Use of endotoxin as a positive (toxic) control in the mouse embryo assay.
OBJECTIVE
The mouse embryo assay (MEA) is used to test media used for in vitro fertilization (IVF). Negative controls usually consist of previously tested media known to support growth of embryos to the blastocyst stage by 72 h. Often, no concurrent positive (toxic) controls are reported. Thus, any unusually hardy cohort of embryos may go undetected. Endotoxin was tested for its suitability as a positive control in the MEA.


RESULTS
Female mice were stimulated with gonadotropins mated with males, and embryos flushed from their oviducts 36 h after HCG injection. Two-cell embryos were pooled and randomly distributed to culture dishes containing media without protein supplement. Endotoxin inhibited blastocyst growth beginning at 50 micrograms/ml, with complete suppression of development at 5000 micrograms/ml. With 500 micrograms/ml endotoxin, an average of 34.8% of the embryos developed to the blastocyst stage for eight separate assays. The interassay coefficient of variation (CV) was 76%, while the intraassay CV was 9.4%. At 48 h the zona pellucida was absent from all of the embryos exposed to the endotoxin. A large difference was found between two lots of endotoxin with the same claimed potency.


CONCLUSIONS
These studies demonstrate the importance for inclusion of a well-defined positive control when performing the mouse embryo assay.
Dubin
N H
NH
Department of Obstetrics and Gynecology, Union Memorial Hospital, Baltimore, Maryland 21218, USA.
Bornstein
D R
DR
Gong
Y
Y
D000818:Animals; D001755:Blastocyst / Q000187:drug effects* / Q000502:physiology; D002455:Cell Division / Q000187:drug effects; D003470:Culture Media; D004305:Dose-Response Relationship, Drug; D005314:Embryonic and Fetal Development / Q000187:drug effects*; D004731:Endotoxins / Q000633:toxicity*; D005260:Female; D005307:Fertilization in Vitro; D006801:Humans; D008070:Lipopolysaccharides / Q000633:toxicity*; D051379:Mice; D008815:Mice, Inbred Strains; D015044:Zona Pellucida / Q000187:drug effects
D003470:Culture Media; D004731:Endotoxins; D008070:Lipopolysaccharides; C044101:endotoxin, Escherichia coli