During this live session, Carolina Andrés, Senior Embryologist at Clínica Tambre, Spain, discussed embryo development stages, why not all embryos reach the blastocyst stage, embryo grading criteria, embryo transfer and its importance, as well as advances in embryo selection.
How are embryos created? Everything starts at the moment of the pickup or the egg retrieval.
The gynaecologist will aspirate the follicles, and the follicular fluid will be passed to the laboratory, where we will examine the liquid in search of the oocytes. At this point, the oocytes have an aspect like the photograph you can see in the left corner. The oocytes are surrounded by granulosa cells, which play very important roles within the follicle.
However, in IVF in vitro, these cells are not acquired, after some time of incubation, they are going to be removed in a process called stripping. This process allows us to check the maturity state of the oocyte.
Why is the maturity state important? Because it tells us whether the oocyte is ready to be fertilized. Oocytes found in metaphase II are the ones that can be microinjected. These are marked by the presence of a polar body, which you can see indicated by the arrow.
Other types of oocytes can also be found— these are immature oocytes, which can be found in two different stages: metaphase I oocytes or germinal vesicles. It’s important to highlight that metaphase I oocytes are susceptible to maturation. This means that after the stripping procedure, while we prepare for microinjection, some oocytes might mature in vitro and transition into metaphase II, meaning they become usable.
Unfortunately, germinal vesicles are not susceptible to maturation. Additionally, we can find atresic oocytes, which cannot be microinjected because they are not normal in formation.
Once the oocytes are ready and classified, the embryologists proceed with microinjection. Oocytes that are going to undergo conventional IVF will be stripped after fertilization is done, one day later. After fertilization, embryos are cultured until day 5 or day 6. At that point, they will either be transferred, vitrified, or biopsied and vitrified while waiting for genetic results.
In some cases, we find oocytes that are not fertilized in a typical manner. We may observe 3 pronuclei. Normally, we cannot assure that the genetic content of these oocytes is correct, so they are discarded. However, in some cases where we perform PGT, they can be considered for culture.
After fertilization is checked, embryos start dividing.
During these first days, we call this the cleavage stage. Embryos at this stage are formed by individual cells.
On day 4, we expect them to transition towards the blastocyst stage. This is a very important point because this is when the genome of the embryo gets activated. Until now, the responsible factor for cell divisions was the oocyte, but from this point onward, the sperm starts playing a role.
At this stage, embryos can be at different points in development:
There are many different grading systems used for embryo evaluation. In Spain, the most typical ones are:
Embryo grading allows for better embryo selection. A better embryo selection helps improve IVF outcomes. Additionally, it helps reduce multiple pregnancies since our goal is to transfer the best embryo to achieve a pregnancy while avoiding transferring more than one embryo.
How are embryos evaluated during the first days? In the cleavage stage, different features are evaluated, including:
Is this always the case? No, this is the ideal situation, but each embryo is different. We will count the number of cells, and we may see a little more or a little less. As long as the embryo is dividing well during these first days, it gives us an idea of its quality.
What is multinucleation, and how does it affect embryo evaluation? Multinucleation is not a definitive factor in embryo evaluation, but it is something to consider. What do we normally expect? On day 2, we expect to see 2 nuclei in each cell. What happens if there are more?
When we observe multinucleation, we know that the prognosis might be poor. However, this does not mean that the embryo cannot form a blastocyst in the end.
What is unequal cleavage? Unequal cleavage occurs when a single cell divides into 3 equal cells instead of 2.
How do embryos normally divide? Embryos divide from 1 cell to 2 cells, then each of those cells divides into 2 more, leading to 3 cells, then 8 cells, and so on. What happens in the case of unequal cleavage? In some cases, one cell divides into three. This is called direct unequal cleavage.
How does this affect the embryo? This is a poor prognosis feature, but it does not mean the embryo cannot form a blastocyst. However, we take note of this for embryo selection at the end of the cycle.
During cell division, we expect a clear division of cells. However, sometimes small cellular debris appear. How is fragmentation graded? It’s graded based on the percentage of fragmented cells in the embryo:
When fragmentation is high, it becomes difficult to differentiate cellular debris from actual cells. This does not mean the embryo cannot form a blastocyst, but the chances are lower.
In a video showing embryo development from the beginning to the end and how it is dividing into 2 cells, 4 cells, 8 cells, and finally reaching the blastocyst stage.
Why are they cultured until day 5 or 6? This is because only embryos that can form a blastocyst will be selected, which means they also have a higher capacity to implant.
There are three aspects to consider when blastocysts are evaluated:
They are graded from A to D based on quality. As the blastocyst forms, it expands, getting bigger and bigger. The level of expansion tells us whether the blastocyst is at an early or advanced stage.
What are the different stages of blastocyst expansion?
Before implantation, embryos must hatch naturally. If needed, assisted hatching can be performed using a laser to help access cells for biopsy.
The first letter we see in embryo grading corresponds to the Inner Cell Mass (ICM). The ICM is graded from A to D, following the Gardner grading system:
What is the difference between Grade A and Grade B? The difference is very small. The ICM in Grade A embryos is more compact, while in Grade B embryos, it is slightly larger. Both are considered very good quality embryos. What about grades C and D? Grade C embryos have an ICM that is not well-defined or is formed by a very small number of cells. Grade D embryos have an ICM with degenerated, nonviable cells.
How is the trophectoderm graded? The trophectoderm is graded similarly to the ICM:
The Grade A trophectoderm has more cells, which are compact, while the Grade B trophectoderm has slightly fewer cells. The difference is very small. Grade C embryos have very few or no cells in the trophectoderm. Grade D embryos have degenerated cells in the trophectoderm.
Embryos graded A or B, either in the ICM or trophectoderm, are considered good quality embryos.
The embryo factor is an important feature in implantation, but it is not the only factor. There are five important factors which affect implantation:
Why do we transfer a blastocyst instead of a day 3 embryo?
A few years ago, day 3 transfer was commonly performed, and some laboratories still do it today. However, technology and embryo culture have advanced significantly. Both the culture system and the culture medium have been optimized, allowing us to extend culture and better synchronize the transfer with embryo development. For higher implantation chances, the embryo should reach the blastocyst stage in the uterus. Therefore, we transfer the embryo when the endometrium is ready and the embryo has reached the appropriate stage.
RI Witness system and advances in embryo selectionThe witnessing system minimizes errors and gives us more control over the process. How does it work? At the moment of transfer, the patient brings a card labeled with their name. The embryo is labeled with the same name, ensuring that only the correct embryo is transferred.
How has embryo selection improved? Embryo culture has improved not only due to better culture medium but also due to advancements in incubators. Time-lapse incubators allow continuous monitoring of embryos, avoiding unnecessary manipulation. Previously, embryos had to be removed from the incubator, evaluated, and returned.
The incubator now takes pictures every five minutes, creating a video of embryo development. This allows us to monitor embryos without disrupting their environment.
How has AI been incorporated into embryo selection? AI tools help us improve embryo selection by evaluating morphokinetic events during development. What does AI analyze? AI evaluates not only the embryo’s appearance but also its division speed. The AI tool provides an implantation potential score, which, combined with the embryologists’ evaluation, helps select the best embryo for transfer.
Embryo grading is essential for proper embryo selection.
Good embryo selection increases success rates, but implantation depends on multiple factors.
A personalized approach is necessary for each patient.
An experienced team of embryologists is crucial in embryo selection.
Yes, embryos graded C have chances of implantation, but the chances are lower. That’s something to keep in mind when considering the transfer. However, once the embryo has implanted, there shouldn’t be any problem throughout the pregnancy. Clinics have transferred lower-grade embryos in some cases, and they have led to healthy pregnancies. So you can feel reassured about that.
Yes, sperm quality can affect embryo development. When we see that embryos stop developing around day three or day four, it can indicate that there is something within the sperm sample affecting embryo growth. ICSI is typically used in male factor cases, but sometimes, intrinsic issues within the sperm, such as fragmentation, can impact blastocyst development. This can reduce the blastocyst formation rate.
In Spain, we don’t recommend transferring 3 embryos due to the risks associated with multiple pregnancies. Each case is evaluated individually in the clinic, but in general, it is very rare to transfer 3 embryos. The maximum we transfer is 2.
Unfortunately, I don’t have experience with these techniques as they are not possible in Spain. They are experimental procedures, and they have started being used in some cases in the UK.
A day-6 blastocyst transfer is completely viable. A grade BB blastocyst is considered good quality. While we prioritize day-5 blastocysts because they reach the stage earlier, there’s no reason not to use day-6 embryos for transfer. So you can feel reassured about that.
I am not very familiar with the C+ grading system as it is not the grading system we use at the clinic. However, the fact that it is a euploid embryo is a positive sign. Grade C embryos do have a chance of implanting. From what I understand, a grade C in this system might be similar to a lower-quality grade B in our evaluation. If it is your only euploid embryo and your clinic has decided to proceed with a biopsy and freezing, they must consider it to have implantation potential.
Yes, we do transfer two embryos, but only under individual considerations. Each case is unique, and we always discuss options with the patient. While our general policy is to transfer one embryo, in some situations, we may consider transferring two, taking the patient’s medical history into account. It’s always a personal decision.
The studies on EmbryoGlue are mixed. Some studies show that it helps significantly, while others do not find a clear difference. It depends on each patient. In IVF, certain treatments work well for some patients but may not be as effective for others. Your clinic will have experience with this type of medium and will be able to recommend whether it’s beneficial in your specific case.
We use it sometimes, but not routinely. We have our own transfer medium, but in cases where there is a history of implantation failure, we consider using a different type of medium.
It is possible, of course. We could freeze 2 embryos at different stages and transfer them, but I don’t think that would increase the chances of implantation. I would more recommend leaving the morula in culture to see if it develops to the blastocyst stage and then transferring both at the blastocyst stage instead of transferring embryos at different stages.
The decision to perform micro-TESE is something that a doctor or urologist must consider because this technique is beneficial only for a specific group of patients. They will be able to let you know if it would be helpful in your case. Regarding the sperm sample, anytime we find sperm in the sample, at our clinic, we centrifuge and concentrate the sample to reduce the volume and increase the chances of finding viable sperm.
PGT is an invasive procedure, so we must consider that some embryos may not survive the procedure itself. That’s why it is very important to select good-quality embryos, as they have a higher probability of undergoing the procedure without being affected. Once the biopsy is done, another risk is that we may end up with no embryos to transfer. However, this can be beneficial because it allows us to identify embryos that are not viable, which otherwise could lead to implantation failure or miscarriage. While this is a risk, it is also a useful outcome.
Exactly. Embryo grading is not related to the genetic content of the embryo. I always tell our patients that a good-quality embryo ensures viability during the PGT procedure, freezing, and thawing, but it does not guarantee that the embryo is genetically normal. We sometimes see beautiful embryos that are aneuploid and must be discarded.
At our clinic, we use vitrification, which is a very rapid freezing process that ensures a high survival rate after thawing. Since we usually transfer only one embryo at a time, we recommend freezing one embryo per straw. However, this depends on each clinic’s policy. It is possible to vitrify 2 embryos in one straw without any problem.
Unfortunately, this is very difficult to determine because each case is different. It is impossible to predict the exact number of embryos needed. However, given the age factor, we know that the more embryos we have, the higher the chances of obtaining a euploid embryo.
This is a good question. Sometimes, we see cases like this where, in the last transfer, the patient was lucky to transfer the right embryo. However, this is something we can never be 100% sure about.
We sometimes see embryos with high fragmentation that develop into high-quality blastocysts. However, if the embryo was not tested with PGT, we must consider that from age 36 onwards, egg quality decreases, affecting the genetic integrity of the embryo. It is possible that, in this case, the embryo was genetically abnormal, which is why implantation did not occur.
We have done this in very limited cases. In some cases, a patient may request a day-three transfer, and we leave the remaining embryos in culture until day five. However, on day five, we froze them instead of transferring them. This is not our routine practice. Our recommendation is to culture all embryos until day five because this allows for better selection. Not all embryos will reach the blastocyst stage, so the longer culture helps us transfer only those with the highest implantation potential, avoiding unfruitful transfers.
Each clinic has its own standardized culture medium optimized for embryo culture. The one we use is the one routinely used in our laboratory, but other clinics may use different media. It’s not that one is better than another; the most important part is that the embryo culture is optimized for the specific medium being used.
For PGT, assisted hatching is helpful because it allows better access to the embryo for biopsy. However, performing assisted hatching just to optimize implantation is not well demonstrated. The literature is diverse—some studies suggest it might help in frozen embryo transfers, while others are inconclusive. Our own research did not show a significant difference in implantation rates with or without assisted hatching. In specific cases, such as when the zona pellucida is very thick, we may consider assisted hatching to help the embryo hatch before transfer, but it is not something we apply to all patients.
No. The difference between conventional IVF and ICSI is the indication for each case, but the embryos generated in both cases are of equally good quality.
Initially, we did not see any differences. We have noticed that in some cases, the second stimulation is slightly better in terms of oocyte recovery than the first one. However, this can vary from patient to patient. Based on the literature, there are no significant differences between stimulations.
Yes. Normally, at our clinic, we recommend between 2 to 4 days of abstinence. This helps to avoid oxidative stress that can occur with a longer abstinence period. If the abstinence period is too short, there is a risk of losing sperm that could be used for treatment. This intermediate timing is recommended to maintain optimal sperm quality.
At our clinic, we only use PICSI, though we apply it differently. It is a medium that helps us select sperm with lower DNA fragmentation. We have seen that this approach benefits certain patients. Each case is individual, but ICSI was originally developed for male factor cases, while PICSI or sperm selection media help in cases with high DNA fragmentation.
Many factors could affect this outcome. Culturing embryos until day 5 allows for better selection. If only one blastocyst forms out of five, possible causes include compromised egg quality, maternal age, or sperm factors. Generally, we expect about 50% of correctly fertilized oocytes to reach the blastocyst stage, but this varies. In cases with lower blastocyst formation rates, we need to investigate further.
It depends on the male factor. PGT-A is primarily indicated based on maternal age, but since donor eggs are used, it is generally not necessary. However, sperm quality must also be considered. If there is a concern about sperm-related factors, PGT-A might be recommended. PGT-A is not a standard requirement for all egg donation cycles.
Poor fertilization can be associated with male factor issues if not properly addressed or compromised oocyte quality. Two pronuclei (2PN) indicate normal fertilization, but if other forms of fertilization occur, egg quality issues should be considered.
It depends on the individual case. Studies suggest that frozen embryo transfers can be as effective as fresh transfers. Some studies indicate better outcomes with frozen transfers, but this may be influenced by the fact that many frozen embryos undergo PGT, leading to better selection. What matters most is a well-prepared endometrium.
The procedure is the same, but the difference is in processing time. With PGT-A 24, results are available the next day instead of 15 days. The embryo remains in culture for 1 extra day, which carries a minimal risk, but the faster results allow for a fresh transfer instead of freezing.
The maximum age for treatment is around 50. This applies to both own egg and egg donation cycles. However, as maternal age increases, the probability of retrieving a good number of eggs decreases, so each case is assessed individually.
With donor eggs, yes. For own eggs, it is very rare because a high number of embryos are needed to find a euploid one. The main limiting factor is embryo quality at that age. In some cases, patients over 40 have had euploid embryos, but success depends on the number of embryos available.
Yes, so at our laboratory, we use the Geri incubator. Both Embryoscope and Geri are very good time-lapse incubators. In the end, each laboratory is used to working with one or the other and will have their workflow and culture optimized for each incubator. Some laboratories even have both.
Both of them work very well. I don’t fully understand the part about one individual incubator, but I believe you might mean the individual camera. It is true that with Embryoscope, they share the same environment, while with Geri, each embryo has its own incubator. That is a big difference between the two.
Maybe you are referring to the device used for culture. But in the Geri incubator, embryos are also separated. In the end, the philosophy behind Embryoscope and Geri is the same. Both are time-lapse incubators, both culture embryos the same way, and both produce a video of the embryo so we can monitor its development daily. In both incubators, the culture dishes have individual wells for each embryo. This is necessary to monitor them with the camera and to ensure that each video corresponds to the correct embryo.
PGT-A is recommended in cases of medical history. However, we also need to consider the age factor. As I mentioned before, age affects genetic content, which impacts embryo quality. From around 38 or 39 years old, we may start considering PGT.
Of course, PGT can be done earlier, but medical history plays an important role. If a patient has a history of implantation failure or miscarriages, PGT becomes very valuable as it provides more information about the genetic state of the embryos.
