Elder K. Human preimplantation embryo selection

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HUMAN PREIMPLANTATION EMBRYO SELECTION

a link between blastomere size and multinucleation.

This is supported by the findings of Hnida et al.

18,24

One possible explanation for the impaired qual-

ity of these embryos could be that the presence of

unequally sized blastomeres indicates that they have

divided in an asynchronous or asymmetrical pattern,

or that one or more of the cells have ceased to divide

(Figure 8.3). However, the fact remains that we are

currently at the stage of having only limited knowl-

edge about the order of magnitude of difference

between blastomere size that is required in order to

compromise the embryo’s developmental potential.

MULTINUCLEATION

Transfer of embryos with multinucleated blastomeres

has been shown to be associated with decreased

implantation, pregnancy, and birth rates.

25–27

Further-

more, multinucleated embryos have increased rates of

chromosomal abnormalities.

28–30

Thus, multinucle-

ated embryos should be excluded from transfer

28,29

and assessment of nuclear status should be included

in embryo scoring systems.

25,26

Van Royen et al

found multinucleation in 34% of a cohort of embryos

from patients undergoing IVF or ICSI treatment.

Hnida et al

showed that the volume of multinucle-

ated blastomeres was significantly larger than their

mononucleated sibling blastomeres (22% in 2-cell

and 30% in 4-cell embryos). These findings support

other studies indicating that an intraembryonic vari-

ation in blastomere diameters of more than approx-

imately 25% is associated with increased rates of

multinucleation and chromosomal abnormalities.

19,30

However, these studies did not measure the precise

blastomere sizes. It has previously been suggested

that multinucleated blastomeres can originate from

an uncoupling of processes that control karyokinesis

and cytokinesis, resulting in duplication of the nucleus

without subsequent cell cleavage.

17,31

The conse-

quence of this would be a multinucleated blastomere

without the size reduction from cell cleavage, thus

retaining the size of the previous cell generation.

These speculations are supported by the findings of

Hnida et al

18,24

demonstrating that a multinucleated

4-cell blastomere was approximately the same size as

a non-multinucleated 2-cell blastomere (Figure 8.6).



of fragmentation based on blastomere sizes or reduc-

tion in cytoplasm.

CELL SIZE CUT-OFF LIMITS

Minimum sizes for biologically competent blas-

tomeres must be defined in order to distinguish

between small blastomeres and large fragments.

Based on the presence of DNA, Johansson et al

suggested a cut-off size limit between blastomeres

and fragments of 45 ␮m in diameter in day 2 embryos

and 40 ␮m in day 3 embryos. However, this study

did not differentiate between different cleavage stages

observed on day 2 or day 3, respectively. Addition-

ally, Hnida et al

analyzed separate blastomeres and

found that none of the analyzed 4-cell blastomeres

smaller than 50 ␮m in diameter contained DNA,

whereas 96% of the blastomeres with a diameter

larger than 50 ␮m contained DNA. However, as the

diameter of the blastomeres in the intact 4-cell

embryos was approximately 3% smaller compared

with the separate blastomeres, Hnida et al suggest a

cut-off diameter of approximately 45–50 ␮m between

blastomeres and fragments in 4-cell stage embryos.

EMBRYOS WITH BLASTOMERES OF UNEVEN SIZE

The impact of unequal-sized blastomeres in an

embryo has long been discussed, as this might be

part of normal embryo development, particularly in

dividing embryos (Figure 8.3). There is also no gen-

eral definition regarding how large the difference

must be in order for blastomeres to be classified as

of unequal size. However, in recent years a number of

studies have demonstrated that the developmental

potential of embryos with blastomeres of unequal

sizes is compromised. Studies have demonstrated

6,30

that implantation and pregnancy rates were both

significantly lowered after transfer of embryos with

unevenly sized blastomeres. Two studies

19,30

found a

highly significant correlation between embryos with

blastomeres having more than 25% difference in

blastomere size and chromosomal abnormality.

Further, the findings by Hardarson et al

indicate

that embryos with unevenly sized blastomeres have

increased rates of multinucleation and that there is

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MORPHOMETRIC ANALYSIS OF HUMAN EMBRYOS

In normal cleaving cells, there is a very close

interaction and timing of the processes that

control karyokinesis and cytokinesis.

32,33

However,

other mechanisms may also be involved in the forma-

tion of multinucleated blastomeres, including errors

in chromosome migration at mitosis, incorrect pack-

aging of chromosomes by the nuclear membrane

after mitosis, or fragmentation of the nuclei.

17,22,31

Additionally, mononucleated blastomeres originat-

ing from multinucleated embryos are on average

smaller in size than the blastomeres from mono-

nucleated embryos (Figure 8.7), and the frequency of

anucleate blastomeres (by definition large fragments)

is higher in multinucleated embryos.

18,24

This indi-

cates that asymmetric cell cleavage may also be asso-

ciated with the occurrence of multinucleation.

17,18,24

In conclusion, detection of nuclear status, espe-

cially in embryos that are otherwise of good mor-

phology is of great importance in order to improve

clinical outcome. However, assessment of multinu-

cleation cannot be evaluated on the basis of blas-

tomere size alone. Detection of embryos with

unevenly sized blastomeres should be used in com-

bination with visual verification of nuclear structures.

Hnida et al

analyzed the nuclear status in a cohort

of embryos and showed that significantly more

embryos were correctly categorized using the

multilevel digital imaging system compared with

traditional evaluation.

SIZE OF NUCLEI

Despite the fact that nuclear : cell volume ratio is

known to control the timing of events in early embry-

onic development in other species,

34,35

very little is

known about nuclear sizes in human embryos. Using

morphometric multilevel measurements to assess the

size of the nuclei in good quality mononucleated

2-cell embryos showed a diameter of 22.1 ␮m and a

volume of 0.006 ⫻ 10

␮m

. This decreased to a

diameter of 18.7 ␮m and a volume of 0.003 ⫻ 10

␮m

in 4-cell embryos. These findings suggest a consis-

tent nuclear : cell volume ratio of approximately 0.2

at least up to the 4-cell stage in human embryos.

KINETICS OF EARLY EMBRYONIC

DEVELOPMENT

Embryo assessment traditionally consists of scor-

ing individual features such as cell number and

fragmentation. However, it is important to bear in



2-cells

3-cells

4-cells

0.1

0.2

0.3

0.4

Blastomere volume (µm

× 10

)

Multinucleated blastomeres

Mononucleated blastomeres

Figure 8.6 It has previously been suggested that multinucle-

ation may originate from an uncoupling of processes that con-

trol karyokineses and cytokinesis, resulting in duplication of the

nucleus without subsequent cell cleavage. These speculations are

supported by the findings that a multinucleated 4-cell blastomere

is approximately the same size as a non-multinucleated 2-cell

blastomere.

Figure 8.7 The volume of multinucleated blastomeres is signifi-

cantly larger than their mononucleated sibling blastomeres.

Further, mononucleated blastomeres from multinucleated

embryos are smaller in size than the blastomeres from

mononucleated embryos.

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HUMAN PREIMPLANTATION EMBRYO SELECTION

analysis also presents many exciting possibilities.

Although this technique is still young and needs

further development, some very promising possi-

bilities are already available today.

REFERENCES

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tation after in-vitro fertilization: based on 957 single embryo transfers.

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4. Van Royen E, Mangelschots K, De Neubourg D et al. Characterization

of top quality embryo, a step towards single-embryo transfer. Hum

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6. Ziebe S, Petersen K, Lindenberg S et al. Embryo morphology or

cleavage stage: how to select the best embryo for transfer after in vitro

fertilization. Hum Reprod 1997; 12: 1545–9.

7. Erenus M, Zouves C, Rajamahendran P et al. The effect of embryo

quality on subsequent pregnancy rates after in vitro fertilization. Fertil

Steril 1991; 56: 707–10.

8. Steer CV, Mills CL, Tan SL, Campbell S and Edwards RG. The cumula-

tive embryo score: a predictive embryo scoring technique to select the

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9. Van Royen E, Mangelschots K, De Noubourg D et al. Calculating the

implantation potential of day 3 embryos in woman younger than 38

years of age: a new model. Hum Reprod 2001; 16: 326–32.

10. Van Blerkom J, Davis P and Alexander S. A microscopic and biochem-

ical study of fragmentation phenotypes in stage-appropriate human

embryos. Hum Reprod 2001; 16: 719–29.

11. Hardarson T, Lofman C, Coull G et al. Internalization of cellular frag-

ments in a human embryo: time-lapse recordings. Reprod Biomed

Online 2002; 5: 36–8.

12. Tsuji K, Sowa M and Nakano R. Relationship between human oocyte

maturation and different follicular sizes. Biol Reprod 1985; 32: 413–17.

13. Goyanes VJ, Ron-Corzo A, Costas E and Maneiro E. Morphometric cat-

egorization of the human oocyte and early conceptus. Hum Reprod

1990; 5: 613–18.

14. Wolf JP, Bulwa S, Rodrigues D and Jouannet P. Human oocyte cytometry

and fertilisation rate after subzonal insemination. Zygote 1995; 3: 101–9.

15. Balakier H and Cadesky K. The frequency and developmental capabil-

ity of human embryos containing multinucleated blastomeres. Hum

Reprod 1997; 12: 800–4.

16. Roux C, Joanne C, Agnani G et al. Morphometric parameters of living

human in-vitro fertilization embryos; importance of asynchronous

division process. Hum Reprod 1995; 10: 1201–7.

17. Hardy K,Winston RML and Handyside AH. Binucleate blastomeres in

preimplantation human embryos in vitro: failure of cytokinesis dur-

ing early cleavage. J Rep Fertil 1993; 98: 549–58.

18. Hnida C, Engenheiro E, Ziebe S. Computer controlled multi-level mor-

phometric analysis of blastomere size as biomarker of fragmentation

and multinuclearity in human embryos. Hum Reprod 2004; 19: 288–93.



mind that embryo development is a dynamic process

and that the kinetics involved can yield additional

information about embryo competence.

A number of studies have demonstrated that the

timing of cell cleavage is a significant indicator of

embryonic competence. The embryo needs not only

to develop to the 4-cell stage, but also it needs to do so

at the correct time. Cleavage that occurs too rapidly

or too slowly is an indication of impaired compe-

tence. Likewise, the onset of mitoses and the appear-

ance/disappearance of the pronuclei after fertilization

need to take place during a narrow time interval

(22–25 hours) in high quality embryos, as suggested

by Fancsovits et al.

It has also been suggested that

the interval between pronuclear breakdown and the

first cleavage division should be relatively constant,

about 3 hours.

37,38

Other studies have demonstrated that the occur-

rence of early cleavage may be a good prognostic fac-

tor. However, the specific timing of early cleavage

seems to be related to the method of fertilization,

suggesting that different kinetics are involved in the

processes of ICSI vs regular IVF.

CONCLUSION

In the past, embryo evaluation has been based mainly

on subjective evaluation of morphological parame-

ters considered to be important markers of quality.

However, a number of drawbacks are associated

with this type of analysis. One example is the differ-

entiation between large fragments and blastomeres,

and another is imprecise estimation of the degree of

fragmentation. The introduction of computer-based

morphometric analysis has allowed us to enter a new

level of embryo evaluation. These techniques open

an array of possibilities for standardization and more

precise measurements, including total cytoplasmic

reduction as a new means of describing fragmenta-

tion, and detection of multinucleation based on

blastomere size.

In the final analysis, the combination of kinetics

and morphometrics that include detailed informa-

tion retrieved over several days is a new and fasci-

nating aspect. The ‘3-dimensionality’ of multilevel

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MORPHOMETRIC ANALYSIS OF HUMAN EMBRYOS

19. Ziebe S, Lundin K, Loft A for the CEMAS II and III Study Group et al.

FISH analysis for chromosomes 13, 16, 18, 21, 22, X and Y in all blas-

tomeres of IVF pre-embryos from 144 randomly selected donated

human oocytes and impact on pre-embryo morphology. Hum Reprod

2003; 18: 2575–81.

20. Ebner T,Yaman C, Moser M et al. Embryo fragmentation in vitro and its

impact on treatment pregnancy outcome. Fertil Steril 2001; 76: 281–5.

21. Alikani M, Cohen J, Tomkin G et al. Human embryo fragmentation in

vitro and its implications for pregnancy and implantation. Fertil Steril

1999; 71: 836–42.

22. Johansson M, Hardarson T, Lundin K. There is a cutoff limit in

diameter between a blastomere and a small anucleate fragment. J Assist

Reprod Genet 2003; 20: 309–13.

23. Hnida C, and Ziebe S. Total cytoplasmic volume as biomarker of frag-

mentation in human embryos. J Assist Reprod Genet 2004; 20: 335–40.

24. Hnida C, Agerholm I and Ziebe S. Traditional detection versus

computer-controlled multilevel analysis of nuclear structures from

donated embryos. Hum Reprod 2005; 20: 665–71.

25. Jackson KV, Ginsburg ES, Hornstein MD, Rein MS and Clarke RN.

Multinucleation in normal fertilized embryos is associated with an

accelerated ovulation induction response and lower implantation

rates in in vitro fertilization-embryo transfer cycles. Fertil Steril 1998;

70: 60–6.

26. Pelinck MJ, De Vos M, Dekens M et al. Embryos cultured in vitro with

multinucleated blastomeres have poor implantation potential in human

in-vitro fertilization and intracytoplasmic sperm injection. Hum

Reprod 1998; 13: 960–3.

27. Van Royen E, Mangelschots K,Vercruyssen M et al. Multinucleation in

cleavage stage embryos. Hum Reprod 2003; 18: 1062–9.

28. Kligman I, Benadiva C, Alikani M, and Munné S. The presence of

multinucleated blastomeres in human embryos is correlated with chro-

mosomal abnormalities. Hum Reprod 1996; 11: 1492–8.

29. Balakier H. and Cadesky K.The frequency and developmental capabil-

ity of human embryos containing multinucleated blastomeres. Hum

Reprod 1997; 12: 800–4.

30. Hardarson T, Hanson C, Sjögren A, and Lundin K. Human embryos

with unevenly sized blastomeres have lower pregnancy and implanta-

tion rates: indications for aneuploidy and multinucleation. Hum

Reprod 2001; 16: 313–18.

31. Pickering SJ, Taylor A, Johnson MH, and Braude PR. An analysis of

multinucleated blastomere formation in human embryos. Hum Reprod

1995; 10: 1912–22.

32. Burke B. and Ellenberg J. Remodelling the walls of the nucleus. Nat Rev

2002; 3: 487–97.

33. Straight AF. and Field CM. Microtubules, membranes and cytokinesis.

Curr Biol 2000; 10: 760–70.

34. Masui M, and Kominami T. Change in the adhesive properties of blas-

tomeres during early cleavage stages in sea urchin embryo. Dev

Growth Differ 2001; 43: 43–53.

35. Masui M, Yoneda M, and Kominami T. Nucleus : cell volume ratio

directs the timing of increase in blastomere adhesiveness in starfish

embryos. Dev Growth Differ 2001; 43: 295.

36. Fancsovits P, Toth L, Takacs Z.F et al. Early pronuclear breakdown is

a good indicator of embryo quality and viability. Fertil Steril 2005;

84: 881–7.

37. Van Wissen B, Wolf JP, Bomsel-Helmreich O, Frydman R, and Jouannet

P. Timing of pronuclear development and first cleavages in human

embryos after subzonal insemination: influence of sperm phenotype.

Hum Reprod 1995; 10: 642–848.

38. Capmany G, Taylor A, Braude PR, and Bolton VN. The timing of

pronuclear formation, DNA synthesis and cleavage in the human 1-

cell embryo. Mol Hum Reprod 1996; 2: 299–306.

39. Van Montfoort APA, Dumoulin JCM, Kester ADM, and Evers JLH

Early cleavage is a valuable addition to existing embryo selection

parameters: a study using single embryo transfers. Hum Reprod 2004;

9: 2103–8.

40. Arce J-C, Ziebe S, Lundin K et al. Interobserver agreement and intra-

observer reproducibility of embryo quality assessments. Hum Reprod

2006; 21: 2141–8.

41. Lehtonen E, et al. Changes in cell dimensions and intercellular contacts

during cleavage-stage cell cycles in mouse embryonic cells. J Embryol

Exp Morphol 1980; 58: 231–9.

42. Massip A. and Mulnard J. Time-lapse cinematographic analysis of

hatching of normal and frozen-thawed cow blastocysts. J Reprod Fertil

1980; 58: 475–8.

43. Aiken CEM, Swoboda PPL, Skepper JN. and Johnson MH. The direct

measurement of embryonic volume and nucleo-cytoplasmic ratio

during mouse pre-implantation development. Reproduction 2004;

128: 527–35.

44. Alikani M, Cohen J, Tomkin G et al. Human embryo fragmentation in

vitro and its implications for pregnancy and implantation. Fertil Steril

1999; 71: 836–42

45. Munne S, Alikani M. and Cohen J. Monospermic polyploidy and

atypical embryo morphology. Hum Reprod 1994; 9: 506–10.



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9. Development rate, cumulative scoring, and

embryonic viability

Christine C Skiadas and Catherine Racowsky



standard of care, even the most rigorous selection

paradigms have limitations, including the inability

to detect genetic disorders or predict pregnancy

with 100% accuracy. Other embryonic markers of

development and metabolic assessment are cur-

rently being explored and these may, in the future,

be used alone or in combination with morphologi-

cal evaluations for improved embryo selection.

This chapter reviews the normal timeline and

sequence of embryonic development from fertiliza-

tion through progression to the blastocyst stage. We

also review the key features of morphological crite-

ria for embryo selection, cumulative grading sys-

tems and their association with implantation rates

and viability. Finally, we consider issues surround-

ing the optimum day for embryo transfer.

DEVELOPMENTAL RATE: NORMAL

TIMELINE OF EVENTS

Preimplantation development follows a programmed

timeline during which an organized series of critical

events take place (Figure 9.1). In vivo, fertilization

and early cleavage occur in the fallopian tube, with

the embryo traversing the uterotubal junction at the

morula stage. The procedure of clinical IVF has

allowed this timeline of events to be observed in

detail and researched. In terms of defining embryo

morphology, one of the first time points that has

been evaluated is that of the zygote or pronuclear

embryo,

12–15

approximately 16–18 hours after fertil-

ization. Key features of the zygote stage are the

development of the two pronuclei (one from the

oocyte and one from the sperm), each containing

multiple nuclear precursor bodies. The two pro-

nuclei (PNs) migrate towards each other and their

INTRODUCTION

Since the inception of clinical in vitro fertilization

(IVF), there has been a drive to optimize pregnancy

rates. Although this was initially achieved by trans-

ferring greater numbers of embryos, transfers of

multiple embryos resulted in the negative side-effect

of increasing the incidence of high-order multiple

gestations.

1–4

In order to reduce high-order multiple

gestations and at the same time maintain pregnancy

rates, there has been a progressive move toward

decreasing the number of day 3 embryos trans-

ferred,

as well as performing day 5 transfers of only

one or two blastocysts.

6–8

However, blastocyst trans-

fer may not be ideal in all cases, and may compro-

mise a successful outcome that would otherwise be

achieved following a day 3 transfer.

8–10

Therefore,

one of the most important challenges in IVF is the

ability to determine which embryos are associated

with the greatest developmental potential, in order

to select optimally only one, or at most two, of these

embryos for transfer.

Ideal methods for embryo selection include: ease

of assessment, standardization among embryologists,

minimal harm to the embryo, and a high correlation

with pregnancy rates; therefore morphological assess-

ment remains the first-line approach, although non-

invasive biomarker methods are currently under

development (Chapters 12 and 20). Since human

embryonic development follows a specifically timed,

coordinated sequence of events, developmental rate

(assessed by certain milestones being reached at

particular points in time) and morphological char-

acteristics (defined at specified intervals after the

day of insemination) provide the two main measures

of embryonic development. Although morphologi-

cal selection of embryos represents the current

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HUMAN PREIMPLANTATION EMBRYO SELECTION

multiple nucleoli align at the pronuclear interface in

preparation for syngamy.

16,17

Specific features of the

PNs have been used in zygote scoring systems,

which are discussed in Chapters 3 and 4.

Following syngamy, the newly formed zygote

undergoes first cleavage between 20 and 27 hours

after insemination,

18–20

and second cleavage to form

the 4-cell stage at approximately 48 hours. The

embryo reaches the 8-cell stage by approximately

72 hours. Early cycles of cell division are thought to

be regulated by the maternal genome, with the

embryonic genome becoming activated to dictate

further cell divisions at approximately day 3, between

the 4- and 8-cell stage.

Following the 8-cell stage,

the cells become increasingly polarized and the

embryo develops cell–cell adhesions and gap junc-

tions during the process of compaction. This is

expected to occur on day 4,

with progression to



Figure 9.1 In vivo embryo maturation. Natural timeline of embryonic development in vivo. Early cleavage stages occur in the

fallopian tube and the embryo enters the uterus once it has reached the blastocyst stage. Reprinted from Figure 2-24, Moore and

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DEVELOPMENT RATE, CUMULATIVE SCORING, AND EMBRYONIC VIABILITY

blastocyst development early on day 5 and comple-

tion of blastulation by late day 5.

Figure 9.2 shows

representative images of embryos at these stages.

EMBRYONIC MORPHOLOGY AND

SCORING SYSTEMS

The optimal timing and method of morphological

assessment has been debated amongst embryologists,

and has been affected by laws governing embryo

transfer and cryopreservation.

ZYGOTE STAGE

Zygote scoring systems allow embryos of optimal

prognosis to be identified immediately after fertil-

ization, which has been particularly useful in countries

where embryo selection at cleavage stages is restricted.

In Germany, the German embryo protection law

dictates that the number of embryos to be trans-

ferred must be selected at the PN stage, with the

remainder being cryopreserved. However, despite the

positive results and practical benefits of PN scoring,

there remains debate over whether such evaluation

is superior to that of standard morphological assess-

ment. Indeed, a recent study

investigated whether

PN scoring was superior to standard day 2 or day 3

morphological assessment; the results failed to

detect a difference in pregnancy rates based on the

scoring system. Although this study was underpow-

ered to conclude a negative result, it suggests that

the ideal method of scoring has yet to be deter-

mined. Zygote scoring systems, and their physiolog-

ical basis, are covered in detail in Chapters 3 and 4.

TWO CELL EMBRYOS

Although very few studies address the morphologi-

cal features of the 2-cell embryo, the time to first cell

division has been extensively studied as a predictor

of improved pregnancy outcomes. Embryos that

undergo ‘early cleavage’ have been postulated to

have a greater degree of developmental competence

than embryos that do not undergo early cleavage.

In 1997, Shoukir et al designated those embryos that

had reached the 2-cell stage by 25 hours postinsem-

ination as having undergone ‘early cleavage,’ and

this occurred in 19% of cycles (see Figure 9.3).

Cycles with early cleavage were associated with sig-

nificantly higher pregnancy rates.

As the timing of

fertilization is often unknown with conventional

insemination, a follow-up study using only ICSI

cycles again confirmed that those embryos under-

going early cleavage were associated with a signifi-

cantly higher pregnancy rate, supporting the idea

that early cleavage is related to developmental com-

petence and not to the timing of fertilization.

Tsai

et al reproduced these findings in a retrospective

study

and Sakkas et al confirmed their original

findings with a prospective study performed in 2001,

where the presence of an increased number of early

cleaving embryos was again associated with increased

implantation rate.

This study assessed early cleavage

on alternate weeks to determine if selecting embryos

on this basis had an impact on implantation rates.



Figure 9.2 Representative embryo images. (A) Pronuclear stage

embryo. In this photograph, the two pronuclei are aligned in the

middle of the zygote with their nucleoli aligned at the pronuclear

interface. (B) 2-Cell embryo. (C) 4-Cell embryo. (D) 8-Cell

embryo. In both the 4-cell and 8-cell embryo, blastomeres

appear symmetrical and display no fragmentation.

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HUMAN PREIMPLANTATION EMBRYO SELECTION

Of note, there was no difference in the total number

of embryos between groups. In the group where

early cleavage was checked, the pregnancy rate was

48%, compared with 31% in the group where this

was not checked.

The fact that checking for early

cleavage did improve the pregnancy rate suggests that

this may be a feature of improved developmental

competence.

In a retrospective analysis, Ciray et al showed an

association between early cleavage, a higher day 3

embryo quality score and increased implantation

rates, demonstrating that there is a link between

early cleavage and improved embryo morphology

at the 8-cell stage.

Therefore, if early cleavage is

another surrogate marker for improved day 3 mor-

phology and implantation, the question arises as to

whether it is necessary to evaluate the embryo at

both stages. A further discussion of multiday embryo

assessment is undertaken later in this chapter.

FOUR-CELL EMBRYOS (DAY 2)

The embryo normally reaches the 4-cell stage on

day 2 after insemination, and many of the embryo

transfers were carried out on day 2 in the earliest

reports of IVF. The features of cell number, degree

of fragmentation, and equal size of blastomeres

have been frequently evaluated at this stage to deter-

mine viability potential. Cummins et al and Puissant

et al were two of the earliest authors to describe such

scoring systems.

1,2

Ideal 4-cell embryos are those

with equal sized blastomeres and minimal fragmen-

tation. Ebner et al also confirmed that day 2

embryos displaying little fragmentation showed an

improved clinical pregnancy rate.

Even in these

early scoring systems, developmental rate was rec-

ognized as an additional marker of improved implan-

tation. Cummins et al reported an ideal embryo

developmental rate (EDR) associated with implan-

tation;

Puissant et al and Steer et al both used

embryo cell number as an approximate marker for

achieving a certain developmental state, with addi-

tional points awarded if the embryo had reached

certain milestones.

1,31

Table 9.1 summarizes studies

that focus on day 2 embryo scoring.

1,2, 31–33

Further

discussion of the individual morphological features

(cell number, fragmentation, symmetry, and com-

paction) is discussed in the section below on 8-cell

embryos.

The nuclear status of the blastomeres and the pres-

ence of mononucleation provide an additional means

of assessment in the 4-cell embryo (for additional



Early

cleavage

No early

cleavage

Insemination

25 h after

insemination

43–47 h after

insemination

2-cells

4-cells

2-cells

4-cells

No distinction between embryo stages

Transfer

Figure 9.3 Timing of assessment of early cleavage. The timeline for assessing early cleavage is crucial, as there is only a certain

window where true early cleavage can be seen. This earlier time point provides differentiation between embryos that may have similar

morphology once they reach the 4-cell stage. Reproduced from Shoukir Y et al. Early cleavage of in-vitro fertilized embryos to the

2-cell stage: a novel indicator of embryo quality and viability. Hum Reprod 1997; 12(7): 1531–6. © European Society of Human

Reproduction and Embryology, with permission from Oxford University Press/Human Reproduction.

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DEVELOPMENT RATE, CUMULATIVE SCORING, AND EMBRYONIC VIABILITY



Table 9.1 Compilation of scoring systems assessing day 2 morphological features

Cummins et al, 1986

Puissant et al, 1987

Steer et al, 1992

Roseboom et al, 1995

Giorgetti et al, 1995

Title of study Embryo Quality (EQ) Embryo The Cumulative Embryo Average Morphology

Development Rating (EDR) Score (CES) Score (AMS)

Type of study Retrospective analysis Retrospective analysis Retrospective analysis Retrospective multivariate Retrospective analysis of

logistic regression single embryo transfers

Timing of embryo Multiple assessments of growth Day 2 Day 2 Day 2 Day 2

assessment rate. Grading performed on day

of transfer

Assessment of EDR: based on growth rates of An additional two points CES was calculated by NA NA

developmental high EQ embryos. Calculated were added if the embryo multiplying grade of

timing a ratio of time observed/time had reached the 4-cell stage embryo ⫻ cell number

expected to predict optimal rate by 48 hours and then summated the

of development scores of all embryos

transferred

Day 2 features EQ Scoring system (Grade 1–4) Grade 4: embryos with clear, Grade 4: equal sized Number of blastomeres, A point scale was

assessed regularity or symmetry regular blastomeres with no blastomeres symmetry of blastomeres, established, which

of blastomeres, presence of fragmentation or a Grade 3: uneven percentage of extracellular assigned a point to

fragments, and quality of maximum of 5 small blastomeres with ⬍10% fragmentation. Symmetrical each feature:

cytoplasm. anucleate fragments fragmentation blastomeres ⫽ 2 points. 1) Achieved cleavage

Ideal being grade 4 embryos: Grade 3: embryos with Grade 2: 10–50% Embryo morphology state

regular blastomeres, no unequal blastomeres, few fragmentation score ⫽ symmetry score/ 2) No fragmentation, or

fragments and clear cytoplasm or no fragments Grade 1: ⬎50% (1 ⫹ % fragmentation). ⬍20% fragmentation

(no granularity) Grade 2: fragments ⬍1/3 of fragmentation Average morphology score 3) No irregular

embryo surface ⫽ embryo morphology blastomeres

Grade 1: fragments over score/# transferred embryos 4) 4-cell stage

⬎1/3 of embryo surface

Day of transfer Did not choose embryos to Day 2 Day 2 Day 2 Day 2

transfer based on scoring

system

Outcome: Both optimal EDR and EQ ‘Good embryos’ scored Pregnancy rates rose as The odds ratio of AMS Pregnancy rate

pregnancy rates were significantly associated 5 or 6 and the number of CES rose to 42. Above was 1.63 (0.990–2.7). increased with each

with pregnancy rates ‘good embryos’ transferred this level, there was no Probability of pregnancy additional point

was significantly associated further increase in increased by 63% when (increase in pregnancy

with pregnancy rates pregnancy rates, but the AMS increased by one rate approx 4% per

only an increase in point. However, this point)

multiple gestation odds ratio crosses one.

rates The AMS was included

in the model, as it

improved the goodness of fit

of the model

NA, not applicable.

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