Human Blastocyst Biopsy and Vitrification

Both blastocyst biopsy and vitrification have been a revolution in IVF, in vitro fertilization, allowing embryologists from all around the world to perform preimplantation genetic testing on human embryos with minimal risk for the embryo. Trophectoderm biopsy has allowed the retrieval of about seven cells from a fully grown embryo, thereby enabling robust downstream genetic analysis. Moreover, no impact has been shown, to date, for this approach.

The clinical efficiency of this workflow allows patients with monogenic conditions and all those patients with increased risk of chromosomal abnormalities within their blastocyst to benefit from preimplantation genetic testing. There is still room for improving the current strategies of embryo selection. For instance, miRNomic and proteomic profiling represent intriguing options to complement preimplantation genetic testing on a single trophectoderm biopsy.

Unexperienced operators often struggle with opening and penetrating the zona pellucida. It's just a matter of focus. The blastocyst, laser objective, and pipettes should be on the same focal plane.

After labeling the biopsy dish with the patient's details, with a permanent, non-toxic marker, number each 10-microliter drop of HEPES-buffered medium with the embryo and cycle ID.Using a 300-micrometer stripping pipette, transfer a viable, fully expanded blastocyst to the first drop of the biopsy dish and rinse the drop to remove the excess culture medium, before moving the blastocyst into the second drop in the dish. Place the dish under an inverted microscope, and prime the biopsy pipette with medium from the third drop of the biopsy dish. Under a 20 times magnification, orient the blastocyst to obtain a clear view of the inner cell mass at seven o'clock, and secure the embryo on the holding pipette.

Focus on the zona pellucida so that both the pipettes and the blastocyst are on the same focal plane. Switch to the laser objective, and position the laser pointer on the zona pellucida at the opposite side of the inner cell mass. Drill the zona pellucida with two to three laser pulses, and gently press the biopsy pipette against the zona pellucida to allow medium to be blown through the breach to detach the trophectoderm cells from the internal surface.

When the trophectoderm is detached, enter through the hole and aspirate seven to eight trophectoderm cells into the biopsy pipette with gentle suction. While applying a moderate suction to stretch the target cells, slightly move the biopsy pipette backwards and direct the laser towards the thinnest part of the aspirated cells. Fire two to five laser pulses at the junctions between the cells to separate the target cells from the body of the embryo.

When the trophectoderm fragment has been separated from the blastocyst, release the fragment into the same biopsy drop far from the blastocyst to prevent the fragment from being sucked back into the biopsy pipette. Release the blastocyst from the holding pipette, and promptly raise both pipettes to prevent the fragment from sticking to pipettes. Then image the biopsied fragment for quality control purposes.

When all of the blastocysts have been biopsied as just demonstrated, move the biopsy dish back to the laminar flow hood and label the post-biopsy culture dish with the couple ID and each drop with the embryo and cycle IDs. In presence of a witness, rinse the blastocyst in clean IVF medium and move the blastocyst to its corresponding drop in the post-biopsy dish. Then place the post-biopsy dish to a 37-degree Celsius, 6%carbon dioxide, and 5%oxygen incubator.

In the presence of the witness and inside a laminar flow hood at room temperature, label the PCR tubes with a permanent, non-toxic marker. Label the lid of a 60 x 15-millimeter tubing culture dish with the biopsied embryo IDs, and add two 10-microliter drops of biopsy washing solution to the dish. Prime a 140-micrometer stripping pipette with biopsy washing solution from the second drop of the tubing dish under the stereo microscope to visualize the trophectoderm fragments.

Gently release some biopsy washing solution upon the trophectoderm fragment, and load the fragment into the stripping pipette. Move the fragment to the second drop of biopsy washing solution, and carefully rinse the tissue two to three times. Transfer the trophectoderm fragment to the bottom of the appropriately labeled PCR tube with loading solution, taking care to avoid touching the walls of the tube with the tip of the stripping pipette.

When all of the fragments have been added to their tubes, spin all of the tubes in a mini centrifuge for a few seconds to sediment the fragments. Then store the samples at minus 20 degrees Celsius until they are shipped to the referring genetic laboratory for testing. Within 30 minutes of trophectoderm biopsy, label a vitrification plate with the patient's details and the IDs of the blastocysts that must be vitrified.

Using special cold temperature-resistant cryolabels, label the vitrification supports with patient's name and surname, couple ID, ID of the embryo that will be loaded on to it, and the date of the procedure. At room temperature, dispense 300 microliters of equilibration solution for each blastocyst that will be vitrified and, in the presence of a witness, use a 300-micrometer stripping pipette to move the blastocyst into one volume of equilibration solution. Leave the blastocyst in the equilibration solution for 13 to 15 minutes.

After an initial shrinkage of the volume, a gradual re-expansion will be observed. At the end of the equilibration, add 300 microliters of vitrification solution to the second well of the vitrification plate and transfer the completely re-expanded blastocyst to the vitrification solution for one minute. At the end of the incubation, rinse the blastocyst to dilute the equilibration solution and, in the presence of a witness, load the blastocyst on the vitrification support.

Remove the excess of vitrification solution. A subtle film of solution should surround the blastocyst. Plunge the vitrification support into liquid nitrogen, and vigorously move the support to reduce the risk of bubble formation close to the specimen.

Then place the protective cap on the support while the vitrification support is still submerged in liquid nitrogen. Of the 1, 544 trophectoderm biopsy procedures conducted over this representative two-year period, between one to four blastocysts were biopsied per procedure for between three to 22 minutes per procedure. In these plots, the mean timing of the biopsy for each operator along the study trimesters are shown, with a mean overall value of 8.24 minutes.

It is helpful to acquire an image of each biopsied fragment for quality control purposes to evaluate whether the cause of inconclusive diagnoses was imputable to the dimension and/or quality of the fragment, to the tubing, or to some issues in the processing of the sample in the genetic laboratory. In this study, 572 euploid blastocysts were warmed to undergo an embryo transfer after a diagnosis of euploidy. All the blastocysts vitrified within 30 minutes, with 2.4%of the blastocysts not re-expanding between 31 and 90 minutes and 4.9%not re-expanding beyond 90 minutes from rewarming.

Especially for poor quality and/or day seven blastocysts, the timing between biopsy and vitrification seems crucial to achieve re-expansion after warming. Take care to focus before opening the zona pellucida when exposing the junction between the trophectoderm cell and firing, and take care also to prevent blastocyst re-expansion before vitrification. Two other blastocyst biopsy approaches exist, both entailing zona pellucida opening and waiting for a spontaneous arching of the trophectoderm cells.

These approaches are easier but require more manipulations and are more time-consuming. The efficacy of this workflow allows the implementation of molecular technologies in IVF in order to try to select the blastocyst with the highest chance to implant before the embryo transfer. Remember not to use any device or solution that might be toxic to the embryos and to prevent DNA contamination by using DNA-free materials.