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>Equipment</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 &#38; 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 0.2ml 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>Consumables</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Biopsy pipette</td>
			<td>RI</td>
			<td>7-71-30FB35720</td>
			<td>30&#181;m ID, flat 35&#176;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&#181;m stripping pipette tip</td>
		</tr>
		<tr>
			<td>Flexipet pipette</td>
			<td>Cook</td>
			<td>G46020</td>
			<td>300&#181;m stripping pipette tips</td>
		</tr>
		<tr>
			<td>Holding pipette</td>
			<td>RI</td>
			<td>7-71-IH35/20</td>
			<td>30&#181;m ID, flat 35&#176;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>60x15mm</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&#181;l</td>
		</tr>
		<tr>
			<td>Tubing Kit</td>
			<td>Provided by the genetic lab</td>
			<td></td>
			<td>PCR tubes (0.2mL), 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

21.9K 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 ...

Three-dimensional Quantification of Dendritic Spines from Pyramidal Neurons Derived from Human Induced Pluripotent Stem Cells

Dendritic spines are small protrusions that correspond to the post-synaptic compartments of excitatory synapses in the central nervous system. They are distributed along the dendrites. Their morphology is largely dependent on neuronal activity, and they are dynamic. Dendritic spines express glutamatergic receptors (AMPA and NMDA receptors) on their surface and at the levels of postsynaptic densities. Each spine allows the neuron to control its state and local activity independently. Spine morphologies have been extensively studied in glutamatergic pyramidal cells of the brain cortex, using both in vivo approaches and neuronal cultures obtained from rodent tissues. Neuropathological conditions can be associated to altered spine induction and maturation, as shown in rodent cultured neurons and one-dimensional quantitative analysis 1. The present study describes a protocol for the 3D quantitative analysis of spine morphologies using human cortical neurons derived from neural stem cells (late cortical progenitors). These cells were initially obtained from induced&#160;pluripotent stem cells. This protocol allows the analysis of spine morphologies at different culture periods, and with possible comparison between induced&#160;pluripotent stem cells obtained from control individuals with those obtained from patients with psychiatric diseases.

Dendritic spines of pyramidal neurons are the sites of most excitatory synapses in mammalian brain cortex. This method describes a 3D quantitative analysis of spine morphologies in human cortical pyramidal glutamatergic neurons derived from induced&#160;pluripotent stem cells.

Dendritic spines are small protrusions that correspond to the post-synaptic compartments of excitatory synapses in the central nervous system. They are ...

Culturing Human Pluripotent and Neural Stem Cells in an Enclosed Cell Culture System for Basic and Preclinical Research

This paper describes how to use a custom manufactured, commercially available enclosed cell culture system for basic and preclinical research. Biosafety cabinets (BSCs) and incubators have long been the standard for culturing and expanding cell lines for basic and preclinical research. However, as the focus of many stem cell laboratories shifts from basic research to clinical translation, additional requirements are needed of the cell culturing system. All processes must be well documented and have exceptional requirements for sterility and reproducibility. In traditional incubators, gas concentrations and temperatures widely fluctuate anytime the cells are removed for feeding, passaging, or other manipulations. Such interruptions contribute to an environment that is not the standard for cGMP and GLP guidelines. These interruptions must be minimized especially when cells are utilized for therapeutic purposes. The motivation to move from the standard BSC and incubator system to a closed system is that such interruptions can be made negligible. Closed systems provide a work space to feed and manipulate cell cultures and maintain them in a controlled environment where temperature and gas concentrations are consistent. This way, pluripotent and multipotent stem cells can be maintained at optimum health from the moment of their derivation all the way to their eventual use in therapy.

Here is a protocol to grow pluripotent stem cells (PSC) and neural stem cells (NSC) in an enclosed cell culture system that permits maximum sterility and reproducibility, replacing the traditional biosafety cabinet and incubator. This equipment meets clinical good manufacturing practice (cGMP) and clinical good lab practice (cGLP) guidelines.

This paper describes how to use a custom manufactured, commercially available enclosed cell culture system for basic and preclinical research. Biosafety ...

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 ...

Cell Aggregation Assays to Evaluate the Binding of the Drosophila Notch with Trans-Ligands and its Inhibition by Cis-Ligands

Notch signaling is an evolutionarily conserved cell-cell communication system used broadly in animal development and adult maintenance. Interaction of the Notch receptor with ligands from neighboring cells induces activation of the signaling pathway (trans-activation), while interaction with ligands from the same cell inhibits signaling (cis-inhibition). Proper balance between trans-activation and cis-inhibition helps establish optimal levels of Notch signaling in some contexts during animal development. Because of the overlapping expression domains of Notch and its ligands in many cell types and the existence of feedback mechanisms, studying the effects of a given post-translational modification on trans- versus cis-interactions of Notch and its ligands in vivo is difficult. Here, we describe a protocol for using Drosophila S2 cells in cell-aggregation assays to assess the effects of knocking down a Notch pathway modifier on the binding of Notch to each ligand in trans and in cis. S2 cells stably or transiently transfected with a Notch-expressing vector are mixed with cells expressing each Notch ligand (S2-Delta or S2-Serrate). Trans-binding between the receptor and ligands results in the formation of heterotypic cell aggregates and is measured in terms of the number of aggregates per mL composed of &#62;6 cells. To examine the inhibitory effect of cis-ligands, S2 cells co-expressing Notch and each ligand are mixed with S2-Delta or S2-Serrate cells and the number of aggregates is quantified as described above. The relative decrease in the number of aggregates due to the presence of cis-ligands provides a measure of cis-ligand-mediated inhibition of trans-binding. These straightforward assays can provide semi-quantitative data on the effects of genetic or pharmacological manipulations on the binding of Notch to its ligands, and can help deciphering the molecular mechanisms underlying the in vivo effects of such manipulations on Notch signaling.

Cell Aggregation Assays to Evaluate the Binding of the Drosophila Notch with Trans-Ligands and its Inhibition by Cis-Ligands

Complexity of in vivo systems makes it difficult to distinguish between the activation and inhibition of Notch receptor by trans- and cis-ligands, respectively. Here, we present a protocol based on in vitro cell-aggregation assays for qualitative and semi-quantitative evaluation of the binding of Drosophila Notch to trans-ligands vs cis-ligands.

Notch signaling is an evolutionarily conserved cell-cell communication system used broadly in animal development and adult maintenance. Interaction of the ...

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 ...

Isolation, Culture, Characterization, and Differentiation of Human Muscle Progenitor Cells from the Skeletal Muscle Biopsy Procedure

The use of primary human tissue and cells is ideal for the investigation of biological and physiological processes such as the skeletal muscle regenerative process. There are recognized challenges to working with human primary adult stem cells, particularly human muscle progenitor cells (hMPCs) derived from skeletal muscle biopsies, including low cell yield from collected tissue and a large degree of donor heterogeneity of growth and death parameters among cultures. While incorporating heterogeneity into experimental design requires a larger sample size to detect significant effects, it also allows us to identify mechanisms that underlie variability in hMPC&#160;expansion capacity, and thus allows us to better understand heterogeneity in skeletal muscle regeneration. Novel mechanisms that distinguish the expansion capacity of cultures have the potential to lead to the development of therapies to improve skeletal muscle regeneration.

We present techniques for isolating, culturing, characterizing, and differentiating human primary muscle progenitor cells (hMPCs) obtained from skeletal muscle biopsy tissue. hMPCs obtained and characterized through these methods can be used to subsequently address research questions related to human myogenesis and skeletal muscle regeneration.

The use of primary human tissue and cells is ideal for the investigation of biological and physiological processes such as the skeletal muscle ...

Determining Bile Duct Density in the Mouse Liver

Mouse is broadly used as a model organism to study biliary diseases. To evaluate the development and function of the biliary system, various techniques are used, including serum chemistry, histological analysis, and immunostaining for specific markers. Although these techniques can provide important information about the biliary system, they often do not present a full picture of bile duct (BD) developmental defects across the whole liver. This is in part due to the robust ability of the mouse liver to drain the bile even in animals with significant impairment in biliary development. Here we present a simple method to calculate the average number of BDs associated with each portal vein (PV) in sections covering all lobes of mutant/transgenic mice. In this method, livers are mounted and sectioned in a stereotypic manner to facilitate comparison among various genotypes and experimental conditions. BDs are identified via light microscopy of cytokeratin-stained cholangiocytes, and then counted and divided by the total number of PVs present in liver section. As an example, we show how this method can clearly distinguish between wild-type mice and a mouse model of Alagille syndrome. The method presented here cannot substitute for techniques that visualize the three-dimensional structure of the biliary tree. However, it offers an easy and direct way to quantitatively assess BD development and the degree of ductular reaction formation in mice.

We present a rather simple and sensitive method for accurate quantification of bile duct density in the mouse liver. This method can aid in determining the effects of genetic and environmental modifiers and the effectiveness of potential therapies in mouse models of biliary diseases.

Mouse is broadly used as a model organism to study biliary diseases. To evaluate the development and function of the biliary system, various techniques ...

Human Egg Maturity Assessment and Its Clinical Application

The optimal timing of intracytoplasmic sperm injection (ICSI) is of a serious concern for fertility programs because untimely sperm entry diminishes the egg&#39;s developmental competence. Presence of the first polar body (PB) together with the meiotic spindle indicates completion of the oocyte maturation and the egg&#39;s readiness for fertilization. In clinical practice, it is customary to assume that all oocytes displaying a PB are mature metaphase (MII) oocytes. However, PB extrusion precedes the formation of the bipolar MII spindle. This asynchrony makes the mere presence of PB an unreliable marker of oocyte maturity. Noninvasive spindle imaging using polarized light microscopy (PLM) allows quick and easy inspection of whether the PB-displaying oocyte actually reassembled a meiotic spindle prior to ICSI. Here, we present a standard protocol to perform human egg maturity assessment in the clinical laboratory. We also show how to optimize the time of ICSI with respect to the oocyte&#39;s developmental stage in order to prevent premature sperm injection of late-maturing oocytes. Using this approach, even immature oocytes extruding PB in vitro can be clinically utilized. Affirmation that MII spindle is present prior to sperm injection and individual adjustment of the time of ICSI is particularly important in poor prognosis in-vitro fertilization (IVF) cycles with a low number of oocytes available for fertilization.

We provide an outline of the clinical protocol for non-invasive assessment of human egg maturity using polarized light microscopy.

The optimal timing of intracytoplasmic sperm injection (ICSI) is of a serious concern for fertility programs because untimely sperm entry diminishes the ...

Generation of Multicellular Human Primary Endometrial Organoids

The human endometrium is one of the most hormonally responsive tissues in the body and is essential for the establishment of pregnancy. This tissue can also become diseased and cause morbidity and even death. Model systems to study human endometrial biology have been limited to in vitro culture systems of single cell types. In addition, the epithelial cells, one of the major cell types of the endometrium, do not propagate well or retain their physiological traits in culture, and thus our understanding of endometrial biology remains limited. We have generated, for the first time, endometrial organoids that consist of both epithelial and stromal cells of the human endometrium. These organoids do not require any exogenous scaffold materials and specifically organize so that epithelial cells encompass the spheroid-like structure and become polarized with stromal cells in the center that produce and secrete collagen. Estrogen, progesterone and androgen receptors are expressed in the epithelial and stromal cells and treatment with physiological levels of estrogen and testosterone promote the organization of the organoids. This new model system can be used to study normal endometrial biology and disease in ways that were not possible before.

A protocol to generate human primary endometrial organoids that consist of epithelial and stromal cells and retain characteristics of the native endometrial tissue is presented. This protocol describes methods from uterine tissue acquisition to the histologic processing of endometrial organoids.

The human endometrium is one of the most hormonally responsive tissues in the body and is essential for the establishment of pregnancy. This tissue can ...