Malformation of cortical development with abnormal cortex: early ultrasound diagnosis between 14 and 24 weeks of gestation

To describe neurosonographic findings diagnostic or highly suggestive of the presence of malformations of cortical development involving the cortex that may be identified before 24 weeks of gestation.


INTRODUCTION
Cortical maturation and gyral formation follow an age-specific temporospatial schedule, with waves of neu-
roblast migration beginning at around the 7 th gestational week and probably lasting until adulthood 1,2 . A six-layered cortex results, with its deeper layers consisting of the first migrating neurons. Anatomical studies show that, while some medial hemispheric sulci are present as early as [16][17][18][19] weeks, those on the outer surface of the hemispheres appear later, starting to form between 20 and 24 weeks 3,4 , mainly as a result of glioblast migration 5 . Until this time, the brain's outer surface is relatively smooth.
Congenital disruption in the proliferation and migration processes results in malformations of cortical development (MCD). These malformations can involve the brain size, cortex, white matter and/or ventricular zone, as well as the sulcation and gyration patterns. The etiology of these disorders may be genetic [6][7][8] , infectious 9,10 or vascular 11,12 and they may result in profound neurological impairment, including intellectual disability, epilepsy and motor dysfunction 13 . The process of formation of a dysplastic cortex and its recognition, either by postmortem histological evaluation of fetuses 14,15 or by imaging, mostly magnetic resonance imaging (MRI) [16][17][18][19][20] , has been described previously. Most reports of prenatal ultrasound diagnosis of these conditions were limited, until recently, to isolated case reports or case series 21 describing hemimegalencephaly 22 , lissencephaly 23,24 or periventricular nodular heterotopias 25 . Our group has described the ultrasound diagnosis of various MCD in 23 fetuses at a mean gestational age of 26 weeks, with earlier mid-trimester diagnosis, before 24 weeks, being achieved mostly in cases of abnormal overdeveloped or underdeveloped sulcation and gyration 26 .
The purpose of the present study was to present our experience regarding early sonographic findings highly suggestive of the presence of MCD involving the cortex, detectable before 24 weeks of gestation.

METHODS
This was a retrospective cohort study of fetuses referred to the Fetal Neurology Clinic in a tertiary referral center in Israel between 2012 and 2019. Considered for inclusion in the analysis were fetuses referred due to a suspected brain anomaly and those identified with suspected MCD during routine brain imaging early in the mid trimester. Patients were studied following standard protocols from the time of diagnosis until delivery or termination of pregnancy (TOP). All patients were evaluated using a transabdominal approach, complemented by transvaginal ultrasound if the fetus was in vertex presentation, as described previously 27 .
Ultrasound examinations were performed according to the detailed neurosonographic examination guidelines provided by the International Society of Ultrasound in Obstetrics and Gynecology (ISUOG) 28 . All examinations were carried using a Voluson 730, E6, E8 or E10 ultrasound machine (GE Healthcare, Zipf, Austria), equipped with a 4-8-MHz and/or 9-MHz transabdominal transducer and a 5-9-MHz or 6-12-MHz transvaginal transducer.
For the purposes of this study, we retrieved from the clinic database patient data and images. Patterns of cortical development from 20 gestational weeks onwards were compared with the normal pattern described by Cohen-Sacher et al. 29 ; before 20 weeks we compared these patterns with those in normal fetuses of similar gestational age, whose data and images were retrieved from the database. During the period up to 20 weeks, the normal fetal brain lacks sulci and gyri, being completely flat ('lissencephalic'). Using a high-resolution transvaginal transducer, the cortical layer is visualized as a hypoechogenic thin line located between the hyperechogenic meninges and the echogenic subplate ( Figure 1). For inclusion in the study cohort, we identified patients with abnormal cortical patterns, including earlier-than-expected development of sulcation and gyration, hemispheric or focal cortical asymmetry with a non-regular focal cortical layer or absence of cortical continuation, or a hyperechogenic cortical rim 29,30 (Figure 2). Their stored images were reanalyzed for other abnormal brain findings and non-central nervous system (CNS) abnormalities.
For each patient, we also collected the following data: the reason for referral and the gestational age at referral; the gestational age at initial suspicion or diagnosis; patient, family and obstetric history; fetal karyotype and chromosomal microarray and exomic sequencing results, when available; details of subsequent sonographic evaluations; and final diagnosis. If the pregnancy ended in live birth, the gestational age at delivery, mode of delivery, Apgar scores and newborn birth weight were recorded. Additionally, parents were contacted to obtain postnatal brain ultrasound and MRI results, as well as neurodevelopmental pediatric evaluations, if available. In cases in which the parents opted for TOP and a postmortem examination was performed, the pathological report was retrieved and reviewed. Pathology evaluations were performed at the Pathology Department of Sapir Medical Center.
Institutional review board approval for this retrospective study was obtained (0704-21-TLV). TOP was performed in compliance with Israeli law and postmortem examinations were performed after obtaining signed informed consent.
On dedicated neurosonographic examination, the premature appearance of abnormal sulci and gyri was observed in 11 fetuses. This pattern was present in
In two fetuses from the same family, the cortical layer was hyperechogenic and a Walker-Warburg pathogenic POMT1 mutation was identified (Figure 3). In another fetus, this finding was less prominent but suggestive of the same disease. This characteristic pattern has been reported previously 31 .
Multiple CNS anomalies were found in 19 of the 20 fetuses (Table 1); in the patient with thanatophoric dysplasia (Case 10) we observed only the characteristic early abnormal development of parietal and temporal sulci and gyri (Figure 4), with a large head circumference and frontal bossing. Interestingly, we were able to diagnose callosal anomalies in four of the 11 fetuses diagnosed < 20 weeks ( Figure 5).
Hemimegalencephaly was suspected in four patients between 16.6 and 23.6 weeks due to the marked asymmetry of the hemispheres, with asymmetric ventricles. These patients presented different brain anomalies, including irregular ventricular walls and cortex, and midline anomalies The karyotype was normal in all four of these patients.

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Krajden Haratz et al. Associated non-CNS anomalies were found in six cases, five of these being in the group diagnosed < 20 weeks (Table 1).
Following counseling, in 18 cases the parents opted for TOP and two were liveborn. A postmortem examination was performed in only three cases; in the other 15 cases, the TOP was performed by dilation and evacuation or the parents declined autopsy. Postmortem and genetic findings are presented in Table 1.

DISCUSSION
Kostovic et al. 32 , using histological techniques and in-vitro fetal MRI specimens, described as early as 15 weeks of gestation the different layers of the fetal brain as being composed of five or six different zones: ventricular zone, periventricular zone, subventricular zone and intermediate zone, subplate zone and cortical plate. Although, based on embryologic and pathological studies, it is clear that most neuroblast migration ends around 24 gestational weeks, current understanding is that the technical capabilities of in-vivo MRI and ultrasound are such that the diagnosis of MCD is not yet feasible 33 . However, our group and others have demonstrated that certain MCD may be diagnosed before 24 weeks 8,16,26,[34][35][36] .
In this retrospective study of fetuses examined using high-resolution transducers in a research setting, we used a pattern recognition model, looking for specific ultrasound signs involving the cortical layer, some of which have been described previously in the late stages of pregnancy 37 .
We demonstrated that, at least in some cases, probably those at the more severe end of the spectrum, it might be possible to visualize certain morphological changes highly suspicious for the presence of MCD and to reach a diagnosis between 14 and 20 gestational weeks. It remains to be demonstrated whether such diagnoses can be made in a clinical setting.
Abnormal sulcation, either delayed or overdeveloped, was the main finding in 11 fetuses in our cohort (Table 1). Early precocious sulci and gyri development was first reported in four stillbirths with agenesis of the corpus callosum 38 ; in two, the examination revealed an abnormal cortex and in both cases there was a history of CNS anomalies in previous pregnancies. Hevner 39 , in 2005, reported abnormal brain gyral and sulcal patterns with associated MCD in fetuses with thanatophoric dysplasia. Later, our group described, using ultrasound, similar findings in fetuses with agenesis of the corpus callosum 40 . Abnormal sulcation has also been observed using MRI in fetuses > 20 weeks 16 . Interestingly, this abnormal pattern is apparently not necessarily associated with the presence of cortical dysplasia 41 .
Fetuses with hemispheric or sulcation asymmetry probably represent incipient cases of hemimegalencephaly or tubulinopathy. In these fetuses, as in those with macrocephaly, it is expected that the abnormal head circumference is amenable to diagnosis during the late stages of the second trimester or in the third trimester 21,26 . In hemimegalencephaly, overactivation of the same pathways described in macrocephaly can occur as focal somatic mosaicism, with normal tau (a microtubule-associated protein) being degraded by a mutant AKT3 or AKT1 gene 42 .
Our study has demonstrated that, in some cases, particularly those at the more severe end of the spectrum, the excellent image resolution and diagnostic capabilities of dedicated neurosonography allow diagnosis of MCD as early as 14 weeks, a time at which other imaging techniques cannot achieve this diagnosis. While in many clinical settings it is not possible to perform such a detailed neurosonographic examination at this early stage, our findings support referral to a center with expertise in this field when a CNS anomaly is suspected.