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
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: freezing/meiotic spindle/metaphase II/Polscope/thawing
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 freezingthawing 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 freezingthawing 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.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
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).
|
|
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 oocytes 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.
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Boiso I, Marti M, Santalo J, Ponsa M, Barri PN and Veiga A (2002) A confocal microscopy analysis of the spindle and chromosome configurations of human oocytes cryopreserved at the germinal vesicle and metaphase II stage. Hum Reprod 17,18851891.
Chen C (1986) Pregnancy after human oocyte cryopreservation. Lancet 19,884886.[CrossRef]
Cobo A, Rubio C, Gerli S, Ruiz A, Pellicer A and Remohi J (2001) Use of fluorescence in situ hybridization to assess the chromosomal status of embryos obtained from cryopreserved oocytes. Fertil Steril 75,354360.[CrossRef][ISI][Medline]
Eichenlaub-Ritter U, Shen Y and Tinneberg HR (2002) Manipulation of the oocyte: possible damage to the spindle apparatus. Reprod Biomed Online 5,117124.[Medline]
Fabbri R, Porcu E, Marsella T, Primavera MR, Seracchioli R, Ciotti PM, Magrini O, Venturoli S and Flamigni C (1998) Oocyte cryopreservation. Hum Reprod 13 (Suppl 4),98108.
Gook DA, Schiewe MC, Osborn SM, Asch RH, Jansen RP and Johnston WI (1995) Intracytoplasmic sperm injection and embryo development of human oocytes cryopreserved using 1,2-propanediol. Hum Reprod 10,26372641.[Abstract]
Gorbsky GJ, Simerly C, Schatten G and Borisy GG (1990) Microtubules in the metaphase-arrested mouse oocyte turn over rapidly. Proc Natl Acad Sci USA 87,60496053.[Abstract]
Johnson MH, Pickering SJ and George M. (1988) The influence of cooling on the properties of the zona pellucida of the mouse oocyte. Hum Reprod 3,383387.[Abstract]
Liu L, Trimarchi JR, Oldenbourg R and Keefe DL (2000) Increased birefringence in the meiotic spindle provides a new marker for the onset of activation in living oocytes. Biol Reprod 63,251258.
Magistrini M and Szollosi D (1980) Effects of cold and of isopropyl-N-phenylcarbamate on the second meiotic spindle of mouse oocytes. Eur J Cell Biol 22,699707.[ISI][Medline]
Mailhes JB, Carabatsos MJ, Young D, London SN, Bell M and Albertini DF (1999) Taxol-induced meiotic maturation delay, spindle defects, and aneuploidy in mouse oocytes and zygotes. Mutat Res 423,7990.[ISI][Medline]
Mandelbaum J, Junca AM, Plachot M, Alnot MO, Salat-Baroux J, Alvarez S, Tibi C, Cohen J, Debache C and Tesquier L (1988) Cryopreservation of human embryos and oocytes. Hum Reprod 3,117119.[ISI][Medline]
Moon JH, Hyun CS, Lee S-W, Son W-Y, Yoon, S-H, and Lim J-H (2003) Visualization of the metaphase II meiotic spindle in living human oocytes using the Polscope enables the prediction of embryonic developmental competence after ICSI. Hum Reprod 18,817820.
Palermo G, Joris H, Devroey P and Van Steirteghem AC (1992) Pregnancies after intracytoplasmic injection of single spermatozoon into an oocyte. Lancet 340,1718.[ISI][Medline]
Pickering S, Braude P, Johnson MH, Cant A and Currie J (1990) Transient cooling to room temperature can cause irreversible disruption of the meiotic spindle in the human oocyte. Fertil Steril 54,102108.[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 13,10141019.[Abstract]
Rienzi L, Ubaldi F, Martinez F, Iacobelli M, Minasi MG, Ferrero S, Tesarik J and Greco E (2003) Relationship between meiotic spindle location with regard to the polar body position and oocyte developmental potential after ICSI. Hum Reprod 18,12891293.
Sathananthan AH, Trounson A, Freemann L and Brady T (1988a) The effects of cooling human oocytes. Hum Reprod 3,968977.[Abstract]
Sathananthan AH, Ng SC, Trounson AO, Bongso A, Ratnam SS, Ho J, Mok H and Lee MN (1988b) The effects of ultrarapid freezing on meiotic and mitotic spindles of mouse oocytes and embryos. Gamete Res 21,385401.[ISI][Medline]
Schalkoff ME, Oskowitz SP and Powers RD (1989) Ultrastructural observations of human and mouse oocytes treated with cryopreservaties. Biol Reprod 40,379393.[Abstract]
Todorow SJ, Siebzehnrubl ER, Koch R, Wildt L and Lang N (1989) Comparative results on survival of human and animal eggs using different cryoprotectants and freezethawing regimens. I. Mouse and hamster. Hum Reprod 4,805811.[Abstract]
Tucker MJ, Morton PC, Wright G, Sweitzer CL and Massey JB (1998) Clinical application of human egg cryopreservation. Hum Reprod 13,31563169.[Abstract]
Ubaldi F, Nagy ZP, Rienzi L, Tesarik J, Anniballo R, Franco G, Menchini-Fabris F and Greco E (1999) Reproductive capacity of spermatozoa from men with testicular failure. Hum Reprod 14,27962800.
VanderElst J, Nerinckx S and Van Steirteghem AC (1992) In vitro maturation of mouse germinal vesicle-stage oocytes following cooling, exposure to cryoprotectants and ultrarapid freezing: limited effect on the morphology of the second meiotic spindle. Hum Reprod 7,14401446.[Abstract]
Wang WH and Keefe DL (2002) Prediction of chromosome misalighment among in vitro matured human oocytes by spindle imaging with the PolScope. Fertil Steril 78,10771081.[CrossRef][ISI][Medline]
Wang WH, Meng L, Hackett RJ, Odenbourg R and Keefe DL (2001a) Limited recovery of meiotic spindles in living human oocytes after coolingrewarming observed using polarized light microscopy. Hum Reprod 16,23742378.
Wang WH, Meng L, Hackett RJ and Keefe DL (2001b) Developmental ability of human oocytes with or without birefringent spindles imaged by Polscope before insemination. Hum Reprod 16,14641468.
Wang WH, Meng L, Hackett RJ, Odenbourg R and Keefe DL (2001c) The spindle observation and its relationship with fertilization after intracytoplasmic sperm injection in living human oocytes. Fertil Steril 75,34853.[CrossRef][ISI][Medline]
Wang WH, Meng L, Hackett RJ, Oldenbourg R and Keefe DL (2002) Rigorous thermal control during intracytoplasmic sperm injection stabilizes the meiotic spindle and improves fertilization and pregnancy rates. Fertil Steril 77,12741277.[CrossRef][ISI][Medline]
Zenzes MT, Bielecki R, Caspera RF and Leibo SP (2001) Effects of chilling to 0°C on the morphology of meiotic spindles in human metaphase II oocytes. Fertil Steril 75,769777.[CrossRef][ISI][Medline]
Submitted on May 30, 2003; resubmitted on October 21, 2003; accepted on October 31, 2003.