Elder K. Human preimplantation embryo selection

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HUMAN PREIMPLANTATION EMBRYO SELECTION

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the time of blastocyst transfers. Hum Reprod 2001; 16: 1115–9.

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Obstet Gynecol 2002; 14: 239–44.

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three- to four-cell stage. Mol Reprod Dev 1997; 48: 442–8.

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results post-thaw? Reprod Biomed Online 2003; 6: 367–74.

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transfers. Clin Obstet Gynecol 2006; 49: 85–92.

31. Papanikolaou EG, D’haeseleer E, Verheyen G et al. Live birth rate is

significantly higher after blastocyst transfer than after cleavage-stage

embryo transfer when at least four embryos are available on day 3 of

embryo culture. A randomized prospective study. Hum Reprod 2005;

20: 3198–203.

32. Papanikolaou EG, Camus M, Fatemi HM et al. Early pregnancy loss is

significantly higher after day 3 single embryo transfer than after day 5

single blastocyst transfer in GnRH antagonist stimulated IVF cycles.

Reprod Biomed Online 2006; 12: 60–5.

33. Schoolcraft WB, Gardner DK. Blastocyst culture and transfer increases

the efficiency of oocyte donation. Fertil Steril 2000; 74: 482–6.

34. Balaban B, Urman B, Alatas C et al. Blastocyst-stage transfer of poor-

quality cleavage-stage embryos results in higher implantation rates.

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35. Levitas E, Lunenfeld E,Har-Vardi I et al. Blastocyst-stage embryo trans-

fer in patients who failed to conceive in three or more day 2–3 embryo

transfer cycles: a prospective, randomized study. Fertil Steril 2004;

81: 567–71.

36. Anderson AR, Weikert ML, Crain JL. Determining the most optimal

stage for embryo cryopreservation. Reprod Biomed Online 2004;

8: 207–11.

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transfer with vitrification using cryoloop: a 4-year follow-up study.

Fertil Steril 2005; 84: 88–92.

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38. Hardy K, Robinson FM, Paraschos T et al. Normal development and

metabolic activity of preimplantation embryos in vitro from patients

with polycystic ovaries. Hum Reprod 1995; 10: 2125–35.

39. Simon C, Garcia Velasco JJ,Valbuena D et al. Increasing uterine recep-

tivity by decreasing estradiol levels during the preimplantation period

in high responders with the use of a follicle-stimulating hormone

step-down regimen. Fertil Steril 1998; 70: 234–9.

40. Van der Auwera I, Pijnenborg R, Koninckx PR. The influence of in-

vitro culture versus stimulated and untreated oviductal environment

on mouse embryo development and implantation. Hum Reprod 1999;

14: 2570–4.

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undernutrition during the preimplantation period of rat development

causes blastocyst abnormalities and programming of postnatal hyper-

tension. Development 2000; 127: 4195–202.

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and protein on bovine follicular dynamics and embryo production in

vitro: associations with the ovarian insulin-like growth factor system.

Biol Reprod 2001; 64: 1624–32.

43. Gardner DK, Stilley K, Lane M. High protein diet inhibits inner cell

mass formation and increases apoptosis in mouse blastocysts devel-

oped in vivo by increasing the levels of ammonium in the reproductive

tract. Reprod Fertil Dev 2004; 16: 190.

44. Gardner DK, Hewitt EA, Linck D. Diet affects embryo imprinting and

fetal development. Hum Reprod 2004; 19(Suppl 1): i27.

45. Gardner DK, Reed L, Linck D, Sheehan C, Lane M. Quality control in

human in vitro fertilization. Semin Reprod Med 2005; 23: 319–24.

46. Bavister BD. Culture of preimplantation embryos: facts and artifacts.

Hum Reprod Update 1995; 1: 91–148.

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during mammalian preimplantation embryo development and their

significance in embryo culture. Theriogenology 1998; 49: 83–102.

48. Gardner DK, Lane M. Development of viable mammalian embryos in

vitro: evolution of sequential media. In: Cibelli J, Lanza RP, Campbell

KHS, West MD, editors. Principles of Cloning. San Diego: Academic

Press, 2002: 187–213.

49. Summers MC, Biggers JD. Chemically defined media and the culture

of mammalian preimplantation embryos: historical perspective and

current issues. Hum Reprod Update 2003; 9: 557–82.

50. Gardner DK, Lane M. Culture systems for the human embryo. In:

Gardner DK, Weissman A, Holwes C, Shoham Z, editors. Textbook of

assisted reproductive technology: laboratory and clinical perspectives,

Second edition. London: Martin Dunitz Press, 2004: 211–34.

51. Lane M, Gardner DK. Understanding cellular disruptions during early

embryo development that perturb viability and fetal development.

Reprod Fertill Dev 2005; 17: 371–8.

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of preimplantation mouse embryos in vitro. J Exp Zool 1978;

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53. Harlow GM, Quinn P. Foetal and placental growth in the mouse after

pre-implantation development in vitro under oxygen concentrations

of 5 and 20%. Aust J Biol Sci 1979; 32: 363–9.

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of oxygen concentration on in-vitro development of preimplantation

sheep and cattle embryos. J Reprod Fertil 1990; 89: 573–8.

55. Batt PA, Gardner DK, Cameron AW. Oxygen concentration and pro-

tein source affect the development of preimplantation goat embryos

in vitro. Reprod Fertil Dev 1991; 3: 601–7.

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ment to the blastocyst of CF1 mouse embryos: role of amino acids,

EDTA and physical parameters. Hum Reprod 1996; 11: 2703–12.

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57. Gardner DK, Lane M. Ex-vivo early embryo development and effects

on gene expression and imprinting. Reprod Fertil Dev 2005; 17:

361–70.

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following the frozen storage of expanding human blastocysts. J In

Vitro Fert Embryo Transf 1985; 2: 59–64.

59. Dokras A, Sargent IL, Barlow DH. Human blastocyst grading: an indi-

cator of developmental potential? Hum Reprod 1993; 8: 2119–27.

60. Kovacic B, Vlaisavljevic V, Reljic M, Cizek-Sajko M. Developmental

capacity of different morphological types of day 5 human morulae and

blastocysts. Reprod Biomed Online 2004; 8: 687–94.

61. Gardner DK, Schoolcraft WB. In-vitro culture of human blastocysts.

In: Jansen R, Mortimer D, editors. Towards Reproductive Certainty:

Fertility and Genetics Beyond 1999. Carnforth: Parthenon Publishing,

1999: 378–88.

62. Balaban B, Yakin K, Urman B. Randomized comparison of two differ-

ent blastocyst grading systems. Fertil Steril 2006; 85: 559–63.

63. Gardner DK, Lane M, Stevens J, Schoolcraft WB. Changing the start

temperature and cooling rate in a slow-freezing protocol increases

human blastocyst viability. Fertil Steril 2003; 79: 407–10.



64. Gardner DK, Lane M, Stevens J, Schlenker T, Schoolcraft WB. Blastocyst

score affects implantation and pregnancy outcome: towards a single

blastocyst transfer. Fertil Steril 2000; 73: 1155–58.

65. Gardner DK, Leese HJ. Assessment of embryo metabolism and viabil-

ity. In: Trounson A, Gardner DK, editors. Handbook of In Vitro

Fertilization, Second Ed. Boca Raton: CRC Press, Inc., 2000: 347–72.

66. Lane M, Gardner DK. Selection of viable mouse blastocysts prior to

transfer using a metabolic criterion. Hum Reprod 1996; 11:1975–8.

67. Gardner DK, Lane M, Stevens J, Schoolcraft WB. Noninvasive assess-

ment of human embryo nutrient consumption as a measure of devel-

opmental potential. Fertil Steril 2001; 76: 1175–80.

68. Katz-Jaffe MG, Gardner DK, Schoolcraft WB. Proteomic analysis of

individual human embryos to identify novel biomarkers of develop-

ment and viability. Fertil Steril 2006; 85: 101–7.

69. Papanikolaou EG, Camus M, Kolibianakis EM et al. In vitro fertiliza-

tion with single blastocyst-stage versus single cleavage-stage embryos.

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70. Adashi EY, Barri PN, Berkowitz R et al. Infertility therapy-associated

multiple pregnancies (births): an ongoing epidemic. Reprod Biomed

Online 2003; 7: 515–42.

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INTRODUCTION

Morphological evaluation of embryonic develop-

mental competence prior to transfer has for many

years been one of the key challenges in assisted repro-

duction. Many studies have shown that morphologi-

cal structures in the embryo can be used as biomarkers

of embryonic quality

1–4

and that embryo selection

based on morphology assessment is important to

improve implantation and pregnancy rates.

3,5–7

Most

of the existing scoring systems are based on combi-

nations of several morphological parameters such

as cleavage stage, embryonic fragmentation, and

blastomere uniformity.

1,3,7,8

However, in recent years

assessing embryo quality has generally moved from

scoring the embryos using a single grade to register-

ing individual parameters considered to be impor-

tant markers of embryo quality. This means that the

embryo selection procedure is based on a balanced

compromise after assessment of individual parame-

ters. This move towards assessment of individual

parameters has underlined the need for more objec-

tive measurements and more precise definitions. One

example is equal blastomere size. When are the

blastomere unequally sized? Some consider even the

slightest difference as unequal size, and others demand

larger differences for a classification of unequal size.

Other examples that require more precise definition

include degree of fragmentation and the size cut-

off limit between a small blastomere and a large

fragment.

Morphometric analysis of embryos is a non-

invasive method of characterizing the biological

properties of the embryo based on measurements of

individual morphological structures such as size of

the nucleus or blastomere, or features of the zona

pellucida. In contrast to biometric analysis, func-

tional parameters such as paracrine production or

nutritional uptake are not included in a morpho-

metric analysis.

A constant drive to find the best markers of

embryo competence and their incorporation into

clinical embryology is very important. This should

be focused not only on selecting the embryos with

the highest developmental competence for transfer,

but also towards improving stimulation regimens

and culture conditions and thus the quality of the

oocytes retrieved, of the embryos, and of the results

after embryo transfer. This increased demand for

more precise measurement has created a requirement

to utilize computers and for adding a third dimension

to the assessment.

A number of new techniques are now emerging

to assist the laboratory in the detailed analysis of

embryos without compromising quality that would

occur as a result of increased handling time outside

the incubator. These techniques include multilevel

digital recording of embryo images and new com-

puter systems that allow detailed analysis of these

pictures.

MONITORING AND MEASURING

HUMAN EMBRYOS

ADVANTAGES OF MORPHOMETRIC

APPROACHES

Embryo evaluation in a clinical setting is a fine bal-

ance between a detailed registration of the individ-

ual parameters and returning the embryos to the

incubator as quickly as possible. Therefore, in order

to avoid compromising embryo quality, methods for

embryo evaluation must be non-invasive and rapidly

carried out. To date, embryo selection has been based

mainly on analysis of morphology at the level of the

8. Morphometric analysis of human embryos

Christina Hnida and Søren Ziebe



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HUMAN PREIMPLANTATION EMBRYO SELECTION

light microscope, evaluating classical parameters

considered as biomarkers of embryonic competence

such as cell number, embryonic fragmentation, and

blastomere uniformity.

1,3,4,9

Embryonic structures can be measured with the

use of a scale mounted within an inverted micro-

scope. Using this technique, the size of embryonic

structures can be read directly while the embryo is

under the microscope.Alternatively, the microscopic

image can be visualized on a computer screen with a

calibrated scale, so that the image can be seen and

measured by more than one observer. However, it is

important to realize that embryo analysis under the

light microscope is based on the subjective judgment

of a single operator. Furthermore, time is a limiting

factor that makes it difficult to investigate embryo

morphology in great detail. Thus, the lack of more

precise, objective, standardized and rapid methods

remains a concern with regard to defining embryo

quality, as well as being able to gain new and more

detailed information about embryo morphology,

and its correlation with developmental competence.

Many of these limitations may be at least partly

overcome by implementing computer assisted embryo

assessment that allows detailed analysis based on high-

resolution digital images of the embryos.Additionally,

monitoring the developing oocyte and embryo by

time lapse recordings may further provide impor-

tant information regarding the kinetics of the early

human embryo.

DIGITAL IMAGE ANALYSIS

SINGLE IMAGE SYSTEM

Morphometric analysis can be improved by record-

ing and visualizing digital images of embryos, which

minimizes handling time and allows more detailed

analysis. For this purpose a digital camera must be

mounted on the microscope, with appropriate soft-

ware to support image recording.

Some systems allow the computer to control image

recording and storing as well as image analysis. Most

of these systems allow morphometric analysis based

on a single image of the embryo. In some applications,

selected morphological structures can be outlined

and subsequently analyzed semiautomatically. Out-

lining the relevant structures allows values describ-

ing the size (diameter and area) of closed structures

such as zona pellucida, oocytes, polar bodies, blas-

tomeres, or nuclear structures to be retrieved. New

parameters can be calculated automatically based

on these values, including the size of enclosed spaces

such as the perivitelline space. Furthermore, the

number of nucleolar precursor bodies and nucleoli

can be registered.

However, analyzing only a single image is associ-

ated with some fundamental problems. Important

information about the embryo may remain unde-

tected, as morphological structures that appear out-

side the focal plane may not be correctly analyzed.

This may present a problem particularly when ana-

lyzing the number of blastomeres and the degree of

fragmentation. In addition, the number of nuclei in

blastomeres outside the focal plane cannot be regis-

tered, and multinucleated embryos may remain unde-

tected. Therefore, an optimal system requires the

capacity to retrieve information from the whole

embryo, in several focal planes.

MULTILEVEL SYSTEM

The FertiMorph from IH-Medical, Denmark, is an

example of a multilevel system. This system is

equipped with a computer controlled, motorized

stepper mounted on the microscope which will auto-

matically focus through the embryo, producing a

sequence of digital images. This enables an automatic

and rapid recording in user-defined steps (Figure 8.1).

The subsequent semiautomatic morphometric

analysis is based on this sequence of images. All the

images of one sequence can be viewed in detail, so

that an image where the different morphological

structures are in focus can be selected.Morphometric

analysis of the embryo is thus based on information

from several images. The morphological structures

are outlined and subsequently analyzed in a similar

manner to that described for the single image system

(Figure 8.2). In addition to retrieving data on size

and volume for all outlined structures, the system



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MORPHOMETRIC ANALYSIS OF HUMAN EMBRYOS

also provides information on their spatial positions

inside the embryo.

TIME-LAPSE MONITORING

It is important to realize that a general problem

when analyzing embryo morphology and calculating

morphometric information at a given time point is

that it reflects a static situation. Such embryo assess-

ment does not consider the kinetics and morpho-

dynamics of the early embryo. Therefore, time-lapse

cinematography set-ups that combine culture sys-

tems and microscopes have been developed.

10,11

Furthermore, such monitoring of development from

the oocyte to the early embryo will help to elucidate

the correct timing and morphology associated with

several developmental key processes such as syn-

gamy, the first cell cleavages, compaction, and blasto-

cyst formation. The morphodynamics of embryonic

structures such as the nucleolar precursor bodies,

fragments, or blastomere size can also be monitored

by time-lapse photography.

10,11,41,42



Figure 8.1 The multilevel system is equipped with a computer controlled, motorized stepper mounted on the microscope which

automatically focuses through the embryo, producing a sequence of digital images of the embryo.

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HUMAN PREIMPLANTATION EMBRYO SELECTION

OOCYTE SIZE

Studies have shown that the diameter of the normal

egg including the zona pellucida is approximately

150–160 ␮m, with a zona pellucida thickness of

about 17–20 ␮m.

12–14

The diameter of an unfertilized

metaphase II oocyte, measured a short time after

oocyte aspiration, is approximately 115 ␮m.

12–14

oocyte of abnormally small or large size may be an

indicator of compromised biological and develop-

mental competence of the egg. A study by Wolf et al

showed abnormally low fertilization rates for oocytes



MORPHOMETRIC ANALYSIS OF THE

OOCYTE AND EARLY EMBRYO

Several studies have applied a variety of morpho-

metric principles to the analysis of oocytes and

embryos. However, very few studies have imple-

mented information from the whole embryo. In the

following section we will try to present the current

status of all types of morphometric analysis on the

parameters considered as important biomarkers of

oocyte and embryo quality.

Figure 8.2 Embryonic structures such as blastomeres or nuclei are outlined in the particular images where the structure is in focus.

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MORPHOMETRIC ANALYSIS OF HUMAN EMBRYOS

with a diameter less than 108 ␮m. Abnormally large

oocytes occur throughout the reproductive life of

most women with a frequency between 0.2 and

1.03%.

The development of giant oocytes during

IVF treatment may be linked to an augmented

response to gonadotropin therapy.

Furthermore,

most giant oocytes have been shown to be chromo-

somal abnormal.

15,45

Based on measurements of oocyte diameter

using a millimeter scale under the microscope, giant

oocytes have been shown to be approximately 30%

larger in diameter than normal oocytes, ranging from

approximately 150 ␮m excluding the zona pellucida

to 200 ␮m including the zona pellucida.

ZYGOTE SIZE

Whether the size of the zygote differs from that of

the oocyte remains uncertain. Wolf et al

found a

significant increase in oocyte diameter from day 0

to day 1. In contrast, Goyanes et al

found that the

volume of the zygote was reduced 10% compared

with the oocyte. Based on the literature, the size of a

morphologically normal human zygote is approxi-

mately 112 ␮m in diameter without the zona pellu-

cida and approximately 150 ␮m including the zona

pellucida.

BLASTOMERE SIZE DURING

CLEAVAGE STAGES

Previous studies have shown that total cytoplasmic

volume remains constant from the zygote stage and

during early embryonic development.

13,16,17

theory, this means that the volume of a blastomere at

any given time will be 50% of that in the previous cell

generation. This is, however, influenced by factors

such as fragmentation, multinucleation, and syn-

chrony and symmetry of cell cleavage (Figure 8.3).

Cleavage stage as well as blastomere uniformity

is involved in most of the existing embryo scoring

systems.

1,3,7,8

However, traditional scoring of blas-

tomere uniformity is intuitive and subjective. Very

little is known about the actual sizes and variations in

blastomeres at different cleavage stages during early

development, and precise and detailed knowledge

about normal blastomere size is necessary in order

to identify deviations from optimal development.

Additionally, minimum sizes for biologically com-

petent blastomeres must be defined in order to dis-

tinguish between blastomeres and large fragments.

Hnida et al

used multilevel morphometric

analysis to show that the average blastomere volume

is approximately 0.3 ⫻ 10

␮m

in the 2-cell embryo

and 0.15 ⫻ 10

␮m

in the 4-cell embryo. The corre-

sponding blastomere diameters were 80 ␮m and

65 ␮m, respectively. This halving in cell volume

during cleavage has been demonstrated right up

to the 16-cell stage,

13,16,17

and supports the general

impression that total cytoplasmic volume remains

constant during early embryonic development.

However, despite the fact that minor asynchrony

in the cleavage process is thought to be normal and

commonly observed,

the extent to which a cell

cleavage can be asynchronous and yet normal is still

unclear (Figure 8.3). Large variations in blastomere

size have been demonstrated to be significantly

associated with increased levels of chromosomal

abnormalities in the embryos.

THE IMPACT OF FRAGMENTATION ON

BLASTOMERE SIZE

The correlation between increasing degree of frag-

mentation, decreasing embryo quality, and clinical

outcome are well documented.

1,3–8,20,44

Fragments

originate from blastomeres, and they may therefore

extract considerable amounts of cytoplasm; two stud-

ies have suggested that this may result in blastomeres

with a quantitative deficiency of important cyto-

plasmic contents such as cell organelles, mRNA, or

proteins.

21,22

The question, therefore is whether the

fragments themselves compromise embryo quality,

or whether it is due to the reduction in blastomere

size is associated with a high degree of fragmentation.

Blastomeres in highly fragmented embryos may be

too small to be biologically competent.Additionally,

two studies

10,11

have demonstrated that some frag-

ments in some of the early embryos may disappear

at later stages by resorption or lysis. However, frag-

mentation was still apparent during late cleavage

stages in other embryos.

These findings underline



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HUMAN PREIMPLANTATION EMBRYO SELECTION

the general assumption that fragments may have

different etiologies and effects on embryo compe-

tence. Traditional analysis of fragmentation using light

microscopy is highly subjective, producing only rough

estimates

and often with huge interobserver varia-

tions.

16,19

A recent paper

indicated that the use of

digital images in embryo assessment may reduce

this variation. The lack of objectivity and standard-

ized methods of assessing fragmentation makes it

difficult to analyze correlations between fragmenta-

tion and embryo quality in detail. However, as

fragments by definition are anucleate structures of



blastomeric origin, the degree of embryonic frag-

mentation should be reflected proportionally in the

size of the blastomeres. This was demonstrated in a

morphometric analysis by Hnida et al

using the

FertiMorph computer system for multilevel analysis

based on recorded embryo sequences of a large con-

secutive cohort of 2-, 3-, or 4-cell embryos. The study

showed that the mean blastomere volume decreased

significantly with increasing degree of fragmentation

(Figure 8.4). This decrease was correlated in a

negative linear fashion for all analyzed cleavage

stages. Two-cell embryos assessed as having more

Light asynchrony

Asymmetry

Theory

Severe asynchrony

Figure 8.3 Examples of variation in blastomere size and cell generations.

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MORPHOMETRIC ANALYSIS OF HUMAN EMBRYOS

than 50% fragmentation showed a 67% reduction in

mean blastomere volume compared to 2-cell embryos

with no fragmentation. The corresponding reduction

was 44% and 43% for the 3- and 4-cell embryos,

respectively. The results from this study demonstrate

the correlation between fragmentation and blasto-

mere size.

TOTAL CYTOPLASMIC VOLUME AS A NEW

TECHNIQUE TO EVALUATE FRAGMENTATION

As the total volume of cytoplasm remains constant

during early embryonic development,

13,16,17,23

it was

suggested that the total cytoplasmic volume of the

early embryo should equal the volume of the corre-

sponding zygote. Thus, the degree of fragmentation

equals the total reduction in cytoplasm calculated as

the cytoplasmic volume of the zygote minus the com-

bined volume of the blastomeres in the embryo. This

method also corrects for inter-embryonic size varia-

tion, as the total volume of the blastomeres in each

particular embryo is correlated to its own zygotic

volume. Assessment of fragmentation based on such

embryo-specific reduction in cytoplasmic volume

would provide a more objective and standardized

method. However, precise assessment of the zygote

and blastomere sizes is a minimum requirement for

successful application of this method

Hnida et al

used multilevel digital imaging

system to take sequential images of all included

zygotes, and of all their resulting day 2 embryos.

Furthermore, the embryos were assessed for frag-

mentation by traditional evaluation. The outer bor-

der of the cell was outlined for all zygotes and for all

embryos, and the outer border of all morphological

structures considered to be blastomeres was also

outlined. The mean reduction in cytoplasmic vol-

ume was found to increase significantly in a linear

manner with increasing degree of fragmentation, as

assessed by traditional evaluation. Comparing these

findings to traditional fragmentation analysis

showed that 27% of the embryos were allocated to a

fragmentation category that did not correspond to

the cytoplasmic reduction analysis (Figure 8.5).

Overall, the data from this study suggest that the

reduction in cytoplasmic volume from the zygote to

the cleaved embryo stage might be used to quantify

the degree of fragmentation in individual early

embryos. This is in line with the finding by Goyanes

et al

showing that the total blastomeric volume is

significantly decreased in fragmented embryos com-

pared with the volume of their zygotes. However,

correct differentiation between blastomeres and frag-

ments is a key parameter when evaluating the degree



0 1–10 11–20 21–50

>50

0.1

0.2

0.3

0.4

% Fragmentation

Blastomere volume (µm

× 10

)

2-cells

3-cells

4-cells

Figure 8.4 Blastomere volume is significantly correlated to the

degree of fragmentation at the early cleavage stages.

–70

–60

–50

–40

–30

–20

–10

% Fragmentation

Cytoplasmic reduction (%)

0 1–10 11–20 21–50 >50

Figure 8.5 The mean reduction in total cytoplasmic volume is

significantly correlated to degree of fragmentation, as assessed

by traditional evaluation.

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