Hum. Reprod. Advance Access originally published online on January 29, 2004
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Human Reproduction, Vol. 19, No. 3, 655-659,
March 2004
© 2004 European Society of Human Reproduction and Embryology
Polscope analysis of meiotic spindle changes in living metaphase II human oocytes during the freezing and thawing procedures
1 Centre for Reproductive Medicine, European Hospital, Via Portuense 700, 00149 Rome, Italy and 2 MAR&Gen, Molecular Assisted Reproduction & Genetics, Granada, Spain
3 To whom correspondence should be addressed. e-mail: rienzi.laura{at}libero.it
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Abstract |
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BACKGROUND: The clinical efficacy of the current methods used for cryopreservation of metaphase II human oocytes is low. Meiotic spindle disorders are thought to be largely responsible for this situation. METHODS: Supernumerary fresh metaphase II human oocytes were cryopreserved in 1,2-propanediol with 0.1 M sucrose using a slow freezing/rapid thawing programme. Meiotic spindles were analysed in these living metaphase II oocytes at sequential steps of the freezing and thawing procedures with the use of a computer-assisted polarization microscopy system (Polscope). RESULTS: The meiotic spindle was detected in all 56 oocytes (from 16 patients) before freezing and remained visible in all these oocytes throughout the preparation for freezing up to the time that they were loaded into cryopreservation straws. Immediately after thawing, the spindle was visible in 35.7% of oocytes, but it disappeared in all of the thawed oocytes during the subsequent washing steps. However, the spindle reappeared in all surviving thawed oocytes after washing (57.4%), by 3 h of incubation at 37°C in culture medium. CONCLUSIONS: The current techniques of oocyte freezing and thawing inevitably cause meiotic spindle destruction. All spindles observed in thawed oocytes result from post-thaw reconstruction.
Key words: freezing/meiotic spindle/metaphase II/Polscope/thawing
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Introduction |
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Oocyte cryopreservation has been used in human assisted reproduction since the late 1980s (Chen, 1986
; Al-Hasani et al., 1987; Fabbri et al., 1998; Mandelbaum et al., 1988; Tucker et al., 1998). However, this technique met with only limited success when conventional IVF was used because the freezing and thawing procedures caused the occurrence of a premature cortical reaction (Schalkoff et al., 1989) leading to hardening of the zona pellucida and reduced sperm penetration (Johnson et al., 1988; Todorow et al., 1989). The introduction of ICSI (Palermo et al, 1992) made it possible to circumvent the negative consequences of zona pellucida hardening for fertilization. However, cryopreservation has also been shown to produce alterations in the meiotic spindle of metaphase II mouse (Magistrini and Szollosi, 1980; Sathananthan et al., 1988a; Van der Elst et al., 1992) and human (Sathananthan et al., 1988b; Pickering et al., 1990; Gook et al., 1995) oocytes, and this problem obviously cannot be overcome by the use of micromanipulation-assisted fertilization.
Abnormalities of the metaphase II meiotic spindle can be expected to lead to abnormal segregation of chromosomes after fertilization of cryopreserved oocytes, resulting in aneuploid embryos. However, a recent study failed to find any significant differences in aneuploidy rate between embryos derived from cryopreserved oocytes as compared with those developing from fresh oocytes (Cobo et al., 2001). These surprising findings could be explained either by the existence of a mechanism preventing complete spindle disintegration during the freezing and thawing procedures or by a rapid reconstruction of the cryo-damaged spindles.
Most studies dealing with the status of the meiotic spindle carried out so far used electron microscopy or immunocytochemistry techniques (reviewed in Eichenlaub-Ritter et al., 2002). However, this approach requires oocyte fixation and thus cannot be used to follow dynamic changes in the same oocytes at different time points. In this study, we employed the Polscope system, which permits non-invasive visualization of spindles in living oocytes (Liu et al., 2000; Wang et al., 2001a; Moon et al., 2003) to follow the behaviour of the metaphase II meiotic spindle at different steps of the freezing and thawing procedures.
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Materials and methods |
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Oocyte source and preparation
This study was approved by the Ethical Committee of the European Hospital.
Metaphase II oocytes were obtained from consenting patients from our IVF-ICSI programme only when an adequate number of oocytes was retrieved.
Ovarian stimulation was conducted in all patients as described previously (Ubaldi et al., 1999) using a combination of GnRH agonist (subcutaneous buserelin acetate 0.2 mg twice daily; Suprefact; Hoechst, Marion Roussel, Milan, Italy) started in late luteal phase of the previous cycle, recombinant FSH (Gonal-F, 75 IU, Serono, Rome Italy or Puregon 100 IU, Organon, Oss, The Netherlands) and HCG (Profasi; Serono).
Oocyte decumulation has been described extensively elsewhere (Rienzi et al., 1998). Briefly, the cumulus and corona radiata were removed immediately after ovum pick-up 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 plastic pipette (Flexipet, 130 µm i.d., COOK, Australia). The denuded oocytes were then evaluated to assess their nuclear maturation stage. Only oocytes that had released the first polar body (metaphase II) were included in this study. Immediately after decumulation, the supernumerary metaphase II oocytes obtained were used for meiotic spindle observation and the freezing procedure.
Freezing and thawing procedures
The freezing solution consisted of phosphate-buffer saline (PBS) with 25 mg/ml human serum albumin (HSA; Cryo-PBS, Vitrolife), freezing solution 1 containing 1.5 M 1,2-propanediol (PrOH) in Cryo-PBS (FS1, Vitrolife) and freezing solution 2 containing 1.5 M PrOH + 0.1 M sucrose in Cryo-PBS (FS2, Vitrolife). A maximum of three oocytes were frozen per straw. Equilibration with the freezing solutions was carried out at room temperature. Oocytes were first incubated for 2 min in Cryo-PBS, then for 10 min in freezing solution 1 and finally in freezing solution 2, and immediately loaded into straws (paillette cristal, Cryo Bio System, IMV Technologies, France). The straws were placed into a freezing chamber (CL-863, Cryologic, Australia) at room temperature. The temperature was decreased to –7°C at a rate of 2°C/min. Seeding was done manually by touching the straw close to the cotton plug with a nitrogen-cooled forceps. After holding for 10 min at –7°C, the temperature was lowered slowly at a rate of 0.3°C/min to –30°C, followed by rapid cooling (–10°C/min) to –110°C. The straws were then plunged into liquid nitrogen (LN2), and stored submerged in LN2 until thawing.
For thawing, the straws were removed from LN2 and rapidly warmed to room temperature. The retrieved oocytes were then transferred to a plastic 4-well dish where they were washed free from the cryoprotectant at room temperature by stepwise dilutions in 1.0, 0.5 and 0.0 M PROH in the presence of 0.1 M sucrose and Cryo-PBS (Thaw Kit 1, Vitrolife). The exposure times for thawing solutions were 5 min (solution 1), 10 min (solution 2), 10 min (solution 3) and 10 min (PBS). All surviving oocytes (with an intact membrane and clear cytoplasm) were cultured further in IVF-medium (Vitrolife) at 37°C, 5% CO2 for 3 h.
Spindle visualization by Polscope
Meiotic spindles were imaged at sequential steps during the freezing–thawing procedure. Briefly, the oocytes were placed in a 5 µl drop of the corresponding medium covered with mineral oil (ovoil, Vitrolife, Gothemburg, Sweeden) in a glass-bottomed culture dish (Willco Wells, Amsterdam, The Netherlands). The meiotic spindle visualization was performed at 20x magnification with LC Polscope optics and controller (SpindleView, CRI, Woburn, MA), combined with a computerized image analysis system (SpindleView software, CRI). For this purpose, each oocyte was rotated with the use of the injection pipette until both the meiotic spindle and the polar body were clearly in focus in the oocyte equatorial plane.
The oocytes were maintained at 37°C on a heated stage (Linkam Scientific instruments Ltd, UK) during the spindle assessment in HEPES-buffered culture medium (Gamete, Vitrolife) performed before starting the freezing procedure and at the end of the thawing procedure. All meiotic spindle observations at different steps of the freezing–thawing procedure were performed at room temperature.
Spindle visualization by immunocytochemistry
All chemicals used were purchased from Sigma (St Louis, MO). Oocytes were treated with 0.5% pronase E from Streptomyces griseus type XIV and fixed with 4% formaldehyde in PBS. The fixed oocytes were then rinsed in PBS and permeabilized using Triton X-100, 0.02% in PBS. After washing in PBS, the oocytes were incubated in blocking solution containing 5% HSA. Oocytes were double-stained for microtubules and chromosomes by incubation in fluorescein isothiocyanate (FITC)-conjugated monoclonal anti-
-tubulin diluted 1:100 in PBS followed by incubation in 10 µg/ml of propidium iodide in PBS. Oocytes were then mounted on poly-L-lysine-treated slides that had a peripheral epoxy ring, covered and sealed with clear nail varnish, and examined in a Nikon Eclipse E600 fluorescence microscope.
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Results |
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The meiotic spindle was visualized in all of the 56 metaphase II oocytes used in this study obtained from 16 different patients. As the oocytes became progressively dehydrated during equilibration with the freezing solutions, the spindles not only remained detectable in all oocytes in spite of the temperature decrease but even became brighter, with the maximum brightness observed in freezing solution 2 (Figure 1). Thereafter, the oocytes were loaded into cryopreservation straws and the behaviour of their spindles could not be evaluated any more.
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Immediately after the removal of the straws from liquid nitrogen and expulsion of the oocytes from the straws, a meiotic spindle was detected in 20 out of the 56 oocytes examined (35.7%). However, 10 (50%) of the oocytes did not survive throughout the subsequent steps of the thawing procedure, during which cryoprotectant was progressively washed out, and these oocytes were discarded from further analysis. When the oocytes that survived the entire thawing procedure were taken into account, the overall percentage of oocytes in which the meiotic spindle was detected was 31.3% in thawing solution 1, 25.0% in thawing solution 2, 0% in thawing solution 3, 0% in PBS and 100% after an additional 3 h of incubation at 37°C in culture medium.
The timing of the changes in meiotic spindle morphology was not uniform in individual oocytes (Table I). In most oocytes in which the spindle was visualized in thawing solution 1 immediately after expulsion from cryopreservation straws, the spindle disappeared during incubation in thawing solution 3 (Figure 2). However, the disappearance of the spindle as early as during incubation in thawing solution 2 was also observed (Table I). At the end of incubation in thawing solution 3, there was not a single oocyte in which the spindle could be detected. However, the spindle reappeared again by 3 h of incubation at 37°C in culture medium in all of the oocytes that survived the entire procedure and in which it was observed immediately after the expulsion from the cryopreservation straws (Table I). Meiotic spindle re-appearance after thawing solution removal was also observed in all oocytes in which the spindle was undetectable in thawing solution 1 immediately after thawing (Figure 3, Table I). The position of the reconstructed spindle with regard to the first polar body was the same as that of the original, pre-freeze spindle position in all oocytes (Figure 2).
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To assess the possible effects of the thawing media on meiotic spindle visualization, fresh oocytes were incubated for 10 min in thawing solutions 1, 2 and 3 (three oocytes in each). The spindle remained clearly visible in all of these oocytes at the end of incubation (Figure 4), and its brightness was even greater than before incubation in all cases. Thus, the disappearance of the spindles during thawing of frozen oocytes was not caused by optical properties of the thawing solutions.
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In all oocytes (n = 12) that were examined by immunocytochemistry with anti-tubulin antibody after previous Polscope imaging, the findings obtained by both methods were consistent. However, immunocytochemistry revealed residual tubulin immunoreactivity in association with chromosomes in most oocytes that lacked the spindle (Figure 3).
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Discussion |
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In this study, the meiotic spindle was detected in 100% of in vivo matured metaphase II oocytes before freezing. This finding is in agreement with our previously published series of observations (Rienzi et al., 2003
) but in disagreement with several other studies using the Polscope, in which the percentage of oocytes with a detectable spindle varied between 60 and 80% (Wang et al., 2001b,c, 2002; Moon et al., 2003). This difference may be due to the fact that we rotated each oocyte with an injection pipette controlled by a micromanipulator during observation until the spindle became visible, which was not done in the other studies. This manipulation made it possible to detect even spindles with relatively low brightness. It cannot be excluded that spindles that can be visualized relatively easily are functionally superior to poorly detectable spindles whose visualization requires extensive oocyte rotation with the injection pipette. This would explain the findings of poor developmental quality of oocytes with non-visualized spindles by the Polscope after ICSI (Wang et al., 2002; Moon et al., 2003).
Interestingly, in all oocytes that showed the meiotic spindle before freezing, the spindle remained detectable throughout the freezing procedure up to the step at which the oocytes were loaded into the cryopreservation straws, a period during which the temperature decreased from 37°C to room temperature. This observation contrasts with the finding that even transient cooling to the room temperature for only 10 min causes irreversible disruption of the meiotic spindle in the human oocyte (Pickering et al., 1990; Wang et al., 2001a; Zenzes et al., 2001). The present observation, showing that the spindle can persist during prolonged incubation of metaphase II human oocytes exposed to laboratory temperature while in the cryoprotecting medium, suggests that some component of this medium also protects the spindle against the temperature-induced disintegration.
In fact, simple incubation of metaphase II human oocytes in cryoprotective solutions without freezing has been shown previously to have no effect on the structure of the meiotic spindle (Boiso et al., 2002). In the absence of any direct deleterious effect of the cryoprotective solutions on the spindle, the progressive replacement of water molecules by those of cryoprotectant, leading to an increase in the concentration of tubulin monomer by reducing the volume of the aqueous solvent, might shift the equilibrium of the temperature-dependent tubulin polymerization/depolymerization reaction in favour of polymerization, thus slowing down the physical, temperature-sensitive tubulin depolymerization process. Accordingly, cryoprotective media may in fact also act as cooling-protective media for metaphase II human oocytes.
Little is known about what happens with the spindles after oocyte loading into the cryopreservation straws, during the rest of the freezing procedure. However, the observation of the absence of spindles in most of the surviving oocytes immediately after thawing suggests that additional destabilization of the meiotic spindle occurs during the final phase of oocyte cooling (after transfer to straws) and subsequent freezing.
This study has shown that, unlike oocyte freezing, the thawing procedure causes severe damage to the oocyte meiotic spindle. In fact, all oocytes lose their spindles during washing from the cryopreservation medium. The loss of the spindles during the thawing procedure was confirmed by immunocytochemistry with anti-tubulin antibody. In fact, immunocytochemistry and Polscope findings were consistent in all oocytes that were examined by both methods. These data are in agreement with those of Wang and Keefe (2002) who found a good correlation between Polscope and immunocytochemistry findings in human in vitro matured oocytes. The disintegration of the meioitic spindle during the thawing procedure may be explained by the fact that, contrary to the freezing sequence, depolymerized tubulin in the oocyte cytoplasm is progressively diluted by water entering the oocyte during this step, thus shifting the equilibrium of the tubulin polymerization/depolymerization reaction in favour of depolymerization. The known rapidity of microtubule turnover in metaphase-arrested oocytes (Gorbsky et al., 1990) explains the rapidity with which the spindle disappears during this period. Consequently, all spindles eventually present in frozen and thawed metaphase II oocytes were products of de novo assembly in the post-thaw period.
These observations have several biological and clinical implications. If all oocytes lose their spindles during the thawing procedures followed by de novo spindle reconstruction, a question arises of whether the thawing methods could be modified so as to protect the original spindle and prevent its disappearance. The use of microtubule-stabilizing agents, such as taxol (Mailhes et al., 1999), might be one possibility, but toxicity by this kind of drug remains a problem. Alternatively, if we accept the destruction of the oocyte’s original spindle as an unavoidable fact, future research might focus on improving the conditions in which the new spindle is formed in the post-thaw period. The Polscope system will be useful in both of these research directions.
From the clinical point of view, the fact that the spindle function in cryopreserved metaphase II oocytes is entirely dependent on spindle reconstruction in the post-thaw period raises some concern about the functional capacity of these newly formed spindles. Thus, further research is needed to evaluate the potential risks of the application of oocyte cryopreservation in assisted reproduction treatment. Since spindle dysfunction may lead to abnormal segregation of chromosomes between the oocyte and the second polar body after oocyte activation, the use of the Polscope system to control the presence and location of the meiotic spindle in cryopreserved oocytes will enable the elimination of oocytes with spindle anomalies that could be at the origin of aneuploidy. In cases in which oocyte cryopreservation is suggested as an alternative to embryo cryopreservation, when the latter is prohibited by law or rejected by the infertile couple for ethical or religious reasons, these uncertainties should be seriously taken into account and explained to the patients.
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Submitted on May 30, 2003; resubmitted on October 21, 2003; accepted on October 31, 2003.
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