Mouse embryo assay for human in vitro fertilization quality control: a fresh look

Abstract

The mouse embryo assay (MEA) has been used in the field of human in vitro fertilization (IVF) for multiple purposes such as developing embryo culture media, quality control within the laboratory, and procedural training and proficiency testing for embryology staff. In addition, manufacturing companies use the MEA as a means of quality control for the development of embryo culture media and medical devices and to meet the standards of testing for FDA approval of new products. It has long been considered by embryologists and laboratory scientists whether the MEA is an accurate or sensitive test in the quality assessment of culture media and medical devices or if use of this testing is more an obligation. There is no uniformly accepted gold standard method for IVF lab quality control or FDA approval. This review aims to revisit the role of the use of mouse embryos in the formulation of IVF media for clinical use and the different methods of employing the mouse embryo assay for quality control. In addition, we will review the use of the MEA as an important adjunct in the training for embryology staff and fellows in training in reproductive endocrinology and infertility (REI), as well as alternatives to the use of the MEA for these purposes.

Introduction and history of MEA

Throughout the history of cell culture and in the development of formulations for IVF culture media, the use of mouse embryos have had a significant impact. In the early 1900s, Hammond was able to retrieve 4-cell and 8-cell mouse embryos by flushing uterine horns and then culturing these divided embryos to the blastocyst stage in a generally simplified media made of Krebs-Ringers bicarbonate with egg white [1]. Whitten later cultured 8-cell mouse embryos to blastocysts in a media with only nine ingredients including glucose, water, and egg white [2]. In 1958, McLaren and Biggers were able to achieve the first healthy mouse offspring from flushing 8-cell embryos from the oviduct, culturing them to blastocyst in a media that contained bovine serum albumin, and subsequently transferring these cultured embryos to the mouse uterus [3]. Finally, in 1962, Biggers cultured 1-cell stage mouse embryos to the blastocyst stage using chemically defined BGJB medium in the presence of mouse fallopian tube cells [4]. In the 1960s, Brinster investigated further into the components of culture media to find out what exactly would be an optimal environment for pre-implantation embryo development. He discovered that factors such as pH, osmolarity, amino acids, albumin, and energy sources have a direct impact on embryo growth in vitro [5]. In 1968, Whitten and Biggers developed the first in vitro culture of mouse embryos from 1-cell stage to blastocysts without the necessity of co-culture with mouse fallopian tube cells [6]. The same year, Whittingham used the first culture medium for in vitro fertilization of mouse oocytes comprised of Krebs-Ringers bicarbonate solution with sodium lactate, sodium pyruvate, glucose, bovine serum albumin, and antibiotics [7]. This formulation of culture media then went on to influence the formulation of culture media for human IVF such as M16 [5].

Moreover, Brinster discovered that different nutrients are critical to proper embryo progression at different points of development. For example, in the mouse, pyruvate and lactate are the main sources of energy for the 2-cell stage embryo, but glucose and malate are the main sources of energy for the 8-cell stage embryo [8]. These findings played a particularly important role in the current practice of using sequential media to culture human embryos in vitro. There are two popular philosophies for how to culture human embryos in vitro: sequential culture and one-step continuous culture. In sequential culture, the culture media mimics the environment in which the embryo would grow in vivo. This principle breaks the embryo life cycle down into two phases of pre- and post-compaction. The pre-compaction media mimics the environment of the fallopian tube, and the post-compaction media mimics the environment of the uterus [9]. The two different media also account for the change in primary energy source through the embryo’s development [5, 8]. In one-step continuous culture medium, all of the elements necessary for embryonic development are available, and the embryo can “choose” to metabolize which nutrients it needs in order to grow at certain points throughout development [9].

Mouse embryo assay applications

Mouse embryo assays have played a key role in establishing the foundational elements for culture media formulation and a critical role in quality control for media systems in the IVF laboratory today. The MEA has also been very central to the scope of development of cryopreservation protocols (slow freezing and vitrification) for human embryos. In the majority of IVF cycles, supernumerary embryos with acceptable quality remain after the embryo transfer (ET), which are often cryopreserved in order to provide a chance for pregnancy in subsequent ET cycles [10]. In 1972, the first mouse live birth occurred following the transfer of slow-frozen embryos proving that the cryopreservation process did not affect viability nor implantation potential of the mouse embryo [11]. The first successful vitrification of mouse embryos did not occur until in 1985 [12], and the protocol for vitrification of mouse embryos that developed by Kasai et al. in 1990 (using ethylene glycol) closely mirrors the solutions used for the vitrification of embryos today [13].

Proficiency testing

Mouse embryos have also been used for proficiency testing in embryology laboratories. According to the College of American Pathologists (CAP), embryology laboratories must perform proficiency testing outlined by CAP, American Association of Bioanalysts (AAB), or another accredited source twice each year [14]. One of the required proficiency tests is the “IVF embryo culture media test” in which the embryologist must culture mouse embryos in two different culture media received from AAB to determine which media supports the in vitro growth of embryos and which one is hostile to the embryos’ development. Since one of the media is purposefully adulterated and is completely embryo toxic, this proficiency test does not seem to properly gauge the quality of the individual laboratory’s culture system. Perhaps a more informative test would have one of the test media samples to be suboptimal, or slightly embryo toxic, compared with the second, or “standard test” media. Thus, the embryologists could observe the differences in embryo development or blastocyst formation rates within the two comparative culture media. We propose that the media provided for proficiency testing should be adulterated to a more limited extent such that unfavorable media is only suboptimal, and not totally embryo toxic.

Embryology training

Mouse eggs and embryos have been widely used in training entry-level embryologists and REI fellows on how to manipulate and culture gametes and embryos. Mouse eggs are also useful in training for micromanipulation and intracytoplasmic sperm injection (ICSI). In addition, the slow freezing and vitrification of mouse embryos have been a useful tool in the education and training of embryologists. The possible downside, however, is that mouse embryos survive freezing/thawing, or vitrification/warming, more readily than human embryos; thus, favorable results with the utilization of mouse embryos might not be a true reflection of the trainee’s competency [15].

Quality control

Currently, there is no widely accepted standard for the bioassay that is used to test culture media, other solutions, and LabWare with which gametes and embryos come to contact. Perhaps the most important use of mouse embryos today is their use in quality control for human IVF products. In the USA, the Food and Drug Administration (FDA) is responsible for the regulation of medical devices and biologic products and ensures that these substances and substrates are safe for public use [16]. IVF products are required to be approved by the FDA before they are made available for clinical use. Therefore, culture media, reagents, consumables, and devices are required to complete quality control testing, such as pH, osmolarity, sterility, endotoxin tests, and the mouse embryo assay (MEA), for FDA approval and for commercial purposes [16]. The FDA states that acceptable criteria for the MEA is the use of either 1-cell or 2-cell stage embryos from hybrid mouse strains (e.g., CBA/B6 hybrid) cultured for 96 h (if starting from 1-cell stage) or 72 h (if starting from 2-cell stage). Acceptance criteria for the assay are ≥ 80% embryos develop to blastocyst upon completion of the assay [17]. As per FDA, “there are no voluntary consensus standards that describe how to conduct the MEA”; therefore, the MEA does not necessarily have to be very sensitive to detect suboptimal or embryo toxic media and/or devices [17]. Based on the vague requirements and the wide range of instructions for a MEA protocol proposed for standardized testing, a laboratory can unintentionally affect the sensitivity of the MEA by the choice of method employed. Thus, the variability in the MEA assay can be falsely reassuring and results in the variability in the detection of levels of embryo toxicity or fail to detect suboptimal quality in proposed IVF products. For example, based on the mouse strain, embryo’s starting stage (1 cell vs 2 cell), time points of morphologic assessment, type of culture, presence of protein or albumin, use of mineral oil to cover culture dishes, and any additional validation tests performed, two laboratories can test the same culture medium and achieve totally different results. This conclusion is supported by a recent survey sent directly to manufacturers by Delaroche et al. These authors found that there is little standardization and a lack of transparency among IVF manufacturers with regard to the characteristics of the MEA testing employed [18].

Factors that affect MEA sensitivity

Many studies have shown that there are ways to increase the MEA’s sensitivity in order to be more effective in the critical evaluation of culture media and medical devices for embryo toxicity. It has been shown that mouse embryos cultured in vitro from 1-cell stage are more sensitive to suboptimal culture than those cultured in vitro from 2-cell stage [19, 20]. Li et al. reported that the 1-cell stage embryos had a significantly lower blastocyst development rate in the same media and culture conditions than the 2-cell stage embryos [19]. Moreover, Davidson et al. showed that the MEA test which specifies the use of embryos grown from 2-cell stage completely failed to demonstrate an effect on embryo development when there was a significant increase in osmolarity and trace amounts of Cidex added to culture media [20]. These studies, among many others, prove that the 1-cell MEA is a more sensitive and useful assay to test culture media for toxicity and suboptimal culture characteristics as compared to the 2-cell MEA.

The strain of the mouse used for the MEA can also affect the assay sensitivity, as other culture characteristics can. Khan et al. found that outbred CF1 mouse embryos are more genetically diverse and more sensitive to toxins than the recommended hybrid embryos [21]. To increase the sensitivity of the assay even further, the MEA should be performed with a simple media, without the addition of albumin, and at atmospheric oxygen concentration to maximize the stress on the embryos [22]. Albumin is known to act as a chelator and aides in embryo development. Therefore, if albumin is present while testing for toxic substances, it can potentially “hide” detrimental effects on the embryo, negating the efficacy of the test [22, 23]. The mouse embryos should also be morphologically assessed at multiple time points throughout the culture rather than just once at the completion of the assay. The extended MEA where embryos are cultured for 144 h instead of the traditional 96 h is another way to increase the assay sensitivity (up to four times higher) for toxins found in mineral oil. In a recent study, the blastocyst development rate was significantly lower in the group cultured with toxins in the oil compared to the control group after 144 h, while there was no significant difference on blastocyst rate between the groups after 96 h [24].

Furthermore, it has been suggested that in addition to the morphologic assessment, an embryo cell count should also be performed at the completion of the 72, or 96, hours of culture [22]. A blastomere cell count reveals any disturbance that could have made the cell cycle slow, which in turn would produce a lower blastocyst total cell count. Blastocysts that have a lower number of cells may appear morphologically normal but may not yield enough cells to produce a viable fetus [25]. Another additional test that may increase sensitivity of MEA, and may reduce variability in subjective morphology scoring, is the mouse embryo genetic assay (MEGA). This assay tracks important genetic markers, such as OCT4 through the embryo’s development to monitor its growth [26]. Morphokinetics is another valuable adjunct for detecting suboptimal culture media. Wolff et al. found that the morphokinetic model they developed detected toxins even when the blastocyst rate was not affected in embryos that were cultured in media with different added concentrations of two known toxins, cumene hydroperoxide and Triton X-100 [27].

Alternatives to MEA

As stated above, the 1-cell MEA, along with a blastomere cell count and MEGA, is more sensitive tests of embryo toxicity. Among other bioassays, human sperm survival assay (HSSA) has been used in human IVF laboratories as an alternative bioassay. The HSSA requires less equipment, does not involve animals, and is less labor intensive than the MEA. Furthermore, the HSSA has a shorter duration of 24 h instead of up to 96 h with the MEA; thus, results are available quicker, and it requires less laboratory work [28]. In order to increase the sensitivity of the HSSA, the laboratory can assess both the motility and the quality of the motility of the sperm [28, 29]. Although Classens et al. showed that the HSSA can be as sensitive as the 2-cell MEA when cultured without albumin [29], Hughes et al. reported that the 1-cell MEA was twenty times more sensitive than the HSSA for certain toxins present in mineral oil [30]. In addition, it is not totally clear if sperm assays can detect any toxicity or suboptimal embryo culture conditions due to the biologic differences of sperm and embryos in terms of metabolism and the fact that sperm do not develop in the culture. Together, Classens et al.’s and Hughes et al.’s results have proven that the 1-cell MEA is more sensitive than the 2-cell MEA, and the HSSA is limited in the detection of embryo toxicity for quality control purposes [29, 30].

Domestic animal embryos (e.g., bovine and porcine) are morphologically very similar to the human embryo in their culture requirements and therefore could be possibly used as models for a quality control assay. The bovine, and porcine embryos cleave until the morula stage at about 32 cells and then compact, cavitate, and the cells differentiate into both trophectoderm cells and an inner cell mass. Embryos from domestic animals also hatch from the zona pellucida the same way the human embryos hatch. In addition, the timing of maternal-zygotic transition of the domestic animal embryo is closer to the human embryo than that of the mouse. This transition occurs at the 4-cell to 16-cell stage in the human and domestic animal but at the 2-cell stage in the mouse [31]. It has, however, not been proven that bovine or porcine embryos will detect embryo toxicity the same way that the mouse embryos do in a MEA. We were unable to find any reports in the published literature about the use of bovine or porcine embryos for human IVF quality control purposes. Another drawback in using a domestic animal model is the cost and space of housing domestic animals which is much greater than mice. Even though the domestic animal embryo seems to mimic the human embryo, at this point, the mouse embryos seems to be the only available mammalian embryos to be used as a measure of quality control.

Conclusion

Throughout the growth and development of the in vitro fertilization field, many advancements were due to integral research conducted with mouse embryos. Mouse embryos have given scientists and embryologists a starting point for media formulation, as well as for a cryopreservation protocol. In addition, mouse embryos have helped to give hands-on training for embryologists and REI fellows. Furthermore, the MEA is widely used as a means for media and IVF product quality control and is a useful tool to detect embryo toxicity if conducted properly. However, it is important to note that not all mouse embryo assays are created equal to detect suboptimal culture conditions, and it is plausible that a reagent or culture media used in an IVF laboratory may contain toxins that were not detected by MEA. In the absence of a gold standard quality control technique, the 1-cell MEA together with other assays seems to be the best option at this point for quality control. In order to optimize the sensitivity of the MEA, the laboratory should expose 1-cell stage embryos from mouse strains with proven sensitivity to embryo toxicity using simplified media without a serum supplement (e.g., albumin), morphologically assess the embryos at multiple points through the 96-h assay, and use a quantitative assay such as embryo cell count and the MEGA when possible to remove subjective scoring.