Elder K. Human preimplantation embryo selection

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HUMAN PREIMPLANTATION EMBRYO SELECTION

90%,

75,85,87,89

but the percentage of abnormal cells was

low compared with cleavage stage mosaic embryos,

being no higher than 30% on average. In addition, the

majority of abnormal cells found in mosaics were

tetraploid, 2N/4N mosaics being very common

(23–86% of all blastocysts), but triploid, haploid,

aneuploid, and chaotic cells were also described.

Although aneuploid cells seem to be detrimental

to embryo development, high levels of mosaicism

and chaotic embryos can still be detected at the

blastocyst stage.

90,91

Compared with lower rates of

2N/4N mosaics on day 3 of development,

this

indicates that the majority of 2N/4N mosaics arise

at morula or later stages. In studies comparing chro-

mosome abnormalities in blastocysts that developed

from good or poor morphology day 3 embryos,

higher rates of abnormal cells per blastocyst were

found in those developing from poor morphology

day 3 embryos.

86,88

A study evaluating chromosome abnormalities

in relation to blastocyst morphology found that 65%

of mosaic blastocysts had good morphology.

Thus,

by itself, morphology is generally not an appropri-

ate tool to screen for chromosome abnormalities.

SURVIVAL OF CHROMOSOMALLY ABNORMAL

EMBRYOS TO BLASTOCYST STAGE

The survival of chromosome abnormalities to the

blastocyst stage has been analyzed.

3,75,90,91

Several

authors have suggested that the fact that many

embryos arrest during the morula stage may act as a

selection against chromosome abnormalities during

extended culture.

82,92

Magli et al

reported that

only 22% of chromosomally abnormal embryos

reached blastocyst stage compared with 34% of

euploid embryos (p 0.001). Of the embryos sur-

viving to blastocyst stage, the majority had a mosaic

inner cell mass, with 2–16 different cell lines. Marquez

et al

compared day 3 and day 4 embryos, and found

that embryos analyzed on day 4 had much higher

rates of polyploidy than those analyzed on day 3,

suggesting that embryos arresting on day 3 become

polyploid by day 4.

MOSAIC EMBRYOS

Bielanska et al

found chaotic mosaics to be more

common in arrested day 3 and day 4 embryos than

in blastocyst stage embryos. They also found that

diploid/polyploid mosaics increased with develop-

mental competence, with an overall decrease in the

number of abnormal cells per mosaic embryo from

cleavage stage to blastocyst stage. Sandalinas et al

observed that embryos with a high frequency of

mosaicism can occasionally develop to blastocyst

stage, although they seldom had more than 60 cells,

compared with an average of 114 for normal

blastocysts.

TRISOMIES

Of trisomies 37%

and 34%

reached blastocyst

stage compared with 66%

and 61%

of normal



Table 18.4 Chromosome abnormalities in unselected blastocyst studies

Number of

blastocysts 2N/aneuploid 2N/chaotic

Reference analyzed Normal 2N/polyploid (and/or polyploid) or chaotic Polyploid Haploid Aneuploid

83 73 43 17 4 0 7 0 2

84 8 1 6 1 0 0 0 0

85 19 1 6 10 (6) 2 0 0 0

86 33 3 24 5 1 0 0 0

304 207 87 0 0 10 0 0

182 162 0 12 0 2 0 6

88 58 30 10 15 (5) 3 0 0 0

87 91 6 60 16 3 3 0 3

Ploidy analysis only;

karyotype analysis.

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CHROMOSOMAL STATUS OF HUMAN EMBRYOS

embryos. The difference was statistically significant

in both studies. Magli et al

also found that abnor-

mal embryos reached blastocysts less often than did

those that were normal (22% vs 34%).

MONOSOMIES AND HAPLOIDY

No haploid embryos, and only 9% monosomies,

restricted to monosomy X and 21, reached blasto-

cyst stage in the Sandalinas study.

Magli et al

and

Rubio et al

also detected a strong selection against

haploidy and autosomal monosomy, but while

some monosomies reached blastocyst stage they did

not re-analyze the whole embryo to confirm the

abnormality; some of the embryos developing to

blastocyst might have been normal embryos that

had been misdiagnosed

or had differences between

inner cell mass and trophectoderm.

Rubio et al

found that 54% of embryos with monosomy X sur-

vived to blastocyst. The fact that only monosomy X

and 21 were found in blastocysts

agrees with pre-

natal diagnosis data, where no other monosomies

are detected in first trimester abortions.

POLYPLOIDY

Polyploid embryos clearly reach blastocyst stage, as

polyploid pregnancies do reach first trimester and

beyond. Sandalinas et al

found that 21% of poly-

ploid embryos developed into blastocysts; this has

been confirmed by others.

EFFECTIVE BUT LIMITED IMPACT OF

MORPHOLOGICAL AND DEVELOPMENTAL

SELECTION

Embryo selection is critical to the success of IVF.

Careful evaluation of embryo morphology per-

formed under powerful inverted microscopes will

detect many abnormalities such as multinucleation

and fragmentation. Dysmorphic and arrested

embryos, 50% of which are chromosomally abnor-

mal, should not be transferred if better embryos are

available. However, this evaluation does not allow

selection against many chromosome abnormalities

that occur with a frequency of 30% in embryos

with apparently normal morphology in women

35–39 years old. This rate increases to about 60%

in women aged 40 years. These frequencies are for

six chromosomes,

2,3

and could be higher with more

chromosomes analyzed.

Assuming that competent IVF centers already

practice appropriate embryo selection on the basis

of morphology and development, a fraction of

chromosomally abnormal embryos have been

selected, but it remains impossible to select against

the majority of aneuploidies and some postmeiotic

abnormalities. As a result, embryos with good mor-

phology and development still show low implanta-

tion rates. Culture to blastocyst stage can further

select against some chromosome abnormalities, but

a number of slow embryos that are chromosomally

normal and could potentially implant will not

survive extended culture.

PREIMPLANTATION GENETIC DIAGNOSIS AS

A SELECTION METHOD

RATIONALE FOR PGD IN INFERTILITY

More than 50% of IVF embryos are chromosomally

abnormal, and this frequency is much higher than

that reported in spontaneous abortions. A sizable

proportion of chromosomally abnormal embryos

are probably eliminated before they are recognized

clinically.

This loss of embryos could account for

the low implantation potential of ART embryos. We

hypothesized that PGD selection against chromo-

somally abnormal embryos predestined to fail

implantation could reverse this trend,

if a sufficient

number of embryos are available. Avoiding the

replacement of embryos with chromosome abnor-

malities should significantly increase implantation,

and reduce both chromosomally abnormal concep-

tions and spontaneous abortions. This should result

in higher take-home baby rates.

APPROPRIATE METHODOLOGY FOR PGD

In general, PGD for numerical chromosome abnor-

malities is indicated for patients aged 35 and older.



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HUMAN PREIMPLANTATION EMBRYO SELECTION

It is also offered to younger patients who are oocyte

donors, have recurrent miscarriages, or a history

of failed implantation. So far, close to 29 000 PGD

cases have been performed by either embryo biopsy

on day 3 of development or polar body biopsy

(Table 18.5). Several studies have shown an increase

in implantation, pregnancy, and take-home baby

rate with a decrease in spontaneous abortion.

95–100

However, others have found little evidence of

similar success.

101

Technical differences can explain

these results: in general, PGD results can be affected

by several parameters (Table 18.6).

CELL TYPE ANALYZED AND NUMBER

OF CELLS ANALYZED

PGD for infertility can be performed after polar

body biopsy (PBs),

104

blastomere biopsy of day-3

cleavage stage,

or blastocyst biopsies.

105

The major-

ity of PGD cases have been carried out after PB or

cleavage stage biopsy. Both procedures have their

advantages and disadvantages. FISH analysis of first

PBs, sometimes in combination with second PB

analysis, was first attempted by Verlinsky et al

104

and Munné et al.

Since autosomal aneuploidy



Table 18.5 Estimate of PGD for infertility procedures

performed worldwide to June 2006

US Total

Reprogenetics 6638

RGI 5010

Integramed 2000

PGDbyART 1533 (until merged with

Reprogenetics)

Others 1500

Europe

IVI, Spain 4300

Centers in ESHRE consortium 1880

SISMER, Bologna, Italy 1710

Reprogenetics, Spain 560

Asia, Oceania, South America

Farah Hospital, Jordan 1629

Abdelmassih, Sao Paulo, Brazil 1000

Sidney IVF, Australia 1000

Others Asia 800

GEN-LAB, Ankara, Turkey 505

Other Turkey 500

Other South America 500

Reprogenetics (Japan) 125

Total 30 665

Most centers in Europe report to the European Society of Human

Reproduction and Embryology (ESHRE) consortium. The largest centers

are reported separatedly.

1788 cases from IVI, and 1320 from SISMER,

although reported to ESHRE consortium, are not included here but in IVI

and SISMER. Data to 2004.

Approximate estimate.

Table 18.6 Summary of studies comparing PGD and control ART outcome

Reference 96 99 97 102 101 98

Types of mosaics

Cells biopsied 1 1 1 1 2 1

Fixation used Carnoid Carnoid Carnoid Twin 20 Several Carnoid

Chromosomes analyzed 4–8** 8 8 8 6 8

Type of study comp comp comp randzd randzd comp

Cycles control 117 127 138 28 141 8706***

Cycles PGD 117 135 138 29 148 562***

Average number of NA 3.0

3.7

NA 2.8

embryos replaced control

Average number of NA 1.8

2.0

NA 2.0

embryos replaced PGD

Implantation rate control (%) 13.7 12.4

10.6

NA 11.5 NA

Implantation rate PGD (%) 17.6 24.2

17.6

NA 17.1 NA

Pregnancy loss rate control (%) 33.8

20.6 NA NA 25.6 21.5

Pregnancy loss rate PGD (%) 15.0

5.4 NA NA 25.0 16.7

Ongoing implant rate* control (%) 10.6

10.2

NA NA 10.4

Ongoing implant rate* PGD (%) 15.9

22.5

NA NA 16.5

Pregnancy rate control (%) 29.9 25.1 NA 20.7 27.7 NA

Pregnancy rate PGD (%) 35.9 29.1 NA 43.0 19.6 NA

comp, prospective non-randomized comparative study; randzd, prospective randomized study; *fetus ongoing 12 weeks/embryos replaced; **36 cycles

with four probes, 50 cycles with five probes, and 31 cycles with eight probes; ***pregnant cycles;

p 0.001;

p  0.06;

p 0.05. Adapted from Cohen et al.

103

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CHROMOSOMAL STATUS OF HUMAN EMBRYOS

occurs predominantly during maternal meiosis

70,106,107

analyzing the first PB for aneuploidy can

detect the majority of autosomal aneuploidies

identified in blastomeres. PB biopsy has three

advantages over embryo biopsy. First, in the case of

a diagnosis error, chromosomally normal oocytes

are discarded rather than embryos, but only if the

diagnosis is known before fertilization is complete.

For example, using a shortened FISH analysis, intra-

cytoplasmic sperm injection (ICSI) can still be per-

formed within 6 hours after retrieval, so that only

oocytes diagnosed as normal are selected for injec-

tion.

68,108

This is a demanding procedure for the

IVF clinic, and very tight time constraints require

the simultaneous collaboration of gynecologists,

embryologists, and biologists from the cytogenetic

laboratory. In countries with ultraconservative rul-

ings regarding IVF (i.e. Chile and Italy), this is the

only form of PGD that is possible.

109

In other coun-

tries such as Switzerland and Germany, it is legal to

inseminate many oocytes and freeze zygotes. The

second PB can be diagnosed followed by zygote

freezing, and only those that are normal are allowed

to proceed to syngamy. Second, the first and second

PBs are not involved in embryo development, and

since their removal does not decrease rates of fertil-

ization, cleavage, and blastocyst formation,

theo-

retically there should be no negative effect on

implantation rates. Third, embryo biopsy has the

disadvantage that about 30% of embryos are

mosaic, and this feature may result in misdiagnosis

in about 5% of cells analyzed (see below). On the

other hand, PB analysis cannot detect mosaic

embryos, whereas single cell embryo biopsy can

screen for a sizable fraction of mosaics.

PGD using PBs also has many disadvantages,

such as difficulties associated with the interpretation

of FISH signals. Univalent chromosomes appear as

double-dotted signals, with one dot per chromatid.

Since predivision chromatids do frequently occur,

single overlapping signals can allow chromatids to

be confused with full chromosomes. In addition,

predivision appears to increase as an artifact of

increasing time in culture, both in first PBs and in

oocytes.

This increase is apparent as early as 6

hours after egg retrieval. The error rate for embryo

biopsy data has been published repeatedly, and in

our hands is manageable, whereas the error rate for

PB biopsy, measured as the full re-analysis of embryos

after PB analysis has not been clearly reported. PB

misdiagnosis can also be due to other artifacts, such as

loss of chromosomes during fixation or the lack of

probe penetration in some forms of chromatin. For

example, hybridization errors or loss of chromo-

somes during fixation can lead to an excess of miss-

ing chromatids in the PB, resulting in an excess of

231/2 oocyte diagnoses

52,111,112

(Table 18.7). Pre-

insemination diagnosis of aneuploidy also has the

disadavantage that paternally inherited aneuploi-

dies and chromosome abnormalities that occur post-

zygotically such as polyploidy, haploidy, and some

mosaics cannot be detected. These account for about

30% of chromosome abnormalities, and they can be

assessed by analyzing blastomeres instead of PBs.

On the other hand, embryo biopsy may jeopardize

the implantation potential of the embryo. However,

to date PGD has been shown to increase implanta-

tion rates and decrease spontaneous abortion rates

in women of advanced maternal age,

96,99

although

the increase in implantation obtained in these stud-

ies is less significant than was originally thought.

One reason could be the effect of embryo biopsy

itself, and this has been the focus of recent investiga-

tion. Studies have shown that the biopsy of 1 cell at

the 8-cell stage of human embryos is not detrimental



Table 18.7 Excess of missing chromatids after polar

body (PB) FISH analysis

Non-disjunction Predivision

231231231/2 221/2

Abnormal oocyte karyoypes

25 (20%) 37 (29%) 26 (21%) 39 (31%)

Abnormal oocyte karyotype

deduced from first PB

105 (11%) 21 (2%) 617 (65%) 208 (22%)

Mamiguchi et al, 1993; Nishino et al, 1994; Dailey et al, 1996; Angell, 1997;

Boiso et al, 1997; Nakaoka et al, 1998; Marquez et al, 1998; Eckel et al,

2000; Mahmood et al, 2000; Rosenbusch et al, in press.

Munné et al,

1995, 2000; Verlinsky et al, 1996, 1999; Dyban et al, 1996. Table adapted

from Rosenbusch et al.

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HUMAN PREIMPLANTATION EMBRYO SELECTION

for embryo development to blastocyst stage,

113

but

the removal of 1/4 of a 4-cell embryo seems to

reduce the ratio of inner cell mass : trophectoderm.

The interpretation of these experiments

15,113

is not

consistent, and biomass reduction is often discussed

in the context of a superfluous cell number and

totipotency. Controlled experiments with ongoing

pregnancy as an endpoint have not been carried

out, so that the effects of biopsy on all stages of

development, not just those with the most rapid

rate of development, can be ascertained. The ques-

tion of whether biopsy has a significant effect on the

embryo remains unresolved.

Cohen et al

103

postulated that embryo biopsy

affects embryo development prior to transfer, and

may have lasting effects after transfer to the uterus.

This suggestion is based on the assumption that cell

loss from biopsy can be compared with cell loss,

after thaw of frozen cleaved embryos. After cryo-

preservation, individual cells are frequently lost,

while remaining cells have the potential to develop

into a viable blastocyst, depending on the quality of

the embryo and the proportion of cells damaged at

thaw. The implantation potential of thawed embryos

that are fully intact and have no degenerate cells

may resemble that of embryos transferred without

cryopreservation.

114,115

Using this analogy, implan-

tation potential becomes a function of cell loss, the

quality of the original embryo, and the number of

cells present at the time of biopsy. According to

our EggCyte database, based on data from 75 000

embryos, the average cell number on day 3 is not 8,

but 6.7 cells. Therefore another factor for consid-

eration is that the proportional effect of cell loss

on implantation potential might be higher than

expected.

Taking the analogy with cryopreservation further,

the removal of a single cell from an 8-cell embryo is

expected to reduce implantation potential by

12.5%.

114

For the purpose of this discussion, if the

expected implantation potential in a hypothetical

8-cell embryo is set at 20%, a single cell biopsy is

predicted to lower its implantation probability to

17.5%. Consequently, for preimplantation diagnosis

to succeed in increasing implantation rates, it must

more than compensate for this initial setback.

The challenge for PGD as a tool in infertility

treatment becomes considerably more difficult to

overcome when 2 cells are removed. The loss of 2

cells from an embryo as described above would

reduce its implantation potential by 25%, resulting

in a 15% chance of implanting versus an initial

probability of 20%. In this case, in order for PGD to

improve embryo selection, it must bridge a larger

deficit if it is to provide any advantage in terms of

embryo implantation. On the basis that the average

cell number at the time of biopsy is only 6.7, rather

than 8.0, an even more pronounced decline in

implantation potential might be expected. With a

theoretical implantation potential of 20% without

biopsy, the implantation potential after a 1- or 2-cell

biopsy would diminish to 17.0% and 14.0%, respec-

tively. It is clear from this analysis that a 2-cell biopsy

significantly impedes embryo development and is

inadvisable in cases where PGD is employed for the

purpose of increasing IVF success rates.

If a PGD study fails to detect a difference in

implantation rate when comparing routine IVF

cycles with cycles that employ 2-cell biopsy, this is

not an indication that chromosome screening has

had no effect. On the contrary, it shows that PGD

has succeeded in compensating for the significant

reduction in implantation caused by the biopsy of

2 cells. This vital distinction has sometimes been

overlooked,

101

with resulting misinterpretations of

data in subsequent reviews.

116,117

A recent study by

the same group has shown no difference in the

error rate of PGD between 1- and 2-cell biopsy.

118

One can conclude from this work that there is little

benefit in biopsy of 2 cells. In theory, trophecto-

derm biopsy at the blastocyst stage should not

affect the inner cell mass, and should be less detri-

mental than the biopsy of 2 cells in cleavage stage

embryos; it should provide all the information that

cleavage stage biopsy can provide, and more infor-

mation than PB analysis. In addition, the results

should not be affected by mosaicism, since five or

more cells are simultaneously analyzed. So far,

very few centers have implemented this technique

clinically,

105

and more data are needed to evaluate

whether these advantages can be confirmed. In

addition, blastocyst culture is not yet able to sustain



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CHROMOSOMAL STATUS OF HUMAN EMBRYOS

some embryos that might result in live births if

replaced on day 3.

37,119

BIOPSY TECHNIQUE

Day 3 embryo biopsy of a single cell is our current

recommendation, since this is the only method that

has consistently shown an improvement in ART

outcome. The technique used for biopsy can be

mechanical; chemical, using acidified Tyrode’s solu-

tion; or physical, by non-contact infrared laser. The

mechanical method was adopted by only a few

groups, while the laser method, being very quick,

precise, and easy to use, has become increasingly

popular and is rapidly replacing the chemical method,

which requires more experience and skill.

120

However,

recent studies have demonstrated that a safe work-

ing distance for the laser is crucial in order to pre-

vent adverse immediate or long term effects on the

development of laser biopsied human embryos.

121

Very few studies in the literature report data that

compare these methods. A recent study comparing

acidified Tyrode’s solution and laser biopsy demon-

strated identical pregnancy rates, but a slight increase

in the number of damaged cells after the acid

technique compared with after the laser.

122

These observations suggest that accurate and

highly specialized training is mandatory for the

correct application of such an invasive procedure,

regardless of the method used. One of the primary

reasons for non-satisfactory results after PGD for

aneuploidy obtained in some centers could be due

to inaccurate performance of the biopsy in labora-

tories with a micromanipulation station that is

equipped with a laser.

123

FIXATION METHOD

The cell must be fixed after biopsy, in a manner that

facilitates proper FISH processing and minimizes

errors. The classical method of cell fixation involves

acetic acid/methanol or Carnoid;

124

the dynamics of

nuclear fixation with these classical techniques have

been studied mostly to improve metaphase chro-

mosome spreading, and not for the purpose of

interphase FISH analysis. However, the principles

are similar, as indicated by Spurbeck et al.

125

‘as the fixative evaporates, the surface tension of

the fixative on the cell makes the cell thinner

from top to bottom and wider from side to side.

The cell continues to get thinner and wider as the

fixative evaporates. As the cell widens and

reaches its widest diameter potential and then

dries, it results in a suitable spread. If the drying

rate is too fast, the cell dries before it has a chance

to reach the optimum diameter. This results in a

tight, compact metaphase where many chromo-

somes are overlapped. On the other hand, if the

drying rate is too slow the cell does not dry and

currents in the fixative solution may move the

cell and it eventually fixes in an uncontrolled

manner.’

These authors found that the proper spreading of

metaphase chromosomes was largely dependent on

humidity and temperature, and they recommend

50% humidity at 25C. In our experience with

interphase blastomere nuclei, 40–50% humidity

and 20C produce optimal blastomere nuclear

spreads. We observed that the ideal diameter is on

average 60 m. In nuclei larger than 80 m in

diameter (which usually happens in an atmosphere

where the humidity is too high) the chromatin is

excessively decondensed, with signals that are more

widely spread and weaker than those found in regu-

lar size nuclei; the signals in these large nuclei are

sometimes imperceptible, leading to misdiagnosis.

Three methods of fixation are currently in use:

method 1: acetic acid/methanol;

124,126

method 2:

Tween 20/HCl;

127

and method 3: Tween 20/HCl and

acetic acid/methanol.

128

Method 1 can be optimized

using the observations of Spurbeck et al.

125

Method

2 involves dissolving the cytoplasmic membrane

without fixative, and results in tight and condensed

nuclei. Method 3 is a combination of methods 1 and

2. Nuclear diameter was previously demonstrated to

be inversely correlated with chromosome overlaps

and FISH misdiagnosis.

129

Velilla et al

130

compared

the three methods, and found that method 1 pro-

duced the largest diameters, and as a result a mini-

mal number of signal overlaps and misdiagnoses,



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HUMAN PREIMPLANTATION EMBRYO SELECTION

while method 2 produced the smallest diameters

and the largest number of overlaps and misdiagnoses

(p 0.005). However, Carnoy’s method (method 1)

can be more difficult to master, and in some hands

produces a larger proportion of lost cells,

131

but at

the same time a larger number of cells that can be

analyzed.

130,131

In conclusion, Carnoy’s method is more difficult

to master, and more cells may initially be lost by

novices with the technique, but once it is mastered it

produces larger nuclear diameters, which translate

into fewer signal overlaps and fewer FISH errors.

130

Not surprisingly, PGD laboratories using method 2

usually report high error rates, unmanageable rates

of discordance between cells, and no improvement

of ART outcome after PGD of infertility.

101,132

PROBES

In order to study as many chromosomes as possible,

several types of FISH protocols have been used to

maximize the use of a limited number of fluo-

rochromes. One approach used ratios of fluoro-

chromes, but this has the disadvantage that

overlapping signals from two different chromo-

somes sharing one or more colors may produce a

misdiagnosis. Therefore, single colors are favored,

but there are only five colors in the visible spectrum

that can be analyzed simultaneously. Thus, in order

to study more than five chromosomes, the re-analy-

sis of the same nucleus with different probes was

implemented.

133

The second set of probes works

with high efficiency (95%). Coupled with fast pro-

tocols this allows ten chromosomes to be analyzed

simultaneously in a single interphase nucleus,

within a time frame that is compatible with routine

IVF.

99,134,135

We have improved the use of a third

consecutive hybridization, which is capable of

simultaneously analyzing up to 15 chromosomes,

with only a 12% error rate.

The question of which chromosomes should be

studied, and whether a minimum number of chro-

mosomes analyzed is needed in order to improve

ART outcome needs to be addressed. Analysis of

thousands of cleavage stage embryos for 16 different

chromosomes (X, Y, 1, 2, 3, 4, 6, 7, 11, 13, 14, 15, 16,

17, 18, 21, 22), demonstrates that the chromosomes

most commonly found in aneuploidies are chro-

mosomes 22 (6.6%), 16 (5.2%), 15 (4.7%), and 21

(4.7%)

13,53

(Table 18.3). Thus, the chromosomes

with the highest proportion of trisomies or risk of

reaching term should be detected by the analysis.

The standard panel of probes that has produced

an improvement in ART results

97,99

contains nine

probes, for chromosomes X, Y, 13, 15, 16, 18, 21, 22

plus one more. Studies with fewer probes have not

been able to show an improvement in implanta-

tion rates, although a reduction in spontaneous

abortions has been found.

MINIMIZING SOURCES OF FISH ERRORS

The use of PGD in infertility has been criticized on

the basis that determining the chromosome com-

plement is unpredictable, due to high levels of

mosaicism in the early human embryo. Whereas

such reservations are sometimes valid, they are not

always based on biological phenomena, and tech-

nology may play a role in artificially amplifying the

true rates of mosaicism. This section deals with the

sources of errors that may be caused by technical

and biological problems during the analysis of sin-

gle blastomeres. Polar body and blastocyst biopsy

errors are not evaluated here.

When a single cell is analyzed, several types of

technical problems may result in a misdiagnosis of

that cell. These problems include unsuitable probe

hybridization, loss of DNA during fixation, signal

overlaps, stretched signals, and double chromatids

giving the appearance of two close signals. In addi-

tion, biological phenomena such as micronucleation

and mosaicism may render the cell not representa-

tive of the rest of the embryo. Criteria for differenti-

ating between mosaicism and false positives and

negatives have been previously described.

These

criteria apply only when all or most of the cells of an

embryo are analyzed, and when all of the remaining

embryo cells are fixed on day 3, or at the latest, early

on day 4. This is due to the fact that some abnormal

embryos arrest and degenerate between day 4 and 5,

so that the remainder are enriched with normal

embryos, and at the same time there is an increase in



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CHROMOSOMAL STATUS OF HUMAN EMBRYOS

polyploidy and production of 2N/4N mosaics

between day 3 and day 5.

Re-analysis performed on

day 5 or later usually results in artificially high rates

of misdiagnosis.

136

In order to keep the proportion of FISH errors

under strict control, every center offering PGD

for aneuploidy should systematically control their

results by re-analyzing the non-transferred embryos,

as recommended by the guidelines on good PGD

practice.

137

Unfortunately, these data are reported

in the literature in only a limited number of publi-

cations.

12,42,45,97,99,101,129,138–140

TECHNICAL SOURCES OF ERRORS

Unsuitable probe hybridization

Reduced hybridization because of poor probe pene-

tration or insufficient probe binding is usually due

to suboptimal fixation, specifically if cytoplasm

remains or the cell did not burst during fixation. This

should not occur with proper protocols and fixation

techniques, and can be foreseen after fixation by

simple phase-contrast observation.

Signal overlaps between homologs

A false diagnosis of monosomy can result from

overlap of chromosome-specific signals for the same

chromosome.As reviewed previously, we found that

poor spread of the nucleus during fixation and the

DNA content in the nucleus increased signal over-

lap,

129

and that some fixation methods produce

more overlaps and misdiagnoses than others.

130

In a

study by Staessen et al,

101

using the error-prone

Tween 20/HCl method, 19/24 detected errors were

false monosomies, even though they analyzed two

cells per embryo.

Signal overlaps between non-homologs

when using color ratios

Misdiagnosis may also occur through overlap of

different chromosomes labeled with mixtures that

share one color. For example, a chromosome labeled

in orange could overlap with another labeled in

orange and aqua, with the result that the first chro-

mosome is masked by the second. The use of a

single color per chromosome analyzed

135

results in a

significant reduction in the error rate of false nor-

mal and abnormal PGD results, from 8% to 4%.

Loss of micronuclei during fixation

Although with correct technique DNA is not lost

from the main nucleus, micronuclei may be lost

during fixation. For example, the FISH error is

higher in multinucleated blastomeres (MNBs)

(11.5%, 13/113) than in those that are mononucleate

(3.1%, 13/415).

45,53

This is probably due to the fact

that many MNBs very often contain micronuclei,

and during fixation, some of them can be more

easily lost than full nuclei, producing false negative

FISH errors. We found a strong correlation between

type of fixation and loss of chromosomes.

126

During

fixation with Carnoid, drops added before the cell

breaks allow the cytoplasm to expand, with increas-

ing expansion with more drops, while adding a drop

postlysis removes cytoplasmic debris, and probably

some anuclear DNA. It is therefore possible that the

loss of DNA is higher after adding a drop postlysis,

when the cell is more expanded (2 drops prelysis

instead of one) as was observed in this study.

Currently we recommend two drops prelysis, fol-

lowed by no drops postlysis; with appropriate humid-

ity conditions, this allow a good spread, minimal

cytoplasmic debris, and minimal loss of DNA.

Stretched signals and double chromatids giving

the appearance of two close signals

Excessive stretching of the DNA during fixation

may cause a signal to split, giving a false positive

result. This occurs more often with some probes

than with others. A false positive result can also

occur if replication is non-synchronous during an

S-phase, so that one chromosome shows a single

signal for its sole chromatid and the other chromo-

some sends two close signals, one for each chro-

matid. When the signals are two or more domains

(individual signals) apart, we score two close signals

as two separate chromosomes: this produced fewer

misdiagnoses in our hands.

141

RESCUE OF TECHNICAL ERRORS

Technical errors can be avoided by using appro-

priate fixation protocols. If there is doubt about a



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HUMAN PREIMPLANTATION EMBRYO SELECTION

possible artifact of the technique, this can be ascer-

tained by analyzing the same chromosome twice at

two different loci, which will then confirm the

correct result.

In a recent study by Colls et al

140

cells with dubious or no results after two rounds of

FISH with nine probes were re-analyzed in a third

hybridization, using probes that bound to different

loci from the original probes used. After analysis

of 34 831 blastomeres, 7.5% showed inconclusive

results. Re-analysis with additional probes reduced

the number of cells with inconclusive results to

3.1% (p 0.001); FISH errors, measured as discrep-

ancies between the PGD diagnosis and the analysis

of the non-transferred embryo, decreased from

13.6% to 4.7% (p 0.001). Thus the use of rescue to

solve dubious signals before transfer proved to be a

powerful tool in reducing the error rate and the

frequency of inconclusive results after PGD.

Alternatively, biopsied embryos with dubious

results may be transferred on day 4,

142,143

allowing

an extra day for the re-biopsy of another cell from

these embryos. An embryo that has been diagnosed

as monosomic for chromosomes other than X and

21 that reaches blastocyst stage contrary to expecta-

tion,

could be biopsied as a blastocyst for a second

PGD analysis.

MOSAICISM AND PGD MISDIAGNOSIS

Published rates of mosaicism vary widely in the

literature, due to several factors.

(1) Mosaicism error rates are center dependent,

and may be influenced by differing intrafollicu-

lar and laboratory conditions.

(2) The frequency of mosaicism is related to the

developmental stage of the embryo. Thus, the

calculation of PGD errors related to mosaicism

should be re-analyzed on the basis of rates in

cleavage stage embryos, not blastocysts.

136

(3) As discussed above, mosaicism errors must be

distinguished from technical errors.

Large studies conducted on cleavage stage embryos

indicate that rates of mosaicism are in the region of

30%.

3,75,144

Munné et al

143

reported that 29% of 1903

discarded embryos analyzed for at least five chro-

mosomes were mosaic. The most common types

of mosaicism were chaotic (48%), diploid/poly-

ploid (26%), and those caused by mitotic non-

disjunction (25%). The number of abnormal cells

per embryo ranged from an average of 44% in

diploid/polyploid to 84% in chaotic mosaics.

Mosaics have been classified as benign if 1/2 cells

from an 8-cell embryo are abnormal, or as detri-

mental if 3 cells are abnormal.

144

This classifica-

tion is based on the reduction in implantation rate

after freeze–thaw cell loss following embryo cryo-

preservation.

145

It is also based on the observation

that mosaic embryos with a sizable number of

abnormal cells do not develop to blastocyst stage,

and that the majority of monosomic, haploid, and

dinucleated cells arrest in culture.

91,146

Thus two

different types of PGD misdiagnosis can be due to

mosaicism, depending on its extent. The first type of

misdiagnosis occurs when a detrimental mosaic is

classified as normal. This is estimated to occur in

4.3% of diagnoses,

144

and is mainly due to the chaotic

and mitotic aneuploid mosaics. The second type of

misdiagnosis occurs when a benign mosaic is classi-

fied as abnormal, which is estimated to happen in

1.3% of diagnoses; this is mainly due to erroneous

classification of a 2N/Pol benign mosaic as normal.

With the implementation of correct methodol-

ogy to eliminate technical errors, and with retesting

to correctly interpret dubious signals to reduce no

results, the overall PGD error rate when results are

obtained, and that can be attributed to mosaicism,

ranges from 5%

140

to 5.6%

144

(Table 16.8).

EMBRYO TRANSFER TECHNIQUE

The protocol used for embryo transfer introduces

another variable to the survival of the biopsied

embryo. Very few reports have explored this issue,

and although the majority of the data are anecdotal,

they indicate that damage can be done very easily

by unsuitable embryo transfer.

147

Numerous studies

have shown that embryo transfer technique is one of

the important factors in ART success, together with

maternal age, egg quality, and quality control in the

laboratory.

148

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CHROMOSOMAL STATUS OF HUMAN EMBRYOS

In addition to technical difficulties during transfer,

other factors have been analyzed, including (in order

of importance) removal of hydrosalpinges, absence

of blood in the mucus, type of catheter (soft better

than stiff), no touching of the uterine fundus, use of

a tenaculum, removal of all mucus, ultrasonogra-

phy of the uterus before and during the procedure,

leaving the catheter in place for 1 minute, trial

transfer, and the administration of prostaglandins

to prevent uterine contractions. The presence of

pathogenic organisms was also reported to be

detrimental, as well as transferring the embryo in a

large volume of media.

149,150

Significant differences

in pregnancy rates have also been reported to depend

on the physician performing the transfer (reviewed

by Schoolcraft et al

149

). One center reporting optimal

results recommends using a continuous fluid col-

umn of 30 mL of transfer media, in a Wallace

catheter, attached to a 1 mL airtight syringe, with the

embryos loaded towards the tip of the catheter.

149

Some of these factors are even more important

when the embryo has been biopsied, such as the

presence of mucus or blood that may occlude the

catheter lumen and squeeze the embryo so that cells

are lost.

149

In conclusion, in order to maximize results and

minimize errors, a single cell should be biopsied on

day 3 of development, fixed using appropriate

techniques such as the modified Tarkowsky

method,

130

with analysis of, at least chromosomes

X, Y, 13, 15, 16, 18, 21, and 22. The signals should be

read and scored by very well qualified personnel,

with each cell analyzed by two people independently;

dubious signals or no results should be re-tested

with a third set of probes binding to a different

locus,

140

and the embryo transfer procedure should

be performed by doctors who are trained in the

transfer of very fragile embryos, such as those with a

large hole in the zona due to recent biopsy. Failure

to clearly follow these guidelines invariably produces

poor PGD results.

The use of appropriate techniques as suggested

above can improve several aspects of ART results.

REDUCTION IN THE INCIDENCE OF

TRISOMIC OFFSPRING

Misdiagnoses have been reported in 2/91

138

and in

5/434 fetuses

after PGD. In all cases, re-analysis of

the misdiagnosed cells with probes binding to a

different locus confirmed prior results, indicating

that the errors were probably due to mosaicism,

with the exception of one misdiagnosis of trisomy

15; this was attributed to the overlap between a Y



Table 16.8 Estimated risk of PGD misdiagnosis due to mosaicism. Embryos were fully trisomic or monosomic

for a specific chromosome and in addition mosaic for the same chromosome

Overall frequency (A) % Normal cells (B) Risk of misdiagnosis (AB)

Risk of classifying an abnormal embryo as normal

2N/POL (detrimental) 3.7% (70/1903) 34.8% 1.3%

Chaotic (detrimental) 12.7% (242/1903) 9.8% 1.2%

Split (detrimental) 0.3% (5/1903) 29.8% 0.1%

Mitotic aneuploid (all) 6.6% (126/1903) 24.2% 1.6%

Meiotic and mitotic aneuploid (all)

0.6% (12/1903) 12.2% 0.1%

Total 23.9% (455/1903) 18.0% 4.3%

Risk of classifying a mostly normal embryo as abnormal

2N/POL (benign) 3.9% (74/1903) 23.1% 0.9%

Chaotic (benign) 1.3% (24/1903) 24.9% 0.3%

Split (benign) 0.2% (3/1903) 26.7% 0.1%

Total 5.3% (101/1903) 23.6% 1.3%

Total misdiagnosis rate due to mosaicism 5.6%

Data from Munné et al.

144

The risk of misdiagnosis was calculated by multiplying column A by column B.

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