Human Reproduction vol.13 no.6 pp.1653–1655, 1998
CASE REPORT
Maturation of a binuclear oocyte from the germinal
vesicle stage to metaphase II: formation of two polar
bodies and two haploid chromosome sets
B.Rosenbusch1,3 and M.Schneider2
1Department
of Gynecology and Obstetrics, University of Ulm,
Prittwitzstrasse 43, 89075 Ulm and 2Laboratory for Medical
Genetics Dr. Tettenborn, Neue Strasse 40, 89073 Ulm, Germany
3To
whom correspondence should be addressed
We report on a binuclear human oocyte that underwent
maturation in vitro from germinal vesicle stage to metaphase II. Following extrusion of two polar bodies, the oocyte
was processed for cytogenetic analysis which revealed two
separate haploid chromosome sets accompanied by the
corresponding polar body chromatin. Tentatively established karyotypes were 23,X and 23,X,ace, respectively.
This condition could have resulted in a tripronuclear
digynic zygote after monospermic fertilization.
Key words: binuclear oocyte/diploidy/germinal vesicle stage/
metaphase chromosomes
Introduction
Triploidy is one of the major chromosomal abnormalities
affecting the human with an incidence of ~1–3% of all
clinically recognized conceptions. Triploid embryos account
for ~15% of spontaneous abortions carrying a chromosomal
aberration (Dyban and Baranov, 1987; Kaufman, 1991). The
birth of a triploid child is an exceptional event and only a few
cases of long survival have been documented (Sherard et al.,
1986). These live births are characterized by multiple anomalies
and malformations.
In at least 75% of human triploids the additional chromosome
set is paternal in origin. This condition, termed diandric
triploidy, is almost exclusively caused by dispermy rather than
by fertilization of a normal oocyte by a diploid spermatozoon
(Kaufman, 1991; Jacobs, 1992). Digynic triploidy, i.e. the
presence of an additional maternal chromosome set, may result
from non-extrusion of either the first or second polar body
followed by monospermic fertilization. Another possible mechanism of origin is the fusion of a haploid spermatozoon with
a diploid ‘giant’ oocyte originating from meiotic division of a
mononuclear or binuclear giant egg (Dyban and Baranov,
1987). Though binucleate ova have repeatedly been found in
human ovaries (Kennedy and Donahue, 1969; Sherrer et al.,
1977; Gougeon, 1981), the probability that these follicles will
ovulate and eventually give rise to digynic triploids has been
considered to be very low (Gougeon, 1981). In fact, it was
not before 1988 that Mahadevan et al. reported on the recovery
© European Society for Human Reproduction and Embryology
of a binucleate oocyte which underwent germinal vesicle
(GV) breakdown in vitro. At the same time, two ‘probably
binucleated oocytes’ remaining unfertilized after in-vitro fertilization (IVF) were described by Eichenlaub-Ritter et al. (1988).
In these three cases a detailed evaluation of the chromosomal
constitution has not been possible.
Here, we wish to demonstrate the maturation of a binuclear
oocyte from the GV stage to metaphase II, characterized by
the extrusion of two individual polar bodies. Cytogenetic
analysis confirmed the presence of two separate haploid
metaphase chromosome sets. This condition could have
resulted in a tripronuclear digynic zygote after monospermic
fertilization.
Case report
The couple was admitted to our IVF programme because of
secondary sterility. The 34 year old woman had given birth to
a child in 1980 but had been unable to conceive again
since then. Gynaecological examination did not reveal any
abnormalities. Repeated semen analyses of her 39 year old
husband showed normal values for sperm concentration and
motility and a borderline level of normal sperm morphology
(35%). Between 1994 and 1996, the couple had unsuccessfully
undergone one gamete intra-Fallopian transfer (GIFT) and four
IVF trials. Because no fertilization could be achieved even
after microdrop insemination with high sperm concentration,
intracytoplasmic sperm injection (ICSI) seemed to be indicated
in the following cycles. The first procedure resulted in a
transient rise in β-human chorionic gonadotrophin (HCG) after
transfer of three embryos but no clinical pregnancy was
established. Likewise, no pregnancy was induced in the second
ICSI cycle following transfer of two embryos whereas on the
third occasion no oocytes could be aspirated.
During the fourth cycle, the patient was treated with
follicle stimulating hormone (FSH, Puregon; Organon,
Dublin, Ireland) after pituitary suppression with a gonadotrophin-releasing hormone agonist (GnRHa, Suprecur;
Hoechst, Frankfurt am Main, Germany). Serum oestradiol
concentration was 1284 pg/ml on the day of injection of
HCG. Two follicles were aspirated transvaginally under
ultrasonographic guidance 36 h after HCG administration.
Each sample of follicular fluid contained one oocyte–
cumulus complex. The cumulus cells were removed by short
incubation (20–30 s) in HEPES-buffered Ham’s F-10 medium
(Biochrom AG, Berlin, Germany) containing 100 IU/ml
hyaluronidase and by aspiration into finely drawn Pasteur
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B.Rosenbusch and M.Schneider
pipettes. The oocytes were then washed several times in
HEPES–Ham’s medium supplemented with 2% human
serum albumin (HSA; Immuno, Copenhagen, Denmark). An
inspection under the phase-contrast microscope revealed that
Figure 1. Maturation of a binuclear oocyte in vitro. (a) Two
germinal vesicles (each one indicated by two arrowheads) were
detected after removal of the cumulus cells. (b) About 27 h after
retrieval, the oocyte had extruded two individual polar bodies.
Their sites of origin are clearly visible as slight depressions in the
plasma membrane (arrowheads). Original magnification 3200.
both oocytes were in the GV stage. However, one of the
cells was ~1.5-fold larger than the other and contained two
nuclei (Figure 1a). The oocytes were incubated separately
in Ham’s F-10 1 2% HSA under mineral oil. Twenty-four
hours after retrieval the oocyte with one GV seemed to be
degenerated. In contrast, the binuclear oocyte had undergone
GV breakdown but a polar body could not yet be detected.
The oocyte was transferred into fresh culture medium and
incubated further. About 3 h later we noted the extrusion
of two individual polar bodies (Figure 1b). Since this
genetically abnormal cell could not be used for ICSI it
was prepared for chromosome analysis. Our cytogenetic
investigations on human gametes have been approved by
the ethical committee of the University of Ulm and consent
was obtained from the patients.
The oocyte was treated with 0.9% sodium citrate in distilled
water containing 3% fetal calf serum for 15 min. Fixation
was accomplished according to the gradual fixation/air-dry
technique recommended by Mikamo and Kamiguchi (1983).
The chromosomes were stained with Giemsa and photographed
at 31000 magnification. Two metaphase chromosome plates
each accompanied by polar body chromatin could be distinguished. Though the chromosomes were highly condensed and
compact, we tentatively established the corresponding karyotypes (Figure 2). One of them was apparently normal [23,X]
whereas the other revealed an acentric fragment [23,X,ace]
that possibly resulted from a chromatid break in the long arm
of the first C-group chromosome.
Discussion
Binuclear oocytes may result from fusion of two individual
oogonia or from nuclear division in an oogonium without
ensuing cytoplasmic cleavage (Austin, 1969). In the human,
binuclear cells were first predominantly seen in neonates and
prepubertal subjects. Therefore, their general fate seemed to
be loss by atresia during fetal and early neonatal life. A
binucleate oocyte surviving to maturation and becoming fertilized was considered a rare event (Austin, 1969). Later, it was
shown that multiovular follicles and multinuclear oocytes are
Figure 2. Tentative karyotypes of the two chromosome sets interpreted as [23,X] and [23,X,ace], respectively. The arrowhead indicates the
acentric fragment. The corresponding polar body chromatin (PBC) is shown on the right.
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Maturation of a binuclear oocyte
present in 98% of 18–52 year old women suggesting that they
can persist throughout the reproductive life span (Gougeon,
1981). By puncturing antral follicles in ovaries removed from
patients for gynaecological indications, Kennedy and Donahue
(1969) found two ova each containing two nuclei in the GV
stage. The authors surmised that binucleate oocytes may be
ovulated, and they further speculated: ‘If each nucleus matured
normally to metaphase II, fertilisation could stimulate release
of a second polar body by each nucleus, and a triploid zygote
would result.’ However, it took almost another 10 years until
Mahadevan et al. (1988) reported on the recovery of a preovulatory binucleate oocyte after induction of ovulation for
IVF. As stated in their communication, this was the first
binuclear oocyte observed to undergo GV breakdown but
extrusion of a polar body did not occur. Subsequent cytogenetic
analysis led to the conclusion that the karyotype contained
.80 chromosomes. In the same year, Eichenlaub-Ritter et al.
(1988) published investigations of unfertilized IVF oocytes
that had been processed for indirect anti-tubulin immunofluorescence. The authors noted two diploid oocytes both containing two sets of chromosomes, each of which was surrounded
by a degenerating spindle. These cells were described as
probably binucleated but their development in vitro, i.e. GV
breakdown and polar body extrusion, could not be monitored
because they had been cultured and inseminated as intact
oocyte-cumulus complexes.
In the present case, we were able to document the in-vitro
maturation of a human binuclear oocyte from the GV stage to
metaphase II. As confirmed by the extrusion of two polar
bodies and subsequent karyotyping, the binuclear state had
been maintained during this time. This behaviour appears
exceptional because it has been assumed that binucleate eggs
usually become mononucleate before chromosome segregation
begins (Dyban and Baranov, 1987). Cytogenetic studies of
giant secondary oocytes from the Chinese hamster revealed
that 17 out of 18 cells had 22 dyads each in a single metaphase
plate (Funaki and Mikamo, 1980). Only one oocyte showed
two clearly separated haploid complements indicating the
retention of the binuclear state.
Once ovulated, binuclear giant Chinese hamster oocytes
were fertilized normally and were able to develop into digynic
triploid 2-cell embryos (Funaki and Mikamo, 1980). Later,
Funaki (1981) demonstrated that the triploid chromosome
constitution was maintained up to the blastocyst stage in the
Chinese hamster. Likewise, the maturation pattern observed
by us could have led to a digynic tripronuclear zygote and
finally to a triploid embryo in the case of monospermic
fertilization but we currently have no evidence for the fertilizability of giant binuclear oocytes in the human. Of note, the
two binucleate ova detected by Eichenlaub-Ritter et al. (1988)
might have remained unfertilized due to impaired sperm quality
because this reason applied to most (21/30) of the oocytes
included in their report.
References
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Received on January 5, 1998; accepted on March 23, 1998
Acknowledgements
We are grateful to Dr Volker Hanf for performing follicular aspiration.
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