Human Blastocyst Biopsy and Vitrification

Summary

Blastocyst biopsy and vitrification are required to efficiently conduct preimplantation genetic testing. An approach entailing the sequential opening of the zona pellucida and retrieval of 7-8 trophectoderm cells in day 5-7 post-insemination limits both the number of manipulations required and the exposure of the embryo to sub-optimal environmental conditions.

Abstract

Blastocyst biopsy is performed to obtain a reliable genetic diagnosis during IVF cycles with preimplantation genetic testing. Then, the ideal workflow entails a safe and efficient vitrification protocol, due to the turnaround time of the diagnostic techniques and to transfer the selected embryo(s) on a physiological endometrium in a following natural cycle. A biopsy approach encompassing the sequential opening of the zona pellucida and retrieval of 5-10 trophectoderm cells (ideally 7-8) limits both the number of manipulations required and the exposure of the embryo to sub-optimal environmental conditions. After proper training, the technique was reproducible across different operators in terms of timing of biopsy (~8 min, ranging 3-22 min based on the number of embryos to biopsy per dish), conclusive diagnoses obtained (~97.5%) and live birth rates after vitrified-warmed euploid blastocyst transfer (>40%). The survival rate after biopsy, vitrification and warming was as high as 99.8%. The re-expansion rate at 1.5 h from warming was as high as 97%, largely dependent on the timing between biopsy and vitrification (ideally ≤30 min), blastocyst morphological quality and day of biopsy. In general, it is better to vitrify a collapsed blastocyst; therefore, in non-PGT cycles, laser-assisted artificial shrinkage might be performed to induce embryo collapse prior to cryopreservation. The most promising future perspective is the non-invasive analysis of the IVF culture media after blastocyst culture as a putative source of embryonic DNA. However, this potential avant-garde is still under investigation and a reliable protocol yet needs to be defined and validated.

Introduction

The main goal of modern human embryology is to maximize the number live births per stimulated cycle and reduce costs, time and efforts to achieve a pregnancy. To accomplish this goal, validated approaches for embryo selection should be employed to identify reproductively competent embryos within a cohort obtained during an IVF cycle. According to the latest evidences, blastocyst culture¹ combined with comprehensive chromosomal testing and vitrified-warmed euploid embryo transfer (ET) is the most efficient framework to increase IVF efficiency². Clearly, aneuploidy testing requires an embryonic specimen, which at present is mostly represented from few cells retrieved from the trophectoderm (TE), i.e., the section of the blastocyst that gives origin to embryo annexes (e.g., the placenta) during pregnancy. Beyond karyotype analysis, also single gene mutations might be assessed from a TE biopsy as part of a clinical strategy known as preimplantation genetic testing (PGT; -A for aneuploidies, -SR for structural chromosomal rearrangements, -M for monogenic diseases). Other oocyte/embryo biopsy methods have been theorized and adopted clinically across the last decades, namely polar bodies biopsy and blastomere biopsy. However, their use is reduced nowadays since their procedural drawbacks (e.g., higher workload and risk for reproductive impact) and diagnostic limitations (e.g., single cell analysis issues) implicitly hinder a sufficient balance between costs, risks and benefits (for a review see³).

In this paper, one of the main protocols for TE biopsy is thoroughly described together with the subsequent vitrification, warming and transfer procedures required. The workflow here outlined is ideal for a busy PGT unit.

As already described previously by our group^4,5, the procedure involves the sequential opening of the zona pellucida of fully-expanded blastocysts and removal of few TE cells (on average 7-8). Compared to the day 3 laser-assisted hatching-based blastocyst biopsy method⁶, this procedure might ease the daily schedule of an IVF unit where delicate procedures, such as blastocyst biopsy and vitrification, must be timely performed. As soon as the blastocyst reaches its full expansion, the biopsy can be carried out by selecting the TE cells to remove, thereby preventing the risk of herniation of the inner cell mass (ICM), which would otherwise render the procedure challenging. In literature, a third protocol of blastocyst biopsy has been also described, which involves laser-assisted hatching being performed once the embryo has already reached the blastocyst stage, few hours before the procedure^5,7. However, this approach is more time-consuming and mainly suits IVF units that are implementing TE biopsy in the hands of limitedly experienced operators and in view of a moderate-low daily workload.

Intracytoplasmatic sperm injection (ICSI)⁸ should be a consolidated technique if aiming at conducting genetic analyses in IVF. Similarly, a proper culture system to safely harvest embryos to the blastocyst stage is crucial for the implementation of TE biopsy strategy. An adequate number of incubators, as well as the use of low oxygen tension are key prerequisites to this end, not to compromise the blastocyst rate⁹. At the same time, an efficient cryopreservation program is needed to safely manage a PGT cycle. In the last decade, the implementation of vitrification has boosted embryo cryo-survival rates even up to >99%^10,11. This provided sufficient time to perform genetic testing and postpone embryo transfer to the following menstrual cycle, on a non-stimulated and probably more receptive endometrium¹².

Both TE biopsy and vitrification are demanding tasks requiring stringent skills and their effectiveness might vary across unexperienced operators. A specific training period is therefore advocated before allowing each operator to perform these procedures clinically; moreover, the maintenance of the operators’ skills should be assessed periodically by monitoring key performance indicators (KPI) for cryopreservation and biopsy procedures. Each IVF clinic should set internal KPIs to this end, which must approximate the ones published by international consortia and/or the outcomes published by reference laboratories.

TE biopsy, vitrification-warming and witnessing procedures are validated techniques at our unit, that have been standardized across all the operators involved as reported in three previous publications^11,13,14.

Protocol

The protocol for human blastocyst biopsy, here described, follows the guidelines of G.EN.E.R.A. Human Research Ethic Committee.

NOTE: Refer to the Table of Materials for materials required. Further material required entails laboratory footwear and outfit, surgical facemask, hair cover, surgical gloves, a permanent non-toxic marker, forceps and disinfectant. The use of surgical gown, disposable surgical gloves, facemask, hair cover is mandatory to prevent risk of contamination. All the working areas, as well as the equipment involved in the process, must be cleaned thoroughly with laboratory disinfectant (e.g., Oosafe) before starting any procedure. All consumables and media used should be sterile and individually packaged or aliquoted. It is suggested to use a dedicated workstation for biopsy and tubing and limit the access of the area only to the operators involved in the procedure (embryologist and witness).

1. Preparation on the Day Before the Biopsy Procedure

1. Prepare the post biopsy culture dish.
   1. Place 6 drops of 20 µL IVF culture medium (Table of Materials) in an IVF culture dish and overlay with 6 mL of prewarmed mineral oil for embryo culture (Table of Materials).
   2. Add another 10 µL of IVF culture medium to each drop. Incubate overnight at 37 °C in a controlled atmosphere (5% O₂, 6% CO₂).
2. Prepare an IVF dish 4-well plate for rinsing the blastocyst after biopsy.
   1. Place 600 µL of IVF culture medium in the first two wells and overlay with 300 µL of pre-warmed mineral oil for embryo culture.
   2. Incubate overnight at 37 °C in a controlled atmosphere (5% O₂, 6% CO₂).
3. Dispense 1.5 µL of loading solution (Table of Materials) in each PCR tube to be used for TE cells tubing, spin it and keep it at 4 °C.

2. Preparation on the Day of the Biopsy Procedure

1. Prepare the biopsy dish.
   1. Place 3 drops of 10 µL HEPES-buffered medium (supplemented with human serum albumin) (Table of Materials) in a row in an IVF culture dish.
   2. Repeat step 1.1.1 according to the number of blastocysts available (up to 4 blastocysts per dish) and overlay with 6 mL of pre-warmed mineral oil for embryo culture.
   3. Incubate the dish at 37 °C for at least 1 h before performing the biopsy procedure.

3. Blastocyst Selection and Grading

1. Grade the blastocyst according to its expansion and the morphological appearance of ICM and TE (Figure 1).
   NOTE: The grading criteria have been adapted from Gardner and Schoolcraft¹⁵ and previously described by Capalbo et al. and Cimadomo et al.^4,11.
   1. Define the size and expansion grade as: A for a fully-hatched blastocyst, B for a hatching blastocyst, C for a fully expanded blastocyst, and D for a not expanded blastocyst (these embryos are biopsied only if they did not expand further up to day 7).
   2. Define ICM grade: 1 for noticeable ICM with several strictly-packed cells; 2 for discernable with several but roughly-packed cells; 3 for difficult to distinguish with very few low-quality cells.
   3. Define TE grade as follows: 1 for well-organized epithelium with several cells; 2 for loose epithelium with few cells; 3 for few and/or large low-quality cells.

4. Trophectoderm Biopsy

1. Perform TE biopsy on all the viable fully-expanded blastocysts (preferably C grade for size and expansion).
2. Set holding and biopsy pipettes for each biopsy procedure. The recommendations for the biopsy pipette are the following: 30 µm internal diameter, 35° bend angle, 0.75 mm distance tip to bend.
3. Label the biopsy dish with patient’s details (woman’s name and surname, date of birth and ID) and then number each drop with embryo and cycle IDs. Use a permanent non-toxic marker.
4. Transfer the blastocyst with a 300 µm stripping pipette to the first drop of the biopsy dish and rinse it in order to remove the excess of culture medium. Then move the blastocyst in the second drop of the biopsy dish.
5. Move the dish to the inverted microscope and prime the biopsy pipette aspirating some medium from the third drop of the biopsy dish.
6. At 20x magnification, orient the blastocyst to have a clear view of the ICM. When it is visualized at 7 o’clock (opposite to the area that will be targeted to remove the TE cells), secure the embryo on the holding pipette (Figure 2a).
7. Focus on the zona pellucida and ascertain that both the pipettes and the blastocyst are on the same focal plane.
8. Switch to the laser objective (pulse time 0.3 ms, 6.5 µm) and position the laser pointer on the zona pellucida at the opposite side of ICM. Drill the zona pellucida through 2−3 laser pulses (Figure 2b).
9. Gently press the biopsy pipette against the zona pellucida and blow some medium through the breach to detach the TE cells from its internal surface and expedite blastocyst collapse (Figure 2c).
10. Once the TE is detached (Figure 2d), enter through the hole (if required, make it wider with a last laser pulse) and aspirate few TE cells (ideally 7−8^13,16) into the biopsy pipette with gentle suction (Figure 2e).
11. Slightly move the biopsy pipette backwards while applying a moderate suction to stretch the target cells (Figure 2f).
12. Direct the laser towards the thinnest part of the aspirated cells and fire 2−5 laser pulses at the junctions between cells to separate the target cells from the body of the embryo (Figure 2g). The timing of laser pulses and the number of pulses can be adjusted according to the quality of the blastocyst; however, try to minimize them to avoid cell lysis.
13. After the separation of the TE fragment from the blastocyst (Figure 2h), release it into the same biopsy drop far from the blastocyst. This is needed to prevent them from being sucked again into the biopsy pipette (Figure 2i).
14. Release the blastocyst from the holding pipette and promptly raise both pipettes to prevent the fragment from sticking to them.
15. Take a picture of the biopsied fragment for quality control purpose (Figure 3).
    NOTE: If more than a single blastocyst is to be biopsied per procedure (up to four per biopsy dish), change the biopsy pipette with a new one to prevent cross-contamination between embryos.
16. Repeat steps 4.1−4.13.
17. Move the biopsy dish back to the laminar flow hood.
18. Label the post-biopsy culture dish with the couple ID, and each drop with the embryo and the cycle ID.
19. In presence of a witness, rinse the blastocyst in clean IVF medium, and lastly move it to its corresponding drop of the post-biopsy dish.
20. Move the post-biopsy dish to the incubator in a controlled atmosphere (37 °C, 6% CO₂, 5% O₂) until vitrification. It is advisable to perform vitrification within 30 min from the biopsy procedure, to prevent blastocyst re-expansion.

5. Tubing

NOTE: The whole procedure must be carried out in the presence of a witness and inside the laminar flow hood at room temperature. During the procedure, keep the PCR tubes in a cold tube rack on ice (Supplementary Figure 1).

1. Label the PCR tubes with a permanent non-toxic marker.
   NOTE: Labeling should be performed as requested by the genetic laboratory. Generally, patient’s name and surname (initials), couple ID, embryo and cycle ID (for instance: JD 12345 1.2 for the embryo N.1 of 2nd cycle belonging to Jane Doe whose couple ID is 12345). Embryo ID should be reported also in letters on the body of the tube.
2. Label the lid of a 60 mm x 15 mm culture dish (tubing dish) with the biopsied embryos’ IDs (Supplementary Figure 1) and prepare two 10 µL drops of biopsy washing solution (Table of Materials) in it.
3. Prime the 140 µm stripping pipette (Supplementary Figure 1) with some biopsy washing solution from the second drop of the tubing dish.
4. Place the biopsy dish under the stereomicroscope to easily visualize the TE fragment(s).
5. Gently release some biopsy washing solution upon the TE fragment; then, load it into the stripping pipette.
6. Move the TE fragment to the second drop of biopsy washing solution in the tubing dish and carefully rinse it 2−3 times.
7. Transfer the TE fragment to the bottom of the PCR tube (previously labeled after it) with loading solution, paying attention to avoid touching its walls with the tip of the stripping pipette.
8. Repeat the procedure from step 5.3 to step 5.7 for each TE fragment, paying attention to use a new capillary for every TE fragment.
9. At the end of the tubing procedure, put all the PCR tubes in a mini centrifuge, and spin them for few seconds.
10. Store the samples at -20 °C until shipping them to the referring genetic laboratory for testing.

6. Blastocyst Vitrification

1. Vitrify collapsed blastocysts within 30 min from TE biopsy to prevent their re-expansion.
2. Label the vitrification plate with woman’s details and the IDs of the blastocysts that must be vitrified.
3. Label the vitrification support(s) with woman’s name and surname, couple ID, ID of the embryo that will be loaded on it, as well as date of the procedure. Special cryolabels are used which preserve their integrity even at very low temperature (-196 °C).
4. At room temperature, dispense 0.3 mL of equilibration solution (ES) (Table of Materials) for each blastocyst that will be vitrified.
5. In the presence of a witness, move the blastocyst in ES using the 300 µm stripping pipette.
6. Leave the blastocyst in the ES for 13−15 min. After an initial shrinkage of the volume, a gradual re-expansion will be observed.
7. Fill a small cooling rack with liquid nitrogen (LN₂) and place it under the laminar flow hood.
8. Dispense 300 μL of vitrification solution (VS) (Table of Materials) in the second well. After the blastocyst complete re-expansion, transfer it in the VS solution for 1 min and rinse it to dilute the ES.
9. In presence of a witness, load the blastocyst on the vitrification support and take care of removing the excess of VS. A subtle film of solution should surround the blastocyst.
10. Plunge the vitrification support into LN₂ and move it energetically in order to reduce the risk of bubble formation close to the specimen.
11. Place the protective cap while keeping the vitrification support submerged in the LN_2.
12. In the presence of a witness, move the vitrification support in a long term LN₂ storage tank.

7. Artificial shrinkage of Non-biopsied Blastocysts

1. If no TE biopsy is conducted, artificially collapse the blastocysts immediately before vitrification (Figure 4).
   1. Move the culture dish from the incubator to the inverted microscope and focus on the selected embryo.
   2. Switch to the laser objective (pre-set pulse: 0.3 ms, 6.5 μm), target the zona pellucida at the opposite side of ICM, then direct 1–2 laser pulses to the junctions between TE cells at a safe distance from the ICM. The blastocysts should collapse in less than 5 min.
2. Proceed with the vitrification of the collapsed blastocyst as described in section 6.

8. Transferable Blastocyst Warming

1. On the day of the ET, prepare the ET 4-well plate by placing 600 µL of pre-equilibrated IVF culture medium for each well. Incubate at 37 °C in a controlled atmosphere (5% O₂, 6% CO₂), while performing blastocyst warming.
2. Label the warming dish with woman’s name and surname, couple ID, ID of the embryo that will be warmed in it.
3. Dispense 1 mL of the thawing solution (TS) (Table of Materials) in an IVF one-well dish (Supplementary Figure 1) and warm it to 37 °C in a thermostat for at least 1 h before starting the procedure.
4. Fill a small cooling support with LN₂ and place it in the laminar flow hood in close proximity of the working area.
5. Before starting the procedure, check the blastocyst information reported on the vitrification support. All the IDs should match the genetic report and the blastocyst to warm must be chosen among the transferable ones. A witness is required during the procedure.
6. Take the one-well dish containing warmed TS out of the thermostat and place it on a heated stage under the stereomicroscope.
7. Pull over the protective cap within the LN₂ using forceps.
8. Plunge quickly the tip of the vitrification support into the 37 °C TS.
9. Under microscopic observation, gently move the vitrification support until the blastocyst is released from the tip.
10. Leave the blastocyst for a total of 1 min in the TS, paying attention to keep it at the bottom of the TS.
11. At room temperature dispense 200 μL of dilution solution (DS) (Table of Materials) on the first well of the vitrification plate.
12. Transfer the blastocyst to DS and place it at the bottom of the well while releasing some TS on the top of it to create a sort of gradient. Leave it for 3 min.
13. Dispense 200 μL of washing solution (WS) in 2 different wells (WS1 and WS2).
14. Transfer the blastocyst to WS1 and place it on the bottom of the well while releasing some DS medium on the top of it. Then transfer the blastocyst to WS2 and leave it undisturbed for 1 min.
15. Label the post-warming ET plate with woman’s name and surname, couple ID, ID of the embryo that will be cultured in it.
16. In the presence of a witness, transfer the warmed blastocyst to pre-equilibrated culture medium in the post-warming ET plate.
17. Rinse the blastocyst in the first well of the ET plate and place it in the second well.
18. Transfer the post-warming culture dish to the incubator in a controlled atmosphere (37 °C, 6% CO₂, 5% O₂) and culture the blastocyst for at least 1.5 h before checking its survival and re-expansion.
    NOTE: Figure 5 shows examples of degenerated, cryo-survived but not re-expanded and cryo-survived and fully-expanded blastocysts.

9. EmbryoTransfer

1. To perform ET, place the dish on the heated stage of the laminar flow hood, so the embryo is visible under the microscope at a low magnification.
2. Take about 0.4 mL of IVF culture medium. Secure the syringe tip firmly into the receiving end of the internal catheter and then release the medium up to 0.1 mL.
3. Aspirate culture medium until observing the embryos entering the catheter (minimal amount of media: 10−15 μL).
4. Place the catheter into the plastic case and go to the operating room and place the internal catheter into the external guide.
5. Once reached the uterus, push the syringe plunger to release the embryo(s). Release very slowly the medium until the plunger is reading 0.1 mL.
6. Take the catheter back to the lab and check under the microscope that the embryo has been transferred.

Results

Figure 6 represents a scheme of all the outcomes of a biopsy procedure that can be adopted to standardize the protocol and monitor the performance of each operator. The main procedural outcome is the timing to complete the biopsy/biopsies; the main technical outcome is the quality of the plot produced after genetic testing that might result in either a conclusive or inconclusive diagnosis, the latter of which requires a re-biopsy of the undiagnosed blastocyst...

Discussion

Only well-experienced skilled embryologists who have completed their training period should perform both TE biopsy and blastocyst vitrification. Furthermore, a witness is required to monitor the procedures and guarantee an efficient traceability during i) the movements of the biopsied blastocyst from the biopsy dish (Supplementary Figure 1) to the post-biopsy dish (Supplementary Figure 1), then to the vitrification plate (Supplementary Figure 1) and lastly to the vitrifi...

Materials

Name	Company	Catalog Number	Comments
Equipment
Cold tube rack	Biocision	XTPCR96
Electronic pipette controller	Fisher Scientific	710931
Flexipet adjustable handle set	Cook	G18674	Stripper holder
Gilson Pipetman	Gilson	66003	p20
IVF Electronic Witness System	CooperSurgical Fertility & Genomic Solutions		RI Witness ART Management System
Inverted microscope	Nikon	Eclipse TE2000-U
Laminar Flow Hood	IVF TECH		Grade A air flow
Laser objective	RI	Saturn 5
Microinjectors	Nikon Narishige	NT-88-V3
Mini centrifuge for PCR tubes	Eppendorf	CSLQSPIN	for 0.2ml PCR tubes
Stereomicroscope	Leica	Leica M80
Thermostat	Panasonic	MCO-5AC-PE
Tri-gas incubator	Panasonic	MCO-5M-PE	02/CO2
Consumables
Biopsy pipette	RI	7-71-30FB35720	30µm ID, flat 35°C
Cryolock	Cryolock	CL-R-CT
CSCM complete	Irvine Scientific	90165	IVF culture medium supplemented with HSA
Embryo Transfer Catheter	Cook	G17934
Flexipet pipette	Cook	G26712	140µm stripping pipette tip
Flexipet pipette	Cook	G46020	300µm stripping pipette tips
Holding pipette	RI	7-71-IH35/20	30µm ID, flat 35°C
Human Serum Albumin	Irvine Scientific	9988
IVF One well dish	Falcon	353653
Mineral Oil for embryo culture	Irvine Scientific	9305
Modified HTF Medium	Irvine Scientific	90126	Hepes-Buffered medium
Nuclon Delta Surface	Thermofisher scientific	176740	IVF dish 4-well plate with sliding lid
Primaria Cell culture dish	Corning	353802	60x15mm
Reproplate	Kitazato	83016
Serological pipette	Falcon	357551	10ml
Sterile disposable Gilson tips	Eppendorf	0030 075.021	200µl
Tubing Kit	Provided by the genetic lab		PCR tubes (0.2mL), loading solution, biopsy washing solution
Vitrification media	Kitazato	VT801	Equilibration and vitrification solutions
Warming media	Kitazato	VT802	Thawing and dilution solutions