Measurement of the nuchal translucency in the first trimester of pregnancy has become a worldwide established method for identifying fetuses at risk for aneuploidy.1,2 Enlarged nuchal translucency is, like aneuploidy, associated with a variety of structural, mainly cardiovascular, defects, genetic syndromes, and also normal outcomes.3–5 The pathogenetic mechanisms causing nuchal translucency enlargement remain insufficiently understood. Different theories have been proposed within the last 10 years, such as cardiac failure, altered extracellular matrix composition, and a disturbance of lymphangiogenesis.6–11 A delayed but completed jugular lymphatic development seems to be a promising explanation because this supposition is able to explain both the temporary and regional character of increased nuchal translucency.12
Former histological studies in trisomy 16 embryos and human fetuses have shown that nuchal edema is associated with distension of the jugular lymphatic sacs.11,13 Ultrasound studies of first-trimester fetuses show a similar association between fetuses with increased nuchal translucency and the presence of enlarged jugular lymphatic sacs, which appeared both in aneuploid fetuses and fetuses with a normal karyotype.12,14,15 Because the jugular lymphatic sacs are not visible under normal circumstances, the ultrasonographic appearance of the jugular lymphatic sacs indicates a distended state.12
Nevertheless, the association between the presence of distended jugular lymphatic sacs and nuchal edema does not inevitably implicate that there is a causal relationship. Therefore, a temporal relationship between the development of the nuchal translucency and the jugular lymphatic sacs must exist. Also, the number of human fetuses with increased nuchal translucency studied in previous studies was small.11,12 In this study we prospectively investigated the relationship between volume of the jugular lymphatic sacs and nuchal translucency thickness during development in fetuses with increased nuchal translucency.
PARTICIPANTS AND METHODS
Seventy-four consecutive pregnant women with singleton pregnancies, who were referred to our hospital for tertiary care because of a nuchal translucency greater than the 95th percentile, were asked to participate in the study from 2003 to 2005. All patients received written information and gave informed consent. The medical ethics committee of the VU University Medical Center approved the study. Gestational age was calculated by the reported last menstrual period and was adjusted to crown-rump length where appropriate.
Ultrasound examination was performed weekly from the initial scan at 11–14 weeks of gestation to a maximum of 17 weeks of gestational age. The number of examinations differed because of different gestational ages at referral date, termination of pregnancies, and patients’ cooperation.
After nuchal translucency measurement, a detailed anomaly scan was performed by one experienced sonologist (M.N.B.) using transvaginal (8–4 MHz probe; HDI-5000) or abdominal (2–4 MHz; ATL HDI-5000; Advanced Technology Laboratories, Seattle, WA) ultrasonography. The nuchal translucency measurement was performed according to the guidelines of the Fetal Medical Foundation.16 The choice between transvaginal and abdominal ultrasonography depended on gestational age, fetal position, and resolution quality. Before 14 weeks of gestation, transvaginal ultrasound examination was performed in all cases.
The neck region was examined for the presence of the jugular lymphatic sacs, which appear as spheroid translucencies in the antero-lateral region of the neck (Fig. 1). The volume of the sacs was calculated by measuring the jugular lymphatic sacs in 3 directions. In the transverse plane, the length and width of the sacs were measured. In the sagittal plane, the height of the sacs were measured. The volume was calculated by using the formula of an ellipsoid (ie, prolate spheroid): length×height×width×¾π. Digital images and videotapes of each examination were stored.
After counseling and election by the parents, karyotyping was performed by chorionic villus sampling or amniocentesis. If the parents requested termination of pregnancy, this was performed through induction of labor following misoprostol administration (after 14 completed weeks of gestation) or by suction aspiration (before 14 completed weeks of gestation).
Postmortem morphological examination was carried out if the parents approved. The whole fetus or aspiration tissue was fixed in formalin 4% and subsequently examined macroscopically or under a dissection microscope. Examination of the whole fetuses was followed by excision of the neck region. The neck region was further analyzed by microscopic examination of paraffin-embedded serial sections stained with hematoxylin and eosin. The morphology and size of the jugular lymphatic sacs were compared with euploid human controls with normal nuchal skin (n=3). These fetuses where obtained at our center after termination of pregnancy because of maternal disease or spontaneous loss of pregnancy due to cervical failure. These patients also received written information and gave informed consent.
Examination of the neck region was not possible in case of suction aspiration. The aspiration tissue was investigated for the presence of fetal organs. If the heart could be identified, further analysis of the morphology was performed.
In ongoing pregnancies a second-trimester ultrasound examination was performed, which is a routine procedure in our center. After delivery the parents completed questionnaires concerning the health of the newborn.
The association between the volume of the jugular lymphatic sacs and gestational age was estimated in the total group of fetuses with increased nuchal translucency. In addition, differences between euploid and aneuploid fetuses and between fetuses with normal hearts and those with cardiac anomalies were studied by multilevel analysis by using the Wald test. To investigate whether the volume of the jugular lymphatic sacs can predict aneuploidy, detection rates (sensitivity) and false-positive rates (specificity) were calculated and plotted in a receiver operating characteristics curve. Subsequently, the area under the curve was calculated. In addition, multiples of the median (MoM) of the jugular lymphatic sac volumes were calculated, and a prediction model for aneuploidy was created by using logistic regression analysis.
In the group of fetuses, which where examined multiple times (minimum of 3 subsequent measurements with a time interval of 7 days), further analysis was performed to study the development of the volume of the jugular lymphatic sacs within each fetus and to determine the relationship between the development of the nuchal translucency and the volume of the sacs during progression of gestation. Therefore, a marginal analysis (nuchal translucency thickness and jugular lymphatic sac volume were modeled simultaneously) was compared with a time-lag analysis (nuchal translucency thickness and jugular lymphatic sac were modeled in time).
The data were analyzed using multilevel analysis (the Wald test).17 Multilevel analysis takes into account that the same patients are repeatedly measured and uses all available data, irrespective of the number of repeated measurements, which indicates that missing observations are allowed. Furthermore, multilevel analysis is capable of dealing with irregularly spaced time intervals. Multilevel analysis was performed with the statistical software package MLwiN 1.0 (Institute of Education, London, UK). The statistical significance level was set at P=.05. Because volume data were skewed to the right, logarithmic transformation was carried out before performing the analysis.
For the cross-sectional comparisons between the two groups, we assumed a standard deviation of approximately 2 mm for the nuchal translucency. We expected to find a difference of about 1.5 mm between the groups (based on previous studies). With a significance level of 5% and a power of 80%, we needed 28 subjects in each group to “detect” this difference. For development over time, we expected the difference between the groups to be less (on average 1.25 mm). Furthermore, we expected a within-person correlation coefficient between measurements of approximately 0.6 (based on previous studies). When we added this information to the sample size calculation, assuming on average of three measurements for each subject, we calculated that we needed approximately 30 subjects in each group to “detect” this difference. To deal with possible violations of the assumptions, we decided to sample a few more subjects.
The characteristics of the 74 fetuses with increased nuchal translucency are listed in Table 1. Invasive tests were offered in all cases but were refused in four cases. In three of these cases, babies with dysmorphic features were born, and additional tests revealed trisomy 21. In the other case, a healthy neonate without dysmorphic features was born. In further analysis, this infant was considered to have a normal karyotype. Figure 2 shows the disposition of the fetuses included in the study. Follow-up was complete. In 40 of the 74 fetuses (54%), a normal karyotype was found; 34 (46%) fetuses were aneuploid. In 26 of the 30 fetuses (86.7%) that underwent pregnancy termination, postmortem examinations were performed. In 20 (29.4%) of the 68 fetuses, in which the cardiac status was known by follow-up or postmortem examination, a cardiac anomaly was identified. The most frequently encountered cardiac malformations were septal defects.
Weekly ultrasound investigation in the 74 fetuses with increased nuchal translucency between 11 and 17 weeks of gestation revealed 159 measurements of the jugular lymphatic sac volume. The distribution of the number of examinations per fetus in euploid and aneuploid fetuses is shown in Table 2. In six of 74 (8.1%) fetuses, the jugular lymphatic sac could not be detected by ultrasonography (Table 3). Postmortem examination confirmed the presence of nuchal edema and enlargement of the jugular lymphatic sacs in all of the fetuses (n=11) that were available for morphological neck examination, compared with three control fetuses (Fig. 2).
The volume of the jugular lymphatic sacs showed a quadratic relation (P<.01) with gestational age, which differed (P<.01) between euploid and aneuploid fetuses (Fig. 3). In aneuploid fetuses the jugular lymphatic sacs reached a larger volume and remained present longer than in euploid fetuses. The mean jugular lymphatic sac volume of fetuses with a cardiac anomaly was larger than that in fetuses with a normal heart, but the relation between jugular lymphatic sac volume and gestational age did not differ significantly.
Estimation of the discriminating value of jugular lymphatic sac volume for aneuploidy revealed an area under the receiver operating characteristics curve of 0.69 (data not shown). Also, the prediction model for the probability of aneuploidy, based on MoM jugular lymphatic sac volumes, shows that, if jugular lymphatic sacs become visible on ultrasonography, the probability of aneuploidy is 30%. This probability increases moderately at higher MoM jugular lymphatic sac volumes (Fig. 4).
In fetuses with a data set of minimal three examinations (n=92 measurements), a similar quadratic relation within each individual fetus was found. With advancing gestation, a progressive increase in jugular lymphatic sac volume was followed by subsequent decrease. However, the gestational age at which the maximum size of the jugular lymphatic sacs was reached differed per fetus, showing a fetus-specific pattern. A similar pattern was also observed for nuchal translucency development. The median gestational age at which the maximum jugular lymphatic sac volume was reached was 96 days, whereas the median gestational age for the maximum nuchal translucency thickness was 88 days.
In addition, the development of jugular lymphatic sac volume and nuchal translucency thickness were significantly related in time (with advancing gestation), whereby nuchal translucency expansion preceded jugular lymphatic sac expansion. The time-lag relationship was twice as strong as the simultaneous relationship (time-lag regression coefficient 0.86, P<.001, versus simultaneous regression coefficient 0.43, P=.004).
All probability values, graphs, and numbers are shown for the right jugular lymphatic sac. The left jugular lymphatic sac showed similar results (data not shown).
This study indicates that a disturbed lymphatic development is a common denominator in the pathophysiology of increased nuchal translucency. Distended jugular lymphatic sacs were visible in the majority (91.9%) of fetuses with increased nuchal translucency. Moreover, the development of the jugular lymphatic sac volume and nuchal translucency thickness were significantly related, whereby nuchal translucency expansion preceded jugular lymphatic sac enlargement. Also, in all fetuses that were available for postmortem neck examination, the presence of distended jugular lymphatic sacs was confirmed, in contrast to controls.
Both nuchal translucency and jugular lymphatic sacs showed a fetus-specific pattern, meaning that each fetus reached a peak nuchal translucency thickness followed by a peak jugular lymphatic sac volume at a specific gestational age, which varied between the examined fetuses. This fetus-specific pattern is already known for the development of nuchal translucency and is considered a normal variation in development.18 The fact that a similar pattern was present in the development of jugular lymphatic sac volume supports the supposition of a common developmental pathway. A limitation of our study was the number of aneuploid fetuses available for time-lag analysis due to pregnancy termination (Table 2).
In six fetuses the jugular lymphatic sacs were not observed. Four of these fetuses were examined once. In these cases the examinations may have been performed out of the expansion phase of the jugular lymphatic sac. As for the fetus with uneventful outcome, the expansion phase might have been before 11 weeks of gestational age. Another explanation is that increased nuchal translucency in these fetuses is caused by another mechanism, especially in case of Turner’s syndrome. Turner’s syndrome is associated with a hypoplasia of the lymphatic system and an extensive nuchal edema, also referred to as cystic hygroma.19,20 We suggest that both a disturbed lymphangiogenesis caused by hyperplasia with enlargement of the jugular lymphatic sacs, or as a result of hypoplasia in the case of Turner syndrome, are able to cause nuchal edema. In case of hypoplasia, however, the jugular lymphatic sacs cannot be visualized by ultrasonography.
The pathophysiology of increased nuchal translucency has been disputed for the last 3 decades. Increased nuchal translucency is associated with a wide range of associated anomalies.3–5 A single origin is therefore not likely. We suggest that the majority of disorders are linked to a common developmental process, which can be delayed or disturbed at different times in development, but eventually leads to increased nuchal translucency. Lymphatic development seems to be a promising candidate for this process because it is able to explain regional and temporal characteristics of nuchal translucency.12 This study further substantiates this hypothesis by showing a temporal relationship between expansion of nuchal translucency and the jugular lymphatic sacs. Nevertheless, there remains a group of rare conditions associated with increased nuchal translucency, like hemodynamic alterations in case of first-trimester monochorionic twin fetuses, that are not likely to be correlated to lymphatic development and are probably caused by a different pathway.21
The disturbed lymphatic development can range from a physiological variation, in which the development is delayed but remains without consequences after completion, to a more severe disturbance. In a morphological study of the jugular lymphatic sacs in aneuploid human fetuses and mouse embryos with nuchal edema, we found an abnormal endothelial differentiation of the jugular lymphatic system.22 The abnormal endothelial development was associated with increased vascular permeability and diminished cell adhesion with the consequence of edema. Abnormal endothelial development might also account for the numerous cardiovascular defects in fetuses with increased nuchal translucency because pathological endothelial processes are said to play an ethological role in the cardiac malformations.23–26 This remains to be investigated.
The initiation of a disturbed lymphatic or blood vascular endothelial development can be either environmental or genetic. For example, hypoxic or nutritional insults or both during gestation can influence endothelial development and cause cardiac defects.27 A disturbed migration of neural crest cells is suggested as one of the causes for abnormal lymphatic and cardiovascular development in fetuses with nuchal edema. Neuronal and endothelial pathways have mutual influences, and this interaction was disturbed in the surrounding regions of jugular lymphatic sacs, aortic arch, and ductus venosus in trisomy 16 mouse embryos, which are an animal model for human trisomy 21.28 An abnormal innervation of the ductus venous and thickening of the endothelium might be attributed to the abnormal flow velocities in fetuses with increased nuchal translucency.28 In a morphological study of human fetuses with nuchal edema and an enlarged jugular lymphatic sac, Miyabara et al29 also suggested the involvement of neural crest cells in lymphatic and cardiovascular malformations. They emphasized that neural crest cells migrate in a hyaluronate-rich matrix and that altered neural crest cell migration can be accompanied by extracellular matrix alterations. This provides an interesting link with the extracellular matrix alterations observed in aneuploid fetuses with increased nuchal translucency.8,30
Aneuploid fetuses seem to form the severe end of the spectrum of anomalies of increased nuchal translucency. This is indicated by the fact that the jugular lymphatic sacs were larger and remained present longer in aneuploid fetuses. The predictive value of jugular lymphatic sac volume for aneuploidy was fair and does not seem to be useful for implementation in a screening model so far. Larger data sets might show better results, as our data set was moderate. On the other hand, the time lag between peak nuchal translucency thickness and jugular lymphatic sac volume requires different optimal times of measurement. Because the probability of aneuploidy was 30% when the jugular lymphatic sacs are detectable on ultrasonography, we suggest that visible jugular lymphatic sacs can serve as a soft marker for aneuploidy. The role for enlarged jugular lymphatic sacs as a sonographic marker for cardiac defects needs further investigation.
The finding that nuchal translucency expansion preceded jugular lymphatic sac enlargement differs from previous histological data of trisomy 16 mouse embryos with nuchal edema.13 In these embryos the appearance of distended jugular lymphatic sacs seemed to coincide with nuchal edema. However, different mouse embryos were compared at subsequent embryonic stages and were not in vivo longitudinally followed as in the present study. The difference can also be explained by the fact that the jugular lymphatic sac has to reach a certain size to become visible on ultrasonography, in contrast to nuchal translucency captured between other anatomical structures, which makes it more difficult to visualize the enlarged jugular lymphatic sac in an early stage. More important is the finding of the present study that the development of nuchal translucency and jugular lymphatic sacs with advancing gestation was significantly related.
In conclusion, this study shows a temporal relationship between distension of the jugular lymphatic system and enlargement of increased nuchal translucency. This finding implies that a disturbed lymphatic development is a common denominator in the spectrum of anomalies associated with nuchal translucency. Also, aneuploid fetuses seem to have a more severe disturbance of the lymph angiogenesis.
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