Article Polar body morphology and spindle imaging as predictors of oocyte quality

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1 RBMOnline - Vol 11. No Reproductive BioMedicine Online; on web 25 April 2005 Article Polar body morphology and spindle imaging as predictors of oocyte quality Dr Lucia De Santis Lucia De Santis graduated in Biological Sciences from the University of Milan, Italy in 1993 collaborating in a research project on the involvement of c-myc oncogene expression in non-small cell lung carcinoma. Concurrently, for 5 years up to 1994, she was associated with the Surgical Pathology Department at the University of Milan, contributing to the Quantitative Pathology Service there and developing extensive experience in DNA flow cytometric analysis of solid tumours and in immunohistochemistry. In 1995 she established the IVF laboratory facilities of S. Raffaele Hospital, also in Milan. Since then she has had a major role in developing a large IVF programme introducing over the years basic and more advanced techniques after attending courses at leading assisted reproduction centres. In January 2005 she was appointed laboratory director. Her current interests concern fundamental and clinical aspects of oocyte cryopreservation. Lucia De Santis 1,3, Ilaria Cino 1, Elisa Rabellotti 1, Federico Calzi 1, Paola Persico 1, Andrea Borini 2, Giovanni Coticchio 2 1 Vita-Salute University, H S. Raffaele, IVF Unit, Milan; 2 Tecnobios Procreazione, Bologna, Italy 3 Correspondence: IVF Unit, Department of Obstetrics/Gynaecology, Vita-Salute University, H S. Raffaele, via Olgettina, Milano, Italy. Tel: Fax: desantis.lucia@hsr.it Abstract It has been suggested that first polar body (PBI) morphology reflects oocyte competence. Oocytes with an intact normalsized PBI have been described as generating better day 2 embryos, higher blastocyst yield, and increased pregnancy and implantation rates. In other studies, PBI morphology was found to be unrelated to fertilization rate, embryo quality, and blastocyst formation. In a prospective analysis, the predictive value of the PBI was investigated by comparing the development of oocytes retrieved from intracytoplasmic sperm injection patients and displaying different PBI morphology, classified according to the following characteristics: normal size and smooth surface (I), fragmented (II), rough surface (III), or large size (IV). Fertilization rates were 59, 57, 64 and 60% respectively. No significant differences were found between the various groups. The proportions of high quality (grade A) day 2 embryos were also comparable among groups I III (14, 12 and 17% respectively), while the low number of grade A embryos in group IV(two embryos) did not allow comparison with the other classes. These data do not suggest that PBI selection can contribute to identification of embryos with high developmental ability. In order to establish alternative criteria for oocyte selection, a metaphase II (MII) spindle analysis was also conducted via Polscope. In oocytes of patients of different age, spindle retardance (which reflects the high order and density of microtubules) was compared with parameters of embryo development. In aged patients, a trend was observed between low retardance and poor embryo quality, although in general the association between retardance and oocyte developmental performance did not reach statistical significance. Keywords: ageing oocytes, first polar body, meiotic spindle, retardance 36 Introduction Advances in fundamental developmental biology and reproductive medicine have indisputably made clear that the single most important factor in determining the outcome of embryo development is the quality of the oocyte. Efficient methods are needed to generate and/or select embryos with superior developmental ability, so as to limit the production of surplus embryos, implement quality control procedures, and ultimately increase clinical efficiency, expressed in terms of rates of implantations, full term pregnancies, and reduction of multiple pregnancies (Gerris and Van Royen, 2000). However, objective criteria for selecting competent oocytes remain elusive, although over 20 years have elapsed since the introduction of IVF for the treatment of human infertility. It is generally accepted that oocyte quality is reflected by easily detectable characteristics, such as the degree of expansion of the cumulus mass, the presence of the PBI, and the absence of cytoplasm and zona pellucida anomalies. However, these features are not sufficient to confer on the oocyte the ability to support a full term pregnancy, appearing unsuitable to establish reliable selection criteria. The acquisition of oocyte competence is a still poorly understood process that unfolds throughout oogenesis (Albertini et al., 2003). In particular, immediately before ovulation, both the nuclear and cytoplasmic compartments are involved in a series of co-ordinated changes. Germinal vesicle breakdown

2 (GVBD), signifying meiotic resumption, and extrusion of the PBI are indicators of such meiotic processes. However, GVBD and PBI emission do not ensure that the oocyte meiotic apparatus has progressed correctly to metaphase II (MII) in preparation for fertilization. Various factors, such as inappropriate in-vitro maturation conditions, can perturb spindle and cytoskeletal organization (Eichenlaub-Ritter et al., 2002; Sanfins et al., 2003), with the consequence that meiosis II segregation and perhaps broader spindle-mediated events may be compromised. At the same time during final maturation, various events take place in the cytoplasmic environment, which may enable the oocyte to support further development. On the basis that PBI extrusion marks an important transition in the acquisition of meiotic competence, various authors have considered the possibility that morphology of this structure may express, at least in part, oocyte competence. Eichenlaub- Ritter et al. (1995) have first proposed that high fragmentation of the PBI could reflect post-ovulatory ageing of the oocyte. A possible link between PBI morphology and oocyte quality is supported also by studies conducted in the mouse, in which genetically modified females are infertile and generate oocytes with abnormally large PBI (Choi et al., 1996). In a series of studies, Ebner et al. extended the hypothesis of a correlation between PBI morphology and oocyte quality, describing that a PBI without signs of fragmentation, with a smooth surface and of normal size, represents a prognostic factor for high fertilization and high-quality embryo rates (Ebner et al., 2000), the ability to develop to blastocyst (Ebner et al., 1999) and increased implantations rates (Ebner et al., 1999, 2002). Other authors have disputed these studies. In particular, Verlinsky (Verlinsky et al., 2003) did not find any correlation between PBI morphology and fertilization rate, average blastomere number in day 2 embryos and blastocyst rate. More recently, Ciotti et al. (2004) have further questioned the evidence produced by Ebner, reporting that embryo quality and clinical outcome are not related to PBI fragmentation. Apart from the polar body, in the last few years the meiotic spindle has also been subjected to investigation in the attempt to establish reliable criteria for oocyte selection. This has been made possible by the optical system referred to as Polscope, a microscopy apparatus that, by using polarized light, allows the observation of highly ordered subcellular structures, such as the spindle microtubules in mammalian oocytes. The Polscope offers the unique advantage of being totally noninvasive, preserving oocyte viability (Keefe et al., 2003). Spindle function is widely recognized with respect to chromosome segregation during meiosis, while it is not always appreciated that this structure is also crucial for fertilization and establishment of oocyte polarity. While the Polscope cannot provide accurate information on the fine structure of the spindle, on the other hand spindle detection can be conducted quantitatively, since retardance, the fundamental quantity measured by the instrument, is directly proportional to the density of microtubules organized in a highly ordered structure (Oldenbourg et al., 1998). Measurements of such a parameter may have clinical relevance because it is conceivable that microtubule density may influence spindle function, with obvious consequences on metaphase chromosomes alignment and segregation during meiosis II. Because evidence on the significance of PBI morphology and spindle detection in relation to oocyte quality remains controversial, this paper aimed to verify the association between PBI morphology with embryological parameters. The study has also investigated possible developmental implications of varying degrees of intensity of meiotic spindle birefringence measured via Polscope analysis. Materials and methods Source of oocytes The oocytes included in this study were obtained after ovarian stimulation induced in 382 consenting patients undergoing oocyte retrieval for intracytoplasmic sperm injection (ICSI) with ejaculated spermatozoa. The study was approved by the local Institutional Ethical Committee. Couples were referred to the ICSI programme for male infertility or previous fertilization failure. The mean patients age was 35.0 ± 4.9. Ovarian stimulation was conducted according to a long protocol using gonadotrophin-releasing hormone agonist (Enantone ; Takeda, Osaka, Japan) and recombinant FSH (Puregon ; Organon or Gonal F ; Serono, Geneve, Switzerland). Ovulation was triggered with human chorionic gonadotrophin (HCG; Ovitrelle or Gonasi ; Serono). Oocytes were collected transvaginally under ultrasound guidance at 36 h after HCG administration. According to Italian IVF law, a maximum of three oocytes per patient were inseminated while spare mature oocytes were cryopreserved if requested by patients (Cheung et al., 2000; Borini et al., 2003, 2004). Oocyte preparation Oocyte and sperm preparation, as well as the ICSI procedure, have been thoroughly described elsewhere (Van de Velde et al., 1997). Briefly, cumulus and corona radiata cells were immediately removed after retrieval by a short exposure to HEPES-buffered medium (Sage IVF Inc., Trumbull, CT, USA) containing 20 IU/ml hyaluronidase (Sage) and gentle aspiration in and out of a hand-drawn pipette (Flexi-PetTM; Cook, Queensland, Australia) and mechanically cleaned from the remaining surrounding cumulus cells by aspiration using a denuding pipette with a µl diameter (Denuding Flexi- PetTM; Cook). The denuded oocytes were then assessed with respect to their meiotic maturation status. In preparation for ICSI, oocytes with an extruded PBI (presumably at the metaphase II stage) were selected for meiotic spindle detection and PBI morphology assessment, while oocytes without a polar body or presenting a germinal vesicle (GVstage) were excluded from the study. Oocyte and embryo classification The classification of PBI morphology was performed using an inverted microscope (1 70 Olympus, Hamburg, Germany). In particular, oocytes were attributed to different groups depending on whether they showed a PBI with normal size and smooth surface (I), rough surface (II), fragmented (III) or large size (IV) (Van de Velde et al., 1997). 37

3 Embryo morphology was assessed h after insemination and classified using the criteria described by Veeck (1999), with minor modifications. Group A included embryos with at least four evenly shaped blastomeres, <20% fragmentation, and absence of multinucleation. Group B included embryos with at least four blastomeres of different size, irregular shape and over 20% fragmentation. Embryos showing fewer than three irregular blastomeres or more than 50% fragmentation were attributed to group C. Meiotic spindle analysis For meiotic spindle observation and ICSI procedure, oocytes were placed in 5 µl drops of HEPES-buffered medium (Sage) covered with mineral oil (Sage) on a glass-bottomed culture dish (Will-Co dish; Intracel, Royston, Herts, UK), which was maintained at 37 C on a heated stage (Labotech, Gottingen, Germany). Meiotic spindle visualization was performed at 200 magnification with LC Polscope optics and controller (SpindleView; CRI, Woburn, MA, USA), combined with a computerized image analysis system (SpindleView software). Oocytes were observed at an equatorial plane and spindle maximum retardance was measured along two, longitudinal and equatorial, lines (Sun et al., 2004). Statistics The chi-squared test was used to compare oocyte fertilization and embryonic development rates. A P-value <0.05 was considered to be statistically significant. Results PBI morphology Eight hundred and seventy-three oocytes derived from 382 patients were classified according to PBI morphology. Fertilization rates were not statistically different between the four PBI classification groups, corresponding to 59, 57, 64 and 60% respectively. Frequencies of high quality (grade A) day 2 embryos were also comparable between groups I, II and III (14, 12 and 17% respectively), while the low number of grade A embryos in group IV(two embryos) did not allow statistical analysis in comparison to the other classes (Table 1). With respect to high quality embryos (A embryos), the contributions of oocytes showing the four PBI classifications were: 39, 25, 34 and 2% respectively (Table 2). Meiotic spindle retardance The meiotic spindle was detected at the time of ICSI in over 80% (n = 78) of the oocytes examined. In one case a spindle structure was found to be partly localized in the cytoplasm and the PBI, indicating that chromosome segregation had not been completed (Figure 1A, B). Average of maximal spindle Table 1. Frequency of fertilization and embryo quality categories compared with polar body I (PBI) morphology groups. Observations represent the number of oocytes assessed for PBI morphology. % Polar body grade (n) I II III IV Embryo quality (n) A (126) 24 (49) 22 (32) 27 (43) 13 (2) B (225) 47 (95) 39 (57) 39 (63) 67 (10) C (174) 29 (58) 39 (58) 34 (55) 20 (3) Fertilization (525) 59 (202) 57 (147) 64 (161) 60 (15) Observations (873) (340) (257) (251) (25) Table 2. Contribution to each embryo quality category by different PBI morphology groups. Polar body grade (n) % Embryo quality (n) A B + C I (202) 39 (49) 38 (153) II (147) 25 (32) 29 (115) III (161) 34 (43) 30 (118) IV(15) 2 (2) 3 (13) 38

4 Figure 1. Human oocytes with an extruded PBI showing (A) a fully formed MII spindle and (B) a spindle in which chromosome segregation has not been completed. Table 3. Relationship between patient age and maximal spindle retardance. Differences between age groups were not significantly different. Age (years) Retardance (nm) Observations (n) Table 4. Relationship between spindle retardance and embryo quality. Embryo quality A B + C Retardance (nm) 2.74 a 2.36 a % Observations (n) 22 (8) 78 (28) a Not significantly different (P = 0.2). retardance in oocytes of patients aged 34, and 40 was 2.41 ± 0.90, 2.36 ± 0.85, 1.94 ± 0.59 respectively. These differences were not statistically significant, but a trend was observed between older age and lower retardance (Table 3). Similarly, no significant relationship was found between average spindle retardance of oocytes and the quality of the ensuing embryos (2.74 ± 1.17 and 2.36 ± 0.74, classes A and B + C respectively, Table 4), although it should be noted that a higher number of oocytes would be required to draw more definite conclusions. Discussion Embryo selection is implemented on day 2 or 3, on the basis of the number, shape and relative size of blastomeres, proportion of anucleated fragments, presence of multinucleated blastomeres and various cytoplasmic abnormalities (Boiso et al., 2002), in order to optimize clinical efficiency while reducing the risk of multiple pregnancies. Considering the pivotal role played by the oocyte in the developmental process, it is understandable that attempts have also been made to identify selection criteria involving stages preceding fertilization. Ovarian stimulation is clearly fundamental in IVF treatment. However, this comes at the cost of recovering oocytes of very heterogeneous nature, some of which are unable to support normal oocyte growth. Oocytes often display various dismorphisms, including non-spherical shape, thick or distorted zona, intense central cytoplasmic granularity, enlarged perivitelline space (PVS), and scattered cytoplasmic fragments in the PVS. Unfortunately, studies aiming at establishing a relationship between morphological features and developmental potential of the oocyte have been inconclusive (Serhal et al., 1997; Balaban et al., 1998). It has been proposed that PBI morphology may reflect oocyte competence. Initial evidence in this respect has been provided by a study in which PBI degeneration has been found to be associated with post-ovulatory ageing in vitro and inadequate fertilization competence, as shown by premature sperm chromosome condensation (Eichenlaub-Ritter et al., 1995). In a prospective randomized study, Ebner et al. (1999) have examined the possibility of predicting embryo developmental potential by observing PBI morphology in oocytes of ICSI patients, finding that implantation and pregnancy rates are higher when embryos for transfer are preferentially chosen among those originating from oocytes with a normal-sized well-shaped PBI. The same authors further expanded their observations, reporting that regular PBI morphology is 39

5 40 indicative of improved embryo quality on day 2 (Ebner et al., 2000), development to blastocyst and implantation rate after blastocyst transfer (Ebner et al., 2002). These conclusions have not found confirmation in other studies published more recently. Verlinsky was unable to ascertain an association between PBI morphology and either day three embryo morphology or development to blastocyst, reporting also that aneuploidy rates in oocytes and embryos is unrelated to PBI status (Verlinsky et al., 2003). In addition, Ciotti et al. (2004) have disputed Ebner s data, providing results that argue against a predictive value of PBI morphology with respect to fertilization ability, embryo quality and clinical outcome. Considering the highly controversial conclusions of these data, it was decided to conduct a prospective analysis of possible relations between PBI morphology and oocyte competence expressed as fertilization ability and generation of high-quality embryos. Rates of fertilization were similar irrespective of PBI size, degree of fragmentation or smoothness of the surface. Similarly, embryo quality on day 2 appeared to be comparable in groups including oocytes with PBI having normal size and smooth surface, showing fragmentation or rough surface. The proportion of high quality embryos in the group of oocytes with large PBI was low, but the small number of embryos available for statistical analysis did not allow objective comparison. This study is being extended to establish whether oocytes belonging to diverse PBI morphology categories can give rise to different clinical outcomes. This will require a large increase in the number of cases, considering that it will be necessary to compare groups characterized by the transfer of homogeneous classes of embryos. Such a study is difficult to pursue in Italy, after the recent introduction of an IVF law that does not allow the insemination of more than three oocytes. The trend indicating a possible link between large PBI and compromised development perhaps deserves particular attention and the accumulation of more data. In fact, the possibility that a large PBI size corresponds to poor day 2 embryo quality had also been suggested by Xia (1997) and Ebner et al. (1999). Experiments conducted in animal models are consistent with the hypothesis that the formation of atypically large PBI may conceal more important developmental perturbances (Verlhac et al., 2000). Asymmetric cell division is a fundamental mechanism contributing to cell diversification during embryonic development. In mammalian oogenesis, unequal cell division that gives rise to PBI formation is functional to the meiotic process as well as the need to ensure that most of the maternal store is preserved in the oocyte rather than being lost in a structure than does not contribute to the formation of the embryo. This mechanism is altered in genetically modified mos / mice (Choi et al., 1996; Verlhac et al., 2000). In these animals, inability to express the mos gene product, a serine threonine kinase, prevents the activation of mitogen-activated protein kinase (MAPK), a cell cycle regulator that promotes metaphase I (MI) to MII transition. Under these conditions, oocytes often extrude an abnormally large PBI, failing also to arrest at the MII stage, with the results that animals bearing this genetic modification are subfertile. Emission of a large polar body is caused by inability of the meiotic spindle to migrate from a slightly off-centre position, where it is assembled to the periphery position, where it normally coordinates PBI extrusion. Because position of the spindle main axis dictates the orientation of the cleavage furrow, a centrally located spindle leads to formation of a large PBI. Also, MAPK deficiency has consequences on the structure of the meiotic spindle that appears abnormally elongated. Failure of the spindle to migrate properly may have far-reaching consequences that go beyond PBI formation, considering that MII spindle position directs also the cleavage plane of the first mitotic division (Cooke et al., 2003). Apart from assessing the significance of PBI morphology, in a proportion of oocytes the possible implications of meiotic spindle retardance were also studied. The application of polarized light microscopy has generated a new dimension in the assessment of oocyte quality (Rienzi et al., 2005). Spindle presence is obviously an essential requisite for chromatid segregation during the second meiotic division. The spindle is also critical for fertilization, as suggested by the fact that mouse oocytes fail to fertilize after treatment with agents that cause spindle depolymerization (Winston et al., 1995). Using the Polscope, several studies have already explored the possible association between spindle presence and location with respect to the oocyte ability to undergo fertilization and generate morphologically normal embryos. Generally, it has been reported that spindle presence coincides with higher fertilization rates (Wang et al., 2001b; Rienzi et al., 2003), although at least one study has not confirmed this relationship (Moon et al., 2003). Spindle presence appears also to be associated with an increased proportion of high quality embryos (Wang et al., 2001a; Moon et al., 2003). However, simple observation in terms of presence or absence of the spindle does not seem to provide a particularly valid advantage for the selection of embryos with higher developmental potential. In fact the proportion of oocytes showing the meiotic spindle has been described to be as high as 90%. So, while spindle absence is probably an important prognostic negative factor, presence of the spindle is of limited significance for the establishment of oocyte quality. It is important to note that different studies have described different rates (61.8 to over 90%) of oocytes displaying the meiotic spindle. Despite the fact that differences in the technique of PBI observation may account for these discrepancies, other procedural diversities in the use of the Polscope may influence the percentage of oocytes in which the spindle can be detected. For example, it has been shown that in oocytes on which spindle analysis is conducted at least 38 h following HCG administration, spindle visualization occurs with higher frequency compared with oocytes in which this analysis is conducted at an earlier time (81.5 versus 61.6%) (Cohen et al., 2004). This may also justify the fact that fertilization can occur in oocytes in which a birefringent spindle is not visible. Oocytes with an extruded polar body may be at a meiotic stage that precedes MII, typically telophase I or prometaphase II (Figure 1B). If cultured for a few hours, they can support spindle organization and normal fertilization. Collectively, these data suggest that simple observation of spindle presence is insufficient to predict oocyte quality accurately. In effect, confocal microscopy studies have demonstrated that the meiotic spindle fine structure, in terms of size and shape, and the distribution of associated proteins can vary considerably between oocytes that express different developmental ability. For example invitro matured mouse oocytes show large barrel-shaped spindles, while oocytes matured in vivo have smaller spindles with highly focused poles (Sanfins et al., 2003). Using the Polscope, it would be difficult if not impossible to detect

6 similar differences in human oocytes, considering also that in humans, the meiotic spindle is relatively small compared with the mouse (Eichenlaub-Ritter et al., 2002; Stachecki et al., 2004). Despite the intrinsic limitations of the Polscope, it has been recently described that also in the case of the human oocyte spindles can be assessed qualitatively, by measuring the retardance, whose value is directly proportional to the density of microtubules organized in a polarized fashion. Sun et al. have been able to shown that an increase in the culture temperature of human oocytes causes a decrease in spindle retardance that is suggestive of partial loss of organization (Sun et al., 2004). This possibility is consistent with the finding that after restoration of normal temperature, spindle shape appears altered. The present study tested the hypothesis that differences in spindle organization existing in oocytes of patients of varying age, as previously ascertained through fluorescence microscopy (Battaglia et al., 1996), may be detected via Polscope analysis and influence the morphological characteristics of day 2 embryos. It was found that despite differences in spindle retardance not being statistically significant in diverse age groups, a trend was observed between low retardance, age and compromised oocyte quality in terms of ability to develop into a morphologically normal embryo. Quantitative spindle analyses conducted on human oocytes through the Polscope are still rare. Recently, Trimarchi et al. have proposed that spindle retardance can predict blastomere number in day 3 embryos (Trimarchi et al., 2004). Bianchi et al. have also suggested that after cryopreservation, the meiotic spindle is reorganized after transient disappearance, but its retardance is significantly decreased in the majority of frozen thawed oocytes (Bianchi et al., 2005). These preliminary attempts of quantitative evaluation of the meiotic spindle using the Polscope are not conclusive, but suggest alternative routes for the identification of novel criteria able to predict oocyte quality. To this end, standard procedures will need to be established to make quantitative Polscope analysis an objective assessment. In conclusion, the present observations confirm the difficulty in determination of oocyte quality. The data do not support the hypothesis that PBI morphology is indicative of developmental potential. However, it cannot be ruled out that more extensive analysis may reveal that oocytes with large PBI are developmentally compromised, a hypothesis that appears to be confirmed by experiments conducted in the mouse. 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