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
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Abstract |
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Key words: cell fusion/chromosomal aberrations/cryopreservation/mosaicism/polyploidy
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Introduction |
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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., 1998). 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., 1991
) or increased mitotic crossing over (Bongso et al., 1988
; Ishida et al., 1997
), 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 cellspossibly as a result of fusion between two blastomereswas 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 (28 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.
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Materials and methods |
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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 24 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 14, 4.0 mg on days 58, and 6 mg on days 911. 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 510 h prior to gradual fixation and staining with Giemsa (Kamiguchi et al., 1993).
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., 1998). 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 515 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).
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Results |
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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 46 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 1E), 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 (9192 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 II. During in-vitro culture after fusion, in two embryos (nos. 1 and 2) the hybrid cells had divided, and in four embryos (nos. 36) 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 2A
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 II
). 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 II
; Figure 2C
). 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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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:23 normal embryos) were performed (12 cases with day 2 embryos, and three cases with day 3 embryos). Two transfers resulted in early spontaneous abortions (68 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.
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Discussion |
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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 I). 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 I
). 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, 1984; Balakier et al., 1991
) 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, 1984
), 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., 1981
; Zimmermann and Vienken, 1982
; Zimmermann, 1996
). 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., 1991
; Mottla et al., 1995
).
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, 1997), this implies that embryos affected by fusion if transferred to the uteruseither 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, 1994
). On the other hand, little information is available regarding the chromosomal aberrations in human abortuses following cryopreservation (Deffontaines et al., 1994
; Wada et al., 1994
). 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., 1991
). 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 (48%) has been reported in human abortive tissues as well as in born infants (Sheppard et al., 1982; Warburton et al., 1991
). 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., 1982
). 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., 1992
).
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Notes |
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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
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References |
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Submitted on May 18, 2000; accepted on July 31, 2000.