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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

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.

Abstract

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.

Introduction

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....

Protocol

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.......

Representative Results

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

Discussion

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.......

Acknowledgements

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

....

Materials

NameCompanyCatalog NumberComments
Inverted microscope Eclipse TE2000-U Nikon, Japan/
Saturn 5 Laser SystemResearch Instruments, Origio, Denmark/
Digital camera DC1Research Instruments, Origio, Denmark/
Digital camera DC2Research Instruments, Origio, Denmark/
Cronus 3.7Research Instruments, Origio, Denmark/microscope recording software
Incubator with 6% CO2, 5% O2Binder, Germany/
Primo Vision microscopeVitrolife, Sweden16600
Primo Vision Capture softwareVitrolife, Sweden16608time-lapse recording software
Adobe Photoshop CS6 Extended softwareAdobe Systems Incorporated, USA/video analysis software
VirtualDub Avery Lee/video editing software
Microsoft Office Excell Microsoft, USA/spreadsheet editor
PrimoVision culture dishVitrolife, Sweden16604
G2-plus mediumVitrolife, Sweden10132cultivation medium for blastocyst stage embryos
Human Serum AlbuminsVitrolife, Sweden10064
Paraffin oilVitrolife, Sweden10029
Equilibration solution mediumIrvine Scientific, Ireland90131
Vitrification solution mediumIrvine Scientific, Ireland90132
Thawing solution mediumIrvine Scientific, Ireland90134
Dilution solution mediumIrvine Scientific, Ireland90135
Washing solution mediumIrvine Scientific, Ireland90136
HSV Vitrification strawsCryoBio System, France025246, 025249, 025250, 025248
Liquid nitrogen/
Cryo vessel Biosafe 120 MD βCryotherm, Germany229286
Cryo tankCryotherm, Germany
Forceps//
Scisors//
Pippete for blastocyst manipulationGynetics, BelgiumID275/10diameter 275 µm
Pipette for oocyte denudationVitromed, GermanyV-DEN-135diameter 135 µm
Pipettor EZ-GripResearch Instruments7-72-2802
Digital interval timer AssistentGlaswarenfabrik Karl Hecht41977010
IBM SPSS Statistics 21IBM, USA/statistical analysis software
Self adjusting wire stripperKnipex, Germany1262180

References

  1. Trounson, A., Mohr, L. Human pregnancy following cryopreservation, thawing and transfer of an eight-cell embryo. Nature. 305 (5936), 707-709 (1983).
  2. Leibo, S. P., McGrath, J. J., Cravalho, E. G.

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