Elder K. Human preimplantation embryo selection

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HUMAN PREIMPLANTATION EMBRYO SELECTION

pregnancy groups in both day 3 and day 5 assays.

Significantly, the 50% group had values that fell

between the 0 and 100% groups (Figure 20.6). Using

the data obtained in the pilot studies, Bayesian prob-

ability statistics were used to establish a predictive

index of embryo viability based on a typical

Gaussian distribution (Figure 20.7).

A total of 432 samples from four different centers

were analyzed (n ⫽ 211 embryo culture media;

n ⫽ 133 seminal plasma; n ⫽ 88 follicular fluid)

with assays that routinely achieved high sensitivity

and specificity of ⬎80%. The results of these

studies demonstrate a clear relationship between the

reproductive potential of human embryos and their

modification of the media in which they have been

cultured.These subtle modifications could be detected

through BSM profiling of OS biomarkers that appear

in the media during embryo culture. Specifically, there

were detectable differences in the metabolomic pro-

files found in culture media obtained from embryos

that resulted in pregnancy compared with those that

did not.

Likewise, significant differences were seen in the

biomarker profiles of normal, healthy semen donors

compared with those with various forms of male

infertility, including idiopathic male factor infertility,



500

1000

1500 2000 2500 3000 3500

–1

–0.5

0.5

–1

–0.5

0.5 1

1.5

0.5

1.5

Implantation index

Wavelength (nm)

Number of samples Relative signal

C=C, SH groups

Figure 20.5 Raman spectral signature of three biomarkers in culture media. (A) A spectrum produced by Raman spectroscopy from

a single embryo media specimen. Three distinct biomarker regions are identified in yellow depicting C苷C, -SH, and -NH bonding at

specific wavelengths. The photon energy detected in these regions was captured and subjected to bioinformatic analysis. (B) The

implantation index score is calculated by the bioinformatic algorithms for each specimen. The distribution of metabolomic data is

shown for each of 20 media specimens: non-viable embryo scores are in blue vs viable embryo scores in red. The sensitivity and

specificity for this group of specimens were 100% and 90%, respectively.

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METABOLOMIC PROFILING OF OXIDATIVE STRESS BIOMARKERS

varicocele, and vasectomy reversal (Figure 20.8).

Profiles characteristic of oocyte quality were also

detectable in follicular fluid and correlated to preg-

nancy outcomes of their respective transferred

embryos (Figure 20.9).

CONCLUSION

This technology offers significant potential as a

tool to allow rapid non-invasive assessment of

embryonic reproductive potential prior to transfer.



–0.6 –0.4 –0.2 0 0.2 0.4 0.6

100

Implantation index

% Implantation

Day 3

–0.6 –0.4 –0.2 0 0.2 0.4 0.6 0.8 1.0 1.2

Implantation index

Day

0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.3

100

Implantation index

% Implantation

Day 5

Figure 20.6 Metabolomic results by NIR of 0, 50%, and 100% implantation rate embryos. Media from embryos that produced 0,

50%, and 100% pregnancies were analyzed in a blinded prospective study; n ⫽ 65 with an equal number of specimens per group.

(A) A statistically significant difference was observed in the metabolomic profiles of day 3 embryos that produced a pregnancy

compared with day 5 embryos. Day 5 embryos showed a greater implantations index score than day 3 embryos. t Statistic ⫺7.86;

two-tailed p ⬍0.0001. (B) A comparison of implantation index scores of three groups of day 5 embryos: 0%, 50%, and 100% pregnancy

outcomes. The 0% and 100% groups are statistically different from each other (t Statistic 3.85; p ⫽ 0.0085).The implantations index

score of the 50% group falls between the 0 and 100% group scores. (C) A comparison of implantation index scores of three groups

of day 3 embryos: 0%, 50%, and 100% pregnancy outcomes. The 0% and 100% groups are statistically different from each other

(t Statistic 3.23; p ⫽ 0.003), while the implantation index score of the 50% group fell between the 0 and 100% group scores.

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HUMAN PREIMPLANTATION EMBRYO SELECTION

Enhanced selection parameters based on metabolomic

profiling might allow a reduction in transfer order,

eventually to single embryo transfer, with the desir-

able effect of lowering multiple gestation rates while

maintaining or raising pregnancy rates.

The application of metabolomic profiling to

assess gamete viability appears to be equally prom-

ising. For example, this technique could be adapted

as a rapid diagnostic screening test for male factor

infertility. The ability to determine oocyte viability

prior to fertilization is expected to increase the num-

ber of viable embryos available for selection, transfer,

and cryopreservation. Conceivably, metabolomics

could work in concert with, or in place of, PGD as

a form of functional genomics testing. In certain

European countries where limitations on embryo

transfer are tightly regulated, viable oocyte selection

could provide a relevant means of sustaining IVF

success rates through improved oocyte fertilization

and cryopreservation techniques. Viability testing

of gametes and embryos used in cryopreservation

cycles is also a logical application of metabolomics

in IVF.



0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

Integrated mean centered CH

Integrated mean centered SH

–2500 –2000 –1500 –1000 –500 0

–100

100

200

300

400

+Pregnancy +Non-pregnancy

Probability

viability

contours

Figure 20.7 Bayesian probability statistics. A distribution of all results from embryos (pregnancy and non-pregnancy) was used to

create this Bayesian probability model. Only results from -SH and -CH biomarker populations were included to construct this curve.

The Bayesian database was then used to assess a single embryo’s metabolomic score, based on comparison with a representative

population of embryo scores between 0 and 100% implantation, to predict that embryo’s relative probability of implantation.

550 600 650 700 750 800

Wavelength (nm)

Relative absorbance

850 900 9501000 1050

–0.02

–0.01

0.01

0.02

0.03

0.04

0.05

Idiopathic

Varicocele

Vasectomy reversal

Normal donors

Figure 20.8 Near infrared spectra from seminal plasma. Seminal

plasma from four groups of men with infertility were studied by

metabolomic profiling: normal healthy donors, patients with

idiopathic male factor infertility, patients with varicocele, and

patients who underwent vasectomy reversal. A qualitative and

quantitative difference in metabolomic profiles could be discerned

between all groups of male infertility compared with normal

donors. The most pronounced difference was observed between

donors and patients with idiopathic male factor infertility. The

groups were statistically different from each other with assay

sensitivity and specificity of ⬎95% (data not shown).

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METABOLOMIC PROFILING OF OXIDATIVE STRESS BIOMARKERS

ADDITIONAL ART STUDIES

Confirmation of these observations is planned through

a large prospective study involving several ART cen-

ters. Currently, a multicenter, multinational study is

underway in nine countries using a common Insti-

tutional Review Board (IRB), approved protocol and

informed consent. IVF programs in the USA, Canada,

Japan, Belgium, Italy, The Netherlands, Finland, Spain,

and Sweden are expected to add up to 500 additional

specimens to the current database. In addition, more

than half of the IVF cycles will be single embryo

transfer cycles. This refined study design will provide

unequivocal data demonstrating the relationship bet-

ween an embryo’s metabolomic profile and its implan-

tation potential.A Metabolomic Study Group for ART

is being formed in Europe to facilitate this and further

investigations in the field using metabolomic profiling.

ADDITIONAL INDICATIONS IN REPRODUCTIVE HEALTH

Beyond the immediate applications of embryo and

gamete viability testing, the Study Group will pursue

the use of metabolomics for functional genomics

testing and to assess endometrial receptivity for

implantation. In the former scenario, metabolomics

is viewed as a possible adjunct or replacement tech-

nology for aneuploidy screening, and perhaps other

genetic testing. With regard to implantation, study

designs employing metabolomics are being reviewed

that allow non-invasive examination of the endome-

trial lining of the uterus just prior to embryo transfer.



Implantation index

Number of samples

Pregnancy

No pregnancy

Relative absorbance

Wavelength (nm)

650

700 750 800 850 900 950

–1

Figure 20.9 Near infrared spectra of follicular fluid. (A) A spectrum produced by NIR spectroscopy from a single follicular fluid

specimen. Six distinct biomarker regions are identified in yellow. The photon energy detected in these regions was captured and

subjected to bioinformatic analysis. (B) The implantation index score was calculated by the bioinformatic algorithms for each

specimen. The distribution of metabolomics data of each of 88 follicular fluid specimens showing the pregnancy outcome data of

oocytes from their respective follicular fluid that did not result in a pregnancy (blue) vs oocytes that produced viable embryos and a

pregnancy (red). The sensitivity and specificity for this group of specimens were 81% and 77%, respectively.

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HUMAN PREIMPLANTATION EMBRYO SELECTION

In the field of maternal fetal medicine, Molecular

Biometrics (Chester, New Jersey, USA), has applied

metabolomics to assess fetal development by exam-

ining biomarkers in amniotic fluid. In several hun-

dred patients studied to date, final fetal birth weight,

intrauterine growth retardation/small for gestational

age, gestational diabetes mellitus, and preterm labor

have been characterized by metabolomic profiling

(Molecular Biometrics, data on file).

INDICATIONS FOR USE IN OTHER FIELDS

This same metabolomics platform has also been

applied to the assessment of neurodegenerative dis-

ease, using biomarker profiles to distinguish between

patients with Alzheimer’s disease, Parkinson’s dis-

ease, mild cognitive impairment, and age-matched

controls. Similarly, pulmonary edema and lactate lev-

els have been accurately diagnosed and monitored

non-invasively using this same metabolomics tech-

nique (Molecular Biometrics, data on file.) These

indications are also entering clinical development

programs for development of their respective med-

ical applications.

ACKNOWLEDGMENTS

The Metabolomics Study Group for ART is compro-

mised of the following investigators: Ashok Agarwal,

The Cleveland Clinic Foundation, Cleveland, Ohio;

Barry Behr, Stanford University, Palo Alto, California;

David Burns, McGill University, Montreal, Quebec,

Canada; Joe B Massey, Reproductive Biology Asso-

ciates, Atlanta, Georgia; Peter Nagy, Reproductive

Biology Associates, Atlanta, Georgia; Denny Sakkas,

Yale University, New Haven, Connecticut; Richard

J Scott, Reproductive Medicine Associates of New

Jersey, Morristown, New Jersey; and Emre Seli, Yale

University, New Haven, Connecticut.

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to challenge by Magnaporthe grisea. Plant J 2006; 46: 351–68.

21. Goodacre R, Timmins EM, Burton R et al. Rapid identification of

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22. Thomas N, Goodacre R, Timmins EM et al. Fourier transform infrared

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31. Seli E, Sakkas D, Behr B et al. Non-invasive metabolomic profiling of

human embryo culture media correlates with pregnancy outcome.

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2006; 86 (Suppl); 117.

32. Scott, RT, Miller K, Picnic S. A prospective blinded evaluation of the

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INTRODUCTION

Large-scale analyses of gene expression during

human oogenesis and embryogenesis has been ham-

pered by a scarcity of material for analysis coupled

with a lack of highly sensitive investigational tools.

Historically, a variety of classical molecular tech-

niques have been employed to examine cellular

mRNA levels.

1–3

However, these methods were gen-

erally crude in nature and thus lacked the precision

to accurately and reproducibly detect or quantify

transcripts in single cells. Reverse transcription-

polymerase chain reaction (RT-PCR) provided a

highly sensitive means to distinguish rare mRNA

species in individual cells.

Together with fluores-

cent dyes, RT-PCR can now be used to reliably

quantify gene expression in individual oocytes.

5,6

Subsequently, PCR-based methods, and, more

recently, analysis tools that provide genome-wide

perspectives such as microarray techniques have

been used to glean a wealth of knowledge regarding

the global expression profile of the oocyte and

embryo.

7–9

However, a great deal of work remains to

be done in order to fully dissect the complex regula-

tory pathways that direct early human development.

This chapter reviews our current understanding of

these developmental programs.

GENE EXPRESSION DURING

FOLLICULOGENESIS

The human follicular cycle culminates with the

expulsion of a single mature oocyte at ovulation,

which is arrested in metaphase II. However, the

process of follicular maturation begins in utero many

years, and even decades earlier. Primordial germ cells

(PGCs) migrate to the genital ridge in the develop-

ing embryo, where they actively engage in mitosis.

The oogonia subsequently initiate meiosis, and the

resulting primary oocytes arrest at the diplotene

phase of prophase I, invest themselves with granu-

losa cells, and thus give rise to primordial follicles.

Concomitantly, those oogonia that do not enter

meiosis I undergo atresia, resulting in the eradica-

tion of all remaining oogonia by the time of birth.

Shortly thereafter, primordial follicle formation

ceases, but the process of primary follicle develop-

ment has already begun. During this process, pri-

mordial follicles develop an extracellular matrix, the

zona pellucida, and a differentiated granulosa layer.

Further proliferation of the granulosa layer gives

rise to a secondary follicle, which then also acquires

a thecal layer.

Although the origin of PGCs and formation of

primordial follicles is not yet well understood at a

molecular level, several factors that are expressed

in adjoining somatic tissues have been identified

which may affect their development. Tumor necro-

sis factor-␣ (TNF␣) expression has been confirmed

in human primordial follicles.

TNF␣ appears to be

involved in the apoptosis of random oocytes, and

this is believed to facilitate primordial follicle

assembly.

Leukemia inhibitory factor (LIF) has

been shown to induce proliferation and differentia-

tion.

Expression profiles of Kit ligand (KL) and its

tyrosine kinase receptor (c-Kit) in the human ovary

suggest that they are involved in human fertility.

While their precise roles in the human are not known,

studies from mouse mutants have revealed that KL

and c-Kit are important in the establishment of the

PGCs within the ovary, oocyte survival and growth,

granulosa cell proliferation, theca cell recruitment,

and the maintenance of meiotic arrest.

21. Gene expression analysis in the human

oocyte and embryo

Nury M Steuerwald

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HUMAN PREIMPLANTATION EMBRYO SELECTION

Genes expressed by the PGCs and oocytes them-

selves are believed to play a role in the develop-

ment of the oocyte and follicle. Human Factor in the

germline alpha (FIGLA)

appears to be associated

with cell–cell interactions and survival during

the process of primordial follicle formation.

The

murine counterpart of FIGLA also regulates the

expression of genes encoding zona pellucida pro-

teins. This role may possibly be conserved among

mammals.

It has been reported that there are four

human zona pellucida genes (ZP1, ZP2, ZP3, and

ZPB), not three as previously predicted by the

murine model.

The zona pellucida serves as the

first point of contact for sperm–egg interactions,

and it is also involved in establishing a block to

polyspermy and providing protection for the pre-

implantation embryo. The human Dazla (deleted in

azoospermia like autosome) gene is the autosomal

homolog of the Daz (deleted in azoospermia) gene

which maps to the long arm of the Y chromosome.

It encodes a cytoplasmic protein with RNA binding

domains and is believed to be involved with trans-

lational regulation. Dazla is expressed in oogonia as

well as in oocytes and granulosa cells of primordial

follicles. Its expression pattern in humans is similar

to that in mouse, where Dazla mutations cause male

and female sterility, suggesting that there may also

be functional parallels during gametogenesis.

The transition from primordial to mature follicle

is further mediated by the expression of several key

genes in the oocyte. Growth differentiation factor

(Gdf-9) and bone morphogenic protein (Bmp-15)

are oocyte-expressed genes that are both members

of the transforming growth factor ␤ superfamily.

In the human, transcripts of these genes are present

in oocytes of primary follicles and persist follow-

ing ovulation.

20,21

GDF-9 is believed to promote

granulosa cell proliferation, as well as induce

cumulus cell differentiation and expansion.

GDF-9

modulates the expression of cumulus granulosa

genes such as hyaluronic acid synthase 2 (Has2),

prostaglandin-endoperoxide synthase 2 (Ptgs2), and



Primordial

germ cell

Primordial

follicle

Primary

follicle

Secondary

follicle

Antral

follicle

Ovarian somatic cells:

Tnf

, Lif, KL,

c-Kit

PGCs: Figla,

ZP genes, Dazla

Oocyte: Gdf9, Bmp15,

bFgf, Nobox

Cumulus cells: Has2,

Ptgs2, Grem1

Figure 21.1 Genes implicated in follicle development. Follicles at various stages of development are depicted. Ovarian somatic

tissues, primordial germ cells (PGC), oocytes, and cumulus cells express various factors throughout folliculogenesis. Genes expressed

by specific cell types are listed.

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GENE EXPRESSION ANALYSIS IN THE HUMAN OOCYTE AND EMBRYO

gremlin (Grem1). The expression of these GDF-9

regulated genes has been shown to correlate with

morphological and physiological criteria consistent

with higher grade embryos, and this may serve as

a biochemical marker for embryo development

during in vitro fertilization procedures.

Bmp-15

shares a high degree of homology with Gdf-9 and is

also thought to regulate critical somatic cell activi-

ties during folliculogenesis, as evidenced by a recent

study implicating Bmp-15 mutations in the patho-

genesis of premature ovarian failure in a large

cohort of women.

Basic fibroblast growth factor

(BFGF) localizes to oocytes, granulosa, and theca

cells in the human ovary.

Human bFgf temporal

and spatial expression patterns hint at a potential

role in primordial follicle activation, as predicted by

studies in rodents.

Likewise, studies in the mouse

have identified another gene, Nobox (newborn

ovary homeobox), expressed in primordial and

growing oocytes, which appears to be essential for

oocyte survival and follicular development.

The

human gene encoding NOBOX has been identified,

but its precise function remains to be elucidated.

Figure 21.1 presents a schematic representation of

selected genes that have been implicated in oogene-

sis and folliculogenesis.

MESSENGER RNA EXPRESSION,

LOCALIZATION, STABILITY,

AND REGULATION DURING

OOGENESIS

Following many years of arrest at meiotic prophase,

the luteinizing hormone (LH) surge attending

ovulation induces the resumption of meiosis by

modulating cAMP levels, which is believed to bring

about the activation of maturation promoting fac-

tor (MPF).

MPF is a highly conserved complex

of proteins consisting of two subunits, one which

serves a regulatory role (cyclin) and another which

possesses kinase activity.

30,31

Activated MPF medi-

ates germinal vesicle breakdown by phosphorylat-

ing several proteins: histone protein phosphorylation

results in chromosomal condensation, and phos-

phorylation of nuclear envelope lamin proteins

results in its depolymerization and breakdown.

However, meiosis is again arrested at metaphase II,

presumably due to the action of a cytostatic factor

(CSF) which contains the protein products of the

c-mos and Cdk-2 genes.

33,34

CSF prevents the degra-

dation of cyclin until fertilization, when the increase

in calcium, probably initiated by the inositol

phosphate pathway,

activates a CSF protease which

allows meiosis to proceed.

It is undeniable that oocytes of several organ-

isms, including certain insects, nematode worms,

and amphibians are polarized with respect to the

distribution of maternal transcripts and proteins,

and this ultimately defines the axes of the embryo.

However, the details regarding the polarized nature

of mammalian oocytes have only recently begun to

emerge. Polarization in mammals is not as dramatic

morphologically as in low-order animals. Never-

theless, it has been described at the molecular level

in human oocytes.

38–40

Non-uniform allocation of

protein products encoded by the leptin and Stat3

genes could play a critical role in axis determination

in the oocyte as well as in the differentiation of the

embryo.

Their ultimate localization to trophecto-

dermal cells has prompted suggestions for their use

as trophectodermal markers. Members of several

classes of proteins serving a variety of functions

have also been found in polarized domains in

human oocytes and embryos.

These include

growth factors (TGF-␤2 and VEGF), growth factor

receptors (C-ERBB and C-KIT), and apopotosis

proteins (BCL-X and BAX). Asymmetric transcript

distributions of ␤-human chorionic gonadotropin

and ␤-LH amongst blastomeres in cleaving embryos

have also been reported.

36,40

Perturbations of the

spatial aspects and/or concentration gradients in

the oocyte could adversely affect its quality and

developmental potential.

Several structural features of eukaryotic mRNA

can affect the stability and may be responsible for

early embryonic message degradation and turnover.

These include polyadenylation and the formation of

secondary structures. The association of mRNAs

with cytoplasmic factors may also result in the

formation of complexes which may confer stability

to the message, or may promote its degradation.

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