Low torcular Herophili position and large brainstem-tentorium angle in fetuses with open spinal dysraphism at 11-13 weeks' gestation.

OBJECTIVE: To evaluate whether in fetuses with open spina bifida (OSB) the tentorium can be seen to be displaced downwards and vertically oriented by the time of the 11-13-week scan and whether this is reflected in an alteration of the brainstem-tentorium (BST) angle. METHODS: The study population was recruited between 2015 and 2020 from three fetal medicine referral centers and comprised a control group and a study group of pregnancies with OSB. The control group was recruited prospectively and included singleton pregnancies with a normal sonographic examination after first-trimester combined screening for chromosomal abnormalities and normal outcome. The study group was selected retrospectively and included all cases with OSB between 2015 and 2020. All cases underwent detailed ultrasound assessment at 11 + 0 to 13 + 6 weeks' gestation. The position of the torcular Herophili (TH) was identified in the midsagittal view of the fetal brain with the use of color Doppler and was considered as a proxy for the insertion of the tentorium on the fetal skull. The BST angle was calculated in the same view and was compared between the two groups. RESULTS: Sixty normal fetuses were included in the control group and 22 fetuses with OSB in the study group. In both groups, the BST angle was found to be independent of gestational age or crown-rump length (P = 0.8815, R2 = 0.0003861 in the controls, and P = 0.2665, R2 = 0.00978 in the OSB group). The mean BST angle was 48.7 ± 7.8° in controls and 88.1 ± 1.18°, i.e. close to 90°, in fetuses with OSB. Comparison of BST-angle measurements between the control group and cases with OSB showed a statistically significant difference (P = 0.0153). In all fetuses with OSB, the downward displacement of the TH and tentorium was clearly visible at the 11-13-week scan. CONCLUSIONS: In fetuses with OSB, the BST angle is significantly larger than in normal controls, with the tentorium being almost perpendicular to the brainstem. This sign confirms the inferior displacement of the tentorium cerebelli with respect to its normal insertion on the occipital clivus as early as the first trimester of pregnancy and is useful in the diagnosis of Chiari-II malformation at this early stage. In fetuses with OSB, the low position of the tentorium and TH is clearly visible, even subjectively, at the 11-13-week scan. © 2021 International Society of Ultrasound in Obstetrics and Gynecology.

Intrauterine fetoscopic closure of myelomeningocele: Clinical case and literature review.

OBJECTIVE: Spina bifida (SB) is a congenital birth defect defined as a failure of the neural tube formation during the embryonic development phase. Fetoscopic repair of SB is a novel treatment technique that allows to close spinal defect early and prevent potential neurological and psychomotor complications. CASE REPORT: We present a case report of a 32-year-old-multigravida whose fetus was diagnosed with lumbosacral myelomeningocele at 23rd week. Fetoscopic closure of MMC was performed at 26 weeks. At 32 weeks, due to premature amniorrhexis and placental abruption, an emergency C-section was performed. Newborn's psychomotor development was within normal limits. CONCLUSION: Although intrauterine treatment has an increased risk of premature labor, placental abruption, prenatal closure is associated with improved postnatal psychomotor development. Prenatal surgery decreases the risk of Arnold-Chiari II malformation development and walking disability. Fetoscopic closure of SB is becoming a choice for treatment with beneficial outcomes for mother and fetus.

Prenatal ultrasound diagnosis of neural tube defects in the era of intrauterine repair - Eleven years' experiences.

OBJECTIVE: To modify the current neural tube defect (NTD) classification for fetal medicine specialists, and to investigate the impact of prenatal ultrasound conus medullaris position screening on the detection rate of closed spinal dysraphism and pregnancy outcomes. MATERIALS AND METHODS: The clinical data of 112 patients prenatally diagnosed with neural tube defects in Taiji clinic from 2008 to 2018 were retrospectively analyzed. All cases were classified following the modified classification. We compared the detection rate before and after introducing the conus medullaris screening and pregnancy outcomes for NTD types. RESULTS: Closed spinal dysraphism type prevailed in our sample (43.8%). The median gestational age at the time of detection for cranial dysraphism was 13.3 weeks, open spinal dysraphism was 22.0 weeks, and closed spinal dysraphism was 22.6 weeks. All cranial dysraphism (n = 43) and open spinal dysraphism cases (n = 20) had pregnancies terminated. For closed spinal dysraphism Class 1, the live-birth rate was 100.0% in the cases without other anomalies and 33.3% in the cases with other anomalies, respectively (X2 = 17.25, p < 0.001). Similarly, for Class 2, pregnancy continuation rate was 50.0% in cases without other anomalies and 20.0% in cases with other anomalies, yet it failed to reach statistical significance (X2 = 0.9, p = 0.524). CONCLUSION: Our case series may help to improve early screening and prenatal diagnosis of NTDs. Modified classification is adjusted for use in ultrasound fetal care facilities, which could be used for predicting pregnancy outcome. We suggest promoting first-trimester anatomical screening in order to make an earlier diagnosis and therefore provide better prenatal care for open spinal dysraphism cases in the era of intrauterine repair. Our findings imply that the use of fetal conus medullaris position as a marker for closed spinal dysraphism improves the detection rate and would unlikely lead to a higher termination rate.

Prenatal and Postnatal Imaging Findings After Fetal Repair of Spinal Dysraphisms Using Cryopreserved Human Umbilical Cord Patch: A Case Series of 4 Patients.

We describe a case series of imaging findings of 4 patients who underwent spinal dysraphisms repair in utero with novel patch material, cryopreserved human umbilical cord, in our institution. In our study, the prenatal and postnatal magnetic resonance imaging and ultrasound are reviewed and showed cord tethering and syrinx progression in all cases. Our report is the first description of magnetic resonance imaging and ultrasound findings in the context of using this novel patch in severe cases of spinal dysraphisms.

Prenatal regenerative fetoscopic interventions for congenital anomalies.

Fetal intervention has progressed in the past two decades from experimental proof-of-concept to practice-adopted, life saving interventions in human fetuses with congenital anomalies. This progress is informed by advances in innovative research, prenatal diagnosis, and fetal surgical techniques. Invasive open hysterotomy, associated with notable maternal-fetal risks, is steadily replaced by less invasive fetoscopic alternatives. A better understanding of the natural history and pathophysiology of congenital diseases has advanced the prenatal regenerative paradigm. By altering the natural course of disease through regrowth or redevelopment of malformed fetal organs, prenatal regenerative medicine has transformed maternal-fetal care. This review discusses the uses of regenerative medicine in the prenatal diagnosis and management of three congenital diseases: congenital diaphragmatic hernia, lower urinary tract obstruction, and spina bifida.

Peri-operative management of percutaneous fetoscopic spina-bifida repair: a descriptive review of five cases from the United Kingdom, with focus on anaesthetic implications.

We present a case-based review of the first five percutaneous fetoscopic in-utero spina bifida repair procedures undertaken in the UK. Our focus is on implications of anaesthesia and analgesia for the mother and fetus, provision of uterine relaxation and fetal immobilisation while providing conditions conducive to surgical access. Minimising risks for fetal acidosis, placental and fetal hypoperfusion, maternal and fetal sepsis and maternal fluid overload were the foremost priorities. We discuss optimisation strategies undertaken to ensure fetal and maternal well-being under anaesthesia, shortcomings in the current approach, and possible directions for improvement.

Non-neural surface ectodermal rosette formation and F-actin dynamics drive mammalian neural tube closure.

The mechanisms underlying mammalian neural tube closure remain poorly understood. We report a unique cellular process involving multicellular rosette formation, convergent cellular protrusions, and F-actin cable network of the non-neural surface ectodermal cells encircling the closure site of the posterior neuropore, which are demonstrated by scanning electron microscopy and genetic fate mapping analyses during mouse spinal neurulation. These unique cellular structures are severely disrupted in the surface ectodermal transcription factor Grhl3 mutants that exhibit fully penetrant spina bifida. We propose a novel model of mammalian neural tube closure driven by surface ectodermal dynamics, which is computationally visualized.

Myelomeningocele sac associated with worse lower-extremity neurological sequelae: evidence for prenatal neural stretch injury?

OBJECTIVE: To determine whether the presence of a myelomeningocele (MMC) sac and sac size correlate with compromised lower-extremity function in fetuses with open spinal dysraphism. METHODS: A radiology database search was performed to identify cases of MMC and myeloschisis (MS) diagnosed prenatally in a single center from 2013 to 2017. All cases were evaluated between 18 and 25 weeks. Ultrasound reports were reviewed for talipes and impaired lower-extremity motion. In MMC cases, sac volume was calculated from ultrasound measurements. Magnetic resonance imaging reports were reviewed for hindbrain herniation. The association of presence of a MMC sac and sac size with talipes and impaired lower-extremity motion was assessed. Post-hoc analysis of data from the multicenter Management of Myelomeningocele Study (MOMS) randomized controlled trial was performed to confirm the study findings. RESULTS: In total, 283 MMC and 121 MS cases were identified. MMC was associated with a lower incidence of hindbrain herniation than was MS (80.9% vs 100%; P < 0.001). Compared with MS cases, MMC cases with hindbrain herniation had a higher rate of talipes (28.4% vs 16.5%, P = 0.02) and of talipes or lower-extremity impairment (34.9% vs 19.0%, P = 0.002). Although there was a higher rate of impaired lower-extremity motion alone in MMC cases with hindbrain herniation than in MS cases, the difference was not statistically significant (6.6% vs 2.5%; P = 0.13). Among MMC cases with hindbrain herniation, mean sac volume was higher in those associated with talipes compared with those without talipes (4.7 ± 4.2 vs 3.0 ± 2.6 mL; P = 0.002). Review of the MOMS data demonstrated similar findings; cases with a sac on baseline imaging had a higher incidence of talipes than did those without a sac (28.2% vs 7.5%; P = 0.007). CONCLUSIONS: In fetuses with open spinal dysraphism, the presence of a MMC sac was associated with fetal talipes, and this effect was correlated with sac size. The presence of a larger sac in fetuses with open spinal dysraphism may result in additional injury through mechanical stretching of the nerves, suggesting another acquired mechanism of injury to the exposed spinal tissue. Copyright © 2019 ISUOG. Published by John Wiley & Sons Ltd.

Spinal Dysraphia, Chiari 2 Malformation, Unified Theory, and Advances in Fetoscopic Repair.

Fetal spina bifida, the most common nonlethal birth defect of the central nervous system, results in substantial neurologic morbidity. The unified theory describes the complex relationship between local spinal lesions and development of Chiari 2 malformation, contributing to hydrocephalus. Prenatal ultrasonography reliably allows diagnosis, but fetal MR imaging is an important complement to identify additional brain abnormalities. Fetal surgery improves neurologic and motor outcomes, but various approaches, either open hysterotomy or minimally invasive to the uterus, carry substantial obstetric risks. Optimization of the fetoscopic approach aims to minimize maternal and obstetric risks, but data regarding longer-term outcomes are awaited.

Dominant negative GPR161 rare variants are risk factors of human spina bifida.

Spina bifida (SB) is a complex disorder of failed neural tube closure during the first month of human gestation, with a suspected etiology involving multiple gene and environmental interactions. GPR161 is a ciliary G-protein coupled receptor that regulates Sonic Hedgehog (Shh) signaling. Gpr161 null and hypomorphic mutations cause neural tube defects (NTDs) in mouse models. Herein we show that several genes involved in Shh and Wnt signaling were differentially expressed in the Gpr161 null embryos using RNA-seq analysis. To determine whether there exists an association between GPR161 and SB in humans, we performed direct Sanger sequencing on the GPR161 gene in a cohort of 384 SB patients and 190 healthy controls. We identified six rare variants of GPR161 in six SB cases, of which two of the variants were novel and did not exist in any databases. Both of these variants were predicted to be damaging by SIFT and/or PolyPhen analysis. The novel GPR161 rare variants mislocalized to the primary cilia, dysregulated Shh and Wnt signaling and inhibited cell proliferation in vitro. Our results demonstrate that GPR161 mutations cause NTDs via dysregulation of Shh and Wnt signaling in mice, and novel rare variants of GPR161 can be risk factors for SB in humans.

Donor mesenchymal stem cell linetics after transamniotic stem cell therapy (TRASCET) for experimental spina bifida.

PURPOSE: We sought to examine donor mesenchymal stem cell (MSC) kinetics after transamniotic stem cell therapy (TRASCET) in experimental spina bifida. METHODS: Pregnant Sprague-Dawley dams exposed to retinoic acid for the induction of fetal neural tube defects received volume-matched intra-amniotic injections on gestational day 17 (E17; term=E22): either amniotic fluid MSCs (afMSCs) labeled with a luciferase reporter gene (n=78), or luciferase protein alone (n=66). Samples from twelve organ systems from each surviving fetus with spina bifida (total n=60) were screened via microplate luminometry at term. RESULTS: Donor afMSCs were identified exclusively in the placenta, umbilical cord, spleen, bone marrow, hip bones, defect, and brain. Luminometry was negative in control fetuses receiving luciferase alone (p<0.001). Signal intensity in relative light units (RLUs) was moderately correlated between the defect and the hip bones (rho=0.38, p=0.048), and between the placenta and the brain (rho=0.40, p=0.038). CONCLUSIONS: Amniotic mesenchymal stem cells engraft to specific sites after concentrated intra-amniotic injection in the setting of spina bifida. A hematogenous route encompassing the bone marrow as well as distant central nervous system homing are fundamental constituents of cell trafficking. These findings must be considered during eventual patient selection for transamniotic stem cell therapy in the prenatal management of spina bifida.

Neural tube closure depends on expression of Grainyhead-like 3 in multiple tissues.

Failure of neural tube closure leads to neural tube defects (NTDs), common congenital abnormalities in humans. Among the genes whose loss of function causes NTDs in mice, Grainyhead-like3 (Grhl3) is essential for spinal neural tube closure, with null mutants exhibiting fully penetrant spina bifida. During spinal neurulation Grhl3 is initially expressed in the surface (non-neural) ectoderm, subsequently in the neuroepithelial component of the neural folds and at the node-streak border, and finally in the hindgut endoderm. Here, we show that endoderm-specific knockout of Grhl3 causes late-arising spinal NTDs, preceded by increased ventral curvature of the caudal region which was shown previously to suppress closure of the spinal neural folds. This finding supports the hypothesis that diminished Grhl3 expression in the hindgut is the cause of spinal NTDs in the curly tail, carrying a hypomorphic Grhl3 allele. Complete loss of Grhl3 function produces a more severe phenotype in which closure fails earlier in neurulation, before the stage of onset of expression in the hindgut of wild-type embryos. This implicates additional tissues and NTD mechanisms in Grhl3 null embryos. Conditional knockout of Grhl3 in the neural plate and node-streak border has minimal effect on closure, suggesting that abnormal function of surface ectoderm, where Grhl3 transcripts are first detected, is primarily responsible for early failure of spinal neurulation in Grhl3 null embryos.

Stage specific requirement of platelet-derived growth factor receptor-α in embryonic development.

BACKGROUND: Platelet-derived growth factor receptor alpha (PDGFRα) is a cell-surface receptor tyrosine kinase for platelet-derived growth factors. Correct timing and level of Pdgfra expression is crucial for embryo development, and deletion of Pdgfra caused developmental defects of multiple endoderm and mesoderm derived structures, resulting in a complex phenotypes including orofacial cleft, spina bifida, rib deformities, and omphalocele in mice. However, it is not clear if deletion of Pdgfra at different embryonic stages differentially affects these structures. PURPOSE: To address the temporal requirement of Pdgfra in embryonic development. METHODS: We have deleted the Pdgfra in Pdgfra-expressing tissues at different embryonic stages in mice, examined and quantified the developmental anomalies. RESULTS: Current study showed that (i) conditional deletion of Pdgfra at different embryonic days (between E7.5 and E10.5) resulted in orofacial cleft, spina bifida, rib cage deformities, and omphalocele, and (ii) the day of Pdgfra deletion influenced the combinations, incidence and severities of these anomalies. Deletion of Pdgfra caused apoptosis of Pdgfra-expressing tissues, and developmental defects of their derivatives. CONCLUSION: Orofacial cleft, spina bifida and omphalocele are among the commonest skeletal and abdominal wall defects of newborns, but their genetic etiologies are largely unknown. The remarkable resemblance of our conditional Pdgfra knockout embryos to theses human congenital anomalies, suggesting that dysregulated PDGFRA expression could cause these anomalies in human. Future work should aim at defining (a) the regulatory elements for the expression of the human PDGFRA during embryonic development, and (b) if mutations / sequence variations of these regulatory elements cause these anomalies.

Apoptosis, Expression of PAX3 and P53, and Caspase Signal in Fetuses with Neural Tube Defects.

BACKGROUND: Neural tube defects (NTDs) are among the most common and severe congenital malformations of the central nervous system. Animal studies have shown that apoptosis is involved in the development of NTDs. However, little evidence is available from human studies. We aim to examine the level of apoptosis and expression of apoptosis-regulating proteins of human terminated fetuses. METHODS: A total of 37 NTD cases and 21 controls from pregnancy terminations were recruited. Tissues of the central nervous system were obtained through autopsy. Apoptosis of neuroepithelial cells was examined by terminal deoxynucleotidyl transferase-mediated deoxyuridinetriphosphate nick end-labeling (TUNEL) assay. Expression of PAX3, p53, and caspase 3/8/9 in central nervous tissue was measured using Western blotting. RESULTS: More TUNEL-positive apoptosis cells were observed in the central nervous tissue of NTD cases than those of controls (p < 0.05). In spinal cord tissue, lower PAX3 expression, higher p53 expression, and increased levels of cleaved caspase 3(17kD) and cleaved caspase 8 (18kD) were found in anencephaly cases but not in spina bifida cases when compared with controls. In brain tissue, levels of PAX3 were significantly reduced in both encephalocele and spina bifida subtypes; the expression levels of cleaved caspase 3(17 kD) of encephalocele cases and cleaved caspase 8(47/45 kD) in spina bifida cases were higher than in controls; no difference was found in the expression of p53 or caspase 9 between NTDs and controls. CONCLUSION: These findings provide some evidence that excessive apoptosis in fetal central nervous tissues may be associated with the development of human NTDs. Birth Defects Research 109:1596-1604, 2017. © 2017 Wiley Periodicals, Inc.

Embryological and clinical implications of the association between anorectal malformations and spinal dysraphisms.

PURPOSE: To describe the association of anorectal malformation (ARM) and spinal dysraphism (SD) in terms of impact on the management of SD and embryogenetic implications. METHODS: Patients with SD associated with (A) or without (B) ARM were included. The two groups were further divided into operated on (A1/B1) or not (A2/B2) for SD. Groups A and B were compared for type of SD (embryogenetic classification) and prevalence of neurosurgery; Groups A1 vs. A2 for type of ARM (Wingspread classification); Groups A1 vs. B1 for age at neurosurgery, neurophysiology, and clinical symptoms. MAIN RESULTS: One hundred twenty-one patients with SD, 83 with and 38 without ARM were consecutively treated (1999-2015). Group A was associated only with SDs developing after primary neurulation, corresponding to the period of cloacal septation and organogenesis (p = 0.0007). Untethering surgery was significantly less frequent in Group A (p < 0.0001 and p = 0.04, respectively). Higher ARMs were not associated with increased risk for neurosurgery. No other significant differences were detected. CONCLUSIONS: In our series, ARMs were associated only with SD developing after primary neurulation, suggesting a single insult leading to both SD and the associated ARM. Neurosurgery is indicated less frequently in patients with ARM-associated SD, despite the similar preoperative clinical features.

Normal intracranial BS/BSOB ratio values in the first trimester of single gestations with live fetuses in a Korean population.

AIM: The purpose of this study was to determine the normal length of the brainstem (BS) in Korean fetuses and to evaluatethe usefulness of the routine measurement of BS size in the first trimester of pregnancy for the early detection of spina bifida. MATERIAL AND METHODS: A total of 2,621 normal singleton pregnant Korean women at 10+6 to 13+6 weeks of gestation were selected for this retrospective cross-sectional study. Ultrasonography was used to measure the length of the longest vertical depth diameter of the BS and brainstem-occipital bone (BSOB) in order to obtain the BS to BSOB ratio. RESULTS: The best indicators for spina bifida ranged from 1.00±0.24 mm to 4.70±0.46 mm for the BS and from 2.90±0.36 mm to 8.50±0.92 mm for the BSOB. For the gestational period, BS (R=0.70) and BSOB (R=0.81) values were considered statistically significant (p<.0001). The value of the BS to BSOB ratio was <1.0 in normal fetuses, and was not correlated with the gestational age. CONCLUSION: Measurement of BS and BSOB diameter in the first trimester is thought to provide the best reference marker for evaluating the posterior brain for diagnosis of spina bifida.

Preliminary investigation of methylation status of microRNA-124a in spinal cords of rat fetuses with congenital spina bifida.

OBJECTIVE: We investigated the expression of microRNA-124a and its methylation status in the spinal cords of rats with congenital spina bifida versus rats with normal fetuses. METHODS: Real-time quantitative reverse transcription-polymerase chain reaction was used to compare the expression of microRNA-124a in the spinal cords of 42 rats with all-trans retinoic acid induced congenital spina bifida and 42 rats with normal fetuses. The DNA methylation status in the promoter region of miRNA-124a was detected using methylation specific-PCR. RESULTS: Compared with rats with normal fetuses, expression of microRNA-124a was significantly decreased in rats with congenital spina bifida fetuses. The percentages of spinal cords with DNA hypermethylation in the microRNA-124a promoter were 81% and 14% in the congenital spina bifida and normal control groups, respectively. The difference was statistically significant. Further apoptosis testing revealed increased apoptosis cell numbers in the congenital spina bifida samples. Meanwhile, the phosphorylated mitogen-activated protein kinase protein expression level dramatically decreased in the congenital spina bifida samples. CONCLUSION: Aberrant DNA methylation was responsible for down-regulation of microRNA-124a by regulating the mitogen-activated protein kinase pathway, suggesting that microRNA-124a is a potential diagnostic biomarker in congenital spina bifida.

Cryopreserved human umbilical cord patch for in-utero spina bifida repair.

OBJECTIVE: To identify a patch system to repair surgically created spina bifida in a sheep model for its efficacy in healing the skin defect, protecting the underlying spinal cord and reducing the Chiari II malformation. METHODS: Spina bifida was created surgically in 16 fetuses from eight timed-pregnant sheep at gestational age of 75 days. Two fetuses did not survive the procedure. Repeat hysterotomy was performed at 95 days' gestation to cover the defect with either biocellulose film with underwater adhesive (BCF-adhesive) (n = 7) or human umbilical cord with suture (HUC-suture) (n = 7). Three fetuses without formation of the defect served as reference controls. The skin healing was examined by direct visualization after a planned Cesarean section at term, followed by histological analysis using hematoxylin and eosin and Masson's trichrome stains. Mid-sagittal sections of the fetal cranium and upper cervical spine were analyzed by a pediatric neuroradiologist who was blinded to the type of patch received. RESULTS: Three fetuses that received the BCF-adhesive and six fetuses that received the HUC-suture survived to term for final analysis. As a result of dislodgment of the BCF-adhesive, all spina bifida defects repaired using BCF-adhesive were not healed and showed exposed spinal cord with leakage of cerebrospinal fluid. In contrast, all spinal defects repaired by HUC-suture were healed with complete regrowth of epidermal, dermal and subdermal tissue components, with no exposed spinal cord. The maximal skin wound width was 21 ± 3.6 mm in the BCF-adhesive group but 3 ± 0.8 mm in the HUC-suture group (P < 0.001). The spinal cord area (P = 0.001) and the number of anterior horn cells (P = 0.03) was preserved to a greater degree in the HUC-suture group than in the BCF-adhesive group, whilst psammoma bodies, signifying neuronal degeneration, were only observed in the BCF-adhesive group. Anatomic changes, indicative of Chiari II malformation, were seen in all three fetuses of the BCF-adhesive group but in none of the HUC-suture group (P < 0.01). CONCLUSION: Cryopreserved umbilical cord graft is a promising regenerative patch for intrauterine repair of spina bifida.

Cell death and cell proliferation in human spina bifida.

BACKGROUND: Spina bifida is a multifactorial congenital malformation of the central nervous system. The aim of this study was to ascertain the relevance of cell death/proliferation balance in human spina bifida and to assess autophagy distribution and levels during embryo-fetal development in neural tissue. METHODS: Five human cases with myelomeningocoele were compared with 10 healthy human controls and LC3 protein expression was also analyzed in mouse embryos. Cell death was evaluated using TUNEL (terminal deoxynucleotidyl transferase-mediated deoxyuridinetriphosphate nick end-labeling) assay; cell proliferation was studied by counting Ki67-positive cells, and autophagy was assessed by observing the presence of LC3 punctuate dots. RESULTS: Comparing human cases and controls (13 to 21 weeks of gestation), we observed a significant increase in TUNEL-positive cells in human spina bifida associated with a significantly decreased proliferation rate, indicating an alteration of the physiological cell rate homeostasis. LC3 distribution was found to be spatiotemporally regulated in both human and murine embryo-fetuses: in early pregnancy a diffuse and ubiquitous LC3 signal was detected. After neural tube closure, an intense LC3-positive signal, normally associated to extra energy requirement, was confined to the Lissauer's tract, the dorsolateral spinal zone containing centrally projecting axons from dorsal root ganglia, at any medullar levels. LC3 signal disappeared from 12 weeks of gestation. CONCLUSION: In conclusion, this study confirms the fundamental role of cell death/proliferation balance during central nervous system development and reports the changing expression of LC3 protein in mouse and human neural tube. Birth Defects Research (Part A) 106:104-113, 2016. © 2015 Wiley Periodicals, Inc.