This study investigated the effects of binucleation on mouse preimplantation embryo development. Embryos were collected and either allowed to develop naturally or treated with cytochalasin B to induce binucleation. Binucleated embryos continued dividing but contained more micronuclei and chromosome segregation errors, as observed using live cell imaging. The results suggest that tetraploidy in mammalian embryos may increase chromosome segregation errors and aneuploidy, which could negatively impact embryo health.

1. Introduc)on
In-­‐vitro
fer,liza,on
(IVF)
is
commonly
used
to
treat
female
and/or
male
infer,lity.
Successful
pregnancy
following
IVF
is
dependent
on
selec,ng
the
healthiest
embryos
for
implanta,on.
Importantly,
IVF
clinics
report
a
rela,vely
high
frequency
of
binucleated,
tetraploid
blastomeres
in
human
preimplanta,on
embryos.
In
soma,c
cells,
tetraploidy
is
known
to
decrease
fitness
and
increase
the
difficulty
of
chromosome
segrega,on,
which
result
in
cell
cycle
arrest
and
ul,mately
apoptosis.
However,
how
binuclea,on
affects
mammalian
embryo
development
and
IVF
success
is
unknown.
Materials
and
Methods
Embryos
from
CD1
mice
were
collected
at
the
two-­‐cell
stage.
Live
Cell
Imaging
experiments
were
visualized
using
long-­‐term,
low
damage
4D
confocal
analysis
Looking
for
Naturally
Occurring
Binuclea)on
• Embryos
were
fixed
and
stained
with
Phalloidin
(plasma
membrane)
and
DRAQ5
(nucleus)
at
the
four-­‐cell,
eight-­‐cell,
morula,
and
blastocyst
stage
Crea)ng
Binucleated
Embryos
• Group
1:
control
embryos
developed
in-­‐vitro
and
were
fixed
approximately
at
the
eight-­‐cell
stage
• To
induce
binuclea,on,
embryos
were
treated
with
cytochalasin
B
immediately
prior
to
the
four-­‐cell
to
eight-­‐cell
transi,on.
AYer
12
hours
in
cytochalasin
B,
embryos
were
washed:
• Group
2:
was
fixed
right
away
aYer
wash
• Group
3:
developed
further
and
was
fixed
12
hours
aYer
wash
Observing
Binucleated
Divisions
• A
method
was
created
to
visualize
binucleated
divisions
• H2B-­‐RFP
RNA
was
microinjected
at
the
two-­‐cell
stage
of
the
embryo
to
visualize
DNA
• Group
1
visualized
the
eight-­‐cell
to
sixteen-­‐cell
division
• Group
2
visualized
the
binucleated
four-­‐cell
to
eight-­‐cell
transi,on
Results
Binucleated
Embryos
Are
Rare
in
Mouse
Embryos
Binucleated
Embryos
Con)nue
to
Divide
But
Contain
More
Micronuclei
Live
Cell
Imaging
Confirmed
that
Binucleated
Divisions
Contain
More
Chromosome
Segrega)on
Errors
Binuclea,on
in
the
Preimplanta,on
Embryo
Cause
a
Higher
Frequency
of
Segrega,on
Errors
Candice
Taguibao1,
Jenna
Haverfield1,
Shoma
Nakagawa1,
Cayetana
Vazquez-­‐Diez1,
Greg
FitzHarris1,2
1Centre
Recherche
CHUM,
Montreal,
Canada,
H2X
0A9.
2Department
of
Obstetrics
and
Gynaecology,
University
of
Montreal,
Montreal,
Canada,
H3T
1J4.
Hypothesis
That
binucleated
blastomeres
will
con,nue
to
divide,
but
chromosome
segrega,on
errors
will
be
common.
Aim
To
inves,gate
the
effect
of
binuclea,on
on
the
preimplanta,on
embryo.
Conclusion
Mouse
embryos
apparently
lack
a
robust
tetraploidy
checkpoint,
and
con,nue
to
divide
even
aYer
binuclea,on
was
induced.
However,
chromosome
segrega,on
errors
are
more
frequent
in
binucleated
embryos.
We
thus
propose
that
tetraploidy
in
the
mammalian
embryo
may
have
the
unexpected
effect
of
inducing
chromosome
segrega,on
errors
and
classical
single-­‐chroma,d
aneuploidy,
which
may
in
term
be
detrimental
to
embryo
health.
Figure
2.
(A)
Image
taken
immediately
aYer
embryos
were
treated
for
12
hours
in
cytochalasin
B;
48
hours
post-­‐collec,on.
Each
arrow
points
to
a
binucleated
blastomere
in
every
embryo
(B)
Image
taken
12
hours
aYer
washing
from
cytochalasin
B
treatment;
60
hours
post-­‐collec,on.
(C)
At
0
hours
aYer
wash,
90%
of
blastomeres
were
binucleated;
at
12
hours
aYer
washing,
9.4%
of
blastomeres
were
binucleated;
at
24
hours,
0%
of
blastomeres
were
binucleated
(D)
The
mononucleated
embryos
that
arose
from
binucleated
embryos
exhibited
a
larger
amount
of
micronuclei
at
%17,
compared
to
%6
seen
in
eight-­‐cell
controls,
and
%8
seen
in
sixteen-­‐cell
controls.
The
eight-­‐cell
controls
(collected
48
hours
post-­‐collec,on)
provide
a
comparison
against
a
similar
cell
number;
the
sixteen-­‐cell
(collected
60
hours
post-­‐collec,on)
provide
a
comparison
against
a
similar
cell-­‐stage
in
development.
Old
Acknowledgements
Candice
Taguibao
was
a
2015
undergraduate
summer
RQR
CREATE
recipient.
Experiments
were
funded
by
Founda,on
Jean-­‐Louis
Levesque.
A
B
D
C
A
B
Figure
3.
(A)
The
top
panel
shows
snapshots
at
NEBD,
metaphase,
anaphase,
and
the
resul,ng
two
daughter
cells.
The
pink
arrows
mark
the
loca,on
of
the
dividing
nuclei
and
metaphase
plate
as
well
as
the
posi,on
of
the
daughter
nuclei.
(B)
This
binucleated
blastomere
forms
a
single
metaphase
plate
with
misaligned
chromosomes.
During
anaphase,
2
lagging
chromosomes
are
seen
to
then
form
micronuclei.
The
green
arrows
indicate
misaligned
chromosomes,
lagging
chromosomes
and
micronuclei.
(C)
This
par,cular
binucleated
division
is
characterized
as
“other
segrega,on
errors’.
The
binucleated
blastomere
forms
2
metaphase
plates
and
divides
into
what
appears
to
be
5
separate
nuclei.
(D)
The
pie
charts
display
the
data
collected.
More
chromosome
segrega,on
errors
occur
in
binucleated
divisions
than
in
the
control
embryos
(p=0.012)
C
D
Figure
1.
(A)
Normal
embryos
shown
are
at
8-­‐cell,
morula,
and
blastocyst
and
do
not
display
binuclea,on
A
36 hrs
12 hrs
Cyt B
12 hrs
Wash:
12 hrs
fix right
away
Group 1: eight-cell controls
Group 3: eight-cell from
Binucleated embryos
Group 2: four-cell
Binucleated embryos
microinject
H2B-RFP
36 hrs
12 hrs
12 hrs
Cyt B
Wash
Group 1: eight-cell to sixteen-cell transition
Group 2: binucleated four-cell to
eight-cell transition
48:00 72:00 96:00
48:00 48:00
60:00
Misaligned
Chromosomes
Lagging
Chromosomes Micronuclei
Series 001
z=13/27
Series 001
z=9/27
Series 002
z=14/28
48:00 49:83 50:17 50:50 51:08 51:17 51:83
49:83 50:08 51:8351:2551:1751:0849:83
49:00 51:9151:7551:5051:3349:33 52:08
CONTROL
BINUCLEATED
BINUCLEATED