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

Abstract

Clinical embryo vitrification evolved with the development of unique vitrification devices in the 21st century and with the misconception that ultra-rapid cooling in an "open" system (i.e., direct LN2 contact) was a necessity to optimize vitrification success. The dogma surrounding the importance of cooling rates led to unsafe practices subject to technical variation and to the creation of vitrification devices that disregarded important quality-control factors (e.g., ease of use, repeatability, reliability, labeling security, and storage safety). Understanding the quality-control flaws of other devices allowed for the development of a safe, secure, repeatable, and reliable µS-VTF method aimed to minimize intra- and inter-technician variation. Equally important, it combined the availability of two existing FDA-compliant devices: 1) a 0.3-mL ionomeric resin embryo straw with internalized, dual-colored, tamper-proof labeling with repeatable weld seal potential; and 2) shortened, commonly-used, 300-µm ID sterile flexipettes to directly load the embryo(s) in order to create a highly-effective global vitrification device. Like other aseptic, closed vitrification systems (e.g., High Security Vitrification (HSV), Rapid-i, and VitriSafe) effectively used in reproductive medicine, microSecure Vitrification (µS-VTF) has proven that it can achieve high post-warming survival and pregnancy outcomes with its attention to simplicity, and reduced technical variation. Although the 0.3-mL embryo straw containing an internal hydrophobic plug was commercially replaced with a standard semen straw possessing cotton-polyvinyl pyrrolidone (PVP) plugs, it maintained its ionomeric resin composition to ensure weld sealing. However, the cotton plugs can wick out the fluid-embryo contents of the flexipettes upon contact. A modified µS-VTF method was adapted to include an additional internal weld seal before the plug on the device loading side. The added technical step to the µS-VTF procedure has not affected its successful application, as high survival rates (> 95%) and pregnancy rates continue today.

Systematic Development, Validation and Optimization of a Human Embryo Culture System

“…Based on our interests to optimize our culture media, we identified and added a highly purified recombinant, high-molecular weight HA product (EAS) which we felt had significant potential merits as a serum-free supplement to LG (mLG). Our studies validate that this EAS was indeed not harmful and is more likely beneficial based on the outstanding implantation and live birth rates we achieved, which has attained elite levels (i.e., upper 5 percentile) as previously demonstrated by our lab [41,44,45]. In addition to its published role as an EAS which enhances implantation [60], we believe strongly that HA promotes membrane plasticity, aiding in cellular healing and protective functions associated with stressful events like trophectoderm biopsy and blastocyst vitrification.…”

“…4), we were even more confident to strive toward a practice of single embryo transfer as advocated by Gardner and Lane [52] years earlier. Certainly, the concurrent development, validation, and implementation of highly reliable vitrification [44,45] and blastocyst biopsy [41,43] procedures has allowed us to confidently implement single ET with unprecedented success, as reflected in the outcome verification of our FET cycles in Figure 2B. Note that implantation success and live birth rates are similar within age groups and each occurs at an equally high level across age groups when transferring a mean of 1 to 1.2 blastocysts/FET.…”

“…Blastocyst formation was documented and typically only those possessing ≥2BB grade were cryopreserved or may have first underwent TE biopsy procedures for PGT-A determination if ≥3BB grade, as previously detailed [41,43]. All fair to excellent quality blastocysts were vitrified using our microSecure vitrification (µS-VTF) procedure [44,45]. Embryos were transferred on Day 3 or 5 depending on the number/quality of embryos available and the IVF history of the patient.…”

Patients of advanced maternal age should only transfer a single euploid blastocyst

“…Aseptic microSecure VTF was performed using a 3-step dilution (5 min/5 min/1 min); individual blastocysts were loaded into 300 μm ID flexipettes (Cook Medical, Spencer, IL; 3 μl volume); flexipettes were then dried and inserted tip first into prelabeled 0.3 ml CBS™ embryo straws; the straws were weld sealed and plunged directly into LN 2 [19,20]. Rapid warming was achieved by direct placement of the vitrified flexipettes into a warm (37°C) 0.5 M sucrose bath [21]. Within 10 seconds, each blastocyst was pipette directly from the flexipette into an open 200 μl droplet of 1.0 M sucrose solution and then transferred into 100 μl droplets under oil for 3 min intervals.…”

Vitrification: the pioneering past to current trends and perspectives of cryopreserving human embryos, gametes and reproductive tissue

“…67 What can be performed to minimize intra-and inter-laboratory procedural variation beyond the essential need for training? Numerous risk factors and safety issues associated with different vitrification methods should be considered (eg, LN 2 device handling; device design flaws; shipment concerns and viral cross-contamination of semen, embryos or ova), 25,36,40,[67][68][69] but are not reviewed in this article. In essence, there are basic quality control (QC) factors warranting implementation to make vitrification a consistent, efficient, reliable and highly effective ART procedure that minimize liability and maximize success.…”