Improved detection of mineral oil toxicity using an extended mouse embryo assay.

Abstract

Successful in vitro fertilization (IVF) relies on sound laboratory methods and culture conditions which depend on sensitive quality control (QC) testing. This study aimed to improve the sensitivity of mouse embryo assays (MEA) for detection of mineral oil toxicity. Five experiments were conducted to study modifications of the standard mouse embryo assay (MEA) in order to improve sensitivity using clinical grade mineral oil with known peroxide concentrations. Assessment of blastocyst development at either 96h or in an extended MEA (eMEA) to 144h was tested in each experiment. In experiment 1, ability to detect peroxides in oil was compared in the MEA, eMEA, and cell number at 96h. In experiment 2, serial dilutions of peroxide in oil were used along with time-lapse imaging to compare sensitivity of the morphokinetic MEA to the eMEA. Culture conditions that may affect assay sensitivity were assessed in experiments 3-5, which examined the effect of group versus individual culture, oxygen concentration, and protein supplementation. Extended MEA and cell counts identified toxicity not detected by the routine endpoint of blastocyst rate at 96h. The eMEA was fourfold more sensitive than the standard MEA, and this sensitivity was similar to the morphokinetic MEA. Group culture had a protective effect against toxicity, while oxygen concentration did not affect blastocyst development. Protein supplementation with HSA had a protective effect on blastocyst development in eMEA. The standard MEA used by manufacturers does not detect potentially lethal toxicity of peroxides in mineral oil. While group culture may mask toxicity, protein supplementation and oxygen concentration have minimal effect on assay sensitivity. The eMEA and time-lapse morphokinetic assessment are equally effective in detection of peroxide toxicity and thus provide manufacturers and end-users a simple process modification that can be readily adopted into an existing QC program.