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

Abstract

Purpose 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. Methods 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 96 h or in an extended MEA (eMEA) to 144 h was tested in each experiment. In experiment 1, ability to detect peroxides in oil was compared in the MEA, eMEA, and cell number at 96 h. 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. Results Extended MEA and cell counts identified toxicity not detected by the routine endpoint of blastocyst rate at 96 h. 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.Conclusions 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.

Mineral oil: safety and use as placebo in REDUCE-IT and other clinical studies

“…7 Reports of mineral oil toxicity are related to non–pharmaceutical-grade mineral oil and mainly due to peroxidation of aromatic and saturated hydrocarbon impurities rather than the mineral oil itself. 31 …”

Off-gassing plasticware to decrease the toxicity effect in embryo culture

“…Unfortunately, there is still no consensus about the procedure. For example, most laboratories and manufactures use different cutoff acceptance criterion: 70%, 75% or 80% blastocyst formation, none based on statistical criteria (Ainsworth et al, 2017;Fleetham et al, 1993;Sathananthan et al, 2003). In the scientific community there is strong disagreement about the use of MEA results to predict human embryos, although several strategies have been reviewed to improve the sensitivity of MEA .…”

Controversies in ART: considerations and risks for uninterrupted embryo culture

“…Therefore, the initial quality of the oil is likely to be important to ensure damaging peroxides are not present before culture. This starting oil quality, including VOC content, peroxide levels and ionic composition, all which may differ between oils , may be more important if using an uninterrupted approach and thorough oil quality testing and improved reporting by manufacturers and distributors is prudent (Hughes et al, 2010;Morbeck et al, 2010;Khan et al, 2012;Wolff et al, 2013;Ainsworth et al, 2017).…”