Elder K. Human preimplantation embryo selection
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HUMAN PREIMPLANTATION EMBRYO SELECTION
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INTRODUCTION
The development of detailed scoring systems for the
pronucleate oocyte and cleavage stage human embryo
have undoubtedly increased the efficacy of human
IVF.
1–9
The advent of more physiological culture
media, together with improvements in embryo cul-
ture systems and the laboratory, has now made it
possible to culture the human embryo to the viable
blastocyst stage as a matter of routine.
10–13
There
appear to be numerous advantages associated with
extended culture and blastocyst transfer (Table 7.1).
The current status of clinical blastocyst transfer has
recently been analyzed,
30
with the conclusion that
extended embryo culture results in an increase in
implantation and clinical pregnancy rates and a
reduction in pregnancy loss in several subpopula-
tions, especially patients under the age of 37
31,32
and
oocyte donors,
33
as well as those patients with poor
quality embryos,
34
or who have experienced multiple
IVF failures.
35
Furthermore, it appears that the blas-
tocyst stage of development more readily maintains
its viability postcryopreservation than the pronucle-
ate oocyte or cleavage stage embryo, making it the log-
ical stage at which to freeze or vitrify embryos.
28,36,37
Taken together, blastocyst culture facilitates a reduc-
tion in the number of embryos transferred, while
increasing the success of cryostorage, thereby increas-
ing the overall efficacy of an IVF cycle.
29
HOW CAN VIABLE EMBRYOS BE OBTAINED
IN AN IVF LABORATORY?
Human embryos can be cultured to the blastocyst
stage in a variety of conditions. However, the result-
ant viability of these blastocysts varies enormously.
Therefore, it is imperative to discuss the development
of viable blastocysts. The ability to grow a viable
embryo involves more than simply the acquisition of
appropriate culture media. Rather, there are many
variables that can have an impact on the outcome of
an IVF cycle,all of which need to be taken into account
in order to optimize pregnancy rates. Figure 7.1 high-
lights the variables associated with an IVF cycle. Time
spent analyzing the variables in Figure 7.1 will not
only assist in improving procedures, but will also
assist in trouble shooting should a problem arise.
Figure 7.1 serves to illustrate the complex and
interdependent nature of human IVF treatment. For
example, the stimulation regimen not only impacts
oocyte quality (hence embryo physiology and via-
bility),
38
but can also affect subsequent endometrial
receptivity.
20,21,39,40
It is also apparent that a patient’s
etiology and genetics will have an impact on their
cycle outcome. Furthermore, the health and dietary
status of the patient can have a profound effect on the
subsequent developmental capacity of the oocyte and
embryo. The dietary status of patients attending IVF
is typically not considered as a compounding vari-
able, but growing data would indicate otherwise.
41–44
7. Analysis of blastocyst morphology
David K Gardner, John Stevens, Courtney B Sheehan, and William B Schoolcraft
Table 7.1 Potential benefits of blastocyst transfer
Embryo selection; ability to identify those embryos with limited, as
well as those with the highest developmental potential
14,15
Synchronization of embryonic stage with the female tract; reduces
cellular stress on the embryo
16,17
Minimize exposure of embryos to a hyperstimulated uterine
environment
18–21
Reduction in uterine contractions; reduces chance of embryos being
expelled
22,23
Ability to undertake cleavage stage embryo biopsy without the need
for cryopreservation when the biopsied blastomere has to be sent to a
different locale for analysis
24
Assessment of true embryo viability; assessing the embryo post
genome activation
25,26
High implantation rates; reduces the need to transfer multiple
embryos
27
Increased potential to maintain viability after cryopreservation
28
Increase in overall efficiency of IVF
29
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HUMAN PREIMPLANTATION EMBRYO SELECTION
A major determinant of the success of a laboratory
and culture system is the level of quality control and
quality assurance in place. For example, one should
never assume that anything coming into the labora-
tory that has not been pretested with a relevant bioas-
say (e.g. mouse embryo assay) is safe merely because
a previous lot has performed satisfactorily. Only a
small percentage of the contact supplies and tissue
culture ware used in IVF is supplied suitably tested.
Therefore, it is essential to assume that everything
entering the IVF laboratory without a suitable pretest
is embryo toxic until proven otherwise.
In our program the 1-cell mouse embryo assay
(MEA) is employed to prescreen every lot of tissue
culture ware that enters the program, i.e. plastics
that are approved for tissue culture. Around 25% of
all such material fails the 1-cell MEA (in a simple
medium lacking protein after the first 24 h).
45
There-
fore, if one does not perform quality control (QC)
to this level, one in four of all contact supplies used
clinically could compromise embryo development. In
reality many programs cannot allocate the resources
required for this level of QC; when embryo quality
is compromised in the laboratory, the culture media
are held responsible, when in fact the tissue culture
ware is more often the culprit.
29,45
Several treatises exist that deal with the details
of embryo culture media composition and their
effects.
12,46–51
In practical terms, there are several key
points to address in order to obtain viable embryo
development. These include the use of microdrops
(20–50 l) for embryo incubation, with a prescreened
paraffin oil overlay. Oils should be pretested with a 1-
cell MEA performed in the absence of protein.
45
Whenever possible, embryos should be cultured in
groups of up to five and the culture media renewed
every 48 h if they contain amino acids.
29,45
A carbon
dioxide concentration of 5–7% should be employed
in order to ensure that the media have a working pH
of between 7.25 and 7.35. Furthermore, the use of a
Stimulation
Laboratory
Embryo
transfer and
luteal support
Air quality
Number of
incubators
Number of
embryologists &
training level
Gas phase (5% or 20% O
2
)
Culture media
Tissue culture ware /
contact supplies
Oil overlay
Number of embryos / drop
Impact on endometrial
receptivity
Oocyte
quality
QC & QA
Outcom
e
Patient
Diet
Etiology
Genetics
Uterus
Culture System
Figure 7.1 A holistic analysis of human IVF. In this schematic, the laboratory has been broken down into its core components, only
one of which is the culture system. The culture system has in turn been broken down into its components, only one of which is the
culture media. Therefore, it would appear rather simplistic to assume that the results of a given laboratory or clinic can be mimicked
by changing only one part of the culture system (i.e. culture media). QC, quality control; QA, quality assurance.
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ANALYSIS OF BLASTOCYST MORPHOLOGY
reduced oxygen concentration of around 5% is highly
recommended (rather than ambient air which con-
tains around 20%). Oxygen at a concentration of 20%
not only has a negative effect on embryo development
and viability,
52–56
but also has detrimental effects on
embryo gene expression.
57
ANALYSIS OF BLASTOCYST MORPHOLOGY
Once a laboratory has validated its culture system,
what is the relationship between embryo morphology
at the blastocyst stage and subsequent developmental
potential post-transfer? Several different morphology
grading systems have been created over the years.
58–60
Such systems are typically based on the degree of blas-
tocoel expansion and amount of necrosis that can be
visualized. A more comprehensive system was there-
fore developed that accounted for both the develop-
ment of the inner cell mass and the ophectoderm.
61
This approach appears to be superior to simply assess-
ing the degree of blastocoel expansion.
62
The system
developed by Gardner and Schoolcraft
61
is represented
in Figure 7.2. Figures 7.3 and 7.4 show photomicro-
graphs of all stages and qualities of human blastocysts.
The relationship between blastocyst morphology,
using the grading system of Gardner and Schoolcraft,
and IVF outcome has been published previously.
64
An update is presented in Table 7.2, with data from
790 IVF (non-donor) cases in which patients had two
blastocysts of documented morphology transferred.
Clearly, those embryos with a good score at the blas-
tocyst stage have high implantation potential (fetal
heart beat/embryo transferred of 66%). It is therefore
evident that single embryo transfer should be advo-
cated for patients who have a blastocyst with a score
of ⭓ 3AA on day 5.
ANALYSIS OF BLASTOCYST PHYSIOLOGY
Although the blastocyst scoring system described and
highlighted in Figures 7.2–7.4 has proved useful in
embryo selection,
62
there is still only a limited amount
of data that can be derived from morphological
assessment. Therefore, the development and imple-
mentation of non-invasive tests of embryo viability
1.
2.
3.
4.
Trophectoderm grading
A. Many cells forming a cohesive
epithelium
B. Few cells forming a loose
epithelium
C. Very few large cells
ICM grading
A. Tightly packed,
many cells
B. Loosely grouped,
several cells
C. Very few cells
Figure 7.2 Scoring system for human blastocysts. Blastocysts are
initially given a numerical score from 1 to 6 based upon their
degree of expansion and hatching status. (1) Early blastocyst, the
blastocoel occupies less than half the volume of the embryo.
(2) Blastocyst, the blastocoel occupies half of the volume of the
embryo or more. (3) Full blastocyst, the blastocoel completely fills
the embryo, but the zona is not thinned. (4) Expanded blastocyst,
the blastocoel volume is now larger than that of the early embryo
and the zona is thinning. (5) Hatching blastocyst, the trophec-
toderm has started to herniate through the zona. (6) Hatched blas-
tocyst, the blastocyst has completely escaped from the zona. The
initial phase of the assessment can be performed on a dissection
microscope. The second step in scoring the blastocysts should be
performed on an inverted microscope. For blastocysts graded as
3–6 (i.e. full blastocysts onwards) the development of the inner cell
mass (ICM) and trophectoderm can then be assessed. Adapted
from reference 61.
have significant merit.
65
Figure 7.5 highlights some of
the known and possible markers that have been linked
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HUMAN PREIMPLANTATION EMBRYO SELECTION
Figure 7.3 Photomicrographs of human blastocysts on day 5 of development. (A)–(C) These blastocysts are scored as 1; the embryos
have begun to cavitate. The cavity (blastocoel) is less than half of the embryo’s volume. (D)–(F) These blastocysts are all scored as 2; the
blastocoel within the embryos is equal to or greater than half the volume of the embryo. (G)–(L) Blastocysts that have a full blastocoel
cavity, and are all graded as 3, but differ in their allocation of cells to the ICM and trophectoderm. (G) Grade 3CA, the ICM is poorly
developed with few cells, the trophectoderm is well formed. (H) Grade 3AB, the ICM is well developed with many cells, the trophecto-
derm has fewer and larger cells than expected. (I) Grade 3BA, the ICM has not formed a tightly packed group, nor does it appear to have
many cells, the trophectoderm is well formed. (J) Grade 3CC, neither the ICM or trophectoderm have many cells. The right hand side of
the embryo consists of just 1 cell. The blastocoel, however, is fully formed, hence the score of 3. (K) Grade 3AC, the ICM is well formed
and has many cells, but the trophectoderm has not formed a competent epithelium. (L) Grade 3AA, both ICM and trophectoderm have
many cells forming a beautiful blastocyst. (M)–(U) Blastocysts that have begun to expand and thin their zona, and are graded as 4,but
differ in their allocation of cells to the ICM and trophectoderm. (M) Grade 4CA, the ICM is hardly evident in this embryo, while the
trophectoderm is clearly formed and functional. (N) Grade 4BB, although at first glance this blastocyst looks good, a critical analysis
reveals that both the ICM and trophectoderm have fewer cells than are required to score AA. (O) Grade 4BC, similar to the blastocyst in
A
D
B
G
C
E
H
F
I
JK
L
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ANALYSIS OF BLASTOCYST MORPHOLOGY
P Q R
S T U
V w X
M N O
photomicrograph (N), this ICM is quite well formed, although there are too few cells for it to score an A. However, by looking at the
right hand side of the embryo the curvature of the trophectoderm indicates that there are very few cells. (P)–(R) Grade 4AA, this series
of images is of the same blastocyst at different focal planes in order to clearly show the ICM and trophectoderm. The ICM is tightly
packed with many cells, and the surrounding cells have formed a cohesive epithelium of many cells. Such an embryo should be trans-
ferred individually. (S)–(U) Grade 4CA, this series of images is of the same blastocyst at different focal planes in order to clearly show the
ICM and trophectoderm. At first glance, the blastocyst in photomicrograph (S) looks of excellent quality. However, when one focuses
through the embryo it can be seen that the ICM has hardly developed at all. Although such an embryo is very likely to implant and give
rise to a high hCG reading, by the time of scan there is little chance of observing a fetal heart. (V) Grade 4, pulsed. The blastocyst in this
photomicrograph is a 4 as evident by the thinned zona. However, the true extent of ICM and trophectoderm differentiation cannot be
assessed as the blastocoel has collapsed. This is a common occurrence in blastocysts, and the cavity will re-expand in the following 1–2
hours. Such an embryo should ideally be re-scored following re-expansion. (W) Grade 5BA, the trophectoderm has begun to herniate
through the surrounding zona, hence the score of 5. However, the ICM is not tightly packed and only has a few cells. (X) Grade 5AA, the
trophectoderm has begun to herniate through the surrounding zona, while in contrast to the blastocyst in (W), the ICM is well formed.
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HUMAN PREIMPLANTATION EMBRYO SELECTION
loss.
32
Furthermore, with the advent of an alpha-
numeric scoring system, it is possible to identify
blastocysts with very high implantation potential
(⬎60%).
64
The ongoing development of non-invasive
means of assessing embryo physiology will augment
the available morphological scoring system, leading to
further increases in implantation rate. As the bene-
fits, to both mother and child, of reducing the num-
ber of embryos transferred are well documented,
70
the day of single embryo, and in particular single
blastocyst transfer, has arrived.
Figure 7.4 Photomicrographs of human blastocysts following preimplantation genetic diagnosis (PGD) or freezing. (A) This
embryo has undergone PGD on day 3 of development. The hole in the zona was formed using a laser, and the resultant blastocyst
(4AA) is hatching through this hole. As a result it is not technically a true grade 5. Furthermore, after hatching on day 3, the zona
does not thin (B) This blastocyst was a 4AA at freezing on day 5.
63
This photomicrograph was taken 2 hours after thawing. (C) This
blastocyst was a 4AA at freezing on day 6. This photomicrograph was taken 3 hours after thawing. Furthermore, after hatching on day
3 the zona does not thin.
to embryo viability post-transfer. This is a tremen-
dously exciting area in modern embryology, and kits
will soon be available that can rapidly quantitate some
of these parameters.
In conclusion, with the development of more
physiological culture conditions, it is now possible to
culture human embryos to the blastocyst stage. Data
to date indicate that embryo transfer at this later stage
of development results in a higher implantation rate
than the transfer of pronucleate oocytes or cleavage
stage embryos,
29,69
and a reduction in pregnancy
ABC
Table 7.2 Effect of blastocyst score on pregnancy outcome in 790 IVF (non-donor) cases where two blastocysts
were scored and transferred
Group 1 Group 2 Group 3
(two blastocysts ⱖ3AA) (one blastocyst ⱖ3AA) (blastocyst ⬍3AA)
No. of embryos transferred 2 2 2
Mean age (⫾SE) (years) 32.8 ⫾ 0.15 33.5 ⫾ 0.3 33 3 ⫾ 0.44
Age range (years) 21–43 23–44 19–43
No. of transfers 560 136 94
Patients with ICSI (%) 62.3 72.1 62.8
Blastocyst development from 2PN (%) 66.7 53.5 42.1
a
Implantation rate (% sacs) 69.6 59.2 35.6
a
Implantation rate (% with FHT) 64.0 57.4 33.0
a
Clinical pregnancy rate (%) 81.8 78.7 50.0
a
Twin (%) 56.6 39.3 25.5
a
PN, pronuclei; FHT, fetal heart tones.
a
Significant differences between groups.
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ANALYSIS OF BLASTOCYST MORPHOLOGY
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Glucose
Pyruvate
Oxygen
Other sugars
Lactate
Ammonium
Enzymes, e.g. LDH
sHLA-G
Novel peptides and proteins
Amino acids
µl drop of defined culture medium
HOXA10 regulator
PAF
Amino acids
Uptake Production
Figure 7.5 Non-invasive analysis of human embryo viability. Individual blastocysts are incubated in 0.5–5.0 l volumes of defined
medium. Serial or endpoint samples of medium can then be removed for analysis.
65
An indirect measurement of metabolic
pathways, i.e. glycolysis and transamination can be obtained by measuring specific nutrients in combination, such as glucose uptake
and lactate production, or amino acid turnover with ammonium production.
66,67
As well as testing for known molecules, surface
enhanced laser desorption/ionization time-of-flight mass spectrometry can also now be used to identify novel peptides and pro-
teins.
68
LDH, lactate dehydrogenase; sHLA-G, soluble histocompatibility antigen class 1 G; HOXA10, homeobox A10; PAF,
platelet activating factor.
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