On the morning of days 5 or 6 after egg retrieval, blastocysts (D5 or D6 stage) are observed under an inverted microscope at 200x magnification. Grading is based on three criteria: the size of the blastocoel, the inner cell mass (ICM), and the trophectoderm (TE). The grading system is as follows:

1. Blastocoel Size

  • 1: Early Blastocyst: The blastocoel occupies less than half of the embryo volume.
  • 2: Blastocyst: The blastocoel occupies more than half of the embryo volume.
  • 3: Full Blastocyst: The blastocoel nearly fills the entire embryo.
  • 4: Expanded Blastocyst: The blastocyst has expanded, the blastocoel has enlarged, and the zona pellucida (outer shell) has thinned.
  • 5: Hatching Blastocyst: Part of the trophectoderm cells have begun to emerge from the zona pellucida.
  • 6: Hatched Blastocyst: The blastocyst has completely emerged from and detached from the zona pellucida.

Blastocysts that reach grade 3 or higher are further evaluated for ICM and TE quality.

2. Inner Cell Mass (ICM) Grading

  • A: The ICM contains many tightly packed cells with a distinct structure.
  • B: The ICM has fewer cells that are loosely packed or slightly clustered.
  • C: The ICM has very few cells, making it difficult to identify a clear structure.

3. Trophectoderm (TE) Grading

  • A: The TE contains many cells forming a continuous epithelial-like layer.
  • B: The TE has fewer cells with a loose or discontinuous epithelial-like structure.
  • C: The TE contains very few cells.

This detailed grading system provides a comprehensive assessment of blastocyst quality to guide embryo selection for transfer or cryopreservation.