Mosaicism, Trauma & Accuracy: Why Day 5 Biopsy is the Gold Standard

Monash Biotech

October 1st, 2025

Preimplantation Genetic Testing (PGT) is a critical tool in the modern ART laboratory, offering invaluable insight into an embryo's chromosomal makeup. However, the accuracy of any genetic test is entirely dependent on the quality of the sample obtained. The biopsy itself—the act of removing cellular material from an oocyte or embryo—is the most technically demanding and high-stakes procedure in the entire process.

The field has witnessed a definitive shift in protocol, moving from the traditional Blastomere Biopsy on Day 3 to the now gold-standard Trophectoderm Biopsy on Day 5/6. This was not merely a change in timing; it was a fundamental evolution driven by a deeper understanding of developmental biology, diagnostic accuracy, and, most importantly, embryo safety.

1. The Original Method: Blastomere Biopsy (Day 3)

This technique involves the biopsy of a cleavage-stage embryo, typically on the third day of development when it has reached the 6- to 8-cell stage.

• The Technical Protocol: The embryologist uses a laser or chemical solution (e.g., Acid Tyrode's) to create a small breach in the zona pellucida. A specialized blastomere biopsy pipette is then introduced to aspirate a single cell (a blastomere) for genetic analysis. In some cases, two blastomeres were removed to improve diagnostic yield.

• The Inherent Risks & Limitations: While pioneering, this method is fraught with significant challenges:

  • High Developmental Trauma: At the 8-cell stage, each blastomere represents 12.5% of the embryo's total cellular mass. Removing one cell is a massive biological insult that has been shown to reduce developmental competence and implantation potential.

  • High Risk of Mosaicism: This is the technique's primary diagnostic flaw. A cleavage-stage embryo can be highly mosaic (containing both aneuploid and euploid cells).A single blastomere is not representative of the entire embryo, leading to a high rate of false positives (diagnosing a healthy embryo as abnormal) or false negatives (diagnosing an abnormal embryo as healthy).

  • Limited Genetic Material: A single cell provides a very small amount of DNA. This increases the risk of "allele dropout" (where one of two alleles fails to be amplified) or total amplification failure, leading to inconclusive or unreliable results.

2. The Gold Standard: Trophectoderm Biopsy (Day 5/6)

This modern technique involves the biopsy of a blastocyst, which has differentiated into two distinct cell types: the Inner Cell Mass (ICM), which becomes the fetus, and the Trophectoderm (TE), which becomes the placenta.

• The Technical Protocol: The biopsy is performed on a Day 5 or Day 6 blastocyst, which contains 100+ cells. A laser is used to assist in "herniating" (or hatching) the blastocyst. The embryologist then carefully aspirates 5-10 cells from the trophectoderm only, meticulously avoiding the ICM.

• The Overwhelming Advantages:

  • Dramatically Reduced Embryo Trauma: The 5-10 TE cells removed represent a much smaller fraction of the total embryo mass. Crucially, these cells are taken from the placental lineage (TE), leaving the fetal lineage (ICM) completely undisturbed. This results in no significant impact on embryo viability or implantation rates.

  • Superior Diagnostic Accuracy: A 5-10 cell sample provides a more comprehensive and reliable genetic "snapshot" of the embryo, significantly reducing the diagnostic errors caused by mosaicism.

  • More DNA, Robust Results: The larger sample size yields more DNA, leading to more robust and reliable genetic amplification and a lower rate of inconclusive results.

The Evolving Role of the Micropipette

This evolution in technique demanded a parallel evolution in the tools used. The micropipettes required for these two procedures are fundamentally different.

• A blastomere biopsy pipette needed a larger inner diameter (e.g., 30-40 µm) and a flat, polished tip to safely aspirate a large, single cell without lysing it.

• A trophectoderm biopsy pipette is a more refined instrument. It typically has a smaller inner diameter (e.g., 20-30 µm) and is often bevelled. This bevelled, angled tip acts like a fine blade, allowing the embryologist to work in concert with the laser to cleanly separate the TE cells without placing traction or stress on the delicate Inner Cell Mass.

The shift to Day 5 biopsy was not just a biological advancement; it was a technological one. It required the manufacturing of more precise, specialized micropipettes to make the procedure safe and efficient.The success of this gold-standard technique is therefore a partnership between the skill of the embryologist and the precision engineering of the pipette in their hand.