Depth of brain fissures in normal fetuses by prenatal ultrasound between 19-30 weeks of gestation Authors: Alonso I, Borenstein M, Grant

G, Narbona I, Azumendi G
Centro Gutenberg, Prenatal Diagnosis and Ultrasound Unit Mlaga, Spain

Correspondance: Dr. Marisa Borenstein Centro Gutenberg, Unidad de Ecografia Calle Gutenberg 5, Mlaga, Mlaga, 29016 Spain Telephone: +34 952 220 944 e-mail: maruboren@gmail.com Fax: +34 952 607 789

Keywords: fetal brain, fissures, migration disorders, neurosonography

This article has been accepted for publication in Ultrasound in Obstetrics & Gynecology and is cu rrently being edited and typeset. Readers should note that this article has been fully refereed, but has not been through the technical editing, copy-editing and proof correction process. WileyBlackwell and the International Society of Ultrasound in Obstetrics and Gynecology cannot be held responsible for errors or consequences arising from the use of information contained in this article; nor do the views and opinions expressed necessarily reflect those of Wiley -Blackwell or the International Society of Ultrasound in Obstetrics and Gynecology

Abstract Objective : To establish a normal range of the depth of the insula (I), Sylvian (SF), parieto -occipital (POF) and calcarine (CF) fissures by prenatal ultrasound between 1930 weeks of gestation. Material and methods: We prospectively selected 15 consecutive normal fetuses per gestational week between 19+0 and 30+6 weeks. We measured the depth of the insula and SF in a standard transventricular axial plane of the fetal head ; the POF, in a plane above and parallel to the previous; the CF in a coronal view of the posterior fossa. All measurements were done transabdominally and during the routine examination. Reproducibility was assessed in 30 cases for each of the parameters in stored images with electronic callipers by two operators. Results: The depth of the four structures increased with gestation. The insula and SF could be seen in all cases from 19 weeks, but the POF and CF fissures could be identified in 93.3% and 6.6% at 19 weeks respectively. After 24 weeks, the calcarine fissure could be seen in all examinations. Intra and inter observer reproducibility analysis showed good results. Conclusions: The examination of these structures is feasible during prenatal ultrasound using standard views of the fetal head, and since the normal ranges increased with gestation, they could be used to estimate the brain development. It is likely that this assessment might be incorporated in the neurosonogram to assess fetuses at risk of maturation disorders or as a complement of other standard evaluations.

Introduction The assessment of the developing fetal brain is still challenging for experienced sonographers. The central nervous system (CNS) is not fully mature until childhood, and at least part of the dynamic process that takes place in the fetal brain can be appreciated by fetal imaging. The increasing use of prenatal transabdominal and transvaginal ultrasound as well as fetal magnetic resonance (MR) has dramatically improved the understanding of the developing fetal brain1-13. Migration defects have diverse sonographic expressions that are usually not visible until the third trimester of pregnancy, unless subtle brain landmarks are looked for. Severa l post-mortem studies confirmed the difference in timing of appearance of the cerebral fissures, sulci and gyri13- 17. The incorporation of MR into autopsy studies facilitated the assessment of the fetal brain and enabled the description and characterization of many fetal intracranial structures16. Several prenatal bidimensional (2D) ultrasound studies carried out at 15 to 40 weeks of gestation indicated that the Sylvian fissure (SF) becomes sonographically evident after 17 to 18 weeks and its shape gradually changes as gestation advances1- 6. Quarello et al7 recently described a score to assess the development of the SF in 200 fetuses at 22-32 weeks of gestation. Despite the increasing number of studies about the fetal CNS development, no objective standardization has been published to assess the fetal brain maturation by routine 2D ultrasound. Three-dimensional (3D) ultrasound 5,8- 10 studies described changes in the sonographic appearance but only Mittal et al10 reported a normal range of the SF in 202 fetuses between 12 and 41 weeks of gestation. Few studies1,2,6 reported that the parieto-occipital (POF) and calcarine (CF) fissures are visible with ultrasound after 18 weeks of gestation with 94% of visualization with transvaginal ultrasound at 26 weeks 1. The aim of this study was to provide an objective method to improve and simplify the early detection of fetal brain migration disorders. We constructed normal ranges of the insula (I), SF, POF and CF between 19 and 30+6 weeks of gestation by 2D transabdominal ultrasound examination using standard views of the fetal head. Methods We prospectively examined all patients who attended our Unit between 19-30 weeks of gestation who had signed a written consent form. We included singleton pregnancies with normally grown fetuses and certain dates, without ultrasound evidence of structural defects. Detailed maternal history was taken and recorded in our database before the scan. We have only included in this study the cases in which no obvious risk factors for abnormal development of the brain were suspected from the maternal history. Standard biometry was recorded and in addition we evaluated four cerebral parameters during the 20 minutes allocated for the second or third trimester scan. In all cases the measurements of the insula, SF, POF and CF were done with electronic callipers on the ultrasound machine and subsequently stored with and without measurements in the hard disk of the ultrasound machine for further review and reproducibility analysis. All the examinations were performed transabdominally (RAB 48-D probe, Voluson E8 and Voluson V730 Expert, GE Healthcare Ultrasound). We prospectively assessed 200 patients between 19 and 30 weeks of gestation during the study period. We selected 15 consecutive cases per ge stational week which fulfilled the inclusion criteria. For standardization purposes we did all the measurements in the brain hemisphere that was more distant to the probe, regardless of the fetal side, to prevent shadows from the fetal skull bones. We eva luated the depth of the Insula and SF, POF and CF. Different steps were followed: -Assessment of the insula and Sylvian fissure (Figure 1):

1) Obtain a standard transventricular axial plane of the fetal head, as described by the ISUOG guidelines 18 to measure the biparietal diameter (BPD) and occipito-frontal diameter in which the cavum septi pellucidi, the atrium and the posterior horns of the lateral ventricles and choroid plexus are identified . The midline of the brain should be equidistant to both sides of the calvarium to ensure that the plane is not oblique. 2) The depth of the insula was measured from the midline, with a perpendicular line towards the upper border of the insular cortex up to the point of maximum prominence. 3) The depth of the SF was measured drawing a line in continuation with the insular line (perpendicular to the midline), from the insular cortex towards the inner table of the parietal bone. -Assessment of the parieto-occipital fissure (Figure 2) 1) Obtain a standard plane for the measurement of the BPD, as described above 18. 2) Slide the probe cranially to obtain a parallel plane to the BPD plane until the full depth of the fissure is visualized. The POF has a triangular shape with the apex pointing away from the midline. 3) The depth was measured from the midline, with a perpendicular line extending towards the apex of the fissure. -Assessment of calcarine fissure (Figure 3): 1) Obtain an axial transcerebellar plane of the fetal head, as used for the measurement of the tran scerebellar diameter in which the cavum septi pellucidi, anterior horns, thalami, cerebellum and cisterna magna are identified 18. 2) Turn the probe 90 degrees to obtain a coronal view of the posterior fossa until the calcarine fissure is completely visualized. 3) The depth of the fissure is measured drawing a line perpendicular to the tentorium or to an imaginary line between the most central portions of the adjacent gyri. In 30 cases paired measurement were carried out by one operator to determine the intra-observer reproducibility and by two operators to determine the inter-observer reproducibility. For this purpose, we used pictures that had been stored in the ultrasound machine for this purpose and performed the measurements with electronic callipers as in the first assessment; we did not rescan the patients. In addition, we examined if there was any different in the depth of the insula and SF, POF and CF according to the fetal gender. Statistical analysis Regression analysis was used to determine the significance of the association between the depth of the insula and SF, POF and CF and weeks of gestation respectively. In order to assess if there was a gender difference, we did linear regression analysis for each of the parameters separately according to the gender, and then calculated the delta values for each of them; we then confirmed the normal distribution of values and applied the T-test for independent samples to compare the means of the delta values from female and male fetuses measurements. The Bland -Altman19 analysis was used to determine the agreement and bias between paired measurements by one operator and by two operators respectively. The data were analyzed using the statistical software SPSS 12.0 (Chicago, Illinois, USA). A p value of less than 0.05 was considered significant. Results We examined 200 patients, but only 180 fetuses were included in the analysis. In 9/200 (4.5%) cases the images were not satisfactory to assess at least one of the structures and 11/200 (5.5%) cases were excluded for fetal abnormalities or discordant biometry.

In the 180 included fetuses, the indications of the scans in our Unit were: routine anomaly scan (60 cases), maternal request of 3D examination (90 cases), routine third trimester growth scan (30 cases). The fetal gender was female in 88 (49%) cases and male in 92 (51%) cases. Insular depth: The insular depth increased significantly with gestation with a mean of 14.4mm at 19.0 weeks to a mean of 24.5mm at 30.6 weeks (insular depth = -1.696 + 0.848 x gestational weeks; r=0.965, p<0.0001; SD=0.804) (Figure 4). Visualization of the insula was possible in all cases. Sylvian fissure: The depth of the Sylvian fissure increased significantly with gestation from a mean of 5.8mm at 19.0 weeks to 14.5mm at 30.6 weeks (SF= -8.080 + 0.732 x gestational weeks; r=0.911, p<0.0001; SD=1.152) (Figure 4). Visualization of the SF was possible in all cases. Parieto - occipital fissure: The depth of the POF increased significantly with gestation from a mean of 0.9 mm at 19 weeks to a mean of 11.4mm at 30.6 weeks (PO= -15.892 + 0.883 x gestational weeks; r=0.914, p<0.0001; SD=1.351) (Figure 5). Visualization of the POF was possible in 93.3% of cases at 19 weeks of gestation and in all cases after 20 weeks (Table 1). Calcarine fissure: The depth of the calcarine fissure increased significantly with gestation from a mean of 1.6mm at 20 weeks to a mean of 12.2mm at 30.6 weeks (-17.724+ 0.969 x gestational weeks; r=0.894, p<0.0001; SD=1.444) (Figure 5). Visualization of the CF according to gestation is shown in Table 1. The reproducibility analysis for intra-observer and inter-observer agreement on the same stored images is shown in Figures 6 and 7 respectively and Table 2. The results show that there is no significant difference in delta values of the insula and the CF between female and male fetuses. In contrast, the differences in the SF and POF are significant; however they are very small and probably clinically not relevant. The results of the comparison of measurements in female and male fetuses, expressed as difference of means in the delta values, are shown in table 3. As expressed in table 3, all measurements are smaller in female fetuses, regardless the statistical significance of such difference. Discussion The findings of our study show that the insula and the Sylvian, parieto-occipital and calcarine fissures increase with gestation between 19 and 30 weeks of gestation. Inter and intra observer reproducibility analysis confirmed that there is a good agreement in the measurement of the structures assessing correlation on stored images and calliper placement. Interestingly, there was a gender difference in the measurements of the Sylvian and parieto-occipital fissures; however it was small and may not be clinically relevant. Our results show that the size of all four structures is smaller in female fetuses compared to male fetuses. Kivilevitch et al recently reported that brain asymmetry can be seen on prenatal ultrasound as a normal phenomena during the brain development and they report that is more common in the male fetus. This asymmetry seems to be more pronounced towards the left side of the brain 20.

These normal ranges were built based on measurements performed in standard ultrasound planes which are employed for routine biometry, facilitating the incorporation into daily practice for sonographers who do not have special training in neurosonography or transvaginal scanning. Furthermore, the landmarks for the measurements are easy to identify in a norma l brain and in contrast to the 3D study by Mittal et al10, the assessment is feasible in 2D ultrasound without increasing the time spent for the scan or the need for special ultrasound equipment. It has been reported that the shape of the fissures change throughout gestation 7 in addition to the increase in depth. The combination of both, the shape and the depth of brain fissures may improve the surveillance of the fetal brain maturation in normal and abnormal cases. According to our data, the calcarine fissure was not visible in all cases until 24 weeks, and this finding is in concordance with other prenatal ultrasound studies 1. However, the insula and Sylvian fissure should be always visible from 19 weeks of gestation and POF from 20 weeks onwards when performing transabdominal assessment. The limitation of this study is that we did not have diagnostic images such as MR or postnatal scans to confirm the prenatal normal evaluation. We could confirm normality in 90% of the newborns according to the pediatricians evaluation before discharge. We opted for the transabdominal approach since it is the method that is mainly used to carry out the scans in the second and third trimester of pregnancy. With this approach it was extremely difficult to visualize clearly all the fissures after 30 -31 weeks of gestation. The transvaginal approach is usually employed in the suspicion of brain defects provided the fetus is in a cephalic presentation, to improve the visualization of the anatomy. This is not the standard procedure in routine ultrasound probably because it is time -consuming and not widely available in terms of technology and training. Another limitation of the present study and of ultrasound as a screening method, is that mild or localized forms of lissencephaly might not be detected with this approach if no other brain abnormality is suspected. Malformations of cortical development are usually diagnosed or suspected in late second or third trimester of pregnancy by ultrasound and MR imaging 21,22, although the abnormal neuronal migration occurs during the third or fourth month of gestation 23. This defect carries a poor prognosis for the newborn, with mental retardation and a high recurrence rate when it is linked to genetic defects 21. We hope that the description of the normal ranges of the insula, and Sylvian, parieto -occipital and calcarine fissures, which are usually abnormal in migration disorders, will improve the timing of diagnosis and therefore the management and understanding of such pathologies. The results of this study need to be confirmed with data reporting cases of abnormal brain development of various etiologies. The finding of abnormal development of the brain fissures should alert the sonographers about a suspicion of migration abnormalities and necessary further steps should be carried out, including transvaginal assessment, referral to specialist in fetal neurosonograhy, MR assessment and/or fetal karyotyping. These second line assessments will improve the management and counselling. It is possible that the nomograms we present in this study may prove helpful to assist in early detection of altered cortical development. Our reference ranges might be employed in the future by experts who carry out neurosonography assessment, and in addition these parameters might prove to be useful as gestational age and brain maturation estimators. We understand these results should be interpreted with care until larger studies became available, but in the meantime sonographers could start their tra ining in such assessment.

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Figure legends 1. Ultrasound picture of an axial view of the fetal head at 24 weeks of gestation demonstrating the measurements of the depth of the insula and Sylvian fissure, including all the necessary landmarks to perform the assessment. Note that it is the standard plane for the measurement of the biparietal diameter. 2. Ultrasound picture of an axial view of the fetal head at 26 weeks of gestation, demonstrating measurement of the parieto-occipital fissure. The plane is at a level above the one used for the measurement of the biparietal diameter. (POF= parietooccipital fissure) 3. Ultrasound picture of the coronal view of the fetal head at 26 weeks of gestation at the level of the posterior fossa, demonstrating the measurement of the calcarine fissure. 4. Depth of the insula (right) and Sylvian fissure (left) in 180 normal fetuses plotted on the normal range (mean, 95Th and 5Th percentiles) with gestational age in weeks respectively. 5. Depth of the parieto-occipital fissure (right) and calcarine fissure (left) in 180 normal fetuses plotted on the normal range (mean, 95 Th and 5Th percentiles) with gestational age in weeks respectively. 6. Mean difference and 95% limits of agreement between paired measurements of the depth of the Insula (A) and Sylvian (B), parieto-occipital (C) and calcarine (D) fissures by the same operator on stored images. 7. Mean difference and 95% limits of agreement between paired mea surements the depth of the insula (A) and Sylvian (B), parieto-occipital (C) and calcarine (D) fissures by two different operators on the same stored images.

Table 1. Success in visualization of the parieto-occipital (POF) and calcarine (CF) fissures
according to gestation. Gestation (weeks) 19 20 21 22 23 24-30 POF n;% 14/15 (93.3) 15/15 (100) 15/15 (100) 15/15 (100) 15/15 (100) 15/15 (100) CF n;% 1/15 (6.6) 11/15 (73.3) 11/15 (73.3) 13/15 (86.6) 14/15 (93.3) 15/15 (100)

Table 2. Mean difference and 95% limits of agreement between paired measurements of
insula, and Sylvian, parieto -occipital and calcarine fissures by the same operator (intraobserver) and by two different operators (inter-observer). The analysis was performed on the same stored images. Structure Insula Sylvian fissure Parieto -occipital fissure Calcarine fissure Intra-observer (mean and 95 CI)
th

-0.15[-1.44 (-1.651 to -1.234) to 1.14 (0.932 to 1.349)] -0.01[-1.07 (-1.244 to 0.901) to 1.05 (0.879 to 1.222)] 0.02 [-1.81 (-2.116 to -1.520) to 1.87 (1.578 to 2.174] 0.16 [-1.17 (-1.395 to -0.960) to 1.51 (1.290 to 1.733)]

Inter-observer (mean and 95 CI)

th

0.27[-1.67 (-1.983 to -1.357) to 2.21 (1.901 to 2.527)] -0.42[-1.92 (-2.168 to -1.683) to 1.08 (0.837 to 1.322)] -0.33[-2.27 (-2.591 to -1.965) to 1.60 (1.290 to 1.917)] -0.31[-1.75 (-1.988 to -1.523) to 1.13 (0.899 to 1.365)]

Table 3. Comparison of delta values of the insula and Sylvian, parieto -occipital and calcarine
fissures according to the fetal gender (female vs male fetuses).

Structure Insula Sylvian fissure Pariet -occipital fissure o Calcarine fissure

Mean difference and 95%CI - 0.13 (-0.36 to 0.10) - 0.60 (-0.93 to -0.27) - 0.52 (-0.91 to -0.13) - 0-28 (-0.72 to 0.15)

P 0.281 >0.001 0.008 0.196

1. Ultrasound picture of an axial view of the fetal head at 24 weeks of gestation demonstrating the measurements of the depth of the insula and Sylvian fissure, including all the necessary landmarks to perform the assessment. Note that it is the standard plane for the measurement of the biparietal diameter.

2. Ultrasound picture of an axial view of the fetal head at 26 weeks of gestation, demonstrating measurement of the parieto-occipital fissure. The plane is at a level above the one used for the measurement of the biparietal diameter. (POF= parietooccipital fissure)

3. Ultrasound picture of the coronal view of the fetal head at 26 weeks of gestation at the level of the posterior fossa, demonstrating the measurement of the calcarine fissure.

4. Depth of the insula (right) and Sylvian fissure (left) in 180 normal fetuses plotted on the normal range (mean, 95Th and 5Th percentiles) with gestational age in weeks respectively.

5. Depth of the parieto-occipital fissure (right) and calcarine fissure (left) in 180 normal fetuses plotted on the normal range (mean, 95 Th and 5Th percentiles) with gestational age in weeks respectively.

6. Mean difference and 95% limits of agreement between paired measurements of the depth of the Insula (A) and Sylvian (B), parieto-occipital (C) and calcarine (D) fissures by the same operator on stored images.

7. Mean difference and 95% limits of agreement between paired measurements the depth of the insula (A) and Sylvian (B), parieto-occipital (C) and calcarine (D) fissures by two different operators on the same stored images.