Preimplantation genetic diagnosis (PGD) has been introduced in clinical practice as a tool for Since the 90s, PGD has been offered to couples undergoing assisted reproduction. In PGD, embryos produced by in vitro fertilization or intracytoplasmatic sperm injection are screened for genetic defects, allowing only ‘healthy’ embryos to be selected for transfer in utero. One component of PGD protocol is embryo biopsy to obtain one or few cells to be genetically screened. Embryo biopsy can be performed on cleaving embryos (blastomere biopsy (BB), consisting in the removal of one or two blastomere from 8-cell embryos) or on blastocyst stage embryos (trophectoderm biopsy – TB, consisting of the removal of few cells from trophectoderm).
Since genetic testing of a single cell obtained by early embryo biopsy was shown to be possible (Monk et al. 1987), much attention has been devoted to optimizing the technique to improve embryo survival. To this aim, Takeuchi et al. (1992) compared three different protocols – enucleation, aspiration and extrusion – for the removal of one blastomere from a developing embryo, showing that the aspiration method was the less harmful protocol for BB when the procedure was performed on both 4- and 8-cell stage embryos. Pierce and coworkers reported that 8-cell stage embryos were less sensitive to the procedure than 4-cell ones (Pierce et al. 1997). Concerning TB, Carson and coworkers showed that the excision of herniating trophectoderm after hatching was the safest way to perform blastocyst biopsy compared to the aspiration method (Carson et al. 1993).
Time of biopsy
Biopsy can be performed by removal of one or two PBs from the unfertilised oocyte or the zygote, respectively, removal of one or two blastomeres at the cleavage stage or removal of several (5–10) TE cells at the blastocyst stage.
Blastocyst biopsy or TE biopsy is at present the most widely used technique (De Rycke et al., 2017). It provides more cells and is at an embryonic stage, more amenable for genetic analysis and less sensitive to possible damage.
Sample collection
After biopsy, cell(s) are washed and either fixed on a slide for FISH analysis or collected in small reaction tubes for amplification-based testing (called ‘tubing’). As genome-wide technologies began to replace the FISH method over the past decade and these technologies require whole-genome amplification (WGA) as a first step, tubing has become the most widely applied method for collection of biopsied samples. General recommendations on tubing have been formulated in this guidance paper.
Rebiopsy of embryos
Rebiopsy of embryos could be considered only in case of failed, incomplete or inconclusive genetic diagnosis, as the impact on further embryo development remains an area of investigation. The rebiopsy policy should be in accordance with local legislation.
What does embryo biopsy involve?
To explain the biopsy procedure we should keep in mind that our point of departure is EMBRYOS. Embryos are retrieved after performing an assisted reproduction cycle, preferably by Intracytoplasmic Sperm Injection (ICSI), and their development is assessed during the culture period until day 3 or day 5.
By day 3 biopsiable embryos should have a number of cells equal or higher than 6 so that their later development is not affected by the fact of having “captured” a cell to do the analysis. To perform the biopsy, a hole has to be drilled in the embryo’s zona pellucida (ZP), either by means of chemical agents (Tyrode’s acid) or laser pulses. A blastomere (an embryo cell) is then collected by aspiration and is subsequently analysed in our molecular and genetic biology laboratories.
Once the biopsy has been performed, embryos are kept under observation to assess their development until the time of transference.
By day 5 biopsiable embryos should have reached the blastocyst stage. If so, a minimum of 4-5 cells from the external cell layer that will form the placental structures (trophectoderm) are biopsied, keeping the inner cell mass that will form the embryo intact. Drilling of ZP is performed by laser, which is the most precise method available, as the thickness of the ZP is reduced by the associated processes of expansion, and the embryo’s development could be jeopardised by using other methods. The embryo will naturally extrude a piece of trophectoderm out of the zona pellucida. This is the piece that can be biopsied. The most common way to biopsy the trophectoderm is by aspiration, using laser pulses to trigger cell separation, although there are other methods.
Embryo biopsy is a delicate procedure that requires high precision and an experienced team of embryologists. Mistakes in performing the technique would jeopardise embryo development or the subsequent genetic diagnosis.
How embryo biopsy may affect embryo development
The preimplantation period is a short window of mammalian development characterized by a well-defined sequence of programming events that will decide the future of the developing embryo. In this period, epigenetic and/or metabolic changes (i.e. adaptations), occurring to promote survival in an inadequate environment (e.g. a suboptimal culture system, maternal undernutrition), may cause permanent changes to developmental programming, thus leading to increased risk of developmental defects and/or metabolic disorders.