How Mosaicism Ended the Day 3 Blastomere Biopsy Era ?

Monash Biotech

October 10th, 2025

In the rapid evolution of assisted reproductive technology, the blastomere biopsy stands as a critical, foundational chapter. For nearly two decades, this Day 3 procedure was the primary method for preimplantation genetic testing (PGT), representing the first-ever glimpse into the genetics of a living human embryo.

Understanding this technique—its protocol, its tools, and ultimately its profound limitations—is essential for appreciating why the field has overwhelmingly transitioned to trophectoderm biopsy. This is a technical review of a pioneering, yet flawed, procedure.

The Rationale: Why Biopsy on Day 3?

The logic of the blastomere biopsy was rooted in the developmental timeline of the early 1990s. The cleavage-stage embryo, typically at the 6- to 10-cell stage on Day 3, was an accessible target. The prevailing theory was that these early blastomeres were totipotent—each cell was identical and capable of developing into a full organism.

Based on this assumption, the thinking was that removing a single cell (or two) would be harmless. The remaining cells would simply compensate and continue development, while the removed cell would provide a complete genetic snapshot of the embryo. This assumption, as we now know, was deeply flawed.

The Technical Protocol

Performing a blastomere biopsy was a high-stakes micromanipulation procedure requiring a steady hand and precise tools.

1. Zona Pellucida Breaching: First, a hole had to be created in the zona pellucida. This was typically done using either a chemical method (a brief, localized exposure to Acid Tyrode's solution) or an early non-contact laser.

2. Pipette Insertion: A specialized blastomere biopsy pipette was introduced through the opening.

3. Cell Aspiration: The embryologist would focus on a single, nucleated blastomere. Gentle negative pressure was applied to aspirate the cell into the pipette. This was often the most difficult step, as adherence between blastomeres could be strong. The cell was then carefully pulled away until it detached from the rest of the embryo.

4. Cell Transfer: The aspirated blastomere was then transferred to a separate medium for genetic processing.

Tool Selection: The Blastomere Biopsy Pipette

The tools for this procedure were fundamentally different from modern biopsy pipettes.

• Inner Diameter (ID): A blastomere is a large, spherical cell.The pipette needed a wide inner diameter, typically between 30µm and 40µm, to aspirate the cell without deforming or lysing it.

• Tip Design: The tip was almost always flat and fire-polished. A bevelled tip (used today for trophectoderm) would have acted like a blade and damaged the blastomere. The flat, smooth tip was designed to create a seal against the cell membrane and hold it securely via negative pressure.

A Critical Review: Why the Field Moved On

The blastomere biopsy was revolutionary, but it was built on a flawed premise. Its decline was driven by two major, unavoidable problems:

1. Significant Embryo Trauma The assumption of harmlessness was incorrect. Removing one cell from an 8-cell embryo means removing 12.5% of the embryo's total cellular mass. Removing two cells (25%) was even more common to improve diagnostic yield. This was a massive biological insult. Studies overwhelmingly showed that biopsied Day 3 embryos had significantly reduced developmental competence and lower implantation potential compared to their non-biopsied siblings.

2. Profound Diagnostic Inaccuracy The most critical flaw was biological, not technical. We now know that cleavage-stage embryos are hotbeds of chromosomal mosaicism.

   • What is Mosaicism? An embryo can have both aneuploid (abnormal) and euploid (normal) cells dividing at the same time.

   • The "Snapshot" was Wrong: The single blastomere that was pulled for testing was not representative of the entire embryo. An embryologist could (and often did) pull an aneuploid cell from a healthy embryo, leading to a false-positive and the discarding of a viable embryo. Worse, they could pull a euploid cell from a mosaic, aneuploid embryo, leading to a false-negative and the transfer of a non-viable embryo.

This high rate of mosaicism-driven misdiagnosis, combined with the significant trauma to the embryo, rendered the blastomere biopsy an unreliable and high-risk procedure.

The Legacy of Blastomere Biopsy

While no longer a front-line protocol, the blastomere biopsy was not a failure. It was a necessary step. The problems it exposed—especially the profound challenge of mosaicism—are what forced the entire field to push for blastocyst culture.

This shift allowed for the development of the trophectoderm biopsy, a technique that solves both of its predecessor's problems: it removes a smaller fraction of cells (<5%) from the placental lineage (TE), leaving the fetal-lineage (ICM) untouched, and provides a 5-10 cell sample that gives a far more accurate genetic picture. The blastomere biopsy was the critical historical lesson that taught us why Day 5 biopsy is the only reliable standard.