Laboratory Procedures

PGD-biopsy procedures

Homapage / PGD-biopsy procedures

Biopsy techniques for Preimplantation Genetic Diagnosis (PGD) cases

Preimplantation Genetic Diagnosis (PGD) refers to procedures that are performed on embryos prior to transfer or on oocytes (eggs) just after fertilization to determine their genetic and chromosomal background. There are currently three sources of genetic material which can be used for PGD:

  • polar bodies (one of the minute cells arising from the divisions of the oocyte at or near the time of fertilization) from oocytes
  • blastomere(s) (any cell(s) produced during embryonic cell division or cleavage) from day3 embryos
  • trophectoderm cells (outer layer of cells of a blastocyst, a day5 embryo) from blastocysts

Day 0 (Oocyte Pick Up day) Polar Body Biopsy

Polar body biopsy can be performed for couples with advanced maternal age, maternal translocation carriers, or maternally transmitted genetic conditions. 

Polar bodies are chromosomal side products of the divisions of an oocyte, reflecting its maturation and fertilization status. They result from the reduction in number of the chromosomes in the maturation process of the oocyte. We perform polar body biopsy post-fertilization (8h post-ICSI) to remove both the first and the second polar bodies simultaneously. Since the polar bodies have no further role in embryo development, in experienced hands their removal has no negative effect on further embryo growth.

As two samples (polar body 1 and 2) are to be analyzed instead of one (blastomere on day 3 or trophectoderm on day5), for cost-effectiveness, we prefer to do polar body biopsy when the number of retrieved mature (metaphaseII) oocytes is below 5. If not, embryos can be incubated in the embryoscope and the best selected according to their quality and the timing of their divisions on day 2 or 3. The corresponding polar bodies can then be genetically analysed. Through this method, the analysis of polar bodies stemming from oocytes that have resulted in poor quality and/ or slow-growing embryos can be avoided.


Day3 blastomere biopsy

Blastomere biopsy is performed on embryos with at least 7 blastomeres on day 3 (Figure 1), which corresponds to 65-72 hours after insemination. The blastomere that will be biopsied is evaluated for the presence of a proper cytoplasmic appearance as well as a nucleus (containing the genetic material) formation. An opening of 20-25 µm in diameter on the zona pellucida (ZP) is made using a laser. The embryo is then held and positioned with a holding pipette (Figure 1). Through the opening created on the ZP, a single blastomere is removed from the embryo (Figure 2). 

Figure 1. An embryo is stabilized with a holding pipette (left) and the ZP is opened by laser shots

 Blastomere biopsy is performed for chromosomal rearrangements (translocations and inversions) if the couple has, on day 3, fewer than 4 good quality embryos (grades 1 and 2). However, since only one cell is removed from the embryo, structural and numerical chromosomal abnormalities found in this unique cell may not represent the embryo as a whole. Also, compared to other biopsy procedures, blastomere biopsy has a greater impact on the implantation potential of the embryo. For this reason the preferred option at our center is trophectoderm biopsy when the number of embryos available for biopsy on day3 is above 4. (See below)
Nevertheless, day3 blastomere biopsy remains a good option to consider for gene analysis in maternally/ paternally inherited single gene disorders and/ or HLA typing.

Figure 2. A blastomere biopsy pipette is placed through the ZP opening; the nucleus-containing and targeted blastomere is gently aspirated into the pipette and slowly released into the medium.



Day 5 trophectoderm biopsy

Trophectoderm cells are extra-embryonic tissue.  This means that they do not become part of the fetus but part of supporting structures, such as the placenta and membranes. Trophectoderm biopsy is performed at the blastocyst stage of development, as the trophectoderm is beginning to protrude through the zona pellucida. Instead of individual blastomeres, several trophectoderm cells are removed by aspiration within the biopsy pipette (Figure 3).

Figure 3. a. Day 5 expanding blastocyst with the trophectoderm protruding through an opening in the zona made on day 3 or 4 with a microlaser. b. Aspiration of the protruding trophectoderm cells through the ZP c. Laser shots are applied to break down the tight junctions between trophectoderm cells d. Aspirated trophectoderm cells (range 2 to 9 cells).



Trophectoderm biopsy has become the method of choice in IVF laboratories since it increases implantation rates. The main advantage of trophectoderm biopsy is the number of cells that can be retrieved. Usually 3 to 6 cells are biopsied on day5, which represents a small proportion of the total cell number of the blastocyst (70-100 cells). Therefore the procedure does not affect embryonic development and, by the biopsy of multiple cells, increases the amount of genetic material that will be analyzed, thus facilitating diagnostic procedures. Furthermore, as the procedure consists of the biopsy of the external tissue (trophectoderm) , which forms the placenta with the maternal endometrium, the  inner cell mass from which the fetus will develop is kept intact. Another advantage is that culturing embryos to blastocyst stage eliminates a portion of chromosomally abnormal embryos, decreasing the number of embryos analyzed and thus reducing the cost of the test.