Frequently Asked Questions
It is possible to freeze them immediately after denuding.
A study has reported that oocytes lose their fertilizing ability every moment after 40 hours following HCG administration. Therefore, please back calculate from it. For example, if oocytes were retrieved 36 hours following HCG administration and cultured for 2 hours each before and after freezing and thawing, the time to ICSI is already 40 hours. Please note that unnecessarily long culture may decrease fertilization rate. We recommend that oocytes be frozen as soon as possible after retrieval and denuding and cultured for about 2 hours after thawing before ICSI.
Since it is recommended that freezing reagents and plates be used at room temperature (25°C-27°C), take them out of the refrigerator at least 1 hour before the start of freezing operation and return them to room temperature before use. If cryopreservation is expected on the next day, it is possible to take them out of the refrigerator in advance in the evening of the previous day and return them to room temperature, as long as the temperature in the lab is maintained at a certain level.
Use the one with a size suitable for the oocyte/embryo to be frozen. We recommend 140 to 150 μm for oocytes/cleavage-stage embryos and 200 to 250 μm for blastocysts.
We recommend up to 3 oocytes/embryos per Cryotec. However, it depends largely on the technique of the manipulator how many oocytes/embryos can be handled safely at the same time. Therefore, the maximum number should be the number of oocytes/embryos which can be manipulated in the same way as manipulating one oocyte/embryo.
In order to freeze oocytes/embryos using the same plate under the same conditions, we recommend up to 15 oocytes per plate be frozen in compliance with the protocol. Pay attention to avoidance of carrying in oil and maintaining appropriate temperature (25°C-27°C). However, if you wish to freeze more than one oocyte/embryo at the same time while identifying each oocyte/embryo, pay attention to identification management by finely adjusting the time interval for immersion in ES. In consideration of hygiene, if freezing is performed after an interval, prepare a new plate even if the number is less than 15.
As long as the protocol is complied with and careful attention is paid to avoidance of carrying in oil and maintaining appropriate temperature (25°C-27°C), there is no limit on the number of use as long as it is used for the same patient and the number of oocytes/embryos per freezing plate is 15 or less. However, if you wish to freeze more than one oocyte/embryo at the same time while identifying each oocyte/embryo, pay attention to identification management by finely adjusting the time interval for immersion in ES.
Yes, GV and MI oocytes can be frozen using the same procedure. At that time, immature oocytes are more susceptible to changes in osmotic pressure. Therefore, please pay more attention to each step of the protocol which requires attention in freezing MII oocytes and embryos.
Yes, they can. At that time, we recommend that at least about 50% of the cumulus cells be removed before cryopreservation to prevent decreased maneuverability and difficulty in seeing changes in oocyte volume.
Yes, they can. However, due to difficulty in determining the grades of morula-stage embryos, we recommend freezing of oocytes, cleavage stage embryos, or blastocysts. At that time, the maximum time for the ES step should be 12 minutes. However, for morula-stage embryos where formation of the blastocoel is beginning, the maximum time for the ES step should be 15 minutes.
The maximum time for the ES step should be 12 minutes even for cleavage stage embryos in which compaction is observed.
In the case of expanded blastocysts (diameter ≥ 220 μm), perform pre-shrinking before the start of freezing. Mix DS and WS in a ratio of 2:3 to prepare a hypertonic solution. When blastocysts are immersed, they will shrink due to difference in osmotic pressure. When the diameter becomes less than 220 μm, you can proceed to the ES step. Subsequent steps are the same.
We recommend 0.2 M because 0.2 M of trehalose is sufficient for pre-shrinking. It may take too much time with 0.1 M, while 0.3 M or higher may impose stress of rapid changes in osmotic pressure. In consideration of this, the effective and safe concentration is 0.2 M.
They can be cryopreserved using exactly the same procedure as usual. As it is difficult to determine the volume of the original embryo and associated volume recovery in the ES step at that time, please immerse them in ES for 15 minutes and then proceed to the VS step.
It is caused by a difference in membrane permeability. Water flows out quickly in some places due to high permeability while water flows out slowly due to low permeability, resulting in irregularities in the cell membrane.
Oocytes of Rank C can be frozen. However, if there is any oocyte that did not shrink at all in the ES step, the cell membrane may not be functioning even if it appears to be alive. Therefore, there is a risk that replacement with a dehydration/cryoprotectant agents (CPAs), that is essential for freezing, cannot be sufficiently achieved. In consideration of the risk, they are out of scope of freezing in our protocol.
If you freeze more than one oocyte/embryo at a time, aspirate them in a pipette at equal intervals (reagent - oocyte - reagent - oocyte -...), place them in a line (not the same location), and put them in ES. However, if you wish to freeze more than one oocyte/embryo at the same time while identifying each oocyte/embryo, pay attention to identification management by finely adjusting the time interval for immersion in ES.
Only the maximum time is fixed; 15 minutes for oocytes and blastocysts and 12 minutes for cleavage-stage embryos. As there is no minimum time, you can proceed to the VS step when the volume of oocyte/embryo recovers to the original volume, the volume before the start of freezing, even if the time is less than the maximum time. On the other hand, even if the volume has not completely recovered, be sure to proceed to the VS step when the maximum time is reached.
It is because the volume per cell is smaller in the cleavage stage as compared with an oocyte. Although blastocysts are much smaller cells, 15 minutes, the same time as oocytes, is required due to the presence of the blastocoel.
If the volume has returned to the original volume at 8 minutes, you can proceed to the VS step. When proceeding to the next VS step without waiting until the maximum immersion time, be sure to wait until the maximum time unless the volume has recovered to the original volume.
As ES contains only permeable CPAs, no shrinkage occurs if the rate of efflux of water from the cells is equivalent to the rate of influx of CPAs. However, as the rate of influx of ES is slower than the rate of efflux of water in reality, efflux of water occurs first and influx of CPAs catches up later, resulting shrinkage of oocytes/embryos.
For blastocysts that were shrunk by pre-shrinking, wait up to 15 minutes before proceeding to the VS step.
No. In the VS1 step, proceed to VS2 when oocytes/embryos stop floating. In the VS2 step, confirm re-shrinking of oocytes/embryos and proceed to loading. You do not need to worry about time in any step, so please focus on observing oocytes/embryos.
Remove the oocytes/embryos and place them slightly away from the previous positions on the bottom of the well again. Perform until the oocytes/embryos stop floating.
Re-shrinking occurs due to increased CPAs concentration and osmotic pressure. VS contains trehalose with a large molecular weight; because it cannot pass through the cell membrane, it does not re-penetrate, leaving oocytes/embryos shrunk. Using this principle, vitrification can be realized even if the CPAs concentration in the solution is not actually 50% but the concentration in the cell is at that level.
Substances with a molecular weight of up to 400 can penetrate the zona pellucida, so trehalose with a molecular weight of 340 will normally penetrate. However, in the case of blastocysts, the zona pellucida is softened by the enzyme secreted at the time of expansion, which can result in deformation at the time of freezing, so there is no problem. However, in the case of oocytes, there may be abnormalities such as that the structure of the zona pellucida is weak and cannot withstand changes in osmotic pressure, or that the permeability is low due to small holes in the zona pellucida. At present, it is not known whether the presence of such zona pellucida affects subsequent culture results and pregnancy rate.
Basically, if shrinkage is seen in ES, shrinkage should also occur in VS. However, it may be difficult to see depending on the angle of oocytes/embryos. So, stir the surroundings with a Pasteur pipette and check them from multiple directions.
Although vitrification freezing itself is possible, osmotic shock is relieved by equilibration with ES once for safer cryopreservation.
If it is possible to freeze them on separate Cryotecs, it is preferable to work on them individually. However, if you freeze oocytes/embryos on the same Cryotec, work on them all at once when the last oocyte/embryo was recovered. If you wish to freeze more than one oocyte/embryo at the same time while identifying each oocyte/embryo, pay attention to identification management by finely adjusting the time interval for immersion in ES.
The Cryotec sheet has no coating, so there is actually no problem in loading on either side. However, since all institutions are instructed to use the side with the logo facing up, it is possible to know the location of oocytes/embryos no matter who performs thawing operation. Please unify within the clinic which side is to be loaded. If the opposite side is used, be sure to inform the transfer destination in case of patient transfer.
A droplet diameter should be 300 to 700 μm. As the width of Cryotec sheet is 1400 μm, it should be approximately 1/3 of it at minimum and half of it at maximum.
One droplet must contain one oocyte/embryo. If more than one oocyte/embryo entered the same droplet, be sure to retrieve the oocyte/embryo and make a different droplet in a different place on the sheet.
On the assumption that the patient for whom more than one oocyte/embryo is loaded on the same sheet requires no identification of each oocyte/embryo, the cooling rate may be reduced because the size of the droplet may become larger than the appropriate size in a state where more than one oocyte/embryo enters the same droplet, and the surface area in contact with the solution per oocyte/embryo may become smaller if more than one oocyte/embryo is placed in the same droplet. Each oocyte/embryo must be placed in a different droplet to optimize the vitrification conditions.
If the droplet size is too small, surface tension causes damage to oocytes/embryos. If it is too large, the cooling and heating rates decrease, which may result in the formation of ice crystals or cracks in the droplet. Please follow the droplet size for safer vitrification freezing.
Retrieve the oocyte/embryo and place a different droplet at a different place on the sheet.
Due to temperature difference from liquid nitrogen, liquid nitrogen bubbles will be generated around Cryotec. To reduce and prevent the decrease in cooling rate, shake Cryotec gently when loading into liquid nitrogen.
We recommend that the cap be grasped and submerged with tweezers after loading and attached after cooling. If you place the cap in liquid nitrogen in advance, submerge it in liquid nitrogen and place it against the wall at an easily visible place (e.g., corner) so that the cap can be found easily and smoothly.
When closing the cover cap, make sure that no bubbles come out from the cover cap. If any bubbles remain, the cover cap can easily come off due to the pressure. After attaching the cover cap with tweezers, close the cover cap tightly with your fingers again so that it will not come off easily.
Since the cover cap is cylindrical, it is difficult to fully tighten the cover cap only at two points of contact with the tweezers, which cannot apply sufficient force in all directions. In addition, if you try to close the cap tightly, excessive force will be applied to these two points, which may damage the cap. Always use your fingers to tighten properly without loosening.
It is possible to tighten the cover cap by pressing it against a cooling rack. However, excessive force may be applied and cause a small crack in the cap without realizing it. In this case, when the cap is removed from liquid nitrogen for thawing, there is a risk that the cap may burst due to minor vibration or temperature change. Basically, please tighten the cap with your fingers for safety reasons. If it is inevitable to use other methods, please perform carefully.
