Session 29: Cryopreservation Triage
Questions for Dr. Jeremy Chang
- Raifa Zody: What is the ideal ICSI time after oocyte thaw?
- How important is the ambient temperature of the lab during Vitrification? And warming? Same temperature?
- Answered live
- Dr. Chang: The temperature is the major factor that can affect osmosis pressure and diffusion rate. If the temperature is higher, then the rate of water movement will also go higher. Therefore, the minimum required time for dehydration and rehydration will be varied, if the ambient temperature changes.
- What is the optimal time after egg retrieval to freeze oocytes? Do you wait or freeze immediately after retrieval and stripping?
- Abdulghaffar Hussin: how do you deal with zona thickness before vitrification
- Dr. Chang: We did not do any special treatment for thicker ZP embryos/eggs.
- Abdulghaffar Hussin what is the best definition of embryo survival rate
- Dr. Chang: For 2PN and cleavage stage embryos, we use intact cell membranes as the indicator to tell the survival. For blastocyst, we also use cell membrane as the major indicator to tell the survival. However, in some occasions, if it is hard to tell the survival by the blastocyst membrane, then we would culture the blastocyst for a while to check the re-expansion for its survival.
- Mitch Schiewe Isn't it true that the critical temperature to be concerned with to ensure that metastable glass transition is maintained is the Tg to Temp. of heterogeneous ice formation (Th; approx. -130 to -55C)?
- Dr. Chang: The -15 degree C freezing/melting point for the vitrification solution was based on the calculation of 30% EG.
- Jayaprakash Divakaran: whether the volume of Equilibration and vitrification media volume affect the survival?
- Dr. Chang: In theory, it may not affect survival. However, practically, there are some other factors that you may want to consider such as evaporations of the ES and VS solutions, the CPA concentration could be diluted by the introduction of the embryos (if high embryo number and carryover media are enough to reduce the CPA concentration).
- Muhammad Anzar: Good talk...Is epigenetic associated with permeant CPAs or process overall?
- Dr. Chang: DNA methylation and histone modifications are two epigenetic modifications that alter the functional state of chromatin and activate or repress gene activation. In animal models, the alteration of epigenetic status of oocyte can be originated from the vitrification and warming process which includes but not limited to the exposure of cryoprotectants and chilling effect (Yan, Yan et al. 2010; Spinaci, Vallorani et al. 2012; Riesco and Robles 2013; Cheng, Fu et al. 2014; Saenz-de-Juano, Penaranda et al. 2014).
- Min Xie: How long should we leave biopsied BC in the ES when using Kitazato vitrification kit? Or how long to wait after biopsy to begin vitrification
- Dr. Chang: There is no standard answer, if you want to have an optimized protocol. Most of the current protocols that commercial companies have usually can provide sufficient time for CPA to dehydrate the embryos. However, they may not be the “best” protocol for every embryo/egg, because the permeability of cell membrane for each embryo/egg may not be the same. Therefore, some of the embryos/eggs may be over or under equilibrated during dehydration, as well some of them may be over or under diluted during rehydration. I am thinking if there is a way to use morphokinetics data with AI to optimize the protocol in the future (in my last slide).
- pentti koivisto: Is active volume reduction of blastocysts beneficial before vitrification ?
- Dr. Chang: The purpose of the volume reduction is mainly to improve the CPA dehydration process and to increase the heat conduction during the vitrification process. So, in theory, yes, it may help. But, is it necessary for every blastocyst? It depends (taking into account the cavity size of blastocoel).
- Fouzia Chouli what is the storage time of the vitrification kit after opening if the storage conditions are well respected; 7 days or more (kitazato or fertipro)...
- Dr. Chang: According to commercial kits recommendations, it is 7 days. The reason for that is the pH of the solutions may have drifted after you opened the bottle. Therefore, you can measure the pH change after you opened the bottle and track the pH change over time. You will find the answer that suits you the best.
- What temperature do you keep your lab at? If you just want to follow the protocols from the packet inserts?
- Dr. Chang: At RBA, we keep at 24-25 degree C as our room temperature.
- Estrella Risemberg: Thank you Dr Chang for your talk. Please can you share which is your thawing protocol
- Dr. Chang: Warming solution 4ml 37 degree C; Dilution solution 1ml 25 degree C; Wash solution 1ml 25 degree C
- Ana Paula Sacur Silvestre: How about the protein, do you add hsa?
- Dr. Chang: Yes, we do add protein in the solutions. We add 20% SPS .
- Mark Larman: Dr Chang-is there any difference between using trehalose and sucrose?
- Dr. Chang: Personally, I have not done the comparison before. But, if we consider sucrose and trehalose both of which cannot permeate through cell membrane, then their major function is to create the concentration gradient to dehydrate the cell. Therefore, it will not be surprising to see the difference indistinguishable (my personal opinion).
Questions for Dr. Ana Cobo
- Teresa: Do thawed oocytes have the same developmental potential as fresh oocytes? What we see sometimes is that the thawed oocytes develop at a much slower rate & form only a few blastocysts compared to fresh oocytes. Do you think this is a technical problem or an oocyte quality problem? The survival rate of the thawed oocytes are good
- Mitch Schiewe: Does IVI have any experience trying to validate an effective aseptic closed vitrification system, similar to reports with VitriSafe or HSV devices? Do you have any comparative experience?
- Peter Nagy: Open v. closed systems?
- Peter Nagy: Fresh v. frozen transfer?
- Peter Nagy: What do you think lies in the future of cryopreservation other than automation?
- Yu Sun: vitrified vs. fresh are similar -- makes sense; what underlying mechanisms can cause better results from vitrification in certain cases?
- Fouzia Chouli: what is the upper and lower limit value of progesterone on the day of the vitrified embryo transfer
- Peter Nagy: most clinics do not measure P in "frozen embryo" transfer cycle; - typically they look at endometrial "lining" / thickness
Questions for Professor Anja Pinborg
- Peter: Birth weight between slow frozen vs vitrified embryos.
- Peter: What are your thoughts on the JAMA study that discussed cancer outcomes in FET cycles?
- Liesl: Is there any data on long term storage in vapor vs liquid nitrogen and the outcome from these eggs and/or embryos?
- What is each speaker's opinion on the advancement of automated systems?