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

Published: February 28th, 2019



1Department of Reproductive Medicine and Gynecological Endocrinology, 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.

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

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 °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....

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All methods described here have been approved by the National Medical Ethics Committee on 19 April 2016 (No. 0120–204/2016–2).

1. Set-up of the Microscope Recording System

  1. Turn off the heating plate of the microscope.
  2. 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.

2. Selection and Prepreparation of Blasto.......

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In a demonstration, we showed blastocyst morphodynamics in only one previtrification and one post-warming phase. A difference in the blastocyst'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

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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.......

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


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

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