P-172 Increment of the Mouse Embryo Assay’s sensitivity by testing the detection of Triton X-100 in culture medium: the role of protein supplementation and medium formulation
Authors: Martínez Casado A , Mestres E , Casals A , Acacio M , Villamar A , Matia-Algué Q , Castelló C , Costa-Borges N , Calderón G 
Published: June 2022

Abstract

Study question
Does the type of culture medium and its supplementation with protein affect the sensitivity of the Mouse Embryo Assay (MEA) to detect Triton X-100 (TX-100)?

Summary answer
HTF medium showed a higher sensitivity than KSOM’s complex formulation, whilst the concentration of protein tested did not have a significant impact on MEA results.

What is known already
TX-100 is a detergent commonly used in the cleaning of production material. Occasionally, small TX-100 traces can remain in the final products, having sublethal effects on embryo development, potentially affecting their viability and implantation potential. The effectiveness of the MEA in detecting low-toxicity levels is not well described due to the lack of methodological standardization or regulation on the appropriateness of different testing protocols.
It is unknown whether media formulation modifies the sensitivity of the MEA. Similarly, media supplementation with albumin is suspected to chelate toxins and decrease the sensitivity of the assay, but no clear relation has been described.

Study design, size, duration
KSOM and HTF were selected and compared as media with complex vs. simple-formulation, respectively. The effect of protein concentration was assessed with both media at 0.5 and 5mg/ml HSA. Both variables were combined to culture embryos in the presence of sublethal concentrations of TX-100, previously established at 0.0005% and 0.0010%. Sensitivity was established by comparing blastocyst formation rates and total cell counts, which were statistically compared between groups to determine the conditions of greatest sensitivity.

Participants/materials, setting, methods
Experiments were performed with freshly-collected mouse zygotes (n = 783). Each TX-100 concentration (0% [control], 0.0005%, 0.0010%) was tested with all the proposed medium-protein combinations: KSOM 0.5mg/ml HSA, KSOM 5mg/ml HSA, HTF 0.5 HSA and HTF 5mg/ml HSA. All tests were performed in triplicate in a big-box humidified trigas incubator. Blastocyst formation was assessed after 96h of culture, and the obtained blastocysts were fixed and stained for cell counting.

Main results and the role of chance
No difference was observed in the blastocyst formation rate, nor the total cell counts between any of the control groups, showing that both studied media and protein concentrations were able to successfully support embryo development in vitro.
Based on the expanded blastocyst formation rates, HTF conferred greater sensitivity than KSOM with 5mg/ml HSA at 0.0010% TX-100 (p = 0.0024), and a similar but non-significant trend was observed with 0.5mg/ml (p = 0.0525). Interestingly, increasing HSA to 5mg/ml in HTF reported a higher sensitivity than 0.5mg/ml (p = 0.0399), which was not observed in KSOM. No medium-protein combination was able to detect TX-100 at 0.0005%.
Regarding the cell counting results, each test group cultured with 0.0005% or 0.0010% TX-100 was compared to its own medium-protein combination control. A statistically lower cell number (p &lt; 0.05) was obtained with 0.0010% TX-100 compared to the control groups, regardless of the culture medium or protein concentration used. By contrast, no statistical differences were detected in any of the groups when reducing TX-100 to 0.0005%. Interestingly, with both media and all TX-100 concentrations, the mean cell number per blastocyst was higher when increasing the concentration of HSA from 0.5 to 5mg/ml, whilst this had no effect on the result of the assays.

Limitations, reasons for caution
HTF proved more sensitive than KSOM with 5mg/ml HSA. The limited number of embryos/group could not confirm the statistical significance of this difference with 0.5mg/ml, but a clear trend was observed (p = 0.0525), which might be confirmed by additional replicas. Moreover, the results applicability to other toxicity sources requires further study.

Wider implications of the findings
Numerous culture variables can influence MEA’s sensitivity. Our results suggest that the type of culture medium can have a direct impact on the ability of the MEA to detect certain types of toxins. This information could help detect sublethal amounts of embryotoxic substances, avoiding potential negative clinical events.

Trial registration number
Not applicable