Download Early embryo cleavage is a strong indicator of embryo quality in

Human Reproduction Vol.16, No.12 pp. 2652–2657, 2001
Early embryo cleavage is a strong indicator of embryo
quality in human IVF
K.Lundin1, C.Bergh and T.Hardarson
Department of Obstetrics and Gynecology, Göteborg University, Sahlgrenska University Hospital, Gothenburg, Sweden
1To
whom correspondence should be addressed at: IVF-lab, Reproductive Medicine, SU/Sahlgrenska, SE-413 45 Göteborg, Sweden.
E-mail: [email protected]
BACKGROUND: In order to decrease multiple birth rates without decreasing birth rates overall, it is important
to increase the capability of selecting the most optimal embryos for transfer. It has been shown that human embryos
which cleave early, i.e. complete the first mitotic division within 25–27 h post insemination, provide higher pregnancy
and implantation rates. METHODS AND RESULTS: In this prospective study, an evaluation of 10 798 scored
embryos showed that early cleavage resulted in a significantly higher proportion of good quality embryos compared
with late cleavage (62.5 versus 33.4%, P < 0.0001). When examining both day 2 and day 3 transfers together, earlycleaving embryos (306 transfers) gave rise to significantly higher rates of pregnancy/transfer (40.5 versus 31.3%,
P ⍧ 0.0049), implantation (28.0 versus 19.5%, P ⍧ 0.0001) and birth/ongoing pregnancy (34.3 versus 24.0%,
P ⍧ 0.0009) than did late-cleaving embryos (521 transfers). A stepwise logistic regression of all data showed that
the total number of good quality embryos and female age were independent predictors of both pregnancies and
birth. For intracytoplasmic sperm injection (ICSI) embryos, early cleavage was found to be an independent
predictor of birth. CONCLUSIONS: Early embryo cleavage is a strong biological indicator of embryo potential,
and may be used as an additional embryo selection factor for ICSI embryos.
Key words: early cleavage/embryo quality/good quality embryos/IVF/pregnancy
Introduction
One of the greatest problems in assisted reproduction today is
the high multiple pregnancy rate, associated mainly with the
number of embryos transferred and embryo quality (Puissant
et al., 1987; Steer et al., 1992; Shulman et al., 1993; Hu et al.,
1998; Strandell et al., 2000). In order to be able to transfer
two, or even one, embryo(s) without markedly lowering the
pregnancy rate, it is important to increase our knowledge
of how to select the optimal cleavage-stage embryos or
blastocysts.
Embryo scoring is performed in different ways, with each
centre having its own scoring system. However, since all
current systems are based on morphological evaluation, most
parameters evaluated are the same between centres, albeit with
different emphases being placed on each parameter.
The current practice in most IVF laboratories is to score
cleavage-stage embryos in connection with embryo transfer,
i.e. on day 2 or 3 after oocyte retrieval, evaluating the grade
of fragmentation, cytoplasmic appearance and number of
blastomeres per embryo. These variables can be collectively
counted as a cumulative embryo score (CES ⫽ grade ⫻
number of blastomeres) (Steer et al., 1992; Visser and Fourie,
1993). In addition, variation in the zona pellucida thickness,
presence of multinucleated blastomeres, location of fragments
and size of blastomeres in relation to each other may be
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analysed (Cohen et al., 1989; Giorgetti et al., 1995; Pickering
et al., 1995; Ziebe et al., 1997; Palmstierna et al., 1998;
Pelinck et al., 1998; Alikani et al., 1999; Hardarson et al.,
2001). Embryo quality has also been reported to correlate
with oocyte and zygote morphology, e.g. appearance of the
cytoplasm, pronuclei and polar bodies (Sadowy et al., 1998;
Scott and Smith, 1998; Manor et al., 1999; Tesarik and Greco,
1999; Ebner et al., 2000).
Another indicator of embryo quality that can easily be
determined is the embryo cleavage rate. Until recently, the
cleavage rate has mainly been documented at day 2 or 3, i.e.
the number of cells at that time has been correlated with
pregnancy and implantation rates. It has been reported (Shoukir
et al., 1997; Sakkas et al., 1998a) that early cleavage, i.e. the
time of the first mitotic division, could also be correlated with
embryo quality and pregnancy rates. However, both these
reports were based on a small number of transfers and had a
biased embryo selection at transfer, since early-cleaving
embryos were chosen whenever possible. In addition, the
transfers were sometimes mixed, with both early- and latecleaving embryos being transferred together. This makes it
difficult to draw any conclusions, since it is impossible to
ascertain which of the embryos actually implanted.
The aim of the present study was therefore to examine, in
a large sample size, whether the time of first cell cleavage
© European Society of Human Reproduction and Embryology
Early cleavage and embryo quality
influenced embryo quality, pregnancy, implantation and/or
birth rates.
Table I. Analysis of all zygotes/embryos screened at 25–27 h post
insemination/microinjection (n ⫽ 10 798)
Early cleavage
Late cleavage
3046 (28.2)
1903 (62.5)a
1909 (34.7)c
1137 (21.5)d
7752
2593
3596
4156
Materials and methods
This prospective study was initiated in January 1997 at the IVF unit,
Sahlgrenska University Hospital, Göteborg, Sweden. Data from this
time until June 2000 were analysed. All zygotes that were normally
fertilized (two pronuclei) and checked for early embryo cleavage
within 25–27 h post insemination/microinjection were included in an
evaluation of cleavage rate and embryo quality. Embryos either
transferred or frozen on day 2 or 3 (grade 1 or 2, with 4–6 blastomeres
on day two, or 6–8 blastomeres on day 3) were considered to be
good quality embryos. Embryos of a lower quality were very seldom
transferred.
Only cycles that led to embryo transfer with embryos of identical
cleavage score at early evaluation (i.e. one or two cells at 25–27 h
post insemination/microinjection) were included in the pregnancy,
implantation and birth rate analysis. Each patient was only included
once (their first cycle during this time period) and no mixed (earlyand late-cleaving embryos together) transfers were included.
Ovarian stimulation was carried out by a desensitizing protocol
using a gonadotrophin-releasing hormone agonist preparation in a
long protocol in combination with recombinant FSH (Gonal-F; Serono,
Geneva, Switzerland or Puregon; Organon, Oss, The Netherlands).
Monitoring was carried out by measuring serum oestradiol concentrations and performing transvaginal ultrasonography. When an adequate
stimulation was achieved (three or more follicles 艌18 mm diameter),
human chorionic gonadotrophin (HCG) (Profasi; Serono) was administered (10 000 IU, s.c.). Oocytes were retrieved 36–38 h following
HCG using transvaginal ultrasound-guided puncture.
Conventional IVF or intracytoplasmic sperm injection (ICSI) was
performed 3–5 h after oocyte aspiration. Only metaphase II oocytes
were used for ICSI. The oocytes were cultured separately in media
droplets under oil (IVF-20 and Ovoil; Scandinavian IVF Science,
Gothenburg, Sweden).
Fertilization was determined by confirmation of two pronuclei
(2PN) 17–19 h after insemination/microinjection. The zygotes were
checked again in the afternoon on day 1 (25–27 h after insemination/
microinjection), and the number of cells (one or two) was documented.
Embryos were transferred routinely on day 2. However, day 3
transfers were occasionally performed, mainly due to slowly cleaving
(fewer than four cells on day 2) or fragmented embryos. It should be
noted that embryos were not selected for transfer on the basis of
early or late cleavage, but on morphology alone.
Pregnancy was defined as a positive HCG test in urine on day 19
post transfer. Implantation rate was defined as number of fetal sacs
at ultrasound in gestational week 7, per number of transferred embryos.
Statistical analysis
Distribution of the variables are given as means, SD and ranges. For
comparison between groups, the Mann–Whitney U-test was used for
ordered and continuous variables, χ2 test for dichotomous variables.
In order to select independent predictors of pregnancy and birth/
ongoing pregnancy, all variables in Table III were entered into
stepwise logistic regression analyses. All significance tests were twotailed and conducted at the 5% significance level. In addition to the
overall analysis including both ICSI and IVF transfers, two separate
stepwise logistic regressions were also performed, for ICSI alone and
for conventional IVF alone. The reason for this was that ICSI and
conventional IVF may be considered as two separate populations in
regard to the time of fertilization.
Cleaved embryos
Good quality embryos
Cleaved embryos from ICSI treatment
Cleaved embryos from IVF treatment
(71.8)
(33.4)b
(65.3)
(78.5)
Values in parentheses are percentages.
aversusb, P ⬍ 0.0001.
cversusd, P ⬍ 0.0001.
Results
During the study period, a total of 18 120 oocytes were
normally fertilized (2PN). Due to laboratory schedules and
availability of personnel, only 10 798 of these zygotes were
checked for early cleavage at 25–27 h post insemination/
microinjection and included in the evaluation of cleaving rate
and embryo quality (Table I).
Of the early cleaving embryos, 1125 were transferred and
778 were frozen, i.e. a total of 62.5% good quality embryos
were obtained from this group. From the late-cleaving embryo
group, 1613 were transferred while 980 were frozen, giving a
total of 33.4% good quality embryos in this group (P ⬍
0.0001). ICSI embryos had a higher rate of early cleavage
than IVF embryos (Table I), although the percentage of good
quality embryos derived with either technique did not differ
(39.7% for IVF versus 42.8% for ICSI).
An analysis of all cycles leading to embryo transfer on day
2 and 3 is shown in Table II. The transfer of early-cleaving
embryos resulted in significantly higher overall pregnancy
(40.5 versus 31.3%, P ⫽ 0.0049) and implantation rates (28.0
versus 19.5%, P ⬍ 0.0001) than the transfer of late-cleaving
embryos (Table II). The twin pregnancy rate (confirmed by
ultrasound in gestational week 7) was 33.3% in the earlycleaving group and 22.3% in the late-cleaving group (P ⫽
0.032). The spontaneous abortion rate was significantly lower
in the early-cleaving group (12.1 versus 20.2%, P ⫽ 0.036),
and the birth rate significantly higher (34.3 versus 24.0%, P ⫽
0.0009) compared with the late-cleaving group (Table II). For
those pregnancies that went to term, the rate of born males
versus females was calculated. No difference was found in the
sex ratio between born children from early-cleaving embryos
compared with late-cleaving embryos (50/50 and 53/47%
respectively).
The results from the univariate correlation analysis are
shown in Table III. Female age, day of transfer, total number
of good quality embryos available and early cleavage were all
correlated with both pregnancy and birth (Table III).
The results from the overall stepwise logistic regression
analyses of all transfers (Table IV) showed that total number
of good quality embryos and female age were independent
predictors of both pregnancy and birth, while early/late
cleavage was not. However, when stepwise analyses were
performed separately for ICSI and IVF, it was found that for
ICSI the variable early/late cleavage was an independent
predictor of birth (Table IV).
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K.Lundin, C.Bergh and T.Hardarson
Table II. Analysis of all cycles with early- or late-cleaving embryos that led to embryo transfer on days 2 or 3
No. of cycles
Aspirated oocytesa
Good quality embryosa
Range
Blastomeres per transferred embryo day 2
Blastomeres per transferred embryo day 3
Mean no. of embryos transferred
Female age (years)a
Range
Pregnancies per transfer
Implantation rate
Spontaneous abortionsb
Birth ratec
Born male/female (%)
Early cleavage
Late cleavage
P
306
13.1 ⫾ 6.31
3.8 ⫾ 2.55
0–18
4.1 (1.00)
7.8 (1.06)
1.92
32.2 ⫾ 4.0
21.9–40.8
124 (40.5)
165/589 (28.0)
15/124 (12.1)
105/306 (34.3)
50/50
521
11.4 ⫾ 5.55
2.3 ⫾ 1.84
0–12
4.0 (0.87)
6.0 (1.46)
1.97
33.2 ⫾ 3.9
22.2–41.4
163 (31.3)
200/1027 (19.5)
33/163 (20.2)
125/521 (24.0)
53/47
0.0004
⬍ 0.0001
0.0010
⬍ 0.0001
NS
⬍ 0.0001
0.0049
⬍ 0.0001
0.036
0.0009
Values in parentheses are percentages.
aMean ⫾ SD; b% per pregnancy; c% per transfer.
NS ⫽ not significant.
Table III. Factors analysed for univariate correlation with pregnancy and birth/ongoing pregnancy
Variable
Pregnancy
Birth/ongoing pregnancy
Yes (n ⫽ 292)
No. of aspirated oocytesa
Female age (years)a
No. of blastomeresa
No. of good quality embryosa
12.32
32.21
4.81
3.51
⫾
⫾
⫾
⫾
5.74
3.78
1.48
2.32
No (n ⫽ 535)
11.86 ⫾ 5.94
33.16 ⫾ 4.02
4.82 ⫾ 1.44
2.57 ⫾ 2.13
Yes (n ⫽ 238)
P
NS
0.0014
NS
⬍ 0.0001
Day of embryo transfer
Day 2
Day 3
223 (38)
69 (29)
363 (62)
172 (71)
–
Fertilization method
ICSI
IVF
160 (36)
132 (35)
290 (64)
245 (65)
Cleavage
Early
Late
124 (41)
163 (31)
182 (59)
358 (69)
12.55
32.3
4.86
3.51
⫾
⫾
⫾
⫾
5.97
3.88
1.43
2.29
No (n ⫽ 589)
11.80 ⫾ 5.83
33.1 ⫾ 4.11
4.80 ⫾ 1.50
2.65 ⫾ 2.18
P
NS
0.0042
NS
⬍ 0.0001
178 (30)
60 (25)
408 (70)
181 (75)
–
–
NS
133 (30)
105 (28)
317 (70)
272 (72)
–
NS
–
105 (34)
125 (24)
201 (66)
396 (76)
–
0.0006
0.0034
0.0050
0.0012
Values in parentheses are percentages.
aMean ⫾ SD.
NS ⫽ not significant.
Discussion
Although many factors influence the result of an IVF cycle
(e.g. stimulation response, endometrial receptivity, oocyte
maturity, culture conditions, paternal contribution), embryo
morphology is regarded as one of the most important. Morphological embryo variables are easy to evaluate, and have thus
been the determining factors when the selection for embryo
transfer is made. Much effort has been made in trying to find
an optimal scoring system. A number of reports have been
published concerning the importance of different factors for
embryo development and pregnancy rate, including the appearance of the cumulus cells, the position of the polar body(ies)
in relation to the pronuclei, the morphology and size of the
pronuclei, and the number and size of nuclei in the blastomeres
(Edwards and Beard, 1997; Scott and Smith, 1998; Manor
et al., 1999; Tesarik and Greco, 1999; Ebner et al., 2000). An
ideal scoring system may start at the oocyte stage, continue
2654
through the zygote stage, and conclude with a morphologybased selection either at the cleavage or blastocyst stage.
However, the advantage of a scoring system with many
observations must be weighed against the impairment of the
environment for the embryo.
When comparing the development of all early- versus
late-cleaving embryos, it was found that significantly higher
numbers of early-cleaving embryos became good quality
embryos (Table I), as well as giving significantly higher
pregnancy, implantation and birth rates (Table II). These results
imply that early cleavage is indeed a good biological indicator
of embryo potential. This was also confirmed in a univariate
analysis where early cleavage, female age, day of transfer and
total number of good quality embryos were shown to be
positively correlated with pregnancy and birth (Table III).
When performing a stepwise multivariate analysis including
the variables of number of aspirated oocytes, female age, mean
Early cleavage and embryo quality
Table IV. Variables independently predictive of pregnancy or birth, adjusted for all other variables
Variable
Pregnancy
Birth
Odds ratio
(95% CI)
Adjusted
P-value
Odds ratio
(95% CI)
Adjusted
P-value
All transfers
Female age
No. of good quality embryos
0.942 (0.91–0.98)
1.240 (1.15–1.33)
0.0023
⬍ 0.0001
0.954 (0.92–0.99)
1.169 (1.10–1.25)
0.017
0.0001
Only ICSI transfers
No. of good quality embryos
Early cleavage
1.297 (1.16–1.45)
–
⬍ 0.0001
–
1.243 (1.12–1.39)
1.744 (1.12–2.73)
⬍ 0.0001
0.015
Only IVF transfers
Female age
No. of good quality embryos
0.929 (0.87–0.99)
1.139 (1.04–1.25)
0.018
0.0045
–
1.106 (1.01–1.21)
–
0.029
CI ⫽ confidence interval.
number of blastomeres per transferred embryo, total number
of good quality embryos, early/late cleavage, day of transfer
and fertilization method, early cleavage was found not to be
an independent predictor of either pregnancy or birth/ongoing
pregnancy (Table IV). However, when performing a similar
stepwise logistic regression analysis with only the ICSI transfers included, early cleavage was found to be an independent
predictor of birth, but not of pregnancy.
In contrast to the results of previous studies which stated
that the time of first cleavage could be used for selection of
embryos at transfer (Shoukir et al., 1997; Sakkas et al., 1998a),
no selection for the early-cleaving embryos was performed at
embryo transfer in the current study. Instead, the embryos
judged to be morphologically best at the time of transfer were
chosen, irrespective of cleavage rate. Also of importance in
the current study is that only results from those transfers where
all embryos in each transfer showed the same cleavage rate,
i.e. either early or late, were included.
Embryos derived after ICSI had a higher rate of early
cleavage than those derived after IVF. This might be expected
since the injected spermatozoa bypass many of the fertilization
steps such as the acrosome reaction and binding to the zona
pellucida, resulting in a shorter fertilization time (van Wissen
et al., 1995; Nagy et al., 1998). However, the percentage of
good quality embryos derived from IVF were similar to those
derived from ICSI. The question therefore arises; if IVF
embryos had been checked for early cleavage at a slightly
later time, would a similar rate of early cleavage as for ICSI
embryos have been found? The reason why the separate
analysis of ICSI/IVF transfers showed that early cleavage was
an independent predictor of birth for ICSI transfers but not
for IVF transfers is not known, but it may possibly be
associated with the higher rate of early cleaved ICSI embryos.
Hence, it may be better to screen IVF embryos later and/or to
inseminate them earlier in relation to ICSI embryos, in order
to make use of the early cleavage rate screening for IVF
embryos. However, the possibility that there is an actual
developmental difference in embryos that stem from different
fertilization methods cannot be excluded.
The reason why early cleavage should yield better quality
embryos is not known. It can be speculated that early-cleaving
embryos stem from oocytes in which cytoplasmic and nuclear
maturation are better synchronized and/or have a higher
‘metabolic fitness’, i.e. the availability and competence of, for
example, ATP, mRNA, mitochondria, etc. Animal studies have
shown (Grisart et al., 1994) that bovine embryos have a ‘lagphase’ (i.e. a lengthened cell cycle) either early (4- to 7-cell
stage) or late (8-cell stage). It was found that embryos with a
late lag-phase had a higher blastocyst rate than those with an
early lag-phase. These authors speculated that the lag-phase is
related to the amount and/or quality of RNA or proteins stored
in the oocyte and to the transcriptional activity of the embryo.
A premature transcription resumption, indicated by a pause in
early growth, might thus harm further development.
Another variable that has to be considered is the contribution
of the spermatozoa, i.e. the addition of paternal factors. In
humans, the centrioles that control the first mitotic divisions
of the oocyte are introduced by the spermatozoa (Sathananthan
et al., 1991; Palermo et al., 1994; Sathananthan, 1998), and
the quality of the spermatozoa could thus be one additional
factor influencing the early cleavage. In addition, the DNA
status of the spermatozoa will influence that of the embryo
(Sakkas et al., 1998b; Obasaju et al., 1999; Larson et al., 2000).
It has been shown that cells having an aneuploid chromosomal status cleave more slowly in general (Paton et al., 1974;
Barrenäs et al., 2000). This was also found in our IVF
programme when investigating 3PN zygotes, which had a rate
of early cleavage of 12.0% (own unpublished observations),
as compared with the overall 26.9% of 2PN zygotes shown in
Table I. The rate of early cleavage might therefore be one
indicator of the chromosomal status of the embryo. This is
supported by the observation that unevenly cleaved embryos
(those with a large difference in blastomere size) have been
shown to have a higher rate of aneuploidy than embryos with
blastomeres of equal size, as well as a significantly lower rate
of early cleavage and lower pregnancy and implantation rates
(Hardarson et al., 2001).
Although the overall results showed significant differences
between early- and late-cleaving embryos in pregnancy,
implantation and birth rates, it is important to note that there
2655
K.Lundin, C.Bergh and T.Hardarson
was a significant difference in blastomere number per embryo
between the groups; many of the later-cleaving embryos were
still slow on the day of transfer (Table II). This indicates that
embryos with a slow start have a lower rate of development
in general, and that this influences the success rate. With
regard to the rate at which cells cleave, a number of reports
have been published showing that embryos having fewer than
four cells at day 2 or fewer than six cells at day 3 have a
significantly lower pregnancy and implantation rate than fastercleaving embryos (Lewin et al., 1994; Söderlund et al., 1998;
Van Royen et al., 1999). This may correlate with the increased
chromosomal abnormality rate for slow/fragmented embryos
(Munne et al., 1995).
It has been found in several studies that mammalian male
embryos may cleave faster than female embryos (Tsunoda
et al., 1985; Xu et al., 1992; Pergament et al., 1994; Ng et al.,
1995; Peippo and Bredbacka 1995; Tarin et al., 1995). These
data originate from studies of in-vitro-cultured embryos
analysed from the 4-cell to the blastocyst stage. In the present
study, no difference was found when analysing the sex ratio
of delivered babies originating from either early- or latecleaving embryos (Table I). This implies that the possible
difference in cleaving rate between male/female human
embryos is something that does not occur until later, possibly
coinciding with the onset of genomic transcription (Braude
et al., 1988).
It can be concluded from the results of the current study
that early cleavage is a highly significant biological indicator
of embryo potential, resulting in overall significantly improved
embryo quality as well as pregnancy, implantation and birth/
ongoing pregnancy rates. Since a separate logistic regression
analysis of ICSI transfers showed that early cleavage was an
independent predictor of birth, it is suggested that early
cleavage may be an additional factor for selecting embryos
with a higher potential for resulting in birth. Further studies
are needed to investigate whether early cleavage might also
be used as a screening variable for embryos derived from
conventional IVF if the time interval from insemination to
screening is increased.
Acknowledgements
The authors thank Nils-Gunnar Pehrsson for advice and statistical
analyses and Graham Coull for valuable criticism of the manuscript.
The study was supported by grants from Hjalmar Svenssons
Forskningsfond and the Marie Curie research training grant to
T.Hardarson.
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Received on February 20, 2001; accepted on August 21, 2001
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