1 Centre for Reproductive Medicine, European Hospital, Rome, Italy and 2 MAR&Gen, Molecular Assisted Reproduction & Genetics, Granada, Spain
3 To whom correspondence should be addressed at: Centre for Reproductive Medicine, European Hospital, Via Portuense 700, 00149 Rome, Italy. e-mail: rienzi.laura{at}libero.it
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Abstract |
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Key words: ICSI/meiotic spindle/oocyte quality/polar body/Polscope
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Introduction |
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In the present study, the position of the oocyte meiotic spindle was assessed at the time of ICSI, and ICSI results (in terms of the percentage of normally and abnormally fertilized oocytes and of the quality of embryos developing from normally fertilized oocytes) were evaluated separately for groups of oocytes with different degrees of deviation of the meiotic spindle from the polar body position.
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Materials and methods |
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Laboratory procedures
Oocyte and sperm preparation, as well as the ICSI procedure, have been extensively described elsewhere (Rienzi et al., 1998). The cells of the cumulus and corona radiata were removed by a brief exposure to HEPES-buffered medium (Gamete; Vitrolife, Gothenburg, Sweden) containing 20 IU/ml hyaluronidase Fraction VIII (Hyase-10X; Vitrolife) and by gentle aspiration in and out of a hand-drawn glass pipette (diameter 135 µm; Sage BioPharma, NJ, USA). The denudated oocytes were then evaluated to assess their nuclear maturation stage. Oocytes that released the first polar body (metaphase II) were immediately used for meiotic spindle observation and ICSI, while oocytes without a polar body (metaphase I) were cultured in vitro for an additional period of 3 h and used for the study only if matured.
For meiotic spindle observation and ICSI procedure the oocytes were placed in a 5 µl drop of HEPES-buffered medium (Gamete) covered with mineral oil (ovoil; Vitrolife) in a glass-bottomed culture dish (Willco Wells, Amsterdam, The Netherlands) which was maintained at 37°C on a heated stage (Linkam Scientific Instruments Ltd, UK). The meiotic spindle visualization was performed at x20 magnification with LC Polscope optics and controller (SpindleView; CRI, Woburn, MA, USA), combined with a computerized image analysis system (SpindleView software). For this purpose, the oocyte was immobilized at the holding pipette, and rotated with the use of the injection pipette until both the meiotic spindle and polar body were clearly in focus in the oocyte equatorial plane, with the polar body in the 6 oclock position and the spindle in the opposite hemisphere to the injection pipette entry site. ICSI was performed immediately after imaging. In the oocytes without detectable spindles, ICSI was simply performed with the polar body placed in the 6 oclock position.
Oocytes in which the meiotic spindle was detected at the time of ICSI were divided into four groups according to the angle of meiotic spindle deviation from the polar body position, measured in the equatorial plane of the oocyte where both the meiotic spindle and the polar body were in focus (Figure 1). The angle of deviation was 05°, 645°, 4690° and >90° for groups I, II, II and IV respectively.
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Statistical analysis
The 2 test was used to compare oocyte fertilization and embryonic development rates. A P value < 0.05 was considered statistically significant.
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Results |
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The percentages of oocytes that were fertilized normally (two pronuclei), and of those showing abnormal fertilization patterns (one pronucleus or more than two pronuclei) was similar in groups I, II and III (Table I). However, oocytes with a higher degree of spindle deviation at the time of ICSI (group IV) showed a significantly lower normal fertilization rate and a higher rate of abnormal fertilization with a more frequent development of three pronuclei (Table I). All of these abnormally fertilized oocytes failed to extrude the second polar body, indicating a failure of the second meiotic anaphase.
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When embryos that developed from normally fertilized oocytes were graded on day 3 after ICSI, the proportions of excellent, good and fair did not differ between groups of embryos developing from oocytes with different degrees of meiotic spindle deviation at the time of ICSI (Table II).
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Discussion |
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The results of the present study have shown that there is no relationship between the displacement of the first polar body with regard to the meiotic spindle position at the time of ICSI and fertilization outcome when the angle of displacement does not exceed 90°. However, when ICSI was performed with oocytes in which the polar body was displaced from the meiotic spindle position by >90°, higher rates of abnormal fertilization ensued. This was mainly due to a higher occurrence of oocytes with three pronuclei that failed to complete the second meiotic division and did not extrude the second polar body. These data suggest that higher degrees of first polar body displacement with regard to meiotic spindle position are associated with oocyte anomalies causing the failure of the second meiotic anaphase.
When investigating the possible mechanism of this oocyte deficiency, two possibilities must be considered. First, the oocytes in question may have been inadvertently exposed to an abnormally strong mechanical deformation during the process of pipetting at the time of the corona radiata removal. In this case, the resulting oocyte abnormality would be purely artifactual, in a sense that even initially good quality oocytes might fall into this category owing to inadequate manipulation. Alternatively, the displacement of the polar body may result from some oocyte characteristics that facilitate this type of reaction, even if the mechanical stress during the corona radiata removal does not exceed normal. If the latter situation is true, the impaired fertilizability of oocytes with high degrees of polar body displacement is an inherent oocyte characteristic, independent of manipulation.
In the present study the meiotic spindle was detected at the time of ICSI in up to 91% of in-vivo-matured oocytes examined. This value was higher than found in two previous studies which utilized metaphase II human oocytes (Wang et al., 2001a;b) and in which the spindle was detected in 61.4 and 82% of oocytes respectively. This difference can be explained by the fact that, in the present study, oocytes were rotated with the ICSI micropipette around the axis connecting the centre of the oocyte with the first polar body during the attempts at spindle visualization, and the spindles sometimes became visible only after rotation, when the orientation of spindle microtubules became favourable for the visualization of the spindle birefringence.
Only 48 oocytes (9%) were identified in which the meiotic spindle could not be visualized even after this manipulation, and only 16 (33.3%) of these were fertilized normally. These findings were in agreement with previously published data (Wang et al., 2001a;b). Nevertheless, in these studies a substantially greater number of oocytes were found to fall into this category. It is possible that the failure to detect the meiotic spindle in some oocytes without oocyte rotation during observation (Wang et al., 2001a
;b) was occasionally due to a high degree of meiotic spindle displacement with regard to the first polar body location. In fact, as shown in the present study, these oocytes also have a reduced ability to undergo normal fertilization.
Unlike the process of fertilization, the further developmental potential of normally fertilized oocytes did not appear to be related to the degree of meiotic spindle versus polar body displacement at the time of ICSI. This observation suggests that, once normal fertilization has occurred, the position of the meiotic spindle with regard to the first polar body at the time of ICSI does not predict embryo developmental potential and should thus not be used as an additional criterion with which to choose the best-quality embryos for transfer. This relationship has not yet been addressed in the literature, but previous studies have reported on an impaired developmental potential of embryos developing from oocytes in which meiotic spindles failed to be visualized (Wang et al., 2001a;b). In the present study, in which the meiotic spindle was not detected in only 9% of oocytes, this group was too small to draw any valid conclusion on this topic.
In contrast to in-vivo-matured oocytes, those oocytes which were still found to be at metaphase I after cumulus oophorus and corona radiata removal and matured during subsequent in-vitro culture always had the meiotic spindle in a location close to the first polar body. This finding further supports the idea that there is no movement of the spindle after the first polar body extrusion. Unlike the in-vivo-matured oocytes, in which the manipulation for the corona radiata removal is performed after extrusion of the first polar body and can thus be at the origin of polar body displacement, in-vitro-matured oocytes have undergone this manipulation before polar body extrusion, and this may explain the meiotic spindle-to-polar body alignment in 100% of these oocytes.
Interestingly, the percentage of in-vitro-matured oocytes in which the meiotic spindle was not detected was much higher when compared with in-vivo-matured oocytes, though in theory the proximity of the spindle to the first polar body should have facilitated visualization. These data are in agreement with the results of another recent study which showed the presence of a birefringent spindle in only 51.9% of human in-vitro-matured oocytes (Wang and Keefe, 2002). These findings explain the relatively high rates of fertilization and cleavage abnormalities when in-vitro-matured human oocytes were used for conventional IVF or ICSI (Cha and Chian, 1998
).
The oocytes (both in-vivo- and in-vitro-matured) in which the meiotic spindle could not be detected, displayed a higher rate of abnormal fertilization when compared with those in which the meiotic spindle could be detected. In particular, a high percentage of these oocytes developed a single pronucleus after ICSI (16.7 and 25.0% of the in-vivo- and in-vitro-matured oocytes respectively). Moreover, all these oocytes failed to extrude the second polar body after activation, suggesting that the single pronucleus visualized is of paternal origin and that the maternal meiotic spindle could in fact be absent from the cytoplasm or be unable to restart the second meiotic division.
In conclusion, the present study has shown that not only the presence or absence of a detectable birefringent meiotic spindle in metaphase II human oocytes but also the position of the meiotic spindle with regard to the first polar body location at the time of ICSI has a predictive value as to the risk of abnormal fertilization and fertilization failure. This is important for couples who wish their ICSI attempts to be performed with a minimum number of oocytes in order to avoid the destruction or cryopreservation of supernumerary embryos. Although the refusal of supernumerary embryo creation is demanded only rarely by infertile couples for religious or ethical reasons, Italian law is likely to impose a limitation on the number of oocytes used for ICSI in the near future. Oocyte selection taking into account the presence and position of the meiotic spindle by using non-invasive examination with the Polscope system will therefore permit the maximum chance of success in this particular situation.
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References |
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Submitted on January 17, 2003; accepted on March 12, 2003.