Selecting embryos for better success

Ana García Sifre

Embryo Implantation, Embryo Transfer, IVF laboratory, Success Rates

From this video you will find out:
  • What are the three main parameters guiding the selection of the best embryos for transfer?
  • What is embryo morphokinetics?
  • What do fragmented embryos mean, and why do they fragment?
  • What are the 4 stages of embryonic development?
  • What is the structure of the blastocyst?
  • Can a blastocyst be abnormal?
  • What percentage of Day 5 blastocysts are euploid?

How embryo selection can increase pregnancy rates?

In this webinar, Ana García Sifre, Embryologist at IVF-Spain, Alicante, has talked about selecting and transferring the best embryos to achieve the highest chances of success. She delved into the significance of identifying the best embryo grade for optimal IVF outcomes.

Ana started with explaining embryo selection, where three main markers are used:

  • embryo morphology
  • embryo kinetics
  • embryo genetics


The morphological criteria are the most commonly used, and it is essential to evaluate the embryo quality. The kinetic criteria talk about the timing, the divisions are taking place at embryo’s development and mixing these. We obtain the morphokinetic criteria, in which we take into consideration the time for the divisions to take place and the quality of the embryos have at the specific point of their development. We also have embryo genetics, which in the latest years has become quite important. By analysing embryo genetics, we can know if the chromosomally normal embryos will increase the chances of achieving positive pregnancy in a short period.


To assess the embryo kinetics, we need the help of time-lapse incubators. These kinds of incubators provide a stable environment for cultural conditions. They have individual chambers for each patient, and pictures of the embryos are taken every 5 minutes. With this information, we can analyse the morphokinetics of the embryo and consider the synchrony of cell division, the cell cycle duration and direct cleavages. These markers help us to predict the implantation potential and make a better embryo selection.

Embryo timing

We say that the time 0 will be when the spermatozoa and the oocyte are fused, this happens the same day as the egg retrieval and normally three or four hours after that. 16 or 18 hours after this union takes place, we will consider it day one of development. On day one, we will see if the oocyte has been fertilized or not and if it is normal or abnormal fertilization. At this stage, we normally contact the patients from one lab and let them know about the number of eggs that we retrieved and are mature and the ones that we have successfully fertilized. This will be the number of all embryos that we will follow during embryo development. Then, they too will take place at 41-45 hours post-insemination We already see embryo lipids, and we can see a certain number of cells and different fragmentation rates depending on the quality of the embryo. It occurs at more or less 70 hours post-insemination. This time can vary between the different labs and due to different culture media conditions. Each lab sets its standard timing considering the conditions that they have. Then, when we have day-4 of development, we can already see the cells compacting. This normally happens 94-98 hours post-insemination. Then we have a day-5 or day-6 embryo, which becomes a wider range because here we have to analyse the particular structure, and the time frame can vary between 110 hours and 134 hours post insemination.


Regarding the morphological assessment at day-2 and day-3 of development, we classify the embryos considering the number of cells they have and the percentage of fragmentation they present. Optimal development is considered to have 4 or 5 cells on day-2 and between 7 and 8 cells on day-3. The fragmentation rate goes from 1 to 5.

  • Grade 1 & 2 — 0% of fragmentation to less than 10%
  • Grade 3 — between 10% and 25%
  • Grade 4 — 25% to 50%
  • Grade 5 — above 50% of fragmentation

When the cells split, they sometimes release remains cell debris with no nucleus that they don’t need. What happens is that the more fragments releases, the more difficult it is for the embryo to keep developing until the blastocyst stage, which is the current destructor.

This means that if there is a grade 3 embryo on day-2 of development, the chances to form a blastocyst are lower, but they also can manage to remove these fragments and still form this structure with the remaining cells.

During these days, we will classify the embryos with the number of cells they have and the number according to the fragmentation rate. We always say that:

  • Grade 1 or 2 — is a good quality embryo
  • Grade 3 or 4 — medium quality
  • Grade 5 — once we arrive at day 5 or 6 of embryo development, we see a completely different structure called a blastocyst, it’s more complex

It is possible to define 3 main parts:

  • the trophectoderm, which is the layer of cells that will become the placenta
  • the internal cell mass, which will be the embryo
  • pellucid zone, which surrounds the oocyte from the beginning and surrounds the embryo, this cell will become thinner and break at some point, so the embryo can come out and implant in the endometrium

Classification of blastocyst

Ana mentioned that they use 3 different markers to assess the morphology, at this point the expansion grade, the quality of the internal cell mass, and the quantity of the true factor.

The expansion rate goes from 1 to 6 when the pellucid zone has broken, and the embryo is completely outside this membrane.
When we have a blastocyst classified as a 1, it means that it’s an early blastocyst, and sometimes it makes it difficult to differentiate from the cell and some bigger cells of the trophectoderm. That’s why we prefer to have a more expanded blastocyst to provide a good assessment of the quality of the trophectoderm and also of the inner cell mass. Then the inner cell mass and the trophectoderm goes from A to D. A being the best quality and D the worst.

In our laboratory, embryos graded as A or AB or B will be transferred and cryopreserved. Embryos graded D will be discarded, whereas C graded embryos have a wide range of subcategories, and the final decision may vary. That’s why every single embryo is assessed individually. It is important to point out that different labs have different criteria and can be more or less strict when classifying embryos. This is just a simplification, and embryos are truly more complex. In the end, it is important to trust in the decision that the embryologist made and not compare it with other patients situations, which probably will not be the same as yours.
Ana emphasized that every embryo needs to be assessed individually.

Case study

Ana also presented a clinical case performed in her lab where the day after the egg collection, 9 oocytes were retrieved, they were placed in the time-lapse incubators, so they were able to follow the divisions of all the embryos. They divided at day -2.
On the slide the qualities of the embryos at day-3 is visible, there were 6 embryos with A quality, 1  was classified as AB quality embryo, and 2 more were classified as C quality embryos.

So how do we select a day-3 embryo? There is timing involved and also the fragmentation rate which will help to select the best quality embryo out of the ones classified as an A, but it’s still difficult to decide which is going to be the best.
What happens if we go to a day — 5 embryo transfer? This decision becomes easier, we get much more information at this stage. Some of the embryos that had good quality on day-3 did not manage to get to the blastocyst stage. As shown on the slide, the embryo day-4 and 4 from this patient. We can decide which embryo will have a priority for transfer. In this case, we have transferred the last embryo, then embryo number 9 and then we also cryopreserved 3 more blastocysts.

Another example shows a woman who had 4 embryos correctly fertilized on day-1 which divided and were classified as day-3  as one quality A embryo, two B qualities, and one C quality embryo.   There’s no discussion in this situation, we will choose the embryo graded as an A to transfer, but what happens if we keep this embryo in culture? Even if they had good quality on day-3 when we kept them in culture until day-5, none of them become a blastocyst. This can also happen, the quality of the embryo on day-3 does not imply that they are going to be of good quality on day-5.
Two examples of presenters help to understand why we strongly recommend doing day-5 culture. The information it provides is relevant and important.

Most of our transfers nowadays are performed on day-5.

PGT-A for embryo selection

We can see morphology and timings, but even if everything looks okay in the embryo, it’s possible that the embryos are not molecularly right, and they can have some sort of chromosomal problems, which is not visible in any way. We can analyse this by doing PGT-A testing.

On the presented graph, you can see that at the age of 35-36, the euploidy percentage dramatically decreases. With the genetic selection, we can choose chromosomally normal embryos to transfer and avoid transferring embryos that may not be euploid. By doing this, we reduce the number of transfers and prevent transferring embryos which will lead to a negative pregnancy or even a miscarriage. How is the biopsy done in the lab?

To perform the biopsy, we need to have an expanded blastocyst, big enough to take some cells out of the trophectoderm without harming the embryo. We do it with a laser that helps us to cut these cells and then this is sent to the genetic lab which will analyse them. Once we finish the biopsy, we will try to preserve them until we have the report from our genetic lab, which usually takes around 10 or 20 days. Then with this information, we can plan the following embryo transfer if the embryo results are normal.

If all the embryos come out as abnormal, in some cases of previous miscarriage or implantation failure, we can presume that this could be the issue. This will give us a lot of information to plan a different treatment for the future and maybe move on to an egg donation program that will be more accurate in this situation.
Considering all these main factors, Ana suggests that:

By doing day-5 embryo culture, having the optimal conditions in the lab with the help of the time-lapse incubator that allows doing morphogenetic analysis, and by doing PGT-A testing, we can help to make a better embryo selection. That way we can transfer the best embryos, and this will be directly related with the successful treatment.


- Questions and Answers

Do you have any experience using embryo culture that uses GM-CSF? EmbryoGen/BlastGen are the ones my clinic uses.

We don’t use this kind of media at our clinic. Each clinic uses different protocols in its daily routine. In our clinic in Alicante, for example, and the one that we have here (Manchester), we always access every media that we use, and we validate them, but unfortunately, I don’t have any experience with the ones you’ve mentioned.

In your experience, do you think PGT-A improves pregnancy rates?

Yes, I do. PGT-A gives us relevant information on what kind of embryos we have, if we have aneuploid embryos, and we transfer them, we know that the result is going to be a failure in the pregnancy. If we have this information, we can just select the embryos that we know are going to have more chances to become a positive pregnancy. Every clinic is going to say that, and it’s really important. It also depends on the age, but we always recommend it.

Is embryo fragmentation bad for the success of an embryo?

If the fragmentation rate is high, the embryo is not going to develop to the blastocyst stage, so it’s not going to be a successful embryo, it’s not going to create this successful structure that we want. If the fragmentation rate is not as big, they can manage to form this structure anyway, and they can also result in a positive pregnancy. We have a lot of cases that it has already happened, but the fragmentation rate will affect the quality of the embryos.

Are frozen embryo transfers more successful than fresh embryo transfers?

We have almost the same success rates with frozen embryo transfer and fresh embryo transfer. I would say we have like a 99%  of the viability of when we thaw the embryos, so they have almost the same quality. The success rates are quite similar, at least in our clinic.

Is an embryo frozen on day-3 not better than one frozen on day-5?

Not better, as I said, it can be the same, but it isn’t better to do it on day-3. If we froze the embryos at day-5, this embryo at this stage has much more cells, they are formed by hundreds and hundreds of cells, whereas  day-3 embryos have normally around 8 cells. If we miss one cell in this one cell is that in this process of cryopreservation, it will imply more damage in the day-3 embryo than it will do on day-5. I would always recommend going to day-5 and then freeze the embryos and then do the transfer.

Do you freeze all embryos or only high-grade embryos?

We just freeze or transfer good quality embryos grade A or grade B, we always freeze them. If we don’t think an embryo has any possibilities of becoming a pregnancy, we will not freeze it.

Could picking a good sperm with PICSI improve the embryo result if the egg is not the best one?

If the egg quality is not the best, PICSI is not going to improve the outcome of the treatment. PICSI is going to help when the quality of the sperm is not so good, but if we have an oocyte problem, it’s not going to help. We can choose a better sperm and try our best, but it’s not going to change the quality of the oocyte.

I heard that embryo development stops on the on day-4 day, and that is often caused by sperm quality, is that true?

The male factor starts to appear on day-3, it becomes part of the embryo on day-3. It’s true that at some point if we, for example, see that the embryo has good fertilization, good quality on day-2, good quality on day-3,  but suddenly something stops on day-3, after day-3, therefore, we can suspect that this can be related to the male factor. This isn’t going to be the only option, but yes, it can be related to the male factor, when we see that all the embryos are stopping on day-3 or day-4 of development.

I have heard that a bad looking embryo could result in a healthy baby. What are your thoughts on that?

I have seen it, not just heard about it. I have seen that embryos that don’t look as they are of perfect quality, let’s say we have a 3 BC embryo, which is not the best, but still, they have the instructors that we need, and we have transferred them, and we have babies, and we have pregnancies. That’s why we don’t discard any embryos. The optimal embryo to have will always be a 5 AA, 4AA, but sometimes we also have 5,3 3, 3, 45 BC quality embryos, which led to a positive pregnancy and a healthy baby. 

There are different criteria between labs in what to keep and want to discard when we assess the embryo, but we always try to get all the information that we get from the embryo and keep the ones that we think are viable, even if they are not of the best quality.

Any advice on improving implantation?

If we’re talking about the embryos to improve the implantation, I would recommend doing PGT-A testing, I don’t know if in your previous attempt you have already done it or not, but the implantation failure most of the time is related to abnormal embryos. If we know that the embryos are chromosomally healthy, we can just try to deal with this problem. 

If we’re talking about endometrium and the capacity of the endometrium to the implant, there are a lot of tests that you can always do such as the ERMAP test. This is a more clinical part, and it’s better to get advice from the doctor, but there are some tests that you can do to improve the implantation potential.

We are in a donor cycle with donor sperm. What is the quality of frozen sperm versus fresh?

When we talk about donor sperm, we cannot compare frozen sperm versus fresh sperm just like that because when we freeze the sperm, some sperm cells are going to die during this process, and if in the fresh sample we have 50 million,  in the frozen one we will have 30 possibly. It doesn’t mean that the quality is worse, sometimes if we just check the numbers, it seems that I have this frozen donor sperm, and it says that it has 10 million, don’t worry because the majority of the banks had wonderful criteria to select the donors that they use, and they have to fulfil this, they have to have normal sperm and this is not going to be a problem to use frozen sperm.

If it comes from the partner’s sperm, it’s a little different. If the fresh sperm quantity or quality is limited, we will always recommend using fresh sperm because we cannot lose these in the cryopreservation procedure.

Do good looking embryos mostly mean good quality, meaning healthy embryos?

The euploidy and the morphology are not related. If we have a good quality embryo, a 5AA quality embryo, it doesn’t guarantee that it’s going to be euploid, we have nice embryos that after the PGT-A test results, we see are aneuploid, so this is not easy to relate. When we talk about health conditions, as I said, the only way to access that is by doing PGT-A testing, we cannot check it with the morphology.   

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Ana García Sifre

Ana García Sifre

Ana Garcia Sifre graduated in Biochemistry and Biomedical Sciences at the University of Valencia (Spain). She specialized in Human Reproduction at the University and Polytechnic La Fe Hospital of Valencia. After her training in 2018, she joined the IVF-Spain team (Former Clinic) in Alicante, where she worked as Embryologist. She speaks fluent English and Spanish.
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Caroline Kulczycka

Caroline Kulczycka

Caroline Kulczycka is managing MyIVFAnswers.com and has been hosting IVFWEBINARS dedicated to patients struggling with infertility since 2020. She's highly motivated and believes that educating patients so that they can make informed decisions is essential in their IVF journey. In the past, she has been working as an International Patient Coordinator, where she was helping and directing patients on their right path. She also worked in the tourism industry, and dealt with international customers on a daily basis, including working abroad. In her free time, you’ll find her travelling, biking, learning new things, or spending time outdoors.
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