Human Reproduction Vol.17, No.5 pp. 1327–1333, 2002
The use of a detailed zygote score after IVF/ICSI to obtain
good quality blastocysts: the German experience
U.Zollner1, K.-P.Zollner, G.Hartl, J.Dietl and T.Steck
Department of Obstetrics and Gynecology, University of Würzburg, Josef-Schneider-Strasse 4, D-97080 Würzburg, Germany
1To
whom correspondence should be addressed. E-mail: [email protected]
BACKGROUND: Due to improvements in embryo culture, it is now possible to transfer embryos 5 days after
oocyte retrieval and IVF/ICSI at the blastocyst stage, giving a better synchronization with the female reproductive
tract. In Germany it is illegal to culture more than three embryos. Therefore, there is need for a sufficient selection
at the pronuclear (PN) stage to select the best zygotes and exclude those of poor quality. METHODS: A prospective
trial was conducted in 168 IVF and ICSI cycles including the size, number and alignment of pronuclei and nucleoli,
cytoplasmic halo effect, the presence of vacuoles and granularity of ooplasm. Based on the above criteria, the best
zygotes were selected (score <15) for embryo transfer on day 5. Blastocysts were classified in eight grades based on
the cleavage speed. RESULTS: A total of 1450 oocytes were collected, of which 1119 reached the pronuclear stage.
Of the zygotes (n ⍧ 424) selected at the PN stage, 46% achieved the blastocyst stage after 5 days (grade
1–5), 26% the morula stage (grade 6–7) and 28% were arrested (grade 8). The mean zygote score showed a
significant positive correlation with the mean blastocyst quality in ICSI, but not in IVF cycles. A cut-off of 15 was
calculated for ICSI cycles giving the best discrimination with blastocyst grades (6 versus 7) and number of arrested
embryos (23 versus 45%) below and above this cut-off. A total of 33 clinical pregnancies was achieved (20%).
Women conceiving had a significantly better mean blastocyst development than those not conceiving. Strong
cytoplasmic vacuolization and an extreme or no halo effect had a negative effect on blastocyst development.
CONCLUSIONS: The data show that PN stage morphology is related to blastocyst development, but the rate of
arrested embryos of almost 30% limits the chance of conception under the conditions of the German Embryo
Protection Law.
Key words: embryo selection/human blastocyst/IVF/ICSI/pronuclear stage score
Introduction
In most IVF programmes, embryos are usually transferred to
the uterus at the cleavage stage on the second or third day
after oocyte retrieval. In the last few years it has been
possible to culture embryos to the blastocyst stage due to the
development of new sequential serum-free culture media
capable of supporting the growth of viable blastocysts (Gardner
and Lane, 1997). Blastocyst culture helps to improve the
synchronization of the embryo with the female reproductive
tract. The implantation rate per cleavage stage embryo transferred has not increased in the last 10 years beyond the range
of 12.5–15% (Edwards and Craft, 1990). The implantation
rates after blastocyst culture are reported to reach 50% in
selected groups of patients (Gardner et al., 1998a, 1998b).
High pregnancy rates, however, can only be achieved when a
cohort of at least eight normal oocytes is available and selection
can be done during the embryo culture and at the blastocyst
stage (Bongso, 1999). The high implantation potential of
blastocysts means that fewer embryos are required for transfer
to achieve clinical pregnancy with a reduction of the incidence
of multiple pregnancies.
© European Society of Human Reproduction and Embryology
One of the main problems in a German IVF unit is that
selection of embryos after conventional IVF or ICSI has to be
performed at the pronuclear (PN) stage, as the German Embryo
Protection Law prohibits both the culture of more than three
embryos and embryo selection. It is only allowed to culture
as many zygotes as will be presumably transferred. Supernumerary zygotes can be cryopreserved or have to be withdrawn. Selection at the PN stage is not only important in
Germany with its restrictive legislation, but also in other
countries in patients with religious or ethical concerns regarding
the destruction of non-selected but viable embryos.
Many studies have proposed a PN scoring system (Payne
et al., 1997; Scott and Smith, 1998; Tesarik and Greco,
1999; Wittemer et al., 2000) with predictive value for the
implantantion rate, but relatively little is known about the
development of selected zygotes after days 2 or 3.
The aim of this prospective study was to describe morphological criteria of zygotes that can predict the development of
high quality blastocysts and therefore maintain a satisfactory
rate of clinical pregnancies even under the conditions of the
German Embryo Protection Law. In comparison with previous
1327
U.Zollner et al.
Ovarian stimulation and sperm preparation
Ovarian stimulation, oocyte recovery, IVF and ICSI procedures were
carried out using standard protocols as described previously (Zollner
et al., 1996, 2001). Ovarian stimulation was induced by a mild stepdown human menopausal gonadotrophin (HMG, Menogon®; Ferring,
Kiel, Germany) or FSH (Gonal F®; Serono, Unterschleißheim,
Germany; Puregon®; Organon, Oberschleißheim, Germany) protocol
starting on day 3 of the cycle after pituitary down-regulation by
0.2 mg intranasal nafarelin 2–4 times a day (Synarela®; Heumann,
Nürnberg, Germany) 6–8 days prior to the expected period. A dose
of 10 000 IU of HCG (Pregnesin®; Serono) was administered late in
the evening of the day during which the mean diameter of the
dominant follicle reached 18 mm after 6–7 days of a steady rise in
serum oestradiol concentrations. Oocyte recovery was performed by
the vaginal route 34–36 h after HCG administration.
All native semen samples and samples after preparation were
evaluated manually according to published guidelines (World Health
Organization, 1992) for volume, total sperm count, concentration,
leukocytes, motility, vitality and morphology. The ejaculates were
prepared by a PureSperm (Nidacon, Sweden) cushion centrifugation
and a swim-up procedure. After several centrifugation and washing
steps the sperm preparation was incubated for 30–60 min at 37°C for
the swim-up.
OvoilTM (Scandinavian IVF). The zygotes were scored on an inverted
microscope at magnification of ⫻400. Photodocumentation of the
embryos were performed during this inspection and on days 3 and 5.
All cells were judged prospectively in detail for the following
criteria: alignment and number of pronuclei and nucleoli, halo effect,
size of pronuclei, appearance of vacuoles and the appearance of the
ooplasma. Zygotes then were assigned a cumulative score as shown
in Table I. For alignment and number of nucleoli, each pronucleus
was evaluated separately. This assessment required the changing of
focus during observation until the whole volume of both pronuclei
could be inspected. With the use of an injection pipette to turn the
oocyte around, an exact evaluation of the juxtaposition of pronuclei
was achieved. The zygote score was calculated as a cumulative score
of all items. The best score thereby was 10 and the worst score was
31 for 2PN stages and 37 for 1PN stages. Embryos with only one
pronucleus were included in this scoring system but were not
selected for culture except when no other pre-embryos were available.
Figure 1 shows some examples of zygote evaluation. According to
the patients’ age, individual history or desire, two or three zygotes
with the lowest score were selected for further culture and transfer.
A mean zygote score was calculated as the sum of all selected zygotes
per number of selected embryos. For each PN stage embryo, the
subsequent development at day 3 and the blastocyst development on
day 5 was evaluated. Embryos were cultured separately to allow for
individual comparisons at different times during the culture.
IVFTM (Scandinavian IVF) medium was used for all oocyte
cultures. Oocytes were cultured at 37°C in 5% CO2 at pH 7.4. At
4 h after recovery the oocytes were inseminated with 10 000–20 000
motile sperm, or, in the case of severe andrological subfertility, the
ICSI procedure was performed as described previously (Zollner et al.,
2001). After injection, each oocyte was transferred separately in a
microdroplet of fresh IVF medium. Fertilized oocytes in excess of
two or three were cryopreserved or withdrawn. The selected embryos
were transferred to G1.2TM medium (Scandinavian IVF), kept in
culture for another 48 h and were then placed into G2.2 medium
(Scandinavian IVF). Cleavage rate and embryo morphology were
evaluated again at that time (day 3). If the embryos showed at least
six blastomeres after 48 h they were placed into G2.2 medium.
Embryos with fewer than six blastomeres were cultured for a further
4 h in G1.2 medium and then checked again. If the 6-cell stage was
achieved, the embryos were transferred to fresh G2.2 medium.
The scoring system used on day 3 was based on the number of
blastomeres and the proportion of anucleate fragments inside the
embryo, allowing calculation of the embryo score (ES) and cumulative
embryo score (CES) (Steer et al., 1992). The embryos were graded
as follows: grade 4, equal-sized symmetrical blastomeres; grade 3,
uneven blastomeres with ⬍10% fragmentation; grade 2, 10–50%
blastomeric fragmentation; grade 1, ⬎50% blastomeric fragmentation
or pronucleate single cell embryos. The morphological grade of the
embryo was then multiplied by the number of blastomeres. The
cumulative scores of all embryos selected at the PN stage per patient
were calculated to obtain the cumulative embryo score (CES).
The mean CES is calculated as CES divided by the number of
transferred embryos.
Zygote and embryo scoring, embryo culture
After incubation for 16–18 h, the oocytes were checked for the
presence of pronuclei as evidence of fertilization. Regular fertilization
was defined as extrusion of the second polar body and presence of
two pronuclei. In IVF cycles, the remaining cumulus and corona cells
were stripped from the pre-embryos and then each zygote was placed
individually into a Petri dish with microdroplets of 5–10 µl of IVFTM
medium (Scandinavian IVF, Gothenburg, Sweden) overlaid with
Blastocyst culture, embryo transfer and pregnancy assessment
After 2 days of culture in G2.2 medium, blastocyst formation was
evaluated. The assessment of embryo viability was based on embryo
morphology and cleavage speed (Bongso, 1999): 1 ⫽ fully expanded
blastocyst (distinct inner cell mass, trophectoderm and blastocoele,
thin zona pellucida, fully expanded diameter); 2 ⫽ expanding blastocyst (distinct inner cell mass, trophectoderm and blastocoele, thin
zona pellucida, substantial increase in embryo diameter, but not
studies, we aimed at a more detailed description of the zygote
morphology than has been done before, including the following
criteria: number and size of pronuclei, juxtaposition of pronuclei, halo effect, alignment and number of nucleoli separately
for both pronuclei, appearance of vacuoles and the appearance
of the ooplasm. The use of this scoring system allows selection
of those zygotes with favourable developmental potential for
further embryo culture, therefore maximizing the chance of
implantation.
Materials and methods
Patients
Between July 1999 and April 2000, 168 infertile couples were
consecutively included in this prospective trial. Patients were unselected for age, sperm parameters or infertility criteria. In 101 patients,
conventional IVF was performed and in 67 cases the ICSI procedure
was applied. Couples were included only if at least one oocyte could
be collected that showed signs of fertilization after 16 to 18 h
(1PN or 2PN stage). All cases of unsuccessful oocyte retrieval or
polyspermic zygotes were excluded. All patients underwent the IVF/
ICSI programme at the University of Würzburg. The indications for
assisted reproduction were one or several of the following: tubal
factor (36%), endometriosis (5%), male factor (46%) or idiopathic
infertility (17%). The indication for ICSI was male subfertility in all
cases. The mean ⫾ SD age of the women was 33.4 ⫾ 4.1 years and
that of their partners 36.1 ⫾ 5.3 and did not differ significantly
between the IVF and ICSI couples. The mean duration of infertility
was 4.2 years.
1328
Blastocyst culture after selection at the pronuclear stage
Table I. Zygote scoring system
1
2
3
4
No. of pronuclei
Juxtaposition of pronuclei
Size of pronuclei
Halo effect
Alignment of nucleoli in PN 1
2PN
Strong together
Equal
Normal
Row
–
Near by
Unequal
Light
Beginning row
–
Disjuncted
–
Extreme
Confused
Alignment of nucleoli in PN 2
Row
Beginning row
Confused
No. of nucleoli in PN 1
3–5
⬎5
⬍3
No. of nucleoli in PN 2
3–5
⬎5
⬍3
Appearance of vacuoles
No vacuoles
Appearance of ooplasma
Homogeneous
granulated
Lightly
vacuolized
Strongly
granulated
Strongly
vacuolized
–
1PN
Only one pronucleus
Only one pronucleus
No halo effect
No nucleoli or
no pronucleus
No nucleoli or no
pronucleus
No nucleoli or
no pronucleus
No nucleoli or
no pronucleus
–
–
PN ⫽ pronucleus.
fully expanded); 3 ⫽ early blastocyst (distinct inner cell mass,
trophectoderm and blastocoele); 4 ⫽ late cavitating embryo with
⬎50% blastocoele (distinct blastocoele, inner cell mass and trophectoderm not laid down); 5 ⫽ early cavitating embryo with ⬍50%
blastocoele (first signs of blastocoele); 6 ⫽ compacted embryo;
7 ⫽ compacting embryo; 8 ⫽ arrested embryo.
A maximum of three embryos were transferred into the uterus
5 days after oocyte recovery. The luteal phase was routinely supported
by vaginal progesterone (Crinone®; Serono) and by at least one
injection of 5000 IU HCG.
A single serum β-HCG measurement was performed 14 days after
embryo transfer. Clinical pregnancy was defined as presence of a
gestational sac on ultrasound scan.
Statistical analysis
Statistical analysis was performed using SPSS 10.0. As not all
parameters were normally distributed, results were expressed as
median and range. The Mann–Whitney test was used to compare
differences between groups while the χ2 analysis was used to compare
proportions. Multiple correlations were calculated using Spearman’s
correlation. P ⬍ 0.05 was considered significant.
Results
Oocyte, zygote, embryo and blastocyst assessment
The oocyte characteristics and the fertilization data of the 168
patients included in this study are presented in Table II,
showing the data from IVF and ICSI cycles separately. A total
of 1450 oocytes were retrieved; 1119 were fertilized and
reached the PN stage after 16–18 h. All oocytes with no
fertilization and polyspermic zygotes were excluded. Only the
data of the pre-embryos selected at the PN stage are presented
here as the surplus zygotes had to be withdrawn or cryopreserved. A total of 424 zygotes was selected, five of which
were at the 1PN stage. In 97 couples three pre-embryos were
selected, in 62 couples two pre-embryos and in nine couples
only one embryo was available.
In ICSI cycles significantly more oocytes were collected
than in IVF cycles, but the fertilization rates were similar.
Although significantly more embryos were transferred in ICSI
than in IVF cycles, the pregnancy rates were not different.
The mean zygote scores, mean and cumulative embryo scores
and blastocyst data are given in Table II showing the data
from IVF and ICSI cycles separately. There were no significant
differences between conventional IVF and microinjection.
All morphological parameters of the PN embryos have been
correlated with the rate of blastocysts and arrested embryos.
If the zygotes showed strong vacuolization, 71% of them
developed into arrested embryos versus only 27% of the
zygotes with no or light vacuolization (P ⫽ 0.019). The
median grade of blastocyst development was 8 (5–8) in strong
vacuolized zygotes and 6 (1–8) in light or not vacuolized
embryos (P ⫽ 0.019). The blastulation rate was 47% in
embryos with normal or light halo effect versus only 29% in
those with extreme or no halo effect (P ⫽ 0.041). The rate
was 49% when pronuclei of equal size were apparent but only
36% when the pronuclei were of unequal size (P ⫽ 0.015).
None of the zygotes with one pronucleus developed into a
blastocyst. The blastulation rate of the 2PN stages was 47%.
There were no further significant differences between the other
morphological parameters of the pronucleate embryos and the
rate of blastocysts and arrested embryos.
The mean zygote score was significantly lower in elective
than in non-elective cycles after IVF as well as after ICSI
(Table III). Patients were allocated to the elective transfer
protocol if more than two or three zygotes were available, to
the non-elective protocol if no surplus zygotes were available.
Of the zygotes (n ⫽ 424) selected at the PN stage, 46%
achieved the blastocyst stage after 5 days. The distribution
into blastocyst grades was as follows: (1) fully expanded
blastocyst 2%, (2) expanding blastocyst 6%, (3) early blastocyst
12%, (4) late cavitating embryo 12%, (5) early cavitating
embryo 14%. The morula stage was achieved by 26%, including
(6) compacted embryo 17%, (7) compacting embryo 9%.
Finally, 28% of the zygotes arrested during their embryonic
development (8). These frequencies were not significantly
different after IVF and ICSI.
A significant correlation between the mean zygote score
1329
U.Zollner et al.
Figure 1. Morphologies of pronuclear human embryos. (a) Two pronuclei (PN); juxtaposition of PN: strong together; size of PN: equal;
normal halo effect; alignment of nucleoli in both PN: row; number of nucleoli in both PN: 3–5; no vacuoles; appearance of the
ooplasma: homogeneous granulated; score ⫽ 10. (b) Light halo effect; score ⫽ 11. (c) Light halo effect; size of PN: unequal; score ⫽ 12.
(d) Juxtaposition of PN: nearby; halo effect: no halo; number of nucleoli in both PN: ⬎5; score ⫽ 16. (e) Juxtaposition of PN: nearby;
size of PN: unequal; halo effect: no halo; alignment of nucleoli in PN 2: beginning row; number of nucleoli in PN 2: ⬎5; score ⫽ 17.
(f) Juxtaposition of PN: disjuncted; halo effect: extreme; alignment of nucleoli in PN 2: beginning row; number of nucleoli in both PN: ⬎5;
score ⫽ 17. Original magnifications ⫽ ⫻400.
and the mean blastocyst development was found in ICSI
(P ⫽ 0.041) but not in IVF cycles (not significant). For the
zygote score, a cut-off value of 15 could be calculated for
ICSI cycles with serial Mann–Whitney tests giving the best
statistical power for the prediction of blastulation. If the PN
score was ⬎15, the blastocyst grade was significantly reduced
(grade 7) compared with zygotes with a score 艋15 (grade 6,
1330
P ⫽ 0.012). With a score below this cut-off, significantly
fewer embryos arrested during the cleavage stages than with
a score ⬎15 (Table IV). If the mean zygote score was ⬎15,
the clinical pregnancy rate was 14%, compared with 22% with
a mean zygote score 艋15 (not significant). The zygote score
also correlated with the female age (P ⫽ 0.017) and the
number of oocytes collected (P ⫽ 0.022).
Blastocyst culture after selection at the pronuclear stage
Table II. Fertilization data
Age (years)
No. of oocytes
No. of oocytes at metaphase II
No. of fertilized oocytes
Fertilization rate (%)
No. of transferred embryos
Implantation rate per embryo transferred (%)
Pregnancy rate (%)
Mean zygote score
Cumulative embryo score
Mean cumulative embryo score
Mean blastocyst grade
Total
(n ⫽ 168)
IVF
(n ⫽ 101)
ICSI
(n ⫽ 67)
P
33 (23–42)
8 (1–23)
–
6 (1–19)
87.5 (17–100)
3 (1–3)
9.2
19.6
12.7 (10–21.5)
60 (12–192)
24 (5.3–64)
5.7 (2–8)
33 (25–41)
7 (1–23)
–
6 (1–19)
85.7 (17–100)
2 (1–3)
9.4
18.8
12.3 (10–21.5)
62 (16–160)
24 (5.3–64)
5.7 (2–8)
33 (23–42)
10 (1–19)
9 (1–18)
7 (1–18)
88.9 (27–100)
3 (1–3)
8.9
20.9
13 (10–19)
59 (12–192)
24 (6–64)
5.5 (2.3–8)
NS
0.004
–
0.042
NS
0.008
NS
NS
NS
NS
NS
NS
Values are given as median and range.
NS ⫽ not significant.
Table III. Fertilization and embryo development data in pregnant and non-pregnant women and in elective versus non-elective embryo transfers
IVF
Age (years)
No. of oocytes
Fertilization rate (%)
No. of transferred embryos
Mean zygote score
Cumulative embryo score
Mean cumulative embryo score
Mean blastocyst grade
ICSI
Pregnant
Not pregnant
P
Pregnant
Not pregnant
P
34 (26–38)
8 (1–19)
83.3 (50–100)
3 (1–3)
12.7 (10–17)
80 (40–109)
30.7 (16.7–38)
4 (2.3–6)
33 (25–41)
7 (1–23)
87.5 (17–100)
2 (1–3)
12.3 (10–21.5)
56 (16–160)
24 (5.3–64)
6 (2–8)
NS
NS
NS
NS
NS
0.024
NS
0.001
32.5 (26–40)
11 (1–18)
88.9 (57–100)
3 (1–3)
12.8 (10–18.3)
76 (43–128)
26 (14.3–64)
4.2 (2.3–7.5)
33 (23–42)
9 (2–19)
89.9 (27–100)
3 (1–3)
13 (10–19)
57 (12–192)
21.7 (6–64)
6 (2.6–8)
NS
NS
NS
NS
NS
NS
NS
0.001
IVF
Age (years)
No. of oocytes
Fertilization rate (%)
No. of transferred embryos
Mean zygote score
Cumulative embryo score
Mean cumulative embryo score
Mean blastocyst grade
Pregnancy rate (%)
ICSI
Elective
transfers
Non-elective
transfers
P
Elective
transfers
Non-elective
transfers
P
33 (25–41)
9 (2–23)
85.7 (38–100)
3 (2–3)
12 (10–15.5)
63 (16–160)
24 (5.3–64)
5.7 (2.3–8)
20.0
34.5 (25–41)
4 (1–13)
92.3 (17–100)
2 (1–3)
13.3 (10–21.5)
58 (16–128)
25.8 (8–64)
5.9 (2–8)
15.0
NS
0.000
NS
0.031
⬍0.001
NS
NS
NS
NS
32 (23–42)
11 (5–19)
88.9 (27–100)
3 (2–3)
12.7 (10–16.3)
59.5 (18–160)
24 (6–64)
5.6 (2.3–8)
23.9
34 (24–42)
6 (1–16)
80 (29–100)
3 (1–3)
14 (11.5–19)
58 (12–192)
23.7 (12–64)
5.3 (2.6–8)
14.3
NS
⬍0.001
NS
NS
0.011
NS
NS
NS
NS
Values are given as median and range unless otherwise stated.
NS ⫽ not significant.
Pregnancy data
The overall pregnancy rate (PR) was 20% and was similar in
IVF and ICSI cycles (Table II). All selected embryos were
transferred, even if they were arrested at the cleavage stage.
In 168 cycles studied, 33 clinical pregnancies occurred, of
which nine ended as abortions, and six pregnancies were twin
pregnancies, giving an overall implantation rate of 9.2%. The
overall PR in non-elective (n ⫽ 61) embryo transfers was
15% (one embryo: PR 22%; two embryos: PR 13%; three
embryos: PR 14%). In elective transfers (n ⫽ 107), it was
22% (two embryos: PR 21%; three embryos: PR 24%). Table III
shows the fertilization and pregnancy data in elective and nonelective transfers.
There was no significant difference between women
conceiving and not conceiving in terms of mean age, semen
quality, number of transferred embryos and fertilization rate
(Table III). Women achieving conception had a significantly
better mean blastocyst grade (4 versus 6, P ⫽ 0.001) than
women who did not.
None of the patients, with transfer of grade 6–8 embryos
1331
U.Zollner et al.
Table IV. Embryo and blastocyst development beyond and below a
pronuclear (PN) score cut-off of 15
Embryo score
Blastocyst grade
Blastocyst development (%)
No. of arrested embryos (%)
Pregnancy rate (%)
PN score 艋15
PN score ⬎15
P
24 (1–64)
6 (1–8)
47.8
22.6
21.7
18 (8–64)
7 (3–8)
30.0
45.0
14.3
NS
0.012
NS
0.03
NS
Only the data of the 67 ICSI cycles are given. Values are given as median
and range unless otherwise stated.
only, conceived. In 12 patients, only arrested embryos were
transferred, resulting in no clinical pregnancy. In all successful
cycles at least one blastocyst was transferred.
Discussion
In this prospective study, morphological parameters of PN
stage pre-embryos that are predictive for blastocyst development were described. The zygote score correlated with subsequent blastocyst development in ICSI cycles. We did not
observe a correlation in IVF cycles, perhaps due to different
cleavage speeds of IVF and ICSI zygotes. With a mean PN
score ⬎15, a poor blastocyst development and a low pregnancy
rate are to be expected. Those zygotes should not be selected
unless they are the only ones available. Furthermore, it could
be shown that the clinical pregnancy rate after selection and
elective embryo transfer exceeds that after non-elective embryo
transfers (not statistically significant), independent from the
number of embryos transferred. As expected, the mean zygote
score was significantly higher in those cycles where only two
or three zygotes were present and therefore no selection was
possible as in the cycles with supernumerary PN stages. We
also correlated all morphological parameters at the PN stage
with the blastulation rate and the rate of arrested embryos. It
could be demonstrated that strong vacuolization or an extreme
or no halo effect are negatively predictive for blastocyst
development. The blastulation rates were also lower when the
pronuclei were unequally sized.
Several authors have attempted to find selection criteria at
the PN stage that are of similar predictive value for the
implantation rate as those of a day 2 or day 3 embryo or a
blastocyst. Scott and Smith published a new scoring system
for zygotes, taking into account the alignment of pronuclei,
the positioning of the nucleoli and the cytoplasm (Scott and
Smith, 1998). Their score also included the time factor of
embryo development as it could be shown that a fast PN
membrane breakdown and a short time until the first cleavage
was correlated with a high pregnancy rate. In Germany, this
score is not applicable because it is illegal to include the speed
of embryo development and because the selection has to be
done at the PN stage.
Nevertheless, one study (Ludwig et al., 2000) applied
the score by Scott and Smith (1998) in a German assisted
reproduction unit considering only the items that can be
evaluated at the PN stage. They found a threshold score of
1332
13. In their study, a mean score of ⬍13 led to a pregnancy
rate of 4% whereas a mean PN score of 艌13 led to a PR of
22%. They also found a significant correlation between embryo
morphology and cumulative PN score.
The tendency to polarization, which is the basis for the
alignment of pronuclei and nucleoli, is already present at the
PN stage. Initially, only the alignment of nucleoli at the
junction of the two pronuclei was considered as a selection
criterion for embryo transfer (Wright et al., 1990), but it could
be shown that the appearance of the cytoplasm is also important.
Tesarik and Greco developed a PN score that can be calculated
by a single observation to predict the potential for implantation
(Tesarik and Greco, 1999). They defined an optimal PN
morphology as pattern 0. The rate of arrested embryos until
the 4–8-cell stage was only 8.5% in pattern 0 embryos. Their
score, however, contains just the number and arrangement of
nucleoli. In contrast, our score is more detailed as we were
aiming at the definition of morphological parameters correlating with the development of blastocysts. Our morphological
description of the PN stage pre-embryos can be translated into
a scoring system which may be evaluated by an experienced
embryologist in ~30 s, so that it can be integrated in the
routine work. Our score may also help to decrease the
occurrence of multiple pregnancies without affecting the overall
success rate, as we did not see a significant difference in
pregnancy rates after the transfer of two versus three embryos.
So in younger patients the choice of only two embryos may
not affect the success rate.
As zygote morphology alone does not consistently identify
embryos with high implantation potential, a graduated embryo
score was developed (Fisch et al., 2001) combining PN
morphology, first cleavage and day 3 morphology. Embryos
with a high score had a significant better blastocyst formation
than embryos with lower scores. In this scoring system, only
the nucleolar alignment along the PN axis was taken into
consideration. Another recent study found that not only the
alignment of nucleoli but also the halo effect is important for
further embryonic development (Salumets et al., 2001). In this
study (Salumets et al., 2001), embryos derived from halopositive zygotes had significantly better morphology compared
to halo-negative-derived embryos.
It is generally accepted that the implantation and pregnancy
rates can be increased when the selection of embryos can be
done at the blastocyst stage. In Germany, however, selection
of embryos and blastocysts is illegal. A significant proportion
of fertilized oocytes is affected by chromosomal abnormalities,
by deficient activation of the embryonic genome or other
disturbances. Fluorescent in-situ hybridization diagnosis of
human embryos in poor prognosis patients revealed that 57%
were chromosomally abnormal (Magli et al., 1998). The
natural selection occurring during development from gamete
to blastocyst can take place in vitro (Menezo et al., 1997).
Although development to the blastocyst stage cannot guarantee
chromosomal normality, the majority of the embryos failing
to continue to develop in extended culture show multiple
aneuploidies for chromosomes X, Y, 16, 18 and 21 (Jones and
Trounson, 1999).
Gardner et al. observed blastulation rates as high as 47%
Blastocyst culture after selection at the pronuclear stage
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Submitted on June 15, 2001; resubmitted on October 8, 2001; accepted on
January 4, 2002
(Gardner et al., 1998b) and 66% (Gardner et al., 1998a), but
it was not possible to predict blastocyst development on the
basis of embryo morphology on day 3. This observation is
confirmed by the findings of our study. We found a positive
correlation between PN and blastocyst morphology, but from
a cohort of zygotes we could not identify those two or three
that will develop into good quality blastocysts with certainty.
In our study we found a blastulation rate of 46% after selection
but only 2% of blastocysts were fully expanded. Only patients
with the transfer of at least one blastulated embryo conceived,
but none of the patients with transfer of only morulae or
arrested embryos. If it is assumed that only blastocysts led to
pregnancies, the implantation rate per transferred blastocyst
would be 20%.
It has been postulated that implantation rates of day 2
embryos is comparable with that of blastocysts when the
selection is done at the PN stage (Banerjee et al., 2000). It
was proposed that prolonged selection of embryos in vitro
until the blastocyst stage eliminates paternal chromosomal
anomalies as only 45% of the fertilized oocytes reach the
blastocyst stage, but they found that phenotypic manifestation
of paternal genomic abnormalities does not occur prior to
implantation. Nevertheless, blastocyst culture will facilitate
the assessment of embryonic development as the genome is
activated at the 8-cell stage, whereas the metabolism of the
cleavage stage embryo reflects that of the oocyte (Gardner
et al., 1998a).
For patients who produce only two or three embryos to
begin with, the extension of culture beyond day 2 or 3 offers
no additional advantage as selection will not be possible at
any stage. Under the German Embryo Protection Law, each
couple is allowed to produce only two or three embryos as
only two or three zygotes are allowed to be cultured. We did
not compare day 3 versus day 5 transfer after selection at the
PN stage, but the pregnancy rates after blastocyst transfer were
not higher than or equal to those during the early years when
the embryo transfers were routinely performed on day 2 or 3
in our programme (~20%). In summary, we think that under
the legal conditions in Germany, blastocyst culture cannot
improve pregnancy rates, in spite of a positive correlation
between some parameters of PN and blastocyst morphology,
when the selection has to be done as early as the PN
stage. Nevertheless, in cases of repeated implantation failure,
extended culture offers the advantage of in-vitro selection and
elimination of chromosomally abnormal embryos and may be
useful as a diagnostic procedure for embryonic development.
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