Here at Kitazato, we are committed to helping you achieve the best survival results that the Cryotop® Method has to offer, so we are taking our training expertise digital.
We are excited to announce our new question and answer series!
These sessions will take place on Instagram Stories every Thursday at 12 pm CET now through early May.
In each one, our experts from the Kitazato Vitrification Training Team will be answering all your questions on the weeks given topic. Follow us on Instagram to join the conversation! Send us your questions, and don’t miss the chance to clear up any doubts you may have with the help our expert embryologists.
The first session was held on Thursday, March 26th, where we discussed high cooling and warming rates and the effects of media composition with our senior embryologist Gisela Maggiotto.
Successful Vitrification Q&A Series
If you missed any of our sessions, click on the links below for a video of each of the Q&A’s from Instagram Stories:
Session 1: High Cooling & Warming Rates and Effects of Media Composition– March 26th
Session 2: Tips & Tricks for Best Survival and Clinical Outcome – April 2nd
Session 3: Social Fertility Preservation – April 9th
Session 4: Preservation for Medical Indication – April 16th
Session 5: Egg Banking for Ovum Donation – April 23rd
Session 6: Contribution to PGT Programs – April 30th
Session 7: Deferred Embryo Transfer & Freeze All Cycles – May 7th
Follow us on Instagram, if you haven’t already!
You will find the frequently asked questions from each of our sessions bellow. We hope to clear up your doubts!
Session 1: High Cooling & Warming Rates and Effects of Media Composition
Why is Kitazato’s protocol done at room temperature?
Permeability and pathway of water and CPAs through the cell membrane depends, among other factors, on the temperature. Kitazato’s media composition work effectively at room temperature allowing a correct exchange between water and CPAs and preserving an intact cell membrane. The only step performed at 37ºC is the very first step of the warming procedure. The difference in temperatures from the liquid nitrogen at -196ºC to the first solution at 37ºC is the so-called “warming rate” which is essential for the later outcome.
What is the function of hydroxipropyl cellulose in the media?
HPC replaced the albumin as the protein source. HPC-supplemented vitriﬁcation solution shows higher viscosity and gives higher survival rates avoiding viral and endotoxin contamination risk. It also reduces mechanical stress during warming and increases survival rate in hatched blastocysts.
Do you think the warming rate is much more important than the cooling rate?
Both parameters are important for the vitrification outcome but being able to guarantee a very high warming rate will avoid lethal recrystallization during the warming procedure. Following Kitazato’s vitrification & warming protocol, you will assure both high cooling and warming rates.
Kitazato media has DMSO and I heard it is toxic for cells
DMSO is nowadays one of the most widely used permeant CPA as it has shown its effectiveness not only for embryo but also for oocyte vitrification. In combination with EG, Kizatato media, assures less toxicity and the best outcome after warming.
SESSION 2: Tips and Tricks for Best Survival and Clinical Outcomes
How many oocytes can be vitrified at the same time?
Following the protocol the maximum number of oocytes that could be vitrified at the same time is 16. All together would be equilibrated in the same Reproplate´s first well and then, at minute 12 we would consecutively start to vitrify taking a group 4 oocytes at a time. We advise to keep under control the time in VS no less than 1 minute and never reuse the VS solution. Use a new line per each group.
What do you think is the most critical step during the vitrification
The most critical step is the time exposure to VS (1’) due to the high CPA concentration.
How much volume is recommended to load on the Cryotop?
The cells have to be loaded with the minimal amount of solution to assure a high cooling rate however, if we exceed the limit, it could be harmful for the cells, which will remain hardly stuck to the Cryotop when warming. We recommend loading minimum volume to assure a high cooling rate but without drying too much to prevent stickiness and mechanical stress of the oocytes/embryos during warming.
When plunging the Cryotop in LN2, why do I have to shake it?
To remove out the bubbles and increase the cooling speed.
Once the specimens are vitrified please pay special attention during storage of the Cryotops, quick expositions to room temperature leads to ice formation and provokes cellular damage.
Do you recommend to artificially collapse blastocysts before freezing?
With Kitazato media there is no need to artificially collapse the blastocysts prior to vitrification. Survival rate for expanded blastocysts is as high as 98%.
How many maximum & minimum oocytes/embryos can be vitrified on a single crytop?
Minimum 1 and maximum 4 per cryotop
How long do I have to keep a blastocyst in culture after warming to perform ET? What parameter do I have to look at?
It is recommended to wait between 2-4 hours after warming. Blastocoele re-expansion degree is the most important morphological predictor of success, even more than the TE or ICM grade.
SESSION 3: Social Fertility Preservation
Which is the best age for fertility preservation?
It has been widely demonstrated that women’s fertility dramatically decrease from the age of 35. Nowadays the women who decide to preserve their eggs are late 30s or early 40s. So, according to many studies, women should be encouraged to vitrify their eggs under 35 years old, due to a better quality of them.
How many eggs are necessary for better results?
According to a large retrospective study, it was demonstrated that oocyte number combined with the age is closely related to the success rate. In fact, in young elective patients (under 35 year) with 10-15 oocytes the cumulative probability of having a baby is between 45%-70%, while in patients over 35 with the same number of oocytes the success rate decreases between 25 and 40%.
How long eggs can remain frozen? Does Kitazato suggest any longevity period?
Many studies have shown that there is no negative relationship between storage time and survival. It was shown that surviving oocytes are able to conserve their potential to develop into competent embryos even after a long storage period.
SESSION 4: Preservation for Medical Indication
Why cancer treatments can cause infertility?
Chemotherapy, radiotherapy, surgery, or a combination of these treatments can induce premature ovarian insufficiency because the ovaries are very sensitive. The damage will be related to the ovarian reserve, which can widely vary from one patient to the other.
Do you recommend to vitrify oocytes or ovarian tissue in oncological patients?
In general, the recommendation will depend on the disease, the age, the timing, and the expertise of the center to perform one technique or the other.
Oocyte vitrification is the standard method for Fertility preservation in women facing benign diseases and for those who can safely postpone the cancer therapy. On the other hand, ovarian tissue cryopreservation is mostly indicated for pre-puberal girls and women who require immediate oncological treatment.
What do you think about Fertility Preservation in women with endometriosis?
Oocyte vitrification is highly recommended in patients with endometriosis before their surgical treatment. Young women are the best candidates for this approach since they will need fewer stimulation cycles due to a better ovarian reserve and better reproductive prognosis. If there is no previous surgery there is more chance to retrieve a higher number of oocytes and finally achieving higher cumulative live births rates.
Can we perform ovarian tissue cryopreservation together with oocyte vitrification?
OTC followed by immediate ovarian stimulation and oocyte retrieval for vitrification, gives a greater chance of success in young patients, as long as chemotherapy can be safely delayed.
SESSION 5: Egg banking for ovum donation
What is the best strategy, using fresh or vitrified oocytes?
There is evidence showing that there are no significant differences between fresh and vitrified oocytes in egg donation programs, however the use of frozen donated oocytes has allowed the following benefits by overcoming the limitations imposed by fresh eggs.
• Easier management of the cycles. No need for synchronization between donor and recipient.
• Reduce the waiting lists
• Set embryo transfer dates. Patients feel much less stressed and more confident and comfortable.
What is the best strategy undergoing an oocyte donation treatment using a cryo-bank?
Two strategies are mainly carried out using cross-border ovum donation cycles:
• Inseminate fresh donor oocytes with the recipient partner´s cryopreserved sperm and produce embryos that then they will be vitrified and shipped back to the IVF centre to conduct vitrified warmed embryo transfer.
• Importation of donated vitrified oocytes, which are then warmed to undergo ICSI with the partner´s fresh sperm and extend culture until blastocyst stage, then set a SET.
Choosing one or the other should be individually considered in each case.
How many donated vitrified oocytes should be allocated to each couple in order to maximize the clinical outcome?
Higher number of oocytes warmed per cycle involves a more confident success strategy however, some studies have shown that an average of 7 up to 9 donated vitrified oocytes was associated with a cumulative live birth delivery rate of >50% per warming cycle. This is a reasonably good result in terms of balancing the costs of the treatment with a thoughtful cycle management plan.
How many embryos are usually obtained and transferred in ovum donation?
After survival, the oocytes should be fertilized correctly with the partner’s or donor’s sperm and develop to good blastocysts and the number of embryos obtained will depend on many factors. To ensure the most suitable and efficient strategy a single blastocyst transfer is strongly recommended for preventing the establishment of multiple pregnancies. The supernumerary viable embryos should be cryopreserved for future attempts if the first one fails or for a second baby.
Is the vitrification process safe for an ovum donation program?
The vitrification procedure in the Egg Banks is performed by highly qualified and trained embryologists using vitrification products certified by the corresponding regulatory authority to ensure efficiency and safety of its use. The Cryotop Method has been widely proved by offering the highest survival outcomes and that is why Kitazato has become the product of reference in the eggs banks worldwide providing high quality service with the maximum guarantee of success.
Ovum donation´s success requires that both the Egg bank and the IVF Center work together to guarantee high survival rates after warming. For that purpose, some Egg Banks offer warming hands-on training to their collaborator clinics in order to guarantee a successful survival rate.
SESSION 6: Contribution to PGT Programs
What about the influence of the blastocyst re-expansion on the outcomes?
The degree of re-expansion is considered one indicator of the recovery status for frozen blastocysts and some studies have shown that fast re-expanding blastocysts have superior pregnancy rates. But, other studies also demonstrated that, correcting for pre-vitriﬁcation features, this predictive power becomes negligible, for untested and euploid blastocysts. So, they support the idea that embryo characteristics before the freezing are the most indicative of their implantation potential.
Can the biopsy affect the blastocyst survival after warming?
Many published data of several important groups which daily implement PGT programs, confirm the high reliability of vitrification and biopsy technique. But it must also be said that a lower blastocyst survival could be associated to a poor quality pre-vitriﬁcation and a slower embryo developmental rate.
Is it safe to biopsy a blastocyst twice in terms of outcomes?
The data reported to date are concordant that no harm seems to derive from a re-biopsy and a following vitrification-warming cycle. The implantation rate of these survived blastocysts, according to several reports, range between 38–57%. This evidence, suggests that human blastocysts are resistant to several sources of stress, supporting also, the safety of vitrification. So, re-vitrification and re-warming is an option and the reproductive outcomes don’t seem significantly compromised. As long as the blastocysts subject to more manipulations have not a poor quality prior to vitrification and slower development.
SESSION 7: Deferred Embryo Transfer & Freeze All Cycles
What is a freeze-all policy about?
Freeze-all policy consist on the cryopreservation of an entire cohort of embryos to be transferred later during a natural cycle, or during a cycle with hormonal replacement for endometrial priming. The aim is to provide a more physiologic environment for ET, free of the adverse effects of gonadotrophins. Additionally, cryopreservation of all embryos allows embryo banking for fertility preservation in women with POR.
For which patients the freeze-all indicated?
The freeze-all strategy can be indicated when a fresh transfer is not advised due to some effects of the stimulation protocol. This is the case of patients at high risk of hyperstimulation syndrome, high progesterone level on the day of trigger and endometrial abnormalities.
It is also an alternative to cycle cancelation among poor responders when a fresh transfer is not advantageous.
Does Freeze-all reduce cancelation cycle?
Cycle cancellation is a major concern in all IVF programs, in terms of emotions and costs to the patients. For poor responders, this risk is much higher than for normo responders, due mainly to the lower number of oocytes retrieved. That is why a freeze-all strategy by preserving a larger number of oocytes or embryos after consecutive stimulation cycles is a good approach for this type of patients.
Freeze-all policy does not affect the survival results due to the vitrification technique?
The risk for cycle cancelation due to cryopreservation injury has become extremely low. Clinical outcomes from frozen-thawed ET have been shown to be higher in terms of pregnancy rates and implantation rates compared to controlled ovarian stimulated cycles. The freeze-all detrimental effects are more related to the stimulation protocol rather than the vitrification technique itself.
Isn´t it better to cryopreserve embryos rather than oocytes?
Even though embryos achieve better survival rates compared to oocytes after vitrification, for patients who benefit from a freeze-all cycle, both strategies, either by accumulation of oocytes or vitrification of all embryos may lead to a higher implantation rate when compared to fresh transfers in stimulated cycles.