RBMOnline - Vol 15. No Reproductive BioMedicine Online; on web 19 July 2007

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1 RBMOnline - Vol 15. No Reproductive BioMedicine Online; on web 19 July 2007 Recent studies of fundamental cryobiology, empirical observations and more systematic clinical experiences have generated a renewed interest in oocyte cryopreservation. Poor survival rate has long been the limiting factor which has prevented widespread adoption of oocyte storage. Slow-cooling and vitrification protocols developed in the last few years have apparently solved this problem, ensuring high recovery of viable oocytes from liquid nitrogen storage. However, the definition of oocyte viability appears rather vague. In fact, post-storage survival as assessed on morphological criteria, indicated by the absence of overt cell degeneration, is not necessarily synonymous with viability. Despite its sensitivity to low temperatures, the meiotic spindle can be preserved after cryopreservation and its constitution after thawing can be monitored non-invasively through polarized light microscopy. Assessment of oocyte cryopreservation via clinical parameters is a daunting task. Most studies are small and difficult to interpret because of confounding factors, such as age, patient selection and quality and strategy of use of the cryopreserved material. Some progress has been made, however, as suggested by recent experiences in which the implantation efficiency of embryos produced from thawed oocytes approaches that reported using cryopreserved embryos directly. Keywords: oocyte cryopreservation, oocyte quality, PolScope, slow freezing Since the mid-1980s and during the successive decade, the cryopreservation of human mature oocytes was reckoned to be unattainable, given the available knowledge and technology. In effect, the first reports of the use of frozen oocytes in the clinical setting were in many respects rather disappointing and overall unable to prove the applicability of this form of fertility preservation. In the last few years, however, studies of fundamental cryobiology, empirical observations and more systematic clinical experiences have generated a renewed interest in this subject. More rational and theoretical approaches could provide the key to the development of methods able to guarantee greater success (Paynter, 2005; Fahy, 2007). Currently, several clinics word-wide offer oocyte cryopreservation as a form of treatment. Italy provides an extreme example where, as an effect of the recent introduction of very restrictive legislation, oocyte cryopreservation has been adopted rather diffusely as an alternative to embryo freezing, and treatment cycles can be counted in their hundreds, if not thousands. This imposes the need for better appreciation of the actual efficiency of oocyte cryopreservation, in order to provide IVF specialists with criteria able to measure their performance and, in the final analysis, guide patients in their choices between well-established and novel treatment options. Poor survival rate has long been the limiting factor that has prevented widespread adoption of oocyte storage. Slow-cooling and vitrification protocols developed in the last few years have apparently solved this problem, ensuring high recovery of viable oocytes from liquid nitrogen storage. However, the definition of oocyte viability appears rather elusive. Paradoxically, in some cases low survival rates based on morphology have been associated with relatively high implantation rates of the 2007 Published by Reproductive Healthcare Ltd, Duck End Farm, Dry Drayton, Cambridge CB3 8DB, UK

2 resulting embryos, while high survival rates have not always coincided with high percentages of clinical success. Therefore, post-storage survival, indicated by the absence of overt cell degeneration, is not necessarily synonymous with functional viability. Embryo cryopreservation poses similar problems, but at least the rate of blastomere loss and the ability to resume cleavage after overnight culture may provide guidance to predict implantation ability. Invasive, non-conservative methods may offer important clues to the degree of preservation of frozen thawed oocytes, but the capacity to assess their quality without affecting their integrity is still limited. During the cryopreservation process, oocytes may be subjected to acute and irreversible damage that manifests as overt degeneration at thawing or shortly afterwards. In other cases, frozen thawed oocytes appear fully intact and visually indistinguishable from their fresh counterparts and as such are considered viable and suitable for treatment. A fraction of thawed material is not classifiable into either of these categories, showing some degree of cytoplasmic anomalies (granularity of the cortical zone, swelling/shrinkage and change in translucency). Fractures in the zona pellucida are thought to be a common cellular alteration, but in reality it is an event rarely observed in experienced hands. Apart from its potential significance in functional terms, the scale of cell damage may be difficult to appreciate. For instance, without information on the original cell size, objective assessment of possible swelling/ shrinkage is a challenging task, also because small changes in diameter implicate large differences in volume. The destiny of these borderline oocytes varies. Depending on the observer, they may be considered viable and suitable for treatment or discarded as abnormal or non-viable. Clearly, this may affect the possibility of comparing data generated from different laboratories. The time point after thawing at which oocytes are assessed is another possible source of bias. Oocytes considered to have survived immediately after thawing may in reality degenerate in the next few hours. This was shown by Gook et al. (1994), who reported that the rate of survival of frozen thawed oocytes decreased from 51% at thawing to 41% following 3 h of culture. A decrease in survival over time perhaps explains why in some studies only a fraction of survived oocytes is in fact microinjected (Yoon et al., 2003, 2007). Therefore, it may be more appropriate to evaluate oocyte survival only after a short culture period (1 2 h) after thawing. Despite the fact that the number of published reports on oocyte cryopreservation has increased progressively over the last few years, many sets of data are of limited value because the origin and/or sample size of the biological material used appear inadequate. The possible influence of the origin, i.e. quality and functional condition, of the starting material on the outcome of cryopreservation is not a problem strictly limited to oocytes. For instance, it is known that cleavage-stage embryos which are morphologically compromised or have been subjected to blastomere biopsy (Jericho et al., 2003) are more sensitive to cryopreservation. In the case of oocyte freezing, this problem can only be enhanced, being reliant on morphological parameters of oocyte quality of rather limited value. Morphology is certainly of no help in predicting the osmotic response to cryoprotectant (CPA) treatment. When oocytes are exposed to a hypertonic solution containing a penetrating CPA, such as ethylene glycol, initially the flux of intracellular water which is drawn out of the cell in response to an osmotic gradient is not counterbalanced by the influx of CPA. This causes a reduction in the cell volume (equivalent to approximately 55% of the original volume in the case of a solution containing 1.5 mol/l ethylene glycol) which can occur homogeneously, preserving the overall cell shape, or rather more irregularly, with loss of the original sphericity (De Santis et al., 2007a). Whether or not the oocyte succeeds in preserving its original shape probably depends on the relative permeability of the oolemma to water and cryoprotectant, as well as on the plasticity of the cytoplasm and the cytoskeleton. These characteristics and the osmotic response which they elicit in the oocyte have been postulated to determine to some extent the later sensitivity to cryopreservation, but unfortunately they cannot be confirmed or predicted by any morphological parameter. This is only one example of the observation that oocytes that appear morphologically comparable respond rather differently to cryopreservation conditions, but is probably sufficient to appreciate how the nature of the biological material can have influence on observation. Possible effects on the cryopreservation outcome of essential patient characteristics, such as aetiology and age, are unknown. Gook et al. (1994) reported that if the number of oocytes recovered at retrieval was greater than 27, after cryopreservation the rate of fertilization was greater than that achieved if the number of oocytes recovered at retrieval was 27 or fewer (68 and 33% respectively). Although it has been suggested that the number of oocytes retrieved affects fertilization rate irrespective of freezing (Pellicer et al., 1989), the fact that oocyte quality can influence the outcome of cryopreservation remains. The current inability to discriminate the intrinsic characteristics which can be relevant to oocyte cryosensitivity make the comparison of different studies very difficult. It is obvious that expansion of sample size somehow would alleviate this problem, but in fact only very few reports described the use of relatively high numbers of frozen thawed oocytes (Borini et al., 2006; Levi Setti et al., 2006; De Santis et al., 2007b). In other cases, the amount of cryopreserved material is much smaller (Table 1). In one extreme case, Kuwayama et al. (2005) in a vitrification versus slow freezing comparative study, included in the slowfreezing group only nine oocytes. It is difficult to appreciate the significance of a reported 22.2% (2/9) survival rate, in the light that other authors described rates in excess of 70% after storing approximately 1000 oocytes with the same protocol (Borini et al., 2006; Levi Setti et al., 2006). This raises the issue of the level of expertise in the application of a given protocol, which increases with experience. Having no previous experience in cryopreservation, in the authors laboratory a progressive improvement has been experienced in post-thaw survival rates over time with different protocols irrespective of their intrinsic efficiency (Figure 1). Obviously, patient selection is another variable which is likely to influence the oocyte cryopreservation outcome. This should be taken into particular consideration in cases where the frozen material derives entirely (Fosas et al., 2003) or in large part (Lucena et al., 2006) from egg donors. The contingent oocyte conditions are also important. For example, excessive extension of the culture period before and/or after

3 Table 1. Sample size (number of patients treated and oocytes thawed) in oocyte freezing studies using different slow cooling protocols. Study No. of No. of No. of enrolled thawed thawed patients cycles oocytes Fosas et al., a 7 88 Borini et al., Chen et al., Li et al., Borini et al., Levi Setti et al., Boldt et al., Bianchi et al., De Santis et al., 2007b Figure 1. Survival rates achieved with different slow cooling protocols at the Vita-Salute IVF Unit, Milan, in the years a Donors. freezing thawing could affect oocyte performance after storage. Ageing in vitro may not only cause perturbances to factors (cell cycle kinases, intracellular calcium stores, cytoskeleton) which are critical for normal fertilization, and lead to deterioration in the original oocyte quality, but more specifically can also alter the overall oocyte response to cryopreservation. This is suggested by the work of Gook et al. (1994) who showed that, after slow freezing, post-thaw survival is higher in human aged oocytes in comparison to non-aged controls (61 and 41% respectively). An effect of in-vitro ageing on post-thaw survival perhaps may be explained with the observation that in human oocytes the oolemma permeability characteristics change over time (Hunter et al., 1992), influencing predictably the exchange of water and CPA during dehydration and rehydration. Oocyte competence is ultimately manifested by the ability to establish and support pregnancy to term, but in part is also expressed by other events occurring during the preimplantation stages of development. Under normal conditions, the ability to undergo fertilization is certainly a major parameter for appraising oocyte competence. This is not always true in the case of stored oocytes. In one of the very first attempts to apply the vitrification methodology to the human field, Hunter et al. (1995) reported a survival rate of 65%. Survived oocytes were mixed with spermatozoa, achieving a fertilization rate of 45%, i.e. not disappointing considering that no sperm microinjection had been employed. However, in all fertilized oocytes, cell division and further development was totally inhibited. Therefore, despite the fact that the fertilization process requires multiple, sophisticated and finely co-ordinated cell functions, its accomplishment is not a guarantee of developmental success. In the case of Hunter et al., it is possible that the cell division block was caused by depolymerization of the cortical actin meshwork which is essential for cytokinesis, but which is known to be sensitive at room temperature to high cryoprotectant concentrations (Younis et al., 1996), conditions that in effect were applied in that study. Newly developed slow-cooling methods generated from the replacement of sodium with choline to prevent the solution effect can give rise to survival and fertilization rates as high as 90 and 74% respectively. However, cleavage rate may be compromised, not exceeding 78% (Stachecki et al., 2006). The same ability to undergo cell cleavage is not always fully informative of the developmental potential of cryopreserved human oocytes. Such a conclusion is suggested by several reports (Borini et al., 2006; Levi Setti et al., 2006; De Santis et al., 2007b). In effect, frozen thawed oocytes may support a high rate of cleavage (90 93%), indicating the ability to undergo at least one cell division by h post-insemination, and regardless may implant with poor efficiency (5 6%). Intriguingly, on the contrary, the ability to resume the mitotic cycle has been recognized to be a factor predicting the implantation ability of frozen thawed embryos (Edgar et al., 2005; Gabrielsen et al., 2006). Little is known about the morphological characteristics of embryos ensuing from cryopreserved oocytes. Although mere morphological criteria, such as proportion of anucleated cytoplasmic fragments, unevenness of blastomere size, multinucleation and cytoplasmic anomalies (small vacuoles, irregular granularity) cannot predict with high accuracy developmental fate and chromosome constitution (Magli et al., 1998), nevertheless they represent general guidance for the selection and transfer of embryos possessing relatively higher chances to implant (Giorgetti et al., 1995; Staessen et al., 1995; Van Royen et al., 1999). Therefore, adequate attention should be paid to embryo morphology in oocyte freezing cases. It seems, however, that mere assessment of embryo morphology does not provide sufficient clues on the implantation ability of frozen thawed oocytes. This may be concluded from a study (Borini et al., 2006) in which, despite the fact that the majority (78%) of embryos developing from frozen oocytes

4 were assigned to the two highest morphological classes (in a scale I IV), implantation rate was disappointing. The inability of sole morphological criteria to predict embryo implantation potential after oocyte cryopreservation is confirmed by results from a recent study (De Santis et al., 2007b), where the comparison of two alternative oocyte slow-cooling protocols led to almost identical patterns of embryo quality but rather different implantation rates. Clearly, further parameters other than morphological normality should be included to adopt preimplantation development as a parameter suitable for assessing the performance of a given oocyte storage protocol. In this respect, the timing of the very first cleavage cycles may be relevant. It has been known for many years that the dynamics of blastomere cleavage are an important signal of implantation potential. Ziebe et al. (1997) established that day 2 embryos with identical morphological score but at different cleavage stages possessed different implantation ability. In particular, the transfer of 4-cell embryos gave rise to a higher implantation rate (23%) in comparison to the transfer of 2- or 3-cell embryos (12 and 7% respectively). It is likely that this is mirrored in two oocyte cryopreservation studies recently conducted by the same group (Borini et al., 2006; Bianchi et al., 2007). Interestingly, two alternative oocyte slow-cooling protocols (including either 0.2 or 0.3 mol/l sucrose in the freezing solution) generated very similar rates of survival (74 versus 75%), fertilization (76 versus 76%), day 2 cleavage (90 versus 93%), and proportion of grade I embryos (45 versus 46%). Nevertheless, the implantation rate of the protocol with lower sucrose concentration was higher (13.5 versus 5.2%). This coincided with a more pronounced representation of 4-cell embryos (42.2 versus 13.6%). In reality, the two studies cannot be rigorously compared because they were not conducted simultaneously and with the intent of prospectively assessing the relative efficiency of the two oocyte freezing protocols. However, they offer the suggestion that a combination of morphological and dynamic criteria of blastomere cleavage could be used as a standard to assess the performance of an oocyte cryopreservation method. Data on embryo development beyond the day 2 stage are even scarcer. Preliminary observations were reported by Gook et al. (1995), but those results are of little current applicative value, having been produced with traditional protocols which are no longer in use. Recently, it was reported by Kuwayama et al. (2005) that variation in the CPA concentration (6.8 versus 5%) in an oocyte vitrification protocol can severely affect the ability to develop to blastocyst (24 versus 50%, respectively), but the significance of these data is uncertain considering that they were derived from very small groups of re-warmed oocytes (23 versus 52). Irrespective of the fact that vitrification data are still incomplete and numerically insufficient, such as methodology is increasingly being perceived as the ultimate answer to the problem of oocyte storage (Vajta and Nagy, 2006). The criteria applied for using the stored material can profoundly influence the clinical outcome of oocyte freezing, and make the comparison between different studies more arduous. Sometimes, religious, ethical or legal restrictions exclude the possibility of generating more embryos than those strictly needed for an embryo transfer. In cryopreserved cycles, the requirement of not producing surplus embryos may be met by thawing only a few oocytes at each attempt, in order to have two or three oocytes viable and suitable for insemination (Borini et al., 2004). Since the introduction in Italy of a new IVF law in 2004, the approach of thawing a small number of oocytes has been adopted in various studies (Borini et al., 2006; Levi Setti et al., 2006; Bianchi et al., 2007; De Santis et al., 2007b). In other circumstances, oocyte thawing cycles may be performed without limitations to the number of embryos which may be transferred and therefore the number of oocytes which may be thawed at each cycle (Chen et al., 2005). The downstream implications of the two situations are clearly different. The thawing of only a few oocytes per cycle involves a higher risk of premature treatment interruption as a consequence of increased incidence of failed fertilization or cleavage (Borini et al., 2004). Besides, the option of embryo selection is not practicable and in some cases the number of transferred embryos may be inadequately low. Obviously, this scenario, which does not occur when an excess of oocytes are thawed in single attempts, can significantly affect the clinical outcome in terms of pregnancy rate per thawing cycle and per transfer, as well as the implantation rate classically defined as the ratio between implantations and embryos transferred. For example, it would be difficult to compare the pregnancy rate of studies in which the mean number of embryos transferred was as different as 1.1 (Borini et al., 2004) and 4.6 (Lucena et al., 2006). An example of the inadequacy of the rate of pregnancy per transfer is offered by a study (De Santis et al., 2007b) in which the conventional slow-freezing method was reported to produce a success rate of 16.7%. In reality, the rate of pregnancy per thawing cycle was much lower (7.7%), with more than 50% of cycles interrupted as a consequence of no oocyte survival after thawing or failed fertilization. Gook and Edgar (1999) proposed a stimulating criterion for the assessment of the relative efficiency of embryo and oocyte cryopreservation cycles. In a previous landmark work, they had shown that in frozen embryo cycles, as a result of cryopreservation-induced total embryo degeneration or partial blastomere loss, about onethird of the expected fresh embryo implantation potential is lost (Edgar et al., 2000). Considering also that, in an IVF/embryo cryopreservation procedure, the original pool of retrieved oocytes is subjected to attrition at various levels (fertilization, cleavage, selection for cryopreservation, thawing), they calculated that about four implantations may be obtained per 100 oocytes collected. They also argued that the same exercise should be made to appraise the efficiency of a given oocyte cryopreservation method in relation to embryo freezing or other storage strategies. Crucially, they stressed the point that the validity of this criterion depends on the condition of including in the calculation all the events of attrition at pre- and post-storage stages. Only under such conditions do certain differences between alternative methods become apparent. For example, it is well known that a major improvement (from to 70 75%) in the survival rate of oocytes frozen and then thawed via slow cooling may be obtained by increasing the sucrose concentration in the freezing solution from 0.1 to 0.3 mol/l. This change also improves the rate of fertilization (Borini et al., 2004, 2006; Levi Setti et al., 2006; De Santis et al., 2007b). However, the different degree of attrition at the steps of survival after thawing and on fertilization in the 0.3 mol/l sucrose protocol is counterbalanced by a higher implantation rate of embryo generated by the protocol involving the lower sucrose

5 concentration (Borini et al., 2004, 2006; Levi Setti et al., 2006; De Santis et al., 2007b). The overall outcome, considered as the proportion of implantations per thawed oocyte, ultimately makes the two methods very similar (2.4 versus 2.6%) and in any case insufficient for competing with embryo freezing. A more recent version of the slow-cooling approach generated a much higher (5.9%) implantation rate per thawed oocyte (Bianchi et al., 2007). It has also been reported that vitrification can produce similar or higher rates (Kuwayama et al., 2005), but failure to report the number of implantations preclude precise assessment of success rate. Implantation rates per thawed oocyte of 5% or higher would outclass the efficiency of embryo freezing. However, they need to be confirmed by much larger numbers, including not hundreds but several thousands of oocytes in multi-centre trials. It is worth noting that one of the most authoritative studies carried out on the efficiency of cryopreserved embryos was based on more than 5000 thawed embryos (Edgar et al., 2000). One of the strongest and more persistent opinions against oocyte cryopreservation resides in the concern that low temperature storage impedes safe preservation of the meiotic spindle. Spindle integrity is obviously functionally of central importance to chromatid segregation during the second meiotic division and represents also the most obvious manifestation of the polarity of the mature oocyte (Albertini and Barrett, 2004). This cytoskeletal component is naturally sensitive to low temperatures. Early evidence showed that murine oocytes transiently cooled to 25 C, 18 C or 4 C undergo spindle disassembly and chromosome dispersal, events that can be reversed in many, but not all, cases after re-incubation at physiological temperature (Pickering and Johnson, 1987). In human oocytes, the spindle appears even more sensitive to low temperatures, considering that over 50% of mature human oocytes lose a recognizable microtubular organization following cooling at room temperature for min and recovery at 37 C (Pickering et al., 1990). Reservations on the possibility of preserving the intact spindle after low temperature storage are therefore well founded, but the actual assessment of the impact of cryopreservation on the oocyte cytoskeletal organization has been so far delayed and complicated by the scarcity of material available for research and the fact that until some years ago spindle studies invariably required the application of destructive techniques, such as immunostaining and fluorescence microscopy. As discussed elsewhere (Coticchio et al., 2005), early fluorescence microscopy studies on the constitution of the meiotic spindle in frozen thawed oocytes generally produced contrasting and inconclusive results, as a consequence of the application of obsolete or inadequate cryopreservation techniques and/or the use of inappropriate material, such as invitro matured oocytes. In more recent years, though, the same kind of microscopy has provided evidence that the overall structure of the meiotic spindle can be safely maintained after cryostorage with protocols specifically designed for the human oocyte. Stachecki et al. (2004), using a protocol involving choline as a substitute for sodium in association with a 0.2 mol/l concentration of sucrose, observed that the frequency of oocytes with a normal barrel-shaped spindle and a regular chromosome alignment was not statistically different between fresh and frozen thawed groups (76.7 versus 69.7% and 76.7 versus 71.2% respectively). Using a slowcooling method including 1.5 mol/l propane-1,2-diol (PrOH) and alternative sucrose concentrations (either 0.1 or 0.3 mol/ l) in the freezing solution, Coticchio et al. (2006) analysed frozen thawed oocytes by confocal microscopy to evaluate the meiotic spindle and chromosome organizations. In the control unfrozen group, 73.1% of oocytes displayed normal bipolar spindles with equatorially aligned chromosomes. Spindle and chromatin organizations were significantly affected (50.8%) after cryopreservation involving lower sucrose concentration, whereas these parameters were unchanged (69.7%) using the 0.3 mol/l sucrose protocol. Therefore, it appears that provided adequate cryopreservation conditions are applied, an intact chromosome segregation apparatus may be retained. The fact that suboptimal cryopreservation conditions may generate damage to the spindle is demonstrated by the finding that after storage using an ethylene glycol-based slow-cooling protocol which does not generate high survival rates, the percentage of normal oocytes is moderately but significantly lower compared with fresh oocytes (53.8 versus 66.7%) (De Santis et al., 2007a). While it is possible to provide detailed information on the meiotic spindle, fluorescence microscopy is characterized by the intrinsic limitation destroying oocyte viability. Therefore, it cannot be adopted in the clinic as a tool for selecting oocytes suitable for treatment. An alternative, non-invasive method which may be employed for detecting the meiotic spindle in oocytes relies on polarized light microscopy. In common with other structures possessing a molecular order, the meiotic spindle is birefringent, i.e. able to cause the resolution of a ray of light into two rays, depending on the polarization of the incident light. This phenomenon, in association with isotropic nature of the surrounding cytoplasm, generates a difference in contrast which makes the spindle potentially visible without fixation and staining. While polarized light has been employed for many decades for the study of mitotic/meiotic spindles of various cell types (Oldenbourg, 1999), its application to human IVF dates back only to recent years (Wang et al., 2001b). This has been made possible after improvement of conventional polarized light microscopy performance with digital imaging and novel electro-optical hardware (commercially developed as PolScope). The non-invasive nature of polarized light microscopy has created new opportunities for the study of the meiotic spindle. In fresh oocytes, several studies have already tested the possible association between spindle presence/location and subsequent fertilization and developmental ability. It has been repeatedly reported that normal spindle morphology coincides with higher fertilization rates (Wang et al., 2001b; Rienzi et al., 2003), although this has not always been confirmed (Moon et al., 2003). Spindle presence appears also to coincide with an increased proportion of high quality embryos (Wang et al., 2001a; Moon et al., 2003). Meiotic spindles can also be assessed using the Polscope quantitatively, by measuring the light wave retardance, the value of which is directly proportional to the density of microtubules organized in a polarized fashion. Quantitative spindle analysis with the PolScope has been attempted only

6 in a few cases. Recently, Trimarchi et al. (2004), reported that spindle retardance is positively associated with blastomere number in day 3 embryos, describing a trend between low retardance, age and compromised oocyte ability to develop into a morphologically normal embryo. In another study, in oocytes of older patients, a trend was observed between low retardance and poor embryo quality (De Santis et al., 2005). Shen et al. (2006), found that mean retardance by spindles in oocytes developing to zygotes with good pronuclear (PN) scores after intracytoplasmic sperm injection (ICSI) was significantly higher compared with spindles of oocytes generating zygotes with a lower PN score and limited developmental potential. Rienzi et al. (2004) reported for the first time the application of the PolScope for the study of oocytes cryopreserved with the conventional slow-cooling protocol (Lassalle et al., 1985) involving a low sucrose concentration (0.1 mol/l) in the freezing solution. Starting from a homogeneous population of fresh oocytes displaying a clearly visible spindle, they observed that immediately after thawing spindle birefringence was retained only in a fraction (35.7%) of oocytes. During the following CPA dilution steps, the spindle was not detectable in any oocytes, reappearing nonetheless after 3 h of culture in all survived oocytes. The fact that the spindle was not visible during the procedure of dehydration may in reality reflect changes in the optical properties of the cytoplasm as an effect of CPA dilution and modifications in ion concentration, rather than a genuine event of microtubule depolymerization. Fluorescence microscopy could contribute to make this matter clearer, being more reliable for the detection of polymerized tubulin. The hypothesis that under the conditions tested all survived oocytes maintain spindle birefringence should be interpreted in the light of a recent confocal microscopy study (Coticchio et al., 2006) which, on the contrary, showed that after freezing-thawing using the same protocol employed by Rienzi et al., 30% of survived oocytes, while still showing polymerized microtubules, do not possess a normally organized spindle. Considering the high degree of detail and reliability of confocal analysis, this inconsistency suggests that polarized light microscopy may not be able to discriminate between microtubular structures with different degree of order, leading to a possible overestimation in a given population of the proportion of oocytes with a normal spindle. Nevertheless, the work of Rienzi et al. (2004) is a valuable example of the possibility to follow via polarized light microscopy the dynamics of spindle polymerization without compromising cell viability. Spindle dynamics and retardance was monitored in frozen thawed oocytes in another study (Bianchi et al., 2005). Fresh oocytes displaying high meiotic spindle birefringence were cryopreserved with a 1.5 mol/l 1,2-propanediol mol/ l sucrose solution. After thawing, spindles were imaged at 0, 3 and 5 h. Spindle birefringence was quantified by classifying microtubule maximum retardance as absent (non-detectable), weak (1.55 ± 0.3 nm) or high (2.50 ± 0.2 nm). Immediately after thawing, only 22.9% of oocytes showed a weak birefringence signal, while only 1.2% of oocytes displayed a high signal. Three hours after thawing, the proportion of oocytes exhibiting a weak or high intensity signal was 49.4 and 18.1% respectively, which did not change significantly after culture for 5 h following thawing. These results confirm that in oocytes stored via slow-freezing protocols, the meiotic spindle undergoes transient partial disappearance immediately after thawing but is reorganized in the majority of oocytes after 3 5 h of culture. The fact that the meiotic spindle retardance was absent (27%) or low (41%) in the majority of thawed oocytes is, again, in partial conflict with confocal microscopy data. In fact, invasive and detailed cytoskeletal analysis (Coticchio et al., 2006) support the hypothesis that after application of the same high sucrose protocol the proportion of oocytes with normal spindles in fresh and frozen thawed oocytes, (the latter observed after 3 h from thawing), is unaltered. The reason for the inconsistency between PolScope and confocal data is not clear. It is conceivable that the use of the PolSscope requires further standardization and refinement. For example, at present, it is not known to what extent different values of retardance, while being suggestive of different degrees of molecular order, in effect can faithfully discriminate between normal and functionally defective spindle conformations. Certainly, polarized light analysis can be particularly valuable for observing changes in microtubular polymerization in real time. After thawing, ascertaining the precise timing of spindle reappearance after possible depolymerization may be crucial in order to set the appropriate time for insemination and avoid oocyte ageing in vitro. In this respect, preliminary evidence has been produced suggesting that after disappearance of the spindle during freezing/thawing, the majority (77.8%) of oocytes regain spindle birefringence within 1 h after completion of CPA dilution steps (De Santis et al., unpublished data). Further culture does not appear to increase significantly the percentage of oocytes with a visible spindle. This adds valuable information to previous studies (Rienzi et al., 2004; Bianchi et al., 2005) concerning the spindle constitution after cryopreservation and suggests more appropriate operational procedures. In particular, it might be appropriate to reconsider the currently accepted procedure to culture frozen thawed oocytes for about 3 h before ICSI (Borini et al., 2004), and to reduce the post-thaw incubation period to a shorter period such as 1 h. Oocyte cryopreservation has been considered with scepticism, essentially because initial attempts conducted more than 2 decades ago produced poor or irreproducible results. Improvements in survival rates, which has represented the major hurdle for many years, have more recently raised new hopes for establishing oocyte storage as a routine treatment. However, in many cases thawed oocytes can survive, fertilize and, cleave with high rates and yet fail to support a high efficiency of implantation. Such a failure is not caused by loss or disruption of the meiotic spindle, which in fact may be found intact after cryopreservation with different protocols. In fact, real time observation after thawing of this cell structure through non-invasive methods is unveiling novel information that could contribute to the optimization of the insemination procedure of thawed oocytes. More in-depth knowledge is needed regarding the preimplantation development of embryos derived from stored oocytes. Concerning this, it has been suggested that a timed pace of day 2 cleavage may underline a better preservation of oocyte quality. Oocyte cryopreservation is becoming an increasingly popular form of treatment, but many studies that claim high success are either small, conducted with selected groups of patients, such as oocyte donors, or carried out with procedures not ubiquitously accepted, such as the transfer of a high number of embryos. Oocyte preservation is an achievable goal, but rigorous and objective criteria for the assessment of its

7 success need to be applied. As was the case with cryopreserved embryos, appraisal of the number of implantations in relation to the original number of oocytes which were frozen and thawed could contribute to the accomplishment of this aim. Albertini DF, Barrett SL 2004 The developmental origins of mammalian oocyte polarity. Seminars in Cell and Developmental Biology 15, Bianchi V, Coticchio G, Distratis V et al Differential sucrose concentration during dehydration (0.2 mol/l) and rehydration (0.3 mol/l) increases the implantation rate of frozen human oocytes. Reproductive BioMedicine Online 14, Bianchi V, Coticchio G, Fava L et al Meiotic spindle imaging in human oocytes frozen with a slow freezing procedure involving high sucrose concentration. 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