Time from insemination to first cleavage predicts developmental competence of human preimplantation embryos in vitro

Abstract

The absence of reliable markers for the identification of viable embryos for transfer at the early cleavage stage is likely to contribute to the generally low implantation rates and high incidence of multiple gestation in IVF treatment. In this study, we investigate the relationship between timing of first cleavage and the incidence of blastocyst formation in vitro.
Couples (n = 70) with at least one embryo remaining after transfer were included in the analyses. All embryos (n = 579) were examined for early cleavage at 25 h after insemination. Following embryo transfer, the remaining embryos (n = 426) were cultured until day 7 of development, and assessed for blastocyst formation.
Eighty-five embryos (14.7%) cleaved to the 2-cell stage within 25 h of insemination; 26 of these were selected for transfer on day 2. Of the 59 embryos remaining in culture, 19 (32.2%) developed to the blastocyst stage; this was a significantly higher number than was observed in embryos (61/367; 16.6%) that failed to cleave within 25 h of insemination (P < 0.01). Within these two groups of embryos the proportion of hatched blastocysts was 11/59 (18.6%) and 26/367 (7.1%) respectively (P < 0.005).
These findings indicate that early cleavage is indicative of increased developmental potential in human embryos and may be useful as an additional criterion in the selection of embryos for transfer.

Full text

Human Reproduction Vol.17, No.2 pp. 407–412, 2002
Time from insemination to first cleavage predicts
developmental competence of human preimplantation
embryos in vitro
J.Fenwick, P.Platteau
1
, A.P.Murdoch and M.Herbert
2
Reproductive Medicine, BioScience Centre, ICFL, Times Square, Newcastle upon Tyne NE1 4EP, UK
1
Present: Centre for Reproductive Medicine, University Hospital, Dutch-speaking Brussels Free University,
Laarbeeklaan 101, B-1090 Brussels, Belgium
2
To whom correspondence should be addressed. E-mail: mary.herbert@ncl.ac.uk
BACKGROUND: The absence of reliable markers for the identification of viable embryos for transfer at the early
cleavage stage is likely to contribute to the generally low implantation rates and high incidence of multiple gestation
in IVF treatment. In this study, we investigate the relationship between timing of first cleavage and the incidence
of blastocyst formation in vitro. METHODS: Couples (n ⍧ 70) with at least one embryo remaining after transfer
were included in the analyses. All embryos (n ⍧ 579) were examined for early cleavage at 25 h after insemination.
Following embryo transfer, the remaining embryos (n ⍧ 426) were cultured until day 7 of development, and
assessed for blastocyst formation. RESULTS: Eighty-five embryos (14.7%) cleaved to the 2-cell stage within 25 h
of insemination; 26 of these were selected for transfer on day 2. Of the 59 embryos remaining in culture, 19 (32.2%)
developed to the blastocyst stage; this was a significantly higher number than was observed in embryos (61/367;
16.6%) that failed to cleave within 25 h of insemination (P < 0.01). Within these two groups of embryos the
proportion of hatched blastocysts was 11/59 (18.6%) and 26/367 (7.1%) respectively (P < 0.005). CONCLUSIONS:
These findings indicate that early cleavage is indicative of increased developmental potential in human embryos
and may be useful as an additional criterion in the selection of embryos for transfer.
Key words: blastocyst formation/early cleavage/first cell cycle/human embryos
Introduction
The ability to identify viable embryos is critical to the success
of IVF treatment. The predominant practice in clinical IVF is
to select embryos for transfer based on an assessment of cell
number and morphological appearance at the time of transfer
on day 2 or 3 of development. This has been shown to
be positively correlated with implantation and pregnancy
(Cummins et al., 1986; Puissant et al., 1987; Staessen et al.,
1992; Giorgetti et al., 1995; Ziebe et al., 1997). However,
with the development of aggressive ovarian stimulation
protocols and improved culture conditions for early embryos,
individual patients may produce multiple good quality embryos,
with equivalent cell numbers and morphological scores. Taken
together with the increasing pressure to reduce the number
of embryos replaced in the uterus, due to the high incidence
of multiple pregnancy (Coetsier and Dhont, 1998; Gerris and
Van Royen, 2000), these developments conspire to make the
selection of embryos for transfer an increasingly important
and difficult task.
A number of strategies, ranging from pronucleate stage
selection to blastocyst transfer, have been devised to help
© European Society of Human Reproduction and Embryology 407
improve the prediction of embryo viability. Studies in which
pregnancy and implantation rates were compared following
transfer of blastocyst or cleavage stage embryos have produced
conflicting results. While some studies have reported that
transfer of blastocysts results in higher implantation rates than
transfer of cleavage stage embryos (Gardner et al., 1998;
Marek et al., 1999; Schoolcraft et al., 1999; Milki et al.,
2000), other studies have found no difference (Coskun et al.,
2000; Huisman et al., 2000). However, the practice of
blastocyst transfer is not in widespread use, partly because
of a general lack of experience in prolonged embryo culture,
as well as anxieties about those patients whose embryos arrest
before blastocyst formation (Van Blerkom, 1997). Furthermore,
an increased incidence of monozygotic twinning after blasto-
cyst transfer has been reported (Behr et al., 2000; da Costa
et al., 2001).
An alternative strategy, which has evolved in situations
where culture beyond the zygote stage is not compatible with
religious beliefs or legal requirements, is to select embryos on
the basis of pronucleate stage morphology. The evidence
indicates that a grading system based on morphological charac-

J.Fenwick et al.
teristics, including the extent of pronuclear apposition and
nucleolar alignment, is predictive of implantation potential
(Scott and Smith, 1998; Tesarik and Greco, 1999; Ludwig
et al., 2000) and blastocyst formation (Scott et al., 2000).
Consistent with this, a recent study, encompassing experimental
evidence and mathematical modelling, supports the idea that
the viability of human embryos is already determined at the
1-cell stage (Hardy et al., 2001).
The 1-cell stage of development represents the return to
mitotic division following completion of maternal meiosis. In
the first cell cycle, S phase, in which the chromosomes replicate,
and M phase, in which the replicated chromosomes segregate,
are separated by two gap phases G1 and G2. Entry into G1 is
marked by the appearance of pronuclei, which persist until the
transition from G2 into M phase. Observations of human zygotes
indicate variability in the timing of all cell cycle transitions
following sperm entry (Balakier et al., 1993; Capmany et al.,
1996; Payne et al, 1997; Nagy et al., 1998). The culmination of
this is that the onset of first cleavage (exit from M phase) is
observed over a time-span of ⬎8 h (between 22 and ⬎30 h after
sperm entry) (Payne et al., 1997; Nagy et al., 1998).
Studies in other species have shown a relationship between
the timing of completion of the first cell cycle and subsequent
developmental potential. In mice and cattle, early onset of first
cleavage is associated with increased blastocyst formation and
implantation (McLaren and Bowman, 1973; Grisart et al., 1994;
Lonergan et al., 1999). The suggestion that such an association
might also exist in human embryos comes from the finding that
patients who produced early cleaving (EC) embryos had higher
pregnancy and implantation rates than those who did not (Shou-
kir et al., 1997; Sakkas et al., 1998). However, an unequivocal
correlation between time from insemination to first cleavage and
embryo development potential has not yet been established,
since the studies to date (Shoukir et al., 1997; Sakkas et al.,
1998) have preferentially replaced EC embryos when available.
Any differences in the implantation potential of EC and non-
early cleaving (NEC) embryos in the population of patients
generating both embryo types were therefore not examined.
Also, it is possible that other aspects of fecundity, such as
improved endometrial receptivity, may account for the improved
treatment success of patients generating EC embryos compared
with patients with only NEC embryos.
Given its ease of application and non-subjective nature, we
were interested to explore further the usefulness of early
cleaving as a marker of embryonic viability. Here, we report
the findings of a study in which we compared blastocyst
formation in vitro of embryos that cleaved within 25 h of
insemination with those that cleaved later. Because the early
cleaving status of each embryo was withheld until after
completion of the embryo transfer, we were able to evaluate
whether there was a natural bias in favour of selecting EC
embryos for transfer based on morphological criteria on day
2 of development.
Materials and methods
Source of embryos
The study was performed on embryos produced by 70 sequential
couples undergoing IVF treatment, with at least one embryo remain-
408
ing after embryo transfer. The culture of embryos remaining after
transfer until day 7 after fertilization to monitor blastocyst develop-
ment was part of routine treatment at the time of the study (January–
June 1998) and no ethical approval was required.
Treatment regime
Ovulation was induced using a standard protocol of GnRH analogue
(Suprefact; Hoechst, Hounslow, UK) and FSH (Metrodin HP or Gonal
F; Serono, London, UK) at a daily dose of 150–300 IU for 10–15
days, followed by 5000 or 10 000 IU HCG, (Profasi; Serono). Follicles
were aspirated 38–40 h after HCG administration, using ultrasound
guidance. Retrieved oocytes were transferred to individual 100 µl
droplets of oocyte culture medium (OCM) under mineral oil (Sigma,
Poole, Dorset, UK). OCM consisted of Earle’s balanced salt solution
(EBSS; Life Technologies, Paisley, UK) supplemented with 25 mmol/
l sodium bicarbonate (Sigma Hybri-max), 0.5 mmol/l sodium pyruvate
(tissue culture grade; Sigma, UK), 10% (v/v) of a 4.5% solution of
human serum albumin (HSA; Immuno Ltd, Sevenoaks, Kent, UK),
10 µg/ml gentamycin sulphate (ICN; Thame, Oxfordshire, UK) and
100 IU/ml benzylpenicillin sodium (Crystapen; Brittania Pharmaceut-
icals, Redhill, Surrey, UK).
Sperm for insemination were isolated by centrifugation at 200 g
on a discontinuous density gradient composed of 90 and 45% Percoll
solutions in HEPES-buffered EBSS (Percoll; Pharmacia, Sweden;
HEPES; Sigma). Between 25 000 and 50 000 sperm were added to
each oocyte at 41–43 h after HCG administration (day 0).
At 18–20 h post-insemination (day 1) the oocytes were mechanically
denuded of their cumulus cells and those showing two pronuclei were
transferred to individual 100 µl droplets of embryo culture medium
(ECM) under mineral oil. ECM comprised EBSS supplemented
with 15% (v/v) of a 4.5% solution of human serum albumin (HSA)
and 0.5 mmol/l sodium pyruvate. All embryos were examined at
24.5–25.5 h post-insemination and those which had divided to become
two cells were termed early cleaving (EC) embryos and those which
had not yet divided were termed non-early cleaving (NEC) embryos.
On day 2, at 42–48 h post-insemination, each embryo was assessed
for cell number and assigned a morphology score between 0 and 1.0.
The latter value was a semi-quantitative assessment of embryo quality,
taking into account the degree of fragmentation, uniformity of
blastomere size and cytoplasmic appearance. Up to three of the fastest
dividing and highest scoring embryos were selected for transfer. The
timing of first cleavage of the embryos was unknown to the operator
at the time of transfer, and therefore did not influence the selection
process.
Blastocyst culture
Following transfer of the best quality embryos, EC and NEC embryos
from individual patients were pooled and cultured in separate wells
of a 4-well dish (Nunclon, Life Technologies, Paisley, UK). The
number of embryos cultured in each well ranged from 1–7 (mean ⫽
2.3) and 1–13 (mean ⫽ 3.3) for EC and NEC embryos respectively.
Each well contained 0.75 ml of Dulbecco’s modified Eagle’s medium
and Ham’s F-12 medium (1:1), supplemented with 2% Ultroser G
(all from Life Technologies), overlaid with 0.25 ml oil. Embryos
were inspected on day 7 for development to the blastocyst stage.
Only expanded, hatching or hatched blastocysts were recorded. All
incubations were performed at 37°C in a humidified environment of
5% CO
2
in air.
Statistical analysis
Statistical evaluations used were contingency table (χ
2
) analyses for
comparison of proportional values and two-sample Student’s t-test
for comparison of mean values.

Early cleavage and blastocyst formation
Table I. Comparison of IVF treatment cycle parameters for group A patients producing both early cleaving
(EC) and non-early cleaving (NEC) embryos and group B patients producing NEC embryos only
Parameter Group A Group B P
No. of cycles 32 38 –
Mean female age (years) ⫾ SD 32.2 ⫾ 4.0 32.8 ⫾ 3.5 NS
Stimulation (mean ampoules ⫾ SD) 39.4 ⫾ 9.2 39.6 ⫾ 7.3 NS
Mean follicles ⫾ SD 15.97 ⫾ 5.78 15.21 ⫾ 7.32 NS
Mean oocytes ⫾ SD 11.16 ⫾ 4.21 11.58 ⫾ 5.36 NS
Mean 2PN embryos ⫾ SD 8.72 ⫾ 3.59 7.89 ⫾ 3.60 NS
Clinical pregnancies (%) 10/32 (31.3) 4/38 (10.5) 0.05
Implantation rate (%) 15/70 (21.4) 5/83 (6.0) 0.005
PN ⫽ pronuclei; NS ⫽ not significant.
Table II. Proportion of early cleaving (EC) and non-early cleaving (NEC)
embryos developing to expanded or hatching blastocyst stage following
extended culture to day 7
EC NEC
Embryos cultured 59 367
Blastocyst (%) 19 (32.2)
a
61 (16.6)
a
Hatched (%) 11 (18.6)
b
26 (7.1)
b
Same letters indicate significant differences, χ
2
-test:
a
P ⬍ 0.01;
b
P ⬍ 0.005.
Results
Incidence of early cleaving in human embryos
A total of 579 zygotes from 70 couples were assessed for
cleavage at 25 h after insemination. Of these, 85 (14.7%)
were at the 2-cell stage (EC), and 494 (85.3%) were still at
the 1-cell stage (NEC). Thirty-two couples (group A, 46% of
patients) produced a mix of EC and NEC embryos while 38
couples (group B) produced only NEC embryos. There was
no difference between the two patient groups in female age,
ovarian response or fertilization rate (Table I). However, group
A patients had a significantly higher incidence of pregnancy
(31.3 versus 10.5%; P ⬍ 0.05) and implantation (21.4 versus
6%; P ⬍ 0.005) than group B patients. This is consistent with
findings in previous studies (Shoukir et al., 1997; Sakkas et al.,
1998), but as with these studies, our implantation data were
insufficient to distinguish between embryonic viability and
other aspects of fecundity. However, we were able to examine
the relationship between embryonic developmental potential
and timing of first cleavage by assessing blastocyst develop-
ment of EC and NEC embryos during culture in vitro.
Early cleaving embryos have a greater potential for blastocyst
formation
Embryos remaining (n ⫽ 426) after transfer were cultured
until day 7; 59 of these were EC and 367 were NEC embryos.
The proportion of EC embryos that developed to the blastocyst
stage by day 7 was 19/59 (32.2%); this was significantly
higher (P ⬍ 0.01) than in NEC embryos (61/367; 16.6%).
The proportions of hatched blastocysts were 18.6 and
7.1% for EC and NEC embryos respectively (P ⬍ 0.005;
Table II). Interestingly, the proportions of hatched blasto-
409
Table III. Proportion of early cleaving (EC) and non-early cleaving (NEC)
embryos developing to expanded or hatching blastocyst stage following
extended culture to day 7 in relation to patient group
Group A Group B
EC NEC NEC
Total no. of 19/59 (32.2)
a,b
27/150 (18.0)
b
34/217 (15.7)
a
blastocysts (%)
Hatching rate (%) 11/59 (18.6)
c
14/150 (9.3) 12/217 (5.5)
c
Same letters indicate significant differences, χ
2
-test:
a
P ⬍ 0.01;
b
P ⬍ 0.05;
c
P ⬍ 0.001.
cysts corresponded closely with the implantation rates for the
two patient groups.
We next asked whether the increased blastocyst formation
of EC embryos was truly a function of the timing of first
cleavage or merely a reflection of a generally superior develop-
mental potential among cohorts of embryos produced by group
A patients. We therefore analysed the incidence of blastocyst
formation and hatching of EC and NEC embryos, according
to whether they were obtained from group A or group B
patients (Table III). Within group A patients, the incidence of
blastocyst formation was significantly higher (P ⬍ 0.05) for
EC than for NEC embryos, and the NEC embryos of group A
patients showed a similar incidence of blastocyst formation to
those of group B patients. The proportions of EC and NEC
embryos from group A patients developing to the hatched
blastocyst stage were 18.6 and 9.3% respectively (borderline
significance: P ⫽ 0.06). While the proportion of NEC embryos
from group B patients developing to hatched blastocyst stage
was 5.5%, this was significantly lower than that of EC embryos
(P ⬍ 0.001) but not NEC embryos from group B patients
(Table III).
Early cleaving is associated with higher mean cell number
and better morphology on day 2
To investigate whether the greater potential for blastocyst
formation of EC embryos was evident at early stages of
development, we compared the grades assigned on day 2 with
the EC and NEC embryos that were subsequently cultured
until day 7, i.e. those embryos not selected for transfer. EC
embryos (n ⫽ 59) had significantly higher mean cell numbers

J.Fenwick et al.
Table IV. Mean cell number and morphology score on day 2 of early
cleaving (EC) and non-early cleaving (NEC) embryos cultured to the
blastocyst stage (day 7)
Group A Group B
EC NEC NEC
No. of embryos 59 150 217
Cell number 3.98 ⫾ 0.29
a,b
3.64 ⫾ 0.99
a,c
3.14 ⫾ 1.10
b,c
Morphology score 0.85 ⫾ 0.11
d,e
0.80 ⫾ 0.15
d
0.81 ⫾ 0.15
e
Results are expressed as means ⫾ SD.
Same letters indicate significant differences, two-sample t-test:
a,b,c
P ⬍ 0.001;
d,e
P ⬍ 0.01.
Table V. Proportions of early cleaving (EC) and non-early cleaving (NEC)
embryos available for transfer and selected for transfer to patients producing
a mix of both embryo types (group A patients)
EC NEC
Available embryos (n ⫽ 279) 85 (30.5) 194 (69.5)
Transferred embryos (n ⫽ 70) 26 (37.1) 44 (62.9)
P-value NS NS
Values in parentheses are percentages.
NS ⫽ not significant.
(P ⬍ 0.001) and mean morphology scores (P ⬍ 0.01) than
NEC embryos (n ⫽ 367). The improved quality of EC embryos
was irrespective of whether the NEC embryos were derived
from group A or group B patients, although NEC embryos
from group A patients had a significantly higher mean cell
number than NEC embryos from group B patients (Table IV).
There is no bias in favour of selection of early cleaving
embryos for transfer
The study protocol dictated that selection of embryos for
transfer was blind with respect to the timing of first cleavage.
On this basis, 5/32 patients in group A had only EC embryos
replaced, 14/32 had a mixture of EC and NEC replaced, and
13/32 had only NEC embryos replaced. Table V shows that
for group A patients, the prevalence of EC embryos in the
cohort of embryos available for transfer (n ⫽ 279) and in
the cohort of embryos transferred (n ⫽ 70) was not significantly
different, indicating that there was no bias in the selection
process. In accordance with this, analysis of mean cell number
and morphology score showed that among the population of
embryos selected for transfer, there was no difference between
EC and NEC embryos (Table VI).
Discussion
In agreement with the findings of others (Shoukir et al, 1997),
the data presented here show that couples who produced
embryos that cleaved within 25 h of insemination yielded
higher pregnancy and implantation rates than those who did
not. A correlation between early cleavage and implantation
was not established in previous studies (Shoukir et al., 1997;
Sakkas et al., 1998) for two main reasons. First, the number
410
Table VI. Mean cell number and morphology score on day 2 of early
cleaving (EC) and non-early cleaving (NEC) embryos selected for transfer
to group A patients
EC NEC P-value
No. transferred 26 44 –
Cell number 4.27 ⫾ 0.72 4.16 ⫾ 0.83 NS
Morphology score 0.92 ⫾ 0.07 0.89 ⫾ 0.09 NS
Results are expressed as mean ⫾ SD.
NS ⫽ not significant.
of cases in which only EC embryos were replaced was small;
second, the preferential replacement of EC embryos, when
available, prevented any possible comparison of the implanta-
tion potential of EC and NEC embryos of equivalent quality
on day 2. By relating the timing of first cleavage to blastocyst
formation and hatching, we have established that the develop-
mental competence of EC embryos in vitro was significantly
higher than their NEC counterparts. Importantly, this difference
was irrespective of whether the NEC embryos were from
patients producing a mix of EC and NEC embryos or NEC
embryos only. The improved development potential of EC
embryos in vitro was therefore a function of the timing of first
cleavage rather than a ‘patient specific effect’.
Our results also indicate that there is no natural bias in
favour of selecting EC embryos for transfer. Consistent with
this, we found that the best quality NEC embryos, i.e. those
selected for transfer, were morphologically indistinguishable
from the best quality EC embryos. However, this was not the
case among the embryos that were not selected for transfer.
Within this population, EC embryos had significantly higher
cell numbers and morphology scores than NEC embryos
regardless of whether they originated from group A or group
B patients. Although NEC embryos produced by group A
patients had significantly more cells than those of NEC embryos
produced by group B patients, this did not lead to improved
blastocyst formation.
A positive correlation between early onset of cleavage and
blastocyst formation has also been reported in mice (McLaren
and Bowman, 1973) and bovine embryos (Grisart et al., 1994;
Lonergan et al., 1999). In these studies, the blastocysts with
early cleavage were found to have more cells than their later
cleaving counterparts (McLaren and Bowman, 1973; Lonergan
et al., 1999). This was found to be attributable to the differences
in the timing of first cleavage rather than to differences in the
rate of progression of subsequent cell cycles (McLaren and
Bowman, 1973).
How might the timing of first cleavage be linked to
blastocyst formation? The transition from fertilized oocyte to
2-cell embryo relies upon a highly regulated sequence of cell
cycle events, which are initiated by sperm-induced, repetitive
transient increases in oocyte free calcium concentration (Kline
and Kline, 1992). There is evidence from studies in which
mammalian oocytes were activated by pulsatile electrical
stimulation that the dynamics of these calcium signals influence
(i) the time course of pronuclear formation (Vitullo and Ozil,
1992), which marks entry into G1 of the first cell cycle, (ii)

Early cleavage and blastocyst formation
the ability to undergo blastocyst formation (Ozil, 1990), and
(iii) implantation (Ozil and Huneau, 2001). It has also been
reported that the G2/M transition in the first cell cycle of
mouse zygotes is dependent upon a calcium-releasing activity
acquired by the pronuclei during fertilization or activation
(Kono et al., 1996). Thus, it could be hypothesized that
asynchrony between zygotes in the timing of first cleavage
and variability in their capacity to undergo blastocyst formation
may be due to differences in the ability of individual sperm
to stimulate calcium transients, and/or differences in the ability
of oocytes to respond to that stimulus. The finding that oocytes
acquire the ability to undergo repetitive calcium transients
during their maturation process (Carroll et al., 1994; Herbert
et al., 1997) suggests that oocyte maturity may be an important
determinant of the timing of first cleavage and subsequent
developmental potential.
A possible alternative or additional mechanistic link
between timing of first cleavage and blastocyst formation lies
in the fidelity of DNA replication. The duration of S phase of
the first cell cycle has been shown to influence blastocyst
formation. A longer S phase in association with a shorter G1
in the case of bovine embryos (Comizzoli et al., 2000), or
shorter G2 in the case of mouse embryos (Schabronath and
Gartner, 1988), gives rise to improved blastocyst formation.
The duration of S phase is paternally regulated (Schabronath
and Gartner, 1988; Comizzoli et al., 2000). However, regula-
tion of the timing of entry into S phase appears to differ
between species, being regulated by maternal factors in mice
(Schabronath and Gartner, 1988) and paternal factors in cattle,
in a manner that, in hamsters at least, is not dependent upon
sperm nuclear decondensation (Naish et al., 1987). It is
conceivable that zygotes with shorter S phases are predisposed
to incomplete or aberrant DNA replication. This is unlikely to
be compatible with normal development to the blastocyst stage
and could impose a delay in progression through G2 and M
phase of the first cell cycle by activating a DNA structure
checkpoint (Nigg, 2001), such as has been identified in mouse
zygotes (Fulka et al., 1999).
In conclusion, our analyses show that in humans, as in other
species (McLaren and Bowman, 1973; Grisart et al., 1994;
Lonergan et al., 1999), early onset of first cleavage is associated
with increased blastocyst formation. Given its ease of applica-
tion and lack of scope for subjectivity, early cleaving could
potentially be used as an additional marker of viability when
selecting embryos for transfer. This would be especially useful
in cases where numerous good quality embryos are produced
and/or the risk of multiple pregnancy is increased. However,
it will be important to establish whether the increased blastocyst
formation of EC embryos equates to increased implantation
potential. While blastocyst formation represents an important
milestone in embryonic development, it is not necessarily
synonymous with viability (Van Blerkom, 1997). Although
our data suggest that ability to develop to the hatched blastocyst
stage may correlate with implantation potential, an unequivocal
correlation between implantation potential and timing of first
cleavage awaits collection and analysis of sufficient homolog-
ous data from group A patients.
411
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Submitted on April 4, 2001; resubmitted on July 2, 2001;
accepted on October 12, 2001