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. 2025 Aug 22;98(3):698–705. doi: 10.1227/neu.0000000000003707

Brain Imaging Findings Show Efficacy of Fetal Endoscopic Third Ventriculostomy as Prenatal Treatment for Induced Congenital Hydrocephalus in Fetal Lambs

Soner Duru , Marc Oria ‡,§,, Blanca Fernandez-Tome , Lucas Peiro ‡,, Jose L Encinas #, Francisco M Sanchez-Margallo , Jose L Peiro ‡,§,
PMCID: PMC12875635  PMID: 40844280

Abstract

BACKGROUND AND OBJECTIVES:

Congenital obstructive hydrocephalus (HCP) causes progressive, irreversible fetal brain damage through ventricular enlargement and increasing fetal cerebral tissue compression. Postnatal treatments of choice include ventriculoperitoneal shunting or endoscopic third ventriculostomy (ETV). Intrauterine treatments, such as ventriculoamniotic shunting, were attempted unsuccessfully 4 decades ago and failed to improve postnatal outcomes, likely due to inadequate fetal patient selection. The aim of this study was to evaluate the efficacy of prenatal ETV for early ventricular decompression and potential prevention of fetal brain damage in hydrocephalic fetal lambs.

METHODS:

HCP was induced in 24 fetal lambs by injecting BioGlue into the cisterna magna at E85. Three weeks later (E105-110), fetal ETV was successfully performed on 8 fetuses using a small rigid cystoscope. Fetal brain lateral ventricular diameters and cerebral mantle thicknesses were monitored by prenatal and postnatal ultrasounds and fetal MRI.

RESULTS:

According to the Cincinnati HCP Severity Scale, moderate and severe HCP subgroups responded positively to fetal ETV with reduced cerebral ventricular diameters. Ten days post-ETV, severe HCP fetal lambs improved to moderate levels, whereas those with moderate HCP normalized by birth. A similar improvement pattern was seen for the mechanical compression threshold (ventricular diameters/biparietal diameter). Biparietal diameter values did not significantly differ among nontreated, treated, and normal control groups during pregnancy. MRI revealed a significant increase in brain mantle thickness in the prenatally treated fetuses.

CONCLUSION:

Prenatal ETV is feasible in hydrocephalic fetal lambs and effectively reverses ventriculomegaly and brain compression in cases of severe or moderate fetal HCP in this ovine model.

KEY WORDS: Congenital hydrocephalus, Obstructive hydrocephalus, Fetal endoscopic third ventriculostomy, BioGlue, Fetal lamb model


ABBREVIATIONS:

ANOVA

Analysis of Variance

BPD

biparietal diameter

CHSS

Cincinnati Hydrocephalus Severity Scale

ETV

endoscopic third ventriculostomy

HCP

hydrocephalus

LVD

lateral ventricular diameters

VAS

ventriculoamniotic shunts.

Congenital obstructive hydrocephalus (HCP) leads to progressive cerebrospinal fluid (CSF) accumulation, causing enlargement of the cerebral lateral ventricles and fetal brain tissue compression, resulting in abnormal brain development. HCP affects 4-6 per 10 000 births.1 Although HCP can occur at any age, congenital HCP manifests early in the intrauterine period and often has poor neurological outcomes despite postnatal treatment.2 The expansion of fetal cerebral ventricles due to accumulated CSF and increased intracranial pressure triggers pathophysiological processes including cerebral white matter damage, ischemia/hypoxia, inflammation, edema, and gliosis.3-5 Congenital HCP is commonly diagnosed prenatally by ultrasound and MRI and treated with postnatal ventriculoperitoneal shunts or endoscopic third ventriculostomy (ETV) to reroute the blocked fluid6-8 but probably too late to prevent brain maldevelopment.4,5 Theoretically, fetuses with isolated obstructive HCP might benefit from intrauterine CSF diversion and cerebral decompression as soon as possible.

Large animal models have been created to assess the feasibility and efficacy of in utero interventions for induced congenital abnormalities. Experimental studies in animal models of induced HCP, mainly in sheep and monkeys, showed improvement in ventriculomegaly after shunting decompression.9-15 Prenatal interventions, such as implanted ventriculoamniotic shunts (VAS), were attempted in humans 40 years ago but were abandoned because of technical complications and poor clinical results.16-21 Current advances in imaging, diagnosis, selection criteria, and surgical techniques warrant a re-evaluation of these treatments, after a long moratorium adopted by fetal therapy groups.22,23

Given the effectiveness of ETV as a postnatal treatment,6-8 we propose evaluating its efficacy in the fetal period. A fetal sheep model was selected for its anatomic and physiological similarity to humans.24 A new fetal obstructive HCP ovine model in which HCP is induced by injecting BioGlue, instead Kaolin, into the fetal cisterna magna was developed.15,25,26 Was demonstrated the technical feasibility of fetal ETV and detailed the surgical technique and anatomical specifics using this model.27 The aim of our study was to test the effectiveness of fetal ETV in reducing or arresting brain ventricular enlargement and cerebral mantle compression in congenital HCP.

METHODS

Animal Husbandry

This study was performed in 31 fetal lambs from a total of 21 young pregnant ewes (<2 years old) with single and twin pregnancies, obtained from the farm at Jesus Uson Minimally Invasive Surgery Centre, in Caceres, Spain. Animals were housed with climate-controlled humidity (55%) and temperature (22°C) with a light/dark cycle of 12 h/12 h. Ewes had ad libitum access to standard sheep chow and drinking water.

The studies performed in this project followed the guidelines for animal research and were approved by the Institutional Animal Care and Use Committee (IACUC) (ES100370001499). They were conducted in the animal facilities of the Jesus Uson Minimally Invasive Surgery Centre, in Caceres, Spain. Data supporting the findings of this study are available within the article and its supplementary information.

In this study, we randomly divided the fetal lambs into 3 groups:

  • Group 1: HCP: Percutaneous injection of 2 mL of BioGlue into the fetal cisterna magna without fetal ETV (n = 16).

  • Group 2: Control: Co-twins’ sham fetal lambs from the same mother with no injection into the fetal cisterna magna and exposed to the same anesthesia. Normal fetal lambs (n = 7).

  • Group 3: HCP + Fetal ETV (HCP + ETV): Fetal ETV after percutaneous injection of BioGlue into the fetal cisterna magna (n = 8).

HCP Induction in Fetal Lambs

We used the BioGlue-induced HCP animal model in fetal lambs previously described by Oria et al.26 We generated fetal HCP in 24 fetal lambs by injecting BioGlue into the cisterna magna at E85, and 3 weeks later (E105-110), fetal ETV was performed using a small rigid cystoscope in 16 of the fetuses with induced HCP. We left 7 fetal lambs as normal controls. The complications and fetal losses rate we had was discussed in the mentioned published study.26

Fetal ETV

Twenty days after the BioGlue injection, at 105-110 days of gestational age, fetal ETV (FETV) was performed in 8 fetuses under general anesthesia and in sterile conditions using small instruments as described previously by Peiro et al.27 The complications and fetal losses rate we had related to this second surgery was discussed in our published study.27

Ultrasound Monitoring

Ultrasound monitoring was conducted on all fetuses using a transabdominal convex transducer (C5-2 at 2-5 MHz, Philips ATL HDI 5000) both before and after cisterna magna injections. After prenatal ETV surgery, weekly ultrasonography (Figure 1) was used to assess the progression of HCP and detect potential complications. Parameters, such as fetal heart rate, remodeling of cerebral structures and ventricles, and measurements of brain and ventricular size, were systematically evaluated up until delivery and euthanasia of the lambs at 140 ± 5 days of gestation. Ventricular size measurements were obtained in the true axial plane at the level of the lateral ventricle atria (lateral ventricular diameters, LVD) and the choroid plexus glomus, as outlined in previous studies.26

FIGURE 1.

FIGURE 1.

A, Prenatal ultrasounds on pregnant ewe monitoring the fetal lambs. B, Severe fetal hydrocephalus on US axial view.

Fetal and Neonatal Biometric Assessment: The LVD was determined by measuring the distance from the inner margin of the medial ventricular wall to the corresponding inner margin of the lateral wall. Biparietal diameter (BPD) was assessed by measuring the maximal transverse distance between the outermost aspects of the parietal bones. The LVD/BPD ratio was subsequently calculated to evaluate relative ventricular size. Measurements were performed blind by different imaging technicians.

MRI Imaging

MRI was conducted on pregnant ewes and newborn lambs to evaluate hydrocephalic changes during the progression of ventricular dilatation and at postnatal day 1 (P1). Imaging was performed using a 1.5 T MR scanner (Intera, Philips Medical Systems) equipped with a Sense body 4-channel coil for pregnant ewes and a Sense Flex small coil for the newborn lambs (Philips Medical Systems). Acquisitions included high-resolution T1-weighted spin-echo sequences (repetition time/echo time/echo train length/acquisition time: 450-650/15 msec) and T2-weighted turbo spin-echo sequences (repetition time/echo time/echo train length/acquisition time: 3778/110 msec) with respiratory gating. Images were obtained in 2-mm-thick slices and reconstructed using a 384 mm × 240 mm acquisition matrix. Image analysis and measurements were independently performed in a blinded manner by different imaging technicians.

Cincinnati HCP Severity Scale

As previously described by Oria et al,26 HCP severity was calculated using the Cincinnati HCP Severity Scale (CHSS). Rated “normal” when LVD is between 2 and 4 mm and LVD/BPD between 0 and 0.08 mm, rated “mild” when LVD ranges between 4 and 6 mm and LVD/BPD is 0.08-01.12, rated “moderate” when LVD is between 6 and 10 mm and LVD/BPD is between 01.12 and 0.2, and rated “severe” when the LVD is more than 10 mm, and the LVD/BPD ratio is over 0.2.

Statistical Analysis

The data are expressed as mean and SD, and P-values of <.05 were considered significant. Intragroup and intergroup comparisons were performed with Student T-Tests for 2 determinations and Analysis of Variance (ANOVA) one-way repeated measures followed by pairwise Tukey multiple comparisons test for multiple determinations. GraphPad Prism 9 package was used for graph and sample size and power analysis statistical calculations.

RESULTS

Cerebral Ventricular Dilatation After BioGlue Injection in Fetuses

As previously described in other studies using this model,26 we observed significant fetal brain lateral ventricular dilatation by ultrasonograms and fetal MRI soon after BioGlue injections into the cisterna magna at E85-90 (Table). This polymer blockage of the space near the fourth ventricle increased the lateral ventricle diameter (LVD) up to 12.63 ± 5.55 mm after 20 ± 5 days (E105), to 13.68 ± 4.34 mm at 30 ± 5 days (E115), and up to 16.78 ± 3.22 mm after 50 ± 5 days (P1), compared with 3.76 ± 0.05 mm, 3.86 ± 0.05 mm, and 5.75 ± 0.07 mm in the control group (ANOVA, *P ≤ .0001, Figure 2A, Table).

FIGURE 2.

FIGURE 2.

Ventricle dilatation in fetal lamb models of HCP and reduction after fetal ETV. A, BioGlue-induced ventriculomegaly 20 days after injection compared with controls (*P < .001). BioGlue-induced severe HCP 20 days after injection, progressing until delivery (*P < .001). ETV reduced ventricle diameter after 10 days (β#P < .001). B, Mechanical compression in fetal lamb model of HCP (LVD/BPD) and C, normal head growth BPD. Values (means ± SD [error bars]). BPD, biparietal diameter; ETV, endoscopic third ventriculostomy; FETV, fetal endoscopic third ventriculostomy; HCP, hydrocephalus; LVD, lateral ventricular diameters.

TABLE.

Summary of Fetal Brain Ultrasound Parameters

Groups # Fetus LVD BPD LVD/BPD
E85 INJ E105 ETV E115 P1 E85 INJ E105 ETV E115 P1 E85 INJ E105 ETV E115 P1
Control 7 3.06 ± 0.09 3.76 ± 0.05 3.86 ± 0.05 5.70 ± 0.07 37.00 ± 2.12 49.63 ± 6.67 49.77 ± 9.65 52.60 ± 5.57 0.066 ± 0.005 0.067 ± 0.004 0.061 ± 0.007 0.106 ± 0.006
HCP 8 3.21 ± 1.36 12.63 ± 5.58 13.68 ± 4.34 16.78 ± 3.22 32.29 ± 4.75 52.20 ± 9.01 55.29 ± 9.55 59.21 ± 4.84 0.111 ± 0.026 0.242 ± 0.080 0.266 ± 0.065 0.263 ± 0.058
Moderate HCP 4 3.60 ± 1.60 7.50 ± 1.4 9.75 ± 2.53 12.19 ± 1.23 32.17 ± 4.79 50.03 ± 11.77 55.70 ± 9.26 59.62 ± 5.15 0.130 ± 0.015 0.182 ± 0.034 0.206 ± 0.014 0.213 ± 0.025
Severe HCP 4 2.83 ± 1.27 15.20 ± 4.94 16.04 ± 3.35 17.93 ± 2.24 32.38 ± 5.27 53.28 ± 8.52 54.98 ± 11.17 58.67 ± 5.66 0.093 ± 0.022 0.288 ± 0.075 0.296 ± 0.057 0.283 ± 0.055
HCP + FETV 16 3,59 ± 0.78 11.79 ± 3.46 8.14 ± 2.96 9.76 ± 3.92 32.91 ± 5.34 49.46 ± 10.06 50.97 ± 7.56 55.26 ± 7.98 0.104 ± 0.018 0.255 ± 0.100 0.164 ± 0.067 0.192 ± 0.09
Moderate HCP + FETV 6 4.35 ± 0.30 7.55 ± 1.29 5.70 ± 0.41 6.68 ± 0.45 34.14 ± 5.58 54.27 ± 11.39 56.50 ± 5.10 58.37 ± 4.56 0.112 ± 0.010 0.143 ± 0.022 0.103 ± 0.008 0.120 ± 0.007
Severe HCP + FETV 10 3.83 ± 0.51 13.90 ± 1.72 9.50 ± 2.88 11.82 ± 3.83 32.35 ± 5.41 47.75 ± 9.38 49.12 ± 7.49 53.71 ± 9.47 0.100 ± 0.020 0.305 ± 0.076 0.195 ± 0.062 0.240 ± 0.087

BPD, biparietal diameter; ETV, endoscopic third ventriculostomy; FETV; fetal endoscopic third ventriculostomy; HCP, hydrocephalus; INJ, injection; LVD, lateral ventricular diameters.

The thickness or degree of mechanical compression of the brain mantle was calculated using the ratio of LVD to the BPD, the diameter of the fetus' head (Table). This degree of tissue compression increased in hydrocephalic fetuses after 20 ± 5 days (E105) to 0.24 ± 0.08, after 30 ± 5 days (E115) to 0.26 ± 0.06, and after 50 ± 5 days to 0.26 ± 0.05 (P1) compared with the only 0.06 ± 0.004, 0.06 ± 0.007, and 0.1 ± 0.006 for the control animals (ANOVA, *P ≤ .0001, Figure 2B, Table).

BPD measurements were not significantly different between the nontreated (HCP) and normal control groups during the pregnancy and suggested a normal evolution of head size (Figure 2C, Table). According to the CHSS that was recently described,26 the fetuses injected with BioGlue acquired severe HCP at 20 ± 5 days postinjection (E105), a condition that worsened until delivery (P1).

LVD Reduction After Fetal ETV

Fetal ETVs were performed in lambs at E105-110, after 20 ± 5 days of the induction of obstructive HCP by BioGlue injection in the fetal cisterna magna. Prenatal ETV was performed as previously described Peiro et al,27 and, subsequently, we observed a statistically significant LVD reduction from 11.79 ± 3.46 at E105 to 8.15 ± 2.96 after 10 days (ANOVA, #P ≤ .01, Figure 2A, Table). We also saw a significant reduction in LVD between the HCP and HCP + ETV groups at E105 from 13.68 ± 4.34 and 8.14 ± 2.96, respectively (ANOVA, β P ≤ .001). Moreover, we observed a statistically significant improvement of the brain mechanical compression degree (LVD/BPD) in the HCP + ETV from 0.25 ± 0.1 at E105 to 0.16 ± 0.06 at 10 days post-ETV (ANOVA, #P ≤ .05, Figure 2B) and from 0.26 ± 0.06 to 0.16 ± 0.06 when comparing the HCP vs HCP + ETV groups at E115 (ANOVA, β P ≤ .001, Figure 1B, Table). BPDs were not significantly different between the HCP, HCP + ETV, and control groups during the pregnancy (Figure 2C, Table). According to the CHSS,26 we observed a reduction from severe to moderate HCP in both parameters analyzed.

Fetal ETV for Moderate and Severe HCP

We observed 2 different hydrocephalic subgroups, moderate and severe HCP, in fetal lambs at 20 + 5 days post-injection (E105).26 One subgroup developed severe HCP (n = 4), in which the LVD increased to 15.2 ± 4.95 mm after 20 ± 5 days (E105), 16.04 ± 3.35 mm after 30 ± 5 days (E115), and 17.93 ± 2.24 mm after 50 ± 5 days (P1). The other subgroup developed moderate HCP (n = 4) in which the LVD increased to 70.2 ± 1.4 mm after 20 ± 5 days (E105), 9.75 ± 2.53 mm after 30 ± 5 days (E115), and 12.19 ± 1.23 mm after 50 ± 5 days (P1), compared with 3.76 ± 0.05 mm, 3.86 ± 0.05 mm, and 5.75 ± 0.07 mm, respectively, for the control group (ANOVA, *P ≤ .0001, Figure 3A, Table).

FIGURE 3.

FIGURE 3.

HCP subgroups in fetal lambs. We observed 2 different hydrocephalic subgroups in fetal lambs at 20 + 5 days after the BioGlue injection (E105). One subgroup developed severe HCP (severe HCP) and another subgroup moderate HCP (moderate HCP). Subgroup ventricle dilatation in fetal lamb models of HCP and reduction after fetal ETV. A, BioGlue-induced ventriculomegaly 20 days after injection, progressing until delivery in both subgroups compared with controls (*P < .001). ETV-reduced ventricle diameter after 10 days (α#P < .001). B, Mechanical compression in fetal lamb model of HCP (LVD/BDP) and C, normal head growth BPD. Values (means ± SD [error bars]). BPD, biparietal diameter; ETV, endoscopic third ventriculostomy; FETV, fetal endoscopic third ventriculostomy; HCP, hydrocephalus; LVD, lateral ventricular diameters.

The degree of brain tissue compression in lambs with severe HCP increased to 0.288 ± 0.07 after 20 ± 5 days (E105), 0.299 ± 0.06 after 30 ± 5 days (E115), and 0.283 ± 0.05 after 50 ± 5 days (P1). In the moderate HCP group, the brain thinning or compression degree increased to 0.182 ± 0.03 after 20 ± 5 days (E105), 0.206 ± 0.01 after 30 ± 5 days (E115), and 0.213 ± 0.02 after 50 ± 5 days (P1), compared with 0.06 ± 0.004, 0.06 ± 0.007, and 0.1 ± 0.006 for the control animals (ANOVA, *P ≤ .0001, Figure 3B, Table). In the treated groups, we observed the same subgroupings, with 1 subgroup developing severe HCP (n = 10) and the other developing moderate HCP (n = 6), reaching 13.9 ± 1.72 mm and 7.55 ± 1.29 mm at E105, respectively (t-Test, *P ≤ .001, Figure 2A, Table).

These 2 subgroups responded positively to ETV treatment with reduced ventricle diameters. Ten days after treatment, LVDs in the severe HCP + ETV subgroup decreased to 9.5 ± 2.88 mm, and they decreased in the moderate HCP + ETV subgroup to 5.7 ± 0.41 mm (ANOVA, α #P ≤ .001, Figure 3B). Fetal lambs with severe HCP dropped to moderate (1 level) in the CHSS scale after ETV (E115), and, interestingly, fetal lambs in the moderate HCP + ETV subgroup returned to their normal LVD values at birth (P1) (Table).

We observed the same pattern of improvement with the degree of mechanical compression of the brain (LVD/BPD). Animals in the severe HCP + ETV subgroup went from 0.30 ± 0.07 at E105 to 0.19 ± 0.06, and animals in the moderate HCP subgroup went from 0.14 ± 0.02 to 0.10 ± 0.008 at 10 days post-ETV (ANOVA, α #P ≤ .01, Figure 3B). Finally, BPD values were not significantly different between the nontreated, treated, and normal control groups during the pregnancy (Figure 3C, Table). Using MRI, we observed an evident increase in brain mantel thickness in the fetal treated animals with ETV, as shown in 1 set of fetal twins, where 1 treated HCP + ETV (Figure 4A) shows clear improvement in brain thickness, and the other nontreated HCP fetal twin appears with very thin brain mantle and corpus callosum agenesis (Figure 4B).

FIGURE 4.

FIGURE 4.

Magnetic resonance imaging of HCP twins A MRI images showed reduced ventricle dilatation and higher fetal brain mantel thickness in the ETV treated group compared with B MRI images of the fetal twin showed severe hydrocephalus and corpus callosum agenesis in the HCP nontreated group at harvest time. ETV, endoscopic third ventriculostomy; HCP, hydrocephalus.

DISCUSSION

Invasive neurosurgical fetal therapy began in the early 1980s with the introduction of shunting procedures for HCP.16-21 During this time, HCP was frequently detected by ultrasound, and its natural progression was observed through serial sonographic examinations of untreated cases.28,29 Since then, fetal imaging has improved significantly, with advancements in MRI and ultrasound techniques enhancing diagnostic accuracy of isolated obstructive HCP and patient selection. Fetal ventriculomegaly can now be detected by ultrasound at 17-21 weeks of gestation and by MRI as early as 8-21 weeks.2,28-31

Ventricular shunting in newborns improves survival and maybe neurological outcomes in obstructive HCP, suggesting that a potential in utero decompression could prevent the progressive brain damage. The first fetal intervention for congenital HCP in humans was performed by Birnholz and Frigoletto,16,17 who used percutaneous cephalocentesis to decompress the cerebral lateral ventricles. However, due to the continuous production of CSF, this method did not reduce ventriculomegaly and increased hemorrhage risk.16,17 Subsequently, VAS were developed for more consistent decompression. Clewell et al18 reported the first VAS in utero in 1982, but these shunts often failed because of obstruction or migration,18,19 besides the inadequate patient selection, leading to a moratorium on fetal HCP treatment.

In 2003, Cavalheiro et al32 attempted fetal endoscopic treatment, performing ventriculoscopy in 3 human fetuses. They found a potential fetal ETV feasible but technically challenging because of fetal positioning. Moreover, the authors did not provide any endoscopic imaging, detailed descriptions, or results in their report.32,33

All these early attempts and failures helped establish indication criteria for fetal ETV, including isolated progressive obstructive ventriculomegaly and the exclusion of fetuses with genetic or other anomalies. Improved imaging technology, such as fetal MRI, and modern genetic testing have enhanced the identification of isolated progressive HCP.

These selected fetuses might benefit from in utero decompression, particularly those with aqueductal stenosis, Chiari II malformations, or Dandy-Walker syndrome. However, studies often have small sample sizes, making it difficult to conclusively determine the benefits of prenatal intervention. Generally, prenatally diagnosed HCP cases have poorer outcomes, suggesting that early brain damage is more severe during that critical period of fetal development. Oi et al indicated that sustained elevated CSF pressures for over a month could cause irreversible brain damage.30,34-36 Similarly, human fetuses that develop HCP during the second trimester are more likely to suffer significant brain damage from progressive HCP compared with those that develop it during the third trimester.5

Recently, a new fetal severe HCP sheep model was created by injecting BioGlue into the cisterna magna of fetal lambs, inducing HCP by obstruction without additional chemical neuroinflammatory responses.26 Normal brain development is likely to be irreversibly impaired by fetal severe HCP, and postnatal shunting or postnatal ETV are unlikely to reverse it. Early onset hydrocephalic patients may therefore have a benefit from early in utero decompression.

This sheep model allowed for the study of intrauterine endoscopic intraventricular intervention in obstructive HCP. We assessed the feasibility of fetal ETV for first time in that translational research large fetal animal model.27 Prenatal ETV was possible overcoming the differences between human and sheep anatomy, such as a narrower foramen of Monro and a more compact third ventricle.

This study demonstrates progressive increasing dimensions of the fetal ventricular system in the hydrocephalic group and the efficacy of prenatal ETV to reduce ventricular size and let the fetal brain to increase thickness. Cerebral ventricular dilatation was evident after BioGlue injections, with varying responses, allowing for subanalysis of fetal ETV effects in moderate and severe cases. While beneficial in both, the procedure had different impacts, with the moderate HCP subgroup returning to normal values at birth. These findings emphasize the importance of treating obstructive HCP in utero to decrease ventricular size before irreversible brain damage occurs.

Limitations

A limitation of this study is that the anatomy of fetal lambs does not fully replicate that of human fetuses. However, due to their larger size and longer gestational period, fetal lambs provide a more relevant comparative model for studying human fetal disease than smaller animals such as rodents. Another potential limitation is the relatively modest sample size, especially when compared with clinical trials or small animal studies. Nonetheless, most fetal large-animal studies assessing prenatal therapies typically use 4-6 subjects; in this study, we have exceeded that number. While this could be viewed as a limitation, it may also be considered a strength given the historical context of similar studies.

In addition, outcome measures in this study were limited to ventricular size and cortical thinning, without volumetric analysis of the brain. We are currently exploring the incorporation of brain volumetry in future studies to broaden our understanding of neurodevelopmental changes. Finally, there is an inherent limitation related to the uncertainty of the long-term implications of the observed ventricular size reduction. Although a transient decrease was documented, its durability over time remains unknown. Extended follow-up is therefore necessary to determine whether these morphological alterations persist and to assess their potential impact on postnatal phenotype.

Further research is needed to define brain injury mechanisms in HCP and, by analyzing cellular and molecular changes, determine the optimal therapeutic window for fetal intervention to prevent or revert brain damage. In addition, long-term postnatal follow-up are planned to assess outcomes and cognitive functions.

CONCLUSION

Prenatal fetal ETV demonstrated to be feasible and now efficient at arresting the progression of the disease by reducing brain lateral ventricular dilatation, that is the main indicator of HCP, in this ovine animal model. Moreover, the degree of brain mantle compression decreases significantly with increased cerebral thickness at delivery when this neuroendoscopic prenatal intervention is performed for severe or moderate fetal HCP in the fetal lamb.

Acknowledgments

We greatly appreciate the MRI technicians and veterinarian support at CCMIJU. Author contributions: Study concept and design: S.D., M.O., JL.P. Acquisition of data: M.O., S.D, B. F-T, FM. S-M., L.P., JL. E., JL.P. Analysis and interpretation of data: S.D., M.O., JL.P. Drafting of the manuscript: M.O., S.D., L.P., JL.P. Critical revision of the manuscript for important intellectual content: M.O., S.D., JL.E., FM.SM, JL.P. Statistical analysis: M.O. Obtained funding: FM. S-M., JL.P. Technical or material support: M.O. Study supervision: S.D., JL.P. M.O. Consent for publication: Authors read and approved the final manuscript.

Footnotes

*

Soner Duru and Marc Oria contributed equally to this work.

**

Francisco M. Sanchez-Margallo and Jose L. Peiro hold equal responsibility for this work.

Contributor Information

Soner Duru, Email: soner.duru@cchmc.org.

Marc Oria, Email: marc.oria@cchmc.org.

Blanca Fernandez-Tome, Email: ccmi@ccmijesususon.com.

Lucas Peiro, Email: lucaspeiro@gmail.com.

Jose L. Encinas, Email: joseluis.encinas@salud.madrid.org.

Francisco M. Sanchez-Margallo, Email: msanchez@ccmijesususon.com.

Funding

This work was supported by Prof. Jose L. Peiro internal CCHMC Pediatric Surgery Division funding, the Rudi Schulte Research Institute (RSRI) (USA), and by the Instituto Carlos III (ISCIII) & European Union (PI21/01886) (Spain).

Disclosures

Soner Duru was supported with a research scholarship by The Scientific and Technological Research Council of Turkey (TUBITAK) (2015/2/2219/1059B191501145). The other authors have no personal, financial, or institutional interest in any of the drugs, materials, or devices described in this article.

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