Relationship between meiotic spindle location with regard to the polar body position and oocyte developmental potential after ICSI

L. Rienzi1,3, F. Ubaldi1, F. Martinez1, M. Iacobelli1, M.G. Minasi1, S. Ferrero1, J. Tesarik2 and E. Greco1

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


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
BACKGROUND: The recent development of a computer-assisted polarization microscopy system (Polscope) with which the meiotic spindle can be visualized in living oocytes on the basis of its birefringence permits analysis of the meiotic spindles of oocytes subjected to ICSI. Previous studies have shown that the meiotic spindle is not always aligned with the first polar body (PB) in metaphase II human oocytes prepared for ICSI. In the present study, the relationship between the degree of meiotic spindle deviation from the first PB location and ICSI outcome was analysed. METHODS: Oocytes were divided into four groups according to the angle of meiotic spindle deviation from the PB position. The angle of deviation was 0–5°, 6–45°, 46–90° and >90° for groups I to IV respectively. RESULTS: The rates of normal [2 pronuclei (PN)] and abnormal (1PN or >2PN) fertilization did not differ between groups I, II and III. However, the rate of normal fertilization was lower among oocytes in which the meiotic spindle deviation angle was >90°; this led to an increased proportion of tripronucleated zygotes that failed to extrude the second PB. When embryos developed from normally fertilized oocytes were evaluated on day 3 after ICSI, no relationship was found between the angle of meiotic spindle deviation and embryo quality. The meiotic spindle was not detected in only 9% of oocytes, and these showed a higher incidence of fertilization and cleavage abnormalities than did oocytes in which the spindle was detected. When oocytes at metaphase I after cumulus oophorus and corona radiata removal were matured in vitro, the meiotic spindle was detected in 53.8% of those that reached metaphase II. In these in-vitro-matured oocytes the meiotic spindle was always aligned with the first PB, suggesting that misalignment seen in those oocytes matured in vivo resulted from PB displacement during manipulations for cumulus and corona removal. CONCLUSION: High degrees of misalignment between the meiotic spindle and the first PB predict an increased risk of fertilization abnormalities. However, when normal fertilization had occurred, the cleavage potential of embryos developing from such oocytes was not impaired. These findings facilitate the selection of oocytes for ICSI in situations when the creation of supernumerary embryos is to be avoided.

Key words: ICSI/meiotic spindle/oocyte quality/polar body/Polscope


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The orientation of the injection channel and the sperm deposition site during ICSI are usually chosen with regard to the position of the first polar body, assuming that the meiotic spindle with metaphase chromosomes is located in a nearby region of the oocyte cytoplasm. However, recent observations made with the use of a novel orientation-independent polarized microscopy system (Polscope), coupled with image processing software enabling the visualization of meiotic spindles in living oocytes, have shown that the first polar body position does not predict accurately the location of the meiotic spindle in metaphase II mammalian oocytes (Hewitson et al., 1999Go; Silva et al., 1999Go). Following these observations, two studies evaluated meiotic spindles of human oocytes at the time of ICSI (Wang et al., 2001aGo;b). In the first of these studies (Wang et al., 2001aGo), meiotic spindles could be imaged in only 61.4% of oocytes, and more of these oocytes fertilized normally as compared with those in which the meiotic spindle was not found. The second study, in which meiotic spindles were detected in 82% of oocytes, confirmed this relationship and, in addition, more oocytes with detectable meiotic spindles were reported to develop to 4- to 11-cell stages than oocytes without detectable meiotic spindles (Wang et al., 2001bGo). However, the relationship between meiotic spindle location with regard to polar body position and ICSI results has not yet been addressed.

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.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Source of oocytes
The oocytes included in this study were obtained after controlled ovarian hyperstimulation in consenting patients undergoing oocyte retrieval for ICSI with ejaculated sperm. Each couple was given extensive information about the procedure envisaged, including its novelty. This study was approved by the Ethical Committee of the European Hospital, Rome.

Laboratory procedures
Oocyte and sperm preparation, as well as the ICSI procedure, have been extensively described elsewhere (Rienzi et al., 1998Go). 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 o’clock 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 o’clock 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 0–5°, 6–45°, 46–90° and >90° for groups I, II, II and IV respectively.



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Figure 1. Schematic representation (upper) and representative micrographs (lower) of metaphase II oocytes showing different angles of deviation of the meiotic spindle, visualized using the Polscope system, with regard to the location of the first polar body (in the 6 o’clock position). These angles, measured between the line connecting the oocyte centre with the meiotic spindle and that connecting the oocyte centre with the first polar body are 0° in panel I, 6° in panel II, 85° in panel III, and 110° in panel IV. These images represent the four oocyte groups (spindle deviation angles of 0–5°, 6–45°, 46–90° and >90° respectively) that were compared as to the fertilization outcomes and the quality of embryos developing from normally fertilized oocytes. The range of the deviation angles characterizing each of the four groups is shown in the upper panel.

 
Embryo culture
The injected oocytes were cultured singly in IVF medium (Vitrolife) up to day 3. Cleaving embryos were evaluated with the use of a cumulative embryo classification scheme as described previously (Rienzi et al., 2002Go). According to this scheme, an embryo was classified as excellent when it scored 0–1, good when it scored 2–3, and fair when it scored >4. The best-scoring embryos were selected for transfer, while the remaining excellent and good quality embryos were cryopreserved.

Statistical analysis
The {chi}2 test was used to compare oocyte fertilization and embryonic development rates. A P value < 0.05 was considered statistically significant.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In-vivo-matured oocytes
A birefringent meiotic spindle was detected in 484 out of 532 oocytes examined (91.0%). The majority of these oocytes (52.5%) showed a minimal angle deviation (0–5°) of the meiotic spindle with regard to the polar body position (group I). In total, 104 oocytes (21.5%) displayed a moderate angle deviation (6–45°) and constituted group II, while group III comprised 102 oocytes (21.1%) showing a medium (46–90°) angle deviation. The final 24 oocytes (4.9%) showed an angle deviation >90° and comprised group IV.

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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Table I. Relationship between the angle of spindle deviation with regard to the first polar body at the time of ICSI and the percentages of in-vivo-matured metaphase II oocytes developing 1, 2 and >2 pronuclei (PN)
 
The 48 oocytes in which the meiotic spindle was not detected at the time of ICSI (9.0%) showed an abnormally low rate of normal fertilization, which was mainly due to the development of a single pronucleus, observed in eight (16.7%) of these oocytes. Another four oocytes of this group (8.3%) developed more than two pronuclei, and only 16 (33.3%) were fertilized.

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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Table II. Relationship between the angle of spindle deviation with regard to the first polar body at the time of ICSI and the percentages of excellent, good and fair quality embryos developing from normally fertilized oocytes
 
In-vitro-matured oocytes
When oocytes (n = 64) that were found to be at metaphase I after cumulus oophorus and corona radiata removal were cultured in vitro for an additional 3 h, the majority of them (52; 81.2%) extruded a polar body. However, a birefringent meiotic spindle was detected in only 28 (53.8%) of these in-vitro-matured oocytes. Interestingly, the meiotic spindle was aligned with the polar body (0–5° deviation angle) in all oocytes in which it could be observed. Consequently, ICSI results were compared only between oocytes with, or without, a detectable meiotic spindle (Table III). As with the in-vivo-matured oocytes, the absence of a detectable meiotic spindle predicted a higher risk of abnormal fertilization (with the development of a single pronucleus) and a lower incidence of normal fertilization as compared with oocytes in which the spindle was visualized.


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Table III. Relationship between the presence of a detectable meiotic spindle at the time of ICSI and the percentages of in-vitro-matured metaphase II oocytes developing 1, 2 and >2 pronuclei (PN)
 
Embryo quality after ICSI with in-vitro-matured oocytes was poorer than that achieved with in-vivo-matured oocytes, and none of the embryos was classified as excellent. However, the failure of meiotic spindle visualization in in-vitro-matured oocytes was associated with a significantly higher proportion of fair embryos as compared with oocytes in which the spindle was detected (Table IV).


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Table IV. Relationship between the presence of a detectable meiotic spindle at the time of ICSI and the percentages of excellent, good and fair quality embryos developing from normally fertilized in-vitro-matured oocytes
 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
When the meiotic spindle is observed in human in-vivo-matured oocytes that have not been exposed to any manipulation, its location always corresponds to the position of the first polar body (Baca and Zamboni, 1967Go; Dvorak and Tesarik, 1980Go). The displacement of the spindle with regard to the first polar body, observed in both human (Wang et al., 2001aGo) and hamster (Silva et al., 1999Go) oocytes after in-vitro removal of the cumulus oophorus and the corona radiata is thus likely to be consequence of the corresponding manipulation. Because the manipulation required for cumulus and corona removal is unlikely to influence intracellular organelle position, artifactual displacement of the first polar body from its original extrusion site is the most acceptable explanation. In agreement with these observations, it is thus more pertinent to talk about the first polar body displacement with regard to the meiotic spindle position than vice versa.

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., 2001aGo;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., 2001aGo;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., 2001aGo;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., 2001aGo;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, 2002Go). 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, 1998Go).

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.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Baca, M. and Zamboni, L. (1967) The fine structure of human follicular oocytes. J. Ultrastruct. Res., 19, 354–381.[ISI][Medline]

Cha, K.Y. and Chian, R.C. (1998) Maturation in vitro of immature human oocytes for clinical use. Hum. Reprod. Update, 4, 103–120.[Abstract/Free Full Text]

Dvorak, M. and Tesarik, J. (1980) Ultrastructure of human ovarian follicles. In Motta, P.M. and Hafez, E.S.E. (eds), Biology of the Ovary. Martinus Nijhoff Publishers, The Hague, Boston, London, pp. 121–137.

Hewitson, L., Dominko, T., Takahashi, D., Martinovich, C., Ramalho-Santos, J., Sutovsky, P., Fanton, J., Jacob, D., Monteith, D., Neuringer, M. et al. (1999) Unique checkpoints during the first cell cycle of fertilization after intracytoplasmic sperm injection in rhesus monkeys. Nat. Med., 5, 431–433.[CrossRef][ISI][Medline]

Rienzi, L., Ubaldi, F., Anniballo, R., Cerulo, G. and Greco, E. (1998) Preincubation of human oocytes may improve fertilization and embryo quality after intracytoplasmic sperm injection. Hum. Reprod., 13, 1014–1019.[Abstract]

Rienzi, L., Ubaldi, F., Iacobelli, M., Ferrero, S., Minasi, M.G., Martinez, F., Tesarik, J. and Greco, E. (2002) Day 3 embryo transfer with combined evaluation at the pronuclear and cleavage stages compares favourably with day 5 blastocyst transfer. Hum. Reprod., 17, 1852–1855.[Abstract/Free Full Text]

Silva, C.P., Kommineni, K., Oldenbourg, R. and Keefe, D.L. (1999) The first polar body does not predict accurately the location of the metaphase II meiotic spindle in mammalian oocytes. Fertil. Steril., 71, 719–721.[CrossRef][ISI][Medline]

Wang, W.H. and Keefe, D.L. (2002) Prediction of chromosome misalignment among in vitro matured human oocytes by spindle imaging with the PolScope. Fertil. Steril., 78, 1077–1081.[CrossRef][ISI][Medline]

Wang, W.H., Meng, L., Hackett, R.J., Odenbourg, R. and Keefe, D.L. (2001a) The spindle observation and its relationship with fertilization after intracytoplasmic sperm injection in living human oocytes. Fertil. Steril., 75, 348–353.[CrossRef][ISI][Medline]

Wang, W.H., Meng, L., Hackett, R.J. and Keefe, D.L. (2001b) Developmental ability of human oocytes with or without birefringent spindles imaged by Polscope before insemination. Hum. Reprod., 16, 1464–1468.[Abstract/Free Full Text]

Submitted on January 17, 2003; accepted on March 12, 2003.