Human Reproduction Vol.22, No.1 pp. 230–240, 2007
doi:10.1093/humrep/del358
Advance Access publication September 18, 2006.
Morphologic parameters of early cleavage-stage embryos
that correlate with fetal development and delivery:
prospective and applied data for increased pregnancy rates
L.Scott1, A.Finn, T.O’Leary, S.McLellan and J.Hill
The Fertility Centers of New England, Reading, MA, USA
1
To whom correspondence should be addressed at: The Fertility Centers of New England, 20 Pond Meadow Drive, Suite 205, Reading,
MA 01978, USA. E-mail: [email protected]
BACKGROUND: Many different embryo selection criteria have been used in assisted reproductive technologies
(ART), but there are no published prospective studies to ascertain their usefulness in predicting implantation.
METHODS: In a prospective trial, 20 early scoring parameters previously reported to influence clinical outcome
were collected, but embryos were selected for transfer by current laboratory protocols; day 1 pronuclear (PN) (Z)
score combined with day 3 or 5 morphology. Data points for each oocyte/embryo were scored independently and
tracked individually. Data were analysed retrospectively for parameters most likely to result in a positive pregnancy test, fetal heartbeat (FHB) and delivery. RESULTS: Results indicated that day 1 PN morphology and nucleolar precursor body (NPB) ratio, day 2 cell number, blastomere symmetry and nucleation and the ability to cleave
from day 2 to day 3 were the six most significant factors in fetal development. This outcome was then applied
prospectively over 8 months. The implantation rate (IR) and clinical pregnancy rate (CPR) increased in each age
group, and the number of embryos used decreased. CONCLUSION: In conclusion, early parameters that include
PN morphology, number and ratio of NPBs per nucleus and the day 2 morphology of cleaving embryos are stronger
positive predictors of implantation than day 3 morphology or the ability to achieve the blastocyst stage of development. Parameters that were most consistently correlated with no delivery were lack of PN symmetry, day 2 multinucleation and uneven cell size. Day 3 and day 5 parameters were not significant compared with the combination of
early parameters.
Key words: day 1 and day 2 scoring/delivery/early embryo morphology/implantation potential
Introduction
The goal of embryo selection in an assisted reproductive technologies (ART) programme is the successful delivery of a
healthy baby while minimizing multiple gestations. Many different embryo selection criteria have been proposed which
include day 1 pronuclear (PN) morphology, early first cleavage, day 2 morphology, especially the state of nucleation, day 3
morphology and rate of development on day 3 and finally
blastocyst development. All methods show adequate correlations with pregnancy although few studies relate scoring to
delivery (for reviews see Scott, 2002, 2003; Ebner et al., 2003;
Rienzi et al., 2005). There are no published prospective studies
where the efficacy of each or in combination is weighed
against each other to ascertain the relative importance of each
or which parameters are best used in gated scoring systems.
Oocyte morphology has not been widely applied in embryo
selection because it necessitates cumulus removal, which is
only done in ICSI cases. Firstly, the contribution of sperm
quality to embryo development has shown negative correlations,
thus embryo assessment after ICSI may negate any oocyte
parameters. Secondly, the number of oocytes that would need
to be scored would be vast, because it is the embryo that is
finally transferred.
The purpose of this study was to first prospectively gather
oocyte and embryo morphology data using conventional selection criteria in good prognosis patients. The relative contribution, both singularly and collectively, of these parameters was
retrospectively correlated with embryo development, selection
for transfer where selection criteria were limited to the laboratories’ current procedures (PN morphology and day 3 morphology), implantation, fetal heartbeat (FHB) and delivery.
Once the most important scoring parameters were elucidated,
further data were gathered in a more diverse group of patients
to validate the original prospective study. The final outcome
results were then applied over the entire programme to determine their usefulness in predicting successful implantation and
pregnancy outcome while reducing the number of embryos
used in transfer.
230 © The Author 2006. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved.
For Permissions, please email: [email protected]
Early embryo scoring is most significant for delivery
Materials and methods
Series 1: prospective data collection
Initially, 120 high-prognosis patients were identified as they entered
the programme. The first 30 patients from each of the four stimulation
protocols used at the time of study were included to eliminate the
influence of stimulation protocol from the outcome data, as stimulation could influence embryo morphology. The stimulation protocols
used were long cycle Lupron (L), short cycle Lupron (S), microdose
long cycle Lupron (M) and antagonist protocol (A). In all protocols,
patients were treated with a minimum of 21 days of oral contraceptives (OCPs) before starting a cycle. A dose of 1 mg or 10 IU twice
daily of Lupron was used for L and 0.5 mg or 10 IU subcutaneous for
microdose Lupron. A baseline ultrasound was performed on day 2 of
the cycle, and if the patient was ready they were started on FSH or
HMG (150–300 IU), the exact dose depending on body mass and age.
When the leading cohort of follicles reached 17–18 mm, 10 000 units of
hCG was administered and an oocyte retrieval performed 36 h later
under conscious sedation.
Series 1: patient inclusion criteria
The criteria were no previous pregnancy, first or second cycle only, no
age >37 years at the time of cycle initiation, no severe male factor
(<10 million motile sperm, <2% normal forms by strict criteria, no
sperm aspirations), no severe female pathology including endometriosis and polycystic ovavain syndrome (PCOS), no donor or PGD or
PGS cycles, no women with BMI >35 kg/m2 or day 2 FSH >12 μ/ml. All
other patients were included. The exclusion of severe male and female
pathology (PCOS, endometriosis and very poor sperm) was to avoid
these variants affecting the outcome data. Of the 120 patients who met
inclusion criteria, 53 had ICSI and 67 had standard insemination.
There was no difference between the scoring parameters and the pregnancy rates of ICSI versus standard insemination; thus, no further
breakdown of these groups is presented. No Institutional Review
Board protocol or informed consent was obtained, because no experimental procedures or techniques outside of the standard operating policies of the unit were employed. Patients were treated according to
physician’s orders, and all patients signed informed consent for the
use and the publication of data gathered during routine procedures.
Oocyte retrievals were performed at 36 h post-hCG and insemination or ICSI at 39–41 h post-hCG, and this day was designated day 0
(zero). Day 1 fertilization checks were at 17–18 h post insemination/
ICSI, day 2 checks at 42–44 h post insemination and day 3 scoring at
64–65 h post-insemination. Day 5 scoring was at 120 h post-insemination.
Timing for hCG, insemination and scoring was kept constant. Oocytes
post-ICSI and all embryos were cultured individually in numbered
drops, and so, they could be followed through all procedures. Each
patient was designated a sequential chronological number as they
began a cycle, and this, plus an embryo number, was used to track
each embryo in a database. It was therefore possible to track all
embryos at each time point and all that were transferred on days 2, 3
or 5. This system was employed routinely in the laboratory over the
entire study period for all retrievals (16 months).
Sperm were prepared using a single-step density gradient (Pureception: Sage BioPharma, NJ, USA) and re-suspended in Sage Q1 fertilization medium (Sage Biopharma) plus 10% human serum albumin
(HSA) (In Vitro Care, San Diego, Ca, USA). Oocytes were inseminated with ∼10 000 sperm in 50 μl drops of Q1 under oil (Sage) or 1
sperm/oocyte with ICSI. After either ICSI or at the time of PN identification, fertilized oocytes were moved to 7–10 μl drops of Q2 plus
10% HSA under oil. These drops were numbered, and this number
corresponded to the oocyte/embryo number. For day 5 culture,
embryos were moved to Q3, extended culture medium plus 10% HSA,
on the afternoon of day 3 of culture. All cleaving embryos not used in
day 2 or 3 transfer and those intended for day 5 transfer were moved
to extended culture. The drops were again numbered appropriately
with the designated embryo number. Good quality blastocysts not
used in transfer were cryopreserved on day 5.
Scoring was performed rapidly on heated stages, and no more than
6 oocytes and embryos were in each dish to facilitate rapid scoring.
Intra-observer variability was reduced with the use of video cameras
and by 2-person scoring on each embryo and analysis of data per
embryologist in the normal laboratory quality assurance programme.
Oocyte scoring
After hyaluronidase treatment, we scored all metaphase II oocytes for
the size and shape of the first polar body, the size of the perivitaline
space, the thickness of the zona pellucida and the texture of the cytoplasm. Polar body morphology was scored as normal, small, large or
fragmented/cleaved. Polar bodies that were indented or not fully
extruded were also noted. The perivitaline space was scored as normal, large (shrunken oocyte) or very small with the oocyte filling the
space completely. Zona pellucida thickness was thin, normal or thick
and/or amorphous. All parameters were subjectively scored, and no
micrometer or other measuring tool was used.
Embryo scoring parameters
Fertilized oocytes were scored for PN morphology using the designations of Scott (2002) (Scott et al., 2000) at 17–18 h post-insemination.
Fertilized oocytes with equal numbers of nucleolar precursor bodies
(NPBs) aligned on the PN junction were designated Z1, those with
equal numbers of NPBs that were scattered were designated Z2, those
with inequality of numbers or alignment were designated Z3 and those
with very unequal-sized PN or PN that were not aligned in a central
position within the oocyte were designated Z4. The presence or absence
of a halo and the alignment of the nuclei in relation to the polar bodies
were recorded (Scott et al., 2000). The state of the polar bodies was
scored as intact, highly fragmented, overly large, not fully extruded or
very far apart (>15°). Early cleavage checks were not performed.
On day 2, embryos were scored for blastomere number, the state of
nucleation: no nuclei visible, multinucleate or one nucleus per blastomere at 42–44 h post-insemination. Embryos with at least one multinucleate blastomere were designated multinucleate. The equality of
cell size was also recorded. Embryos in which there was no more than
a 20% difference in size/volume between the blastomeres were designated ‘even’, and those in which there was unequal blastomere sizes
were designated ‘uneven’.
On day 3, 64–65 h post-insemination, embryos were scored for cell
number, degree of fragmentation and cell size. They were scored on a
5-point scoring system as follows: Grade 1, 6–8 cell, correct cell size
[remembering that a 6-cell embryo has 2-cell sizes, Roux et al.,
(1995)], no fragmentation, no multinucleation visible; Grade 2, equivalent to grade 1 except with 10–15% fragmentation; Grade 3, <6 cell
but with no multinucleation, fragmentation and with correct cell size;
Grade 4, any cleavage stage with >20% fragmentation or multinucleation or very uneven, incorrect cell size and Grade 5, arrested, atretic or
totally fragmented.
Day 5 blastocysts were selected for transfer or for cryopreservation
if they had an adequate number of even-sized cells, visible inner cell
mass, an even trophectoderm with sufficient cells to form a continuous
cell layer, no atretic areas and no excluded cells.
Embryos were selected for transfer by the standard procedures in
the laboratory, which included both day 1 PN score (Z score) and day 3
embryo morphology or day 5 blastocyst morphology. Day 2 scoring
was not used in the laboratory at this stage, but the data were gathered
for retrospective analysis. A Z1, 8-cell grade 1 was the most desired.
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L.Scott et al.
The PN score was weighted over day 3 score if embryos were at least
6-cell and grade 3 and above. If embryos were not progressing, cleavage and development were used in preference to day 1 score. On day 5,
day 1 plus day 5 score was used unless there was limited blastocyst
development, in which case blastocyst morphology was weighted. An
hCG level was obtained on day 12 after transfer and a second one 48 h
later. A clinical pregnancy with active fetal activity (FHB) was confirmed with ultrasound at 6 and 8 weeks after transfer. Delivery was
confirmed verbally from the patient (date of birth, weight and sex of
infant) for series 1 and 2 patients.
Embryo morphology data were collected as discreet units, and no
attempt was made to analyse the sequence or the correlation of PN
morphology to day 2 morphology to day 3/5 morphology. The aim of
the study was to gather embryo morphometrics prospectively on all
embryos and retrospectively analyse each parameters’ contribution to
implantation, fetal development and delivery and weigh these against
each other to understand which of these singularly or in combination
was most predictive of delivery.
Data were analysed by analysis of variance (ANOVA) and Wilcox
Sign Rank for the contribution of the 20 factors to establishing a
pregnancy, FHB and a successful delivery.
Series 2: expanded prospective data collection
Once the significant morphometric parameters were elucidated from
the initial group of patients, embryo data were again prospectively
collected on a broader group of patients who entered the programme
over a 4-month period (January through April 2005). These data were
again analysed for contribution to a positive outcome event that is
delivery. In this group, the inclusion criteria were expanded to include
anyone <38 years of age, on treatment cycle 1–3, regardless of previous pregnancy, infertility diagnosis, semen characteristics, day 3 FSH
or BMI. Excluded were donor, PGD/PGS and surrogate cycles. By
expanding the inclusion criteria to include women and men with
pathologies (sperm and ovarian), the true impact of the embryo morphometrics on implantation and delivery could be realized. In this
group, a further parameter, the number of NPBs per nucleus, was also
collected. Alignment was still scored as before, and the lower number
of NPBs arbitrarily scored as ‘left’ and the higher as ‘right’. The data
were again analysed by ANOVA to elucidate the relative impact of
each collected data point on fetal development and delivery.
Series 3: application of morphometric parameters of early scoring
From May through December 2005, the data from series 1 and 2 were
prospectively applied to all included patients entering the programme
who met the expanded criteria of series 2. Here, embryos were prospectively selected by the six most significant parameters elucidated in the
initial studies (series 1 and 2). These included PN score, NPB ratio in the
nuclei, day 2 embryo state of nucleation, cell stage and evenness of cell
size and finally day 3/5-cell morphology. Days 1 and 2 were weighted
heavily over day 3 and 5 morphology, with a sequential system of Z1
with 5–7 NPBs, 4-cell with one nucleus per blastomere and even cell
size and at least a 6-cell on day 3 being an ideal embryo. Day 2 morphology was slightly weighted over day 1 unless this did not allow at least a
6-cell embryo to be selected. In this group, only FHB and implantation
are reported as the final outcome, delivery, is not yet available. However, extrapolation from pregnancy loss rates observed in series 1 and 2
allows overall estimations of the effectiveness of the method.
All data were calculated per embryo transfer and not per cycle start
or oocyte retrieval, because the morphometrics of the embryos and
their contribution to fetal development and delivery were the objectives of the study, not infertility aetiology, stimulation or other compounding factors. Once the embryo parameters are elucidated, the
impact of infertility pathologies, ovarian reserve, stimulation pattern,
232
age and other factors can be correlated with these morphometrics in an
attempt to obtain more embryos with the desired parameters.
Statistical analysis
Chi-squared and ANOVA (one- and two-way ANOVA), and Wilcox
Sign Rank were performed using StatView (for Mac). A multivariate
analysis (20 way) of all the embryo factors was performed utilizing
GraphPad (for Mac). All data were considered significant at a P value
of 0.01.
Results
Series 1: prospective study
One hundred and twenty patients, 30 from each stimulation
protocol, who started treatment and who met inclusion criteria,
were selected for the prospective study. Of these, all had
oocyte retrieval, six had all embryos frozen on day 1, PN stage
for clinical reasons (thin lining, hyperstimulation) with 114
patients proceeding to embryo transfer. There was no difference in the mean ages of the four stimulation groups (34.2 ±
1.8 years). Ninety-five women had day 3 and 19 day 5 embryo
transfer. There was no difference in pregnancy rates (day 3:
38% versus day 5: 44%, FHB; NS) between the 2 days of
embryo transfer; thus, the data were combined. A total of 59
patients had a positive pregnancy test (52%); 47 with a FHB
(41%) and 46 continuing until at least 12 weeks of gestation
(40%), and there were 43 successful deliveries (38% of embryo
transfer). A total of 256 embryos were transferred, for a mean
of 2.2 embryos per transfer. Overall, there were 70 fetal sacs
[(27% implantation rate (IR)] with 68 FHBs (26.7%) and 54
babies born (21% of embryos transferred). Of the embryos
transferred, 34 were involved in a 100% implantation and
delivery event (13%). The data are presented in Table 1.
The mean number of oocytes was different between stimulation groups, L: 16; S: 12 (P < 0.05); F: 16; A: 8.5 (P < 0.05),
which reflects the use of A on patients who are likely to be
poor responders due to previous intrauterine insemination
cycle history or lowered antral follicle count. However, the
percentage of mature oocytes (80%) and fertilization rates of
mature oocytes (±72%) was the same in all groups. There was
no difference in pregnancy rate between standard insemination
and ICSI (45 versus 37% FHB, P = 0.34, NS). There was also
no difference in pregnancy rate or FHB events between any
infertility diagnoses, days of stimulation or day, of embryo
Table I. Clinical data from the prospective trial of 120 patients on cycle 1 or 2
meeting inclusion criteria: series 1
Cycles
Transfers
Clinical pregnancies (FHB)
Deliveries
Number of embryos (ET)
Mean number of ET
FHBs on ultrasound
Delivered babies (%)
<35 years
35–37 years
Total
87
84
40 (48%)
37 (44%)
181
2.1
59 (32.5%)
46 (25%)
33
30
7 (23%)
5 (17%)
75
2.4
9 (12%)
8 (11%)
120
114
47 (40%)
42 (37%)
256
2.2
68 (27%)
54a (21%)
FHB, Fetal heartbeat.
1 triplet, 15 twin, 21 singleton deliveries; no abnormalities.
a
Early embryo scoring is most significant for delivery
transfer. There was a difference in pregnancy rate between the
stimulation protocols with L having a high twin rate (47%),
which resulted in a 40% IR and an increased delivery rate
(55%: 16/29; P < 0.01). The other stimulation protocols gave
similar delivery and IRs (S: 38 and 20%; M: 30 and 28% and
A: 28 and 24%). However, there was no difference in the
number of good grade embryos on day 1 (Z1 + Z2: mean 69%)
or on day 3 or 5 between the different stimulation protocols in
this selected group of patients; thus, for the morphometric
analysis, all embryo data were combined.
Oocyte morphology
A total 1714 oocytes were retrieved of which 596 (35%) were
stripped for ICSI. There was no impact of the recorded oocyte
morphologies on fertilization, embryo morphology or development, selection for embryo transfer or subsequent implantation. The only significant features of oocyte morphology were
very aberrant polar body and cytoplasmic morphology. Polar
bodies that were very large, indented or so fragmented that it
was hard to assign exact PB location, resulted in embryos that
arrested in development, were commonly multinucleate on day 2
and were never selected for transfer. The same was true of
oocytes that had deep central pits, almost vacuoles, in the cytoplasm. However, these morphologies accounted for ∼5% of all
oocytes, and, as such, the data were not significant when the
embryo morphometrics and contribution to fetal development
were concerned. However, it is appreciated that oocyte morphology is important as poor oocytes cannot produce embryos
with high potential. Further studies on oocyte morphology and
their contribution to embryo development are needed in a
much larger and wider group of patients than presented here.
Fertilized oocyte morphology
Of the 1714 oocytes retrieved, 1394 were mature (81%) with
1193 (85%) fertilized normally (2 PN) by a combination of
ICSI and standard insemination. Overall, PN scores were not
different to those previously reported (Scott et al., 2000) with
an equitable distribution between Z1: 37%, Z2: 32%, Z3: 26%
and Z4: 4%. There was also an equal distribution of embryos
showing a halo (52%) and no halo (48%) and with PN alignment (49 versus 51%, aligned versus non-aligned). Using
ANOVA (2 way ANOVA), neither the presence nor the
absence of a halo, or the nuclei alignment, impacted early
embryo development, the 6- to 8-cell or blastocyst stage of
development or implantation after embryo transfer. Thus, no
further data are presented on these two parameters.
There was a bias in the selection of embryos for embryo
transfer, with 46 and 38% of embryos scored Z1 or Z2, and
only 16% that were Z3 (41) and no Z4 used in transfer (256
embryos transferred). However, of these only 15% that were
Z3 (6) were involved in a positive pregnancy event, 7% (3) in a
transfer that resulted in a FHB event and none in a delivery
event. Of the embryos that were scored Z3, many used in
embryo transfer were involved in a negative pregnancy test
with all 54 babies successfully delivered originating from Z1
or Z2 fertilized oocytes (Figure 1).
Day 2 embryo morphology
Day 2 morphology was not used prospectively for embryo
selection but proved to be very important in retrospective data
analysis. Of the 1193 fertilized oocytes scored, 58 were frozen
as on day 1 (two PN) (5%) and 59 either fragmented or did not
cleave (5%) leaving 1076 embryos for continued development
and scoring. Data were analysed for all embryos, those used in
embryo transfer, those contributing to a positive or negative
pregnancy test or FHB and delivery event. It was found that the
data were highly significant by using 2-way ANOVA and multivariate analysis.
The data for the presence or absence of nuclei and multinucleation in all embryo groups are shown in Figure 2. Overall,
there was an equal spread of embryos in each nucleation state,
with a significant (P < 0.01) increase in one nucleus per blastomere in the embryos selected for embryo transfer (based on
day 1 and three parameters) and positive pregnancy test and
FHB events. Only embryos with one nucleus per blastomere,
or those in which nuclei were not visible, resulted in a successful delivery. No embryos presenting with multinucleation, and
60
P<0.01
50
P<0.01
P<0.01
P<0.001
Z1
Z2
Z3
Z4
40
30
20
10
0
Total
Embryos:
P a t ien t s :
11 9 3
1 20
ET
256
114
Negative
13 2
55
Positive
FHB
12 4
59
68
47
All Implant
34
17
Delivered
54
43
Figure 1. Pronuclear (PN) score. The percentage of each PN score for 1193 fertilized oocytes depending on their fate: series 1.
233
L.Scott et al.
120
P<0.001
100
1n/b
nnv
MN
P<0.001
80
P<0.01
60
P<0.01
40
20
0
All
Embryos:
P a tien t s :
1 0 76
114
ET
2 56
11 4
Negative
Positive
132
55
1 24
59
FHB
68
47
All Implant
34
17
Delivered
54
43
Figure 2. Day 2 state of nucleation. The percentage of each nucleation score for 1076 day 2 embryos depending on their fate: series 1. 1n/b, 1 nucleus
per blastomere; nnv, nuclei not visible and MN, multinucleation.
120
P<0.001
100
80
P<0.001
P<0.001
Even
Uneven
P<0.01
60
40
20
0
Embryos:
Pa tien ts :
All
ET
1076
114
256
11 4
Negative
132
55
Positive
124
59
FHB
68
47
All Implant Delivered
34
17
54
43
Figure 3. Day 2 cell symmetry. The percentage of even or uneven blastomeres from 1076 day 2 embryos depending on their fate: series 1.
which were selected for embryo transfer based on their day 1
and day 3 or 5 parameters, resulted in a successful delivery.
There was an equal distribution of even and uneven sized
blastomeres in all embryos (47 versus 53%; Figure 3), with an
equal number used in embryo transfer and for those involved in
a negative pregnancy event (43 versus 57%). However, there
was a significant increase of even cell size in embryos selected
for embryo transfer and which resulted in a positive pregnancy
event (71 versus 29%; P < 0.001, ANOVA) and a greater significance when FHB was involved (93 versus 7%, P<0.001).
Embryos with uneven cell size that did implant resulted in a
pregnancy loss. Deliveries were only from embryos that had
even cell sizes.
The cell number of embryos on day 2, at 42 h post-insemination, was also significant (even when not used as a selection
criterion). The data are presented in Figure 4. Most significant
was that both 2-cell and 4-cell embryos contributed equally to
positive pregnancy events, whereas 3-cell and >4-cell embryos
did not (P < 0.001, ANOVA). When FHBs, 100% implantation
and delivery events were considered, the majority of embryos
234
selected for embryo transfer were at the 4-cell stage on day 2 at 42
h post-insemination (P < 0.001; ANOVA).
Day 3 and 5 morphology and embryo transfer
It was found that, on day 3 or 5, cell number and fragmentation
pattern or blastocyst morphology were not significant for outcome (data not shown), by using the multivariate analysis
when including day 1 and day 2 morphology. However, the
data were biased as ≥6-cell embryos with minimal fragmentation and which originated from a Z1 or Z2 fertilized oocyte
were preferentially selected. Blastocysts were selected first by
day 1 and day 3 morphology and then by blastocyst morphology (see Materials and methods). It must also be realized that
the patients included in this study were likely to have at least 2
good grade embryos on day 3 as they were high-prognosis
patients, and thus again, the data are biased for these selection
criteria.
Sixteen pregnancy losses occurred in this population of
patients [70 sacs, 68 FHBs and 54 (Table I) babies]. All
Early embryo scoring is most significant for delivery
80
P<0.001
70
P<0.01
P<0.001
P<0.01
60
2
3
4
>4
50
40
30
20
10
0
Embryos:
P a t i en t s :
All
1076
1 14
ET
256
1 14
Negative
13 2
55
Positive
124
59
FHB
68
47
All Implant
34
17
Delivered
54
43
Figure 4. Day 2 cell number. The percentage of each cell stage for 1076 day 2 embryos depending on their fate: series 1.
embryos from this group were transferred as either 6- to 8-cell
embryos or blastocysts; yet, 5 (from sac group) were Z3, 11
(from sac group) were multinucleated on day 2 and 3 FHB
losses occurred after embryo transfer of good grade day 3
embryos developing from Z3 day 1 embryos. The balance of
the losses originated from multinucleated and uneven day 2
embryos. These data imply that selection using day 3 morphology criteria might contribute to implantation but that day 1 and
day 2 morphologies have more weight in outcome.
Table II. Clinical data from all cycles 1–3, January–April 2005, which
includes wider inclusion criteria of patients: series 2
Cycles
Transfers
Clinical pregnancies (FHB)
Deliveries
Number of embryos (ET)
Mean number of ET
FHBs on ultrasound
Delivered babies (%)
<35 years
35–37 years
Total
119
112
50 (45%)
46 (41%)
232
2.1
73 (31.5%)
72 (31%)
47
43
12 (28%)
12 (28%)
98
2.3
15 (15%)
15 (15%)
166
155
62 (40%)
58 (37%)
330
2.2
88 (27%)
87 (26%)
Series 2: extended prospective data collection
A total of 166 of patients met the criteria (<38 years of age, on
cycles 1–3, regardless of previous pregnancy, infertility diagnosis, semen characteristics, day 3 FSH or BMI) and were
included in the data collection and analysis. As series 1 data
showed no difference in embryo morphometrics between stimulation protocols, these data were not accounted for in series 2.
Of these, 155 proceeded to embryo transfer with 11 having no
embryo transfer due to failed fertilization (3), thin
endometrium (5) and a day 3 freeze of all embryos due to
fibroids or a polyp that prevented embryo transfer (3). Within
this group, a further parameter was collected, the ratio of NPBs
in each nucleus at the PN stage.
A total of 2263 oocytes were retrieved, of which 1837 (81%)
were mature and 1335 (73%) were fertilized normally and
scored for development. Of these, 330 were used in embryo
transfer (mean number per embryo transfer, 2.2). Overall, the
data for early parameters were not different to that collected in
series 1, and the clinical outcomes based on early embryo morphometrics were also not different. Day 1 and day 2 morphology were again shown to be most helpful in selecting an
embryo resulting in a successful delivery. The clinical outcome
data are shown in Table II.
Of the 330 embryos used in transfer, 88 resulted in a FHB
and 87 were live born (26%). Of the embryos used in transfer,
54 (16%) were Z3 and the remaining Z1 or Z2. A total of 43
embryos had definitive implantation (number of embryo
transfer = number of babies) and resulted in a successful delivery
Table III. Nucleolar precursor body (NPB) numbers and ratio in the two
nuclei in fertilized oocytes according to fate: series 2
NPB
All
ET
Negative Positive Fetal
Percentage Delivered
heartbeat
L/R mean
L/R range
Ratio
P
4/9
1–15
0.44
NS
5/8
2–10
0.63
NS
4/9
2–10
0.44
NS
5/7
4–9
0.71
0.05
6/7
5–9
0.86
0.01
6/7
5–7
0.86
0.01
6/7
6–7
0.86
0.01
in 16 women. For Z score, 34 (79%) were Z1/Z2 and 9 (20%) Z3.
Of these, six were Z3-1, Z3-3 and Z3-4. Five non-Z1/2 embryos
were transferred on days 2 and four on day 3. All Z1/Z2
embryos had a NPB ratio that was ∼1, with three having four to
six NPBs per nucleus and four with greater than eight (Z3-4).
All embryos that resulted in a live birth had even or notrecorded (n = 3) cell size, and only 1 of 43 had multi-nucleation
in one blastomere. The remaining had one nucleus per blastomere or no nuclei visible. This was significant (P < 0.001).
There was no significance of cell number on day 3 in this group.
When the NPB ratio per nucleus was analysed by both
chi square and ANOVA, the ratio was significant (P < 0.01;
Table III). Embryos that had six or seven NPBs per nucleus
were more likely to implant the ones that had more or less, or
ratios that deviated from 1.0 by more than 0.3. When this
parameter was combined with the other aspects of PN and day 2
235
L.Scott et al.
Table IV. Combined embryo outcome data for series 1 and 2 showing the percentage of each morphology type that contributes to fetal heartbeat and delivery
Number of ET
Negative
Positive
Fetal heartbeat
Delivered
% of embryos deliveredc
n
Z1 + Z2 (%)
Z3 (%)
MN (%)
NNV (%)
1N/B (%)
2 cell (%)
4 cell (%)
Other (%)
Even (%)
Uneven (%)
586
300
286
156
141
493 (84)a
252 (51)b
241 (48)
140 (28)
132 (27)
94
93 (16)a
48 (52)
45 (48)
16 (17)
9 (10)
6
111 (19)a
96 (86)
15 (14)
5 (5)
0 (0)
0
186 (32)a
78 (42)
108 (58)
25 (13)
16 (9)
11
289 (49)a
126 (44)
163 (56)
126 (43.5)
125 (43)
89
180 (31)a
106 (59)
69 (38)
37 (20.5)
36 (20)
25
325 (55)a
55 (17)
187 (58)
98 (30)
97 (30)
69
81 (14)a
68 (83)
30 (37)
21 (25)
8 (10)
6
317 (54)a
156 (49)
225 (71)
151 (48)
141 (44)
100
269 (46)a
144 (53)
61 (23)
5 (2)
0 (0)
0
MN, multi nucleation; NNV, nucleus not visible; 1N/B, one nucleus per blastomere.
a
Percentage of each morphological type of those transferred.
b
These percentages represent the proportion of each morphology type that contributes to a negative or positive pregnancy test, a fetal heartbeat or delivery event.
They are the percentage of what was transferred with the particular morphology, not of all that were transferred.
c
The final percentage is the contribution of each morphology type to delivery.
scoring, NPB ratio was proved to be significant in contributing
to a FHB and delivery (P < 0.01; multivariate analysis).
These data suggest that early morphometrics has a significant
impact on embryo implantation and delivery rates and demonstrated more significance than day 3 or day 5 development in a
larger/expanded group of patients. When ranked for significance
on multivariate analysis, day 2 state of multinucleation and evenness of cell size ranked at P < 0.001 and PN morphology and NPB
ratio at a P < 0.01 level. Day 3 or 5 morphology ranked at P < 0.05.
Combining all embryo transfer data from series 1 and 2
(Table IV), the significance of each of the early morphometric
parameters can be seen. Although there was a bias in the selection of Z1 and Z2 embryos for embryo transfer, only 10% of Z3
embryos selected contributed to a delivery event whereas 27%
of Z1 and Z2 embryos did (P < 0.01). The higher significance of
the day 2 morphologies is well demonstrated in the table. It can
be seen that multinucleation clearly impacted day 3 development as only 19% of transferred embryos were multinucleated
on day 2 (when this parameter was not used in embryo selection), but of those embryos, none (0%) contributed to a delivery.
Again, it is clear that 4-cell embryos on day 2 are more likely to
be good grade embryos on day 3 (55% selected for embryo
transfer) and that 3-cell and greater than 4-cell embryos have
poor developmental potential. The same is true for even/uneven
embryos. No uneven embryos contributed to a delivery event.
Series 3: application of early morphometrics
From May through December 2005, embryo data were again
prospectively collected for all patients who were on cycles 1–3
and <38 years, regardless of infertility diagnosis, BMI or FSH.
The embryos were prospectively selected for transfer using the
six most significant early parameters in a sequential/gated system:
PN score, NPB ratio, day 2 size, nucleation and cell number
and then by day 3 or 5 morphology. All data were computed
per embryo, and the clinical data followed for the embryos
used in transfer. The clinical results are presented in Table V.
A total of 330 patients met the criteria, 318 had oocyte retrievals and 299 had a transfer and were included in the data set. Of the
19 patients not having an embryo transfer, 4 had failed fertilization
and 13 had all embryos frozen due to polyps/fibroids at the time of
embryo transfer (5), hyperstimulation (4), thin endometrium at
embryo transfer (4) and 2 for social reasons.
There was an increase in both IR and CPR when compared
with series 2 (IR: 27 versus 30% and CPR: 40 versus 44%;
236
Table V. Results of the application of day 1 and 2 morphometrics for embryo
selection in all cycles 1–3, May–December 2005 (delivery data not available):
series 3
Cycles
Transfers
Clinical pregnancies (FHB)
Number of embryos (ET)
Mean number of ET
FHBs on ultrasound
<35 years
35–37 years
Total
210
199
94 (47%)*
398
2.0
135 (34%)
108
100
38 (38%)**
213
2.1
47 (22%)***
318
299
132 (44%)*
611
2.05
182 (30%)*
*P < 0.05 compared with series 2.
**P < 0.01 compared with series 2.
***P < 0.001 compared with series 2.
P < 0.05). The most significant increase was in the 35- to 37year-old group for IR (12 versus 22%, P < 0.001) and CPR (23
versus 38%, P < 0.01). There was little increase in IR or CPR
in younger women (<35), which can be explained by the fact
that in these women the majority of embryos used in embryo
transfer had the desired parameters required for selection in all
series. Furthermore, most good grade day 3 and 5 embryos did
originate from good grade day 1 and day 2 embryos in these
patients.
Applying the early selection criteria, we were able to reduce
the number of embryos used in transfer and found that implantation and fetal development increased. In series 1, there was 1
set of triplets (2%), 15 twins (36%) and 21 singleton (62%)
deliveries. In series 2, there were 3 sets of triplets of which 2
were monozygotic (2 embryos embryo transfer), 19 twins
(31%) and 40 singleton deliveries (64.5%). Deliveries for
series 3 were not available. However, of the 132 patients discharged at 8 weeks of gestation with a FHB, 4 patients were
discharged with triplets (4/132, 3%, all third cycle patients, 3
embryos used in transfer per unit policy) and 44 with twins
(33%). The remaining (84, 64%) was singleton. In series 3, 9
elective single embryo transfers were performed on day 3 or 5
based on the early selection criteria resulting in 5 singleton
FHBs at 8 weeks of gestation (55.5%). The mean number of
embryos transferred in this series was 2.05 (a downward trend).
Discussion
In this study, the most common early, and many of the current
later, points of embryo scoring were correlated with implantation
Early embryo scoring is most significant for delivery
and delivery in a combined prospective trial. The data showed
that early scoring is highly efficacious and correlates strongly
with fetal development and delivery, more so than later scoring
parameters, both retrospectively and prospectively.
Preimplantation morphologic parameters are the standard
criteria used in embryology laboratories as a means of selecting
the assumed most viable embryos for transfer. Many different
selection criteria and systems exist, all of which have been
reported to have correlations with implantation and fetal development. However, none are totally definitive and none can
offer anything greater than a 30–40% IR in high-prognosis
patients. This is the first study that brings together all the current scoring techniques in a single prospective trial as a means
of testing their efficacy to the final outcome, delivery. The
most significant data elucidated were that early scoring on days
1 and 2, were the key to fetal development and delivery of a
health infant and should be used in a sequential or gated system
for embryo selection, with weighting for day 2 and then day 1
parameters. However, certain features of day 1 (NPB ratio
deviations from 1 and asymmetry) and day 2 embryos (multinucleation and cell unevenness) were not consistent with delivery,
even if day 3 or day 5 scoring was good, or a positive pregnancy test or even FHB was recorded.
Oocyte morphology was not found to be a meaningful
parameter because an embryo selection criterion in the initial
group of patients was used for this data collection, in contrast
to previously published data (Hill et al., 1989; Ebner et al.,
1999; Ebner et al., 2000; Gabrielsen et al., 2001). This outcome is not unexpected because this group of patients were
selected for high probability of positive outcome and were
meant to have the lowest incidence of ovarian, oocyte and
male/sperm pathogenesis. The reasoning for this selection was
that gametes are the key to any ART treatment success, and to
evaluate embryo morphometrics that contributes to outcome,
gamete pathologies must be excluded, because no in vitro technique can transform or correct a fundamentally flawed gamete
and produce a viable embryo. This is true of both oocyte and
sperm, hence the exclusion of severe female pathology and
male factor from the initial series of patients. Furthermore, the
end-point of delivery would be hard to correlate with any one
oocyte factor as it would require a vast number of scored
oocytes. A more effective initial method of elucidating the
contribution of oocyte morphology to outcome would be to
correlate oocyte morphometrics with the six key parameters
indicated in this study.
The only oocyte parameters that were significant as a negative indicator of continued development in this group of
patients were large or major polar body abnormalities and
severe cytoplasmic abnormalities. Large, small, indented and
fragmented polar bodies have all been associated with spindle
abnormalities, non-disjunction events and aged oocytes (Ortiz
et al., 1983; Hill et al., 1989; Van Blerkom, 1990; Maro and
Verlhac, 2002; Scott, 2002, 2003). Oocytes with central pits
and vacuoles have also been associated with high estradiol,
post-maturity and over-stimulation (Hill et al., 1989). However, these abnormalities were rare in the initial high-prognosis
group and in the entire study group, accounting for ∼10% of all
oocytes, which makes it not significant. In some patients, the
prevalence was higher, which may explain the aetiology of
their infertility; however, there was no pattern to this observation.
If ICSI patients do present with a large number of abnormal
oocyte morphologies, combined male–female factor infertility
factors should be considered.
On day 1, the PN morphology was shown to be significant.
Alignment of NPBs on the first mitotic spindle (Z1) has been
correlated with both continued development and outcome
(Sadowy et al., 1998; Scott and Smith, 1998; Garello et al.,
1999; Tesarik and Greco, 1999; Ludwig et al., 2000; Scott
et al., 2000; Wittemer et al., 2000; Balaban et al., 2001; Kahraman
et al., 2002; Rossi-Ferragut et al., 2003; Zollner et al., 2003;
Borini A, 2005; Senn et al., 2005; Chen and Kattera, 2006).
This was again validated with these data, with the majority of
embryos used in transfer and resulting in a fetal development
and delivery originating from Z1 fertilized oocytes. Although
the data were biased for selection, only 48% of embryos used
were Z1, with 25% Z2 and the remaining, 27%, Z3 over all
series. Of the Z3 embryos that resulted in a live offspring
(series 1 and 2), all were either Z3-1, where there is equality in
numbers but not in alignment or where there were multiple
small equally distributed NPBs (Z3-4), or Z3-3. Fertilized
oocytes that present with a score of Z3-1 correlate with the
original scoring of ‘2’ (Scott and Smith, 1998) where it was
assumed that alignment would occur but was slightly delayed.
If one considers a 3D ball, and the alignment of NPBs on the
spindle, it may also be that Z3-1 embryos are actually becoming aligned, as the spindle may be in a plane that is not easy to
assign. In Z3-4 fertilized oocytes, many small, scattered NPBs
may represent slow and not necessarily aberrant development,
and in some cases can result in viable offspring.
Another significant factor, presented for the first time in this
data, is that the ratio of NPBs per nucleus is also very important for continued viability. This has to be coupled with alignment. The significance of NPB ratio makes biological sense,
because in PN scoring, it is the NPBs that are being counted
and scored. These are the functional centres of what will ultimately become the nucleoli (Schwarzacher and Mosgoeller,
2000; Scott, 2003), once they have reformed. Nucleoli are the
protein powerhouses of any cell and are the site of rRNA production. They are also responsible for the production of some
growth factors and developmental regulatory proteins (Pedersen,
1998). Nucleoli develop on DNA at sites where the genes for
ribosomal RNA are located (rDNA), at areas known as nucleolar organizing regions (NORS) (Goessens, 1984). Once transcribed, the rDNA results in three functional components that
are assembled into the nucleolus: the dense fibrillar component
(DFC) that is required for transcription, the fibrillar component
(FC) that is surrounded by the DFC and that stores inactive
transcription factors and is the centre of the nucleolus and the
granular or cytoplasmic component (GC) (Goessens, 1984;
Schwarzacher and Mosgoeller, 2000). The FC region is what is
observed during PN scoring as the NPBs. Human cells,
including embryos and sperm, have between two and seven
nucleoli per cell which dissociate into their component parts
during mitosis (cells, sperm) and in the final maturation of the
oocyte, as the chromatin disperses. When chromatin begins to
condense onto the mitotic spindle, the NPBs will be seen to
237
L.Scott et al.
also condense in this plane. If the NPBs are not evenly distributed,
or there are unequal numbers or random location in the nuclei
relevant to the mitotic spindle, it should follow that the cell
cycle, karyo- and cyto-kinesis will be abnormal, which will
lead to an abnormal embryo with little developmental potential. To extrapolate, chromatin needs to condense into the chromosomes, which need to line up on the spindle in a set time
frame, dictated by the LH surge or hCG injection in order for
normal development to proceed (Eppig, 1990; Albertini et al.,
2001). Analysis of the FC regions of the disassociated nucleoli
(NPBs) provides a glimpse into the condensation and the
organization of chromatin onto the first mitotic spindle. Lack
of organization or synchrony between the nuclei is most likely
responsible for the poor development of these embryos.
In addition to the current data, in which the PN score was
significant in a prospective data collection series and delivery
was recorded from tacked embryos, a large body of literature
has shown the correlation of outcome with PN score. It has
also been demonstrated that the incidence of chromosomally
normal embryos is highest with equal-sized nuclei and equal
numbers of NPB that are aligned (Kahraman et al., 2002; Chen
et al., 2003; Gamiz et al., 2003; Gianaroli et al., 2003; Edirisinghe
et al., 2005). The data of Gamiz et al. (2003) showed a collation
with age, the PN score positively correlating with chromosomal normality only in patients <37 years of age. Gianaroli
et al. (2003) did not break the data down by age but showed a
complex correlation with both the NPB numbers and their
alignment and with nuclei position/rotation relating to the polar
bodies. They concluded that PN scoring enables the selection
of embryos with both the greatest potential to develop to the
blastocyst stage and, in patients <37 years, the lowest incidence of aneuploidy, which the data presented here would also
suggest. All of these data indicate that PN score can aid in
embryo selection, especially in countries where limited embryo
culture is allowed. The addition of NPB ratio will allow yet a
further selection point for increased implantation success.
A highly relevant criterion that emerged from the data in this
prospective study was the impact of day 2 morphometrics on
developmental potential. Day 2 scoring is not a new concept
and has been shown to increase IR and pregnancy rate, regardless of day of transfer. The data from this study were gathered
prospectively and the scores were not used in embryo selection
which are different from previous studies. However, they were
highly significant when outcome was retrospectively considered.
Considering cell number at a set time (42–44 h post-hCG), it
is interesting that even (two and four cells) rather than uneven
(three, five and greater) cell numbers were significant in positive outcome. The transition from 2-cell to 4-cell requires that
the embryo proceeds through a 3-cell phase first and then rapidly cleaves again to form a 4-cell tetrahedron embryo (Roux et
al., 1995; Hiiragi and Solter, 2005; Gardner and Davies, 2006). If
an embryo is an even-sized 3-cell, or a 3-cell that is not progressing fast to a 4-cell, it is most likely abnormal or has
cytokinetic delays. This is borne out from the data (series 1 and
2) in which no embryo scored as 3-cell on day 2 resulted in a
successful delivery. If an embryo is cleaving too fast and is 5cell and greater at a set time point, relevant to all other embryos,
it is also most likely abnormal (Racowsky et al., 2000). The most
238
important point when assessing embryo evaluations is time of
scoring, because embryos move rapidly and even a 4–5 h
difference in scoring could affect the data significantly.
The evenness of cell size on day 2 was also highly significant. This is again not a unique observation (Hardarson et al.,
2001; Van Royen et al., 2001; Hnida et al., 2004), but this is
the first report where this parameter was assessed prospectively and independently of transfer. The concept of oocyte and
embryo polarity has been gaining credence in the last few
years, although the view of when true determination through
polarity is established is still in question (Edwards and Beard,
1997; Gardner, 1997; Hiiragi and Solter, 2005; Gardner and
Davies, 2006). What is clear from this data is that if the fertilized oocyte does not follow a very strict equal division process
that can distribute the cytoplasmic content of the cells at the
first and second mitotic division equally to the resulting daughter cells (equal to even versus uneven cell size), the embryo has
little developmental capacity. This division could be seen as an
elementary form of polarity and of cellular-content distribution. In both series 1 and 2, no deliveries were recorded when
transferred embryos presented with unequal cell size on day 2,
regardless of day 1, 3 or 5 morphology. In the applied series,
only 12 of 132 clinical pregnancies (9%) were from the transfer of at least one embryo with uneven cell size on day 2 (26
embryos, 15 with uneven cell size). Nine of these pregnancies,
at 8 weeks of gestation, are singleton and three are twin, where
two are from the transfer of one equal and one unequal and the
third from the transfer of two unequal-sized embryos on day 2.
The definitive data will come with delivery. Clearly, this is a
highly significant scoring parameter that has biological significance and should be incorporated into any gated/sequential
embryo selection system.
Blastomere cell size has been well studied both by computercontrolled multilevel morphometric analysis (Hnida et al.,
2004) and by conventional microscopy (Hardarson et al., 2001).
Cell size was also linked to fragmentation (Hnida et al., 2004)
where the degree of fragmentation was correlated with the
extent of unevenness of blastomeres. This was not a phenomenon noted here, where on day 2 uneven cell size appeared to be
more inherent in the embryo and related to cleavage rather than
to fragmentation. There are also reports of increased aneuploidy rates with uneven-sized blastomeres, which can also be
linked to decreased implantation and developmental potential
(Hardarson et al., 2001).
Day 2 state of nucleation was another parameter that proved
to be highly significant in terms of fetal development and
delivery. There are many reports on this subject (Pickering
et al., 1995; Pelinck et al., 1998; Hardarson et al., 2001; Van
Royen et al., 2003; Hnida et al., 2004; Meriano et al., 2004;
Moriwaki et al., 2004; Ciray et al., 2005), most retrospective in
nature and showing very high correlations with positive outcome. An early report (Pickering et al., 1995) indicated that two
mechanisms might be functioning to produce multi-nucleated
blastomeres in embryos proceeding through the second and
third mitotic event. They disaggregated embryos and allowed
the blastomeres to cleave in culture. Some of the blastomeres
went through karyokinesis without cytokynesis, accounting for
about 30% of multinucleated blastomeres. The remainder were
Early embryo scoring is most significant for delivery
multinucleated by means most likely dictated by errors in
chromosome segregation or other mitotic errors. However, this
does not explain multi-nucleation occurring during the first
mitotic event, leading to the 2-cell stage. It is more likely that a
chromosome segregation error and/or mitotic error is responsible in the first cleavage. The data of Kligman et al. (1996),
where >70% of embryos displaying multi-nucleation had aneuploidy when analysed by FISH, would support this hypothesis.
The prospective data of Meriano et al. (2004) showed that
binucleated and micronucleated embryos had different implantation potential, with embryos displaying micronucleated/
multinucleated blastomeres having little developmental potential, as did that of Ciray et al. (2005). What is clear from the
current prospective data and the published literature is that if an
embryo presents on day 2 with multi-nucleation in any blastomere its developmental potential is highly compromised and
it is unlikely to contribute to a delivery. This fact should be
used as a selection criterion regardless of day 3 or 5 morphology, to avoid aneuploidy and increase implantation potential,
and indicates that a screen on day 2 can increase selection ability
with little extra effort or harm to the embryo.
In conclusion, in a trial in which embryo morphometrics was
gathered and applied prospectively, over a 16-month period,
and analysed retrospectively in a multivariate analysis, the six
most significant embryonic factors contributing to a successful
delivery were elucidated. These included NPB numbers (ratio)
and alignment, day 2 cell size, state of nucleation and cell
number and the ability to progress from day 2 to day 3 through
at least one further cleavage division (or to the blastocyst). The
exact cell number on day 3 or the extent of fragmentation was
outweighed by day 1 and 2 morphology. Day 2 morphology
outweighed everything else. Because a normal embryo cannot
develop from an abnormal gamete, the early stage of development, the fertilized oocyte and the day 2 embryo, which is the
closest to the gametes, may offer windows into gamete normality. The more developed embryo, which has begun to rely on
the embryonic genome, is more reflective of developmental
ability in vitro and will represent gene expression and development control. Selecting embryos with the highest potential
actually means selecting normal gametes, which requires looking for early markers of the expression of that normalcy. The
data presented here suggest that day 1 and 2 scoring in a gated
system will allow this and result in increased implantation.
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Submitted on June 13, 2006; resubmitted on July 31, 2006; accepted on August 14,
2006