Human Reproduction, Vol. 17, No. 7, 1892-1894,
July 2002
© 2002 European Society of Human Reproduction and Embryology
A human oocyte with two sets of MII/PB-structures: Case report
1 Department of Obstetrics and Gynaecology, Göteborg University, SU/Sahlgrenska, 13 45 Gothenburg, Sweden
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Abstract |
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This paper reports a case where a normal-sized human oocyte has been documented having two polar bodies and two metaphase spindles. This finding suggests that the chromosomal content in oocytes of normal size may occasionally be duplicated and offers an additional explanation for the origin of triploid zygotes in humans. We speculate that this duplication may be caused by oocyte fusion at an early stage.
Key words: digyny/oocyte/oocyte fusion/triploid
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Introduction |
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One phenomenon frequently observed in routine IVF/ICSI is triploid (3PN) zygotes (Plachot et al., 1987
; Sachs et al., 2000). The embryos developing from such zygotes are in general unable to support normal fetal development, although triploid embryos can develop up to the blastocyst stage (Lopata et al., 1997) and even to term. The main explanations for the occurrence of 3PN zygotes have been that their extra DNA content arises from the entry of either two haploid or one diploid spermatozoon, or that the oocyte fails to extrude the second polar body (PB). Here we report the observation of a of normal-sized human oocyte with two sets of MII/PB-structures that can offer an additional explanation for the origin of digynic 3PN zygotes, namely that the chromosomal content in oocytes also of normal size may occasionally be duplicated. |
Case report |
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The oocyte came from a 35 year-old woman who underwent IVF treatment at the Sahlgrenska University Hospital, Gothenburg, Sweden. The treatment indications were low sperm count with tubal factor and this was the second treatment cycle for the couple. Ovarian stimulation was carried out by a desensitizing protocol using a short-acting GnRH-agonist preparation in combination with recombinant FSH. Follicular aspiration was performed 38 h after HCG administration using vaginal ultrasonography and follicle puncture. Of the 12 aspirated oocytes 10 were metaphase II (MII) and two were germinal vesicle (GV) oocytes with a clear single GV nucleus and of normal size as compared with their sibling oocytes. The oocytes were treated with 80 IU/ml hyaluronidase for 30 s, followed by mechanical removal using a thin denudation pipette that removed most of the cumulus cells surrounding the oocyte. The GV oocytes were to be included in a study of cytoskeletal changes in in-vitro matured GV oocytes and were thus cultured further for 24 h in IVF-20 (Scandinavian IVF Science, Göteborg, Sweden) to observe PB extrusion. The oocyte was then fixed and attached to a glass slide and thereafter stained for both chromosomes and cytoskeletal proteins as previously described (Hardarson et al., 2000
). Briefly the oocyte was fixed and extracted in a formaldehyde-based buffer, washed and labelled with fluorescent antibodies specific for microtubules followed by chromosome identification by DAPI counterstaining. The oocyte was photographed using a Nikon epifluorescence microscope equipped with appropriate filters. Images were transferred via a video camera using a data-imaging program and stored in a computer.
The fluorescence visualization showed that the oocyte had two polar bodies and two metaphase spindles (Figure 1). The first set of MII/PB-structures was oriented perpendicular to the viewer (Figures 1 and 3, upper set) while the second set was on top of the oocyte facing towards the viewer. The sibling GV oocyte arrested at the GV stage and degenerated quickly. Nine of the ten MII oocytes were fertilized, seven showing 2PN and two showing 3PN at 20 h post-insemination. The embryos developed slowly and resulted in a day 3 transfer where only three embryos of 4–6 cells were available. Two embryos were transferred, not resulting in a pregnancy.
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Discussion |
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To our knowledge this is the first report of two sets of MII/PB-structures in a human oocyte of normal size. This finding suggests an additional explanation to the aetiology of three pronuclear zygotes aside from the traditional ones, i.e. failed PB extrusion (Flaherty et al., 1995
) or dispermy (Austin and Braden, 1953). If we hypothesize that both observed sets of MII/PB-structures have a normal chromosomal set-up, the chromosomal number had to already be doubled when meiosis was commenced during oogenesis. This means that the first meiosis was then normal, including two separate metaphase I spindles, each extruding its first PB and resulting in two normal separate sets of MII/PB-structures. The question then arises—how can an oocyte with a double set of chromosomes be created?
One mechanism might be the fusion of two primary oocytes (follicles) creating a single oocyte containing two germinal vesicles. After the migration of primordial germ cells to the ovary in early embryogenesis, it may be that two primary oocytes become surrounded by a common sheet of follicle cells, thus making the fusion of two oogonia possible. In our laboratory, two GV oocytes with a common zona pellucida have been observed (Figure 2) supporting the theory that one of the mechanisms behind a `double' oocyte could be fusion of two oocytes inside a single follicle. It is possible that the result of this oogonial fusion depends on where in the developmental progression the follicle/oocyte is, i.e. an oocyte of normal size will arise from fusion of two small, immature oocytes, at an early stage, while enlarged oocytes would develop from fusion at later developmental stages. Enlarged oocytes have been reported in human IVF and their chromosomal content cytologically determined as diploid (Rosenbusch et al., 2001). Such giant oocytes have also been observed in the hamster (Funaki and Mikamo, 1980). These giant oocytes occur infrequently and upon fertilization often produce digynic triploid zygotes. In contrast, the oocyte reported here was of normal size and had two separate sets of MII/PB, something that was not reported in the other studies.
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Another possible mechanism behind the creation of oocytes with diploid MII/PB-structures might be mitotic nuclear division at the oogonial stage without any cytoplasmic cleavage, something that should result in two GV structures. A parallel can be drawn with the occurrences of binucleate blastomeres in cleavage stage embryos, as strong evidence suggests that nuclear division, unaccompanied by cytoplasmic division is the main cause of binuclear blastomeres (Hardy et al., 1993
). However, in this study only a single GV was observed which is not consistent with this theory. The occurrence of two 3PN zygotes in the sibling oocytes from this patient suggests that there might be a common factor causing these observed abnormalties. An additional explanation for this observation that cannot be excluded is that the oocyte had gone through a normal meiosis and was actually haploid. In such cases the metaphase plates observed would be haploid with unreplicated chromosomes and hence the oocyte would be in a state of meiotic arrest. Since the images were obtained by DAPI-staining of the chromosomes it is very difficult to determine whether the DNA content of the spindle is normal or not.
To conclude, our finding shows that human MII oocytes can have two sets of PB/MII structures. Assuming that the chromosomal content of both PB/MII sets is diploid, it may offer an additional explanation for the origin of triploid zygotes in humans.
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Notes |
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1 To whom correspondence should be addressed. E-mail: thorir.hardarson{at}obgyn.gu.se
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References |
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Austin, C.R. and Braden, A.W.H. (1953) An investigation of polyspermy in the rat and rabbit. Aust. J. Biol. Sci., 6, 674–692.
Flaherty, S.P., Payne, D., Nicholas, J., Swann, M. and Colin. D. (1995) Aetiology of failed and abnormal fertilization after intracytoplasmic sperm injection. Hum. Reprod., 10, 2623–2629.
Funaki, K. and Mikamo, K. (1980) Giant diploid oocytes as a cause of digynic triploidy in mammals. Cytogenet. Cell Genet., 28, 158–168.[Web of Science][Medline]
Hardarson, T., Lundin, K. and Hamberger, L. (2000) The position of the metaphase II spindle cannot be predicted by the location of the first polar body in the human oocyte. Hum. Reprod., 15, 1372–1376.
Hardy, K., Winston, R.M. and Handyside, A.H. (1993) Binucleate blastomeres in preimplantation human embryos in vitro: failure of cytokinesis during early cleavage. J. Reprod. Fertil., 98, 549–558.
Lopata, A., Oliva, K., Stanton, P.G. and Robertson, D.M. (1997) Analysis of chorionic gonadotrophin secreted by cultured human blastocysts. Mol. Hum. Reprod., 3, 517–521.
Plachot, M., Junca, A.M., Mandelbaum, J., de Grouchy, J., Salat-Baroux, J. and Cohen, J. (1987) Chromosome investigations in early life. II. Human preimplantation embryos. Hum. Reprod., 2, 29–35.
Rosenbusch, B., Schneider, M., Kreienberg, R. and Brucker, C. (2001) Mechanisms leading to triploidy: the possible role of binucleate giant oocytes. Hum. Reprod. (Abstract Bk 1), 16, 183.
Sachs, A.R., Politch, J.A., Jackson, K.V., Racowsky, C., Hornstein, M. D. and Ginsburg, E. S. (2000) Factors associated with the formation of triploid zygotes after intracytoplasmic sperm injection. Fertil. Steril., 73, 1109–1114.[Web of Science][Medline]
Submitted on December 3, 2001; resubmitted on February 2, 2002; accepted on March 25, 2002.
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