“Zygote and embryo morphology scoring”.

Welcome to chapter 5.
The following chapter is called “Zygote and embryo morphology scoring”.
The author is Lisbet Van Landuyt.
1
The aim of this chapter is to learn how to score the zygotes on day 1 after oocyte
retrieval, the cleavage-stage embryos from day 1 (early cleavage) until day 4 of
embryo development and finally to learn how to score blastocysts on day 5 and day
6 of development.
In this chapter you will learn
•How to evaluate the different zygote parameters and how to distinguish oocytes
with normal, abnormal and non fertilization on day 1 of development (this is one day
after oocyte retrieval)
•How to score cleavage stage embryos and the different additional embryo
parameters that define the final embryo quality (day 1, 2, 3, 4)
•How to score compacted and compacting embryos on day 4
•How to distinguish the different blastocyst stages on day 5 and day 6 and how to
score the quality of the inner cell mass and trophectoderm
2
The fertilization process involves the fusion of an oocyte and a sperm, followed by
fusion of the haploid chromosomal content of both gametes. The sperm nucleus
decondenses, influenced by factors within the ooplasm. The second meiotic division
is completed and the second polar body is extruded. The chromosomes of the
oocyte are surrounded by a membrane and form the female pronucleus (near the
second polar body). The decondensing chromatin of the sperm head forms the male
pronucleus.
When is the right time to assess fertilization?
In case of ICSI, assessment of fertilization is done preferably between 16–18 hours
after microinjection of oocytes when the two pronuclei are visible in the cytoplasm of
a normally fertilized egg. In case of in vitro fertilization, the appearance of the
pronuclei is slightly delayed by approximately 4 hours compared to ICSI oocytes.
Nagy ZP et al., 1994
Nagy ZP et al., 1998
3
To assess the fertilization and quality of the zygote, different parameters can be
assessed. The most important parameter is the evaluation of the pronuclei.
Furthermore, zygote scoring systems are often based on the different patterns of
the nucleolar precursor bodies within the pronuclei.
Evaluation of fertilization also involves the assessment of polar bodies. Finally,
evaluation of the cytoplasmic quality can be performed.
4
A pronucleus contains a haploid set of chromosomes derived from the oocyte
(female pronucleus) or from the sperm (male pronucleus).
Pronuclear assessment involves the evaluation of the number of pronuclei, the size
of each pronucleus and the position of the pronuclei within the cytoplasm.
5
A normally fertilized oocyte contains two pronuclei and two polar bodies.
When no pronuclei and only one polar body are present, the oocyte is not fertilized.
However, it sometimes occurs that the pronuclei are not visible and two polar bodies
or fragmented polar bodies are present. In this case, when the two pronuclei are
‘missed’ and already disappeared, the oocyte is probably fertilized. When checking
fertilization, the time period between time of injection and time of evaluation has to
be considered. Pronuclei are often missed after early microinjections with a timeinterval of more than 20 hours between injection and evaluation.
6
After ICSI or IVF, a considerable number of oocytes (approximately 30 %) do not
contain pronuclei and are not fertilized.
After IVF insemination, possible reasons can be
•that no sperm cell penetrated into the cytoplasm of the oocyte;
•that the oocyte was still immature at the moment of insemination;
•that the oocyte is chromosomally abnormal and finally
•that there is premature chromosome condensation.
In case ICSI was performed, the possible reasons can be the absence of oocyte
activation by the sperm or chromosomal aberrations of the oocyte and/or the sperm.
7
When only one pronucleus, or three or more pronuclei are visible, the oocyte is
abnormally fertilized.
8
In approximately 3–6 % of oocytes after IVF and ICSI, an oocyte with one pronucleus is
found (Staessen et al., 1993).
In the study of Staessen et al (1997), the chromosomal constitution of embryos
developing from mono- (1PN) and tripronuclear (3PN) oocytes was analyzed.
After IVF, the possible etiology can be:
•That there is no decondensation of a penetrated sperm head (in about 70–75 % of
monopronuclear oocytes).
•That there is spontaneous oocyte activation in 25–30 % of monopronuclear oocytes
(parthenogenetic activation).
•That there is asynchronous appearance of male and female pronucleus and the oocyte
is normally fertilized. In the study of Staessen et al. (1997), 48.7 % of monopronuclear
oocytes after IVF were found diploid, with an equal ratio of XX- and XY-bearing embryos.
In another study, in 25 % of monopronuclear oocytes, a second pronucleus could be
observed when the oocyte was examined a few hours later (Staessen et al. 1993).
•It is also possible that the female and male pronucleus fused into one large pronucleus.
After ICSI, the possible etiologies can be
•Oocyte activation.
•That no decondensation of the injected sperm occurred.
•Fusion of female and male pronucleus. After ICSI, 27.9 % of monopronuclear oocytes
were found diploid and resulted from a normal fertilization process. A second observation
of monopronuclear oocytes after ICSI could also be considered (Staessen et al., 1997).
9
Approximately 5 % of injected or inseminated oocytes are tripronuclear. After IVF,
this occurs mostly (in 90 % of cases) by penetration of one (dispermy) or more
(polyspermy) additional sperm cells into the oocyte. The embryos can be triploid
XXX or XXY or XYY
Another reason can be the non-extrusion of the second polar body and that the
polar body forms a nucleus instead. This is also the main reason of tripronuclear
oocytes after ICSI. In this case, the embryos are XXX or XXY. XYY is not possible!
Tripronuclear oocytes are not selected for transfer.
Staessen et al., 1997
10
A second parameter to assess the pronuclei and thus the quality of the zygote is the
relative size of each pronucleus.
Normal pronuclei are equally sized or slightly different in size. A large difference in
size can be correlated with chromosomal defects in the embryo such as aneuploidy
(Munné and Cohen, 1998).
11
Pronuclei are in normal position when they are located centrally in the cytoplasm
and are in apposition (in alignment) with each other. Non-alignment may lead to
absence of syngamy or aberrations during fertilization.
12
Nucleolar precursor bodies (NPB) are defined as follows:
They are organelles in the nucleus in which pre-ribosomal RNA is synthesized, this
is essential for meiosis and cell cleavage.
During fertilization, rRNA synthesis increases, and actively synthesizing nucleolar
precursor bodies are then called nucleoli.
They undergo a time-dependent process of fusion and polarization, which is ideally
synchronous in the two pronuclei.
13
For the scoring of nucleoli, the following aspects are evaluated.
First, the number of pronuclei is assessed. Ideally, each pronucleus contains three
to ten nucleoli. The maximum difference in the number of nucleoli between the two
pronuclei should be one to three.
Secondly, the size is recorded: they should be small, and then fusion follows. The
size is abnormal when there are very small pinpoint nucleoli.
Also the distribution of the nucleoli within the ooplasm is assessed. A polarized
position in both pronuclei is preferred.
Finally, we look at the synchronization of the nucleoli. A synchronized fusion and
polarization in both pronuclei is preferred.
14
In literature, there are several studies on zygote scoring systems that are based on
the different patterns of nuclear precursor bodies: Tesarik and Greco (1999), Scott
et al. (2000), Montag et al. (2001), Scott et al. (2003), Balaban et al. (2004).
From these studies we can conclude that nuclear precursor body scoring can
predict embryo development, blastocyst formation, pregnancy, and implantation
rates. Furthermore, pronuclear morphology predicts chromosome constitution.
15
The Center for Reproductive Medicine at UZ Brussel uses five categories for
pronuclear scoring, based on the ‘revised scoring system’ published by Scott et al.
in 2000. These categories are type two-zero, type two-one, type two-two, type twothree and finally type two-four.
The best zygote score is type two-zero in which the pronuclei and the nucleoli are
equally sized, and the nucleoli are polarized and present in equal numbers.
The second best score is type two-one in which the pronuclei and nucleoli are
equally sized and nucleoli are present in equal numbers. However, the nucleoli are
not polarized but distributed in both pronuclei.
Zygote score type two-two groups different types of asynchronous and abnormal
nucleolar morphologies: pronuclei that contain a different number of non-polarized
nucleoli, pronuclei with asynchronous polarization of nucleoli or pronuclei with very
low number of large nucleoli.
Pronuclei types two-three and two-four are considered as bad quality zygotes. In a
two-three pronucleus, the pronuclei are separated and are not in apposition.
Zygotes with pronuclei that are not centrally located in the cytoplasm are also
defined type two-three. A type two-four zygote has pronuclei with a considerable
difference in size.
16
From the study of Scott et al. (2000) it was concluded that
1. Pronuclei that are not in apposition (type two-three) are indicative for an
abnormal function of the centriole and abnormal formation of the sperm aster.
2. Polarization of the nucleolar precursor bodies (NPB) is time-dependent and is not
always visible after a single observation.
3. Asynchronous observations (type two-two, two-three and two-four in our zygote
grading system) are correlated with lower blastocyst formation rate and lower
implantation rate.
17
The aspect of the polar bodies is also evaluated when fertilization is assessed.
In a fertilized oocyte, two distinct polar bodies or fragmented polar bodies can be
seen. They are mostly located next to each other (in close apposition) but can also
be located in a distant position.
According to the study of Rienzi et al. (2003), dislocation of polar bodies results
from a high dislocation of the meiotic spindle from the first polar body and is
correlated with an increased risk for impaired embryo quality.
18
After IVF and ICSI, the cytoplasmic quality of the oocytes can be assessed.
Oocytes can have a damaged cytoplasm both after ICSI or IVF. In case of ICSI, this
degeneration can occur immediately after injection or afterwards during overnight
culture. The oocyte’s cytoplasm develops a dark-brownish color and loses its
integrity. These degenerated oocytes will not be evaluated further the following days
and can immediately be discarded.
After ICSI, approximately 5–8 % of oocytes are degenerated but this is only rarely
seen after IVF insemination.
The cytoplasm can also contain one or several vacuoles. Vacuoles are membranebound cytoplasmic inclusions filled with fluid that is virtually identical with
perivitelline fluid. They can vary in size and in number. Zygotes with large vacuoles
may fail to initiate first cleavage or display a rather abnormal cytokinesis pattern
(Van Blerkom, 1990).
19
During further development in vitro, three different scoring systems are used to
evaluate the embryo quality
•A scoring system for cleavage stage embryos. The quality of cleavage stage
embryos is primarily based on the cell stage (number of blastomeres) and the
presence or absence of fragmentation. Furthermore, additional parameters such as
compaction, blastomere size, presence of vacuoles, granulation of the cytoplasm,
and multinucleation of blastomeres are assessed. This scoring system can be used
first on day 1 to assess early cleavage but it is primarily used on day 2 and day 3
and up to day 4 of development.
•For embryos that are compacting or fully compacted on day 4, another scoring
system is used to differentiate between 2 stages of compaction. This can also be
used when the embryo is showing early compaction on day 3 or late compaction on
day 5.
•The scoring system to evaluate blastocyst quality is based on three parameters:
blastocyst stage, quality of the inner cell mass and quality of the trophectoderm.
This can be used from day 4 onwards when there is early cavitation and blastocoel
formation, but it is primarily used on day 5 and day 6 of embryo culture.
20
The most important observation for cleavage stage embryo scoring is the cell stage
of the embryo. This is evaluated by counting the number of blastomeres present in
the embryo. To assess the cell number correctly, the embryo must be visualized in
different focal planes to be able to count all cells of the three-dimensional embryo.
On day 1 in the afternoon the fertilized oocyte starts to divide in two cells. This is
called early cleavage and the importance of this second evaluation on day 1 is
discussed in a later slide.
An ideal cleavage pattern would be an embryo with four cells on day 2, eight cells
on day 3, compaction on day 4 and blastocyst formation on day 5. However, all
different cell stages (from two cells up to 16 cells) can be observed depending if the
cleavage rate is advanced or rather slowly.
It is therefore possible to have, for example, a six or eight cell embryo on day 2 and
an embryo with more than eight cells or compact embryo on day 3. It is also
possible to observe embryos with an uneven number of blastomeres, for example a
three cell embryo on day 2 or a five cell embryo on day 2 or day 3. This is called
asynchronous cleavage and such an embryo is preferentially not chosen for
transfer.
21
Fragmentation or the presence of small anucleated fragments between the
blastomeres is the second most used parameter to score the quality of an embryo.
Embryos with no or little fragmentation are considered to have a better implantation
potential than embryos with many anucleated fragments. In our center, we use five
different categories according to the percentage of fragmentation that is present in
the embryo.
In the first category are the embryos with 0 % fragmentation and this is called F0.
Also embryos with one or two small fragments that are probably the remnants of the
polar bodies are considered as type F0.
F1 embryos have fragments below or equal 10 % of the total volume within the zona
pellucida.
F2 embryos show a fragmentation between 10 and 20 %.
Staessen C et al., 1992
22
F3 embryos have between 20 and 50 % fragmentation.
In F4 embryos, more than half of the volume within the zona is fragmented or the
embryo is totally fragmented.
23
Blastomere size is the third parameter to characterize a cleavage stage embryo.
Blastomere size should always be assessed in relation to the cell stage. In our
center, we use two different types of blastomere size scores.
In a normal synchronous cleavage pattern such as 2-4-8, the blastomeres are
expected to be of equal size. When the blastomere size is in accordance with the
cleavage pattern, the embryo is scored as B0 (B-zero) and this is the best
blastomere size score. It is possible that a type B0 embryo has unequal
blastomeres, for instance in the case of a six cell embryo with two large and four
smaller cells. In the latter case, the six cells originated from a four cell embryo in
which two cells already underwent the third cleavage division.
When the size or the volume of the blastomeres is not in accordance with a normal
developmental pattern, the embryo is scored as B1. For instance when a four cell
embryo has two large cells and two smaller cells (see picture), it developed from a
three cell embryo and implies an asynchronous cleavage pattern. Also a three cell
embryo (see picture) or a six cell embryo with all blastomeres of the same size are
scored as B1.
24
Another parameter that is scored for cleavage stage embryos is the grade of
compaction.
In C0 embryos, early signs of compaction are present but the number of cells can
still be counted.
In C1 embryos there is no compaction present.
Both pictures are showing embryos that start to compact.
25
Another important parameter that should be scored is the possible multinucleation
of the blastomeres. In theory, each blastomere of an embryo should contain one
single nucleus. However, multinucleated blastomeres (MNB) can be present from
the first cleavage division onwards.
Different mechanisms can result in multinucleation (described by Staessen et al.,
1998): nuclear division (karyokinesis) without cell division (cytokinesis), partial
fragmentation of nuclei and defective migration of chromosomes during mitotic
anaphase.
In our center, we introduced five different types to distinguish between
multinucleated and non-multinucleated embryos and to assess the different grades
of multinucleation.
The best score is M0 which represents an embryo without multinucleation and in
which at least one nucleus in one blastomere is visible. The second type, M1, is
given when no nuclei are visible in any blastomere. M2 for multinucleation is given
to an embryo in which up to 50 % of multinucleated blastomeres are present and no
more than two normal nuclei are visible per multinucleated blastomere. In M3
embryos, several normal nuclei or smaller fragmented nuclei are visible in up to 50
% of blastomeres. M4 embryos are multinucleated in more than 50 % of cells. It is
our policy not to transfer or to freeze M4 embryos.
26
In the first two pictures an eight cell and a two cell embryo are shown in which all
blastomeres have one single nucleus that is clearly visible.
The third picture presents a two cell embryo without visible nuclei.
27
The M3 score is demonstrated in the left picture in which one blastomere of a five
cell embryo has several nuclei.
The M4 score is shown in a two cell embryo with both cells having many
fragmented nuclei.
28
Another additional parameter that is assessed routinely is the grade of
vacuolization. Vacuoles can be present in all different cleavage stages, even in
blastocysts.
There are five different grades of vacuoles. An embryo without vacuoles is given the
best score and is called V0.
V1 embryos contain one or few vacuoles in only one single blastomere. V2 embryos
contain one or few vacuoles in several blastomeres. V3 embryos have many
vacuoles in several blastomeres and V4 embryos are fully vacuolized and are not
selected for transfer or freezing.
29
For evaluation of granulation, we have three grades. Grade G0 are embryos with a
homogenic cytoplasm without granulation. Embryos with slight granulation of the
cytoplasm are classified as grade G1. G2 embryos have very strong granulation or
have a degenerative aspect of the cytoplasm.
30
Evaluation of early cleavage of the two cell stage on day 1 is used as an additional
selection parameter to differentiate between embryos with morphological equivalent
quality on the day of transfer.
The studies of Lundin et al. (2001) and Sakkas et al. (1998 and 2001) demonstrated
that early cleavage is correlated with better embryo morphology and a higher cell
number on the day of transfer. It was concluded that assessment of early cleavage
can improve embryo selection. Van Montfoort et al. (2004) found that early cleavage
is a significant predictor of both pregnancy and blastocyst development.
The assessment of early cleaving embryos is preferably done at 23 to 26 hours post
ICSI and at 25 to 28 hours post IVF.
31
In the left picture, a two cell embryo is shown with no nuclei visible in the
blastomeres. In the picture in the middle, a two cell embryo is shown with both cells
having one clear nucleus.
The third picture on the right shows an oocyte that was normally fertilized. The two
pronuclei disappeared but the oocyte did not cleave yet.
32
On day 4 of embryo development, embryos start to compact or are already fully
compacted. When it is not possible to count the exact cell number anymore, the
embryo is given a stage of compaction instead of a cell stage. A compacting embryo
is scored as stage C1, a fully compacted embryo is scored as stage C2.
In a compacting embryo (C1), compaction is not complete yet, some cell borders
can still be observed. In a C2 embryo, cell borders are not visible anymore.
Occasionally, it is possible that already on day 3, embryos with this stage of
compaction are observed. However, in most day 3 embryos, it is still possible to
count the number of cells and the compaction is scored as an additional parameter
and not as a cell stage.
33
These pictures are examples of how to score all parameters for a given embryo.
The upper picture shows an eight-cell embryo without fragments, equal
blastomeres, no compaction is visible yet, one nucleus per blastomere, no vacuoles
and no granulation.
The picture below shows a four-cell embryo without fragmentation but with unequal
blastomeres. Since a four-cell embryo is expected to have four equal sized cells,
this is a B1 embryo. There is no compaction, one nucleus in each blastomere, no
vacuoles and no granulation of the cytoplasm.
34
The first picture of this slide shows a fully compacted embryo with less than 10 %
fragmentation. There are no vacuoles and no granulation of the cytoplasm. In
compacted embryos, it is neither possible to score the number of nuclei in the cells,
nor the size of the blastomeres.
Since the compaction is already obvious from the cell stage (because it is a C2), it
is not necessary to score it as an additional parameter.
The second picture shows an embryo with eight cells without fragmentation, equal
blastomeres but first signs of compaction between the cells. Therefore the
additional parameter for compaction is scored as C0. There is one nucleus visible in
each blastomere, vacuoles and granulation are not detectable.
35
To score the quality of blastocyst stage embryos, we use a scoring system which is
deducted from the grading system of Gardner and Schoolcraft (1999).
Three different blastocyst parameters are assessed. First, the developmental stage
of the blastocyst is evaluated. Secondly, the quality of the inner cell mass is scored
and finally, the quality of the trophectoderm cells is graded.
36
According to the type of blastocoel formation and grade of expansion of the
blastocyst, the following blastocyst stages can be distinguished and categorized
from early to advanced developmental stage:
BL1: Early blastocyst in which the volume of the blastocoel is less than half the
volume of the embryo. Embryos in which a cavity is starting to form (indicated by
the presence of sickle-shaped cells) are also considered as BL1 blastocysts.
BL2: Early blastocyst in which the cavity is larger than half the volume of the
embryo.
BL3: Full blastocyst: the blastocoel fills the embryo completely. In this stage of
blastocyst formation the inner cell mass and trophectoderm can be distinguished.
BL4: Expanded blastocyst: the volume of the blastocoel is larger than the initial
volume of the embryo. The zona pellucida is significantly thinned.
37
BL5: Hatching blastocyst: part of the trophectoderm cells are expelled from the zona
pellucida.
BL6: Hatched blastocyst: the blastocyst has completely escaped from the zona
pellucida.
BL7: Blastocyst that is artificially hatching through the hole of the embryo biopsy in
case of preimplantation genetic diagnosis. The zona is not thinned.
BL8: collapsed blastocyst. This stage indicates that the blastocyst did not hatch
after it has expanded, but has collapsed instead. However, it is possible that the
blastocyst re-expands afterwards and transforms into a blastocyst with a blastocoel.
38
These pictures demonstrate the different blastocyst stages from early to hatching
and artificially hatching blastocysts.
39
From the full blastocyst stage onwards (BL3), the inner cell mass and
trophectoderm can be scored.
The type of inner cell mass (ICM) is recorded as the first digit after the blastocyst
stage. Four ICM types can be distinguished:
•A: ICM cells are tightly packed, many cells are present
•B: The ICM cells are loosely grouped, several cells are present
•C: Very few ICM cells are visible
•D: No cells are visible, the ICM is not present or is degenerative
The type of trophectoderm (TE) is recorded as the second digit after the blastocyst
stage. Four types of trophectoderm qualities are possible:
•A: Many cells forming a cohesive epithelium
•B: Few cells forming a loose epithelium
•C: Very few large cells are visible
•D: The trophectoderm is degenerative or abnormal with no cells visible
40
These pictures demonstrate blastocysts with inner cell masses type A, B, C and D
(inner cell mass typing is the first digit after the blastocyst stage).
41
These pictures demonstrate blastocysts with trophectoderm qualities type A, B, C
and D (trophectoderm typing is the second digit after the blastocyst stage)
42
From this chapter we can conclude that
For the zygote morphology scoring the most important parameters to assess fertilization are
the number and size of the pronuclei. Additionally, the zygote morphology scoring is based
on different patterns of nucleoli. An optimal zygote has equally sized and centrally located
pronuclei with polarized position of the nucleoli.
For the embryo scoring the most important parameters are cell number and percentage of
fragmentation. Secondly, the blastomere size should be assessed in relation to the cell
stage. Synchronous cleavage is preferred when selecting an embryo for transfer. Additional
parameters to score are compaction, granulation, vacuolization and multinucleation.
Assessment of early cleavage can improve embryo selection especially in cases of
morphologically equivalent embryo quality.
43
For the blastocyst scoring different stages are described based on blastocoel formation,
expansion of the blastocyst volume, zona thinning and hatching status. Different qualities
are described based on size of inner cell mass and trophectoderm quality.
44
45
46
47