NAD+ deficiency in human congenital malformations and miscarriage: A new model of pleiotropy.

Pleiotropy is defined as the phenomenon of a single gene locus influencing two or more distinct phenotypic traits. However, nicotinamide adenine dinucleotide (NAD+) deficiency through diet alone can cause multiple or single malformations in mice. Additionally, humans with decreased NAD+ production due to changes in pathway genes display similar malformations. Here, I hypothesize NAD+ deficiency as a pleiotropic mechanism for multiple malformation conditions, including limb-body wall complex (LBWC), pentalogy of Cantrell (POC), omphalocele-exstrophy-imperforate anus-spinal defects (OEIS) complex, vertebral-anal-cardiac-tracheoesophageal fistula-renal-limb (VACTERL) association (hereafter VACTERL), oculoauriculovertebral spectrum (OAVS), Mullerian duct aplasia-renal anomalies-cervicothoracic somite dysplasia (MURCS), sirenomelia, and urorectal septum malformation (URSM) sequence, along with miscarriages and other forms of congenital malformation. The term Congenital NAD Deficiency Disorder (CNDD) could be considered for patients with these malformations; however, it is important to emphasize there have been no confirmatory experimental studies in humans to prove this hypothesis. In addition, these multiple malformation conditions should not be considered individual entities for the following reasons: First, there is no uniform consensus of clinical diagnostic criteria and all of them fail to capture cases with partial expression of the phenotype. Second, reports of individuals consistently show overlapping features with other reported conditions in this group. Finally, what is currently defined as VACTERL is what I would refer to as a default label when more striking features such as body wall defects, caudal dysgenesis, or cloacal exstrophy are not present.

Hyperemesis gravidarum and vitamin K deficiency: a systematic review.

Hyperemesis gravidarum (HG), severe nausea and vomiting in pregnancy, can lead to vitamin deficiencies. Little is known about HG-related vitamin K deficiency. We aimed to summarise available evidence on the occurrence of HG-related vitamin K deficiency and corresponding maternal and neonatal complications. A systematic review was conducted, searching Medline and EMBASE from inception to 12 November 2020. We identified 1564 articles, of which we included fifteen in this study: fourteen case reports (n 21 women) and one retrospective cohort study (n 109 women). Nine out of twenty-one women reported in case reports had a prolonged prothrombin time (PT). The cohort study measured PT in 39/109 women with HG, of whom 10/39 women (26 %) had prolonged PT. In total, 30-50 % women received vitamin K supplementation after vitamin K deficiency had been diagnosed. Four case reports (n 4 women) reported corresponding maternal complications, all consisting of coagulopathy-related haemorrhage. Nine case reports (n 16 neonates) reported corresponding neonatal complications including intracranial haemorrhage (n 2 neonates) and embryopathy (n 14 neonates), which consisted of Binder phenotype (n 14 neonates), chondrodysplasia punctata (n 9 neonates) and grey matter heterotopia (n 3 neonates). In conclusion, vitamin K deficiency and related complications occur among women with HG. In our systematic review, we were unable to assess the incidence rate.

Maternal SARS-CoV-2 infection during pregnancy: possible impact on the infant.

The risk and potential consequences of mother-to-child transmission of severe acute respiratory syndrome-coronavirus type 2 (SARS-CoV-2) during pregnancy are still a matter of debate. We studied the impact of SARS-CoV-2 infection on 56 complete households, including 27 newborns whose mothers were pregnant when exposed to the virus. Two PCR-confirmed perinatal SARS-CoV-2 transmissions with mild symptoms in affected neonates were recorded. In addition, we observed a severe eye malformation (unilateral microphthalmia, optic nerve hypoplasia, and congenital retinopathy) associated with maternal SARS-CoV-2 infection in weeks 5 and 6 of embryonic development. This embryopathy could not be explained by other infectious agents, genetic factors, drug use, or maternal disease during pregnancy. Eight other women with a history of SARS-CoV-2 infection prior to gestational week 12, however, delivered healthy infants.Conclusion: The repeated occurrence of mother-to-child transmission in our cohort with risks that remain incompletely understood, such as long-term effects and the possibility of an embryopathy, should sensitize researchers and stimulate further studies as well as support COVID-19 vaccination recommendations for pregnant women. Trial registration number: NCT04741412. Date of registration: November 18, 2020 What is Known: •Materno-fetal transmission of severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) during pregnancy has rarely been reported so far, but was demonstrated in isolated cases. What is New: •In a study of complete households with documented SARS-CoV-2 infection, including a cohort of pregnant women, we observed perinatal coronavirus transmission at a higher frequency than expected. •We also describe a newborn boy with an eye malformation reminiscent of rubella embryopathy but associated with early gestation SARS-CoV-2 infection of his mother. •A coronavirus-related embryopathy, reported here for the first time, is a finding that requires further investigation.

[Thalidomide and unilateral limb defects: the Italian chapter of a neverending story]. / Talidomide e difetti monolaterali degli arti: il capitolo italiano di una storia infinita.

Fifty years after the event, Italy introduced legislation to compensate malformations in children - now in their sixties - born to mothers who had been prescribed the antiemetic drug thalidomide for morning sickness. However, compensation has been denied to people 'only' damaged in one half of their body as opposed to those with bilateral malformations. The present study reviews the papers describing case series of children born with 'thalidomide embryopathy' in the UK, Germany, and other countries around 1960. Most clinical series were not organized on the basis of inclusion/exclusion criteria, thus allowing for probable selection and information biases on maternal use of thalidomide. In any case, they included a sizable number of children with a unilateral limb defect born from mothers certainly exposed to thalidomide during the relevant pregnancy. In many of these children, limb defects were associated with visceral malformations, as frequently observed following exposure to thalidomide in utero. Similarly, later literature reviews were not bias-free in their choice of articles, as is the case of a recently proposed 'diagnostic algorithm' for thalidomide-caused specific malformations and of the advice by the Italian National Institute of Health ruling out the possibility of thalidomide producing unilateral limb defects. Overall, the scientific evidence suggests that thalidomide can cause unilateral limb defects.

Continued use of Warfarin in lower dose has safe maternal and neonatal outcomes in pregnant women with Prosthetic Heart Valves.

BACKGROUND AND OBJECTIVE: There has been concerns regarding the safety of Warfarin in pregnant females due to its teratogenic potential. At the same time warfarin provides best anticoagulation in patients with prosthetic valves. Various dosage regimes have been tried to strike a balance between safety of mother and the avoidance of congenital anomalies in the newborn. This study was conducted to observe the effect of Warfarin in pregnant mothers taking different doses of warfarin, and their neonatal outcome, in our outdoor patients. METHODS: This is a cross sectional observational study conducted at the Faisalabad Institute of Cardiology. The pregnant mothers taking warfarin for prosthetic valve replacement who presented to our specialized clinic between November 2016 to April 2017 were included in the study. These included a total of 75 females between the age of 20-35 years. To compare the dose related effect of warfarin, two groups of the patients were formed. One group comprised of patients taking warfarin ≤5mg while the other group consisted of those who were taking >5mg of warfarin daily. These patients were followed till their delivery. The information was collected about the maternal and fetal outcomes. The maternal outcomes including mode of delivery/miscarriage, peripartum bleeding and any valve related thromboembolic complications. The fetal outcomes included birth weight, maturity, embryopathy and congenital anomaly in the baby. RESULTS: Patient's mean age was 29.25±3.75 years. The mitral valve replacement was present in 60% patients (n=45) while 25.3% patients (n=19) had aortic valve replacement and 14.7% patients (n=11) had double valve replacement. In this group 30 patients (40%) had taken <5 mg warfarin and 45 patients (60%) had received >5 mg warfarin medicine. Miscarriages, cesarean sections, low birth weight and prematurity were more common in patients receiving warfarin >5 mg with p-values 0.005, 0.046, 0.01 and 0.033 respectively. No case of fetal embryopathy was found in both groups. CONCLUSION: No case of embryopathy was found in each group which signifies that warfarin in lower doses is safe anticoagulant in patients with prosthetic valve replacements.

Mechanisms of Congenital Malformations in Pregnancies with Pre-existing Diabetes.

PURPOSE OF REVIEW: Fetuses of diabetic mothers are at increased risk for congenital malformations. Research in recent decades using animal and embryonic stem cell models has revealed many embryonic developmental processes that are disturbed by maternal diabetes. The aim of this review is to give clinicians a better understanding of the reasons for rigorous glycemic control in early pregnancy, and to provide background to guide future research. RECENT FINDINGS: Mouse models of diabetic pregnancy have revealed mechanisms for altered expression of tissue-specific genes that lead to malformations that are more common in diabetic pregnancies, such as neural tube defects (NTDs) and congenital heart defects (CHDs), and how altered gene expression causes apoptosis that leads to malformations. Embryos express the glucose transporter, GLUT2, which confers susceptibility to malformation, due to high rates of glucose uptake during maternal hyperglycemia and subsequent oxidative stress; however, the teleological function of GLUT2 for mammalian embryos may be to transport the amino sugar glucosamine (GlcN) from maternal circulation to be used as substrate for glycosylation reactions and to promote embryo cell growth. Malformations in diabetic pregnancy may be not only due to excess glucose uptake but also due to insufficient GlcN uptake. Avoiding maternal hyperglycemia during early pregnancy should prevent excess glucose uptake via GLUT2 into embryo cells, and also permit sufficient GLUT2-mediated GlcN uptake.

Deficiency of the oxidative stress-responsive kinase p70S6K1 restores autophagy and ameliorates neural tube defects in diabetic embryopathy.

BACKGROUND: Autophagy is highly active in neuroepithelial cells of the developing neuroepithelium, and impairment of autophagy leads to neural tube defects. In this study, we have found that maternal diabetes suppresses autophagy that leads to neural tube defects and consequent cellular imbalance in the endoplasmic reticulum where critical events occur, leading to the induction of diabetic embryopathy. Because the mammalian target of rapamycin pathway suppresses autophagy, we hypothesized that 70 kDa ribosomal protein S6 kinase 1 (p70S6K1), a major downstream effector of mammalian target of rapamycin, mediates the inhibitory effect of maternal diabetes on autophagy in the developing neuroepithelium. OBJECTIVE: We investigated whether p70S6K1 mediates the inhibitory effect of maternal diabetes on autophagy during neurulation. We also examined whether p70S6K1 deficiency restores autophagy and therefore relieves endoplasmic reticulum stress and inhibits maternal diabetes-induced apoptosis, which leads to reduction in neural tube defect incidence in diabetic embryopathy. STUDY DESIGN: Female p70S6K1 heterogeneous knockout (p70S6K1+/-) mice were bred with male p70S6K1 heterogeneous knockout (p70S6K1+/-) mice to generate wild-type (WT), p70S6K1+/- and p70S6K1 knockout (p70S6K1-/-) embryos. Embryos at embryonic day 8.5 were harvested for the assessment of indices of autophagy, endoplasmic reticulum stress, and apoptosis. Neural tube defect incidence in embryos was determined at embryonic day 10.5. For in vitro studies, small interfering RNA knockdown of p70S6K1 in C17.2 mouse neural stem cells was used to determine the effect of p70S6K1 deficiency on autophagy impairment and endoplasmic reticulum stress under high glucose conditions. RESULTS: Knockout of the Rps6kb1 gene, which encodes for p70S6K1, ameliorated maternal diabetes-induced NTDs and restored autophagosome formation in neuroepithelial cells suppressed by maternal diabetes. Maternal diabetes-suppressed conversion of LC3-I (microtubule-associated protein 1A/1B-light chain 3) to LC3-II, an index of autophagic activity, in neurulation stage embryos was abrogated in the absence of p70S6K1. p70S6K1 knockdown in neural stem cells also restored autophagosome formation and the conversion of LC3-I to LC3-II. The activation of the major unfolded protein response, indicated by phosphorylation of inositol-requiring enzyme 1 alpha, and protein kinase R-like endoplasmic reticulum kinase, and eukaryotic translation initiation factor 2α, and the increase of the endoplasmic reticulum stress marker, C/EBP homologous protein, were induced by maternal diabetes in vivo and high glucose in vitro. Unfolded protein response and endoplasmic reticulum stress induced by maternal diabetes or high glucose were reduced by Rps6kb1 deletion or p70S6K1 knockdown, respectively. Rps6kb1 knockout blocked maternal diabetes-induced caspase cleavage and neuroepithelial cell apoptosis. The superoxide dismutase mimetic Tempol abolished high glucose-induced p70S6K1 activation. CONCLUSION: The study revealed the critical involvement of p70S6K1 in the pathogenesis of diabetic embryopathy.

Treatment of Graves' hyperthyroidism with thionamides: a position paper on indications and safety in pregnancy.

CONTEXT: Graves' disease affects 3% of women and 0.5% of men in the general population. The first line treatment of Graves' hyperthyroidism is based on the administration of antithyroid drugs (ATD), propylthiouracil (PTU), methimazole (MMI) and carbimazole. A recent warning from the Italian Drug Agency (Agenzia Italiana del Farmaco AIFA) reported the risk of MMI-induced acute pancreatitis. In addition, AIFA highlighted the possible association of MMI treatment during the first trimester of pregnancy with congenital malformations, thus recommending the use of effective contraceptive methods in women of childbearing age treated with MMI. METHODS AND RESULTS: Revision of literature reported less than ten cases of the alleged MMI pancreatitis, allowing the inclusion of MMI in class III drug regarding the relative risk for drug-induced pancreatitis. Data available on the effect of hyperthyroidism per se on the risk of fetal malformations, although scanty, are sufficient to recommend treatment with ATD of the hyperthyroid pregnant woman. Case reports and population studies either suggesting or not suggesting MMI-induced fetal malformations do not allow unquestionable conclusions on this matter. CONCLUSIONS: This consensus by experts from Italian Endocrine and Gynecologic Scientific Societies has edited recommendations derived form the available data and published guidelines of International Scientific Societies.

Formation of neurodegenerative aggresome and death-inducing signaling complex in maternal diabetes-induced neural tube defects.

Diabetes mellitus in early pregnancy increases the risk in infants of birth defects, such as neural tube defects (NTDs), known as diabetic embryopathy. NTDs are associated with hyperglycemia-induced protein misfolding and Caspase-8-induced programmed cell death. The present study shows that misfolded proteins are ubiquitinylated, suggesting that ubiquitin-proteasomal degradation is impaired. Misfolded proteins form aggregates containing ubiquitin-binding protein p62, suggesting that autophagic-lysosomal clearance is insufficient. Additionally, these aggregates contain the neurodegenerative disease-associated proteins α-Synuclein, Parkin, and Huntingtin (Htt). Aggregation of Htt may lead to formation of a death-inducing signaling complex of Hip1, Hippi, and Caspase-8. Treatment with chemical chaperones, such as sodium 4-phenylbutyrate (PBA), reduces protein aggregation in neural stem cells in vitro and in embryos in vivo. Furthermore, treatment with PBA in vivo decreases NTD rate in the embryos of diabetic mice, as well as Caspase-8 activation and cell death. Enhancing protein folding could be a potential interventional approach to preventing embryonic malformations in diabetic pregnancies.

Disturbed intracellular calcium homeostasis in neural tube defects in diabetic embryopathy.

Pregnancies complicated by preexisting maternal diabetes mellitus are associated with a higher risk of birth defects in infants, known as diabetic embryopathy. The common defects seen in the central nervous system result from failure of neural tube closure. The formation of neural tube defects (NTDs) is associated with excessive programmed cell death (apoptosis) in the neuroepithelium under hyperglycemia-induced intracellular stress conditions. The early cellular response to hyperglycemia remains to be identified. We hypothesize that hyperglycemia may disturb intracellular calcium (Ca2+) homeostasis, which perturbs organelle function and apoptotic regulation, resulting in increased apoptosis and embryonic NTDs. In an animal model of diabetic embryopathy, we performed Ca2+ imaging and observed significant increases in intracellular Ca2+ ([Ca2+]i) in the embryonic neural epithelium. Blocking T-type Ca2+ channels with mibefradil, but not L-type with verapamil, significantly blunted the increases in [Ca2+]i, implicating an involvement of channel type-dependent Ca2+ influx in hyperglycemia-perturbed Ca2+ homeostasis. Treatment of diabetic pregnant mice with mibefradil during neurulation significantly reduced NTD rates in the embryos. This effect was associated with decreases in apoptosis, alleviation of endoplasmic reticulum stress, and increases of anti-apoptotic factors. Taken together, our data suggest an important role of Ca2+ influx in hyperglycemia-induced NTDs and of T-type Ca2+ channels as a potential target to prevent birth defects in diabetic pregnancies.

Aberrant DNA methylation as a diagnostic biomarker of diabetic embryopathy.

PURPOSE: Maternal diabetes is a known teratogen that can cause a wide spectrum of birth defects, collectively referred to as diabetic embryopathy (DE). However, the pathogenic mechanisms underlying DE remain uncertain and there are no definitive tests to establish the diagnosis. Here, we explore the potential of DNA methylation as a diagnostic biomarker for DE and to inform disease pathogenesis. METHODS: Bisulfite sequencing was used to identify gene regions with differential methylation between DE neonates and healthy infants born with or without prenatal exposure to maternal diabetes, and to investigate the role of allele-specific methylation at implicated sites. RESULTS: We identified a methylation signature consisting of 237 differentially methylated loci that distinguished infants with DE from control infants. These loci were found proximal to genes associated with Mendelian syndromes that overlap the DE phenotype (e.g., CACNA1C, TRIO, ANKRD11) or genes known to influence embryonic development (e.g., BRAX1, RASA3). Further, we identified allele-specific methylation (ASM) at 11 of these loci, within which 61.5% of ASM single-nucleotide variants are known expression quantitative trait loci (eQTLs). CONCLUSIONS: Our study suggests a role for aberrant DNA methylation and cis-sequence variation in the pathogenesis of DE and highlights the diagnostic potential of DNA methylation for teratogenic birth defects.

The increased activity of a transcription factor inhibits autophagy in diabetic embryopathy.

BACKGROUND: Maternal diabetes induces neural tube defects and stimulates the activity of the forkhead box O3 (Fox)O3a in the embryonic neuroepithelium. We previously demonstrated that deleting the FOXO3a gene ameliorates maternal diabetes-induced neural tube defects. Macroautophagy (hereafter referred to as "autophagy") is essential for neurulation. Rescuing autophagy suppressed by maternal diabetes in the developing neuroepithelium inhibits neural tube defect formation in diabetic pregnancy. This evidence suggests a possible link between FoxO3a and impaired autophagy in diabetic embryopathy. OBJECTIVE: We aimed to determine whether maternal diabetes suppresses autophagy through FoxO3a, and if the transcriptional activity of FoxO3a is required for the induction of diabetic embryopathy. STUDY DESIGN: We used a well-established type 1 diabetic embryopathy mouse model, in which diabetes was induced by streptozotocin, for our in vivo studies. To determine if FoxO3a mediates the inhibitory effect of maternal diabetes on autophagy in the developing neuroepithelium, we induced diabetic embryopathy in FOXO3a gene knockout mice and FoxO3a dominant negative transgenic mice. Embryos were harvested at embryonic day 8.5 to determine FoxO3a and autophagy activity and at embryonic day 10.5 for the presence of neural tube defects. We also examined the expression of autophagy-related genes. C17.2 neural stem cells were used for in vitro examination of the potential effects of FoxO3a on autophagy. RESULTS: Deletion of the FOXO3a gene restored the autophagy markers, lipidation of microtubule-associated protein 1A/1B-light chain 3I to light chain 3II, in neurulation stage embryos. Maternal diabetes decreased light chain 3I-positive puncta number in the neuroepithelium, which was restored by deleting FoxO3a. Maternal diabetes also decreased the expression of positive regulators of autophagy (Unc-51 like autophagy activating kinase 1, Coiled-coil myosin-like BCL2-interacting protein, and autophagy-related gene 5) and the negative regulator of autophagy, p62. FOXO3a gene deletion abrogated the dysregulation of autophagy genes. In vitro data showed that the constitutively active form of FoxO3a mimicked high glucose in repressing autophagy. In cells cultured under high-glucose conditions, overexpression of the dominant negative FoxO3a mutant blocked autophagy impairment. Dominant negative FoxO3a overexpression in the developing neuroepithelium restored autophagy and significantly reduced maternal diabetes-induced apoptosis and neural tube defects. CONCLUSION: Our study revealed that diabetes-induced FoxO3a activation inhibited autophagy in the embryonic neuroepithelium. We also observed that FoxO3a transcriptional activity mediated the teratogenic effect of maternal diabetes because dominant negative FoxO3a prevents maternal diabetes-induced autophagy impairment and neural tube defect formation. Our findings suggest that autophagy activators could be therapeutically effective in treating maternal diabetes-induced neural tube defects.

Life situation of women impaired by Thalidomide embryopathy in North Rhine-Westphalia - a comparative analysis of a recent cross-sectional study with earlier data.

BACKGROUND: Between 1957 and 1961 the substance Thalidomide was sold in West Germany and taken by many women as a sedative during pregnancy. This lead to miscarriages and infants been born with several severe malformations. The aim of this study was to describe the current situation of women impaired by Thalidomide induced embryopahty in North Rhine-Westphalia (Nordrhein-Westfalen), Germany, in comparison with the results found in a study done in 2002 by Nippert et al. METHODS: Questionnaires as well as examinations were performed. Data were compared using descriptive and inductive statistical methods. RESULTS: Both studies show that women impaired by Thalidomide embryopathy face a poorer health status than women their age in the general population and live in fear of further deteriorating health. The majority can only work reduced hours or are already retired due to poor health. Most of those who need assistance are being assisted by their social environment, while professional care is still utilized in only few cases. CONCLUSIONS: An obvious need for a shift in the provision of assistance and/or care provided was found as the social environment supporting the impaired women is also aging and therefore in high danger of breaking apart. TRIAL REGISTRATION: The study has been registered at German Clinical Trials Register, DRKS00010593 , on 07.06.2016 retrospectively.

Warfarin-exposed zebrafish embryos resembles human warfarin embryopathy in a dose and developmental-time dependent manner - From molecular mechanisms to environmental concerns.

Warfarin is the most worldwide used anticoagulant drug and rodenticide. Since it crosses placental barrier it can induce warfarin embryopathy (WE), a fetal mortality in neonates characterized by skeletal deformities in addition to brain hemorrhages. Although the effects of warfarin exposure in aquatic off target species were already described, the particular molecular toxicological mechanisms during early development are still unclear. Here, we used zebrafish (Danio rerio) to describe and compare the developmental effects of warfarin exposure (0, 15.13, 75.68 and 378.43 mM) on two distinct early developmental phases (embryos and eleuthero-embryos). Although exposure to both developmental phases induced fish mortality, only embryos exposed to the highest warfarin level exhibited features mimicking mammalian WE, e.g. high mortality, higher incidence of hemorrhages and altered skeletal development, among other effects. To gain insights into the toxic mechanisms underlying warfarin exposure, the transcriptome of embryos exposed to warfarin was explored through RNA-Seq and compared to that of control embryos. 766 differentially expressed (564 up- and 202 down-regulated) genes were identified. Gene Ontology analysis revealed particular cellular components (cytoplasm, extracellular matrix, lysosome and vacuole), biological processes (mainly amino acid and lipid metabolism and response to stimulus) and pathways (oxidative stress response and apoptosis signaling pathways) being significantly overrepresented in zebrafish embryos upon warfarin exposure. Protein-protein interaction further evidenced an altered redox system, blood coagulation and vasculogenesis, visual phototransduction and collagen formation upon warfarin exposure. The present study not only describes for the first time the WE in zebrafish, it provides new insights for a better risk assessment, and highlights the need for programming the rat eradication actions outside the fish spawning season to avoid an impact on off target fish community. The urge for the development of more species-specific anticoagulants for rodent pest control is also highlighted.

Progress in Development of Interventions to Prevent Birth Defects in Diabetic Pregnancies.

Diabetic embryopathy is a diabetic complication, in which maternal hyperglycemia in early pregnancy causes birth defects in newborn infants. Under maternal diabetic conditions, hyperglycemia disturbs intracellular molecular activities and organelles functions. These include protein misfolding in the endoplasmic reticulum (ER), overproduction of reactive oxygen species (ROS) in mitochondria, and high levels of nitric oxide (NO). The resultant ER, oxidative, and nitrosative stresses activate apoptotic machinery to cause cell death in the embryo, ultimately resulting in developmental malformations. Based on the basic research data, efforts have been made to develop interventional strategies to alleviate the stress conditions and to reduce embryonic malformations. One of the challenges in birth defect prevention is to identify effective and safe agents to be used in pregnancy. One approach is to search and characterize naturally occurring phytochemicals, including flavonoids, curcuminoids and stilbenoids, for use in prevention of diabetic embryopathy.

CPS49-induced neurotoxicity does not cause limb patterning anomalies in developing chicken embryos.

Thalidomide notoriously caused severe birth defects, particularly to the limbs, in those exposed in utero following maternal use of the drug to treat morning sickness. How the drug caused these birth defects remains unclear. Many theories have been proposed including actions on the forming blood vessels. However, thalidomide survivors also have altered nerve patterns and the drug is known for its neurotoxic actions in adults following prolonged use. We have previously shown that CPS49, an anti-angiogenic analog of thalidomide, causes a range of limb malformations in a time-sensitive manner in chicken embryos. Here we investigated whether CPS49 also is neurotoxic and whether effects on nerve development impact upon limb development. We found that CPS49 is neurotoxic, just like thalidomide, and can cause some neuronal loss late developing chicken limbs, but only when the limb is already innervated. However, CPS49 exposure does not cause defects in limb size when added to late developing chicken limbs. In contrast, in early limb buds which are not innervated, CPS49 exposure affects limb area significantly. To investigate in more detail the role of neurotoxicity and its impact on chicken limb development we inhibited nerve innervation at a range of developmental timepoints through using ß-bungarotoxin. We found that neuronal inhibition or ablation before, during or after limb outgrowth and innervation does not result in obvious limb cartilage patterning or number changes. We conclude that while CPS49 is neurotoxic, given the late innervation of the developing limb, and that neuronal inhibition/ablation throughout limb development does not cause similar limb patterning anomalies to those seen in thalidomide survivors, nerve defects are not the primary underlying cause of the severe limb patterning defects induced by CPS49/thalidomide.

Overexpression of p53 explains isotretinoin's teratogenicity.

The precise molecular basis of retinoid embryopathy is yet unknown. This hypothesis predicts that isotretinoin (13-cis retinoic acid), the prodrug of all-trans retinoic acid (ATRA), exaggerates neural crest cell (NCC) apoptosis via upregulation of the pro-apoptotic transcription factor p53, the guardian of the genome. Increased p53 signalling is associated with Treacher Collins-, CHARGE- and fetal alcohol syndrome, which exhibit dysmorphic craniofacial features resembling retinoid embryopathy. In addition, developmental studies of NCC homeostasis in the zebrafish support the pivotal role of p53. Translational evidence implies that isotretinoin-stimulated overactivation of p53 during embryogenesis represents the molecular basis of isotretinoin's teratogenicity.

Congenital lumbar hernia-A feature of diabetic embryopathy?

Congenital lumbar hernia is a rare anomaly consisting of protrusion of abdominal organs or extraperitoneal tissue through a defect in the lateral abdominal wall. The majority of affected patients have additional anomalies in a pattern described as the lumbocostovertebral syndrome. We report four patients born to mothers with poorly controlled diabetes with congenital lumbar hernia. All patients exhibited features of lumbocostovertebral syndrome with lumbar hernia, multiple vertebral segmentation anomalies in the lower thoracic and/or upper lumbar spine, rib anomalies, and unilateral renal agenesis. Additional anomalies present in the patients included preaxial hallucal polydactyly, abnormal situs, and sacral dysgenesis, anomalies known to be associated with diabetic embryopathy. At least 11 other patients have been previously reported with the lumbocostovertebral syndrome in the setting of maternal diabetes. We suggest that congenital lumbar hernia and the lumbocostovertebral syndrome are related to diabetic embryopathy.

Oxidative stress-induced miR-27a targets the redox gene nuclear factor erythroid 2-related factor 2 in diabetic embryopathy.

BACKGROUND: Maternal diabetes induces neural tube defects, and oxidative stress is a causal factor for maternal diabetes-induced neural tube defects. The redox gene nuclear factor erythroid 2-related factor 2 is the master regulator of the cellular antioxidant system. OBJECTIVE: In this study, we aimed to determine whether maternal diabetes inhibits nuclear factor erythroid 2-related factor 2 expression and nuclear factor erythroid 2-related factor 2-controlled antioxidant genes through the redox-sensitive miR-27a. STUDY DESIGN: We used a well-established type 1 diabetic embryopathy mouse model induced by streptozotocin for our in vivo studies. Embryos at embryonic day 8.5 were harvested for analysis of nuclear factor erythroid 2-related factor 2, nuclear factor erythroid 2-related factor 2-controlled antioxidant genes, and miR-27a expression. To determine if mitigating oxidative stress inhibits the increase of miR-27a and the decrease of nuclear factor erythroid 2-related factor 2 expression, we induced diabetic embryopathy in superoxide dismutase 2 (mitochondrial-associated antioxidant gene)-overexpressing mice. This model exhibits reduced mitochondria reactive oxygen species even in the presence of hyperglycemia. To investigate the causal relationship between miR-27a and nuclear factor erythroid 2-related factor 2 in vitro, we examined C17.2 neural stem cells under normal and high-glucose conditions. RESULTS: We observed that the messenger RNA and protein levels of nuclear factor erythroid 2-related factor 2 were significantly decreased in embryos on embryonic day 8.5 from diabetic dams compared to those from nondiabetic dams. High-glucose also significantly decreased nuclear factor erythroid 2-related factor 2 expression in a dose- and time-dependent manner in cultured neural stem cells. Our data revealed that miR-27a was up-regulated in embryos on embryonic day 8.5 exposed to diabetes, and that high glucose increased miR-27a levels in a dose- and time-dependent manner in cultured neural stem cells. In addition, we found that a miR-27a inhibitor abrogated the inhibitory effect of high glucose on nuclear factor erythroid 2-related factor 2 expression, and a miR-27a mimic suppressed nuclear factor erythroid 2-related factor 2 expression in cultured neural stem cells. Furthermore, our data indicated that the nuclear factor erythroid 2-related factor 2-controlled antioxidant enzymes glutamate-cysteine ligase catalytic subunit, glutamate-cysteine ligase modifier subunit, and glutathione S-transferase A1 were down-regulated by maternal diabetes in embryos on embryonic day 8.5 and high glucose in cultured neural stem cells. Inhibiting miR-27a restored expression of glutamate-cysteine ligase catalytic subunit, glutamate-cysteine ligase modifier subunit, and glutathione S-transferase A1. Overexpressing superoxide dismutase 2 reversed the maternal diabetes-induced increase of miR-27a and suppression of nuclear factor erythroid 2-related factor 2 and nuclear factor erythroid 2-related factor 2-controlled antioxidant enzymes. CONCLUSION: Our study demonstrates that maternal diabetes-induced oxidative stress increases miR-27a, which, in turn, suppresses nuclear factor erythroid 2-related factor 2 and its responsive antioxidant enzymes, resulting in diabetic embryopathy.