Department of Obstetrics and Gynaecology, Göteborg University, SU/Sahlgrenska, 413 45 Gothenburg, Sweden
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Abstract |
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Key words: aneuploidy/embryo/fluorescence in-situ hybridization/FISH/IVF
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Introduction |
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The study of a single parameter is very valuable when trying to understand better which parameters are most important in an embryo scoring system that includes multiple factors (Steer et al., 1992; Scott and Smith, 1998
). Based on previous observations (Hardarson et al., 1998
) indicating that uneven embryo cleavage negatively affected pregnancy rates, the objective of the present study was to compile results from a database in which information regarding embryo morphology and cleavage rates has been collected for many years. Comparison was then made between transfers where only embryos with evenly cleaved blastomeres were replaced, and those where only embryos with unevenly cleaved blastomeres were replaced. Furthermore, in a small group of good quality embryos that were donated by IVF patients, it was investigated whether a difference in the degree of multinuclear content and/or aneuploidy could be found between these groups, as both of these genetic aberrations have been reported to reduce embryo development (Plachot et al., 1988
; Hardy et al., 1993
). It was postulated that uneven cellular cleavage may result in an uneven distribution of genetic material, and thereby negatively affect both pregnancy and implantation rates in human IVF.
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Materials and methods |
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For statistical analyses, the 2 and Student's t-tests were used. A P value < 0.05 was considered to be significant. The volume of each blastomere was calculated using the average radius (r), according to the following formula: 4/3 r3
.
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Results |
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The clinical results in this study were based on 378 embryo transfers. When comparing the pregnancy and implantation results from grades I, IIA, IIB and IIAB, it was found that there was no significant difference within the even groups (I + IIA) or the uneven (IIB + IIAB) groups. No significant difference was seen in maternal age or number of aspirated oocytes between the groups.
It was found that the unevenly cleaved group of embryos had statistically significant lower pregnancy (P = 0.013) and implantation rates (P = 0.003) than the evenly cleaved group (Table I).
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For the multicolour FISH analyses, a total of 24 unevenly and evenly cleaved human embryos was fixed. It was observed that the unevenly cleaved embryos had a higher aneuploidy rate when compared on the blastomere level, but not per embryo. In contrast, the multinuclear rate was significantly higher, both per blastomere and per embryo (Table II). Furthermore, the rate of anucleate blastomeres in the uneven group (11%) was found to be higher when compared with the even group (2%) (Table III
), though this difference did not reach statistical significance (P = 0.057). The percentage of nuclei not analysed by FISH as a result of failure during fixation and/or the FISH procedure was similar in the even (9%) and uneven groups (11%). It was also noted that all the multinuclear blastomeres but one had nuclei with an abnormal chromosomal number (Table III
).
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Discussion |
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The clinical results in the present investigation are partly supported by those of a previous study in which a significant difference was found when comparing embryos displaying inconsistencies in size and/or abnormalities in shape (Giorgetti et al., 1995). The latter authors, however, did not distinguish between irregularity in shape and size. Also, in line with our results, it has been shown previously (Ziebe et al., 1997
) that uneven embryo cleavage results in a lower implantation rate. However, in that study embryos at different cleavage stages were pooled (2 to >4 cells), while only embryos at the 4-cell stage were included in the present study. Four-cell embryos are expected to have equally sized blastomeres, whereas 3- or 5-cell embryos might well be expected to have one or two larger or smaller cells.
It is interesting to note that slight fragmentation (<20%) does not seem to affect human embryonic development. Using time-lapse photography (own unpublished data), it has been shown that fragments can be reabsorbed into newly cleaved blastomeres, indicating that a slight fragmentation may indeed be normal. The degree of fragmentation observed may thus vary with the time of embryo scoring in the IVF laboratory.
A significant difference was found between the two embryo groups with regard to early embryo cleavage (Table I), this being another indicator of embryo quality and developmental capacity (Shoukir et al., 1997
; Lundin and Söderlund, 1999
). A connection between delayed cell cycle and aneuploidy has been documented in human somatic cells where both monosomic (Nielsen, 1976
; Nielsen and Krag-Olsen, 1980
) and trisomic (Paton et al., 1974
) cells have been shown to cleave at a slower rate than their euploid `counterparts'. This indicates that these embryos have a slightly slower cell cycle already at the zygote stage, possibly due to aneuploidy/multinuclear rate.
It was found that blastomeres originating from embryos with uneven cleavage are more affected by numerical chromosomal aberrations and multinuclear rate than embryos with evenly cleaved blastomeres (Tables II and III). This pattern was not obvious when the overall degree of embryo aneuploidy was compared between the groups, but on examining aneuploidy at a blastomere level it was found not only that there was a higher number of blastomeres affected in each uneven embryo but also that the severity of the aneuploidy and multinuclear rate was higher. Both aneuploidy and multinuclear rate have been shown to have a negative effect on the developmental capacity of the human embryo (Plachot et al., 1988
; Jackson et al., 1998
; Pelinck et al., 1998
). Bi- or abnormal nuclei contribute to cleavage arrest, and these cells are expected to be developmentally incompetent (Hardy et al., 1993
), with only 30% of them cleaving (Pickering et al., 1995
). Possible mechanisms of multinucleation include karyokinesis in the absence of cytokinesis (Hardy et al., 1993
), partial fragmentation of nuclei, or defective migration of chromosomes at the mitotic anaphase (Tesarik et al., 1987
; Winston et al., 1993
), and these might be induced either by changes in temperature or by suboptimal culture conditions (Winston et al., 1991
). It has been suggested (Munné et al., 1995
) that cytoplasmic impairment may produce both mosaicism and polyploidy, through cytoskeletal and spindle malfunction, cellular division block or other mechanisms. It has also been shown that impairment of the spermatozoa, where abnormal zygote centrioles were generated, could produce chromosome abnormalities (Palermo et al., 1994
). Furthermore, aneuploidy has been shown by many investigators to be detrimental to embryo development (Angell et al., 1986
; Bongso et al., 1991
; Munné et al., 1994
, 1995
; Pellestor et al., 1994
). It is therefore suggested that at least part of the explanation as to why uneven embryos implant at a lower rate than evenly cleaved ones lies in the severity of the aneuploidy of the embryo. That is, when aneuploidy is found in an uneven embryo it is more severe, and thus the embryo might be less likely to continue with normal development. Favouring the evenly cleaved embryos when performing the selection for embryo transfer might therefore reduce the possibility of replacing a chromosomally abnormal embryo.
In this study, a total aneuploidy rate of 41.7% (10/24) was found, which supports previous observations that human embryos have a relatively high degree of aneuploidy (Harper et al., 1995; Delhanty et al., 1997
; Munné et al., 1998
; Gianaroli et al., 1999
; Iwarsson et al., 1999
). The fact that such a high percentage (30.8%) of morphologically `good looking' embryos have only one aneuploid blastomere, while the remainder are normal diploids (as far as can be determined by the probes used in the present study), is important to keep in mind when applying methods that try to ascertain the normality of an embryo by single blastomere biopsy, for example in preimplantation genetic diagnosis (Bahce et al., 1999
). A single aneuploid blastomere in a 4-cell embryo (Table III
) could be the result of blastomere fragmentation. If a fragment that is not reabsorbed contains a chromosome, it will be lost when the fragment degenerates, or it may be that a chromosome-containing fragment fuses with another blastomere, leading to trisomy in that cell.
It can be speculated that when a blastomere divides unequally, the two resulting sister cells will receive unequal amounts of, for example, proteins, mRNA, mitochondria and/or different cell organelles. In addition, recent studies suggest that certain proteins and gene products are not evenly distributed, but polarized in the oocyte/embryo (Antczak and Van Blerkom, 1997, 1999
; Edwards and Beard, 1997
), possibly amplifying the effect of such an uneven cleavage. Despite the difficulty in determining whether or not uneven distribution has a negative effect on either the bigger and/or the smaller cell, it seems plausible that a blastomere needs a certain amount of cytoplasmic constituents to sustain itself.
In conclusion, it has been found that when scoring human embryos for embryo transfer, blastomere size is of more importance than slight fragmentation, and that aneuploidy and multinuclear rate may partially explain why unevenly cleaved embryos have lower implantation and pregnancy rates than embryos cleaving evenly. It is suggested therefore that current scoring systems should be modified, perhaps by a reduction to four scoring categories: grade I, embryos with evenly sized blastomeres, with or without fragments (<20%); grade II, embryos with unevenly sized blastomeres with or without fragments (<20%); grade III, embryos with 2050% fragmentation and/or non-homogeneous cytoplasm and/or multinucleate blastomeres; and grade IV, embryos with >50% fragmentation.
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Acknowledgments |
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Notes |
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References |
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Submitted on July 10, 2000; accepted on October 9, 2000.