Name: human blastocyst biopsy and vitrification
Uploaded: 2019-07-26T12:30:00.000+00:00
Duration: 10 min 59 s
Description: Watch this Scientific Journal Video about Human Blastocyst Biopsy and Vitrification at JoVE.com

AG and RM collected the data and drafted the manuscript. DC analyzed the data, drafted the representative results, performed the statistics and revised the manuscript. FMU and LR provided critical discussion of the results and of the whole manuscript.

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
<ol>
	<li>Prepare the post biopsy culture dish.
	<ol>
		<li>Place 6 drops of 20 &#181;L 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).</li>
		<li>Add another 10 &#181;L of IVF culture medium to each drop. Incubate overnight at 37 &#176;C in a controlled atmosphere (5% O2, 6% CO2).</li>
	</ol>
	</li>
	<li>Prepare an IVF dish 4-well plate for rinsing the blastocyst after biopsy.
	<ol>
		<li>Place 600 &#181;L of IVF culture medium in the first two wells and overlay with 300 &#181;L of pre-warmed mineral oil for embryo culture.</li>
		<li>Incubate overnight at 37 &#176;C in a controlled atmosphere (5% O2, 6% CO2).</li>
	</ol>
	</li>
	<li>Dispense 1.5 &#181;L of loading solution (Table of Materials) in each PCR tube to be used for TE cells tubing, spin it and keep it at 4 &#176;C.</li>
</ol>
2. Preparation on the Day of the Biopsy Procedure
<ol>
	<li>Prepare the biopsy dish.
	<ol>
		<li>Place 3 drops of 10 &#181;L HEPES-buffered medium (supplemented with human serum albumin) (Table of Materials) in a row in an IVF culture dish.</li>
		<li>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.</li>
		<li>Incubate the dish at 37 &#176;C for at least 1 h before performing the biopsy procedure.</li>
	</ol>
	</li>
</ol>
3. Blastocyst Selection and Grading
<ol>
	<li>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.
	<ol>
		<li>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).</li>
		<li>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.</li>
		<li>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.</li>
	</ol>
	</li>
</ol>
4. Trophectoderm Biopsy
<ol>
	<li>Perform TE biopsy on all the viable fully-expanded blastocysts (preferably C grade for size and expansion).</li>
	<li>Set holding and biopsy pipettes for each biopsy procedure. The recommendations for the biopsy pipette are the following: 30 &#181;m internal diameter, 35&#176; bend angle, 0.75 mm distance tip to bend.</li>
	<li>Label the biopsy dish with patient&#8217;s details (woman&#8217;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.</li>
	<li>Transfer the blastocyst with a 300 &#181;m 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.</li>
	<li>Move the dish to the inverted microscope and prime the biopsy pipette aspirating some medium from the third drop of the biopsy dish.</li>
	<li>At 20x magnification, orient the blastocyst to have a clear view of the ICM. When it is visualized at 7 o&#8217;clock (opposite to the area that will be targeted to remove the TE cells), secure the embryo on the holding pipette (Figure 2a).</li>
	<li>Focus on the zona pellucida and ascertain that both the pipettes and the blastocyst are on the same focal plane.</li>
	<li>Switch to the laser objective (pulse time 0.3 ms, 6.5 &#181;m) and position the laser pointer on the zona pellucida at the opposite side of ICM. Drill the zona pellucida through 2&#8722;3 laser pulses (Figure 2b).</li>
	<li>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).</li>
	<li>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&#8722;813,16) into the biopsy pipette with gentle suction (Figure 2e).</li>
	<li>Slightly move the biopsy pipette backwards while applying a moderate suction to stretch the target cells (Figure 2f).</li>
	<li>Direct the laser towards the thinnest part of the aspirated cells and fire 2&#8722;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.</li>
	<li>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).</li>
	<li>Release the blastocyst from the holding pipette and promptly raise both pipettes to prevent the fragment from sticking to them.</li>
	<li>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.</li>
	<li>Repeat steps 4.1&#8722;4.13.</li>
	<li>Move the biopsy dish back to the laminar flow hood.</li>
	<li>Label the post-biopsy culture dish with the couple ID, and each drop with the embryo and the cycle ID.</li>
	<li>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.</li>
	<li>Move the post-biopsy dish to the incubator in a controlled atmosphere (37 &#176;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.</li>
</ol>
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).
<ol>
	<li>Label the PCR tubes with a permanent non-toxic marker. 
	NOTE: Labeling should be performed as requested by the genetic laboratory. Generally, patient&#8217;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.</li>
	<li>Label the lid of a 60 mm x 15 mm culture dish (tubing dish) with the biopsied embryos&#8217; IDs (Supplementary Figure 1) and prepare two 10 &#181;L drops of biopsy washing solution (Table of Materials) in it.</li>
	<li>Prime the 140 &#181;m stripping pipette (Supplementary Figure 1) with some biopsy washing solution from the second drop of the tubing dish.</li>
	<li>Place the biopsy dish under the stereomicroscope to easily visualize the TE fragment(s).</li>
	<li>Gently release some biopsy washing solution upon the TE fragment; then, load it into the stripping pipette.</li>
	<li>Move the TE fragment to the second drop of biopsy washing solution in the tubing dish and carefully rinse it 2&#8722;3 times.</li>
	<li>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.</li>
	<li>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.</li>
	<li>At the end of the tubing procedure, put all the PCR tubes in a mini centrifuge, and spin them for few seconds.</li>
	<li>Store the samples at -20 &#176;C until shipping them to the referring genetic laboratory for testing.</li>
</ol>
6. Blastocyst Vitrification
<ol>
	<li>Vitrify collapsed blastocysts within 30 min from TE biopsy to prevent their re-expansion.</li>
	<li>Label the vitrification plate with woman&#8217;s details and the IDs of the blastocysts that must be vitrified.</li>
	<li>Label the vitrification support(s) with woman&#8217;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 &#176;C).</li>
	<li>At room temperature, dispense 0.3 mL of equilibration solution (ES) (Table of Materials) for each blastocyst that will be vitrified.</li>
	<li>In the presence of a witness, move the blastocyst in ES using the 300 &#181;m stripping pipette.</li>
	<li>Leave the blastocyst in the ES for 13&#8722;15 min. After an initial shrinkage of the volume, a gradual re-expansion will be observed.</li>
	<li>Fill a small cooling rack with liquid nitrogen (LN2) and place it under the laminar flow hood.</li>
	<li>Dispense 300 &#956;L 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.</li>
	<li>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.</li>
	<li>Plunge the vitrification support into LN2 and move it energetically in order to reduce the risk of bubble formation close to the specimen.</li>
	<li>Place the protective cap while keeping the vitrification support submerged in the LN2.</li>
	<li>In the presence of a witness, move the vitrification support in a long term LN2 storage tank.</li>
</ol>
7. Artificial shrinkage of Non-biopsied Blastocysts
<ol>
	<li>If no TE biopsy is conducted, artificially collapse the blastocysts immediately before vitrification (Figure 4).
	<ol>
		<li>Move the culture dish from the incubator to the inverted microscope and focus on the selected embryo.</li>
		<li>Switch to the laser objective (pre-set pulse: 0.3 ms, 6.5 &#956;m), target the zona pellucida at the opposite side of ICM, then direct 1&#8211;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.</li>
	</ol>
	</li>
	<li>Proceed with the vitrification of the collapsed blastocyst as described in section 6.</li>
</ol>
8. Transferable Blastocyst Warming
<ol>
	<li>On the day of the ET, prepare the ET 4-well plate by placing 600 &#181;L of pre-equilibrated IVF culture medium for each well. Incubate at 37 &#176;C in a controlled atmosphere (5% O2, 6% CO2), while performing blastocyst warming.</li>
	<li>Label the warming dish with woman&#8217;s name and surname, couple ID, ID of the embryo that will be warmed in it.</li>
	<li>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 &#176;C in a thermostat for at least 1 h before starting the procedure.</li>
	<li>Fill a small cooling support with LN2 and place it in the laminar flow hood in close proximity of the working area.</li>
	<li>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.</li>
	<li>Take the one-well dish containing warmed TS out of the thermostat and place it on a heated stage under the stereomicroscope.</li>
	<li>Pull over the protective cap within the LN2 using forceps.</li>
	<li>Plunge quickly the tip of the vitrification support into the 37 &#176;C TS.</li>
	<li>Under microscopic observation, gently move the vitrification support until the blastocyst is released from the tip.</li>
	<li>Leave the blastocyst for a total of 1 min in the TS, paying attention to keep it at the bottom of the TS.</li>
	<li>At room temperature dispense 200 &#956;L of dilution solution (DS) (Table of Materials) on the first well of the vitrification plate.</li>
	<li>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.</li>
	<li>Dispense 200 &#956;L of washing solution (WS) in 2 different wells (WS1 and WS2).</li>
	<li>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.</li>
	<li>Label the post-warming ET plate with woman&#8217;s name and surname, couple ID, ID of the embryo that will be cultured in it.</li>
	<li>In the presence of a witness, transfer the warmed blastocyst to pre-equilibrated culture medium in the post-warming ET plate.</li>
	<li>Rinse the blastocyst in the first well of the ET plate and place it in the second well.</li>
	<li>Transfer the post-warming culture dish to the incubator in a controlled atmosphere (37 &#176;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.</li>
</ol>
9. EmbryoTransfer
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>Aspirate culture medium until observing the embryos entering the catheter (minimal amount of media: 10&#8722;15 &#956;L).</li>
	<li>Place the catheter into the plastic case and go to the operating room and place the internal catheter into the external guide.</li>
	<li>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.</li>
	<li>Take the catheter back to the lab and check under the microscope that the embryo has been transferred.</li>
</ol>

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The authors have nothing to disclose.

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...

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 group4,5, 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 procedure5,7. 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 &#62;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&#8217; 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 publications11,13,14.

<table><tbody><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Cold tube rack</td><td>Biocision</td><td>XTPCR96</td><td></td></tr><tr><td>Electronic pipette controller</td><td>Fisher Scientific</td><td>710931</td><td></td></tr><tr><td>Flexipet adjustable handle set</td><td>Cook</td><td>G18674</td><td>Stripper holder</td></tr><tr><td>Gilson Pipetman</td><td>Gilson</td><td>66003</td><td>p20</td></tr><tr><td>IVF Electronic Witness System</td><td>CooperSurgical Fertility &amp;amp; Genomic Solutions</td><td></td><td>RI Witness ART Management System</td></tr><tr><td>Inverted microscope</td><td>Nikon</td><td>Eclipse TE2000-U</td><td></td></tr><tr><td>Laminar Flow Hood</td><td>IVF TECH</td><td></td><td>Grade A air flow</td></tr><tr><td>Laser objective</td><td>RI</td><td>Saturn 5</td><td></td></tr><tr><td>Microinjectors</td><td>Nikon Narishige</td><td>NT-88-V3</td><td></td></tr><tr><td>Mini centrifuge for PCR tubes</td><td>Eppendorf</td><td>CSLQSPIN</td><td>for 0.2 mLl PCR tubes</td></tr><tr><td>Stereomicroscope</td><td>Leica</td><td>Leica M80</td><td></td></tr><tr><td>Thermostat</td><td>Panasonic</td><td>MCO-5AC-PE</td><td></td></tr><tr><td>Tri-gas incubator</td><td>Panasonic</td><td>MCO-5M-PE</td><td>02/CO2</td></tr><tr><td>&lt;strong&gt;Consumables&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Biopsy pipette</td><td>RI</td><td>7-71-30FB35720</td><td>30 &amp;micro;m ID, flat 35 &amp;deg;C</td></tr><tr><td>Cryolock</td><td>Cryolock</td><td>CL-R-CT</td><td></td></tr><tr><td>CSCM complete</td><td>Irvine Scientific</td><td>90165</td><td>IVF culture medium supplemented with HSA</td></tr><tr><td>Embryo Transfer Catheter</td><td>Cook</td><td>G17934</td><td></td></tr><tr><td>Flexipet pipette</td><td>Cook</td><td>G26712</td><td>140&amp;micro;m stripping pipette tip</td></tr><tr><td>Flexipet pipette</td><td>Cook</td><td>G46020</td><td>300&amp;micro;m stripping pipette tips</td></tr><tr><td>Holding pipette</td><td>RI</td><td>7-71-IH35/20</td><td>30 &amp;micro;m ID, flat 35 &amp;deg;C</td></tr><tr><td>Human Serum Albumin</td><td>Irvine Scientific</td><td>9988</td><td></td></tr><tr><td>IVF One well dish</td><td>Falcon</td><td>353653</td><td></td></tr><tr><td>Mineral Oil for embryo culture</td><td>Irvine Scientific</td><td>9305</td><td></td></tr><tr><td>Modified HTF Medium</td><td>Irvine Scientific</td><td>90126</td><td>Hepes-Buffered medium</td></tr><tr><td>Nuclon Delta Surface</td><td>Thermofisher scientific</td><td>176740</td><td>IVF dish 4-well plate with sliding lid</td></tr><tr><td>Primaria Cell culture dish</td><td>Corning</td><td>353802</td><td>60 mm x15 mm</td></tr><tr><td>Reproplate</td><td>Kitazato</td><td>83016</td><td></td></tr><tr><td>Serological pipette</td><td>Falcon</td><td>357551</td><td>10ml</td></tr><tr><td>Sterile disposable Gilson tips</td><td>Eppendorf</td><td>0030 075.021</td><td>200 &amp;micro;L</td></tr><tr><td>Tubing Kit</td><td>Provided by the genetic lab</td><td></td><td>PCR tubes (0.2 mL), loading solution, biopsy washing solution</td></tr><tr><td>Vitrification media</td><td>Kitazato</td><td>VT801</td><td>Equilibration and vitrification solutions</td></tr><tr><td>Warming media</td><td>Kitazato</td><td>VT802</td><td>Thawing and dilution solutions</td></tr></tbody></table>

human blastocyst biopsy and vitrification

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 (&#62;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 &#8804;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.

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...

Watch this Scientific Journal Video about Human Blastocyst Biopsy and Vitrification at JoVE.com

Human Blastocyst Biopsy and Vitrification

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.

Blastocyst biopsy is performed to obtain a reliable genetic diagnosis during IVF cycles with preimplantation genetic testing. Then, the ideal workflow ...

human-blastocyst-biopsy-and-vitrification

Research

JoVE Journal

Developmental Biology

22.1K Views.  G.EN.E.R.A. Centers for Reproductive Medicine. 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.

Video: Human Blastocyst Biopsy and Vitrification

Isolation and Quantitative Immunocytochemical Characterization of Primary Myogenic Cells and Fibroblasts from Human Skeletal Muscle

The repair and regeneration of skeletal muscle requires the action of satellite cells, which are the resident muscle stem cells. These can be isolated from human muscle biopsy samples using enzymatic digestion and their myogenic properties studied in culture. Quantitatively, the two main adherent cell types obtained from enzymatic digestion are: (i) the satellite cells (termed myogenic cells or muscle precursor cells), identified initially as CD56+ and later as CD56+/desmin+ cells and (ii) muscle-derived fibroblasts, identified as CD56&#8211; and TE-7+. Fibroblasts proliferate very efficiently in culture and in mixed cell populations these cells may overrun myogenic cells to dominate the culture. The isolation and purification of different cell types from human muscle is thus an important methodological consideration when trying to investigate the innate behavior of either cell type in culture. Here we describe a system of sorting based on the gentle enzymatic digestion of cells using collagenase and dispase followed by magnetic activated cell sorting (MACS) which gives both a high purity (&#62;95% myogenic cells) and good yield (~2.8 x 106 &#177; 8.87 x 105 cells/g tissue after 7 days in vitro) for experiments in culture. This approach is based on incubating the mixed muscle-derived cell population with magnetic microbeads beads conjugated to an antibody against CD56 and then passing cells though a magnetic field. CD56+ cells bound to microbeads are retained by the field whereas CD56&#8211; cells pass unimpeded through the column. Cell suspensions from any stage of the sorting process can be plated and cultured. Following a given intervention, cell morphology, and the expression and localization of proteins including nuclear transcription factors can be quantified using immunofluorescent labeling with specific antibodies and an image processing and analysis package.

The main adherent cell types derived from human muscle are myogenic cells and fibroblasts. Here, cell populations are enriched using magnetic-activated cell sorting based on the CD56 antigen. Subsequent immunolabelling with specific antibodies and use of image analysis techniques allows quantification of cytoplasmic and nuclear characteristics in individual cells.

The repair and regeneration of skeletal muscle requires the action of satellite cells, which are the resident muscle stem cells. These can be isolated ...

Modified MicroSecure Vitrification: A Safe, Simple and Highly Effective Cryopreservation Procedure for Human Blastocysts

Clinical embryo vitrification evolved with the development of unique vitrification devices in the 21st century and with the misconception that ultra-rapid cooling in an &#34;open&#34; system (i.e., direct LN2 contact) was a necessity to optimize vitrification success. The dogma surrounding the importance of cooling rates led to unsafe practices subject to technical variation and to the creation of vitrification devices that disregarded important quality-control factors (e.g., ease of use, repeatability, reliability, labeling security, and storage safety). Understanding the quality-control flaws of other devices allowed for the development of a safe, secure, repeatable, and reliable &#181;S-VTF method aimed to minimize intra- and inter-technician variation. Equally important, it combined the availability of two existing FDA-compliant devices: 1) a 0.3-mL ionomeric resin embryo straw with internalized, dual-colored, tamper-proof labeling with repeatable weld seal potential; and 2) shortened, commonly-used, 300-&#181;m ID sterile flexipettes to directly load the embryo(s) in order to create a highly-effective global vitrification device. Like other aseptic, closed vitrification systems (e.g., High Security Vitrification (HSV), Rapid-i, and VitriSafe) effectively used in reproductive medicine, microSecure Vitrification (&#181;S-VTF) has proven that it can achieve high post-warming survival and pregnancy outcomes with its attention to simplicity, and reduced technical variation. Although the 0.3-mL embryo straw containing an internal hydrophobic plug was commercially replaced with a standard semen straw possessing cotton-polyvinyl pyrrolidone (PVP) plugs, it maintained its ionomeric resin composition to ensure weld sealing. However, the cotton plugs can wick out the fluid-embryo contents of the flexipettes upon contact. A modified &#181;S-VTF method was adapted to include an additional internal weld seal before the plug on the device loading side. The added technical step to the &#181;S-VTF procedure has not affected its successful application, as high survival rates (&#62; 95%) and pregnancy rates continue today.

MicroSecure Vitrification was developed as a non-commercial, aseptic closed vitrification device system that is compliant with FDA good manufacturing and tissue-handling practices. Due to the withdrawal of hydrophobic plugged embryo straws from the industry, the vitrification procedure was modified to include an inner seal before the standard internal cotton plug.

Clinical embryo vitrification evolved with the development of unique vitrification devices in the 21st century and with the misconception that ultra-rapid ...

Chromosome Screening of Human Preimplantation Embryos by Using Spent Culture Medium: Sample Collection and Chromosomal Ploidy Analysis

In clinical in vitro fertilization (IVF), the prevailing method for PGT-A requires biopsy of a few cells from the trophectoderm (TE). This is the lineage that forms the placenta. This method, however, requires specialized skills, is invasive, and suffers from false positives and negatives because the chromosome numbers in the TE and the inner cell mass (ICM), which develops into the fetus, are not always the same. NICS, a technology requiring sequencing of DNA that released into the culture medium from both TE and ICM, may offer a way out to these problems but has previously been shown to have limited efficacy. The present study reports the full protocol of NICS, which includes culture medium sampling methods, whole genome amplification (WGA) and library preparation, and NGS data analysis by analysis software. Considering the different cryopreservation times in different embryo laboratories, embryologists have two methods for collecting embryo culture medium that can be selected according to the actual conditions of the IVF laboratory.

The present study reports a protocol for chromosome screening of human embryos that uses spent culture medium, which avoids embryo biopsy and enables chromosome ploidy identification using NGS. The present article presents the detailed procedure, including the preparation of culture medium, whole genome amplification (WGA), next-generation sequencing (NGS) library preparation, and data analysis.

In clinical in vitro fertilization (IVF), the prevailing method for PGT-A requires biopsy of a few cells from the trophectoderm (TE). This is the lineage ...

Genetics

Minimally Invasive Embryo Transfer and Embryo Vitrification at the Optimal Embryo Stage in Rabbit Model

Assisted reproductive techniques (ARTs), such as in vitro embryo culture or embryo cryopreservation, affect natural development patterns with perinatal and postnatal consequences. To ensure the innocuousness of ART applications, studies in animal models are necessary. In addition, as a last step, embryo development studies require evaluation of their capacity to develop full-term healthy offspring. Here, embryo transfer to the uterus is indispensable to perform any ARTs-related experiment.
The rabbit has been used as a model organism to study mammalian reproduction for over a century. In addition to its phylogenetic proximity to the human species and its small size and low maintenance cost, it has important reproductive characteristics such as induced ovulation, a chronology of early embryonic development similar to humans and a short gestation that allow us to study the consequences of ART application easily. Moreover, ARTs (such as intracytoplasmic sperm injection, embryo culture, or cryopreservation) are applied with suitable efficiency in this species.
Using the laparoscopic embryo transfer technique and the cryopreservation protocol presented in this article, we describe 1) how to transfer embryos through an easy, minimally invasive technique and 2) an effective protocol for long-term storage of rabbit embryos to provide time-flexible logistical capacities and the ability to transport the sample. The outcomes obtained after transferring rabbit embryos at different developmental stages indicate that morula is the ideal stage for rabbit embryo recovery and transfer. Thus, an oviductal embryo transfer is required, justifying the surgical procedure. Furthermore, rabbit morulae are successfully vitrified and laparoscopically transferred, proving the effectiveness of the described techniques.

Assisted reproductive techniques (ARTs) are in continuous evaluation to improve outcomes and reduce the associated risks. This manuscript describes a minimally invasive embryo transfer procedure with an efficient cryopreservation protocol that allows the use of rabbits as an ideal animal model of human reproduction.

Assisted reproductive techniques (ARTs), such as in vitro embryo culture or embryo cryopreservation, affect natural development patterns with perinatal ...

Analysis of Transforming Growth Factor &#223; Family Cleavage Products Secreted Into the Blastocoele of Xenopus laevis Embryos

The two arms of the Transforming Growth Factor &#223; (Tgf&#223;) superfamily, represented by Tgf&#223;/Nodal or Bone morphogenetic protein (Bmp) ligands, respectively, play essential roles in embryonic development and adult homeostasis. Members of the Tgf&#223; family are made as inactive precursors that dimerize and fold within the endoplasmic reticulum. The precursor is subsequently cleaved into ligand and prodomain fragments. Although only the dimeric ligand can engage Tgf&#223; receptors and activate downstream signaling, there is growing recognition that the prodomain moiety contributes to ligand activity. This article describes a protocol that can be used to identify cleavage products generated during activation of Tgf&#223; precursor proteins. RNA encoding Tgf&#223; precursors are first microinjected into X. laevis embryos. The following day, cleavage products are collected from the blastocoele of&#160;gastrula stage embryos and analyzed on Western blots. This protocol can be completed relatively quickly, does not require expensive reagents and provides a source of concentrated Tgf&#223; cleavage products under physiologic conditions.

Analysis of Transforming Growth Factor &#223; Family Cleavage Products Secreted Into the Blastocoele of Xenopus laevis Embryos

When Transforming Growth Factor &#223; family precursor proteins are ectopically expressed in Xenopus laevis embryos, they dimerize, get cleaved and are secreted into the blastocoele, which begins at the late blastula to early gastrula stage. We describe a method for aspirating cleavage products from the blastocoele cavity for immunoblot analysis.

The two arms of the Transforming Growth Factor &#223; (Tgf&#223;) superfamily, represented by Tgf&#223;/Nodal or Bone morphogenetic protein (Bmp) ligands, ...

Protocol for Human Blastoids Modeling Blastocyst Development and Implantation

A model of the human blastocyst formed from stem cells (blastoid) would support scientific and medical advances. However, its predictive power will depend on its ability to efficiently, timely, and faithfully recapitulate the sequences of blastocyst development (morphogenesis, specification, patterning), and to form cells reflecting the blastocyst stage. Here we show that na&#239;ve human pluripotent stem cells cultured in PXGL conditions and then triply inhibited for the Hippo, transforming growth factor- &#946;, and extracellular signal-regulated kinase pathways efficiently undergo morphogenesis to form blastoids (&gt;70%). Matching with developmental timing (~4 days), blastoids unroll the blastocyst sequence of specification by producing analogs of the trophoblast and epiblast, followed by the formation of analogs of the primitive endoderm and the polar trophoblasts. This results in the formation of cells transcriptionally similar to the blastocyst (&gt;96%) and a minority of post-implantation analogs. Blastoids efficiently pattern by forming the embryonic-abembryonic axis marked by the maturation of the polar region (NR2F2+), which acquires the specific potential to directionally attach to hormonally stimulated endometrial cells, as in utero. Such a human blastoid is a scalable, versatile, and ethical model to study human development and implantation in vitro.

A protocol outlining the formation of human blastoids that efficiently, timely, and sequentially generate blastocyst-like cells.

A model of the human blastocyst formed from stem cells (blastoid) would support scientific and medical advances. However, its predictive power will depend ...

Morphometric Protocol for the Objective Assessment of Blastocyst Behavior During Vitrification and Warming Steps

This article describes the noninvasive method of blastocyst morphometry based on time-lapse microphotography for the accurate monitoring of a blastocyst&#39;s volume changing during individual phases before and after vitrification. The method can be useful in searching for the most optimal timing of blastocyst exposure to different concentrations of cryoprotectants by observing blastocyst shrinkage and re-expansion in different pre- and post-vitrification phases. With this methodology, the blastocyst vitrification protocol can be optimized. For a better demonstration of the usefulness of this morphometric method, two different blastocyst preparation protocols for vitrification are compared; one with using an artificial blastocoel collapsing and one without this intervention before vitrification. Both blastocysts&#39; volume changes are followed by time-lapse microphotography and measured by photo-editing software tools. The measurements are taken every 20 seconds in previtrification phases and every 5 minutes in the post-warming period. The changes of the blastocyst dimensions per time unit are presented graphically in line diagrams. The results show a long equilibration previtrification phase in which the intact blastocyst first shrinks and then slowly refills the blastocoel, entering vitrification with a fluid-filled blastocoel. The artificially collapsed blastocyst remains in its shrunken stage through the entire equilibration phase. During the vitrification phase, it also does not change its volume. Since the blastocyst morphometry shows a constant volume of the artificially collapsed blastocysts during the previtrification step, it seems that this stage could be shorter. The described protocol provides many additional comparative parameters of blastocyst behavior during and after cryopreservation on the basis of the speed and intensity of the volume changes, the number of partial blastocoel contractions or total blastocyst collapses, and the time to a total blastocoel re-expansion or the time to hatching.

Here we present a time-lapse morphometric protocol to follow the intensity of blastocyst shrinkage and re-expansion during previtrification interventions and post-warming recovery. The application of the protocol is possible in in vitro fertilization laboratories equipped with time-lapse microscopes and is recommended in the development of an optimal blastocyst vitrification method.

This article describes the noninvasive method of blastocyst morphometry based on time-lapse microphotography for the accurate monitoring of a ...

Bioengineering

Ablation of a Single Cell From Eight-cell Embryos of the Amphipod Crustacean Parhyale hawaiensis

The amphipod Parhyale hawaiensis is a small crustacean found in intertidal marine habitats worldwide. Over the past decade, Parhyale has emerged as a promising model organism for laboratory studies of development, providing a useful outgroup comparison to the well studied arthropod model organism Drosophila melanogaster. In contrast to the syncytial cleavages of Drosophila, the early cleavages of Parhyale are holoblastic. Fate mapping using tracer dyes injected into early blastomeres have shown that all three germ layers and the germ line are established by the eight-cell stage. At this stage, three blastomeres are fated to give rise to the ectoderm, three are fated to give rise to the mesoderm, and the remaining two blastomeres are the precursors of the endoderm and germ line respectively. However, blastomere ablation experiments have shown that Parhyale embryos also possess significant regulatory capabilities, such that the fates of blastomeres ablated at the eight-cell stage can be taken over by the descendants of some of the remaining blastomeres. Blastomere ablation has previously been described by one of two methods: injection and subsequent activation of phototoxic dyes or manual ablation. However, photoablation kills blastomeres but does not remove the dead cell body from the embryo. Complete physical removal of specific blastomeres may therefore be a preferred method of ablation for some applications. Here we present a protocol for manual removal of single blastomeres from the eight-cell stage of Parhyale embryos, illustrating the instruments and manual procedures necessary for complete removal of the cell body while keeping the remaining blastomeres alive and intact. This protocol can be applied to any Parhyale cell at the eight-cell stage, or to blastomeres of other early cleavage stages. In addition, in principle this protocol could be applicable to early cleavage stage embryos of other holoblastically cleaving marine invertebrates.

Ablation of a Single Cell From Eight-cell Embryos of the Amphipod Crustacean Parhyale hawaiensis

The amphipod Parhyale hawaiensis is a promising model organism for studies of crustacean embryology and comparative arthropod development and evolution. This protocol describes a method for manual removal of single blastomeres from early cleavage stage embryos of Parhyale.

The amphipod Parhyale hawaiensis is a small crustacean found in intertidal marine habitats worldwide. Over the past decade, Parhyale has emerged as a ...

Biology

Single Cell Collection of Trophoblast Cells in Peri-implantation Stage Human Embryos

Human implantation, the apposition and adhesion to the uterine surface epithelia and subsequent invasion of the blastocyst into the maternal decidua, is a critical yet enigmatic biological event that has been historically difficult to study due to technical and ethical limitations. Implantation is initiated by the development of the trophectoderm to early trophoblast and subsequent differentiation into distinct trophoblast sublineages. Aberrant early trophoblast differentiation may lead to implantation failure, placental pathologies, fetal abnormalities, and miscarriage. Recently, methods have been developed to allow human embryos to grow until day 13 post-fertilization in vitro in the absence of maternal tissues, a time-period that encompasses the implantation period in humans. This has given researchers the opportunity to investigate human implantation and recapitulate the dynamics of trophoblast differentiation during this critical period without confounding maternal influences and avoiding inherent obstacles to study early embryo differentiation events in vivo. To characterize different trophoblast sublineages during implantation, we have adopted existing two-dimensional (2D) extended culture methods and developed a procedure to enzymatically digest and isolate different types of trophoblast cells for downstream assays. Embryos cultured in 2D conditions have a relatively flattened morphology and may be suboptimal in modeling in vivo three-dimensional (3D) embryonic architectures. However, trophoblast differentiation seems to be less affected as demonstrated by anticipated morphology and gene expression changes over the course of extended culture. Different trophoblast sublineages, including cytotrophoblast, syncytiotrophoblast and migratory trophoblast can be separated by size, location, and temporal emergence, and used for further characterization or experimentation. Investigation of these early trophoblast cells may be instrumental in understanding human implantation, treating common placental pathologies, and mitigating the incidence of pregnancy loss.

Here, we describe a method for warming vitrified human blastocysts, culturing them through the implantation period in vitro, digesting them into single cells and collecting early trophoblast cells for further investigation.

Human implantation, the apposition and adhesion to the uterine surface epithelia and subsequent invasion of the blastocyst into the maternal decidua, is a ...

Follicular Viability Assessment in Vitrified Human Ovarian Cortex Tissues

Vitrification of Ovarian Cortex Tissue to Achieve a Glassy State of Aggregation

Ovarian tissue cryopreservation (OTC) is an important option for fertility preservation. For patients whose gonadotoxic treatments cannot be postponed or for pre-pubertal girls, it is often the only option for fertility protection. Cryopreservation can be performed either by vitrification or by slow freezing. Slow freezing is currently the standard approach. An increasing number of studies indicate that vitrification can replace slow freezing in the state-of-the-art in vitro fertilization (IVF) laboratories, significantly improving thawing survival rates and simplifying the technical aspects of cryopreservation. A metal grid-based, high-throughput protocol for rapid vitrification of ovarian cortex tissue, suitable for clinical routine, is described. The sterilization of metal grids and liquid nitrogen ensures high quality, meeting good manufacturing practice (GMP) standards. Vitrification was conducted to ensure ultra-rapid cooling rates. Instead of slowly thawing, samples were rapidly warmed. To assess follicular viability, calcein staining was performed both prior to cryopreservation and after rapid warming. The successful application of vitrification and rapid warming using metal grids is reported. No significant differences in follicular viability were observed prior to vitrification and after rapid warming. These results substantiate the high capacity of tissue vitrification for clinical routine applications as a potential substitute for the widely used slow-freezing method.

Author Spotlight: Advancing Fertility Preservation in Young Female Cancer Patients Through Ovarian Tissue Vitrification and In Vitro Follicular Growth

A protocol for the vitrification of ovarian tissue, as an alternative cryopreservation method to the widely used slow freezing protocol, is presented.

Ovarian tissue cryopreservation (OTC) is an important option for fertility preservation. For patients whose gonadotoxic treatments cannot be postponed or ...

Human Blastocyst Biopsy and Vitrification

Summary

Explore More Videos

Chapters in this video

Isolation and Quantitative Immunocytochemical Characterization of Primary Myogenic Cells and Fibroblasts from Human Skeletal Muscle

Modified MicroSecure Vitrification: A Safe, Simple and Highly Effective Cryopreservation Procedure for Human Blastocysts

Chromosome Screening of Human Preimplantation Embryos by Using Spent Culture Medium: Sample Collection and Chromosomal Ploidy Analysis

Minimally Invasive Embryo Transfer and Embryo Vitrification at the Optimal Embryo Stage in Rabbit Model

Analysis of Transforming Growth Factor ß Family Cleavage Products Secreted Into the Blastocoele of Xenopus laevis Embryos

Protocol for Human Blastoids Modeling Blastocyst Development and Implantation

Morphometric Protocol for the Objective Assessment of Blastocyst Behavior During Vitrification and Warming Steps

Ablation of a Single Cell From Eight-cell Embryos of the Amphipod Crustacean Parhyale hawaiensis

Single Cell Collection of Trophoblast Cells in Peri-implantation Stage Human Embryos

Vitrification of Ovarian Cortex Tissue to Achieve a Glassy State of Aggregation