Elder K. Human preimplantation embryo selection

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HUMAN PREIMPLANTATION EMBRYO SELECTION

anaerobic respiration, producing an energy output

(⌬G) of ⫺686 kcal/mol glucose compared with ⌬G

of ⫺47 kcal/mol glucose for anaerobic respiration.

Therefore, it is logical to suggest that aerobic

respiration is the preferred choice when sufficient

oxygen is available.

Mitochondria are membrane-bound intracel-

lular organelles, and also contain a second inner

membrane. The outer mitochondrial membrane is

permeable to molecules with molecular weights of

up to 10 000 Da.

The inner mitochondrial mem-

brane is relatively impermeable and surrounds the

‘matrix’. The inner mitochondrial membrane con-

tains most of the enzymes required for the respira-

tory chain, and has foldings, or ‘cristae’, which give

this membrane a greater surface area for the electron

transport chain. The number of cristae in a mito-

chondrion indicates the degree of oxidative phospho-

rylation taking place, and corresponds to its capacity

to produce ATP. In fact, local mitochondrial config-

urations often represent local energy requirements

in the body.

The mitochondrial matrix also contains a small

amount of DNA termed ‘mitochondrial DNA’

(mtDNA), 16 560 kb of double stranded DNA that

codes for 13 proteins, 22 transfer RNA (tRNA) species,

and two ribosomal RNA (rRNA) species.

16–19

Only a

small portion of the enzymes required for the respi-

ratory chain are coded for by mtDNA; the majority

are derived from the nuclear DNA. The current con-

sensus is that one copy of mtDNA is present for

every single mitochondrion in the mammalian oocyte,

and this therefore provides a useful means of esti-

mating the density of mitochondria within these

cells.

Mitochondrial DNA is subject to degenera-

tion via the action of free radicals produced within

the mitochondrial matrix during oxidative phos-

phorylation.

21,22

It is thought that mtDNA has little

in the way of DNA repair or protection mecha-

nisms.

23,24

Free radical attack can influence the pro-

duction of some of the proteins involved in the

respiratory chain, and over long periods this could

result in reduced efficiency of the respiratory path-

way.

13,25,26

The respiratory chain consists of a series of enzy-

mes that catalyze a cyclic pathway (‘Krebs’ cycle)

in which carbon dioxide and energy are produced

from substrates of oxygen and carbon atoms in mol-

ecules such as pyruvate and glucose. The energy pro-

duced through the respiratory chain is converted to

ATP via an indirect pathway, in which H

⫹

ions pro-

duced by the respiratory chain are expelled from the

matrix. This process creates an electrophysio-

logically negatively charged environment within the

matrix, causing the expelled H

⫹

ions to be attracted

towards the mitochondrial matrix. The H

⫹

ions

return to the matrix through the action of an ATPase

enzyme, thus linking ATP production to the activity

of the respiratory chain. Therefore, according to

Mitchell’s chemiosmotic hypothesis,

28,29

the H

⫹

potential (⌬⌿) of the inner mitochondrial

membrane is highly correlated with the capacity

of individual mitochondria to produce ATP.

MITOCHONDRIA IN MAMMALIAN OOCYTES

AND EMBRYOS

The mitochondrial ‘bottleneck’ theory states that

the mitochondrial content of primordial oocytes is

progressively reduced to a pool of 3–4 copies of

mitochondria which then replicates to form the com-

plement of mature oocytes.

30,31

Therefore, the rela-

tive ‘fitness’ of these original components strongly

determines the quality of mitochondria within the

mature oocyte. Although some paternal mitochon-

dria may survive after fertilization,

the majority of

spermatozoal mitochondria are either targeted for

destruction after fertilization,

or simply become

dysfunctional, so that inheritance of mitochondria

is nearly 100% maternal. By measuring the mtDNA

content of individual oocytes, it has been estimated

that mature oocytes contain between 20 000 and

800 000 mitochondria.

26,33,34

Interestingly, oocytes

from patients with fertilization failure for unknown

causes were often found to contain low copy

numbers of mtDNA, suggesting that mtDNA copy

number, and therefore number of mitochondria,

may be indicative of fertilization potential.

Mitochondria in human oocytes and preimplan-

tation embryos undergo distinct changes in local-

ization during each phase of development, and are



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MITOCHONDRIA IN REPRODUCTION: FUTURE ASSAYS FOR EMBRYO SELECTION

spatially reorganized during mammalian oocyte mat-

uration. In germinal vesicle (GV) stage oocytes, the

cytoplasm surrounding the germinal vesicle is very

dense (Figure 22.1A), and our data confirm previous

reports demonstrating that this clustering represents

the localization of mitochondria

26,36

(Figure 22.1A

and B).After GV breakdown (BD), mitochondria reor-

ganize and tend to distribute more evenly throughout

the cytoplasm (Figure 22.1C and D). Some groups

have observed a higher density of mitochondria in

the region of the metaphase I and II meiotic appara-

tus,

26,36

but this is not always visible (Figures 22.1



and 22.2); we have observed a mitochondria-free

region of cytoplasm directly surrounding the meiotic

apparatus (Figure 22.2C and D). Human metaphase

II oocytes have a wide variety of morphological con-

figurations, and this appears to be reflected in the den-

sity and morphology of mitochondria, as visualized

under fluorescence microscopy (Figure 22.2). Using

a variety of mitochondria specific fluorescence dyes

and confocal microscopy, we have observed that not

Figure 22.1 Mitochondrial morphology in human immature

oocytes. Confocal microscopy images of immature human

oocytes donated for research (donations were made prior to

2004, after which legislation forbade research on human

embryos)

loaded with the mitochondrial specific fluorescence

dye 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolyl-

carbocyanine iodide (JC-1). (A) and (B) Images of a germinal

vesicle (GV) stage human oocyte. (C) and (D) Images of a

metaphase I (MI) stage human oocyte. (A) and (C) show the

bright-field images of the oocytes (non-confocal) and (B) and

(D) show a single confocal section of the mitochondrial mem-

brane potential-insensitive channel (green fluorescence output).

‘I’ shows mitochondrial type I morphology. ‘II’ shows mito-

chondrial type II morphology (bright-field visualization in (A)

and (C'), and fluorescence images in (B) and (D)). White bars

represent 20 ␮m.

Figure 22.2 Mitochondrial morphology in human mature

oocytes. Confocal microscopy images of fresh mature human

oocytes (metaphase II stage) donated for research (donations

were made prior to 2004, after which legislation forbade

research on human embryos).

Oocytes were loaded with the

mitochondrial specific fluorescence dye JC-1. (A) and (B)

Images of a fresh metaphase II (MII) stage human oocyte with

polarized granularity. (C) and (D) Images of a MII stage human

oocyte with a ‘smooth’ (non-polarized) cytoplasm. (A) and (C)

show the bright-field images of the oocytes (non-confocal) and

(B) and (D) show a single confocal section of the mitochondrial

membrane potential-insensitive channel (green fluorescence

output). ‘I’ shows mitochondrial type I morphology. ‘II’ shows

mitochondrial type II morphology (bright-field visualization in

(A) and (C), and fluorescence images in (B) and (D)). ‘A’

denotes an area surrounding the meiotic apparatus in which no

mitochondria are observed (see (D)). White bars represent

20 ␮m.

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HUMAN PREIMPLANTATION EMBRYO SELECTION

Type II mitochondria are small and compact and

exist at a very high density. This morphological type

appears to correlate with a smooth appearance of

the oocyte cytoplasm.

Whereas GV oocytes are char-

acterized by type I morphology towards the periph-

ery of the oocyte, and some small areas of type II

mitochondrial morphology surrounding the GV

(Figure 22.1B), the cytoplasm of metaphase I and II

oocytes is often polarized for the two distinct

morphologies

(Figures 22.1 and 22.2). Attempts

have been made to correlate these morphological

observations with developmental characteristics (see

Table 22.1 and below). After fertilization in mam-

malian oocytes, changes in the distribution of mito-

chondria occur in synchrony with each round of cell

division. In the zygote, mitochondria may be

densely packed around the pronuclei, although this

is often absent

26,36

(Figure 22.3). This morphology

often appears when the blastomere nucleus re-forms

after each round of division is completed, alternating

with a more even distribution of mitochondria at

the time of nuclear membrane breakdown. Inter-

estingly, mitochondria cluster around the presumed

location of the centrosome in the preimplantation



only the density, but also the morphological appear-

ance of mitochondria correlate with features observed

under the light microscope

(Figures 22.1–22.4). Two

distinct mitochondrial morphologies can be seen

throughout human oogenesis and preimplantation

embryo development

(Figures 22.1–22.4). Type I

mitochondria appear as loosely packed clusters,

usually towards the periphery of the cytoplasm

(Figures 22.1–22.4). This morphology appears to

correlate with a granular appearance of the oocyte

cytoplasm when visualized under light microscopy.

Figure 22.3 Mitochondrial morphology in human zygotes.

Confocal microscopy images of human zygotes donated for

research, 19–20 hours after insemination (donations were made

prior to 2004, after which legislation forbade research on

human embryos).

Zygotes were loaded with the mitochondrial

specific fluorescence dye JC-1. (A) and (B) Images of a human

zygote with contracted cytoplasm surrounding the pronuclei.

(no cytoplasmic contraction) surrounding the pronuclei.

(A) and (C) show the bright-field images of the oocytes (non-

confocal) and (B) and (D) a single confocal section of the mito-

chondrial membrane potential-insensitive channel (green

fluorescence output). ‘I’ shows mitochondrial type I morphol-

ogy. ‘II’ shows mitochondrial type II morphology (bright-field

visualization in (A) and (C), and fluorescence images in (B) and

(D)). White bars represent 20 ␮m.

Figure 22.4 Mitochondrial morphology in human embryos.

Confocal microscopy images of a human six blastomere embryo

67 hours after insemination (embryos were donated for research

prior to 2004, after which legislation forbade research on

human embryos).

Embryos were loaded with the mitochon-

drial specific fluorescence dye JC-1. (A) Shows the bright-field

image of the embryo (non-confocal) and (B) a single confocal

section of the mitochondrial membrane potential-insensitive

channel (green fluorescence output). ‘I’ shows mitochondrial

type I morphology. ‘II’ shows mitochondrial type II morphol-

ogy (bright-field visualization in (A), and fluorescence images

in (B)). White bars represent 20 ␮m.

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MITOCHONDRIA IN REPRODUCTION: FUTURE ASSAYS FOR EMBRYO SELECTION

embryo, suggesting that this structure requires a high

energy input (Figure 22.4). Both active (as revealed

by Rh123, Mitotracker, or JC-1 staining)

37,39,40

and

inactive mitochondria (as visualized with nonyl

acridine orange (NAO) staining),

are reorganized in

the same manner during development, suggesting

that mitochondrial relocalization is not activity dep-

endent, although highly active mitochondria may

be localized to peripheral regions.

The role of

mitochondrial reorganization during each phase of

development may reflect the energy requirement of

distinct regions of the cytoplasm,

26,36

and therefore

the normal physiology of oocyte and embryo

development.

RELATIONSHIP BETWEEN MITOCHONDRIAL

LOCALIZATION, ACTIVITY, AND HUMAN

EMBRYO DEVELOPMENT

The above data demonstrate that mitochondria loc-

alize to distinct regions of the oocyte and embryo

cytoplasm during preimplantation embryo devel-

opment and that they are active during this period.

However, evidence regarding the role of these two

features of mitochondria in mammalian preimplan-

tation embryo development is less well characterized.

MITOCHONDRIAL LOCALIZATION

Observations on human metaphase II oocytes prior

to intracytoplasmic sperm injection (ICSI) reveal a

wide variety of morphologies in the oocyte cyto-

plasm, and that the cytoplasm may be smooth or

granular in appearance under the light microscope.

The distribution of these two types of cytoplasm is

highly diverse, but is often polarized to distinct

regions of the cytoplasm. Our group and others have

recorded the appearance of the human oocyte prior

to ICSI in an attempt to correlate these distinct types

of morphology with fertilization, development, and

implantation potential of individual oocytes.

41–43

Human oocytes present a wide variety of morpho-

logical features, even within cohorts of oocytes from

individual patients. Data from fluorescence labeling

suggest that these granular features of oocytes rep-

resent distinct zones of mitochondrial localization.

In our laboratory, we characterized the different

oocyte morphologies into three groups for simplicity:

granularity towards one side of the oocyte (type I),

granularity in the center of the oocyte (type II), and

complete absence of granularity (type III, Table 22.1).

Patients may have a mixture of morphologies within

a cohort of oocytes, but in some cases have oocytes

with a single morphological type. When a single



Table 22.1 Correlation between oocyte morphology and developmental characteristics

during intracytoplasmic sperm injection (ICSI). Data refer to ICSI cycles where all oocytes

observed prior to microinjection were of a single morphological type

Type I Type II Type III

Number of patients 56 44 32

Age in years (mean ⫾ SD) 33.4 ⫾ 5.5 32.8 ⫾ 4.9 33 ⫾ 4.0

Number of ICSI cycles 56 44 32

Number of oocytes retrieved (mean ⫾ SD) 634 (11.1 ⫾ 3.5) 515 (10.8 ⫾ 4.0) 305 (9.2 ⫾ 3.6)

Number of oocytes with normal fertilization 480 (75.8%) 284 (55.1%) 122 (40%)

(% fertilization)

Number of embryos transferred (mean ⫾ SD) 152 (2.7 ⫾ 0.4) 128 (2.9 ⫾ 0.3) 89 (2.8 ⫾ 0.5)

Number of grade I embryos transferred

135 (88.8%) 91 (71.1%) 65 (73.3%)

(% of transferred)

Number of pregnancies 22 (39.3%) 16 (36.4%) 11 (34.4%)

Number of gestational sacs (implantation 36 (23.7%) 28 (21.8%) 13 (14.6%)

rate per embryo transferred, %)

Type I, oocytes with granularity to one side of the cytoplasm; type II, oocytes where granularity was observed in the center

of the oocyte; type III, oocytes where granularity was not observed.

Grade I embryos were scored by both morphology and growth rate using previously described methods.

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HUMAN PREIMPLANTATION EMBRYO SELECTION

morphological type is present, we can correlate clin-

ical and biological parameters after fertilization and

embryo transfer. In 132 patients from a total of 190

observed to have a single morphological type within

the analysis period (Table 22.1), correlations between

these oocyte types and clinical and biological param-

eters suggest that fertilization rates and embryo devel-

opment do not show obvious differences. However,

there was a significant difference in the clinical out-

come based on oocyte morphology. Therefore, fea-

tures of oocyte morphology may provide an insight

into the developmental history, and in consequence

the implantation potential of each individual oocyte.

An accumulation of cytoplasm around the pro-

nuclei can also be observed in the zygotes of human

and other mammalian species, and this and other

features have been applied to selection criteria in the

IVF laboratory. Staining with mitochondrial dyes

has shown that this accumulated material includes

a large proportion of mitochondria. However, this

effect is not always seen in human zygotes. Zygotes

which have abnormal, or no cytoplasmic accumula-

tion around the pronuclei show slower rates of

development, abnormal distributions of mitochon-

dria within the embryo, and significant differences

in ATP content between blastomeres

40,44

(Table 22.2).

Therefore, the zygote stage could be considered an

essential stage in the developmental programming

of the fertilized oocyte.Again,the cytoplasm of human

preimplantation embryos is often characterized by

granularity observed in the nuclear region. These

morphological features continue through embryo

development and may be highly indicative of human

embryo implantation potential.

MITOCHONDRIAL ACTIVITY

As described above, mitochondrial activity, meas-

ured by either mitochondrial membrane potential

or oxygen consumption, is a measure of the level

of aerobic respiration within oocytes. Correlations

between the state of perifollicular vascularity of

developing follicles, the dissolved oxygen content of

follicular fluid, and the quality of oocytes retrieved

from these follicles suggests that oxidative phospho-

rylation plays an important role in oogenesis,

44,45

and aerobic respiration appears to be a vital source

of energy during oocyte maturation. After the lute-

inizing hormone (LH) surge, the available evidence

also suggests that mitochondrial metabolism has a

fundamental role in the final maturation of the oocyte,

since the rate of oxygen consumption, a measure of

mitochondrial metabolism, increases dramatically

in the isolated oocyte after the LH surge.

Although

an increase in lactic acid is also observed after the

LH surge, an indication of anaerobic respiration,



Table 22.2 Correlation between zygote morphology and developmental characteristics

during ICSI. Data refer to ICSI cycles where zygotes of a single morphological type were

observed 19–20 hours after microinjection and selected for transfer on day 2.

Type I Type II Type III

Number of patients 64 55 13

Age in years (mean ⫾ SD) 32.3 ⫾ 5.0 33.5 ⫾ 7.6 32.8 ⫾ 6.0

Number of ICSI cycles 64 55 13

Number of zygotes selected (mean ⫾ SD) 189 (2.9 ⫾ 0.4) 143 (2.6 ⫾ 0.6) 37 (2.8 ⫾ 0.3)

Number of grade I embryos

157 (83.1%) 109 (76.2%) 25 (67.6%)

(% of zygotes)

Number of embryos transferred 189 143 37

Number of pregnancies 25 (39.1%) 21 (38.2%) 3 (23.1%)

Number of gestational sacs (implantation 47 (24.9%) 26 (18.2%) 4 (10.8%)

rate per embryo transferred, %)

Type I, zygotes with pronuclei surrounded by granular cytoplasm in the center of the cell; type II, zygotes with pronuclei

surrounded by granular cytoplasm to one side of the cell; type III, zygotes where granularity was not observed.

Grade I embryos were scored by both morphology and growth rate using previously described methods.

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blocking anaerobic respiration with iodoacetate

does not inhibit oocyte maturation, suggesting that

anaerobic respiration is not necessary for oocyte

maturation.

In contrast, suppressing aerobic res-

piration does block oocyte maturation.

However,

the aerobic respiration measured here probably

reflects the activity of granulosa cells surrounding

the oocyte within the follicle.

After release from the follicle and fertilization,

mammalian oocytes and embryos are no longer

dependent on helper cells, and start to produce and

utilize their own energy. Although aerobic respira-

tion is clearly present in human preimplantation

embryos, its role in development is controversial.

Mitochondria within human oocytes have few matrix

membrane foldings and appear relatively inactive.

In fact, estimates of the contribution of mitochon-

drial respiration to the energetic requirement of mam-

malian embryo development suggest that as little as

10% of glucose is metabolized through aerobic res-

piration in the early stages of development, although

this rises to 85% in the blastocyst.

However, since

aerobic respiration is 14-fold more efficient than

anaerobic respiration with respect to ATP produc-

tion, the 10% of glucose passing through aerobic

respiration produces more energy in the form of

ATP than the 90% of glucose metabolized without

mitochondria. Mammalian oocytes and preimplan-

tation embryos prefer pyruvate as an energy source,

and this preference for pyruvate does not eliminate

either aerobic or anaerobic respiration as a possible

route to ATP production. Lactic acid is present in

fluid sampled from human fallopian tubes,

and is

also produced during mammalian embryo develop-

ment in vitro,

suggesting that there is a compo-

nent of anaerobic respiration in the ATP-generating

mechanism. Mitochondria within early cleavage stage

preimplantation embryos do not replicate, and con-

tain poorly formed cristae, so the level of mitochon-

drial respiration may be low during this stage.

Aerobic respiration appears to be upregulated at the

stage of blastocyst development: the mitochondrial

cristae become more compact, mitochondria start to

replicate, and the utilization of glucose as a carbo-

hydrate substrate increases.

These data suggest that

both aerobic and anaerobic respiration pathways

are active during oocyte maturation and embryo

preimplantation development, but aerobic respira-

tion is upregulated during blastocyst development

and implantation.

Despite the fact that anaerobic respiration can

permit some degree of mammalian embryo devel-

opment in the absence of aerobic respiration, current

evidence suggests that a minimum level of aerobic

respiration is required to enable embryo implanta-

tion and subsequent development of the mammalian

fetus.

This hypothesis may explain the ‘maternal

age effect’ observed in human fertility.

Oocyte mito-

chondria replicate from a small initial pool of organ-

elles residing in primordial follicles created in the

fetus; some primordial follicles reside in the human

ovary for up to 40 years, and are therefore subject to

the degeneration of the mtDNA through free radi-

cal attack. It has therefore been suggested that the

reduction in fertility associated with advanced mat-

ernal age is caused by a slow loss in the capacity of

mitochondria to provide the energy required.

Although a precise relationship is yet to be defined,

there are some molecular data to support this

hypothesis.

50–52

Physiological data also suggest that

there is a relationship between mitochondrial activ-

ity and maternal age;

recent data suggest that the

level of aerobic respiration may be directly corre-

lated with maternal age,

with a significant influ-

ence on embryo quality.

Mitochondrial activity

decreases linearly with increasing age and influences

both embryo development and IVF outcome,

13,26,37,53

and therefore appears to exert a strong influence on

the inherent developmental potential of individual

oocytes. Our previous data using fresh oocytes and

embryos donated to research suggest that mitochon-

drial activity does not change between the maturing

oocyte and the first stages of human embryo devel-

opment,

and this indicates that measuring the level

of aerobic respiration in individual human oocytes

could provide a strong indication of the implantation

potential of developing embryos.

37,53

Experimental

elimination of mitochondrial activity in human and

mammalian embryos does not immediately block

embryo development. Evidence from mice suggests

that embryos can develop, and even implant ade-

quately in the absence of mitochondrial respiration,



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HUMAN PREIMPLANTATION EMBRYO SELECTION

although fetal development does not continue until

birth.

54–56

These data suggest that anaerobic respira-

tion can support preimplantation embryo develop-

ment, at least in the mouse. In the human, eliminating

energy substrates in the culture medium lowers the

mitochondrial membrane potential and slows devel-

opment.

It must be assumed that the lack of aero-

bic respiration can be compensated for, at least in the

short term, by anaerobic respiration, enabling suffi-

cient energy production to permit the completion

of the cell cycle. Our present hypothesis suggests that

aerobic and anaerobic respiration are coupled, and

despite the fact that embryo development can per-

sist in the absence of aerobic respiration,

implanta-

tion requires a minimum level of aerobic respiration.

CAN MITOCHONDRIAL MORPHOLOGY OR

ACTIVITY BE USED AS A PARAMETER TO

ASSIST EMBRYO SELECTION?

The above data demonstrate that human oocyte and

embryo morphology is related to the morphology

of mitochondria within the oocyte cytoplasm, and

suggest that the activity of mitochondria is impor-

tant for embryo development. Could these parame-

ters then be used to aid in embryo selection? Although

we do not suggest that a single criterion can be used

in selection, it is possible that the selection of embryos

for transfer may be assisted by adding parameters

related to mitochondria. Currently, embryo selection

criteria include the rate of embryo development in

vitro, the morphology of these embryos (blastomere

volumes, presence of fragments, etc.)

, and to a lesser

extent zygote morphology.

Although some analysis

of oocytes is included during ICSI protocols,

propose that oocyte cytoplasmic morphology could

be added as a selection criterion. Our data suggest

that the cytoplasmic morphology of human oocytes

is an indicator of development potential (Table 22.1),

and the presence of ‘polarized’ cytoplasm in the

oocyte seems to be a positive indicator of implanta-

tion potential. However, the current evidence suggests

that oocyte morphology alone is not highly predic-

tive, as high quality embryos and pregnancy often

result from a variety of cytoplasmic morphologies

(Table 22.1). Therefore, assessment of oocyte

cytoplasmic morphology should be added to the

embryology routine, but not applied uniquely.

The level of mitochondrial activity is not currently

tested, but this could prove to be a highly useful tool

in embryo selection criteria, even before fertilization.

However, technical difficulties limit the application of

these assays in the clinical laboratory. At present,

oxygen consumption and fluorescence techniques can

be used to measure the activity of mitochondria in

mammalian oocytes and embryos. Fluorescence tech-

niques measure the electrophysiological membrane

potential across the mitochondrial matrix, and are

therefore an indirect measure of mitochondrial

activity. However, this technique involves the intro-

duction of potentially hazardous dyes into the oocyte

cytoplasm, and therefore it cannot be applied clini-

cally. The advantage of the technique is that meas-

urements can be made quickly in single oocytes and

embryos, permitting individual variations in activity

to be observed. Data compiled from fluorescence

techniques have demonstrated that the activity of

mitochondria in oocytes and preimplantation

embryos is both patient and oocyte specific.

37,58

The

measurement of oxygen consumption is a direct

measure of mitochondrial activity, and this can be

done via two techniques. The first uses an oil soluble,

non-toxic quarternary benzoid compound, pyrene

(Sigma-Aldrich, St. Louis, MO, USA), to measure

fluorescence that is reversibly quenched by oxygen.

Pyrene’s oil solubility is an advantage, as this facili-

tates the technique’s application in IVF culture, where

an oil overlay is often employed. However, in order to

measure oxygen consumption, oocytes and embryos

must be cultured in an air-tight apparatus for

appreciable lengths of time (up to 4 hours) and in

minute quantities of culture media in the absence of

air.

Even under these circumstances, the minute

amounts of oxygen consumed by individual oocytes

and embryos (19.6 pmol/embryo/ hour)

mean that

a minimum of three embryos are required to allow

the measurement of oxygen consumption.

There-

fore, the oxygen consumption of individual human

oocytes and embryos has not yet been determined.

A second technique utilizes the fluorescent dye

tris-1,7-diphenyl-1,10-phenanthroline ruthenium (II)



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chloride (BD Biosciences, Bedford, MA, USA) the

fluorescence of which is also reversibly quenched by

oxygen. The advantage of this technique is that the

dye can be embedded in a gas-permeable silicone

polymer and used to coat standard cell culture plates

for analysis. Theoretically, the set-up and analysis

of the material can be performed inside a standard

incubator, although minute quantities of cell cul-

ture media and sealed conditions are again required.

This system has not been tested on human oocytes

or embryos at the present time.

A third technique for measuring mitochondrial

activity is through the use of coenzymes such as

nicotinamide adenine dinucleotide (NAD), nicotin-

amide adenine dinucleotide phosphate (NADP),

or flavine adenine dinucleotide (FAD), which are

vital to metabolic pathways through their properties

of reversible oxidation. The relative proportion of

these compounds in the cytoplasm can be directly

measured by non-invasive fluorescence techniques.

NAD(P)H emits fluorescence at 445 nm when excited

at a wavelength of 340 nm, whereas FAD

⫹⫹

emits

fluorescence at 520 nm when excited at a wavelength

of 458 nm. Both NAD(P) and FAD are linked to the

aerobic and anaerobic metabolic pathways.

There-

fore, the level of fluorescence derived from individ-

ual oocytes could be used to determine the relative

levels of aerobic and anaerobic metabolism in cells.

However, excitation of NAD(P)H requires ultravio-

let light (340 nm), and this unfortunately poses risks

for clinical use. These risks could theoretically be

overcome by the use of newer techniques such as

two-photon fluorescence microscopy.

FAD

⫹⫹

flu-

orescence can be measured with visible light wave-

lengths, and this is therefore safe. Both NADH and

FAD

⫹⫹

fluorescence have been successfully meas-

ured in mouse eggs,

but calibrating the fluores-

cence output in terms of mitochondrial activity is a

problem that remains to be solved. Genetically, the

measurement of mitochondrial DNA mutations or

the expression of mitochondrial genes could be used

to determine the health of an embryo. Both large

mitochondrial DNA mutations and point muta-

tions have been detected in human oocytes and

preimplantation embryos.

50,52,63

Although no defi-

nite relationship has yet been defined between these

mutations and reproductive capacity, the fact that

the incidence of point mutations in human oocytes

was correlated with maternal age appears signifi-

cant.

The expression of several mitochondrial genes

has also been measured in unfertilized oocytes and

arrested embryos from assisted reproduction pro-

grams.

Data suggesting a correlation may in the

future serve as a diagnostic tool for the selection of

embryos for transfer.

CONCLUSIONS

Physiological techniques that can be applied to the

selection of viable embryos for transfer during IVF

cycles are currently scarce. In contrast, morphologi-

cal and chromosomal data are abundant. Although

morphological and chromosomal analyses are easily

applied in the IVF laboratory, they do not reliably

determine an important parameter – the implanta-

tion potential of the human embryo. Identifying the

implantation potential of the human preimplanta-

tion embryo from the stage of the unfertilized human

oocyte could prove to be a highly effective tool in

embryo selection, especially in combination with

diverse techniques applied to the embryology

routine in order to assist selection.

We suggest that

measuring an aspect of oocyte physiology such as

mitochondrial activity could prove to be a very

useful addition to the complement of selection

techniques available. Although our data suggest that

characteristics of oocyte and embryo cytoplasm are

related to mitochondrial morphology, this obser-

vation is limited by the fact that mitochondrial

morphology, at the level revealed by fluorescence

microscopy, is not related to mitochondrial activity.

In recent years, there has been an increased interest

in the role of mitochondrial metabolism during

preimplantation human embryogenesis. Current data

suggest that measuring mitochondrial metabolism

may be useful as an aid in selecting human oocytes

and embryos for transfer, if the technical difficulties

inherent in measuring mitochondrial metabolism can

be overcome to the extent that analyses can be per-

formed during routine IVF cycles. We feel that this

could become an important tool for successful in

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HUMAN PREIMPLANTATION EMBRYO SELECTION

vitro fertilization. In combination with morpholog-

ical assessment and chromosomal analysis, the assess-

ment of mitochondria may add an important step

towards the goal of IVF – the transfer of a single

embryo.

ACKNOWLEDGMENTS

We thank the Fondazione Nuovi Orizzonti, Naples,

Italy and Vincenzo Monfrecola for technical support.

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