We have investigated the functional characteristics of term human placental villous explants kept in long-term (7-11 days) culture. Fragments of placental villous tissue (approximately 5-10 mg wet wt) were cultured in supplemented CMRL-1066 culture medium for up to 11 days. After the first day of culture, the syncytiotrophoblast appeared vacuolated and eventually degenerated. However, a new syncytiotrophoblast developed by day 4, being indistinguishable from that of a fresh placenta by 11 days. Release of human chorionic gonadotrophin increased and activity of lactate dehydrogenase in culture medium decreased with culture time. Transport variables were measured over the first 7 days of culture. Basal (86)Rb efflux was reduced with time in culture and was inhibited by Ba2+, suggesting the efflux was mediated by K+ channels. At all stages of culture, (86)Rb efflux was stimulated by ATP, hyposmotic medium, and ANG II. A complex pattern of efflux changes with culture time and type of stimulator was observed, suggesting that several compartments of the tissue contributed to stimulated efflux. This culture system provides opportunities for studies of chronic regulation of placental function.

The aim was to investigate whether dietary supplementation of a combination of the two antioxidants, vitamin E and vitamin C, would protect the fetus in diabetic rat pregnancy at a lower dose than previously used. Normal and streptozotocin-induced diabetic rats were mated and given standard food or food supplemented with either 0.5% vitamin E ϩ 1% vitamin C or 2% vitamin E ϩ 4% vitamin C. At gestational d 20, gross morphology and weights of fetuses were evaluated. Vitamins E and C and thiobarbituric acid reactive substances were measured in maternal and fetal compartments. In addition, protein carbonylation was estimated in fetal liver. Maternal diabetes increased the rate of malformation and resorption in the offspring. High-dose antioxidant supplementation decreased fetal dysmorphogenesis to near normal levels. The low-dose group showed malformations and resorptions at an intermediate rate between the untreated and the high-dose groups. Thiobarbituric acid reactive substances were increased in fetal livers of diabetic rats and reduced to normal levels already by low-dose antioxidative treatment. Protein carbonylation rate was also increased in fetal liver of diabetic rats; it was normalized by high-dose treatment but only partially reduced by low-dose antioxidants. We conclude that combined antioxidative treatment with vitamins E and C decreases fetal malformation rate and diminishes oxygen radicalrelated tissue damage. However, no synergistic effect between the two antioxidants was noted, a result that may influence future attempts to design antiteratogenic treatments in diabetic pregnancy. Oxidatively modified proteins may be teratogenically important mediators in diabetic embryopathy. Maternal type-1 diabetes during pregnancy causes an increased rate of malformation in the fetus despite insulin treatment and glucose monitoring (1-3). The incidence of fetal malformations in type-1 diabetic pregnancies is estimated to be 5-10% in recent studies (4 -8). Several clinical studies have demonstrated that high maternal HbA1c levels during early pregnancy are associated with an increased risk for malformations (4, 9, 10). It is, however, likely that the pathogenesis of diabetic embryopathy is multifactorial (11), and in experimental work not only maternal serum levels of glucose but also levels of triglycerides, ␤-hydroxybutyrate, branched-chain amino acids, and creatinine correlate positively with increased resorption and malformation rates (12). Also, genetic factors may predispose for the development of malformations in maternal diabetes (13,14).The putative role of ROS in the development of diabetic complications has been investigated for several decades (15-17). Evidence of ROS involvement in hyperglycemia-induced embryopathy was first obtained in studies in which antioxidant enzymes proved to be protective in vitro (18,19). Increased ROS production (20, 21) and lipid peroxidation (22) have subsequently been found in rat embryos cultured in high glucose and in embryos of diabetic rat mothers. Enzymatically prod...

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Although improved metabolic control of maternal diabetes has reduced the rate of complications during pregnancy, the incidence of congenital malformations is still increased in children of insulin-dependent-diabetic (IDDM) mothers [1±4]. The mechanisms by which maternal diabetes disrupts embryonic development are not completely understood, but the risk of fetal malformation is strongly correlated to the maternal glucose concentration [2±4]. Maternal levels of triglycerides, b-hydroxybutyrate and branched chain amino acids are also of importance for the occurrence of fetal dysmorphogenesis [5]. In the embryo, disturbed metabolism of sorbitol, inositol, arachidonic acid and prostaglandins has been demonstrated in experimental diabetic pregnancy and Diabetologia (1997 Vitamin C supplementation of the maternal diet reduces the rate of malformation in the offspring of diabetic ratsDepartment of Medical Cell Biology, Uppsala University, Uppsala, SwedenSummary An excess of reactive oxygen species (ROS) has been associated with the increased rate of congenital malformations in experimental diabetic pregnancy. Previous in vitro and in vivo studies show that antioxidants can protect the embryonic development in a diabetic environment. In the present investigation we examined the antiteratogenic capacity of vitamin C, an antioxidative agent not previously evaluated as a dietary supplement in diabetic pregnancy. Normal and streptozotocin diabetic rats were either fed a standard diet or a diet enriched with 0.9, 1.8 or 4 % sodium ascorbate throughout pregnancy. On gestational day 20, the litters of normal and diabetic rats without vitamin C supplement contained 9 and 12 % early resorptions, 2 and 17 % late resorptions and 1 and 27 % malformations, respectively. Vitamin C treatment reduced the rates of late resorptions and malformations in the diabetic groups in proportion to the dose administered. Thus, in the diabetic group with 4 % ascorbate treatment we found unchanged numbers of early resorptions, but only 7 % late resorptions (p < 0.05 vs untreated diabetic pregnancy) and 8 % malformations (p < 0.05 vs untreated diabetic pregnancy). Maternal diabetes did not alter tissue levels of ascorbic acid in the fetuses at term, whereas vitamin C treatment caused accumulation of ascorbic acid in the placenta, maternal and fetal liver. Vitamin C supplementation yielded increased a-tocopherol concentration in the placenta and caused a reduction of the high concentrations of thiobarbituric acid reactive substances (TBARS) in serum of pregnant diabetic rats. Vitamin C treatment reduces the rates of congenital malformations and late resorptions, thereby supporting that ROS are involved in the embryonic dysmorphogenesis of diabetic pregnancy. [Diabetologia (1997 Keywords Diabetic pregnancy, streptozotocin, rat, embryo, fetus, congenital malformation, vitamin C, antioxidant therapy, TBARS, oxidative stress. Corresponding author: C. M. Sima  n, Department of Medical Cell Biology, Uppsala University, Biomedicum, P. O. Box 571, S-751 23 Up...

The increased rate of fetal malformation in diabetic pregnancy represents both a clinical problem and a research challenge. In recent years, experimental and clinical studies have given insight into the teratological mechanisms and generated suggestions for improved future treatment regimens. The teratological role of disturbances in the metabolism of inositol, prostaglandins, and reactive oxygen species has been particularly highlighted, and the beneficial effect of dietary addition of inositol, arachidonic acid and antioxidants has been elucidated in experimental work. Changes in gene expression and induction of apoptosis in embryos exposed to a diabetic environment have been investigated and assigned roles in the teratogenic processes. The diabetic environment appears to simultaneously induce alterations in several interrelated teratological pathways. The complex pathogenesis of diabetic embryopathy has started to unravel, and future research efforts will utilize both clinical intervention studies and experimental work that aim to characterize the human applicability and the cell biological components of the discovered teratological mechanisms.