Human Reproduction Vol.16, No.11 pp. 2362–2367, 2001
Cytogenetic analysis of human zygotes displaying three
pronuclei and one polar body after intracytoplasmic sperm
injection*
B.Rosenbusch1,3, M.Schneider2, R.Kreienberg1 and C.Brucker1
1Department
of Gynecology and Obstetrics, University of Ulm, Prittwitzstrasse 43, D-89075 Ulm and 2Gregor Mendel Laboratories,
89231 Neu-Ulm, Germany
3To
whom correspondence should be addressed. E-mail: [email protected]
BACKGROUND: Digynic zygotes with three pronuclei and one polar body obtained after intracytoplasmic sperm
injection (ICSI) were studied cytogenetically to elucidate the frequency and origin of chromosomal abnormalities
at the earliest stage of conception. METHODS: Uncleaved, single-cell zygotes were incubated with podophyllotoxin
and vinblastine and fixed by a gradual fixation air drying method. The chromosomes were stained with Giemsa.
RESULTS: Twenty-two (50%) out of 44 informative zygotes revealed cytogenetic alterations, including aneuploidy
(six cells, 13.6%), structural aberrations (10 cells, 22.7%) and combinations of numerical and structural abnormalities
(two cells, 4.5%). In one case (2.3%), double aneuploidy or an effect of chromosomal translocation could not be
distinguished and one zygote (2.3%) turned out tetraploid due to injection of a diploid spermatozoon. Two zygotes
(4.5%) showed an irregular chromatid segregation between the two maternal complements. In completely analysable
cells, the sex chromosome ratio XXX:XXY was 17:15. CONCLUSIONS: Digynic ICSI zygotes carry a high rate of
cytogenetic abnormalities that obviously have been transmitted by the participating oocytes and spermatozoa. We
also confirmed the previously reported, possibly ICSI-induced irregular oocyte chromatid segregation. The results
suggest that aneuploidy in the oocytes must have been caused by predivision instead of non-disjunction.
Key words: chromosomal abnormalities/digynic triploidy/ICSI/irregular chromatid segregation/tripronuclear zygotes
Introduction
An inspection of pronucleus formation is routinely performed
after IVF and intracytoplasmic sperm injection (ICSI) in order
to detect and isolate abnormally fertilized oocytes. A common
deviation from the regular bipronuclear state is the occurrence
of three pronuclei (PN). Cytogenetically, this means the gain
of a whole haploid chromosome complement. Subsequent
cleavage of the affected zygotes can lead to the development
of a triploid embryo. Triploidy plays an important role in human
reproductive loss and accounts for ~15% of chromosomally
abnormal spontaneous abortions (Kaufman, 1991).
Tripronuclear zygotes observed after conventional IVF
mostly arise from dispermic fertilization including regular
extrusion of the second polar body (PB). Due to the additional
paternal chromosome set, this condition is termed diandric
triploidy. During ICSI, only one spermatozoon is injected into
a mature metaphase II oocyte, but nevertheless, occasionally
three PN are formed while the presence of only one (the first)
PB is maintained. It has been assumed that corresponding
*The results of this study have been presented in part at the 16th
Annual Meeting of ESHRE, Bologna, Italy, 2000.
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zygotes represent examples for digynic triploidy, the supernumerary pronucleus being a female one that results from nonextrusion of the second PB (Palermo et al., 1995). Analyses
of uncleaved one-cell zygotes (Grossmann et al., 1997) and
of embryos originating from tripronuclear zygotes (Staessen
and Van Steirteghem, 1997) supported this assumption since
no XYY chromosome complement was found.
Further information on the cytogenetic constitution of
digynic ICSI zygotes has only been provided by Macas
et al. who demonstrated a possibly ICSI-induced irregular
chromatid segregation between the two maternal complements
(Macas et al., 1996a,b). These earlier investigations largely
relied on chromosome counts and the occurrence of structural
chromosome abnormalities was not considered. Using threecolour fluorescent in-situ hybridization (FISH), the same
research group reported an increase in numerical chromosome
abnormalities in the paternal pronuclei of tripronuclear zygotes
(Macas et al., 2001). However, the authors admitted that an
exact evaluation of the overall rate of aneuploidy was not
possible by this approach.
We have already shown that karyotyping of multipronuclear
zygotes can serve as a model for drawing conclusions on
the incidence of both numerical and structural chromosome
© European Society of Human Reproduction and Embryology
Chromosomes of tripronuclear ICSI zygotes
Materials and methods
(1% sodium citrate in distilled water containing 2% HSA) for 10 min
and fixed according to a gradual fixation air drying method described
previously (Rosenbusch et al., 1998a). The preparations were stained
with Giemsa and karyotypes were established from photographs taken
at ⫻1000 magnification.
Our cytogenetic investigations of unfertilized or abnormally
fertilized human oocytes have been approved by the ethical committee of the University of Ulm.
The present study includes 40 couples allocated to ICSI because of
impaired sperm quality (n ⫽ 31) or unsuccessful previous IVF
attempts despite normal semen parameters (n ⫽ 5). Four men
were azoospermic and underwent microsurgical epididymal sperm
aspiration (n ⫽ 3) or testicular sperm extraction (n ⫽ 1). The mean
age of the female patients was 32.5 years (range 23–43), while that
of the partners was 36.2 years (range 23–61). Ovarian stimulation
was performed with human menopausal gonadotrophin or FSH after
pituitary suppression with a gonadotrophin-releasing hormone agonist.
During one and the same treatment cycle, 29 patients provided one
tripronuclear zygote, eight patients provided two and two patients
provided three cells. Another three cells were obtained from one
patient during two consecutive cycles. All of these zygotes were
derived from oocytes injected with initially motile spermatozoa.
Our ICSI technique has been described elsewhere (Rosenbusch
et al., 1998b; Rosenbusch, 2000). IVF medium (Medi-Cult,
Copenhagen, Denmark or IVF Science Scandinavia, Vitrolife
Productions AB, Gothenburg, Sweden) was used for incubating
untreated and injected oocytes as well as pronuclear and cleavage
stages. Pronucleus formation was assessed 18–20 h after ICSI.
Presumed multipronuclear zygotes were examined once more to avoid
confusion of PN and cytoplasmic vacuoles. Oocytes with three PN
and one PB were incubated for 24–28 h in culture medium supplemented with podophyllotoxin and vinblastine (Sigma, St Louis, MO,
USA) at a final concentration of 0.15 µg/ml each. Prior to fixation,
the zona pellucida was digested in a 0.1% protease solution (type
XIV, pronase E; Sigma). The zygotes were left in hypotonic solution
Results
Of 54 tripronuclear zygotes which were processed for cytogenetic analysis, 10 preparations could not be evaluated because
of insufficient chromosome spreading and/or poor chromosome
quality (seven cells). Two zygotes revealed incompletely condensed metaphase chromosomes whereas another cell was
excluded due to presumed cell rupture during fixation and
excessive chromosome scattering.
The varying arrangement of the chromosome sets was taken
into consideration during slide analysis (Table I). Seventeen
zygotes showed three separate haploid metaphases (distribution pattern n/n/n). Here, 11 cells could be completely
karyotyped whereas six presented with one or two uninterpretable metaphases. Five zygotes had one haploid and
one diploid chromosome set (n/2n). They included one cell in
which chromosome quality of the diploid metaphase only
allowed counting and two zygotes in which either the haploid
or the diploid component was not analysable. In 21 zygotes
the individual sets were not distinguishable (3n). In this group,
one cell had chromosomes of poor quality and therefore they
were only counted. Finally, one zygote with three pronuclei
was in fact tetraploid. It could be karyotyped but the individual
chromosome sets were not clearly separated (4n).
abnormalities, at the earliest stage of conception (Rosenbusch
et al., 1997; 1998a). The present report summarizes our data
on tripronuclear one-cell ICSI zygotes. For this purpose, two
previously published single observations (Rosenbusch and
Sterzik, 1996; Rosenbusch et al., 1998b) have been included.
Table I. Cytogenetic analysis of 44 tripronuclear zygotes obtained after intracytoplasmic sperm injection (ICSI)
Chromosome distribution
Normal (no. of cells)
Abnormal
Numerical and structural anomalies
Karyotype: 3 PN–3n
3n
69 counted (1)
69,XXX (6)
69,XXY (4)
n/2n
23,X/?? (1)
23,X/46 counted (1)
n/n/n
23,X/?/? (2)
23,X/23,X/? (1)
23,X/23,X/23,X (3)
23,Y/?/? (1)
23,X/23,X/23,Y (2)
Karyotype: 3 PN–4n
4n
68,XXX,-D
68,XX,-C or -Y
70,XXX,⫹C
69,XXX,chtb(1)
69,XXX,chrb(1)(p)
69,XXX,chrb(C)(cen),ace
69,XXX,ace
69,XXX,del(1)(q?)
69,XXY,chrb(2)(p)
70,XXY,chrb(C)(cen),⫹16
?/47,XX,⫹C
23,X/45,XY,-E
23,Y,chrb(C)(cen)/47,XX,⫹16
23,X,⫹C,-D/?/?
23,X/23,X/23,X,del(B)(p)
23,X/22,X,-G/23,Y
23,X/23,X,chrb(3)/?
23,X/23,X,ace/23,Y
23,X/23,X/23,Y,chrb(C)(cen)
Irregular chromatid segregation
20,X,-2E,-F/26,X,⫹2E,⫹F/23,Y
22,X,-C/24,X,⫹C/23,Y
92,XXYY,end3,-18,-18,end18
? ⫽ not analysable haploid chromosome set; ?? ⫽ not analysable diploid chromosome set.
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B.Rosenbusch et al.
Within 44 fully or partially analysable zygotes we detected
six cases (13.6%) with missing or extra chromosomes whereas
two cells (4.5%) had a combination of numerical and structural
abnormalities. Thus, there was a total of eight (18.2%) aneuploid cells, four (9.1%) of them being hypertriploid and
four (9.1%) being hypotriploid. One zygote (2.3%) with an
obviously abnormal (23,X,⫹C,-D) and two uninterpretable
metaphases should be considered separately, since beside the
given interpretation we cannot exclude that the supposed C
group chromosome is in fact a product of translocation on
the missing D. In total, 12 cells (27.3%) carried structural
chromosome abnormalities. These included chromosome
breaks (chrb) on the chromosome arms (Figure 1a) or in the
centromeric region (cen) (Figure 1b), a chromatid break
(chtb), acentric fragments (ace) and two deletions (del). As
stated above, in two of these cells an additional numerical
abnormality was present (Figure 1b). A particular case is the
tetraploid zygote (2.3%) that has been discussed in detail
earlier (Rosenbusch et al., 1998b). Here, we assumed injection
of a diploid spermatozoon due to the presence of two Y
chromosomes. Moreover, this cell revealed missing and
endoreduplicated chromosomes. Finally, we discovered two
unambiguous examples (4.5%) of irregular chromatid segregation between the two female PN (Figure 2). As suggested
earlier (Rosenbusch, 1997), this possibly ICSI-induced phenomenon was distinguished in Table I from chromosomal
abnormalities transmitted by male and/or female gametes. In
total, 22 zygotes (50.0%) revealed some kind of cytogenetic
aberration in the present material. Excluding the tetraploid and
the hypotriploid (68,XX,-C or -Y) zygotes, as well as all
cases with uninterpretable or counted metaphases, the sex
chromosome ratio XXX:XXY was 17:15.
Figure 1. Two examples of chromosomally aberrant zygotes.
(a) 3n ⫽ 69,XXY,chrb(2)(p): break in the short arm of
chromosome 2 (arrow). (b) 3n ⫽ 70,XXY,chrb(C)(cen),⫹16: a
centromeric break in a chromosome belonging to group C (arrow)
and a supernumerary chromosome 16 (asterisk). Only partial
karyotypes are given.
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The karyotypes 23,Y,chrb(C)(cen)/47,XX,⫹16 and 23,X/
23,X/23,Y,chrb(C)(cen) indicate that the structural chromosome abnormality in both zygotes originated from the
spermatozoon whereas the supernumerary chromosome 16
must have been transmitted by the oocyte. Moreover, a
female origin of the cytogenetic abnormality is evident in two
completely karyotyped zygotes (23,X/22,X,-G/23,Y and
23,X/ 23,X,ace/23,Y) and may be assumed in another two
cells with one uninterpretable metaphase (?/47,XX,⫹C and
23,X/23,X,chrb(3)/? respectively).
Discussion
Both the oocyte and its first PB contain a haploid chromosome
set (n ⫽ 23) with chromosomes consisting of two chromatids
held together at the centromere. Sperm penetration triggers
longitudinal division of the oocyte chromosomes followed by
inclusion of 23 chromatids in the maternal pronucleus while
the corresponding halves are extruded into the second PB. The
spermatozoon also contributes 23 chromatids in the male
pronucleus (Figure 3a). Subsequent DNA replication and
disintegration of the pronuclear membranes restore diploidy
(2n ⫽ 46).
Tripronuclear zygotes with one PB observed after ICSI
result from non-extrusion of the second PB, i.e. its chromatids
remain in the ooplasm giving rise to an additional female
pronucleus (Figure 3b). After DNA replication, three haploid
chromosome sets will be present leading finally to triploidy
(3n ⫽ 69). The reasons for a retention of the second PB are
poorly understood and may involve the orientation or position
of the second meiotic spindle in the oocyte cortex, damage to
the cytoskeleton during ICSI, or reorientation of the spindle
during injection (Flaherty et al., 1995). Possibly, clinical
parameters may affect maturation of some oocytes rendering
them susceptible to abnormal fertilization. Palermo et al. found
a significantly higher incidence of digyny in oocytes with a
specific membrane behaviour, i.e. sudden breakage without
funnel formation, upon penetration of the injection needle
(Palermo et al., 1996). These authors suggested a link between
ovarian stimulation and oolemma characteristics because a
significantly shorter length of stimulation and lower serum
oestradiol concentrations were found in the sudden breakage
group when compared with oocytes with different breakage
patterns. It is conceivable that such membrane irregularities
impair the oocyte metabolism and thus the function of microtubular structures needed for chromatid segregation and
formation of the second PB.
Recently, Sachs et al. concluded that patients who produce
at least one digynic zygote are high responders to ovarian
stimulation since this group was characterized by more follicles
and oocytes and higher oestradiol concentrations compared
with cycles without tripronuclear oocytes (Sachs et al., 2000).
In their study, the occurrence of three PN did not correlate
with sudden breakage of the oolemma, but the number of
corresponding oocytes was very small. It is evident that the
factors and mechanisms responsible for a non-extrusion of the
second PB need further investigation.
The postulated digynic origin of tripronuclear ICSI zygotes
Chromosomes of tripronuclear ICSI zygotes
Figure 2. Metaphase spread (above) and karyotypes of a zygote with irregular chromatid segregation. The supernumerary chromosomes
(arrowheads) of the first maternal (I.mat) set are missing (asterisks) in the second maternal (II.mat) metaphase. PBC ⫽ first polar body
chromosomes; pat ⫽ paternal complement.
excludes the occurrence of an XYY sex chromosome constitution. In fact, FISH analyses failed to detect the XYY karyotype
in uncleaved zygotes (Grossmann et al., 1997) and in uniformly
triploid or mosaic embryos developing from tripronuclear
zygotes (Staessen and Van Steirteghem, 1997). The XXX:XXY
ratios were 7:8 and 34:38 respectively, compared with 17:15
in the present study. The tetraploid XXYY chromosome set
observed by us can be ascribed to injection of a diploid
spermatozoon and does not contradict a digynic origin of
this particular zygote. It has been shown that infertile men
with oligoasthenoteratozoospermia have significantly higher
frequencies of diploid spermatozoa than the control groups
(Aran et al., 1999; Ushijima et al., 2000). In our case,
ICSI had been performed with spermatozoa from a patient
with oligoteratozoospermia and borderline sperm motility
(Rosenbusch et al., 1998b) and therefore the occurrence of a
diploid male gamete is not completely unexpected. Of note,
the corresponding zygote would have appeared regularly
fertilized and the resulting (possibly triploid) embryo would
have been transferred if the oocyte had managed to extrude
its second PB. Just recently, further examples for tripronuclear
zygotes carrying a diploid sperm pronucleus have been reported
by Macas et al. who applied triple colour FISH with probes
for chromosomes X, Y and 18 (Macas et al., 2001).
The parental origin of the cytogenetic abnormality can be
determined in some of the aberrant zygotes studied by us. A
conspicuous example is the karyotype 23,X/22,X,-G/23,Y
which implies that chromosome loss has affected the oocyte.
Artefactual loss during fixation appears highly improbable
since we use a technique that keeps the cytoplasm intact. Also,
the chromosomes within the three sets were lying close together
and repeated screening of the slide yielded no indication
for scattered chromosomal elements. We therefore surmise
participation of an aneuploid oocyte. However, this particular
oocyte could not have been hypohaploid for a whole chromosome (karyotype 22,X,-G) since this constitution would have
caused two hypohaploid (22,X,-G) female PN. Instead, the
abnormal zygote may have developed from an oocyte with a
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B.Rosenbusch et al.
Figure 3. Schematic representation of regular fertilization, digyny and development of a hypotriploid digynic zygote. (a) Normal
fertilization is characterized by extrusion of the second PB and the formation of one male (m) and one female (f) pronucleus. (b) Digynic
triploidy results from non-extrusion of the second PB. The corresponding chromatids remain in the ooplasm and will form an additional
female pronucleus. (c) The abnormal (23,X/22,X,-G/23,Y) digynic zygote most probably results from an oocyte with a pre-divided G group
chromosome. The oocyte has received only one chromatid of this chromosome because the other chromatid has been distributed into the
first PB. Consequently, one of the female PN will lack this particular chromatid. For further explanations see text.
single G group chromatid resulting from predivision (Angell,
1997). According to our recently suggested nomenclature
(Rosenbusch and Schneider, 2000b), the oocyte’s karyotype
would have been 23,X,-G,⫹Gcht indicating that one whole G
group chromosome has been replaced by a single chromatid.
The chromatid was distributed into one female pronucleus
while the other pronucleus was lacking the corresponding half
(Figure 3c). Thus, after DNA replication one maternal set will
be normal (23,X) whereas the other will be hypohaploid
(22,X,-G).
The karyotype 23,Y,chrb(C)(cen)/47,XX,⫹16 points to an
aneuploid oocyte characterized by the presence of an additional
chromatid (24,X,⫹16cht). Moreover, the zygote described
as ?/47,XX,⫹C could have arisen from an oocyte with ‘predivided’ chromosomes (24,X,⫹Ccht) if we assume a paternal
origin of the uninterpretable metaphase. Considering all
numerical deviations from the triploid count (hypo- and
hypertriploidy) in our material, it is interesting to note that
without exception only one chromosome was involved. This
observation excludes non-disjunction of whole chromosomes
in all maternally transmitted cases of aneuploidy since the
affected zygotes would then have revealed two missing or
additional chromosomes.
Concerning structural aberrations, the karyotype
23,Y,chrb(C)(cen) has been detected twice and clearly
indicates a transmission of the breakage event by the
spermatozoa. This is not unexpected since structural abnormalities, in particular centromeric breaks and acentric fragments,
are frequently found in sperm chromosomes (Rosenbusch,
1995). However, we also obtained two karyotypes [23,X/
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23,X,ace/23,Y and 23,X/23,X,chrb(3)/?] that suggest a female
origin of the structural abnormality. In the second case, we
ascribed the uninterpretable metaphase to the spermatozoon
because its appearance resembled one of the patterns of
prematurely condensed sperm chromatin found in unfertilized
oocytes after ICSI (Rosenbusch, 2000).
Two of the zygotes with an unambiguous separation of
all participating metaphases showed an irregular chromatid
segregation between the female chromosome sets. This
phenomenon was first reported by Macas et al. and has been
attributed to alterations of the oocyte cytoskeleton (Macas
et al., 1996a,b). Besides a purely mechanical impact of the
injection procedure, other causal mechanisms may comprise
oocyte ageing, changes in temperature and an effect of calcium
ions or hydrostatic pressure during injection. However, intrinsic
errors during meiotic maturation of the oocytes prior to ICSI
cannot be excluded with certainty (Macas et al., 1996a,b).
The relevance of irregular chromatid segregation lies in the
fact that it could also occur in normally fertilized oocytes, i.e.
those that manage to extrude the second PB. An unequal
distribution of the chromatids, e.g. 24 into the female pronucleus and 22 into the second PB or vice versa, would
render the oocyte aneuploid with serious consequences for the
developing embryo. To our knowledge, this topic has not yet
been studied in regularly fertilized ICSI oocytes. Of note, the
chromosomal situation may become rather complicated as
soon as an aneuploid oocyte undergoes irregular chromatid
segregation and one should be able to observe such an
event in digynic zygotes. Indeed, Macas et al. described a
chromosome distribution pattern of 23/22/20 that can be
Chromosomes of tripronuclear ICSI zygotes
attributed to a normal spermatozoon and irregular chromatid
segregation in an oocyte with 21 chromosomes (Macas et al.,
1996b). A comparable case was not detected in our material.
In summary, our study provides evidence for a high rate of
chromosomal abnormalities in gametes capable of undergoing
IVF. Moreover, the digynic situation allowed us to trace back
the transmission of some chromosomal abnormalities and to
confirm the previously described phenomenon of irregular
chromatid segregation. Multipronuclear zygotes therefore
appear as a valuable model to investigate the kind, frequency
and origin of cytogenetic abnormalities at the earliest stage of
conception. Finally, apart from oocytes with three PN and one
PB, there are other patterns of abnormal fertilization, e.g. a
combination of three PN and two PB. Some of these cells
have been studied by us and they always revealed a severely
hypotriploid count of ~56–59 chromosomes. This led us to
suggest that the corresponding zygotes might be products of
an incomplete chromatid segregation into the second PB
(Rosenbusch and Schneider, 2000a). It will be a challenging
task to analyse further, classify and explain these phenomena
observed after ICSI.
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Received on March 3, 2001; accepted on July 24, 2001
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