Name: morphometric protocol for the objective assessment of blastocyst behavior during vitrification and warming steps
Uploaded: 2019-02-28T13:30:00.000+00:00
Duration: 8 min 28 s
Description: Watch this Scientific Journal Video about Morphometric Protocol for the Objective Assessment of Blastocyst Behavior During Vitrification and Warming Steps at JoVE.com

This work is a part of research program P3-0327 and research project J3-7177, founded by the Slovenian Research Foundation.

All methods described here have been approved by the National Medical Ethics Committee on 19 April 2016 (No. 0120&#8211;204/2016&#8211;2).
1. Set-up of the Microscope Recording System
<ol>
	<li>Turn off the heating plate of the microscope.</li>
	<li>Before any treatment, take a snapshot of a blastocyst under a camera-equipped inverted microscope. Remember to note the magnification at which the blastocyst was recorded.</li>
</ol>
2. Selection and Prepreparation of Blastocysts for Vitrification
<ol>
	<li>Select only fully expanded day-5 or day-6 blastocysts for cryopreservation with at least a few inner cell mass (ICM) cells and cohesive trophectoderm.</li>
	<li>For laser-treated blastocysts with a collapsed blastocoel, subject a blastocyst to a short laser pulse (0.4 - 0.7 ms) with a hole diameter ranging from 4.3 to 9.4 &#181;m, tangentially directed at the zona pellucida and at a junction of two adjacent trophectodermal cells. Allow the blastocyst to fully or partially collapse for 5 min.</li>
	<li>For untreated blastocysts with an intact blastocoel, perform no particular treatment before vitrification. 
	Note: These blastocysts are considered intact.</li>
</ol>
3. Preparation of Cryoprotectant and a Petri Dish for the Equilibration Phase
<ol>
	<li>Use pithe pipette for oocyte denudation (diameter 135 &#181;m) to aseptically fill equilibration media (M-199 HEPES-buffered medium with 7.5% DMSO, 7.5% ethylene glycol, 20% DSS) into the holes of the microdroplet area of a 9-well Petri dish specially designed for time-lapse microscopy and recording. 
	Note: Oocyte denudation is a process of removing cumulus cells surrounding the oocyte. The use of a pipette for oocyte denudation will help to avoid the creation of air bubbles.</li>
	<li>Overlay the microdroplet area with 30 &#181;L of equilibration media.</li>
</ol>
4. Transfer of the Blastocyst in Equilibration Solution
<ol>
	<li>Submerge a laser-treated or intact blastocyst into the equilibration medium to the bottom of the microdroplet area of the microdroplet dish for 10 min at room temperature (equilibration phase).</li>
	<li>Start a 10-min countdown as soon as the blastocyst is transferred to the equilibration medium.</li>
</ol>
5. Recording of the Blastocyst at the Equilibration Phase
<ol>
	<li>Transfer the microdroplet dish with the blastocyst to a camera-equipped microscope. Use the same magnification as previously for taking a snapshot of the same blastocyst. Position the microdroplet dish so that the blastocyst is positioned approximately in the center of the recording.</li>
	<li>Start recording with the microscope recording software. Note the countdown time at which the recording is started. 
	Note: See representative results of recordings of an intact (Figure 1, Video 1) and a collapsed blastocyst (Figure 2, Video 2) during the 10-min exposure to an equilibration solution.</li>
</ol>
6. Vitrification of the Blastocyst
<ol>
	<li>Aseptically dispense a 50-&#181;L drop of vitrification solution (M-199 HEPES-buffered medium with 15% DMSO, 15% ethylene glycol, 20% DSS, 0.5 M sucrose) in a Petri dish 2 min before the countdown ends.</li>
	<li>Stop recording when the 10-min countdown ends and quickly transfer the blastocyst to vitrification solution for 30 s at room temperature.</li>
	<li>Gently pipette the blastocyst at least 1x within the vitrification solution.</li>
	<li>Use a vitrification straw for loading the blastocyst. Load, seal, and plunge the vitrification straw into liquid nitrogen (LN) within 80 s, not exceeding 110 s after the initial exposure to the vitrification solution.</li>
</ol>
7. Video Editing of the Recorded Blastocyst During the Equilibration Phase
<ol>
	<li>Import a recorded video file into video editing software.</li>
	<li>Move the timeline slider to 20 s. Note the frame number at this time. 
	Note: This number will be used to decimate unnecessary video frames. Only frames every 20 s will be used.</li>
	<li>Click the Video tab, then Frame rate.... Under Frame rate conversion, check the Decimate by field and enter the previously noted frame number. Confirm by clicking OK. Click File &#8594; Export &#8594; Image sequence.</li>
	<li>Choose JPEG as the output format, set the directory to hold the saved sequence of images, and click OK. 
	Note: This will create a directory with up to 30 images of the recorded blastocyst in sequence during the equilibration phase of vitrification.</li>
</ol>
8. Measurements of the Blastocyst&#8217;s Cross-sectional Area from Images Created During the Equilibration Phase
<ol>
	<li>Open the video analysis software. Click the Window tab and select Timeline.</li>
	<li>In the Timeline pane, click on the film strip sign and choose Add Media... to add an image of the blastocyst before the equilibration phase of vitrification&#8212;and before the laser intervention, if there was any. 
	Note: This image will show the blastocyst at its most expanded state and its cross-sectional area will serve as a reference.</li>
	<li>Add the image sequence of the corresponding blastocyst generated previously with video editing software (of the equilibration phase of vitrification).</li>
	<li>Go to Image &#8594; Analysis &#8594; Select Data Points &#8594; Custom to open the Select data points window.</li>
	<li>Deselect all data points, then select only Area, and confirm by clicking OK.</li>
	<li>Move the timeline slider in the Timeline window to the first image.</li>
	<li>Click the Window tab and choose Tools. 
	Note: This will activate the Tools panel on the left side.</li>
	<li>Choose the Quick Selection Tool.</li>
	<li>Position the tool marker inside the blastocyst to touch the edge of the blastocyst. Outline the blastocyst by placing the tool marker on the blastocyst&#8217;s outer edge, clicking and holding the left mouse button, and dragging the tool marker along the blastocyst&#8217;s outer edge until the whole blastocyst&#8217;s cross-sectional area is selected. 
	Note: Zona pellucida is not a part of the measurement, so it must be excluded (Figure 5).</li>
	<li>In the Timeline window, click the Measurement Log and press the Record Measurements button. 
	Note: This will record the selected area.</li>
	<li>Return back to the Timeline, right-click on the image, and choose Deselect.</li>
	<li>Move the timeline slider to the next image and click on the corresponding tile beneath it.</li>
	<li>Outline the blastocyst in the new image with the Quick Selection Tool. Repeat the measurement.</li>
	<li>Repeat this procedure (steps 8.9 - 8.13) image by image until all the images&#8217; cross-sectional areas are measured and recorded.</li>
	<li>In the end, go to the Measurements Log, select all the measurements under the Area variable, and export them in a .txt file. 
	Note: The units of the cross-sectional area are square pixels.</li>
</ol>
9. Editing of the Data File with the Recorded Blastocyst&#8217;s Cross-sectional Areas from Images Created During the Equilibration Phase
<ol>
	<li>Transfer the data of the recorded blastocyst&#8217;s cross-sectional areas from the .txt file to a spreadsheet editor.</li>
	<li>Use the first cross-sectional area value as reference value 1 and express (transform) all the other values to be a fraction of that reference value.</li>
	<li>Create a new variable Area_r and paste the transcoded area values (except the reference value) under it.</li>
	<li>Next to the Area_r variable, create a Time variable and add the first time value. 
	Note: The first time value represents the time which passed from the moment the blastocyst was exposed to the equilibration solution to the onset of recording under a camera-equipped microscope.</li>
	<li>Calculate each next consecutive time value by adding 20 s to the previous time value. 
	Note: This is the time interval used at decimating unnecessary video frames created during the recording.</li>
</ol>
10. Warming of the Blastocyst
<ol>
	<li>Prepare media for warming.
	<ol>
		<li>A day before warming the blastocyst, aseptically dispense three 150-&#181;L drops of recovery medium in a 4-well dish. Also, aseptically dispense recovery medium in a microdroplet 9-well dish specially designed for time-lapse microscopy and recording. Cover the recovery medium with paraffin oil and preincubate it at 37 &#176;C with 6% CO2 and 5% O2. 
		Note: Recovery medium is a cultivation medium for blastocyst-stage embryos with added human serum albumin (HSA). The HSA in the recovery medium should be 12 mg/mL. To fill the holes in the 9-well dish, use a pipette for oocyte denudation to avoid the creation of air bubbles, then overlay the microdroplet area with 30 &#181;L of recovery medium.</li>
		<li>On the day of warming the blastocyst, aseptically dispense 500 &#181;L of thawing solution (TS) in a single well of a 4-well dish and allow it to warm to 37 &#176;C in an incubator without CO2.</li>
		<li>At room temperature, aseptically dispense one 50-&#181;L drop of dilution solution (DS) and two 50-&#181;L drops of washing solution (WS) in a sterile Petri dish. Cover the DS and the WS1 and WS2 drops with paraffin oil. 
		Note: Instead of a Petri dish, a 4-well dish can also be used.</li>
	</ol>
	</li>
	<li>Warm the blastocyst.
	<ol>
		<li>Identify the HSV straw with the vitrified blastocyst to be removed from LN storage and quickly transfer the straw to an LN-filled portable reservoir in preparation for the warming procedure.</li>
		<li>Lift the straw with forceps, just enough to expose the colored handling rod. Use a self-adjusting wire stripper to cut the straw at the height of the colored handling rod.</li>
		<li>Grab the handling rod and extract it from the straw with a swift, yet controlled movement, and immediately plunge the curved spatula of the handling rod into the 37 &#176;C TS.</li>
		<li>Gently swirl the handling rod to detach the blastocyst and leave it for a total of 1 min in TS.</li>
		<li>At room temperature, transfer the blastocyst consecutively to DS, WS1, and WS2 for 4 min in each medium.</li>
		<li>After the warming procedure, transfer the blastocyst to a 4-well dish with recovery medium and wash it in all three drops by gently pipetting it. 
		Note: The washing is carried out in approximately 1 min.</li>
		<li>Transfer the blastocyst to the time-lapse 9-well dish with recovery medium. Place the 9-well dish under a time-lapse camera in an incubator (at 37 &#176;C with 6% CO2 and 5% O2). 
		Note: Thre warming procedure, which continues with placing the 9-well dish under a time-lapse camera in an incubator, lasts approximately 17 min.</li>
	</ol>
	</li>
</ol>
11. Time-lapse Recording of the Blastocyst Re-expansion During Recovery Post-warming
<ol>
	<li>Run time-lapse recording software.</li>
	<li>Choose a recording camera.</li>
	<li>Press the Live mode button.</li>
	<li>Place the mouse cursor on the image and use the scroll button to enlarge it. Click and hold the left mouse button and move the cursor to place the well with the blastocyst in the middle of the screen.</li>
	<li>Under Focusing, use the up-down green arrows to focus the recording plane of the blastocyst. Under Light intensity, set the light intensity.</li>
	<li>Click the Microscope parameters button to set the Exposure time and Gamma.</li>
	<li>Press Close live mode.</li>
	<li>Press the Start project button and enter the project data, select the culture dish type (3 x 3 or 4 x 4) and uncheck all positions except the one to be recorded.</li>
	<li>Set the capture timing to take a picture every 5 min.</li>
	<li>Start recording by pressing the Approve button. Record at least 150 min.</li>
	<li>Stop recording by pressing the Stop project button. 
	Note: See the representative results of the recording of an intact (Figure 3) and a collapsed blastocyst (Figure 4) during the post-warming recovery period.</li>
</ol>
12. Video Editing of the Recorded Blastocyst During the Re-expansion Post-warming
<ol>
	<li>Go to the project folder and locate recorded images that are roughly 80 KB in size.</li>
	<li>Create another folder and transfer these images into it. Rename the images in numbers according to the time created. Import them into the video editing software as an image sequence.</li>
	<li>Go to Video tab &#8594; Filter &#8594; Add and select Null transform filter.</li>
	<li>Click the Cropping button on the right side and crop the image, leaving visible only the field with the recorded blastocyst. Confirm with OK and again with OK.</li>
	<li>Click File &#8594; Export &#8594; Image sequence. Choose JPEG as the output format, set the directory to hold the saved sequence of images, and click OK. 
	Note: This will create resized (cropped) images prepared for further measurements in the video analysis software.</li>
</ol>
13. Setting of a Measurement Scale in Video Analysis Software for the Images Created with the Time-lapse Recording Software
<ol>
	<li>Run the Analyzer of the time-lapse recording software.</li>
	<li>Open the project with the recorded blastocyst.</li>
	<li>Click the Analyze button on the field with the recorded blastocyst. 
	Note: A new Analyze window will open. Click on the Measurement button to show the measurement tool.</li>
	<li>Use the measurement tool to measure the width of the whole image.</li>
	<li>Right-click on the image and save it. In the properties, check the image&#8217;s width in pixels.</li>
	<li>Divide the image&#8217;s actual width with its width in pixels. 
	Note: This calculates the actual length of a single pixel in this image.</li>
	<li>Run the video analysis software.</li>
	<li>Click the Window tab and select Timeline.</li>
	<li>In the Timeline pane, click on the film strip sign and choose Add Media... to add the image sequence of the post-warm re-expanding blastocyst.</li>
	<li>Click Image &#8594; Analysis &#8594; Set Measurement Scale &#8594; Custom to open the Measurement Scale window. For Pixel length, enter 1; for Logical length, enter the previously calculated actual length of a pixel (step 13.6); for Logical units, enter &#181;m. Save the preset.</li>
</ol>
14. Measurements of the Blastocyst&#8217;s Cross-sectional Area from Images Created During the Re-expansion Post-warm
<ol>
	<li>Once the Measurement scale is set and the image sequence imported, measure the blastocyst&#8217;s cross-sectional areas the same way as described in step 8, with the only difference being that the measured cross-sectional areas in these measurements have units in &#181;m2.</li>
</ol>
15. Editing of the Data File with the Recorded Blastocyst&#8217;s Cross-sectional Areas from Images Created during the Re-expansion Post-warm
<ol>
	<li>Transfer the data of the recorded blastocyst&#8217;s cross-sectional areas from the .txt file to a spreadsheet editor.</li>
	<li>Create a Time variable next to the Area variable and add the first time value. 
	Note: In this case, the first time value is set to 0 minutes, which is the onset of the time-lapse recording. Each next consecutive time value is calculated by adding 5 minutes to the previous time value. Five minutes is the time interval used between two consecutive time-lapse recordings.</li>
</ol>
16. Creation of a Line Diagram
<ol>
	<li>Import the spreadsheet data file in statistical analysis software for creating a time plot of the blastocyst&#8217;s cross-sectional area changes in time during the equilibration phase of the vitrification or re-expansion post-warming.</li>
</ol>

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The authors have nothing to disclose.

The protocol for the observation of blastocyst morphodynamics during and after cryopreservation can also be carried out by using similar instruments and software tools from other manufacturers. Time-lapse systems adjusted for embryology allow the continuous monitoring of embryo development. The purpose of this work was to introduce the quantification of blastocyst behavior during the preparation of blastocysts for vitrification and after their warming. This was done by the objective measurement of changes in the morpholo...

Cryopreservation of human preimplantation embryos from the in vitro fertilization program (IVF) is nowadays a routine practice in most IVF laboratories. The slow embryo freezing method began to be clinically used in 1985, with the introduction of specific cryoprotectants and computer-controlled freezers, which enabled the controlled cooling of embryos down to -7 &#176;C, when ice nucleation (seeding) was induced in the surrounding cryoprotective medium1. By continuous cooling, the ice crystals would grow, causing the hyperosmolality of the remaining liquid fraction and, consequently, the dehydration and shrinkage of the embryonic cells. At -30 &#176;C or -80 &#176;C, the embryos would be plunged into the liquid nitrogen for longer storage. What was happening with the embryos or oocytes during cooling could be observed only by specially adapted cryomicroscopes, which helped to improve cryopreservation protocols2. The freezing of blastocysts, a higher-volume and more fluid-contained embryonic stage, gave less promising clinical results in those days3.
The breakthrough in the cryopreservation of the blastocyst was the introduction of the vitrification method, in which the dehydration of the cells took place before cooling by using high concentrations of cryoprotectants4. The removal of the fluid from the blastocoel before vitrification can also be achieved by making a mechanical opening between two trophectoderm cells5. Although the immediate blastocyst survival rate after vitrification and warming is above 90%, and the clinical outcome following the transfer of vitrified/warmed blastocyst into the uterine cavity is almost comparable to results after the transfer of fresh embryos, this cryopreservation method has not yet been standardized6,7. Vitrification protocols vary according to (a) the type and concentration of the cryoprotectants, (b) the number of previtrification steps, (c) the duration of individual steps, (d) the use of an artificial blastocoel collapsing before vitrification or not, (e) collapsing methods, (f) the blastocyst expansion stage, and (g) the equilibration/vitrification temperature at which the embryos should be vitrified8. Since cryoprotectants can be toxic for cells, the blastocyst exposure time to these solutions has to be well defined. However, some cryopreservation media manufacturers allow very flexible protocols.
The interest of scientists is usually focused on studying the blastocyst re-expansion ability with the aim to find new biomarkers with a better prediction of implantation6,9,10. How human blastocysts dehydrate at different steps of adding cryoprotectants before vitrification and what happens with blastocysts after vitrification and warming, when cryoprotectants have to be removed from the cells, and how the blastocysts rehydrate and re-expand after warming, is not well described nor understood. The development of a methodology for the objective and quantified monitoring of blastocyst behavior during different cryopreservation steps is, thus, rationale.
With time-lapse microscopes of various manufacturers, it is now possible to monitor the behavior of the blastocyst in pre- and post-vitrification phases. By including additional computer tools, measurements of their size (morphometry) can also be performed. By measuring the decrease or increase in embryo size at a given time, it is possible to objectify the evaluation of morphodynamics of embryos during dehydration and rehydration.

<table><tbody><tr><td>Inverted microscope Eclipse TE2000-U&amp;nbsp;</td><td>Nikon, Japan</td><td>/</td><td></td></tr><tr><td>Saturn 5 Laser System</td><td>Research Instruments, Origio, Denmark</td><td>/</td><td></td></tr><tr><td>Digital camera DC1</td><td>Research Instruments, Origio, Denmark</td><td>/</td><td></td></tr><tr><td>Digital camera DC2</td><td>Research Instruments, Origio, Denmark</td><td>/</td><td></td></tr><tr><td>Cronus 3.7</td><td>Research Instruments, Origio, Denmark</td><td>/</td><td>microscope recording software</td></tr><tr><td>Incubator with 6% CO2, 5% O2</td><td>Binder, Germany</td><td>/</td><td></td></tr><tr><td>Primo Vision microscope</td><td>Vitrolife, Sweden</td><td>16600</td><td></td></tr><tr><td>Primo Vision Capture software</td><td>Vitrolife, Sweden</td><td>16608</td><td>time-lapse recording software</td></tr><tr><td>Adobe Photoshop CS6 Extended software</td><td>Adobe Systems Incorporated, USA</td><td>/</td><td>video analysis software</td></tr><tr><td>VirtualDub&amp;nbsp;</td><td>Avery Lee</td><td>/</td><td>video editing software</td></tr><tr><td>Microsoft Office Excell&amp;nbsp;</td><td>Microsoft, USA</td><td>/</td><td>spreadsheet editor</td></tr><tr><td>PrimoVision culture dish</td><td>Vitrolife, Sweden</td><td>16604</td><td></td></tr><tr><td>G2-plus medium</td><td>Vitrolife, Sweden</td><td>10132</td><td>cultivation medium for blastocyst stage embryos</td></tr><tr><td>Human Serum Albumins</td><td>Vitrolife, Sweden</td><td>10064</td><td></td></tr><tr><td>Paraffin oil</td><td>Vitrolife, Sweden</td><td>10029</td><td></td></tr><tr><td>Equilibration solution medium</td><td>Irvine Scientific, Ireland</td><td>90131</td><td></td></tr><tr><td>Vitrification solution medium</td><td>Irvine Scientific, Ireland</td><td>90132</td><td></td></tr><tr><td>Thawing solution medium</td><td>Irvine Scientific, Ireland</td><td>90134</td><td></td></tr><tr><td>Dilution solution medium</td><td>Irvine Scientific, Ireland</td><td>90135</td><td></td></tr><tr><td>Washing solution medium</td><td>Irvine Scientific, Ireland</td><td>90136</td><td></td></tr><tr><td>HSV Vitrification straws</td><td>CryoBio System, France</td><td>025246, 025249, 025250, 025248</td><td></td></tr><tr><td>Liquid nitrogen</td><td>/</td><td></td><td></td></tr><tr><td>Cryo vessel Biosafe 120 MD &amp;beta;</td><td>Cryotherm, Germany</td><td>229286</td><td></td></tr><tr><td>Cryo tank</td><td>Cryotherm, Germany</td><td></td><td></td></tr><tr><td>Forceps</td><td>/</td><td>/</td><td></td></tr><tr><td>Scisors</td><td>/</td><td>/</td><td></td></tr><tr><td>Pippete for blastocyst manipulation</td><td>Gynetics, Belgium</td><td>ID275/10</td><td>diameter 275 &amp;micro;m</td></tr><tr><td>Pipette for oocyte denudation</td><td>Vitromed, Germany</td><td>V-DEN-135</td><td>diameter 135 &amp;micro;m</td></tr><tr><td>Pipettor EZ-Grip</td><td>Research Instruments</td><td>7-72-2802</td><td></td></tr><tr><td>Digital interval timer Assistent</td><td>Glaswarenfabrik Karl Hecht</td><td>41977010</td><td></td></tr><tr><td>IBM SPSS Statistics 21</td><td>IBM, USA</td><td>/</td><td>statistical analysis software</td></tr><tr><td>Self adjusting wire stripper</td><td>Knipex, Germany</td><td>1262180</td><td></td></tr></tbody></table>

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.

In a demonstration, we showed blastocyst morphodynamics in only one previtrification and one post-warming phase. A difference in the blastocyst&#39;s volume at the end of the equilibration phase and at the beginning of recovering in culture medium showed the intensity of embryo shrinkage, which is, in fact, the intensity of embryo preservation against ice crystallization.
As it can be noticed from Figure 1</...

Watch this Scientific Journal Video about Morphometric Protocol for the Objective Assessment of Blastocyst Behavior During Vitrification and Warming Steps at JoVE.com

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

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 ...

morphometric-protocol-for-objective-assessment-blastocyst-behavior

Research

JoVE Journal

Bioengineering

8.6K Views.  University Medical Centre Maribor. 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.

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

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 ...

Developmental Biology

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 ...

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.

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 ...

Minimum Volume Vitrification of Immature Feline Oocytes

In wild animals&#8217; conservation programs, gamete banking is crucial to safeguard genetic resources of valuable individuals and rare species and to promote biodiversity preservation. In felids, most species are threatened with extinction, and domestic breeds are used as a model to increase the efficiency of protocols for germplasm banking. Among oocyte cryopreservation techniques, vitrification is more and more popular in human and veterinary assisted reproduction. Cryotop vitrification, which was at first developed for human oocytes and embryos, has demonstrated to be well-suited for cat oocytes. This method offers several advantages, such as the feasibility in field conditions and the speed of the procedure, which can be helpful when several samples need to be processed. However, the efficiency is strongly dependent on the operator&#8217;s skills, and intra- and inter-laboratory standardization are needed, as well as personnel training. This protocol describes minimum volume vitrification of immature feline oocytes on a commercial support in a step by step field-friendly protocol, from oocyte collection to warming. Following the protocol, preservation of oocyte integrity and viability at warming (as high as 90%) can be expected, although there is still room for improvement in post-warming maturation and embryonic development outcomes.

This manuscript describes a protocol for the minimum volume vitrification of immature cat oocytes with laboratory-made media on commercial supports. It covers every step from oocyte isolation from ex vivo gonads to vitrification and warming.

In wild animals&#8217; conservation programs, gamete banking is crucial to safeguard genetic resources of valuable individuals and rare species and to ...

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 ...

Cryopreservation of Mouse Embryos by Ethylene Glycol-Based Vitrification

Cryopreservation of mouse embryos is a technological basis that supports biomedical sciences, because many strains of mice have been produced by genetic modifications and the number is consistently increasing year by year. Its technical development started with slow freezing methods in the 1970s1, then followed by vitrification methods developed in the late 1980s2. Generally, the latter technique is advantageous in its quickness, simplicity, and high survivability of recovered embryos. However, the cryoprotectants contained are highly toxic and may affect subsequent embryo development. Therefore, the technique was not applicable to certain strains of mice, even when the solutions are cooled to 4&deg;C to mitigate the toxic effect during embryo handling. At the RIKEN BioResource Center, more than 5000 mouse strains with different genetic backgrounds and phenotypes are maintained3, and therefore we have optimized a vitrification technique with which we can cryopreserve embryos from many different strains of mice, with the benefits of high embryo survival after vitrifying and thawing (or liquefying, more precisely) at the ambient temperature4.
Here, we present a vitrification method for mouse embryos that has been successfully used at our center. The cryopreservation solution contains ethylene glycol instead of DMSO to minimize the toxicity to embryos5. It also contains Ficoll and sucrose for prevention of devitrification and osmotic adjustment, respectively. Embryos can be handled at room temperature and transferred into liquid nitrogen within 5 min. Because the original method was optimized for plastic straws as containers, we have slightly modified the protocol for cryotubes, which are more easily accessible in laboratories and more resistant to physical damages. We also describe the procedure of thawing vitrified embryos in detail because it is a critical step for efficient recovery of live mice. These methodologies would be helpful to researchers and technicians who need preservation of mouse strains for later use in a safe and cost-effective manner.

An ethylene glycol-based vitrification method for mouse embryos is described. It is advantageous to other methods in its simplicity and low embryonic toxicity, and therefore can be broadly applicable to many strains of mice, including inbred and gene-modified mice.

Cryopreservation of mouse embryos is a technological basis that supports biomedical sciences, because many strains of mice have been produced by genetic ...

Biology

Fertility Preservation Through Oocyte Vitrification: Clinical and Laboratory Perspectives

Preserving female fertility is crucial in a multifunctional healthcare system that takes care of patients' future quality of life. Oocyte cryopreservation is recognized by several international scientific societies as the gold standard for fertility preservation in postpubertal women, for both medical and non-medical indications. The main medical indications are oncologic diseases, gynecologic diseases such as severe endometriosis, systemic diseases compromising the ovarian reserve, and genetic conditions involving premature menopause. This paper describes the whole clinical and laboratory work-up of a fertility preservation treatment by outlining recommendations for objective and evidence-based counseling. Furthermore, it focuses on the effectiveness of the procedure and describes the most appropriate strategies to fully exploit the ovarian reserve and maximize the number of oocytes retrieved in the shortest possible time. The evaluation of the ovarian reserve, the definition of an ideal stimulation protocol, as well as oocyte retrieval, denudation, and vitrification procedures have been detailed along with approaches to maximize their efficacy, efficiency, and safety.

Oocyte cryopreservation is recognized by several international scientific societies as the gold standard for fertility preservation in postpubertal women. Appropriate clinical and laboratory strategies ensure maximum efficacy, efficiency, and safety of fertility preservation treatments.

Preserving female fertility is crucial in a multifunctional healthcare system that takes care of patients' future quality of life. Oocyte cryopreservation ...

Medicine

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 ...