Implication of chromosomal microarray analysis prior to in-utero repair of fetuses with open neural tube defects

Abstract

Objective:

In-utero repair of open neural tube defects (ONTD) is an accepted treatment option with demonstrated superior outcomes for eligible patients. While current guidelines recommend genetic testing by chromosomal microarray analysis (CMA) when a major congenital anomaly is detected prenatally, the requirement for an in-utero repair, based on the Management of Myelomeningocele Study (MOMS) criteria, is a normal karyotype. In this study, we aimed to evaluate if CMA should be recommended as a prerequisite for in-utero ONTD repair.

Methods:

This was a retrospective cohort study that included pregnancies complicated by ONTD for which laparotomy-assisted fetoscopic repair or an open hysterotomy fetal surgery was performed at a single tertiary center between September 2011 and July 2021. All patients met the MOMS eligibility criteria and had a normal karyotype. For a subset of the pregnancies (n=77), CMA testing was also conducted. We reviewed the results of the CMA and divided the cohort into two groups: group A – no detected CNV, and group B – reportable CNV detected. We then compared the groups’ surgical characteristics, complications, and maternal and early neonatal outcomes. Standard parametric and non-parametric statistical methods were employed as appropriate.

Results:

One hundred and forty-six fetuses with ONTD were eligible and underwent in-utero repair during the study period. CMA results were available for 77 (52.7%) patients. Of those, 65 (84%) had a normal CMA, and 12 (16%) had a reportable CNV, 2 (2.6%) of which were classified as pathogenic. Maternal demographics, clinical characteristics, operative data, and post-operative complications were similar between those with normal CMA results and those with reportable CNVs. There were no significant differences in gestational age at delivery or any obstetrical and early neonatal outcome between the study groups.

Conclusions:

Standard diagnostic testing with CMA should be offered when an ONTD is detected prenatally, as this approach has implications for counseling about prognosis and recurrence risk. Our results indicate that the presence of a reportable CNV should not a priori affect eligibility for in-utero repair, as overall outcomes for pregnancies are similar to those with normal CMA. Nevertheless, significant CMA results will require a case-by-case multidisciplinary discussion to evaluate eligibility. To generalize the conclusion of this single-center series, a larger long-term study should be considered.

INTRODUCTION

Neural tube defects (NTD) are the second most common major congenital anomalies, with an annual worldwide incidence of approximately 300,000 infants.^1,2 The majority of NTDs occur sporadically, and have been associated with environmental insults and genetic factors.³ Established risk factors include maternal exposure to teratogens, poorly controlled diabetes, folic acid deficiency, hyperthermia, obesity, ethnic and geographic differences, and family history.^4–9 Nevertheless, 90% of children with NTD are born to mothers without known risk factors.¹⁰

Although most NTDs are multifactorial in origin, there is considerable evidence for genetic contributions, especially when there are other congenital anomalies.^2,3,11 Chromosomal abnormalities have been reported in 2.6% to 16.3% of fetuses or newborns with NTDs, with the most common ones being trisomy 13, 18, and triploidy.^12–17 Some microdeletions and microduplications have also been reported to be associated with NTD.^18–21

Spina bifida results from the failure of primary neurulation of the caudal portion of the neural tube. The neurological damage in open spina bifida is thought to be due to sequential processes, the first being the primary developmental abnormality, followed by a combination of inflammation, stretch and direct trauma to the exposed neural elements in the intrauterine milieu.^22,23 Thus, in-utero repair of ONTD was introduced to reduce the progressive intrauterine injury.^24,25 Currently, the in-utero repair can be conducted by an open hysterotomy approach or a fetoscopic repair.^26–28

The American College of Obstetrics and Gynecology (ACOG) guidelines state that fetal repair should be offered to selected patients at experienced facilities, but one of the eligibility criteria for fetal surgery is a normal chromosomal analysis (karyotype).²⁹ These recommendations are based on the inclusion criteria of the Management of Myelomeningocele Study (MOMS),²⁶ but they are inconsistent with ACOG’s and other professional societies’ recommendations for prenatal genetic diagnosis which state that invasive prenatal diagnosis with chromosomal microarray analysis (CMA) should be offered for prenatally detected major congenital anomalies, including NTDs.^2,11,30 The aim of this study was to evaluate if fetal CMA testing should be required before in-utero ONTD repair by examining the frequency of copy number variants (CNVs) reported on CMA and the association of such results with changes in management and counseling.

METHODS

Study design and population

This retrospective cohort study includes all patients with ONTD who underwent in-utero repair using either an open hysterotomy approach or laparotomy-assisted fetoscopic approach at a highly specialized single tertiary referral center, between September 2011 and July 2021. All fetuses had an isolated ONTD and a normal karyotype. CMA was conducted for a subset of the pregnancies. Not all CMA results were available before the in-utero repair. We reviewed the results of the CMA and summarized all cases with reportable CNVs, including pathogenic, likely pathogenic, and variants of uncertain significance (VUS)³¹. The study population comprised two groups according to the CMA finding: (Group A) no detected CNV (normal CMA group); and (Group B) reportable CNV detected (reportable CNV group) that included all cases with abnormal CMA result.

Intervention

The standard open hysterotomy in-utero ONTD repair has been conducted at our institution since 2011. Fetoscopic myelomeningocele repair has been offered at our institution since July 2014. The fetoscopic repair was performed under the US Food and Drug Administration (Investigational Drug Exemption #G140201) and was overseen by the Baylor College of Medicine Institutional Review Board (IRB) (protocols H-34680, H-43359, H-33264), the Baylor College of Medicine Fetal Therapy Board, and a Data Safety Monitoring Board. The fetoscopic repair became standard of care at Texas Children’s in May 2021. For each patient, once they were considered to be eligible for in-utero repair, applying MOMS trial criteria (expanding maternal BMI to 40 kg/m²), the type of procedure performed was decided based on the patient’s preference following counseling on the risks and benefits of available repair options (postnatal repair, open hysterotomy, and laparotomy-assisted fetoscopic approach). The therapeutic window for in-utero repair was between 19 0/7 and 25 6/7 weeks gestation.

Preoperative evaluation

Preoperative assessment included maternal health evaluation, including mental health and social support. Lab work and diagnostic imaging modalities were utilized for each candidate as elaborated in prior publications.^27,32,33 Genetic analysis was done by amniocentesis. The extracted amniotic fluid was sent for chromosomal analysis for all fetuses. Most cases (n=89, 61%) also had Fluorescence In Situ Hybridization (FISH) for chromosomes 13, 18, 21, X and Y. The FISH was considered instead of the chromosomal analysis in cases presented at an advanced gestational age that did not allow the completion of karyotype prior to the closure of the surgical window (26 weeks gestation). A subset of patients had CMA (n=77, 52.7%), and three also had prenatal trio whole-exome sequencing. These tests were done at the discretion of the referring physician. When the karyotype was normal, but a reportable CNV was detected before in-utero repair, there was a multidisciplinary team discussion, involving fetal intervention, prenatal genetics, fetal radiology, neurosurgery, pediatric surgery, neonatology, and the ethics board. Fetal surgery was offered when the detected CNV was not associated with an early life-limiting phenotype, the fetus was otherwise an excellent candidate expected to benefit from prenatal surgery, and the potential benefits were deemed to outweigh potential risks from the procedure. After extensive consideration and fully transparent counseling of risks and benefits, the parents then opted and consented to proceed with a prenatal repair.

Definitions

The anatomic level of the lesion was determined through prenatal ultrasound and magnetic resonance imaging (MRI) examinations. Ultrasound video clips of lower extremity movements were reviewed at the initial evaluation to determine the functional level according to the nerve distribution (Table S1). Fetal cerebral ventriculomegaly was defined as a lateral ventricle atrial diameter > 10 mm on prenatal ultrasound.

MRI was conducted at the time of referral and six weeks after the procedure to evaluate the fetal brain and spinal repair site. Reversal of hindbrain herniation was considered positive if the most caudal portion of the cerebellum was seen above the foramen magnum. Cases that underwent open fetal surgery were scheduled for elective cesarean delivery at no later than 37 weeks gestation. Cases that underwent fetoscopic repair, if there were no contraindication for vaginal delivery, they were scheduled for induction of labor by 39–40 weeks gestation.³⁴

All neonates were admitted to the neonatal intensive care unit (NICU) for multidisciplinary evaluation and treatment. Transcranial ultrasound was conducted within the first 48 hours of life, the skin closure was inspected for cerebrospinal fluid (CSF) leakage and dehiscence, and the newborn’s bowel and bladder function were assessed. Postnatal follow-up (at 3, 6 and 12 months) was done at the Spina Bifida Program and Neurosurgery Clinic at Texas Children’s Hospital. For patients unable to travel to our center, local providers assessed the motor function of the neonates, and records were obtained. Motor function in the lower extremities was evaluated by a pediatric neurosurgeon after birth, at 12 months and 30 months of age. Motor function was determined using the lowest myotomes involved in active motor function (better than antigravity). Walking status at 30 months was categorized into four levels, walking independently, walking with orthotics, walking with assistive devices, and non-ambulatory.

Outcome measures

We compared the characteristics and outcomes of patients with normal CMA results to those with reportable CMA findings. The primary outcomes were fetal or neonatal death, hydrocephalus defined by the placement of a ventriculoperitoneal shunt or performance of an endoscopic third ventriculostomy by 12 months, the child’s motor function at 12 months, and walking status at 30 months.

The secondary outcomes included surgical and pregnancy complications, obstetric outcomes, and adverse neonatal outcomes. The obstetric outcomes were the rate of maternal pulmonary edema, post-operative blood transfusion, preterm premature rupture of membranes (PPROM), placental abruption, clinical chorioamnionitis, chorioamniotic membrane separation (CAS), gestational age at delivery and mode of delivery. PPROM was diagnosed when a positive pooling, nitrazine test, or ferning were observed before 37 weeks of gestation. CAS was diagnosed by ultrasound, while severe CAS was defined by the separation of >50% of the membranes. Clinical chorioamnionitis was diagnosed using the standard criteria,^35,36 and placental abruption was suspected clinically and confirmed during delivery.

Fetal and neonatal outcomes included the rate of hindbrain herniation reversal diagnosed at a fetal MRI 6 weeks after the in-utero repair and the rate of CSF leakage at birth. Other neonatal outcomes were the rate of respiratory distress syndrome, necrotizing enterocolitis, sepsis, periventricular leukomalacia, retinopathy of prematurity, and the length of stay in the NICU.

Statistical analysis

Continuous variables were presented as median and range. Categorical variables were reported as numbers and percentages. Comparison between continuous variables was conducted with Student’s t-test and Mann-Whitney U. Qualitative variables were compared using the Chi-square test or Fisher’s exact test, as appropriate. All tests were 2-tailed, and the significance threshold was set as a p-value<0.05. Statistical analyses were conducted using the IBM Statistical Package for the Social Sciences (IBM SPSS Statistics for windows V28.0; IBM Corporation Inc, Armonk, NY, USA).

RESULTS

Demographic, clinical characteristics and intraoperative data of study population

One hundred and forty-six patients underwent in-utero ONTD repair at our center during the study period. Of them, 40 (27.4%) underwent the standard open hysterotomy approach, and 106 underwent the laparotomy-assisted fetoscopic repair. Seventy-seven (52.7%) had CMA testing, 70 of which were done prenatally on DNA extracted from amniotic fluid, and seven were done postnatally on DNA from a neonatal blood sample. Sixty-five (84%) had a normal CMA and comprised study group A. Reportable CNVs were recorded in 12 fetuses (16%; Table 1) who comprised study group B. Of them, two (2.6%) were classified as pathogenic (figure 1). The first fetus with a pathogenic CNV was diagnosed with a 749 Kb “central” 22q11.21 deletion spanning Low-Copy-Repeat regions B-D of chromosome 22 (LCR22B-D). Common features of this deletion include developmental delay, intellectual disability, cardiac defects, and dysmorphic features, but with reduced penetrance and variable expressivity.^37,38 The lesion level at MRI was L4, and no other structural anomalies were identified by prenatal imaging that included targeted ultrasound, fetal echocardiogram and MRI. Due to gestational age limitations, the repair was conducted following normal FISH results, and the CMA results became available after the procedure. The patient had PPROM at 28 6/7 weeks and was delivered at 34 3/7 weeks gestation. There were no neonatal complications, and the baby had no dysmorphic features. The child is currently 31-months-old, walks independently, without hydrocephalus, but she has a speech delay. The second fetus with a pathogenic CNV was diagnosed with a 1.3 Mb interstitial deletion at 1q21.1q21.2. Common features of this deletion include microcephaly, intellectual disability, mild dysmorphic features, cardiac defects, skeletal malformations, seizures, and psychiatric and behavioral differences. This recurrent microdeletion also has reduced penetrance and variable expressivity.³⁹ In this patient, the lesion level at MRI was L3, with no other structural anomalies identified. After a multidisciplinary discussion regarding eligibility for the procedure, the team decided to offer fetoscopic repair, and after counseling, the parents opted to proceed with this prenatal repair. The patient had no post-procedure complications and was delivered at term with no neonatal complications. The baby is currently 10-months-old, with intact motor function at birth, no dysmorphic features, and no hydrocephalus to date. There were two fetuses with an absence of heterozygosity (AOH) on CMA of 36.6 Mb and 12 Mb, which increases the risk for autosomal recessive disorders. The fetus with an AOH of 36.6 Mb had a normal prenatal trio whole exome sequencing result. For the other eight fetuses, the reportable CNVs were classified as VUS, and among those, one was annotated as a VUS that is most likely benign.

Table 1.

Fetuses with significant copy number variants in chromosomal microarray analysis

Case ID	The phenotype	Chromosomal microarray analysis results	ISCN nomenclature	Size in Mb	Inheritance	Interpretation	Gestational age at delivery
Case 1	Myelomeningocele in lumbar region (L4). Ventriculomegaly, no talipes equinovarus. Fetoscopic repair	A 749 kb “central” 22q11.21 deletion, spans Low-Copy-Repeat regions B-D of chromosome 22 (LCR22B-D). Common features include developmental delay, intellectual disability, cardiac defects, and dysmorphic features. Reduced penetrance and variable expressivity	arr 22q11.21(20,716,902–21,465,659)X1	0.749	Not known	Pathogenic	34w3d
Case 2	Myeloschisis in lumbar region (L3). No ventriculomegaly, no talipes equinovarus. Fetoscopic repair	1.3 Mb interstitial copy number loss at 1q21.1q21.2. Common features include microcephaly, mild intellectual disability, mild dysmorphic features, eye abnormalities, cardiac defects, skeletal malformations, seizures, and psychiatric and behavioral differences	arr 1q21.1q21.2(146470887–147832190)X1	1.3	De novo	Pathogenic	39w3d
Case 3	Myeloschisis in lumbar region (L3). No ventriculomegaly, no talipes equinovarus. Fetoscopic repair	Absence of heterozygosity of 36.6 Mb at 11q12.3q22.1. The region does not include imprinted genes. Negative exome sequencing	arr 11q12.3q22.1(62,166,234–98,723,216)X2 hmz	36.6 AOH	N/A	No CNV identified, AOH was noted	39w3d
Case 4	Myelomeningocele in high lumbar region (L1). Ventriculomegaly, no talipes equinovarus. Fetoscopic repair	Absence of Heterozygosity of 12 Mb at 5q14.2q15. The region does not include imprinted genes	arr 5q14.2q15(81909354–93907889)x2 hmz	12 AOH	N/A	No CNV identified, AOH was noted	34w1d
Case 5	Myelomeningocele in lumbar region (L4). No ventriculomegaly, no talipes equinovarus. Open repair	Copy number gain of 1.928 Mb at 22q11.22q11.23, distal to the DiGeorge syndrome region, between LCR22-E and LCR22-H. Duplications within this region are often inherited and have been associated with a variable phenotype	arr 22q11.22q11.23 (23063664–24991856)X3	1.928	Inherited from mother	VUS	32w0d
Case 6	Myelomeningocele in high lumbar region (L2). Ventriculomegaly, talipes equinovarus. Fetoscopic repair	Copy number loss of 611 Kb at 2p24.3 of uncertain clinical significance, encompassing the entire NBAS gene and a portion of DDX1. Biallelic variants in NBAS have been associated with two autosomal recessive disorders: infantile liver failure syndrome type 2, and short stature, optic nerve atrophy with Pelger-Huet anomaly	arr 2p24.3(15128971–15739810)x1	0.611	Not known	VUS, carrier status	31w1d
Case 7	Myelomeningocele in lumbar region (L3). Ventriculomegaly, no talipes equinovarus. Fetoscopic repair	A 302 Kb interstitial duplication at 2p24.3 that includes the genes MYCNUT, MYCNOS, MYCN, and GACAT3. Only MYCN is associated with a disease, but it is a haploinsufficient gene. This duplication has been associated with an increased risk of tumors, specifically renal tumors and neuroblastoma	arr 2p24.3(16057692–16359561)X3	0.302	Not inherited from mother, father was not tested	VUS	37w1d
Case 8	Myelomeningocele in lumbar region (L4). Ventriculomegaly, no talipes equinovarus. Fetoscopic repair	Copy number gain of 0.165 Mb in a non-disease associated region on 19p13.2	arr 19p13.2(7070410–7235466)x3	0.165	Not known	VUS	36w0d
Case 9	Myelomeningocele in high lumbar region (L2). Ventriculomegaly, talipes equinovarus. Open repair	Copy number gain of 399 Kb at 8p11.31p11.23, not known to be associated with a clinical phenotype	arr 18p11.31p11.23 (6908018–7307000)X3	0.399	Inherited from mother	VUS	36w3d
Case 10	Myelomeningocele in lumbar region (L4). Ventriculomegaly, no talipes equinovarus. Open repair	Copy number loss of 1.458 Mb at 7q32.3q33, not known to be associated with a clinical phenotype	arr 7q32.3q33(132523117–133981103)x1pat	1.458	Inherited from father	VUS	37w0d
Case 11	Myeloschisis in lumbar region (L4). No ventriculomegaly, no talipes equinovarus. Open repair	A 372 Kb deletion associated with probable carrier status of 9q22.32 that encompasses FBP1, associated with fructose-1,6-bisphosphatase deficiency	arr 9q22.32(97208231–97580551)X1	0.372	Not known	VUS, carrier status	33w6d
Case 12	Myelomeningocele in lumber region (L4). Ventriculomegaly, no talipes equinovarus. Fetoscopic repair	A 1.69 Mb interstitial duplication of Xp22.31-> p22.31. The duplication includes five OMIM genes (VCX3A, PUDP, STS, VCX, and PNPLA4), but is considered to be a normal variant of the X chromosome	arr Xp22.31(6446579–8135644)X2 (male)	1.69	Not known	VUS/Likely benign	39w3d

CNV, copy number variants; N/A, not applicable; VUS, variant of unknown significance.

Figure 1.

Enrollment and outcomes. The figure describes the number of patients who underwent in-utero ONTD repair, the surgical approach, the number of fetuses who had chromosomal microarray analysis, and a summary of obtained results.

Table 2 displays the demographic and clinical characteristics of the study groups. Maternal age, ethnicity, BMI, gravidity and parity, family history of NTD, prior uterine surgery, diabetes, and hypertensive disease of pregnancy were comparable between patients with normal CMA to those with reportable CMA finding. The preoperative data were also similar between the groups, including the percentage of myeloschisis versus myelomeningocele, the anatomical level of the lesion, the estimated functional level, and the rates of clubfoot and ventriculomegaly. The groups did not differ in their operative characteristics, such as median gestational age at repair (25.1 weeks for the normal CMA group vs. 25.4 weeks for the reportable CNV group, p=0.13), the surgical approach (standard open hysterotomy vs. laparotomy-assisted fetoscopic repair), number of layers used for closure, total operative time, and fetal neurosurgery time (Table S2).

Table 2.

Demographic and clinical characteristics of study group

	Normal CMA (n=65)	Reportable CMA finding (n=12)	P-value
Maternal age (years)	29 (19–45)	29 (23–41)	0.33
Ethnic group			0.7
White	49 (75.4)	7 (58.3)
Hispanic	14 (21.5)	3 (25)
Black	2 (3.1)	2 (16.7)
Gravidity	2 (1–7)	2.5 (1–6)	0.09
Parity	1 (0–5)	1 (0–5)	0.14
Nulliparity	20 (30.8)	1 (8.3)	0.16
Male neonate	28 (43. 1)	6 (50)	0.72
Maternal BMI at screening (kg/m2)	26.5 (18.5–38.1)	29.5 (17.4–38.7)	0.38
Prior uterine surgery	15 (23.1)	3 (25.0)	1.0
Diabetes			0.34
None	62 (95.4)	11 (91.7)
Pre-gestational	1 (1.5)	1 (8.3)
Gestational	2 (3.1)	0 (0)
Hypertensive disease of pregnancy			0.58
None	61 (93.8)	11 (91.7)
Gestational HTN	2 (3.1)	1 (8.3)
Preeclampsia	2 (3.1)	0 (0)
Family history of NTD	2 (3.1%)	0	1

Data are presented as median (range) for continuous variables and as n (%) for categorical variables. BMI, body mass index; HTN, hypertension; NTD, neural tube defects.

Primary outcomes

There were two demises in the normal CMA group. One intrauterine fetal death (IUFD) occurred at 28 weeks’ gestation following membrane separation and a possible umbilical cord-related accident. There was one neonatal death after an open in-utero repair of myelomeningocele at 25 6/7 weeks’ gestation. This patient had PPROM at POD#3 and underwent an emergency cesarean section due to a non-reassuring fetal heart rate at 27 2/7 weeks’ gestation. The baby was small for gestational age and passed away at day of life #3 following respiratory distress, pulmonary hemorrhage, and sepsis.

Similar motor function level at birth was found in the two groups. At 12 months of age, no significant difference was noted in motor function or the rate of hydrocephalus between the study groups. Only 40.6% (26/64) of neonates in the normal CMA group and 58.3% (7/12) of neonates in the reportable CNV group have reached 30 months by the conclusion of this study. For those, there was no difference in the walking status between the groups (Table 3).

Table 3.

Primary outcome

	Normal CMA (n=65)	Reportable CMA finding (n=12)	P-value
Fetal or neonatal death	2 (3.1)	0 (0)	1.0
Hydrocephalus*	18/46 (39.1)	2/9 (22.2)	0.6
Motor function at birth
L1 – hip flexion	64 (100)†	12 (100)	1.0
L3 – knee extension	62 (96.6)	11 (91.7)	0.41
L4 – ankle dorsiflexion	58 (84.1)	11 (91.7)	1.0
S1 – ankle plantarflexion	48 (75.0)	7 (58.3)	0.3
Motor function at 12 months*
L1 – hip flexion	45/45 (100)	7/7 (100)	1.0
L3 – knee extension	45/45 (100)	6/7 (85.7)	0.14
L4 – ankle dorsiflexion	39/45 (86.7)	5/7 (71.4)	0.29
S1 – ankle plantarflexion	34/45 (75.6)	5/7 (71.4)	1.0
Walking status at 30 months‡			0.283
Independently	3/26 (11.5)	1/5 (20)
Walking using braces/orthotics	6/26 (23.1)	2/5 (40)
Walking with assistive devices	12/26 (46.2)	0 (0)
Non-ambulatory	5/26 (19.2)	2/5 (40)

Data are presented as n (%) or n/N (%).

^*Available data at 12 months are presented.

^†The fetal demise was excluded.

^‡Available data at 30 months are presented. VP, ventriculoperitoneal.

Obstetric outcome

A similar proportion of patients in both groups presented with post-operative complications (Table 4). The incidence of any degree of CAS was comparable between the normal CMA (36.9%) and reportable CNV (41.7%) groups (p=0.76). The rate of severe CAS was also similar between the groups (23.1% versus 25%, p=1). The median gestational age at delivery was 37.3 (25.1–40.6) weeks in the normal CMA group and 36.2 (31.1–39.3) weeks in the reportable CNV group, p=0.15. Although 34.4% of the normal CMA group and 58.3% of the reportable CNV group were born prior to 37 weeks of gestation (p=0.12), only 9.4% and 8.3%, respectively, were born prior to 32 weeks of gestation (p=0.91). The median time from repair to delivery and the proportion of women who delivered vaginally were similar between the two groups (Table S3).

Table 4.

Postoperative complications

	Normal CMA (n=65)	Reportable CMA finding (n=12)	P-value
PPROM*	15 (23.1)	5 (41.7)	0.3
GA at PPROM	32.4 (25–36.1)	30.3 (27.4–36.0)	0.9
PPROM to delivery (days)	2 (0–17)	5.5 (0–38)	0.6
Placental abruption	2 (3.1)	0 (0)	1.0
Chorioamnionitis	2 (3.1)	0 (0)	1.0
CAS	24 (36.9)	5 (41.7)	0.76
GA at CAS	27.1 (25–33)	28.4 (26.3–28.6)	0.77
Severe	15/24 (23.1)	3/12 (25.0)	1.0
Pulmonary edema	6 (9.2)	3 (25.0)	0.14
Postoperative blood transfusions	2 (3.1)	0 (0)	1.0

Data are presented as median (range) for continuous variables and as n (%) or n/N (%) for categorical variables. GA, gestational age; CAS, chorioamniotic membrane separation; PPROM, preterm premature rupture of membranes.

^*Rupture of membranes between 24+0 and 36+6 weeks of gestation.

Neonatal outcome

In terms of neurologic outcomes, reversal of hindbrain herniation at six weeks post-procedure and CSF leakage were comparable between the study groups (Table S3). No significant differences were found between the groups in the prevalence of neonatal complications, including the rate of respiratory distress syndrome, necrotizing enterocolitis, sepsis, periventricular leukomalacia, retinopathy of prematurity, and the length of stay in the NICU.

DISCUSSION

We reported a 16% rate of CNVs in fetuses with isolated ONTD that were eligible for in-utero repair. A pathogenic CNV was diagnosed in only 2 fetuses (2.6%) who underwent in-utero repair; all had comparable outcomes to a control group with normal CMA results.

There are limited data on CMA findings associated with isolated ONTD. Available literature focuses on chromosomal abnormalities, with trisomy 13, 18, and triploidy being the most common ones,^11,16,17,40 hence the minimal requirement of normal karyotype when considering an in-utero repair.^26,29 In a review of nine case series with a combined 547 patients with spina bifida, 9.7% were diagnosed with chromosomal abnormalities,¹⁶ and the rate reached 24–38% when additional structural anomalies were reported.^14,15 When isolated, the rate was 2.6%.¹⁵ In the past decade many centers worldwide have abandoned basic chromosome analysis for next-generation testing, such as CMA and exome sequencing. This is now included in the ACOG/SMFM recommendations for prenatal genetic diagnosis.³⁰ Nevertheless, the association of CNVs with NTD is not well established.⁴¹ A 2q35–36.2 deletion containing the PAX3 gene has been reported in patients with NTD^18,42. A 22q11.2 deletion syndrome has also been reported, but the link to NTD is unclear.^19,43 Similarly, a SOX duplication (Xq27.1) has been diagnosed in a few patients with myelomeningocele.²⁰ The association between NTD and monogenic disorders is also incompletely defined. The process of neural tube closure involves multiple cellular and molecular processes, and changes in any of the genes involved could result in a NTD.⁴⁴ Classes of implicated genes include those in the folate one-carbon metabolism pathways, Wnt signaling genes, in particular those involved in planar cell polarity, and genes involved in the development of cilia, which are essential for cell signaling.^3,44–48

We acknowledge that a positive CNV finding further complicates the already complex decision-making process for in-utero ONTD repair.^28,32,49 For this study, we considered a positive CMA result any reported CNV that is classified as pathogenic, likely pathogenic, and of uncertain significance, as well as increased regions of AOH which could predispose to recessive disorders.⁵⁰ Although several of these findings, in particular VUS and regions of AOH, cannot be conclusively associated with a phenotypic anomaly, their detection makes counseling more difficult and potentially confusing to parents. Reassuringly, in our study, these results were not associated with a higher rate of adverse outcomes after in-utero ONTD repair.

There was one case with a pathogenic “central” 22q11.21 deletion, but the inheritance pattern of the deletion could not be tested due to insurance concerns. There is a description of two siblings with myelomeningocele and the same microdeletion of chromosome 22q11 (~751 kb between LCR22B-D) that was inherited from a mother who denied having any significant medical problems.⁵¹ These siblings and the fetus in our study may support a possible link between a “central” deletion of 22q11 and NTDs, but further confirmation is needed. A second pathogenic finding was a de novo 1.3 Mb interstitial deletion at 1q21.1q21.2. To our knowledge, there is no known association between this microdeletion and NTD. Nevertheless, we evaluated another fetus with ONTD and the same microdeletion in our center, possibly representing an expansion of the phenotype resulting from this deletion. While fetuses with reportable CNVs had comparable outcomes to those with normal CMA results following in-utero NTD repair, such abnormal CMA results have implications for patient counseling regarding prognosis and recurrence risk of the genetic finding. In an era when we strive for comprehensive and complete counseling and a fully informed consent process, it is important that all data, including CMA results, are available to patients to empower their decision regarding in-utero ONTD repair. Furthermore, certain CNVs, such as those associated with poor neurodevelopmental prognosis, may influence eligibility for in-utero repair. Another important aspect is postnatal treatment planning. While all neonates in our cohort underwent a postnatal neurodevelopmental evaluation, those with significant CNV results required additional multidisciplinary evaluation and treatment. Informing the parents of what lays ahead is important for their decision-making and planning of resources for the postnatal period.

Our study has some limitations, due to its retrospective nature we had to exclude patients who underwent in-utero repair and had no CMA testing and we were not able to include a control group of patients with an isolated ONTD and a reportable CNV who underwent postnatal repair. As our institution is a referral center for in-utero ONTD repair, the rate of pathogenic CNVs does not necessarly correlate with the entire population of patients with NTD. Some patients who are ineligible for a fetal intervention due to abnormal CMA may not have been referred for evaluation. CMA was performed at the discretion of the referring physician and the patient which may conceal differences between the groups. Nonetheless, the demographic and clinical characteristics were comparable between patients with normal CMA to those with reportable CMA finding, and the groups had similar preoperative and operative features. Our data lacks long-term outcomes beyond 30 months of age but it is the largest cohort of fetuses with CMA results who underwent an in-utero repair in a single center following a strict treatment protocol. While there were two fetuses with pathogenic CNVs in our series, we recognize that our study was likely underpowered to demonstrate significant differences in the surgical outcomes of in-utero repair. To generalize our conclusions, a long-term prospective multi-center study could be considered.

In conclusion, we found reportable CNVs in 16%, including pathogenic CNVs in 2.6%, of fetuses with isolated ONTD who had in-utero repair. This strengthens the overall recommendation to complete this genetic evaluation whenever a major congenital anomaly is encountered. Although our study was relatively small, we found comparable complication rates and outcomes regardless of the CMA result, and none of the reported CMA findings resulted in exclusion from fetal surgery. This supports the need to adopt a case-by-case approach for each ONTD repair candidate to evaluate the benefit of such surgery. Finally, providing CMA results to the parents will allow a more informed consent prior to choosing prenatal versus postnatal repair.

Supplementary Material

SUPINFO

SUPPORTING INFORMATION ON THE INTERNET

Table S1. Motor function classification

Table S2. Pre- and intraoperative evaluation

Table S3. Secondary outcome: maternal and neonatal outcomes

CONTRIBUTION.

What are the novel findings of this work?

A CNV was reported for 16% of fetuses with isolated ONTD considered eligible for in-utero repair. Of the reported CNVs, 2.6% were classified as pathogenic. Fetuses with reportable CNVs who underwent in-utero repair had comparable outcomes to a control group with normal CMA results.

What are the clinical implications of this work?

Standard diagnostic testing with CMA should be offered when an ONTD is detected prenatally, as this approach has implications for counseling about prognosis and recurrence risk, but detection of a reportable CNV should not a priori affect eligibility for in-utero repair because overall outcomes for pregnancies are similar to those with normal CMA.