St Vincent's Declaration 10 years on: outcomes of diabetic pregnancies.

AIMS: To monitor pregnancies in women with pregestational Type 1 diabetes for pregnancy loss, congenital malformations and fetal growth parameters, in a geographically defined area in the north west of England. METHODS: Population cohort study of 547 pregnancies in women with Type 1 diabetes from maternity clinics in 10 centres over a 5-year period (1995-1999 inclusive). Main outcome measures were numbers and rates of miscarriages, stillbirths, neonatal and post-neonatal deaths; prevalence of congenital malformations; birth weight in relation to gestational age. RESULTS: Among 547 pregnancies, there were six (1.1%) pairs of liveborn twins, 439 (80.3%) liveborn singletons; 72 (13.2%) spontaneous abortions, 14 (2.6%) stillbirths and 16 (2.9%) terminations. Four of the terminations were performed because of congenital malformations. Both the stillbirth rate (30.1/1000 total births (95% confidence interval (CI) 16.6-50.0)), and prevalence of congenital malformations (84.3/1000 live births (95% CI 60.3-113.8)) were significantly higher than the local population (P < 0.001). When corrected for gestational age, mean birth weight in the sample was 1.3 sd greater than that of infants of non-diabetic mothers (P = 0.12). Infants with congenital malformations weighed less than those without. CONCLUSION: In an unselected population, the infants of women with pregestational Type 1 diabetes mellitus have 6.4 times the reported risk of a congenital malformation and 5.1 times the reported risk of perinatal mortality than infants in the general population. Further improvements in the management of diabetes and pregnancy in these women are needed if the St Vincent's Declaration target is to be met.

Outcomes of pregnancy in insulin dependent diabetic women: results of a five year population cohort study.

OBJECTIVE: To monitor pregnancies in women with pre-existent insulin dependent diabetes for pregnancy loss, congenital malformations, and fetal growth in a geographically defined area of north west England. DESIGN: Population cohort study. SETTING: 10 maternity units in Cheshire, Lancashire, and Merseyside which had no regional guidelines for the management of pregnancy in diabetic women. SUBJECTS: 462 pregnancies in 355 women with insulin dependent diabetes from the 10 centres over five years (1990-4 inclusive). MAIN OUTCOME MEASURES: Numbers and rates of miscarriages, stillbirths, and neonatal and postneonatal deaths; prevalence of congenital malformations; birth weight in relation to gestational age. RESULTS: Among 462 pregnancies, 351 (76%) resulted in a liveborn infant, 78 (17%) aborted spontaneously, nine (2%) resulted in stillbirth, and 24 (5%) were terminated. Of the terminations, nine were for congenital malformation. The stillbirth rate was 25.0/1000 total births (95% confidence interval 8.9 to 41.1) compared with a population rate of 5.0/1000, and infant mortality was 19.9/1000 live births (5.3 to 34.6) compared with 6.8/1000. The prevalence of congenital malformations was 94.0/1000 live births (63.5 to 124.5) compared with 9.7/1000 in the general population. When corrected for gestational age, mean birth weight in the sample was 1.3 standard deviations greater than that of infants of non-diabetic mothers. Infants with congenital malformations weighed less than those without. CONCLUSION: In an unselected population the infants of women with pre-existent insulin dependent diabetes mellitus have a 10-fold greater risk of a congenital malformation and a fivefold greater risk of being stillborn than infants in the general population. Further improvements in the management of pregnancy in diabetic women are needed if target of the St Vincent declaration of 1989 is to be met.

Adolescents' ideas about the health of the fetus.

OBJECTIVE: To determine the preponderance of ideas in adolescents about the relationship between maternal habits and the health of the fetus. DESIGN: Quantitative survey using a precoded questionnaire, the content of which was derived from the transcripts of interviews and the responses to open-form questionnaires. SETTING: North West Region Health Authority area, UK. PARTICIPANTS: 674 adolescents in British National Curriculum Year 10 (age 14/15) from 6 Community Comprehensive Schools. FINDINGS: Most of the adolescents were aware of the dangers to the fetus of alcohol and smoking, including passive smoking. However, they were less aware of the potential hazards during pregnancy of eggs (Salmonella), soft cheeses (Listeria), liver (Vitamin A excess) or handling cats (Toxoplasmosis). Most of the respondents thought that the optimum time to initiate actions for a healthy pregnancy was when pregnancy had been confirmed, suggesting that the benefits of preconceptual care are not well known. KEY CONCLUSIONS: Adolescents lack knowledge about some of the specific hazards to the fetus of maternal diet and behaviour during pregnancy and are unaware of the importance of the early stages of pregnancy in this context. IMPLICATIONS: There is a need for the provision of education about the importance of a healthy maternal lifestyle before conception and during early pregnancy for adolescents. Midwives may have a proactive role in such education, but should be aware that young people may have specific areas of ignorance and misconceptions which will need addressing.

Effect of propylthiouracil treatment during prenatal and early postnatal development on the neocortex of rat pups.

The effects of a pre- and postnatal hypothyroid environment on the development of the neocortex have been determined. Rats were treated with propylthiouracil (PTU) with or without thyroid hormone supplementation during pregnancy and throughout the early development of the pups. The Cavalieri method was used to provide an estimate of the volume of the neocortex, and a combination of the Cavalieri method and the 'disector' particle-counting method provided an unbiased estimate of the numbers of glia and neurons in the neocortex. In pups from non-treated rats the mean volume of the neocortex increased from 31.3 mm3 at 5 days postnatally to 191.5 mm3 at 20 days and then remained constant to day 48. Similarly, the mean number of glial cells increased from 5.2 x 10(6) at day 5 to 12.0 x 10(6) on day 20 and then remained constant to day 48. The mean number of neurons in the neocortex in the control condition was constant at about 14.6 x 10(6) from day 5 to day 48. PTU treatment during pregnancy and postnatal development significantly decreased the mean volume of the neocortex at all of the stages studied, for example from 31.3 to 23.3 mm3 (p less than 0.001) on day 5 and from 191.5 to 155.0 mm3 (p less than 0.001) on day 20. Supplementation with thyroxine (T4) or tri-iodothyronine partially reversed this at certain stages. For example, the mean volume of the neocortex at 48 days was 197.5 mm3 in control rats, 118.1 mm3 in PTU-treated rats and 169.3 mm3 in PTU-treated rats supplemented with T4. Within the neocortex, the volume of the neuropile was more severely affected than was the volume of the neuron cell bodies.(ABSTRACT TRUNCATED AT 250 WORDS)

Computer modelling of neural tube defects.

Neurulation, the curling of the neuroepithelium to form the neural tube, is an essential component of the development of animal embryos. Defects of neural tube formation, which occur with an overall frequency of one in 500 human births, are the cause of severe and distressing congenital abnormalities. However, despite the fact that there is increasing information from animal experiments about the mechanisms which effect neural tube formation, much less is known about the fundamental causes of neural tube defects (NTD). The use of computer models provides one way of gaining clues about the ways in which neurulation may be compromised. Here we employ one computer model to examine the robustness of different cellular mechanisms which are thought to contribute to neurulation. The model, modified from that of Odell et al (Odell, G.M., Oster, G., Alberch, P. and Burnside, B., (1981)) mimics neurulation by laterally propagating a wave of apical contraction along an active zone within a ring of cells. We link the results to experimental evidence gained from studies of embryos in which neurulation has been perturbed. The results indicate that alteration of one of the properties of non-neural tissue can delay or inhibit neurulation, supporting the idea, gained from observation of embryos bearing genes which predispose to NTD, that the tissue underlying the neuroepithelium may contribute to the elevation of the neural folds. The results also show that reduction of the contractile properties of a small proportion of the neuroepithelial cell population may have a profound effect on overall tissue profiling. The results suggest that the elevation of the neural folds, and hence successful neurulation, may be vulnerable to relatively minor deficiencies in cell properties.

Effects of thyroid antagonists on rat embryos cultured in vitro.

A literature review of individual pregnancies and recent surveys involving large cohorts reveal an association between congenital malformation and maternal hyperthyroidism, suggesting that some aspect of hyperthyroidism or its treatment might compromise the development of the fetus. Experiments have shown that the thyroid antagonist, ethylenethiourea (ETU), causes fetal malformations when administered to pregnant rats, but it is not known whether it is ETU or the imbalance in maternal thyroid hormone which it causes which is the teratogenic agent. Here we employ in vitro culture to determine the possible direct effects on rat embryos of two thyroid antagonists, ETU and methimazole (MMI), the latter being one which is used for treatment of thyrotoxicosis in humans. It was found that ETU can compromise the development of rat embryos in vitro, confirming that ETU has a direct effect on the rat embryo. It was also found that MMI can cause abnormal development of rat embryos in vitro, although the concentration at which MMI disturbs rat embryogenesis is higher than that which is reached in hyperthyroid patients treated with clinical doses of MMI or carbimazole.

Morphological and physiological effects of beta-hydroxybutyrate on rat embryos grown in vitro at different stages.

Diabetic women are more likely to give birth to infants with congenital malformations than are nondiabetic women. Rodent embryos have been used as a model for the study of abnormal fetal development associated with maternal diabetes, and some of the metabolic factors which are altered in diabetes, such as raised glucose and ketones, have been shown to cause abnormal development of rodent embryos in vitro. The present work explores further the teratogenicity of beta-hydroxybutyrate to rat embryos. To determine the sensitivities of rat embryos at different stages of their development, rat embryos at 9.5 days of gestation have been cultured in vitro for 24 or 48 h, with or without 4 x 10(-2) M beta-hydroxybutyrate for all or part of the culture period. The embryos have been examined by scanning electron microscopy, and a detailed morphometric analysis of one tissue, the neuroepithelium, has been undertaken. The results confirm that beta-hydroxybutyrate causes abnormal development of rat embryos. The results of experiments in which embryos were exposed to beta-hydroxybutyrate for only part of a 48 h culture show that embryos exposed to beta-hydroxybutyrate for a complete 48 h culture are more severely affected than embryos exposed to beta-hydroxybutyrate for only part of the culture and that embryos are more vulnerable to beta-hydroxybutyrate during the first half of a 48 h culture (equivalent to 9.5 to 10.5 days of gestation) than during the second half of a 48 h culture (10.5 to 11.5 days of gestation). The results of experiments in which embryos were cultured with beta-hydroxybutyrate from 9.5 days of gestation for 24 h (equivalent to 9.5 to 10.5 days of gestation) showed that some effects of beta-hydroxybutyrate are already apparent after 24 hours in culture. Many of the abnormalities produced by beta-hydroxybutyrate can be classified as embryonic retardations rather than malformations--that is, embryos show features characteristic of normal, but younger, embryos. Embryos exposed to beta-hydroxybutyrate for the complete 48 h culture period consume less glucose and produce less lactate than control embryos on a per embryo basis, but not on a per microgram protein basis, suggesting that the reduced metabolism is an effect of beta-hydroxybutyrate-induced developmental delay rather than a cause of it.(ABSTRACT TRUNCATED AT 400 WORDS)

Perturbation of in vitro development of rodent embryos by calcium antagonist Quin-2.

Tissue shaping during embryogenesis is driven, at least in part, by changes in the shapes of the cells of the tissue undergoing morphogenesis. These cell shape changes are effected by microfilaments, and appear, as do microfilament-mediated events in non-embryonic cells, to be calcium-dependent. To explore the possible role of calcium in morphogenesis in mammalian embryos, the effects of Quin-2, a calcium-binding agent which can be loaded into cells as the acetomethyl tetraester (Quin-2-AM), on rat embryos cultured in vitro were determined. Quin-2-AM at 5 x 10(-5) M concentration delays the development of, and subsequently causes the abnormal development of, rat embryos cultured in vitro. In addition, Quin-2-AM inhibits the normal re-elevation of the cephalic neural folds which have been caused to collapse by exposure to medium containing a low concentration of calcium. These results suggest that morphogenetic movements, such as neurulation, depend upon an intracellular relocation of calcium, which can be perturbed by the calcium-binding capacity of Quin-2-AM.

Morphometric analyses of changes in cell shape in the neuroepithelium of mammalian embryos.

During neurulation the elevation of the neural folds is accompanied by, and thought to be partly driven by, changes in the shapes of the neuroepithelial cells. This paper explores the use of form factors for the measurement of changes in cell shape during normal and perturbed neural fold elevation in rat embryos; it is the first attempt to quantify changes in the shapes of cells during neurulation in mammalian embryos. Rat embryos at 9.9 days of gestation, when the neural epithelium is a biconvex plate, and at 10.4 days of gestation, when the cervical neural epithelium has formed the neural tube and when the cephalic neural folds have elevated but not fused, were used. In addition, embryos in which the cephalic neural folds had been caused to re-open by culture in saline with low calcium were examined. Embryos were fixed and then fractured across the neural axis. A montage of the fractured edge of the neuroepithelium was prepared from a series of scanning electron micrographs. Cells of the neuroepithelium were classified according to the shapes of their profiles as rectangular, round, apically tapered, basally tapered or spindle-shaped, the last three categories being considered as 'tapered'. The outlines of the cells were digitised using a computer-linked graphics tablet to provide estimations of the perimeters (p) and areas (a) of the cell profiles, and the form factors, f. of the cell profiles were calculated by f = 4 pi a/p2. This factor is a unitless ratio which is unity for a circle and less than unity for any other shape. It provides a measure of the efficiency with which a perimeter encloses an area, and thus indicates the degree of deformation of a shape from circular. From 9.9 to 10.4 days the cervical neuroepithelium shows a decrease in the percentage of rectangular cells and an increase in the percentage of spindle-shaped cells. The mean form factors of cells in the apically tapered and spindle-shaped categories decrease, showing that these cells are more elongated and tapered in later embryos. When embryos at 10.4 days are cultured in saline with low calcium the elevated cephalic neural folds collapse. Concomitantly the percentage of round cells in the cephalic neural folds increases and the percentage of apically tapered and spindle-shaped cells decrease.(ABSTRACT TRUNCATED AT 400 WORDS)

The effects of mannose on rat embryos grown in vitro.

Rat embryos have been cultured in vitro from 9.5 days of gestation for different times in serum containing mannose, and the embryos have been observed by scanning electron microscopy. Embryos cultured in 3 mg/ml (1.7 X 10(-2) M) or 6 mg/ml (3.3 X 10(-2) M) mannose for 48 h showed inhibition of the expansion of the yolk sac and were smaller than the control embryos. Mannose-treated embryos also showed delayed development according to morphological criteria, and a range of abnormalities including abnormalities of the neural tube. Embryos cultured in 6 mg/ml mannose for 24 h also showed significant inhibition of yolk-sac expansion and were smaller and less advanced than the control embryos. Abnormalities were seen, including a delay in the closure of the neural folds. Abnormalities were also observed in embryos cultured in mannose for 10 h; embryos at the neural groove stage showed irregularities in the neural groove. Mannose did not inhibit the re-elevation of neural folds which had been caused to collapse by exposure to medium containing low calcium. These results are compatible with the idea that mannose retards development and thereby perturbs the morphogenesis of the neural tube.

Effect on rat embryos of in vitro culture in sera from human diabetic patients.

Recently conditions have been established whereby rat embryos can be cultured in vitro in human serum over the period of their major morphogenesis. By standardizing the amount of glucose in sera from diabetic subjects prior to culture, it has been possible to investigate factors other than hyperglycemia which might account for the increased incidence of congenital defects seen in infants of diabetic mothers. The incidence of abnormalities in rat embryos cultured in sera from chlorpropamide-treated diabetics was not significantly different from that using control sera from normal subjects. On the other hand, sera from insulin-dependent diabetics produced more defects in rat embryos than sera from either normal subjects or diabetics on oral agents. The incidence of abnormal rat embryos does not appear to be correlated with patient age, with the original blood glucose concentration or with the concentration of hemoglobin A1 or, in the insulin group, with the total daily insulin dosage.

Calcium and neurulation in mammalian embryos. II. Effects of cytoskeletal inhibitors and calcium antagonists on the neural folds of rat embryos.

The role of calcium in neurulation in mammalian embryos has been studied by culturing rat embryos at 10.4 days of gestation, when the cephalic neural folds have elevated but not fused, in serum containing cytoskeletal inhibitors or calcium antagonists. The effects of these antagonists on the morphology of the cephalic neural folds have been examined by scanning electron microscopy. The different agents caused the cephalic neural folds to part to varying degrees. The neural folds were classified as intact (normal), open (folds parted up to 90 degrees with each other), flattened (folds parted from 90 degrees to 180 degrees) or collapsed (folds parted more than 180 degrees). The microtubule inhibitors colchicine and nocodazole at 10(-4) M respectively cause the cephalic neural folds of 10.4-day embryos to collapse after 60 min. At 5.2 X 10(-6)M the microfilament inhibitor cytochalasin B causes the folds to open after 60 min. Longer term culture of 9.5-day embryos for 24 h in diazepam, which is reported to inhibit myosin synthesis, causes general developmental retardation including a delay in the closure of the neural tube. Culture of 10.4-day rat embryos for 60 min in papaverine at 2.4 X 10(-4) M or gallopamil (D-600) at 5.0 X 10(-4) M, agents which reduce the entry of calcium into cells, causes opening of the elevated cephalic neural folds. In contrast TMB-8, which is purported to perturb some intracellular calcium-dependent functions, does not cause opening of the elevated cephalic neural folds, even at high concentrations. The results suggest that both microtubules and microfilaments are essential to the maintenance of the elevated cephalic neural folds in rat embryos. The results are also compatible with the idea that calcium ion flux across the membranes of the neuroepithelial cells might be important for the elevation of the neural folds, and thus for successful neurulation.

Ionophore-induced cell shape changes in Xenopus early embryos.

Local application of the Ca++ ionophore A23187 to the intact lateral ectoderm of Xenopus early neurulae causes changes in the shapes of the cells; ectoderm cells lose their relatively flat surfaces and become rounded. Some of the affected cells form microvilli. Ionophore was found to induce cell shape changes in ectoderm in the presence of cytochalasin-B, suggesting that microfilaments are not involved. Ionophore was also found to induce cell shape changes in neurula ectoderm when it was applied to embryos cultured in Ca++- and Mg++-free medium containing EDTA, suggesting that extracellular Ca++ is not utilized in the ionophore-induced cell shape changes. Similarly, the Ca++ antagonists D-600, which reduces the entry of Ca++ into cells, and TMB-8, which antagonises certain intracellular Ca++-dependent functions, did not inhibit the effects of A23187 on amphibian embryos.

Effects of calcium antagonists and calcium-buffered salines on wound healing in Xenopus early embryos.

The role of calcium in the process of wound closure in Xenopus early embryos was studied. Embryos were wounded in the presence of the calcium antagonists D-600 and TMB-8 or in calcium-buffered salines, and the effects on wound healing were observed by scanning electron microscopy. D-600 and TMB-8 inhibit wound closure and these antagonists appear to act synergistically since their combined effect is greater than their individual effects. Experiments with calcium-buffered salines suggest that wound closure can proceed in the presence of low extracellular calcium. In all conditions there is a correlation between the degree of wound closure and the shapes of the cells at the wound margin; closing wounds are accompanied by cells elongated radial to the wound, gaping (non-closing) wounds are accompanied by cells stretched tangential to the wound. Thus the results suggest that calcium influx may not be a requirement for the changes in cell shape which accompany, and probably effect, wound closure in Xenopus early embryos.

Calcium requirement for neural fold elevation in rat embryos.

To investigate the calcium requirement for the maintenance of the elevation of the neural folds in mammalian embryos 10.4-day-old-rat embryos, at a stage when the cephalic neural folds had elevated but not yet fused, were cultured in salines containing different concentrations of calcium. At 3 x 10(-3) M calcium the neural folds remained intact. As the concentration of calcium was reduced the elevated cephalic neural folds re-opened, flattened and then, at 10(-5) M calcium, collapsed. Replacement of calcium by strontium at 5 x 10(-3) M did not prevent the collapse of the cephalic neural folds, although at higher concentrations strontium partially prevented the collapse of the neural folds. In the cervical region most of the neuroepithelial cells were apically tapered. In intact cephalic neural folds nearly half of the cells were apically tapered. In cephalic neural folds which had been induced to collapse by culture in saline with low calcium there was an increase in the proportion of round cells and a decrease in the proportion of apically tapered cells.

Calcium and neurulation in mammalian embryos.

The role of calcium in neurulation in rat embryos has been studied. Rat embryos at 10 X 4 days of gestation, when the cephalic neural folds have elevated but not fused, have been cultured in various media, and the effects of these media on the morphology of the cephalic neural folds have been observed by scanning and transmission electron microscopy. Embryos cultured in serum containing EDTA or EGTA, or in saline without divalent cations exhibit opening, then folding back ('collapse') of the cephalic neural folds. The neural cells lose their elongated shape and become rounded. Older embryos in which the cephalic neural folds have already fused do not show collapse of the neural tube. Culture of 10 X 4-day rat embryos with elevated but unfused cephalic neural folds in calcium- and magnesium-free saline to which either calcium or magnesium has been restored shows that calcium is the divalent cation which is essential for the maintenance of the elevated neural folds. In the presence of calcium, lanthanum, which competes for calcium sites, causes opening but not collapse of the elevated cephalic neural folds. Embryos treated with trypsin show dissociation of the lateral (non-neural) ectoderm but the neural folds remain elevated. If embryos in which the cephalic neural folds have been caused to collapse are further cultured in serum the folds re-elevate, although normal neural tube morphology is not completely regained. The possible implications of these observations to the understanding of the cellular mechanisms of normal neurulation, and of neural tube malformations are discussed.