Classification of Embryos According to Their Quality

By BSc, MSc (senior clinical embryologist), MD, PhD (gynecologist), BSc, MSc, PhD (senior clinical embryologist), BSc, MSc, PhD (senior clinical embryologist), BSc, MSc (embryologist), BSc, MSc (embryologist) and (invitra staff).
Last Update: 10/16/2019

In in vitro fertilization (IVF) treatments, whether conventional IVF or ICSI, embryos are cultured in the laboratory for a few days to decide which will be transferred to the uterus and which will be frozen (vitrified). In order to be able to make the decision, the quality of these must be evaluated during their in vitro development.

Embryologists analyze different characteristics in each embryo to determine if it is of quality and, therefore, susceptible to implantation to end up giving rise to a pregnancy. If, on the other hand, it is of poor quality, it could stop its development, cause an implantation failure or cause an abortion in the first months of pregnancy.

The different sections of this article have been assembled into the following table of contents.

How and why is embryo quality measured?

In IVF-ICSI processes, in which egg fertilization takes place in the laboratory, it is important to assess the quality of the resulting embryos.

It is common for more than one embryo to be obtained, so its classification helps to choose which of the embryos in culture are most likely to implant and result in pregnancy. Thus, the embryo classification will be taken into account for the transfer, since the highest quality embryos will be those that will be introduced into the uterus.

Evaluation of the quality of embryos requires taking into account different characteristics of their morphology, i.e. their shape or appearance, and the evolution over the days they remain in culture. In order to do this, there are two options:

  • Remove the embryos from the incubator every day for a few minutes so that they can be evaluated under a microscope.
  • Valuate the embryos by means of a time-lapse system, which allows images to be obtained every few minutes in order to evaluate their complete development without removing them from the incubator.

The first alternative is the traditional method, while the second is a recent addition to assisted reproduction laboratories. The use of time-lapse systems allows the embryologist to observe the entire embryonic development, not just the specific moments when the observation would be made under the microscope. It also avoids the temperature and gas changes that occur when embryos are removed from the incubator, reducing stress and improving embryo viability.

Embryo on day 1: zygote

It is important to evaluate this time of development to see if fertilization has occurred. If this is the case, a zygote, i.e. a "newly fertilized" egg, will be observed. The following is taken into account when assessing this stage of development:

  • The presence of two pronuclei (PN), one from the egg and the other from the sperm.
  • The presence of two polar corpuscles (CP), indicating that after fertilization the meiosis of the ovum has been completed.
  • The appearance of the cytoplasm of the zygote, which should be uniform and clear.

To avoid misinterpretation, it should be observed between 16 and 18 hours after in vitro insemination or microinjection (postinsemination). If they are observed later, the pronuclei may have disappeared, an event necessary for the first division to take place, which will give rise to the two-cell embryo.

The existence of the 2 PNs confirms that there has been fertilization. If 1 or 3 pronuclei are observed, the embryo should be discarded, as it indicates that the genetic endowment of the embryo is not adequate. Their development in the next few days occurs in the same way as in viable embryos, so it is important to visualize them before PN fusion occurs and we are not able to distinguish viable from non-viable embryos.

Embryo on day 2: four cells

After the first two divisions (day 2 of development), the embryo must have 4 cells, called blastomeres. Both the number of blastomers and their appearance at this point in time will be decisive in the classification. Observing fewer than 4 cells or many more is indicative of delayed or accelerated development.

The observation of embryos at this stage of development should be done between 44 and 45 hours postinsemination paying attention mainly to the following aspects:

  • Size and symmetry of the blastomeres: the 4 cells should be approximately equal in size.
  • Number of cells: each cell must have only one nucleus. If they have two, they would be binucleated, and if they have more than two, they would be multinucleated. If they have more than one nucleus they are considered abnormal and are associated with cell division errors.
  • Percentage of fragmentation: the fragments are small traces of cytoplasm from an abnormal division of the blastomeres. The quantity, distribution and volume of the fragments are important, as they can compromise the development of the embryo.
  • Presence of vacuoles: vacuoles are like "sachets" filled with liquid. If they are large or numerous, they can have a negative influence on embryo quality.
  • Shape and thickness of the zona pellucida: it should be round and not too thick or too thin. Alterations in the zona pellucida are associated with low implantation rates, as it will be difficult for the embryo to detach from it in order to implant.

Some of the embryonic alterations influence the implantation capacity of the embryo to a greater or lesser extent, so that one quality category or another will be assigned according to this.

Embryo on day 3: eight cells

To analyze embryo quality on day 3, embryos are assessed between 68 and 69 hours postinsemination. At this moment, the same parameters that have been considered in day 2 of development are analyzed, as well as the rhythm of division. The best quality embryos would be those with 7-8 cells proceeding from 4-cell embryos on day 2.

Embryos can be transferred at this time of development or kept in the incubator until day 5 or 6 for blastocyst stage transfer. Also, embryos that are not transferred can be vitrified at this time or on day 5 or 6.

Embryo on Day 4: Morula

Compaction is the process by which embryonic cells form tight joints between themselves to form the morula. It is usually produced from the fourth day of development, although some embryos may show signs of the beginning of early compaction (on day 3).

The morula can be observed between 90 and 94 hours postinsemination. This moment provides little information about the state of the embryo, since all its cells have been compacted and it is not possible to observe distinctive features in these embryos. Nevertheless, the following aspects are valued:

  • Number of cells: the embryo must have more than 8 cells.
  • Degree of compaction: if compaction takes place at an early stage, cell membrane junctions are observed but can be distinguished from each other, while if it is advanced it is no longer possible to distinguish one cell from another. The compaction must be complete, i.e. it must affect all the cells of the embryo. If it is partial it means that some cell has been excluded and it is a sign of bad prognosis.
  • Fragments and vacuoles: If one of these two structures is observed, the embryo or part of it may be degenerating.

The optimal embryo on day 3 is the one with the following characteristics: it has more than 8 cells, it is compacted or compacting and the compaction affects the whole volume of the embryo.

Embryo on day 5 or 6: blastocyst

Between 114 and 118 (day 5) or 136-140 hours postinsemination (day 6) we are faced with a blastocyst, the last stage of embryonic development that can take place in the laboratory.

The formation of the blastocyst is essential for the implantation of the embryo in the uterus to take place, so its formation in culture is considered to have a good prognosis.

These embryos present two key structures in their morphology: the internal cell mass (ICM), which will originate the embryonic layers that will give rise to the baby's organs, and the trofoectoderm or external cell mass, which will originate the placenta. They are distinguished by the appearance of the blastocele, the fluid-filled central cavity.

Parameters similar to those proposed by Gardner in 1998 are taken into account to assess the quality of blastocysts:

  • The degree of expansion: from lowest to highest degree, from 1 to 5.
  • The state of ICM: size, shape and compaction of the internal cell mass. Valuation is done by assigning 4 letters (A, B, C, D).
  • The state of the trofoectoderm: structure and number of cells. Valuation is done by assigning 4 letters (A, B, C, D).

Thickness of the zona pellucida is also taken into account. It must become thinner to allow the blastocyst to expand and exit for implantation into the endometrium. A fine zona pellucida is related to good embryonic quality and high probability of implantation.

Categories of embryo quality

Both Day 3 embryos and blastocysts are currently categorized. Because the embryonic structure is different in both stages, the cataloguing is also different.

Quality of early embryos

The following quality grades are assigned to the embryos on day 2 and day 3 according to the different parameters mentioned in the previous section:

  • Category A or 1: embryos of excellent quality, with maximum implantation capacity.
  • Category B or 2: embryos of good quality, with high implantation capacity.
  • Category C or 3: embryos of intermediate quality with medium implantation capacity.
  • Category D or 4: poor quality embryos with low implantation capacity.

Quality of blastocysts

In order to assess blastocysts, as we have already seen, the usual embryonic classification has a number and 2 letters: the number (from 1 to 5) indicates the degree of expansion of the blastocyst; the first letter (A, B, C or D) indicates the quality of the internal cell mass; and the second (A, B, C or D), that of the trofoectoderm. Thus, the blastocysts with the best morphology and the highest implantation capacity would be the 3AA.

The numerical categories assigned according to the expansion are the following:

  • Grade 1 or early blastocyst: the blastocele begins to become visible.
  • Grade 2 or cavitated blastocyst : the different parts of the blastocyst are perfectly visualized.
  • Grade 3 or Expanded Blastocyst : The blastocyst has increased in size and the zona pellucida is thin.
  • Grade 4 or blastocyst hatching: the blastocyst begins to leave the zona pellucida.
  • Grade 5 or blastocyst hatching: the blastocyst begins to leave the zona pellucida.

As for the internal cell mass, the 4 categories would be as follows:

  • Category A: numerous cells forming a compact structure.
  • Category B: numerous non-compacted cells.
  • Category C: few cells.
  • Category D: cells with signs of degeneration.

On the other hand, the trofoectoderm must have only one layer. According to the state of its cells, it is classified as follows:

  • Category A: homogeneous, cohesive and with many cells.
  • Category B: homogeneous and with fewer cells.
  • Category C: few cells.
  • Category D: cells with signs of degeneration.

The embryologist Jose Luis De Pablo tells us what is taken into account when assessing blastocysts:

In the blastocyst, two fundamental parts are valued: the internal cell mass, which will give rise to the embryo, and the trofoectoderm, the layer of cells that will give rise to the placenta.

Depending on the number of cells, the compaction of the internal cell mass and the disposition of these cells, the final category of the blastocyst will be given.

New cataloguing of blastocysts

The Association for the Study of Reproductive Biology (ASEBIR) has proposed a new embryonic classification that gives greater weight to the morphology of the trofoectoderm with respect to that of the internal cell mass.

In it, the quality is assigned with a single letter (A, B, C or D) that encompasses the state of both the internal cell mass (ICM) and the trophectoderm. Thus, if the ICM is of quality A and the trofoectoderm is of quality B, the overall assessment of the blastocyst would be B.

In both the conventional and the new cataloguing, if embryos are taken to long culture for blastocyst stage transfer, it will be necessary to take into account the early stage classification (days 2 and 4) as well as that presented on day 5/6 for its overall assessment.

It is also important to bear in mind that embryo assessment is often very subjective, so there may be variations between laboratories.

FAQs from users

Can you pregnant with C and D scored embryos?

By José Muñoz Ramírez BSc, MSc, PhD (senior clinical embryologist).

Yes, although the chances are quite low.

When we classify embryos according to their quality at the lab, we do so by evaluating their implantation potential, that is, trying to "guess" which ones have greater chances for attaching tot he uterus, and which don't. C and D scored embryos are embryos of moderate-to-low quality, which means that a C or D scored embryo has a reduced chance of implantation if compared to a B or A scored embryo. In any case, however, whenever we select an embryo for the transfer, it's because it has been observed that its implantation potential is a good one.

How much likely is pregnancy with 2 embryos of A quality?

By Zaira Salvador BSc, MSc (embryologist).

Reply from the fertility clinic Institut Marquès

It is a high probability as long as the other factors involved are correct (endometrium, transfer...). We can conclude that the pregnancy rate for these embryos can exceed 70% when the conditions are correct.

Which quality does an embryo with 8 cells have on day 8?

By Zaira Salvador BSc, MSc (embryologist).

It would correspond to an embryo of 8 cells of category II or B, so it is of good quality, although not excellent. The implantation rates of these embryos are high, so there would be a high probability of pregnancy.

Is it possible to do a D-grade embryo transfer?

By Zaira Salvador BSc, MSc (embryologist).

These types of embryos have a low probability of implanting, as their quality is poor. They have signs of degeneration, serious morphological alterations such as high fragmentation or vacuolization, or an abnormal division rhythm. It is advisable to avoid transferring this type of embryos but if there is no other of better quality they can be transferred. Even if the possibilities are low, they could lead to pregnancy.

What quality does an embryo have to have in order to be frozen?

By Zaira Salvador BSc, MSc (embryologist).

Not all embryos are capable of surviving the processes of freezing and thawing. The best quality embryos have higher survival rates, so it is preferable to freeze those of quality A and B, although it is also possible to freeze those of type C. However, if the couple does not have better quality embryos, even type D embryos can be frozen, but this is not done in all centers because of their low survival chances. In most cases what is done with poor quality embryos is to leave them until day 5-6 in culture (under "observation") to see how they evolve.

Is the quality of an 8-cell or 6-cell embryo higher?

By Zaira Salvador BSc, MSc (embryologist).

The optimal embryos on day 2 are those with 4 cells, while on day 3 are those with 8 cells. Having more or fewer cells, as we have seen throughout the article, assumes that the embryo is not of excellent quality.

In the case of having 6 cells, the embryo would be classified in category C, whether it is day 2 or day 3. In the case of having 8 cells, it will be necessary to assess how many there were on day 2. Thus, if it comes from an embryo with 4 cells on day 2, it will be of quality A, while if it had 6 cells it will be of category C. If it comes from an embryo with 4 cells on day 2, it will be of quality A, while if it had 6 cells it will be of category C.

Will AA quality embryos always lead to pregnancy?

By Zaira Salvador BSc, MSc (embryologist).

This type of classification corresponds to a blastocyst of excellent quality, with an internal cell mass and a good looking trofoectoderm. It has a high implantation capacity and, therefore, a high probability of giving rise to a pregnancy. However, there is no guarantee that a woman will become pregnant. The fact that the embryo quality is good has a great influence on the implantation, but other factors such as the state of the endometrium at the time of the transfer, the age of the mother or the cause of infertility are also affected.

What causes poor embryo quality?

By Zaira Salvador BSc, MSc (embryologist).

There are several factors, both intrinsic and extrinsic, that can influence embryo quality:

That being said, it is difficult to know the specific cause that could affect a particular case in women with good prognosis and good reproductive health.

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References

Authors and contributors

 Edurne Martínez Sanz
Edurne Martínez Sanz
BSc, MSc
Senior Clinical Embryologist
Bachelor's Degree in Biochemistry from the University of Navarra and Master's Degree in Biotechnology from the Valencian Infertility Institute (IVI) and the University of Valencia (UV). More than 10 years' experience working as an embryologist for several fertility clinics, including IVI, Grupo Hospitalario Quirónsalud, and currently, Reproducción Bilbao. More information about Edurne Martínez Sanz
 Gorka Barrenetxea Ziarrusta
Gorka Barrenetxea Ziarrusta
MD, PhD
Gynecologist
Bachelor's Degree in Medicine & Surgery from the University of Navarra, with specialty in Obstetrics and Gynecology from the University of the Basque Country. He has over 30 years of experience in the field and works as a Titular Professor at the University of the Basque Country and the Master's Degree in Human Reproduction of the Complutense University of Madrid. Vice-president of the SEF. More information about Gorka Barrenetxea Ziarrusta
License: 484806591
 José Luis de Pablo
José Luis de Pablo
BSc, MSc, PhD
Senior Clinical Embryologist
Double Degree in Biology and Biochemistry from the University of Navarra. Master’s Degree about the Theoretical Basis and Laboratory Procedures in Assisted Reproduction from the University of Valencia (UV). Senior Clinical Embryologist from the ESHRE and ASEBIR Certification in Clinical Embryology. More than 15 years of experience as the director of IVF labs. More information about José Luis de Pablo
 José Muñoz Ramírez
José Muñoz Ramírez
BSc, MSc, PhD
Senior Clinical Embryologist
Bachelor's Degree in Biology from the University of Malaga. Master's Degree in Genetics by the University of Alcalá, and Master's Degree in Assisted Reproduction from the University of Valencia. He works as a clinical embryologist at Clínica Tambre (Madrid, Spain), in addition to being an Associate Professor at the University of Murcia. More information about José Muñoz Ramírez
 Neus Ferrando Gilabert
Neus Ferrando Gilabert
BSc, MSc
Embryologist
Bachelor's Degree in Biology from the University of Valencia (UV). Postgraduate Course in Biotechnology of Human Assisted Reproduction from the Miguel Hernández University of Elche (UMH). Experience managing Embryology and Andrology Labs at Centro Médico Manzanera (Logroño, Spain). More information about Neus Ferrando Gilabert
 Zaira Salvador
Zaira Salvador
BSc, MSc
Embryologist
Bachelor's Degree in Biotechnology from the Technical University of Valencia (UPV). Biotechnology Degree from the National University of Ireland en Galway (NUIG) and embryologist specializing in Assisted Reproduction, with a Master's Degree in Biotechnology of Human Reproduction from the University of Valencia (UV) and the Valencian Infertility Institute (IVI) More information about Zaira Salvador
License: 3185-CV
Adapted into english by:
 Romina Packan
Romina Packan
inviTRA Staff
Editor and translator for the English edition of inviTRA. More information about Romina Packan

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