of the ovum^14–16. However, advanced maternal age could not be the only factor, because, in some cases, there appeared to be a hereditary factor as well. For instance, dizygotic twins were unequally affected whereas monozygotic twins were equally affected¹⁷. It was also noticed that the condition could be transmitted from mother to baby, and, when more than one member of a family was affected, the dependence on the mother’s age was weakened^18–21. The concept of non-dysjunction in Down’s syndrome was suggested by Waardenburg in 1932²². In 1934, Bleyer proposed that an unequal migration of chromosomes during cell division may result in trisomy¹⁶.

In 1956, Tjio and Levan, working with improved techniques on cultures of lung fibroblasts, established that the normal diploid chromosome number is 46²³. In the same year, Ford and Hamerton found that the haploid number was 23 in human spermatocytes²⁴. These discoveries led a number of laboratories to study the karyotype in various pathological conditions and in 1959 Lejeune et al. and Jacobs et al. demonstrated that an extra acrocentric chromosome was present in persons with Down’s syndrome, resulting in an aneuploid chromosome number of 47^25,26.

There were familial cases of Down’s syndrome which were not the result of trisomy. In 1960 Polani et al. examined the chromosomes of a child with Down’s syndrome from a 21-year-old mother, there were 46 chromosomes with a centric fusion of two chromosomes (15:21)²⁷. Familial transmission of this type of translocation was demonstrated by Penrose et al. in 1960 in a family with two Down’s syndrome sibs²⁸. In 1961, Clarke et al. reported on a 2-year-old girl with normal intelligence but some physical features suggestive of Down’s syndrome; she was discovered to be a mosaic for normal and trisomic cells²⁹.

Today we know that Down’s syndrome occurs when either the whole or a segment of the long arm of chromosome 21 is present in three copies instead of two. This can occur as a result of three separate mechanisms: non-dysjunction, found in about 95% of cases, translocation and mosaicism. In 1991, Antonarakis et al. examined DNA polymorphisms in Down’s syndrome infants and demonstrated that 95% of non-dysjunction trisomy 21 is maternal in origin³⁰. The region that codes for most of the Down’s syndrome phenotype is the distal portion of band q21.1 and bands q22.2 and q22.3. This region determines the facial features, heart defects, mental retardation and probably the dermatoglyphic changes in affected individuals³¹.

In 1966, 100 years after the original essay of Langdon Down, it became possible to diagnose trisomy 21 prenatally by karyotyping of cultured amniotic fluid cells^32,33.

The first method of screening for trisomy 21, introduced in the early 1970s, was based on the observation of Shuttleworth on the association with advanced maternal age⁶. It was apparent that amniocentesis carried a risk of miscarriage and this in conjunction with the cost implications, meant that prenatal diagnosis could not be offered to the entire pregnant population. Consequently, amniocentesis was initially offered only to women with a minimum age of 40 years. Gradually, as the application of amniocentesis became more widespread and it appeared to be ‘safe’, the ‘high-risk’ group was redefined to include women with a minimum age of 35 years; this ‘high-risk’ group constituted 5% of the pregnant population.

In the last 20 years, two dogmatic policies have emerged in terms of screening. The first, mainly observed in countries with private healthcare systems, adhered to the dogma of the 35 years of age or equivalent risk; since the maternal age of pregnant women has increased in most developed countries, the screen-positive group now constitute about 10% of pregnancies. The second policy, instituted in countries with national health systems, has adhered to the dogma of offering invasive testing to the 5% group of women with the highest risk; in the last 20 years, the cut-off age for invasive testing has therefore increased from 35 to 37 years. In screening by maternal age with a cut-off age of 37 years, 5% of the population are classified as ‘high risk’ and this group contains about 30% of trisomy 21 babies.

In the late 1980s, a new method of screening was introduced that takes into account not only maternal age but also the concentration of various fetoplacental products in the maternal circulation. At 16 weeks of gestation the median maternal serum concentrations of a-fetoprotein, estriol and human chorionic gonadotropin (hCG) (total and free-b) in trisomy 21 pregnancies are sufficiently different from normal to allow the use of combinations of some or all of these substances to select a ‘high-risk’ group. This method of screening is more effective than maternal age alone and, for the same rate of invasive testing (about 5%), it can identify about 60% of the fetuses with trisomy 21.

In the 1990s, screening by a combination of maternal age and fetal nuchal translucency thickness at 11–14 weeks of gestation was introduced. This method has now been shown to identify about 75% of affected fetuses for a screen-positive rate of about 5%.

Recent evidence suggests that maternal age can be combined with fetal nuchal translucency and maternal serum biochemistry (free b-hCG and pregnancy-associated plasma protein (PAPP-A)) at 11–14 weeks to identify about 90% of affected fetuses. Furthermore, the development of new methods of biochemical testing, within 30min of taking a blood sample, has now made it possible to introduce One-Stop Clinics for Assessment of Risk (Figure 3).

Figure 3 - Assessment of risk for chromosomal defects can be achieved by the combination of maternal age and history of previously affected pregnancies, ultrasound measurement of fetal nuchal translucency and biochemical measurement of maternal serum free b-hCG and PAPP-A in an OSCAR at 11-14 weeks of gestation. After counseling, the patient can decide if she wants fetal karyotyping, which can be carried out by chorionic villus sampling in the same visit.

Figure 4 - Maternal age-related risk for chromosomal abnormalities	Figure 5 -Gestational age-related risk for chromosomal abnormalities. The lines represent the relative risk according to the risk at 10 weeks of gestation.

Similar methods were used to produce estimates of risks for other chromosomal abnormalities⁴⁰. The risk for trisomy 18 and trisomy 13 increases with maternal age and decreases with gestation; the rate of intrauterine lethality between 12 weeks and 40 weeks is about 80% (Table 2 and Table 3). Turner syndrome is usually due to loss of the paternal X chromosome and, consequently, the frequency of conception of 45,X embryos, unlike that of trisomies, is unrelated to maternal age. The prevalence is about 1 per 1500 at 12 weeks, 1 per 3000 at 20 weeks and 1 per 4000 at 40 weeks. For the other sex chromosome abnormalities (47,XXX, 47,XXY and 47,XYY), there is no significant change with maternal age and since the rate of intrauterine lethality is not higher than in chromosomally normal fetuses the overall prevalence (about 1 per 500) does not decrease with gestation. Polyploidy affects about 2% of recognized conceptions but it is highly lethal and thus very rarely observed in live births; the prevalences at 12 and 20 weeks are about 1 per 2000 and 1 per 250000, respectively⁴⁰.

Crown–Rump Length - CRL 6 to 14 weeks +6days	Nuchal translucency normally increases with gestation (crown–rump length)

Figure 6 - Maternal age-related risk for trisomy 21 at 12 weeks of gestation and the effect of fetal nuchal translucency thickness (NT)

Figure 7 - Maternal age-related risk for trisomy 21 at 12 weeks of gestation and the effect of maternal serum free b-hCG (left) and PAPP-A (right)

Nuchal translucency can be measured successfully by transabdominal ultrasound examination in about 95% of cases; in the others, it is necessary to perform transvaginal sonography. The equipment must be of good quality (about £30000–50000), it should have a video-loop function and the calipers should be able to provide measurements to one decimal point. The average time allocated for each fetal scan should be at least 10 minutes. All sonographers performing fetal scans should be capable of reliably measuring the crown–rump length and obtaining a proper sagittal view of the fetal spine. For such sonographers, it is easy to acquire, within a few hours, the skill to measure nuchal translucency thickness. Furthermore, it is essential that the same criteria are used to achieve uniformity of results among different operators (Figure 8):

1. The minimum fetal crown–rump length should be 45mm and the maximum 84mm. The optimal gestational age for measurement of fetal nuchal translucency is 11 to 13⁺⁶ weeks. The success rate for taking a measurement at this gestation is 98–100%, falling to 90% at 14 weeks; from 14 weeks onwards, the fetal position (vertical) makes it more difficult to obtain measurements⁴⁷.

2. The results from transabdominal and transvaginal scanning are similar but reproducibility may be better with the transvaginal method⁴⁸.

3. A good sagittal section of the fetus, as for measurement of fetal crown–rump length, should be obtained.

4. The magnification should be such that the fetus occupies at least three-quarters of the image. Essentially, the magnification should be increased so that each increment in the distance between calipers should be only 0.1mm. A study, in which rat heart ventricles were measured initially by ultrasound and then by dissection, has demonstrated that ultrasound measurements can be accurate to the nearest 0.1–0.2mm⁴⁹.

5. Care must be taken to distinguish between fetal skin and amnion because, at this gestation, both structures appear as thin membranes. This is achieved by waiting for spontaneous fetal movement away from the amniotic membrane; alternatively, the fetus is bounced off the amnion by asking the mother to cough and/or by tapping the maternal abdomen.

6. The maximum thickness of the subcutaneous translucency between the skin and the soft tissue overlying the cervical spine should be measured by placing the calipers on the lines as shown in Figure 8. During the scan, more than one measurement must be taken and the maximum one should be recorded.

7. The nuchal translucency should be measured with the fetus in the neutral position. When the fetal neck is hyperextended the measurement can be increased by 0.6mm and when the neck is flexed, the measurement can be decreased by 0.4mm⁵⁰.

8. The umbilical cord may be round the fetal neck in 5–10% of cases and this finding may produce a falsely increased nuchal translucency, adding about 0.8mm to the measurement⁵¹. In such cases, the measurements of nuchal translucency above and below the cord are different and, in the calculation of risk, it is more appropriate to use the smaller measurement.
   

Figure 8 - In all the figures there is a good sagittal section of the fetus. In images (a) and (b), the magnification is too small for accurate measurements. The correct magnification is shown in images (c) and (d), where the fetuses are in the neutral position and the amniotic membrane can be clearly seen separated from the nuchal membrane. In image (e), the umbilical cord is round the neck, producing an indentation in the nuchal membrane and the translucency that is larger above than below the cord. The correct placement of the calipers is indicated in diagram (f)

The distribution of nuchal translucency measurements as well as the quality of the images in terms of magnification, section (sagittal or oblique), caliper placement, skin line (nuchal only or nuchal and back) and visualization of the amnion separate from the nuchal membrane are taken into account in the audit of results⁵².

A potential criticism of screening by ultrasound is that scanning not only requires highly skilled operators but it is also prone to operator variability. This issue was addressed by a prospective study at 10–14 weeks of gestation in which the nuchal translucency was measured by two of four operators in 200 pregnant women⁵⁷. This study demonstrated that, after an initial measurement, the second one made by the same (intra-) observer or another (inter-) observer varies from the first by less than 0.54mm and 0.62mm, respectively in 95% of the cases. Additionally, the study demonstrated that the caliper placement repeatability was similar to the intra-observer and inter-observer repeatability, suggesting that a large part of the variation in measurements can be accounted for by the placement of the calipers rather than the generation of the image⁵⁷. Subsequent studies have reported that the intra-observer and inter-observer differences in measurements were less than 0.5mm in 95% of cases^58,59.

Digital image processing and automation of caliper placement may reduce the variation of measurements⁶⁰. In the meantime, it is best to rely on the mean of two good measurements for the calculation of risk, rather than on a single one.

Fetal nuchal translucency thickness increases with crown–rump length^49,61, and therefore it is essential to take gestation into account when determining whether a given translucency thickness is increased. In a study involving more than 100000 pregnancies, the median increased from 1.2mm at 11 weeks to 1.9mm at 13⁺⁶ weeks⁶². Figure 9 illustrates the increases in the 5th, 25th, 50th, 75th and 95th centile of nuchal translucency with crown–rump length; the 99th centile is about 3.5mm throughout this gestational range.

Figure 9 - Reference range of fetal nuchal translucency with crown–rump length showing the 5th, 25th, 50th, 75th and 95th centile

Snijders, Nicolaides

Table 4 - Summary of reported series on first-trimester fetal nuchal translucency (NT) providing data on gestational age (GA) in weeks, criteria for diagnosis of increased NT thickness and the presence of associated chromosomal defects

1. It is possible to measure nuchal translucency successfully during a routine first-trimester scan in 96–100% of cases, provided that, first, the gestation is 11–14 weeks and, second, the sonographers are motivated to take such a measurement. Thus, in the two studies that examined the feasibility of measuring nuchal translucency but in which (a) they included patients from as early as 8 weeks, and (b) no action was taken on the results of the translucency measurement, such a measurement was obtained in only 58% and 66% of cases, respectively^83,84.

2. The false-positive rate varied from as low as 0.8⁸⁶ to as high as 6.3%^54,84, demonstrating the need for unifying the criteria in (a) obtaining the appropriate image, (b) caliper placement, and (c) using the same normal range and same cut-off.
   

Table 5 Studies examining the implementation of fetal nuchal translucency (NT) screening

Figure 10 -Nuchal translucency measurement in 326 trisomy 21 fetuses plotted on the normal range for crown–rump length (95th and 5th centiles)62

Figure 11 - Distribution of fetal nuchal translucency thickness expressed as deviation from expected normal median for crown-rump length in chromosomally normal fetuses (open bars) and 326 with trisomy 21 (solid bars)62

Figure 12 - Likelihood ratios for trisomy 21 in relation to the deviation in fetal nuchal translucency thickness from the expected normal median for crown-rump length62

Table 11 - Harris Birthright Research Centre for Fetal Medicine 10–14-week Ultrasound Study. The fetal heart rate in 842 chromosomally abnormal pregnancies is presented as a percentage of cases above the 95th and 50th and below the 5th centile of the normal range for crown–rump length, derived from 10,083 normal pregnancies

In a study of 10083 normal pregnancies at the Harris Birthright Research Centre for Fetal Medicine, the mean fetal heart rate decreased with gestation from 169bpm at a fetal crown–rump length of 38mm to 154bpm at a crown–rump length of 84mm. The data were normally distributed and the 95th and 5th centile were 10bpm above and below the appropriate normal mean for crown–rump length, respectively. In trisomy 13, Turner syndrome and trisomy 21, the respective mean fetal heart rates were 14bpm, 11.4bpm and 1.4bpm above the normal mean for crown–rump length, whereas, in trisomy 18 and triploidy, the fetal heart rates were 3.4bpm and 4.8bpm below the normal mean, respectively.

Figure 16 - Fetal heart rate (FHR) in trisomy 21 (top left), trisomy 13 (top right), Turner syndrome (bottom left) and triploidy (q) and trisomy 18 (r) (bottom right) plotted on the normal range for gestation (median, 95th and 5th centile)

The tables 11a and 11b shows the effects of the chromosomal defects on fetal heart rate at 10-14 weeks. (Liao et col, Ultrasound Obstet Gynecol 2000; 16: 610-611)

Doppler ultrasound studies have demonstrated that impedance to flow (measured as pulsatility index) decreases with gestation^123,124. This decrease is believed to be a consequence of the increase in the number of vessels (and their relative volume) within the chorionic villi and the expansion of the intervillous circulation¹²⁵.

Doppler sites of interest in the first trimester

Uterine Artery	Spiral Artery
Umbilical Artery (6-12 weeks) Note the absence of the end diastolic velocity in the umbilical artery and the umbilical vein pulsatility - normal findings in the 1o trimester.	Umbilical Artery (10-14 weeks) Note the positive of the end diastolic velocity in the umbilical artery and the absence of umbilical vein pulsatility - normal findings in the 1o trimester.
Middle Cerebral Artery	Descending Aorta
Circle of Willis (13-14 weeks)	Ductus Venosus

There is contradictory evidence on the possible association of trisomy 21 at 11–14 weeks of gestation and increased umbilical artery pulsatility index. Martinez et al. reported that the umbilical artery pulsatility index was above the 95th centile in 55% of their nine cases of trisomy 21 and this was not always associated with an increased nuchal translucency; they estimated that the measurements of both factors might allow detection of up to 89% of cases of trisomy 21¹²⁶. In contrast, Jauniaux et al. examined 11 cases of trisomy 21 and reported that there was no significant difference in umbilical artery pulsatility index compared to normal fetuses and that in none of their cases was the pulsatility index above the 95th centile¹²⁷. Similarly, Brown et al. examined 19 trisomy 21 fetuses with increased nuchal translucency at 11–14 weeks and reported that the umbilical artery pulsatility index was not significantly different from normal; the pulsatility index was above the 95th centile in only two of the cases¹²⁴.

Umbilical artery and Vein	Umbilical artery and Vein
Umbilical artery: high impedance to flow and absent end diastolic flow (normal).	Umbilical artery: high impedance to flow and presence of diastolic flow (normal).
Umbilical Vein: pulsatile flow pattern,	Umbilical Vein: presence of continuous flow pattern, sometimes the pulsatile pattern could persist a little longer, bu must be absent before complete 20 weeks

In normal second- and third-trimester fetuses, pulsatile umbilical venous flow is observed only during fetal breathing. Pulsatile venous flow is also observed in fetuses with growth restriction and in non-immune hydrops and is considered to be a late and ominous sign of fetal compromise^128,129. Evidence from growth-restricted human fetuses and animal models suggests that pulsatile venous flow may result from an increased reversal of flow in the inferior vena cava during atrial contraction, associated with heart failure and abnormal cardiac filling^129,130.

A Doppler study at 11–14 weeks of gestation reported the presence of pulsatile flow in the umbilical vein in about 25% of 302 chromosomally normal fetuses and in 90% of 18 fetuses with trisomy 18 or 13; in 18 fetuses with trisomy 21, the prevalence of pulsatile venous flow was not significantly different from that in chromosomally normal fetuses, but in trisomies 13 and 18 the prevalence was increased¹³¹.

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. It appears to be the most useful vessel in assessing disturbed cardiac function¹³². 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). In cardiac failure, with or without cardiac defects, there is absent or reversed A-wave (see Chapter 3, page 102)¹³³.

Color Doppler Energy "Arteriography" showing the anatomy of the vessels at mid-sagittal plane of the fetal trunk.

It is possible to assess ductus venosus blood flow at 11–14 weeks of gestation by Doppler ultrasound, both transabdominally and transvaginally. A right ventral mid-sagittal plane of the fetal trunk is first obtained during fetal quiescence and the pulsed Doppler gate is placed in the distal portion of the umbilical sinus. The inferior vena cava, left and medial hepatic veins and the ductus venosus drain into a common sub-diaphragmatic vestibulum and therefore, when attempting to obtain flow velocity waveforms from the ductus, care should be taken to avoid contamination from the other veins.

Normal Ductus venosus sonogram. Positive A wave.	Abnormal Ductus venosus sonogram. Reverse A wave
S= systole; D= diastole; A= atrial contraction

A study, examining ductal flow at 11–14 weeks in fetuses with increased nuchal translucency, reported absent or reversed flow during atrial contraction in 57 of 63 (90.5%) chromosomally abnormal fetuses and in only 13 of 423 (3.1%) chromosomally normal fetuses¹³⁴. In seven of the 13 chromosomally normal fetuses with absent or reversed flow, an ultrasound scan at 14–16 weeks demonstrated a major cardiac defect¹³⁴.

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. However, the data suggest that the 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 nuchal translucency and maternal serum free b-hCG and PAPP-A at 11–14 weeks. A policy of reserving invasive testing only for those with abnormal ductal flow could reduce the overall need for chorionic villus sampling from 5% to less than 0.5%, with a small reduction (5–10%) in the estimated sensitivity of 90%¹³⁴.

In trisomy 21 during the first trimester of pregnancy, the maternal serum concentration of free b-hCG is higher than in chromosomally normal fetuses (Table 12), whereas PAPP-A is lower (Table 13). Pregnancy-specific b-1 glycoprotein (SP1), a-fetoprotein and inhibin-A do not provide a useful distinction between affected and normal pregnancies^135–137.

Table 12 - Median MoM in published studies of free b-hCG in trisomy 21 pregnancies

Table 13 - Median MoM in published studies of PAPP-A in trisomy 21 pregnancies

Maternal serum free b-hCG normally decreases with gestation after 10 weeks. In trisomy 21 pregnancies, the levels are increased and the difference between these and those of normal pregnancies increases with advancing gestation. This may account for the variation in the reported median MoM between the various studies (Table 12)^138–158, because there was a considerable variation in the gestational age range of the populations that were examined. Consequently, population parameters derived from studies using a wide gestational age range are not appropriate if screening is to be focused on the optimal time for nuchal translucency measurement (11–14 weeks). The increase with gestation in the difference between trisomy 21 and normal pregnancies has also been shown in studies of paired samples from trisomy 21 pregnancies collected in the first and second trimesters¹⁵³. In a study involving 210 trisomy 21 pregnancies that were examined at 10–14 weeks, the median free b-hCG was 2.15MoM (95% CI, 1.94–2.33); at a 5% screen-positive rate, the detection rate using free b-hCG alone is about 35% and, in combination with maternal age, the detection increases to about 45%¹⁵⁸.

Maternal serum PAPP-A normally increases with gestation. In trisomy 21 pregnancies, the levels are lower but the difference between trisomy 21 and normal pregnancies decreases with advancing gestation. This may account for the variation in the reported median MoM between the various studies (Table 13)^{140,143,145,146,150,152,153,155–166}. In a study involving 210 trisomy 21 pregnancies that were examined at 10–14 weeks, the median PAPP-A was 0.51MoM (95% CI, 0.44–0.56); at a 5% screen-positive rate, the detection rate using PAPP-A alone is about 40% and, in combination with maternal age, the detection increases to about 50%¹⁵⁸.

When considering to combine biochemical markers, it is necessary to take into account the degree of correlation between the markers. In our study, involving 210 trisomy 21 and 946 chromosomally normal controls, the correlations were 0.216 and 0.160, respectively¹⁵⁸. Additionally, each marker showed a small but significant negative correlation with maternal weight (PAPP-A, r = -0.278; free b-hCG, r = -0.146). After combining free b-hCG and PAPP-A with maternal age in mathematical models, it has been estimated that the detection rate of trisomy 21 is about 60% at a 5% screen-positive rate (Table 14) ^{150,152,153,155–158,167,168}.

Table 14 - Estimated detection rate for trisomy 21 by a combination of maternal age and first-trimester maternal serum PAPP-A and free b-hCG at a 5% fixed screen-positive rate

There is no significant association between fetal nuchal translucency and maternal serum free b-hCG or PAPP-A in either trisomy 21 or chromosomally normal pregnancies ^147,158,164. The estimated detection rate for trisomy 21 by a combination of maternal age, fetal nuchal translucency and maternal serum PAPP-A and free b-hCG is about 90% for a screen-positive rate of 5% (Table 15)^{148,157,158,164,167,169}. Alternatively, at a fixed detection rate of 70%, the screen-positive rate would be only 1%¹⁵⁸. The performance of the combined test now requires assessment in prospective studies.

Table 15 - Estimated detection rate for trisomy 21 by a combination of maternal age, fetal nuchal translucency and first-trimester maternal serum PAPP-A and free b-hCG at a 5% fixed false-positive rate

An important development in biochemical analysis is the introduction of a new technique (random access immunoassay analyzer using time-resolved-amplified-cryptate-emission), which provides automated, precise and reproducible measurements within 30 minutes of obtaining a blood sample¹⁵⁸. This has made it possible to combine biochemical and ultrasonographic testing as well as to counsel in one-stop clinics for early assessment of fetal risk (OSCAR).

At 16 weeks of gestation, the median maternal serum concentrations of a-fetoprotein, estriol, hCG (total and free b) and inhibin A in trisomy 21 pregnancies are different from normal. The risk for trisomy 21 can be derived by multiplying the background maternal age and gestational age-related risk by the likelihood ratios for these substances, after corrections for the interrelations between them. The risk of trisomy 21 is increased if the levels of hCG and/or inhibin A are high, and the levels of a-fetoprotein and/or estriol are low. The estimated detection rates are 50–70% for a screen-positive rate of about 5%.

In women having second-trimester biochemical testing following first-trimester nuchal translucency screening (with or without maternal serum biochemistry), the background risk needs to be adjusted to take into account the first-trimester screening results. Since first-trimester screening identifies almost 90% of trisomy 21 pregnancies, second-trimester biochemistry will identify – at best – 6% (60% of the residual 10%) of the affected pregnancies, with doubling of the overall invasive testing rate (from 5% to 10%). It is theoretically possible to use various statistical techniques to combine nuchal translucency with different components of first-trimester and second-trimester biochemical testing. One such hypothetical model has combined first-trimester nuchal and PAPP-A with second-trimester free b-hCG, estriol and inhibin A, claiming a potential sensitivity of 94% for a 5% false-positive rate¹⁷⁰. Even if the assumptions made in this statistical technique are valid, it is unlikely that it will gain widespread clinical acceptability¹⁷¹.

Two studies have reported on the impact of first-trimester screening by nuchal translucency on second-trimester serum biochemical testing. In one study, the proportion of affected pregnancies in the screen-positive group (positive predictive value) of screening by the double test in the second trimester was 1 in 40; after the introduction of screening by nuchal translucency, 83% of trisomy 21 pregnancies were identified in the first trimester and the positive predictive value of biochemical screening decreased to 1 in 200¹⁷². In the second study, first-trimester screening by nuchal translucency identified 71% of trisomy 21 pregnancies for a screen-positive rate of 2%, and the positive predictive value of second-trimester screening by the quadruple test was only 1 in 150¹⁷³.

In women who had first-trimester screening by a combination of fetal nuchal translucency and maternal serum PAPP-A and free b-hCG, it is clearly advisable that second-trimester biochemical testing is avoided because, first, the sensitivities of first- and second-trimester biochemical screening are similar; second, the main component of the second-trimester biochemical screening is free b-hCG, and, third, there is a good correlation between first- and second-trimester maternal serum hCG levels. If both first- and second-trimester biochemical testing have been carried out, then the likelihood ratio from the measurement of nuchal translucency can be multiplied with the results of either first- or second-trimester serum testing. This is certainly valid for second-trimester programs that are mainly based on free b-hCG because the interrelation between nuchal translucency and this metabolite has been established¹⁴⁸.

Figure 17 - Incidence of chromosomal abnormalities in relation to number of sonographically detected defects174

These defects are common and they are not usually associated with any handicap, unless there is an associated chromosomal abnormality. Routine karyotyping of all pregnancies with these markers would have major implications, both in terms of miscarriage and in economic costs. It is best to base counseling on an individual estimated risk for a chromosomal abnormality, rather than the arbitrary advice that invasive testing is recommended because the risk is ‘high’. The estimated risk can be derived by multiplying the background risk (based on maternal age, gestational age, history of previously affected pregnancies and, where appropriate, the results of previous screening by nuchal translucency and/or biochemistry in the current pregnancy) by the likelihood ratio of the specific defect^175–177. For the following conditions, there are sufficient data in the literature to estimate the likelihood ratio for trisomy 21.

This is the second-trimester form of nuchal translucency. It is found in about 0.5% of fetuses and it may be of no pathological significance. However, it is sometimes associated with chromosomal defects, cardiac anomalies, infection or genetic syndromes46. For isolated nuchal edema, the risk for trisomy 21 may be 15 times the background178,179.

If the femur is below the 5th centile and all other measurements are normal, the baby is likely to be normal but rather short. Rarely is this a sign of dwarfism. Occasionally, it may be a marker of chromosomal defects. On the basis of existing studies, short femur is found four times as commonly in trisomy 21 fetuses compared to normal fetuses180–185. However, there is some evidence that isolated short femur may not be more common in trisomic than in normal fetuses178.

This is found in about 0.5% of fetuses and is usually of no pathological significance. The commonest cause is intra-amniotic bleeding, but occasionally it may be a marker of cystic fibrosis or chromosomal defects. For isolated hyperechogenic bowel, the risk for trisomy 21 may be three times the background178,186,187.

These are found in about 4% of pregnancies and they are usually of no pathological significance. However, they are sometimes associated with cardiac defects and chromosomal abnormalities. For isolated hyperechogenic foci the risk, for trisomy 21 may be four-times the background178,189–191.

These are found in about 1–2% of pregnancies and they are usually of no pathological significance. When other defects are present, there is a high risk of chromosomal defects, usually trisomy 18 but occasionally trisomy 21. For isolated choroid plexus cysts, the risk for trisomy 18 and trisomy 21 is about 1.5 times the background177.

This is found in about 1–2% of pregnancies and is usually of no pathological significance. When other abnormalities are present, there is a high risk of chromosomal defects, usually trisomy 21. For isolated mild hydronephrosis, the risk for trisomy 21 is about 1.5 times the background176,192,193.

There are no data on the interrelation between these second-trimester ultrasound markers and nuchal translucency at 11–14 weeks or first- and second-trimester biochemistry. However, there is no obvious physiological reason for such an interrelation and it is therefore reasonable to assume that they are independent. Consequently, in estimating the risk in a pregnancy with a marker, it is logical to take into account the results of previous screening tests. For example, in a 20-year-old woman at 20 weeks of gestation (background risk of 1 in 1295), who had a 11–14 week assessment by nuchal translucency measurement that resulted in a 5-fold reduction in risk (to about 1 in 6475), after the diagnosis of mild hydronephrosis at the 20-week scan, the estimated risk has increased by a factor of 1.5 to 1 in 4317. In contrast, for the same ultrasound finding of fetal mild hydronephrosis in a 40-year-old woman (background risk of 1 in 82), who did not have nuchal translucency or biochemistry screening, the new estimated risk is 1 in 55.

There are some exceptions to this process of sequential screening, which assumes independence between the findings of different screening results. The findings of nuchal edema or a cardiac defect at the mid-trimester scan cannot be considered independently of nuchal translucency screening at 11–14 weeks. Similarly, hyperechogenic bowel (which may be due to intra-amniotic bleeding) and relative shortening of the femur (which may be due to placental insufficiency) may well be related to serum biochemistry (high free b-hCG and inhibin-A and low estriol may be markers of placental damage) and can therefore not be considered independently in estimating the risk for trisomy 21. For example, in a 20-year-old woman (background risk for trisomy 21 of 1 in 1295), with high free b-hCG and inhibin-A and low estriol at the 16-week serum testing resulting in a 10-fold increase in risk (to 1 in 129), the finding of hyperechogenic bowel at the 20-week scan should not lead to the erroneous conclusion of a further three-fold increase in risk (to 1 in 43). The coincidence of biochemical and sonographic features of placental insufficiency makes it very unlikely that the problem is trisomy 21 and should lead to increased monitoring for pre-eclampsia and growth restriction, rather than amniocentesis for fetal karyotyping.

About 1 in 10³–10⁷ nucleated cells in maternal blood are fetal^194–196. The proportion of fetal cells can be enriched to about 1 in 10–100 by techniques such as magnetic cell sorting (MACS) or fluorescence activated cell sorting (FACS) after attachment of magnetically labeled or fluorescent antibodies on to specific fetal cell surface markers^{194,197–199}. The most commonly used antibody is anti-CD71, which is directed against the transferrin receptor present on the surface of all cells actively incorporating iron^198,200. Other cell types in maternal blood, such as activated lymphocytes, have this receptor but anti-CD71 provides a reasonable level of enrichment once maternal lymphocytes have been removed. Magnetic cell sorting is cheaper, quicker and requires less expertise to perform than FACS. The technique utilizes metallic beads labeled with an antibody specific for the target cell. The antibody is incubated with the sample and the cell–antibody–bead complex is isolated by placing on a magnet. Successful use of MACS involves prior separation of cells by triple density centrifugation. Essentially, the maternal blood sample is placed in a tube containing three sugar-based reagents of different density and, after centrifugation, the middle layer containing erythroblasts and neutrophil granulocytes is separated. These cells are incubated with magnetically labeled CD71 antibody and MACS is then carried out (Figure 18).

Figure 18 - Triple density centrifugation and magnetic cell sorting techniques, using magnetically labeled anti-CD71 (antibody against transferrin receptor antigen)

Figure 19 - Fluorescence in situ hybridization analyzed cells, in maternal blood enriched for fetal cells, from trisomy 21 (A), trisomy 18 (B) and trisomy 13 (C). Pregnancies demonstrating three-signal nuclei with the appropriate chromosome-specific probe

Al-Mufti et al. took maternal peripheral blood immediately before chorionic villus sampling from 230 women with singleton pregnancies at 11–14 weeks of gestation¹⁹⁹. These pregnancies had been identified as being at high risk for trisomies after screening by a combination of maternal age and fetal nuchal translucency thickness. Triple density gradient centrifugation, followed by incubation of the erythroblast-rich fraction with magnetically labeled CD71 antibody, MACS and FISH were carried out. In 3% of cases, no fetal hemoglobin-positive cells were observed. In the chromosomally abnormal group, the percentage of cells demonstrating three-signal nuclei was higher than in the normal group but there was an overlap in values between the two groups (Figure 20).

Figure 20 - Percentage of cells with three-signal nuclei using the 21 chromosome-specific probe in maternal blood enriched for fetal cells from chromosomally normal pregnancies and those with trisomy 21

Table 16 - Sensitivity and false-positive rate for the various cut-off percentages of cells with three-signal nuclei in the chromosomally abnormal and normal groups using fluorescent probes for chromosomes 21, 18 and 13

On the basis of currently available technology, examination of fetal cells from maternal peripheral blood is more likely to find an application as a method for assessment of risk, rather than the non-invasive prenatal diagnosis of chromosomal defects. First-line screening by a combination of maternal age, fetal nuchal translucency and maternal serum free b-hCG with PAPP-A could detect 90% of trisomy 21 pregnancies for an invasive testing rate of about 5%¹⁵⁸. One option in the management of the high-risk group is to carry out FISH on maternal blood enriched for fetal cells and reserve chorionic villus sampling only for those pregnancies where no fetal hemoglobin-positive cells are recovered and those where at least 3% of the cells demonstrate three signals with the 21-chromosome specific probe. Such a policy could potentially reduce the need for invasive testing to less than 1% of the whole population with a minor loss (about 3%) in the sensitivity for detection of trisomy 21.

The only randomized trial was performed in Denmark²⁰⁶. In this study, 4606 low-risk, healthy women, 25–34 years old, at 14–20 weeks of gestation, were randomly allocated to amniocentesis or ultrasound examination alone. The total fetal loss rate in the patients having amniocentesis was 1% higher than in the controls. There were significant associations between spontaneous fetal loss and (1) puncture of the placenta, (2) high maternal serum a-fetoprotein and (3) discolored amniotic fluid. The Danish study also reported that amniocentesis was associated with an increased risk of respiratory distress syndrome and pneumonia in neonates. Some studies have reported an increased incidence of talipes and dislocation of the hip after amniocentesis, but this was not confirmed by the Danish study²⁰⁶.

Table 17 - Total fetal loss rate in four randomized studies comparing first-trimester chorionic villus sampling with second-trimester amniocentesis

In 1991, severe transverse limb abnormalities, micrognathia and microglossia were reported in five of 289 pregnancies that had undergone chorionic villus sampling at less than 10 weeks of gestation²¹⁴. Subsequently, a series of other reports confirmed the possible association between early chorionic villus sampling and fetal defects; analysis of 75 such cases demonstrated a strong association between the severity of the defect and the gestation at sampling²¹⁵. Thus, the median gestation at chorionic villus sampling was 8 weeks for those with amputation of the whole limb and 10 weeks for those with defects affecting the terminal phalanxes. The background incidence of terminal transverse limb defects is about 1.8 per 10000 live births²¹⁶, and the incidence following early chorionic villus sampling is estimated at 1 in 200–1000 cases. The types of defects are compatible with the pattern of limb development, which is essentially completed by the 10th week of gestation. Possible mechanisms by which early sampling may lead to limb defects include hypoperfusion, embolization or release of vasoactive substances, and all these mechanisms are related to trauma. It is therefore imperative that chorionic villus sampling is performed only after 11 weeks by appropriately trained operators. The data from the International Registry on chorionic villus sampling are disputing the association between this procedure and limb reduction defects²¹⁷.

Chapter 1 References

1. Langdon Down J. Observations on an ethnic classification of idiots. Clin Lectures and Reports, London Hospital 1866; 3:259–62

2. Crookshank FG. In The Mongol in our Midst. London: Kegan Paul, Trench and Trubner Ltd, 1924

3. Fraser J, Mitchell A. Kalmuc idiocy: report of a case with autopsy with notes on 62 cases by A. Mitchell. J Ment Sci 1876;22:169–79

4. Goddard HH. In Feeble-mindedness, its Causes and Consequences. New York: Macmillan and Co, 1914

5. Sutherland GA. Mongolian imbecility in infants. Practitioner 1899;63:632

6. Shuttleworth GE. Mongolian imbecility. Br Med J 1909;2:661–5

7. Cafferata JF. Contribution a la literature du mongolisme. Arch Med Enf 1909;12:929

8. Caldecott C. Tuberculosis as a cause of death in mongolism. Br Med J 1909;2:665

9. Tredgold AF. In Mental Deficiency (Amentia). London: Bailliere, Tindall and Cox, 1908

10. Stoeltzner W. Zur Atiologie des Mongolismus. Munich Med Wschr 1919;66:1943

11. Vas J. Beitrage zur Pathogenese und Therapie der Idiotia Mongoliana. Jb Kinderheik 1925;111:51

12. Benda CE, Bixby EM. Function of the thyroid and the pituitary in mongolism. Am J Dis Child 1939;58:1240

13. Barnes NP. Mongolism – importance of early recognition and treatment. Ann Clin Med 1923;1:302

14. Jenkins RL. Etiology of mongolism. Am J Dis Child 1933;45:506

15. Rosanoff AJ, Handy LM. Etiology of mongolism with special reference to its occurrence in twins. Am J Dis Child 1934;48:764

16. Bleyer A. Indications that mongoloid idiocy is a gametic mutation of degenerative type. Am J Dis Child 1934;47:342

17. Halbertsma T. Mongolism in one of twins and the etiology of mongolism. Am J Dis Child 1923;25: 350

18. Lelong M, Borniche P, Kreisler L, Baudy R. Mongolien issu de mere mongolienne. Arch Franc Pediat 1949; 6:231

19. Rehn AT, Thomas E. Family history of a mongolian girl who bore a mongolian child. Am J Ment Defic 1957;62:496

20. Penrose LS. Maternal age in familial mongolism. J Ment Sci 1951;97:738

21. Penrose LS. Mongolian idiocy (mongolism) and maternal age. Ann NY Acad Sci 1953;57:494

22. Waardenburg PJ. In Das menschliche Auge und seine Erbalagen. Haag: Martinus Nijhoff, 1932

23. Tijo JH, Levan A. The chromosome number of man. Hereditas 1956;42:1

24. Ford CE, Hamerton JL. The chromosomes of man. Nature 1956;168:1020

25. Lejeune J, Gautier M, Turpin R. Etudes des chromosomes somatiques de neuf enfants mongoliens. C R Acad Sci 1959;248:1721

26. Jacobs PA, Baikie AG, Court Brown WM, Strong JA. The somatic chromosomes in mongolism. Lancet 1959;1:710

27. Polani PE, Briggs JH, Ford CE, Clarke CM, Berg JM. A mongol girl with 46 chromosomes. Lancet 1960;1:721

28. Penrose LS, Ellis JR, Delhanty JDA. Chromosomal translocations on mongolism and in normal relatives. Lancet 1960;2:409

29. Clarke CM, Edwards JH, Smallpiece V. 21 trisomy/normal mosaicism in an intelligent child with mongoloid characters. Lancet 1961;1:1028

30. Antonarakis SE, Lewi JG, Adelsberger PA, Petersen MB, Schinzel AA, Cohen MM, Roulston D, Schwartz S, Mikkelsen M, Tranebjorg L, Greenberg F, How DI, Rudd NL. Parental origin of the extra chromosome in trisomy 21 as indicated by analysis of DNA polymorphisms. N Engl J Med 1991;324:872–6

31. Korenberg JR. Toward a molecular understanding of Down syndrome. Prog Clin Bio Res 1993;384: 87–115

32. Steele MW, Breg WR.Chromosome analysis of human amniotic-fluid cells. Lancet 1966;i:383–5

33. Valenti C, Schutta EJ, Kehaty T. Prenatal diagnosis of Down’s syndrome. Lancet 1968;ii:220

34. Snijders RJM, Nicolaides KH. Assessment of risks. In Ultrasound Markers for Fetal Chromosomal Defects. Carnforth, UK: Parthenon Publishing, 1996:109–13

35. Koulisher L, Gillerot Y. Down’s syndrome in Wallonia (South Belgium), 1971–1978: cytogenetics and incidence. Hum Genet 1980;54:243–50

36. Hook EB, Lindsjo A. Down syndrome in live births by single year maternal age interval in a Swedish study: comparison with results from a New York State study. AmJ Hum Genet 1978;30:19–27

37. Hecht CA, Hook EB. The imprecision in rates of Down syndrome by 1-year maternal age intervals: a critical analysis of rates used in biochemical screening. Prenat Diagn 1994;14:729–38

38. Snijders RJM, Sundberg K, Holzgreve W, Henry G, Nicolaides KH. Maternal age and gestation- specific risk for trisomy 21. Ultrasound Obstet Gynecol 1999;13:167–70

39. Snijders RJM, Holzgreve W, Cuckle H, Nicolaides KH. Maternal age-specific risks for trisomies at 9–14 weeks’ gestation. Prenat Diagn 1994;14:543–52

40. Snijders RJM, Sebire NJ, Cuckle H, Nicolaides KH. Maternal age and gestational age-specific risks for chromosomal defects. Fetal Diag Ther 1995;10:356–67

41. Halliday JL, Watson LF, Lumley J, Danks DM, Sheffield LJ. New estimates of Down syndrome risks at chorionic villus sampling, amniocentesis, and livebirth in women of advanced maternal age from a uniquely defined population. Prenat Diagn 1995; 15:455–65

42. Morris JK, Wald NJ, Watt HC. Fetal loss in Down syndrome pregnancies. Prenat Diagn 1999;19: 142–5

43. Macintosh MC, Wald NJ, Chard T, Hansen J, Mikkelsen M, Therkelsen AJ, Petersen GB, Lundsteen C. Selective miscarriage of Down’s syndrome fetuses in women aged 35 years and older. Br J Obstet Gynaecol 1995;102:798–801

44. Snijders RJM, Sundberg K, Holzgreve W, Henry G, Nicolaides KH. Maternal age and gestation- specific risk for trisomy 21: effect of previous affected pregnancy. In press

45. Azar G, Snijders RJM, Gosden CM, Nicolaides KH. Fetal nuchal cystic hygromata: associated malformations and chromosomal defects. Fetal Diagn Ther 1991;6:46–57

46. Nicolaides KH, Azar G, Snijders RJM, Gosden CM. Fetal nuchal edema: associated malformations and chromosomal defects. Fetal Diagn Ther 1992;7:123–31

47. Whitlow BJ, Economides DL. The optimal gestational age to examine fetal anatomy and measure nuchal translucency in the first trimester. Ultrasound Obstet Gynecol 1998;11:258–61

48. Braithwaite JM, Economides DL. The measurement of nuchal translucency with transabdominal and transvaginal sonography – success rates, repeatability and levels of agreement. Br J Radiol 1995; 68:720–3

49. Braithwaite JM, Morris RW, Economides DL. Nuchal translucency measurements: frequency distribution and changes with gestation in a general population. Br J Obstet Gynaecol 1996;103: 1201–4

50. Whitlow BJ, Chatzipapas IK, Economides DL. The effect of fetal neck position on nuchal translucency measurement. Br J Obstet Gynaecol 1998;105:872–6

51. Schaefer M, Laurichesse-Delmas H, Ville Y. The effect of nuchal cord on nuchal translucency measurement at 10–14 weeks. Ultrasound Obstet Gynecol 1998;11:271–3

52. Herman A, Maymon R, Dreazen E, Caspi E, Bukovsky I, Weinraub Z. Nuchal translucency audit: a novel image-scoring method. Ultrasound Obstet Gynecol 1998;12:398–403

53. Braithwaite JM, Kadir RA, Pepera TA, Morris RW, Thompson PJ, Economides DL. Nuchal translucency measurement: training of potential examiners. Ultrasound Obstet Gynecol 1996;8:192–5

54. Bower S, Chitty L, Bewley S, Roberts L, Clark T, Fisk NM, Maxwell D, Rodeck CH. First trimester nuchal translucency screening of the general population: data from three centres [abstract]. Presented at the 27th British Congress of Obstetrics and Gynaecology. Dublin: Royal College of Obstetrics and Gynaecology, 1995

55. Roberts LJ, Bewley S, Mackinson AM, Rodeck CH. First trimester fetal nuchal translucency: Problems with screening the general population 1. Br J Obstet Gynaecol 1995;102:381–5

56. Monni G, Zoppi MA, Ibba RM, Floris M. Results of measurement of nuchal translucency before and after training. (Letter in reply to: Assessment of fetal nuchal translucency test for Down’s syndrome. Lancet 1997, 350:745–55). Lancet 1997;350:1631

57. Pandya PP, Altman D, Brizot ML, Pettersen H, Nicolaides KH. Repeatability of measurement of fetal nuchal translucency thickness. Ultrasound Obstet Gynecol 1995;5:334–7

58. Schuchter K, Wald N, Hackshaw AK, Hafner E, Liebhart E. The distribution of nuchal translucency at 10–13 weeks of pregnancy. Prenat Diagn 1998;18:281–6

59. Pajkrt E, de Graaf IM, Mol BW, van Lith JM, Bleker OP, Bilardo CM. Weekly nuchal translucency measurements in normal fetuses. Obstet Gynecol 1998;91:208–11

60. Bernardino F, Cardoso R, Montenegro N, Bernardes J, de Sa JM. Semiautomated ultrasonographic measurement of fetal nuchal translucency using a computer software tool. Ultrasound Med Biol 1998; 24:51–4

61. Pandya PP, Snijders RJM, Johnson SJ, Brizot M, Nicolaides KH. Screening for fetal trisomies by maternal age and fetal nuchal translucency thickness at 10 to 14 weeks of gestation. Br J Obstet Gynaecol 1995;102:957–62

62. Snijders RJM, Noble P, Sebire N, Souka A, Nicolaides KH. UK multicentre project on assessment of risk of trisomy 21 by maternal age and fetal nuchal translucency thickness at 10–14 weeks of gestation. Lancet 1998;351:343–6

63. Johnson MP, Johnson A, Holzgreve W, Isada NB, Wapner RJ, Treadwell MC, Heeger S, Evans M. First-trimester simple hygroma: cause and outcome. Am J Obstet Gynecol 1993;168:156–61

64. Hewitt B. Nuchal translucency in the first trimester. Aust NZ J Obstet Gynaecol 1993;33:389–91

65. Shulman LP, Emerson D, Felker R, Phillips O, Simpson J, Elias S. High frequency of cytogenetic abnormalities with cystic hygroma diagnosed in the first trimester. Obstet Gynecol 1992;80:80–2

66. Nicolaides KH, Azar G, Byrne D, Mansur C, Marks K. Fetal nuchal translucency: ultrasound screening for chromosomal defects in first trimester of pregnancy. Br Med J 1992;304:867–89

67. Pandya PP, Kondylios A, Hilbert L, Snijders RJM, Nicolaides KH. Chromosomal defects and outcome in 1,015 fetuses with increased nuchal translucency. Ultrasound Obstet Gynecol 1995;5:15–19

68. Szabo J, Gellen J. Nuchal fluid accumulation in trisomy-21 detected by vaginal sonography in first trimester. Lancet 1990;336:1133

69. Wilson RD, Venir N, Faquharson DF. Fetal nuchal fluid – physiological or pathological? – in pregnancies less than 17 menstrual weeks. Prenat Diagn 1992;12:755–63

70. Ville Y, Lalondrelle C, Doumerc S, Daffos F, Frydman R, Oury JF, Dumez Y. First-trimester diagnosis of nuchal anomalies: significance and fetal outcome. Ultrasound Obstet Gynecol 1992;2:314–16

71. Trauffer ML, Anderson CE, Johnson A, Heeger S, Morgan P, Wapner RJ. The natural history of euploid pregnancies with first-trimester cystic hygromas. Am J Obstet Gynecol 1994;170:1279–84

72. Brambati B, Cislaghi C, Tului L, Alberti E, Amidani M, Colombo U, Zuliani G. First-trimester Down’s syndrome screening using nuchal translucency: a prospective study. Ultrasound Obstet Gynecol 1995;5: 9–14

73. Comas C, Martinez JM, Ojuel J, Casals E, Puerto B, Borrell A, Fortuny A. First-trimester nuchal edema as a marker of aneuploidy. Ultrasound Obstet Gynecol 1995;5:26–9

74. Szabo J, Gellen J, Szemere G. First-trimester ultrasound screening for fetal aneuploidies in women over 35 and under 35 years of age. Ultrasound Obstet Gynecol 1995;5:161–3

75. Nadel A, Bromley B, Benacerraf BR. Nuchal thickening or cystic hygromas in first- and early second-trimester fetuses: prognosis and outcome. Obstet Gynecol 1993;82: 43–8

76. Savoldelli G, Binkert F, Achermann J, Schmid W. Ultrasound screening for chromosomal anomalies in the first trimester of pregnancy. Prenat Diagn 1993;13:513–18

77. Schulte-Vallentin M, Schindler H. Non-echogenic nuchal oedema as a marker in trisomy 21 screening. Lancet 1992;339:1053

78. Van Zalen-Sprock MM, Van Vugt JMG, Van Geijn HP. First-trimester diagnosis of cystic hygroma – course and outcome. Am J Obstet Gynecol 1992;167; 94–8

79. Cullen MT, Gabrielli S, Green JJ, Rizzo N, Mahoney MJ, Salafia C, Bovicelli L, Hobbins JC. Diagnosis and significance of cystic hygroma in the first trimester. Prenat Diagn 1990;10: 643–51

80. Suchet IB, Van der Westhuizen NG, Labatte MF. Fetal cystic hygromas: further insights into their natural history. Can Assoc Radiol J 1992;6:420–4

81. Pandya PP, Brizot ML, Kuhn P, Snijders RJM, Nicolaides KH. First trimester fetal nuchal translucency thickness and risk for trisomies. Obstet Gynecol 1994;84:420–3

82. Pandya PP, Goldberg H, Walton B, Riddle A, Shelley S, Snijders RJM, Nicolaides KH. The implementation of first-trimester scanning at 10–13 weeks’ gestation and the measurement of fetal nuchal translucency thickness in two maternity units. Ultrasound Obstet Gynecol 1995;5:20–5

83. Bewley S, Roberts LJ, Mackinson M, Rodeck C. First trimester fetal nuchal translucency: problems with screening the general population. II. Br J Obstet Gynaecol 1995;102:386–8

84. Kornman LH, Morssink LP, Beekhuis JR, DeWolf BTHM, Heringa MP, Mantingh A. Nuchal translucency cannot be used as a screening test for chromosomal abnormalities in the first trimester of pregnancy in a routine ultrasound practice. Prenat Diagn 1996;16:797–805

85. Zimmerman R, Hucha A, Salvoldelli G, Binkert F, Acherman J, Grudzinskas JG. Serum parameters and nuchal translucency in first trimester screening for fetal chromosomal abnormalities. Br J Obstet Gynaecol 1996;103:1009–14

86. Taipale P, Hiilesmaa V, Salonen R, Ylostalo P. Increased nuchal translucency as a marker for fetal chromosomal defects. N Engl J Med 1997;337:1654–8

87. Hafner E, Schuchter K, Liebhart E, Philipp K. Results of routine fetal nuchal translucency measurement at 10–13 weeks in 4,233 unselected pregnant women. Prenat Diagn 1998;18: 29–34

88. Pajkrt E, van Lith JMM, Mol BWJ, Bleker OP, Bilardo CM. Screening for Down’s syndrome by fetal nuchal translucency measurement in a general obstetric population. Ultrasound Obstet Gynecol 1998;12:163–9

89. Economides DL, Whitlow BJ, Kadir R, Lazanakis M, Verdin SM. First trimester sonographic detection of chromosomal abnormalities in an unselected population. Br J Obstet Gynaecol 1998; 105:58–62

90. Thilaganathan B, Slack A, Wathen NC. Effect of first-trimester nuchal translucency on second- trimester maternal serum biochemical screening for Down’s syndrome. Ultrasound Obstet Gynecol 1997;10:261–4

91. Theodoropoulos P, Lolis D, Papageorgiou C, Papaioannou S, Plachouras N, Makrydimas G. Evaluation of first-trimester screening by fetal nuchal translucency and maternal age. Prenat Diagn 1998;18:133–7

92. Biagiotti R, Periti E, Brizzi L, Vanzi E, Cariati E. Comparison between two methods of standardization for gestational age differences in fetal nuchal translucency measurement in first-trimester screening for trisomy 21. Ultrasound Obstet Gynecol 1997;9:248–52

93. Orlandi F, Damiani G, Hallahan TW, Krantz DA, Macri JN. First-trimester screening for fetal aneuploidy: biochemistry and nuchal translucency. Ultrasound Obstet Gynecol 1997;10:381–6

94. Pandya PP, Snijders RJM, Johnson S, Nicolaides KH. Natural history of trisomy 21 fetuses with fetal nuchal translucency. Ultrasound Obstet Gynecol 1995;5:381–3

95. Hyett JH, Sebire NJ, Snijders RJM, Nicolaides KH. Intrauterine lethality of trisomy 21 fetuses with increased nuchal translucency. Ultrasound Obstet Gynecol 1996;7:101–3

96. Sherrod C, Sebire NJ, Soares W, Snijders RJ, Nicolaides KH. Prenatal diagnosis of trisomy 18 at the 10–14-week ultrasound scan. Ultrasound Obstet Gynecol 1997;10:387–90

97. Snijders RJM, Sebire NJ, Nayar R, Souka A, Nicolaides KH. Increased nuchal translucency in trisomy 13 fetuses at 10–14 weeks of gestation. Am J Med Genet 1999:in press

98. Sebire NJ, Snijders RJ, Brown R, Southall T, Nicolaides KH. Detection of sex chromosome abnormalities by nuchal translucency screening at 10–14 weeks. Prenat Diagn 1998;18:581–4

99. Jauniaux E, Brown R, Snijders RJ, Noble P, Nicolaides KH. Early prenatal diagnosis of triploidy. Am J Obstet Gynecol 1997;176:550–4

100. Reisman IE. Chromosomal abnormalities and intrauterine growth retardation. Pediatr Clin North Am 1970;17:101–10

101. Chen ATL, Chan YK, Falek A. The effects of chromosomal abnormalities on birth weight in man. Hum Hered 1972;22:209–24

102. Snijders RJ, Sherrod C, Gosden CM, Nicolaides KH. Fetal growth retardation: associated malformations and chromosomal abnormalities. Am J Obstet Gynecol 1993;168:547–55

103. Lynch L, Berkowitz RL. First trimester growth delay in trisomy 18. AmJ Perinatol 1989;6:237–9

104. Drugan A, Johnson MP, Isada NB, Holzgreve W. Zador IE, Dombrowski MP, Sokol RJ, Hallak M, Evans MI. The smaller than expected first-trimester fetus is at increased risk for chromosome anomalies. Am J Obstet Gynecol 1992;167:1525–8

105. Kuhn P, Brizot ML, Pandya PP, Snijders RJ, Nicolaides KH. Crown-rump length in chromosomally abnormal fetuses at 10 to 13 weeks’ gestation. Am J Obstet Gynecol 1995;172: 32–5

106. Macintosh MC, Brambati B, Chard T, Grudzinskas JG. Crown–rump length in aneuploid fetuses: implications for first-trimester biochemical screening for aneuploidies. Prenat Diagn 1995; 15:691–4

107. Bahado-Singh RO, Lynch L, Deren O, Morotti R, Copel J, Mahoney MJ, Williams J. First- trimester growth restriction and fetal aneuploidy: the effect of type of aneuploidy and gestational age. Am J Obstet Gynecol 1997;176:976–80

108. Schemmer G, Wapner RJ, Johnson A, Schemmer M, Norton HJ, Anderson WE. First-trimester growth patterns of aneuploid fetuses. Prenat Diagn 1997;17:155–9

109. Campbell S, Warsof SL, Little D, Cooper DJ. Routine ultrasound screening for the prediction of gestational age. Obstet Gynecol 1985;65:613–20

110. Bergsiø P, Denman III DW, Hoffman J, Meirik O. Duration of human singleton pregnancy. Acta Obstet Gynecol Scand 1990;69:197–207

111. Geirsson RT. Ultrasound instead of last menstrual period as the basis of gestational age assignment. Ultrasound Obstet Gynecol 1991;1:212–19

112. Schats R, Jansen CAM, Wladimiroff JW. Abnormal embryonic heart rate pattern in early pregnancy associated with Down’s syndrome. Hum Reprod 1990;5: 877–9

113. Van Lith JMM, Visser GHA, Mantingh A, Beekhuis JR. Fetal heart rate in early pregnancy and chromosomal disorders. Br J Obstet Gynaecol 1992;99:741–4

114. Martinez JM, Echevarria M, Borrell A Puerto B, Ojuel J, Fortuny A. Fetal heart rate and nuchal translucency in detecting chromosomal abnormalities other than Down syndrome. Obstet Gynecol 1998;92:68–71

115. Hyett JA, Noble PL, Snijders RJ, Montenegro N, Nicolaides KH. Fetal heart rate in trisomy 21 and other chromosomal abnormalities at 10–14 weeks of gestation. Ultrasound Obstet Gynecol 1996;7: 239–44

116. Robinson HP, Shaw-Dunn J. Fetal heart rates as determined by sonar in early pregnancy. J Obstet Gynaecol Br Commonw 1973;90:805–9

117. Rempen A. Diagnosis of viability in early pregnancy with vaginal sonography. J Ultrasound Med 1990; 9:711–16

118. Wisser J, Dirschedl P. Embryonic heart rate in dated human embryos. Early Hum Dev 1994;37: 107–15

119. Wladimiroff JW, Seelen JC. Fetal heart action in early pregnancy. Development of fetal vagal function. Eur J Obstet Gynecol 1972;2:55–63

120. Hyett JA, Brizot ML, Von-Kaisenberg CS, McKie AT, Farzaneh F, Nicolaides KH. Cardiac gene expression of atrial natriuretic peptide and brain natriuretic peptide in trisomic fetuses. Obstet Gynecol 1996;87:506–10

121. Teitel D, Rudolph AM. Perinatal oxygen delivery and cardiac function. Adv Paediatr 1985;32: 321–47

122. Rudolph AM, Heymann MA. Cardiac output in the fetal lamb: the effects of spontaneous and induced changes of heart rate on right and left ventricular output. Am J Obstet Gynecol 1976;124: 183–92

123. Wladimiroff J, Huisman T, Stewart P. Fetal and umbilical blood flow velocity waveforms between 10 and 16 weeks gestation: a preliminary study. Obstet Gynecol 1991;78:812–14

124. Brown R, Di Luzio L, Gomes C, Nicolaides KH. The umbilical artery pulsatility index in the first trimester: is there an association with increased nuchal translucency or chromosomal abnormality? Ultrasound Obstet Gynecol 1998;12:244–7

125. Jauniaux E, Jurkovic D, Campbell S. In vivo investigation of the placental circulation by Doppler echography. Placenta 1995;16:323–31

126. Martinez JM, Borrell A, Antonin E, Puerto B, Casals E, Ojuel J, Fortuny A. Combining nuchal translucency and umbilical Doppler velocimetry for detecting fetal trisomies in the first trimester of pregnancy. Br J Obstet Gynaecol 1997;104:11–14

127. Jauniaux E, Gavrill P, Khun P, Kurdi W, Hyett J, Nicolaides KH. Fetal heart rate and umbilico-placental Doppler flow velocity waveforms in early pregnancies with a chromosomal abnormality and/or an increased nuchal translucency thickness. Hum Reprod 1996;11:435–9

128. Gudmundsson S, Huhta JC, Wood DC, Tulzer G, Cohen AW, Weiner S. Venous Doppler ultrasonography in the fetus with nonimmune hydrops. Am J Obstet Gynecol 1991;164:333–7

129. Reed KL, Appleton CP, Anderson CF, Shenker L, Sahn DJ. Doppler studies of vena cava flows in human fetuses. Insights into normal and abnormal cardiac physiology. Circulation 1990;81: 498–505

130. Reuss ML, Rudolph AM, Dae MW. Phasic blood flow patterns in the superior and inferior venae cavae and umbilical vein of fetal sheep. Am J Obstet Gynecol 1983;145:70–8

131. Brown RN, Di Luzio L, Gomes C, Nicolaides KH. First trimester umbilical venous Doppler sonography in chromosomally normal and abnormal fetuses. J Ultrasound Med 1999;18:543–6

132. Kiserud T. In a different vein: the ductus venosus could yield much valuable information. Ultrasound Obstet Gynecol 1997;9:369–72

133. Montenegro N, Matias A, Areias JC, Castedo S, Barros H. Increased fetal nuchal translucency: possible involvement of early cardiac failure. Ultrasound Obstet Gynecol 1997;10:265–8

134. Matias A, Gomes C, Flack N, Montenegro N, Nicolaides KH. Screening for chromosomal abnormalities at 11–14 weeks: the role of ductus venosus blood flow. Ultrasound Obstet Gynecol 1998;12:380–4

135. Brizot ML, Bersinger NA, Zydias G, Snijders RJ, Nicolaides KH. Maternal serum Schwangerschafts protein-1 (SP1) and fetal chromosomal abnormalities at 10–13 weeks of gestation. Early Hum Dev 1995;30:31–6

136. Brizot ML, Kuhn P, Bersinger NA, Snijders RJM, Nicolaides KH. First trimester maternal serum alpha-fetoprotein in fetal trisomies. Br J Obstet Gynaecol 1995;102:31–4

137. Noble PL, Wallace EM, Snijders RJM, Groome NP, Nicolaides KH. Maternal serum inhibin-A and free beta hCG concentrations in trisomy 21 pregnancies at 10–14 weeks of gestation. Br J Obstet Gynaecol 1997;104:367–71

138. Ozturk M, Milunsky A, Brambati B, Sachs ES, Miller SL, Wands JR. Abnormal maternal levels of hCG subunits in trisomy 18. Am J Med Genet 1990;36:480–3

139. Spencer K, Macri JN, Aitken DA, Connor JM. Free beta hCG as a first trimester marker for fetal trisomy. Lancet 1992;339:1480

140. Isles RK, Sharma K, Wathen NC, et al. hCG, free subunit and PAPP-A composition in normal and Down’s syndrome pregnancies. In Fourth conference: Endocrinology and Metabolism in Human Reproduction. London: RCOG, 1993

141. Macri JN, Spencer K, Aitken DA, Garver K, Buchanan PD, Muller F, Boue A. First trimester free beta-hCG screening for Down syndrome. Prenat Diagn 1993;13:557–62

142. Pescia G, Marguerat PH, Weihs D, The HN, Maillard C, Loertscher A, Senn A. First trimester free beta-hCG and SP1 as markers for fetal chromosomal disorders: a prospective study of 250 women undergoing CVS. In Fourth Conference: Endocrinology and Metabolism in Human Reproduction. London: RCOG, 1993:45

143. Brambati B, Tului L, Bonacchi I, Shrimanker K, Suzuki Y, Grudzinskas JG. Serum PAPP-A and free beta hCG are first-trimester screening markers for Down syndrome. Prenat Diagn 1994;14: 1043–7

144. Kellner LH, Weiss RR, Weiner Z, Neur M, Martin G. Early first trimester serum AFP, UE3, hCG and free beta-hCG measurements in unaffected and affected pregnancies with fetal Down syndrome. Am J Hum Genet 1994;55:A281

145. Forest J-C, Masse J, Moutquin J-M. Screening for Down syndrome during the first trimester: a prospective study using free b-human chorionic gonadotropin and pregnancy associated plasma protein-A. Clin Biochem 1997;30:333–8

146. Macintosh MCM, Iles R, Teisner B, Sharma K, Chard T, Grudzinskas JG, Ward RHT, Muller F. Maternal serum human chorionic gonadotrophin and pregnancy associated plasma protein A, markers for fetal Down syndrome at 8–14 weeks. Prenat Diagn 1994;14:203–8

147. Biagiotti R, Cariati E, Brizzi L, D’Agata A. Maternal serum screening for Down syndrome in the first trimester of pregnancy. Br J Obstet Gynaecol 1995;102: 660–2

148. Brizot ML, Snijders RJM, Butler J, Bersinger NA, Nicolaides KH. Maternal serum hCG and fetal nuchal translucency thickness for the prediction of fetal trisomies in the first trimester of pregnancy. Br J Obstet Gynaecol 1995;102:127–32

149. Noble PL, Abraha HD, Snijders RJM, Sherwood R, Nicolaides KH. Screening for fetal trisomy 21 in the first trimester of pregnancy: maternal serum free b-hCG and fetal nuchal translucency thickness. Ultrasound Obstet Gynecol 1996;6:390–5

150. Krantz DA, Larsen JW, Buchanan PD, Macri JN. First trimester Down syndrome screening: free ??human chorionic gonadotropin and pregnancy associated plasma protein A. Am J Obstet Gynecol 1996;174:612–16

151. Scott F, Wheeler D, Sinosich M, Boogert A, Anderson J, Edelman D. First trimester aneuploidy screening using nuchal translucency, free beta human chorionic gonadotrophin and maternal age. Aust NZ Obstet Gynaecol 1996;36:381–4

152. Wald NJ, George L, Smith D, Densem JW, Petterson K, on behalf of the International Prenatal Screening Research Group. Serum screening for Down’s syndrome between 8 and 14 weeks of pregnancy. Br J Obstet Gynaecol 1996;104:407–12

153. Berry E, Aitken DA, Crossley JA, Macri JN, Connor JM. Screening for Down’s syndrome: changes in marker levels and detection rates between first and second trimester. Br J Obstet Gynaecol 1997; 104:811–17

154. Spencer K, Noble P, Snijders RJM, Nicolaides KH. First trimester urine free beta hCG, beta core and total oestriol in pregnancies affected by Down’s syndrome: implications for first trimester screening with nuchal translucency and serum free beta hCG. Prenat Diagn 1997;17:525–38

155. Haddow JE, Palomaki GE, Knight GJ, Williams J, Miller WA, Johnson A. Screening of maternal serum for fetal Down’s syndrome in the first trimester. N Engl J Med 1998;338:955–61

156. Wheeler DM, Sinosich MJ. Prenatal screening in the first trimester of pregnancy. Prenat Diagn 1998;18: 537–43

157. de Graaf IM, Pajkrt E, Bilardo CM, Leschot NJ, Cuckle HS, Van Lith JM. Early pregnancy screening for fetal aneuploidy with serum markers and nuchal translucency. Prenat Diagn 1999;19: 458–62

158. Spencer K, Souter V, Tul N, Snijders R, Nicolaides KH. A screening program for trisomy 21 at 10–14 weeks using fetal nuchal translucency, maternal serum free ?-human chorionic gonadotropin and pregnancy-associated plasma protein-A. Ultrasound Obstet Gynecol 1999;13:231–7

159. Wald N, Stone R, Cuckle HS, Grudzinskas JG, Barkai G, Brambati B, Teisner B, Fuhrmann W. First trimester concentrations of pregnancy associated plasma protein A and placental protein 14 in Down’s syndrome. Br Med J 1992;305:28

160. Brambati B, Macintosh MCM, Teisner B, Maguiness S, Shrimanker K, Lanzani A, Bonacchi I, Tului L, Chard T, Grudzinskas JG. Low maternal serum levels of pregnancy associated plasma protein A (PAPP-A) in the first trimester in association with abnormal fetal karyotype. Br J Obstet Gynaecol 1993;100:324–6

161. Hurley PA, Ward RHT, Teisner B, Isles RK, Lucas M, Grudzinskas JG. Serum PAPP-A measurements in first trimester screening for Down’s syndrome. Prenat Diagn 1996;13:903–8

162. Muller F, Cuckle HS, Teisner B, Grudzinskas JG. Serum PAPP-A levels are depressed in women with fetal Down’s syndrome in early pregnancy. Prenat Diagn 1993;13:633–6

163. Bersinger NA, Brizot ML, Johnson A, Snijders RJM, Abbott J, Schneider H, Nicolaides KH. First trimester maternal serum pregnancy-associated plasma protein A and pregnancy-specific ?1-glycoprotein in fetal trisomies. Br J Obstet Gynaecol 1994;101:970–4

164. Brizot ML, Snijders RJM, Bersinger NA, Kuhn P, Nicolaides KH. Maternal serum pregnancy associated placental protein A and fetal nuchal translucency thickness for the prediction of fetal trisomies in early pregnancy. Obstet Gynecol 1994;84:918–22

165. Spencer K, Aitken DA, Crossley JA, McGaw G, Berry E, Anderson R, Connor JM, Macri JN. First trimester biochemical screening for trisomy 21: the role of free beta hCG, alpha fetoprotein and pregnancy associated plasma protein A. Ann Clin Biochem 1994;31:447–54

166. Casals E, Fortuny A, Grudzinskas JG, Suzuki Y, Teisner B, Comas C, Sanllehy C, Ojuel J, Borrell A, Soler A, Ballesta AM. First trimester biochemical screening for Down syndrome with the use of PAPP-A, AFP and beta-hCG. Prenat Diagn 1996;16:405–10

167. Orlandi F, Damiani G, Hallahan TW, Krantz DA, Macri JN. First trimester screening for aneuploidy: biochemistry and nuchal translucency. Ultrasound Obstet Gynecol 1997;10:381–6 168. Tsukerman GL, Gusina NB, Cuckle HS. Maternal serum screening for Down syndrome in the first trimester: experience from Belarus. Prenat Diagn 1999;19:499–504

168. Tsukerman GL, Gusina NB, Cuckle HS. Maternal serumscreening for Down syndrome in the first trimester: experience from Belarus. Prenat Diagn 1999;19:499–504

169. De Biasio P, Siccardi M, Volpe G, Famularo L, Santi P, Canini S. First trimester screening for Down’s syndrome using nuchal translucency measurement with free beta-hCG and PAPP-A between 10 and 13 weeks of pregnancy: the combined test. Prenat Diagn 1999;19:360–3

170. Wald NJ, Watt HC, Hackshaw AK. Integrated screening for Down’s syndrome based on tests performed during the first and second trimesters. N Engl J Med 1999;341:461–7

171. Copel J, Bahado-Singh RO. Prenatal screening for Down’s syndrome – a search for the family’s values. N Engl J Med 1999;341:521–2

172. Kadir RA, Economides DL. The effect of nuchal translucency measurement on second trimester biochemical screening for Down’s syndrome. Ultrasound Obstet Gynecol 1997;9:244–7

173. Thilaganathan B, Slack A, Wathen NC. Effect of first-trimester nuchal translucency on second- trimester maternal serum biochemical screening for Down’s syndrome. Ultrasound Obstet Gynecol 1997;10:261–4

174. Nicolaides KH, Snijders RJM, Gosden CM, Berry C, Campbell S. Ultrasonographically detectable markers of fetal chromosomal abnormalities. Lancet 1992;340:704–7

175. Snijders RJM, Nicolaides KH. Assessment of risks. In Ultrasound Markers for Fetal Chromosomal Defects. Carnforth, UK: Parthenon Publishing, 1996:63–120

176. Snijders RJ, Sebire NJ, Faria M, Patel F, Nicolaides KH. Fetal mild hydronephrosis and chromosomal defects: relation to maternal age and gestation. Fetal Diagn Ther 1995;10:349–55

177. Snijders RJM, Shawa L, Nicolaides KH. Fetal choroid plexus cysts and trisomy 18: assessment of risk based on ultrasound findings and maternal age. Prenat Diagn 1994;14:1119–27

178. Nyberg DA, Luthy DA, Resta RG, Nyberg BC, Williams MA. Age-adjusted ultrasound risk assessment for fetal Down’s syndrome during the second trimester: description of the method and analysis of 142 cases. Ultrasound Obstet Gynecol 1998;12:8–14

179. Donnenfeld AE, Carlson DE, Palomaki GE, Librizzi RJ, Weiner S, Platt L. Prospective multicenter study of second trimester nuchal skinfold thickness in unaffected and Down syndrome pregnancies. Obstet Gynecol 1994;84:844–7

180. Biagiotti R, Periti E, Cariati E. Humerus and femur length in fetuses with Down syndrome. Prenat Diagn 1994;14:429–34

181. Cuckle H, Wald N, Quinn J, Royston P, Butler L. Ultrasound fetal femur length measurement in the screening for Down’s syndrome. Br J Obstet Gynaecol 1989;96:1373–8

182. Grandjean H, Sarramon MF. Femur/foot length ratio for detection of Down syndrome: results of a multicenter prospective study. The Association Francaise pour le Depistage et la Prevention des Handicaps de l’Enfant Study Group. Am J Obstet Gynecol 1995;173:16–19

183. Johnson MP, Michaelson JE, Barr M, Treadwell MC, Hume RF, Dombrowski MP, Evans MI. Combining humerus and femur length for improved ultrasonographic identification of pregnancies at increased risk for trisomy 21. Am J Obstet Gynecol 1995;172:1229–35

184. Owen J, Wenstrom KD, Hardin JM, Boots LR, Hsu CC, Cosper PC, DuBard MB. The utility of fetal biometry as an adjunct to the multiple-marker screening test for Down syndrome. Am J Obstet Gynecol 1994;17:1041–6

185. Vintzileos AM, Egan JF, Smulian JC, Campbell WA, Guzman ER, Rodis JF. Adjusting the risk for trisomy 21 by a simple ultrasound method using fetal long-bone biometry. Obstet Gynecol 1996;87: 953–8

186. Bromley B, Doubilet P, Frigoletto FD, Jr, Krauss C, Estroff JA, Benacerraf BR. Is fetal hyperechoic bowel on second-trimester sonogram an indication for amniocentesis? Obstet Gynecol 1994;83: 647–51

187. Corteville JE, Gray DL, Langer JC. Bowel abnormalities in the fetus – correlation of prenatal ultrasonographic findings with outcome. Am J Obstet Gynecol 1996;175:724–9

188. Muller F, Dommergues M, Aubry MC, Simon-Bouy B, Gautier E, Oury JF, Narcy F. Hyperechogenic fetal bowel: an ultrasonographic marker for adverse fetal and neonatal outcome. Am J Obstet Gynecol 1995;173:508–13

189. Homola J. Are echogenic foci in fetal heart ventricles insignificant findings?. Ceska Gynekologie 1997;62:280–2

190. Simpson JM, Cook A, Sharland G. The significance of echogenic foci in the fetal heart: a prospective study of 228 cases. Ultrasound Obstet Gynecol 1996;8:225–8

191. Vibhakar NI, Budorick NE, Sciosia AL, Harby LD, Mullen ML, Sklansky MS. Prevalence of aneuploidy with a cardiac intraventricular echogenic focus in an at-risk patient population. J Ultrasound Med 1999;18:265–8

192. Vintzileos AM, Campbell WA, Guzman ER, Smulian JC, McLean DA, Ananth CV. Second- trimester ultrasound markers for detection of trisomy 21: which markers are best? Obstet Gynecol 1997;89:941–4

193. Wickstrom EA, Thangavelu M, Parilla BV, Tamura RK, Sabbagha RE. A prospective study of the association between isolated fetal pyelectasis and chromosomal abnormality. Obstet Gynecol 1996; 88:379–82

194. Bianchi DW, Flint AF, Pizzimenti MF, Knoll JHM, Latt SA. Isolation of fetal DNA from nucleated erythrocytes in maternal blood. Proc Natl Acad Sci USA 1990;87:3279–83

195. Price JO, Elias S, Wachtel S, Klinger K, Dockter M, Tharapel A, Shulman LP, Phillips OP, Meyers CM, Shook D, Simpso JL. Prenatal diagnosis with fetal cells isolated from maternal blood by multiparameter flow cytometry. Am J Obstet Gynecol 1991;165:1731–7

196. Hamada H, Arinami T, Kubo T, Hamaguchi H, Iwasaki H. Fetal nucleated cells in maternal peripheral blood: frequency and relationship to gestational age. Hum Genet 1993;91:427–32

197. Ganshirt-Ahlert D, Burschyk M, Garritsen HSP, Helmer L, Miny P, Horst J, Schneider HPG, Holzgreve W. Magnetic cell sorting and the transferrin receptor as potential means of prenatal diagnosis from maternal blood. Am J Obstet Gynecol 1992;166:1350–5

198. Wachtel S, Elias S, Price J, Wachtel G, Phillips O, Shulman L, Meyers C, Simpson JL, Dockter M. Fetal cells in the maternal circulation: isolation by multiparameter flow cytometry and confirmation by polymerase chain reaction. Hum Reprod 1991;6:1466–9

199. Al-Mufti R, Hambley H, Farzaneh F, Nicolaides KH. Investigation of maternal blood enriched for fetal cells: Role in screening and diagnosis of fetal trisomies. Am J Med Genet 1999;85:66–75

200. Durrant LG, MeDowall KM, Holmes RA, Liu DTY. Screening of monoclonal antibodies recognizing oncofetal antigens for isolation of trophoblasts from maternal blood for prenatal diagnosis. Prenat Diagn 1994;14:131–40

201. Pandya PP, Kuhn P, Brizot M, Cardy DL, Nicolaides KH. Rapid detection of chromosome aneuploides in fetal blood and chorionic villi by fluorescence in situ hybridisation (FISH). Br J Obstet Gynaecol 1994;101:493–7

202. Bianchi DW, Mahr A, Zickwolf GK, House TW, Flint AF, Klinger KW. Detection of fetal cells with 47XY,+21 karyotype in maternal peripheral blood. Hum Genet 1992; 90:368–70

203. Ganshirt-Ahlert D, Borjesson-Stoll R, Burschyk M, Dohr A, Garritsen HSP, Helmer L, Miny P, Velasco M, Walde C, Patterson D, Teng N, Bhat NM, Bieber MM, Holzgreve W. Detection of fetal trisomies 21 and 18 from maternal blood using triple gradient and magnetic cell sorting. Am J Reprod Immunol 1993;30:194–201

204. Simpson JL, Elias S. Isolating fetal cells in maternal circulation for prenatal diagnosis. Prenat Diagn 1994;14:1229–42

205. Ager RP, Oliver RW. In The Risks of Mid-trimester Amniocentesis, Being a Comparative, Analytical Review of the Major Clinical Studies. Salford University, 1986

206. Tabor A, Philip J, Madsen M, Bang J, Obel EB, Norgaard-Pedersen B. Randomised controlled trial of genetic amniocentesis in 4,606 low-risk women. Lancet 1986;i:1287–93

207. Nicolaides KH, Brizot M, Patel F, Snijders R. Comparison of chorionic villus sampling and amniocentesis for fetal karyotyping at 10–13 weeks’ gestation. Lancet 1994;344:435–9

208. Sundberg K, Bang J, Smidt-Jensen S, et al. Randomised study of risk of fetal loss related to early amniocentesis versus chorionic villus sampling. Lancet 1997;350:697–703

209. CEMAT Group. Randomised trial to assess safety and fetal outcome of early and mid-trimester amniocentesis. Lancet 1998;351:242–7

210. Canadian Collaborative CVS–Amniocentesis Clinical Trial Group. Multicentre randomised clinical trial of chorion villus sampling and amniocentesis. Lancet 1989;i:1–6

211. Smidt-Jensen S, Permin M, Philip J, Lundsteen C, Zachary JM, Fowler SE, Gruning K. Randomised comparison of amniocentesis and transabdominal and transcervical chorionic villus sampling. Lancet 1992;340:1238–44

212. Ammala P, Hiilesmaa VK, Liukkonen S, Saisto T, Teramo K, Von Koskull H. Randomized trial comparing first trimester transcervical chorionic villus sampling and second trimester amniocentesis. Prenat Diagn 1993;13:919–27

213. European study: MRC working party on the evaluation of chorion villus sampling. Medical Research Council European trial of chorion villus sampling. Lancet 1991;337:1491–9

214. Firth HV, Boyd PA, Chamberlain P, MacKenzie IZ, Lindenbaum RH, Huson SM. Severe limb abnormalities after chorion villous sampling at 56–66 days’ gestation. Lancet 1991;337:762–3

215. Firth HV, Boyd PA, Chamberlain PF, MacKenzie IZ, Morriss-Kay GM, Huson SM. Analysis of limb reduction defects in babies exposed to chorion villus sampling. Lancet 1994;343:1069–71

216. Froster-Iskenius UG, Baird PA. Limb reduction defects in over one million consecutive livebirths. Teratology 1989;39:127–35

217. Foster UG, Jackson L. Limb defects and chorionic villus sampling; results from an international registry 1992–94. Lancet 1996;347:489–94