Nonspecific stimulation of the maternal immune system. II. Effects on gene expression in the fetus

BACKGROUND: Maternal immune stimulation reduces malformations caused by chemical teratogens. Mechanisms for this effect are not known. Altered expression of regulatory molecules (e.g., transforming growth factor [TGF-beta], tumor necrosis factor-alpha [TNF-alpha]) has been reported in fetuses from immunostimulated mice, which may affect gene expression. Expression of selected genes that function to control proliferation, differentiation, or apoptosis was evaluated in chemical-exposed fetuses, with or without maternal immunostimulation. METHODS: Ethyl carbamate (urethane) was given to pregnant ICR mice on day 10 of gestation to induce cleft palate. Before teratogen administration, the immune system of the female mice was stimulated by footpad injection with Freund's complete adjuvant (FCA) or by intraperitoneal injection with interferon-gamma (IFN-gamma). RESULTS: Maternal immunostimulation with interferon-gamma (IFN-gamma) decreased severity of the cleft palate lesion caused by urethane, while FCA decreased both incidence and severity of cleft palate. Gestation day 14 fetuses from urethane-exposed mothers displayed decreased expression of cell cycle/apoptotic genes bcl2alpha, bcl2beta, pkCalpha, and p53 in fetal heads. Immune stimulation with IFN-gamma-normalized expression of bcl2alpha, bcl2beta, and pkCalpha to control levels. Urethane also decreased the ratio of expression of bclalpha/p53, bclbeta/p53, and pkCalpha/p53, while maternal injection with IFN-gamma restored these expression ratios to control levels. Maternal immunization with FCA also significantly increased bcl2alpha/p53, bcl2beta/p53, and pkCalpha/p53 gene expression ratios. CONCLUSIONS: These results suggest that (1) the maternal immune system may possess heretofore unrecognized regulatory activity in fetal development, and (2) protection against urethane-induced cleft palate may be mediated through maternal immune regulation of fetal gene expression.     

Reduced birth defects caused by maternal immune stimulation in methylnitrosourea-exposed mice: association with placental improvement

BACKGROUND: Methylnitrosourea (MNU) is a potent carcinogen and teratogen that is associated with central nervous system, craniofacial, skeletal, ocular, and appendicular birth defects following transplacental exposure at critical time points during development, and preliminary studies have suggested that nonspecific maternal immunostimulation may offer protection against development of these birth defects. METHODS: Our study examined morphologic alterations in fetal limb and digital development and placental integrity following maternal exposure to MNU on GD 9 in CD-1 mice, and characterized the improvement in placental integrity and abrogation of fetal defects following maternal immune stimulation with interferon-gamma (IFN-gamma) on GD 7. RESULTS: Fetal limbs were significantly shortened (p < 0.0001) and incidence of limb and digital defects (syndactyly, polydactyly, oligodactyly, clubbing, and webbing) was dramatically increased following mid-gestational maternal MNU exposure. Maternal immune stimulation with IFN-gamma on GD 7 lessened incidence of fetal limb shortening and maldevelopment on GD 12 and 14. Further, disruption of placental spongiotrophoblast integrity, increased cell death in placental trophoblasts with increased intercellular spaces in the spongiotrophoblast layer and minimal inflammation, and increased loss of fetal labyrinthine endothelial cells from MNU-exposed dams suggested that MNU-induced placental breakdown may contribute to fetal limb and digital maldevelopment. MNU + IFN-gamma was associated with diminished cell death within all layers of the placenta, especially in the labyrinthine layer. CONCLUSIONS: These data verify improved distal limb development in MNU-exposed mice as a result of maternal IFN-gamma administration, and suggest a link between placental integrity and proper fetal development.     

Aortic and ventricular dilation and myocardial reduction in gestation day 17 ICR mouse fetuses of diabetic mothers

BACKGROUND: Maternal diabetes mellitus is associated with increased fetal teratogenesis, including cardiovascular defects. Information regarding cardiovascular changes in late-gestation fetal mice, related to maternal hyperglycemia, is not present in the literature. METHODS: Late-gestation fetal heart and great vessel morphology were analyzed in fetuses from control and diabetic mice. Female ICR mice were injected with streptozocin (200 mg/kg IP) prior to mating to induce diabetes (n = 8). Nonhyperglycemic females were used as controls (n = 8). At day 17 of gestation, females were euthanized and one fetus was arbitrarily selected per litter to analyze the heart and great vessels. Six additional fetuses from different litters, showing external malformations (spina bifida and/or exencephaly), were also evaluated from the diabetic group. Fetal thoraxes were processed using routine histopathologic techniques, and 7-mum transversal sections were stained with hematoxylin-eosin. Digital images of sections were made and analyzed using NIH Image J software to compare regional cardiac development. Student's t tests for means were performed to determine differences between groups (p < .05). RESULTS: Maternal hyperglycemia caused a dilation of late-gestation fetal ventricular chambers, a reduction of total ventricular myocardial area, and an increase in transversal ascending thoracic aortic area. Three of six fetuses that displayed external malformations showed an overt cardiac defect, beyond the ventricular and myocardial changes. CONCLUSIONS: Maternal hyperglycemia altered morphology of the late-gestation fetal mouse heart. Postnatal persistence or consequences of late-gestation heart chamber dilation and myocardial reduction are not yet known.     

Nonspecific stimulation of the maternal immune system. I. Effects On teratogen-induced fetal malformations

BACKGROUND: Maternal immune stimulation has reported, but unconfirmed, efficacy for reducing chemical-induced morphologic defects in mice. METHODS: Teratogenic chemicals (2,3,7, 8-tetrachlorodibenzo-p-dioxin [TCDD], ethyl carbamate [urethane], methylnitrosourea [MNU], or valproic acid [VA]) were given to pregnant mice to induce cleft palate (TCDD, urethane), digital defects (urethane, MNU), or exencephaly (VA). Before teratogen administration, the immune system of female mice was stimulated by intraperitoneal (IP) administration of pyran copolymer or attenuated bacillus Calmette Guérin (BCG), or by footpad injection with Freund's complete adjuvant (FCA). RESULTS: Fetal defects caused by all four chemicals studied were reduced by maternal immunostimulation, sometimes dramatically. In addition to reducing VA-induced exencephaly, immunostimulation with FCA resulted in fetal mice displaying anury (absence of tails). Activated maternal immune cells could not be detected in fetal circulation using flow cytometry and a fluorescent cell-tracking probe. CONCLUSIONS: For the chemicals tested, maternal immune stimulation has efficacy in reducing fetal defects. Immune protection against teratogenesis may be an indirect effect of maternal immune cell activation.     

Fetal cardiac effects of maternal hyperglycemia during pregnancy

Maternal diabetes mellitus is associated with increased teratogenesis, which can occur in pregestational type 1 and type 2 diabetes. Cardiac defects and with neural tube defects are the most common malformations observed in fetuses of pregestational diabetic mothers. The exact mechanism by which diabetes exerts its teratogenic effects and induces embryonic malformations is unclear. Whereas the sequelae of maternal pregestational diabetes, such as modulating insulin levels, altered fat levels, and increased reactive oxygen species, may play a role in fetal damage during diabetic pregnancy, hyperglycemia is thought to be the primary teratogen, causing particularly adverse effects on cardiovascular development. Fetal cardiac defects are associated with raised maternal glycosylated hemoglobin levels and are up to five times more likely in infants of mothers with pregestational diabetes compared with those without diabetes. The resulting anomalies are varied and include transposition of the great arteries, mitral and pulmonary atresia, double outlet of the right ventricle, tetralogy of Fallot, and fetal cardiomyopathy. A wide variety of rodent models have been used to study diabetic teratogenesis. Both genetic and chemically induced models of type 1 and 2 diabetes have been used to examine the effects of hyperglycemia on fetal development. Factors such as genetic background as well as confounding variables such as obesity appear to influence the severity of fetal abnormalities in mice. In this review, we will summarize recent data on fetal cardiac effects from human pregestational diabetic mothers, as well as the most relevant findings in rodent models of diabetic cardiac teratogenesis. Birth Defects Research (Part A), 2009. © 2009 Wiley‐Liss, Inc.     

Late-gestation ventricular myocardial reduction in fetuses of hyperglycemic CD1 mice is associated with increased apoptosis

BACKGROUND : Previous work in our laboratory showed reduced myocardium and dilated ventricular chambers in gestation day (GD) 17 hearts that were collected from hyperglycemic CD1 mouse dams. Pre-breeding maternal immune stimulation, using Freund's complete adjuvant (FCA), diminished the severity of these fetal heart lesions. The following experiments were performed to detect possible changes in fetal heart apoptotic cell death, under hyperglycemic conditions and with or without maternal immune stimulation. METHODS : Female CD1 mice were injected with 200 mg/kg of streptozocin (STZ) to induce insulin-dependent diabetes mellitus. Half of these mice received prior FCA injection. Fetal hearts were collected on GD 17 and myocardial apoptotic cells were quantified using flow cytometry. A panel of apoptosis regulatory genes (Bcl2, p53, Casp3, Casp9, PkCe) was then examined in the fetal myocardium using RT-PCR. RESULTS : Early apoptotic cells and late apoptotic/necrotic cells were significantly increased in fetal hearts from STZ or STZ+FCA dams. Pre-treatment with FCA reduced late apoptotic/necrotic cells to control level, suggesting some cell death protection was rendered by FCA. Paradoxically in the face of such increased cell death, the expression of pro-apoptotic genes Casp3 and Casp9 was decreased by diabetes, while the anti-apoptotic gene Bcl2 was increased. CONCLUSIONS : Maternal hyperglycemia causes dys-regulated apoptosis of fetal myocardial cells. Such effect may be prevented by maternal immune stimulation. Birth Defects Res (Part B) 86:409–415, 2009. © 2009 Wiley-Liss, Inc.     

Fetal rat islet insulin deficiency following maternal administration of streptozotocin

Pancreatic islets were isolated from the fetuses of normal rats and rats made diabetic by the iv administration of streptozotocin (STZ) on either Day 3 or 5 of pregnancy. Of the rats made diabetic on Day 3, one group also received insulin injections at the appearance of glucosuria. Maternal blood glucose on Day 20 of gestation was significantly different in the diabetic rats (405 +/- 27 mg/dl) from the normal (97 +/- 1 mg/dl) and insulin-treated diabetic rats (69 +/- 9 mg/dl). While fetal weight was significantly decreased in the STZ-treated rats (2.64 +/- 0.13 g vs 3.52 +/- 0.05 g for the control group, P less than 0.005), fetal glucose was significantly higher in the STZ-treated than in normal pups (342 +/- 11 vs 35 +/- 1 mg/dl, P less than 0.005). Both fetal weight and glucose were normalized by insulin treatment: 3.16 +/- 0.18 g and 31 +/- 7 mg/dl, respectively. Insulin release from fetal islets of diabetic dams was blunted after a week in culture both in basal and stimulated conditions. After 2 weeks in culture, there was partial recovery in the insulin response to glucose but it did not equal to that measured in fetal islets from the normal and insulin-treated diabetic rats. These data suggest maternal hyperglycemia severely impairs fetal weight and insulin release from fetal rat islets in vitro, and correction of the hyperglycemia by insulin treatment not only improves fetal weight and glucose concentrations, but it also normalizes insulin release from fetal rat islets in vitro.     

Maternal alpha-fetoprotein screening: two years' experience in a low-risk district.

Over a two-year period, 3479 pregnant women in the Kings'' Lynn Health District were screened for neural tube defects by estimation of maternal serum alpha-fetoprotein. Most pregnancies were scanned by sonar for fetal maturity. Eight women had fetuses with open neural tube defects; four with anencephaly were associated with very high alpha-fetoprotein values. Of the four with open neural tube defects without anencephaly, only one was detected by screening and confirmed after amniocentesis. One other had a raised serum alpha-fetoprotein but a normal amniotic fluid value. The other two affected fetuses were missed. This disappointing outcome was attributed to the poor predictive value of alpha-fetoprotein in detecting open neural tube defects (anencephaly apart) rather than to errors in its estimation or in assessment of fetal maturity by sonar scan. We question the validity of screening, particularly in areas of intermediate or low incidence.     

Interferon-gamma differentially regulates monocyte matrix metalloproteinase-1 and -9 through tumor necrosis factor-alpha and caspase 8

Tumor necrosis factor-alpha (TNFalpha) and granulocyte macrophage colony-stimulating factor (GM-CSF) individually enhance monocyte matrix metalloproteinase-9 (MMP-9) but induce MMP-1 only when added in combination. Because interferon-gamma (IFNgamma) is also found at inflammatory sites, we determined its effect on monocyte MMPs in the presence or absence of TNFalpha and GM-CSF. IFNgamma alone did not stimulate monocyte MMP-9 or MMP-1; however, in the presence of GM-CSF it induced MMP-1 and enhanced MMP-1 stimulated by GM-CSF and TNFalpha. IFNgamma induced MMP-1 in the presence of GM-CSF through the stimulation of TNFalpha production through a mechanism involving both p38 and ERK1/2 MAPKs, in which GM-CSF stimulated ERK1/2 whereas IFNgamma activated p38. In support of this conclusion TNFalpha neutralizing antibody and antibodies against TNF receptor I and -II blocked the induction of MMP-1 by GM-CSF and IFNgamma. In contrast to its effects on MMP-1, IFNgamma inhibited TNFalpha-induced MMP-9 through a caspase 8-dependent pathway as demonstrated by the restoration of MMP-9 with caspase 8 inhibitors. Moreover, the phosphorylation of STAT1 by IFNgamma was blocked by an inhibitor of caspase 8, indicating that STAT1 had a suppressive effect on MMP-9. Caspase 8-mediated phosphorylation of STAT1 through p38 MAPK as shown by the inhibition of IFNgamma-induced phosphorylation of p38 by caspase 8 inhibitors. Activation of caspase 8 by IFNgamma did not result in increased apoptosis. Thus IFNgamma in the presence of GM-CSF and/or TNFalpha differentially regulates monocyte MMPs through induction of TNFalpha and a novel mechanism involving caspase 8 that is independent of apoptosis.     

Oxidative stress during diabetic pregnancy disrupts cardiac neural crest migration and causes outflow tract defects

BACKGROUND: Maternal diabetes increases risk for congenital malformations, particularly cardiac outflow tract defects. Maternal diabetes inhibits expression of Pax3 in neuroepithelium through hyperglycemia‐induced oxidative stress. The neuroepithelium gives rise to the neural crest, and Pax3 expression in cardiac neural crest (CNC) is required for CNC migration to the heart and for outflow tract septation. Here we tested whether maternal diabetes, through hyperglycemia‐induced oxidative stress, before the onset of CNC delamination, impairs CNC migration and cardiac outflow tract septation. METHODS: CNC migration was mapped in mouse embryos whose mothers were diabetic, or transiently hyperglycemic, or in which oxidative stress was transiently induced, using reporters linked to Pax3 expression. CNC apoptosis was examined by TUNEL assay. Outflow tract septation was examined histologically and by gross inspection. RESULTS: Few, if any, migrating CNC cells were observed in embryos of diabetic mice, and this was associated with increased apoptosis along the path of CNC migration. Outflow tract defects were significantly increased in fetuses of diabetic mice. Notably, induction of hyperglycemia or oxidative stress on the day prior to the onset of Pax3 expression and CNC migration also impaired CNC migration, increased apoptosis, and caused outflow tract defects. However, antioxidants administered on the day prior to the onset of Pax3 expression and CNC migration prevented these effects of hyperglycemia or oxidative stress. CONCLUSIONS: In diabetic pregnancy, oxidative stress, which inhibits expression of genes required for CNC viability, causes subsequent CNC depletion by apoptosis during migration, which leads to outflow tract defects. Birth Defects Research (Part A), 2008. © 2008 Wiley‐Liss, Inc.     

Oxidative stress and diabetes in pregnant rats

A considerable amount of clinical and experimental evidence now exists suggesting the involvement of free radical-mediated oxidative processes in the pathogenesis of diabetic complications. If the diabetic state is associated with a generalized increase in oxidative stress, it might well be reflected in the alterations in embryonic and fetal development during pregnancy. In the present study, incidence of the malformed fetuses, biochemical parameters and antioxidant system activity of streptozotocin (STZ)-induced diabetic pregnant rats was investigated and the results obtained were compared with those of the control group (non-diabetic). Virgin female Wistar rats were injected with 40 mg/kg streptozotocin (STZ) before mating. All the females were killed on Day 21 of pregnancy and the fetuses were analyzed. A maternal blood sample was collected by venous puncture and the maternal liver was removed for biochemical measurement. The diabetic dams presented hyperglycemia, hyperlipemia, hypertriglyceridemia, hypercholesterolemia, hyperuricemia, decreased reduced glutathione (GSH), hepatic glycogen and superoxide dismutase (SOD) determinations. There was an increased incidence of skeletal and visceral malformation in fetuses from diabetic rats. Our findings suggest that oxidative stress occurs in the diabetic pregnant state, which might promote maternal homeostasis alterations. These diabetic complications might be a contributory factor to conceptus damage causing embryonic death (abortion/miscarriage) or the appearance of malformations in the fetuses of diabetic dams. Antioxidant treatment of women with diabetes may be important in future attempts to prevent congenital malformations.     

Malformations in offspring of diabetic rats: morphometric analysis of neural crest-derived organs and effects of maternal vitamin E treatment

BACKGROUND: We have previously reported on a malformation-prone Sprague-Dawley rat substrain (U), which presents a high frequency of micrognathia in the offspring of diabetic mothers. This malformation is related to impaired development of the cranial neural crest cells (NCC); the defect may be prevented by antioxidative treatment of the mother. METHODS: We have therefore investigated whether fetuses of diabetic rats display other malformations associated with altered cranial NCC development and whether maternal vitamin E supplementation may affect such malformations. RESULTS: Fetuses of diabetic rats showed low-set external ears, severely malformed Meckel's cartilage, small thyroid and thymus, and absence of parathyroid glands. Cardiac anomalies were frequently observed, including rightward displacement of the aorta, double outlet right ventricle (DORV), persistent truncus arteriosus (PTA) combined with ventricular septal defects due to a malaligned outlet septum. The malformations in the outflow tract included abnormalities of the great arteries; right-sided aortic arch/descending aorta, and double aortic arches. These defects tended to occur together within individual fetuses. Maternal dietary treatment with 2% vitamin E markedly reduced the severity of the malformations. CONCLUSIONS: The phenotypic appearance of these defects is strikingly similar to the DiGeorge anomaly in humans, which has been found in children of diabetic mothers together with an overrepresentation of PTA and DORV. The malformations associated with defective NCC development in the offspring of diabetic U rats show several morphological similarities to those in humans; hence the teratogenic mechanisms may be similar and accessible for study.     

Placental improvement and reduced distal limb defects by maternal interferon-gamma injection in methylnitrosourea-exposed mice

BACKGROUND: Methylnitrosourea (MNU), an alkylating agent derived from creatinine metabolism, is cytotoxic, genotoxic, and mutagenic. Mid-gestational exposure to MNU leads to distal limb defects in mice. Previous studies have shown that nonspecific maternal immune stimulation protects against MNU-induced teratogenesis. A role for immune-mediated placental improvement in this effect remains uncertain. METHODS: The immune system of timed-pregnant C57BL/6N and CD-1 mice was stimulated by GD 7 intraperitoneal (IP) injection with the cytokine interferon-gamma (IFN-gamma). A teratogenic dose of MNU was then administered by IP injection on the morning of GD 9 to disrupt distal limb formation. Fetal limb length, body length, digital deformities, and placental integrity were evaluated on GD 14. RESULTS: The incidence of syndactyly, polydactyly, and interdigital webbing in MNU-exposed mice was decreased by maternal IFN-gamma treatment. In C57BL/6N mice, these defects were reduced by 47, 100, and 63%, respectively, as compared to previous reports on CD-1 mice, by 39, 71, and 20%, respectively. Administration of IFN-gamma significantly diminished MNU-induced endothelial and trophoblast placental damage in both strains of mice. CONCLUSIONS: These findings support a possible link between maternal immunity, placental integrity, and fetal distal limb development. Further, these results suggest that IFN-gamma might act through placental improvement to indirectly protect against MNU-induced fetal limb malformations.     

Developmental toxic effects of antifungal flusilazole administered by gavage to mice

BACKGROUND: The developmental toxicity of flusilazole was studied in CD-1 mice after oral administration. METHODS: Pregnant mice were given flusilazole at doses of 0 (corn oil), 10, 20, and 40 mg/kg/day, by gavage, on gestational days (GD) 6-15. RESULTS: Maternal toxicity, as evidenced by reduction in body weight gain and signs of toxicity, was observed at the middle- and high-dose groups. No significant incidence of resorptions or death was observed in any of dose groups. There was a pronounced reduction in fetal weight, which was significantly lower than control from 20 and 40 mg/kg/day. There was no significant increase in the incidence of fetuses with external or visceral malformations in any of dose groups, but there was a significant increase in the incidence of skeletal malformations was observed at 20 and 40 mg/kg/day. CONCLUSIONS: The results of this study reported marked maternal toxicity, growth retardation, and skeletal abnormalities in the mid- and high-dose groups. It seems likely that marked maternal toxicity contributed to the observed alterations in fetal growth retardation and skeletal development. The no-observed-effect level in the present study for maternal and developmental toxicity was 10 mg/kg/day.     

Oxidative stress promotes the increase of matrix metalloproteinases-2 and -9 activities in the feto-placental unit of diabetic rats

Maternal diabetes increases the risk of congenital malformations, placental dysfunction and diseases in both the neonate and the offspring's later life. Oxidative stress has been involved in the etiology of these abnormalities. Matrix metalloproteases (MMPs), involved in multiple developmental pathways, are increased in the fetus and placenta from diabetic experimental models. As oxidants could be involved in the activation of latent MMPs, we investigated a putative relationship between MMPs activities and oxidative stress in the feto-placental unit of diabetic rats at midgestation. We found that H₂O₂ enhanced and that superoxide dismutase (SOD) reduced MMPs activities in the maternal side of the placenta and in the fetuses from control and diabetic rats. MMPs were not modified by oxidative status in the fetal side of the placenta. Lipid peroxidation was enhanced in the maternal and fetal sides of the placenta and in the fetus from diabetic rats when compared to controls, and gradually decreased from the maternal placental side to the fetus in diabetic animals. The activities of the antioxidant enzymes SOD and catalase were decreased in the maternal placental side, catalase activity was enhanced in the fetal placental side and both enzymes were increased in the fetuses from diabetic rats when compared to controls. Our data demonstrate changes in the oxidative balance and capability of oxidants to upregulate MMPs activity in the feto-placental unit from diabetic rats, a basis to elucidate links between oxidative stress and alterations in the developmental pathways in which MMPs are involved.     

Prenatal diagnosis and outcome of pregnancy in 2036 women investigated by amniocentesis

Summary The report presents the indications for prenatal diagnosis, the results from amniocentesis and details of outcome of pregnancy in 2036 women. Aneuploidy was found in 26 fetuses (1.3%) including 16 with trisomy 21 and 9 sex chromosome abnormalities. There were 38 balanced chromosomal rearrangements (1.9%): 23 of these (1.1%) were pericentric inversions of a number 9 chromosome. Only twoof the chromosomal abnormalities were found in other than those mothers referred for maternal age of 35 or over. Concern is expressed at the low referral rate for older mothers in the population served (only 25% of those over 40 years). Failure of amniotic cell culture occurred in 2.8% of cultures. Maternal cell contamination was detected in 23 cultures (1.1%) with four errors in reported fetal sex. Total error estimate was 0.5%. There were 20 in vitro artefacts (1.0%) with no reporting errors. Neural tube defects were identified in 28 fetuses and there were three false-positive and one false-negative results. Data on outcome of pregnancy was available from 1805 pregnancies (96.5%): 1295 were normal (71.7%) and 510 (28.3%) showed some abnormality. Pregnancy was terminated for fetal abnormality in 53 cases (2.9%) and fetal loss occurred in 65 (3.7%). Methods, quality control, safety and service considerations are discussed. It is suggested that amniocentesis should be restricted to centres where the greatest expertise is available. The service should be improved to meet the needs of a greater number of patients. The series is compared with other studies of over 1500 cases.     

Diabetes in the Cohen Rat Intensifies the Fetal Pancreatic Damage Induced by the Diabetogenic High Sucrose Low Copper Diet

Intrauterine hyperglycemic environment could harm the fetus making it more susceptible to develop postnatal glucose intolerance. A possible mechanism is compromise of the fetal pancreatic development. We previously found that a high sucrose low copper diabetogenic diet induces type 2 diabetes in the Cohen diabetic sensitive rats, but not in the Sabra control rats. However, oxidative stress was observed in the placenta and term fetal liver of diabetic and nondiabetic controls. We now investigated whether the fetal pancreas is affected by this diet and whether the effects result from oxidative stress, maternal hyperglycemia, or both. Term fetal pancreases were evaluated for morphology, beta cells, oxidative stress, apoptosis, and DNA methylation. There were no microscopic changes in hematoxylin and eosin stained sections and beta cells immunostaining in the pancreas of fetuses of both strains. Fetuses of the sensitive strain fed diabetogenic diet had significantly higher activity of superoxide dismutase and catalase, elevated levels of low molecular weight antioxidants, and more intense immunostaining for nuclear factor kappa‐B and hypoxia inducing factor‐1α. Both strains fed diabetogenic diet had increased immunostaining for Bcl‐2‐like protein and caspase 3 and decreased immunostaining for 5‐methylcytosine in their islets and acini. Our data suggest that maternal diabetogenic diet alters apoptotic rate and epigenetic steady states in the term fetal pancreas, unrelated to maternal diabetes. Maternal hyperglycemia further increases pancreatic oxidative stress, aggravating the pancreatic damage. The diet‐induced insults to the fetal pancreas may be an important contributor to the high susceptibility to develop diabetes following metabolic intrauterine insults     

Reduction in Valproic Acid–Induced Neural Tube Defects by Maternal Immune Stimulation: Role of Apoptosis

Teratogenic deregulation of apoptosis during development is a possible mechanism for birth defects. Administration of valproic acid (VA) during first trimester of pregnancy causes neural tube defects (NTDs). Nonspecific stimulation of the mother's immune system has been shown to reduce various teratogen‐induced fetal malformations including NTDs in rodents. This present study investigated the role of reduced apoptosis by maternal immune stimulation in prevention of VA‐induced NTDs in CD‐1 mice. Prevention of VA‐induced NTDs by nonspecific maternal immune stimulation using IFNγ was employed to evaluate the role of reduced apoptosis by IFNγ in this protective mechanism. Apoptosis was quantified using flow cytometry. Terminal Transferase dUTP Nick End Labeling assay was used to localize the apoptosis. Increased apoptosis, suggesting involvement in VA teratogenicity, was observed along the neural tube in both normal and abnormal embryos from VA‐exposed dams. Increased apoptosis in normal VA‐exposed embryos suggests that VA may alter other cellular processes such as cell proliferation and differentiation in addition to apoptosis. Apoptotic levels in embryos with closed neural tubes from IFNγ + VA dams were similar to controls indicating resistance to VA‐induced apoptosis and protection against teratogenicity of VA. In IFNγ + VA exposed embryos with open neural tubes, maternal immune stimulation failed to regulate apoptosis resulting in an NTD. Overall, these results suggest that VA alters several biological processes including apoptosis in the developing embryos to induce fetal malformations. Resistance to VA‐induced apoptosis in embryos resulting from maternal immune stimulation may be involved in protective mechanism.     

Oxidative stress promotes the increase of matrix metalloproteinases-2 and -9 activities in the feto-placental unit of diabetic rats

Maternal diabetes increases the risk of congenital malformations, placental dysfunction and diseases in both the neonate and the offspring's later life. Oxidative stress has been involved in the etiology of these abnormalities. Matrix metalloproteases (MMPs), involved in multiple developmental pathways, are increased in the fetus and placenta from diabetic experimental models. As oxidants could be involved in the activation of latent MMPs, we investigated a putative relationship between MMPs activities and oxidative stress in the feto-placental unit of diabetic rats at midgestation. We found that H₂O₂ enhanced and that superoxide dismutase (SOD) reduced MMPs activities in the maternal side of the placenta and in the fetuses from control and diabetic rats. MMPs were not modified by oxidative status in the fetal side of the placenta. Lipid peroxidation was enhanced in the maternal and fetal sides of the placenta and in the fetus from diabetic rats when compared to controls, and gradually decreased from the maternal placental side to the fetus in diabetic animals. The activities of the antioxidant enzymes SOD and catalase were decreased in the maternal placental side, catalase activity was enhanced in the fetal placental side and both enzymes were increased in the fetuses from diabetic rats when compared to controls. Our data demonstrate changes in the oxidative balance and capability of oxidants to upregulate MMPs activity in the feto-placental unit from diabetic rats, a basis to elucidate links between oxidative stress and alterations in the developmental pathways in which MMPs are involved.     

Fetal lung development in streptozotocin-induced experimental diabetes: cytidylyl transferase activity, disaturated phosphatidyl choline and glycogen levels

The influence of streptozotocin-induced maternal diabetes on choline phosphate cytidylyltransferase activity (EC.2.7.7.15) glycogen content and disaturated phosphatidyl choline in fetal lung was studied between 19 and 21 days of gestation. In this experimental model, induction of maternal diabetes two days after mating, resulted in fetal hyperglycemia and hyperinsulinemia; the fetuses were neither macrosomic nor showed any evidence of fetal growth retardation. The glycogen content of lungs on days 19 and 20, but not on day 21 of gestation was significantly higher in fetuses of diabetic rats than in controls. The pulmonary cytosol cytidylyltransferase activity was similar in the two groups of fetuses on days 19 and 20. On day 21 of gestation the enzyme activity was significantly lower in fetuses of diabetic rats than in those of controls. On day 21 of gestation and in newborns of diabetic mothers, although there was no difference in the total pulmonary phospholipids, the levels of disaturated phosphatidyl cholines were significantly lower than in controls.