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Chromosomal Microarray Analysis in Pregnancies With Corpus Callosum or Posterior Fossa Anomalies

Objective We investigated the detection rate of clinically significant chromosomal microarray analysis (CMA) results in pregnancies with sonographic diagnosis of fetal corpus callosum anomalies (CCA) or posterior fossa anomalies (PFA). Methods All CMA tests in pregnancies with CCA or PFA performed between January 2015 and June 2020 were retrospectively evaluated from the Israeli Ministry of Health database. The rate of CMA with clinically significant (pathogenic or likely pathogenic) findings was calculated and compared to a local Israeli cohort of 5,541 pregnancies with normal ultrasound. Results One hundred eighty-two pregnancies were enrolled: 102 cases with CCA and 89 with PFA (9 cases had both). Clinically significant CMA results were found in 7/102 of CCA (6.9%) and in 7/89 of PFA (7.9%) cases. The CMA detection rate in pregnancies with isolated CCA (2/57, 3.5%) or PFA (2/50, 4.0%) was lower than in nonisolated cases, including additional CNS and/or extra-CNS sonographic anomalies (CCA-5/45, 11.1%; PFA-5/39, 12.8%), but this was not statistically significant. However, the rate among pregnancies that had extra-CNS anomalies, with or without additional CNS involvement (CCA-5/24, 20.8%; PFA-5/29, 17.2%), was significantly higher compared to all other cases (p = 0.0075 for CCA; p = 0.035 for PFA). Risk of CMA with clinically significant results for all and nonisolated CCA or PFA pregnancies was higher compared to the background risk reported in the control cohort (p < 0.001), but was not significant for isolated cases. Conclusions Our findings suggest that CMA testing is beneficial for the genetic workup of pregnancies with CCA or PFA, and is probably most informative when additional extra-CNS anomalies are observed.


Results
One hundred eighty-two pregnancies were enrolled: 102 cases with CCA and 89 with PFA (9 cases had both). Clinically significant CMA results were found in 7/102 of CCA (6.9%) and in 7/89 of PFA (7.9%) cases. The CMA detection rate in pregnancies with isolated CCA (2/57, 3.5%) or PFA (2/50, 4.0%) was lower than in nonisolated cases, including additional CNS and/or extra-CNS sonographic anomalies (CCA-5/45, 11.1%; PFA-5/39, 12.8%), but this was not statistically significant. However, the rate among pregnancies that had extra-CNS anomalies, with or without additional CNS involvement (CCA-5/24, 20.8%; PFA-5/29, 17.2%), was significantly higher compared to all other cases (p = 0.0075 for CCA; p = 0.035 for PFA). Risk of CMA with clinically significant results for all and nonisolated CCA or PFA pregnancies was higher compared to the background risk reported in the control cohort (p < 0.001), but was not significant for isolated cases.

Conclusions
Our findings suggest that CMA testing is beneficial for the genetic workup of pregnancies with CCA or PFA, and is probably most informative when additional extra-CNS anomalies are observed.
Chromosomal microarray analysis (CMA), aimed at detecting microscopic and submicroscopic chromosomal aberrations, is an important tool for the genetic analysis of fetuses with abnormal sonographic findings. 1-3 CMA is considered a primary test for women undergoing invasive prenatal diagnosis due to one or more fetal structural anomalies. 4,5 In the presence of a normal karyotype, CMA can detect clinically relevant deletions or duplications in 6% of pregnancies with structural anomaly in ultrasound (US). 3 Fetal CNS anomalies detected by prenatal US are an important referral indication for genetic counseling. In the current study, we focused on 2 groups of CNS anomalies: corpus callosum anomalies (CCA) and posterior fossa anomalies (PFA). In addition to various environmental factors, genetic causes such as copy number variants (CNVs) or point mutations in multiple genes have been reported to play a role in the etiology of these anomalies. [6][7][8][9][10] The corpus callosum (CC) is the largest white matter tract in the human brain, connecting the 2 cerebral hemispheres. 11 Generally, CCA include complete or partial agenesis, hypoplasia, thickening, irregular shape, and other findings. [8][9][10]12 Each may be isolated or combined with additional CNS and extra-CNS findings, leading to a wide range of clinical manifestations. 8,13,14 A population-based study in California found that the combined prevalence of CC agenesis and hypoplasia was 1.8 per 10,000 live births. 15 However, in developmentally disabled individuals, the prevalence of CC agenesis, determined by CT, reaches to 2.3%. 16 PFA encompasses a heterogeneous group of morphologically defined cystic and noncystic conditions. 6,7,[17][18][19] Among them are cerebellar hypoplasia, vermian hypoplasia or agenesis, Dandy-Walker malformation, Blake pouch cyst, arachnoid cyst, and mega cisterna magna. 7,17,19 The prevalence, involved pathophysiology, and future outcomes are variable, depending on the specific anomaly type. 20,21 For example, the estimated birth prevalence of cerebellar hypoplasia was 1.3 per 100,000 persons. 22 For Dandy-Walker Malformation, the overall and live birth prevalence were 6.79 and 2.74 per 100,000 births, respectively. 23 There are relatively limited data regarding the yield of CMA in prenatal diagnosis of CCA [24][25][26][27][28] and PFA, 26,[28][29][30][31][32] which vary between studies and are influenced by several factors, such as the specific anomaly type and involvement of additional CNS or extra-CNS anomalies.
The Israeli Ministry of Health (MOH) cohort of CMA performed due to fetal sonographic anomalies was previously described and used to evaluate the risk of clinically significant CMA results in various types of anomalies. [33][34][35][36][37][38][39][40][41][42][43][44][45][46] In this study, we investigated the frequency of CMA with clinically significant findings in pregnancies with CCA or PFA and compared it to pregnancies with normal US.

Data Acquisition
A retrospective search was conducted in a computerized database of all CMA tests reported to the Israeli MOH between January 2015 and June 2020 (inclusive). Generally, every pregnant woman in Israel is recommended to undergo 2 routine sonographic anatomic surveys for the fetus (at 14-16 and at 20-23 weeks of gestation). If abnormal US findings are observed, the woman is referred to genetic counseling. For major anomalies (including CCA and PFA), invasive prenatal testing (usually amniocentesis) for CMA is advised, which is reimbursed by the MOH. Testing reports are transferred to the department of community genetics at the MOH. Data are recoded in an electronic database, including information about the indication for testing, summary of genetic counseling, and CMA results.
In the present study, we enrolled only CMA performed for pregnancies with fetal diagnosis of CCA or PFA detected by prenatal US (few cases had both CCA and PFA). CCA included hypoplastic (thin) CC, short or short and thick CC, complete or partial agenesis of the CC, large or hyperplastic CC, and other or unspecified CCA. PFA included cerebellar and vermian anomalies (such as agenesis, hypoplasia, and dysplasia), Dandy-Walker malformation, Blake pouch cyst, mega cisterna magna, arachnoid cyst, and other or unspecified PFA.
All cases were retrieved and classified as isolated or nonisolated. The nonisolated cases were accompanied by other CNS and/or extra-CNS sonographic anomalies (including structural abnormalities, increased nuchal translucency, intrauterine growth restriction, oligohydramnios, and polyhydramnios). We also divided cases according to the involvement of additional extra-CNS anomalies (with or without other CNS anomalies), to compare this group to all other cases (isolated or with additional CNS anomalies only).
In fetuses with CCA, coexisting brain midline findings (e.g., in the cavum spectrum pellucidum) and colpocephaly were not classified as an additional anomaly because they probably Glossary CC = corpus callosum; CCA = corpus callosum anomalies; CI = confidence interval; CMA = chromosomal microarray analysis; CNV = copy number variant; LCR = low copy repeat; MOH = Ministry of Health; NIPS = noninvasive prenatal screening; PFA = posterior fossa anomalies; US = ultrasound; VUS = variant of unknown significance. constitute a part of CCA and not a separate brain anomaly. 24,47 The presence of soft markers alone (e.g., choroid plexus cyst, echogenic cardiac focus, short femur, single umbilical artery, or mild dilatation of the renal pelvis) was not considered as additional CNS or extra-CNS anomalies. We excluded pregnancies in which indications for CMA testing were missing or unclear.
Following the American College of Medical Genetics and Genomics standards and guidelines for the interpretation and reporting of constitutional CNVs, 48 CMA findings were classified as pathogenic, likely pathogenic, variants of unknown significance (VUSs), likely benign and benign. Categorization was based on the original laboratory reports, which were reviewed according to updated knowledge in medical literature and the author's experience. Of note, based on the guidelines of the Israeli Medical Genetic Association in the prenatal setting, VUS deletions sized less than 1 Mb and duplications sized less than 2 Mb are considered below the obligatory report threshold.
We defined pathogenic and likely pathogenic findings (including both gross chromosomal aberrations and submicroscopic CNVs) as clinically significant. We excluded benign and likely benign CNVs, among them CNVs with high frequency and low penetrance, not expected to be relevant for the sonographic phenotype.
Karyotype-detectable alterations were defined as at least 10 Mb in size. Noninvasive prenatal screening (NIPS)-detectable CNVs refer to trisomies 13, 18, 21, XXX, XXY, XYY, and monosomy X. 49 Comparison to Control Cohort The overall frequency of clinically significant CMA results in pregnancies with CCA or PFA was compared to a cohort of 5,541 pregnancies with normal US from a single, large medical center in Israel. 49 In this cohort, 78 clinically significant CMA results (1.4%) were found. 49 A similar analysis was conducted on previous studies based on the Israeli MOH CMA database, [33][34][35][36][37][38][39][40][41][42][43][44][45][46] aimed to assess the risk of clinically significant CMA results in various US anomalies, compared to background risk.
We further performed an additional analysis using a subgroup of 2,752 low-risk pregnancies from this cohort (women younger than 35 years with normal serum screening, for which CMA was performed with no medical indication), with 21 clinically significant CMA findings (0.76%). 49 Statistical Analysis Continuous data are described with mean and SD. Categorical data are presented as numbers and proportions. To analyze differences in CMA detection rate between groups (isolated vs nonisolated cases, and according to involvement of extra-CNS anomalies), we employed the Fisher exact test, using IBM SPSS Statistics version 25 (IBM Corp, Armonk, NY).
In comparing results to pregnancies with normal US, effect estimates were calculated as relative risk with 95% confidence interval (CI). All statistical tests were 2 sided, and a p value <0.05 was considered statistically significant.

Standard Protocol Approvals, Registrations, and Patient Consents
The study was approved by the Israeli MOH Review Board Committee (Helsinki committee) for Human Subjects. Informed consent was not required due to the retrospective data acquisition.

Data Availability
The data set analyzed during the current study is available from the corresponding author on reasonable request.

Results
From January 2015 to June 2020 (inclusive), a total of 13,266 CMA tests were reported to the MOH due to abnormal US findings during pregnancy (excluding soft signs markers). Overall, 182 cases were included in the final analysis: 102 pregnancies with CCA and 89 with PFA (9 cases had both CCA and PFA). CMAs with clinically significant (pathogenic or likely pathogenic) results were found in 7/102 of CCA cases (6.9%) and similarly in 7/89 of PFA cases (7.9%) (tables 1 and 2). Information about these CMA findings and the main sonographic characteristics is presented in table 3. None of the genomic alterations recurred. Of interest, clinically significant findings were found in 2/9 (22.2%) of the cases with both CCA and PFA. The CMA detection rates by specific type of CCA and PFA are presented in tables 1 and 2.
CCA Among 102 pregnancies with CCA, 57 cases were isolated (55.9%), and 45 (44.1%) had additional sonographic anomalies (nonisolated). As presented in table 1, 21 of the nonisolated cases had additional CNS anomalies only (20.6% of all cases), and 24 had extra-CNS anomalies (23.5%), with or without additional CNS involvement. Mean maternal age was 30.4 (SD ± 4.6) years, and mean gestational age at amniocentesis was 27.4 (SD ± 5.3) weeks. CMA detected 7 cases with clinically significant results in the whole group (7/102, 6.9%) and a total of 9 pathogenic or likely pathogenic genomic alterations (in 2 cases, 2 findings were observed in each). Of them, only 4/9 (44.4%) could have been detected by standard karyotype, and none by NIPS. VUS was observed in a single case.
A higher rate of pregnancies with clinically significant CMA results was observed in nonisolated CCA cases (5/45, 11.1%) compared to the isolated CCA group (2/57, 3.5%), but this was not statistically significant (p = 0.236). There was no significant difference in CMA detection rate between cases with isolated CCA and cases with CCA and additional CNS anomalies only (0/21, 0%) (p = 1.0). However, the detection rate was significantly higher in pregnancies that had extra-CNS anomalies (with or without additional CNS involvement [5/24, 20.8%]) than in isolated cases (p = 0.022). This effect was even more robust (p = 0.0075) when the detection rate in the 24 CCA pregnancies that had extra-CNS anomalies was compared to all pregnancies without extra-CNS involvement (CCA with or without additional CNS anomalies; 2/78, 2.6%).
As shown in table 2, CMAs with clinically significant results were found in 7 pregnancies in the whole group (7.9%). Nine pathogenic or likely pathogenic genomic alterations were noticed (in 2 cases, 2 findings were reported). Three of them were karyotype detectable (33.3%), and only 1 (case 10) was NIPS detectable (11.1%). VUSs were found in 2 cases (2/89, 2.3%), 1 in addition to other pathogenic finding in the same fetus.
The rate of CMA with clinically significant results in pregnancies with nonisolated PFA (5/39, 12.8%) was higher than in pregnancies with isolated PFA (2/50, 4.0%), but was not statistically significant (p = 0.233). Comparing the CMA detection rate among isolated PFA to 2 subgroups of pregnancies with nonisolated PFA (additional CNS anomalies only [0/10, 0%] and extra-CNS anomalies with or without additional CNS involvement [5/29, 17.2%]), the difference was not significant (p = 1.0 and p = 0.093, respectively). However, the detection rate in the latter group of 29 PFA pregnancies that had extra-CNS anomalies was significantly higher compared to all pregnancies without extra-CNS anomalies (PFA with or without additional CNS involvement; 2/60, 3.3%) (p = 0.035).

Comparison of CMA Detection Rate to Pregnancies With Normal US (Relative Risk)
Among all and nonisolated CCA or PFA cases, the risk for CMA with clinically significant findings was higher than for pregnancies with normal US in the whole control cohort and in a subgroup of low-risk pregnancies (p < 0.001) (table 4). Similar results were noticed for CCA or PFA cases with extra-CNS anomalies.
The risks in isolated CCA or PFA cases, as well as of those without extra-CNS anomalies, were higher than in the whole control cohort (table 4), but were not statistically significant. However, compared with the low-risk pregnancy subgroup, the relative risks were significant (p < 0.05), except for CCA without extra-CNS anomalies.
As seen in table 4, the 95% CIs around the relative risk are wide. This is probably due to the small sizes of the CCA and PFA groups (compared to the much larger control cohort).
Therefore, these results and the estimated sizes of the relative risk should be interpreted with caution.

Diagnostic Landscape
As presented in table 3, interpretation of CMA results in many cases was rather straightforward. This refers to pregnancies with chromosomal aberrations such as 9p arm duplication (case 2), part of isochromosome 18 (6), Pallister-Killian syndrome (9), Down syndrome (10)   In case 13 (a female fetus with partial agenesis of the CC, cerebellar hypoplasia, polymicrogyria, choanal atresia, and frontal bossing), we detected a paternally inherited deletion in Xq13.1, encompassing the EFNB1 gene. Heterozygous mutations in EFNB1 cause craniofrontonasal syndrome (MIM #304110), which may include partial or complete agenesis of CC. e4-e6 This disorder manifests more severely in females than males e7,e8 (potentially explaining the self-reported healthy father), and the CNV was classified as likely pathogenic. The pregnancy was terminated, and therefore we cannot assess the presence of other postnatal characteristics of this disorder.
In other cases, the association of the reported CNV and the brain anomaly is less well established. In case 1, a de novo distal 16p11.2 deletion was detected in a fetus with CC   hypoplasia. This finding, classified as pathogenic, is mainly associated with neurodevelopmental phenotypes with incomplete penetrance, and according of our knowledge, CCA were not previously reported in affected individuals. e9,e10 On the other hand, this CNV has been associated (negative doseresponse) with intracranial volume and volume of basal ganglia structures. e11 A 1q21 distal microdeletion was observed in a fetus with PFA (probably Dandy-Walker malformation), persistent left superior vena cava, and right heart ventricular hypertrophy (case 11). The syndrome caused by the CNV has a wide range of manifestations, including cardiac and brain anomalies, e12 but PFA has not yet been described as part of it.
In some pregnancies, more than 1 finding was observed. In case 7, with Blake pouch cyst, we found a distal 6p25.3 deletion of approximately 4 Mb. The 6p terminal deletion syndrome is described in the literature, and PFA (in particular Dandy-Walker malformation) is one of its features, potentially due to deletion of the FOXC1 gene. e13-e15 Additional CNV noticed in this female fetus was a deletion in Xp22.33, which harbored the SHOX gene, located in a pseudoautosomal region. Although probably incidental and nonrelated to the US phenotype, we classified this CNV as pathogenic due to SHOX involvement in a known syndrome with a wide clinical spectrum. e16,e17 Three CNVs were classified as VUS, 2 of them involving duplication of the 22q11 region: partial proximal 22q11 duplication (low copy repeats (LCR)-B to LCR-D) in case 10 and more distal duplication (LCR-G to LCR-H) in case 12. These CNVs are associated with 2 different syndromes with incomplete penetrance and mainly neurodevelopmental or neuropsychiatric manifestations. e18,e19 Consistent with previous reports, e20,e21 and since the relevance of these CNVs (within their specific positions) to the prenatal US anomalies was unclear, they were classified as VUS (in case 10, the phenotype is probably better explained by a diagnosis of Down syndrome).
A male fetus (case 3) with partial agenesis of the CC had a 216 kb duplication in Xp22.2, including part (exons 4-10) of the MID1 gene, inherited from a self-reported healthy mother. Mutations in MID1 cause X-linked Opitz G/BBB syndrome (MIM #300000). e22 At least some of MID1 mutations are related to loss of function mechanisms, e23,e24 but duplications of exons 1 or 2 were reported as pathogenic. e25,e26 However, because the functional role of the duplication is not known, the fetus had no other features of this syndrome (except CCA), and familial segregation was not completed, we regarded this finding as VUS.

Discussion
We investigated the frequency of CMA with clinically significant results in fetal CCA or PFA, in a nationwide cohort of pregnant women who underwent invasive testing. The overall rate of CMA with clinically significant findings was 6.9% for pregnancies with CCA and 7.9% for pregnancies with PFA. Although the rate was higher in nonisolated than in isolated cases, it was not statistically significant. However, the detection rate among pregnancies that had extra-CNS sonographic anomalies (with or without other CNS involvement) was significantly elevated compared to all other cases (isolated or with additional CNS anomalies only). The risk of CMA detection rate in pregnancies with CCA or PFA (all, nonisolated and cases with extra-CNS anomalies) was significantly higher than in pregnancies with normal US from a control cohort and subgroup of low-risk pregnancies (p < 0.001). Results were not significant for isolated pregnancies, as well as for cases without extra-CNS anomalies, compared to the whole cohort. Only 40% and 6.67% of the clinically significant genomic alterations were karyotype and NIPS detectable, respectively. Taken together, our findings support the benefit of CMA in pregnancies with CCA or PFA, and it is probably most informative in cases with extra-CNS anomalies.
The contribution of chromosomal aberrations and CNVs to CCA has been studied mainly in postnatal settings. For example, a meta-analysis of 27 studies found chromosomal anomalies in 4.81% and 7.45% of complete and partial agenesis of the CC, respectively. 14 Among 149 patients with CCA and intellectual disability, 13% had at least 1 CNV that likely contributed to the phenotype. e27 In the prenatal setting, however, data are more limited, and CMA yield is variable. Microarray (performed pre-or postnatally) detected a causal chromosomal anomaly in 2 of 18 pregnancies (11.1%) with isolated complete agenesis of the CC on US examination. 24 In 16 fetuses with isolated agenesis of the CC, She et al. 25 found 2 cases with pathogenic CNV (12.5%). In additional study of 69 pregnancies with agenesis of the CC and normal karyotype (when performed), the diagnostic rate of microarray analysis was 5.8% (4.4% in isolated and 8.3% in cases with additional structural anomalies in other systems). 26 On the other hand, no pathogenic CNVs were found in 32 fetuses with CCA in 8 years observational study from a tertiary care university hospital. 28 Finally, a study of 65 fetuses with abnormalities of the CC identified pathogenic CNV in 3 cases (4.5%), whereas exome sequencing found a pathogenic single-nucleotide variant in 12 (18%). 27 The role of CMA in fetuses with PFA was also investigated previously with variable results, but the detection rate was generally higher than in our study. 26,[28][29][30][31][32] Among 77 pregnancies with PFA that underwent CMA, Zou et al. 30 found chromosomal aberrations (including pathogenic CNVs and VUSs) in 31.2% of all cases, and in 18.5% of fetuses with normal karyotypes. In another study of 144 pregnancies with PFA tested by microarrays, the detection rate of clinically significant genomic alterations was 14.6% (6.8% in isolated and 22.9% in cases with additional structural anomalies in other systems). 26 Lei et al. 29 found no chromosomal aneuploidies or pathogenic CNVs in 30 fetuses with isolated PFA or in 13 PFA cases accompanied by other CNS abnormalities. However, the rate of these findings in fetuses with PFA and extra-CNS structural abnormalities was 54.8% (17 out 31). Santirocco et al. 28 reported that among 31 fetuses with PFA, prevalence of pathogenic CNVs was 12.9% (11.8% in isolation and 15.4% in fetuses with complex abnormalities), a higher rate than for other types CNS anomalies that were investigated. Last, in fetuses with cerebellar vermis defects, CMA provided diagnosis in 10/ 43 cases (23.3%), significantly higher in fetuses with multiple malformations compared to isolated, whereas exome sequencing yielded diagnostic genetic variants in 8/19 (42.1%). 31 In general, involvement of additional CNS or extra-CNS anomalies probably increases the likelihood to detect genomic disorder in the affected fetus, compare to isolated CCA or PFA. The lack of significant differences in part of our analyses may stem from the limited number of cases. Our study encompassed a relatively large number of CMA tests from pregnancies with a rather rare condition of fetal CCA or PFA, collected from a nationwide database. However, it is likely that a larger sample size with increased power would have facilitated reaching more definitive conclusions, including narrower CIs and more accurate estimates of relative risk.
One should keep in mind that this study sample does not include all pregnancies with CCA or PFA in Israel during the investigated time period. Part of the pregnant woman do not undergo the recommended sonographic surveys, whereas others avoid invasive testing. Moreover, the rising use of NIPS at earlier stages of pregnancy and the recommendation to have first and second trimester Down syndrome screening, facilitate early detection of pregnancies at risk for chromosomal aneuploidies. These factors decrease the rate of abnormal pregnancies, which could be later sonographically diagnosed with CCA or PFA. It is also plausible that in some pregnancies with suspected diagnosis of common trisomies, early prenatal diagnosis by rapid tests (like quantitative fluorescent PCR) eliminated the need to further perform CMA. Last, when multiple anomalies are observed in the fetus in early sonographic evaluation, parents may decide to terminate the pregnancy without having an invasive prenatal testing.
The specific types of anomalies included in the CCA and PFA groups are heterogeneous, with a wide spectrum of severity. For example, the CCA group includes fetuses with complete agenesis of the CC, as well as fetuses with only short but complete CC or other unspecified CC anomaly. The definition of the short CC is also variable and considered by some authors as a CC length below the fifth percentile e28 . In some cases, the distinction between hypoplastic, short, and CC within the normal range is challenging, especially when fetal MRI is not performed. The heterogeneity of CCA and PFA types in our analysis may explain the difference in CMA detection rate compared to the studies discussed above.
Notably, CMA testing did not lead to molecular diagnosis in more than 90% of pregnancies, and the etiology remained unexplained. Further genetic evaluation, including fetal whole-exome sequencing, could assist in resolving an additional part of the undiagnosed cases. 27,31 Several additional limitations of the current study should be acknowledged. The clinical data are based on referral indications for invasive testing and CMA, and were collected retrospectively. Sonographic evaluations were taken by multiple gynecologists across Israel, using different equipment, which may lead to inter-rater variability. Follow-up data of US or fetal brain MRI from later stages of pregnancy were unavailable. All these may have influenced the accuracy of anomaly classification and categorization to isolated and nonisolated cases. For example, in some conditions, CCA and ventriculomegaly (mainly of a mild degree) may represent a single structural anomaly rather than 2 separate findings. We considered cases with this combination as nonisolated CCA, due to a lack of further follow-up imaging information and for the sake of simplicity, but in some cases, this classification is probably not accurate.
A further drawback is lack of data regarding the outcomes of the pregnancies, including termination or postnatal developmental outcomes and imaging follow-up (when pregnancies were continued). Other limitation is primarily technical, due to the fact that CMA tests were conducted and interpreted by several different laboratories rather than a single one. To overcome this, we revised the classification of all reported CMA findings based on current knowledge.
In conclusion, our findings demonstrate that in pregnancies with sonographic diagnosis of CCA or PFA, CMA testing may be beneficial for the genetic workup and contribute to the prenatal counseling. Future studies in larger samples with both prenatal and postnatal assessment are required to better define the CMA detection rate in isolated and nonisolated CCA and PFA pregnancies, as well as in specific anomaly types.

Study Funding
The invasive tests, as well as CMA, were funded as part of the public health services in Israel.