of five cleaving embryos from tripronuclear human oocytes were triploid, and two .... blastomeres. Figure. 1(b) shows a 3-cell human embryo, with one large and.
BIOLOGY
OF
REPRODUCTION
37,
395-401
(1987)
Tripronuclear Human Oocytes: Subsequent Karyotypic ISMAIL
Altered Cleavage Patterns Analysis of Embryos
KOLA,”2
GAREY
ALAN
DAWSON,3
and
and
TROUNSON,2 PETER
ROGERS4
Centre for Early Human Development2 Cytogenetics Laboratory, Division of Laboratory
Medicine3
and Department
of Obstetrics
Monash Melbourne,
& Gynecology4
University Australia
3000
ABSTRACT Between
1 and
4%
of
human
oocytes
fertilized
tripronuclear oocytes can develop to grossly could be triploid, diploid, or severely depleted. embiyos deriving from tripronuclear that most (18 of 29) tripronuclear
human human
embryos
(but
have
tripronuclear (7 of 29)
a severely
abnormal
human oocytes cleave cleave to 2-cells, and these
in vitro
normal-appearing The etiology
are
oocytes oocytes
is unknown. cleave directly
triploid)
chromosomal
not
to 2-cells embryos
trip ronuclear.
morulae and and proportion
plus an extrusion, are triploid after
This study to 3-cells
Although
into
the
1-4%
mechanisms
by which
these
oocytes
fail
to develop
50.5
(Mahadevan
severely
Trounson,
1984)
into
h
after
about
subsequent
chromosomal
nuclear
oocytes
embryos
and
with
4,6-diamidino-2-phenylindole
blastomere Rudak
contained et
Accepted Received 1 Reprint ment,
Monash
3168,
Australia.
al. (1984)
February December requests: University,
(DAPI),
46
chromosomes.
reported
that
25, 1987. 5, 1986. Dr. Ismail Monash
Kola,
Centre
Medical
that
for Early Centre,
Human Clayton,
some
(4 of 29)
one
complement
blastomere of
33
et al. (1986) evaluated of 5 embryos derived and
two
found
1 diploid,
embryos
with
two
near
triploid
and
39
chromo-
per
evaluated by DNA microfluorimetry, Most of the embryos in the Angell
We apparent
oocytes,
evaluated
respectively.
after
Two
the second
a
the chromofrom tripro-
bryos, diploid.
were
blastomere,
38
had
chromosomes.
somes
study
other
em-
were near et a!. (1986)
cleavage
division.
were interested in the derivation of the diploidy and aneuploidies in tripronuclear especially as electron microscopic observa-
tions of tripronuclear oocytes had confirmed the presence of three pronuclei arising through dispermy
one
(Sathananthan and Trounson, 1985). Zenzes et a!. (1985) have shown that oocyte was hypotriploid (had 64
In addition,
in an embryo
these
is also provided.
insemination,
Recently, Angell somal constitution
composition of preimplantation embryos that derive from these oocytes, and the data that does exist suggest a complex picture. We (Trounson, 1982) previously found that three of five cleaving embryos from tripronuclear human oocytes were triploid, and two were near diploid. Van Blerkom et al. (1984) showed that two such embryos may develop to at least the 8-cell stage and demonstrated, by serialsectioning a 12-cell embryo and fluorescent staining
embryos
abnormal
tripronuclear information
and
Furthermore,
triploid
oocytes fertilized in vitro are 3 pronuclei), there is little development
that
these embryos and aneuploid
pattern at the first cleavage division, that into triploid embryos, and that a correlasubsequent karyotype of these embryos.
of human (contain the
reported
provides evidence for the first time at the first cleavage division. These
complement.
INTRODUCTION
and
been
and these embryos are diploids, whereas some the first cleavage division. These findings demon-
strate that most tripronuclear human oocytes have an altered cleavage most tripronuclear human oocytes (76% in this study) do not develop tion exists between the pattern of the first cleavage division and the Insight
It has
that chromosomally, of apparently diploid
fixed
before the tripronuclear chromosomal
Develop-
division occurs
Victoria,
395
first
Furthermore, a tripronuclear chromosomes)
cleavage division. Thus, it appears that oocytes have a triploid or near triploid complement before the first cleavage
and that if deviation from at later stages of embryogenesis.
triploidy occurs, Theoretically,
it
396
KOLA
for
a tripronuclear
oocyte
(consisting
of 69
chromo-
or
cleavage vestigated
the
oocyte
cleaves
to
3 cells
tripronuclear
human
constitution
oocytes
these
of
cleavage
division
trip!oid
and
the
embryos both
oocytes to gain
MATERIALS
Oocytes undergoing
obtained treatment
number
of
microscopy pronuclear and/or
12-20 oocyte interference
pronuclei
were
develop into the
an
of
oocytes
and fertilized and Mohr, 1983).
scored
which
under
as The
dissecting
In addition, indicates
state of these to dispermy.
human
investigation,
4
initial
human
being with with
all oocytes that the
oocytes
is almost
oocytes
tripronuclear three clearly colcemid (0.1
that
(Fig. visible pg/mI)
were
la
shows a pronuc!ei) just before
the first cleavage division (24 h after insemination). The oocytes were assessed for chromosomal constitution by using a modification of the technique previously described (Kola et a!., 1986). Briefly, embryos
were
incubated
in hypotonic
solution
(1%
trisodium citrate) for 3-5 mm at room temperature. The embryos were then transferred onto clean glass slides One 3:1)
in
a minimal
drop was
amount
of
hypotonic
of fixative (methanol/glacial dropped onto the embryo
from
solution. acetic acid, behind it.
Immediately drop of
after adherence to the slide, a second fixative was dropped onto the embryo. Finally, 3 more drops of fixative were added 1 at a time to the side of the embryo. Chromosomes were stained with 10% Giemsa (BDH, Poole, England) in buffer,
pH
6.8,
In subsequent were monitored and
cleavage.
for
10 mm.
experiments, tripronuclear constantly for dissolution Three
trip ronuclear
oocytes
oocytes of pronuclei were
time-lapse incubated
dissolution
filmed
and
cinematography. with colcemid
first
cleavage
fixed
The
the
first
of embryos did fixed earlier.
conducted
8
embryos division
not have any Chromosome
independently
by at least
using
embryos were 12 h after the
remaining cleavage
by
embryos were 6-8 h after the
The first constitutions
division.
14 h after
cleavage
The beginning
first cleavage division. assessed for chromosomal
into mech-
h after insemination. Each tnwas reexamined under phase contrast microscopy and
to be tripronuclear. polar bodies,
evaluated as human oocyte were incubated
second
from superovulated patients of infertility by in vitro fertil-
(IVF) were collected described (Trounson
tripronuclear certainly due
the
pronuclear
number when
METHODS
ization previously
confirmed had two
inof
proportion
that insight
AND
first
chromosomal
the
anism by which these tripronuclear fail to develop into triploid embryos.
to to is
we division
before
to determine
human embryos and
tripronuclear
In
at the
division. Against this background, the pattern of the first cleavage
AL.
for
somes before the first cleavage division) to develop a diploid embryo, one of two mechanisms have operate. Either a haploid set of chromosomes ejected,
ET
were
because
a
cells in mitosis scoring was
two
investigators.
RESULTS
The cleavage
4
oocytes division
that each
had
were fixed before 69 chromosomes.
the
first
Of 29 tripronuclear oocytes allowed to cleave, (62%) cleaved directly to 3-cells (Table 1, Fig. We found that the dissolution of the pronuclei cleavage both
to 3-cells microscopic
was
cinematography). varied; 6 embryos whereas smaller embryo,
The had
synchronous evaluation regularity 3 equally
18 ib). and
(as determined and time
by lapse
of the blastomeres sized blastomeres,
the remaining 12 embryos had 1 large and 2 blastomeres. Figure 1(b) shows a 3-cell human with one large and two smaller blastomeres
that had resulted from cleavage of a tripronuclear blastomere
size
was
the not
direct oocyte. related
and synchronous The variation
of
to
of
synchrony
cleavage, since those embryos that had 1 large and 2 smaller blastomeres had also cleaved synchronously to 3-cells. Chromosoma! analysis of 10 of these 3-cell embryos just before the second cleavage division revealed that all of these embryos had a severely abnormal (Table
1).
an embryo
but near-diploid Furthermore, different
of chromosomes.
It in
that
cleave directly to 3-cells. a 3-cell embryo, but
numbers
constitution 3-cells within
also appeared that the number of chromosomes individual cells was random. This is the first demonstrating
had
chromosomal in all cases the
tripronuclear
human
study oocytes
Angell et al. (1986) observed were unable to differentiate
between direct cleavage to 3-cells or cleavage to 2-cells and a rapid second division. We found that 14% (4 of 29) of tripronuclear human oocytes cleaved to 2-cells plus an extrusion (a mass that is extruded from the embryo simultaneously with cleavage of the embryo) (Table 1). Figure 2a shows a 2-cell-plus-extrusion embryo, with the extrusion indicated by an arrow. Three of these 4 embryos were successfully karyotyped, the cells of these embryos were found Figure 2b shows a chromosomal spread
and both to be diploid. of 1-cell of a
TRIPRONUCLEAR
HUMAN
397
OOCYTES
#{149}.
‘..
$
.
a FIG.
1. (a)
to the permy.
Photomicrograph
of
center
of the oocyte. (b) Photomicrograph
2-cell-plus-extrusion diploid shown
that
two
interphase
somes,
had
not
been
nuclei,
possibly
Twenty-four oocytes in normal
(46XY) 2b. The
this
2-cell
Figure
one
from
(7 of cleaved (Fig.
(X 200).
2c shows
the
spread in this
karyotyped,
haploid the
oocyte
bodies are also seen, 2 smaller blastomeres)
metaphase embryo
successfully
percent study
embryos
and of the remaining
and
derived
human
and second polar embryo (1 large,
embryo,
karyotype in Figure
group
a tripronuclear
The first of a 3-cell
had
set of chromo-
extrusion. 29) to
Figure the
near-triploid embryos (Table a metaphase spread of a 2-cell 3c shows the triploid karyotype
spread
or
shown
in Figure
1). Figure
3a is
1). Figure embryo, (69XXX)
3b and of
3b.
DISCUSSION
The findings large majority develop cleavage
in this study of tripronuclear
have demonstrated human oocytes
into triploid embryos, division is a critical stage
and that
photomicrograph
that a do not
that the influences
subsequent chromosomal constitution of tripronuclear human oocytes. We have shown that three types
first the
clearly
demonstrating derived from
that the the direct
events occur at pronuclear human cells,
2)
they
the
the
the
three
first oocytes:
cleave
to
pattern
cleavage 1) they
triploid
For spindle
an
that
have
oocyte oocyte
that of
migrated
is due to dis(X200).
division cleave
of to
2-cells-plus-extrusion,
at the
chromosomal
tnthree
and
triploid embryos. a correlation
cleavage
first
3)
Our exists division
composition
the embryos that abnormal chromosomal
the 2-cell-plus-extrusion whereas the morphologically were
pronuclei
2-cell
subsequent
these embryos-all had a severely
of
cleaved to 3-cells composition;
embryos were regular-looking
diploid embryos
or near-tniploid. embryo
formed
to at the
cleave first
from
cleavage
1- to
3-cells,
division
should
the be
tripolar.
The data in this study indicate that the movement of chromosomes on such a tripolar spindle is disorganized, resulting in the cells in an embryo having different numbers of chromosomes in each cell, and the cells thus having an abnormal chromosomal constitution. Tn- and/or quadripolar spindles have been observed in the sea urchin (Wilson, 1925) and
in rat The
oocytes
finding
plus-extrusion,
of
shows
tripronuclear state of the cleavage of a tripronuclear
they cleave to regular findings also demonstrate and
a photomicrograph of a morphologically normal 2-cell embryo. Chromosomal analysis of 4 of these embryos at the second cleavage division revealed triploid shows
The
between
of tripronuclear morphologically
3a, Table
b
suggest
that
(Austin
and
Walton,
1960).
that some embryos cleave to 2-cellswhich are karyotypically diploid, in this situation one haploid set of
398
KOLA
TABLE
1. Cleavage
details
and
Number of cells after first cleavage division
Oocyte
3-cell
+
3 fragments
2-cells
+ extrusion
2-cells
*ND *
tThe
=
Not
chromosome
findings
of tripronuclear
human
ET
AL. oocytes.
Time of first cleavage division (h after insemination)
code
Number
of
metaphase
Chromosome findings
cells
57,51
MCK MUL DEM7 DEM1 MIT JAN CHR SHA4 TS1 BAL GAN SUM COP FRA3 HOL COA OTT MAR
29 32 28 27 30 28 33 26 28 26 33 28 28 33 33 27 29 30
2 0 1 0 0 1 ND* 3 1 ND ND ND 1 2 1 ND
COC THE DOM COR
30 30 27 27
2 2 2 2 nuclei
DEM4 SM1 BRO HOO SHAI AT!’ FRA4
34 24 33 31 36 27 29
2 0 0 ND I 1 1
-
57 -
54 ND 42,44,5 2 34 ND ND ND 51 52,39 38 ND 34 34,42,32,9,12,6**
-
3
46,46 46,46 46,46 + 23 chromosomes 69,69 -
ND 69 72 69
done.
derivation
chromosomes at the first
of these
6 groups
of chromosomes
is unclear.
is excluded from the metaphase cleavage division, and that this
plate division
participate
in
mitosis.
the
only involves 46 chromosomes and a normal bipolar spindle. It may be that the chromosomes of one pronucleus has a different degree of condensation
triploid
chromosomal
complement.
study,
two of the embryos fixed at the 2-cell
from the other two; thus, a haploid set of chromosomes do not gather on the metaphase plate. However, we have not yet elucidated whether the subsequent development of these embryos is normal nor deter-
found to be triploid. This study has
mined the fate of the haploid set of chromosomes. Van Blerkom et al. (1984) found that one tnipronuclear oocyte that cleaved to 2-cells was diploid at the 8-cell stage, but overlooked. The situation
logically these
it is possible
data
in
this
that study
(tripronuclear
extrusion
suggest
2-cell are
tniploid),
was
that in the third cleaving to morphoembryos; chromosomally,
oocytes
normal embryos
a nucleated
all
the
chromosomes
a!. (1986) 2-cells
and
tnipronuclear
production
These embryos have a regular of daughter cells having a full
division,
were
human
In
and
recently
a!., 1986).
one
that
cleaved to were also
that
only
develop
into
tniploid
division. One conceptions
percent in man
thus demonstrating do develop to have also been survived from
that stage
et
oocytes
are tniploid (Jacobs et a!., 1978). (1985) have reported the live
There have
Angel!
demonstrated
embryos after the first cleavage of all in vivo clinically recognized
infants, embryos
the
Uchida birth that stages
later
24%
of
and Freeman of 7 triploid some tniploid of pregnancy.
reports of 5 tniploid infants who 2 to 7 mo (Borgaonkar, 1984), survived
to
10#{189} mo
(Sherard
et
TRIPRONUCLEAR
HUMAN
OOCYTES
399
‘I
a
B
a
1W
-‘-I-i-’F
b FIG. 2. (a) Photomicrograph of 2-cells-plus-extrusion meres are clearly visible. The extrusion is indicated embryo (X 1000). These photomicrographs demonstrate
by
G
C
embryo, derived an arrow. (b) that the cell had
from the direct cleavage of a tripronuclear Metaphase spread and (c) karyotype of a diploid (46 XY) karyotype.
oocyte (X 200). The two blasto1-cell from a 2-cell-plus-extrusion
400
KOLA
ET
AL.
a b
icu’
*r3MV E
2-s-I--
F
FIG. logically that the
The
3. (a) Photomicrograph normal 2-cell embryo. cell was triploid (69
findings
in this
-$r*-j
C of
2-cell
(b) Metaphase
xxx
embryo spread
derived from the and (c) karyotype
and
the
direct cleavage of 1-cell from
of a tripronuclear a 2-cell embryo
oocyte (X 1000).
(X200). This embryo These photomicrographs
is a morphodemonstrate
karyotype).
study
have
demonstrated
that
the vast majority of tripronuclear human oocytes do not develop into triploid embryos and that a correlation exists between the pattern of the first cleavage division
C
subsequent
karyotype
of
these
embryos. This study has also provided mechanisms by which tnipronuclear to develop into triploid embryos
insight into the oocytes fail in the human.
However, microtubules
on staining the 1986), centro-
research is being (Schatten et
focused a!., 1985,
TRIPRONUCLEAR
somes,
and
mechanisms fail
chromosomes whereby
to develop
into
to
further
tnipronuclear triploid
HUMAN
investigate human
the
Rudak
oocytes
OOCYTES
E, Dor J, Mashiach S, Nebel L, Goldman B, 1984. Chromosome analysis of multipronuclear human oocytes fertilized in vitro. Fertil Steril 41:538-45
Sathananthan AH, fine structure
embryos.
401
Trounson AO, of monospermic
1985. and
The human polyspermic
pronuclear ovum: fertilization in
Gamete Res 12:385-98 H, Schatten G, Mazin D, Balezan R, Simerly C, 1986. Behavior of centrosomes during fertilization and cell division in mouse oocytes and in sea urchin eggs. Proc NatI Acad Sci (USA) 83:105-09 Schatten G, Simerly C, Schatten H, 1985. Microtubule configurations during fertilization, mitosis, and early development in the mouse and the requirement for egg microtubule-mediated motility during mammalian fertilization. Proc NatI Acad Sci (USA) 82:4152-56 Sherard J, Bean C, Bove B, Delduca V, Esterly KL, Kavish HJ, Munshi G, Reamer iF, Suazo G, Wilmoth D, Dahlke MB, Weiss C, Borgaonkar DS, 1986. Long survival in a 69XXY triploid male. AmJ Med Gen 25:307-12 Trounson AO, 1982. Discussion on risks of triploidy arising through polyspermy in vitro. In: Edwards RG, Purdy JM (eds.), Human Conception in Vitro. London: Academic Press, p. 227 vitro.
ACKNOWLEDGMENTS We would like to thank C. King, du Plessis, G. Jones, and all the members program for support and encouragement; the film; Ian Barker for photographic manuscript; and Dr. Jill Shaw for critical
Schatten L.
Chiappazzo, C. Thomas, V of the Monash-Epworth IVF Chris Hale for assistance with work; Jan Ryan for typing the comments on the manuscript.
REFERENCES AngeIl
RR, spermy
Templeton in man.
AA, Cytogenet
Messinis Cell
IE, Genet
1986. 42:1-7
Austin
CR, Walton A, 1960. Fertilisation. In: Physiology of Reproduction. London:
Consequences
ParkesAS Longmans
of
poly-
(ed.), Marshall’s Green & Co.,
pp. 310-416 Borgaonkar DS, 1984. Chromosomal Variation in Man-A Catalog of Chromosomal Variants and Anomalies, 4th Ed. New York: Alan R. Liss, pp. 778-85, 953 Jacobs PA, Angell RR, Buchanan JM, Hassold TJ, Matsuyama AM, Manuel B, 1978. The origins of human triploids. Ann Hum Genet 42:49-57 Kola I, FoIb P1, Parker Ml, 1986. Maternal administration of cyclophosphamide induces chromosomal aberrations and inhibits cell number, histone synthesis, and DNA synthesis in preimplantation mouse embryos. Teratog Carcinog Mutagen 6:115-27 Mahadevan MM, Trounson AO, 1984. The influence of seminal characteristics on the success rate of human in vitro fertilization. Fertil Steril 42:400-05
Trounson AO, Mohr LR, 1983. Human pregnancy following cryopreservation, thawing and transfer of an eight-cell embryo. Nature 305:707-09 Iichida IA, Freeman VC, 1985. Triploidy and chromosomes. Am J Obstet Gynecol 151:65-69 Van Blerkom J, Henry G, Porreco R, 1984. Preimplantation human embryonic development from polypronuclear eggs after in vitro fertilization. Feral Steril 41:686-96 Wilson EB, 1925. The Cell in Development and Heredity. New York: Macmillan, pp. 416 zenzes MT, Belkien L, Bordt J, Kan I, Schneider HPG, Nieschlag E, 1985. Cytologic investigation of human in vitro fertilization failures. Fertil Steril 43:883-91