Elder K. Human preimplantation embryo selection

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HUMAN PREIMPLANTATION EMBRYO SELECTION

to this a process of zona hardening in vitro has

been described, which might impede the blastocyst’s

attempt to hatch. Failed implantation may be due at

least in part to entrapment of the embryo as a conse-

quence of impaired zona-hatching.

Indeed, imple-

mentation of the assisted hatching (AH) technique

was demonstrated to be effective in patients where

chemical or physical changes in the zona pellucida

had occurred as a consequence of advanced reproduc-

tive age or elevated basal follicle stimulating hormone

(FSH) concentrations.

24,25

From these considerations

it might be speculated that the structure and function

of the zona is associated with specific changes within

the oocyte, and this may therefore also affect the

oocyte’s potential for successful embryo development.

The morphology of the zona pellucida can also

be influenced by extrinsic factors. The periovulatory

hormonal environment may have an impact on the

thickness of the zona pellucida. Studies by Bertrand

et al

demonstrated that the thickness of the zona

pellucida is also influenced by hormonal stimula-

tion in assisted reproductive technology (ART) cycles.

Therefore, ovulation induction might be an impor-

tant modulator of zona function in the process of

fertilization in vitro.

There is also increasing evi-

dence that suggests a further influence of ovulation

induction on the hatching procedure. This will be

discussed below.

MORPHOLOGICAL CHANGES IN THE ZONA

AS PREDICTORS OF ART OUTCOME

Criteria that can be assessed by routine microscopy

prior to fertilization or embryo transfer would be

useful in selecting embryos for transfer during ART

procedures; however, very few reliable, predictive and

non-invasive markers for oocyte quality have thus

far been identified. Nearly two decades ago it was

described that the zona pellucida of a proportion of

cleaving embryos had thinned areas in their zonae

pellucidae.

This could be expressed as a percentage

variation of zona pellucida thickness and was con-

sidered an important factor associated with implan-

tation of embryos.When the ‘best’ embryo had a zona



pellucida that varied more than 25%, 24 of 60 (40%)

resulted in pregnancy; pregnancies were not induced

(0/21) when the ‘best’ embryo had less than 10%

variation. The thinned areas appeared to be part of

a dynamic process that was already noticeable in

zygotes, but can increase during preimplantation

development, particularly in embryos with a good

prognosis.

Early studies showed that zona thinning

of the expanding blastocyst is not a unique process,

as it is preceded, at least in the human, by a gradual

thinning process correlated with increased viability

starting as early as the zygote and cleavage stages.

Several other studies have since reported on the

relationship between the thickness and morphology

of the human zona pellucida and embryo quality,

embryo development and pregnancy rates. Three

major approaches have been used to assess morpho-

logical criteria in the human zona, including direct

imaging with camera or video using an inverted

microscope with Hoffman modulation optics fol-

lowed by processing the film for manual measure-

ments.

This method has advantages in terms of

reliability but is slow and not helpful in real-time

prediction of viability. A second approach offered

by other investigators

uses a digitized imaging sys-

tem to store images, which can be measured with the

aid of a computer. Many different criteria have been

measured, but the two that have been most commonly

used are zona pellucida thickness (ZPT) and zona

pellucida thickness variation (ZPTV). In the majority

of studies, the zona was measured at three points as

suggested by Cohen and co-workers,

with the fol-

lowing calculations applied. Mean ZPT value and

ZPTV value computed as:

An example of this type of measurement is shown

in Figure 2.1, with multiple data sets used to increase

the accuracy of the measurements.

28–30

Zona pellucida thickness variation is associated

not only with better outcome in terms of pregnancy,

ZPT

mean

(ZPT

ZPT

)/3

ZPTV (ZPT

max

ZPT

mean

⫽⫹⫹

⫽⫺ ZZPT

mean

100%.⫻

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THE ZONA PELLUCIDA AND MARKERS OF OOCYTE AND EMBRYO VIABILITY

but also with better overall embryo morphology.

One might therefore speculate as to which variable

might be the better predictor. We investigated this

question in a smaller series, and found no significant

impact of the ZPTV in embryos that already had

optimal morphology measured as a function of devel-

opment rate and fragmentation, among other para-

meters. The ZPTV did predict viability of embryos

when other morphology criteria were inferior.

In reports where pregnancy rate was the meas-

ured outcome,

27–32

ZPT was not associated with an

improved pregnancy rate for all groups of patients,

but was found to be generally correlated with preg-

nancy in all of the studies, which included biometric

analysis of the zona pellucida at the time of embryo

transfer. The data suggest that assessment of ZPTV

should be included in criteria used to select

embryos of optimal quality prior to transfer.

A third exciting and real-time technique for eval-

uating zona pellucida morphology can be achieved

with the use of a Polscope.

With this system,

the retardance magnitude and the thickness of the

inner, middle and outer layers of the ZP are meas-

ured before embryo transfer. The authors found

that the magnitude of light retardance by the inner

layer of the zona pellucida appears to represent a

unique non-invasive marker for the developmental

potential of the oocyte. We were able to confirm this

observation in a small series of 20 oocytes prepared

for ICSI (Lindenberg, unpublished data).



Figure 2.1 The outer and inner zona is delineated and the zona pellucida thickness has been calculated.

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HUMAN PREIMPLANTATION EMBRYO SELECTION

CONCLUSION

The human zona pellucida is not static, but is a highly

dynamic structure that serves several known and

unknown functions during oogenesis and embry-

onic development. Specifically, the zona seems to

have a vital impact on co-ordinating the influence of

the somatic cell compartment in the ovary on oocyte

maturation. Fertilization and remodelling of the zona

after cortical granule release are also classical features

of the zona pellucida’s role. With the use of conven-

tional microscopy to measure ZPTV, it has been

found that remodelling of the zona after fertilization

seems to influence both the immediate in vitro, and

later in vivo development of the human embryo.

ZPTV can be seen as early as the zygote stage and

increases in a subset of embryos over time. It is our

firm conviction that assessment of ZPTV should be

an integral part of the selection criteria for embryo

transfer. We therefore conclude that ZPTV meas-

urement is a well documented but clearly under-

appreciated tool that can be used to select embryos for

transfer on days 2–3 after fertilization. Furthermore,

this criterion appears to be even more important

when only embryos of suboptimal morphology are

available for transfer.

The ZPTV could also be used to select those

embryos that might benefit from assisted hatching.

These observations are a valuable addition to criteria

that may be of benefit in a policy of selected single

embryo transfer.

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17. Hoodbhoy T, Dean J. Insights into the molecular basis of sperm-egg

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18. Soupart P, Strong PA. Ultrastructural observations on polyspermic

penetration of zona pellucida-free human oocytes inseminated in vitro.

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19. Gardner AJ, Evans JP. Mammalian membrane block to polyspermy:

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21. Willadsen SM. A method for culture of micromanipulated sheep

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human embryos. Ann Acad Med Singapore 1992; 21: 565–70.

25. Cohen J. Assisted hatching of human embryos. J Assist Reprod Genet

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preservation. Hum Reprod 1990; 5: 109–15.

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30. Gabrielsen A, Lindenberg S, Petersen K. The impact of the zona pellu-

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31. Garside WT, Loret D, Mola JR et al. Sequential analysis of zona thick-

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INTRODUCTION

The process of fertilization is a complex sequence of

events the signs of which are perceptible as early as

several seconds after sperm–oocyte fusion.

However,

the demonstration of very early signs of fertilization

requires recourse to special techniques, most of

which are destructive for the fertilized oocyte. Hence,

since the first successful clinical application of human

in vitro fertilization (IVF),

two signs of fertilization

that are detectable by non-invasive inspection of

inseminated oocytes have been used: the extrusion

of the second polar body and the development of

pronuclei.

The extrusion of the second polar body is a much

earlier sign of fertilization than is the development

of pronuclei. However, the high incidence of first

polar body division or fragmentation compromises

the accuracy of second polar body detection in many

cases. Consequently, the development of pronuclei

after IVF has become the gold standard of fertiliza-

tion assessment. The possibility of distinguishing

between normal fertilization (2 pronuclei), partheno-

genetic oocyte activation (1 pronucleus), and poly-

sper

mic oocyte penetration (⬎2 pronuclei) is an

addi

tional advantage of pronucleus-based evalua-

tion systems, because parthenogenetically activated

and polyspermically penetrated oocytes both show

the same pattern of second polar body extrusion as

does a normally fertilized oocyte. There are a few

exceptions to these common observations: firstly,

eggs can be activated by a sperm cell without

decondensation. This is apparently quite common,

and may be overlooked as abnormal fertilization,

activation or failed fertilization.

Secondly, a single

pronucleus can be the result of the close association

of male and female genomes in human zygotes.

Thirdly, some eggs may activate without extruding

the second polar body, rendering the zygote digynic.

The idea that analysis of pronuclei can provide

something more than simply evidence of fertiliza-

tion arose from studies performed in the late 1980s

which showed that pronuclear development reflects

the activity of developmentally important oocyte

cytoplasmic factors,

and is also related to an early

period of RNA synthesis in the zygote.

6,7

At the same

time, the relationship between characteristics of

pronuclear development, nucleolar distribution/

movement and embryo viability/implantation poten-

tial, was established by Wright et al;

this was con-

firmed and expanded upon by our team and others

some years later.

9–11

Here we outline the biological basis that under-

lies pronuclear development, as well as the patho-

logical conditions that lead to abnormal pronuclear

development. This information should help to under-

stand the relationship between pronuclear mor-

phology and IVF outcomes. It can also serve as a

background for further studies aimed at refining the

existing pronuclear scoring systems and defining

new clinical applications and relationships.

THE PHYSIOLOGY OF THE HUMAN ZYGOTE

The beginning of the zygote’s existence is marked

by sperm–oocyte fusion. At this point in time, the

fertilizing spermatozoon triggers a cascade of cell sig-

nalling events in the oocyte, collectively termed oocyte

activation. A series of repetitive increases in free intra-

cellular calcium concentration (calcium oscillations)

has become the most well studied aspect of human

oocyte activation.

Relatively less is known about

downstream elements of the oocyte-activating sig-

nal transduction cascade, which can be expected to

affect regulatory elements that control the oocyte’s

cell cycle checkpoints and the function of cytoskele-

tal elements; these promote the exit of the oocyte

3. Morphology and kinetics of human pronuclei

Jan Tesarik, Raquel Mendoza-Tesarik, Ermanno Greco, and Carmen Mendoza



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HUMAN PREIMPLANTATION EMBRYO SELECTION

from metaphase II (MII) arrest and its entry into

the regular mitotic cell cycle.

13,14

Studies performed in different mammalian species

have shown that early embryonic development

occurs in the absence of active gene expression

during a period which is species dependent. In the

human, a major activation of nuclear RNA synthesis

occurs at the 4-cell stage,

15,16

and the first biochemi-

cal

and morphological

signs of embryonic gene

expression are detectable between the 4- and the 8-

cell stage. The messages required to guide develop-

mental processes that take place prior to embryonic

gene activation are derived from a pool of mRNAs

that have been synthesized during oocyte growth and

are stored in the oocyte cytoplasm as polyadenylated

mRNA. It is tempting to speculate that the drawn-out

and spatially differentiated series of periodic calcium-

driven signalling events observed throughout human

zygote development

may determine the timing of

deadenylation and expression of specific maternal

mRNAs stored in different regions of the oocyte and

zygote. In fact, spontaneous

and drug-induced

disturbances of calcium oscillations in the human

zygote were shown to be associated with abnormal-

ities of pronuclear morphology and developmental

arrest.

From the developmental point of view, the main

function of the zygote is to ensure a timely start for

the embryonic cell cycle after the long period of arrest

that both the sperm- and the oocyte-derived genomes

have experienced in their respective gametes. This

function includes the correct timing of DNA syn-

thesis during S-phase and the subsequent fusion

of paternal and maternal genomes to merge into a

unique genome for the future embryo.

THE DEVELOPMENT AND FUNCTION OF

PRONUCLEI

EXPERIMENTAL APPROACHES

Most of the basic knowledge about the structure

and function of human pronuclei is derived from

invasive studies in which either normal zygotes were

sacrificed, or polypronuclear eggs were formed for

research purposes by inseminating zona-free human

oocytes. These studies used electron microscopy and

autoradiography to analyze changes in pronuclear

ultrastructure and nucleic acid synthesis.

5–7,21

More recently, a number of non-invasive studies

were carried out, based on observations of pro-

nuclei in living human zygotes. These studies, nicely

reviewed by Scott,

could not use the same structural

detail and experimental rigor as the earlier invasive

studies. However, they have the merit of having

established the existence of relationships between

the appearance of pronuclei and further embryonic

development, including data about implantation

and post-implantation events. Both types of studies

are thus complementary.

THE ESTABLISHMENT OF PRONUCLEI

Soon after sperm–oocyte fusion, the nuclear enve-

lope of the fertilizing spermatozoon disintegrates,

and both sperm- and oocyte-derived chromatin is

thus directly accessible to oocyte cytoplasmic fac-

tors.

However, sperm chromatin differs from the

oocyte telophase chromosomes by its higher degree

of condensation, due to its association with sperm-

specific nuclear protamines. Sperm nuclear prota-

mines are removed and replaced with oocyte-derived

histones before the male pronucleus is formed.

This step is obviously dependent on the availability

of histones in the oocyte. Relative insufficiency of

oocyte histones may thus slow down the transforma-

tion of the sperm nucleus into the male pronucleus,

and consequently cause asynchrony of pronuclear

development.

The establishment of pronuclei is not completed

until a new nuclear envelope is formed around the

sperm- and oocyte-derived chromatin. This process

has been studied in human polyspermically pene-

trated zona-free oocytes,

and was shown to

involve alignment and stepwise fusion of vesicles

and tubules of endoplasmic reticulum around the

chromatin (Figure 3.1). Once nuclear envelope for-

mation is completed, the pronuclei can be easily

visualized in living zygotes with the use of phase con-

trast, Nomarski differential interference contrast or

Hoffman modulation contrast optics.



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MORPHOLOGY AND KINETICS OF HUMAN PRONUCLEI

DEVELOPMENTAL CHANGES IN

PRONUCLEAR STRUCTURES

Newly formed pronuclei contain finely dispersed

chromatin with disseminated small electron-dense

bodies.

The latter progressively aggregate and fuse

with each other, and are closely associated with large

chromatin clusters (Figure 3.2). This process leads

to the formation of still larger aggregates, consisting

of material derived from the electron dense intra-

nuclear bodies and chromatin, merged in compact

structures that develop into functionally active nucle-

oli three cell cycles later.

16,23

Thus, these structures

were given the name of nucleolar precursor bodies

(NPBs).

In the experimental system based on polysper-

mically penetrated human zona-free oocytes, the

process of NPB formation began as early as 4 hours

after in vitro insemination, and the first fully devel-

oped NPBs appeared 12 hours after in vitro insemi-

nation.

This transformation coincides with the

development of typical nuclear pores all around the

pronuclei, and with the appearance of characteristic

vesicles between the two membranes of the

nuclear envelope.

The function of these vesicles,

also observed in blastomeres of cleaving human

embryos,

is unknown.

DEVELOPMENTAL CHANGES IN

PRONUCLEAR FUNCTION

One of the main functions of pronuclei is to ensure

adequate conditions for the first phase of DNA

synthesis after fertilization so that the sperm- and

oocyte-derived chromatin is eventually mixed. An

experiment in which

H-thymidine was incorpo-

rated into the pronuclei of polyspermically pene-

trated human zona-free oocytes, evaluation and

analysis by autoradiography, showed the first signs of

DNA synthesis no earlier than 12 hours after in vitro

insemination, and only in those pronuclei that had

completed ultrastructural differentiation of NPBs.

Interestingly, human paternal pronuclei can only com-

plete their NPB differentiation when they have previ-

ously carried out a limited RNA synthesis, detected in

polyspermically penetrated human zona-free oocytes



Figure 3.1 Electron micrograph of a fully decondensed sperm

nucleus in the course of nuclear envelope assembly, occurring

in an early phase of male pronucleus development, 4 hours

after in vitro insemination. Chromatin (ch) is surrounded

by a discontinuous series of vesicles (v) and cisternae (c) of

endoplasmic reticulum. Arrows indicate intranuclear dense

bodies. ⫻20 000. Reprinted from Tesarik et al.

Figure 3.2 Electron micrograph showing a part of a developing

male pronucleus with nucleolar precursors (np) at different

phases of their assembly. ⫻44 000. Reprinted from Tesarik et al.

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HUMAN PREIMPLANTATION EMBRYO SELECTION

as early as 4 hours after in vitro insemination.

Consequently, delayed NPB development is likely to

signal a failure or delay of the early pronuclear RNA

synthesis, a condition which will in turn cause a

delay in the onset of DNA synthesis.

The nature of the transcripts resulting from pronu-

clear RNA synthesis is largely unknown. However, it

has been shown that paternal Y-linked transcripts are

expressed in paternal pronuclei of human zygotes.

Early RNA synthetic activity has also been observed

in mouse pronuclei, similar to that observed in

humans.

Interestingly, chromatin-mediated repres-

sion of promoter activity that is almost total is

imposed on maternal pronuclei, but not on paternal

pronuclei.

27,28

Consequently, the ongoing rate of tran-

scription by endogenous genes in the mouse zygote is

four to five times greater in the paternal pronucleus

than in the maternal pronucleus.

The existence of

similar differences between transcriptional activity

in male and female pronuclei in the human zygote

still remains to be determined, although the male

pronucleus in general contains more nucleoli than

the female pronucleus.

PRONUCLEAR MOVEMENT

The male and female pronuclei are initially separate

spatially, and they then move in the zygote cytoplasm

to approach each other. This process culminates in a

close apposition of both pronuclei.

It appears that

apposition of the two pronuclei is achieved near the

spindle if a spermatozoon has penetrated close by, and

in the center of the zygote if the spermatozoon has

entered the oocyte through a region opposite the

spindle.

30,31

It was hypothesized that the movement of the

pronuclei is integrated within an overall cytoplasmic

movement (rotation) in the oocyte and zygote.

32,33

Direct evidence for zygote cytoplasmic rotation comes

from time-lapse video recordings of human sperm-

injected oocytes showing that the sperm centro-

some can organize contraction waves, resulting in

clockwise rotations of cortical granulated cyto-

plasm. This was noted in 95% of mature human MII

oocytes, beginning 2–3 hours before second polar

body extrusion following ICSI, and ending as the

second polar body was extruded.

Indirect evidence

for oocyte cytoplasmic rotation near the time of fer-

tilization comes from the observation that second

polar body extrusion often occurs distant from the

first polar body after fertilization by ICSI.

The correct function of the sperm-derived cen-

trosome, acting as a microtubule-organizing center

(MTOC), is a necessary prerequisite for pronuclear

apposition.

If a sperm-derived centrosome (aster)

has an inherent defect that prevents it from nucleat-

ing the formation of microtubules, the apposition

of pronuclei and syngamy in the human zygote

fail.

This mechanism also functions between the

pronuclei of the same gamete (sperm) origin in

polyspermically penetrated human zona-free oocytes

(Figure 3.3). A similar mechanism may be in place

in parthenogenetic human embryos, where mosaicism

is commonly observed.

POLARIZATION OF INTRAPRONUCLEAR

STRUCTURES AND INTERPRONUCLEAR

SYNCHRONY

Concomitantly with the apposition of the male and

female pronuclei, chromatin and NPBs in both

pronuclei polarize, and rotate to face the adjacent

pronucleus. Moreover, in both pronuclei, chromatin

that faces the other pronucleus becomes highly con-

densed during apposition.

This mechanism seems

to work only when pronuclei originate from different

gametes (male and female), because only an insignif-

icant, if any, intrapronuclear polarization can be

observed in multiple male pronuclei developing in

polyspermically penetrated human oocytes, even

though the pronuclei lie in close apposition to each

other (Figure 3.3).

Circumstantial evidence suggests that the pater-

nal pronucleus imposes, or collaborates in, the

formation of polar axes in mammalian eggs.

Chromatin located near the sperm tail becomes polar-

ized facing the oocyte interior as the differentiating

paternal pronucleus rotates, and this occurs even

while the sperm head lies distant from the maternal

pronucleus.

The failure of pronuclei to come into apposition

is usually associated with a failure of cleavage and



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MORPHOLOGY AND KINETICS OF HUMAN PRONUCLEI

arrest at the zygote stage. Zygotes with pronuclei of

uneven size nearly all develop into mosaic embryos.

On the other hand, failure of NPB polarization can

occur even if pronuclei are in apposition, and this

usually involves only one of the two pronuclei. Such

interpronuclear asynchrony was shown to be associ-

ated with abnormalities of further embryonic devel-

opment.

Based on the finding that intrapronuclear

RNA synthesis is required for NPB development

and growth,

together with the observation that the

failure of NPBs to polarize is typically associated with

a reduced NPB size,

interpronuclear asynchrony

seems to be caused by abnormal gene expression in

the pronucleus that is lagging behind.

ANALYSIS OF PRONUCLEI IN LIVING

HUMAN ZYGOTES

VISUALIZATION OF PRONUCLEI

Using Hoffman modulation contrast optics, the most

widely used optical system in the current IVF and

ICSI practice, pronuclei can be easily observed in liv-

ing human zygotes only after completion of pro-

nuclear envelope formation (Figure 3.4). According

to electron microscopic studies,

this occurs about

10–12 hours after fertilization. When pronuclear

differentiation and DNA synthesis are completed,

the pronuclear envelopes disintegrate, the chromatin

of both pronuclei forms a single spindle, and the

zygote enters the first cleavage division. This can

occur as early as 18–20 hours after fertilization. The

optimal time window for pronuclear observation is

thus between 12 and 16 hours after in vitro insemi-

nation or ICSI.

Both immature and fully developed NPBs can be

visualized by Hoffman modulation contrast optics.

Since the fully developed NPBs are formed by fusion

between several NPBs that are immature with the

participation of chromatin,

6,21

with advancing pro-

nuclear development, fewer NPBs can be visualized,

and the size of each NPB is greater. This process

coincides with NPB migration towards the area of

interpronuclear contact. Hence, the number, size, and

position of NPBs are key elements of any pronuclear

scoring system used in living human zygotes.Changes

in the intrapronuclear distribution of chromatin,

which is also a part of pronuclear developmental

changes (see above), are inaccessible to observation

in the living state.

POSSIBILITIES AND LIMITS OF NON-INVASIVE

PRONUCLEAR EVALUATION

The development of pronuclear scoring systems was

initially largely motivated by the need to select the

best embryos for fresh transfer and to determine

which embryos were to be cryopreserved, as early as

the zygote stage.

The first clinical study to describe a

coherent pronuclear scoring system

was performed



Figure 3.3 Electron micrograph showing multiple

sperm-derived pronuclei in a polyspermically penetrated

human oocyte. The pronuclei are in close apposition with each

other. ⫻12 000.

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