Elder K. Human preimplantation embryo selection

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HUMAN PREIMPLANTATION EMBRYO SELECTION

In mammals, altered polyadenylation is critical in

controlling the translation of the oocyte’s mRNA,

the efficiency of which is regulated by the 3´ untrans-

lated region (UTR).

Indeed, it has been demon-

strated that this is a fairly generalized phenomenon

in the mammalian oocyte and embryo.

Messages

with shortened poly(A) tails are repressed until

meiotic maturation and fertilization. At the appro-

priate time, an inversion occurs and those messages

that had been actively translated lose their poly(A)

tails and cease to be expressed, whereas the messages

that had been stored in a quiescent form become

functional.

Two signals in the UTR are required

to regulate poly(A) length, the hexanucleotide

AAUAAA and the sequence known as the cytoplas-

mic polyadenylation element (CPE).

46,47

A variety

of proteins may bind the CPE (CPE binding pro-

teins, CPEB) and thus exert translational control

of specific messages throughout development.

48,49

human gene encoding CPEB protein has been iden-

tified, and its expression in immature oocytes has

been confirmed.

This human gene, by extension,

may be involved in modulating expression through-

out early embryonic development.

The advent of microarray techniques has allowed

the human oocyte to be even more extensively ana-

lyzed.

The study of Bermudez et al

identified a

dataset of 1361 transcripts expressed in common

among all of the human oocytes examined, and 406

of these were independently confirmed by other

work. The detection of these commonly expressed

genes suggests that specific physiological pathways

may be functional in human oocytes. Conversely,

7432 transcripts were not found to be present in

oocytes, which could be indicative of pathways that

are not active during oogenesis. Alternately, the genes

may not have been activated due to patient-specific

responses to ovarian stimulation protocols, diverse

in vitro conditions, and selection criteria among study

samples. More recently, these investigators deter-

mined by microarray analysis of oocytes from

younger versus older patients that several major func-

tional categories of genes are influenced by maternal

age including genes involved in cell cycle regulation,

energy pathways, stress responses, cytoskeletal

structure and transcription.

108

A comparison of gene

expression in immature and mature human oocytes

using microarray techniques revealed 52 genes with

progressively increasing expression during oocyte

maturation.

In addition, genes were found to be

differentially expressed in cumulus cells versus

oocytes. In particular, cumulus cells were found to

over-express cell-to-cell signaling genes, numerous

complement components, semaphorins, and CD

antigen genes. Likewise, another study found

evidence of differential expression between human

oocytes and embryos.

Transcripts detected by these

genome-wide studies included those involved in

apoptosis (Bcl), cell cycle regulation (cyclins, cyclin-

dependent kinases, and checkpoint components),

signaling pathways (GTPases), and enzyme activa-

tion (casein, calmodulin, and serine/threonine

kinases), demonstrating that many conserved regu-

lators of basic cellular processes are active at early

stages of oocyte maturation. Bioinformatics tools

provide a means of carefully scrutinizing datasets

obtained by microarray methods, and this may yield

valuable information regarding processes governing

oocyte maturation; in turn, this may lead to

improvements in in vitro methods and diagnostics.

CHECKPOINTS DURING MEIOSIS: SPINDLE

AND DNA DAMAGE

Following germinal vesicle breakdown (GVBD),

maintaining the orderly segregation of chromo-

somes during meiosis is critical for oocyte health

and viability. The signal transduction machinery

which regulates the resumption of meiosis during

oocyte maturation must remain in close communi-

cation with defect sensing systems in the oocyte at

all times, otherwise errors in chromosome distribu-

tion may result. However, the very existence of these

surveillance mechanisms in the oocyte has been a

matter of controversy.

A multitude of genetic and biochemical experi-

ments have identified a signal transduction cascade

that is activated in response to spindle damage, and

this halts further cell cycle progression.

54–60

The

spindle assembly checkpoint ensures that chromo-

somes are accurately segregated by preventing

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GENE EXPRESSION ANALYSIS IN THE HUMAN OOCYTE AND EMBRYO

anaphase onset until bipolar attachment to the

spindle is achieved.

Several human genes encod-

ing kinetochore-associated proteins were initially

identified as members of this regulatory apparatus,

including mitotic arrest deficient (Mad) and bud-

ding uninhibited by benomyl (Bub).

57,62,63

In the

absence of appropriate kinetochore–microtubule

interactions, the spindle checkpoint is activated,

resulting in cell cycle arrest. Checkpoint gene prod-

ucts are believed to broadcast a wait signal from the

unattached kinetochore. The checkpoint proteins

could then potentially become modified and released

to act as soluble inhibitors.

58,64

The primary target

of this inhibitory signal is the anaphase promoting

complex (APC), the enzyme responsible for the reg-

ulated proteolysis of cyclins.

54–56

However, once

proper chromosomal alignment is achieved, the

checkpoint is quenched, bringing about the

initiation of anaphase.

57,60,62,63

Conflicting results have been reported concerning

checkpoint mechanisms in oocytes, arguing both

for

65–67

and against

68–71

their existence. The high

rate of chromosomal aberrations routinely observed

during preimplantation development would certainly

tend to support the latter conclusion.

However,

these contradictory findings could possibly be

explained by surveillance systems in oocytes that are

more permissive than those present in somatic cells.

Thus it would follow that oocytes may be more

susceptible to cell cycle error, environmental assault,

or cytoplasmic manipulations.

Additional studies have bolstered the argument

that checkpoint surveillance is indeed functional in

mammalian oocytes.

73,74

These investigators con-

firmed the expression of checkpoint proteins during

the metaphase-anaphase transition and demon-

strated that the mouse oocyte responded to spindle

aberrations in a checkpoint competent manner.

Compelling evidence supporting checkpoint activity

during meiosis came from work in yeast.

75,76

These

researchers hypothesized that an age-dependent

loss of the spindle checkpoint may be the causative

factor in the occurrence of birth defects such as Down

syndrome. This premise led to the partly successful

attempt to rescue aging-associated meiotic chromo-

some misalignment in senescence-accelerated mouse

oocytes by nuclear transplantation to the ooplasm

of young mice of another strain.

77,78

Their findings

indicate a potential cytoplasmic deficiency in the

senescence-accelerated mouse oocyte, including

extensive mitochondrial dysfunction, which may

impair spindle checkpoint function. Likewise, a

possible link between the incidence of age-related

aneuploidy and the decline in spindle checkpoint

function led to the quantification of checkpoint

gene expression during oocyte maturation.

The

results of these experiments appear to indicate that

the steady-state levels of these transcripts within

stages of maturation decrease as the oocyte ages,

perhaps due to degradation over time. Microarray

analysis by these investigators confirmed that the

expression of spindle checkpoint genes is affected

by maternal age.

108

Another study also revealed that

postovulatory aging results in a quantitative decrease

in Mad2 transcripts and an increased incidence of

premature centromere separation (PCS), leading to

a predisposition to aneuploidy.

Considerable data

have shown that PCS represents a predisposition to

chromosome missegration resulting in aneuploidy.

All in all, these studies substantiate the view that

oocytes do possess a surveillance system, albeit not

necessarily a robust one.

Securin is another key substrate of the APC, a

target of checkpoint regulation.

In addition to

cyclin degradation, the proteolysis of securin must

occur to allow further progression through the cell

cycle. Securin regulates the protein separase, which

in turn mediates cleavage of the chromosomal

cohesion complex. (Separase becomes active only

following securin destruction.) Sister chromatids

are held together by the cohesion protein complex;

when chiasmata or crossover sites form between

homologous chromosomes, cohesion is instrumental

in keeping the paired homologs together long enough

for them to orient properly with respect to the mei-

otic spindle. Consequently, cohesions are critical in

ensuring the accurate segregation of chromosomes

and averting the production of aneuploid oocytes.

The mammalian cohesin complex formed during

meiosis has not been fully characterized.

However,

it is believed to include meiosis-specific variants of

their mitotic counterparts, encoded by Smc1

␤

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HUMAN PREIMPLANTATION EMBRYO SELECTION

in the incidence of age-related aneuploidy. These

investigators used a mouse model with a mutated

Smc1

␤

gene to examine the role of this cohesin sub-

unit in oogenesis. Coincidentally, they observed an

age-dependent increase in the percentage of univalent

or unpaired chromosomes and single chromatids,

as well as a reduced number of chiasmata per oocyte

in SMC1␤ deficient mice. Thus, they postulated that

the cohesin complex containing SMC1␤ protein is

required for the maintenance of cohesion. Their

findings support the hypothesis that a progressive

loss of sister chromatid cohesion may underlie

the age-related increase in aneuploidy observed in

human female meiosis.

Double-stranded DNA breaks (DSBs) precede

recombination initiation, which is necessary to

ensure faithful chromosome segregation during

meiosis I in yeast. DSBs also probably promote

meiotic recombination in mammals.

DSB repair

genes mediate recombination repair events, with the

creation of recombination intermediates. Meiosis-

specific MutS homologs such as Msh4 and Msh5 are

believed to be involved in this process. It has been

hypothesized that the products of these genes

process nascent recombination events by stabilizing

recombination intermediates.

MutL homologs,

Mlh1 and Mlh3, act downstream of these genes by

resolving these intermediates into crossovers.

86–88

Atm (ataxia-telangiectasia-mutant) encodes a pro-

tein kinase which is also believed to be involved in

DSB repair and cell cycle control.

Its expression in

human oocytes and embryos has been examined,

and was found to be increased in fragmented

embryos.

It should be noted that differences have

been observed between male and female gametes

with regards to faulty DSB repair during recombi-

nation. Oocytes tend to be more tolerant of failed

progression or completion of recombination than

sperm.

GENE EXPRESSION DURING

PREIMPLANTATION DEVELOPMENT

The zygote relies on maternally derived messages to

direct the initial cleavage divisions, until its genome



(structural maintenance of chromosome),

Rec8,

and Stag3 (stromal antigen 3).

SMC1␤ is a cohesin

subunit that is concentrated at the arms and cen-

tromeric regions of chromosomes during meiosis I.

It remains associated with the centromeric regions

through metaphase II. REC8 and STAG3 co-localize

in fibers between sister chromatids which eventu-

ally associate with synaptonemal complex, the

proteinaceous structure that forms between synapsed

chromosomes during prophase I. Rec8 is believed to

promote sister chromatid cohesion during meiosis I

and II in a variety of species. It is also thought to

be involved in maintaining centromeric cohesion.

However, its precise role in humans has yet to be

described. Figure 21.2 provides an illustration of

selected genes involved with cell cycle regulation in

oocytes.

A recent study by Hodges et al

presented com-

pelling evidence for the involvement of cohesins

APC

Spindle checkpoint

(Mad, Bub)

DNA damage

checkpoint

(Atm, Tp53, Brca1)

MPF

(Cyclin,

Cdk1/Cdc2)

CSF

(c-mos, Cdk2)

Securin

Separase

Cohesin complex

(Smc1b, Rec8, Stag3)

Anaphase onset

Figure 21.2 Diagram of selected genes thought to be involved

in cell cycle regulation in oocytes. The anaphase promoting

complex (APC) is responsible for the proteolysis of cyclin (a

component of maturation promoting factor, MPF) and securin

(which is bound to and regulates separase). Securin degradation

activates separase, resulting in the cleavage of the chromosomal

cohesion complex and triggering the onset of anaphase. The

activity of the APC is inhibited by the spindle checkpoint which

is activated in response to the improper attachment of the

chromosomes on the spindle. The cell cycle can also be halted in

response to DNA damage. The oocyte is arrested at metaphase

II at ovulation due to the action of cytostatic factor (CSF).

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GENE EXPRESSION ANALYSIS IN THE HUMAN OOCYTE AND EMBRYO

is activated in later cleavage stages.

92,93

The transi-

tion between the dependence on maternally inher-

ited transcripts and the initiation of embryonic

gene expression does not appear to be absolute, as

maternal RNAs and their products persist in

advanced phases of preimplantation development.

Nevertheless, the switch from maternal to embry-

onic genome control can be characterized by the

selective degradation of maternally derived macro-

molecules and by the initiation of embryonic gene

expression, as well as by a shift in protein synthesis

patterns.

92,95

By postponing translation of stored

maternal transcripts, the temporal coordination of

gene expression required for mammalian oocyte–

embryo transition can be achieved.

A group of genes collectively referred to as

maternal effect genes are of particular interest.

These genes encode factors that are stored in the

oocyte, and are required for proper embryonic

development. A variety of maternal effect genes

have been identified in insects, nematodes, and

amphibians, but relatively few have been identified

in mammals. Zygote arrest 1 (ZAR1) is an ovary-

specific maternal factor that functions during the

oocyte–embryo transition, and is necessary for the

completion of fertilization.

This gene displays an

evolutionarily conserved expression pattern, and its

protein exhibits a high degree of structural homol-

ogy among divergent vertebrate species, including

human, suggesting that it plays a critical role in

development. MATER (maternal antigen that

embryos require), a maternal effect gene expressed

solely in the oocyte, was initially identified as an

autoantigen in a mouse model of autoimmune pre-

mature ovarian failure.

It is believed to be involved

in embryonic genome activation due to the devel-

opmental arrest noted in 2-cell mouse embryos

lacking MATER.

A homolog of Mater has been

identified in human.

Several other maternal effect

genes with human homologs have been identified,

all of which are believed to be master controllers in

development (Table 21.1).

Interest in human embryonic gene expression

has intensified in recent years, as the search for

accurate clinical tools that can be used to predict

embryo viability and health continues. For years,

clinicians have relied on morphological criteria as a

primary basis for embryo selection. Typically, the

embryo has been evaluated by assessing the num-

ber of cells present and the degree of fragmenta-

tion, However, a variety of additional parameters

have been examined, including the presence of

cytoplasmic inclusions, refractile bodies and/or

vacuoles, degree of granularity, symmetry of the

blastomeres, and multinucleation. Although careful

morphological scrutiny of the embryo has met with

some success, there continues to be a significant

degree of embryonic wastage in in vitro fertiliza-

tion. As a result, embryologists have resorted to the

transfer of multiple embryos, risking the incidence

of high-order births in order to increase pregnancy

rates. Extended culture of human embryos to the

blastocyst stage has also been used as a means of

providing a longer period of evaluation and selec-

tion.

100

This is an intuitively attractive approach,

since it allows embryos to be transferred at a stage

that is in synchrony with the recipient uterus.

However, development in culture to blastocyst stage

does not guarantee that the embryo is genetically

normal.

101

Furthermore, extended culture places the

embryo at increased risk of exposure to potentially

suboptimal conditions for a longer period of time.

Chromosomal screening through preimplantation

genetic diagnostic (PGD) methods has been used

successfully for years to identify embryos harboring



Table 1.1 Maternal effect genes with human

homologs

Gene symbol Gene name Reference

Zar1 zygote arrest 1 Wu et al, 2003

Mater maternal antigen Tong and Nelson, 1999;

that embryos Tong et al, 2002

require

Spin Spindlin Oh et al, 1997

109

Fmn2 Formin-2 Ryley et al, 2005;

110

Leader et al, 2002

111

Hsf1 heat shock factor-1 Christians et al, 2000;

112

Rallu et al, 1997

113

Dnmt DNA methytransferases Hamatani et al,

2004

114

Nom2 Nucleoplasmin 2 Burns et al, 2003

115

Maternal effect genes encode RNAs and proteins which are stored in the

oocyte for later use. The genes listed above all have human homologues

and are believed to influence early developmental processes.

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HUMAN PREIMPLANTATION EMBRYO SELECTION

lethal genetic anomalies.

102

However PGD has its

limitations, since it can only detect gross chromo-

somal aberrations. A technique capable of detecting

subtle yet critical irregularities is still lacking.

The search for novel diagnostic tools prompted

the analysis of gene expression and its relationship

to abnormal morphology in embryos.

Wells et al

examined the expression of multiple genes in 50

embryos at various stages of preimplantation

development. Using cluster analysis, the embryos

were sorted into distinct groups consistent with

their stage of development. However, subgroups

were identified which contained embryos display-

ing abnormal features. The embryos were also

grouped according to their abnormal characteris-

tics in order to determine if a specific abnormality

was associated with any particular expression pat-

tern, and a correlation was found between increased

fragmentation and altered expression of several

genes. The strongest correlation found was between

fragmentation and the expression of Tp53 (tumor

protein p53), a transcription factor involved in cell

cycle regulation, programmed cell death (apopto-

sis), and DNA repair. Similarly, a relationship

between the expression of Brca1 (breast cancer 1,

early onset) gene and fragmentation was also

revealed. Like TP53, Brca1 is also involved in the

regulation of the cell cycle and repair of damaged

DNA. Finally, as previously mentioned, an increase

in Atm (ataxia telangiectasia mutated) expression

was observed in embryos with moderate amounts

of fragmentation, providing further evidence for

a role for DNA damage in the etiology of this

morphological characteristic. Atm is believed to play

a central role in the control of cell cycle checkpoint

signaling pathways that are required for genome

stability and for cellular response to DNA damage.

The search for clinically relevant molecular

markers has prompted more comprehensive analy-

ses of gene expression in human embryos. A recent

study by Dobson et al

103

revealed that microarray

analysis could clearly identify stage-specific patterns

of gene expression in human embryos. Wells et al

104

confirmed this finding by real-time PCR analysis

of selected genes. Both studies reported a sharp

decrease in gene expression postfertilization, followed

by an increase after the presumptive activation of

the embryonic genome. However, Dobson’s group

found that even arrested embryos displayed expres-

sion patterns consistent with their day of develop-

ment, as long as they had cleaved at least once.

Surprisingly, these investigators observed compar-

able levels of expression for markers of normal

embryonic genome activation between normal and

arrested 2-, 3- and 4-cell embryos. They concluded

that embryonic arrest in human embryos may not

be linked to a failure in global activation of the

genome, as is the case in other mammalian

species.

96,99,105

Dobson et al

103

instead hypothesized

that the loss of embryos without gross chromoso-

mal abnormalities may be attributed to the aberrant

expression of individual genes.

Following embryonic genome activation, the

cleavage stage embryo becomes compacted and the

blastomeres are no longer distinct, giving rise to

the morula. Shortly thereafter, mammalian embryos

enter a key developmental phase characterized mor-

phologically by the formation of two distinct cell

lineages, the inner cell mass (ICM) and the trophec-

toderm (TE) of the blastocyst. Differential gene

expression is believed to be responsible for these

morphological transitions. However, the precise

nature of these stages has yet to be defined at the

molecular level. A recent study examining global

expression patterns has been undertaken to expose

the molecular cues that signal these transformations

in human embryos.

106

These researchers detected

ICM specific (i.e. Oct4/Pou5F1, Nanog, Hmgb1 and

Dppa5) and TE specific (i.e. Cdx2, Atp1b3, Sfn and

Ipl) marker transcripts, and they hypothesized that

complex metabolic and signaling pathways were

responsible for the emergence of ICM and TE cell

lineages. Another study analyzed the proteome of

individual human blastocysts using mass spectrom-

etry.

107

Consistent with the previous studies that

examined gene expression patterns in embryos,

these investigators observed differential protein

expression profiles corresponding to the stage of

development and morphology. Additional work will

be necessary to permit the thorough molecular dis-

section of the pathways responsible for blastocyst

formation.



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GENE EXPRESSION ANALYSIS IN THE HUMAN OOCYTE AND EMBRYO

CONCLUDING REMARKS

All of the studies reviewed here have produced an

immense repository of information, enabling massive

parallel mining of biological data. Further inspec-

tion of the results may form the basis for selective

analysis, using more traditional hypothesis-driven

approaches. In the process, it is anticipated that a

better explanation of how regulatory networks are

activated throughout human development may be

obtained. Likewise, insight into these physiological

pathways may provide new tools for use in clinical

diagnostics.

The complex interplay between the developing

oocyte and its surroundings is only now beginning

to be exposed, and a few key players during human

embryo development have been uncovered. Many

genes have been identified that are involved in fol-

liculogenesis, oocyte maturation, and early preim-

plantation development. Undoubtedly, many more

remain to be discovered. A better understanding of

these processes could lead to further advances in the

management and treatment of human infertility.

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22. Mitochondria in reproduction: future

assays for embryo selection

Brian Dale, Loredana Di Matteo and Martin Wilding



INTRODUCTION

Although techniques such as preimplantation genetic

diagnosis (PGD) have been used to aid in the selec-

tion for transfer of human embryos developing in

vitro,

the technology and expertise required limit

their use to specialized laboratories; selection cri-

teria in the routine IVF laboratory are largely based

on parameters that the biologist can observe

under the light microscope. These parameters include

oocyte morphology (position and size of polar

body, morphology of metaphase II plate, presence

of imperfections in the oocyte cytoplasm),

zygote

morphology (the size and position of pronuclei,

presence of a ‘cytoplasmic flare’, and nucleolar mor-

phology),

the rate of development (number of cell

cleavages/day), and morphology of embryos (such as

inequalities in cleavage and the presence of cell frag-

ments). These morphological parameters are based

on cellular physiology, and research in recent years has

revealed many aspects of embryonic physiology that

shed light on the basis for these observations.

Current data suggest that the mitochondrial

physiology of preimplantation embryos may play an

important role in human preimplantation embryo

development, and in determining embryo quality.

In this chapter, we discuss the role of mitochondria,

and in particular mitochondrial physiology in human

embryology. Some aspects of morphological analy-

sis are determined by the morphology of mitochon-

dria within the cytoplasm, and we suggest that assays

of mitochondrial activity and analysis of mitochon-

drial morphology may become important parame-

ters in the analysis of developing human embryos

and their selection for transfer into the uterus. Unfor-

tunately, assays for mitochondrial respiration that

can be incorporated into the IVF laboratory routine

are not yet available, and we look at future possibilities

for mitochondrial assessment in embryos developing

in vitro.

MITOCHONDRIA

All eukaryotic cells contain mitochondria as intra-

cellular organelles. Mitochondria are now known to

have a wide variety of critical physiological roles,

including: the triggering of apoptosis; synthesis of

pyrimidines, heme, and steroid hormones; redox

regulation; protein glycosylation; and metabolism

of neurotransmitters, calcium, and iron.

3–6

Many of

these mitochondrial pathways are tissue specific, and

all are essential for human health and development.

In yeast, mitochondrial defects prevent sporulation –

a complex developmental program that involves

cellular co-operation, pattern formation, and differ-

entiation.

In humans, disorders of mitochondrial

function are now known to contribute to maladies

such as childhood developmental disorders, seizures,

diabetes, hearing loss, blindness, heart disease, kid-

ney disease, liver disease, gastrointestinal dysmotility

disorders, antiviral drug toxicity, delayed wound heal-

ing, multiorgan system failure during sepsis, cancer,

stroke, dementia, and possibly even infertility.

6,8–12

A major mitochondrial role is to supply energy

to the cell cytoplasm through the production of aden-

osine trisphosphate (ATP). Mitochondria produce

ATP through aerobic respiration; ATP can also be

produced by anaerobic respiration in mammalian

cells. In the oocyte cytoplasm, the two processes ini-

tiate with the conversion of glucose to pyruvate, and

the production of ATP in all mammalian cells, includ-

ing human embryos, probably includes components

of both aerobic and anaerobic respiration at any time.

Aerobic respiration is significantly more efficient than

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