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.
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.
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.
The development of the µS-VTF procedure was conducted as part of an approved Institutional Review Board (IRB) study in 2008-2009 (Aspire IRB, Santee, CA) on human oocyte and embryo cryopreservation. The procedure has since been routinely applied to the clinical treatment of infertility patients who have signed informed consent forms.
1. Quality-control Considerations
2. Cryopreservation Procedure
1,341 vitrified blastocysts were warmed between 2012 to June 2014, and 1,341 embryos recovered (100%) while 1,316 survived (98.1%). Biopsied blastocysts experienced over 99.5% survival. Upon transferring predominantly single, genetically tested, euploid, vitrified embryos, we were able to achieve some of the highest implantation rates and live birth rates in the USA, independent of age (Figure 3)20, according to recent national statistics reported by the Center for Disease Control and the Society for Assisted Reproductive Technologies (Tables 1 and 2). Evaluating a good prognosis patient population (less than 35 years old) for cryopreserved cycles alone (Table 1), our laboratory outperformed the national average for both implantation rates (i.e., the efficiency of an embryo to establish a pregnancy) and ultimately live birth rates by over 30%. Additionally, the initial validation/verification of our modified storage straws in mid-2014 using 70 research-consented, re-vitrified aneuploid blastocysts resulted in 100% recovery and 100% survival.
Figure 1. MicroSecure VTF Setup. The VTF dish (A) is assembled with 3 distinct rows of vitrification solutions, which utilize 3 wash droplets before placement in distinct, numbered holding droplets. Additionally, individual pipettes with shortened VTF tips (i.e., 300-µm ID flexipettes) are secured and organized in a styrofoam tube rack (B), which can be rotated for orderly use. Note that a representative disposable micropipette bulb pipetting assembly is resting on the styrofoam rack. Please click here to view a larger version of this figure.
Figure 2. Modified Blastocyst Grading System. Our modified Gardner blastocyst grading system accounts for the induced herniation of TE cells to facilitate blastocyst biopsy. The modification considers the premature hatching of full blastocysts (A: Grade 3 = less than 10% herniation) and expanded blastocysts (B: Grade 4 = up to 50% herniation), while a hatching blastocyst (C) has greater than 50% herniation16. Please click here to view a larger version of this figure.
Figure 3. Comparative Pregnancy Outcomes. Over a cumulative 1.5-year interval, pregnancy data were compared to assess the effects of transferring vitrified-warmed euploid blastocysts (n = 172 cycles/144 transfers) compared to non-PGS cycles (n = 160 cycles/153 transfers) involving predominantly fresh transfers18. For our experimental purposes, this data clearly reveals that biopsied blastocysts vitrified by the µS-VTF procedure maintain their viability. Please click here to view a larger version of this figure.
Data Source | # Cycles | Mean # of embryos transferred | Implantation Rates (%) | Live Birth Rates (%) |
NB Lab * | 144 | 1.2 | 74.00% | 74.50% |
National Average | 20,423 | 1.7 | 39.60% | 44.10% |
* Data were averaged based on the 2014 performance of Orange County Fertility, Southern California Fertility Center and the Southern California Center for Reproductive Medicine clinics using the current Ovation Fertility laboratory (NB Lab) which founded the microSecure vitrification procedure in 2008. |
Table 1. 2014 CDC Assisted Reproductive Statistics. Frozen embryo transfer pregnancy data of women under 35 years old from three reporting physician clinics using our NB Lab compared to the US 2013 national average of over 450 reporting clinics.
SART National Averages | ||||
Clinic – State | Women | Women | Women | Women |
< 35 yrs | 35-37 yrs | 38-40 yrs | 41-42 yrs | |
SCCRM-CA * | 63.1% | 58.3% | 40.6% | 32.3% |
CCRM-CO * | 64.7% | 61.5% | 40.4% | 32.2% |
RMA-NJ * | 63.2% | 59.7% | 34.6% | 18.7% |
National Average | 48.6% | 38.3% | 24.3% | 12.3% |
SCCRM—Southern California Center for Reproductive Medicine; CCRM—Colorado Center for for Reproductive Medicine; and RMA—Reproductive Medicine Associates | ||||
* These leading clinics have all predominantly implemented vitrified embryo transfer cycles, in association with preimplantation genetic testing, into their standard practice of patient care to optimize live birth success per embryo transplanted. |
Table 2. 2014 Cumulative live birth rates, as reported by the Society for Assisted Reproductive Technologies (SART), for the SCCRM Clinic using our Ovation Fertility Lab in Newport Beach, CA, contrasted with several other respected programs, as well as with the SART national averages.
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. |