Mouse strain and quality control testing improved sensitivity of the mouse embryo assay with embryos from outbred mice

Capsule:
Embryos from outbred mice are sensitive to in vitro toxins and may provide a more reliable measure of quality for undefined culture components such as mineral oil.

Authors:
Zaraq Khan, M.B.B.S., Heather S. Wolff, M.D., Jolene R. Fredrickson, M.S., David L.Walker, M.S., Gaurang S. Daftary, M.D., Dean E. Morbeck, Ph.D.

Volume 99, Issue 3, Pages 847-854.e2, 1 March 2013

Abstract:

Objective:
To determine the relative sensitivities of embryos from different strains of mice to in vitro stress.

Design:
Laboratory experiment with embryos from different mouse strains

Setting:
University hospital-based fertility clinic.

Animals:
Mice.

Interventions:
Fresh one-cell embryos from outbred (CF1), inbred (FVB), F1 hybrid (B6/CBA) and cryopreserved F2 hybrid embryos (bcl/B6 x B6/bcl) were compared in a mouse embryo assay (MEA) using six doses of each of three in vitro stressors. Treatments included cumene hydroperoxide (CH) in mineral oil, Triton X-100 (TX-100) in media, and hyperosmolality.

Main Outcome Measures:
Blastocyst rate (BR) at 96 hours was considered the primary outcome. All studies were conducted in triplicate; data were analyzed with Chi Square analysis based on fitting a logistic regression model.

Result(s):
Both CH and TX-100 affected blastocyst formation in the outbred strain at concentrations that were less than half of the concentration that affected the other strains. Total number of cells was affected by treatments in all strains.

Conclusion(s):
CF1 embryos are genetically diverse and more sensitive to toxins than either inbred or hybrid mouse embryos. Outbred embryos provide an additional tool for effective quality control testing.

  • Dean Morbeck

    Gametes and human embryos in particular are probably the most sensitive biological specimens handled therapeutically, making the search for a suitable, sensitive surrogate critical for assurance of optimal conditions. While many different assays have been studied and their merits are still debated, the prevailing opinion was that outbred mice were the most sensitive, and yet other than one company, few are using them and since there was no scientific evidence of superiority, advancement of this model seemed unlikely.

    When Dr. Khan, an OB/GYN resident at the time, was looking for a project, I mentioned this one and said, while not overly glamorous, it was a question needing an answer. Two people deserve credit for the genesis of this idea. First, through many discussion about merits of different QC assays with Eric Dorman from EmbryoTech, he always complained that good toxicity studies have not been done to compare assays. Based on these discussions and prior to this mouse strain study, we did exactly that. First with formaldehyde (Gada et al., 2012 FS) and then with peroxides (Hughes et al., 2010 JARG) (I’ve listed these in order of when they were performed, not when they were published!). The second individual who influenced this work is Kim Pomeroy, Ph.D., HCLD. I organized a Madison Embryologist Summit on QC and invited Dr. Pomeroy to speak. It was at this or a subsequent meeting, likely over a game of foosball or darts at State Street Brats, that we discussed the need for a good comparison of mouse strains. As often is the case, good ideas come over a good social time and perhaps over a pint or two of beer….

    Dr. Khan put in many hours to make this project a reality, which is no small task given the work-hours residents log here in the US, where only recently were they restricted to 80 hours! In the end, we were pleased to see that embryos from outbred mice are indeed more sensitive to toxins, though we learned first-hand how challenging they can be to work with. Due to these challenges, it remains to be seen if more manufacturers use outbred mice for QC testing.

    This story is perhaps still evolving, though, as a recent study by Schwarzer et al., (2012, HR) illustrates that embryos with different paternal genotypes, in this case inbred C57/B6 vs outbred CD1males with F1 females, develop cell lineages differently in response to different ART culture media, suggesting that quality control sensitivity could be enhanced further with the resilient F1 female paired with a less resilient male genotype.

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