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

This morphometric protocol allows monitoring of the intensity of blastocyst shrinkage and re-expansion during prior vitrification interventions and post-warming recovery and provides insights into the effectiveness of virus vitrification methods. The main advantage of this technique is that it can be applied to in vitro fertilization laboratories equipped with time-lapse microscopes and computers with advanced image editing software. On day five or six of expansion, subject a blastocyst with at least a few inner cell mass cells and a cohesive trophectoderm to a 0.4 to 0.7 millisecond laser pulse with a hole diameter ranging from 4.3 to 9.4 micrometers tangentially directed at the zona pellucida and the junction of two adjacent trophectodermal cells.

Then, allow the blastocoel to fully or partially collapse for five minutes. Next, use a tiny pipette to aseptically add equilibration medium into the holes of the microdroplet area of a nine-well petri dish, specially designed for time-lapse microscopy and recording. When all the of the medium has been added, overlay the microdroplet area with approximately 30 microliters of equilibration medium.

And submerge the laser-treated blastocyst in the equilibration medium in the bottom of one microdroplet well of the microdroplet dish. As soon as the blastocyst is transferred, start a countdown timer for 10 minutes. And transfer the microdroplet dish on the stage of a camera-equipped microscope.

Using a 200 X magnification, adjust the microdroplet dish so that the blastocyst is positioned in approximately the center of the recording window. Then, begin recording with the microscope recording software noting the countdown time at which the recording is started. To store the blastocyst, after loading the embryo into a vitrification straw, seal the straw, and plunge the straw into liquid nitrogen between 80 to 110 seconds of loading, before placing the blastocyst at negative 196 degrees Celsius.

To measure the cross-sectional area of the monitored blastocyst during the equilibration phase, position the quick selection tool marker inside the blastocyst until it touches the edge of the blastocyst and repeatedly click and hold the left mouse button to outline the outer edge of the blastocyst until the entire cross-sectional area of the blastocyst has been selected. Then, in the timeline window, click the measurement log and record measurements. For blastocyst re-expansion, quickly transfer the HSV straw with the vitrified blastocyst from storage to a liquid nitrogen-filled portable reservoir.

And use forceps to lift the straw just high 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. And extract the handling rod from the straw with a swift controlled movement.

Immediately plunge the curved spatula of the handling rod into a container of 37 degrees Celsius thawing solution. And gently swirl the handling rod to detach the blastocyst. After one minute, wash the blastocyst with sequential four minute immersions and dilution solution.

Washing solution one, and washing solution two. Next, transfer the warmed blastocyst into a four well dish containing fresh recovery medium and use a pipette to wash the blastocyst in all three drops of medium. After the last wash, transfer the blastocyst to a nine well time-lapse dish, containing recovery medium.

And place the nine well dish under a time-lapse camera in a 37 degree Celsius, 6%CO2 and 5%O2 incubator. Now, open the time-lapse recording software and select a recording camera. Select live mode and place the cursor on the image.

Scroll to enlarge the image and use the left mouse button to position the well containing the blastocyst in the middle of the screen. Under focusing, use the arrows to focus the recording plane of the blastocyst and under light intensity set the light intensity of the image. Click microscope parameters to set the exposure time and gamma, and click close live mode.

Click start project to enter the project data. Select the culture dish type and uncheck all of the positions except the one to be recorded. Then, set the capture timing to take a photo every five minutes and click approve to record the blastocyst re-expansion for at least 150 minutes.

In this representative, continuously monitored, intact embryo, the intact blastocyst did not collapse completely in the equilibration solution and re-expanded to about 70%of the original size, after 10 minutes of exposure to equilibration solution. In contrast, this artificially collapsed blastocyst was completely emptied of the blastocoel immediately after laser treatment and no significant re-expansion was observed during the 10 minutes of exposure to equilibration solution. Upon treatment with vitrification medium, containing a high concentration of cryoprotectants, this equilibrated intact blastocyst underwent a step-wise reduction of the blastocoel.

After warming, it re-expanded again upon exposure to recovery medium. This laser-pulsed blastocyst however, demonstrated an immediate collapse of the blastocoel upon treatment with the laser, that was not recovered during the equilibration or vitrification steps, bringing into question the necessity of such a long equilibration phase for collapsed blastocysts. The protocol also gives insight into the ability of blastocysts to recover blastocoel after warming.

For example, blastocoel re-expansion after warming can be linear, or can be interrupted with several bigger or smaller contractions. While attempting this procedure it's important to remember that the embryos must remain at the bottom of the petri dish during the recording, for as little time as possible. Don't forget that high concentrations of cryoprotectants can be toxic for embryos.

After each development, this technique paved the way for researchers in the field of cryobiology, to explore the effectiveness of cryopreservation protocols in an in vitro fertilization program.