Modified MicroSecure Vitrification: A Safe, Simple and Highly Effective Cryopreservation Procedure for Human Blastocysts

MicroSecure Vitrification was developed as a non-commercial, aseptic closed vitrification device system that is compliant with FDA good manufacturing and tissue-handling practices. Due to the withdrawal of hydrophobic plugged embryo straws from the industry, the vitrification procedure was modified to include an inner seal before the standard internal cotton plug.

The overall goal of the MicroSecure embryo vitrification method is to provide a safe, secure, simple yet effective aseptic closed vitrification procedure for human embryos. This method was developed to minimize the quality control variables in the vitrification of human embryos, thus ensuring reliable and reproducible success. The main advantage of this technique is that it strives to eliminate technical variations between biologists and laboratories.

Our technique is so simple to perform that we find the biggest challenge for our trainees is learning how to pipette embryos without losing them. The idea behind our method was to eliminate the need for specialized devices by adapting two commonly used FDA-approved items. An internally labeled ionomeric storage straw, and a sterile flexipet.

Visual demonstration of this technique is extremely helpful as it highlights the basic tips that ensure its successful application. Before beginning the procedure, place the sterile 0.3ml semen straw onto the teflon surface of the electrode of a basic tabletop impulse sealer, just in front of the plug, to create and internal seal. Depress the handle to activate the heat, releasing the handle when the red light is off and a beep is heard.

Then push the cotton plug forward against the inner seal before completing the setup. For cryo-dilution and blastocyst loading of the samples, remove the patient culture dish from the incubator and confirm all specimen identifications with a secondary source. Under a stereomicroscope, use a vitrification flexipet, referred to as a vit tip, to move the blastocysts into individual holding droplets in the top row of a corresponding cryo dish.

Next, prefill the flexipet with 3 microliters of V1 solution and dispense half of the contents onto the first embryo. Aspirate the embryo and transfer it to the first V1 wash droplet. Then pipette the embryo two to three times around the perimeter of the droplet, and clear the residual pipette contents outside of the droplet onto the dish surface.

Then move the blastocyst to an individual V1 holding drop and preload the vit tip with next V2 solution before pipetting the additional embryos with a new vit tip. After washing all of the blastocysts in the V2 and V3 droplets as just demonstrated, clear the residual solution and bubbles from the vit tip outside of the V3 holding droplet, and completely fill the tip with clean V3 solution from around the first embryo. Expel approximately 1 microliter of the volume, and aspirate the blastocyst and the remaining V3 solution completely into the vit tip.

Now, remove the tip from the pipette, and use a sterile gauze pad to dry the residual V3 solution from the outer surface of the vit tip. Drying the outer surface of the tip is critical and does not incur any additional risk of embryo loss. Then insert the tip end first into the open end of the pre-sealed straw and invert the straw, label end down.

Observe the tip at the inner plug. Leave a 1 cm airspace on the open end and then safely weld seal it closed. When all of the straws have been sealed, plunge them directly into liquid nitrogen in a stainless steel dewar flask and store the straws in the open goblet attached to the patient's cane.

For cryo-solution elution and dilution, place the corresponding six well dilution dish to the center of a stereomicroscope stage and a 60 mm petri dish containing a 37 degree Celsius sucrose warming bath to one side of the microscope. Next, isolate the straw to be warmed, and hold the upper straw seal above the liquid level to confirm the identity of the sample. Use mayo scissors to secure the straw below the internal plug, to keep the vit tip submerged in the liquid nitrogen.

Firmly tap the scissors against the dewar flask several times to dislodge the tip from the inner straw side wall as a quality control precaution, and lift the straw into the ambient air in a horizontal orientation next to, or below, the stereoscope surface. Grasping the non-labeled end of the straw, cut the straw at the inner plug. Then lift the straw above the warming bath dish and pour the vit tip into the bath at a 60 degree angle, until the tip is completely extruded.

The base of the flexipet should rest above the dish side wall. After 5 to 10 seconds, transfer the pipette base into a microcap pipetting device. Start a five minute timer.

While viewing the microscope, use an index finger to cover the bulb hole, and gently squeeze the bulb to release the vitrified blastocyst from the vit tip into the T1 wash. After confirming the release of the blastocyst, clear the residual V3 solution and bubbles by positive pressure, evacuating the contents into the center of an unused discard well. Transfer the vit tip onto a sterber pipette, moving the embryo into the second T1 droplet under oil for the remaining four minutes.

During the incubation, prefill the flexipet with the next solution, then dilute the blastocyst in the T2, 3, and 4 droplets at three minute intervals. Finally, equilibrate the blastocysts in the T5 droplet for five minutes on a 37 degree Celsius surface, before returning them to the incubator. The Modified Gardner Blastocyst Grading System considers full blastocysts as having less than 10%herniation of the trophectoderm cells, while blastocysts with up to 50%herniation are classified as expanded.

Blastocysts that demonstrate a greater than 50%herniation are considered to be hatching. Using the demonstrated cryo-preservation and warming methods, high levels of live births per cycle start have been consistently achieved with or without pre-implantation genetic testing. Although these successes are appreciably higher, using pre-selected euploid blastocysts.

Indeed, evaluating a good prognosis patient population for cryo-preserved cycles alone, these frozen embryo transfer data demonstrate better success rates than the national average for both implantation and live birth rates by over 30%By transferring predominantly single genetically tested euploid vitrified blastocysts, some of the highest implantation rates and live birth rates in the United States, independent of age, have been attained, compared to recent national statistics reported by the Center for Disease Control for two other well-respected fertility clinics, and the data released by the Society for Assisted Reproductive Technologies. MicroSecure vitrification is a novel aseptic procedure developed for technical ease, reliability, and cryo-security in mind. This simple, non-commercial vitrification system offers a highly effective low-cost alternative to vitrification devices.

Since its development, the MicroSecure method has ensured an over 99.9%embryo recovery and over 95%complete blastocyst survival. This has enabled the transformation of our clinical practice into nearly all cryo-preserved embyro transfer cycles over the past three years. We have effectively modified our procedure to include an inner seal in front of the cotton plug of ionomeric semen straws to still produce reliable weld seals and outstanding post-warming outcomes.

There are many different device systems used in the assistive reproductive technology industries today, but no other device offers the simplicity and technical repeatability to essentially eliminate user variation and provide optimized safety, like MicroSecure vitrification.