**3. The microSecure‐VTF (μS‐VTF): a quality control solution**

vapor conditions (described above), these straws will allow LN2 seepage into the container to occur. If these containers are warmed too rapidly, the vaporization pressure could be excessive and damaging. One other noteworthy example here is a device requiring two different heat settings to optimally seal two different size openings (e.g., Cryotips). The latter was simply a formula for repeated errors, frequently resulting in bent and burnt tips. It is also possible that the overheating of the fine tip ends may have been partially responsible for the excessive and

Another problem experienced by many inexperienced user of the Cryotip device was the overcrowding of devices within a given storage goblet. Unlike a compact arrangement of straws, the tight opposition of Cryotips could cause their protective shields to rise, leaving their delicate tips exposed to damage (e.g., bending, breakage). Similarly, although Cryop‐ ettes were not as delicate, they were completely unexposed in storage without support. The potential for breakage or fracturing its bulb connection, if accidentally compressed in frozen storage, was a real risk. Another important practical factor to consider is the LN2 storage capacity of a device. If we consider that 0.25‐ml straws (e.g., HSV, Vitrisafe) or perhaps Cryotop devices in large goblets is an optimal standard of 10 units/goblet, then the storage of 8 units of 0.5‐ml straw‐size devices (e.g., rapid‐i, μS‐VTF) or square‐capped Cryolock de‐ vices is very good. However, the safe storage of five Cryotips per goblet begins to become inefficient, while one or two Cryoleaf devices are completely impractical. Lastly, we have already discussed the ability to safely access and visualize samples in storage/LN2‐filled dewar flasks or specialized bathes, but what about the safety of the handler. Most open‐sys‐ tem methods require the placement of a protective straw cover (e.g., Cryotop, Cryotech) or a plastic cap (e.g., Cryolock) over the supercooled device end for storage protection. Likewise, these protective covers must be removed in LN2 prior to warming to facilitate high warming rates. However, these covers can be difficult to unlock and remove under freezing condi‐ tions. Any miscues in the insertion or removal of the protective covers could adversely in‐ fluence the stability of the embryo/oocyte(s) on the surface of the open blade. Both vitrification and warming events entail the coordinated handling of device components, with fingers and forceps, in close proximity to LN2, thus creating reoccurring safety issues. Although the use of protective liners provides delicate finger agility in handling and re‐ duces potential contact burns, it does eliminate a mishandling event (e.g., connecting or sealing hybrid devices) that could result in the accidental LN2 spillage of a full dewar flask. In short, the unnecessary handling and manipulation of devices in LN2 creates biosafety is‐ sues for the user. By contrast, the assembly and sealing of aseptic closed devices under am‐

Our final end point consideration is the recovery and survival rate potential of a given de‐ vice. As we have already discussed above, there are several quality control factors that can ultimately influence the final outcome. One issue we have not touched on is the advantage of being able to visualize the embryo or oocyte(s) upon warming. This is particularly impor‐ tant with oocytes as they do become highly translucent during their initial exposure to the T1 sucrose solution. Therefore, methods that allow you to distinctly image and account for the expected number of embryo(s)/oocyte(s) present (e.g., Cryoloops, rapid‐i and pipetting

problematic bubbling experienced in these devices.

190 Cryopreservation in Eukaryotes

bient conditions eliminates similar safety concerns.

Having a firm grasp of the cryobiological principles of vitrification, we developed an aseptic closed vitrification device aimed at insuring the simplicity, efficacy, and reliability of vitrifica‐ tion success [57]. It was developed in 2008 as an inexpensive, noncommercial, FDA‐compliant method which optimized quality control aspects of vitrification to reduce or eliminate technical variation. Using the CBS™ 0.3‐ml embryo straw (with hydrophobic plug) as our model, our system uniquely offers tamperproof internalized, dual‐colored labeling. The use of different label and rod colors allows for quick identification of patient samples based on day of cryopreservation, whether blastocyst biopsy was performed, or blastocyst quality, for example. In contrast to the HSV system, we maintained secure labeling by not reducing the straw diameter. Having internalized labels allows us to use nonpermanent adhesion labels (GA International, USA) that can be easily removed postwarming and placed onto the patients' Cryo‐data sheet record to confirm identification with the patient at the time of ET. Furthermore, in the case of a preimplantation genetic screening (PGS) cycle with discard aneuploidy embryos, the placement of removed labels onto the Cryo record is an excellent quality assurance practice. Finally, in terms of labeling it is essential that an accurate description of the patient sample is conveyed, including the last and first name, secondary ID, embryo descrip‐ tion (#, stage, quality grade; Ex: 1x4AA or 1x8cB), and the cryopreservation date. Upon receiving other devices in our laboratory, it is so surprising to witness how little information some programs actually provide on a device. Out of respect to all IVF laboratories, proper labeling is essential to avoid possible liability issues.

Since μS‐VTF uses shorten sterile flexipettes to pipette, load, and directly store embryo(s) or oocytes, there is no secondary device surface to introduce technical variation. Thus, μS‐VTF embryos and oocytes are simply loaded and easily visualized upon removal to insure >99.9% recovery rates. To achieve rapid warming after safe patient sample identification in a dewar flask, the straw is cut below the plug/seal (below the ID rod) and quickly tipped (60° angle) and tapped to promote the free fall of the flexipette into a warm sucrose bath (see You Tube video "microSecure vitrification warming"). On rare occasion, if an embryo is missing upon pipetting into T1 solution it has invariably been found in the sucrose bath, due to it having been loaded to close to the tip. From this rare experience, we have learned that although there is capillary drift into flexipettes while resting on the sidewall of a 60‐mm dish in the sucrose bath (for 5–10 s, as the pipette fluid volume will attempt to equilibrate to the sucrose level), the initial plunge into the bath may create an initial force that pulls a fraction of fluid from the tip. It is important that biologists remain mindful to load the embryo(s)/oocytes approximately mid‐way in the fluid column. Again, we control this by aspirating a full, fresh 3‐μl column of vitrification solution into the pipette (i.e., plunger released, no technical variation) and then expel one‐third to a half of the fluid upon picking up the embryo(s)/oocytes, followed by controlled plunger release (to preset fill volume). Upon pipette removal and tip drying (i.e., sterile gauze wiping), the capillary volume in the flexipette is stable during handling proce‐ dures. Our rare loss of an embryo has been exclusively related to hatched blastocysts post‐ biopsy. These embryos can be extremely adherent on contact with any plastic (i.e., charged surface) and potentially difficult to ID in their completely collapsed state. Thus, as with our standard blastocyst biopsying of trophectoderm cells, we suggest pre‐coating the surface of all pipettes with human serum albumin (HSA) before handling to minimize cellular stickiness and possible loss of hatched blastocysts.

Next, the μS‐VTF system uses CBS™ ionomeric‐resin straws that completely weld seal us‐ ing an automated sealer, which again effectively eliminates technical variation. By not wor‐ rying about the quality of the seal, our system offers repeatability and reliability only found in CBS™ straw products (e.g., HSV). Prior to sealing, we make sure the tip of the flexipette has dropped down to the plug end, insuring at least 1 cm of air space to safely seal the straw. Next, we suggest supporting the straw at the point of sealer contact (as opposed to the natural instinct to hold the end of the straw) to minimize any abrupt vibration stimulat‐ ed by the automatic sealer. Upon inverting the straw label‐end up, we check the quality of the seals and whether any fluid remnant/discharge appears in the upper straw air space (as the flexipette base should now be resting against the bottom seal). The upper air space near the plug/labeling rod insures safety to cut the straw postwarming. If any fluid was visual‐ ized, we check to make the flexipette did not accidentally get sealed into the straw. If on a rare occasion this happened: (1) if the seal is incomplete then you must extract the flexipette and attempt to find the embryo in the residual fluid droplet before reloading; or (2) if the seal is complete, simply make a note on the record (for that straw #) of the situation, so that proper care is taken postwarming to rinse the inner straw for possible extruded oocytes/ embryo(s). Upon storing the straws in LN2 on canes with large open goblets, up to eight straws can be stored/cane (i.e., good storage capacity). Furthermore, there is no need for an upper cover on the cane, as each straw is weighted, unless they are transported and suscep‐ tible to not maintaining their upright position. Coincidentally, if a straw is ever to acciden‐ tally drop into an LN2 tank, they are easily recovered as the weighted rod drops the tank bottom and sticks straight upward (due to air buoyancy in the straw), as opposed to lying on the bottom somewhere in the residual N2 debris.

As an aseptic closed system whose vitrification device (i.e., a sterile flexipette) is stored in an outer straw container, the μS‐VTF device achieves a cooling rate of 1391°C/min and corresponding warming rate of over 6000°C/min. As an insulated device with lower cool‐ ing rates than an open device system, it has proven to be more resilient to accidental room temperature exposures (Ovation Fertility, unpublished data). Overall, the μS‐VTF device has been systematically validated to be a simple and reliable approach that minimizes in‐ tra‐ and inter‐laboratory technical variation, while providing maximum cryosecurity using sterile products [41]. In addition, it has been developed without commercial influence and marketing pressure, thus providing the added benefit of substantial cost‐savings. In to‐ day's IVF industry, which is increasingly reliant of biopsying and vitrifying every fair to excellent quality blastocyst to optimize pregnancy success [58], costs are an increasingly important factor to consider. This is especially true when one realizes that 50–75% of the genetically tested blastocysts will be aneuploidy and destine to be discarded after short‐ term storage. In conclusion, the μS‐VTF system has proven to be a highly effective proce‐ dure that may offer "universal" acceptance to alleviate current quality control concerns with the handling, storage, and shipment of vitrified oocytes and embryos.
