1 CReATe Program, Inc. and 2 Department of Obstetrics and Gynecology, Sunnybrook and Women's College Health Sciences Center, University of Toronto, Toronto and the 3 University Health Network and Department of Laboratory Medicine and Pathobiology, and Medical Biophysics, Faculty of Medicine, University of Toronto, Toronto, Canada
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Abstract |
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Key words: digynic triploidy/digyny/diploid oocytes/human triploidy/ploidy
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Introduction |
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The recovery of sporadic giant oocytes appears to be possible after hormonal stimulation for human assisted reproductive technologies. The occurrence of few individual cases of giant binucleated immature or mature oocytes with double sets of chromosomes has been described recently (Eichenlaub-Ritter et al., 1988; Mahadevan et al., 1988
; Lim et al., 1995
; Rosenbusch and Schneider, 1998
; Veeck, 1999
). Nevertheless, there is limited knowledge concerning the developmental capability, cytogenetics and chromosomal complements of human giant cells. In this study, we took an advantage of a unique opportunity to collect a number of giant oocytes and zygotes during human IVF procedures. The objective of this study was to examine the morphology and numerical chromosomal constitution of such abnormal cells during their maturation and early development.
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Materials and methods |
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Giant oocytes and embryos
Giant fertilized and unfertilized oocytes were identified either during the fertilization assessment of in-vitro inseminated oocytes or during a course of cumulus cell removal just before the ICSI procedure. The abnormally large oocytes were examined for the presence and number of the nuclei and polar bodies under an inverted microscope at x320 magnification (Leitz DMIL, relief contrast; Leica, Richmont Hill, Ontario, Canada). The diameters of giant oocytes and their normal siblings, serving as a control, were also measured. Additionally the volumes of cells were calculated according to the formula; V = (4/3)r3 (results in microns3, µm3). Patients consented to the chromosomal investigation of their giant cells. When the consents were not obtained, the abnormal oocytes of those patients were not submitted for further analysis.
Chromosomal preparations of unfertilized giant oocytes were performed usually 5 h and in some cases 24 h after ovum retrieval, according to a published method (Kamiguchi et al., 1993). The zona pellucida was removed by acid Tyrode solution (pH 2; Sigma) and the oocytes were treated with hypotonic sodium citrate (1%), followed by gradual fixation and staining with Giemsa.
Two-colour interphase fluorescence in-situ hybridization (FISH) analysis was applied to investigate the ploidy of embryos derived from giant zygotes. The fixation and FISH techniques used were as described previously (Liu et al., 1998). Two commercial probe sets were used to evaluate the genetic constitution of giant embryos. The first probe set (Ventana Medical Systems, Tucson, AZ, USA) was a cocktail that hybridizes with the ABL, gene on chromosome 9 at band 9q34 (green fluorescent) and also hybridizes with the BCR gene on chromosome 22 at band 22q11.2 (red fluorescence). The second probe set used to evaluate the sex chromosome constitution of the embryos was a commercially available cocktail probe (Vysis, Inc., Downers Grove, IL, USA) that hybridizes with the centromeric region of chromosome Y (green fluorescence) and with the centromeric region of chromosome X (red fluorescence). The slides were denatured at 70°C in 70% formamide/2xSSC for 515 s and hybridized overnight. Then they were washed in 0.4xstandard saline citrate (SSC)/0.3% NP-40 at 70°C for 2 min followed by 2xSSC/0.1% NP-40 at room temperature for 1 min and subsequently counterstained with 4,6-diamino-2-phenylindole (1.5 mg/ml). The probe sets were applied sequentially. Slides were evaluated on an epifluorescence microscope (Zeiss; Don Mills, Ontario, Canada) with software and hardware provided by Applied Imagining (Santa Clara, CA, USA). Hybridization sensitivities with all four probes demonstrated an excellent signal:noise ratio, low background and an efficiency of >98% using normal control lymphocyte preparations.
Statistical analysis
Statistical analysis was performed using two-sample t-test to compare the diameters of giant and control, normal oocytes. Two-way ANOVA was also applied to evaluate the significant differences in the estradiol (E2) levels and the number of retrieved eggs between the patients that possessed and those that did not have giant oocytes. P < 0.05 was considered statistically significant.
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Results |
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Table II presents the stage of chromosomal constitution of giant oocytes at retrieval, and after fertilization/in-vitro culture. Among 29 unfertilized giant oocytes, 12 were immature and had either two germinal vesicles (GV; n = 7; Figure 1A
) or were at metaphase of the first meiotic division (MI; n = 5) and did not display any nuclei and polar bodies. The remaining 17 giant cells were classified as mature oocytes that were arrested at the second meiotic division (MII). Seven of them possessed one polar body, which is considered to be normal, and the others had two polar bodies (Figure 1B, C
). The polar body size was similar to those observed in the control mature oocytes. Chromosomal preparations from four MI oocytes indicated the presence of two separate metaphase plates in each oocyte. In two cases, individual plates contained haploid number of 23 chromosomes, and in two others the chromosomes were compact and because of overlaps accurate numerical counts were not possible. Analysis of four MII mature oocytes that contained only one polar body revealed the presence of a single diploid metaphase plate comprising 46 chromosomes (Figure 2A
). Among seven, MII oocytes displaying two polar bodies, four oocytes showed double sets of 23 chromosomes (Figure 2B
), one oocyte had two compact groups of chromatin, and two oocytes contained a common group with 46 chromosomes.
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Discussion |
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Our findings also revealed the existence of two different morphological types of human giant unfertilized and fertilized oocytes. All giant, immature GV oocytes were binucleated and MI/MII oocytes appeared to be diploid. About half of MII oocytes contained a single, diploid group of chromosomes while another half displayed two separate haploid sets of chromosomes, resulting in a diploid state as well. Consequently, giant fertilized oocytes, obtained after standard in-vitro insemination, showed normal or typical polyploid number of pronuclei. In the first case, giant zygotes resembled normal ones with two pronuclei and two polar bodies. In contrast, polyploid types of zygotes contained three or four pronuclei and four or two polar bodies. Thus, this indicates that giant zygotes showing normal nuclear morphology derive from fertilization of giant MII oocytes with a single set of chromosomes, whereas atypical polyploidy zygotes are formed due to monospermic or dispermic fertilization of giant oocytes possessing either one (3PN, 2PB, dispermic) or two (3 or 4PN, 4PB) groups of meiotic chromosomes. Interestingly, cytogenetic studies on hamster have shown that although both mononuclear and binuclear giant immature eggs can be found, the binucleate state is rarely maintained to MII (Funaki and Mikamo, 1980; Funaki, 1981
). For this reason, the majority of hamster giant MII oocytes contain a diploid set of chromosomes arranged in a single spindle and accordingly most giant zygotes exhibit a normal number of pronuclei comprising one female and one male pronucleus. This behaviour contrasts with that of human giant oocytes in which both chromosomal anomalies occurred with similar frequency while binuclear germinal vesicle oocytes were exclusively observed. Interestingly, mouse giant oocytes produced by cell fusion (Karnikova et al., 2000
) revealed distribution patterns of metaphase plates and polar bodies (one or two plates and, respectively, one or two polar bodies) similar to our observations in human giant oocytes.
In this study, human giant oocytes demonstrated full viability during the preimplantation period. Fertilization was compromised (15 giant oocytes from IVF procedure) and thereafter giant zygotes cleaved normally and some were capable of reaching blastocyst stage (Figure 3). On the other hand, giant embryos showed numerical chromosomal abnormalities and FISH analysis suggested the presence of triploid or tetraploid nuclei as well as different types of mosaics. It should be noted that, in a previous report on humans, four abnormally large embryos were identified as triploid or triploid mosaic by means of FISH involving chromosomes 18, X and Y, and it was concluded that these embryos most likely originated from diploid oocytes (Munné et al., 1994
). Indeed, based on our observations, it is evident that those atypical embryos could develop from giant zygotes containing 2PN, 2PB. The preimplantation development of giant oocytes into triploid 4-cell or blastocyst stage embryos has also been described in the past in hamster and rat (Funaki, 1981
). Taking into consideration the obvious chromosomal abnormalities present in giant oocytes and embryos, we conclude that all giant cells obtained during IVF procedures should be excluded from uterine transfers.
Although there is no a single proven human digyny originated in vivo from giant oocytes, the contribution of diploid oocytes to human triploidy cannot be completely excluded. First, giant oocytes exist in human ovaries and show full developmental capability when retrieved and cultured in vitro. Second, in contrast to early studies that were based on the analysis of cytogenetic heteromorphisms (Kajii and Nikawa, 1977; Jacobs et al., 1982; Procter et al., 1984
; Uchida and Freeman, 1985
), several recent reports on the aetiology of human aborted material have revealed that digyny rather than diandry is the most common cause of human triploidy (Dietzsch et al., 1995
; Miny et al., 1995
; Baumer et al., 2000
; McFadden and Langlois, 2000
). However, in the light of the complex allelic segregation patterns possible for giant oocytes, it is apparent that to distinguish between triploids caused by meiotic failure and those derived from giant diploid gametes would require comprehensive analysis with various genetic markers. Otherwise triploids could be mistakenly interpreted as suppression of the first or second polar bodies because a similar distribution of maternal alleles could take place. The investigation of the involvement of giant diploid oocytes in the development of human triploids remains an intriguing challenge for future genetic studies.
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Acknowledgements |
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Notes |
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4 To whom correspondence should be addressed at: CReATe (Canadian Reproductive Assisted Technology) Program, Inc., 790 Bay Street, suite 1020, Toronto, Ontario M5G 1N8, Canada. E-mail: hbalakier{at}sympatico.ca
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Submitted on February 8, 2002; accepted on May 10, 2002.