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.
Klinische Embryo Vitrifikation mit der Entwicklung von einzigartigen Vitrifikation Geräte im 21. Jahrhundert und mit der falschen Vorstellung entwickelt , dass extrem schnelle Abkühlung in einem "offenen" System (dh direkte LN 2 Kontakt) war eine Notwendigkeit Vitrifikation Erfolg zu optimieren. Das Dogma die Bedeutung der Kühlraten rund um führte zu unsicheren Praktiken vorbehaltlich der technischen Veränderung und zur Schaffung von Vitrifikation Geräte , die wichtige Qualitätskontrollfaktoren (zB einfache Bedienung, Wiederholbarkeit, Zuverlässigkeit, Kennzeichnung Sicherheit und Lagersicherheit) nicht berücksichtigt. Das Verständnis der Qualitätskontrolle Mängel anderer Geräte für die Entwicklung eines sicheren, reproduzierbaren erlaubt und zuverlässige uS-VTF Methode zur Minimierung richtet intra- und inter Techniker Variation. Ebenso wichtig ist, kombiniert es die Verfügbarkeit von zwei bestehenden FDA-konformen Geräten: 1) ein 0,3-ml-Ionomerharz Embryo Stroh mit internalisiert, zweifarbige, manipulations pDach Kennzeichnung mit wiederholbaren Dichtschweißung Potential; und 2) verkürzt, häufig verwendete, 300 & mgr; m ID sterile flexipettes, um direkt den Embryo zu laden (s), um eine hocheffiziente globale Vitrifikation Gerät zu erstellen. Wie andere aseptisch, geschlossen Vitrifikation Systeme (zB High Security Vitrifikation (HSV), Rapid-I und VitriSafe) effektiv in der Reproduktionsmedizin verwendet, microSecure Vitrifikation (uS-VTF) hat bewiesen , dass es eine hohe post-Erwärmung Überleben und Schwangerschaft erreichen können Ergebnisse mit seiner Aufmerksamkeit auf Einfachheit und geringere technische Variation. Obwohl die 0,3-ml Embryo Stroh eine interne hydrophobe Stecker enthalten, im Handel mit einem Standard-Samen Stroh ersetzt wurde besitzen Baumwoll Polyvinylpyrrolidon (PVP) Stecker, behielt er seine Ionomerharz Zusammensetzung Schweiß Abdichtung zu gewährleisten. Jedoch können die Wattestopfen fluid embryo Inhalte der flexipettes bei Kontakt Docht aus. Eine modifizierte uS-VTF Verfahren wurde angepasst, um eine zusätzliche innere Schweißnaht zu schließenversiegeln, bevor die Stecker auf dem Gerät Ladeseite. Der zusätzliche technische Schritt zum uS-VTF Verfahren hat seine erfolgreiche Anwendung nicht betroffen, da hohe Überlebensraten (> 95%) und die Schwangerschaftsraten heute fortsetzen.
Vitrification is the single most impactful assisted reproductive technology in the in vitro fertilization (IVF) industry since the development of intracytoplasmic sperm injection. Today, blastocysts are cryopreserved without the loss in embryo viability previously associated with conventional slow-freezing methods1. With reliable post-warming embryo survival, the infertility industry is transforming into the preferred use of cryopreserved embryo transfer cycles, which yield similar or higher pregnancy outcomes than traditional fresh embryo transfer. In association with blastocyst biopsy and preimplantation genetic screening (PGS), vitrification has become a vital clinical tool to optimize healthy live birth outcomes via euploid single embryo transfer2,3.
Murine embryo vitrification was developed in the mid-1980s4,5 and adapted to animal agriculture by 19906. Based on the premise that vitrification solutions form a metastable glasseous state, free of damaging ice crystal formation, it has proven to more efficiently preserve the complete cellular integrity of embryos. Interestingly, the promising acceptance of vitrification into human embryology did not begin to be realized until the 21st century. Early publications promoting the use of vitrification coincided with the development of unique "open" system devices7,8,9. However, the adoption of vitrification into clinical practice was slow, as it came at a time when improvements in slow blastocyst freezing were also occurring. Successful conventional slow-rate freezing, in addition to vitrification, were aligned with improvements in embryo culture systems, as well as with the incorporation of blastocoele-collapsing approaches, which enhanced both the overall survival of trophectoderm and, subsequently, implantation10.
In the last decade, vitrification technology has rapidly supplanted conventional freezing practices. To a great extent, this was due to the development of specialized vitrification devices. Some of these devices have handicapped the overall safety, efficiency and effectiveness of clinical vitrification by introducing inherent design flaws to devices used in the IVF industry11. Indeed, the nuances of different devices introduce significant technical variation between programs, commonly referred to as "technical signatures"12. Thus, scientific journals, like the Journal of Visualized Experiments (JoVE), can serve as a valuable resource for demonstrating technical details, which will help to reduce outcome variation. Another ongoing problem is that some embryologists continue to be misinformed, even today, based on claims that the "ultra-rapid cooling of embryos or oocytes in an 'open vitrification system' (i.e., direct embryo contact with liquid nitrogen (LN2)) is a prerequisite to optimizing success rates." Clearly, this belief is inaccurate, based on the proven success of aseptic closed systems13,14,15.
Based on the cryobiological principles of vitrification, the efficacy of vitrification is more highly dependent upon warming rates than on cooling rates16,17,18. In general, independent of the vitrification device used, the warming rate must exceed the cooling rate to insure high survival rates. High warming rates minimize the opportunity for any ice growth (i.e., the recrystallization of nucleated impurities in cryo-solutions) during the devitrification phase of warming. Granted, the stability of the vitrification solution (i.e., the type and concentration of cryoprotective agents used) may have a confounding effect, but this is addressed in a separate publication11. Considering the cooling-warming rate issues, MicroSecure Vitrification (µS-VTF) was developed in 2008 as an inexpensive, non-commercial, FDA-compliant method that optimized the quality-control aspects of vitrification. It was unique in that it offered tamper-proof, internalized, dual-colored labeling. Furthermore, by loading and storing the embryos directly in the sterile flexipette used for pipetting (i.e., without pipetting to a secondary device surface) and by using ionomeric-resin straws that completely weld seal using an automated sealer, technical variation has been effectively eliminated.
When assessing the completeness of vitrification devices for potential use, there are several quality-control factors that should be taken into account, including: 1) Labeling potential—Can labels be securely adhered? Are they tamperproof? Do they offer dual-color identification potential? Does it require a secondary label, and can the label be easily removed for record-keeping purposes (i.e., patient verification) post-warming? 2) Technical ease—Can embryos be easily loaded into/onto the device in a timely manner and simply identified and tracked post-warming? 3) Procedural simplicity/Repeatability—Does the vitrification method offer simplicity and reliability that easily allows for repeatability, which minimizes the variation between technicians (internal) and programs (external)? 4) LN2 storage capacity—Can the devices be easily and safely handled and identified? Is their storage potential space efficient? Does the device offer security and safety from physical damage or possible contaminants as an aseptic closed system? 5) Recovery potential /Survivability—Is the device design prone to potential problems in the guaranteed recovery of embryos, and will they reliably vitrify and maintain complete cellular integrity post-warming? The latter specific quality concern, the recovery rate, has actually been surprisingly minimized in published reports; this is done by generally hiding the unfavorable outcome (i.e., lost embryo or egg) in typically-good survival rates. Any device prone to inconsistent recovery (<99%) is seriously flawed and constitutes a procedural liability.
Our aseptic, closed µS-VTF method has been strategically developed to account for each quality-control measure. However, after 5 years of superior clinical success and validation14, the procedure had to be modified. The original 0.3-mL embryo straws (possessing a hydrophobic plug) were removed from the IVF industry and replaced with a 0.3-mL semen straw possessing a standard cotton/PVP plug (i.e., relabeled as a semen/embryo straw). This procedural paper outlines the specific steps and strategies needed to implement µS-VTF safely, simply, and effectively. Furthermore, we highlight the modification(s) needed to reliably account for supply limitations, until such time as an alternative ideal straw container is reintroduced back into the clinical laboratory.
Today, there is a high expectation of attaining complete blastocyst survival (> 95%) and achieving implantation success similar to that of fresh embryos. Some groups have suggested that the live birth rates of vitrified embryo transfer cycles are perhaps even higher than fresh blastocysts when the intact cryopreserved embryo is transplanted into a healthy, non-hormonally-stimulated uterus. Our data clearly indicate that vitrification effectively and reliably maintains the viability of the fresh embryo. Furthermore, we have proven that our aseptic, closed method, called MicroSecure Vitrification (µS-VTF), can achieve an optimal outcome comparable to or greater than the commercial standards (i.e., open device systems) used in the IVF industry.
Variation is associated with technical repeatability and reliability between individuals using a multitude of vitrification devices/methods. In turn, this has resulted in inconsistencies between programs applying vitrification. Therefore, it is not surprising that device familiarity is an important factor regarding laboratory proficiency and successful outcomes. It is this concept of "technical signature"11 that explains why repeatability between programs may be problematic. Not only has the development of more than a dozen commercial devices complicated this phenomenon, it has created quality-control concerns. Fortunately, closed vitrification systems, like µS-VTF, Rapid-I, and VitriSafe, are now proving to be equally effective to open device systems12,13,14.
MicroSecure VTF is a novel, aseptic vitrification technique developed with technical ease, reliability, and cryo-security in mind. By combining the use of two previously-approved, FDA-compliant devices, it is a non-commercial vitrification system with the distinct advantage of having an established low cost, in contrast to specialized devices. In addition, its unique tamperproof and internalized dual-colored labeling system, as well as numerous other quality-control advantages, have made µS-VTF an attractive global option11. As a non-commercial VTF device, however, its widespread industry application is evolving slowly. Only through the continued publication of its safety, security, and clinical effectiveness will µS-VTF gain growing utilization.
In August 2014, when faced with the inability to acquire the original 0.3-mL embryo straw manufactured with an inner, non-wicking hydrophobic plug, we were able to reliably modify the µS-VTF method. In short, the new 0.3-mL semen/embryo straws with their cotton-PVP plugs were effectively adapted. This was simply achieved by creating an inner seal before the cotton plug, thus preventing contact and fluid wicking with the open-ended VTF tip (i.e., the flexipette containing the embryo or eggs). Furthermore, if 40-mm ID rods are not accessible, the buoyancy issue associated with the lighter 30-mm rods can be counterweighted using two ball bearings.
These additional steps have made the technique slightly less simple, but still highly effective. Today, economics and efficacy are becoming increasingly important concerns, as biopsied blastocysts are typically vitrified individually until their euploidy status is confirmed on a genetics report. Blastocysts with a confirmed non-viable aneuploidy status typically get discarded, with patient consent, within weeks. Thus, a majority (> 50%) of VTF devices are discarded after short-term storage, causing an escalating annual cost when using commercial devices. Overall, µS-VTF is a highly-effective, reliable, and repeatable procedure for the cryopreservation of human blastocysts. The superior quality-control design securely labels and safely stores embryos/oocytes while eliminating recovery failure and optimizing post-warming survival and viability, which justifies the system's use as a universal approach to embryo vitrification.
The authors have nothing to disclose.
M.C. Schiewe would like to thank Mr. Forest Garner at the Fertility Center of Las Vegas for his statistical expertise in analyzing and evaluating annual CDC and SART data. Also, the authors wish to thank their Medical Director, Dr. Robert E. Anderson, for his dedicated support and faith in our technical abilities and expertise.
Aluminum Cane | IVM | XC055 | ||||
Ball bearings, 3/32" | VXB.com | KIT15977 | stainless steel | |||
CBS semen/embryo straw, 0.3ml | CryoBioSystems | 25292 | individual sterile | |||
Color, ID rods, 30 mm | CryoBioSystems | 019021-26 | weighted | |||
Culture tubes, 15ml | Falcon | 352099 | Conical | |||
Culture tubes, 10ml | Falcon | 352057 | Snap-cap | |||
Cryosleeves | Nalgene | 5016-001 | ||||
Filter, 250ml | Fisher Sci. | 09-740-2A | 0.22 μm | |||
Flasks, Tissue Culture 50ml | Falcon | 353014 | ||||
Flexipettes, 300μm ID | Cook Med. | K-FPIP-1300-10BS-5 | Sterile, 20/pack | |||
Forcep, Large | Miltex | 6-30TC | ||||
Forcep,Splinter – fine | Miltex | 17-305 | ||||
Goblet | IVM | PA003 | ||||
Heat Sealer, SYMS 1 | CryoBioSystems | 16399 | 110V or 220V with adapter | |||
Hepes-buffered media | Life Global or | LGGH-100; 100ml, or | stored at 2-8ºC | |||
Irvine Scientific | H-HTF; 90126; 100ml | with non-essential AA's | ||||
Labels, Cryo | GA International | CL-23T1 | Various colors | |||
Liquid Nitrogen Tank, 40L | MVE or Taylor Warton | various | liquid storage | |||
LN2 Dewar flask, 0.5L | Hampton Research | HR4-695 | Stainless steel | |||
6-well Custer Dishes | Biogenics | 015/020 | plasticware by case | |||
Pipette Bulb, Micro Cap | Drummond | Fisher#13681451 | Hole on bulb apex | |||
Petri Dishes, 35mm | Falcon | 351006 | ||||
Petri Dishes, 58mm | Nunc | 150288 | ||||
Petri Dishes, 100mm | Falcon | 351029 | ||||
Pipette Tips, ART long | Fisher Sci. | 02-707-80 | 10-100μl | |||
Pipet Aid | Drummond or Falcon | various | rechargeable | |||
Pipetting Device, Stripper | Cooper Surgical | MXL3-STR | ||||
Pipettes, Serological 1ml | Falcon | 357521 | ||||
Pipettes, Serological 2ml | Falcon | 357507 | ||||
Pipettes, Serological 5ml | Falcon | 357543 | ||||
Pipettes, Serological 10ml | Falcon | 357551 | ||||
Scissors, Surgical Mayo | Miltex | 5-SC-16 | ||||
Stereomicroscope | Nikon, Olympus, Leica | various | ||||
Sterile Gauze pads, 4"x4" | Kendall Healthcare | 6939 | ||||
Synthetic serum | Life Global or | LGPS-20 ; 20ml, or | stored at 2-8ºC | |||
Irvine Scientific | SS-99193; 12 x 10ml | purchase low endotoxin lot | ||||
Sucrose | Sigma Chemical Co. | #S9378 | Aliquot into 50ml flasks, 1year | |||
17.1g/flask +Medium to 50ml | ||||||
makes a 1M solution | ||||||
Filter with 0.22µm unit | ||||||
Timer | Nalgene | 5016-001 | ||||
Thawing Solution | Innovative | BL-TS | T1, T2, T3, T4 | |||
Cryo Enterprises | (≤1.0M Sucrose) | stored at 2-8ºC for ≤1 month after opening | ||||
Vitrification Solution*,** | Innovative | BL-VS | V1, V2, V3 | |||
Cryo Enterprises | (≥7.9M [Glycerol/EG]) | stored at 2-8ºC for ≤1 month after opening | ||||
* Non-permeating cryoprotective additives may include: sucrose, ficoll and sodium hyaluronate | ||||||
** other commercial preparations are typically ethylene glycol (EG)/dimethyl sulfoxide (DMSO; 30% v/v; 4.8M), but could be EG/propylene glycol (32% v/v; 5.2M). Mixed solutions are typically used to reduce cryo-toxicity concerns of a high molar solution. Commercial solutions typically include an ES and VS solution. The formulation of commercial preparation is typically proprietary property. |