Quality Control Mouse Bioassay

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The preimplantation mouse embryo is the most widely used bioassay for medium components, culture media, and equipment used in clinical IVF. Using mice for testing media for human embryos has been the focus of much discussion due to conflicting reports in the literature of its suitability as a bioassay (310,311). Fukuda et al. (312) reported that for the mouse, in vitro fertilization and the development of zygotes and 2-cell embryos in culture was positively correlated with the purity of the water source used in the preparation of media. In contrast, George et al. (313) and Silverman et al. (314)

found that media prepared with tap water could support adequate development of both 1-cell and 2-cell mouse embryos to the blastocyst stage respectively, compared to media prepared with ultrapure water. The apparent contradiction of these studies can be resolved by taking into account the different stages of development used at the start of culture, the types of media used, and the supplementation of medium with protein. Fukada et al. (312) used BWW, a "simple-type" medium, whereas Silverman et al. (314) used Ham's F-10. The latter medium contains amino acids, which may chelate any possible toxins present in the tap water, e.g., heavy metals. George et al. (313), included high levels of BSA in their zygote cultures to the blastocyst. Albumins can chelate potential embryotoxins and thereby mask the effect of any present in the culture medium (154,201). Furthermore, all studies used blastocyst development as the sole criterion for assessing embryo development. Blastocyst development is a poor indicator of embryo quality and does not reflect developmental potential (49,50,114). A far more sensitive and quantitative parameter is blastocyst cell number (38,274).

The sensitivity of mouse embryos to their environment is inversely proportional to the age of the embryo at recovery, i.e., 1-cell embryos are more susceptible to toxins in the medium than embryos collected at the 2-cell stage (315). Removal of the zona pellucida from the zygote may further increases the sensitivity of the embryo to the culture conditions (316), and endotoxins (317). Furthermore, the type of mouse bioassay performed depends upon the strain of mice used. Inbred strains and their F1 hybrids are less sensitive to their environment and, therefore, the embryos are collected at the zygote. Embryos from outbred strains of mice are more sensitive to environmental factors, however, such embryos exhibit the 2-cell block in culture media such as HTF and therefore are collected at the 2-cell stage. However, with the recent development of more optimized culture conditions, it is now possible to routinely culture such embryos from the zygote stage (16,19,24,25,40,50,105,130).

A practical mouse embryo bioassay is to culture the zygote for 96 hours in protein-free medium. The rationale for using protein-free medium is that serum or serum albumin can chelate toxins, such as heavy metal ions, present in the medium. The presence of proteins would therefore hide any potential detrimental effects of the medium. Multiple ovulations are induced by injecting four to six week old virgin females with 5-10 i.u. pregnant mares serum (PMS), followed 48 hours later with 5-10 i.u. human chorionic gonadotrophin (HCG). Females are placed with males immediately following the second injection and mating assessed the following morning by the presence of a vaginal plug. Embryos are cultured in groups of 10 in 20 ml drops of medium under an oil overlay at 37° C. Culture dishes should be set-up and allowed to equilibrate overnight in a 6% CO2 atmosphere. To overcome any donor variation, embryos from each female should be allocated equally to each treatment group. Around 10 am on the day of plug, females are sacrificed and the oviducts excised and placed into warm collecting medium in a petri dish. The ampullary region of the oviduct is torn open close to the cumulus mass, which is then expelled under positive pressure into the medium. The cumulus is disaggregated by the addition of hyaluronidase (1mg/mL) in collecting medium. After around 1min, the cumulus disperses leaving denuded zygotes. The embryos are then washed twice in collecting medium and once in the culture medium and placed into culture. Zygotes are cultured in a protein free medium and cultured for 96 hours. Rather than a single end point of blastocyst development, it is important to determine appropriate on time development at set time points, and that there is no signs of necrosis in the blastocyst (this can be visualized easily on an inverted but not a stereo microscope, (258). Furthermore, it is most important that the cell number of the resulting blastocysts is determined. One can readily obtain 80% blastocyst development on day 5, but it is important also to note how embryos formed blastocysts in the afternoon of day 4 (i.e., on time development), and what the cell number is of the resultant blastocysts. There is a significant difference in the viability of blas-tocysts with 40 versus 80 cells.

Using such an approach to testing, one can pick up subtle problems that can exist with any media, or more typically with any contact supplies. It is important to note that just because a lot number of culture dishes has been approved safe for use in somatic cell tissue culture, does not automatically mean that it can support gametes or embryos.

With regards to alternative assays, such as hybridoma cell lines, although such cells in culture can be particularly sensitive to toxins in the medium, they are not embryonic cells and may therefore not detect potential embryo toxins. Although the mouse embryo bioassay does have a role in clinical IVF, at best it is only a test of the ability of the mouse embryo to develop. There is no guarantee that factors that do not affect the mouse will not be detrimental to the human. The ultimate quality control on all media for IVF is their ability to support human embryo growth. It is obviously unethical to use human embryos for this purpose. A possible solution would be the use of triploid embryos as an assay of media quality, as these cannot be replaced in the prospective mother. Therefore vigilant monitoring of embryo development within the IVF laboratory is not only essential, but should be considered as part of the laboratory's overall quality control system.

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