Until a few years back, it felt as if knowing how to do good ICSI, is all that matters in embryology. That, to disappoint some of you is the ‘biggest myth’. This does not mean ICSI is not important; there are other aspects to embryology which can make or break even the most perfect ICSI (if there is such a thing) Working in an embryology laboratory is a multi-dimensional network of activities inter-dependent all moving to the same goal. Every step in the process needs to be done with equal focus so the next step is fruitful. There is no use of a good OPU, denudation and ICSI, if the media is not equilibrated or contaminated due to lapse in preparation. You will have no good embryos. Similarly if you do not do a fertilization check, you would be questioning the probability of the embryos being normal. So thus, if you notice all is linked and you have to look at it as one big picture with many acts, but, some require attention to great detail. One such step is the grading of embryos and deciding the fate of these embryos. We weigh grading of embryos a notch higher than ICSI and do not be surprised, over your years of experience hopefully you will learn the same. Always remember, whether you chose to consult any laboratory as a freelancer or you are working full time with a laboratory, the outcome of your cycle will largely corelate to what quality embryos you chose to transfer or freeze for a couple, if, all clinical parameters are normal. You can only assess external features of an embryo and judge it based on morphological basis, thus taking important calls of regarding or disregarding any embryo.
In addition, remember there may be multiple brands available of a ICSI machine, media to denude or store, but the technique of using these machines and media remains the same. This does not hold true for grading. There have been large studies done by multiple laboratories and clinics to understand how to grade. This is the reason you will notice unlike other processes in embryology, there are multiple methods, how you can grade embryos. No two methods are the same. This is the reason we emphasise on this step and the need to not take it lightly. Today we are in an era of information and technology often referred to as “OMICS” and thus we are trying to find out as much as possible from the appearance of human embryos at various stages of development, trying to figure out what the final morphology or grade of the embryo can be. Why do you think development of embryoscope is considered as an important stage of development in IVF. That is because without altering any conditions, we can study any embryo from the time of insemination to transfer or vitrification. Not just that, but we can have data to then, apply mathematical algorithms and understand exactly how embryos will behave the way they do. Such is the importance given on grading globally, and thus, so is by us, since we understand the reason.
As suggested there are many ways of grading embryos and each laboratory choses the type which suits their practice the best, it is also decided as per suggestions and acceptance by the embryology team. Through our journey, we have practiced many such methods and the most effective way of grading we decided a well as apply in our laboratories, is a mix of two different grading methods. Until Day 3, we use the Spanish grading system (also adopted by the famous IVI group) called ASEBIR and for Day 5, we use Gardner’s grading principles. A combination of these 2 techniques has allowed us to adopt the best suggested by both these great principles. And for those of you who are wondering, these 2 stages from a grading perspective are independent and don’t precede each other. This is because the best grade on Day 3 may not become the best blastocyst and the best Day 5 blastocyst could be an average grade embryo on Day 3.
Grading of embryos is a continuous process through the cycle and this process begins with the onset of fertilization. The reason we adopted ASEBIR grading system is because, it makes you observe certain attributes even before the embryo development starts. The ASEBIR system is an alphabetical grading system which is bifurcated in 2 sections; for cleavage stage (day 3) and the other for blastocysts (day 5/6). As suggested embryos are analysed on multiple parameters, with every parameter having an alphabetical order from A to D. A being best, B similar, C average and D poor grade. The beauty and important aspect is, that, the final grade of the embryo is assigned by taking the lowest order making it an extremely stringent system.
- Assessment done 16-19 hours post ICSI/insemination
- Important to observe development of pro-nuclei, symmetry of pro-nuclei and NPB distribution E.g
|1||3-7 polarised or mirrored NPB||1st zygote in above image|
|2||Anything that is not point type 1 or 3||2nd zygote in above image|
|3||Only 1 or 2 NPB in at least 1 pro-nuclei||3rd zygote in above image|
|1||Same size||1st zygote in above image|
|2||‘similar’ PN size but not same||2nd zygote in above image|
|3||Different PN size||3rd zygote in above image|
In the above image and tables, lets take an example and understand. As suggested on fertilization, we don’t grade but note attributes and hence we do not give an alphabetical order to fertilization, but just separate the fertilized from the unfertilized zygotes. If we see a fertilized zygote with 2 identical pro-nuclei but 1 to 2 NPB in the pro nuclei, then we denote the fertilized zygote as 2-1-3.
2 = presence of 2 pro-nuclei, 1 = symmetry of pro-nuclei and 3 = distribution of NPBs.
Similarly if we have a fertilized zygote with different size pro-nuclei but identical/ polarised or mirrored NPB, then we denote it as 2-3-1. 2 = for 2 pro-nuclei, 3 = different or uneven symmetry and 1 = polarised NPB. We have done a study couple of years back and seen a correlation between presence of NPB and implantation, suggesting fertilizations with category 3 NPB distribution have a lower implantation rate. Thus adopting this method is optional and if you think this is very intense, you could do away with documenting attributes of fertilization and focus only on grading which is what we will see in the next section.
Cleavage stage grading: Day 2 and Day 3
- Timeline for grading – Fertilization check post 16-19 hours of ICSI, Day2 and Day3 (approximately same time as fertilization check)
- The reason this stage is referred to as cleavage stage is, since the cells in the embryo are dividing (cleaving) but the embryo itself is not growing
- The genetic material replicates and the cells divide, however, the volume of the embryo does not differ
- The ideal division would mean one cell becomes two, two becomes four and four turns into eight. However, embryos not always divide synchronously, and hence various parameters are assessed.
Grading of Day 2/3 is based on the following parameters:
- Cell number, cell symmetry
- Percentage (%) of fragmentation in the embryos (fragmentation is nothing but part cell which is left behind and does not divide
- Multi-nucleation and Vacuoles/li>
As suggested earlier, ASEBIR system is an alphabetical order and based on each parameter the embryo is given an alphabetical order and total or final grade is the lowest grade of them all. Similar to fertilization, lets first understand the order suggested for these parameters.
- Cell Number : As per text book description, the perfect and best cell number for day 2 embryos is 4, however it is not necessary and embryos can have any cells starting from 2 cells to 6 or more cells on day 2.
Similarly, for day 3 embryos, the alphabet assigned will depend on the grade and alphabet assigned on day 2. Please refer the chart below to see how a grade A embryo could be a grade B, C or D on day 3 or a grade B embryo become a grade C or D. So from the chart below, if a day 2 embryo is 4 cells then as per the chart, you till assign it an A. However on day 3 if the same embryo cleaves into 6 cells then 4 to 6 cells will be C. Similarly, if the embryo has developed to 7 or 8 cells then you would continue giving it an A and if it grows to more than 8 cells then you would give it a B. This sounds tricky at the beginning but day after day, you not only find it easy, but also start to acknowledge its importance.
- Symmetry: Remember the PN observation ? symmetry is documented in the same way
Displayed above are 4 images; image 1 has identical symmetry is given the order of A, image 2 has a similar symmetry but not same and is given B, image 3 has uneven shape and is given C, whereas, image 4 has very uneven or all uneven and is given D.
An additional point to remember here; If the embryo has 3 or 7 cells with all cells even except 1 cell then as well it is assigned A. This is often missed and forgotten
- Fragmentation: Fragmentation is nothing but, mass left behind by cells when they cleave.
|% Fragmentation||Alphabetical order|
- Multinucleation and vacuoles: Studies have indicated that multinucleation may result in increased rate of aneuploidy or chromosomal abnormality as also lower implantation rates. If possible and the cycle can permit, embryos with multinucleation should be avoided
Multinucleation and vacuoles are graded the same on way Day 2 or Day 3:
|% Multinucleation seen||Alphabetical order||Vacuoles Seen||Alphabetical order|
|None seen||A||No vacuoles||A/B|
|< 25%||B||Low Number||B|
If you have followed the above association, then let us take an example and grade a few embryos.
- Day 2 grading Embryo 1: 5 cells, similar symmetry with 25% fragmentation and multinucleation in 2 blastomeres or cells.
- As per the chart, 5 cell will point at B, similar symmetry again is B.
- However fragmentation is 25%, so as per the chart, this indicates C
- Multinucleation in 2 cells indicates less than 25% so again it’s a B.
But overall, since the least alphabet assigned was C, the grade of this embryo will be C
- Similarly, this embryo growing on Day 3 has 9 cells with 15% fragmentation and no multinucleation or vacuoles
- Day 2 to Day 3, the embryo has moved from 5 cells to 9 cells, so as per grading chart it is B
- Fragmentation of 15% denotes B
- No multinucleation means A
Although multinucleation is A, lowest assigned alphabet is B, so it’s a B grade. Thus, if you notice, this embryos was a grade C on day 2, but due to proper development, on Day 3, is a grade C.
Sometimes, you might be surprised to find the cells of an embryos are fusing together on day3 and appear like a large shapeless mass. This stage is called compaction and is a very good sign as the embryo has already begun its journey of forming a blastocyst. If you come across such an embryo, grade it ‘A’.
Thus, grading of Day 2 to Day 3 can be tell us a lot about how an embryos is developing as well as it will make you understand that until the final day, nobody can predict how an embryo will look like. What’s interesting is, that, the story doesn’t end at Day3. The laboratories who culture embryos up to Day 5, are awaiting further surprises. Before we go ahead to grading blastocysts, one golden rule to remember is, you should not check day 4 embryos.
Blastocyst or Day5/6 grading:
Not all day 3 embryos will reach the blastocyst stage, hence, do not get disheartened if you have 8 embryos and only 4 become blastocysts. If 50% of your day 3 embryos reach blastocysts rate, it is considered to be a good rate. The journey from day 3 to blastocyst is a dynamic journey and one in which the embryo self selects itself. Formation of blastocyst happens when all the cells fuse together and then separates into 2 cell lines. These two cells lines make the ICM and trophectoderm and grading of the blastocyst will depend on the pattern of how the ICM and trophectoderm (TE) appear. The ICM will eventually grow into the fetus and the TE will form tissues needed during pregnancy like the placenta. Blastocyst are typically classified into 5 types in order of expansion from a to e and are denoted by a numerical representing the degree of their expansion ;
- EB or early blastocyst denoted by numerical 2
- CB or cavity blastocyst denoted by numerical 3
- EB or expanded blastocyst denoted by numerical 4
- HB or hatching blastocyst denoted by numerical 5
- iHB or hatched blastocyst denoted by numerical 6
From left to right: 1st image is early blastocyst or grade 2, 2nd image is cavity blastocyst or grade 3, 3rd image is expanded blastocyst or grade 4, 4th image is hatching blastocyst or grade 5 and 5th image is hatched blastocyst or grade 6.
The greater the expansion the better will be the grade. We however, do not grade an early blastocyst since the differentiation between the ICM and TE is not evident and for this reason Gardner has not started his classification from 1, but from 2 as the degree of expansion. Sometimes, this may also hold true for a cavity blastocyst wherein, the ICM and TE is not clearly separated but we should note the degree of expansion which is 3 , as this helps us in making a decision about when to re-check the dish. However, from the cavity blastocyst onwards, if the ICM and TE is not visible, then it doesn’t mean that it is not evident, but the blastocyst lacks either of them. The grading system helps in suggesting the expected implantation potential into four categories:
A : blastocyst with optimal quality and high implantation potential
B : good quality blastocyst with high implantation potential
C : acceptable blastocyst with an average chance of implantation
D : poor quality blastocyst with a low chance of implantation
Like discussed earlier, the pattern of ICM and TE will further define the grade of the embryo. The cells of the ICM and TE have to show the following character to be assigned an alphabetical grade:
|A : many cells,tightly packed||A : many cells forming a cohesive layer|
|B : several cells, loosely grouped||B : few cells forming a loose epithelium|
|C : very few cells||C : very few large cells|
|D : ICM hardly visible||D : smooth TE|
Thus, if the ICM has many cells tightly packed, we grade the ICM as ‘A’, however, if the TE is made of few cells forming a loose epithelium, then we grade the TE as ‘B’. Similarly, if the ICM is not compact and is loosely formed, then we assign it ‘B’, but if, the TE is formed tightly as a uniform cohesive layer we assign it ‘A’. The overall grade of the blastocyst will be a combination of the expansion grade as well as the grade of the ICM and TE. E.g if the expansion grade is 4, wherein, the ICM is A and TE is C, then the overall grade will be 4AC.
Let us understand using a few examples:
- Combining the blastocyst developmental form and ICM/TE formation, how would we grade a cavity blastocyst?
- Figure 1: We need to first see the expansion. It is not a fully expanded blastocyst, and neither is it an early blastocyst. We can differentiate the two cell lines; ICM and TE as well as see a cavity in the embryo. Thus it is a cavity blastocyst and we can grade the expansion as 3. Now let us focus on the ICM and TE format. The TE has many cells forming a cohesive layer and hence as per our chart we mark it as A. Similarly, ICM displays tightly packed cells and corresponding this to our grading chart, suggests, we mark it A again. Hence the final grade of this blastocyst is 3AA.
- Figure 2: This embryo, also suggests, it has a cavity and we can see two separate cell lines. So it is a grade 3 blastocyst. If you see the TE, the cells in certain areas are extremely weak showing very few large cells and hence we will mark it as C. The ICM is very loosely grouped and not well separated, so, we will mark it B or sometimes we also internally mark it B- (minus) for our reference. Thus the overall grade will be 3BC
- Combining the blastocyst developmental form and ICM/TE formation, how would we grade an expanded blastocyst?
- Figure 3; an interesting observation in this embryos is, at the top right side there is something oozing out. This is the indication, that the embryo has begun its journey of hatching. Thus considering this aspect, it is a grade 5 blastocyst. Both cell lines are well separated with a healthy TE, but a small though compact ICM. The TE has nice cohesive large cells making it grade A and the ICM though small, is compact thus again making it grade A. Thus the overall, grade is 5AA.
- Figure 4; this is classic grade 4AA blastocyst with good expansion, perfect ICM and a well-defined TE.
It is important we also see an well expanded blastocyst with no features of ICM and minimal TE. Below image represents such an embryo:
We can see the embryo has expanded very well, but the TE does not appear as several separate cells but as a layer and there is not visibility of an ICM. Thus the ICM is of grade D and the TE is smooth and hence it is also grade D. Thus the overall grade is 4DD. Such an embryo is not to be transferred or frozen.
Grading is a very important parameter in embryology, affecting the outcome of an IVF cycle. Some might say ICSI is important, freezing is important etc; but please remember, choosing the right embryo and giving the best chance at every embryo transfer is an extremely critical aspect in embryology, which is sometimes not focussed on as much as it should be.
We hope this article has been of interest and please feel free to ask questions or comment on any section.