Human Reproduction vol.12 no.4 pp.800–804, 1997
The frequency and developmental capability of human
embryos containing multinucleated blastomeres
Hanna Balakier1 and Ken Cadesky
S.T.A.R.T. Inc., In Vitro Fertilization Clinic, 655 Bay, PO Box 4,
Toronto, Ontario M5G 2K4, Canada
1To
whom correspondence should be addressed
The frequency of multinucleated blastomeres (MNB) in
2- and 4-cell stage human embryos was recorded
immediately before embryo transfer using a high-power
inverted microscope. About 44% of patients (150/338)
possessed embryos exhibiting MNB. The appearance of
this nuclear abnormality was not correlated with maternal
age. Overall, 15% of the otherwise good quality embryos
(274/1885) that developed after monospermic fertilization
contained several multinuclei (from two to seven) in at
least one cell. Quite often MNB were found within all
cells of the embryo (50% in 2-cell embryos). Blastomere
multinucleation was significantly higher in 2-cell than 4cell embryos (P ,0.0001). This suggests that a considerable number of human embryos become abnormal during
the first embryonic division. The embryos containing
MNB were usually excluded for uterine transfers, with
the exception of 19 cases when only such embryos
could be replaced (6%; 19/338 patients). The results
demonstrated that embryos with MNB may implant (4/19
cases; 21%) and they can lead to both spontaneous
abortions and the successful birth of healthy infants (two
cases). The fact that in the successful cases, 2-cell stage
embryos with a mononucleated and a binucleated blastomere were transferred also suggests that due to the cell
totipotency, development of a healthy baby is possible from
one normal blastomere. Since multinucleation in early
embryos may reflect gross chromosomal abnormalities or
development of mosaic embryos, it is advisable not to
replace embryos with MNB. Occasional transfers, however,
can be considered because defective embryos may sometimes develop normally.
Key words: developmental potential/embryo selection/human
embryos/multinucleation
Introduction
Multinucleation of human blastomeres was first thought to be
related only to polyspermic fertilization (Van Blerkom et al.,
1984). However, later observations under the electron microscope (Lopata et al., 1983; Tesarik et al., 1987) and other
studies based on fluorescent staining (Winston et al., 1991;
Hardy et al., 1993; Pickering et al., 1995), indicated that this
nuclear abnormality may also develop in embryos derived
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from normally fertilized oocytes exhibiting two pronuclei and
two polar bodies. Especially high incidences of blastomere
multinucleation were found in poor quality embryos which
had fragmented or arrested in cleavage during prolonged invitro culture (Munné and Cohen, 1993; Munné et al., 1994).
Interestingly, it has also been suggested that such nuclear
defect may frequently occur in good quality embryos too
(Hardy et al., 1993; Munné and Cohen, 1993).
Multiple nuclei that are clearly defined on fixed and stained
preparations (as in the above mentioned studies) cannot be
seen in living embryos under low power microscopes and the
widely used morphological system of embryo selection does
not include examination for their presence prior to the embryo
transfer procedure. This implies that many embryos chosen as
suitable for replacement may contain undetected multinucleated
blastomeres (MNB). Such nuclear abnormality, which is most
likely associated with chromosomal aberrations, may contribute
to embryonic failures and lower pregnancy rates. However,
due to the lack of systematic observations, the impact of
blastomere multinucleation in good quality embryos on embryo
development and in-vitro fertilization (IVF) success still
remains unexplored.
Considering the importance of maximizing the quality of
embryo selection for uterine replacement and to advance our
understanding of early human embryology, the aim of the
present study is to: (i) detect the MNB in living embryos
immediately before their transfer by performing detailed
observations under a high power microscope, (ii) to record the
frequency of their appearance and (iii) to investigate the
developmental potential of embryos containing MNB during
in-vitro culture and after exclusive transfers of such embryos
in situations when other, apparently normal, embryos without
MNB did not exist. The paradoxical finding that the transfer
of embryos containing MNB leads to both developmental
failures and successful live birth of healthy infants is discussed.
Materials and methods
The procedures for IVF and embryo culture have been described
previously (Balakier and Stronell, 1994). Briefly, pituitary downregulation (long protocol) was achieved by treatment with the
gonadotrophin-releasing hormone (GnRH) analogue leuprolide acetate
(Lupron®; Abbott Laboratories Ltd, Montreal, Quebec, Canada) and
ovarian stimulation was induced with human menopausal gonadotrophin (Pergonal®; Serono Canada Inc., Mississauga, Ontario, Canada
or Humagon®; Organon Canada Ltd, Scarborough, Ontario, Canada).
Transvaginal follicular aspiration was performed at 36 h after human
chorionic gonadotrophin injection (10 000 IU HCG; Profasi, Serono)
and retrieved oocytes were incubated for 6–7 h before insemination
with 200 000–500 000 motile spermatozoa/ml. About 16–18 h later
© European Society for Human Reproduction and Embryology
Human multinucleated blastomeres
Table I. The frequency of multinucleated blastomeres (MNB) in living 2-, 3-, and 4-cell stage human embryos (41–43 h post insemination)
Age of
patients
(years)
26–35
No. of
patients
164
MNB
103
a40
ù40
MNB
71
a35
Total
Embryos with MNB
Total no.
with MNB
a75
36–39
Total no. of
embryos
338
MNB
1021
100%
556
100%
308
100%
1885
100%
160
b(15.6)
68
b(12.2)
46
b(14.9)
274
b(14.5)
No. pregnancies
per no. of
embryo transfers
2-cell
3-cell
4-cell
102
13
45
43
7
18
39
3
4
184
23
67
60/164c
(37%) (10)f
23/103d
(22%) (5)f
8/71e
(11%) (2)f
91/338
(27%) (17)f
aNo. of patients with embryos containing MNB.
bPercentage of overall total no. of embryos.
cTen transfers with defective embryos (three
dThree transfers with defective embryos.
eSix
fNo.
pregnant, one delivered).
transfers with defective embryos (one pregnant, delivered).
of spontaneous abortions.
polyspermic zygotes were separated from normally fertilized oocytes.
The oocytes and embryos were cultured in human tubal fluid (HTF)
medium (Quinn et al., 1985) supplemented with 10% human albumin
under mineral oil (Sigma) in 5% CO2 in the air (22% O2 concentration).
A maximum of three to four embryos was transferred about 41–43 h
after insemination. During morphological assessment, the embryos
were carefully examined for the presence of multinuclei using inverted
microscopes at 3320–400 magnification (Leitz DM IL relief contrast,
Leica or Axiovert 100, Zeiss with Hoffman modulation contrast).
The embryos containing visible MNB were usually excluded for
uterine replacement, except sporadic cases (x 5 19) when apparently
normal embryos did not exist. The possibility of chromosomal
abnormalities within defective embryos was discussed with the
patients and preneonatal diagnosis was suggested. The final decision
to transfer potentially defective embryos was given to the patients.
The luteal phase was supported by four injections of 2500 IU HCG,
given on days 2, 5, 8 and 11 after embryo transfer, and by progesterone
(vaginal suppositories; 100 mg twice daily after HCG injection;
Medicine Shoppe, Toronto, Ontario, Canada).
Results
The 1885 embryos from 338 patients were examined for the
presence of MNB during morphological assessment immediately
before the embryo transfer procedure (Table I). About 44% of the
patients (150/338) possessed embryos exhibiting MNB (87 patients
with one, 54 patients with two to four and nine patients with five to
eight embryos). The presence of MNB was not correlated with
patient age. The proportion of defective embryos among three age
groups (26–35, 36–39 and .40 years) was not statistically significant
(χ2 5 2.1, P .0.1, with two degrees of freedom). Overall, 15% of
good quality embryos at 2- , 3- and 4-cell stage contained several
multinuclei of similar or variable sizes (two to seven nuclei/cell)
within one or more cells (Figure 1A,B). Multinucleated blastomeres were detected more often in 2-cell (67%, 184/274) than 4-cell
(25%, 67/274) embryos. Such a difference appeared to be statistically
highly significant (χ2 5 107.8, P ,0.0001, with one degree of
freedom). Approximately half of the 2-cell defective embryos contained only one abnormal blastomere (54%, 100/184), while in the
other half, both cells were defective (46%, 84/184). Similarly in
3-cell stage embryos about 50% contained one MNB (11/23, 48%)
and the other 50% contained two or three MNB (12/23, 52%). Within
4-cell defective embryos, multinuclei were visible more frequently
in 1- (46%, 31/67) and 2-cell embryos (46%, 31/67) than within
3- or 4-cell embryos (8%, 5/67).
In 19 exceptional cases (6%, 19/338 patients; Table I), normalappearing embryos were not available and only one (nine patients)
or two (10 patients) abnormal embryos with MNB could be transferred.
The majority of those embryos developed into regular appearing 2cell embryos in which one blastomere displayed two different-sized
nuclei and the second blastomere was mononucleated (14 patients,
Figure 1A). In the other five cases, the 4-cell stage embryos contained
one or two MNB. Of these 19 patients, four became pregnant (4/19;
21%) with serum β-HCG concentration ~1000 IU/l at 3 weeks after
embryo transfer, and one showed chemical pregnancy (53.9 IU/l).
Spontaneous abortion took place in one case during the fifth week of
gestation. Another pregnancy was terminated at 17th week, when
trisomy 18 with fetal abnormalities related to this syndrome were
discovered. On the other hand, two other pregnancies were successful
and healthy baby boys were delivered. In the course of these two
pregnancies, one patient declined amniocentesis because of fear of
miscarriage. However, triple-screen test suggested low risk of spina
bifida (1:732) and Down’s syndrome (1:800). The second patient had
undergone amniocentesis which revealed the presence of a normal
male fetus.
Development of 147 embryos containing MNB (117 3 2-cell and
30 3 4-cell) was followed during in-vitro culture (96–120 h in HTF
medium supplemented with 10% fetal calf syndrome instead of human
albumin). Regardless of the number of MNB per embryo (from one
to three), the majority of embryos were arrested in development at
the stage of 2–15 cells (n 5 84; 57%, Figure 1C). The remaining
embryos (n 5 63; 43%) were capable of forming blastocysts. However,
only 20 blastocysts (14%) were expanded and contained a normallooking inner cell mass (ICM; five from 2-cell embryos with one
MNB, 15 from 4-cell embryos with one to two MNB). The other 43
embryos formed trophoblast vesicles in which the ICM was not
visible (eight from 2-cell embryos with two MNB), or else they
developed into small abortive blastocysts (~10–20 cells) that contained
poor ICM, sometimes with large vacuolized cells (27 from 2-cell
embryos with one to two MNB; eight from 4-cell embryos with one
to three MNB; Figure 1D,E).
Discussion
The appearance of MNB in human embryos is considered to be
a pathological event, probably causing significant chromosomal
801
H.Balakier and K.Cadesky
Figure 1. Human embryos that developed from monospermic zygotes examined under inverted microscope with Hoffman modulation
contrast. (A) 2-cell stage embryo containing a mononucleated and a binucleated blastomere. (B) 2-cell stage embryo with both heavily
multinucleated blastomeres (MNB) (about six to seven nuclei/cell). (C) 8-cell embryo which at the 2-cell stage contained one binuleated
blastomere. Two nuclei are visible in one cell. The other cells contain single nuclei (majority of them are out of focus). (D and E) Abortive
blastocysts that originated from 2-cell stage embryos containing both MNB (three to four nuclei/cell; 96–120 h of culture). The inner cell
mass is composed of large cells in D and is poorly developed in E.
aberrations. Although there are no detailed investigations on
chromosome constitution of such embryos, the recent analysis
of chromosomes X, Y, 18 and 13/21 have indicated that MNB
are generally abnormal as well as some of their mononucleated siblings (Munné and Cohen, 1993; Kligman et al.,
1996). Defective embryos may certainly be abnormal for other
chromosomes. For this reason, it is generally assumed that
embryos containing MNB are incompetent in producing viable
fetuses and their transfers result in developmental failures.
Therefore, it would be important to identify such abnormal
embryos and reject them for replacement. In this respect our
efforts to detect MNB in living embryos led us to the conclusion
that visualization of multiple nuclei under a high-power
inverted microscope can be a valuable additional parameter in
selecting the embryos for uterine transfer. Detailed examination
has revealed a relatively high incidence of blastomere multinucleation within 2- and 4-cell stage good quality embryos
(15%) developing after normal, monospermic fertilization. It
802
also appeared that a large number of patients (45%) possessed
a minimum of one embryo, but quite often from two to eight,
which exhibited this nuclear abnormality. Considering the fact
that nuclei may sometimes remain undetected due to the course
of mitosis (nuclei not seen) or due to difficulties in their
visualization (granular cytoplasm, presence of cytoplasmic
fragments, overlapping blastomeres), the frequency of multinucleation is likely higher. Nevertheless, as applied here, a
non-invasive method of detecting MNB in living embryos
seems to be quite reliable since the present observations
correspond with the previous findings obtained from fixed,
fluorescent stained preparations which have indicated a similar
percentage (9–24%) of multiple nuclei in human 2- and 4-cell
stage embryos of good quality (Hardy et al., 1993; Munné
and Cohen, 1993; Pickering et al., 1995).
The present observations have also shown that the incidence
of blastomere multinucleation was significantly higher in 2cell embryos than 4-cell embryos. This suggests that a relatively
Human multinucleated blastomeres
large portion of human embryos become abnormal during the
first mitotic division (multinucleation in 4-cell embryos may
start at the first or the second cleavage), which could be a
consequence of inadequate oocyte maturation or abnormalities
in sperm function. This is especially interesting in light of
recent reports indicating that the first embryonic spindle is
organized by the sperm centriole, which must be present for
proper embryo cleavage (Palermo et al., 1994; Sultan et al.,
1995). Preliminary experiments have also shown that the
disturbance of sperm structure created by injection of mechanically isolated sperm heads or tails into human oocytes induced
normal fertilization and cleavage but resulted in complete
aneuploid distribution of chromosomal material (Palermo
et al., 1996). Furthermore, the fact that 2-cell stage embryos
frequently exhibit nuclear abnormalities may explain why in
the other IVF studies, in which MNB were not scored,
significantly more pregnancies have been documented after
transfers of fast-dividing 4-cell embryos than transfers of slowdividing 2-cell embryos (Erenus et al., 1991; Staessen et al.,
1992). This seems very likely because development of defective
embryos can be delayed, and for this reason MNB are more
frequently seen in slower and poorer quality embryos than in
faster and presumably better ones.
In contrast to the general assumption that embryos with
MNB are developmentally incompetent, the present findings
clearly demonstrated that although defective embryos contribute to pregnancy losses, in certain cases they may also
retain full developmental capability and give rise to healthy
babies. Since the number of cases was low, the frequency of
failures versus successful results cannot be determined but it
still remains intriguing that about 11% (2/19) of the transfers
involving defective embryos ended in full development. More
observations are certainly needed to answer these questions,
but it must be emphasized that collection of such specific data
can rarely be performed (only 6% of our transfers were
exceptional) and the intentional randomized replacement of
defective embryos does not appear ethical. The mechanism by
which certain defective embryos may retain full developmental
potential is obscure. However, it seems logical that embryo
viability can depend on the degree of chromosomal abnormalities and on an unknown embryonic system of correcting or
eliminating the aberrant cells. Therefore, it can be suggested
that in our successful cases, healthy babies had most likely
developed from normal-looking, mononucleated blastomeres
of the 2-cell stage embryos (Figure 1A), while their abnormal,
binucleated siblings (or their descendants) became arrested
and excluded from fetal development. This would mean that
at the 2-cell stage, only one normal blastomere is required for
full development to occur. However, it is impossible to
recognize chromosomal normality of the mononucleated
blastomere in such an abnormal embryo. Perhaps the lower
the number of nuclei in the MNB, the better the chance for
normality in the other blastomere (majority of transferred
embryos had binucleated cells). On the other hand, a different
explanation for the successful results could be that, on occasion,
MNB had been restored to the diploid state and participated
in embryo development. Nevertheless, based on previous
findings, this seems unlikely and the concept of arrest and
elimination of aberrant cells appears more probable. For
instance, ultrastructural studies have implied that accumulation
of abnormalities within human MNB might lead to irreparable
cleavage arrest (Tesarik et al., 1987). Also, recent experiments
on mouse embryos have shown that chromosomally abnormal
cells are preferentially allocated to the trophectoderm rather
than the inner cell mass, possibly allowing normal embryo
development (James and West, 1994). It should be pointed out
that the fact that human babies may develop from only one
blastomere suggests that at the 2-cell stage both blastomeres
are totipotent. This property of human embryos would then
be similar to the attribute of other mammalian embryos, in
which individual blastomeres up to about the 4-cell stage could
develop to live offspring (Papaioannou and Ebert, 1986).
In contrast to the successful cases, two other pregnancies
which developed from transfer of embryos with MNB resulted
in abortions. One occurred early after implantation, but the
other had to be induced in the second trimester because of
development of a chromosomally abnormal fetus. Undoubtedly,
this illustrates the potentially detrimental consequences of
transferring embryos that contain MNB. The substantially
reduced viability of defective embryos expressed by early
cleavage arrest and the formation of abortive blastocysts during
in-vitro culture, may also indicate the highly lethal nature of
these embryos. Especially gross abnormalities can be expected
in the cases when most or all cells are multinucleated (for
instance 2-cell embryos with two MNB; Figure 1B). In spite
of reduced viability, abnormal embryos containing MNB may
have a strong ability to implant since four out of 19 patients
became pregnant (21%). It is also tempting to suggest that
due to allocation of aberrant cells into the trophoblast layer
(James and West, 1994), embryos with MNB that are capable
of forming trophoblast vesicles or abortive blastocysts with
poor inner cell mass may develop to blighted ova when
implanted.
In summary, we conclude that a non-invasive technique of
detecting MNB in live human embryos may prevent replacement of chromosomally abnormal or mosaic embryos. Since
defective embryos can progress in development up to blastocyst
stage and in practice MNB cannot be detected beyond the 4–
6-cell stage, it is advisable that, regardless of the day of
embryo transfer (second, third or fourth day), examination for
the presence of MNB should be performed on the second day
after insemination. In certain cases, embryos with MNB
may retain full developmental potential, therefore occasional
transfers of such embryos can be considered when other
embryos without MNB are not available. It seems that isolation
of defective embryos should decrease the incidence of
implantation of chromosomally abnormal embryos. This may
be difficult to prove because implantation and successful
pregnancy depend on multiple factors. In this respect, although
our results are not unequivocal, they have indicated an overall
low abortion rate (19%, 17/91; Table I) with only 8% (three)
abortions in the last 39 (43%) pregnancies in the series. More
observations are definitely needed to determine the frequency
of implantation and normal development of embryos with
MNB, as well as to investigate the reasons for the appearance
of MNB.
803
H.Balakier and K.Cadesky
Acknowledgements
The authors appreciate the excellent technical assistance of Agata
Sojecki and Oliver Cabaca.
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Received on September 25, 1996; accepted on January 24, 1997
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