Full text of DOI:10.1093/humrep/16.11.2384

Human Reproduction Vol.16, No.11 pp. 2384–2389, 2001
Evaluation of pronuclear morphology as the only selection
criterion for further embryo culture and transfer:
results of a prospective multicentre study
M.Montag1 and H.van der Ven on behalf of the German Pronuclear Morphology Study
Group*
1To whom correspondence should be addressed at: Universita¨ts-Frauenklinik Bonn, Sigmund-Freud-Strasse 25, D-53105 Bonn,
Germany. E-mail: m.montag@uni-bonn.de
BACKGROUND: The aim was to study zygote evaluation based on pronuclear morphology under the German
embryo protection law, according to which only a maximum of three zygotes are allowed to be cultured for embryo
transfer. METHODS: In this prospective multicentre study, a total of 512 treatment cycles was performed at 10
centres, between November 1999 and October 2000. Zygotes were classified into seven patterns (0A, 0B and 1–5).
Pattern 0A and 0B zygotes were preferentially used for further culture and transfer. RESULTS: Cycles with
transfer of at least one embryo derived from pattern 0B, but not pattern 0A, resulted in significantly higher
pregnancy (37.9%) and implantation rates (20.5%) compared with non-pattern 0B cycles (26.4 and 15.7%;
P < 0.05 and P < 0.01 respectively). In younger patients (aged ≤35 years), significantly more 0B zygotes were
available for transfer than in older patients (34.2 versus 25.8%; P < 0.005). CONCLUSIONS: From these data, it
is concluded that evaluation of pronuclear morphology is beneficial, especially for countries with legal restrictions
regarding embryo selection.
Key words: ICSI/implantation/IVF/maternal age/pronuclear morphology
Introduction
and Smith, 1998) which proposed that the implantation poten-
tial of an embryo can be predicted at the pronuclear stage, based
on morphological criteria. This idea was further supported by
others (Tesarik and Greco, 1999), who showed that a single
examination of pronuclear morphology, namely the number
and distribution of nucleolar precursor bodies (NPB) in each
pronucleus, can predict abnormal preimplantation develop-
ment. The same group reported that following intracytoplasmic
sperm injection (ICSI), embryos derived from a specific
pronuclear pattern gave higher implantation rates compared
with other patterns (Tesarik et al., 2000); these observations
were confirmed by another group (Wittemer et al., 2000).
More recently, it has been demonstrated that the morphology
of human pronuclear stage embryos is related to blastocyst
development (Scott et al., 2000), which may explain the high
implantation potential of embryos developing from distinct
pronuclear morphology pattern.
The selection of embryos with the highest implantation poten-
tial for transfer is a crucial part of assisted reproduction. The
introduction of sequential culture media enables the culture of
a maximum number of embryos under optimal conditions to
the blastocyst stage and consequently, high implantation rates
have been reported following the transfer of selected blastocysts
(Gardner et al., 1998). Another approach was presented (Scott
*Participating centres: V.Blumenauer, J.Schuster, F.A.Hmeidan,
Zentrum f. Reproduktionsmedizin, Goldschmidstr. 30, D-04103
Leipzig; C.Balko, R.Sudik, Klinik f. Gyna¨kologie u. Geburtshilfe,
Salvador-Allende-Str. 30, D-17036 Neubrandenburg; E.Schanze,
K.Marx, R.J.van Kooij, U.Weidner, Praxis Verhoeven Scholtes Marx
Wohlers Schulte, Neusser Str. 111, D-40219 Dusseldorf;
J.Hirchenhain, J.S.Kru¨ssel, P.Bielfeld, Universita¨ts-Frauenklinik,
¨
Moorenstr. 5, D-40225 Dusseldorf; B.Herrmann, J.Tigges, Praxis Dres
¨
Tigges Doerner Tuchel, Rheydter Str. 143, D-41515 Grevenbroich;
The approach to identify human embryos with the highest
implantation potential at the pronuclear stage is extremely
interesting for countries with a strict embryo protection law,
such as Germany and Switzerland. In these countries, the
selection of cleavage stages is prohibited by law, and therefore
no other embryos are available for later transfer than those
selected at the pronuclear stage. At the pronuclear stage it has
to be decided which cells are cultured and used for embryo
transfer, and which are cryopreserved or discarded. A prelimin-
P.Gaβner, A.Schepers, H.M.Behre, Institut f. Reproduktionsmedizin,
Universita¨t Munster, Domagkstr. 11, D-48129 Munster; M.Montag,
¨
¨
M.Kupka, K.van der Ven, H.van der Ven, Abt. Gyn. Endokrinol.
u. Reproduktionsmedizin, Universita¨ts-Frauenklinik, Sigmund-Freud-
Str. 25, D-53105 Bonn; M.Greuner, S.Roth, M.Thaele, Praxis Happel
Thaele Happel, Kaiserstr. 7, 66111 Saarbrucken; I.Eberhardt,
¨
D.Seehaus, T.Strowitzki, Abt. Gyn. Endokrinol. u. Fertilita¨tssto¨-
rungen, Universita¨ts-Frauenklinik, Voβstr. 9, D-69115 Heidelberg;
D.Ecke, C.Keck, Breckwoldt, Universita¨ts-Frauenklinik, Hugstetter
Str. 55, D-79106 Freiburg
2384
© European Society of Human Reproduction and Embryology

Pronuclear morphology in embryo culture and transfer
Evaluations
ary study on the use of a pronuclear score under these
conditions has been published (Ludwig et al., 2000). These
authors assessed several morphological criteria in 74 non-
selected ICSI cycles and found a higher pregnancy rate above
a certain pronuclear score threshold. In accordance with
German embryo protection law, the aim of the present study
was to evaluate the potential use of pronuclear selection in IVF
and ICSI cycles based on a single morphological observation in
a prospective, non-randomized multicentre study.
For the calculation of the pregnancy rate only cycles with proven
implantation, documented by ultrasound, were considered. The
implantation rate was calculated from the number of gestational sacs
divided by the total number of embryos transferred. For each
pronuclear morphology pattern the mean embryo score was calculated
based on all embryos derived from that specific pattern. Clinical
pregnancy and implantation rates were calculated for all transfer
cycles with at least one embryo derived from pattern 0B versus all
remaining transfer cycles without embryos derived from pattern 0B.
For all other patterns, pregnancy and implantation rates were calcu-
lated for all transfer cycles with at least one embryo derived from
that specific pattern X (X ϭ 0A, 1, 2, 3, 4, 5) versus all remaining
Materials and methods
Study design and patients
transfer cycles without embryos derived from pattern
X
([0Aϩ1ϩ2ϩ3ϩ4ϩ5] – X). For both of these calculations, all transfer
cycles with at least one embryo derived from pattern 0B were excluded.
An evaluation of the data was performed for the number of
presumable good pronuclear morphology pattern 0A/0B in relation
to maternal age (ഛ35 years/Ͼ35 years) and in relation to assisted
reproduction technique (IVF/ICSI).
This study was performed between November 1999 and October
2000 at 10 IVF centres in Germany. In total, 512 patients receiving
either IVF (n ϭ 247) or ICSI (n ϭ 265) treatment were enrolled in
this study. Criteria for inclusion were normal FSH concentrations
during early follicular phase, fewer than four previous treatment
cycles, and a negative test for hepatitis and HIV. Exclusion criteria
were polycystic ovaries, endometriosis and poor ovarian response in
previous stimulation cycles. Stimulation was performed by down-
regulation in a long protocol in all centres, whereas the embryo
culture system was not the same for all centres. On the day after
follicular puncture, a maximum of three pronuclear stage oocytes
were selected (see below) for further culture and transfer on days
2–5. All other pronuclear-stage oocytes were either cryopreserved or
discarded, based on the policy of each centre. In order to fulfil the
criteria of a prospective study, each centre had to register a treatment
cycle for the study at the central study office on the day of pronuclear
morphology assessment. On that day, detailed information on the
pronuclear morphology pattern of those pronuclear-stage oocytes
which were selected for further culture and transfer were reported to
the study office. The study office requested for each cycle a second
report form, containing data on human chorionic gonadotrophin
(HCG) measurement, number of gestational sacs and fetal heart beat
following the induction of a pregnancy.
Statistical analysis
Statistical evaluation was performed with ANOVA and a χ2 test
where appropriate; a P-value Ͻ 0.05 was considered significant.
Results
Study characteristics
A total of 512 treatment cycles (variation between centres 1 to
180) were prospectively registered at the central study office.
Among these, 495 treatment cycles fulfilled all criteria and
were included in the final evaluation. In all, 495 transfers
(IVF, n ϭ 239; ICSI, n ϭ 256) gave rise to 160 pregnancies
(IVF, n ϭ 69; ICSI, n ϭ 91) with a mean clinical pregnancy
rate per transfer of 32.3% and a variation of pregnancy rates
between centres from 0–41% (0% pregnancy rate in the centre
which reported one treatment cycle), with 203 out of 1114
transferred embryos implanting (implantation rate per embryo
18.2%). The mean maternal age was 33.2 Ϯ 4.2 years.
Assessment of pronuclear and embryo morphology
Pronuclear morphology was classified 16–20 h after IVF or ICSI
according to patterns previously described (0–5; Tesarik and Greco,
1999) with the following exception. In the original description,
pattern 0 comprized two sub-patterns, one with Ͼ7 NPB showing an
equal/symmetric distribution in each pronucleus and another with Ͻ7
NPB with polarization in each pronucleus at the area of contact. In
this study, these two patterns were specified as pattern 0A (Ͼ7 NPB,
equally distributed) and 0B (ഛ7 NPB, polarized). Each centre selected
preferentially pattern 0A and/or 0B for further culture and transfer.
When sufficient pattern 0 pronuclear stage oocytes were not available,
other patterns were selected. Each pronuclear-stage oocyte was
cultured individually. Embryo morphology was assessed on day 2,
and each embryo was given an embryo score according to a published
system (Steer et al., 1992). Embryo transfers were performed on days
2–5 after follicular aspiration, depending on the transfer policy of
each centre. According to the German embryo protection law, no
embryos other than those derived from the pronuclear-stage oocytes
selected for further culture and transfer were available.
Embryo development and implantation potential
Out of 1114 embryos which were transferred, 489 were derived
from a pronuclear morphology pattern with a presumable good
prognosis for implantation (patterns 0A/0B), and 625 from a
pronuclear morphology pattern with a presumable poor pro-
gnosis for implantation (patterns 1–5; Table I). Embryos
derived from a pronuclear morphology pattern with a presum-
ably good prognosis showed a significant higher embryonic
score (12.7 Ϯ 5.0) compared with embryos derived from poor
prognosis patterns (11.7 Ϯ 5.3; P Ͻ 0.001).
Clinical pregnancy and implantation rates in transfer cycles
involving at least one embryo derived from pattern 0B were
compared with transfer cycles involving no embryos derived
from pattern 0B (Table II). The pregnancy and implantation
rates were significantly higher in transfer cycles with embryos
derived from pattern 0B (37.9 and 20.5% respectively)
compared with all remaining transfer cycles (26.4 and 15.7%;
P Ͻ 0.01 and P Ͻ 0.05 respectively). In cycles where only
two embryos were transferred, the pregnancy rate of two
Based on previously published data on the assessment of pronuclear
morphology (Scott and Smith, 1998; Tesarik and Greco, 1999), it
was considered unethical to perform a randomization with one group
of patients receiving embryos from good prognosis pronuclear-stage
oocytes and another group of patients receiving embryos from
pronuclear-stage oocytes with a poor prognosis (Ludwig et al., 2000).
2385

M.Montag et al.
pattern 0B for calculations of pregnancy and implantation rates
of other patterns.
Transfer cycles involving pattern 3 gave significantly higher
pregnancy and implantation rates. No significant differences
were found for patterns 0A, 1, 2 and 4. Transfer cycles
involving pattern 5 showed significantly lower implantation
rates compared with cycles without pattern 5.
Maternal age and pronuclear morphology
The relationship between maternal age and pronuclear morpho-
logy was analysed (Table III). Significantly more pronuclear-
stage oocytes of pattern 0B and 3 were available in younger
patients (aged ഛ35 years; 34.2 and 9.2%) than in older patients
(aged Ͼ35 years; 25.8 and 5.4%; P Ͻ 0.005 and P Ͻ 0.05
respectively). For pattern 0A, 4 and 5 no significant differences
were found, whereas significantly more pattern 1 and 2 oocytes
were observed for transfer in older patients.
Compared with older patients, younger patients presented
with significantly higher pregnancy rates (35.4 versus 25.5%;
P Ͻ 0.05) and implantation rates (20.4 versus 13.9%;
P Ͻ 0.001). Therefore, subgroups of younger and older patients
were analysed and the number of transfer cycles involving at
least one embryo derived from pattern 0B versus cycles without
embryos from pattern 0B compared. No significant differences
were found with regard to the number of cycles. The pregnancy
rate in younger patients with transfer involving at least one
embryo from a 0B pattern oocyte was significantly higher
compared with all other subgroups. For the implantation rate,
a significant difference was found only between younger
patients with transfer of at least one embryo from 0B compared
with older patients without pattern 0B (Table III). Older
patients with pattern 0B attained the same pregnancy rate
(28.8%) as younger patients without 0B (28.4%).
Figure 1. The different patterns of pronuclear morphology were
adapted from Tesarik and Greco (Tesarik and Greco, 1999);
however, the original pattern 0 was subclassified into pattern 0A
and 0B as shown.
Pronuclear morphology and assisted reproductive technique
The distribution of pronuclear morphology patterns selected
for transfer in IVF and ICSI treatment cycles is shown in
Table IV. Significantly more 0B pattern were available in ICSI
(36.3%) compared with IVF (27.9%; P Ͻ 0.025). For pattern
0A and pattern 1, significantly more oocytes were available
in IVF, whereas for patterns 2–5 no significant differences
were found. In ICSI, significantly more transfer cycles were
noted involving pattern 0B (144/256) compared with IVF
(109/239; P Ͻ 0.025); however, despite this trend the corres-
ponding pregnancy (35.5 versus 28.9%) and implantation rates
(19.3 versus 17.1%) were not significantly different. There
was also no significant difference in maternal age (33.0 Ϯ 4.3
versus 33.5 Ϯ 4.1 years).
Table I. Numbers of pronuclear morphology patterns and corresponding
embryo scores
Pattern
n (%)
Embryo scorea
0A
0B
139 (12.5)
350 (31.4)
489 (43.9)
108 (9.7)
118 (10.6)
89 (8.0)
44 (3.9)
266 (23.9)
625 (56.1)
12.7 Ϯ 4.7
12.7 Ϯ 5.1
12.7 Ϯ 5.0*
11.7 Ϯ 5.8
12.1 Ϯ 5.0
11.7 Ϯ 5.3
10.1 Ϯ 4.9
11.6 Ϯ 5.3
11.7 Ϯ 5.3*
Total 0A ϩ 0B
1
2
3
4
5
Total 1–5
Discussion
aValues are mean Ϯ SD.
*P Ͻ 0.001.
In this prospective multicentre study, the possibility of selecting
a maximum of three pronuclear-stage oocytes with a presum-
able high implantation potential for further culture and transfer
was evaluated. The selection of only a limited number of
pronuclear stages for subsequent embryo transfer (ഛ3) is a
requirement of current German national legislation. This study
differed from all previous investigations, as it is the first to be
embryos derived from pattern 0B was 43.3% (26/60), compared
with 26.8% (41/153; P Ͻ 0.025) for two embryos derived
from other patterns (data not shown). On the basis of these
results, it was decided to exclude all mixed cycles involving
2386

Pronuclear morphology in embryo culture and transfer
Table II. Clinical pregnancy and implantation rates of pronuclear morphology patterns
Pattern
Cycles with at least one embryo derived from pattern Xa
Remaining cycles without pattern Xa
P (Pregnancy/
Implantation)
Cycles (n)
Pregnancy rate (%) Implantation rate (%)
Cycles (n)
Pregnancy rate (%) Implantation rate (%)
0B
0A
1
2
3
253
58
66
63
44
37.9
29.3
30.3
28.6
36.4
21.1
22.8
20.5
15.9
17.2
14.4
23.4
13.9
12.5
242
184
176
179
198
223
106
26.4
25.5
25.0
25.7
24.2
26.9
31.1
15.7
15.6
15.1
16.2
14.0
15.9
20.2
Ͻ 0.01/Ͻ 0.05
NS/NS
NS/NS
NS/NS
Ͻ 0.01/Ͻ 0.025
NS/NS
4
5
19
136
NS/Ͻ 0.025
aCycles involving pattern 0B were excluded for all calculations in both groups (except for calculation of cycles with at least one embryo derived from pattern
0B).
NS ϭ not significant.
this multicentre study clearly showed that transfer cycles
involving embryos derived from pattern 0B gave significantly
Table III. Relationship between pronuclear morphology and maternal age
higher pregnancy and implantation rates compared with those
Pattern
ഛ35 years n (%)
Ͼ35 years n (%)
P
cycles in which selection of pattern 0B for transfer was not
possible. In contrast to pattern 0B, transfer cycles with or
without pattern 0A showed no difference with regard to
pregnancy and implantation rates. Hence, these data imply that
the subclassification of pattern 0 into 0A and 0B is a useful
selection criterion.
In the current study, transfer cycles involving at least one
embryo with pattern 3 versus transfer cycles without pattern
3 gave significantly higher pregnancy and implantation rates.
As the primary aim of the study was the selection of pronuclear
morphology pattern 0, the number of cycles involving pattern
3 are rather small and cannot be compared with transfer cycles
with pattern 0B. It cannot be excluded that pattern 3 pronuclear-
stage patterns can develop into pattern 0B with time however,
and this point deserves further attention.
Another interesting point with regard to the subclassification
of pattern 0 into 0A and 0B is embryo quality. Pattern 0A-
derived embryos exhibited on day 2 an embryonic score
comparable with that of pattern 0B, and significantly different
from patterns 1–5. However, despite the higher embryonic
score, transfers involving pattern 0A gave no higher implanta-
tion or pregnancy rates compared with transfers without pattern
0A. This suggests that a possible selection based on embryo
quality on day 2 would not necessarily guarantee selection of
embryos with the best implantation potential. In other words,
at this stage of embryo development there are good quality
embryos (derived from pattern 0B) with a high implantation
potential, and also good quality embryos with a lower implanta-
tion potential (derived from pattern 0A). Consequently, embryo
selection should not rely only on embryonic morphology, and
other evaluation criteria such as pronuclear morphology or
the developmental progression of the embryos must also be
considered (Huisman et al., 2000; Racowsky et al., 2000;
Shapiro et al., 2000).
0A
0B
1
2
3
4
5
96 (12.9)
255 (34.2)
60 (8.0)
69 (9.2)
69 (9.2)
27 (3.6)
170 (22.8)
746
43 (11.7)
95 (25.8)
48 (13.0)
49 (13.3)
20 (5.4)
17 (4.6)
96 (26.1)
368
NS
Ͻ 0.005
Ͻ 0.01
Ͻ 0.05
Ͻ 0.05
NS
NS
Total 1–5
Assisted reproduction technique results
ഛ35 years
ജ1 0B
Ͼ35 years
No 0B
ജ1 0B
No 0B
Cycles
180
403
162
73
183
80
No. of embryos
Pregnancy rate (%)
Implantation rate (%)
343
185
41.7a,b
28.4a
17.8c
28.8a
15.8c
22.5b
22.6b,c
11.9b
aP Ͻ 0.05; bP Ͻ 0.005; Not significant for differences within rows.
c
Table IV. Relationship between pronuclear morphology and assisted
reproduction technique
Pattern
IVF n (%)
ICSI n (%)
P
0A
0B
1
2
3
4
5
80 (14.7)
152 (27.9)
66 (12.1)
51 (9.4)
42 (7.7)
19 (3.5)
135 (24.8)
545
59 (10.4)
198 (36.3)
42 (7.7)
67 (11.8)
47 (8.3)
25 (4.4)
131 (23.0)
569
Ͻ 0.05
Ͻ 0.025
Ͻ 0.01
NS
NS
NS
NS
Total 1–5
multicentre in nature, and because IVF and ICSI treatment
cycles were included. The evaluation was based on a single
observation of pronuclear morphology similar to that proposed
by others (Tesarik and Greco, 1999) except that pattern 0 was
subclassified into pattern 0A (Ͼ7 NPB showing an equal/
symmetric distribution in each pronucleus) and 0B (ഛ7 NPB
aligned at the contact of the two pronuclei). The results of
During the course of this study, another scoring system was
published (Scott et al., 2000) which is in fact a revision of
the original zygote scoring system established previously by
the same group (Scott and Smith, 1998). The revised system
is mainly defined by alignment, number and distribution of
2387

M.Montag et al.
NPB. The different zygote morphology patterns were classified
as Z1, Z2, Z3 and Z4. Scott and co-workers showed that Z1-
scored zygotes possess a high potential to develop to blastocysts
on days 5–6 and that these blastocysts exhibited a high
implantation potential. Patterns different from Z1 showed
significantly lower blastocyst formation rates and implantation
rates. Interestingly, pattern 0B corresponds to zygote morpho-
logy Z1, patterns 0A and 1 correspond to Z2, and Z3–Z4
(Scott et al., 2000) may be compared with patterns 2–5
classified by others (Tesarik and Greco, 1999) and by the
current study. The results from the current prospective
multicentre study correlate well with the data presented else-
where (Scott et al., 2000) in that preselection by a zygote
score can give high implantation rates, although in the current
study no double selection was possible by day 3 morphology.
However, in contrast to results of the previously cited study
(Scott et al., 2000), a difference was found in the pattern
distribution with regard to age and assisted reproduction
technique. Therefore, the approach of pronuclear selection is
a good option for countries with legal restriction of embryo
selection at the blastocyst stage. However, from the patient’s
viewpoint, a better strategy would be pre-selection of pronuc-
lear morphology patterns 0B/Z1, followed by selection of the
best embryos derived from these pronuclear stages on the day
of transfer, as proposed by others (Scott et al., 2000). This
treatment option offers undoubtedly the best basis towards
single embryo transfer without compromising pregnancy rates.
The current study revealed that for patients with advanced
maternal age (Ͼ35 years) significantly fewer pronuclear mor-
phology pattern 0B were available for further culture and
transfer. It should be noted that this analysis did not include
all day 1 pronuclear oocytes, but only those selected for further
culture and transfer. The reason for choosing the age of 35
years was due to an existing guideline in Germany, according
to which only two embryos should be transferred in patients
aged ഛ35 years, and three embryos in those aged Ͼ35 years.
A similar evaluation in choosing a cut-off at 38 years has
already been made for 262 transfer cycles (Wittemer et al.,
2000). These authors found no significant difference for
pattern 0 (0A ϩ 0B) with regard to maternal age, although a
trend was detectable towards reduced numbers of pattern 0
(39.3 versus 52.3%) in older patients (aged Ͼ38 years). The
higher number of transfer cycles in this study (495 versus
262) may explain why statistical significance was reached.
The current data also show that the implantation rate of cycles
with pattern 0B is lower for older patients (Ͼ35 years; 15.8%)
compared with younger patients (ഛ35 years; 22.6%). These
differences were not significant, but such a trend shows that
in older patients the success of implantation is also influenced
by other factors.
occur in the nucleus which involve polarization of chromatin
(Van Blerkom et al., 1995) and NPB (Tesarik and Kopecny,
1989; Payne et al., 1997). NPB are markers for chromosomes
hosting rDNA-genes (Goessens, 1984; Tesarik and Kopecny,
1990), and consequently pattern 0B/Z1 characterizes the most
advanced stage of nuclear polarization, which is also part of
embryonic axis formation in the zygote (Edwards and Beard,
1997). Zygotes reach this stage earlier after ICSI than after
IVF. All centres participating in this study usually screened
the pronuclear stages at 16–20 h after insemination of oocytes,
independently of the type of assisted reproductive technique
used. It may be worthwhile exploring whether the difference
observed would still exist if zygotes derived from IVF were
to be screened later (18–20 h) than those from ICSI (16–18 h).
In conclusion, the current study provides further evidence
that pronuclear morphology is an important addition to current
assisted reproduction technique practice. In countries with
legal restrictions of embryo selection, pronuclear morphology
screening appears to be a useful and easy technique. In all
other countries, pronuclear morphology offers an additional
screening method which, in combination with extended embryo
culture and a second selection based on blastocyst evaluation,
represents a major step towards single embryo transfer.
Acknowledgements
The Pronuclear Morphology Study Group acknowledges the help and
support of all colleagues in the participating centres who, due to
limited space, cannot be acknowledged individually. Special thanks
go to the Arbeitsgemeinschaft Reproduktionsbiologie des Menschen,
to Prof. Dr Geistho¨vel (Freiburg) who co-initiated this study, and to
Prof. Dr Rabe (University of Heidelberg, Germany) for his help in
recruiting centres.
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Received on May 15, 2001; accepted on August 10, 2001
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