Spontaneous blastomere fusion after freezing and thawing of early human embryos leads to polyploidy and chromosomal mosaicism*

Hanna Balakier1,4, Oliver Cabaca1, Derek Bouman2, Alan B. Shewchuk1, Carl Laskin1,3 and Jeremy A. Squire2

1 Success Through Assisted Reproductive Technologies (START), 2 University Health Network and Department of Laboratory Medicine and Pathobiology, and Medical Biophysics, Faculty of Medicine, University of Toronto, and 3 Department of Medicine, Immunology and Obstetrics and Gynecology, University of Toronto, Toronto, Ontario, Canada


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The incidence of blastomere fusion after cryopreservation of early human embryos (day 2 and day 3) was investigated using the standard propanediol technique. The process of fusion was observed in all developmental stages (from 2 to 10 cells) and the frequency of this event was 4.6% in day 2 (41/889) and 1.5% in day 3 (10/646) embryos that survived the thawing (embryos with 50–100% intact cells). Fusion of two, and occasionally of several, blastomeres resulted in the formation of multinucleated hybrid cells, which clearly indicated that the ploidy of these newly created cells had been altered. This event, depending on the number of fused cells per embryo, transformed the embryos into either entirely polyploid embryos (complete fusion at 2- or 3-cell stage) or into mosaics being a mixture of polyploid and normal cells. Chromosomal preparations of embryos affected by blastomere fusion indicated the presence of tetraploid mitotic plates. Also, fluorescence in-situ hybridization (FISH) analysis using DNA probes targeting unique sequences on chromosomes 9, 15, 17 and 22 indicated the existence of tetraploid and diploid fluorescence signals in the interphase nuclei within mosaics. Therefore, observations on live and fixed embryos suggested that tetraploid (4n) or hexaploid (6n) and tetraploid–diploid or more complex aberrations of ploidy might be formed as a consequence of blastomere fusion. Furthermore, this demonstrates that freezing and thawing may induce numerical chromosomal changes in human embryos.

Key words: cell fusion/chromosomal aberrations/cryopreservation/mosaicism/polyploidy


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
It is well documented that cryopreservation of human embryos may enhance the cumulative pregnancy rate for in-vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI) patients who have chosen to freeze their excess embryos (for review, see Wood, 1997). However, freezing and thawing significantly reduces embryo viability and the number of embryos available for uterine transfers (Selick et al., 1995Go). Some embryos do not survive freezing and thawing (17–70%), and in some cases the majority of blastomeres undergo degeneration (Wood, 1997Go). The adverse effects of cryopreservation may also lead to the formation of cracks in the zona pellucida, or injuries to the cell membranes and intracellular components (Ng et al., 1988Go; Dumoulin et al., 1994Go). In spite of the negative impact, freezing and thawing is not considered to be harmful to the potential development of babies. Although our present knowledge is limited to only a few follow-up studies, there is no evidence that cryopreservation of embryos increases the frequency of birth defects. In some reports major chromosomal anomalies such as trisomy of chromosome 13, 18 and 21, as well as major and minor congenital malformations, have been identified in fetuses and babies conceived from frozen embryos (Rizk et al., 1991Go; Deffontaines et al., 1994Go; Sutcliffe et al., 1995Go; Wennerholm et al., 1997Go). On the other hand, a comparison of babies resulting from the transfer of frozen–thawed embryos with those conceived normally or from fresh IVF cycles showed a similar or even decreased incidence of congenital abnormalities after cryopreservation (Wada et al., 1994Go; Wood, 1997Go). Also, two studies focusing on the development of children at 1 to 9 years of age did not reveal any major differences between children born from frozen embryos and children of natural conception (Sutcliffe et al., 1995Go; Olivennes et al., 1996Go).

A recent study of aneuploidy and mosaicism of chromosomes X, Y and 1 in human frozen embryos (day 2 and 3 of development) using fluorescence in-situ hybridization (FISH) showed that a large proportion of thawed embryos (57%) exhibiting cleavage arrest during the first 24 h of in-vitro culture carried numerical chromosomal abnormalities (Laverge et al., 1998Go). However, it remains uncertain whether those aberrations were induced by the cryopreservation technique, or were already present in chromosomally abnormal embryos before thawing. Although several reports on mouse embryos have shown that rapid freezing (vitrification) may cause some chromosomal damage (Shaw et al., 1991Go) or increased mitotic crossing over (Bongso et al., 1988Go; Ishida et al., 1997Go), there is presently no evidence that cryopreservation of human embryos could induce structural or numerical chromosomal changes.

On the other hand, our previous observations have suggested that cryopreservation of early mouse embryos, using the cryoprotectant propanediol, may cause the formation of polyploid cells by the mechanism of spontaneous blastomere fusion (fusion in 16/932 thawed embryos; 1.7%; Balakier et al., 1991). In this experiment, numerical chromosomal abnormalities were not increased in the mouse blastocysts that developed from frozen 4-cell embryos. However, formation of tetraploid cells—possibly as a result of fusion between two blastomeres—was found in some thawed embryos, while similar polyploid cells were not observed in the control group of embryos without cryopreservation. Blastomere fusion in early cleavage stage human embryos (2–8 cells) has also been reported in the past during preliminary work on establishing the cryopreservation technique with glycerol as a cryoprotectant (Trounson et al., 1984). However, this technique appeared to be highly lethal for embryonic development and was abandoned for early embryo freezing.

Based on these findings, and on our occasional observations of cell fusion in thawed human embryos, it was interesting to explore the link between chromosomal aberrations and blastomere fusion. Therefore, the purpose of this study was to determine the incidence of blastomere fusion after freezing and thawing of early human embryos, and to characterize numerical chromosomal changes such as ploidy using a standard cytogenetic technique and the FISH method. The implications of the formation of polyploid and mosaic embryos that may arise from such blastomere fusion are discussed.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The freezing and thawing of day 2 and day 3 human embryos was performed according to the standard technique that involves 1,2-propanediol (PROH; ICN Biomedicals Inc., Mississagua, Canada) and sucrose (BDH Inc., Toronto, Canada) as cryoprotectants (Testart et al., 1986Go). The solutions were prepared using Q-HTF HEPES medium (human tubal fluid from In Vitro Care Inc., San Diego, CA, USA) supplemented with 20% synthetic serum substitute (SSS; Somagen, Canada). For freezing, the embryos were equilibrated at room temperature in 1.0 mol/l PROH for 5 min and in 1.5 mol/l PROH containing 0.1 mol/l sucrose for 15 min. The embryos were loaded into 0.25 ml straws, and cooled in a biological freezer (Bio-Cool 80, FTS-System Inc., Toronto, Canada) from 22°C to –7°C at the rate 2°C/min, at which temperature manual seeding was performed. Freezing was continued to –30°C at rate of 0.3°C/min, after which the straws were plunged into liquid nitrogen.

The thawing procedure was rapid (30 s at room temperature and 1 min at 37°C), and cryoprotectants were removed by exposing the embryos to stepwise decreasing PROH concentrations (1.0 mol/l PROH + 0.2 mol/l sucrose, 0.5 mol/l PROH + 0.2 mol/l sucrose, 0.2 mol/l sucrose; each step 5 min). Immediately after thawing, embryos were evaluated for morphological appearance and for the number of survived blastomeres using an inverted microscope at x320 magnification (Leitz DMIL relief contrast; Leica, Wellowdale, Canada). Embryos were cultured for 2–4 h in IVC-One medium (In Vitro Care Inc.) containing 20% SSS, and were examined hourly before transfer to the uterus. One to six embryos per patient were transferred during stimulated cycles (average 4.3 embryos/patient). The endometrium was prepared by graduated dosage of oestradiol valerate (Roberts, Oakville, Ontario, Canada); 2.0 mg on days 1–4, 4.0 mg on days 5–8, and 6 mg on days 9–11. When the endometrium was >=0.8 cm, the luteal phase was supported by progesterone in the form of vaginal suppositories (100 or 200 mg, twice daily; Medicine Shoppe, Toronto, Canada) or in the form of intramuscular injections in oil (50 or 100 mg; Cytex Pharmaceuticals Inc., Halifax, Canada).

Chromosomal preparations were made from representative embryos in which fusion of two blastomeres had been observed after thawing. To obtain metaphase plates, the embryos were exposed to a mitotic inhibitor (Demecolcine, 10 µg/ml; Sigma-Aldrich Canada Ltd, Oakville, Canada) for 5–10 h prior to gradual fixation and staining with Giemsa (Kamiguchi et al., 1993Go).

FISH interphase analysis was performed to investigate further any numerical chromosomal changes in embryos affected by fusion. All preparations were made at 18 h post fusion, except embryo no. 2, which was fixed at 40 h post fusion. The fixation and FISH techniques used were as described previously (Liu et al., 1998Go). The FISH technique utilized a two-colour FISH combination of single-copy probes that hybridize to DNA on the long arm of chromosomes 9, 15, 17 and 22. The two-colour FISH probe cocktail for chromosome 9 (chr.9), was the ABL gene locus at band 9q34 (green-labelled) and for chromosome 22 (chr.22), was the BCR gene locus at band 22q11.2 (red-labelled) (Ventana Medical Systems, Tucson, AZ, USA). The two-colour FISH probe cocktail to chromosome 15 (chr.15) was the PML gene locus at band q22 (red-labelled), and for chromosome 17 (chr.17) was the RARA gene locus at band q21.1 (green-labelled) (Vysis, Downers Grove, IL, USA). The slides from embryos were denatured at 70°C in 70% formamide/2xSSC for 5–15 s, then hybridized and washed using standard procedures for FISH, and subsequently counterstained with 4,6-diamino-2-phenylinole (DAPI). Slides were evaluated on an epi-fluorescence microscope using imaging hardware and software provided by PSI (Perspective Scientific Instruments, League City, Texas, USA).


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
A total of 1141 embryos frozen on day 2 (2- to 5-cell stage) and 873 embryos frozen on day 3 (5- to 10-cell stage) of in-vitro culture, were thawed for 492 patients (266 IVF and 226 ICSI patients; Table IGo). The overall survival rate was 76%, including fully intact embryos (53%) and embryos containing at least 50% live cells (23%). The other embryos were either totally degenerated or had the majority of their cells lysed. Among the embryos that survived the thawing (embryos with 50–100% intact cells), 51 cases of blastomere fusion were detected within the first 2 h of in-vitro culture (Figure 1AGo–F). About 70% of those affected embryos were of good quality (regular cells, no or few fragments), while the others exhibited poor morphology (20–30% fragmentation, uneven cells or granular cytoplasm). The process of fusion was observed in all developmental stages (from 2 to 10 cells), and the frequency of this event was 4.6% in day 2 (41/889) and 1.5% in day 3 (10/646) embryos (Table IGo). An especially low incidence of blastomere fusion was found in 7- to 10-cell embryos frozen on day 3 of development. In total, a slightly higher incidence of fusion was observed in embryos obtained after IVF than ICSI procedure (33 IVF versus 18 ICSI cases of fusion). However, further conclusions on any relationship between fusion and type of procedure cannot be drawn because the observations were distorted by the fact that on day 2 the majority of cases were represented by IVF (72%, 188/262 patients; 31 cases of fusion) and on day 3 by ICSI procedures (66%, 152/230 patients; eight cases of fusion).


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Table I. The survival rate and frequency of blastomere fusion after thawing of day 2 and day 3 embryos with 50–100% intact cells
 







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Figure 1. Frozen–thawed human embryos in which two (AE) or three (F) blastomeres have undergone fusion. Two or three nuclei are visible in the large hybrid cells. (A) A 2-cell stage embryo in the course of fusion. The nuclei in this particular embryo are not visible because just before cell fusion both blastomeres have entered mitosis. (E) This embryo contains two binucleated hybrid cells due to `double' fusion of two blastomeres at the 4-cell stage. (C,D,F) Embryos with some degenerated blastomeres that did not survive thawing. Original magnification (AF), x340.

 
In the vast majority of cases (47/51) two blastomeres of an embryo were fused, resulting in a hybrid cell that contained two nuclei (Figure 1BGo–D). In the remaining four embryos (4/51), very large three- or four-nucleated hybrid cells were formed after fusion of three or four blastomeres (Figure 1FGo). When blastomere fusion occurred to all cells of an embryo—for example, in the case of complete fusion at the 2- or 3-cell stage—the whole embryo was transformed into a single multinucleated hybrid cell (Figure 1AGo, D and F). In contrast, fusion of only some blastomeres of an embryo had created mosaics, being a mixture of hybrid and apparently normal cells (Figure 1B and CGo). In two unusual embryos, double fusion of two blastomeres took place at the 4-cell stage, which resulted in the formation of two sets of binucleated hybrid cells within each embryo (Figure 1EGo).

To compare if similar rates of cell fusion occurred within control embryos that had not been subjected to cryopreservation, day 2 and day 3 fresh embryos were examined every 2 h during a period of 4–6 h. Among 2315 day 2 embryos, only one fusion of two blastomeres was observed in a 4-cell stage embryo (0.04%). On day 3 of development, fusion was not recorded in any 5- to 6-cell or 7- to 10-cell embryos (1795 embryos examined). However, in the group of arrested 3- or 4-cell embryos that did not divide from the previous day (from day 2 to day 3), blastomere fusion was recorded in eight embryos (8/323; 2.4%). Except for two embryos that underwent double cell fusion (similar to that shown in Figure 1EGo), the other six arrested embryos became mosaics, with one binucleated hybrid cell.

In order to examine the ploidy of the embryos that underwent fusion of two blastomeres, metaphase chromosomal spreads were made from three day 2 embryos. In the first, a 3-cell embryo, tetraploidy was observed in one mitotic plate (91–92 chromosomes), and two compact groups of chromosomes were also seen. In the other two embryos, near-tetraploid mitotic plates were also found. In the second embryo (two cells + fragments), 76 chromosomes, one compact metaphase plate and seven condensed pieces of chromatin were seen. In the third embryo (two cells + fragments), one large nucleus, 89 chromosomes in a metaphase spreading and seven small fragments of condensed chromatin were observed.

Results from the FISH technique applied to six representative embryos that underwent fusion of two blastomeres are presented in Table IIGo. During in-vitro culture after fusion, in two embryos (nos. 1 and 2) the hybrid cells had divided, and in four embryos (nos. 3–6) they remained unchanged as binucleated cells. In the first two embryos, hybridization with both chromosome sets showed the signal pattern expected for the tetraploid and diploid nuclei. For example, in the tetraploid nucleus from embryo no. 1, four chr.9 and four chr.22 signals and four chr.15 and four chr.17 signals were seen (Figure 2AGo and B). In diploid nuclei, two chr.9 and two chr.22 signals or two chr.15 and two chr.17 signals were present. Embryo no. 1 also exhibited mixed configuration of tested chromosomes such as diploid/haploid (two chr.9 and one chr.22; one chr.15 and two chr.17) and haploid (one chr.15 and one chr.17), or showed loss of one or more chromosomes (especially in small subnuclei, Table IIGo). In embryo no. 2, which developed to morula stage after 40 h post fusion, 14 nuclei were found and, with the exception of two, all contained two signals for chromosome 22 and only one signal for chromosome 9 (Table IIGo; Figure 2CGo). This is consistent with a diploid clone that has a loss of chromosome 9 (monosomic for chr.9). Therefore, the presence of the nucleus with two and four hybridization signals for chromosomes 9 and 22 respectively indicates tetraploidization of the diploid cell, with chromosome 9 loss. In another nucleus of embryo no. 2 only a single signal for both chromosomes was seen (one chr.9 and one chr.22).


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Table II. Fluorescence in-situ hybridization on human embryos that were affected by blastomere fusion
 


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Figure 2. Fluorescence in-situ hybridization (FISH) analysis of nuclei from human embryos affected by fusion of two blastomeres after freezing and thawing (detailed description in Table IIGo). (A,B) Sequential application of probes to the nuclei from embryo no. 1. (A) Green signals indicate chromosome 9, and red signals indicate chromosome 22. The tetraploid nucleus in the centre contains four signals. (B) The probe from the preparation shown in (A) was removed and FISH was performed with the second probe cocktail. Red signals indicate chromosome 15, and green signals indicate chromosome 17. Note the same tetraploid nucleus with four signals for both chromosomes. The normal diploid nuclei are showing two FISH signals for each probe. (C) FISH with chromosome 9 and 22 in nuclei from embryo no. 2. Twelve nuclei with monosomy for chromosome 9 and diploidy for chromosome 22. The tetraploid nucleus with two green (chr.9) and four red (chr.22) signals can be seen in the centre.

 
In the case of embryos nos. 3, 4, 5 and 6, in which hybrid cells did not divide and remained arrested at binucleated phase during in-vitro culture, polyploid nuclei were not found (Table IIGo). About half of the nuclei (57%) were normal diploid, having two signals for both chromosomes 9 and 22, while the remaining nuclei (43%) showed abnormal chromosome configurations. In the case of embryo no. 6, signal configurations indicated two nuclei with trisomy 22 and one nucleus with octosomy of chromosome 9. It seems likely that amplification of individual chromosomes rather than blastomere fusion per se was a cause of such numerical changes.

The overall clinical pregnancy rate in the 492 thawed cycles was 19%. The embryos containing fused blastomeres were usually not transferred. However, in 15 cases mixed transfers of embryos affected by fusion and normal-looking embryos (ratio 1 mosaic:2–3 normal embryos) were performed (12 cases with day 2 embryos, and three cases with day 3 embryos). Two transfers resulted in early spontaneous abortions (6–8 weeks), one pregnancy was chemical, and one pregnancy resulted in the birth of a healthy baby girl, while no implantation occurred in the other 11 cases.


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The present study has clearly demonstrated that cryopreservation of early human embryos using the standard propanediol technique may induce blastomere fusion resulting in chromosomal aberrations. Simple observations on the number of fused blastomeres indicated that the ploidy of newly created hybrid cells had been altered (Figure 1Go). It was apparent that embryos affected by fusion were transformed into either polyploid embryos or into mosaics consisting of a mixture of polyploid and normal cells. Therefore, it can be suggested that tetraploid (4n) or hexaploid (6n) embryos might be formed due to complete fusion of all blastomeres at the 2- or 3-cell stage or after double fusion in 4-cell embryos (4n; Figure 1EGo). The creation of entirely polyploid embryos was less frequent (8/51 embryos; 16%), and the majority of affected embryos were converted into tetraploid–diploid (4n/2n; 40/51, 78%) or other complex mosaics (6n/2n or 8n/2n, 6%) when only some blastomeres of an embryo had fused. To reinforce these conclusions based on our observations of live embryos, some mosaics obtained by fusion were examined on fixed preparations. Although a number of logistical factors invariably limit these investigations (low number of donated embryos, and limited number of samples well suited for FISH procedures), the FISH and cytogenetic results confirmed the morphological appearance suggestive of altered ploidy in the embryos affected by fusion. Mitotic tetraploid chromosomal plates as well as a mixture of tetraploid and diploid FISH signals within intact nuclei were found in the fixed mosaics, indicating the presence of polyploid cells.

The present observations may also suggest that early human embryos are more susceptible to cryodamage and blastomere fusion when compared with older, more advanced embryos. It is likely that the properties of cell membranes, for instance fluidity, are changing during embryo development, and perhaps for this reason more fusion has been observed in day 2 than in day 3 thawed embryos (4.6% versus 1.5%; Table IGo). Similarly, the frequency of hybrid formation appeared to be higher in 5- to 6-cell than in 7- to 10-cell embryos examined on the third day of development (1.2% and 0.3% respectively; Table IGo). Also, an interesting observation was that in the control group of unfrozen embryos, the only fusion that was found had occurred in early-arrested 2- to 4-cell stage embryos. Alternatively, the occurrence of blastomere fusion could be associated with existing membrane abnormalities that may promote fusion either after freezing and thawing or in unfrozen fresh embryos due to other factors such as pH, temperature, osmotic pressure, etc.

From our present and previous studies (Trounson, 1984Go; Balakier et al., 1991Go) it seems that freezing and thawing is responsible for blastomere fusion, and this may occur regardless of the type of cryoprotectants used. These studies also suggest that blastomere fusion is not only attributed to fair and poor quality embryos, as was previously thought (Trounson, 1984Go), but it can also affect morphologically good embryos, as was shown by our observation (70% of affected embryos were of good quality). Although the molecular mechanism of cell fusion has not yet been elucidated, the general studies have proven that a defect in the cell membrane is required for initiation of fusion, which can be induced by many membrane-disrupting agents such as a virus, polyethylene glycol or electric field, as well as freezing and thawing (Hui et al., 1981Go; Zimmermann and Vienken, 1982Go; Zimmermann, 1996Go). Based on these reports, it seems that cryoprotectants may also contribute to the process of fusion by causing cell dehydration and osmotic swelling which induce other changes that are necessary to induce fusion (for instance, changes in the cytoskeleton, tight contact between cells, etc.). It can also be assumed that cytoplasmic bridges may play a role in blastomere fusion; however, research on early human embryos did not detect them when embryos were examined in electron microscope serial sections or after injection of specific dyes into single blastomeres of 2- to 8-cell stage embryos (Dale et al., 1991Go; Mottla et al., 1995Go).

The transfer of embryos containing numerical chromosomal aberrations due to the process of fusion may potentially have clinical relevance; at present, the developmental result of such transfers remains unclear. Since there is no evidence that cryopreservation increases the incidence of birth defects (Wood, 1997Go), this implies that embryos affected by fusion— if transferred to the uterus—either fail to implant or are spontaneously aborted, and for this reason are not seen among the babies born from the frozen cycles. The fact that in our study cleavage arrest and fragmentation of the nuclei were observed in the hybrid cells also suggests that embryos affected by fusion are either eliminated before clinical recognition, or they are rescued by protective mechanisms that correct embryonic errors. It is possible that abnormal cells resulting from fusion may be sequestered to the trophoblast and later to the placenta, since it has been shown that polyploid cells are frequently found in these extraembryonic tissues (James and West, 1994Go). On the other hand, little information is available regarding the chromosomal aberrations in human abortuses following cryopreservation (Deffontaines et al., 1994Go; Wada et al., 1994Go). There has been, however, one report on a twin pregnancy of two empty tetraploid sacks that had resulted from a transfer of frozen zygotes (Ginsburg et al., 1991Go). It is probable that in such cases mitotic spindles of the zygotes were destroyed, resulting in the formation of tetraploid embryos. Therefore, it cannot be ruled out that similar abnormal abortuses may also arise from frozen thawed embryos by means of blastomere fusion. It is worth noting that in the present study, out of 51 embryos affected by fusion, 27 embryos that were not transferred or used for preparations, although being left in suboptimal culture conditions (IVC-One medium), reached the blastocyst stage in 7% of the cases, cleaved in 56% (5- to 12-cell stage), and were totally arrested in 37%.

Although our observations cannot be conclusive based on our limited sample size of 15 mixed transfers that involved chromosomal mosaics (possibly 4n/2n) and normal-looking embryos, it should also be mentioned that two transfers resulted in early spontaneous abortions, and one pregnancy was chemical. More extensive studies are required from abortive tissues to determine whether chromosomal abnormalities are associated with embryo cryopreservation. It is important to note that in natural conceptions the incidence of tetraploidy (4–8%) has been reported in human abortive tissues as well as in born infants (Sheppard et al., 1982Go; Warburton et al., 1991Go). It is generally assumed that such a chromosomal anomaly is formed due to genome duplication and suppression of the early cleavage divisions (Sheppard et al., 1982Go). However, from our observations it can be suggested that blastomere fusion may be an alternative mechanism by which human embryos acquire polyploidy. This concept is supported by the observations that blastomere fusion can be induced by freezing and thawing, or in rare circumstances may occur spontaneously in fresh/unfrozen embryos. Furthermore, it has been shown that tetraploid mouse embryos experimentally induced by means of cell fusion are capable of advanced post-implantation development, which demonstrates that polyploid embryos obtained by this mechanism could be viable (Henery et al., 1992Go).


    Notes
 
* Presented at the 2000 Canadian Fertility and Andrology Society (CFAS) Annual Meeting, St. John's Newfoundland, Canada, September 14–16, 2000. Back

4 To whom correspondence should be addressed at: Success Through Assisted Reproductive Technologies (START), 655 Bay Street, 18th Floor, PO Box 4, Toronto, Ontario, M5G 2K4, Canada. E-mail: hbalakier{at}startclinic.com Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Submitted on May 18, 2000; accepted on July 31, 2000.





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