First-trimester cardiac scan and study - Fetal Heart Ultrasound: How, Why and When; 3 Steps and 10 Key Points

Fetal Heart Ultrasound: How, Why and When; 3 Steps and 10 Key Points, 3th Ed.

5. First-trimester cardiac scan and study

This chapter is also covered by accompanying online material

Claudio Lombardi

CHAPTER CONTENTS

Introduction

Technical aspects: equipment

Technical aspects: settings

Examination: risk factors

Anatomic correlation and its limitations

Introduction

Why?

A major aim of prenatal diagnosis is to provide parents with relevant information on the health of the fetus as early as possible during the pregnancy. Sonographic measurement of the nuchal translucency (NT) thickness at 11–13 weeks is a well-established screening method for aneuploidies¹ and other major anomalies.² Other noninvasive methods are already being used to identify aneuploidies,³ and they will probably replace nuchal scanning for this purpose in the near future. At that point, the main goal of the 11–13 week scan will be to identify major structural anomalies in the fetus.

The most likely site of such anomalies is the fetal heart, which is already fully developed at this stage of gestation.⁴ Effective screening and accurate diagnosis of cardiac malformations is already feasible during the 12th week of gestation.⁵^–⁷ Several markers of aneuploidy, including increased NT,⁸ tricuspid valve regurgitation (TR),⁹ and abnormal flow through the ductus venosus (DV),¹⁰ have also proved to be effective in identifying congenital cardiac anomalies. With the aid of new tools—e.g., genomics arrays, which can identify submicroscopic deletions, duplications, or other rearrangements¹¹—early detection and diagnosis of major cardiac malformations in the fetus can provide parents with prognostic information that will enable them to make timely, informed choices about the management of the pregnancy.¹²

Who?

The first-trimester examination of the fetal heart begins—and usually ends—with the US practitioner. If the suspicion of cardiac abnormalities arises during the screening examination, however, the patient must be referred to a pediatric cardiologist for a specific diagnosis and prognostic assessment. Later, if the decision is made to interrupt the pregnancy, a key role will also be played by the pathologist, whose job it is to confirm and shed additional light on the antenatal diagnosis. For the latter two figures, earlier assessment of the fetal heart has some obvious drawbacks. The size of the organ at 11–13 weeks (cross sectional diameter 4–6 mm) is clearly a challenge for the cardiologist, who must diagnose the specific abnormality and assess the fetus' prognosis, but studies have shown that over 70% of major congenital heart diseases (CHDs) can be defined early with this method.¹³^–¹⁸ For the pathologist, the obstacles are even more daunting since dissection of an organ this size is out of the question.

For US practitioners, however, the objective is merely to screen the fetus for signs of possible cardiac abnormalities during the NT scan. In their view, the first-trimester cardiac examination is a way to add diagnostic value to a well-accepted screening program without significantly increasing its costs. Studies have demonstrated that, in approximately 90% of all low-risk cases, the addition of fetal cardiac screening prolongs the routine NT scan by no more than ten minutes or so, and the rate of false-positive results is <5%.¹⁹^,²⁰

A high index of suspicion at screening is still the major determinant of successful detection of major cardiac defects at this stage of gestation. The presence of an increased NT can predict 30% of the major CHDs, and the percentage increases if this marker is also associated with TR or abnormal DV flow.²¹ Single-marker positivity is much more common, however, and early fetal echocardiography will probably not be available for all patients with findings of this type. Consequently, US practitioners themselves should be able to provide additional information based on a basic study of the fetal heart aimed at distinguishing cases that require prompt attention by a pediatric cardiologist.

What?

Screening of the fetal heart during the NT scan should include verification of all of the following elements:

Step 1: Abdominal situs and position (axis) of the heart within the chest

Step 2: The presence of four properly proportioned heart chambers; the AV-valve offset, with pulsed-wave Doppler imaging of flow across the tricuspid valve; and equal filling of the right and left ventricles with different color flow mapping techniques. Assessment of the four chambers of the fetal heart early in pregnancy (or at midgestation) can identify 45% of all CHDs. Consequently, it is the single most important indicator of risk!²²

Step 3: Intersection of the aorta and the main pulmonary artery (X or b sign) with color flow modalities; forward flow and equal size of the aortic arch and the ductus arteriosus at their confluence (V sign).

The pulmonary arterial branches, the pulmonary veins, and the systemic veins, which are examined during the mid-gestational study, are not assessed during the first-trimester examination. The order in which the above elements are assessed will vary: be flexible and seize the moment! If the fetal position is obstructing your view of one structure, examine something else until the position changes. Each component should be examined and the findings documented photographically in the report.

!!!Attention!!!

This screening protocol is associated with >90% sensitivity in the detection of major CHDs present at 11–13 weeks. Use of nonoptimal technology or a hurried examination will reduce sensitivity, and the examination will yield only very basic information. However, even this information adds value to the NT screening procedure ordered by the patient's physician.

In all cases, the examination findings will have to be followed up on at 20 weeks.

This must be explained to the parents!

When?

Ideally, the first-trimester cardiac examination is done during the NT scan, between the 11th and 13th weeks of gestation, when crown–rump lengths (CRLs) range from 46–84 mm, and the heart itself has a cross-sectional diameter of 4–6 mm. The optimal period for assessing NT and the fetal heart is about the 12th week. During this period, CRLs range from 55–65 mm, and the fetus frequently assumes a transverse, dorsoposterior position that greatly facilitates visualization of the nuchal area and the heart. Later, a vertical position is more likely. With the technology currently being used by most practitioners, it is undeniably easier to examine the fetal heart at 13 instead of 12 weeks. However, if this approach is used, only a fraction of the patients who come in for NT scanning will benefit from the early cardiac screening.

How?

For US practitioners familiar with second-trimester cardiac screening and diagnosis, the main obstacles to a successful first-trimester examination are technical. Equipment settings for the second-trimester cardiac study are well known, and most scanners come with a preinstalled program (or preset) for this examination. In contrast, settings for the study of the first-trimester fetal heart have to be configured by the operator. For those practitioners who are put off by the fact that there is no single “magic” button to press, this chapter should be of particular value: it will provide indispensable information on the technical aspects of cardiac imaging that supplements information provided elsewhere in this book. The image quality offered by a given ultrasound system is the result of a unique balance between the specific features of the scanner and the transducers, so any exam preset that is offered is inevitably machine-specific. Understanding the specific goals and problems of early fetal cardiac imaging allows operators to choose equipment more effectively and to adjust the settings to obtain the best images possible in each case.

!!!Remember!!!

Before undertaking a first-trimester study of the fetal heart:

• Be fully familiar with fetal heart anatomy and with techniques used in the midgestation study

• Understand specific technical requirements for the early study: machine and transducer properties, settings

• Define how you plan to manage cases with suspicious findings

• Make sure the patient understands the objectives and limitations of the study (see Table 5.4)

Technical aspects: equipment

Question: Transabdominal vs. transvaginal approach?

Suggestion: The transabdominal approach should always be the first choice.

Sonographic studies of fetal heart disease were initially attempted by skilled obstetricians using the transvaginal route.²³^–²⁵ Over the years, continuous improvements in US resolution have made it possible to carry out these studies with a transabdominal approach. This advance enabled cardiologists to perform a complete evaluation of the fetal heart that included diagnosis and prognostic assessment of abnormalities.

NT studies are usually performed during transabdominal scans, which are not only more acceptable to patients than the transvaginal route: they are also less dependent on the position of the fetus, do not require manipulations, and can be mastered more rapidly with a more affordable training period.

Question: Transducer design?

Suggestion: Use a specific linear-array transducer

Ultrasound image resolution depends on several factors, including the composition of the body wall and the depth of the structure being examined. Thus far, no studies have been conducted that specifically examined the position of the fetus in the first trimester. Research carried out by our group has shown that the hearts of fetuses between the ages of 11 and 13 weeks lie anywhere from 4–11 cm below the maternal skin surface (Fig. 5.1).

FIGURE 5.1 Distribution of fetal heart depths at the time of the nuchal scan (based on studies of 1388 fetuses) with a median CRL of 60 mm (range 46–74 mm). Depths, expressed as distance from the maternal skin surface to the crux of the fetal heart, ranged from 40–110 mm (median 63 mm).

According to the model we studied, the actual depth will be 4–6 cm in about 60% of the cases and up to 11 cm in the others. At this stage, the depth of the fetal heart is not significantly influenced by the maternal BMI (as it is at mid or late gestation) because at 12 weeks, the transducer is placed over the symphysis pubis, where the thickness of the fat layer is limited. Problems are more likely to arise from the presence of a retroverted uterus (Fig. 5.2).

FIGURE 5.2 Distribution of fetal heart depths as a function of maternal BMI (based on the study of 1388 fetuses at 12 weeks of gestation). The number of fetuses studied is admittedly quite limited, but on the basis of this experience, there do not appear to be significant differences in the depth of the fetal heart in mothers who are underweight, normal weight, overweight, and obese.

The transabdominal probes used by obstetricians are usually curved-array transducers designed for second-trimester imaging, which requires a wide field of view and a penetration depth of up to 20 cm. Each ultrasound beam emitted radiates at a 90° angle from the face of the transducer, and if the face is convex, the beams diverge progressively with increasing imaging depth. Consequently, the distance between adjacent beams increases with the distance from the transducer, and resolution deteriorates as a function of imaging depth (Fig. 5.3).²⁶ The image quality provided by curved-array transducers is acceptable during a second-trimester study of the fetal heart, which is relatively large, but high resolution is essential for examining the tiny anatomical features of the first-trimester heart. With conventional convex-array transducers, resolution begins to diminish substantially at a depth of 6.5 cm (Fig. 5.4). This decline will affect visualization of the heart, which lies >6.5 cm below the surface in approximately 40% of all 12-week fetuses (Fig. 5.5).

FIGURE 5.3 (A) With a conventional convex transducer (6 MHz), image resolution diminishes with distance because of the divergence of the ultrasound beams. (B) With a linear array transducer of the same frequency (6 MHz), the beams travel parallel to one another, and there is no loss of resolution with increasing depth. (C) Higher frequency linear transducers (12 MHz) can be used to obtain higher resolution images of the same area.

FIGURE 5.4 Lateral resolution (i.e., the capacity for distinguishing two adjacent points) offered by conventional convex and linear array transducers as a function of imaging depth (based on examination of phantom models). The areas studied lay at depths of 4–11 cm (where the fetal heart is expected to be found at 12 weeks). Both convex and linear transducers provide adequate lateral resolution for imaging structures approximately of 6–7 cm below the maternal skin surface, but at greater depths the resolution of the 6 MHz convex transducer (red line) decreases progressively. In contrast, with a 9 MHz linear probe, resolution remains stable (white line), and it is further enhanced by use of higher frequency linear probes (12 MHz) (yellow dotted line)

FIGURE 5.5 (A) With conventional convex-array transducers, resolution begins to decline at a depth of 6–7 cm and therefore affects optimal visualization of the heart (shaded area). (B) According to the model we studied, the hearts of approximately 40% of all 12-week fetuses will be in this area (shaded area).

For this reason, I recommend using a linear transducer, which maintains high spatial resolution with distance. It can also be used at higher frequencies than convex-array transducers. For examining features like the AV-valve offset (which measures 0.2 mm in a 12-week fetus), the difference is impressive (Fig. 5.6). With recently developed linear-array systems, one can shift back and forth between a linear format (when higher resolution is required) and a virtual trapezoidal format (similar to that of a curvilinear transducer) when a wider field of view is needed (Fig. 5.7). The efficiency of high-frequency linear transducers has improved dramatically in recent years. Advances in crystal design and piezoelectric materials, improved pulse shape and disposition, and increases in the number of elements have greatly enhanced penetration and image resolution.²⁷^,²⁸

FIGURE 5.6 Resolution of traditional convex and linear transducers in a 12-week fetus. The fetal heart lies 7 cm below the maternal skin surface, and the AV-valve offset at this age is only 0.2 mm. (A) When the study is done with a 6 Mhz convex probe, the loss of spatial resolution makes it impossible to assess the AV-valve offset. (B) When the same fetus is imaged with a 9 Mhz linear probe, the AV valve offset is clearly visible.

FIGURE 5.7 Use of linear array transducers at 11-13 weeks. (A) Modern linear array transducers can be used in a virtual trapezoidal format, which resembles that of a convex-array transducer. (B) Most US practitioners are more comfortable using a wide field of view for imaging the fetus. (C) The switch to linear format is done in real time. (D) Higher B-mode and color flow anatomical details will be available at all depths.

Question: Transducer frequency?

Suggestion: An optimal range is 6–12 Mhz

Currently available transducers used for a first-trimester fetal heart scan should have frequency range of 6–12 Mhz and an effective penetration depth of 12 cm. Power management techniques can be used to design special pulses that enhance penetration. We have successfully used coded excitation (i.e., coded pulse trains) in our studies. The effects resemble that of a power increase, but this approach has no impact on the mechanical index.²⁹^,³⁰ Coded excitation allows high-frequency (9–12 MHz) linear transducers to compensate for the natural decrease in penetration caused by the attenuation of the ultrasound signal. Structures at depths of up to 11 cm—typical for the first-trimester fetus—can thus be clearly depicted. Coded signals are also used to amplify low-intensity waves produced by blood cells and allow B-mode-based visualization of flow (B-flow) (Fig. 5.8).

FIGURE 5.8 Higher frequencies translate into higher image resolution. A general consequence of increasing the imaging frequency is that the depth of penetration decreases because high frequencies are subjected to greater attenuation than lower frequencies. (A) A high-frequency linear transducer (9–15 Mhz) usually has an effective penetration depth of up to 6–7 cm, which is adequate for its intended purpose, i.e., the study of small parts. (B) Penetration can be enhanced by increasing the acoustic power, but in medical imaging, and fetal imaging in particular, the rule of thumb is to keep the power or the mechanical index (MI) as low as possible. (C) Another option is to apply more power to the ultrasound pulse along the length of its path, i.e., increase the pulse duration. This is the principle underlying coded excitation. With this technique, the effective penetration of a high-frequency linear probe can be extended. Coded signals can also be used to amplify low intensity waves produced by blood cells in the vessels and provide visualization of flow based on B-mode (B-flow).

Reverberation artifacts are common in a moving small liquid-filled areas like those of the fetal heart. They can be overcome by using a scanner equipped for the use of the following techniques:

Tissue Harmonic Imaging (THI)

THI suppresses the weak echoes caused by artifacts. Compared with fundamental-frequency imaging, it reduces reverberation noise, improves border delineation, and increases spatial, axial, and contrast resolution.³¹ For early studies of the fetal heart, recent generation THI technology should be used whenever possible: ongoing development of this technology has dramatically improved spatial resolution in the near-medium field of view, where the fetus is located at the time of the NT scan (Fig. 5.9).³²

FIGURE 5.9 (A) In conventional B-mode imaging (or fundamental imaging), the ultrasound beam is transmitted and received over the same frequency bandwidth. However, reflected echoes in human tissue generate higher-frequency components (the tissue harmonic response) whose directional performance and spatial resolution are better than those of the fundamental wave because their frequency is twice as high and they pass through the tissue only once, reducing the number of overlapping signals. (B) Because the second harmonic component has much less energy than the fundamental component, it can be picked up only by a high-sensitivity receiving system with a broad dynamic range. Harmonic imaging constructed with the pulse-inversion technique can dramatically improve resolution.

Compound Imaging

Spatial compounding allows one to view a structure from different angles and combine the information thus acquired to obtain a single sharper image. The temporal resolution is somewhat diminished because the frame displayed is the average of several sweeps, but this loss is offset by increased contrast resolution and reduced acoustic noise,³³ which improves border definition. In some cases, spatial compounding can be used with tissue harmonics to better demonstrate the AV-valve offset (approximately 0.2 mm) and septoaortic continuity in the fetal heart (Fig. 5.10).

FIGURE 5.10 (A) In conventional modality, the ultrasound beam is transmitted at a 90° angle from the face of the transducer. (B) With compound imaging, the ultrasound beams are transmitted from several different angles and are thus associated with different artifact patterns. When frames do not overlap, the patterns are considered artifacts and suppressed; where frames overlap, the patterns are considered real structures and reinforced. It is geometrically obvious that a linear-array transducer will be able to generate more beams from different angles than a curved-array transducer. Spatial compounding enhances contrast resolution and border definition. (C) Compounding can be useful for assessing specific cardiac features, like septoaortic continuity. As this modality considerably reduces the frame rate, its use at first trimester is limited.

Post-Processing

Different post-processing settings should be tested to minimize speckle artifacts, ultrasound signals caused by variations in the positions and signal strength of the various scattering within the beam. The first-trimester heart has all the features of a structure that produces speckle. Not all speckle is noise: some represents true tissue information. The ultrasound machine should be able to distinguish between the two and eliminate only the useless, artifactual subset.³⁴^,³⁵

Post-processing settings designed to make a sense of speckle signals have been marketed under different names. Optimizing these functions to reduce noise and improve conspicuity requires patience, perseverance, and, ideally, the assistance of an application specialist. The best image quality is achieved by fine tuning.

Technical aspects: settings

Image quality reflects the characteristics of the transducer, those of the ultrasound machine, and the balance between the two components. General aspects of the exam presets are described in Chapter 2, which should be consulted before reading this chapter. Settings for the first-trimester fetal heart study should be defined by the practitioner, ideally with the aid of an application specialist.

To create a first-trimester 2D package, the operator should image a fetus with a CRL of 6 cm whose heart is no more than 4–6 cm beneath the maternal skin surface. The examination begins with an apical four-chamber view of the heart, and spatial resolution should be optimized to allow demonstration of the AV-valve offset while viewing the crux of the heart. Noise suppression should then be adjusted so that the image of a ventricular chamber includes no spurious signals (Fig. 5.11).

FIGURE 5.11 (A) Heart chambers contain no spurious signals. (B) Crux of the heart: the AV valve offset at 12 weeks is 0.2 mm.

!!!Attention!!!

Image quality = transducer characteristics × scanner characteristics

The balance between these two components is different for each system.

Table 5.1 contains practical tips for creating a system-specific preset for 2D imaging of the first trimester fetal heart, starting with the preset configured by the manufacturer for the mid-gestational study. It briefly describes each parameter's effect on image quality and how it should be adjusted for a first-trimester study.

Table 5.1

Practical tips for creating a system-specific preset for 2D imaging of the first-trimester fetal heart

Color Flow Modalities

Color flow imaging is essential for first-trimester cardiac studies: even the great vessels are too small to be examined exclusively in the 2D mode. In addition to the anatomic features and relationships of these vessels, color images are often used to evaluate heart chamber morphology. Different color modes can be selected to obtain full information on blood flow events (color flow mode) or spatial resolution of blood vessels and heart cavities (power Doppler, B/E-Flow).

Color Doppler

For first or second trimester cardiac studies, color Doppler ultrasound provides the most informative images. Using the original preset for the second-trimester study, the operator should select an apical four-chamber view and then narrow the field so that the box includes only the heart or only the aorta and pulmonary artery in the three-vessel view.

Table 5.2 contains practical tips for creating a system-specific color imaging preset.

Table 5.2

Practical tips for creating a system-specific color imaging preset

Power Doppler

When a short pulse of transmitted ultrasound is scattered from a small sample volume, the transducer receives information on flow velocity (pulsed-color Doppler) and on the amplitude of the echo signal. The power (or energy) of the echoes, which reflects the number of moving cells in the sample volume, is used to produce the power Doppler images. For identifying and defining vessels and assessing myocardial cavity filling, power Doppler is more sensitive than conventional color Doppler because the color image depends on the amplitude of the signal generated by erythrocyte movement within the vessel, independently from the insonation angle.³⁶ Unlike color Doppler, power Doppler imaging does not display the flow velocity, but this type of evaluation can be postponed until midgestation. Some newer scanners offer directional power Doppler modes, but full-resolution, unidirectional power Doppler is probably better for the first-trimester fetal heart study. Power Doppler is not used at all for the second-trimester scan, so it will have to be set up ex novo.

Power doppler settings:

Frequency = high

PRF = low

Directional modality = monodirectional

Persistence = high

However, power Doppler resolution is still inferior to that of B-mode imaging because the frequency used is lower. The resulting image overlap can make blood vessels appear thicker than they actually are.

B/E-flow ultrasound

Other color flow modes that are not based on the Doppler shift offer spatial resolutions closer to that of B-mode imaging. In B/E-flow imaging, for example, digital encoded ultrasound is used to enhance the backscatter signals from blood cells, while echoes from stationary tissues are cancelled.³⁷ Compared with Doppler-based imaging, B/E-flow imaging discriminates more accurately between blood flow and the borders of the vessel lumen because it provides direct visualization of blood echoes with the same frequency of B-mode imaging.

Technical Aspects: Summary

First-trimester fetal heart screening can be performed during the NT scan, which is already widely accepted by pregnant women and their physicians, but its use is less widespread than one would imagine, probably for technical reasons. The NT scan is usually performed transabdominally with transducers designed for second- or third-trimester screening, but these instruments are not the best tools for studying the fetal heart at 11–13 weeks of gestation. Experts agree that this examination can be done transabdominally only if the CRL exceeds 6 cm (i.e., a fetus of approximately 12.6 weeks).³⁸ For smaller fetuses, the endovaginal approach is necessary to achieve adequate spatial resolution. Consequently, if the mother presents for NT screening at 11–12 weeks, inclusion of the fetal heart examination will require the addition of an endovaginal scan. This is a more complicated procedure, which requires specific training and experience that the operator may not have. It is also likely to prolong the screening examination significantly, and it may also reduce the rate of patient acceptance of the supplemental screening.

On the other hand, scheduling the NT scan for 12.5–13 weeks also has its drawbacks. The fetus is more likely to be lying in a vertical position, which complicates assessment of NT. There is also the question of depth. When the fetus is located more than 6–7 cm below the maternal skin surface, B-mode examination of the crux is appreciably less reliable for reasons that have been outlined in the previous pages.

Our solution is to use a high-frequency linear transducer. As demonstrated in three studies,¹⁹^,³⁹^,⁴⁰ this approach resolves the problems related to size and depth. All scanners can be equipped with linear transducers, even though they are designed for other purposes. Linear transducers and scanners used for a first-trimester cardiac study must have specific characteristics; otherwise, the results will be inferior to those that are obtainable with well-configured, convex-array transducers used for midgestational studies.⁴¹

This approach is not widely used. The information provided in the previous pages are, in our opinion, useful for understanding the limitations and technical difficulties associated with currently available equipment.

With the convex transducers being produced for midgestational studies, only fetuses with a CRL of >6 cm should be studied transabdominally. In these fetuses, structures lying at a depths of >6 or 7 cm should be examined with the scanner's color imaging modality. In particular, power Doppler or B/E-flow ultrasound should be used for morphological studies, not only of the great vessels but also of the heart chambers.

• Do not be intimidated by a high maternal BMI: it's not likely to cause problems.

• When the distance between fetus and transducer is excessive, be patient: examine something else and see if the position changes.

• Make sure the patient is relaxed: turn her on her side, let her walk a bit, offer her a snack.

Examination: risk factors

Conventional prenatal screening for CHD is still associated with low detection rates:⁴² the approach based on family or maternal risk factors identifies only 5–10% of affected fetuses.⁴³^,⁴⁴ Consequently, interest has been focused on alternative methods for identifying high-risk patients for specialist referral. Certain markers used to screen for aneuploidies, for example, have also proved to be effective for the detection of major cardiac defects.

Nuchal Translucency (NT)

A major advance in screening for fetal cardiac defects came with the realization that many affected fetuses had an increased NT thickness at 11–13 weeks,⁵^,⁴⁵ and the incidence of CHD increased with the thickness of the NT (Table 5.3). Values at or above the 95th percentile (2.5 mm) are found in 30% of euploid fetuses with major cardiac defects and 5% of those with normal hearts. Given the limited availability of fetal cardiology services needed to provide a diagnosis, this false-positivity rate is still too high. Early fetal echocardiography is indicated when NT thicknesses >3.5 mm (> the 99th percentile).

Table 5.3

Increased NT thickness and risk of CHD in euploid fetuses

NT (mm)	RISK OF MAJOR CHD
2.5–3.4	~ 1 : 50
3.5–4.4	~ 1 : 35
4.5–6.5	~ 1 : 10
6.6–8.5	~ 1 : 5

It is important to note that fetuses with NT thicknesses of 2.5–3.5 mm (95th–99th percentile) are four times more common than those with values >99th percentile (>3.5 mm). This group has a background of risk of major CHD (1 : 50) usually higher than the risk of a chromosomal disorder. When the NT thickness is in this range, the mother's understandable anxiety will lead the practitioner to offer an early scan of the fetal heart itself to identify any direct signs of major abnormality.

NT assessment is of limited value in primary screening for major cardiac abnormalities, but its performance improves when it is combined with evaluation of flow through the DV and flow across the tricuspid valve. When the increased NT (95th–99th percentile) is associated with TR and/or an abnormal DV flow pattern, the parents should be referred promptly for fetal echocardiography.

Tricuspid Valve Regurgitation

TR at 12 weeks is a powerful marker of major cardiac defects although the mechanism underlying this association is uncertain. TR is present in ~1% of euploid fetuses with normal hearts (false positivity) and 30% of those with major cardiac defects (~40% if the NT thickness is also increased).⁴⁶ Pulsed-wave Doppler imaging is used to detect TR. This technique should be used sparingly during screening: the ultrasound beam is focused on a very small amount of tissue, and excessive exposure of this type can be harmful to the fetus. Box 5.1 shows the requirements for performing the TR study.

Box 5.1

Tricuspid valve presets

The following pulsed-wave Doppler parameters should be stored in the scanner:

• Velocity: >60 cm/sec

• Sample volume: 2–3 mm

• Sweep speed: 2–3 cm/sec

• Angle between beam and flow: <30°

The examination should be carried out as follows:

• The fetus should not be moving

• The image should be magnified so that the fetal thorax occupies the whole screen

• An apical four-chamber view of the fetal heart should be obtained

• Color flow mapping should not be used because it is unreliable for the diagnosis of tricuspid regurgitation in the first trimester

At least three attempts should be made because the tricuspid valve can be insufficient in one or more of its three cusps next.

The transducer should be moved across the valve from one side to the other.

Nicolaides 2012 Fetal medicine Foundation: Tricuspid flow; www.fetalmedicine.com. Extensive information and courses can be found online at this site.

The cusp of the tricuspid valve can produce a sharp click: this is physiological (Fig. 5.12). Tricuspid regurgitation is diagnosed if regurgitation into the right atrium is observed for at least half the duration of systole and has a velocity of more than 60 cm/sec. (The velocity of aortic or pulmonary arterial blood flow at this stage of gestation never exceeds 50 cm/sec.) The regurgitation may be mild or holosystolic: both are significantly associated with CHD. Exclusion of TR is especially important when low resolution precludes reliable assessment of the AV-valve off-set: the absence of TR in these cases is a good indicator that there is no AVSD.

FIGURE 5.12 (A) Normal flow across the tricuspid valve. High image magnification is required, and the insonation angle is almost parallel (7°) to the axis of the interventricular septum. (B) Normal flow across the tricuspid valve. Closure of a valve cusp generates a short reverse spike representing retrograde flow, but it does not occupy half of the systolic interval. (C) Regurgitation. The retrograde flow associated with regurgitation is present for half of systole and its velocity exceeds 60 cm/sec.

!!!Attention!!!

TR is a powerful early marker for CHD: the detection rate is 30% with a false-positivity rate of 1%. TR assessment is an essential part of the early fetal heart scan.

Ductus Venosus

The DV is a trumpet-shaped structure that shunts blood from the umbilical vein to the right atrium. Abnormal blood flow through this structure is associated with major cardiac defects (Fig. 5.13).⁴⁷

FIGURE 5.13 Ductus venosus flow. The fetal thorax and abdomen occupy the whole screen.The sample volume is reduced to 0.5 mm to avoid contamination from adjacent veins. The insonation angle should be less than 30 degrees. The filter should be set at a low frequency (50-70 Hz) to allow visualization of the whole waveform. The sweep speed should be high (2-3 cm/sec) so that the waveforms are widely spread for better assessment of the a-wave. The pulsatility index is measured by the machine after manual tracing the outline of the waveform.

Reliable Doppler assessment of DV flow requires extensive supervised training.⁴⁸ Waveforms in the ductus can be assessed qualitatively or quantitatively. In the former case, abnormality is defined by the absence or reversal of the a-wave; in the latter, by an increase in the DV pulsatility index (DVPI). Contamination from adjacent vessels is common. Box 5.2 lists the requirements for performing the DV study.

Box 5.2

Ductus venosus presets

The following pulsed-wave Doppler parameters should be stored in the scanner:

• Velocity: 20 cm/sec

• Sample volume: 0.5–1.0 mm

• Sweep speed: 2–3 cm/sec

• Filter frequency: 50–70 Hz

• Angle between beam and flow: <30°

The examination should be carried out as follows:

• The fetus should be in a state of quiescence

• Magnify the image so that the fetal thorax and abdomen occupy the entire screen

• Obtain a right ventral mid-sagittal view of the fetal trunk

• Position the pulsed Doppler sample in the aliasing area, which is above the umbilical sinus

Nicolaides 2012 Fetal medicine Foundation: Tricuspid flow; www.fetalmedicine.com. Extensive information and courses can be found online at this site.

In most studies on the association between abnormal DV blood flow and CHD, the Doppler studies have been performed as second-line studies in fetuses with documented increases in the NT thickness.⁴⁹However, qualitatively abnormal DV flow during the first trimester is also an independent marker of cardiac defects in euploid fetuses (detection rate 10%).⁵⁰ Consequently, the CHD detection rate for the NT study increases from approximately 30% to ~40% when it is combined with assessment of DV flow. Qualitatively abnormal DV flow is observed in 2% of normal hearts (false positivity). Quantitative assessment based on the DVPI reportedly reduces the false-positivity rate by approximately half.⁵¹^,⁵²

Problems have been reported in obtaining a DV tracing to interpret, mainly because during the scan the tracing may vary (from normal to reversed) or even disappear.⁵³ In some cases, the DV cannot be visualized connecting the portal sinus with the subphrenic confluence. In these cases, the umbilical venous drainage may be intra- or extrahepatic. Absence of the DV is not associated with structural or chromosomal abnormalities or with functional cardiac overload. The prognosis appears to be good.⁵⁴

Risk Factors: In Summary

As practitioners' access to the necessary equipment increases, a basic cardiac scan can be gradually incorporated into the early fetal study. As with the midgestational study, the operator should assume that the heart is abnormal until proven otherwise: suspicion of abnormality is the best indication for referring the patient to a fetal cardiologist. The morphology of the four chambers of the fetal heart early in pregnancy (and at midgestation) is the single most reliable indicator of risk! In the presence of certain markers of risk, more detailed cardiac studies should be done to determine whether specialist referral for early echocardiography is indicated. These include (1) an NT thickness in the 95th to 99th percentile with TR or abnormal DV flow; (2) an NT thickness above the 99th percentile; and (3) findings in the NT scan that are suggestive of cardiac anomalies or other structural defects. We consider assessment of TR to be an essential part of the routine cardiac scan because of its low false-positivity rate and because it can be used as a substitute for studies of the AV-valve offset when the latter cannot be adequately visualized.

Anatomic correlation and its limitations

The cardiac architecture is examined with the approach described in Chapter 4 for studies performed later in gestation, but there are certain important differences.

Step 1: Verification Of The Position Of The Heart

To the left, the position of the stomach should also be confirmed in a longitudinal view. Diaphragmatic hernias can be more difficult to visualize at this stage than at midgestation.

The inferior vena cava should lie anterior to and on the right of the spine; the aorta will be on the left side of the spine. On the left, the apex of the heart and one vessel will be seen. The normal cardiac axis at 11 weeks is approximately 50°, but by 12 weeks it is closer to 45°, i.e., the same as that observed later in gestation. The size of the heart is estimated on the basis of the cardiothoracic circumference ratio (normal range 0.38–0.40).

When the fetus is in a suitable position for measurement of the NT, a 90° rotation and slight caudal translation of the transducer will provide a reliable plane for an apical view. The cephalic translation needed to arrive at the level of the four chamber view is very small.

To assess the normal situs of the heart, we use abdominal organs as landmarks and color modalities to verify the arrangement of the abdominal vessels. The junction of the inferior vena cava with the right atrium—a major landmark in the midgestational study—is too small to be of much help in the first trimester. However, the right atrial appendage can be used instead. It is larger than it will be later in gestation, and it has a typical quadrangular shape, which is easy to distinguish from the tiny tubular appearance of the left atrial appendage. Both appendages are better visualized with power Doppler or B/E-flow imaging (Fig. 5.14).

FIGURE 5.14 Color power Doppler image of the atrial appendages at 12 weeks acquired with a 9-MHz linear transducer. The right appendage (R) is quadrangular when filled and larger than the tubular left appendage (L).

Step 2: Verification Of The Inlet

The relative proportions of the four chambers in a first-trimester fetal heart are the same as those observed at mid to late gestation. Velocity or power Doppler will sometimes be necessary to assess chamber morphology. AV valve patency can be easily verified with color modalities. Clear visualization of the crux and the AV-valve offset—a major goal for the practitioner—is the best indication that the 2D settings have been optimized. If the offset cannot be properly visualized with the equipment being used, pulsed Doppler verification of normal flow across the tricuspid valve is essential.

Step 3: Verification Of The Outlet Tract

The great vessels are studied with color flow modalities. It is easier at this stage to demonstrate the crossing of the aorta and pulmonary artery (x or b sign) and to compare their sizes (V sign) to exclude transposition than it is later on in gestation (Fig. 5.15).

FIGURE 5.15 (A) The pulmonary artery passes over the aorta producing the “X” sign. (B) The “b” sign: the straight back of the b represents the pulmonary outflow and duct; the curved face is the aortic arch. (C) The “V” sign: confluence of ductus arteriosus at the aortic isthmus. The size of the two vessels can be compared, without formal measurement.

The three-vessel-trachea view is also easier to obtain because the rib cage and spine of the first-trimester fetus cause no interference. The aorta and pulmonary artery are very tiny so both will be imaged in the same view. Therefore, there is no need to tilt the transducer to study the point where the great vessels cross (whereas at midgestation different transducer orientations have to be used for this purpose) (Fig. 5.16).

FIGURE 5.16 The images shown here can be easily and quickly produced by reducing overall B-mode gain to zero when high-definition flow imaging modalities are available. The angiography-like images clearly reveal the relationship between the great arteries with visualization similar to 3D–volume images. (A) Because the outflow tracts are so small (scale bar: 1 mm), the point where they cross can be clearly visualized without tilting the transducer to obtain favorable planes (necessary during the midgestational study). (B) The ductal and aortic arches: at 12 weeks the ductal arch forms an angle close to 90°. At midgestation the angle will be slightly less acute, resembling that of a hockey stick.

Verification of septal–aortic continuity is often difficult, even for specialists. Two-dimensional images are not very reliable for this purpose at this stage, so color modalities must be used. They provide lower resolution than B-mode imaging (with the exception of B/E-flow) and may fail to detect some cases of tetralogy of Fallot, particularly those associated with a relatively normal-sized pulmonary artery (Fig. 5.17).

FIGURE 5.17 The small dimensions of the heart make it difficult to obtain reliable 2D images documenting the continuity of ventricular septum with the aortic root. Color flow modalities are useful for this purpose. (A) Normal heart. (B) ToF with no discrepancy between the size of the vessels at 12 weeks. Cases like this can be missed on the first-trimester examination. The only abnormality is a slight difference in angulation of the aorta and septum—a finding that would be regarded as suspicious only by a fetal cardiologist.

An aberrant right subclavian artery (ARSA) is a common variant observed at the level of the aortic arch (1%). It is reported to be more frequently associated with cardiac anomalies in euploid fetuses, especially conotruncal defects. In expert hands, the first-trimester examination can detect ARSA.⁵⁵

• Take your time!

• Examine systematically and use a check list to make sure all findings are documented.

• Screen opportunistically: when you see it, assess it!

• Be patient if visibility is low.

Fetal Cardiologists

Pioneering studies of the first-trimester fetal heart were done in the early 1990s, mainly by experts in the field of obstetrics using transvaginal probes. Later in this decade, as ultrasound technology improved, researchers in fetal cardiology began to perform these studies transabdominally.¹⁷^,¹⁸ By pure coincidence, clinical interest in first-trimester sonography was increasing at this time in light of the diagnostic importance of NT. The fact that increased NT was also associated with CHDs also increased the demand for early assessments of the fetal heart.

During the first trimester, fetal cardiologists can reliably diagnose major CHDs with a sensitivity of 85% and specificity of 99%.⁵⁶ As shown in Table 5.4, the 12-week scan has well-established limitations, which have been documented by detailed follow-up during the second and third trimesters. Disproportion between the right- and left-sided heart structures raises the possibility of CHD, but it may also represent a transient anomaly in an otherwise normal heart. Unfortunately, in these cases fetal cardiologists cannot provide sufficient information to allow families to decide to interrupt the pregnancy because of a fetal cardiac abnormality alone. A normal scan at 12 weeks is rarely followed by evidence of an important defect at 20 weeks, but the possibility cannot be excluded. For these reasons, the heart must be re-examined at midgestation.⁵⁷ If a CHD is diagnosed in the first trimester, microarray analysis can be a valuable tool in prenatal genetic testing of fetuses when conventional cytogenetic methods fail.¹¹

Table 5.4

Major CHDs that are missed on first-trimester scans

Pathologists

The post-mortem examination is essential not only for confirming or refuting the antenatal diagnosis: it also provides valuable details on the defect that are important for counseling parents on future pregnancies. However, conventional (i.e., invasive) autopsy of a first-trimester fetus is simply not feasible because the heart at this stage is only about 4–6 mm wide.

The first problem is to obtain the specimen. After the pregnancy has been terminated, the products of conception must be carefully inspected. Locating and identifying the fetal heart requires a certain degree of expertise, but experienced hands can usually retrieve a satisfactory specimen in nearly 70% of all cases.⁴⁵ As alternatives to dissection, several imaging techniques are being developed to collect ex vivoinformation on the first-trimester heart structures.

Histological Imaging

High-resolution episcopic microscopy and episcopic fluorescence image capturing are histological imaging-related techniques based on volume data sets composed of digital images obtained while a plastic- or paraffin-embedded anatomic specimen is being sectioned with a microtome. Thousands of consecutive images, each with histological quality, can then be adjusted to create a virtual 3D model of the structure in toto. These models can be used for detailed morphological investigation of the fetal heart to produce a “virtual” dissection.⁵⁸ These techniques offer high (histological) image resolution (0.5–5.0 µm), but they are extremely time-consuming, and they also result in the destruction of the physical specimen.

Recent studies have shown that magnetic resonance imaging (MRI) and computed tomography (CT) can also be used to obtain information on the tissues and organs of human embryos.

Magnetic resonance imaging

Conventional MRI machines are designed to contain the whole body of an adult patient, and they typically offer an image resolution of about 1 mm. In contrast, micro-MRI machines are small, high-field (7-Tesla) scanners designed originally for imaging studies of rats, mice, and other small rodents. They offer image resolution (20–25 µm) that is ideal for examining the heart of a 12-week fetus. MRI-rendered data can provide a digitally resectioned view of the specimen from different angles (Fig. 5.18).

FIGURE 5.18 Micro MRI of the fetal heart at 12 weeks. Acquired data are digitally resectioned for viewing the aortic valve within the specimen. The aorta is viewed in the (A) longitudinal and (B) axial planes.

The advantage of this approach is that the specimen requires no specific treatment before data acquisition (as MRI can analyze the soft tissue contrast of heart structures). The main disadvantages are the time it requires (data acquisition takes 24–48 hours), its high cost, and the difficulties involved in setting up an MRI room within a pathology laboratory.⁵⁹^,⁶⁰

Computed tomography

The basic principles of X-ray microcomputed tomography (or micro-CT) are identical to those of conventional medical CT. Micro-CT has proven to be a powerful technique for imaging bone structures in small animals, but with X-ray-absorbing contrast agents, it can also be used to characterize other types of tissues. Soft tissues in biological specimens can be imaged with contrast and spatial resolution of 6–8 µm.⁶¹

Our group initially explored the feasibility of micro-CT imaging for examining postmortem specimens of the first-trimester fetal heart (11–14 weeks). It is much less time consuming than micro-MRI (data can be acquired in few minutes) and CT systems are much cheaper to set up and maintain than micro-MRI systems (Fig. 5.19).

FIGURE 5.19 Micro-CT of the fetal heart at 12 weeks. (A) In this volume rendered micro-CT of the whole heart, details like coronary arteries are visible. (B) Virtual section of the same heart demonstrates the appearance of the ventricular cavities. Anatomical details can be in the scale of microns.

Virtual autopsies based on 3D micro-MRI or micro-CT imaging save time and labor, and they also eliminate the need to section the specimen.

References

1. Nicolaides, KH. Screening for fetal aneuploidies at 11 to 13 weeks. Prenat Diagn. 2011; 31:7–15.

2. Syngelaki, A, Chelemen, T, Dagklis, T, et al. Challenges in the diagnosis of fetal non-chromosomal abnormalities at 11-13 weeks. Prenat Diagn. 2011; 31(1):90–102.

3. Chiu, RWK, Lo, YMD. Noninvasive prenatal diagnosis empowered by high-throughput sequencing. Prenat Diagn. 2012; 32:401–406.

4. Trines, J, Hornberger, LK. Evolution of heart disease in utero. Pediatr Cardiol. 2004; 25:287–298.

5. Clur, SA, Mathijssen, IB. Structural heart defects associated with an increased nuchal translucency: 9 years experience in a referral centre. Prenat Diagn. 2008; 28:347–354.

6. Clur, SAJ, Ottenkamp, CM. Bilardo The nuchal translucency and the fetal heart: a literature review. Prenat Diagn. 2009; 29:739–748.

7. Volpe, P, Ubaldo, P, Volpe, N, et al. Fetal cardiac evaluation at 11-14 weeks by experienced obstetricians in a low-risk population. J Ultrasound Med. 2012; 31(4):563–568.

8. Makrydimas, G, Sotiriadis, A, Ioannidis, JP. Screening performance of first-trimester nuchal translucency for major cardiac defects: a meta-analysis. Am J Obstet Gynecol. 2003; 189:1330–1335.

9. Pereira, SR, Ganapathy, A, Syngelaki, N, et al. Nicolaides Contribution of fetal tricuspid regurgitation in first trimester screening for major cardiac defects. Obstet Gynecol. 2011; 117:1384–1391.

10. Maiz, N, Plasencia, W, Dagklis, T, et al. Ductus venosus Doppler in fetuses with cardiac defects and increased nuchal translucency thickness. Ultrasound Obstet Gynecol. 2008; 31:256–260.

11. Schmid, M, Stary, S, Blaicher, W, et al. Prenatal genetic diagnosis using microarray analysis in fetuses with congenital heart defects. Prenat Diagn. 2012; 32:376–382.

12. Allan, LD, Huggon, IC. Counselling following a diagnosis of congenital heart disease. Prenat Diagn. 2004; 24(13):1136–1142.

13. Allan, L. Fetal cardiac scanning today. Prenatal Diagnosis. 2010; 30(7):639–643.

14. Smrcek, JM, Berg, C, Geipel, A, et al. Detection rate of early fetal echocardiography and in utero development of congenital heart defects. J Ultrasound Med. 2006; 25(2):187–196.

15. Becker, R, Wegner, RD. Detailed screening for fetal anomalies and cardiac defects at the 11-13-week scan. Ultrasound Obstet Gynecol. 2006; 27(6):613–618.

16. Yagel, S, Cohen, SM, Messing, B. First and early second trimester fetal heart screening. Curr Opin Obstet Gynecol. 2007; 19(2):183–190.

17. Carvalho, JS, Moscoso, G, Ville, Y. First-trimester transabdominal fetal echocardiography. Lancet. 1998; 351:1023–1027.

18. Huggon, IC, Ghi, T, Cook, AC, et al. Fetal cardiac abnormalities identified prior to 14 weeks’ gestation. Ultrasound Obstet Gynecol. 2002; 20:22–29.

19. Lombardi, CM, Bellotti, M, Fesslova, V, et al. Fetal echocardiography at the time of the nuchal translucency scan. Ultrasound Obstet Gynecol. 2007; 29:249–257.

20. Abu-Rustum, RS, Ziade, MF, Abu-Rustum, SE. Learning curve and factors influencing the feasibility of performing fetal echocardiography at the time of the first-trimester scan. J Ultrasound Med. 2011; 30(5):695–700.

21. Mogra, R, Alabbad, N, Hyett, J. Increased nuchal translucency and congenital heart disease. Early Hum Dev. 2012; 88(5):261–267.

22. Eleftheriades, M, Tsapakis, E, Sotiriadis, A, et al. Detection of congenital heart defects throughout pregnancy; impact of first trimester ultrasound screening for cardiac abnormalities. J Matern Fetal Neonatal Med. 2012.

23. Bronshtein, M, Zimmer, EZ, Milo, S, et al. Fetal cardiac abnormalities detected by transvaginal sonographyat 12–16 weeks’ gestation. Obstet Gynecol. 1991; 78:374–378.

24. Gembruch, U, Knopfle, G, Bald, R, et al. Early diagnosis of fetal congenital heart disease by transvaginal echocardiography. Ultrasound Obstet Gynecol. 1993; 3:310–317.

25. Achiron, R, Rotstein, Z, Lipitz, S, et al. First trimester diagnosis of fetal congenital heart disease by transvaginal ultrasonography. Obstet Gynecol. 1994; 84:69–72.

26. Browne, J, Watson, A, Gibson, N, et al. Objective measurements of improvements in image quality with new imaging techniques. Ultrasound Med Biol. 2004; 30:229–237.

27. Fukada, E. History and recent progress in piezoelectric polymers. IEEE Trans Ultrason Ferroelectr Freq Control. 2000; 47:1277–1290.

28. Jie, Chen, Panda, R. Commercialization of piezoelectric single crystals for medical imaging applications. IEEE Ultrasonics Symposium. 2005; 1:235–240.

29. Chiao, RY. Xiaohui Hao Coded excitation for diagnostic ultrasound: a system developer's perspective. IEEE Trans Ultrason, Ferroelect, Freq Contr. 2005; 52(2):160–170.

30. Misaridis, T, Jensen, JA. Use of modulated excitation signals in medical ultrasound. Part I: basic concepts and expected benefits. IEEE Trans Ultrason, Ferroelect, Freq Contr. 2005; 52(2):177–191.

31. Mesurolle, B, Helou, T, El-Khoury, M, et al. Tissue harmonic imaging, frequency compound imaging, and conventional imaging: use and benefit in breast sonography. J Ultrasound Med. 2007; 26(8):1041–1051.

32. von Kaisenberg, CS, Kuhling-von Kaisenberg, H, Fritzer, E, et al. Jonat Fetal transabdominal anatomy scanning using standard views at 11 to 14 weeks’ gestation. Am J Obstet Gynecol. 2005; 192(2):535–542.

33. Jespersen, SK, Wilhjelm, JE, Sillesen, H. Multi-angle compound imaging. Ultrason Imaging. 1998; 20(2):81–102.

34. Michailovich, OV, Tannenbaum, A. Despeckling of medical ultrasound images. IEEE Trans Ultrason, Ferroelect, Freq Contr. 2006; 53(1):64–78.

35. Goodman, JW. Some fundamental properties of speckle. J Opt Soc Amer. 1976; 66(11):1145–1150.

36. McDicken, WN. Anderson The Difference Between Colour Doppler Velocity Imaging and Power Doppler Imaging. and T. Eur J Echocardiography. 2002; 3:240–244.

37. Chiao, RY, Mo, LY, Hall, AL, et al. B-mode blood flow (B-flow) imaging Ultrasonics Symposium. 2000 IEEE. 2000; 2(2):1469–1472.

38. Huggon, IC, Ghi, T, Cook, AC, et al. Fetal cardiac abnormalities identified prior to 14 weeks’ gestation. Ultrasound Obstet Gynecol. 2002; 20(1):22–29.

39. Persico, N, Moratalia, J, Lombardi, CM, et al. Fetal echocardiography at 11–13 weeks by transabdominal high-frequency ultrasound. Ultrasound Obstet Gynecol. 2011; 37:296–301.

40. Bellotti, M, Fesslova, V, De Gasperi, C, et al. Reliability of the first trimester cardiac scan by ultrasound-trained obstetricians with high-frequency transabdominal probes in fetuses with increased nuchal translucency. Ultrasound Obstet Gynecol. 2010; 36:272–278.

41. Votino, C, Kacem, Y, Dobrescu, O, et al. Use of a high-frequency linear transducer and MTI filtered color flow mapping in the assessment of fetal heart anatomy at the routine 11 to 13 + 6-week scan: a randomized trial. Ultrasound Obstet Gynecol. 2012; 39(2):145–151.

42. Garne, E, Stoll, C, Clementi, M, The Euroscan Group. Evaluation of prenatal diagnosis of congenital heart diseases by ultrasound:experience from 20 European registries. Ultrasound Obstet Gynecol. 2001; 17:386–391.

43. Nora JJ, ed. Congenital Heart Disease: Causes and Processes. Futura Publishing Co: New York, 1984; 339–358.

44. Allan, LD. Echocardiographic detection of congenital heart disease in the fetus: present and future. Br Heart J. 1995; 74(2):103–106.

45. Hyett, JA, Moscoso, G, Nicolaides, KH. Abnormalities of the heart and great arteries in first trimester chromosomally abnormal fetuses. Am J Med Genet. 1997; 69:207–216.

46. Pereira, S, Ganapathy, R, Syngelaki, A, et al. Contribution of fetal tricuspid regurgitation in first-trimester screening for major cardiac defects. Obstet Gynecol. 2011; 117(6):1384–1391.

47. Matias, A, Huggon, I, Areias, JC, et al. Cardiac defects in chromosomally normal fetuses with abnormal ductus venosus blood flow at 10–14 weeks. Ultrasound Obstet Gynecol. 1999; 14:307–310.

48. Maiz, N, Kagan, KO, Milovanovic, Z, et al. Learning curve for Doppler assessment of ductus venosus flow at 11 + 0 to 13 + 6 weeks’ gestation. Ultrasound Obstet Gynecol. 2008; 31(5):503–506.

49. Favre, R, Cherif, Y, Kohler, M, et al. The role of fetal nuchal translucency and ductus venosus Doppler at 11–14 weeks of gestation in the detection of major congenital heart defects. Ultrasound Obstet Gynecol. 2003; 21:239–243.

50. Chelemen, T, Syngelaki, A, Maiz, N, et al. Contribution of ductus venosus doppler in first-trimester screening for major cardiac defects. Fetal Diagn Ther. 2011; 29:127–134.

51. Timmerman, E, Oude Rengerink, K, Pajkrt, E, et al. Ductus venosus pulsatility index measurement reduces thefalse-positive rate in first-trimester screening. Ultrasound Obstet Gynecol. 2010; 36:661–667.

52. Wald, NJ, Bestwick, JP, Borrell, A. Adding ductus venosus blood flow as a categorical variable to the Combined and Integrated tests in Down's syndrome screening. J Med Screen. 2012; 19(1):49–50.

53. Bilardo, CM, Muller, MA, Zikulnig, L, et al. Ductus venosus studies in fetuses at high risk for chromosomal or heart abnormalities: relationship with nuchal translucency measurement and fetal outcome. Ultrasound Obstet Gynecol. 2001; 17:288–294.

54. Thomas, JT, Petersen, S, Cincotta, R, et al. Absent ductus venosus - outcomes and implications from a tertiary centre. Prenat Diagn. 2012; 32(7):686–691.

55. Rembouskos, G, Passamonti, U, De Robertis, V, et al. Aberrant right subclavian artery (ARSA) in unselected population at first and second trimester ultrasonography. Prenat Diagn. 2012; 31:1–8.

56. Rasiah, SV, Publicover, M, Ewer, AK, et al. A systematic review of the accuracy of first-trimester ultrasound examination for detecting major congenital heart disease. Ultrasound Obstet Gynecol. 2006; 28(1):110–116.

57. Yagel S, Weissman A, Rotstein Z, et al. Congenital heart defects: natural course and in utero development.

58. Gindes, L, Matsui, H, Achiron, R, et al. Comparison of ex-vivo high-resolution episcopic microscopy with in-vivo four-dimensional high-resolution transvaginal sonography of the first-trimester fetal heart. Ultrasound Obstet Gynecol. 2012; 39(2):196–202.

59. Thayyil, S, Cleary, JO, Sebire, NJ, et al. Post-mortem examination of human fetuses: a comparison of whole-body high-field MRI at 9. 4 T with conventional MRI and invasive autopsy. Lancet. 2009; 374:467–475.

60. Votino, C, Jani, J, Verhoye, M, et al. Postmortem examination of human fetal hearts at or below 20 weeks’ gestation: a comparison of high-field MRI at 9. 4 T with lower-field MRI magnets and stereomicroscopic autopsy. Ultrasound Obstet Gynecol. 2012; 40(4):437–444.

61. Schambach, SJ, Bag, S, Schilling, L, et al. Application of micro-CT in small animal imaging. Methods. 2010; 50(1):2–13.