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Use of endotoxin as a positive (toxic) control in the mouse embryo assay
N H Dubin1, D R Bornstein, Y Gong
1Department of Obstetrics and Gynecology, Union Memorial Hospital, Baltimore, Maryland 21218, USA.
Purpose:
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.
Insights
Endotoxin serves as a suitable positive control in the mouse embryo assay (MEA) for in vitro fertilization (IVF) media testing. Its inclusion helps detect unusually robust embryo development, ensuring accurate assessment of media quality.
Area of Science:
- Reproductive biology
- Developmental toxicology
Background:
- The mouse embryo assay (MEA) is crucial for evaluating in vitro fertilization (IVF) media.
- Standard MEA protocols often lack concurrent positive (toxic) controls, potentially masking outlier embryo development.
- A reliable positive control is needed to validate MEA results.
Purpose of the Study:
- To assess the efficacy of endotoxin as a positive control in the MEA.
- To establish a benchmark for detecting unusually hardy embryo development during IVF media testing.
Main Methods:
- Two-cell mouse embryos were collected and cultured in media with varying concentrations of endotoxin.
- Embryo development to the blastocyst stage was monitored over 72 hours.
- Assays were repeated to evaluate inter- and intra-assay variability.
Main Results:
- Endotoxin inhibited blastocyst development starting at 50 µg/ml, with complete suppression at 5000 µg/ml.
- At 500 µg/ml, 34.8% of embryos reached the blastocyst stage, with significant inter-assay variability (CV 76%).
- Endotoxin exposure led to zona pellucida loss by 48 hours and revealed lot-to-lot variability in endotoxin potency.
Conclusions:
- A well-defined positive control, such as endotoxin, is essential for accurate MEA performance.
- The inclusion of a positive control enhances the reliability of IVF media assessment.
- Further standardization of positive control agents is warranted.
