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

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.......

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.......

Materials

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