Introduction

Morphological evaluation of embryo quality is performed for three major reasons. First reason is to differentiate between embryos and unfertilized ova. Careful evaluation of harvested embryos/ova is necessary to ensure that viable embryos are not discarded or, conversely, that unfertilized ova are not transferred or cryopreserved. The second reason to perform morphological assessment of embryos harvested from a donor is to determine if the developmental stage of the embryos is consistent with the expected developmental stage based on the days since estrus that the embryos were collected. There is often a high degree of variability in observed stage of embryonic development among embryos obtained during embryo recovery from a single donor female, and this variation in stage of development should be considered when selecting a suitable recipient female. The third reason to assess embryo quality is to enable technicians to have sufficient information on which to base the decision to transfer or to cryopreserve the harvested embryos. Poorer-quality embryos that may result in pregnancies if transferred fresh usually do not survive the cryopreservation process.

Despite the importance of this topic, embryo evaluation is a difficult aspect of the overall embryo transfer process to learn, especially if attempting to self-train. Although this chapter will provide numerous helpful photographs of embryos and unfertilized ova, beginners would benefit from joining an experienced embryo transfer mentor for intensive and repetitive training sessions on morphological embryo evaluation. Proper training, proper equipment, and extensive experience are the three major factors affecting one’s proficiency with embryo evaluation.

History of bovine embryo evaluation

The very earliest stages of bovine embryonic development (ovum and 2-cell embryo) were first described in 1931 by Hartman et al.¹ In the following decade two other research groups^2,3 described and illustrated various stages of bovine embryonic development. These very early descriptions of embryonic development served as the foundation of the morphological embryo evaluation method that subsequently evolved and became widely used in the commercial bovine embryo transfer industry.⁴

The morphological embryo evaluation method depends on the human eye to critically evaluate subtle differences among harvested embryos. During the early years of its use, the method was criticized as being too subjective, inconsistent, and prone to error. As a result, several alternative methods for assessing embryo quality were investigated for their potential to be less subjective and more consistent than morphological evaluation. Some of these methods included the ability of an embryo to continue its development during in vitro culture,^5,6 dye exclusion test,⁷ measurement of embryonic metabolic activity,⁶ glucose uptake,⁸ live–dead stains,⁹ computerized image analysis,¹⁰ fluorescein diacetate vital stain,^11,12 DAPI (4′,6′-diamidino-2-phenyl-indole) staining for dead cells,¹³ and in vivo culture in rabbit oviducts.¹⁴

The importance of a good microscope

Before delving into the specific methodology used for morphological evaluation of embryos, it is important to remind readers that proper equipment is needed for accurate embryo evaluation. Although many beginners in embryo transfer are tempted to purchase an inexpensive microscope as a means to reduce start-up costs, doing so will likely create difficulties in finding and evaluating embryos. A poor-quality microscope can make it extremely challenging for a practitioner to tell the difference between an unfertilized ovum (UFO) and a compact morula. Similarly, a practitioner will be hampered by a poor-quality microscope when attempting to discern the various embryo quality grades.

Unlike semen evaluation, where a compound microscope is used, embryo transfer requires the use of a stereomicroscope. Stereomicroscopes, also called dissecting microscopes, are designed for use with three-dimensional specimens. Preimplantation bovine embryos, although microscopic in size, are round like a basketball and thus are best evaluated using a stereomicroscope.

There are several important features of a stereomicroscope that can directly influence one’s ability to easily and accurately evaluate embryos. One important feature is the overall magnification. Overall magnification of a stereomicroscope is a function of the magnification of the eyepieces, objective lens, and zoom changer. For example, total magnification of a stereomicroscope equipped with 10× eyepieces, a 1× objective lens, and a 2× zoom changer would be approximately 20× (10 × 1 × 2 = 20). Many technicians prefer to search for embryos at a total magnification between 10× and 15×, but to effectively evaluate the fine details of an embryo and to identify any abnormalities, such as cracks in the zona pellucida, embryos should be observed under a good optical quality stereomicroscope with a magnification range that reaches at least 50×. It should be noted that the IETS-approved procedure for sanitary handling of embryos requires that embryos be inspected at a magnification of at least 50 × .

Most embryo transfer technicians prefer that the eyepieces not exceed 10× in order to maintain an adequate field of view and image resolution. A microscope equipped with a diopter adjustment on one eyepiece (if not both) is very useful for compensating differences in eyesight between a technician’s left and right eyes. An adjustable binocular tube will also enable multiple technicians with varying interpupillary distances to use the same stereomicroscope.

A second important feature of a stereomicroscope for embryo handling and evaluation is the working distance (distance from the embryo to the objective lens). The working distance should be long to give maximal flexibility when searching for embryos and the microscope stage should be large and free of obstacles such as stage clips, power buttons, or similar items that make sliding of Petri dishes on the stage difficult. A large and clear stage is important for providing adequate space to move Petri dishes and to operate the embryo handling device without accidently spilling the Petri dish and losing the embryos.

A third important feature of a stereomicroscope used for embryo evaluation is the illumination. The illumination should come from underneath the stage plate and it should be bright and uniform across the entire stage plate. A clear stage plate is needed; frosted stage plates are not suitable for embryo work. Brightfield illumination is standard for most stereomicroscopes, but darkfield illumination can be helpful when searching through “cloudy” fluids in a search dish.