**4. Conclusion**

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

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

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

and possible loss of hatched blastocysts.

192 Cryopreservation in Eukaryotes

on the bottom somewhere in the residual N2 debris.

Vitrification is the single most impactful assisted reproductive technology in the IVF industry since the development of intracytoplasmic sperm injection (ICSI). Today, we faithfully cryopreserve blastocysts and oocytes without regard to possible loss. We have had to reeducate ourselves, and our infertility patients, that fresh ET is no longer better than vitrified ET cycles, especially in combination with blastocyst biopsying and preimplantation genetic screening. By adhering to strict quality control standards and quality assurance practices, we can continue to improve the reliability of our laboratory outcomes, and help avoid future liability issues together.
