1. family and parenting
2. motherhood
3. pregnancy

Hum. Reprod. Advance Access published June 26, 2012
Human Reproduction, Vol.0, No.0 pp. 1– 9, 2012
doi:10.1093/humrep/des210
ORIGINAL ARTICLE Embryology
The impact of pronuclei morphology
and dynamicity on live birth outcome
after time-lapse culture
A. Azzarello*, T. Hoest, and A.L. Mikkelsen
The Fertility Clinic, Holbaek Regional Hospital, Copenhagen University, Smedelundsgade 60, DK-4300 Holbaek, Denmark
Correspondence address. E-mail: aaz@regionsjaelland.dk
Submitted on February 16, 2012; resubmitted on May 11, 2012; accepted on May 15, 2012
summary answer: In comparison to embryos resulting in no live birth, PNB occurred significantly later in embryos resulting in live
birth and never earlier than 20 h 45 min. None of the tested scoring systems were shown to predict the live birth outcome in a time-lapse
set-up.
what is known already: The PN morphology is supported as a prominent embryo selection parameter in single light microscopy
observations, although controversial results have been reported.
study design, size, duration: This was a prospective study of 159 embryos, all of which were later transferred. The PN
morphology of 46 embryos which resulted in live birth was compared with that of 113 embryos which resulted in no live birth.
participants, setting: From 1 March 2010 to 30 August 2011, 130 couples underwent fertility treatment by ICSI. Embryo culture
was performed in a time-lapse set-up from fertilization to intrauterine transfer. PN morphological assessment was performed on every
embryo replaced, using six different scoring systems at different times.
main results and the role of chance: No embryo with PNB earlier than 20 h 45 min resulted in live birth. All six PN
assessment models showed no significant distribution of scores (P ¼ NS) between the live birth and no live birth groups at 16 h post-fertilization (PF), 18 h PF and 40 min before PNB. The outcomes of assessments changed significantly (P , 0.001) over time and the time of
PNB was found to be the optimal stage to evaluate the PN morphology.
limitations, reasons for caution: The study includes only embryos reaching the 4-cell stage after ICSI, and transferred
at 44 h PF.
wider implications of the findings: The PN morphology changes over time, indicating that the single light microscopy
observation approach is deficient in comparison to time-lapse. Although the assessment of the PN morphology does not improve
embryo selection, the timing of PNB should be included in embryo selection parameters.
study funding/competing interest(s): None.
trial registration number: Approval number from the National Ethical Committee of Medical Science of Denmark: SJ-250.
Key words: pronuclei dynamics / zygote scoring / live birth / pregnancy / preimplantation embryo
Introduction
Elective single embryo transfer has been suggested as the most efficient approach to minimize multiple pregnancies resulting from
assisted reproduction treatments (Cutting et al., 2008).
The embryo selection routine in IVF clinics is based on a single observation by light microscopy at pre-set times (ALPHA and ESHRE, 2011).
Among the different parameters evaluated, pronuclei (PN) scoring has
been a subject of debate. While some studies have shown a prognostic
effect of PN scoring (Scott and Smith, 1998; Tesarik and Greco, 1999;
Scott et al., 2000; Tesarik et al., 2000; Balaban et al., 2001; Nagy
et al., 2003; Scott, 2003), some have identified a correlation between
PN scoring and aneuploidy of embryos (Sadowy et al., 1998; Gianaroli
et al., 2003; Edirisinghe et al., 2005) and some were not able to
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study question: Can the pronuclei (PN) morphology and the time of PN breakdown (PNB) predict the potential of embryos to
result in live birth?
2
Materials and Methods
Study group
This study included 159 zygotes obtained from 130 couples who underwent fertility treatment by ICSI in the Holbaek Fertility Clinic between 1
March 2010 and 30 August 2011.
The inclusion criteria for this study were couples using their own
gametes where the female partner was aged ≤39 years and the reason
for infertility was the male factor with a motile sperm count in the
range of 1–5 million spermatozoa per ejaculate.
We included all transfers of 4-cells embryos, with equal blastomeres
and fragmentation ,25% and no multinucleation, cultured in a time-lapse
culture system, from fertilization (Day 0) or from Day 1.
The National Ethical Committee of Medical Science of Denmark
approved the study (approval number: SJ-250).
Oocyte recruitment and recovery
ICSI was performed in women with regular menstruation and normal
ovaries detected by ultrasound. Ovarian stimulation was performed with
recombinant follicular-stimulating hormone (rFSH; Gonal-Fw, Merck
&
&
Serono , Denmark or Puregonw, Organon , Denmark) using an agonist
protocol (Gardner et al., 2004).
Follicle growth was monitored by ultrasound examination and recom&
binant hCG (rhCG; Ovitrellew, Merck Serono , Denmark) was given
when at least three follicles reached a diameter of 17 mm. Oocyte aspiration was performed 36 h after rhCG administration. Embryo transfer was
performed on Day 2 following ICSI. To support the luteal phase, the
&
women initiated progesterone support (Crinone , Watsonw, Denmark
&
or Lutinusw, Ferring , Denmark) on the day of transfer. Progesterone
was continued until the pregnancy test. Transvaginal ultrasound visualization of a gestational sac with the evidence of heart activity 7 weeks
after embryo transfer indicated a clinical pregnancy. Live birth was
defined as the delivery of a living fetus with heartbeat and respiration, regardless of gestational age.
Fertilization and embryo culture
Cumulus –ocyte complexes were washed and cultured for 2 h in Fertiliza&
tion Medium (Cookw, Australia) and then cumulus cells were removed
by hyaluronidase treatment (Gardner et al., 2011). The denuded
&
oocytes were placed individually in droplets of Cleavage Medium
w
(Cook , Australia) covered by mineral oil. The metaphase II oocytes
were immediately microinseminated and cultured individually
(Cruz et al., 2011) in an atmosphere of 5.0% O2, 5.5% CO2, 89.5% N2
controlled by the time-lapse incubator, EmbryoScopeTM (Unisensew,
Denmark),
Embryo analysis
The zygotes were retrospectively divided into two groups: (i) live birth
group, if the transferred embryo resulted in a live birth and (ii) no live
birth group. Transfers of two embryos with only one fetus delivered
were excluded from the study. The no live birth group included nonimplanted embryos, defined as no detection by ultrasound of a heartbeat
after 7 weeks, as well as biochemical pregnancies, defined as a positive
hCG test without a gestational sac detected by ultrasound, and spontaneous abortions, defined as the spontaneous interruption of the pregnancy
after detection by ultrasound of a heartbeat after 7 weeks of gestation.
The live birth group included 37 embryos observed from Day 0 and 9
embryos from Day 1. The no live birth group included 108 embryos
observed from Day 0 and 5 embryos from Day 1. Embryos cultured in
a time-lapse set-up from fertilization were evaluated at three different
times: (i) 16 h PF, (ii) 18 h PF and (iii) 40 min prior to PNB. Embryos cultured from Day 1 were evaluated only 40 min prior to PNB. The assessment of PNB was performed 40 min beforehand in favor of a recognizable
PN morphology. The criteria for accepting fertilization as normal were the
presence of 2 PN as well as second polar bodies. PNB was defined as the
end of the PN envelope fading. Embryo selection for transfer was performed according to our routine protocol criteria: early cleavage at 26 h
PF, development to at least four equal blastomeres at 44 h PF, ,25% fragmentation at 44 h PF and absence of multinucleation at 44 h PF. PN evaluation was not included in the routine selection criteria.
Time-lapse images were acquired every 20 min on seven focus planes
with focal intervals of 15 mm, from fertilization until transfer on Day 2
(44 h PF).
We defined the fertilization process as the time of the first recorded
frame of the second polar body extrusion, and PNB was defined as the
first picture frame where PN were not observable.
PN evaluation
All zygotes were assessed in six different models:
(1) Z-score: According to the Z-score system (Z1, Z2, Z3, Z4), from Z1,
suggested as the best, to Z4 (Scott, 2003).
(2) Consensus score: According to ALPHA and ESHRE consensus
(ALPHA and ESHRE, 2011).
(3) PN envelope score: The assessment of zygotes into two categories: (i)
PN with high-quality envelopes, in contact, and of equal size (difference ≤1/3) and centrally positioned (≤1/3 of the zygote diameter),
versus (ii) PN envelope with low quality, without these characteristics.
(4) Nuclear precursor body pattern score: The assessment of zygotes
into three categories, respectively, from the best to the worst: (a)
PN with ≤6 aligned and equal (difference ≤2) NPB, (b) PN
with less than or equal to six non-aligned but equal (difference ≤2)
NPB and (c) PN without these characteristics.
(5) Nuclear precursor body quantity score: The assessment of zygotes
into four categories, according to the difference in the number of
NPB in each pronucleus, from the best to the worst: (a) equal
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demonstrate such associations (Salumets et al., 2001; James et al., 2006;
Weitzman et al., 2010; Bar-Yoseph et al., 2011).
In previous studies and recommendations, PN assessment has been
performed by a single observation between 16 and 18 h postfertilization (PF; Tesarik et al., 2000; Scott, 2003; ALPHA and
ESHRE, 2011).
Time-lapse technology has offered the opportunity to make multiple observations of embryo developmental rates (Lemmen et al.,
2008; Wong et al., 2010) and of the PN development (Payne et al.,
1997; Montag et al., 2011). Since PN assessment describes PN development, a dynamic process reported as PN abutting, growing and coalescence of nuclear precursor bodies (NPB; Wright et al., 1990;
Payne et al., 1997), this multiple observation approach seems to be
more suitable.
The aim of the present study was to explore PN development in
embryos after ICSI by two previously used criteria and by four
newly suggested criteria. Evaluation was performed at the extremes
of the recommended lapse, 16 and 18 h PF and before PN breakdown
(PNB). The PN morphology was related to the live birth outcome of
the transferred embryos. Furthermore, the timing of PNB and second
polar body extrusion after fertilization were evaluated.
Azzarello et al.
3
PN morphology and the time of PNB in zygotes
number (when the divergance was ≤2), (b) low difference (when 3 ≤
5), (c) high difference (when ≥6) and (d) high number when NPB
exceeded 10 in both PN.
(6) Nuclear precursor body polarization score: The assessment of
zygotes, according to the position of NPB within each PN, into
three categories. Each PN was evaluated as (i) when all NPB were
aligned on the edge of PN membrane, (ii) when NPB were distributed
only in the PN hemisphere in contact with the opposite PN and (ii)
when NPB were scattered. Considering equal polarization as a positive indicator of quality (Tesarik and Greco, 1999; Gianaroli et al.,
2003; Scott, 2003), the equally distributed combination (AA, BB,
CC) were grouped and compared with those with unequal combinations (AB, AC, BC). Considering NPB alignment as an indicator of development (Tesarik and Greco, 1999; Gianaroli et al., 2003),
combinations were scored from the best to the worst as following:
AA, AB, BB, AC, BC, CC.
Every evaluation was strictly performed at the selected time, supported
only by the change in the focus plane.
We used a standard Student’s t-test to evaluate the average patient age
and to assess time of two PB extrusion and PNB. Every time value,
described as hour (h) and minutes (m), was reported with the standard
error of the mean (+SEM) and P , 0.05 was considered significant. x 2
table contingency tests were performed to evaluate differences in distributions. For PNB distribution, we pooled PNB results from Days 0 and 1
zygotes, while for 16 h PF and 18 h PF distributions we used only Day 0
zygotes. Statistical significance corresponds to P , 0.05. We analyzed
the changes in the PN morphology with time by linear regression test,
assigning a progressive unit value to each score grade and predicting the
time-conditional means with ordinary least squares. We then associated
the pattern trend to the time of observation and we compared the two
groups, evaluating differences at 0.01 confidence level.
All results were obtained using statistic software Stataw 11 (StataCorp
&
&
LP , USA) and Prismw 5 (GraphPad Software , USA).
Results
Timing of second polar body extrusion
and PNB
The average time interval from fertilization to second polar body extrusion did not differ significantly (NS; P ¼ 0.607) between zygotes
resulting in live birth (3 h 47 min + 0 h 17 min) and zygotes that did
not result in live birth (3 h 37 min + 0 h 11 min; Fig. 1).
In contrast, the PNB time was associated with live birth, since the
PNB time of zygotes resulting in live birth (24 h 52 min + 0 h
35 min) was significantly higher (P ¼ 0.022) than the PNB time of
the no live birth group (23 h 10 min + 0 h 23 min; Fig. 1). No live
birth was obtained if PNB was observed earlier than 20 h and
45 min PF, but this was observed in 20.3% of embryos from no live
birth group.
PN evaluation
Z-score
The distribution of the four Z groups (Z1, Z2, Z3, Z4) did not differ
between the live birth and no live birth zygotes in any of the three evaluations (16 h PF, NS; P ¼ 0.333; 18 h PF, NS; P ¼ 0.331; PNB, NS;
P ¼ 0.916; Fig. 2A).
Figure 1 Time of second polar body extrusion and pronuclei
breakdown in 4-cell embryos resulting in live birth or no live birth.
Each data set shows data range, whiskers and mean value (+); L,
live birth; NL, no live birth; 2nd PB, second polar body; PNB, pronuclei breakdown; X̄, mean time in hours (h) and minutes (m); the
same letters indicates a significant difference: (a) P ¼ 0.022.
In the zygotes that resulted in live birth, the distribution of Z groups
differed significantly (P ¼ 0.001) between 16 h PF and PNB observations and significantly (P ¼ 0.043) between 18 h PF and PNB observations. In the zygotes that gave no live birth, the distribution of Z groups
differed significantly (P ¼ 0.001) between 16 h PF and PNB observations and significantly (P ¼ 0.047) between 18 h PF and PNB (Fig. 1;
Table I).
A significant improvement (P , 0.001) in the Z score over time was
observed in zygotes (Fig. 2B) but no significant differences (NS; P ¼
0.867) were observed when comparing the live birth and no live
birth zygotes.
Consensus score
The distribution of the three groups according to the consensus score
(symmetrical, non-symmetrical and abnormal) did not differ between
zygotes resulting in live birth and no live birth in any of the three evaluations (16 h PF, NS, P ¼ 1.000; 18 h PF, NS, P ¼ 0.740; PNB, NS,
P ¼ 0.893; Fig. 2C).
The mean value of the consensus score improved significantly (P ,
0.001) during the observation interval (Fig. 2D) and this did not differ
between the live birth and no live birth embryos (NS; P ¼ 0.520).
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Statistical analyses
4
Azzarello et al.
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Figure 2 The distribution and mean value at the three different times of the Z score (A, B), the consensus score (2C, 2D) and the PN envelope
score (2E, 2F) in transferred 4-cell embryos. (A) The distribution of the Z score: Z1 ¼ PN with ≤6 aligned and equal (difference ≤2) NPB; Z2 ¼ PN
with ≤6 non-aligned and equal (difference ≤2) NPB; Z3 ¼ PN without Z1 and Z2 characteristics; Z4 ¼ PN not in contact or unequal size (difference
≤1/3) or peripheral positioned (≤1/3 of the zygote diameter). Columns with the same letters were significantly different: (a) P ¼ 0.001, (b)
P ¼ 0.001, (c) P ¼ 0.043 and (d) P ¼ 0.047. (B) Cumulated mean of all Z score values versus time (average score), tested by a linear regression
test (linear prediction); the curve increment was seen to rise significantly over time (P , 0.001). (C) The distribution of the consensus score: S, symmetrical (equivalent of Z1 and Z2). N, non-symmetrical (equivalent of Z3 and Z4); A, abnormal (1 or 0 NPB in one pronucleus). Columns with the
same letters were significantly different: (a) P ¼ 0.019, (b) P ¼ 0.001 and (c) P ¼ 0.047. (D) The cumulated mean of all consensus score values versus
time (average score), tested by a linear regression test (linear prediction); the curve increment was seen to rise significantly over time (P , 0.001). (E)
The distribution of the PN envelope score: A ¼ PN in contact with equal size (difference ≤1/3) and centrally positioned (≤1/3 of the zygote diameter); B ¼ PN without these characteristics. Columns with the same letters were significantly different: (a) P ¼ 0.004, (b) P ¼ 0.002 and (c) P ¼ 0.041.
(F) The cumulated mean of all PN envelope score values versus time (average score), tested by a linear regression test (linear prediction); the curve
increment was seen to rise significantly over time (P , 0.001). PN, pronuclei; NPB, nuclear precursor bodies; 16 h PF, 16 h post-fertilization; 18 h PF,
18 h post-fertilization; PNB, pronuclei breakdown; L, live birth embryos; NL, no live birth embryos.
5
PN morphology and the time of PNB in zygotes
Table I Z score results of transferred 4-cells embryos.
16 h post fertilization
18 h post fertilization
Pronuclei breakdown
Live birth (a)
Live birth (c)
Live birth (a,c)
...................................................
No live birth (b)
...................................................
No live birth (d)
........................................................
No live birth (b,d)
.............................................................................................................................................................................................
Z1
0 (0%)
0 (0%)
0 (0%)
1 (0.9%)
4 (8.7%)
7 (6.2%)
Z2
1 (2.7%)
4 (3.7%)
4 (10.8%)
11 (7.4%)
6 (13.0%)
14 (12.4%)
Z3
27 (73.1%)
89 (82.4%)
27 (73.0%)
90 (83.3%)
35 (76.1%)
88 (77.9%)
Z4
9 (24.3%)
15 (13.9%)
6 (16.2%)
9 (8.3%)
1 (2.2%)
Tot
37
108
37
108
46
4 (3.5%)
113
Comparison of the Z score at different times between the live birth and no live birth embryos. Tot, Total number of embryos assessed in the group at each time.
Columns with same letters were significantly different: (a) P ¼ 0.001, (b) P ¼ 0.001, (c) P ¼ 0.043 and (d) P ¼ 0.047.
PN envelope score
Nuclear precursor body pattern score
The distribution of NPB pattern did not differ between the live birth
and no live birth groups in any of the three evaluations (16 h PF,
NS, P ¼ 1.000; 18 h PF, NS, P ¼ 0.533; PNB, NS, P ¼ 0.841; Fig. 3A).
The average score of NPB pattern increased significantly (P ,
0.001) over time (Fig. 3B), but was not different between the live
birth and no live birth zygotes (NS; P ¼ 0.560).
Nuclear precursor body quantity score
The number of NPB did not differ between the live birth and no live
birth zygotes in any of the three evaluations (16 h PF, NS, P ¼ 0.402;
18 h PF, NS, P ¼ 0.939; PNB, NS, P ¼ 0.510; Fig. 3C).
The average score of NPB quantity increased significantly (P ,
0.001) over time (Fig. 3D) in both the groups, but was not different
between the live birth and no live birth groups (NS; P ¼ 0.899).
Nuclear precursor body polarization score
The NPB polarization did not differ between the live birth and no live
birth zygotes in any of the three evaluations (16 h PF, NS, P ¼ 0.198;
18 h PF, NS, P ¼ 0.718; PNB, NS, P ¼ 0.783; Fig. 3E).
Zygotes with equal polarization (AA, BB, CC) were aggregated and
compared with the unequal ones (AB, AC, BC). At any time, the two
groups were almost identical (NS; P ¼ 0.603).
NPB polarization alignment increased significantly (P , 0.001) over
time in both the groups (Fig. 3F), without any difference between the
live birth and no live birth groups (NS; P ¼ 0.494).
Gonadotrophin exposure, BMI and age
distribution
Gonadotrophin exposure, expressed as units FSH per oocyte, did not
significantly differ (NS; P ¼ 0.692) between the live birth group
(436.6 + 82.5) and the no live birth group (401.6 + 45.4).
Discussion
This is the first study that evaluates the PN morphology at three distinct intervals (16 h PF, 18 h PF and close to PNB) by six different
systems, in contrast to previous studies that have analyzed PN development by a single observation between 16 h and 18 h (Scott et al.,
2000; Montag and van der Ven, 2001; Gianaroli et al., 2003). We
found that the time of observation was a determining factor since outcomes changed over time in all six models used.
The interval time from fertilization to second body extrusion
showed no difference between the live birth and no live birth
embryos and this may indicate a uniform activation of the second
meiotic cleavage.
PNB, however, occurred later in the live birth embryos compared
with no live birth embryos. The significant difference (P ¼ 0.022)
was of 1 h and 42 min + 0 h 12 min. Moreover, we observed that
none of the embryos resulting in live birth showed a PNB before
20 h 45 min. In contrast, 20.3% of the no live birth embryos
showed PNB before 20 h 45 min. This is in contrast to previous
studies by Lemmen et al. (2008) that has shown that earlier PNB
was associated with a higher embryo potential. However, a recent
morphokinetic study, focusing on timing of cell division, showed that
too early cleavage may not be beneficial and maybe this supports
our finding (Meseguer et al., 2011). A possible explanation of our
results could be an impairment of the checkpoints regulating the
embryo development path.
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Assessing PN by their envelope morphology, no difference in distribution was observed at any time between the live birth and no live birth
groups (16 h PF, NS, P ¼ 0.205; 18 h PF, NS, P ¼ 0.238; PNB, NS,
P ¼ 1.000; Fig. 2E).
Zygotes with a normal PN envelope position and size increased significantly (P , 0.001) from 16 h PF to PNB (Fig. 2F), but no differences
were observed between the live birth and no live birth zygotes (NS;
P ¼ 0.209).
The maternal body mass index was not significant different (NS;
P ¼ 0.075) between the two groups (live birth 23.6 + 0.6 versus no
live birth 24.9 + 0.4).
Maternal age was lower in the live birth group (30.4 + 0.6 years)
than that in no live birth group (31.9 + 0.5 years), but was not significantly different (NS; P ¼ 0.067).
Nonetheless, maternal age had no impact on PN scoring. When
testing age subgroup ≥35 years old (live birth 37.1 + 0.6 years; no
live birth 37.3 + 0.2 years; NS, P ¼ 0.676) and subgroup ≤29 years
old (live birth 27.2 + 0.3 years; no live birth 27.3 + 0.3 years; NS,
P ¼ 0.868), Z score distributions between the live birth and no live
birth groups were not significantly different (P ¼ NS). The mean
values of Z score for the two age subgroups improved over time
(P , 0.001) without difference between the live birth and no live
birth groups.
6
Azzarello et al.
We recommend not transferring embryos with PNB earlier than
20 h, as a security interval, if there is the opportunity to select
between embryos. However, further studies should be performed
to confirm our findings.
During this study of transferred embryos, no embryos were found
to be in syngamy before 18 h PF, in contrast to previous studies of
transferred and surplus embryos (ALPHA and ESHRE, 2011). As
our study groups included only good morphology embryos, the
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Figure 3 The distribution and mean value at the three different times in the nuclear precursor body pattern score (A, B), nuclear precursor body
quantity score (C, D) and nuclear precursor body polarization score (b, F) in transferred 4-cell embryos. (A) The distribution of nuclear precursor
body pattern score: A ¼ PN with ≤6 aligned and equal (difference ≤2) NPB; B ¼ PN with ≤6 non-aligned and equal (difference ≤2) NPB; C ¼ PN
without A and B characteristics. Columns with the same letters were significantly different: (a) P ¼ 0.008. (B) The cumulated mean of all nuclear precursor body pattern score values versus time (average score), tested by a linear regression test (linear prediction); the curve increment was seen to
rise significantly over time (P , 0.001). (C) The distribution of nuclear precursor body quantity score: EN, equal number with ≤2 NPB; LD, low
difference with 3 ≤ 5 NPB; HD, high difference with ≤6 NPB; HN, high number with .10 NPB in both PN. (D) The cumulated mean of all
nuclear precursor body quantity score values versus time (average score), tested by a linear regression test (linear prediction); the curve increment
was seen to rise significantly over time (P , 0.001). (E) The distribution of nuclear precursor body polarization score: A, aligned; B, distributed in
juxtaposed hemisphere; C, Scattered. Columns with the same letters were significantly different: (a) P , 0.001. (F) The cumulated mean of all
nuclear precursor body polarization score values versus time (average score), tested by a linear regression test (linear prediction); the curve increment
was seen to rise significantly over time (P , 0.001). PN, pronuclei; NPB, nuclear precursor bodies; 16 h PF, 16 h post-fertilization; 18 h PF, 18 h postfertilization; PNB, pronuclei breakdown; L, live birth embryos; NL, no live birth embryos.
PN morphology and the time of PNB in zygotes
Equal distribution and alignment of NPB have been suggested as
indicators of zygote quality (Scott and Smith, 1998). In the present
study, we could not demonstrate this assumption.
To investigate polarization, we tested a hypothetical ranking assuming more polarization as a good quality indicator. A strong tendency
toward polarization has been observed in our study, with a definite increase in the most polarized categories, especially from 16 h PF to
PNB. This may support the theory (Wright et al., 1990) that PN development involves NPB polarization toward the juxtaposed side of
PN, although alignment cannot be regarded a necessary step to
achieve a pregnancy. Indeed aligned PN, considered the best configuration, increased by time, but remained a minority even at the time of
PNB (10.9% in live birth versus 8.0% in no live birth) in both groups,
while a scattered configuration, although slight decreasing over time,
continued to be dominant in both groups (26.1% in live birth versus
25.7% in no live birth).
Since no PN morphological parameter at any time was shown to
predict development of the zygote to live birth, a rethinking of PN assessment has to be considered. Moreover, maternal age, as well as
BMI and dose of FSH per MII oocyte, were not significantly associated
with embryo development potential to result in a live birth.
In numerous studies, NPB have been defined as nucleoli (Wright
et al., 1990; Payne et al., 1997, 2005; Scott and Smith, 1998; Scott
et al., 2000) although substantial differences are well established
(Tesarik et al., 1986). Nucleoli are sub-cellular organelles, constituted
by three different compartments: fibrillar centers (FCs), surrounded
by a dense fibrillar component (DFC), embedded in the granular component (GC; Boisvert et al., 2007; Sirri et al., 2008). Nucleoli play a
central role in cell cycle progression and proliferation, as well as ribosome synthesis, as ribosomal DNA situated and transcripted in FCs, is
processed in DFC while final maturation and ribosomes assemblage
occurs in GC compartment (Boisvert et al., 2007; Sirri et al., 2008).
In nucleoli several morphological alterations, such as size and
number, are indicators of neoplasia, like prostatic adenocarcinoma,
certain breast cancers and salivary glands tumors (Boisvert et al.,
2007; Sirri et al., 2008).
Nucleoli are disassembled during mitosis and reassembled in interphase, when transcription is reactivate (Hernandez-verdun et al.,
2002; Boisvert et al., 2007; Sirri et al., 2008). Nucleoli reassembly is
supported by pre-nuclear bodies, small structures well conserved in
plants and animals, that appear in late anaphase on the chromosomes
surface, recruiting nucleolar-processing components, such as ribonuclear proteins, pre-rRNA and fibrillarin, released by the dissolving perichromosomal regions (Hernandez-verdun et al., 2002; Boisvert et al.,
2007; Sirri et al., 2008). Pre-nuclear bodies release early- and lateprocessing proteins that move to form nucleoli (Savino et al., 2001;
Boisvert et al., 2007; Sirri et al., 2008).
In early embryos, NPB are considered the precursor of nucleoli,
instead of pre-nuclear bodies that have never been recognized at
this stage. While pre-nuclear bodies are tiny (0.2 mm) with a loose
and fibrillogranular structure, NPB are larger, with a tight homogeneous fibrillar organization, persisting along interphase and disassembling during cleavage (Zatsepina et al., 2003; Romanova et al.,
2006). NPB do not contain ribosomal DNA, but some of them interact with ribosomal DNA on the NPB surface, supporting the idea that
they function as structural support to nucleate nucleoli, when the
embryo resumes transcription (Zatsepina et al., 2003; Romanova
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absence of embryos in syngamy at 18 h PF could be consistent with
the idea that embryos cleaving earlier than 20 h PF have poor development (ALPHA and ESHRE, 2011).
Concerning the Z score, we have reported for the first time that
there is a limited but significant development of the PN during the observation time. Moreover, we have reported that a low rate of highquality configurations was detected, in contrast to previous studies
(Scott, 2003; Scott et al., 2007; Weitzman et al., 2010).
Within each group, no significant changes in the distribution of the Z
score occurred between 16 h PF and 18 h PF, while a significant improvement was observed from 16 and 18 h PF to the PNB. Similar
changes of distribution were observed in the other five PN scoring
models. These results are consistent with the idea that PN goes
through a development process (Tesarik and Kopecny, 1989; Payne
et al., 1997; Scott et al., 2000), associated with the ability to result
in live birth. Since all these changes occurred in a short time interval,
reliability of a single observation is questionable in this context.
The reason that underlies the differences between our scoring
results and previous studies could be the evaluation approach.
Based on recorded images, our assessments should be considered
more objective, since repeatable and safer, in contrast to live single
observations, which require assessment of the embryos in the shortest
possible time, since microscopy light, as well as extra-incubator conditions, are harmful to the zygotes. Beside these considerations, our
score distribution is consistent with previous studies (Payne et al.,
2005; Bar-Yoseph et al., 2011).
The abnormal category, according to the consensus score (ALPHA
and ESHRE, 2011), was extremely infrequent in our study. Interestingly, a single NPB, considered as abnormal in mice (Svarcova et al.,
2009), was observed once in the live birth group and three times in
no live birth group, suggesting that these embryos still have some development potential in this subpopulation of early embryos.
We evaluated the rate and the outcome of PN juxtaposed, of equal
size and in a central position. In both the live birth and no live birth
groups, we saw a higher number of zygotes without those characteristics at 16 h PF, but decreasing at 18 h PF and almost disappearing at
the PNB. This suggest that 18 h PF and even more 16 h PF could be
too early for PN envelope evaluation, since most of the abnormal
PN were likely to achieve proper morphology, without affecting the
embryo potential. These envelope abnormalities have been suggested
as associated with poor quality (Scott, 2003), while in this study we
have not shown any difference between the two groups.
The differences in NPB number in each PN showed a progressive
reduction, consistent with previous time-lapse observations describing
NPB coalescence (Payne et al., 1997). Zygotes with an NPB difference
≤2, suggested to be an indicator of zygote competence (Scott, 2003),
increased by time although remained low in both groups even at the
final stage of development (41.3% of live birth zygotes versus 48.7%
of no live birth zygotes), without a significant difference between the
groups. Despite the suggestion that development is determinate by
fusion of NPB (Tesarik and Kopecny, 1989), 4.3% of embryos from
the live birth group kept more than 10 small NPB in both PN, and
8.7% presented a difference over 5, indicating that in at least one
PN the NPB fusion was incomplete. These findings could suggest
that NPB have a strong tendency to coalescence, but do not predict
embryo quality.
7
8
et al., 2006). Due to their different functions and their heterogenic activities (Zatsepina et al., 2003; Romanova et al., 2006), NPB do not
reflect nucleoli potential, as previously suggested (Scott, 2003) and
may not be connected to neoplasia.
Despite this, these morphological features have been chosen as a
quality indicator of the fertilized oocyte (Wright et al., 1990), but
the function of NPB fusion and polarization, as well as PN juxtaposition, are at present not explainable. This strongly suggests further
studies are required to clarify the role and dynamics of the PN and
NPB before using these characters in embryo selection.
Conclusion
Acknowledgements
We gratefully acknowledge Alessandro Martinello, M.Econ, SFI (The
Danish National Centre for Social Research) and the Department of
Economics, University of Copenhagen, Denmark, for statistical assistance in the analysis of the data.
Authors’ roles
A.A. and T.H. were involved in the study design, data acquisition and
analysis, drafting and final approval of the article. A.L.M. was involved
in the study design, revision and final approval of the article.
Funding
No external funding was either sought or obtained for this study.
Conflict of interest
None declared.
References
Alpha Scientists in Reproductive Medicine and ESHRE Special Interest
Group of Embryology. The Istanbul consensus workshop on embryo
assessment: proceedings of an expert meeting. Hum Reprod 2011;
26:1270 –1283.
Balaban B, Urman B, Isiklar A, Alatas C, Aksoy S, Mercan R, Mumcu A,
Nuhoglu A. The effect of pronuclear morphology on embryo quality
parameters and blastocyst transfer outcome. Hum Reprod 2001;
16:2357 –2361.
Bar-Yoseph H, Levy A, Sonin Y, Alboteanu S, Levitas E, Lunenfeld E,
Har-Vardi I. Morphological embryo assessment: reevaluation. Fertil
Steril 2011;95:1624–1628.
Boisvert FM, Van Koningsbruggen S, Navascués J, Lamond AI. The
multifunctional nucleolus. Nat Rev Mol Cell Biol 2007;8:574 – 585.
Cruz M, Gadea B, Garrido N, Pedersen KS, Martı́nez M, Pérez-Cano I,
Muñoz M, Meseguer M. Embryo quality, blastocyst and ongoing
pregnancy rates in oocyte donation patients whose embryos were
monitored by time-lapse imaging. J Assist Reprod Genet 2011;
28:569 – 573.
Cutting R, Morroll D, Roberts SA, Pickering S, Rutherford A. Elective
single embryo transfer: guidelines for practice British Fertility Society
and Association of Clinical Embryologists. Hum Fertil 2008;11:131– 146.
Edirisinghe WR, Jemmott R, Smith C, Allan J. Association of pronuclear Z
score with rates of aneuploidy in in-vitro fertilised embryos. Reprod Fertil
Dev 2005;17:529– 534.
Gardner DK, Weissman A, Howles CM, Shoham Z. Textbook of Assisted
Reproductive Techniques: Laboratory and Clinical Perspectives, 2nd edn.
London, UK: Informa Healthcare, 2004.
Gardner DK, Wale PL, Collins R, Lane M. Glucose consumption of single
post-compaction human embryos is predictive of embryo sex and live
birth outcome. Hum Reprod 2011;8:1981 – 1986.
Gianaroli L, Magli MC, Farraretti AP, Fortini D, Grieco N. Pronuclear
morphology and chromosomal abnormalities as scoring criteria for
embryo selection. Fertil Steril 2003;80:341– 349.
Hernandez-Verdun D, Roussel P, Gébrane-Younès J. Emerging concepts of
nucleolar assembly. J Cell Sci 2002;115:2265 – 2270.
James AN, Hennessy S, Reggio B, Wiemer K, Larsen F, Cohen J. The
limited importance of pronuclear scoring of human zygotes. Hum
Reprod 2006;21:1599 – 1604.
Lemmen JG, Agerholm I, Ziebe S. Kinetic markers of human embryo
quality using time-lapse recordings of IVF/ICSI-fertilized oocytes.
Reprod Biomed Online 2008;17:385 – 391.
Meseguer M, Herrero J, Tejera A, Hilligsøe KM, Ramsing NB, Remohı́ J.
The use of morphokinetics as a predictor of embryo implantation.
Hum Reprod 2011;10:2658 – 2671.
Montag M, Van der Ven H. Evaluation of pronuclear morphology as the
only selection criterion for further embryo culture and transfer:
results of a prospective multicentre study. Hum Reprod 2001;
16:2384 – 2389.
Montag M, Liebenthron J, Köster M. Which morphological scoring system
is relevant in human embryo development? Placenta 2011;32:252– 256.
Nagy ZP, Dozortsev D, Diamond M, Rienzi L, Ubaldi F, Abdelmassih R,
Greco E. Pronuclear morphology evaluation with subsequent
evaluation of embryo morphology significantly increases implantation
rates. Fertil Steril 2003;80:67 – 74.
Payne D, Flaherty SP, Barry MF, Matthews CD. Preliminary observations
on polar body extrusion and pronuclear formation in human oocytes
using time-lapse video cinematography. Hum Reprod 1997;12:
532 – 541.
Payne JF, Raburn DJ, Couchman GM, Price TM, Jamison MG, Walmer DK.
Relationship between pre-embryo pronuclear morphology (zygote
score) and standard day 2 or 3 embryo morphology with regard
to assisted reproductive technique outcomes. Fertil Steril 2005;
84:900 – 909.
Romanova L, Korobova F, Noniashvilli E, Dyban A, Zatsepina O. High
resolution mapping of ribosomal DNA in early mouse embryos by
fluorescence in situ hybridization. Biol Reprod 2006;74:807 – 815.
Sadowy S, Tomkin G, Munné S, Ferrara-Congedo T, Cohen J. Impaired
development of zygotes with uneven pronuclear size. Zygote 1998;
6:137 – 141.
Salumets A, Hydén-Granskog C, Suikkari AM, Tiitinen A, Tuuri T. The
predictive value of pronuclear morphology of zygotes in the
assessment of human embryo quality. Hum Reprod 2001;16:
2177 – 2181.
Savino TM, Gébrane-younès J, Mey JD, Sibarita J-baptiste,
Hernandez-verdun D. Nucleolar assembly of the rRNA processing
machinery in living cells. J Cell Biol 2001;153:1097 – 1110.
Scott L. Pronuclear scoring as a predictor of embryo development. Reprod
Biomed Online 2003;6:201– 214.
Downloaded from http://humrep.oxfordjournals.org/ by guest on June 11, 2014
In conclusion, PNB occurred significantly later in embryos resulting in
live birth than in embryos not resulting in live birth. PNB in live birth
embryos never occurred before 20 h 45 min. However, the development of the PN does not predicte the quality of the zygote, in terms of
live birth potential.
Azzarello et al.
PN morphology and the time of PNB in zygotes
ultrastructural and autoradiographic analysis. Dev Biol 1986;1:
193 – 203.
Tesarik J, Junca AM, Hazout A, Aubriot FX, Nathan C, Cohen-Bacrie P,
Dumont-Hassan M. Embryos and high implantation potential after
intracytoplasmic sperm injection can be recognized by a simple,
non-invasive examination of pronuclear morphology. Hum Reprod
2000;15:1396 – 1399.
Weitzman VN, Schnee-Riesz J, Benadiva C, Nulsen J, Siano L, Maier D.
Predictive value of embryo grading for embryos with known
outcomes. Fertil Steril 2010;93:658 –662.
Wong CC, Loewke KE, Bossert NL, Behr B, De Jonge CJ, Baer TM, Reijo
Pera RA. Non-invasive imaging of human embryos before embryonic
genome activation predicts development to the blastocyst stage. Nat
Biotechnol 2010;28:1115 – 1121.
Wright G, Wiker S, Elsner C, Kort H, Massey J, Mitchell D, Toledo A,
Cohen J. Observations on the morphology of pronuclei and nucleoli
in human zygotes and implications for cryopreservation. Hum Reprod
1990;5:109 – 115.
Zatsepina O, Baly C, Chebrout M, Debey P. The step-wise
assembly of a functional nucleolus in preimplantation mouse
embryos involves the cajal (coiled) body. Dev Biol 2003;253:
66 – 83.
Downloaded from http://humrep.oxfordjournals.org/ by guest on June 11, 2014
Scott LA, Smith S. The successful use of pronuclear embryo transfers the
day following oocyte retrieval. Hum Reprod 1998;13:1003 – 1013.
Scott L, Alvero R, Leondires M, Miller B. The morphology of human
pronuclear embryos is positively related to blastocyst development
and implantation. Hum Reprod 2000;15:2394–2403.
Scott L, Finn A, O’Leary T, McLellan S, Hill J. Morphologic parameters of
early cleavage-stage embryos that correlate with fetal development and
delivery: prospective and applied data for increased pregnancy rates.
Hum Reprod 2007;22:230 –240.
Sirri V, Urcuqui-Inchima S, Roussel P, Hernandez-Verdun D. Nucleolus:
the fascinating nuclear body. Histochem Cell Biol 2008;129:13 – 31.
Svarcova O, Dinnyes A, Polgar Z, Bodo S, Adorjan M, Meng Q,
Maddox-Hyttel P. Nucleolar re-activation is delayed in mouse
embryos cloned from two different cell lines. Mol Reprod Dev 2009;
76:132–141.
Tesarik J, Greco E. The probability of abnormal preimplantation
development can be predicted by a single static observation on
pronuclear stage morphology. Hum Reprod 1999;14:1318– 1323.
Tesarik J, Kopecny V. Development of human male pronucleus:
ultrastructure and timing. Gamete Res 1989;24:135–149.
Tesarik J, Kopecny V, Plachot M, Mandelbaum J, Da Lage C, Flechon JE.
Nucleologenesis in the human embryo developing in vitro:
9