“Embryo selection and transfer”.

Welcome to chapter 7.
The following chapter is called “Embryo selection and transfer”.
The author is Lisbet Van Landuyt.
1
The aim of this chapter is to explain how the best embryo for transfer is selected.
The embryo scoring is extensively explained in the chapter on zygote and embryo
morphology scoring. In this chapter, a schematic presentation shows how to
calculate the embryo score on day three or on day five of embryo culture. Beyond
that, the impact of the different additional scoring parameters will be discussed.
Furthermore, the Belgian transfer policy concerning the number of embryos for
transfer is discussed and on which day the transfer should be carried out.
Finally, a short description of the transfer procedure itself is given.
2
Obviously, the best quality embryos derived from normally fertilized oocytes are
selected for transfer. The embryo quality can be classified into four categories of
embryos: excellent or top (EQ1), good (EQ2), poor or moderate quality (EQ3) and
bad quality (EQ4) embryos.
Embryos are transferable if they are at least of EQ3. EQ4 embryos are not
transferable. In our center, supernumerary embryos can be selected for freezing if
they are at least EQ2.
The embryo quality is mostly determined according to a combination of cell stage
and percentage of fragmentation. Furthermore, additional parameters such as
blastomere size, compaction, granulation, vacuolization, and multinucleation are
also taken into consideration to determine the embryo quality and to select the best
embryo for transfer.
3
In our center, embryo transfer is performed on day three or on day five of embryo
culture. This table shows how the embryo quality is determined for multicellular
embryos on day three of embryo culture, taking into consideration the cell stage and
fragmentation on day three.
According to our scoring system a top quality embryo, is a normally fertilized
embryo with at least seven cells on day three and a fragmentation up to 10 %.
Good quality embryos have at least six cells and a fragmentation up to 20 %. When
an embryo with at least eight cells has between 20 % and 50 % fragmentation, it is
also considered a good quality embryo.
Four and five cell embryos are considered as poor quality embryos but can be
transferred.
All embryo stages with a fragmentation of more than 50 % are considered bad
quality embryos and are not transferred.
4
Furthermore, additional parameters (compaction, blastomere size, vacuolization,
granulation and multinucleation) are taken into account to select the best embryo for
transfer. The different parameters are explained in the chapter on zygote and
embryo morphology scoring. The embryo quality decreases according to the
following table if the embryos show deficits in one of these parameters (indicated by
a higher value than zero or one),
For instance, a six cell embryo in which the cell size is not in accordance with a
normal developmental pattern (grade B1), is given a lower embryo score than a six
cell with a normal cell size symmetry.
In case of vacuolization, only embryos that are completely vacuolized (grade V4)
are not transferable.
Embryos with severe granulation are also given a lower embryo quality.
Embryos having multinucleated blastomeres are transferrable if not more than 50 %
of the blastomeres are multinucleated.
5
For blastocysts in which an inner cell mass and trophectoderm can be
distinguished, blastocyst quality is calculated according to the four tables presented
on the next slides.
Early cavitating type 1 and 2 blastocysts on day five are considered as good quality
blastocysts (EQ2).
Compact or compacting embryos on day five without any signs of blastulation are
considered as poor quality blastocysts (EQ3).
6
According to this table, which presents embryo quality for blastocysts on day five,
top quality blastocysts are defined as those of at least blastocyst stage three, an
inner cell mass (ICM) type A and at least a trophectoderm (TE) type B. Further
information on blastocyst scoring can be found in chapter 5 of this module.
7
Full or expanded blastocysts with inner cell mass type B and trophectoderm type A
or B are considered as good quality embryos (EQ2). Blastocysts with ICM type B
but with a lower quality of trophectoderm are defined as poor quality blastocysts
(EQ3).
8
Blastocysts with ICM and/or TE quality type C and at least trophectoderm quality B
are poor quality blastocysts. Blastocysts with ICM type C and lower quality of
trophectoderm are also considered as bad quality blastocysts.
9
All blastocysts with ICM type D are defined as bad quality embryos and are not
transferred.
10
How can we select the best embryo for transfer?
1) First of all, the cell stage is evaluated.
2) Beside the selection of embryos based on their cell stage, fragmentation and
additional parameters, the developmental pattern of the embryo throughout embryo
culture (from day 1 to day 3 or to day 5) is taken into account, especially when a
cohort of good quality embryos is available.
3) In case of morphologically equivalent embryo quality, the zygote morphology
scoring as well as the early cleavage can be taken into account to select the best
embryo.
11
The following slides will provide information about which embryos can be
transferred on day three or day five of development and which of these embryos are
preferably transferred.
Day three embryos are selected for transfer and freezing in the morning of day
three (68 hours ± 2 hours after IVF/ICSI). They are transferable if they have
reached the five-cell stage and have fragmentation up to grade 3 (meaning up to 50
% fragmentation). A four cell embryo with a fragmentation up to 50 % can also be
transferred if it has evolved compared to its cell stage on day two.
On day three, preference for transfer is given to embryos with at least seven cells
with no or little fragmentation (with or without signs of compaction). The selection of
good quality compacting (C1) or completely compacted embryos (C2), in which the
cell number can no longer be distinguished, can also be considered.
Rapid cleaving embryos that already have more than eight blastomeres but with an
irregular cleavage pattern are given second preference for transfer.
12
The following figure presents the data from 1,969 single embryo transfers
performed at UZ Brussel between 2004 and 2007 (presented at ESHRE 2009 by
Van de Velde Hilde, invited lecture ”Totipotency”). In this diagram, the implantation
rate with fetal heartbeat is correlated with the developmental stage at the moment of
transfer which is performed on day three in the afternoon. It frequently occurs that
an eight cell stage embryo selected in the morning of day three has cleaved further
and has 9 to 16 cells at the moment of transfer.
This figure shows that four cell embryos on day three are capable of implantation
(11.3 % per embryo transferred) and give better results than five cell embryos (1.6
%).
The highest implantation rates were obtained in the group of 12 and 16 cell embryos
(34.7 %) and compacting or compact embryos (32 %).
Eight cell embryos (26.0 %) give better implantation rates than embryos with 9, 10,
11, 13 or 14 cells (19.9 %).
After transfer of seven and six cell embryos, an implantation rate of 16.3 % and 8.7
% was obtained.
13
The additional parameter of cell size symmetry (blastomere size scoring) can be
given a higher priority than percentage of fragmentation. For instance, it is probably
better to select an eight cell embryo with equal blastomere size and a higher
percentage of fragmentation than an eight cell embryo with lower percentage of
fragmentation but with very irregular blastomeres.
This slide presents data from UZ Brussel from single embryo transfers performed
between 2004 and 2007. The implantation rate of eight cell embryos is shown in
relation to the percentage of fragmentation and to the blastomere size. An eight cell
embryo with a blastomere size score (B0) has equally sized blastomeres (normal
blastomere symmetry). An eight cell embryo with unequally sized blastomeres is
given a blastomere size score B1 (abnormal blastomere symmetry). This slide
clearly demonstrates that transfer of eight cell embryos with higher percentage of
fragmentation (F2 and F3) but with normal blastomere symmetry can result in
similar implantation rates as in case of an eight cell embryo with abnormal
blastomere symmetry and lower percentage of fragmentation (F0 and F1).
14
Secondly, when selecting the embryo for transfer, preference is given to a normal
developmental pattern with a two cell stage on day one (early cleavage), a four cell
stage on day two and an eight cell stage on day three. Asynchronous cleavage (e.g.
three-six-nine) is given a lower priority for transfer. With this in view, the evaluation
of the blastomere size in relation to its cell stage becomes very important. For
instance, a four-seven cell pattern can be a better choice than a four-nine cell
pattern when the seven cell embryo has clearly one larger cell that did not cleave
compared to the other six cells, whereas the nine cell embryo has nine blastomeres
of equal size.
Van Royen et al. performed a retrospective analysis of the implantation potential of
day three embryos according to embryo morphology and cleavage kinetics in 2001
and published their results in Human Reproduction.
They found out that what is generally considered as an optimal cleavage pattern
(four cells on day two and 8 cells on day three) was indeed associated with the best
ongoing implantation rate which was 47 % per transfer.
A high implantation rate (35 %) was also obtained with embryos having two cells on
day two and seven cells on day three.
A poor outcome of only 13 % was found for embryos with three blastomeres on day
two.
15
According to the transfer policy of our center, day five embryos are transferable if
they have at least reached the stage of compaction (C1 or C2). All blastocyst stages
are transferable except those with an inner cell mass grading type D. Blastocysts
with a trophectoderm grading type D can be considered for transfer because it is
sometimes difficult to evaluate the quality of the trophectoderm cells (personal
observations). It can occur that blastocysts with a very poor trophectoderm scoring
on day five can progress on day six to a well-developed trophectoderm.
Preference for transfer on day five is given to full or expanded blastocysts with an
inner cell mass type A.
Second preference is given to blastocysts with an inner cell mass type B. For
instance, a blastocyst of type 3AA is selected for transfer instead of a blastocyst of
type 4BA.
Blastocysts type 1 and 2 with good morphology are the third choice for transfer and
are given priority upon blastocysts with an inner cell mass with very few cells (type
C) and/or poorly developed trophectoderm (type C).
Finally, compact or compacting embryos can also be transferred. Multi-cellular
embryos without signs of compaction (e.g. a sixteen cell without compaction) are
not transferred.
16
It is important to keep in mind, that there exist many strategies for the selection of
viable embryos for transfer. All have shown some correlations with implantation.
In the early years of in vitro fertilization embryo evaluation was based largely on
morphological criteria at one certain point of embryo development. Today most
advanced embryologists evaluate the embryos on the basis of the so called
Sequential Embryo Assessment. This approach includes multiple parameters and
multiple observations of the embryo at different developmental stages.
The future lies in an advanced sequential evaluation of the embryo, which is a
dynamic morphometric evaluation, including a continuous assessment of embryo
development.
Continuous progress in Genomics and Proteomics will as well allow identifying the
best embryo with high implantation potential.
17
In reproductive medicine, the birth of a single healthy child is considered to be the
primary goal.
The only strategy to prevent multiple pregnancies is performing a single embryo
transfer (SET). Several studies on SET demonstrated that young women with good
embryo quality are most suitable for SET (Gerris et al. 1999 and 2002, Martikainen
et al. 2001), in at least one treatment cycle.
Thurin et al. (2004) found a similar live birth rate when patients received a fresh
SET in combination with a frozen transfer in the subsequent cycle compared to a
fresh double embryo transfer. However, lower multiple pregnancy rates were found
when choosing the first transfer strategy.
18
In July 2003, a new Belgian law was passed, which restricts the number of embryos
that can be transferred, in order to limit the risk of having a multiple pregnancy.
When this new law was drawn up, the age of the patient and the rank of trial were
taken into account. In all Belgian IVF centers, the following rules have to be strictly
respected:
For patients up to 35 years of age:
•
1st cycle: only one embryo per IVF cycle is allowed
•
2nd cycle: one or two embryos, depending on the embryo quality. Two embryos
are transferred when only poor quality embryos are available.
•
3rd–6th cycle: maximum two embryos
For patients between 35 and 39 years of age:
•
1st–2nd cycle: maximum two embryos
•
3rd–6th cycle: maximum three embryos
For patients between 39 and 42 years of age there are no restrictions in the number
of embryos transferred.
Frozen embryo transfer cycles for all age categories are restricted to a maximum of
two embryos per transfer.
19
The impact of the new Belgian embryo transfer policy was evaluated in the following
studies.
Gordts et al. (2005) found a drop in twin gestation rate from 19 % in 2002 to 3 % in
a 6-months period after the start of the new legislation.
Van Landuyt et al. (2006) compared two 15-months periods before and after the
new legislation and observed a significant reduction of the multiple pregnancy rate
from 29.1 % to 9.5 % (in all patient groups) and from 28.9 % to 6.2 % in patients
younger than 36 years. Similar clinical pregnancy rates were found for both periods.
De Brock et al. (2006) reported a threefold decrease in multiple pregnancies without
a significant reduction in pregnancy rates.
References
Gordts S et al. (2005) Belgian legislation and the effect of elective single embryo
transfer on IVF outcome. Reproductive Biomedicine Online; 10: 436-441.
Van Landuyt L et al. (2000) New Belgian embryo transfer policy leads to sharp
decrease in multiple pregnancy rate. Reproductive Biomedicine Online; 13: 765771.
Debrock S et al. (2006) New Belgian legislation regarding the limitation of
transferable embryos in in vitro fertilization cycles does not significantly influence
the pregnancy rate but reduces the multiple pregnancy rate in a threefold way in the
Leuven University Fertility Center. Fertility and Sterility; 83: 1572-1574.
20
Intrauterine embryo transfer can be carried out on day two, three, four or five of
embryo culture. In our center, embryo transfer is carried out either on day three or
on day five of development. However, the choice between day three and day five
transfers is still debatable.
One advantage of day five transfer is the better synchronization with the
endometrium which can result in higher implantation rates. Hence, the increased
implantation potential of a day five embryo allows us to perform single embryo
transfers (SET) in order to decrease the number of multiple pregnancies. A second
advantage of the prolonged culture is the better selection of a viable embryo.
However, the question still remains if the viability of the embryos is not
compromised by this prolonged in vitro culture. Performing day five transfer and
culturing the embryos up to day five is of course a more labor intensive approach
and requires optimal culturing conditions.
21
In literature, many conflicting reports have been published about the benefits of
blastocyst culture and transfer.
Reports on higher implantation rates after day five transfer have been published by
the groups of Marek et al. in 1999; Milki et al. in 2000; Garnder et al. in 2000; and
Langley et al in 2001.
Comparable pregnancy and implantation rates for both day three and day five
transfers were reported by Scholtes and Zeilmaker in 1996; Coskun et al. in 2000;
and Huisman et al. in 2000.
References
Marek D et al. (1999) Introduction of blastocyst culture and transfer for all patients in
an IVF program. Fertility and Sterility; 72: 1035-1040.
Milki AA et al. (2000) Comparison of blastocyst transfer with day 3 embryo transfer
in similar patient populations. Fertility and Sterility; 73: 126-129.
Gardner DK et al. (2000) Culture and transfer of viable blastocysts: a feasible
proposition for human IVF. Human Reproduction; 15(6): 9-23.
Langley MT et al. (2001) Extended embryo culture in human assisted reproduction
treatments. Human Reproduction; 16: 902-908.
Scholtes MC, Zeilmaker GH (1996) A prospective, randomized study of embryo
transfer results after 3 or 5 days of embryo culture in in vitro fertilization. Fertility and
Sterility; 65(6): 1245-1248.
Coskun S et al. (2000) Day 5 versus day 3 embryo transfer: a controlled
randomized trial. Human Reproduction; 15: 1947-1952.
22
In 2002 Blake et al. published a review titled “Cleavage stage versus blastocyst
stage embryo transfer in assisted conception” in the Cochrane Database of
Systematic Reviews. It was updated in August 2005.
In this review Blake et al. concluded that:
•There is no evidence for a difference in live birth rates between day two or day
three embryo transfers (34.3 %) and day five or day six embryo transfers (35.4 %).
•There is no difference in clinical pregnancy rates per couple for day two or day
three embryo transfers (38.8 %) and day five or day six embryo transfers (40.3 %).
•A lower embryo freezing rate of 41 % was obtained after day five or day six embryo
transfers compared to 60 % for day two or day three embryo transfers.
•Finally, a higher embryo transfer failure rate of 10.1 % was obtained for day five or
day six embryo transfers compared to 3.5 % for day two or day three embryo
transfers.
However, the Cochrane review found significantly higher implantation rates for day
five or day six embryos which were 33 % compared to 26 % for day two or day
three embryos.
The implantation rate for blastocysts is expected to be higher because of the better
selection of embryos on day five and day six of embryo culture. However, this did
not result in higher clinical pregnancy rates due to a higher transfer failure rate after
blastocyst culture.
23
In the next slides, the experience with blastocyst transfer in our center is
demonstrated by the following three randomized trials.
The first study was published by Kolibianakis et al. in 2004. In this study the
outcomes of day three and day five transfers in an unselected patient population
were compared. Patients were under the age of 43 years and were included into the
study and randomized at consultation.
The results of the second study were published by Papanikolaou et al. in 2005. Day
three and day five transfers were compared in a selected patient population.
Patients were included at consultation but the assignment to day three or day five
transfer and randomization were made on day three.
In the third study of Papanikolaou et al. in 2006, a selected young patient population
receiving single embryo transfer was included. Inclusion and randomization were
made at consultation.
24
In the study by Kolibianakis et al. in 2004, 234 day three transfers and 226 day five
transfers were compared.
No difference was found in the ongoing pregnancy rate per started cycle or per
transfer. The rates were 32.1 % for day three transfer and 33.2 % for day five
transfer per started cycle and 34.4 % for day three and 39.5 % for day five per
transfer.
There was also no significant difference in the implantation rates per embryo
transferred between the two groups. After day three an implantation rate of 24.5 %
was obtained compared to 26.6 % after day five transfer.
However, there was a difference in transfer rates which were higher for day three
transfers (93.2 %) compared to day five transfers (84.1 %).
The authors concluded that for the general population of fertility patients seen at
consultation no advantage of day five transfer compared to day three transfer could
be demonstrated.
25
Papanikolaou et al included a selected population of IVF or ICSI patients younger
than 37 years old in their first or second trial. Two embryos were transferred in each
cycle and only patients with ejaculated semen were included.
Patients were allocated and randomized for the study if at least four good quality
embryos (EQ1 and EQ2) were present in the morning of day three. In total, 273
patients were included at the start of the study but only 164 fulfilled the criteria on
day three and were randomized.
26
This table represents the results of the study by Papanikolaou et al. in 2005.
Significantly higher clinical and ongoing pregnancy rates were obtained for day five
transfer compared to day three transfer. Also a higher implantation and live birth
rates were found after day five double embryo transfer. The multiple pregnancy
rates were not significantly different.
The authors concluded that in a selected patient population with allocation on day
three based on the number of good quality embryos, day five transfer has a higher
likelihood of ongoing pregnancy and live birth than a transfer on day three.
27
In the study by Papanikolaou et al. in 2006, only those IVF or ICSI patients were
included who were younger than 36 years old and in their first or second cycle. In
this study only single embryo transfers were performed. 171 patients were
randomized for day three transfer and 169 patients for day five transfer. The
randomization was performed at consultation.
28
This table shows the results of the study by Papanikolaou et al. No significant
difference in transfer rates were found for day three or day five single embryo
transfer. Significantly higher clinical and ongoing pregnancy rates and delivery rates
were obtained after day five single blastocyst transfer. Two monozygotic twins were
born after day three single embryo transfer. This was not significantly different from
the day five group. The authors concluded that the data support the transfer of a
single day five blastocyst in women under the age of 36.
29
In the following slides, the actual transfer procedure will be discussed briefly.
The transfer of human in vitro cultured embryos into the uterus was first described
by Edwards et al. in 1980.
The procedure is generally performed without anesthesia. However anesthesia can
be applied when it is difficult to pass through the cervical canal.
The success rate of an embryo transfer is mainly determined by the technique
applied and the embryo transfer skill of the surgeon.
During the transfer procedure, it is important to keep the temperature and osmolality
optimal and to work in a sterile environment. Therefore, all disposables, media, and
devices are kept at 37°C. Petri dishes are pre-warmed on a warming plate and
syringes/catheters are stored in an oven at 37°C. The selected embryos are
collected and put together just before the surgeon is ready to start the transfer
procedure.
30
The following steps are performed at the UZ Brussel:
Selected embryos are put into a droplet of transfer medium (about 0.5 ml).
The Petri dish is kept in a small table incubator at 37°C with 5 % O2, 6 % CO2 and
89 % N2 until the moment of transfer. This time period should not exceed five
minutes in order to prevent a high osmolality of the transfer medium.
The embryologist fills a syringe with transfer medium and the syringe is put onto the
soft catheter (inner catheter). After that, the catheter is rinsed with the transfer
medium and loaded with the embryos as follows:
•first, an air bubble is aspirated (2–3 mm);
•then, embryos are aspirated in the smallest volume possible (this is approximately
15–25 µl);
•after that, a second air bubble is aspirated;
•finally, a small volume of medium is aspirated to seal the catheter.
The inner catheter is put into a warm sterile towel and is brought into the transfer
room once the gynecologist is ready for the transfer.
When the outer catheter is placed into the uterus, the gynecologist puts the inner
catheter with the embryos into the uterus at the right position.
31
Then, the embryologist injects a small volume of the transfer medium with the
embryos. Finally, the gynecologist gently pulls back the catheter.
As a final check, the embryologist empties the content of the catheter into a Petri
dish to make sure that no embryos are still present in the medium.
32
From this chapter we can conclude that:
The most commonly used parameters to define embryo quality are cell stage and
fragmentation in combination with a normal developmental pattern. Additional
parameters are scored to distinguish between embryos of almost the same quality.
Multiple pregnancies are a complication of assisted reproduction technology and
should be prevented as much as possible. For young women with good embryo
quality single embryo transfer should be the first choice. Day of transfer is chosen
according to the local transfer policy. However, day five transfers can result in
higher implantation rates in young women. During transfer, it is important to keep
the temperature and osmolality optimal.
33
34
35
36