Human Reproduction vol.12 no.10 pp.2257–2262, 1997
The chromosomal constitution of multipronuclear
zygotes resulting from in-vitro fertilization
B.Rosenbusch1,3, M.Schneider2 and K.Sterzik1
1Department
of Gynecology and Obstetrics, University of Ulm,
Prittwitzstrasse 43, 89075 Ulm/Donau, Germany and 2Laboratory
for Medical Genetics Dr. Tettenborn, Friedenstrasse 1, 89073 Ulm/
Donau, Germany
3To
whom correspondence should be addressed
We have attempted to analyse the chromosome constitution
of 77 multipronuclear uncleaved zygotes obtained from
our in-vitro fertilization programme. Complete karyotypes
could be established for 51 tripronuclear cells and eight
zygotes with four pronuclei. When compiling the results,
the varying arrangement of the chromosome sets was taken
into consideration. Eighteen tripronuclear zygotes showed
three separate haploid metaphases (distribution pattern n/
n/n), 16 cells had one haploid and one diploid chromosome
set (n/2n), and in 15 zygotes the individual sets were not
distinguishable (3n). Two zygotes were in fact tetraploid,
the distribution of metaphases on the slide being n/3n
and n/n/2n, respectively. In tripronuclear zygotes the sex
chromosome ratio XXX:XXY:XYY was 14:16:18, excluding
the two tetraploid cells and one zygote with a 23,X/23,X/
22,-C or -Y karyotype. Chromosome abnormalities were
found in 16 zygotes (31.4%) and included numerical (six
cells), structural (four cells) as well as combinations of
numerical and structural alterations (six cells). Four of the
zygotes with four pronuclei (50%) had numerical and/or
structural chromosome aberrations. Excluding two cells
with one uninterpretable metaphase and a 22,-C or -Y
karyotype, respectively, the sex chromosome distribution
XXXX:XXXY:XXYY:XYYY was 1:1:2:1 in zygotes with
four pronuclei. Another zygote was found to be pentaploid
after fixation. These results suggest that analysis of multipronuclear zygotes yields valuable information about cytogenetic abnormalities occurring at the earliest stage of
conception.
Key words: multipronuclear zygotes/chromosomes/in-vitro
fertilization
Introduction
During the past decade, two techniques gained considerable
importance to elucidate the incidence of chromosome abnormalities at the time of conception. First, sperm chromosome
complements were demonstrated after interspecific penetration
of zona-free hamster eggs. Second, oocytes that failed to
become fertilized in in-vitro fertilization (IVF) programmes
were used for investigating the female chromosomes. The
© European Society for Human Reproduction and Embryology
information provided by these sources improved our understanding of the impact of chromosomal disorders on human
reproduction and contributed to developing a comprehensive
model for natural selection against abnormal embryos (Plachot
et al., 1988). However, the interpretation of data obtained from
direct germ cell analyses is accompanied by some uncertainty
and reservations. For instance, the high rate of structural
aberrations in sperm chromosomes has been considered to be
merely artefactual (Jacobs, 1992). Among other factors, the
hamster egg cytoplasm has been suspected to induce an
incorrect ‘processing’ of human sperm chromatin. Consequently, conclusions on the reproductive relevance of the
observed chromosome alterations remain questionable (Rosenbusch, 1995). In contrast, the compact appearance of human
oocyte chromosomes hardly allows the recognition of structural
aberrations. Furthermore, pooling the data from unfertilized
oocytes has been criticized because this would be based on two
unproven assumptions: (i) that the chromosome constitution of
the oocyte does not affect its fertilization capacity and (ii)
that there is no difference between oocytes unfertilized after
insemination with either high or low quality sperm
(Ben-Shlomo, 1990).
These shortcomings prompted us to look for additional
information on the chromosome constitution of human gametes.
As suggested earlier (Rosenbusch, 1995), multipronuclear
zygotes obtained from IVF programmes might be studied
cytogenetically before cleavage. Their incidence is ~5–7% of
all fertilized oocytes (Pieters et al., 1992; Plachot and Crozet,
1992; Yie et al., 1996), the majority being triploid. Polyploidy
mostly results in spontaneous abortion (Jacobs, 1992) but birth
of tetraploid and long survival of triploid children have been
reported (Pitt et al., 1981; Sherard et al., 1986). These live
births are characterized by severe malformations and multiple
anomalies. Therefore, multipronuclear zygotes are excluded
from replacement in IVF programmes. Their analysis, however,
seems to offer some advantages in view of the criticism on
germ cell studies listed above: (i) the gametes involved can
undergo fertilization in vitro (Rudak et al., 1984); (ii) the
shape of oocyte chromosomes will be improved; and (iii)
condensation of sperm chromosomes takes place in homologous cytoplasm.
Information on the cytogenetic constitution of uncleaved
polyploid zygotes has remained rather scarce (Rudak et al.,
1984, 1985; Macas et al., 1988; Martin-Pont et al., 1991;
Pieters et al., 1992; Ma et al., 1995; Macas et al., 1996) and
a detailed analysis was not always performed. Here, we present
the karyotypes of 59 multipronuclear zygotes which reveal a
variety of chromosomal abnormalities occurring at the earliest
stage of conception.
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B.Rosenbusch, M.Schneider and K.Sterzik
Materials and methods
This study includes couples with female factor infertility treated
with a standard IVF procedure. Ejaculates were normozoospermic
according to WHO apart from a few cases with increased abnormal
sperm morphology. The majority of the women received human
menopausal gonadotrophin (HMG) after pituitary suppression with a
gonadotrophin-releasing hormone agonist (GnRHa, Suprecur;
Hoechst, Frankfurt am Main, Germany). In some cases, follicular
growth was induced with HMG and clomiphene citrate (Sterzik et al.,
1988). Oocytes were retrieved transvaginally under ultrasonographic
guidance 36 h following administration of human chorionic gonadotrophin (HCG). Oocyte–cumulus complexes were incubated for 4–6 h
in Ham’s F-10 medium (Biochrom, Berlin, Germany) supplemented
with 2% human serum albumin (HSA) and then inseminated with
motile spermatozoa prepared by the swim-up technique or a twolayer Percoll gradient centrifugation. The number of motile sperm
used for insemination varied between 50 000 and 100 000 per oocyte.
About 18–20 h after insemination the cumulus cells were removed
mechanically to assess fertilization. Presumed multipronuclear zygotes
were examined under the phase contrast microscope to exclude the
presence of cytoplasmic vacuoles (‘pseudo-pronuclei’; Van Blerkom
et al., 1987) and to determine the number of polar bodies. Cells with
three or four pronuclei were transferred to 1.5 ml of culture medium
supplemented with podophyllotoxin and vinblastine (Sigma, St Louis,
MO, USA) at a final concentration of 0.15 µg/ml each. The zygotes
were incubated for 24–28 h and then treated with 1% sodium citrate
in distilled water containing 2% HSA for 15 min. Fixation was
accomplished according to the gradual fixation/air-dry technique by
Mikamo and Kamiguchi (1983). The preparations were stained with
Giemsa and karyotypes were established from photographs taken at
31000 magnification. Zygotes with insufficient chromosome
spreading or excessive chromosome scattering were excluded from
analysis.
The present report is part of our project entitled ‘Cytogenetic
analysis of unfertilized or abnormally fertilized human oocytes after
in-vitro fertilization’ which has been approved by the ethics committee
of the University of Ulm. Consent was obtained from the patients
before starting a treatment cycle.
Results
Out of 77 multipronuclear zygotes which were processed for
cytogenetic analysis, 18 preparations (16 triploids and two
tetraploids) could not be evaluated because of the following
reasons: (i) no metaphase chromosomes present (three cells),
(ii) insufficient chromosome spreading (13 cells), (iii) excessive
chromosome scattering due to presumed cell rupture during
fixation (two cells). In total, 51 tripronuclear zygotes and eight
cells with four pronuclei were informative, giving a success
rate of karyotyping of 76.6%. These cells were obtained from
40 women with a mean age of 32.7 years (range 24–42). The
mean corresponding age of the husbands was 35.3 years (range
25–44). During one and the same treatment cycle, 27 patients
provided one zygote, seven patients provided two and two
patients provided three cells. In one case, four abnormally
fertilized oocytes were detected. The remaining eight cells
were obtained on different occasions: two patients provided
two and one patient provided a total of four zygotes during
two consecutive cycles. The rate of polyspermic fertilization
during the study period was 6.3% of all fertilized eggs, which
2258
was within the range reported by most other authors (5–7%,
see above).
Tripronuclear zygotes
The majority of these cells (39/51 5 76.5%) revealed two
polar bodies. Nine zygotes showed one and one cell had no
detectable polar body. Two zygotes could not be evaluated
unambiguously because the determination was impaired by
large numbers of attached spermatozoa and remaining cumulus
cells. In order to prevent the zygotes from being damaged,
further mechanical treatment was discontinued.
When compiling the cytogenetic results (Table I), the varying
arrangement of the chromosome sets was taken into consideration. Eighteen zygotes showed three separate haploid metaphases (distribution pattern n/n/n), 16 cells had one haploid and
one diploid chromosome set (n/2n), and in 15 zygotes the
individual sets were not distinguishable (3n, Figure 1). Two
zygotes with three pronuclei were in fact tetraploid, the
distribution of metaphases on the slide being n/3n and n/n/2n,
respectively. The sex chromosome ratio in the tripronuclear
zygotes (XXX:XXY:XYY) was 14:16:18, excluding the two
tetraploid cells and one zygote with a 23,X/23,X/22,-C or -Y
karyotype. Chromosomal abnormalities were found in 16
zygotes (31.4%) and included numerical (six cells), structural
(four cells) as well as combinations of numerical and structural
aberrations (six cells). Six zygotes were hypertriploid, five
were hypotriploid, and one cell had an additional C-group
chromosome but three missing chromosomes from other
groups. Chromosome breaks (chrb) appeared on the chromosome arms, e.g. chrb(2)(q), or in the centromeric region, e.g.
chrb(16)(cen). Further anomalies included acentric fragments
(ace) of unknown origin, dicentric chromosomes (dic), and a
chromatid exchange (chte). In one case, chromosome no. 2
had an elongated arm (q1) but it could not be determined
whether this was a product of translocation. In three separate
metaphases the origin of the abnormal pronucleus could be
ascribed to the spermatozoon due to the presence of a Y
chromosome.
Zygotes with four pronuclei
Six cells were characterized by the presence of two polar
bodies. No polar body was detected in one case. Another
zygote which was found to be pentaploid after fixation had
only one polar body. Cytogenetic analysis revealed that four
zygotes (50%) had chromosomal abnormalities (Table II)
which included supernumerary and missing chromosomes, an
acentric fragment (ace) and two chromosome breaks (chrb) in
the centromeric region (cen). One cell was characterized by
an increase in length of the short arms of G-group chromosomes
(Gp1) which has been considered a normal chromosome
variant (Vogel and Motulsky, 1996). Two abnormal pronuclei
could be attributed to spermatozoa due to the presence of a
Y chromosome (Figure 2). Excluding two cells with one
uninterpretable metaphase and a 22,-C or -Y karyotype, respectively, the sex chromosome distribution in the zygotes with
four pronuclei (XXXX:XXXY:XXYY:XYYY) was 1:1:2:1.
Chromosomes of multipronuclear zygotes
Table I. Cytogenetic analysis of 51 tripronuclear zygotes
Chromosome distribution
Normal (no. of cells)
Abnormal
Karyotype: 3 PN – 3n
3n
69,XXX (1)
69,XXX,chrb(16)(cen)
70,XXX,chrb(3),1D
71,XXX,chrb(1),chrb(2)(q),12C
69,XXY (2)
69,XYY (5)
n/2n
23,X/46,XX (2)
23,X/46,XY
23,X/46,YY
23,Y/46,XX
23,Y/46,XY
n/n/n
(2)
(1)
(4)
(1)
23,X/23,X/23,X (5)
23,X/23,X/23,Y (5)
69,XYY,chrb(2)(cen)
70,XYY,1F,ace
70,XYY,chrb(2)(q),1E
76,XYY,13C,13E,1F
23,X/45,XX, -3
21,X,-1,-F/46,XX
23,X/44,X,dic?(C;C),dic?(C;E),ace
21,Y,chte(2;C):dic,tr,-B,-D/44,XX,2q-,-2G
23,Y/46,XY,chrb(C)(cen),2ace
22,Y,-E/46,XY
23,X/23,X/22,-C or -Y
23,X/21,X,-B,1C,-E,-G/23,Y,2q1
23,X/24,X,1E/23,Y
23,X/23,Y/23,Y (5)
Karyotype: 3 PN – 4n
n/3n
n/n/2n
23,X/69,XXX (1)
23,X/23,Y/46,YY (1)
PN 5 pronuclei; n 5 haploid chromosome set; chrb 5 chromosome break; cen 5 centromere; dic 5
dicentric chromosome; q1 5 elongated arm; chte 5 chromatid exchange; tr 5 triradial.
Figure 1. Chromosome spread and karyotype (3n 5 69,XYY) of a
triploid zygote.
Discussion
Approximately 50% of all spontaneous abortions carry chromosomal aberrations and polyploidy (10%) is one of the main
classes of abnormality (Jacobs, 1992). In about 80% of triploids
the additional chromosome set is of paternal origin. This socalled diandric triploidy mostly results from penetration of
two haploid spermatozoa into a metaphase II oocyte (dispermy)
whereas fertilization by a single diploid spermatozoon is
considered a rare event (Kaufman, 1991; Jacobs, 1992). In
accordance with normal monospermic fertilization, diandric
zygotes should be characterized by the presence of two
polar bodies (Dyban and Baranov, 1987). In contrast, digynic
triploids arise from union of a haploid spermatozoon with a
diploid oocyte. Binuclear ova can be found in various mammalian species including man (Kennedy and Donahue, 1969;
Sherrer et al., 1977) and have been suggested as one possibility
for producing diploid female gametes. Other conceivable
mechanisms are blockage of either the first or second meiotic
division, i.e. non-extrusion of the first or second polar body,
respectively (Dyban and Baranov, 1987). Accordingly, a maternal origin of the supernumerary chromosome set may be
presumed for nine (17%) of our triploid zygotes with a single
polar body. A comparable rate (12%) has been reported by
Balakier (1983). However, this author indicates the possibility
that the first polar body had already degenerated at the time
of inspection. The presence of only one, i.e. the second, polar
body would then lead to an incorrect classification for cells
that are actually diandric. A degenerative process could also
explain our observation of a triploid and a tetraploid zygote
with no obvious polar body. Moreover, the first polar body of
a digynic cell may divide or become fragmented, thus mimicking a diandric zygote. From this it becomes evident that
2259
B.Rosenbusch, M.Schneider and K.Sterzik
Table II. Cytogenetic analysis of eight zygotes with four pronuclei
Chromosome distribution
Normal (no. of cells)
Karyotype: 4 PN – 4n
4n
92,XXXY,chrb(G)(cen)
92,XXYY,chrb(2)(cen)
n/n/2n
23,X/23,Y/46,YY (1)
n/n/n/n
23,X/23,X/23,X/23,X (1)
23,X/23,X/23,Y/? (1)
Karyotype: II. 4 PN – 5n
n/4n
Abnormal
23,Y,1ace/25,Y,1B1C/50,XX,13,1D,1E,1G
23,X/23,X,Gp1/23,X,Gp1/22,-C or -Y,Gp1a
23,Y/92,XXYY (1)
PN 5 pronuclei; n 5 haploid chromosome set; ? 5 not analysable metaphase; chrb 5 chromosome break;
cen 5 centromere; ace 5 acentric fragment.
aGp1 has been considered a variant.
Figure 2. Abnormal karyotypes of a tetraploid zygote according to
their distribution on the slide (n/n/2n). Arrow head 5 acentric
fragment; asterisks 5 supernumerary chromosomes.
determination of the presence and number of polar bodies
provides no absolute certainty about the origin of polyploidy.
Possible mechanisms giving rise to tetraploid spontaneous
abortions include the fertilization of a digynic oocyte by either
two haploid spermatozoa or a diploid male gamete (Dyban
and Baranov, 1987) but inhibition of the first cleavage division
after monospermic fertilization of a normal metaphase II
oocyte appears to be the most important event (Jacobs, 1992).
2260
On the other hand, there is evidence for polyspermic fertilization of a normal oocyte by three independent haploid spermatozoa (Sheppard et al., 1982; Surti et al., 1986; Vejerslev et
al., 1987).
A variety of factors has been investigated in an attempt to
predict the occurrence of polyspermic zygotes but the findings
appear rather inconsistent. For instance, the increased incidence
of polyspermic fertilization in immature oocytes reported by
Van der Ven et al. (1985) could not be confirmed in other
studies. Instead, an equal or even higher proportion of polyploidy was found in mature and post-mature oocytes (Wentz
et al., 1983; Rudak et al., 1984; Diamond et al., 1985; Golan
et al., 1992; Yie et al., 1996). Most authors except Lowe et
al. (1988) and Plachot et al. (1988) failed to detect an
association between polyploidy and the type of ovarian stimulation. In a recent study by Yie et al. (1996), the rate of
polyploidy was not correlated with sperm concentration, the
percentages of normal morphology or progressive motility in
the insemination medium. This contradicts earlier observations
that higher sperm concentrations will increase the risk of
polyspermic fertilization (Van der Ven et al., 1985; Englert et
al., 1986). Furthermore, an influence of sperm quality has
been suggested by Plachot et al. (1988) and Dandekar et al.
(1990) because polyploidy was less common with male factor
infertility compared with other indications. We have also found
a low incidence of multipronuclear zygotes in cases of impaired
sperm quality even when insemination was performed in a
microdrop with high sperm density (unpublished observations).
From these discrepancies, we conclude that polyspermy is a
multifactorial event involving the functional competence of
both male and female gametes. As to the oocyte, an important
aspect may be an absent, incomplete or delayed release of
cortical granules and consequent impairment of the zona
reaction resulting in a failure to protect against multiple sperm
entry. Finally, it is conceivable that accidental fractures of the
zona pellucida created by syringe suction during follicular
aspiration (Lowe et al., 1988) will contribute to polyploidy.
This mechanism could be compared with partial zona dissection
for the alleviation of male infertility which is known for
causing an elevated rate of multipronuclear zygotes.
Kola et al. (1987) have shown that most tripronuclear
zygotes develop directly into 3-cell embryos which possess a
Chromosomes of multipronuclear zygotes
highly abnormal but not triploid chromosome constitution.
Nowadays, there is evidence that this happens in dispermic
zygotes because two centrosomes are introduced by the penetrating spermatozoa leading to the formation of a tripolar
spindle. The sperm centrosome controls the first mitotic
divisions after fertilization (Palermo et al., 1994; Asch et al.,
1995). The observation of diploid embryos arising from
tripronuclear zygotes has been explained by the extrusion of
a haploid nucleus (Kola et al., 1987; Pieters et al., 1992).
Thus, a dispermic zygote may develop normally if a male
pronucleus is extruded or it may produce a hydatidiform mole
in the case of exclusion of the female chromosome set.
A detailed classification of chromosome distributions after
cleavage divisions of tripronuclear zygotes has been provided
by Pieters et al. (1992). Confusion of a pronucleus with a
cytoplasmic vacuole may also lead to erroneous recording of
a diploid embryo arising from a tripronuclear zygote. Appearance of a pseudo-pronucleus (Van Blerkom et al., 1987) besides
two real pronuclei is conceivable in a hypertriploid zygote
with an endoreduplicated 46,XX chromosome set detected
during our cytogenetic investigations. This peculiar cell has
been described separately (Rosenbusch et al., 1997).
The distribution of sex chromosomes in multipronuclear
zygotes has been documented best by Pieters et al. (1992)
who found an XXX:XXY:XYY ratio of 8:11:5 in 24 triploid
cells. Assuming that triploidy is mostly caused by polyspermy,
this would correspond to the theoretical value of 1:2:1 (Pieters
et al., 1992). The ratio of 14:16:18 noted in our study contrasts
with these theoretical expectations because of an increase in
the XYY constitution. It remains to be clarified whether this
finding reflects an advantage for Y-bearing spermatozoa to
achieve fertilization. On the other hand, our data support the
assumption of an early selection of XYY triploids because
this karyotype appears at the zygote stage but is rarely
encountered in abortuses (Kaufman, 1991).
A review of 120 tetraploid spontaneous abortions indicated
that they either belonged to the XXXX or XXYY class with
a ratio of 75:45 (Sheppard et al., 1982). Later, two partial
moles with a triple paternal contribution and a 92,XXXY
karyotype have been reported (Surti et al., 1986). Vejerslev et
al. (1987) found one tetraploid specimen with a 91,XXXX,-4
karyotype and suggested fertilization by three haploid spermatozoa or by one haploid and one diploid sperm. Concerning
karyotyped tetraploid zygotes, few data are currently available.
Besides the XXXX and XXYY constitution, two cells with an
XXXY complement could be demonstrated (Rudak et al., 1984
and present study). Moreover, we observed one XYYY zygote
which had to be expected in the case of one maternal and
three paternal contributions.
Polyploidy per se represents a cytogenetic anomaly that may
be superimposed with numerical and/or structural chromosome
alterations originating from the gametes involved. All of the
previously reported abnormalities in uncleaved multipronuclear
zygotes have been numerical, leading to hypo- or hyperpolyploid chromosome complements (Rudak et al., 1984, 1985;
Macas et al., 1988, 1996; Martin-Pont et al., 1991; Pieters et al.,
1992). An interesting observation is the unexpected presence of
an additional haploid set, e.g. the tripronuclear zygotes that
are in fact tetraploid and those cells with four pronuclei that
turn out to be pentaploid. Possible explanations are that either
(i) one of the pronuclei arose from a diploid gamete or (ii)
another spermatozoon penetrated the oocyte during or after
the assessment of fertilization and the resulting pronucleus
escaped detection (Pieters et al., 1992). The only structural
chromosome aberrations have been reported by Rudak et al.
(1984) who found 33 chromosomes plus eight fragments after
the first cleavage division of a tripronuclear zygote. An analysis
of embryos derived from multipronuclear cells has also been
included in other studies (Macas et al., 1988; Martin-Pont et
al., 1991; Pieters et al., 1992; Ma et al., 1995). However, it
appears that the uncleaved zygote is the most suitable model
for the detection of structural alterations because anomalies
such as acentric fragments are at risk of being lost during
cell division (Rosenbusch, 1995). Using a reliable fixation
technique is another important prerequisite to prevent loss of
chromosomes or chromosome fragments. In this way, we were
able to demonstrate the occurrence of chromosome breaks,
acentric fragments, dicentrics, and a chromatid exchange
(Tables I and II). Since these abnormalities are frequently
encountered in sperm chromosomes examined after heterospecific penetration into zona-free hamster eggs (Rosenbusch and
Sterzik, 1994; Rosenbusch, 1995), we suppose a predominant
transmission by the male gametes. This view is supported by
the detection of abnormal zygotes with individually analysable
pronuclear metaphases carrying a Y chromosome such as the
23,Y,1ace complement shown in Figure 2. However, a few
unstable chromosome abnormalities such as chromatid gaps,
chromatid breaks, and fragments have been observed in
oocytes. Considering investigations employing the more reliable fixation technique by Mikamo and Kamiguchi (1983), the
mean incidence of structural abnormalities is between 2.8%
(Lim et al., 1995) and 4.9% (Kamiguchi et al., 1993). Thus,
a transmission into the zygote by the female gametes must be
taken into account.
The distinction between paternally and maternally transmitted chromosome aberrations is a critical point during cytogenetic studies of multipronuclear zygotes. We and previous
authors noted that the participating pronuclear chromosome
complements do not always appear as clearly separated entities
and, if so, the paternal origin can only be determined by the
presence of a Y chromosome. Moreover, an unambiguous
distinction of all metaphases, e.g. in a tripronuclear zygote,
will only be possible in the case of a n,X/n,Y/n,Y distribution
pattern. Even then, chromosome drift during slide preparation
cannot be excluded with absolute certainty. This may cause
an incorrect classification of the parental origin of chromosomal
alterations. Nevertheless, there are possibilities of calculating
at least the minimum incidence of aberrations per gamete. Let
us make a simplification and assume that all tripronuclear
zygotes (excluding the two tetraploid cells) are dispermic. We
have analysed 49 zygotes and therefore 4933 5 147 haploid
metaphases. Let us further assume that the aberrations in nonseparated metaphases have been transmitted by only one
gamete. Then we arrive at a mean incidence of 18/147 5
12.2% abnormal (male 1 female) germ cells. Rudak et al.
(1985), who did not describe structural abnormalities, reported
2261
B.Rosenbusch, M.Schneider and K.Sterzik
a minimum level of aneuploidy of 16.7% without specifying
parental origin. It is evident that more data on individual
pronuclei are still needed to trace the origin of chromosome
aberrations back to the male or female gametes.
On the other hand, multipronuclear zygotes represent a
valuable model to assess the occurrence of cytogenetic abnormalities at the earliest stage of conception that avoids ethical
problems arising from investigations of cleaved embryos.
Furthermore, its usefulness to detect possible irregularities in
chromosome segregation after intracytoplasmic sperm injection
has been shown recently (Macas et al., 1996; Rosenbusch and
Sterzik, 1996).
References
Asch, R., Simerly, C., Ord, T. et al. (1995) The stages at which human
fertilization arrests: microtubule and chromosome configurations in
inseminated oocytes which failed to complete fertilization and development
in humans. Mol. Hum. Reprod., 1, Hum. Reprod., 10, 1897–1906.
Balakier, H. (1993) Tripronuclear human zygotes: the first cell cycle and
subsequent development. Hum. Reprod., 8, 1892–1897.
Ben-Shlomo, I. (1990) Oocyte karyotyping. [Letter.] Fertil. Steril., 53,
954–955.
Dandekar, P.V., Martin, M.C. and Glass, R.H. (1990) Polypronuclear embryos
after in vitro fertilization. Fertil. Steril., 53, 510–514.
Diamond, M.P., Rogers, B.J., Webster, B.W. et al. (1985) Polyspermy: effect
of varying stimulation protocols and inseminating sperm concentrations.
Fertil. Steril., 43, 777–780.
Dyban, A.P. and Baranov, V.S. (1987) Cytogenetics of Mammalian Embryonic
Development. Clarendon Press, Oxford, pp. 40–67.
Englert, Y., Puissant, F., Camus, M. et al. (1986) Factors leading to
tripronucleate eggs during human in-vitro fertilization. Hum. Reprod., 1,
117–119.
Golan, A., Nachum, H., Herman, A. et al. (1992) Increased fertilization and
pregnancy rate in polypronuclear fertilization cycles in in vitro fertilization–
embryo transfer. Fertil. Steril., 57, 139–142.
Jacobs, P.A. (1992) The chromosome complement of human gametes. In
Milligan, S.R. (ed.), Oxford Reviews of Reproductive Biology, Vol. 14.
Oxford University Press, Oxford, pp. 47–72.
Kamiguchi, Y., Rosenbusch, B., Sterzik, K. and Mikamo, K. (1993)
Chromosomal analysis of unfertilized human oocytes prepared by a gradual
fixation–air drying method. Hum. Genet., 90, 533–541.
Kaufman, M.H. (1991) New insights into triploidy and tetraploidy, from an
analysis of model systems for these conditions. Hum. Reprod., 6, 8–16.
Kennedy, J.F. and Donahue, R.P. (1969) Binucleate human oocytes from large
follicles. Lancet, April 12, 754–755.
Kola, I., Trounson, A., Dawson, G. and Rogers, P. (1987) Tripronuclear human
oocytes: altered cleavage patterns and subsequent karyotypic analysis of
embryos. Biol. Reprod., 37, 395–401.
Lim, A.S.T., Ho, A.T.N. and Tsakok, M.F.H. (1995) Chromosomes of oocytes
failing in-vitro fertilization. Hum. Reprod., 10, 2570–2575.
Lowe, B., Osborn, J.C., Fothergill, D.J. and Lieberman, B.A. (1988) Factors
associated with accidental fractures of the zona pellucida and
multipronuclear human oocytes following in-vitro fertilization. Hum.
Reprod., 3, 901–904.
Ma, S., Kalousek, D.K., Yuen, B.H. and Moon, Y.S. (1995) The chromosome
pattern of embryos derived from tripronuclear zygotes studied by cytogenetic
analysis and fluorescence in situ hybridization. Fertil. Steril., 63, 1246–1250.
Macas, E., Suchanek, E., Grizelj, V. et al. (1988) Chromosomal preparations
of human triploid zygotes and embryos fertilized in vitro. Eur. J. Obstet.
Gynecol. Reprod. Biol., 29, 299–304.
Macas, E., Imthurn, B., Roselli, M. and Keller, P.J. (1996) The chromosomal
complements of multipronuclear human zygotes resulting from
intracytoplasmic sperm injection. Hum. Reprod., 11, 2496–2501.
Martin-Pont, B., Selva, J., Bergere, M. et al. (1991) Chromosome analysis of
multipronuclear human oocytes after in vitro fertilization. Prenat. Diagn.,
11, 501–507.
Mikamo, K. and Kamiguchi, Y. (1983) A new assessment system for
chromosomal mutagenicity using oocytes and early zygotes of the chinese
2262
hamster. In Ishihara, T. and Sasaki, M.S. (eds), Radiation-induced
Chromosome Damage in Man. Alan R.Liss, Inc., New York, pp. 411–432.
Palermo, G., Munné, S. and Cohen, J. (1994) The human zygote inherits its
mitotic potential from the male gamete. Hum. Reprod., 9, 1220–1225.
Pieters, M.H.E.C., Dumoulin, J.C.M., Ignoul-Vanvuchelen, R.C.M. et al.
(1992) Triploidy after in vitro fertilization: cytogenetic analysis of human
zygotes and embryos. J. Assist. Reprod. Genet., 9, 68–76.
Pitt, D., Leversha, M., Sinfield, C. et al. (1981) Tetraploidy in a liveborn
infant with spina bifida and other anomalies. J. Med. Genet., 18, 309–311.
Plachot, M. and Crozet, N. (1992) Fertilization abnormalities in human invitro fertilization. Hum. Reprod., 7 (Suppl. 1), 89–94.
Plachot, M., Veiga, A., Montagut, J. et al. (1988) Are clinical and biological
IVF parameters correlated with chromosomal disorders in early life: a
multicentric study. Hum. Reprod., 3, 627–635.
Rosenbusch, B. (1995) Cytogenetics of human spermatozoa: what about the
reproductive relevance of structural chromosome aberrations? J. Assist.
Reprod. Genet., 12, 375–383.
Rosenbusch, B. and Sterzik, K. (1994) Cytogenetics of human spermatozoa.
The frequency of various chromosome aberrations and their relationship to
clinical and biological parameters. Arch. Gynecol. Obstet., 255, 81–89.
Rosenbusch, B. and Sterzik, K. (1996) Irregular chromosome segregation
following ICSI? [Letter.] Hum. Reprod., 11, 2337–2338.
Rosenbusch, B., Schneider, M. and Sterzik, K. (1997) Triploidy caused by
endoreduplication in a human zygote obtained after in-vitro fertilization.
Hum. Reprod, 12, 1059–1061.
Rudak, E., Dor, J., Mashiach, S. et al. (1984) Chromosome analysis of
multipronuclear human oocytes fertilized in vitro. Fertil. Steril., 41, 538–545.
Rudak, E., Dor, J., Mashiach, S. et al. (1985) Chromosome analysis of human
oocytes and embryos fertilized in vitro. Ann. NY Acad. Sci., 442, 476–486.
Sheppard, D.M., Fisher, R.A., Lawler, S.D. and Povey, S. (1982) Tetraploid
conceptus with three paternal contributions. Hum. Genet., 62, 371–374.
Sherard, J., Bean, C., Bove, B. et al. (1986) Long survival in a 69,XXY
triploid male. Am. J. Med. Genet., 25, 307–312.
Sherrer, C.W., Gerson, B. and Woodruff, J.D. (1977) The incidence and
significance of polynuclear follicles. Am. J. Obstet. Gynecol., 128, 6–12.
Sterzik, K., Dallenbach, C., Schneider, V. et al. (1988) In vitro fertilization:
the degree of endometrial insufficiency varies with the type of ovarian
stimulation. Fertil. Steril., 50, 457–462.
Surti, U., Szulman, A.E., Wagner, K. et al. (1986) Tetraploid partial
hydatidiform moles: two cases with a triple paternal contribution and a
92,XXXY karyotype. Hum. Genet., 72, 15–21.
Van Blerkom, J., Bell, H. and Henry, G. (1987) The occurrence, recognition
and developmental fate of pseudo-multipronuclear eggs after in-vitro
fertilization of human oocytes. Hum. Reprod., 2, 217–225.
Van der Ven, H.H., Al-Hasani, S., Diedrich, K. et al. (1985) Polyspermy in
in vitro fertilization of human oocytes: frequency and possible causes. Ann.
NY Acad. Sci., 442, 88–95.
Vejerslev, L.O., Dissing, J., Hansen, H.E. and Poulsen, H. (1987) Hydatidiform
mole: genetic origin in polyploid conceptuses. Hum. Genet., 76, 11–19.
Vogel, F. and Motulsky, A.G. (eds) (1996) Human Genetics. Problems and
Approaches, 3rd edn. Springer, Berlin, pp. 31–37.
Wentz, A.C., Repp, J.E., Maxson, W.S. et al. (1983) The problem of
polyspermy in in vitro fertilization. Fertil. Steril., 40, 748–754.
Yie, S.-M., Collins, J.A., Daya, S. et al. (1996) Polyploidy and failed
fertilization in in-vitro fertilization are related to patient’s age and gamete
quality. Hum. Reprod., 11, 614–617.
Received on March 31, 1997; accepted on July 8, 1997