Cytogenetic analysis of giant oocytes and zygotes to assess their relevance for the development of digynic triploidy*

B. Rosenbusch1,4, M. Schneider2, B. Gläser3 and C. Brucker1

1 Department of Gynecology and Obstetrics, University of Ulm, Prittwitzstrasse 43, D-89075 Ulm, 2 Gregor Mendel Laboratories, Wegenerstrasse 15, D-89231 Neu-Ulm and 3 Department of Human Genetics, Parkstrasse 11, D-89073 Ulm, Germany


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
BACKGROUND: In order to assess the role of binucleate giant oocytes for generating digynic triploidy, we studied their frequency, maturation patterns and chromosomal complements at metaphase II (MII) or after fertilization. METHODS: Uncleaved, giant zygotes were incubated with podophyllotoxin and vinblastine, treated with hypotonic solution and fixed by a gradual fixation method. Giant MII oocytes were directly subjected to hypotonic treatment. The chromosomes were stained with Giemsa. RESULTS: A total of 7065 oocytes were collected during the study period, of which 18 (0.26%) were classified as giant cells. When considering only those patients in whom giant cells were identified (among other normal sized cells) a giant cell frequency of 18/237 (7.6%) was found. Nine cells underwent a union of the nuclei during maturation to MII and four of them became fertilized showing two pronuclei. Seven oocytes maintained the binucleate state to MII and one of them was fertilized showing three pronuclei. Ten unfertilized cells were available for cytogenetic analysis and proved to be diploid. All five giant zygotes revealed triploidy. CONCLUSIONS: The data suggest that giant oocytes may play an important, yet underestimated role in causing digynic triploidy. We recommend the exclusion of giant oocytes from IVF trials and that giant cells should be discarded, even if they carry the regular number of two pronuclei.

Key words: binucleate oocytes/cytogenetic analysis/digyny/giant oocytes/triploidy


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Among chromosomal causes of human reproductive loss, triploidy is the third most prevalent with a frequency of ~15–18% of all abnormal, aborted fetuses (Dyban and Baranov, 1987Go). Approximately one in 10 000 triploid conceptions results in a live birth (Jacobs et al., 1982Go). Full triploids occasionally survive up to several months (Niemann-Seyde et al., 1993Go; Hasegawa et al., 1999Go) but they normally die soon after birth due to a variety of anomalies.

Depending on the origin of the supernumerary haploid chromosome set, diandric (two paternal contributions) and digynic triploidy (two maternal complements) can be distinguished. In general, digynic triploids survive longer than those with two paternal complements (Hasegawa et al., 1999Go). It has been suggested that the vast majority of triploids arises through diandry, with 66.4% being the result of dispermy and 23.6% being caused by a diploid spermatozoon. The remaining 10% were attributed to a diploid oocyte due to a failure in the first meiotic division (Jacobs et al., 1978Go). Whereas the study of Zaragoza et al. also revealed a diandry rate of ~66% (Zaragoza et al., 2000Go), others have provided evidence for a preponderance of digynic triploidy (Dietzsch et al., 1995Go; Miny et al., 1995Go; Baumer et al., 2000Go; McFadden and Langlois, 2000Go) or found a nearly equal distribution of diandric and digynic specimens (Daniel et al., 2001Go). One reason for such discrepancies might be differences in ascertainment leading to variable gestational ages of the samples.

Digynic triploidy may also develop after fertilization of a diploid giant oocyte (Dyban and Baranov, 1987Go) but this has been considered rather improbable (Jacobs et al., 1978Go; Kaufman, 1991Go). Giant female gametes have about twice the volume of normal oocytes and are tetraploid before meiosis due to their presumed origin, i.e. nuclear but no cytoplasmic division in an oogonium or cytoplasmic fusion of two oogonia (Austin, 1960Go). These mechanisms explain the binucleate state of immature giant cells. Giant oocytes have been observed in the rabbit, rat, mouse and cotton-rat (Austin and Amoroso, 1959Go) and their development has been extensively studied in the Chinese hamster (Funaki and Mikamo, 1980Go; Funaki, 1981Go). With the advent of IVF techniques, giant oocytes (Angell et al., 1993Go; Lim et al., 1995Go; Munné and Cohen, 1998Go; Veeck, 1999Go; Mandelbaum, 2000Go) and giant embryos (Munné et al., 1994Go) have also been described in the human. However, these single observations were not always accompanied by an examination of the chromosomal complements and little seems to be known about the incidence and development of giant oocytes during assisted reproduction. Therefore, we investigated the frequency, maturation patterns and fertilizability of binucleate giant oocytes in our IVF programme. Unfertilized metaphase II (MII) oocytes and pronuclear stages were analysed cytogenetically to assess their contribution to the generation of digynic triploidy. Some of the results have been presented in preliminary communications (Rosenbusch and Schneider, 1998Go; Rosenbusch, 2000Go, 2001Go).


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
This study includes data on 18 giant oocytes obtained from 16 patients who underwent conventional IVF or ICSI because of tubal infertility (n = 3), male factor infertility (n = 8), female (tubal) + male factors (n = 4) or idiopathic infertility (n = 1). The mean age of the female patients was 33.1 years (range 23–40), while that of the partners was 38.9 years (range 28–55). Ovarian stimulation was performed with hMG (seven patients) or recombinant FSH (nine patients) after down-regulation with a GnRH analogue. Transvaginal, ultrasound-guided follicular aspiration took place 36 h after administration of hCG. During one and the same treatment cycle, one patient (no. 8) provided two giant oocytes. Another two giant cells were obtained from one patient (no. 6) during two consecutive cycles.

IVF medium (IVF Science Scandinavia, Vitrolife Productions AB, Gothenburg, Sweden) was used for incubating untreated and injected or inseminated oocytes as well as pronuclear and cleavage stages. Details on our IVF and ICSI techniques have been given previously (Rosenbusch et al., 1997Go, 1998bGo). Pronucleus formation was assessed 18–20 h after IVF/ICSI. Fertilized giant oocytes were incubated overnight in 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 left for 5–8 min in hypotonic solution consisting of 1% sodium citrate in distilled water with 2% human serum albumin. They were fixed by successive application of three different fixatives (gradual fixation, air drying method) (Mikamo et al., 1994Go). Preceding removal of the zona pellucida with protease (Rosenbusch et al., 1998aGo, 2001Go) has been abandoned. Unfertilized MII oocytes were directly subjected to hypotonic treatment. The chromosomes were stained with Giemsa and karyotypes were established from photographs taken at x1000 magnification. The distribution of the metaphases on the slide was taken into consideration. For example, 23,X/23,X,ace indicates two separate haploid chromosome sets and 69,XXX a metaphase in which the single participating sets could not be distinguished with certainty.

Our cytogenetic investigations of human oocytes were approved by the ethical committee of the University of Ulm.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Among 237 oocytes obtained from 16 patients, we identified 18 (7.6%) giant cells. Thirteen of these were unfertilized in different stages of maturity (group I) and five showed formation of pronuclei (PN) (group II). The total number of oocytes recovered during the observation period was 7065 and thus the frequency of giant gametes was 0.26%.

Group I: unfertilized giant oocytes
Five cells were in germinal vesicle (GV) stage and had two nuclei as previously illustrated (Rosenbusch and Schneider, 1998Go). Three of these progressed to MII and the remaining two degenerated in culture. Three cells were in metaphase I (MI) and all of them reached MII after further incubation. Five gametes were already at MII at the time of their detection (Figure 1Go). A cytogenetic analysis was attempted in 10 giant MII oocytes (Table IGo) and diploidy was unequivocally demonstrated in six cases by karyotyping (Figure 2aGo). Four cells that could not be karyotyped due to insufficient chromosomal quality had chromosome counts in the diploid range.



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Figure 1. Examples for giant oocytes. (a) An unfertilized giant oocyte at MII (right) in comparison with a normal MII oocyte from the same patient. Both cells have one fragmented first PB. (b) A fertilized giant oocyte (right) with 2PN in comparison with an immature (MI) oocyte from the same patient. (c) Higher magnification of the zygote shown in (b). The 2PN apparently do not differ in size.

 

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Table I. Stage of maturity, further development, and results of cytogenetic analysis of 18 giant oocytes observed in our IVF programme
 



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Figure 2. (a) Diploid karyotype of a giant oocyte with an additional chromatid in chromosome group G (arrow). (b) Triploid karyotype of a fertilized giant oocyte with 2PN. Since giant MII oocytes have been shown to be diploid, the 46,XX complement of this zygote can be ascribed to the oocyte, whereas the haploid set must originate from the spermatozoon.

 
Group II: giant zygotes
Three zygotes were detected after IVF and appeared to be normally fertilized with 2PN and two polar bodies (PBs) (Figure 1Go). Two oocytes at MII were unintentionally injected during ICSI. The first of these zygotes revealed 2PN and two PBs while the second had developed 3PN and four PBs. Since giant embryos have been reported to be triploids or triploid mosaics (Munné et al., 1994Go) and since we already knew about the diploid state of giant oocytes from our own investigations, all five giant zygotes were excluded from transfer or cryopreservation and prepared for cytogenetic analysis irrespective of an apparently normal number of PN in four of them. Triploidy was confirmed in all cases (Table IGo). One of the zygotes with 2PN showed two well-separated haploid (n) and diploid (2n) chromosome sets respectively (Figure 2bGo). The three haploid complements of the tripronuclear zygote could also be demonstrated separately, whereas the metaphases were mixed in the remaining three cases.

Maturation patterns
Binucleate giant oocytes in the GV stage have two possibilities of maturation to MII, which are depicted in Figure 3Go. First, the two haploid chromosome sets can unite during formation of the meiotic spindle. The corresponding MII oocytes are characterized by a single diploid chromosome set (2n) and a diploid first PB (Figure 3aGo). Monospermic fertilization of this oocyte will lead to the formation of a haploid male and a diploid female pronucleus as observed (Figures 1c and 2bGoGo). Second, the binucleate state may be maintained, resulting in extrusion of two haploid first PBs and the presence of two haploid complements in the oocyte (Figure 3bGo). This distribution was found in two MII oocytes with two PBs, whereas chromosome spreading did not allow distinction of the individual sets in the third case. Fertilization of such an oocyte will result in three haploid PN (Figure 3bGo). From the number of PBs and PN, the maturation pattern of the oocyte can be concluded. In summary, nine cells underwent a union of the chromosomes during maturation to MII and seven oocytes maintained the binucleate state.



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Figure 3. Schematic representation of the two possibilities of maturation from GV stage to MII of a binucleate oocyte and the corresponding behaviour after sperm penetration. Each chromosome or chromatid represents a haploid complement. (a) The two haploid chromosome sets have united and the giant MII oocyte possesses a single diploid chromosome complement and a single diploid first PB. After fertilization, maternal chromosomes separate into their chromatids. Half of these chromatids are extruded into a diploid second PB. The other half will form a diploid female pronucleus (f) besides the haploid male pronucleus (m). (b) The binucleate state has been maintained and the giant oocyte possesses two separate haploid metaphases and accordingly, two haploid first PBs. After fertilization, two haploid second PBs will be extruded while two separate haploid female PN will be formed besides the haploid male pronucleus. In both cases, the resulting zygotes will have a triploid chromosome count after DNA replication.

 
Chromosomal abnormalities
In karyotyped, unfertilized oocytes only one had a diploid 46,XX complement without aberrations (Table IGo). Three giant gametes were aneuploid, including hypodiploidy, an additional chromatid and one case with premature separation of three chromosomes into their chromatids. One of the corresponding chromatids was missing. The nomenclature for karyotypes with predivided chromosomes followed our recent suggestions (Rosenbusch and Schneider, 2000Go). Two cells showed the presence of acentric fragments accompanied by hypodiploidy in one case. In group II, one metaphase of the tripronuclear zygote carried a variety of aberrations, including a chromosome break, a chromosome gap and a possible translocation.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The present study has demonstrated that giant oocytes occur at different stages of maturity in our IVF programme and that maturation to MII can be achieved either by maintenance of the binucleate state or union of the nuclei. Moreover, giant oocytes can become fertilized and both theoretically possible types of zygotes, i.e. 2PN or 3PN, have been observed. Cytogenetic analysis has shown that giant MII oocytes are diploid and that triploid zygotes will result after monospermic fertilization. Of note, giant oocytes with 2PN appear regularly fertilized if normality is only defined by the number of PN. Cytogenetically, however, the corresponding cells are abnormal since the female pronucleus is diploid.

The earliest observation of a binucleate oocyte recovered after ovarian stimulation came from Mahadevan et al. This particular cell underwent GV breakdown and reached MI and was shown to carry a roughly tetraploid chromosome complement. The authors discussed its formation by fusion of two adjacent oocytes (Mahadevan et al., 1988Go) but did not specify its size and, therefore, it is not sure whether this was a giant gamete. Likewise, an indication of size is missing for two unfertilized oocytes with two sets of chromosomes each that were characterized as probably binucleated by Eichenlaub-Ritter et al. (Eichenlaub-Ritter et al., 1988Go). A normal sized oocyte with two GV has been shown by Veeck and attributed to abnormal division of an oogonium (Veeck, 1999Go). However, all GV stage oocytes with two nuclei from our IVF programme have been giant cells. Others have also provided unambiguous photographic evidence of immature binucleate giant oocytes or giant MII oocytes (Munné and Cohen, 1998Go; Veeck, 1999Go; Mandelbaum, 2000Go).

Judging from a comment by Mandelbaum who supposed that giant oocytes are diploid (Mandelbaum, 2000Go), uncertainty on the cytogenetic constitution of these cells still appears to exist. This is surprising in view of previous data. For instance, Lim et al. observed two giant gametes showing two distinct haploid chromosome sets or a diploid 46,XX complement respectively (Lim et al., 1995Go). Previously, Angell et al. had described two `morphologically giant oocytes' one of which had two chromosome sets each accompanied by its own polar body chromosomes, and the second cell was even tetraploid (Angell et al., 1993Go). Munné et al. found that four embryos developing from giant oocytes were triploid or triploid mosaics (Munné et al., 1994Go).

The present study has constantly demonstrated diploidy in a number of giant cells and, comparable with the situation in normal sized haploid oocytes, we detected numerical and/or structural chromosome abnormalities. Moreover, our investigation of 1-cell zygotes fills in the gap between oocyte and embryo analyses, documenting the different patterns of pronuclear formation and the development of digynic triploidy after fertilization. Of note, corresponding data had been accumulated >20 years ago for the Chinese hamster. Here, unfertilized giant oocytes always revealed diploidy and giant zygotes, embryos or blastocysts were digynic triploids (Funaki and Mikamo, 1980Go; Funaki, 1981Go). Concerning the maturation patterns, Funaki and Mikamo reported that out of 18 giant MII oocytes, only one had two haploid chromosome plates. The authors therefore concluded that the binucleate state is rarely maintained during maturation (Funaki and Mikamo, 1980Go). In contrast, as deduced from the number of PBs and PN, maintenance of the binucleate state (n = 7) was nearly as common as union of the nuclei (n = 9) in our study.

More recently, Karnikova et al. succeeded in fusing immature mouse oocytes and obtained 116 giant cells containing two germinal vesicles (Karnikova et al., 2000Go). During further culture, 38% did not extrude the first PB, 31% had two first PBs and two metaphase plates, and 31% had one PB and one metaphase plate. In the second group, parthenogenetic activation resulted in the extrusion of two second PBs and formation of 2PN, whereas oocytes of the third group responded with one second PB and 1PN. These experimental data are in full agreement with our outline of the different maturation and fertilization patterns of human giant oocytes (Figure 3Go).

Karnikova et al. also observed two fertilized giant mouse oocytes that both contained two female PN and one male pronucleus. One of these zygotes was fixed and the other was kept in culture and appeared to develop normally to the 4-cell stage (Karnikova et al., 2000Go). In the Chinese hamster, giant zygotes showed normal viability (Funaki and Mikamo, 1980Go) and development to the blastocyst stage (Funaki, 1981Go). These findings led to the assumption that the nuclear–cytoplasmic ratio of giant zygotes might be more favourable for embryonic development than that of other triploids (Funaki, 1981Go). In the human, four giant embryos with 3–7 blastomeres have been reported (Munné et al., 1994Go). The zygotes from which they originated had two PBs and 2PN, thus corresponding to the mechanism of maturation and fertilization described in Figure 3aGo. However, to our knowledge implantation of a giant embryo has not been documented in the human. In addition, nothing seems to be known about the developmental anomalies of such embryos that could be related to their increased cytoplasmic volume.

The incidence of giant oocytes is ~0.1% in rats and mice and 0.45% in rabbits (Austin and Braden, 1954Go). In the Chinese hamster, 0.44% of MII oocytes recovered from oviducts were giant gametes, whereas their frequency increased to 1.37% in mature follicles missing ovulation. After fertilization, giant 1- and 2-cell zygotes constituted 0.47% of the cells analysed (Funaki and Mikamo, 1980Go). In human ovaries, multiovular follicles and multinuclear ova can be regularly found (Bacsich, 1949Go; Sherrer et al., 1977Go; Gougeon, 1981Go) and among the latter, binucleate oocytes are the most common abnormality (~96%) (Gougeon, 1981Go). Primordial follicles containing binucleate oocytes have twice the volume of normal follicles (Gougeon, 1981Go), suggesting that further maturation will also lead to an altered size of the corresponding oocytes. This gives support to a previous assumption (Austin, 1960Go) that, `It may well be that the binuclear oocytes recorded in the literature were in fact giant oocytes in the making'. However, the probability that these follicles will ovulate and contribute to digynic triploidy was considered very low (Gougeon, 1981Go). In contrast, Kennedy and Donahue observed two oocytes each containing two nuclei in the GV stage in human ovaries and speculated that binucleate oocytes might be ovulated and give rise to triploid zygotes after fertilization (Kennedy and Donahue, 1969Go).

Gougeon reported that multinuclear oocytes and multiovular follicles are found in 98% of 18- to 52-year-old women with a frequency between 0.06 and 2.44% of the total follicular population of the ovary (Gougeon, 1981Go). Their incidence was independent of age, gonadotrophic hormones, oral contraceptives, pregnancy or day of the menstrual cycle. In our study, the frequency of giant oocytes recovered by follicular aspiration was 0.26%. These cells were obtained from women 23–40 years of age who underwent varying protocols for ovarian stimulation because of different indications for infertility treatment. Though the number of oocytes analysed by us is too small for statistical analysis, it appears that in accordance with the findings of Gougeon, the generation of such abnormalities is an inherent phenomenon of female gametogenesis without any influence from specific factors (Gougeon, 1981Go).

The true distribution of diandry and digyny among triploid pregnancies is still at issue, possibly depending on differences in ascertainment (McFadden and Langlois, 2000Go) and digynic triploidy is almost exclusively attributed to meiotic irregularities. For instance, it has been estimated that 0.2% of the oocytes have a failure of meiosis (Pergament et al., 2000Go). However, the incidence of giant oocytes and their capability of maturation and fertilization in our IVF programme lead us to assume that their role in causing triploidy in the human deserves further attention. Based on our results and previous observations, exclusion of giant oocytes from IVF trials appears to be justified. In case of fertilization, giant oocytes with 2PN should be treated like multipronuclear zygotes, i.e. they should be discarded and not considered for transfer or cryopreservation.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
We thank Mrs B.Mattheis, Mrs M.Kopp and Mrs A.Jäger for technical assistance.


    Notes
 
* The results of this study have been presented in part at the 17th Annual Meeting of ESHRE, Lausanne, Switzerland, 2001 Back

4 To whom correspondence should be addressed. E-mail: bernd.rosenbusch{at}medizin.uni-ulm.de Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
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Submitted on February 4, 2002; accepted on May 1, 2002.