VALIDATION OF AN EMBRYOTOXICITY ASSAY

PROBLEM: Culture of mouse blastocysts has served as a tool for identif ying various embryotoxic factors in human serum, While inactivated, se ra from recurrently aborting women inhibit mouse blastocyst developmen t in vitro. Variation in results from individual serum samples has lim ited the usefulness of this assay in establishing a new classification of idiopathic recurrent spontaneous abortion (RSA). METHOD: Two-cell embryos were collected from superovulated mated CB6F1/J mice and cultu red in Ham's F-10 media supplemented with 10% fetal bovine serum (FBS) or tested human serum at 37 degrees C with 5% CO2 and high humidity. Each sample was assayed in triplicate using three mice with at least f ive embryos from the same mouse per dish. Development was evaluated at 72 h and the frequency of atretic embryos was recorded, RESULTS: Intr asample (interassay) variation yielded a coefficient of variation of 9 %. When repeated, samples from a given individual were evaluated and t he coefficient of variation was 8.7%. Interoperator variability was 4% interassay and 2% intraassay. Atresia of embryos was 23% when incubat ed with FBS (N=122), 21% in FC (N=122), and in the sera of patients wi th RSA 34.6% (N=95). Results of percentage of atresia from the fertile control group had a nonparametric distribution. Using 2.2 multiples o f the median to determine the 95% confidence interval, a threshold at 44.0% of atresia was established. CONCLUSIONS: The critical step in ma intaining low variability in this bioassay is to control mouse variabi lity by averaging the percentage atresia from different mice as embryo donors for each tested serum. A subgroup of 24% (23/95) RSA patients who displayed embryotoxic activity was identified with a specificity o f 95% and positive predictive value of 83%, P=0.001.