Elder K. Human preimplantation embryo selection

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HUMAN PREIMPLANTATION EMBRYO SELECTION

possible differences in viability of embryos with

chromosomal abnormalities of meiotic and mitotic

origin.

Testing for errors in meiosis may also be useful for

predicting developmental abnormalities in embryos

that originate from uniparental disomies. Recent

reviews suggest that uniparental disomies of chro-

mosomes 6, 7, 11, 15, 19, and 20 can potentially lead

to imprinting disorders.

Sufficient data on the

prevalence of uniparental disomies in preimplanta-

tion development is not available, because FISH

techniques in current use cannot distinguish between

uniparental disomies and normal sets of chromo-

somes. Uniparental disomy may be suspected in

euploid embryos that were predicted as trisomic

by PB1 and PB2 testing, because one-third of the

embryos originating from trisomic zygotes may

become uniparentally disomic as a result of trisomy

rescue. Uniparental disomy may also be accurately

detected by DNA fingerprinting.

More than half of IVF patients are 35 years and

older, and more than half of their oocytes may be

aneuploid. Therefore, avoiding the transfer of

embryos resulting from oocytes diagnosed as aneu-

ploid through PGD should be clinically useful, by

potentially improving implantation and pregnancy

rates and avoiding uniparental disomies. Although

the biological significance of uniparental disomies

in preimplantation development is not known, it is

possible that the detection and avoidance of uni-

parental disomies may also contribute to an improve-

ment in implantation and pregnancy rates.

CONCLUSION

The above data indicate that more than half of the

oocytes or embryos that were tested are aneuploid,

and PGD is therefore of practical relevance for poor

prognosis patients. Aneuploidy clearly can affect the

embryo’s developmental competence and potential

to implant following transfer. In contrast to the data

obtained from traditional studies of meiosis, direct

testing of meiotic outcomes in patients of advanced

reproductive age shows that chromosomal abnor-

malities originate to a similar extent from meiosis I

and meiosis II, and are predominantly of chromatid

origin. Although isolated errors in meiosis I and

meiosis II were also observed, overall 42.7% of

oocytes with meiosis I errors also had sequential

errors in meiosis II, resulting in apparently balanced

zygotes in 32.5% of cases. This may represent a phe-

nomenon of aneuploidy rescue in female meiosis.

However, the embryos resulting from such apparently

balanced zygotes were predominantly aneuploid,

suggesting that these zygotes may be inherently pre-

disposed to postzygotic chromosomal errors follow-

ing sequential errors in meiosis I and meiosis II.

Patterns of errors in meiosis I and meiosis II dif-

fered according to the chromosome tested, and these

patterns were not in agreement with previously

reported data based on DNA polymorphism in

liveborn trisomies or spontaneous abortions. Com-

paring the types of chromosomal aneuploidies and

the prevalence of each chromosome specific error

detected by FISH and DNA fingerprinting in oocytes

and embryos suggests that the majority of chromo-

somal aneuploidies in embryos originate from female

meiosis. This error can predispose the oocyte to

further sequential postzygotic errors, and this may

explain the high rate of mosaicism in preimplanta-

tion embryos. PB and blastomere testing has also

demonstrated that uniparental disomies can occur,

and the possible impact of this phenomenon is still

to be documented.

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10. Dyban A, Fredine M, Severova E et al. Detection of aneuploidy in

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first polar bodies and metaphase II oocytes for the detection of

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13. Verlinsky Y, Cieslak J, Ivakhnenko V et al. Preimplantation diagnosis of

common aneuploidies by the first and second polar body FISH analy-

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14. Verlinsky Y, Cieslak J, Ivakhnenko V et al. Prepregnancy genetic testing

for common age-related aneuploidies by polar body analysis. Genet

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15. Verlinsky Y, Cieslak J, Ivakhnenko V et al. Prevention of age-related

aneuploidies by polar body testing of oocytes. J Assist Reprod Genet

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16. Verlinsky Y, Cieslak J, Ivakhnenko V et al. Chromosomal abnormalities in

the first and second polar body. Mol Cell Endocrinol 2001; 183: S47–9.

17. Gianaroli L, Magli MC, Ferraretti AP. The in vivo and in vitro effi-

ciency and efficacy of PGD for aneuploidy. Mol Cell Endocrinol 2001;

183: S13–18.

18. Munne S. Preimplantation genetic diagnosis of numerical and structural

chromosome abnormalities. Reprod BioMed Online 2002; 4: 183–96.

19. Kuliev A, Verlinsky Y. Current feature of preimplantation genetic

diagnosis. Reprod BioMed Online 2002; 5: 296–301.

20. Kahraman S, Bahce M, Samli H et al. Healthy births and ongoing

pregnancies obtained by preimplantation genetic diagnosis in patients

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Reprod 2000; 15: 2003–7.

21. De Boer KA, Catt JW, Jansen RPC et al. Moving to blastocyst biopsy

for preimplantation genetic diagnosis and single embryo transfer at

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22. Verlinsky Y, Munne S, Cohen J et al. Over a decade of preimplantation

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82: 292–4.

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ities in a series of 6733 human oocytes in preimplantation diagnosis of

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24. Kuliev A, Verlinsky Y. Meiotic and mitotic nondisjunction: lessons from

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Am J Hum Genet 1997; 65: 23–32.

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mosome behaviour in intact mammalian oocytes: monitoring the seg-

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27. Fisher JM, Harvey JF, Morton NE, Jacobs PA. Trisomy 18: studies of the

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28. Kuliev A, Cieslak J, Zlatopolsky Z et al. Origin of aneuploidies in

preimplantation embryos. 2003 Fifth International Symposium on

Preimplantation Genetics, 5–7 June, Antalya, Turkey. P. 16–17.

29. Kuliev A, Cieslak J, Zlatopolsky Z et al. Aneuploidy rescue after female

meiosis I and follow up analysis of its outcome in resulting preimplan-

tation embryos. Am J Hum Genet 2003; 73 (Suppl): 189.

30. Verlinsky Y, Kuliev A. Practical Preimplantation Genetic Diagnosis.

Springer, London: New York, 2006.

31. Kim NH, Chung HM, Cha KY, Chung KS. Microtubule and microfila-

ment organization in maturing human oocytes. Hum Reprod 1998;

13: 2217–22.

32. Barrit J, Brenner C, Cohen J, Matt D. Mitochondrial DNA rearrange-

ment in human oocytes and embryos. Mol Hum Reprod 1999; 5:

927–33.

33. Perez G, Flaherty S, Barry M, Matthews C. Preliminary observations of

polar body extrusion and pronuclear formation in human oocytes

using timelapse video cinematography. Hum Reprod 1997; 12: 532–41.

34. Gianaroli L, Magli MC, Ferraretti AP et al. Pronuclear morphology

and chromosomal abnormalities as scoring criteria for embryo selec-

tion. Fertil Steril 2003; 80: 837–44.

35. Munne S, Bahce M, Sandalinas M. Differences in chromosome suscep-

tibility to aneuploidy and survival to first trimester. Reprod BioMed

Online 2004; 8: 81–90.

36. Munne S, Sandalinas M, Escudero T et al. Some mosaic types increase

with maternal age. Reprod BioMed Online 2002; 4: 223–32.

37. Silber S, Sadowy S, Lehahan K et al. High rate of chromosome mosaicism

but not aneuploidy in embryos from karyotypically normal men

requiring TESE. Reprod BioMed Online 2002; (Suppl 2), 20.

38. Lucifero D, Chaillet JR, Trasler M. Potential significance of genomic

imprinting defects for reproduction and assisted reproductive tech-

nology. Hum Reprod Update 2004; 10: 3–18.

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INTRODUCTION

Data from SART and ASRM

indicate that only

38.1% of cycles in women younger than 34 years

reach delivery of a healthy child after assisted repro-

duction. In the age group 35–37 years, this figure is

32%, 23% in ages 38–40 years, and 11% in women

older than 40 years. Assuming an average of three

embryos transferred in women 35 and older, these

figures indicate that very few of their embryos implant.

Irrespective of whether this is a result of chromo-

some abnormalities or other factors, the high rate of

chromosome abnormalities in cleavage stage embryos

itself can largely explain the high degree of implanta-

tion failure observed in assisted reproductive tech-

nology (ART), since 60–80% of human embryos are

chromosomally abnormal, depending on maternal

age (see Table 18.1).

2–4

The chromosome abnormal-

ities seen in human IVF embryos create three imme-

diate broad questions that are addressed in this

chapter:

(1) What causes these abnormalities, and can they

be prevented?

(2) How reliable is morphological selection in

selecting against chromosome abnormalities?

(3) How effective is preimplantation genetic diag-

nosis (PGD) as a selection tool to improve ART

outcome?

Before answering these questions, it is necessary to

understand what type of data we are working with,

and how it was obtained. Several requirements must

be met in order to study numerical chromosome

abnormalities in preimplantation human embryos.

First, cleavage arrested embryos must be evaluated

by interphase analysis, because their cells are seldom

at metaphase stage when fixed. Second, distinguish-

ing differences in aneuploidy rates among different

chromosomes requires analysis of individual chro-

mosomes. Third, all blastomeres of non-transferred

embryos should be analyzed to distinguish mosaicism

from other abnormalities. To date, fluorescence in

situ hybridization (FISH) is the most efficient method

used to fulfill these requirements. FISH can be used to

study the chromosome constitution of cleavage stage

blastomeres in interphase, with efficiencies of over

90% per cell.

5,6

Simultaneous analysis of more than

three chromosome pairs permits differentiation of

polyploidy, haploidy, and aneuploidy in arrested

human embryos.

When most or all cells of an embryo

are analyzed, mosaicism can be differentiated from

FISH failure, as well as from aneuploidy, and their

mechanism of formation ascertained.

However, FISH

has its disadvantages: FISH supplies information only

on the chromosomes for which specific probes are

used, and only five fluorochromes of the visible spec-

trum can be used simultaneously. Thus, sequential

FISH must be applied, and even then only a maxi-

mum of three rounds of FISH can be used reliably;

this reduces the potential number of chromosomes

that can be studied to 15, so far. Other techniques

such as spectral karyotyping (SKY),

or comparative

18. Chromosomal status of human embryos

Santiago Munné and Luca Gianaroli



Table 18.1 Frequency (%) of chromosome abnormal-

ities in cleavage stage human embryos

Age (years)

20–34 35–39 40–47

Aneuploidy 24 27 39 p 0.001

(9 chromosomes)

Other aneuploidy 5 6 8 Unknown

(by CGH)

Postmeiotic 35 36 35 NS

abnormalities

Total abnormal 64 69 82 p 0.001

CGH, comparative genomic hybridization; NS, not significant.

From Munné et al,

Marquez et al,

and

Gutierrez-Mateo et al.

Mosaics, polyploid, haploid, chaotic.

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HUMAN PREIMPLANTATION EMBRYO SELECTION

genome hybridization (CGH)

9–11

have also been

used. However, studies using CGH

indicate that the

nine-chromosome probe FISH test currently used in

PGD

does detect about 75–85% of chromosome

abnormalities that can be detected by CGH, both

because the nine chromosomes are the ones most

frequently involved in aneuploidy, and also because

the other chromosomal aneuploidies tend to occur

simultaneously with those involving the nine chro-

mosomes tested.

10,13

FISH therefore remains the

most suitable method for preimplantation genetic

analysis of human embryos.

CAUSES OF CHROMOSOME ABNORMALITIES

IN ART EMBRYOS

HORMONAL STIMULATION

Aneuploidy rates in infertile women undergoing

ovarian stimulation with gonadotropins have been

uniformly reported as high. Whether these rates are

‘natural’, or are the consequence of gonadotropin

stimulation is, however, unknown. There are virtually

no reports comparing aneuploidy rates in natural

and stimulated cycles. In a study that compared the

rate of spontaneous abortions between IVF patients

and patients conceiving naturally, it was found that

IVF patients had a 1.2 greater risk of miscarriage

than did the control group.

However, there is mounting evidence to suggest

that different types of hormonal stimulation may

have a different impact on the frequency of euploid

embryos. Tarin et al

reported that cycles generating

slow-cleaving embryos occurred in women who

were treated with gonadotropin releasing hormone

agonist (GnRH-a) before ovarian stimulation for a

shorter period of time than in women whose cycles

generated fast-cleaving embryos. Slowly developing

embryos have a higher frequency of chromosomal

abnormalities than do those that develop normally.

Another study demonstrated that embryos

obtained from several laboratories that used differ-

ent stimulation protocols had very diverse rates of

mosaicism (p 0.001).

Specifically, cycles resulting

from clomiphene citrate stimulation showed 40%

mosaicism, compared with 25% mosaicism in

embryos from down regulated cycles (p

0.05). The

chromosome abnormality rates in the two different

hormonal regimens are probably higher, since that

study was performed using FISH with only five probes.

The fact that embryos with multinucleated blas-

tomeres (MNBs) have higher rates of chromosome

abnormalities than non-MNB embryos is well doc-

umented,

and it also seems that the incidence of

embryos with MNBs is related to hormonal stimu-

lation response. Jackson et al

also found that in

cycles containing MNBs, the level of estradiol on the

day of human chorionic gonadotropin (hCG) was

doubled (2401 vs 1270 pg/ml, p 0.001), twice as

many oocytes were collected (22 vs 10, p 0.001), and

fewer ampoules of gonadotropins (p 0.001) were

used than in cycles without MNBs. Follicular under-

oxygenation has also been correlated with multi-

nucleation seen at the 2-cell stage of development.

In addition, cycles with large numbers of oocytes

retrieved have significantly more MNB embryos,

18,20

and lower implantation rates.

Another study

demonstrated that cycles containing MNB embryos

have double the level of estradiol on the day of

hCG administration, and need fewer ampoules of

gonadotropins than cycles without MNBs.

However, this was not confirmed by other studies

(see Chapter 5).

The duration of hormonal stimu-

lation has also been associated with multinucle-

ation; MNB occur more commonly in embryos of

patients who required fewer days of gonadotropins

before hCG (Chapter 5).

Patients with low (1 mIU/ml) levels of follicle

stimulating hormone (FSH) on day 3 tend to have

a higher incidence of multinucleation (Chapter 5).

According to Van Royen et al

the higher rates of

multinucleation observed in patients that require a

higher dose of gonadotropins, as well as in patients

with shorter cycles, suggests that the incidence of

MNB might be related to a high number of immature

follicles at the time of ovulation induction.Immature

follicles may reach metaphase II, but either their

nucleus or cytoplasm might be inadequately mature,

so that they are unable to achieve correct cleavage.

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CHROMOSOMAL STATUS OF HUMAN EMBRYOS

Nogueira et al

found that embryos developing

from in vitro matured oocytes were mostly multi-

nucleated, and Van Royen et al.

therefore sug-

gested that the frequency of MNB embryos might

be minimized by modifying hormonal stimulation.

Recently, Weghofer et al

evaluated the impact

of controlled ovarian hyperstimulation with gona-

dotropins on euploidy rates in human cleavage

stage embryos. Three different protocols of ovarian

hyperstimulation with gonadotropins were com-

pared: (1) long agonist stimulation; (2) short agonist

stimulation; and (3) the use of a GnRH antagonist.

Their preliminary results suggested that higher

gonadotropin dosages result in more euploid embryos

for transfer, but the percentage of euploid embryos

is lower overall. Therefore, high dose gonadotropin

stimulation, resulting in large numbers of embryos,

may represent a potential indication for PGD. Rates

of euploidy appear to be improved by exogenous

luteinizing hormone (LH) supplementation in long

agonist protocols, especially in younger women.

Euploidy rates after ovarian stimulation thus appear

to be affected both quantitatively and qualitatively

by stimulation protocols and doses of medication.

Differing responses to hormonal stimulation may

also have an impact on the frequency of euploid

embryos per cycle. For example, patients with reduced

ovarian reserve and a lower total number of follicles

in their ovaries have an increased risk of trisomy.

24,25

Indeed, Gianaroli et al

described a high rate of

chromosome abnormalities in embryos of women

who had a poor response to hormonal stimulation.

High ovarian response is considered to be detri-

mental to implantation, because this has a negative

effect on the physiological relationship between

estradiol levels and endometrial receptivity, reducing

implantation potential.

27,28

Reis Soares et al

also

observed that patients showing high ovarian response

produce more chromosomally abnormal embryos.

Oocyte donors were compared with patients under-

going PGD for X-linked diseases, and these two

groups were found to have a significant difference

in oocytes retrieved (25 and 15, respectively), in

chromosome abnormalities (56% and 37%, res-

pectively) (p 0.01), and in implantation rate (25%

and 36.4%, respectively). High ovarian response has

been associated with multinucleation,

and uneven

pronuclei,

both features that correlate with

chromosome abnormalities.

Patients with polycystic ovarian syndrome tend

to have HOR and higher miscarriage rates,

but

they do not have a higher proportion of chromoso-

mally abnormal embryos.

OTHER FACTORS

Although controlled ovarian hyperstimulation may

be the principal factor responsible for the high rate

of chromosome abnormalities in cleavage stage

embryos created by ART, other factors may also have

a detrimental effect, such as exposure of gametes

and embryos to microscope or room light, changes

in temperature when opening and closing incuba-

tors or while handling dishes, air impurities, and

unsuitable culture media.

Spindle microtubules are thermosensitive, and

even a small change in temperature can deeply dis-

turb oocyte spindle structure.

32,33

Pickering et al

found that after 10 minutes at room temperature,

human oocytes have a disassembled spindle in 50%

of cases, and 100% have a disassembled spindle after

30 minutes at room temperature, accompanied by

chromosome malsegregation.

Cohen et al

showed that volatile compounds

negatively affect mouse embryo development, and

also decrease human pregnancy and implantation

rates in IVF programs. More recently, Boone et al

demonstrated that fertilization rates, embryo quality,

and pregnancy rates increased significantly after

the installation of air filters in IVF laboratories. The

effect of volatile organic compounds on chromosome

abnormalities has not been studied.

Identifying the factors that produce an increase

in chromosomally abnormal embryos will lead to

better ART methodology, with the creation of a

higher proportion of euploid embryos. However, the

current reality is that more than 50% of embryos

are chromosomally abnormal. Can these chromoso-

mally abnormal embryos be selected morphologi-

cally or developmentally?

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HUMAN PREIMPLANTATION EMBRYO SELECTION

MORPHOLOGICAL SELECTION AGAINST

CHROMOSOME ABNORMALITIES

DYSMORPHISM AND CHROMOSOME

ABNORMALITIES

Morphological and developmental variants in

cleavage stage embryos have recently been review-

ed in relation to chromosome abnormalities.

Several types of dysmorphic embryos were com-

pared with non-dysmorphic embryos, with respect

to chromosome abnormalities. Some of the fea-

tures associated with an increased incidence of

chromosomal abnormality were fragmentation,

multinucleation, and asymmetric blastomeres.

However, decreasing morphological quality was

not related to a higher rate of aneuploidy, but

instead to an increase in postmeiotic abnormali-

ties, such as mosaicism, monospermic polyploidy,

haploidy, and chaotic embryos. In contrast, aneu-

ploidy increases only with maternal age and is

not related to morphology, at least up to day 3 of

development.

Most dysmorphisms (fragmentation, MNB, asym-

metry, etc.) tend to occur in the same embryos. For

example, Hardarson et al

found that asymmetric

embryos have higher rates of multinucleation, and

Van Royen et al

found that highly fragmented

embryos also have more multinucleated blastomeres.

In terms of cleavage rate, embryos with 4 cells on day

2 and 8 cells on day 3 had a lesser chance of being

multinucleated than those at other stages.

FRAGMENTATION

It is well known that fragmented embryos have a

lower implantation potential than those that are not

fragmented.

37–39

Fragmentation rate has also been

associated with chromosome abnormalities.

40–43

Chromosome abnormalities increase from 50–60%

in non-fragmented embryos to 70–90% in embryos

with 35% fragmentation, but while fragmenta-

tion is strongly correlated with mosaicism and other

postzygotic abnormalities, aneuploidy does not

appear to increase with fragmentation.

41,42

MULTINUCLEATION

The frequency of multinucleated embryos per

patient ranges from 15% to 33.6% (Chapter 5).

18,20,44

The different rates reported by different centers are

significant, and could be attributed to differences in

hormonal stimulation and culture conditions.

The effect of multinucleation on implantation is

very clear. Alikani et al

reported that only 16% of

embryos with one or more multinucleated cells on

day 2 or day 3 reached blastocyst stage, compared

with non-multinucleated embryos (32%, p 0.001).

Similar low rates of implantation for embryos with

multinucleation have been reported by others

(Chapter 5).

18,20,44

As with fragmented embryos, the

implantation potential of MNB embryos was higher

if they were transferred on day 3 (13%) than on day

5 (7%), compared with non-dysmorphic and nor-

mally developing embryos (40.1% and 49%, respec-

tively) (Chapter 5). FISH studies on MNBs showed

that the chromosomal content of each MNB nucleus

was not always the same as the chromosomal content

in nuclei of sibling blastomere MNBs.

41,45

Several

studies have analyzed MNBs observed at the 2-cell

stage, and all have detected high rates of abnormali-

ties, ranging from 55 to 100%; the differences

between studies depend mostly on the number of

chromosomes analyzed by FISH (Chapter 5).

42,46–48

EMBRYOS WITH ASYMMETRIC BLASTOMERES

Blastomere asymmetry has been linked to reduced

embryo competence.

36,38,49

Compared with embryos

that have symmetric blastomeres, embryos with

asymmetric blastomeres had more chromosome

abnormalities occurring postmeiotically (mosaicism,

polyploidy, and haploidy) (35% vs 21%) (p 0.001),

fewer normal embryos (32.5% vs 40%, p 0.05), but

similar rates of aneuploidy.

GIANT OOCYTES

Giant oocytes and embryos have an average diameter

of 200 m, including the zona pellucida, and occur

at a frequency of 0.3%.

51,52

Embryos developing from

giant oocytes were found to be invariably triploid or

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CHROMOSOMAL STATUS OF HUMAN EMBRYOS

triploid mosaics, with XXX or XXY gonosome

constitutions, which suggests a higher contribution

from the maternal genome (Munné et al

(n  6),

Balakier et al

(n  4)). Similar oocytes karyotyped

at MII by Rosenbusch et al

(n  6) and Balakier

et al

(n  9) were also found to be triploid. Accor-

ding to Balakier et al

embryos developing from

giant eggs can reach blastocyst stage, indicating that

they can be a source of digynic triploid fetuses.

EMBRYOS WITH A DOMINANT BLASTOMERE

Embryos with only one large cell surrounded by

smaller blastomere-sized extracellular fragments were

found to be invariably polyploid, and frequently

polyploid mosaics.

42,53

EMBRYOS WITH CYTOPLASMIC IRREGULARITIES

According to Magli et al,

embryos with cytoplas-

mic aggregates had 86% chromosome abnormalities

compared with 63% in embryos with regular cyto-

plasm (p 0.001).

EMBRYOS WITH ELONGATED SHAPE

Magli et al

analyzed the chromosome constitution

of 18 elongated embryos, and found that they have

the same proportion of chromosome abnormalities

as non-elongated embryos. These embryos have

been known to produce babies.

Table 18.2 sum-

marizes the relationships found between embryo

morphologies and chromosome abnormalities.

PRONUCLEAR MORPHOLOGY AND

CHROMOSOMAL ABNORMALITIES

Previous studies have demonstrated that some

morphological characteristics of pronuclear zygote

morphology are an expression of events occurring

in the oocyte after fertilization, strictly related to the

design of the embryonic axis.

57,58

Any alteration in

these events may cause abnormalities in embryo

cleavage which are often related to chromosomal

abnormalities (see next section). Based on these con-

siderations, an association was postulated between

pronuclear morphology and the chromosomal

condition of preimplantation embryos.

According

to these results, the combination of patterns related

to pronuclear morphology indicates that some con-

figurations are associated with a higher proportion

of euploid embryos, while the opposite is true for

other configurations.

59–61

These results are in full

agreement with those derived from the insemina-

tion of euploid oocytes, as defined by first polar body

testing.

The current observations confirm that

some patterns of pronuclear morphology are asso-

ciated with a higher proportion of euploidy, not

only reaffirming the relevance of oocyte quality in

determining its fate, but also suggesting that this

scoring system may be beneficial for the prediction

of zygote viability. However, it was recently shown

that cohorts of zygotes with poor pronuclear score

have similar outcomes to cohorts of zygotes with

very poor scores.

The similarity in outcomes was

measured from cohorts that were identical in scoring

leaving little doubt that chromosomal anomalies are

not absolute even in the poorest zygote cohorts.

COMMENT

CLEAVAGE STAGE PATTERNS AND

CHROMOSOME ABNORMALITIES

In addition to the dysmorphisms mentioned above,

the cleavage patterns of embryos from day 1–3 are at



Table 18.2 Summary of morphological abnormalities

and their relation to chromosomal abnormalities

Embryo morphology FISH analysis Reference

Normal morphology

20–34 years old 16% abnormal 2

35–39 years old 37% abnormal 2

40–45 years old 53% abnormal 2

Dysmorphic 2pn embryos:

Uneven 2PN 73–87% abnormal 30,55

Abnormal NPB distribution 71–81% abnormal 55,56

Giant embryos (220 mm) Triploid 7,51

Dominant single blastomere Polyploid 7

35% fragments 70–90% abnormal 42,45

Multinucleated embryos 74–100% abnormal 42,45

Asymmetric blastomeres 67% 45

PN, pronuclei.

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HUMAN PREIMPLANTATION EMBRYO SELECTION

least as important in the selection of embryos with

the highest implantation potential. Developmental

day 3 embryos can be classified into four large groups

according to their rate of cleavage: ‘arrested’, ‘slow’,

‘normal’, or ‘fast’ developing embryos. Arrested

embryos are those that have not cleaved during a 24-

hour period. Slow embryos have not reached the

7-cell stage on day 3 of development but have cleaved

during a 24-hour period. Normal embryos reach 7–9

cells on day 3, with 15% fragmentation and without

multinucleation, and have cleaved in the preceding

24 hours (‘normally developing’ embryos with

abnormal morphology are here grouped with slow

embryos).Accelerated embryos have 9 cells by day 3.

Several articles have reported a poor clinical out-

come and/or development for embryos with slow

or accelerated cleavage rates.

37,38,49,64

In contrast to

Alikani et al,

other studies have found that the

major and almost sole indicator for an embryo to

result in offspring was blastocyst morphology, and

not day 3 morphology.

38,65,66

Three studies now report the analysis of more

than 500 embryos per study.

42,67

Study I analyzed

524 embryos,

study II analyzed 721 embryos,

and

study III analyzed a single cell from each of 1596

embryos.

Study III analyzed chromosome abnor-

malities in relation to cell number, and found that

normal day 3 embryos with 7–8 cells showed the

lowest rate of abnormalities (55%), while slow

embryos with 4 cells or less had 74% (p 0.001);

accelerated embryos with 9 or more cells were simi-

lar, with 79% abnormal (p

0.005).

Studies I and

3,67

classified the embryos in three maternal age

groups: 20–34; 35–39; and 40–47 years old. Pooled

results from a total of 1255 embryos from these two

studies demonstrated a highly significant relation-

ship between maternal age and aneuploidy (p

0.001).

Individual chromosomes involved in non-disjunc-

tion were also analyzed using pooled results from

the two studies, and chromosomes 16, 18, and 21

showed significantly higher frequencies of aneu-

ploidy with increasing maternal age (p 0.05, p 0.05,

and p 0.01, respectively).

The chromosomes most

frequently involved in aneuploidy at the cleavage

stage were found to be, in order of frequency,

chromosomes 22, 16, 15, and 21 (Table 18.3).

A recent study analyzing 4665 embryos confirmed

that the incidence of chromosomal abnormalities is

significantly higher in arrested or slow-cleaving

embryos as well as in accelerated embryos, compared

to embryos with 8 cells at 62 hours postinsemina-

tion.

This study also reported that the presence of

an uneven number of blastomeres or fragments scat-

tered in the perivitelline space was associated with

an increased incidence of chromosomal abnormali-

ties. Following these considerations, the study by

Magli et al

focused on the question of whether it is

preferable to transfer a slow-cleaving embryo with

no fragmentation, or a regularly cleaving embryo,

i.e. with 7 or 8 cells on day 3, with a variable propor-

tion and type of fragmentation. According to the

reported data, when considering embryos with no

fragments or with a concentrated pattern of frag-

ments, the incidence of chromosomal abnormali-

ties is dependent on the cellular stage, irrespective of

the percentage of fragmentation. Conversely, in the

presence of scattered fragmentation, the incidence



Table 18.3 Specific chromosome aneuploidy rates.

Double aneuploidies counted twice, once for each

chromosome. Tetrasomies and nulisomies were

counted as two trisomies and two monosomies,

respectively

Number of aneuploid

embryos analyzed

Chromosome Number of embryos n %

XY 1741 21 1.2

1 559 14 2.5

2 426 11 2.6

3 426 11 2.6

4 753 17 2.3

6 194 3 1.5

7 244 7 2.9

11 426 9 2.1

13 2227 76 3.4

14 280 3 1.1

15 1492 91 6.1

16 2091 134 6.4

17 1035 33 3.2

18 2484 69 2.8

21 2437 132 5.4

22 1700 118 6.9

Total 2484 750

From Munne et al

and Abdelhadi et al.

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CHROMOSOMAL STATUS OF HUMAN EMBRYOS

of chromosomal abnormalities is significantly higher

in 7- or 8-cell embryos compared with an embryo

with fragments concentrated in one area. For

slow-cleaving embryos (4–6 cells on day 3) there is

apparently no correlation between fragmentation

and the total amount of chromosomal abnormali-

ties. Altogether, these observations suggest that 8-cell

embryos on day 3 are always preferable for transfer,

even in the presence of fragmentation, especially if

fragments are concentrated in one area of the peri-

vitelline space. However, the large difference in the

number of aneuploid embryos detected at cleavage

stage compared with prenatal data from spontaneous

abortions

69–71

indicates a strong selection against

aneuploid embryos before or shortly after implantation.

Aneuploidy does not necessarily lead to develop-

mental arrest in the first 3 days of culture, since the

embryonic genome is not fully active until day 2 or

3 of development.

72,73

Indeed, aneuploidy does not

increase with decreasing developmental potential

and cannot be reduced by selecting day 3 embryos

of normal appearance.

3,42,67

In contrast, postmeiotic abnormalities do increase

with decreasing embryonic competence and are more

common in arrested, slow, and dysmorphic embryos

than in those that are normally developing.

3,42,67

These postmeiotic chromosome abnormalities

account for more than half of the abnormalities

detected in cleavage stage embryos. Although post-

meiotic abnormalities significantly decrease with

embryonic competence (polyploidy p 0.001, exten-

sive diploid mosaicism p

0.01), they are not

affected by maternal age.

3,67

Polyploidy is found mostly in arrested embryos,

and increases with decreasing embryo compe-

tence.

3,67,74,75

As previously noted, it is unlikely

that this represents polyspermy, since all of these

embryos were derived from dipronucleated zygotes

with two polar bodies.

The most likely explana-

tion is that their DNA synthesis continued in the

absence of cellular division.

76,77

Excluding the 40 years age group, extensive

(3/8 abnormal) diploid mosaicism is the major

chromosome abnormality in IVF-generated human

embryos. For the age group 35–39, mosaicism was

found in 23.3% of the embryos,

followed by

polyploidy (21.8%), aneuploidy (10.2%), and

haploidy (3.6%). Even in the group of normally

developing embryos, which are closer in quality to

embryos that are transferred, aneuploidy (19.3%)

contributes to less than half of the chromosome

abnormalities detected, with extensive diploid

mosaicism (14.7%), polyploidy (4.5%), and hap-

loidy (4%) together contributing to a greater extent

than aneuploidy.

Similarly, another study by Bielanska et al

detected 2N/mosaicism (extensive and limited) and

chaotic mosaics in 55% of surplus embryos, fol-

lowed by 30% normal embryos, and the remainder

were aneuploid, polyploidy, and haploid.

Because the embryonic genome is not fully active

until day 3 of development,

72,73

mosaicism, poly-

ploidy, and haploidy cannot produce dysmorphism

originating in the first and second meiotic divisions.

However, cytoplasmic impairment could produce

both mosaicism and polyploidy, through cytoskele-

tal and spindle malfunction leading to a block in cell

division, or other mechanisms. For example, abnor-

malities of the centriole in a fertilizing spermato-

zoon may produce mosaicism or other chromosome

abnormalities in the resulting zygote.

78–80

In addi-

tion, intrinsically low mitochondrial activity has

been associated with chaotic mosaicism, but not

with other types of chromosome abnormalities.

The majority of chromosome studies on human

embryos have focused on meiotic irregularities as the

principal source of chromosome abnormalities.

However, the reviewed data

indicate that other

sources of chromosome abnormalities are more

important, and further investigation of other factors

such as culture conditions, hormonal stimulation,

centriole abnormalities, and cytoplasmic factors is

justified.

BLASTOCYST FORMATION AND

CHROMOSOME ABNORMALITIES

STUDIES IN UNSELECTED BLASTOCYSTS

Many studies have assessed the chromosome com-

position of surplus blastocysts (Table 18.4).

76,82–89

The rate of mosaicism detected by FISH is as high as



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