Elder K. Human preimplantation embryo selection

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HUMAN PREIMPLANTATION EMBRYO SELECTION

in relation to embryo transfer at the zygote stage.

Although transfer at the zygote stage is rather

unusual nowadays, the issue of pronuclear scoring

was revisited in studies aiming at zygote selection

for fresh transfer and for cryopreservation. The

policy of freezing embryos at the zygote stage was

considered as an alternative to freezing embryos at

cleavage stages in the context of legal restrictions

prohibiting embryo cryopreservation beyond the

zygote stage.

37–39

More recently, it was noted that neither zygote

evaluation nor cleaving embryo evaluation in

isolation can yield an optimal prediction of embryo

developmental potential. In combination, however,

zygote and cleaving embryo evaluation yield a highly

efficient embryo selection system which offers an

implantation rate after embryo transfer on day 3 after

ICSI that is comparable to transfer of day-5 embryos

at the blastocyst stage.

The potential usefulness of

pronuclear scoring as a means of selection against

embryos carrying chromosomal abnormalities has

also been reported.

36,41–43

However, pronuclei are highly dynamic struc-

tures, and essential features may sometimes escape

attention due to the limited time period during

which they can be analyzed by non-invasive means.

Moreover, the ability of a zygote showing various

pronuclear abnormalities to recover and develop nor-

mally may be conditioned by independent factors,

such as maternal age, ovarian reserve or sperm qual-

ity, which still remain to be analyzed in this context.

These caveats may be responsible for the inconsisten-

cies sporadically reported in the literature as to the

value of pronuclear scoring for prediction of embryo

developmental potential.

PRONUCLEAR SCORING SYSTEMS

Most of the current studies evaluating pronuclear

morphology in living human zygotes use one of the

two most popular scoring systems, described by Scott

and Smith

and by Tesarik and Greco.

The former

scoring system for evaluation of living human

pronuclear-stage zygotes

was based on the defini-

tion of five patterns, graded 1–5 according to nuclear

size, nuclear alignment, nucleolar alignment and dis-

tribution, and position of the nuclei withinthe zygote.

Grade 1 zygotes had equal numbers of nucleoli aligned

at the pronuclear junction. The absolute number was

not counted but was between three and seven. Grade 2

had equal numbers of nucleoli of equal sizes in the

same nuclei but with one nucleus having alignment

at the pronuclear junction andthe other with scattered



Figure 3.4 Micrographs showing the size and distribution of

nucleolar precursor bodies (NPB) in pronuclei of human zygotes

at different phases of pronuclear development. (A) Relatively

early phase of pronuclear development, characterized by a high

number of NPB in both pronuclei (only part of them

is visible at this focal level). The NPB are relatively small at this

phase and are distributed randomly in the pronuclei. (B) Later

phase of pronuclear development, characterized by a low

number of NPB in both pronuclei. The NPB are larger at this

phase and show a polarized distribution, with accumulation

near that pole of each pronucleus at which it makes contact

with the other one. (Hoffman modulation contrast, original

magnification ⫻200.) Reprinted from Tesarik and Kopecny.

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MORPHOLOGY AND KINETICS OF HUMAN PRONUCLEI

nucleoli. Grade 3 zygotes had equal numbers and

sizes of nucleoli (between three and seven) which

were equally scattered in the two nuclei. Grade 4

zygotes had unequal numbers (a difference of more

than one nucleolus) and/or sizes of nucleoli. Grade 5

zygotes were those with pronuclei that were not

aligned, were of grossly different sizes or were not

located in the central part of the zygote.

This system

was later refined and simplified by grouping grade

2 and 4 together (Z3 score). The most desirable

pronuclear patterns in this new Z-scoring system are

Z1 and Z2.

The appearance of cytoplasm around

the pronuclei, forming a characteristic ‘halo’ was

considered an additional quality parameter in this

scoring system.

The other widely used pronuclear scoring sys-

tem

has the advantage of simplicity, as it requires

only a single static observation of the zygotes, 12–16

hours after in vitro insemination or ICSI. It is also

very simple because it does not include cytoplasmic

appearance as an additional zygote evaluation

parameter. In fact, a subsequent study has shown that

cytoplasmic appearance has little importance in the

prediction of zygote and embryo developmental

potential.

The scoring system proposed by Tesarik

and Greco

is based on the evaluation of the syn-

chrony of developmental changes (with regard to

the size and position of NPBs) in both pronuclei

rather than the timing of these changes. Accordingly,

zygotes in which differences between both pronuclei

can be observed at this stage have a greater risk of

cleavage arrest, poor cleaving-embryo morphology,

and the appearance of blastomere multinucleation

as compared with zygotes in which pronuclear

differentiation progresses in synchrony.

ETIOLOGY OF PRONUCLEAR DEVELOPMENTAL

PERTURBATIONS

PATERNAL FACTOR

In view of the complex transformations the sperm

nucleus must undergo in the oocyte cytoplasm

before it forms the male pronucleus, the paternal con-

tribution to pronuclear developmental perturbations

is highly probable. However, an unequivocal demon-

stration of such a contribution was made difficult

by the frequent co-existence of sperm and oocyte

abnormalities in current IVF and ICSI programs.

The first direct evidence for the role of a sperm

factor in the etiology of pronuclear developmental

perturbations came from studies in which donor

oocytes were shared.

Consequently, it is evident

that ICSI with spermatozoa from some men repeat-

edly results in the formation of zygotes with pronu-

clear abnormalities, and these patients achieve lower

pregnancy and implantation rates than couples who

share oocytes from the same donors. This adverse

paternal effect is often associated with abnormal

basic sperm parameters, but is also observed in men

with normal sperm.

OOCYTE FACTORS AND OVARIAN STIMULATION

The finding that poor oocyte quality can also cause

abnormalities of pronuclear development came

from a study analyzing the relationship between

serum luteinizing hormone (LH) levels and embryo

quality in an oocyte donation program.

When

serum LH levels during ovarian stimulation were too

low or too high, the frequency of pronuclear abnor-

malities in the resulting zygotes was higher as com-

pared with oocytes from donors whose LH level was

between 0.5 and 1.0 IU per liter.

Further studies

are needed to obtain more information about the

effect of different aspects of ovarian stimulation on

pronuclear development.

SUMMARY

The development of pronuclei is a complex sequence

of events that involves the nuclear envelope, chro-

matin, and nucleolar precursor bodies (NPBs). The

timely occurrence of these events is a prerequisite

for correct pronuclear function and further embry-

onic development. These events were analyzed with

the use of electron microscopy and autoradiogra-

phy in polyspermically penetrated human zona-free

oocytes. Some of these events, namely those concern-

ing the development of NPBs, can also be visualized



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HUMAN PREIMPLANTATION EMBRYO SELECTION

by non-invasive examination of living human

zygotes with the use of Hoffman modulation con-

trast optics. Pronuclear scoring systems, based on the

determination of the number, size, and distribution of

NPBs, have been suggested to predict further embryo

development and implantation potential. The etiol-

ogy of abnormal pronuclear development can be

related to both sperm and oocyte quality.

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

INTRODUCTION

Age-corrected implantation rates with the use of

assisted reproductive technologies (ART) have been

on the increase over the past 25 years. Despite this

success, supernumerary embryos are still transferred

simultaneously, with the risk of increased multiple

pregnancy rates. Although enforced restrictive poli-

cies of single embryo transfer have yet to be enacted

in most countries, the desire to select the best embryos

and predict implantation has aided the develop-

ment of several evaluation methods to enhance

selection of embryos with optimal prognosis for

implantation. Embryo selection is most commonly

based on morphological criteria that are assessed

postfertilization, including both pronuclear and

cleavage stage embryo evaluation. Studies of phe-

nomena related to development rate, pronuclear and

nucleolar behavior, blastomere fragmentation, and

multinucleation have been of particular interest.

1–8

Events related to pronuclear and nucleolar move-

ments were first described by Wright et al

and Van

Blerkom.

Recently, these phenomena have been

expressed as more distinct pronuclear scoring systems

that can be used for the purpose of selecting embryos.

The scores have been correlated with improved

embryo development

5,10–13

as well as with increased

pregnancy and implantation rates.

5,10,12–20

A number of different pronuclear scoring systems

have been proposed by different ART laboratories,

and are in use to select high quality embryos; however,

no standardized scoring system for zygote grading is

currently in use. Unfortunately, the same holds true

for other systems used to evaluate embryo morphol-

ogy. As a result, comparing success rates between

laboratories while controlling for embryo quality is

a challenge. A consistent scoring system, such as a

pronuclear scoring system in combination with other

embryo development markers and patient status,

may be useful in determining the optimal number of

embryos to transfer. This could lead to an increase in

pregnancy and implantation rates, while decreasing

the number of high order multiple pregnancies.

PRONUCLEAR SCORING METHODOLOGY

Pronuclear scoring is a non-invasive examination that

relies upon one static observation of simple mor-

phological parameters, and is routinely performed

at 16–18 hours postinsemination with both standard

IVF and ICSI. However, pronuclear examination for

scoring purposes can be more time-consuming than

the usual cursory examination used for determining

fertilization. Caution should always be used when-

ever embryos are removed from incubators for pro-

longed periods to perform any type of examination.

Cumulus cells must first be removed from zygotes

that result from oocytes that were inseminated with

standard IVF procedures, to allow an unobstructed

view of the pronuclei during analysis.

All pronuclear scoring systems attempt to clas-

sify zygotes based on the characteristics of the two

pronuclei as well as the nucleoli. Pronuclei are scored

based on the following criteria: symmetry (equal vs

unequal), position (in apposition vs at a distance),

and location (central vs non-central).The nucleoli are

scored based on number (3 to 7), symmetry (equal vs

unequal sizes), and location (polarized or aligned vs

non-polarized or non-aligned). Other observations

factored into the score might include polar body mor-

phology and alignment, cytoplasmic morphology

(presence vs absence of halo; i.e., cytoplasmic con-

traction), and finally a second embryo evaluation

at 25–27 hours postinsemination for pronuclear

4. Human pronuclei as a mode of

predicting viability

Aidita N James

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HUMAN PREIMPLANTATION EMBRYO SELECTION

morphology (presence vs absence of pronuclei)

and/or early cell division.

Evaluation of pronuclear morphology is most effi-

cient or accurate under phase-contrast microscopy

utilizing either Hoffman modulation contrast (HMC)

or differential-interference contrast (DIC) optics.

During this assessment, the technician should visu-

alize the zygote through several focal planes to ensure

the best possible evaluation of morphology. Visuali-

zation in only one focal plane can be misleading and

result in an incorrect assessment of zygote morphol-

ogy. Pronuclear morphology is not a static event, and

therefore all embryos should be assessed at the same

time point postinsemination in order to avoid incor-

rect assessment due to a bias based on timing.

PRONUCLEAR SCORING PARAMETERS

AND THEIR IMPORTANCE

A suspicion that major abnormalities of pronuclear

development (i.e. pronuclei of unequal sizes, at a

distance or not centrally located within the zygote

cytoplasm) are incompatible with normally progress-

ing development is not a novel concept. A number

of studies have confirmed that zygotes exhibiting

unequal pronuclear sizes are associated with an

increased risk of embryonic chromosomal anom-

alies.

4,8,21

The sperm-derived centriole and associated

microtubule organizing region function to reposition

the pronuclei into apposition,

2,22,23

and visual changes

in the pronuclei may thus correlate with functional

abnormalities in chromosome segregation.

While parameters of pronuclear size, location,

and position are obvious and easily assessed, other

morphological parameters are less distinct, includ-

ing polarization of the nucleoli or nucleolar precur-

sor bodies (NPB) along the pronuclear junction.

Nucleolar precursor body alignment has been related

to the chromatin rotation that occurs in developing

pronuclei,

14,24

and therefore anomalies in this param-

eter potentially have severe consequences. Chromatin

rotation plays an important role in establishing the

embryonic axis, an essential part of cell determination

for preimplantation embryos.

Tesarik and Kopecny

reported that NPB alignment is not evident at the



beginning of pronuclear formation but progresses

over time as the NPB coalesce to form fewer, larger

nucleolar structures within the pronuclei. This again

demonstrates the fluidity of pronuclear morphology.

Different numbers and dimensions of nucleoli are

compatible with development, although assessments

may be inaccurate because of light microscopic

limitations. It is likely that there is considerable

biological variation within normal development.

The alignment of the pronuclei and polar bodies

is another phenomenon in pronuclear morphology

assessment that has been less well studied. This

relationship is critical, because it eventually corre-

lates with the polar axis of the first cleavage division

as defined by the second polar body.

The align-

ment that is currently hypothesized to be most ideal

is for the pronuclei to be parallel to the polar axis

(Figure 4.1). Perpendicular placement of the pronu-

clei would instead position one pronucleus on the

opposite side of the cleavage plane. This misalign-

ment could subsequently result in incorrect nuclear

division followed by chromosomally abnormal

embryo development.

The appearance of a cytoplasmic halo has been

associated with the redistribution of mitochondria

in the human zygote after fertilization, and has been

positively correlated with blastocyst quality.

26–28

This redistribution should place the majority of the

Figure 4.1 Graphical representation of pronuclear orientation

as it relates to the second polar body.

Perpendicular configuration Parallel configuration

Pronuclei

First and second polar body

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HUMAN PRONUCLEI AS A MODE OF PREDICTING VIABILITY

mitochondria around the pronuclei, producing a

cytoplasmic clearing around the periphery of the

ooplasm and thereby creating the appearance of a

halo.

Incorrect distribution of mitochondria and

associated ATP could lead to unequal cellular divi-

sion and abnormal embryo metabolism.

Finally, early embryonic cleavage at 25–27 hours

postinsemination has been associated with increased

embryo implantation potential and live birth

rates.

7,30,31

This correlation may be due to cyto-

plasmic and/or nuclear maturity. It is plausible that

oocytes with optimal cytoplasmic/nuclear maturity

may lead to more appropriate metabolism (ATP,

mitochondria, etc.) in the embryo, so that it is better

able to perform cellular functions.

PRONUCLEAR SCORING SYSTEMS

Scott and Smith

proposed a Z score system for

assessing pronuclear morphology, and this was later

modified by Scott et al

and Scott.

Tesarik and

Greco

developed another system for scoring pro-

nuclei, which was further modified by Tesarik et al

to create scores based on pattern 0 vs non-pattern

0 groups. In response to pressure from German and

Swiss law to aid in the prediction of pregnancy and

blastocyst development Ludwig et al,

Zollner et al

and Senn et al

developed further systems for

scoring pronuclear morphology. Both Germany and

Switzerland have extremely restrictive IVF laws that

allow culture only of embryos that will be transferred,

which thereby limits embryo selection to choosing

zygotes prior to the first cellular division. All of the

different scoring systems are based on a common

theme, that is the value of symmetry in all of the

morphological parameters that are examined.

THE Z SCORE

One of the early pronuclear scoring systems, pro-

posed by Scott and Smith,

was based on a total score

derived from values awarded for specific parameters

of morphology, and the sum of these values was used

to determine which embryos would have a higher

implantation potential. The parameters assessed were

pronuclear size and alignment, alignment of nucleoli

within the pronuclei, and cytoplasmic morphology

(Table 4.1). An additional score of 10 points was

awarded to embryos for early cell division observed

at a later embryo evaluation (24–26 hours postin-

semination). The total sum yields a score ranging

from 7 to 25, with a higher score related to greater

implantation potential. Scott and Smith

reported

that a pronuclear morphology score of ⱖ15 was

associated with a pregnancy rate of 71%.

Scott et al

then revised the scoring system to

include only parameters that could be observed at

the time of fertilization assessment. This system clas-

sified zygotes into five basic groups, denoted as a

Z score (Figure 4.2). The system was subsequently

revised to classify zygotes into four basic categories

(Figure 4.2, Table 4.2). Numerous reports have con-

firmed the zygote scoring system known as Z scoring



Table 4.1 The pronuclear scoring system created by Scott and Smith.

A cumulative point system

with values ranging from 3 to 35 with an ideal of ⱖ15 correlating to increased pregnancy rates

Points awarded for various morphological parameters

Parameters 5 4 3 1

Pronuclear position In apposition NA NA At a distance or very

unequal in size

NPB position Aligned Coming into Unaligned NA

alignment

Cytoplasmic morphology Clear halo NA Non-homogenous NA

and/or darkened

NPB, nucleolar precursor bodies (nucleoli); NA, not applicable.

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HUMAN PREIMPLANTATION EMBRYO SELECTION



Figure 4.2 Comparison of the various pronuclear morphology scoring systems by Scott’s group and by Tesarik’s group and a

representative illustration of pronuclei in each scoring group.

Scott et al

Series 1

Scott et al

Series 2

Scott

Tesarik and

Greco

Tesarik et

Pronuclear

morphology

Grade 1 Z1 Z1 Pattern 0 Pattern 0

Grade 3 Z2 Z2 Pattern 0 Pattern 0

Grade 2 Z3 Z3-2 Pattern 2

Non-pattern 0

Grade 4 Z3 Z3-1 Pattern 5 Non-pattern 0

Grade 4 Z3 Z3-4 Pattern 1 Non-pattern 0

Grade 4 Z3 Z3-4 Pattern 3 Non-pattern 0

Grade 5

NA, not applicable.

Z3 Z3-1 Pattern 4 Non-pattern 0

Grade 5 Z4 Z4-2 Pattern 4 Non-pattern 0

Grade 5 Z4 Z4-1 NA Non-pattern 0

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HUMAN PRONUCLEI AS A MODE OF PREDICTING VIABILITY

to be useful in the selection of embryos with improved

implantation rates

and therefore this system has been

widely accepted in ART.

The Z score system was later revised

in order to

incorporate further data and new evaluation param-

eters, the presence of a cytoplasmic halo and the ori-

entation of the pronuclei in relation to the second

polar body, as outlined in Figure 4.2. This version

was then adapted to define Z3 and Z4 scores, which

included these parameters.

The presence of a halo

was correlated to faster development and better mor-

phological scores on day 3 and to higher blastulation

rate on day 5 of culture. Pronuclear/polar body ori-

entation was not observed to correlate with embryo

development. Consistent with the older versions of

the Z scoring system, embryos that were derived from

zygotes scored as Z1 or Z2 had a better blastocyst

development rate and a higher implantation potential.

0 PATTERNS

Tesarik and Greco

proposed yet another pronuclear

scoring system, based upon one static observation

made on an unobstructed view of the entire volume

of both pronuclei at the time of fertilization assess-

ment; this assessment noted the number and distribu-

tion of the NPBs within each pronucleus (aligned vs

unaligned). The size (equal vs unequal) and position

(in apposition vs at a distance) of both pronuclei was

also evaluated during this assessment. The zygotes

were categorized into six different groups (patterns

0 to 5) (Figure 4.2, Table 4.3). This system did show

some difference in the percentage of embryos that

developed into good morphology embryos on day 3 of

culture,based upon their respective pattern grouping.

Tesarik et al

then further simplified their scoring

system to one that placed embryos into two groups,

pattern 0 and non-pattern 0. Non-pattern 0 comprised

all of the other pattern groupings, from 1 to 5. The use

of this scoring system increased the prediction of

pregnancy and implantation rates with the use of a

single, static observation at the zygote stage. The

authors reported pregnancy and implantation rates

of 45% and 30%, respectively, for embryos derived

from pattern 0 zygotes, and 22% and 11%, respec-

tively, for those derived from non-pattern 0 zygotes.

CUMULATIVE POINT SCORING

In response to the constraints of the German IVF

laws, Ludwig et al

adapted the pronuclear scoring



Table 4.2 The simplified pronuclear scoring system

developed by Scott et al

with scores of Z1 and Z2

considered morphologically normal zygotes

Z score Morphological description

Z1 Equal PN

Equal number and size of nucleoli (ranging between

3 and 7)

All nucleoli aligned at the pronuclear junction in

both PN

Z2 Equal PN

Equal number and size of nucleoli (ranging between

3 and 7)

Nucleoli unaligned in both PN

Z3 Equal PN

Equal number and even and/or uneven size of nucleoli

(ranging between 3 and 7)

Nucleoli aligned at the pronuclear junction in one PN

and unaligned in the other PN

Z4 Unequal or

separated or

not centrally located PN

PN, pronuclei.

Table 4.3 The pronuclear scoring system devel-

oped by Tesarik and Greco,

with normal zygotes

assigned to the pattern 0 group

Pattern Morphological description

0 Equal number and size of NPB (ranging between

3 and 7)

All NPB either polarized or non-polarized in both

pronuclei

1 Unequal number (difference ⬎ 3) of NPB between

both pronuclei

2 Small number (⬍ 7) of NPB

Non-polarized in at least one pronuclei

3 Large number (⬎ 7) of NPB

Polarized in at least one pronuclei

4 Very small number (⬍ 3) of NPB in at least one

pronuclei

5 Polarized NPB in one pronucleus and non-polarized

NPB in the other pronucleus

NPB, nucleolar precursor bodies (nucleoli).

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