Elder K. Human preimplantation embryo selection

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HUMAN PREIMPLANTATION EMBRYO SELECTION

system developed by Scott and Smith,

using the

same point scoring system. However, further assess-

ment of pronuclear membrane breakdown and the

first cellular division were excluded because of the

constraints of the IVF laws. They reported that the use

of this scoring system provided a negative predictive

value of 92% when the threshold pronuclear score

was set at 13. Embryos transferred from zygotes that

had earned a score of ⱖ13 produced a pregnancy

rate of 22%, whilst those with a score of ⬍13 resulted

in a pregnancy rate of 4%.

The pronuclear morphology scoring system sug-

gested by Senn et al

is also based upon a point sys-

tem to determine the implantation potential of zygotes

prior to assignment of embryo status (for transfer or

for cryopreservation). According to Swiss law, embryo

selection is prohibited, forcing practitioners to select

zygotes for immediate cryopreservation or fresh

transfer. Like ART facilities in Germany, a scoring

system at the pronuclear stage is beneficial in aiding

practitioners to select the zygotes with the greatest

implantation potential. Senn et al

developed the

cumulative pronuclear score (CPNS) as a sum of

points assigned to six morphological parameters,

scored from 1 (best) to 3 (worst) with a CPNS, rang-

ing from 6 to 18: pronuclear position, pronuclear

orientation in relation to the second polar body,

pronuclear location (centering), cytoplasmic halo,

number of nucleoli,and nucleolar polarization. Lower

CPNS scores were positively correlated with embryo

implantation. They found that polarization of nucle-

oli and the appearance of a halo were the most impor-

tant predictive factors of implantation potential.

Zollner et al

developed a pronuclear scoring

system that is also similar to that of Scott and Smith.

Zygotes were given points for ten different criteria,

with a lower total morphology score (ranging from 10

to 30) correlating to better zygote quality. The ten cri-

teria used to assess zygote quality were the number

of pronuclei, position of pronuclei, size of pronuclei,

halo effect, alignment of nucleoli in pronucleus num-

ber one, alignment of nucleoli in pronucleus num-

ber two, number of nucleoli in pronucleus number

one, number of nucleoli in pronucleus number two,

appearance of vacuoles, and appearance of ooplasm.

Points awarded for each characteristic are described

in Table 4.4. Zollner et al

reported that a mean

zygote score of ⱕ15 was positively correlated to

increased blastocyst quality for ICSI derived embryos.

Interestingly, this was not found to be the case with

IVF derived embryos.

PRONUCLEI MORPHOLOGY SCORING SYSTEMS

Kahraman et al

created a pronuclei morphology

scoring (PNMS) system that considered the effect of



Table 4.4 The pronuclear scoring system developed by Zollner et al.

A cumulative

point system with values ranging from 10 to 30 with a lower value indicative of

better morphology

Points awarded for various morphological parameters

Parameters 1 2 3 4

PN number 2 PN NA NA 1 PN

PN position IA Nearby Not IA NA

PN size Equal Unequal NA NA

Halo effect Normal Light Extreme None

NPB polarity (PN1) Aligned CIA Unaligned No NPB/no PN

NPB polarity (PN2) Aligned CIA Unaligned No NPB/no PN

NPB number (PN1) 3–5 ⬎ 5 ⬍ 3 No NPB/no PN

NPB number (PN2) 3–5 ⬎ 5 ⬍ 3 No NPB/no PN

Vacuoles No vacuoles Lightly Vac Strongly Vac NA

Ooplasm Homogenous Gr Strong Gr NA NA

PN, pronucleus; IA, in apposition; NPB, nucleolar precursor bodies (nucleoli); CIA, coming into alignment; NA, not

applicable; Vac, vacuolated; Gr, granulation.

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HUMAN PRONUCLEI AS A MODE OF PREDICTING VIABILITY

sperm source (testicular sperm and round spermatids

vs ejaculated sperm) on corresponding score and

subsequent chromosomal status. The morphological

parameters evaluated were pronuclear position and

size, nucleolar polarity and size, and the halo effect

(Table 4.5). The zygotes were placed into eight groups

labeled as A, B, C, D, E, F, G, and J, and after further

review categorized as group I (A–D) and group II

(E–J). Group I zygotes from this scoring system are

similar in morphology to Z scores of 1 and 2 as well

as pattern 0 from previously discussed scoring sys-

tems, and these would be considered the more normal

of the zygote morphologies. This study found a corre-

lation between sperm source and resulting pronuclear

morphology, as well as with PNMS score and further

embryo development and chromosomal abnormali-

ties. Zygotes in group II were more likely to be derived

from testicular sperm or round spermatids (32.1%)

versus ejaculated sperm (22.7%). A higher proportion

of good quality blastocysts were derived from group I

(50.4%) versus group II (28.2%). Finally, chromoso-

mal abnormalities were lower in embryos from group

I (37.6%) versus embryos from group II (56.2%).

Gianaroli et al

proposed another PNMS system

based on three parameters, and used this to correlate

chromosomal status with zygote morphology. The

three parameters were location and position of

pronuclei (A through E), size and polarity of nucleoli

(1 through 4), and pronuclear orientation in relation

to the second polar body (␣, ␤, and ␥) (Table 4.6).

Upon completion of chromosomal analysis it was

determined that four configurations had a greater

chance of producing euploid embryos (A1␣,A2␤,

A3␤, and A3␣) which correlated to better embryonic

development.

STUDIES UTILIZING ZYGOTE

SCORING SYSTEMS

Testing of these pronuclear morphology scoring sys-

tems by various ART facilities reported inconsistent

results in their usefulness in predicting embryonic

implantation potential. This inconsistency is two-

fold; first, it can be attributed to the multifactorial

situations in which nearly all of these studies were

performed, and second, a static observation cannot

be used as the sole predictive source of embryonic

implantation potential. The predictive value of zygote

scoring can be increased by combining several mor-

phological factors, evaluated at different time points

during embryonic development.

Tesarik and Greco

reported a 50% (22/44)

pregnancy rate following the transfer of at least one

embryo that was derived from a zygote of good mor-

phology (pattern 0) versus a 9% (2/23) pregnancy

rate for the transfer of embryos derived from abnor-

mal zygotes (patterns 1–5). Another study corre-

lated pattern 0 zygotes with higher quality embryos

of greater implantation potential than embryos



Table 4.5 Pronuclear morphology scoring (PNMS) system created by Kahraman et al.

Zygotes

in subgroups A through D are considered to have normal morphology that correlates to embryo

development and the incidence of chromosomal abnormalities

Nucleolar Nucleolar size

Group Sub-group Halo PN position PN size alignment

I A Distinct IA Equal Aligned Large

B Distinct IA Equal Aligned Small

C Distinct IA Equal Scattered Large

D Distinct IA Equal Scattered Small

II E Distinct IA Equal Aligned 1 Large, 1 Small PN

F Distinct IA Equal Scattered 1 Large, 1 Small PN

G Blurred IA Equal or Unknown Unknown

unequal

J Blurred Not IA Equal Syngamic Large or Small

PN, pronucleus; IA, in apposition.

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HUMAN PREIMPLANTATION EMBRYO SELECTION

originating from abnormal zygote patterns.

Pattern

0 embryos

correspond to Z1 and Z2 embryo scores

as developed by Scott et al

and Scott

(Figure 4.2).

Scott et al

associated clinical pregnancies with day 3

transfers only when embryos derived from a zygote

with a score of Z1 or Z2 were included in the transfer.

In another study, Scott

reported no live births from

transfers involving only Z3- or Z4-derived embryos.

Unlike Tesarik and Greco,

Scott et al

or Wittemer

et al

, James et al

demonstrated an equal chance of

pregnancy and live birth from transfers consisting

of only Z3-derived embryos replaced on day 3.

Taking this one step further, Jaroudi et al

reported

absolutely no correlation between the selection of

higher quality zygotes and pregnancy rates when

embryos were selected for transfer using Tesarik and

Greco’s system,

or when retrospectively assessed

using the Scott et al

revised Z scoring system. Similar

results were observed by Salumets et al

who also

found no difference in pregnancy or implantation

rates between pattern 0 and non-pattern 0 zygotes

in single embryo transfer patients. Payne et al

reported a similar finding for embryo morphology

and pregnancy rate from Z1- and Z3-derived

embryos.A multicenter study completed in Germany

showed that embryos selected for transfer based

upon pronuclear morphology exhibited some differ-

ences in pregnancy and implantation rates exhibited

according to the morphological scoring group.

Germany is one of the few countries in which embryo

transfer is limited by legislation so that embryos must

be selected prior to development beyond the pronu-

clear stage. Despite the reported differences, the study

showed that pregnancy and implantation still occur

even if the pronuclear morphology was suboptimal,

or less than a Z1 or Z2 score

or a pattern 0.

Balaban et al

reported that embryos were more

likely to develop into high quality blastocysts as well

as achieve higher implantation and pregnancy rates

if they derived from a zygote with an ideal pronuclear

pattern as compared with those embryos derived

from a non-ideal pronuclear pattern. Despite the bet-

ter developmental prognosis of the embryos with

ideal pronuclear pattern, they also found that blas-

tocysts from abnormal pronuclear patterns would

produce pregnancies, but at a lower rate. On the other

hand, Scott et al

reported that when only Z3-

derived blastocysts were available for transfer on day

5, there were no pregnancies (n ⫽ 6). In direct con-

trast to this study, James et al

reported a live birth

rate of 100% when only Z3-derived blastocysts were

transferred (n ⫽ 4).

Kattera and Chen

reported on the predictive

value of pronuclear orientation in relation to the

second polar body upon embryo development and

implantation and pregnancy rates. There was a pos-

itive correlation with early embryo cleavage when the

pronuclei were perpendicular to the second polar

body. However, there was no clear difference for

pronuclear orientation in relation to implantation or

pregnancy rates.

A number of investigators have correlated chro-

mosomal status to zygote pronuclear morphology.

Sadowy et al

reported an increased incidence of

mosaicism associated with zygotes that showed

pronuclei of unequal sizes. Kahraman et al

also

showed an increased number of chromosomally

abnormal embryos derived from zygotes with subop-

timal morphology. Another study also reported that

zygotes with normal pronuclear patterns, evaluated

using the method of Tesarik and Greco,

had a greater

chance of producing genetically normal embryos.



Table 4.6 The pronuclear morphology scoring

(PNMS) system developed by Gianaroli et al,

with

normal morphology configurations being A1␣, A2␤,

A3␤, and A3␣

Parameter

Parameter Configuration description

Pronuclei A Central, in apposition

(location, B Non-central, in

position, apposition

other) C Central, at a distance

D Unequal sizes

EFragmented

Nucleoli 1 Large, aligned

(size, alignment) 2 Large, unaligned

3 Large, 1 PN aligned

4 Small, unaligned

Pronuclear

␣ Parallel

orientation

␤ Perpendicular

␥ Other

PN, pronucleus.

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HUMAN PRONUCLEI AS A MODE OF PREDICTING VIABILITY

Gamiz et al

stated that in patients ⱕ37 years of age,

zygotes with equal sized pronuclei and polarized

nucleoli were positively correlated with embryo

development and a lesser incidence of chromosomal

abnormalities.

CONCLUSIONS

Timing is a critical issue in the evaluation of early

embryos, and this creates a challenge when assessing

a dynamic event such as pronuclear morphology. To

date, laboratories have evaluated this parameter

within a time range that is based on the timing of the

insemination procedure, with the rationale that there

is a limited time range during which pronuclei can be

visualized for evaluation. Furthermore, the timing of

fertilization can depend upon the method of insem-

ination as well as the actual oocyte undergoing treat-

ment. The exact time of insemination is known with

ICSI because each sperm is injected into individual

oocytes at a precise time. This is not the case with

IVF, in which the fertilization time can only be spec-

ulated. The amount of time sperm and oocytes are

together during the insemination procedure for stan-

dard IVF can vary between laboratories, and can range

from 4 to 18 hours. There is no way of knowing the

time of sperm–oocyte fusion within this window.

Montag and van der Ven

reported that a greater

number of higher quality embryos were derived from

zygotes with a high score after ICSI versus IVF. This

difference is probably related to a difference in the

timing of insemination between the two groups and

not to inherent differences in IVF itself.

Pronuclear development is a dynamic

event

14,20,24,33

and to disregard an embryo based on a

single time assessment can be misleading. Zygotes that

are assigned a poor pronuclear score are not necessar-

ily chromosomally aberrant, and have been shown to

become high grade zygotes when assessed at a later

time. Sadowy et al

analyzed chaotic mosaicism in

zygotes with uneven pronuclei and found no indica-

tion that all such zygotes were abnormal. This was

further demonstrated by Wright et al

and Demirel

et al

who observed morphological changes over

time. They reported that the nucleoli were small,

numerous, and distributed in a random pattern early

in pronuclear development, but that as time pro-

ceeded, the nucleoli coalesced and began to align near

the furrow between the pronuclei. Adding to the pre-

vious literature showing the dynamic nature of pro-

nuclear development, they demonstrated that

timing of embryonic evaluation can have a drastic

effect on the assigned quality when static observa-

tions are used.

6,14,39,45

Jaroudi et al

suggested that zygote scoring used

as the only selection criteria may not be sufficient to

select the most viable embryos. This has been rein-

forced by recent studies stating that evaluation of

pronuclear morphology is useful for embryo selec-

tion only when used in conjunction with other meth-

ods of evaluation (i.e. day 3 morphology).

46,47

Thus,

a single observation may not reveal the true status of

a zygote, because this cannot determine whether

nuclear development is in progress, or has yet to

progress when syngamy is initiated. It is important

to use pronuclear morphology scoring carefully when

determining the fate of embryos, because their poten-

tial can easily be misdiagnosed based upon a single

point in time.

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HUMAN PREIMPLANTATION EMBRYO SELECTION

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5. Multinucleation and mosaicism in the

human preimplantation embryo

Renee Walmsley



INTRODUCTION

Human embryos that are cultured in vitro sometimes

have two or more nuclei visible within a single blas-

tomere, even after apparently normal fertilization.

This phenomenon, referred to as multinucleation,

occurs after the completion of meiosis, and can be

seen during interphase throughout the stages of

in vitro culture. Multinucleated blastomeres (MNB)

may be observed most readily on day 2 and 3 of

development, but on day 3 definitive identification

may be compromised by overlap between cells,

compaction and/or fragmentation. Although multi-

nucleation continues to occur through blastocyst

development, it is very difficult to observe micro-

scopically after day 3.

A high incidence of multi-

nucleation is associated with other types of embryo

dysmorphism, including fragmentation, impaired

embryo development and arrest,

2–5

but it does not

always correlate with poor embryo morphology.

2,6

Consequently, care should be exercised when decid-

ing the fate of multinucleated embryos (MNE) that

are otherwise apparently of good quality. MNE do

have the potential to implant, but their implanta-

tion and subsequent pregnancy rates are lower than

those of non-MNE.

4,7–10

Several studies have con-

firmed the birth of healthy babies that originated

from MNE,

4,8,10

and therefore, if no other embryos

are available for transfer, it is preferable to transfer

these embryos unless other developmental informa-

tion such as the presence of chromosomal anomalies

is known.

The use of fluorescence in situ hybridization

(FISH) has allowed a greater understanding of the

developmental potential of MNE,

2,11,12

and several

studies have demonstrated that multinucleation is

associated with higher rates of chromosomal

abnormalities.

13–15

At our center, the Institute

for Reproductive Medicine and Science at Saint

Barnabas (IRMS), a large retrospective study using

FISH to analyze 498 non-arrested MNE demon-

strated abnormal chromosome compositions in

71.9% of MNE. It is important to bear in mind that

the accuracy of FISH is limited by the number of

chromosomes that can be analyzed for a given cell,

and therefore rates of abnormality for MNE could

potentially be higher if the entire complement of

chromosomes were to be assessed.

TYPES OF MULTINUCLEATION

Multinucleation can be observed in one or more of

the blastomeres of two- to eight-celled embryos

(Figure 5.1). Blastomeres can be binucleated, with two

nuclei (Figure 5.1B and 5.1E), or micronucleated

(Figure 5.1C, 5.1D and 5.1F) with ⱖ3 nuclei in a single

cell. The impact of specific types of multinucleation

on clinical outcomes has recently been studied and

is discussed in detail later in this chapter.

FREQUENCY OF MULTINUCLEATION

The frequency of multinucleation has been analyzed

in several comprehensive studies, where MNE were

observed in 14–79% of all patient cycles, with

15–33% of all embryos multinucleated.

4,8–10,13,16

The differences found between studies may be due

to variation in hormonal stimulation protocols, as

well as to diverse laboratory culture conditions.

IRMS, 57 015 monospermic non-arrested embryos

from 6012 standard IVF/ICSI patient cycles were

assessed, and at least one MNE in the cohort of

embryos was found in 63.6% of these cycles. In

agreement with previous studies,

a higher rate of

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HUMAN PREIMPLANTATION EMBRYO SELECTION

MNE was observed on day 2 than on day 3: multi-

nucleation first presented on day 2 of development

in 12.6% of embryos, whereas only 5.0% of

embryos demonstrated multinucleation for the first

time on day 3 of development (Table 5.1). By 72

hours of culture, 17.5% of embryos showed signs of

multinucleation.

Maternal age has been shown to correlate with

the frequency of multinucleation, although the data

are conflicting: younger patients had slightly higher

rates of multinucleation than older patients,

and a

slight correlation was also seen at IRMS, specifically

for patients ⱕ30 years old (Table 5.1). In contrast,

other studies found no correlation between MNE

and maternal age.

8,10

The role of maternal age with

respect to the frequency of multinucleation is there-

fore still unclear, although these contradicting data

sets may simply be related to differences in stimula-

tion protocols for the centers studied. Those protocols

which ‘push’ younger patients into multifollicular

development with high doses of gonadotropins

equivalent to those administered to older patients

may have a possible correlation related to increased

estrogen levels in younger patients.

Culture conditions within the laboratory may

influence the incidence of multinucleation. For

example, embryos from IRMS patients that were

cultured in human tubal fluid (HTF) supplemented

with maternal serum had a significantly higher rate

of multinucleation than those cultured in HTF

supplemented with human serum albumin (HSA)

(Table 5.2). HSA was used as a protein supplement

during culture either for poor prognosis patients (as

an effort to change culture conditions in the hope of

encouraging better overall embryo development),

or when maternal serum was not available. Interes-

tingly, patients who exhibited multinucleation in

their first cycle showed lower rates of multinucle-

ation in a subsequent second cycle (Table 5.3). This

finding supports the suggestion that multinucleation



AB C

DE F

Figure 5.1 Types of multinucleation commonly observed in human embryos cultured in vitro: (A) day 2 non-MNE; (B) day 2 MNE,

one binucleate blastomere (1w2); (C) day 2 MNE, one micronucleate blastomere (1w3); (D) day 2 MNE, two blastomeres with

multinucleation (2wⱖ2); (E) day 3 MNE, one binucleated blastomere (1w2); (F) day 3 MNE, one micronucleated blastomere (1w3).

HPE_Chapter05.qxp 7/13/2007 4:45 PM Page 42

is not exclusively a patient-specific phenomenon,

but may be influenced by culture and/or stimula-

tion protocols. The lower rates of multinucleation

for repeat cycles at IRMS were encouraging, sug-

gesting that modification of cycle protocols and/or

culture conditions were effective in improving

patient treatment. Indeed, pregnancy and implanta-

tion rates were collectively higher for second cycles

of repeat patients where multinucleation was

observed during initial cycles (Table 5.3).

MECHANISMS OF MULTINUCLEATION

The mechanisms that underlie embryo multinucle-

ation during early cleavage divisions are still relatively

unclear; it may be a result of karyokinesis (specifically

DNA synthesis) continuing in the absence of cyto-

kinesis.

6,18

Cells of this type were thought to be

arrested, without participating in further embryo

development, and therefore multinucleated embryos

were felt to be developmentally (possibly terminally)

compromised by multinucleation. Because cleavage

stage embryos have on average more cells on day 3

than on day 2, the transfer of multinucleated embryos

on day 3 in preference to those where multinucle-

ation was witnessed on day 2 was recommended

when no other embryos were available.

13,19

In con-

trast, higher pregnancy and implantation rates for

embryos where multinucleation appeared first on

day 2 rather than on day 3 were recently observed at

IRMS (Figure 5.2). Furthermore, in this study, 15

patient cycles in which all embryos transferred were

MULTINUCLEATION AND MOSAICISM IN THE HUMAN PREIMPLANTATION EMBRYO



Table 5.1 The incidence of multinucleation in relation to age

Age group Cycles (n) Total 2PN Total day 2 MNE Total day 3 MNE Total MNE

All 6012 57 015 7178 (12.6%) 2901 (5.0%) 10 079 (17.6%)

ⱕ30 years 719 7912 1068 (13.4%) 403 (5.0%) 1471 (18.5%)

ⱖ31 years 5293 49 103 6110 (12.4%) 2498 (5.0%) 8608 (17.5%)

*p ⫽ 0.022

PN, pronuclei; MNE, multinucleated embryos.

Table 5.2 Incidence of multinucleation in relation to culture media supplementation

Protein Cycles (n) Total 2PN Total day 2 MNE Total day 3 MNE Total MNE

HSA 133 1069 102 (9.5%) 46 (4.3%) 148 (13.8%)

Maternal serum 2350 20 884 3066 (14.6%) 1342 (6.6%) 4408 (21.1%)

*p⬍ 0.001

PN, pronuclei; MNE, multinucleated embryos; HSA, human serum albumin.

Table 5.3 Incidence of multinucleation in patients with repeated IVF cycles

No. Age Implantation Pregnancy

Attempt of cycles (years) Total 2PN Total day 2 MNE Total day 3 MNE Total MNE rate (%) rate (%)

1 617 35.3 5611 1079 (19.2%) 483 (8.6%) 1562 (27.8%) 12.8 31.7

2 617 36.3 5879 893 (15.1%) 344 (6.0%) 1237 (21%) 28.0 55.4

PN, pronuclei; MNE, multinucleated embryos.

HPE_Chapter05.qxp 7/13/2007 4:45 PM Page 43

HUMAN PREIMPLANTATION EMBRYO SELECTION

MNE exhibiting multinucleation in all cells on day 2

(i.e. every blastomere in all transferred embryos

displayed multinucleation on day 2) resulted in four

pregnancies. In view of this, multinucleated cells

must have the potential to give rise to normal diploid

daughter cells at some stage in their development. A

recent and significant study using time-lapse photo-

graphy showed that multinucleated blastomeres

(MNB) undergo normal cell division, providing

visual evidence that MNB are capable of normal

mitotic cell division, and are not necessarily arrested.

The mechanisms that are involved in cleavage stage

multinucleation may occur even prior to oocyte

retrieval or ovulation, and may be related to the

response to stimulation. Specifically, follicular under-

oxygenation has been correlated with multinucle-

ation seen at the two-cell stage of development.

This finding supports the evidence for higher rates of

multinucleation in patients who responded aggres-

sively to hormonal stimulation during their IVF

cycles,

4,10,16

a trend that was also confirmed in our

center (Table 5.4). Another interesting study demon-

strated that oocytes matured in vitro (IVM) fre-

quently presented with multinucleation during later

development,

suggesting that incomplete nuclear

and cytoplasmic remodeling in an oocyte may result

in higher incidences of multinucleation.

Nuclear fragmentation and dysfunctional spindle

tubules can cause improper separation of chromo-

somes during mitotic divisions, thereby leading to

multinucleation.

6,22,23

The oocyte cytoskeleton can

be damaged by substandard laboratory conditions

and suboptimal culture conditions, such as sudden

changes in temperature; this can result in improper

cleavage, inducing multinucleation,

20,24,25

and may

also play a role in causing multinucleation during

later cleavage divisions.

6,23

IMPACT OF MULTINUCLEATION ON

EMBRYO MORPHOLOGY

An embryo’s potential to establish a viable pregnancy

can be diagnosed to a certain degree by assessing

gross morphology and identifying anomalies pres-

ent within the embryo.

26–29

Total blastomere count

and phenotypic dysmorphisms, including degree and

type of fragmentation,

appearance and severity of

multinucleation, blastomere organization, contracted

or granular cytoplasm, vacuoles, dense bodies and

abnormal zona pellucida are recorded during

morphological assessments on day 2 and 3, and the

majority of studies show a clear tendency for MNE

to be compromised morphologically. The presence

of nucleoli in the nucleus distinguishes multinucle-

ation from vacuoles. On average, MNE have lower

cell counts, higher rates of fragmentation, uneven

blastomere sizes and division, and lower rates of

blastocyst formation.

1,4,5,8,29,31

Micronucleated

embryos are more frequently associated with poor

pronuclear morphology than binucleated embryos,

and they have lower rates of blastocyst formation.

In support of this finding, IRMS observed higher

implantation and pregnancy rates from the exclu-

sive and mixed transfer of binucleated embryos than

the exclusive and mixed transfer of micronucleated

embryos (Table 5.5).

CHROMOSOMAL STATUS OF

MULTINUCLEATED EMBRYOS

Preimplantation genetic diagnosis is an important

tool for analyzing the chromosomal status of

embryos in vitro. When applied to specific types of



Cell #

Fragmentation (%)

Implantation (%)

Pregnancy (%)

non-MNE (n = 5573)

MNE (n = 86)

Day 2 MNE (n = 63)

Day 3 MNE (n = 23)

Figure 5.2 Comparison of clinical outcomes for transfer of

non-multinucleated embryos (MNE) exclusively, all MNE, day 2

MNE and day 3 MNE exclusively. Cell#, the average number of

cells in the embryo.

HPE_Chapter05.qxp 7/13/2007 4:45 PM Page 44

MULTINUCLEATION AND MOSAICISM IN THE HUMAN PREIMPLANTATION EMBRYO

dysmorphic embryos, FISH analysis revealed higher

rates of chromosomal abnormalities in MNE than

in non-multinucleated embryos (non-MNE). Speci-

fically, extensive mosaicism and/or polyploidy was

observed in 44–75% of MNE embryos.

13,14,32

Mosai-

cism in an embryo can only be accurately detected

when at least two cells are analyzed;

when a single

cell is analyzed, there is a risk that a mosaic embryo

may be misdiagnosed as aneuploidy.

Mosaicism,

haploidy and polyploidy occur during mitotic cell

division after fertilization, and although rates of

aneuploidy in cleavage stage embryos are known to

increase with maternal age, this is not the case for

embryo mosaicism.

3,34

Polyploidy in monospermic

MNE probably results from continued DNA synthe-

sis (and possibly karyokinesis) without cytokine-

sis.

In the studies carried out in our center, the

embryos were analyzed on day 3, 4 or 5, and some

may have been continuing DNA replication in the

absence of cleavage, leading to polyploidy.

22,36

We investigated correlations between chromo-

some anomalies and the incidence and severity of

multinucleation by fixing and analyzing disaggre-

gated blastomeres of MNE according to previously

published protocols.

1,37,38

Individual blastomeres

from multinucleated embryos were analyzed by

FISH, simultaneously using probes specific for

chromosomes 13, 16, 18, 21 and 22. In the majority

of cases, chromosomes X, Y, 15, and 17 were also

analyzed with additional probes. The chromosomal

status of all MNE and non-MNE embryos was

analyzed and recorded in the EggCyte™ database,

and the results were separated into groups accord-

ing to the following criteria: aneuploid (ANE),

aneuploid/mosaic (ANE-MOS), aneuploid/poly-

ploid (ANE-POL), haploid (HAP), normal (NORM),

polyploid (POL), high rate of mosaicism (MOS-HI),

and low rate of mosaicism (MOS-LO). In order to

compensate for the effect of age on FISH results,

wholly aneuploid embryos were then discounted.



Table 5.4 Incidence of multinucleation in relation to length of stimulation

Days on GND No. of cycles Total 2PN Total day 2 MNE Total day 3 MNE Total MNE

6–7 775 7647 1050 (13.7%) 457 (5.9%) 1507 (19.7%)

8–11 4811 46 051 5721 (12.4%) 2261 (4.9%) 7982 (17.3%)

*p ⬍ 0.001

GND, gonadotropin; PN, pronuclei; MNE, multinucleated embryos.

Table 5.5 Comparison of the severity of multinucleated embryos (MNE) in exclusive transfers after transfer of

2 embryos only

MNB Age Total embryo Total FHB Procedures Embryos Implantation

severity MNE repl (%) Procedures (years) repl FHB ⬎0 repl (%) Pregnancy (%)

None 0 1023 34.3 806 577 541 2 35.3 52.8

Any MNE 50 163 35.7 326 75 59 2 23.0 36.2

Any MNE 100 36 36.0 72 9 8 2 12.5 22.2

1w2 50 87 35.4 174 44 36 2 25.2 41.3

1 w2 100 11 36.2 22 4 4 2 18.1 36.3

1wⱖ3 50 23 36.1 46 10 7 2 21.7 30.4

1wⱖ3 100 11 35.7 22 1 0 2 4.5 9.1

2wⱖ2 50 15 36.1 30 4 3 2 13.3 20.0

2wⱖ2 100 14 35.8 28 4 4 2 14.2 28.5

MNB, multinucleated blastomere; FHB, fetal heart beat; 1w2, one binucleated blastomere; 1wⱖ3, one micronucleated blastomere; 2wⱖ2, two blastomeres

with multinucleation; Repl, replaced.

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