From mouse embryo assay to bovine embryo assay: towards an ethical and scientifically superior quality control standard in assisted reproduction technologies.

Since its inception, the number of voices proposing improvements or changes to MEA testing in the ART industry and within the scientific community has been increasing, particularly since the publication of the FDA Modernization Act 2–0 which indicates that “animal testing should be phased out with the exception of appropriate allowances” [ 121 ]. However, there has been a paucity of implementation for realistic alternatives. One of the primary criticisms of the MEA is its lack of standardisation, which consequently impacts the consistency of the results. The protocols outlined in the FDA Guidelines [ 4 ] are not universally defined, and ambiguity exists with regard to subjective analysis of morphology in developing embryos, culture conditions, media, O 2 concentration, size of medium drop, number of embryos in each drop, pH, osmolality, and other factors. This variability among laboratories can introduce inconsistencies and potentially compromise the reliability of the results. A 2018 survey by Delaroche [ 72 ] of several leading IVF device manufacturers revealed significant differences in MEA methodology and thresholds for toxicity testing. Some of these parameters influence test sensitivity. In the authors’ words, their “study confirms the high degree of heterogeneity of the embryotoxicity tests performed by manufacturers when validating their IVF disposable devices. Future recommendations are urgently awaited to improve the sensitivity and reproducibility of embryotoxicity assays over time.” A number of eminent scientists have also expressed reservations about MEA, citing various reasons. One such contention refers to the evident disparity in the reproductive physiology of mice when compared with that of humans [ 7 ]. Indeed, mice ovulate multiple oocytes per cycle (≈10) while human is a single ovulation species. The final stages of oocyte maturation appear to be subject to more stringent regulation in humans, where a minimum follicle size is required for in vitro maturation to occur [ 7 ]. Furthermore, the developmental timeline of the mouse embryo is characterised by the attainment of the morula stage on day 2.5, followed by the progression to the blastocyst stage by day 3.5–4 [ 122 ]. Conversely, the human embryo reaches the morula stage on day 4, and the blastocyst develops by day 5 [ 123 ]. With regard to embryonic genome activation (EGA), this occurs at the 4–8 cell stage in the human [ 124 ] and the 1–2-cell stage in the mouse [ 125 ]. Finally, it is noteworthy that the gestation period is shorter in mice, spanning less than 1 month, and longer in humans, extending to approximately 9 months. As Chronopoulou and Harper [ 20 ] pointed out, there are other reasons why people have criticised MEA. These include the fact that the results can be different depending on the strain [ 59 , 126 , 127 ], the ability of mouse embryos to develop into blastocysts when cultured in tap water without any protein supplement [ 128 , 129 ], and the finding that mouse embryos that lost one blastomere at the 2-cell stage and therefore had reduced inner cell mass (ICM) developed into blastocysts and achieved implantation [ 130 ]. Nonetheless, the primary impediment to the continued utilisation of MEA for regulatory and testing purposes within the domain of ART, as perceived by the author, is ethically grounded. Contemporary alternatives have emerged that no longer substantiate the continued employment of MEA. Among them, the use of bovine model [ 7 ], functional molecular biomarkers [ 41 ], time-lapse imaging, human embryonic stem cell-based assays [ 91 ], “organ-on-a-chip” technology, organoids, digital artificial intelligence, and machine learning [ 121 ] have been proposed. Of course, the most similar test to MEA from all the above referred is that using the cow as an animal model, that is the bovine embryo assay (BEA). The advantages of this model begin with the similarities between the reproductive cycle of the cow and the woman, being the cow a single ovulation species, where the final stages of oocyte maturation are similarly regulated to humans [ 7 ], the developing embryo reaches morula stage at day 5, blastocyst stage at day 7–8, EGA occurs at 8–16 cells [ 131 ], and the gestation takes 9 months as it happens in humans. Also, the availability of biological material is unlimited, since the oocyte donors are not experimental animals bred with the only purpose of being hormonally stimulated, mated, and sacrificed to collect their oocytes, zygotes, or embryos, but animals come from the dairy or meat industry, are bred to provide food for the human consumption and sacrificed in slaughterhouses under strict sanitary and welfare controls. Two major problems can be deduced from the bovine model: the variety of breeds and genetic background at abattoirs, and the lack of standardised protocols. The former can be regarded as a benefit, however, as referenced in the recent studies by Korchivaia et al. [ 28 ], which propose that only a pooled sample of different mouse strains can be used for comprehensive media MEA testing. Interestingly, despite the significant variations in slaughterhouse materials and embryo production protocols between laboratories, the blastocyst yield at the end of the culture period remains consistent at 30–40% [ 132 – 135 ]. This figure can therefore be used as a reference standard for comparison in an embryotoxicity assay. The second issue may be resolved through the implementation of appropriate laboratory practices, such as those outlined in ISO 17025:2017, thereby ensuring the reliability and reproducibility of the results obtained. Given the increasing number of laboratories and industries producing bovine embryos worldwide (nearly 1.6 million in vitro-derived cow embryos were transferred in [ 50 ], the development of standardised and authorised BEA protocols, accepted by regulatory agencies in each country, does not seem utopian. Efforts to achieve this goal are already underway in our laboratory and others, with comparable data between MEA and BEA [ 6 ]. Among other advantages (see Table  4 ), BEA enables the toxicity of devices and culture media to be assessed at various stages of the in vitro embryo growth process, from the collection and manipulation of oocytes to in vitro fertilisation, or development until the two-cell stage, the point at which standard MEA begins. Therefore, unlike the standard MEA described by FDA, not only is the sensitivity of two-cell embryos to developing into blastocysts in a specific culture medium or device assessed, but also the sensitivity of oocytes and spermatozoa to being manipulated or fertilised in such devices. Table 4 Merits and drawbacks of replacing the mouse embryo assay with the bovine embryo assay when assessing the functionality and toxicity of medical devices in the assisted reproduction industry Advantages Disadvantages • Single offspring (like humans) •Long reproductive cycle (21 days) and gestation (9 months) •No need of animal sacrifice to run the tests •More similar to humans in terms of biochemical and intrinsic paternal and maternal regulatory processes •Similar regulation of the final stages of oocyte maturation to humans •Embryo development chronology and EGA more similar to human •Gastrulating/neurulating processes are closer to humans •Unlimited availability of biological material •Big group size, hence high statistical power and less variability across groups •Obtaining semen is an easy process thanks to the numerous specialized companies dedicated to commercializing it for artificial insemination •No need of the establishment of dedicated animal care facilities or the allocation of extra personnel for this purpose •Bovine embryos are more sensitive to environmental conditions (concerns about mouse embryo sensitivity) •Mouse embryos not requiring amino acids to develop to the blastocyst stage, in contrast to bovine and human embryos •Mouse embryos are less sensitive to and recover more easily from changes in pH than either human or bovine embryos •Non ethical issues •Greater social acceptance •Contribution to sustainability and organic waste recovery • Single offspring (more difficult to perform long term studies with the progeny) •Long reproductive cycle (21 days) and gestation (9 months) •High background pregnancy loss •Limited availability of pure breed animals •Expensive for offspring studies •Less historical control data and laboratory experience/capability •Highly variable age, weight and pregnancy history at the start Merits and drawbacks of replacing the mouse embryo assay with the bovine embryo assay when assessing the functionality and toxicity of medical devices in the assisted reproduction industry • Single offspring (like humans) •Long reproductive cycle (21 days) and gestation (9 months) •No need of animal sacrifice to run the tests •More similar to humans in terms of biochemical and intrinsic paternal and maternal regulatory processes •Similar regulation of the final stages of oocyte maturation to humans •Embryo development chronology and EGA more similar to human •Gastrulating/neurulating processes are closer to humans •Unlimited availability of biological material •Big group size, hence high statistical power and less variability across groups •Obtaining semen is an easy process thanks to the numerous specialized companies dedicated to commercializing it for artificial insemination •No need of the establishment of dedicated animal care facilities or the allocation of extra personnel for this purpose •Bovine embryos are more sensitive to environmental conditions (concerns about mouse embryo sensitivity) •Mouse embryos not requiring amino acids to develop to the blastocyst stage, in contrast to bovine and human embryos •Mouse embryos are less sensitive to and recover more easily from changes in pH than either human or bovine embryos •Non ethical issues •Greater social acceptance •Contribution to sustainability and organic waste recovery • Single offspring (more difficult to perform long term studies with the progeny) •Long reproductive cycle (21 days) and gestation (9 months) •High background pregnancy loss •Limited availability of pure breed animals •Expensive for offspring studies •Less historical control data and laboratory experience/capability •Highly variable age, weight and pregnancy history at the start Some other advantages and disadvantages of BEA versus MEA are shown in Table  4 .

A comprehensive search of the PubMed database using the search term “mouse embryo assay” yielded 48 articles that focused primarily on this test, which were then selected and analysed. Information on the medical devices (preferably culture media) used in assisted reproduction that had been tested by the MEA was gathered from company websites, advertising materials, instructions for use and certificates of analysis. To investigate the current legislation regarding the use of the mouse embryo assay in assisted reproduction in the EU, the websites of the European Medicines Agency (EMA), the European Commission and the European Society of Human Reproduction and Embryology (ESHRE) were explored. Legislation in China (National Medical Products Administration, NMPA), the USA (FDA), and Australia (Therapeutic Goods Administration, TGA) was also investigated. Realistic estimates of the number of animals slaughtered for MEA purposes, the number of animal facilities involved in rearing these animals, and the amount of waste and residues generated were obtained through thorough searches of PubMed and specific websites. The same approach was used to gather data on the historical development of social movements committed to pursuing animal rights, as well as the ethical principles that became universal under the 3Rs premise.

Assisted reproduction technologies (ART) encompass a growing set of tools whose demand is increasing exponentially as clinical indications continue to expand and diversify. Today, millions of individuals worldwide rely on ART to address fertility challenges. With the global proliferation of infertility clinics, the implementation of robust quality management systems and regulatory controls has become increasingly necessary. The mouse embryo assay (MEA) is currently the reference test used to assess functionality and embryotoxicity in ART devices. It is estimated that hundreds of thousands of mice are sacrificed annually for this purpose. However, MEA has been subject to growing criticism due to concerns about its sensitivity, lack of standardization, and ethical implications. A thorough review of international regulations reveals that the use of MEA for toxicity and functionality testing of ART-related medical devices is not mandatory in most regions. On the contrary, many regulatory agencies actively promote the use of alternative methods that reduce or replace animal use, in line with the 3Rs principle (Replacement, Reduction, and Refinement) and the broader goal of advancing animal welfare. This article proposes the gradual replacement of MEA with the bovine embryo assay (BEA) and recommends that BEA be incorporated into the regulatory frameworks of relevant institutions worldwide. BEA does not require the use of laboratory animals, relying instead on by-products from slaughterhouses, where animals are already processed for food production. Coordinated efforts among research institutions, cattle reproduction laboratories, regulatory bodies, and ART device manufacturers will be essential to support this transition and to establish BEA as a reliable, ethical, and scientifically robust alternative to MEA. Assisted reproduction technologies (ART) encompass a growing set of tools whose demand is increasing exponentially as clinical indications continue to expand and diversify. Today, millions of individuals worldwide rely on ART to address fertility challenges. With the global proliferation of infertility clinics, the implementation of robust quality management systems and regulatory controls has become increasingly necessary. The mouse embryo assay (MEA) is currently the reference test used to assess functionality and embryotoxicity in ART devices. It is estimated that hundreds of thousands of mice are sacrificed annually for this purpose. However, MEA has been subject to growing criticism due to concerns about its sensitivity, lack of standardization, and ethical implications. A thorough review of international regulations reveals that the use of MEA for toxicity and functionality testing of ART-related medical devices is not mandatory in most regions. On the contrary, many regulatory agencies actively promote the use of alternative methods that reduce or replace animal use, in line with the 3Rs principle (Replacement, Reduction, and Refinement) and the broader goal of advancing animal welfare. This article proposes the gradual replacement of MEA with the bovine embryo assay (BEA) and recommends that BEA be incorporated into the regulatory frameworks of relevant institutions worldwide. BEA does not require the use of laboratory animals, relying instead on by-products from slaughterhouses, where animals are already processed for food production. Coordinated efforts among research institutions, cattle reproduction laboratories, regulatory bodies, and ART device manufacturers will be essential to support this transition and to establish BEA as a reliable, ethical, and scientifically robust alternative to MEA.

It has been estimated that 111.5 million rats and mice were used for various research and toxicity control purposes in the USA alone in 2017 [ 1 ], even assuming the fulfilment of the principles of the 3Rs (Replacement, Reduction, and Refinement), developed over 50 years ago [ 2 ]. These principles are embedded in national and international legislation and regulations on the use of animals in scientific or regulatory procedures, as well as in the policies of organisations that fund or conduct animal research. While recognising the necessity of animal use in various fields, nine countries have incorporated language safeguarding animals into their constitutions, and the majority of countries currently have fundamental legal prohibitions against animal cruelty. Only eleven countries worldwide lack legislation to protect animals [ 3 ]. In this context, the widespread use of the mouse embryo assay (MEA) in the assisted reproduction industry, which is projected to reach USD 45.06 billion by 2026, is paradoxical. The MEA involves the sacrifice of mice and is the accepted method for assessing the toxicity and functionality of media, laboratory equipment, consumables, and any device that may come into contact with gametes or embryos. While the Food and Drug Administration (FDA) of the USA has issued guidance outlining recommendations for conducting the MEA to support premarket submissions and the release of device lots [ 4 ], the agency has also recently proposed the goal of phasing out animal toxicity testing within 3 to 5 years [ 5 ]. Different experimental work shows that the information provided by the MEA is equivalent to, or worse than, that provided by similar tests using livestock gametes collected at slaughterhouses [ 6 ]. These animals are not killed specifically for the purpose of the test, but rather for food. Furthermore, the metabolic differences between mice and human embryos are far greater than those between cattle and humans. This makes cattle a more appropriate model for testing the embryotoxicity of medical devices [ 7 , 8 ]. Assuming this premise is valid, it can be deduced that incorporating the MEA test into the medical device regulatory framework is inappropriate. A similar argument can be made regarding the necessity of mouse breeding facilities worldwide for this purpose, as these would consequently become superfluous, representing an economic advantage. Furthermore, the substantial amount of waste generated by these facilities could be eliminated. This article provides substantiation for this perspective.