Elder K. Human preimplantation embryo selection

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HUMAN PREIMPLANTATION EMBRYO SELECTION

stages.

38,64,70,71

Overall, amino acid depletion increases

from early cleavage stage up to the blastocyst stage.

In human embryos, the amino acid profile differed

between embryos that reached the blastocyst stage

compared with those that arrested.

39,72

Developing

embryos depleted amino acids from the culture

medium to a lesser extent than did those that arrested,

and the profile was also different, in that developing

embryos took up significantly more alanine,

glycine, and serine.

However, the amino acid pro-

file could not be related to embryo morphology.

Appearance of alanine in the culture medium dur-

ing early cleavage could be used to select embryos

that will arrest their development. Houghton et al

reported that a higher concentration of alanine in

the culture medium was observed for arrested

embryos, and suggested that alanine is used to

metabolize ammonium production by embryos. The

presence of high levels of alanine in the culture

medium can therefore reflect the level of amino

acid turnover and ammonium production. As

developing embryos have a low turnover level, the

amount of alanine in the culture medium can be

related to embryo viability and therefore may help

to select embryos before transfer.

Leucine is

another amino acid that could be useful to assay, as

leucine is the only amino acid that is significantly

depleted from the culture by early cleavage stage

human embryos.

39,72

OXYGEN CONSUMPTION

Oxygen is essential for the conversion of ADP to

ATP in oxidative phosphorylation through its role

as an electron acceptor in the mitochondrial elec-

tron transport chain. Compared with the extensive

analysis of the effects of carbohydrates or amino

acids on early embryo development, few data are

available for oxygen consumption. However, oxygen

consumption reflects embryo metabolism and via-

bility more closely. Indeed, oxidative phosphoryla-

tion requires both oxygen and mitochondrial

integrity. Oxygen uptake by mouse embryos is pres-

ent from the 1-cell stage, and increases drastically at

the blastocyst stage.

A similar pattern is observed

for bovine embryos, although the consumption of

oxygen during oocyte maturation is as high as that

during blastocyst formation.

OTHER MARKERS OF EMBRYO VIABILITY

HUMAN CHORIONIC GONADOTROPIN

The culture of embryos up to the blastocyst stage is

a means of improving embryo selection prior to

transfer, as it is considered that grossly abnormal

embryos will arrest their development before reach-

ing the blastocyst stage. Blastocyst embryos are also

more metabolically active. Dokras et al

therefore

tested the secretion of human chorionic gonado-

tropin (hCG) by individual postcompaction stage

human embryos. Unfortunately, although secretion

of hCG correlated well with blastocyst morphology,

it could not be detected until day 8, and therefore is

not detectable early enough to assist in the selection

of embryos for transfer.

CONCLUSIONS

Early embryo development is a complex process

involving many different metabolic reactions that

require the production of energy. Deficiencies in

ATP production can be responsible for alterations

in gene expression, chromosomal segregation, and

signal transduction cascades. Alterations in the meta-

bolic status of early embryos can therefore impair

embryo viability. Embryo viability is dependent on

both the oocyte from which it derives and in vitro

culture conditions. In this respect intrafollicular

and ooplasmic conditions may also have an

important influence on the chromosomal status or

cytoplasmic reserve necessary for the embryos to

undergo the first cellular division and to survive

in vitro conditions. Indeed, these conditions impose

a great deal of cellular stress on preimplantation

embryos. Although early cleavage stage embryos

possess a great plasticity, their adaptation to in vitro

conditions impairs their viability as reflected by

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lower cleavage rates, cleavage arrest, poor morphology,

retarded genome activation, abnormal gene

expression, or energy production. The more an

embryo has to adapt, the less likely it is to be viable.

Similar to the rat,

in the mouse and human there

is little vasculature in the vicinity of the implanta-

tion site for several hours. Glycolysis will therefore

be the only available means of energy production

for the blastocyst during this period.

Analysis of

variations in metabolic activity during the different

stages of development could be of great help in the

evaluation of embryo viability and its potential to

form a neonate.

In summary, different metabolic studies show

that embryos are relatively metabolically quiescent

prior to compaction, and oxidative phosphoryla-

tion is the major pathway for energy production.

After activation of the embryonic genome, embryos

are more active and a shift to energy production

occurs with the suppression of phosphofructokinase

inhibition, the enzyme that converts fructose

6-phosphate to fructose 1,6-diphosphate, or at the

level of hexokinase, where it is converted into glu-

cose 6-phosphate (Figure 15.1). This adaptation is

thought to be necessary for embryos to survive in

hypoxic conditions at the time of implantation,

although oxidative phosphorylation is still active.

The formation of the blastocoel cavity and the

first two lineages are processes that require a large

amount of energy. They are accompanied by a

drastic increase in gene expression and protein syn-

thesis.

6,22,74

Pyruvate, glucose, and amino acid con-

sumption and lactate production are all related to

embryo viability. Oxygen, which is one of the most

important substrates in energy production, has

been relatively less studied. All experiments seem to

agree that embryos with a relatively low metabolic

activity, i.e. low level of lactate production and

amino acid turnover, are those with the highest

potential.Viable embryos that do not have to strug-

gle to develop in vitro are probably those with a

metabolic activity comparable with that of those

developed in vivo.

1,75

Indeed, transformation of

high amounts of glucose into lactate is associated

with a general suppression of respiration and

oxidative phosphorylation (Crabtree-like effect)

and modification of the redox status of the embryo.

Glucose phosphorylation into glucose 6-phosphate

by hexokinase depletes the stock of cellular and

mitochondrial ATP. An excessive production of

lactate decreases the level of NADH necessary for

oxidative phosphorylation. Both mechanisms impair

the function of the mitochondrial electron trans-

port chain reactions, which in turn decreases the

amount of energy available for biosynthetic

processes.

14,22

The ratio of ATP/ADP decreases,

raising phosphofructokinase inhibition and result-

ing in the activation of the glycolytic pathway. This

excessive production of lactate during the cleavage

stage could therefore impair embryo viability, by

altering mitochondrial energy production.

However, the lack of prediction shown by meta-

bolic measurements could be due to different factors.

First, those performed during early cleavage stages

represent oocyte competence more than that of the

future embryos. Although correct oocyte matura-

tion and preservation of the integrity of cellular

components and metabolic pathways are important

to sustain future development, it is probably not

sufficient to predict viability of later stages. Second,

carbohydrates and amino acids represent only a

fraction of embryo metabolism. Little is known

about lipid metabolism, which can be a valuable

source of energy substrate. Third, the majority of

nutrient uptake and production studies have been

performed in suboptimal culture media. Further

experiments should be performed in better adapted

culture media, for example a sequential salt solution

supplemented with human serum albumin, pyru-

vate, low level of glucose (0.5 mmol/l), so-called

‘non essential’ amino acids with glycine, taurine, and

leucine for cleavage stage embryos and all amino

acids with 1 mmol/l glucose for later embryo develop-

ment. Such culture media should probably contain

some growth factors, but this remains to be ana-

lyzed in detail. Fourth, human embryos display a

large range of values for pyruvate, lactate, glucose, or

amino acids, which renders it difficult to use a single

value to predict viability. Finally, all the methods

actually employed to measure metabolic activity –

even if simplified – are too time consuming for

routine use in an IVF program.

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HUMAN PREIMPLANTATION EMBRYO SELECTION

ACKNOWLEDGMENTS

I thank Professor Kate Hardy for welcoming me into

her laboratory. With great patience and kindness,

she trained me to handle preimplantation embryos

in culture in vitro as well as in the different tech-

niques used in this work. She was most generous

with her time in teaching me indispensable analyti-

cal methods; her comments were always pertinent

and most helpful. I am grateful to Professor Yvon

Englert for enabling me to pursue my research suc-

cessfully and his constant encouragement and many

constructive discussions. The work was supported

by the Belgian Funds for National Research.

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INTRODUCTION

The goal of in vitro fertilization (IVF) and embryo

culture is to provide high quality embryos capable

of continued development and implantation, which

will result in the birth of healthy babies. Considerable

progress has been made in culturing preimplantation

embryos since the initial studies were undertaken. We

began to design and define new, more complex culture

media in the early 1970s, which were based on the

composition of genital tract secretions.

During the

initial stages of zygote formation and early cleavage

divisions, the cells carry out only a minimal level of

transcription, since early preimplantation develop-

ment, i.e. up to the stage of maternal to zygotic tran-

sition (MZT), is maternally driven. A mature oocyte

must contain a storage pool of proteins and/or

mRNA transcripts in order to maintain its viability

during these early stages: all of the enzymes required

for metabolic pathways must be present and in har-

mony with the components of the culture medium.

During and after the cycle of MZT, the longest cycle

of preimplantation development, transcription of

the new zygote genome then results in an increase

in mRNA levels. The requirements of the embryo

before and after MZT differ, and the environment,

i.e. culture conditions, will have a direct impact on

transcription and translation. Moreover, epigenetic

reprogramming throughout early preimplantation

development is also important, and this has generated

concerns regarding the role of culture conditions in

assisted reproductive technologies.

2–6

In this chap-

ter we describe the basis of embryo metabolism, and

the impact of culture media composition on embryo

quality and viability.

16. Preimplantation embryo metabolism

and embryo interaction with the

in vitro environment

Yves J R Ménézo and Pierre Guérin



IN VITRO CULTURE CONDITIONS AND

ENVIRONMENTAL FACTORS

The physical conditions used during in vitro culture

differ significantly from conditions in vivo, in terms

of light, variations in pH, pCO

,temperature,

static medium, etc. Physiological pH is regulated

by a HCO

/CO

buffer according to the equation

pH = pKa ⫹ log (HCO

)/(CO

). This Henderson-

Hasselbach equation allows pH to be calculated under

a 5% CO

atmosphere, in relation to the concentra-

tion of bicarbonate. The embryo has an alkaline pH

(7.4), and is not able to deal with an acidic pH before

the stage of MZT. The gas phase may be from 5% to

6.5% CO

in air, or 5% CO

,5% O

and 90% N

Based on the genital tract environment and an

increased potential to decrease free radical forma-

tion, a reduced O

atmosphere might appear to be

more physiological. However, no clear-cut data

support the superiority of reduced oxygen tension in

human embryo culture in terms of ongoing pregnancy

rates, particularly if the medium is well protected by

the addition of agents that counteract the reactive

oxygen species (ROS). Gas phase contaminants

such as NO and CO are deleterious, as are volatile

organic compounds (VOCs). The effect of ammonia

is discussed in the paragraph describing amino acid

metabolism.

An osmolarity between 280 and 300 mosmol

allows fertilization and early embryonic development.

Redox potential is difficult to evaluate, and is there-

fore rarely considered. Based on oviduct and uterine

secretions, redox potential should be calibrated to

⫺0.1 mV. In culture conditions, antioxidants can

turn into pro-oxidants, this is the case for vitamin C,

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HUMAN PREIMPLANTATION EMBRYO SELECTION

especially in the presence of ferric ions. On the other

hand, an excessively reducing redox potential may

have harmful effects on protein tertiary structure.

Glutathione is sometimes added to culture media

as a reducing agent, but it should be noted that this

cannot enter the embryo. The addition of serum

destabilizes the culture media, by contributing

enzymes as well as unknown metabolites and catabo-

lites. Serum does not make a useful contribution, and

may even be deleterious by potentially increasing

pathologies, in particular those linked to imprint-

ing.

7,8

GLUCOSE AND ITS METABOLITES

(FIGURE 16.1)

High levels of glucose have been considered to be a

major factor in precipitating embryonic develop-

mental arrest during in vitro culture. The present

trend towards removing glucose and phosphate from

mammalian embryo culture media is not physiolog-

ical, and merely replaces one artifact with another.

The mouse model should be interpreted with care

as glucose metabolism in rodents may be impaired

due to a metabolic cul de sac that gives glucose poor

entry into the TCA cycle, with a block at the level of

glucose 6-phosphate isomerase that leads to a useless

accumulation of glycogen. However, there is a species

specific difference between rodents and humans,

and the human zygote has a completely different fea-

ture in its enzyme activity: hexokinase activity is high,

and glycogen synthase is low.

The activity of the

pentose phosphate pathway is equally high in human

and in rodents. High levels of glucose also have a

deleterious effect through an increase in free radical

formation. This aspect is particularly obvious in dia-

betic mammals, and is more than probably the case

in humans. Glucose itself is not toxic per se, and its

metabolism is necessary for the synthesis of ATP.



Hexokinase

G6Pi

Nucleic

acids

+ H

O + ATP

G6PDH

Glycoproteins,

glycolipids…

Amino

acids

Tricarboxylic

cycle

Amino

acids

Fructose 6-phosphate

Fructose 1,6-diphosphate

Pyruvate

LDH

Glycogen

Amino

acids

Lactate

Hexokinase

Fructose

Glucosamine-6-

phosphate

Ribose

Deoxyribose

Glucose

Glu

Glucose 6-phosphate

Gln

Figure 16.1 Glucose and fructose metabolism in the embryo. Glucose metabolism leads to the synthesis of nucleic acids and amino acids,

and produces energy. G6Pi, glucose 6-phosphoisomerase; G6PDH, glucose 6-phosphate dehydrogenase; LDH, lactate dehydrogenase.

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EMBRYO METABOLISM AND INTERACTION WITH IN VITRO ENVIRONMENT

The problem arises when too much glucose is present

in a medium that has an inadequate balance of sub-

strates. This is the case for several metabolic pathways,

including purine salvage in bovine (Figure 16.2),

where excess of glucose also induces shifts in the sex

ratio towards female

as the female embryo has a

more effective antiapoptotic mechanism. A relation-

ship between glucose-related apoptosis and monozy-

gotic twinning in humans has been proposed.

Pyruvate, on the other hand, is an interesting com-

pound, in that it acts not only as an energy source,

but can also detoxify ammonia in the embryo, through

transamination and export of alanine formed as a

result (Figure 16.3). It also plays a role in protection

against oxidative stress by preventing peroxide

induced injury. Lactate is an end product of metab-

olism, and therefore adding it to culture media is of

questionable value. In order to be re-introduced into

metabolism, lactate must be converted (oxidized) to

pyruvate, with generation of NADH. These short

chain carboxylic acids require monocarboxylate trans-

porters (MCTs) and their chaperone protein basigin

to allow their entry into metabolic pathways; both of

these are present in human oocytes and embryos.

These C2/3 carbon chains are also used for the syn-

thesis of lipids. Other glucose-derived metabolites

(such as acetate, citrate, malate, etc.) are sometimes

added to media, although no real impact on devel-

opment has been established; they will be used for

the synthesis of amino acids.

LIPID METABOLISM

The embryo needs to synthesize ‘sophisticated’

lipids at a very early stage. For this purpose, it is able

to ‘pick up’ saturated and unsaturated fatty acids,

free or bound to proteins, from its environment. The

ratio of individual fatty acids is much more impor-

tant than the presence of any single one. Phospholipid

synthesis can be carried out with exogenous choline

and glucose (metabolized toward fatty acid synthesis).

In the mouse oocyte, cholesterol levels increase

(three fold) from the 1-cell to the blastocyst stage.

Cholesterol can be taken up from the environment,

and it can be synthesized from the precursors meval-

onate and lanosterol, as well as from acetate that is

present as a result of pyruvate (and probably lactate)

decarboxylation. Blocking the metabolic pathway

for cholesterol synthesis with specific inhibitors

such as compactine or diosgenine leads to develop-

mental arrest before the blastocyst stage (Figure 16.4).

Cholesterol and fatty acids bound to albumin can also

be directly incorporated into the embryo.

AMINO ACIDS

Amino acids are necessary very early after fertiliza-

tion, and even for short-term embryo handling.

Active amino acid synthesis is grafted onto the tricar-

boxylic acid (TCA) cycle. Amino acids are used for

protein synthesis after translation of the mRNA stored

during maturation, and again for messages that are

translated after MZT. Moreover, there is an accelerated

protein turnover under in vitro conditions. Micro-

array technology has demonstrated that expression

of 114 genes is affected in culture without amino

acids, versus 29 genes with affected expression in cul-

ture with amino acids.

Glycine is present at a higher



HPRT

G6PDH

–

Glucose

Pentose

phosphate

pathway

Purines

Hypoxanthine

Xanthine

2233

Glucose 6-phospate

Figure 16.2 Glucose and purine metabolism. Excess glucose

will inhibit the purine salvage pathway and thus increase

reactive oxygen species (ROS) formation. (1) Glucose inhibits

hypoxanthine phosphoribosyl transferase (HPRT). (2) Purine

catabolism. (3) Purine salvage. HK, hexokinase.

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HUMAN PREIMPLANTATION EMBRYO SELECTION

concentration than any other amino acid in the

female genital tract.It can reach 5 mmol,10–50 times

higher than the other amino acids that are present

during the embryo’s transition through the tract.

Glycine, glutamine, alanine, and taurine may act as

organic osmolytes, allowing endogenous osmolarity

and volume regulation, and preventing an eventual

‘salting out’ effect. Amino acid uptake takes place

through active transport, and the overall scheme is

complex. Different amino acids will compete for the

same class of transporters, and individual affinities

will allow a higher or lower incorporation. Moreover,

some transporters are subject to a kinetic catabolism

before genomic activation, while others appear after

MZT. It is impossible to have a clear idea of the quan-

titative exchanges for each amino acid in the develop-

ing embryo, as the efficiency of uptake may differ

widely, as is the case for glycine and methionine

(Met). Glycine transport in the embryo is severely

reduced in the presence of Met (Figure 16.5). It

should also be noted that the Met concentration in

vivo is much lower than the concentration of glycine.

The concentrations of the different amino acids



2 Acetyl CoA

Acetoacetyl CoA

β HMG-CoA

MEVALONATE

β HMG-CoA reductase

NADPH

NADP

CoA

CHOLESTEROL

NADPH

Fatty acid

synthesis

Diosgenine

Figure 16.4 Cholesterol synthesis and its inhibition in the

mouse embryo. Inhibition of this pathway will cause the embryo

to arrest before blastocyst stage. HMG, hydroxymethyl-glutaryl.

Glucose

Glycine

Glyoxylate

GPT

GOT

pyruvatepyruvate

Pyruvate

glutamine

Glutamine

TCA

Cycle

TCA

cycle

Alanine

Lactate

Pyruvate Glutamate

Oxoglutarate

Aspartate

Acetyl CoA

Oxaloacetate

Alanine

Malate

Figure 16.3 Transamination reactions in the embryo. GPT, glutamate-pyruvate transaminase; GOT, glutamate-oxaloacetate

transaminase.

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EMBRYO METABOLISM AND INTERACTION WITH IN VITRO ENVIRONMENT

found in vivo are probably an important considera-

tion for the composition of culture media. Several

points requiring clarification are discussed below.

ESSENTIAL AMINO ACIDS AND

SULFUR AMINO ACIDS

It has been suggested that ‘essential amino acids’

may be toxic during preimplantation development

before the stage of MZT.

However, the distinction

between essential and non-essential amino acids is

somewhat unclear, as some amino acids can be pro-

duced from others. Phenylalanine, valine, trypto-

phan, threonine, isoleucine, methionine, histidine,

arginine, lysine, leucine, cysteine, and tyrosine were

described by Waymouth in a particular context

(diploid cell culture).

Leucine uptake is consid-

ered to be a good marker of embryo quality before

genomic activation (MZT), indicating that this ‘essen-

tial amino acid’ may be important for human

embryo construction.

Methionine is classified as an essential amino

acid and is thus omitted in the majority of first phase

sequential media. This is a questionable concept,

because in mouse embryos, silent paternal alleles of

H19, Igf2, Grb10, and Grb7 are aberrantly expressed

and hypomethylated in simple media, but not in

media with amino acids. In reality, the balance

between Met and the other amino acids is of major

importance. Met has such a high affinity for the

transporter molecules that if it is present in too high

a concentration, it may prevent the uptake of other

amino acids, thus disrupting the equilibrium so that

the endogenous pool becomes unbalanced. Met is

required for the initiation of all protein synthesis

through Met-tRNA (Figure 16.6), and is incorpo-

rated by mouse, bovine, and human embryos. Equally

important, Met is the fuel of methylation through the



Glycine

free

Glycine

in proteins

Glycine uptake

With methionine

Without methionine

Export (ala)

(2) AA competition

(4) Protein

catabolism

Carrier

Glycine

(1) AA external

concentration

(3) Protein

synthesis

Methionine

100 µmol/l

250 µmol/l

AA uptake

Time

50 µmol/l

Amino acid

pool

Figure 16.5 Factors influencing the endogenous pool of amino acids (AA) in the embryo. (1) The external concentration of each

amino acid affects its entry into the embryo. (2) Competition between amino acids for the same carriers will facilitate the entry of

some and reduce uptake of others. (3) Protein synthesis will decrease the endogenous pool. (4) Protein turnover will partly increase

the endogenous pool.

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