Rengasamy Padmanabhan

ABSTRACT  Epileptic women do not withdraw antiepileptic drug (AED) therapy during pregnancy, therefore, exposure to AED during preimplantation stages might result in considerable embryonic concentrations endangering development. Neither clinical nor experimental research has addressed this important issue adequately. Vigabatrin (VGB), a second generation AED, is both effective and well tolerated as an add‐on therapy in epilepsy with partial seizures. However, there is little data on the possible reproductive toxicity of this widely used drug. The objective of the present study was to evaluate the effects of VGB on pregnancy and pregnancy outcome in an experimental model. VGB was administered in single doses of 450 mg/kg intraperitoneally (IP) to groups of mice on one of gestation days (GD) 1, 3, or 5. The treated animals consumed moderately reduced amounts of food and water on the day of treatment, so the controls were saline‐injected and food and water‐restricted to match the amounts consumed by the experimental animals. All animals were killed on GD 18. VGB treatment did not interfere with implantation, nor did it cause significant embryo resorption. However, it caused significant reduction in fetal bodyweight and increased frequency of growth restricted fetuses which weighed two standard deviations (SD) less than the mean of the controls. The VGB group fetuses also had retarded development of the skeletal system in terms of delay in maturity of the suproccipital bone development, cervical and coccygeal vertebral hypoplasia, and poor ossification of the bones of the fore and hind paws. Another major finding was the increased incidence of minor malformations, such as the presence of cervical ribs and sternal anomalies. The results of this study show that VGB administered at preimplantation stages of development causes intrauterine growth restriction (IUGR) and augments minor malformation rates in mice. Future studies must address the mechanisms of VGB‐induced IUGR and minor malformations.

Abstract:  Uncontrolled diabetes mellitus (DM) is an etiological factor for recurrent pregnancy loss, fetal growth disorders, and major congenital malformations in the offspring. Antioxidant therapy has been advocated to overcome the oxidant–antioxidant disequilibrium inherent in diabetes. The objective of this article was to evaluate the beneficial effects of α‐lipoic acid (LA) on fetal outcome in a mouse model of streptozotocin (STZ)‐induced DM. Timed pregnant mice were made diabetic by intraperitoneal (IP) injection of a single dose of STZ (200 mg/kg) on gestation day (GD) 2. Diabetic animals were supplemented daily with an IP injection of 15 mg/kg of LA starting on GD 4 and continued through GD 12. Fetuses were examined on GD 18 for malformations and growth restriction. Some diabetic mice injected with Evans blue were examined on GD 3.5 and GD 6.5 to evaluate frequency of implantations. STZ‐treated mice had all cardinal signs of DM. LA treatment did not normalize blood glucose levels of DM mice. Rates of pregnancy in saline control, DM, and DM + LA groups were 90%, 28%, and 64%, respectively, indicating that LA promotes pregnancy in DM animals. However, postimplantation resorption showed a threefold increase in the DM + LA group. Rates of intrauterine growth restriction and major congenital malformations were also augmented thus indicating that the interaction between DM and LA has deleterious effects on postimplantation embryos.

Hyperthermia is teratogenic to human and animal embryos and induces mainly anomalies of the nervous system. However, the teratogenic mechanism is poorly understood. Mammalian embryos are known to switch from anaerobic to aerobic metabolism around the time of neural tube closure. This critical event might be sensitive to hyperthermia. The objective of the present study was to evaluate the ultrastructural changes of the mitochondria of the neuroepithelium (NE) of rat embryos following maternal exposure to hyperthermia. Pregnant rats were heat stressed for an hour on gestation day (GD) 9 and embryos were examined by electron microscopy on GD 10. NE presented extensive apoptosis. Intercellular junctions were weakened and copious cellular debris projected into the ventricle. The mitochondria were of diverse size and shape. Most of them were swollen and had short cristae and electron dense matrix. Hydropic changes were also observed in numerous mitochondria. Lipid-laden mitochondria were found in the apical portions of neuroblasts. The mesenchyme (ME) of heat-treated embryos showed paucity of cells and only as frequent apoptosis as the controls. Their mitochondria also showed changes similar to those of the NE. Additionally extensive lipid accumulation was observed in and in the vicinity of mitochondria, often surrounded by short strands of endoplasmic reticulum. Whereas mitochondrial pathology was associated with profound apoptosis in the NE, growth restriction and lipid accumulation accompanied mitochondrial changes in the ME. The results of this study indicate that the embryonic response to maternal heat shock is tissue-specific and morphologically distinct in this species.

ABSTRACT  Spina bifida, anencephaly, and encephalocele are commonly grouped together and termed neural tube defects (NTD). Failure of closure of the neural tube during development results in anencephaly or spina bifida aperta but encephaloceles are possibly post‐closure defects. NTD are associated with a number of other central nervous system (CNS) and non‐neural malformations. Racial, geographic and seasonal variations seem to affect their incidence. Etiology of NTD is unknown. Most of the non‐syndromic NTD are of multifactorial origin. Recent in vitro and in vivo studies have highlighted the molecular mechanisms of neurulation in vertebrates but the morphologic development of human neural tube is poorly understood. A multisite closure theory, extrapolated directly from mouse experiments highlighted the clinical relevance of closure mechanisms to human NTD. Animal models, such as circle tail, curly tail, loop tail, shrm and numerous knockouts provide some insight into the mechanisms of NTD. Also available in the literature are a plethora of chemically induced preclosure and a few post‐closure models of NTD, which highlight the fact that CNS malformations are of hetergeneitic nature. No Mendelian pattern of inheritance has been reported. Association with single gene defects, enhanced recurrence risk among siblings, and a higher frequency in twins than in singletons indicate the presence of a strong genetic contribution to the etiology of NTD. Non‐availability of families with a significant number of NTD cases makes research into genetic causation of NTD difficult. Case reports and epidemiologic studies have implicated a number of chemicals, widely differing therapeutic drugs, environmental contaminants, pollutants, infectious agents, and solvents. Maternal hyperthermia, use of valproate by epileptic women during pregnancy, deficiency and excess of certain nutrients and chronic maternal diseases (e.g. diabetes mellitus) are reported to cause a manifold increase in the incidence of NTD. A host of suspected teratogens are also available in the literature. The UK and Hungarian studies showed that periconceptional supplementation of women with folate (FA) reduces significantly both the first occurrence and recurrence of NTD in the offspring. This led to mandatory periconceptional FA supplementation in a number of countries. Encouraged by the results of clinical studies, numerous laboratory investigations focused on the genes involved in the FA, vitamin B12 and homocysteine metabolism during neural tube development. As of today no clinical or experimental study has provided unequivocal evidence for a definitive role for any of these genes in the causation of NTD suggesting that a multitude of genes, growth factors and receptors interact in controlling neural tube development by yet unknown mechanisms. Future studies must address issues of gene‐gene, gene‐nutrient and gene–environment interactions in the pathogenesis of NTD.

Summary Background Neural tube defects (NTD) are mainly of multifactorial origin. Maternal treatment with valproic acid (VPA) during pregnancy induces NTD in susceptible fetuses. Elevated levels of homocysteine are observed in pregnancies with NTD. The mechanism by which homocysteine might cause NTD is unknown. Aim of the Study The aim of this study was to determine if homocystine would augment VPA-induced exencephaly in an experimental model. Methods Groups of mice were injected (IP) on gestational day 8 (GD) with a single dose of 75 mg/kg of l-Homocystine (HC) or a proportionate volume of saline, followed by a single dose of 600 mg/kg of VPA or an equal volume of saline. In a second experiment, mice were treated with a daily dose of 75 mg/kg of HC or an equal volume of saline (IP) from GD 5 and continued through GD 10. These animals had a single exposure to 600 mg/kg of VPA or saline (IP) on GD 8. All animals were killed by cervical dislocation on GD 18. Plasma homocysteine, folate and vitamin B12 were determined on GD 8 and GD 10 from single and multiple dose groups of mice, respectively, from additional experiments. Results The VPA and HC+VPA induced significantly higher rates of embryonic resorption and intrauterine growth retardation (IUGR) than HC or saline alone. HC + VPA groups had significantly more numerous fetuses with severe IUGR than HC alone or VPA alone groups. Both single and multiple doses of HC augmented VPA-induced reduction in fetal body weight. Successive doses of HC did not augment the rate of IUGR more significantly than a single dose of HC. Incidence of exencephaly was significantly enhanced in the HC + VPA groups compared to that in the HC or VPA alone groups. HC alone was not teratogenic. Plasma homocysteine levels increased several fold both in HC and HC + VPA groups and the increase was not particularly more marked in multiple dose groups than in the single dose groups. VPA did not elevate homocysteine concentration. Both FA and vitamin B12 concentrations were reduced by VPA, HC and HC + VPA, but HC and VPA when combined did not produce an additive effect on vitamin levels. Conclusion These data indicate that HC and VPA interact in neurulation stage embryos, affect fundamental processes of closure of the neural tube and lead to enhanced incidence of NTD.

Both clinical and experimental investigations have shown that maternal hyperthermia during critical stages of embryo development can induce malformations in the offspring. Studies of the effect of heat stress on the placental functions are limited to the ewes, but that on microscopic structure is unknown. In the present study, rats were exposed to 41 or 42°C for 1 h on gestation day (GD) 9. The controls were sham treated. Fetuses and placentas were collected on GD 20. Intrauterine growth retardation (IUGR) and several craniofacial malformations were observed in the fetuses of the heat-treated group. The placentas of the 42°C group were significantly lighter in weight than those of the control. Light microscopy (LM) revealed thickening, hyalinization and occasional lymphocytic infiltration of the decidua basalis. Giant cells were prominent and glycogen cells had degenerated, leaving behind large cysts in the basal (spongy) zone. Best’s carmine stain with or without diastase indicated the reduction in number and degeneration of glycogen cells and cyst formation. The labyrinthine zone was relatively thin in comparison to that of the controls. Perivascular fibrosis and paucity of vascularization were other features of the placentas of the hyperthermia group. Electron microscopy (EM) revealed lipid droplet accumulation in the trophoblast, the presence of myelin bodies and an increased production of collagen in the basal zone. Perivascular fibrosis appeared to have contributed to placental barrier thickening. EM also revealed accumulation of glycogen and lipid droplets in the trophoblasts and fibrin secretion into the extracellular space of the labyrinthine zone. These data suggest that placental pathology possibly contributes to fetal growth retardation in maternally heat-stressed rat fetuses.

ABSTRACT  In humans, the presence of cervical and lumbar ribs is of particular clinical significance. However, the relevance of their occurrence in the offspring of experimental animals in reproductive toxicologic studies is poorly understood. Maternal toxicity has been implicated in the etiology but conclusive evidence is lacking. The present study was undertaken to determine the incidence of supernumerary ribs (SNR) in mouse fetuses prenatally exposed to valproic acid (VPA) and retinoic acid (RA), and to compare their differential developmental susceptibility and morphological association with other axial skeletal anomalies. Single doses of valproic acid (VPA) or retinoic acid (RA) were administered to groups of mice on one of gestation days (GD) 7–12. Fetuses were collected on GD 18 and their skeletons examined for SNR. VPA treatment on GD 7 and GD 8 resulted in a high incidence of cervical and lumbar ribs, respectively. Cervical neural arch anomalies in the GD 7 group, and eight pairs of sternal ribs and seven sternebrae in the GD 8 group were observed in excess of the background SNR suggesting a direct effect of VPA on the developing mouse skeletal system. In the RA groups, GD 8–12 were susceptible for lumbar rib induction but increased incidence of cervical ribs was observed only from GD 9–12. Peak incidence of cervical ribs was found in the GD 10 and 11 groups and that of the lumbar ribs in the GD 8 and 11 groups. Although SNR incidence generally increased with increasing dose of RA, a strict dose–response relationship was lacking. Cervical arch anomalies were observed in as many embryos as those with cervical ribs, but eight pairs of sternal ribs and seven sternebrae did not correlate well with the lumbar ribs in the peak day groups. Interrupted cervical neural arches correlated well with lumbar ribs. The reduction in the frequency of presacral vertebrae from 26 to 25 in the VPA groups was limited to GD 7 (30%) and 8 (18%) groups. RA‐induced reduction in presacral vertebral number extended to GD 9 and was greater in the GD 8 than in the GD 9 groups. Sternal anomalies occurred both in VPA and RA experiments and did not strictly correlate with the frequency of SNR. VPA had a narrow window of susceptibility, whereas RA effects on sternum extended from GD 9–12. The incidence of sternal anomalies generally increased with increasing dose and advancing developmental stage at which RA exposure occurred. These developmental susceptibility windows and associated malformations, when considered in the context of the ability of these drugs to induce alterations in gene expression in mouse embryos suggest that SNR are polygenic in origin and greatly influenced by environmental toxicants.

The aim of this study was to determine the possible fetal effects of interaction between maternal diabetes and acute doses of alcohol. Pregnant TO mice were made diabetic by a single injection of...