Rat embryo development on human sera is related to numbers of previous spontaneous abortions and nutritional factors.

OBJECTIVES: The objectives were to determine (1) if sera from women with histories of spontaneous abortions were teratogenic to cultured embryos more often than were sera of nonaborters, (2) if the teratogenicity could be corrected by adding nutrients to the sera, and (3) if these findings were relevant to reproductive outcomes. STUDY DESIGN: Rat embryos were cultured for 48 hours on sera from 102 subjects who had experienced spontaneous abortions. Samples from 48 were retested with nutrients added and 10 took dietary supplements, were again tested with embryo cultures, and reported on their pregnancy outcomes. RESULTS: The frequencies of teratogenic sera increased with numbers of spontaneous abortions (0 to > or = 5) in a manner that did not deviate from linearity (27% to 89%) (chi 2 p > 0.957). Nutrient supplements were added to 48 samples, and 40 were corrected and 10 subjects were given dietary supplement. Sera from six showed improved embryo cultures, and these women completed their pregnancies. CONCLUSIONS: Rat embryo cultures may provide unique insights into the causes and treatment of spontaneous abortions.

Methionine decreases the embryotoxicity of sodium valproate in the rat: in vivo and in vitro observations.

Methionine provided in the drinking water of pregnant rats injected with sodium valproate reduced the frequency of resorptions but did not improve embryo growth. Rats drinking methionine supplemented water had approximately twice the level of serum-free methionine and consumed only one-half the volume of water of controls. Using whole rat embryo cultures, the simultaneous addition of methionine and sodium valproate to the medium provided no protection from neural tube defects, nor did the addition of methionine to a medium of serum obtained from rats previously dosed with sodium valproate. However, protection from the teratogenic effects of sodium valproate was afforded by methionine when the culture medium was sera from rats consuming methionine and was particularly striking when embryos for culture were taken from pregnant rats that had been consuming methionine. These observations along with those of others indicated the importance of dietary and culture media methionine levels in evaluating experimental and regulatory teratology studies and suggested the possibility that methionine may play an important role in human teratology where multifactorial causes have been implicated in problems such as neural tube closure defects.

Whole rat embryos require methionine for neural tube closure when cultured on cow serum.

Headfold-stage rat embryos, when cultured on cow serum without supplemental methionine, failed to close their neural tubes, lacked eyes and branchial arches, were abnormally shaped and were reduced in protein content compared to methionine-supplemented embryos. Methionine was essential during the first 18 h of culture, a period in which neural tube closure was initiated in supplemented cultures. All cow serum samples tested were found to require methionine addition, and the methionine was not replaced by other amino acids or vitamins, including folate. Methionine was not toxic to cultured rat embryos at concentrations up to at least 500 micrograms/ml. Analyses of serum free amino acids revealed lower levels of free methionine in cow serum compared to rat serum, and cow serum proteins contained less methionine relative to other amino acids than did rat serum proteins. Dialysis of cow serum reduced but did not eliminate the requirement for methionine. This suggested either that the free amino acids of cow serum were imbalanced or that a dialyzable component in serum interfered with the availability/utilization of methionine. Dietary supplementation of cows with rumen-protected DL-methionine increased the serum methionine level, and serum drawn from supplemented cows supported normal rat embryo development without additional methionine.

Methionine and iron as growth factors for rat embryos cultured in canine serum.

Development of headfold-staged rat embryos cultured in canine serum containing various supplements was compared with development in rat serum to seek suitable alternatives to rat serum in rodent embryo culture and to identify nutritional factors for cultured rodent embryos that may have relevance for normal mammalian embryonic growth and development. Supplementation of canine serum with glucose, methionine, and a lipophilic iron chelate allowed growth and development of cultured rat embryos, approximating those obtained with rat serum. These findings suggest that properly supplemented canine serum can serve as a suitable rodent embryo culture medium and that glucose, iron, and methionine may be important nutrients in mammalian embryonic development.

Human serum teratogenicity studied by rat embryo culture: epilepsy, anticonvulsant drugs, and nutrition.

Epileptic women have a greater risk for spontaneous abortions and children with birth defects than do nonepileptics. In a unique approach to identifying causes of these problems, we have cultured whole rat embryos for 48 h on blood sera from epileptics. In the first part of the study, three embryos were cultured on each serum sample from 128 different epileptics being treated with either single anticonvulsants or no drug to compare the teratogenicity of these drugs. Sera from subjects receiving either phenobarbital or no drug had comparable frequencies of cultured embryo abnormalities, which were lower than those from subjects taking phenytoin, valproic acid, or carbamazepine. In the second phase of the study, attempts to identify causes for serum teratogenicity led to the finding that the abnormalities and reduced embryo growth produced by many serum samples could be completely overcome by adding vitamins and/or amino acids to the serum. Of 53 samples tested, 32 (60%) were corrected by supplementation (17 of 23 phenytoin, seven of nine phenobarbital, six of 12 carbamazepine, none of six valproic acid, and two of three no drug). Although the results of this study provided a general assessment of drug teratogenicity that agreed with other studies and emphasized the role of nutrition in fetal defects, the importance of individual differences in causes of teratogenicity was also noted.

Successful culture of rat embryos on human serum: use in the detection of teratogens.

Growth of head-fold-stage rat embryos cultured with human serum for 48 hours was enhanced by supplementation with glucose. Embryo growth (protein and DNA contents) varied with the source of the serum. Serum from 16 of 19 untreated subjects produced normal embryos. Serum from five subjects undergoing cancer chemotherapy and six subjects receiving anticonvulsants was either lethal or teratogenic.