Fetal anomalies, skeletal examination

A case in point would be a study in which the doses were such that an increase in minor anomalies or skeletal variants was found at the lower doses and a high incidence of in utero death at the top dose (Doses A, B, and C in Fig. 1). It is possible that there are malformations occurring at a dose intermediate between those with anomalies and that resulting in embryo-fetal lethality (B and C). This is another instance where additional work should be done to ascertain whether this is indeed the case. In the author s experience, this pattern of findings can occur with various teratogens, particularly cardiovascular teratogens, and cardiovascular malformations should be suspected if they have not been found at the initial examination. 

Viable fetuses should be labelled for identification, examined for visible malformations, visceral and skeletal anomalies and variations, sexed and weighed. Since there is an inverse correlation between number of fetuses per litter and individual fetal weight, total litter weight is also a useful measurement. 

Kuna and Kapp (1981) found decreased fetal weight after exposure to 50 ppm and demonstrated that there was only 1 exencephalic rat in a group of 151 pups examined after in utero exposure to 500 ppm benzene. In the same study, of 98 pups examined for skeletal effects after in utero exposures of 500 ppm, only 1 pup had angulated ribs and 2 others had nonsequential ossification of the forefeet. These anomalies were not statistically significant and may have resulted from maternal nutritional stress. 

In contrast to primates, which abort dead embryos, dead rodent embryos are resorbed and the implantation site is recorded as a resorption site. The numbers of living and dead fetuses, and the number of resorption sites, are counted, and fetal weight, sex, and external malformations are recorded. Fetuses can either be inspected fresh in evaluation of internal soft tissues (known as the Staples technique) or can be placed in a fixative (usually Bouin s solution) and sectioned at a later time (Wilson technique). Other fetuses are fixed in ethanol, cleared in potassium hydroxide, and the cartilage and bone are stained with Alcian Blue and Alizarin Red, respectively. The US FDA recommends that one-third of the rodent fetuses be subjected to visceral examination and that two-thirds be studied for abnormalities of cartilage and bone. The US Environmental Protection Agency (EPA) recommends that one-third to one-half of each litter be examined for skeletal anomalies. For rabbits, all fetuses are to be examined for both visceral and skeletal malformations. 

---