Maria Infertility Hospital, Seoul, Korea
1 To whom correspondence should be addressed at: In Vitro Fertilization Laboratory, Maria Infertility Hospital, 10311, Sinseol-dong, Dongdaemun-gu, Seoul, Korea e-mail: mjhee{at}yahoo.co.kr
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Abstract |
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Key words: embryo development/ICSI/MII oocytes/Polscope/spindle
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Introduction |
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The Polscope was recently developed to study spindles in living cells non-invasively (Liu et al., 2000) including in human oocytes (Wang et al., 2001a,b,c
). The Polscope, which combines innovations in polarization optics with novel image-processing software, enables us to image bi-refringent spindles regardless of their orientation (Oldenbourg et al., 1995
). It had no detrimental effects on oocyte maturation and subsequent embryonic development when the Polscope was used to image mouse oocytes (Liu et al., 2000
); so this technique has been successfully used in human assisted reproduction technology (Wang et al., 2001a
,b
,c
). During ICSI, the location of the metaphase II (MII) spindle is commonly assessed in relation to the location of the first polar body. The ICSI needle must avoid the metaphase spindle during sperm injection. However, recent reports showed that the location of the first polar body does not predict accurately the location of the MII meiotic spindle in mammalian oocytes (Silva et al., 1999
) including human oocytes (Wang et al., 2001a
,b,c). Damage to the spindles may happen in some oocytes if the spindles are away from the first polar body. Therefore, the use of a Polscope may be an alternative approach for decreasing spindle damage during ICSI in human IVF.
Wang et al. (2001b,c
) also suggested that the presence of a bi-refringent spindle in the human oocyte predicts higher fertilization rate as well as embryo developmental compe tence after ICSI. They explained the possibility that oocytes without bi-refringent spindles may have abundant chromosomal abnormalities, which in turn might induce cell cycle arrest. Also, they suggested that changes in spindle structure might reflect cytoplasmic dysfunction or other damage to the oocytes (Wang et al., 2001a
,b,c). Although Wang et al., (2001a
,b,c) reported that the spindles were located in various areas during MII, it has not reported whether fertilization and embryo development vary depending on the location of meiotic spindle. In the present study, we examined the embryo developmental competence in the oocytes with various spindle positions and in the oocytes without visible spindles after ICSI.
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Materials and methods |
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Statistics
Statistical analysis was performed by the 2-test.
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Results |
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Discussion |
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We confirm that there was no significance difference in the rate of fertilization, multipronuclear formation and embryonic development between total MII oocytes injected post imaging with the Polscope and controls which were routinely injected with polar body held at 12 oclock or 6 oclock without Polscope. It indicated no detrimental effects of imaging with the Polscope in human.
The rates of fertilization and embryonic development were significantly lower in oocytes without visible spindle than oocytes with visible spindle. Wang et al., (2001a,b,c) suggested that certain environmental changes or stimuli that induce microtubule depolymerization cause spindle damage. For example, a change in ambient temperature can induce temporary disappearance of the spindle and this could also result in poor quality of embryos due to chromosomal abnormalities. They also reported limited spindle recovery after coolingrewarming (Wang et al., 2001a
,b,c). In this study, we examined the bi-refringent spindles in MII oocytes while maintaining the temperature at 37°C for 34 hr. This implies that other factors other than temperature have influenced the presence of spindle birefringence in MII oocytes without spindles.
In this study, we observed that the rate of multipronuclear formation appeared to be higher in oocytes without a bi-refringent spindle than in oocytes with bi-refringent spindle although this failed to be statistically significant. This result suggests that the high rate of multipronuclear formation in oocytes without a spindle may be the result of dispersion of chromosomes into clusters (e.g. in the absence of a spindle) followed by the formation of multipronuclei, each with haploid set of chromosomes. However, the relationship between absence of a spindle and multipronuclear formation after ICSI requires further investigation.
Furthermore, as Silva et al. (1999) and Hewitson et al. (1999
) proposed, it seems that location of the first polar body does not accurately predict the position of meiotic spindle in the hamster oocytes. Wang et al. (2001a
,b,c) also found that the first polar body does not predict the exact spindle position in living human oocytes. As in previous reports, in the present study, we found that 48.2% of oocytes had spindles directly beneath or adjacent to the first polar body, but 11.2% of oocytes had their spindles located in the path of the injection needle if the first polar body was at the 6 or 12 oclock position during conventional ICSI and other oocytes (24.1%) had spindles in different locations. One possible explanation for the various spindle positions is that the first polar body does not remain attached to the oocyte due to removal of cumulus cells and this can cause free rotation of the polar body. Another possibility is that spindle positioning changes due to the fluidity of the membrane area to which the spindle is attached. Further study is required to confirm this suggestion.
There is no difference in fertilization rates and quality of embryos among groups (AE) of oocytes with spindle. This might be due to minimizing the potential for the spindle damage during ICSI at the moment of injection.
In this study, the rate of high quality embryos was higher in oocytes with visible spindle than that in oocytes without visible spindle (P < 0.01). Some explanations are suggested for the lower developmental ability of oocytes without a bi-refringent spindle. It is possible that oocytes without bi-refringent spindles might have abundant chromosomal abnormalities, which in turn might induce cell cycle arrest, although direct confirmation of the association between aneuploidy and spindle structure is needed (Wang et al., 2001a,b,c). It also was reported that in a telophase I or an early prometaphase I stage of meiosis the spindle is still unordered and therefore not bi-refringent and hence cannot be imaged by using the Polscope (Eichenlaub-Ritter et al., 2002
). Furthermore the damage to the spindle caused by ICSI might possibly be one reason for the poor quality of oocytes without bi-refringent spindle compared with those with bi-refringent spindles (Wang et al., 2001a
,b,c), although due to the narrow gauge of the ICSI needle, the chance of damaging the spindle is probably very small. In conclusion, our results indicate oocytes in which a bi-refringent spindle can be visualized may have a higher embryonic developmental competence than oocytes without a bi-refringent spindle and that spindle position does not influence the rate of fertilization or embryonic development. Our results also indicate that the use of a Polscope in combination with ICSI may minimize the mechanical damage to the spindle caused during the ICSI procedure and thus reduce multipronuclear formation.
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References |
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Submitted on September 9, 2002; accepted on December 20, 2002.