Raman spectroscopy analysis of human amniotic fluid cells from fetuses with myelomeningocele.

Prenatal surgery for the treatment of spina bifida (myelomeningocele, MMC) significantly enhances the neurological prognosis of the patient. To ensure better protection of the spinal cord by large defects, the application of skin grafts produced with cells gained from the amniotic fluid is presently studied. In order to determine the most appropriate cells for this purpose, we tried to shed light on the extremely complex amniotic fluid cellular composition in healthy and MMC pregnancies. We exploited the potential of micro-Raman spectroscopy to analyse and characterize human amniotic fluid cells in total and putative (cKit/CD117-positive) stem cells of fetuses with MMC in comparison with amniotic fluid cells from healthy individuals, human fetal dermal fibroblasts and adult adipose derived stem cells. We found that (i) the differences between healthy and MMC amniocytes can be attributed to specific spectral regions involving collagen, lipids, sugars, tryptophan, aspartate, glutamate, and carotenoids, (ii) MMC amniotic fluid contains two particular cell populations which are absent or reduced in normal pregnancies, (iii) the cKit-negative healthy amniocyte subpopulation shares molecular features with human fetal fibroblasts. On the one hand we demonstrate a different amniotic fluid cellular composition in healthy and MMC pregnancies, on the other our work confirms micro-Raman spectroscopy to be a valuable tool for discriminating cell populations in unknown mixtures of cells.

Immunohistochemical and Histopathological Characterization of Spina Bifida Defect Tissues Removed After Prenatal and Postnatal Surgical Repair.

Background: Myelomeningocele or spina bifida is an open neural tube defect that is characterized by protrusion of the meninges and the spinal cord through a deformity in the vertebral arch and spinous process. Myelomeningocele of post-natal tissue is well described; however, pre-natal tissue of this defect has no known previous histologic characterization. We compared the histology of different forms of pre-natal myelomeningocele and post-natal myelomeningocele tissue obtained via prenatal intrauterine and postnatal surgical repairs. Methods: Pre-and post-natal tissues from spina bifida repair surgeries were obtained from lipomyelomeningocele, myeloschisis, and myelomeningocele spina bifida defects. Tissue samples were processed for H&E and immunohistochemical staining (KRT14 and p63) to assess epidermal and dermal development. Results: Prenatal skin near the defect site develops with normal epidermal, dermal, and adnexal structures. Within the grossly cystic specimens, histology shows highly dense fibrous connective tissue with complete absence of a normal epidermal development with a lack of p63 and KRT14 expression. Conclusion: Tissues harvested from prenatal and postnatal spina bifida repair surgeries appear as normal skin near the defect site. However, cystic tissues consist of highly dense fibrous connective tissue with complete absence of normal epidermal development.

Folic acid supplementation rescues bladder injury in fetal rats with myelomeningocele.

BACKGROUND: Bladder dysfunction has been linked to the progression of renal failure in children with neurogenic bladder (NB) dysfunction. The purpose of this study was to determine whether bladder injuries in fetal rats with myelomeningocele (MMC) may be treated with folic acid. METHODS: Pregnant Sprague-Dawley rats were randomly divided into three groups. On the 10th day of gestation, pregnant rats were intragastrically injected with all-trans retinoic acid (ATRA) (60 mg/kg) to induce MMC fetal rats. The same amount of olive oil was put into the control group to create normal fetal rats. The rats in the rescue group were given folic acid (40 mg/kg) by gavage 0.5 and 12 hr after ATRA therapy. Bladders were obtained via cesarean section on embryonic day E20.5 and examined for MMC. The histology of the fetuses was examined using hematoxylin and eosin staining, and immunohistochemistry (IHC) was utilized to determine the expression of α-smooth muscle actin (α-SMA) and neuron-specific nuclear-binding protein (NeuN). Furthermore, the levels of neuromuscular development-related and apoptotic proteins were determined by western blotting. RESULTS: The incidence of MMC in the model group was 60.6% (20/33) while it was much lower in the rescue group (21.4%). In comparison to the model group, the weight and crown-rump length of the fetal rats in the rescue group were significantly improved. IHC revealed that there was no significant difference in the expression of α-SMA and NeuN between the control and ATRA groups, while the expression levels decreased significantly in the MMC group. Western blot analysis showed that there was no significant difference between the model and ATRA groups, but the expression of the α-SMA protein and the ß3-tubulin was much lower in the MMC group than in the control group. After the administration of folic acid, the α-SMA and ß3-tubulin proteins considerably increased in the folic acid-rescued MMC group and folic acid-rescued ATRA group. Meanwhile, in the control group, the expression of cleaved caspase-3 in the bladder tissue was significantly higher, and the expression of poly (ADP-ribose) polymerase (PARP) protein was significantly lower compared to the control group. Folic acid therapy reduced cleaved caspase-3 expression while increasing PARP expression in comparison to the MMC group. CONCLUSIONS: NB in MMC fetal rats is associated with the reduction of bladder nerve and smooth muscle-related protein synthesis. However, folic acid therapy can help improve these functional deficiencies. Folic acid also exhibits strong anti-apoptotic properties against NB in MMC fetal rats.

Intrauterine neuromuscular and stromal dysplasia of the bladder in retinoic acid-induced myelomeningocele fetal rats.

OBJECTIVES: To observe the changes in the bladder of fetal rats with myelomeningocele (MMC) induced by all-trans retinoic acid (atRA) during the embryonic development stages. METHODS: The fetal rat model of MMC was induced by intragastric administration of atRA to pregnant rats on embryonic day 10 (E10). Fetal rats were harvested at E16, E18, E20, and E21 for observation and further testing. Those with MMC were classified as the MMC group, while those without MMC as the RA group. The areas of MMC skin defect, the crown-rump length (CRL), and body weight in different groups were compared. The histopathological changes in the bladder were compared. The expression levels of alpha-smooth muscle actin (αSMA), smooth muscle myosin heavy chain (SMMHC), connexin 43 (Cx43), desmin, ß3 tubulin, and vesicular acetylcholine transporter (VAChT) in the bladder were investigated by immunohistochemical staining and Western blotting. Pregnant rats given intragastric administration with olive oil (OIL group) at E10 were set as the blank control group. RESULTS: A total of 415 fetal rats of different gestational ages were harvested with an MMC incidence of 56.05 % (139/248). The incidence rate increased with embryonic days (p < 0.001). Compared with the other two control groups, the CRL and bodyweight of MMC fetal rats were significantly delayed at E21 (p < 0.001). The expression levels of αSMA, SMMHC, Cx43, desmin, ß3 tubulin and VAChT in the bladder of MMC fetal rats were significantly decreased at E21 (p < 0.05). CONCLUSIONS: In atRA-induced MMC fetal rats, there is neural, muscular, and stromal dysplasia in the bladder at an early gestational age. Further investigations on neurogenic bladder secondary to MMC are applicable using this animal model.

Biodistribution of allogenic umbilical cord-derived mesenchymal stromal cells after fetal repair of myelomeningocele in an ovine model.

BACKGROUND: Myelomeningocele (MMC) is a spinal cord congenital defect that leads to paraplegia, sphincter disorders and potential neurocognitive disabilities. Prenatal surgery of MMC provides a significant benefit compared to surgery at birth. Mesenchymal stromal cell (MSC) therapy as an adjuvant treatment for prenatal surgery showed promising results in animal experiments which could be considered for clinical use in human fetuses. Despite numerous reassuring studies on the safety of MSCs administration in humans, no study focused on MSCs biodistribution after a local MSCs graft on the fetal spinal cord. AIM: The purpose of our study was to assess the biodistribution of umbilical cord-derived mesenchymal stromal cells (UC-MSCs) at birth in lambs who had a prenatal myelomeningocele repair using a fibrin patch seeded with allogenic UC-MSCs. METHODS: After isolation, UC-MSCs were tagged using a green fluorescent protein (GFP)-containing lentiviral vector. MMC defects were surgically created at 75 days of gestation and repaired 15 days later using UC-MSCs patch. Lambs were delivered at 142 days and sacrificed. DNA extraction was performed among biopsies of the different organs and q-PCR analysis was used to detect the expression of GFP (GFP DNA coding sequence). RESULTS: In our 6 surviving lambs grafted with UC-MSCs, GFP lentivirus genomic DNA was not detected in the organs. CONCLUSION: These reassuring data will support translational application in humans, especially since the first human clinical trial using mesenchymal stromal cells for in-utero treatment of MMC started recently in U.S.A.

Fetal and Perinatal Expression Profiles of Proinflammatory Cytokines in the Neuroplacodes of Rats with Myelomeningoceles: A Contribution to the Understanding of Secondary Spinal Cord Injury in Open Spinal Dysraphism.

The cellular and molecular mechanisms that presumably underlie the progressive functional decline of the myelomeningocele (MMC) placode are not well understood. We previously identified key players in post-traumatic spinal cord injury cascades in human MMC tissues obtained during postnatal repair. In this study, we conducted experiments to further investigate these mediators in the prenatal time course under standardized conditions in a retinoic acid-induced MMC rat model. A retinoic acid MMC model was established using time-dated Sprague-Dawley rats, which were gavage-fed with all-trans retinoic acid (RA; 60 mg/kg) dissolved in olive oil at E10. Control animals received olive oil only. Fetuses from both groups were obtained at E16, E18, and E22. The spinal cords (SCs) of both groups were formalin-fixed or snap-frozen. Tissues were screened by real-time reverse transcription polymerase chain reaction for the expression of cytokines and chemokines known to play a role in the lesion cascades of the central nervous system after trauma. MMC placodes exhibited inflammatory cells and glial activation in the later gestational stages. At the messenger RNA (mRNA) level, interleukin-1 beta, tumor necrosis factor alpha, and tumor necrosis factor receptor type 1 exhibited significant induction at E22. interleukin-1 beta receptor type 1 mRNA was induced significantly at E16 and E22. Double labeling experiments confirmed the co-staining of these cytokines and their receptors with ionized calcium-binding adapter molecule 1 (i.e., inflammatory cells), vimentin, and nestin in different anatomical SC areas and neuronal nuclear protein in ventral horn neurons. C-X-C motif chemokine 12 mRNA was elevated in control and MMC animals at E16 compared with E18 and E22. C-X3-C motif ligand 1 mRNA was lower in MMC tissues than in control tissues on E16. The presented findings contribute to the concept that pathophysiological mechanisms, such as cytokine induction in the neuroplacode, in addition to the "first hit," promote secondary spinal cord injury with functional loss in the late fetal time course. Further, these mediators should be taken into consideration in the development of new therapeutic approaches for open spinal dysraphism.

Preclinical stem cell therapy in fetuses with myelomeningocele: A systematic review and meta-analysis.

OBJECTIVE: We performed a systematic review to summarize the efficacy and safety of in utero stem cells application in preclinical models with myelomeningocele (MMC). METHODS: The study was registered with PROSPERO (CRD42019160399). We searched MEDLINE, Embase, Web of Science, Scopus and CENTRAL for publications articles on stem cell therapy in animal fetuses with MMC until May 2020. Publication quality was assessed by the SYRCLE's tool. Meta-analyses were pooled if studies were done in the same animal model providing similar type of stem cell used and outcome measurements. Narrative synthesis was performed for studies that could not be pooled. RESULTS: Nineteen and seven studies were included in narrative and quantitative syntheses, respectively. Most used mesenchymal stem cells (MSCs) and primarily involved ovine and rodent models. Both intra-amniotic injection of allogeneic amniotic fluid (AF)-MSCs in rat MMC model and the application of human placental (P)-MSCs to the spinal cord during fetal surgery in MMC ovine model did not compromise fetal survival rates at term (rat model, relative risk [RR] 1.03, 95% CI 0.92-1.16; ovine model, RR 0.94, 95% CI 0.78-1.13). A single intra-amniotic injection of allogeneic AF-MSCs into rat MMC model was associated with a higher rate of complete defect coverage compared to saline injection (RR 16.35, 95% CI 3.27-81.79). The incorporation of human P-MSCs as a therapeutic adjunct to fetal surgery in the ovine MMC model significantly improved sheep locomotor rating scale after birth (mean difference 5.18, 95% CI 3.36-6.99). CONCLUSIONS: Stem cell application during prenatal period in preclinical animal models is safe and effective.

Study of serum and urinary markers of the reninangiotensin-aldosterone system in myelomeningocele patients with renal injury detected by DMSA

ABSTRACT Introduction: The Renin-Angiotensin-Aldosterone System (RAAS) has been suggested as a possible marker of renal injury in chronic diseases. This study proposes to analyze the serum and urinary markers of the RAAS in myelomeningocele patients with renal function abnormalities detected on DMSA. Material and Methods: Seventeen patients followed in our institution that presented with renal injury on DMSA. We review nephrologic and urologic clinical aspects and evaluated ultrassonagraphy, voiding urethrocystography and urodynamics. Urinary and serum samples were collected to evaluate possible correlations of renal lesions with RAAS. Control group urine and serum samples were also sent for analysis. Results: Serum ACE 2 activity means in relation to urodynamic findings were the only values that had a statistically significant difference (p = 0.040). Patients with normal bladder pattern presented higher ACE 2 levels than the high risk group. Statistical analysis showed that the study group (SG) had a significantly higher mean serum ACE than the CG. The means of ACE 2 and urinary ACE of the SG and CG were not statistically different. The ROC curve for serum ACE values had a statistically significant area for case and non-case differentiation, with 100% sensitivity and 53% specificity for values above 60.2 mg/dL. No statistically significant areas were observed in relation to ACE 2 and urinary ACE values between SG and CG. Conclusion: The analysis of serum ACE, ACE 2 and urinary ACE were not significant in patients with myelomeningocele and neurogenic bladder with renal injury previously detected by renal DMSA.

Study of serum and urinary markers of the renin-angiotensin-aldosterone system in myelomeningocele patients with renal injury detected by DMSA.

INTRODUCTION: The Renin-Angiotensin-Aldosterone System (RAAS) has been suggested as a possible marker of renal injury in chronic diseases. This study proposes to analyze the serum and urinary markers of the RAAS in myelomeningocele patients with renal function abnormalities detected on DMSA. MATERIAL AND METHODS: Seventeen patients followed in our institution that presented with renal injury on DMSA. We review nephrologic and urologic clinical aspects and evaluated ultrassonagraphy, voiding urethrocystography and urodynamics. Urinary and serum samples were collected to evaluate possible correlations of renal lesions with RAAS. Control group urine and serum samples were also sent for analysis. RESULTS: Serum ACE 2 activity means in relation to urodynamic findings were the only values that had a statistically significant difference (p = 0.040). Patients with normal bladder pattern presented higher ACE 2 levels than the high risk group. Statistical analysis showed that the study group (SG) had a significantly higher mean serum ACE than the CG. The means of ACE 2 and urinary ACE of the SG and CG were not statistically different. The ROC curve for serum ACE values had a statistically significant area for case and non-case differentiation, with 100% sensitivity and 53% specificity for values above 60.2 mg/dL. No statistically significant areas were observed in relation to ACE 2 and urinary ACE values between SG and CG. CONCLUSION: The analysis of serum ACE, ACE 2 and urinary ACE were not significant in patients with myelomeningocele and neurogenic bladder with renal injury previously detected by renal DMSA.

Using poly(l-lactic acid) and poly(ɛ-caprolactone) blends to fabricate self-expanding, watertight and biodegradable surgical patches for potential fetoscopic myelomeningocele repair.

Our study focuses on the development and characterization of a self-expanding, watertight and biodegradable patch for fetoscopic myelomeningocele (MMC) prenatal repair. We fabricated poly(l-lactic acid) (PLA) and poly(ɛ-caprolactone) (PCL) blend films by solution casting. Formulation c with average glass transition temperature of 37.6 ± 1.2°C was chosen for temporospatial recovery. Favorable results from surface studies reflected homogeneous dispersion of polymers in the blend. The cytotoxicity was studied in human foreskin fibroblasts. The blend film was cytocompatible, evidenced by matching percentage of live cells in exposed and control solutions. Subsequently, liquid water permeability experiments confirmed watertight nature of films. Finally, in vitro degradation was investigated in phosphate buffered saline (PBS) and amniotic fluid (AF) separately for 16 weeks. Similar weight loss (n = 6, p = 0.912) and significantly different (n = 3, p = 0.025) surface roughness was observed in PBS and AF, respectively, at 16 weeks. Functional group analysis displayed increasing carbonyl and hydroxyl bonds in PBS and AF, respectively, over time, indicating progression of hydrolytic degradation. Favorable characterization results provide strong evidence to employ PLA-PCL blend films as surgical patches in fetoscopic MMC repair. Designed patch serves as standalone system to successfully tackle impending hurdles of MMC repair and proves to be a superior alternative compared to existing patches. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 107B: 295-305, 2019.

Altered Amniotic Fluid Levels of Hyaluronic Acid in Fetal Rats with Myelomeningocele: Understanding Spinal Cord Injury.

Myelomeningocele (MMC) is a devastating congenital neural tube defect that results in the exposure of spinal cord to the intrauterine environment, leading to secondary spinal cord injury and severe impairment. Although the mechanisms underlying the secondary pathogenesis are clinically relevant, the exact cause of in utero-acquired spinal cord damage remains unclear. The objective of this study was to determine whether the hyaluronic acid (HA) concentration in amniotic fluid (AF) in the retinoic acid-induced model of MMC is different from that in normal controls and whether these differences could have an impact on the viscosity of AF. Our data shows that the concentration of HA in AF samples from fetuses with MMC (MMC-AF) and normal control samples (Norm-AF) were not significantly different at embryonic day 18 (E18) and E20. Thereafter, the HA concentration significantly increased in Norm-AF but not in MMC-AF. Compared with Norm-AF, the concentration of HA in MMC-AF and the viscosity of MMC-AF were significantly lower at E21. Agarose gel electrophoresis confirmed a significant reduction in the HA level of MMC-AF compared with Norm-AF at E21. No HA-degrading activity was detected in MMC-AF. In summary, we identified a deficiency in the AF level of HA and the viscosity of AF in fetal rats with MMC. These data are discussed in relation to a potential role the reduction in the AF viscosity due to the low level of HA may play in the exacerbating effects of mechanical trauma on spinal cord damage at the MMC lesion site.

Human Amniotic Fluid Stem Cells: Therapeutic Potential for Perinatal Patients with Intractable Neurological Disease.

Mesenchymal stem cells (MSCs) have generated great interest in the fields of regenerative medicine and immunotherapy because of their unique biological properties. Among MSCs, amniotic fluid stem cells (AFS) have a number of characteristics that make them attractive candidates for tissue engineering and cell replacement strategies, particularly for perinatal medicine. If various neonatal conditions, including birth asphyxia, preterm birth, and congenital abnormalities, which result in long-lasting severe impairments, could be predicted during pregnancy, it would allow collection of small samples of amniotic fluid cells by amniocentesis. In vitro culture of these autologous AFS during pregnancy would make them available for use soon after birth. Hypoxic-ischemic encephalopathy (HIE) and myelomeningocele (MMC) are neonatal conditions that cause permanent neurological disability, for which the treatment options are extremely limited. Experiments using animal models of HIE and MMC and human clinical trials have demonstrated that MSCs, including AFS, have beneficial effects on the central nervous system through paracrine influences, indicating that autologous AFS treatment may be applicable for intractable neurological diseases, including HIE and MMC, during the perinatal period. In this review, we focus on recent research related to the therapeutic potential of AFS for perinatal neurological diseases such as HIE and MMC.

Composición corporal y gasto energético en población de niños y adolescentes con mielomeningocele / Body composition and energy expenditure in a population of children and adolescents with myelomeningocele

Introducción: El mielomeningocele es un defecto congénito con cierre incompleto del tubo neural. Presenta alteraciones en la composición corporal y alta prevalencia de obesidad. Es difícil detectar el indicador más apropiado para diagnóstico nutricional por impresición de las medidas antropométricas. Objetivo: Describir en una población de pacientes con mielomeningocele seguidos en el "Hospital Garrahan", la composición corporal, gasto energético en reposo y trastornos metabólicos, comparando los pacientes con mielomeningocele obesos con una población control con obesidad multifactorial. Población y Métodos: Se realizó antropometría, impedanciometría, pliegues cutáneos, perímetro braquial, calorimetría indirecta y determinaciones bioquímicas a todos los pacientes con mielomeningocele entre junio/2013-abril/2014, previa firma del Consentimiento Informado. Resultados: Se evaluaron 131 pacientes de 0,718,6 años, clasificados según Score-Z de Índice de Masa Corporal en 15% bajo peso, 42% normopeso, 12% sobrepeso y 31% obesidad. Se encontró alta correlación (r²0,74) entre %masa grasa por impedanciometría vs calculado con pliegues cutaneos. Los pacientes con % masa grasa elevada vs %masa grasa normal tuvieron mayor score-Z de Indice de Masa Corporal (1,07 vs -0,27 p0,0001) aunque ambos valores se encontraban dentro de parámetros normales. Hubo menor gasto energético en reposo en los pacientes con mielomeningocele obesos comparado con el esperado y con obesos multifactoriales. Conclusiones: Se encontró alto porcentaje de sobrepeso/obesidad en la población con mielomeningocele. Los pliegues cutáneos serían más apropiados para detectar obesidad. Los pacientes con mielomeningocele obesos presentaron gasto energetico en reposo menor al esperado y a los controles. La indicación de energía debe ser personalizada.

Introduction. Myelomeningocele is a congenital defect that occurs when the neural tube fails to close completely. It causes body composition alterations and a high prevalence of obesity. It is difficult to detect the most adequate indicator for a nutritional diagnosis due to the impossibility of recording accurate anthropometric measurements. Objective. To describe body composition, resting energy expenditure and metabolic disorders in a population of patients with myelomeningocele managed at "Hospital Garrahan" by comparing obese patients with myelomeningocele and a control population with multifactorial obesity. Population and methods. An anthropometry, an impedance analysis, skinfold equations, arm circumference equations, indirect calorimetry, and biochemical determinations were done to all patients with myelomeningocele between June 2013 and April 2014, once the informed consent had been signed. Results. 131 patients aged 0.7-18.6 years were assessed; they were classified according to their body mass index Z-score into low weight (15%), normal weight (42%), overweight (12%), and obese (31%). A high correlation (r: 20.74) was observed between the fat mass % measured by impedance analysis versus that estimated using skinfolds. Patients with a high fat mass % had a higher body mass index Z-score than those with a normal fat mass % (1.07 versus -0.27, p: 0.0001) although both values were within normal parameters. A lower resting energy expenditure was observed among obese patients with myelomeningocele than predicted and in comparison with multifactorial obese controls. Conclusions. A high percentage of overweight/obesity was found in the population with myelomeningocele. Skinfold equations would be more adequate to detect obesity. Obese patients with myelomeningocele had a lower resting energy expenditure than predicted and in comparison with controls. Energy indication should be customized.

Body composition and energy expenditure in a population of children and adolescents with myelomeningocele. / Composición corporal y gasto energético en población de niños y adolescentes con mielomeningocele.

INTRODUCTION: Myelomeningocele is a congenital defect that occurs when the neural tube fails to close completely. It causes body composition alterations and a high prevalence of obesity. It is difficult to detect the most adequate indicator for a nutritional diagnosis due to the impossibility of recording accurate anthropometric measurements. OBJECTIVE: To describe body composition, resting energy expenditure and metabolic disorders in a population of patients with myelomeningocele managed at "Hospital Garrahan" by comparing obese patients with myelomeningocele and a control population with multifactorial obesity. POPULATION AND METHODS: An anthropometry, an impedance analysis, skinfold equations, arm circumference equations, indirect calorimetry, and biochemical determinations were done to all patients with myelomeningocele between June 2013 and April 2014, once the informed consent had been signed. RESULTS: 131 patients aged 0.7-18.6 years were assessed; they were classified according to their body mass index Z-score into low weight (15%), normal weight (42%), overweight (12%), and obese (31%). A high correlation (r: 20.74) was observed between the fat mass % measured by impedance analysis versus that estimated using skinfolds. Patients with a high fat mass % had a higher body mass index Z-score than those with a normal fat mass % (1.07 versus -0.27, p: 0.0001) although both values were within normal parameters. A lower resting energy expenditure was observed among obese patients with myelomeningocele than predicted and in comparison with multifactorial obese controls. CONCLUSIONS: A high percentage of overweight/obesity was found in the population with myelomeningocele. Skinfold equations would be more adequate to detect obesity. Obese patients with myelomeningocele had a lower resting energy expenditure than predicted and in comparison with controls. Energy indication should be customized.

INTRODUCCIÓN: El mielomeningocele es un defecto congénito con cierre incompleto del tubo neural. Presenta alteraciones en la composición corporal y alta prevalencia de obesidad. Es difícil detectar el indicador más apropiado para diagnóstico nutricional por impresición de las medidas antropométricas. OBJETIVO: Describir en una población de pacientes con mielomeningocele seguidos en el "Hospital Garrahan", la composición corporal, gasto energético en reposo y trastornos metabólicos, comparando los pacientes con mielomeningocele obesos con una población control con obesidad multifactorial. POBLACIÓN Y MÉTODOS: Se realizó antropometría, impedanciometría, pliegues cutáneos, perímetro braquial, calorimetría indirecta y determinaciones bioquímicas a todos los pacientes con mielomeningocele entre junio/2013-abril/2014, previa firma del Consentimiento Informado. RESULTADOS: Se evaluaron 131 pacientes de 0,718,6 años, clasificados según Score-Z de Índice de Masa Corporal en 15% bajo peso, 42% normopeso, 12% sobrepeso y 31% obesidad. Se encontró alta correlación (r20,74) entre %masa grasa por impedanciometría vs calculado con pliegues cutaneos. Los pacientes con % masa grasa elevada vs %masa grasa normal tuvieron mayor score-Z de Indice de Masa Corporal (1,07 vs -0,27 p0,0001) aunque ambos valores se encontraban dentro de parámetros normales. Hubo menor gasto energético en reposo en los pacientes con mielomeningocele obesos comparado con el esperado y con obesos multifactoriales. CONCLUSIONES: Se encontró alto porcentaje de sobrepeso/obesidad en la población con mielomeningocele. Los pliegues cutáneos serían más apropiados para detectar obesidad. Los pacientes con mielomeningocele obesos presentaron gasto energetico en reposo menor al esperado y a los controles. La indicación de energía debe ser personalizada.

Amniotic Fluid Concentrations of Glial Fibrillary Acidic Protein Do Not Correlate with Prenatal Metrics in Fetuses with Myelomeningocele.

INTRODUCTION: We investigated the correlation of amniotic fluid (AF) concentrations of glial fibrillary acidic protein (GFAP) with prenatal features of myelomeningocele (MMC) and neurodevelopmental outcome after fetal MMC (fMMC) surgery. MATERIALS AND METHODS: AF was collected during fMMC surgery between December 2012 and November 2015. AF-GFAP concentration was determined by ELISA. Retrospective chart review identified the characteristics of the defect. Data regarding delivery and 1-year neurodevelopmental outcome was collected from The Children's Hospital of Philadelphia fMMC Registry. RESULTS: Eighty-two AF samples were collected from fMMC surgeries. Perinatal data were obtained from 77 subjects, and 1-year follow-up data from 65 subjects. GFAP concentrations were significantly elevated in MMC compared to myeloschisis (24.1 ± 2.9 and 10.3 ± 1.5 ng/mL; p < 0.0001). A larger percentage of subjects with myeloschisis defects delivered before their scheduled due date (myeloschisis 88.5%; MMC 55.0%; p = 0.003) and delivered at an earlier mean gestational age (34.6 ± 0.4 weeks, n = 26) compared to those with MMC defects (35.2 ± 0.4 weeks, n = 51) (p = 0.04). DISCUSSION: AF-GFAP levels differentiate between MMC and myeloschisis, and raise interesting questions regarding the clinical significance between the 2 types of defects.

Isolation, characterization, and differentiation of multipotent neural progenitor cells from human cerebrospinal fluid in fetal cystic myelomeningocele.

Despite benefits of prenatal in utero repair of myelomeningocele, a severe type of spina bifida aperta, many of these patients will still suffer mild to severe impairment. One potential source of stem cells for new regenerative medicine-based therapeutic approaches for spinal cord injury repair is neural progenitor cells (NPCs) in cerebrospinal fluid (CSF). To this aim, we extracted CSF from the cyst surrounding the exposed neural placode during the surgical repair of myelomeningocele in 6 fetuses (20 to 26weeks of gestation). In primary cultured CSF-derived cells, neurogenic properties were confirmed by in vitro differentiation into various neural lineage cell types, and NPC markers expression (TBR2, CD15, SOX2) were detected by immunofluorescence and RT-PCR analysis. Differentiation into three neural lineages was corroborated by arbitrary differentiation (depletion of growths factors) or explicit differentiation as neuronal, astrocyte, or oligodendrocyte cell types using specific induction mediums. Differentiated cells showed the specific expression of neural differentiation markers (ßIII-tubulin, GFAP, CNPase, oligo-O1). In myelomeningocele patients, CSF-derived cells could become a potential source of NPCs with neurogenic capacity. Our findings support the development of innovative stem-cell-based therapeutics by autologous transplantation of CSF-derived NPCs in damaged spinal cords, such as myelomeningocele, thus promoting neural tissue regeneration in fetuses.

Maternal gene-micronutrient interactions related to one-carbon metabolism and the risk of myelomeningocele among offspring.

BACKGROUND: Few studies have evaluated interactions between maternal genetic variation in 5,10-methylenetetrahydrofolate reductase (MTHFR) and micronutrient intake on the risk of myelomeningocele (MM) in offspring. Therefore, we sought to determine if the role of maternal MTHFR C677T and A1298C on MM risk is altered by maternal intake of micronutrients related to one-carbon metabolism. METHODS: The study consisted of 220 MM case-parent trios recruited from 1996 to 2006. A dietary questionnaire was used to obtain information on maternal dietary intake on eight micronutrients including folate and cobalamin. TaqMan assays were used to generate MTHFR C677T and A1298C genotypes. Log-linear models were used to evaluate the joint effects of maternal genotype and micronutrient intake dichotomized as at or above versus below the United States Recommended Dietary Allowance (US RDA) on MM. RESULTS: There was little evidence to suggest maternal MTHFR genotypes interacted with micronutrient intake to influence the risk of MM. For instance, the effect of MTHFR 677T was similar for mothers with cobalamin intake below US RDA (relative risk [RR] = 0.97) versus at or above US RDA cobalamin intake (RR = 0.81, interaction p = 0.87). However, some differences were noted. For example, the effect of MTHFR 1298C appeared to be different between those mothers below US RDA folate intake (RR = 0.98) versus those at or above US RDA folate intake (RR = 0.68), but the interaction was not statistically significant (interaction p = 0.27). CONCLUSION: There did not appear to be strong effects of maternal micronutrient intake on the role of maternal genetic polymorphisms in MTHFR C677T and A1298C on MM risk. Birth Defects Research 109:99-105, 2017. © 2016 Wiley Periodicals, Inc.

Does arsenic increase the risk of neural tube defects among a highly exposed population? A new case-control study in Bangladesh.

BACKGROUND: Neural tube defects are debilitating birth defects that occur when the developing neural plate fails to close in early gestation. Arsenic induces neural tube defects in animal models, but whether environmental arsenic exposure increases risk of neural tube defects in humans is unknown. METHODS: We describe a new case-control study in Bangladesh, a country currently experiencing an epidemic of arsenic poisoning through contaminated drinking water. We plan to understand how arsenic influences risk of neural tube defects in humans through mechanisms that include disruption of maternal glucose and folate metabolism, as well as epigenetic effects. We also investigate whether sweat chloride concentration, a potential new biomarker for arsenic toxicity, can be used to identify women at higher risk for having a child affected by neural tube defect. We will collect dural tissue from cases, obtained at the time of surgical closure of the defect, and believe investigation of these samples will provide insight into the epigenetic mechanisms by which prenatal arsenic exposure affects the developing nervous system. CONCLUSION: These studies explore mechanisms by which arsenic may increase risk of neural tube defects in humans and use a unique population with high arsenic exposure to test hypotheses. If successful, these studies may assist countries with high arsenic exposure such as Bangladesh to identify populations at high risk of neural tube defects, as well as direct development of novel screening strategies for maternal risk.Birth Defects Research 109:92-98, 2017.© 2016 The Authors Birth Defects Research Published by Wiley Periodicals, Inc.

Genetic association of the glycine cleavage system genes and myelomeningocele.

BACKGROUND: Neural tube defects (NTDs) are one of the most common congenital birth defects, with myelomeningocele (MM) being the most severe form compatible with life. Recent studies show a link between mitochondrial folate one carbon metabolism and NTDs by means of the glycine cleavage system (GCS). We hypothesize that single nucleotide polymorphisms and novel variants in the coding regions of the GCS genes increase the risk for MM. METHODS: DNA was obtained from 96 subjects with MM born before the United States mandated folic acid fortification of grains in 1998. Primers were designed for polymerase chain reaction amplification and sequencing of all exons in the AMT gene, one of four genes in the GCS, followed by identification of single nucleotide polymorphisms and novel variants. An additional 252 MM subjects underwent whole exome sequencing to examine all four GCS genes (aminomethyltransferase, glycine dehydrogenase, glycine cleavage system protein-H, and dihydrolipoamide dehydrogenase). RESULTS: We identified six novel, heterozygous variants in the AMT gene with three predicted to be deleterious to AMT function (p.Val7Leu, p.Pro251Arg, and p.Val380Met). Five extremely rare, known heterozygous variants were found in the AMT gene and one in the GLDC gene. No novel variants in the exons of the other two GCS genes (DLD and GCSH) were identified. CONCLUSION: We identified novel and rare, known variants in two of the four GCS genes that may contribute to the development of MM. Consistent with previous findings, the current study provides additional support that genetic variations in GCS genes contribute to the risk of NTDs. Birth Defects Research (Part A) 106:847-853, 2016. © 2016 Wiley Periodicals, Inc.

Cellular Profiles and Molecular Mediators of Lesion Cascades in the Placode in Human Open Spinal Neural Tube Defects.

Myelomeningoceles (mmc) are clinically challenging CNS malformations. Although improvement in their management has been achieved with respect to antenatal diagnosis, prevention, and fetal surgery, the cellular mechanisms of damage in the neural placode are poorly understood. We aimed to identify cellular and molecular factors in lesion amplifying cascades in mmc placodes. Seventeen mmc specimens obtained during reconstructive surgery that harbored sufficient neuroepithelial tissue were investigated. Normal adult and stillborn spinal cord tissue served as controls. Placodes exhibited similar cellular profiles with consistent neuronal marker expression, elevated GFAP-/vimentin immunoreactivity in all, and CD3/CD11b/CD68-immunolabeling in some cases. Increased expression of pro-inflammatory (tumor necrosis factor, interleukin-1ß [Il-1ß]/IL-1 receptor type 1 [IL-R1]) and neuroprotective erythropoietin/erythropoietin receptor (Epo/EpoR) cytokines was detected by immunohistochemistry, double-fluorescence labeling, and real-time RT-PCR. In all cases, there was a multi-cellular induction of IL-1ß and IL1-R1. EpoR and Epo immunoreactivity was elevated in some cases with neuronal expression patterns. Epo was further co-expressed with HIF-1/-2α, which paralleled Epo induction in the corresponding placodes. These observations confirm the induction of cellular and molecular alterations in human mmc placodes that resemble the secondary lesion cascades induced by spinal cord injury. The pro-inflammatory and neuroprotective cytokine expression in mmc placodes may represent new targets for the treatment of open neural tube defects.