Name: morphometric protocol for the objective assessment of blastocyst behavior during vitrification and warming steps
Uploaded: 2019-02-28T13:30:00
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 Blasto...

<ol>
	<li>Trounson, A., Mohr, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+pregnancy+following+cryopreservation,+thawing+and+transfer+of+an+eight-cell+embryo.">Human pregnancy following cryopreservation, thawing and transfer of an eight-cell embryo.</a> Nature. 305 (5936), 707-709 (1983).</li><li>Leibo, S. P., McGrath, J. J., Cravalho, E. 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A., Choi, A., Orief, Y., Agarwal, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Factors+affecting+the+outcome+of+human+blastocyst+vitrification.">Factors affecting the outcome of human blastocyst vitrification.</a> Reproductive Biology and Endocrinology. 16 (7), 99 (2009).</li><li>Ebner, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphokinetics+of+vitrified+and+warmed+blastocysts+predicts+implantation+potential.">Morphokinetics of vitrified and warmed blastocysts predicts implantation potential.</a> Journal of Assisted Reproduction and Genetics. 34 (2), 239-244 (2017).</li><li>Coello, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+the+morphological+dynamics+of+blastocysts+after+vitrification/warming:+defining+new+predictive+variables+of+implantation.">Analysis of the morphological dynamics of blastocysts after vitrification/warming: defining new predictive variables of implantation.</a> Fertility Sterility. 108 (4), 659-666 (2017).</li><li>Huang, T. T. F., Chinn, K., Kosasa, T., Ahn, H. J., Kessel, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphokinetics+of+human+blastocyst+expansion+in+vitro.">Morphokinetics of human blastocyst expansion in vitro.</a> Reproductive Biomedicine Online. 33, 659-667 (2016).</li><li>Vanderzwalmen, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lower+intracellular+concentration+of+cryoprotectants+after+vitrification+than+after+slow+freezing+despite+exposure+to+higher+concentration+of+cryoprotectant+solutions.">Lower intracellular concentration of cryoprotectants after vitrification than after slow freezing despite exposure to higher concentration of cryoprotectant solutions.</a> Human Reproduction. 28, 2101-2110 (2013).</li></ol>

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&#160;</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&#160;</td>
			<td>Avery Lee</td>
			<td>/</td>
			<td>video editing software</td>
		</tr>
		<tr>
			<td>Microsoft Office Excell&#160;</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 &#946;</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 &#181;m</td>
		</tr>
		<tr>
			<td>Pipette for oocyte denudation</td>
			<td>Vitromed, Germany</td>
			<td>V-DEN-135</td>
			<td>diameter 135 &#181;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 ...

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