Thomas H. Shepard

Teratogenic bioactivation of cyclophosphamide in vitro

Abstract Day 10 rat embryos grown as cultured explants in vitro developed characteristic defects when culture media contained cyclophosphamide (6.25 micrograms per milliliter or more), an hepatic microsomal fraction, and cofactors for a monooxygenase system. Cyclophosphamide concentrations as high as 250 micrograms per milliliter were innocuous when either the microsomal material or cofactors were omitted from the medium. These experiments represent the first direct demonstration of bioactivation of a proteratogen.

MATERNAL HYPERTHERMIA AS A POSSIBLE CAUSE OF ANENCEPHALY

Mothers of anencephalic infants were asked about febrile illness and sauna bathing during their pregnancies. In 7 of 63 affected pregnancies (11%), a history of maternal hyperthermia near the presumed time of anterior neural-groove closure was given. 5 had fever with a maximum recorded temperature ranging from 38.9 to 40.0 degrees C, each case being secondary to a different type of illness. The other 2 had possible hyperthermia episodes as a consequence of sauna bathing, with no infectious agent involved. The frequencies of hyperthermia at the same period of gestation in two control groups were 0% and 0.1%. This excess frequency of maternal hyperthermia at the period of anterior neural-groove closure, and the differing natures of its causes, imply that hyperthermia itself may be an aetiological factor in anencephaly.

The development of hemostasis in the human fetus and newborn infant

Fragmentary understanding of normal perinatal hemostasis limits the clinician in his evaluation and therapy of neonatal hemorrhage. This report attempts to outline current concepts of hemostasis and to summarize the data obtained from fetal and perinatal studies, including investigations by the authors on human embryos and fetuses. The developmental events in human hemostasis are described and normal values for vascular, platelet, and clotting factor determinations are tabulated. Ontogenetically, the sequential development of vessel contractibility, platelet aggregation, and fibrin formation appears to recapitulate the phylogenetic pattern.

Glucose Metabolism By Rat Embryos in Vitro

Summary During the somite stages of rat embryogenesis glucose metabolism was high. Its sharp decline with advancing age was associated with reduced lactate production. By comparison of 14CO2 evolution rates from I-14C and 6-14C-glucose the pentose shunt pathway was shown to be active.

A selection of candidate compounds for in vitro teratogenesis test validation

The Consensus Workshop on In Vitro Teratogenesis Testing recommended that test validation be facilitated by a listing of agents with defined teratogenicity; subsequently, a panel was convened to review and select such agents. This communication established a list of 47 compounds or conditions which demonstrate a wide range of teratogenicity in vivo. The agents were chosen primarily on the strength of the literature base denoting their in vivo effects. The tables note a number of general biological and toxicological characteristics for each agent, and the details of representative in vivo teratology studies are summarized and referenced. This list is intended to serve as a base for in vitro teratogenesis test validation and should prove useful in developing and identifying those systems which will contribute to a more effective testing program.

Teratogenicity of cyclophosphamide metabolites: Phosphoramide mustard, acrolein, and 4-ketocyclophosphamide in rat embryos cultured in vitro

Abstract Phosphoramide mustard, acrolein, and 4-ketocyclosphosphamide, known stable metabolic products of bioactivated cyclophosphamide, were tested for their teratogenicity against rat embryos grown in vitro from Day 10 to Day 11 of gestation. Results indicate that phosphoramide mustard is the teratogenic metabolite of cyclophosphamide, since the effects of phosphoramide mustard exactly parallel those of bioactivated cyclophosphamide. These effects are reductions in total embryo protein content, crown-rump length and number of somites, characteristic malformations, and pattern of cell necrosis. Acrolein at a dose equimolar to a dose of bioactivated cyclophosphamide which produced malformations in 100% of treated embryos had no effect on any of the parameters measured. An equimolar dose of 4-ketocyclophosphamide had no effect on total embryo protein content or crown-rump length and number of somites, but consistently produced some malformed embryos. The kinds of malformations observed, however, are not seen in embryos treated with phospharamide mustard or bioactivated cyclophosphamide.

Chromosomal abnormalities in fetuses with omphalocele. Significance of omphalocele contents.

Twenty‐six consecutive fetuses with a sonographically detectable omphalocele and known karyotype were reviewed to identify risk factors that might be associated with chromosomal abnormalities. Risk factors that were analyzed included contents of the omphalocele sac, maternal age, fetal sex, sonographically detectable concurrent anomalies, and any major concurrent anomaly. Chromosomal abnormalities were found in 10 cases (38%) from trisomy 18 (n = 4), trisomy 13 (n = 4), trisomy 21 (n = 1), or 45, X (n = 1). The absence of liver from the omphalocele sac (intracorporeal liver) was strongly associated with an abnormal karyotype; chromosomal abnormalities were present in all 8 fetuses with an intracorporeal liver compared to 2 of 18 fetuses with an extracorporeal liver (p less than .0001, two‐tailed Fisher exact test). Other risk factors that were statistically associated with chromosomal abnormalities included advanced maternal age (greater than or equal to 33 years, p = .03) and sonographically detectable concurrent malformations (p = .05). We conclude that sonographic findings can help determine the relative risk of chromosomal abnormalities in fetuses with omphalocele; abnormal karyotypes were significantly associated with the absence of liver from the omphalocele sac and sonographically detectable concurrent malformations in this series. Sonographers should also be aware that omphaloceles that contain bowel alone tend to be small and can be missed or mistaken for other abdominal wall defects (gastroschisis or umbilical hernia).

Long-bone growth in fetuses with Down syndrome

Short stature is a well-recognized component of Down syndrome. The femur lengths of affected fetuses have been observed to be shorter than normal, with a ratio of actual to expected femur length of less than 0.91 indicating a high risk of trisomy. To further evaluate this finding we have determined the relationship between limb lengths and gestational age in 37 postmortem fetal specimens with trisomy 21. Control values were obtained from 174 normal fetuses. Measurements of the femur, tibia, fibula, humerus, radius, and ulna were made from roentgenograms or by direct measurement of the bone. Most measurements for each bone of the affected fetuses fell below the normal regression line for that bone, but only 3/37 femurs, 4/32 tibias, 5/32 fibulas, 9/32 humeri, 10/32 radii, and 7/32 ulnas fell more than 2 SDs below the mean. The ratios of actual to expected femur lengths were computed and six fetuses with Down syndrome (16.2%) had ratios less than 0.91. The bones of the extremities of fetuses with trisomy 21 are shorter than normal, but the differences are relatively small. The ratio of actual/expected femur lengths was a less efficient predictor of Down syndrome than were either maternal age or maternal serum alpha-fetoprotein tests. The upper extremity bones were shorter than normal more often than were the bones of the lower extremity, and this finding should be explored further in a prospective study.

Teratogenicity of vitamin B6 deficiency: omphalocele, skeletal and neural defects, and splenic hypoplasia.

Vitamin B6 deficiency was induced in pregnant rats with a deficient diet and with 4-deoxypyridoxine, a B6 antagonist. Treated animals developed typical skin changes of B6 deficiency. Fetuses were small and appeared anemic. Major fetal malformations were omphalocele, exencephaly, cleft palate, micrognathia, digital defects, and splenic hypoplasia. This teratologic system was developed as a model for human syndromes that exhibit combined immunologic and neurologic or skeletal defects.

Organ Weight Standards for Human Fetuses

Five hundred fifty-eight fresh human embryos and fetuses were obtained from the universities of Washington and Michigan following spontaneous loss, elective termination, or neonatal death within 2 days of delivery. The body weights ranged from 1.5 to 1500 g. Each of these autopsied specimens was morphologically normal. Specimens from diabetic or hypertensive mothers were not included. Correlations between fetal body weight and weights of adrenal, brain, kidney, liver, lung, spleen, and thymus were established. For analysis, regression curves were calculated as quadratic equations of best fit by the weighted least squares. The relation of the weights of brain, heart, and liver to body weight appeared linear. The ratios of thymus, spleen, and kidney to body weight were nonlinear and gradually increased. The ratios of lung and adrenal weights to body weight were also nonlinear and gradually decreased. Ninety-five percent prediction intervals were generated for each of the eight organs using a computerized statistical package. The results compare closely with smaller studies in the literature.