In Vitro Fertilization (IVF) technology frequently results in more viable embryos than can be safely transferred in a single cycle. Cryopreservation, or the process of freezing these surplus embryos, allows for future attempts without the need for another full ovarian stimulation and egg retrieval procedure. Not every embryo created in the laboratory is suitable for this long-term storage. Embryologists use a standardized grading system to assess the morphological quality of each embryo, which serves as a prediction of its potential for successful implantation and survival after freezing and thawing. The grade assigned to an embryo directly informs the decision to freeze, transfer, or discontinue culture.
The Two Stages of Embryo Development and Grading
Embryos are typically evaluated at two distinct developmental milestones during their time in the laboratory culture before transfer or freezing. The first point of assessment is the Cleavage Stage, which occurs on Day 3 following fertilization. At this stage, a healthy embryo is expected to consist of between six and ten cells, referred to as blastomeres, that are actively dividing inside the outer shell called the zona pellucida. Cleavage stage grading focuses on the number of cells present, the uniformity of their size, and the percentage of cellular fragmentation.
A high degree of fragmentation suggests poorer cell integrity and is associated with a reduced chance of continued development. Most clinics prioritize culturing embryos to the Blastocyst Stage, reached on Day 5 or Day 6. This later-stage evaluation is preferred because it represents a natural developmental checkpoint, as only the most vigorous embryos successfully differentiate into the complex structure of a blastocyst.
The blastocyst structure is characterized by a fluid-filled cavity called the blastocoel. It contains the Inner Cell Mass (ICM), a compact cluster of cells that will eventually develop into the fetus itself. Surrounding the ICM is the Trophectoderm (TE), a layer of cells that will form the placenta and other supportive tissues. Grading at this stage provides a better morphological prediction of an embryo’s developmental potential and suitability for cryopreservation.
Detailed Criteria for Blastocyst Grading
The most common method for evaluating this complex structure is the Gardner grading system, which assigns a three-part score to the blastocyst. The first component is a number from one to six, representing the degree of expansion and the status of the blastocyst’s hatching from the zona pellucida. A score of ‘1’ indicates an early blastocyst where the blastocoel is just forming, while a ‘4’ signifies a fully expanded blastocyst with a thinned shell. The highest scores, ‘5’ and ‘6,’ denote a blastocyst that is actively hatching or has completely escaped the zona, respectively.
The next two parts of the grade are letter scores, typically ‘A,’ ‘B,’ or ‘C,’ which reflect the quality of the ICM and the TE, in that order. The first letter refers to the Inner Cell Mass. An ‘A’ indicates many tightly packed cells, which is the desired morphology. A ‘B’ grade denotes a more loosely grouped collection of cells, and a ‘C’ grade signifies very few cells.
The second letter grades the Trophectoderm, the cells responsible for placental development. An ‘A’ for the TE means there are many cells forming a cohesive, uniform layer, suggesting robust potential for implantation. A ‘B’ grade describes a thinner or slightly more irregular layer, while a ‘C’ indicates a sparse layer. An embryo with a score of 4AA, for example, is fully expanded with excellent quality ICM and TE, representing the highest morphological grade. Conversely, a score like 3BC suggests a partial expansion with a poor quality ICM and TE.
Selection Criteria for Cryopreservation
The grade assigned to a blastocyst serves as the primary determinant for the decision to proceed with cryopreservation. While a 4AA is the morphological ideal, many embryos with slightly lower but still respectable grades are candidates for freezing. Most clinics establish a minimum morphological threshold, which generally requires the ICM and TE to have at least a ‘B’ grade, or at least one ‘A’ and one ‘B’ grade, to be considered viable for freezing. Common grades that meet this standard include 4AB, 5BA, and 5BB, among others.
Embryos with an expansion score of three or higher are preferred for cryopreservation because they have already demonstrated significant developmental progress. Lower-grade blastocysts, such as those with two ‘C’ scores (e.g., 4CC), are often not frozen because their predicted implantation rate is lower, and they are less likely to survive the subsequent warming. Modern practices prioritize the freezing of high-quality blastocysts because this stage has already demonstrated the necessary competence to progress past the early cleavage stage.
Factors Influencing Embryo Survival After Thaw
The initial morphological grade is a strong predictor of viability, but it is not the only factor that influences the success of a frozen embryo. The method of cryopreservation itself plays a role in the embryo’s ability to recover. Vitrification, a technique that involves ultra-rapid cooling to prevent the formation of damaging ice crystals, is now the standard practice and has largely replaced the older slow-freezing method. This flash-freezing process yields high survival rates, typically above 95% for good-quality blastocysts.
The embryo’s ability to recover from the thaw is a separate metric from its initial grade. Even a high-quality embryo may experience some cellular damage during the warming process, and embryologists assess the percentage of intact cells immediately following the thaw. Embryos that are fully hatched (‘6’ grade) may be slightly more fragile and less likely to survive the process compared to those that are fully expanded or hatching (‘4’ or ‘5’ grades). The experience and consistent technique of the embryology laboratory personnel are also factors that contribute to optimal survival rates and subsequent implantation potential.