Elder K. Human preimplantation embryo selection

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HUMAN PREIMPLANTATION EMBRYO SELECTION

review, see Chapter 5). Embryos with multinucle-

ated blastomeres have been associated with a higher

rate of chromosomal abnormalities

and lower

implantation rates, when compared with embryos

with mononucleated blastomeres.

Saldeen et al

added the number of mononucleated blastomeres

to traditional morphological features of cell number,

fragmentation, and equivalence in the size of blas-

tomeres in order to select embryos. The ideal embryo

for transfer contained four mononucleated blas-

tomeres, and embryos with multinucleated blas-

tomeres were excluded from transfer.

Interestingly,

although standard morphological features were used

to guide embryo grading, mononucleation of all four

blastomeres was the only morphological sign that

was associated with implantation in this study.

A shift to transferring embryos on day 3 led to a

further opportunity for evaluation, and this led to a

refinement in criteria for assessing embryo mor-

phology, while maintaining the original features of

day 2 embryo assessment.

EIGHT-CELL EMBRYOS (DAY 3)

Evaluation of day 3 embryos typically focuses on cell

number, degree of fragmentation, and blastomere



symmetry. Each of these features has been shown to

correlate with implantation and pregnancy rates

after day 3 transfer.

37–39

More recent attention has

also been given to early compaction as a further pre-

dictor of developmental competence.

40,41

We briefly

review some of the studies that have analyzed the

individual elements of morphology as they relate to

implantation potential.

CELL NUMBER

Several studies have shown that a proportional rela-

tionship exists between the number of blastomeres

on day 3 (up to 8 cells) and implantation rates fol-

lowing day 3 transfer.

1,31

When specifically evaluat-

ing the day 3 embryo, Carillo et al demonstrated

that embryos with ⱖ8 cells on day 3 resulted in sig-

nificantly higher pregnancy rates when compared

with embryos with ⬍8 cells,

and Racowsky et al

demonstrated that those embryos with exactly 8 cells

on day 3 had the highest implantation rates (see

Figures 9.4).

Some authors have also used progression to the

blastocyst stage as a surrogate marker of develop-

mental competence, and these studies have also

revealed a relationship between the number of cells

n = 512 246 722 343 287 734 560 238 744 826 292

<7 7 8 >8 0 1–9 10–25 >25 None

Cell number % Fragmentation Asymmetry

% Viable

Severe

Some

Figure 9.4 Association of individual day 3 features (cell number, fragmentation, and asymmetry) with implantation rates. Embryo

viability according to morphological parameters: cell numbers (left panel), degree of fragmentation (middle panel), and asymmetry

(right panel). Total numbers (N) of embryos analyzed in each group are indicated. Different letters denote significant differences:

(left panel) a vs b, p ⬍0.0001; b vs c, p ⬍0.0001; c vs d, p ⫽ 0.016; (middle panel) a vs b, p ⬍0.0001; b vs c, p ⬍0.0001; (right panel)

a vs b, p ⫽ 0.042; b vs c, p ⬍0.0001. Reprinted from figure 2, Racowsky et al. Day 3 and Day 5 Morphological predictors of embryo

viability. Reprod Biomed Online 2003; 6(3): 323–331. With permission from Reproductive Healthcare Ltd.

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DEVELOPMENT RATE, CUMULATIVE SCORING, AND EMBRYONIC VIABILITY

on day 3 and blastocyst conversion,

suggesting

that an optimal number of blastomeres on day 3 is a

key developmental feature. Alikani et al demon-

strated that embryos with 7–9 cells on day 3 have a

significantly higher blastocyst conversion rate, com-

pared with day 3 embryos with ⬍7 cells or ⬎9 cells.

The fact that embryos with exactly 8 cells have

both higher implantation rates and blastocyst con-

version rates points to an interaction between mor-

phological assessment and developmental timing.

The idea that there is an ideal rate of development

for embryonic growth has been postulated since the

early studies of embryo assessment

and as we learn

more about developmental progression, embryo grad-

ing may focus more on overall growth and multiday

assessment rather than the static evaluation of day 3

blastomere number.

FRAGMENTATION

Blastomere fragmentation in embryos has been

extensively described, but the exact significance and

cause of fragmentation remains a topic of debate.

Several types and degrees of fragmentation have been

identified, with varying impact on implantation and

pregnancy rates, as described in detail in Chapter 6.

SYMMETRY

Blastomere symmetry has been included in the

majority of standard systems that assess both day 2

and day 3 morphology (see Table 9.1), but it has

infrequently been evaluated as an independent marker

of implantation potential. Generally, asymmetry

has been thought to be less important than either

cell number or fragmentation; however, embryos

with marked cellular asymmetry have demonstrated

substantially reduced implantation rates. Hardarson

reported that embryos displaying uneven cleavage

had lower implantation rates than embryos with even

cleavage (23.9% vs 36.4%).

Racowsky et al also

described that the implantation rates significantly

decreased as the degree of asymmetry increased (no

asymmetry 22.4%, some asymmetry 13.3%, and

severe 1.4%). The detrimental effects of asymmetry

were seen even after controlling for cell number and

percentage fragmentation.

For additional review,

see Rienzi et al, 2005.

COMPACTION

Few studies have focused on the degree of embryo

compaction, or the utility of assessing early com-

paction (i.e. the presence of compaction on day 3).

Tao et al devised a grading system for the day 4

embryo, when compaction is typically initiated,

and concluded that the degree of compaction on

day 4 was associated with implantation potential.

Desai et al also used compaction grading as part of a

combined embryo grading score on day 3. In this

scoring system, the pregnancy rate was found to

increase with the transfer of a compacting embryo,

but this observation did not reach statistical signifi-

cance.

We have recently shown that early com-

paction is related to implantation potential, but the

degree of fragmentation has an influence on this

effect. In embryos (ⱖ8 cells) displaying ⬍10% frag-

mentation, early compaction is associated with a

significantly higher implantation rate, whereas, in

embryos with ⱖ10% fragmentation, early com-

paction is negatively associated with implantation

potential.

COMBINING DAY 3 CHARACTERISTICS

Most embryologists have tried to weigh these indi-

vidual factors in a combined manner in order to

determine the ‘best’ embryo for transfer. Several

studies have evaluated the implantation potential of

these ‘top quality’ embryos. Volpes et al determined

that an increased number of embryos of ‘good qual-

ity’ (defined as 8-cell stage with ⬍20% fragmenta-

tion) were associated with increased pregnancy rates.

In addition, embryos transferred from cohorts hav-

ing at least one good quality embryo resulted in sig-

nificantly greater pregnancy rates than those arising

from cohorts with zero good quality embryos.

Van

Royen et al, also attempted to identify ‘top quality

embryos,’ defined as: absence of multinucleated

blastomeres, presence of 4 or 5 blastomeres on day 2

and 7 or more cells on day 3, with ⱕ20% fragmen-

tation. In this study, transfer of two top quality



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embryos was significantly more likely to result in a

twin pregnancy, and the presence of a top quality

embryo was associated with a higher implantation

rate.

Clearly, there are interactions between these fea-

tures of morphology. Racowsky et al investigated

the relative weight of cell number, fragmentation,

and degree of asymmetry, proposing that the opti-

mal embryo for day 3 transfer is an 8-cell embryo

with ⬍10% fragmentation and no asymmetry; these

embryos were associated with the highest percent-

age viability.

Desai et al expanded the number of

features involved in day 3 morphological grading

and developed a day 3 embryo quality score (D3EQ)

that assessed: cell number, presence of equal sized

blastomeres, blastomere expansion (blastomeres

touching the zona with minimal perivitelline space),

cellular cytoplasm clear of vacuoles, presence of

cytoplasmic pitting, signs of compaction, and the

pattern of fragmentation.

Using the D3EQ scores

of the two highest scoring embryos transferred, the

scoring system had a sensitivity of 83% for predict-

ing pregnant versus non-pregnant cycles.

CRITICISMS OF DAY 3 MORPHOLOGICAL

GRADING

Despite the improved implantation rates seen with

certain features of day 3 embryo selection, there have

been criticisms of morphological grading. Some

authors have argued that day 3 embryo quality is a

poor predictor for progression to the blastocyst

stage or for prediction of blastocyst quality.

47,48

Furthermore, experienced embryologists were only

able to pick the embryos that would develop into

the two best blastocysts in 23% of cycles.

Finally,

normal morphology does not guarantee euploidy or

a normal pregnancy. Gianaroli et al biopsied day 3

blastomeres to assess chromosomes X, Y, 13, 18, and

21 using multicolored FISH (fluorescence in-situ

hybridization) in a study population of 75 embryos.

Of 61 embryos (81%) with normal morphology, 50

were successfully biopsied, and 24 were diagnosed

as chromosomally abnormal.

Although blastomere

biopsy is a potential tool for embryo selection, it

also risks potential harm to the embryo and has

been associated with misinformation due to the

errors inherent in single-cell FISH. For review of

the interaction between embryo morphology and

chromosomal abnormalities see Munne et al

and Chapter 18.

BLASTOCYST DEVELOPMENT

Improvements in culture media and the subsequent

ability to culture embryos to the blastocyst stage has

made blastocyst transfer possible, and this has been

proposed as a means of selecting embryos with

improved developmental competence and implan-

tation potential. Blastocyst transfer also mirrors the

natural timeline of development in vivo, potentially

improving synchrony between uterine receptivity

and embryonic development. Blastocyst development

is influenced by patient factors, including sperm

quality

23,51

and maternal age

52,53

as well as factors

associated with the earlier stages of embryo devel-

opment. The number of oocytes collected, number

of oocytes inseminated, number of two pronucleate

zygotes, and number of embryos cleaving to at least

the 8-cell stage by day 3 in culture also influence

blastocyst formation.

BLASTOCYST MORPHOLOGY

Assessment of blastocyst morphology focuses on

characteristic features: extent of cavitation, the num-

ber of cells in the trophectoderm and inner cell mass

(ICM), and the shape of the ICM (see Figure 9.5).

Please refer to Chapter 7 for a detailed discussion of

blastocyst morphology. The blastocyst stage of devel-

opment is usually reached by day 5, although some

slow developing blastocysts do not reach this stage

until day 6 or 7.

Hardarson et al evaluated the differences in

blastocyst morphology and chromosomal abnor-

malities in blastocysts derived from surplus embryos

versus those derived from good quality embryos,



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DEVELOPMENT RATE, CUMULATIVE SCORING, AND EMBRYONIC VIABILITY

using FISH to identify chromosomes 13, 16, 18, 21,

22, X, and Y.

Grade A blastocysts were defined as

expanded or expanding with a distinct trophecto-

derm and eccentrically located ICM. Grade B blas-

tocysts were defined as those blastocysts that were

poorly expanded and/or with less defined trophec-

toderm and ICM cells, but without signs of degen-

erative foci. Grade C blastocysts exhibited a number

of degenerative foci in the ICM and trophectoderm

and a poorly developed blastocyst cavity. Good mor-

phology blastocysts had a significantly higher cell

number and a significantly higher number of chro-

mosomally normal cells, compared with poor qual-

ity blastocysts.

As described in Chapter 7, Gardner

used a similar scoring system, but evaluated the

ICM and the trophectoderm independently.

There is also a relationship between the size and

shape of the ICM, as well as the arrangement of

ICM cells and implantation potential. Richter et al

demonstrated that blastocysts with an ICM size

⬎4500 ␮m

had significantly higher implantation

rates than those with a size ⱕ4500 ␮m

(measured

as length ⫻ width). These authors also identified an

optimal shape of the ICM, using what they phrased

as ‘the roundness index.’ This value was calculated

by dividing length by the width of the ICM. The

optimal shape (associated with the highest implan-

tation rates) was a roundness index of 1.04–1.20.

When they combined these factors together, the

implantation rates were highest for blastocysts that

demonstrated both optimal size and shape.

CUMULATIVE SCORING: MULTIDAY

ASSESSMENTS

In an effort to improve embryo selection, several

investigators have combined key morphological cri-

teria for predicting the developmental competence

of cleavage stage embryos. Given the multitude of

scoring systems available, many of which have focused

on individual time points in embryo development,

several authors have turned to multi-day scoring as

a means of enhancing embryo selection (see Tables

9.2 and 9.3 for review of multiday scoring systems).

Early investigators of embryo scoring systems also

foresaw the utility of combining aspects of both

developmental rate and morphological assessment:

Cummins, Puissant and Steer all included some

assessment of developmental rate in their respective

scoring systems.

1,2,31

In 1998, Scott and Smith devised the corrected

embryo score, which was based on a combination of

pronuclear morphology and assessment of early

cleavage.A retrospective analysis found that the cor-

rected embryo score was associated with improved



Figure 9.5 Representative blastocyst images.These photographs depict the typical development of the blastocyst from early (A),

expanding (B) to fully expanded (C).

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

Table 9.2 Retrospective studies using multiday embryo scoring

Scott and Smith, 1998

Fisch et al, 2001

De Placido et al, 2002

Lan et al, 2003

Ciray et al, 2005

Title Corrected Embryo Score The Graduated Embryo Score

Type of study Retrospective analysis Retrospective analysis Retrospective analysis Retrospective analysis Retrospective analysis

Timing of Two assessments:

embryo 1) Assessment of pronuclear Three assessments: Three assessments: Two assessments: Three assessments:

assessment morphology 1) Pronuclear scoring 1) Pronuclear morphology 1) Combined Z-score 1) Early cleavage

2) Early cleavage 2) Early cleavage 2) Assessment of day 2 and 2) Day 3 morphology 2) Day 2 mononucleation

3) Weighted assessment day 3 embryo morphology assessment 3) Day 3 features

of day 3 morphology 3) Growth rate

Assessment of Alignment of pronuclei, Alignment of pronuclei, 1) Position of pronuclei Zygotes scoring NA

pronuclear (PN) nucleolar, and cytoplasmic nucleolar, and cytoplasmic 2) Position and type of according to Scott et al,

morphology features features. Alignment of the nucleoli 2000

(16–18 hours) Pronuclear alignment: nucleoli were given 3) Cytoplasmic morphology Z-1: equal number and

Score 5 additional weight (See Equally divided among three size of pronuclei, equal

Nucleolar scoring: Score 5: Table 9.4, definition factors (rated 1–5, with 5 numbers of nucleoli

nucleoli aligned in a row at of GES) being the best score). Max. aligned at pronuclear

the PN junction. Score 4: score 15 junction

nucleoli beginning to align. Z-2: equal number and

Score 3: nucleoli scattered size of nuceloli, but

Cytoplasmic scoring: scattered

Score 5: clear area around Z-3: equal numbers of

the pronuclei with a nucleoli with one

darkened ring or halo in the pronucleus displaying

middle. Score 3: Embryos alignment and the other

with a pitted or darkened with scattered nucleoli,

cytoplasm. Max. 15 points. OR unequal numbers or

size of nucleoli

Z-4 pronuclei not aligned

or of grossly different size

Assessment of If the embryo had progressed Additional points were Embryos were inspected

early cleavage to nuclear membrane awarded if cleavage was 26 hours after ICSI (all

(24–27 hours) breakdown or had cleaved present. Symmetrical cleavage cycles ICSI in this study).

to the 2-cell stage, it received and ⬍20% fragmentation at 2-cell embryos were

an additional 10 points the first cell division were also regarded as ‘early cleaved’

given additional weight

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

Day 2 features NA NA Additional points awarded NA A 4-cell embryo with a

(44–48 hours) for advanced growth single nucleus in all cells

(optimal being ⬎4 was given the designation

blastomeres on day 2 and ⬎7 of ‘mononucleated’

blastomeres on day 3)

Day 3 features NA Cell number and grade were Embryos were scored on Grade I: 8 cells, Grade 1: (4 points) ⫽

(64–72 hours) assessed. Additional weight three parameters (rated 1–5, blastomeres of equal size even and clear

was given to embryos with 5 being the best) and no cytoplasmic blastomeres with

containing 7–9 cells with 1) Blastomere volume and fragments no fragments

Grade I morphology synchrony of cleavage Grade II: 8 cells, Grade 2: (3 points)

Grade I: symmetrical 2) Observation of a single blastomeres of equal size uneven and irregular

blastomeres and absent nucleus within individual and ⬍20% fragments blastomeres, moderately

fragmentation blastomeres Grade III: 8 cells with clear cytoplasm and

Grade II: slightly uneven 3) Extent of fragmentation uneven blastomere sizes fragmentation ⬍20%

blastomeres and ⬍20% Max. score of 15 and no cytoplasmic Grade 3: (2 points)

fragmentation fragments 20–50% fragmentation

Grade III: uneven Grade IV: 4 or 8 cells Grade 4: (1 point)

blastomeres and ⬎20% with ⬎20% fragmentation ⬎50%

fragmentation fragmentation Embryos with the highest

Grade V: few blastomeres scores were transferred

of any size or major

fragmentation

Maximum 25 per embryo. CS was 100 Weighted score ⫽ (embryo Combined Embryo score was

embryo score calculated by adding the score ⫻ number of calculated by multiplying

scores for each embryo and blastomeres) ⫻ blastomere number by

then dividing by no. zygote score the given point value.

embryos transferred Embryos were divided into Two or three embryos

three groups on the basis of with the highest scores

score were transferred.

Embryos were transferred

on the basis of day

3 scores

Day of transfer Day 1 Day 3 and day 5 Day 2 or day 3. Embryos Day 3, 4, or 5 Day 3

were selected for transfer

based on

1) Number of blastomeres

2) Embryo morphologic

score

3) Zygote score

(Continued)

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

Table 9.2 (Continued)

Scott and Smith, 1998

Fisch et al, 2001

De Placido et al, 2002

Lan et al, 2003

Ciray et al, 2005

Outcome: NA Blastocyst conversion was NA Embryos derived from NA

blastocyst correlated with GES. Z-1 zygotes and grade

conversion Embryos with GES ⬎70 I day 3 morphology were

had 44% blastocyst defined as ‘top quality’

conversion compared with These top quality

9% conversion in embryos embryos had a 92%

scoring 0–65 day 5 ‘survival’ rate

Outcome: When CS ⱖ15, the GES 70–100 had an Implantation rates The implantation rate

implantation implantation rate was 28%, implantation rate of 39% increased as the number was the highest in the

rates compared with 2% when compared with 24% of of top quality embryos group of embryos that

the CS ⬍15 GES 0–65 for transfer increased had both early cleavage

(p ⬍0.001) and mononucleation.

(45% versus 15%

compared with embryos

without early cleavage or

mononucleation)

Outcome: When CS ⬎15, the The pregnancy rates were The weighted day 3 score Pregnancy rates increased Pregnancy rates were

pregnancy pregnancy rate was 71%, significantly correlated with had the highest correlation as the number of top highest in the cohort of

rates compared with 8% when GES. If the best embryo with pregnancy rates. Using quality embryos available embryos displaying at

the CS ⬍15. In addition, transferred had GES ⬎70, the weighted day 3 scores, for transfer increased early cleavage and

patients with CS ⬎15 had pregnancy rates were 59% the top group of embryos (p ⫽ 0.008). The mononucleation (69%

a multiple pregnancy compared with 34% if the had a 71% pregnancy rate, multiple pregnancy versus 33% compared

rate of 29% best scoring embryo compared with 45% rate also increased with embryos without

was ⬍65 pregnancy rate in group significantly as well early cleavage or

2 and 12.5% pregnancy rate mononucleation)

in group 3

NA, not applicable; CS, cytoplasmic scoring; GES, graduated embryo score.

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Table 9.3 Prospective studies using multiday embryo scoring

Scott et al, 2000

Fisch et al, 2003

Nagy et al, 2004

Chen et al, 2006

Title Z-Score The Graduated Embryo Score

Type of study Prospective cohort: Prospective cohort: Prospective randomized trial Prospective randomized trial

Comparison of selection with Comparison of GES versus Group 1: selection (for transfer) Group 1: evaluation of pronuclear

Z-score ⫹ day 3 morphology traditional day 3 morphology based only on PN morphology morphology combined with day 3

versus selection based on Group 2: selection based only on morphology and progression

day 3 morphology alone day 3 morphology Group 2: evaluation of early cleavage

Group 3: selection based on PN ⫹ combined with day 3 morphology

day 3 morphology and progression

Timing of Two assessments: Three assessments: Two assessments: Two assessments:

embryo 1) Assessment of pronuclear 1) Pronuclear scoring and 1) Pronuclear morphology 1) Pronuclear morphology or

assessment morphology and 2) Early cleavage and 2) Day 3 morphology 2) Early cleavage combined with

2) Day 3 morphology 3) Weighted assessment of 3) Day 3 morphology

day 3 morphology

Assessment of Zygote scoring: Assessment of nucleolar Pronuclear assessment occurred Group 1: Three subgroups:

pronuclear (PN) Z-1: equal number and size alignment. If nucleoli were 20 hours following ICSI. Subgroup A: nucleoli large or

morphology of pronuclei, equal numbers aligned along the pronuclear 1) Position and size of pronuclei medium sized and nucleolar

(16–18 hours) of nucleoli aligned at axis, 20 points were awarded 2) Distribution of nucleoli alignment seen

pronuclear junction (polarized or non-polarized) Subgroup B: Nucleoli large or

Z-2: equal number and size medium without alignment

of nuceloli, but scattered Subgroup C: Nucleoli small or

Z-3: equal numbers of pinpoint without any nucleolar

nucleoli with one pronucleus alignment

displaying alignment and the

other with scattered nucleoli

OR unequal numbers or size

of nucleoli

Z-4 pronuclei not aligned or

of grossly different size

Assessment of NA 30 points were awarded if NA Group 2: Three subgroups

early cleavage early cleavage was present. Subgroup A: zygotes that cleaved

(24–27 hours) Cells with ⬍20% into 2 cells at 26 h

fragmentation at the first cell Subgroup B: zygotes where PN

division were also given breakdown had occurred but

additional points cleavage had not occurred

Subgroup C: PN still intact

Day 3 features

(64–72 hours) Grade 1: 8 cells, ⬍10% Cell number and grade were Cell number and size of The exact criteria for ideal day 3

fragmentation, no assessed. Additional weight blastomeres, presence of embryo morphology is not

multinucleation. was given to embryos fragments were assessed. discussed.

Grade 2: 8-cell, 10–20% containing 7–9 cells with Grade A: ⬎4 blastomeres on

fragmentation or lacking ⬍20% fragmentation. day 2, ⬎6 on day 3 with ⬍10%

cell–cell contact, no (See Table 9.4) fragmentation

(Continued)

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

Table 9.3 (Continued)

Scott et al, 2000

Fisch et al, 2003

Nagy et al, 2004

Chen et al, 2006

multinucleation Grade B: 10–30% fragmentation

Grade 3: 6–7 cells or 8 cells Grade C: 30–50% fragmentation

with 20% fragmentation or

uneven blastomeres, no

multinucleation

Grade 4: ⬎8 cells or 4–6 cells

or 8-cells with ⬎20%

fragmentation or uneven

blastomeres or

multinucleation

Grade 5: ⬍4 cells or grossly

fragmented or with ⬎50%

multinucleated blastomeres

Day of transfer Day 3 or day 5 Day 3 and day 5 Day 3 Day 3

Outcome: Zygotes displaying equality NA NA

blastocyst between the nuclei

conversion (Z1 or Z2) had a significantly

higher blastocyst conversion

rate than those zygotes

without nuclear equality

(49.5% versus 28%)

Outcome: Using Z-score ⫹ day 3 Using GES to select embryos Implantation rates were higher for No difference in implantation seen

implantation morphology resulted in for transfer was associated patients randomized to have between group 1 and group 2 or

rates significantly higher with a higher implantation combined pronuclear and day 3 between corresponding subgroups.

implantation rates than day 3 rate than traditional scoring, compared with either Among both group 1 and group 2

morphology alone. (Day 3 morphology alone (36% parameter alone (21%, compared patients, embryos in subgroups A

transfers 31% versus 19%, implantation versus 27%) with 12% and 15% respectively) showed significantly higher

day 5 transfers 52% implantation rates

versus 39%)

Outcome: Using Z-score ⫹ day 3 With ⱖ1 embryo with GES Pregnancy rates were higher for No difference in implantation seen

pregnancy rates morphology resulted in ⱖ70, pregnancy rates were patients randomized to have between group 1 and group 2 or

significantly higher pregnancy 62%. With ⱖ1 grade A combined pronuclear and day 3 between corresponding subgroups.

rates than day 3 morphology embryo, pregnancy rate was scoring, compared with either Among both group 1 and group 2

alone. (Day 3 transfers 57% 50%. Transferring ⱖ1 parameter alone (39%, compared patients, embryos in subgroup A

versus 33%, day 5 transfers embryo with GES ⱖ70 did with 23% and 26% respectively) showed significantly higher

73% versus 58%) not improve pregnancy rates, pregnancy rates

but increased rates of

multiples

NA, not applicable; GES, graduated embryo score.

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

implantation and pregnancy rates.

In 2000, the

same group designed a prospective cohort study in

which they compared the utility of selecting embryos

based on combining Z-score (modified from their

original work) with day 3 embryo morphology, versus

day 3 morphology alone. These results indicated

that the combination of pronuclear and day 3 assess-

ment resulted in significantly higher implantation

and pregnancy rates.

Using similar principles of multiday assessment,

Fisch et al devised the graduated embryo score

(GES), see Table 9.4.

The GES is comprised of three

total embryo assessments (two interval evaluations

of early developmental milestones, combined with a

weighted assessment of conventional morphologi-

cal characteristics on day 3). The first assessment

was carried out at 16–18 hours and evaluated cyto-

plasmic halo and vacuoles, pronuclear size and

juxtaposition, nucleolar alignment and polar body

apposition and fragmentation. The embryos were

then evaluated at 25–27 hours postinsemination for

dissolution of the pronuclear membrane, blastomere

cleavage, symmetry, and degree of fragmentation. A

final evaluation was done at 64–67 hours postin-

semination for blastomere morphology and number,

and then a total GES was calculated and correlated

with blastocyst development and implantation rate.

In their initial retrospective analysis, a GES ⬎70 was

significantly associated with higher pregnancy rates

when compared to scores 0–65 (59% versus 34%,

p ⬍0.0015) and this also helped to predict blasto-

cyst conversion rate.

A follow-up study compared

the outcomes of embryos selected using the GES

versus those chosen for transfer based on morpho-

logical features alone. IVF outcomes using the GES

to select embryos for transfer resulted in a statisti-

cally significant increase in ongoing pregnancy rate

at 12 weeks compared with embryos graded by day

3 morphological assessment alone (62% vs 50%).

Several additional studies using combined assess-

ment of pronuclear morphology and day 3 embryo

assessment have followed the publication of the com-

bined Z-score assessment and the graduated embryo

score. Nagy et al. randomized patients to one of three

groups for embryo selection (selection based on PN

morphology alone, day 3 morphology alone, or com-

bined assessment). In this randomized study, those

patients assigned for combined assessment had sig-

nificantly higher implantation and pregnancy rates.

Lan et al., also used the approach of combined

Table 9.4 Graduated embryo score. Reprinted from Fisch et al. The graduated

embryo score predicts the outcome of assisted reproductive technologies better

than a single day 3 evaluation and achieves results associated with blastocyst trans-

permission from The American Society for Reproductive Medicine.

Hours after

Evaluation insemination Developmental milestone Score

1 16–18 Nucleoli aligned along pronuclear axis 20

2 25–27 Cleavage regular and symmetrical 30

Fragmentation

Absent 30

⬍20% 25

⬎20% 0

3 64–67 Cell number and grade

7CI, 8CI, 8CII, 20

9CI, 7CII, 9CII, 10CI, 11CI, compacting I 10

Total score 100

If the embryo was not cleaved at 25–27 hours, grading of fragmentation should occur at the 64–67 hour evaluation,

if the embryo reached the 7-cell stage and had ⬍20% fragmentation.

Grade I ⫽ symmetrical blastomeres and absent fragmentation. Grade II ⫽ slightly uneven blastomeres and ⬍20%

fragmentation. Grade III ⫽ uneven blastomeres and ⬎20% fragmentation. Grade A embryos are 7 or more cells

with ⬍20% fragmentation.

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