Update on new developments in the study of human teratogens.

BACKGROUND AND METHODS: The purpose of this annual article is to highlight and briefly review new and significant information on agents that may be teratogenic in pregnant women. Various sources of on-line and printed information are given. RESULTS: The following topics have been discussed: 1) lithium medication: decreased estimate of risk; 2) cigarette smoking and genotype as contributors to oral-facial clefts and clubfoot; 3) trimethoprim; 4) methimazole syndrome?; 5) glucocorticoids and oral-facial clefts; 6) binge drinking; 7) fetal valproate syndrome; and 8) carbamazepine. CONCLUSIONS: We have highlighted several maternal exposures during pregnancy that are associated with small but increased rates of birth defects, generally only a few cases per 1,000 infants. These exposures include cigarette smoking, and treatment with lithium, trimethoprim, methimazole, or corticosteroids. This weak teratogenic effect was usually identified by the linkage of an uncommon treatment with an unusual birth defect outcome. The use of modern epidemiologic techniques, especially prospective multicenter studies that provide increased numbers, has helped to strengthen the evidence for these associations. We discuss how teratogenic risks that are small in comparison to the background risk can be presented to at-risk women and their doctors. We have briefly listed some elements that might be used in prioritizing further studies of suspected teratogenic exposures. Various existing methods for expressing the strength of evidence for human teratogenicity are also given.

A history of the Teratology Society.

BACKGROUND: The 39-year history of the Teratology Society is reviewed. An abbreviated history is outlined in table form, along with listings of the Warkany Lectures, the postgraduate courses, and officers of the Society. METHODS: A year-by-year description of the events, including the scientific and social content of the annual meetings and changes in the business of the Society, is given, in many cases using comments from the past presidents. The valuable and unique diversity of the members is discussed and illustrated, presenting the disciplines and main research area of the presidents. The number of submitted abstracts and the various categories are tabulated, averaging the number and type over four periods. Within the past 10 years, a significant increase in the number of abstracts dealing with epidemiology and developmental biology is evident. The Society's development is compared with that of a human, and the question is asked: Have we reached the maturational stage of old age or senescence, or is the Society still maturing gracefully? This question needs further discussion by all the members. RESULTS: During the past 40 years, we have developed the scientific basis to prevent birth defects caused by rubella, alcoholism, and folate deficiency, as well as many other prenatal exposures. CONCLUSIONS: We must now engage in the political battles to obtain the resources needed to conduct further research and to implement the prevention programs, as well as to provide care and rehabilitation for persons with birth defects.

Mitochondrial ultrastructure in embryos after implantation.

Information on the morphology of mitochondria during embryogenesis is scattered in the literature, but there appears to be a consistent pattern. During early organogenesis, the embryo is in a state of relative hypoxia associated with a major decrease in terminal electron transport system activity and a marked increase in anaerobic glycolysis. Ultrastructural studies of a 14-somite monkey embryo and day 10 and 12 rat embryos, together with a review of the literature, led us to determine that this hypoxic stage is characterized by vesiculation of the mitochondrial inner membranes, or cristae. Starting in the late morula stage and continuing during early postimplantation embryogenesis, the cristae increase but appear tubular or vesicular. After the end of neurulation, and with the onset of vascular perfusion of embryonic tissues, the cristae gradually become lamellated; by the limb bud stage they appear more mature. We suggest that new cristae derive from blebs of the inner mitochondrial membrane and that with maturation these blebs collapse, giving them a lamelliform appearance. The delamellated state of the cristae might inactivate oxidative phosphorylation to protect the embryo from toxic respiratory end-products that could accumulate in an embryo before there is vascular perfusion. Consistent with this hypothesis, mitochondrial diameters in the developing heart of monkey and rat embryos were approximately twice those found in skin and neural tube.

Ultrastructural study of mitochondria and their cristae in embryonic rats and primate (N. nemistrina).

Information on the morphology of mitochondria during embryogenesis is scattered in the literature but there appears to be a developmental pattern characterized by vesiculation of the mitochondrial cristae. During early organogenesis, the embryo is in a relative state of hypoxia and this is associated with decrease of terminal electron transport system activity and a marked increase in glycolysis. Ultrastructural studies of a 14 somite monkey embryo, and day 10 and 12 rat embryos, along with a review of the literature led us to determine that this hypoxic stage is characterized by vesiculation of the mitochondrial cristae. Starting in the late morula stage and continuing during early postimplantation embryogenesis the cristae increase and appear tubular or vesicular. After the end of neurulation, and with onset of vascular perfusion, the cristae gradually become lamellated and by the limb bud stage appear more mature. We suggest that new cristae form from blebs of the inner mitochondrial membrane and that subsequently with maturation these blebs collapse giving them a lamelliform appearance. The delamellated state of the cristae may protect the embryo from toxic respiratory end-products of oxidative respiration which could accumulate in an embryo lacking vascular perfusion. In the heart of monkey and rat embryos, the mitochondria had diameters which were approximately twice those found in skin and neural tube.

The teratogenicity of N(omega)-nitro-L-ariginine methyl ester (L-NAME), a nitric oxide synthase inhibitor, in rats.

Exposure of gravid rats to the nitric oxide synthase inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME) in drinking water or by implanted osmotic minipumps significantly elevates maternal blood pressure, reducing uteroplacental perfusion. Administration by either route causes fetal growth retardation, but oral exposure also causes hind limb reduction malformations. The present study employed both oral and intraperitoneal routes to determine the period of sensitivity to developmental toxicity, dose-response, and possible fetotoxic mechanisms. Hind limb hemorrhage occurred only in litters from dams exposed to oral doses of 1 to 2 mg/mL from gestational days 15 through 17. In contrast to oral exposure, single intraperitoneal injections caused both fore and hind limb reductions at doses of 25 mg/kg and above administered on gestational day 16 and later. Many other exposures that reduce uteroplacental perfusion have been associated with vascular disruptive dysmorphogenesis. These exposures include phenytoin, calcium channel inhibitors, cocaine, and uterine vascular clamping. Limb hemorrhage induced by these exposures is usually limited to distal structures, typically phalanges, and the incidence of affected fetuses rarely exceeds 50%. By contrast, hemorrhage caused by L-NAME frequently involves entire limbs, extending into adjacent flank in severe instances, and 100% of fetuses from treated dams may be affected. The basis of this difference and the differing defect patterns associated with the various routes of exposure are unclear, but the generation of reactive oxygen species during resumption of normal perfusion may play a role in this vascular disruption.

Glucose absorption and utilization by rat embryos.

There is little doubt that glucose plays a significant nutritional role in early somite embryos. The high glucose utilization of anaerobic glycolysis drops as the activity of the Kreb's cycle and terminal electron transport increase. Concurrently, maturation of mitochondrial cristae and dependence on oxygen supply are taking place. The neuroepithelium of the early somite rat embryo responds in vitro during culture by microvilliar lengthening when exposed to glucose levels of 50 mg/dl or more. At lower glucose concentrations both in whole embryo culture and inside the closed neural tube the microvilli are shorter. Lengthening of the microvilli at room temperature is produced only by d-glucose and 2-deoxyglucose, two hexoses that are absorbed and phosphorylated. Cytochalasin D which disrupts actin polymerization causes ballooning of the microvilli. A role of this microvillar elongation in degenerative changes seen in uncontrolled diabetes and on function of the immune system is proposed. The amniotic cavity is one major portal of entry for glucose during the early somite embryo stage. The 7-fold increase in volume of the amniotic cavity after day 10 allows the rat embryo to convert its axis from dorsal to ventral flexion.

Uterine contraction in the development of Möbius syndrome.

We report a patient with Möbius syndrome. The birth defect in this child is probably the result of both vasoconstriction and uterine contractions brought on by ergotamine taken during the sixth week of pregnancy. We propose that vasoconstrictive or mechanical effects, or both, of abortifacient drugs such as ergotamine and misoprostol may account for other cases of Möbius syndrome, and we suggest that uterine contraction from any cause, at about the sixth or seventh week of pregnancy, may cause this birth defect. Further observational studies are needed to verify this hypothesis.

Assessment of reproductive disorders and birth defects in communities near hazardous chemical sites. I. Birth defects and developmental disorders.

Members of the workgroup on birth defects and developmental disorders discussed methods to assess structural anomalies, genetic changes and mutations, fetal and infant mortality, functional deficits, and impaired fetal and neonatal growth. Tier 1 assessments for all five adverse reproductive outcomes consist of questionnaires and reviews of medical records rather than laboratory testing of biologic specimens. The work-group members noted a role for neurodevelopmental testing and for limited genetic studies, such as karyotyping in Tier 2 assessments. Emerging methodologies to identify chromosomal aberrations, DNA adducts, and repair inhibition were reserved for Tier 3.

Neural plate microvillus lengthening in rat embryos grown in various concentrations of glucose and further studies of the mechanism.

Glucose is an important cellular nutrient, and in the early embryo, which is dependent mostly on anaerobic glycolysis, it is even more essential. Based on tissue culture cells in which glucose utilization has become membrane-limited, a concept has been developed that the tip of the microvilli is the entrance compartment for glucose and that the shaft sets up a diffusion barrier. An increase in length of the microvillus is associated with decreased entry of phosphorylated hexose into the cells. Our previous findings of lengthening of the microvilli of the neural plate cells after 40 min exposure to glucose at room temperature have been extended to a 17 hr whole embryo culture system. In cultures where the final concentration of glucose was 20 and 24 mg/dl there was embryonic death. In those cultures ending with 29-137 mg/dl of glucose the embryos developed normally. Those grown in dialyzed serum supplemented with B vitamins and glucose grew equally as well as those in whole rat serum. Somite numbers attained did not change with increasing glucose concentration but a modest increase in micromoles of glucose used per embryo was found, suggesting the presence of another source of energy at lower glucose concentrations. The average glucose utilization per gram of protein per hour was 844 mumol in these day 9.5-10 embryos and this compares to 733 mumol previously found using uniformly labeled 14C glucose on day 10.3. Lactate production averaged 85% of the glucose utilized. Pyruvate did not support growth in the absence of glucose.(ABSTRACT TRUNCATED AT 250 WORDS)

Staurosporine does not prevent adrenergic-induced situs inversus, but causes a unique syndrome of defects in rat embryos grown in culture.

Staurosporine, an alkaloid isolated from Streptomyces species, is commonly used as a protein kinase C (PKC) inhibitor in animal investigations. In the present study, we used this compound to determine whether alpha 1-adrenergic stimulation-induced situs inversus in rats is mediated by PKC. Embryos were explanted at 8 A.M. on day 9 of gestation. Those with a neural groove but with no visible neural folds (Stage 11a) were selected and were cultured in medium containing various concentrations of staurosporine with or without 50 microM of phenylephrine, an alpha 1-adrenergic agonist. At 10 A.M. on day 11 of gestation, embryos were examined for situs inversus and other abnormalities. Staurosporine, tested at 0.0001, 0.001, 0.01, 0.1, 0.375, and 0.5 microM (lethal concentration), did not block phenylephrine-induced situs inversus at any concentration. However, staurosporine alone produced situs inversus at concentrations above 0.1 microM. At 0.5 and 1.0 microM, staurosporine also caused cyst-like lesions projecting dorsally from the mesencephalon that we named "mesencephalic vesicles" and the formation of secondary somites. To confirm and further examine these unique effects of staurosporine both grossly and histologically, we conducted additional experiments using staurosporine from another source. Embryos were explanted between 6 A.M. and 9 P.M. on day 9 of gestation and were placed in one of the following groups according to their stage of development: 10b, 11a, 11b, 11c, 12/s1-2, 12/s3-4, and 12/s5-6. Embryos were then cultured with various concentrations of staurosporine. Those cultured from Stage 11a exhibited similar lesions to those seen in the initial experiment but at somewhat higher concentrations of staurosporine. Embryos cultured from Stage 10b showed a similar pattern of lesions as seen at Stage 11a, except that higher concentrations of staurosporine were required to cause mesencephalic vesicles and secondary somites formation. Embryos cultured from Stage 11b showed similar effects to those cultured from younger stages except that maximum incidences of situs inversus were much lower. Those cultured from Stage 11c showed similar dose-response to those cultured from Stage 11b except that the incidence of secondary somites formation was much higher. In addition, in approximately 40% (n = 25) of embryos treated with greater than 1.0 microM of staurosporine, the growing end of the allantois did not reach the chorion and remained unattached in the exocoelomic cavity.(ABSTRACT TRUNCATED AT 400 WORDS)

Growth parameters in mid-trimester fetal Turner syndrome.

Growth failure is a consistent finding at birth in infants with Turner syndrome. However, the time of onset and pattern of growth deficiency is unknown. To determine the presence of growth failure in the second trimester in fetuses with Turner syndrome, second trimester fetuses that had a complete autopsy at the Central Laboratory for Human Embryology at the University of Washington were studied. A control group of specimens with normal findings was selected and compared with a study group with Turner syndrome documented by karyotype. Footlength and crown-rump length were measured directly with a ruler and femur, tibia, fibula, humerus, ulna and radius were measured from X-rays. Crown-rump length was used as the indicator of gestational age. Statistical comparisons between the normal and study groups were performed by multiple regression. Long bone measurements were made on 105 normal and 13 Turner fetuses. Footlength and the six long bones showed evidence of statistically significant growth failure. Fetuses with 45,X/46,XX mosaic Turner syndrome may demonstrate a lesser degree of growth retardation, at least for footlength, than those with a 45,X karyotype, but small numbers limited the analysis. We conclude that the growth failure consistently demonstrated in newborns with Turner syndrome begins early in gestation and is well-established by mid-pregnancy.

Volume and glucose concentration of rat amniotic fluid: effects on embryo nutrition and axis rotation.

In order to pursue our previous studies of the changes in neural tube microvilli produced by glucose, we developed a micro method of measuring glucose concentration in the very small volumes of amniotic fluid during neurulation. The volume of amniotic fluid was found to increase nearly 10-fold during major neurulation (day 10 to day 11 in the rat). This increase in volume and our repeated observations that physical removal of the restraining amnion initiates embryonic rotation leads us to propose that the growth of the amniotic cavity is essential for conversion from the ventral- to dorsi-flexion of the embryonic axis. Amniotic fluid volume continues to increase until day 18 but dropped by day 20. A method for glucose determination was developed using the color reaction on glucose oxidase indicator paper. The intensity of the color was analyzed with a color scanner. Amniotic glucose was 27.1 +/- 1.6 mg/dl on day 10 and continued in this range with some fluctuation until day 20 when it decreased. We isolated days 10, 11, and 16 embryonic sites from their decidua and incubated them at 0 degrees C and 38 degrees C while measuring glucose concentration. The glucose concentration did not show significant decrease at 0 degrees C on day 10 or 11 or on day 16 at 38 degrees C. At 38 degrees C the day 10 embryo amniotic fluid glucose disappeared after 22 minutes and the day 11 amniotic fluid glucose was gone in 34 minutes. These depletion times were statistically different. The magnitude of glucose depletion on day 10 was shown by calculation to be approximately 323 mumoles/gm protein per hour which is a substantial portion of the glucose utilized by the embryo as determined in previous experiments (731 mumoles/gm protein per hour). This model may serve as a way to study glucose utilization by embryos after their exposure to various teratogens.

Reversed circulation in acardiac fetuses is associated with anatomic inversions in the aortic wall.

Blood vessel anatomy is probably related to hemodynamic factors which change during development. This principle has been postulated as the basis for differences in the numbers of elastic lamellae in the tunica media of the proximal and distal human aorta. Recent studies of human fetuses at varying stages confirmed that the number of elastin lamellae in the aortic wall varied along its length (spatially) and with age (temporally). These findings suggest that hemodynamic influences during prenatal development induce structural changes in the aortic wall. Acardiac fetuses provide a model for studying the effects of hemodynamic changes on the structure of elastic arteries because blood flow through the aorta in such fetuses is reversed and greatly reduced. Also, analysis of the vascular structure of acardiac fetuses would further define the characteristic features of this congenital disorder. In the present study, we have examined the gross and histological anatomy of the aorta from seven acardiac fetuses. In each case, the microscopic architecture of the aorta was deranged and the normal proximal-distal differences in arterial caliber and number of elastin lamellae were reversed. In the proximal aorta, medial lamellar units were thin, fragmented, and irregular. In some segments, only traces of medial lamellar units existed. We believe that these anatomic abnormalities represent degenerative or dysplastic responses to reversed flow and pressure gradients in the aorta. These findings are consistent with the hypothesis that aortic wall structure is influenced by hemodynamic factors during development.