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Effects of cryopreservation on the meiotic spindle of human oocytes
European Journal of Obstetrics & Gynecology and Reproductive Biology 113S (2004) S17–S23
Effects of cryopreservation on the meiotic spindle of human oocytes J. Mandelbauma,b,*, O. Anastasioua,b, R. Le´vyd, J.F. Gue´rind, V. de Larouzie`rea,b, J.M. Antoinec a
Service d’Histologie, Biologie de la Reproduction et Cytoge´ne´tique (Pr. Vendrely), Hoˆpital Tenon, 4 rue de la Chine, 75020 Paris, France b EA1533, Faculte´ de Me´decine Saint-Antoine, Paris, France c Service de Gyne´cologie–Obste´trique (Pr. S. Uzan), Hoˆpital Tenon, Paris, France d De´partement de Me´decine de la Reproduction, Hoˆpital Edouard Herriot, Lyon, France
Abstract The microtubular meiotic spindle of most mammals, including humans, is very sensitive to cooling [Hum. Reprod. 16 (2001) 2374; Fertil. Steril. 54 (1990) 102; Fertil. Steril. 75 (2001) 769; Zygote 3 (1995) 357] and is rapidly depolymerised even after a slight reduction in temperature to 33 8C. Spindle disassembly is dependent on the extent of temperature decrease and its duration. After rewarming, the recovery is far from complete. Cryoprotectants themselves may alter the spindle structure, depending on the duration and temperature of exposure, the duration of recovery at 37 8C and the species [Hum. Reprod. Update 2 (1996) 193]. Damage to the meiotic spindle is considered to be the cause of aneuploid embryos, by inducing chromatid non-disjunction and chromosome scattering and by disturbing the sequence of events leading to the completion of meiosis and fertilisation. Nevertheless, a consensus arose from all the studies: appropriate exposure to cryoprotectants and appropriate rates of cooling and thawing allow the cryopreservation of mature oocytes without any significant changes in their second meiotic spindle organisation and without any increase in the rate of aneuploid embryos [Mol. Hum. Reprod. 2 (1996) 445; Hum. Reprod. 8 (1993) 1101; Hum. Reprod. 9 (1994) 684; Microsc. Res. Technol. 27 (1994) 165; Fertil. Steril. 75 (2001) 354]. These fundamental studies in humans, showing good preservation of cell structures after freeze–thaw procedures opened the way to new successful clinical trials with embryos derived from cryopreserved mature oocytes [Fertil. Steril. 68 (1997) 724]. Considering immature oocyte freezing at prophase I (germinal vesicle (GV) stage), a stage which was thought to be less sensitive to cryoinjury, pooled data from the literature showed no advantage in terms of survival rates, fertilisation rates of in vitro matured oocytes and developmental ability of the resulting embryos, especially in unstimulated cycles. Moreover, conflicting results are reported on the effects of freezing on the spindle-chromosome configuration of immature oocytes or in vitro matured oocytes, highlighting the need for large scale studies [Hum. Reprod. 10 (1995) 1816; Hum. Reprod. 13 (Suppl. 3) (1998) 161; Hum. Reprod. 17 (2002) 1885; Microsc. Res. Technol. 27 (1994) 165; Fertil. Steril. 68 (1997) 920]. One child has been born after the use of cryopreserved immature oocytes at GV stage, matured in vitro and fertilised by ICSI [Hum. Reprod. 13 (1998) 3156], demonstrating at least the feasibility of this technique. Improvements are required so as to make mature and immature oocyte cryopreservation an established and safe technique for ART. # 2003 Elsevier Ireland Ltd. All rights reserved. Keywords: Oocyte; Cryopreservation; Meiotic spindle; Human
1. Introduction Whereas frozen storage of human semen and embryos has become routine, consistent cryopreservation of oocytes has remains an elusive goal. Routine oocyte cryopreservation would greatly benefit assisted reproductive technologies in general, by offering an alternative to embryo freezing, allowing donated eggs to be used in a more flexible manner and providing a means of preserving women’s fertility after *
Corresponding author. Tel.: þ33-1-56016832; fax: þ33-1-56017803. E-mail address: [email protected] (J. Mandelbaum).
pelvic diseases, surgery or radio-/chemotherapy and consequent ovarian damage. The initial reports by Chen [1] and Van Uem et al. [2] of the first births following human mature oocyte cryopreservation were highly encouraging. However, their results could not be reproduced until 1997 [3], since all the teams who subsequently attempted oocyte cryopreservation failed or obtained only short-lived pregnancies. The combined data indicate that 383 mature oocytes had been thawed, resulting in the birth of four babies, i.e. 1% of living babies per thawed oocyte (see [4], for review). Various factors were responsible for this lack of success: low survival rates (25–40%), low fertilisation rates after classical insemination, a high inci-
0301-2115/$ – see front matter # 2003 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.ejogrb.2003.11.005
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dence of polyploidy and poor developmental ability of embryos. This led several teams to reinvestigate the effects of cooling, freezing and cryoprotectants on oocyte structures, especially on the meiotic spindle.
2. Structure of the meiotic spindle (1) The mature mammalian oocyte is arrested at the metaphase II (MII) stage of the second meiotic division with the chromosomes equatorially located on a microtubular spindle which is associated with the oocyte cortex and its subcortical microfilamentous net. The meiotic spindle is a temporary and dynamic structure of microtubules (MT). Two types of microtubule are observed: those running from pole to pole, passing between the chromosomes, through the metaphase plate and those attached to the kinetochores of chromosomes. (2) At the pachytene stage of oocyte prophase I, centrioles disappear while there remains a filamentous pericentriolar material (PCM) containing microtubule organising centres (MTOC). When oocyte meiosis is reinitiated in vivo (after the ovulatory gonadotropic discharge) or in vitro (after removal from the inhibitory follicular environment), tubulin polymerisation begins as the first breaks appear in the envelope of the oocyte nucleus, the germinal vesicle (GV). After germinal vesicle breakdown GVBD), microtubules emanate from the MTOCs and join the kinetochores of the condensing chromosomes. As the microtubules polymerise, the MTOCs migrate to form the poles of this acentriolar spindle and this determines the architecture of the first meiotic spindle The detailed behaviour of MTOCs during anaphase I, telophase I and the very brief prophase II is poorly known but they are also present at both poles of the second meiotic spindle. Other foci of PCM material can be seen in the ooplasm but they lack any MTOC activity, except in the mouse. (3) Human meiotic spindles are symmetrical, barrelshaped with anastral poles, slightly pointed at each pole, with the pole adjacent to the oolemma being smaller than that directed towards the centre of the ooplasm. The second meiotic spindle is narrower and more pointed as the components of the MTOCs are more closely assembled. Both spindles are peripheral, radially oriented towards the oocyte plasma membrane, unlike rodents in which the spindle is parallel to the surface and must rotate by 908 to extrude the polar body. The size of the human spindle from pole to pole, has been evaluated around 18–21 mm by Kim et al. [28] and 11 mm by Wang et al. [5]. (4) The microtubules, forming the meiotic spindle, are constituted by the assembly of heterodimeric units of a- and b-tubulin composing 13 protofilaments arranged
side by side to form a cylindrical wall. These polymers are labile and polarised. Associated proteins Microtubule Associated Proteins (MAPs) regulate the assembly of microtubules and participate in interactions with other cell components. The polymerised tubulin is in equilibrium with the free tubulin pool in the ooplasm. Microtubules are therefore highly sensitive to any physical (cooling, exposure to cryoprotectants) or physiological (ageing) changes which may trigger tubulin depolymerisation and microtubular disassembly. Chemical and physical stresses have been shown, in several species, to affect the microtubular structure of the oocyte meiotic spindle with deleterious consequences on chromosomal organisation (see [6], for review). Similar studies have been performed in humans.
3. Assessment of the integrity of the meiotic spindle Most of our knowledge about spindle structure is obtained from analysis of fixed samples imaged by classical staining on serial sections (Fig. 1), by immunocytochemistry or confocal microscopy after immunostaining of tubulin and chromatin (Figs. 2 and 3) and by electron microscopy. These techniques provide static images and do not allow study of the dynamic behaviour of spindles in individual oocytes. The Polscope, an orientation independent polarised light microscope, gives a non-invasive opportunity to analyse spindle architecture kinetics in the same living oocytes, based on the birefringence of the microtubular spindle (Fig. 4). Intensity of light is similar to differential interference contrast, used safely in IVF for two decades. Moreover, it is possible to measure microtubule density [5]. It is also possible to indirectly assess spindle integrity by analysing the chromosomal consequences of damage to the meiotic spindle. It is considered to be the cause of aneuploid embryos by inducing non-disjunction, unbalanced disjunction or premature disjunction of chromatids and chromo-
Fig. 1. Serial semi-thin sections of a human oocyte cryopreserved at the GV stage, matured in vitro to the MII stage, and stained with hematoxylin–eosin. The spindle and chromosome configuration appears totally preserved.
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Fig. 2. Confocal microscopy analysis of an oocyte stained immunocytochemically with anti a-tubulin monoclonal antibody and FITC to visualise the spindle (green) and counterstained with propidium iodide to visualise the chromosomes (red). After freezing at the GV stage and in vitro maturation, the spindle configuration appears normal with chromosomes equatorially arranged on a regular plate. On the left, the first polar body can be seen.
some scattering and by disturbing the sequence of events leading to the completion of meiosis and fertilisation. Cytogenetic analysis of resulting embryos or children gives the ultimate proof of the normality of the meiotic spindle.
4. Effects of cooling on the human meiotic spindle Human oocytes (n ¼ 576) have been cooled to various temperatures from room temperature (RT) to 0 8C in order to
Fig. 3. Confocal microscopy analysis of a GV oocyte, cryopreserved at the MII stage, after in vitro maturation, thawed and cultured for 4 h before fixation. Complete destabilisation of the spindle is associated with a still compact metaphase plate.
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oocytes were kept for 10 min at 33, 28 or 25 8C and then warmed, spindle recovery occurred in 5/5, 2/5, 0/5 oocytes, respectively. Spindle disassembly is dependent on the extent of temperature decrease and its duration. 4.2. Cooling to 0 8C
Fig. 4. Spindle image in a human oocyte at 37 8C, using the Polscope (picture by K. Rink).
assess spindle changes [5,7–11]. They were either donated to research at recovery or obtained after in vitro maturation or assessed after fertilisation failure (unfertilised aged oocytes). 4.1. Cooling to room temperature In the study by Pickering et al. [8], 52 freshly recovered oocytes were analysed by immunocytochemistry: 50% of spindles were abnormal after 10 min at room temperature as compared to 8% in control oocytes. Thirty minutes at RT caused disruption in all oocytes (5/5) and some chromosomal dispersal. Return to 37 8C for 1 or 4 h restored normal morphology in 28% of oocytes only while 11% displayed chromosomal dispersal. Human oocytes are rapidly affected by brief exposure to room temperature. Almeida and Bolton [9] showed that abnormalities were identical when oocytes were exposed to 32 8C (2 min at RT) or 27–25 8C (10–30 min at RT). In cooled oocytes, 77–89% of spindles displayed anomalies and 50% had chromosomal dispersal versus 69 and 13%, respectively, in control oocytes. These high rates of abnormalities resulted from the use of unfertilised aged oocytes but, nevertheless, control results were significantly different in this large series (n ¼ 340). The effects were reversed when oocytes returned to 37 8C, but only after exposure to RT for 2 min. Microtubule depolymerisation begins even with a fall in temperature of only 5 8C in humans and seems irreversible when oocytes are exposed to 27 8C. The study by Wang et al. [5], using the Polscope in living human oocytes, confirmed the results of Pickering and Bolton: depolymerisation begins at 32 8C and complete disassembly is observed after 5 min at 27 8C or 10 min at 33 8C. Rewarming to 37 8C restores the spindle integrity by 20 min if started soon after RT has been reached. When
Not surprisingly, exposure to 0 8C is highly detrimental to the meiotic spindle. The study by Zenzes et al. [11], on 55 in vitro matured oocytes, again confirmed that damage to the spindle in metaphase II stage (MII) oocytes is time dependent: negligible after 1 min, complete disappearance after 10 min. In control unchilled oocytes, 19% of spindles only, were abnormal after in vitro maturation. Chromosomes remained close together, a usual feature suggesting that chromosome separation triggered by spindle dynamics does not occur after the rapid disassembly of the spindle. Achievement of meiosis may, however, be impaired in the absence of spindle restoration. Another possible explanation might be the fact that microtubules attached to the kinetochores appeared less sensitive to chilling than pole to pole MT which were damaged within 3 min. In the report by Sathananthan et al. [7], cooling to 0 8C was performed in the presence of a cryoprotectant (DMSO, 1.5 mol) which did not provide substantial stabilisation of the spindle. Although no rewarming had been performed, the studies of both Zenzes et al. and Sathananthan et al. are highly disturbing. These conclusions may be reappraised after a study by George et al. [10]. They used a protocol that had been developed to improve the survival and developmental potential of cryopreserved mouse oocytes. Donated fresh human oocytes were cooled to 4 8C with or without DMSO (1.5 mol). Exposure of oocytes to 4 8C in the absence of DMSO caused dismantling of the spindle. In the presence of DMSO, only a slight reduction in size was observed, indicating depolymerisation of some microtubules, especially when oocytes were stripped enzymatically of their cumulus cells prior to DMSO exposure. Restoration to control medium at 37 8C for 3 h restored normal spindle structure. The consensus from all these studies including studies in mice [6,12,13,29] appears to be that inappropriate exposure to cryoprotectants and/or cooling may induce deleterious anomalies in the spindle microtubules. It seems possible, however, to use protocols which minimise these potentially dangerous alterations. One must also be aware of the numerous biases that may lead to overestimation of the true rate of damage caused by cooling and/ or cryoprotectants to the oocyte spindle. Among these biases, one can find interspecies variation suggesting that the human oocyte should be its own model; inappropriate freezing protocols or lack of adequate rewarming, leading to spindle alterations. Varying criteria of what is an abnormal spindle-chromosome configuration and varying techniques of analysis make comparisons between studies difficult. These difficulties are increased by the methodology of
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existing reports: small numbers of oocytes, inadequate material (aged unfertilised oocytes, in vitro matured oocytes) and low efficiency of fixation and staining procedures, rarely clearly evaluated by authors. The age of the patients included in the studies is an important parameter, as it has been shown that spindle and chromosomal anomalies are significantly and positively correlated to increasing age. More non-disjunction and predivision of chromatids are found in oocytes of patients older than 35 years [14]. Battaglia et al. [15] reported that the meiotic spindle in older women (40–45 years) is frequently abnormal: 79% of the oocytes exhibit abnormal tubulin placement and one or more chromosomes are displaced from the metaphase II plate. In contrast, 83% of the oocytes of younger patients (20–25 years) possess well ordered meiotic spindles with fully aligned chromosomes. Similar results were also reported by Volarcik et al. [16].
5. Oocyte cryopreservation and effects on the meiotic spindle 5.1. The mature human oocyte In the 1990s, reappraisal of the effects of freezing on human oocyte structures gave a more optimistic outlook [17,18]. Cryopreservation by a slow freeze–rapid thaw method, using propanediol as cryoprotectant allowed 64% of MII oocytes to survive after thawing and 60% of surviving oocytes had normal spindle and chromosome configurations. Moreover, there was no evidence of an increased incidence of freezing-associated aneuploidy, as assessed by fluorescence and cytogenetics [19]. After fertilised cryopreserved oocytes were analysed for lack of incorporation of chromosomes into the pronuclei or the second polar body; no stray chromosomes or micronuclei were detected using DNA staining by Hoechst dye. Van Blerkom and Davis [18] performed karyotyping on 268 control mature human oocytes and 182 oocytes cryopreserved at the metaphase II stage, with the propanediol– sucrose, slow cooling protocol. Survival rates reached 65%. Seven percent of control oocytes were aneuploid and there was no increase in abnormal karyotypes in cryopreserved mature oocytes, despite a severe impairment in early embryonic development after in vitro fertilisation of these thawed oocytes. Cobo et al. [20] reported the first study in which the rate of chromosomal abnormalities in embryos resulting from frozen oocytes was determined by blastomere biopsy and analysis by FISH of chromosomes X, Y, 13, 18 and 21, and compared to a control group of patients undergoing preimplantation genetic diagnosis for sex chromosomelinked disease. The overall survival rate was 59%. There was no significant difference between the control group (n ¼ 18) and the cryopreserved group (43 donated oocytes) in fertilisation rates (90% versus 76%) or blastocyst devel-
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opment (35% versus 30%). Aneuploid embryos were observed at the same rate in both groups (26% and 29%). Fundamental studies in humans showing good preservation of oocyte structures after freeze–thaw procedures opened the way to new clinical trials and to several live births of normal children suggesting that these studies were reliable. Taking together the normal births after mature oocyte cryopreservation [1–3] and the euploid embryos derived from frozen oocytes [19,20], almost 40 conceptuses displayed no chromosomal abnormalities and no aneuploid embryos had arisen from oocyte freezing, although large scale studies are needed to confirm these reassuring data. 5.2. The immature human oocyte cryopreservation Nevertheless, the concerns that had been raised over mature oocyte cryopreservation prompted some teams to turn to immature oocyte freezing at prophase I (GV stage), which was thought to be less sensitive to cryoinjury, lacking a mitotic spindle and possessing different membrane permeability characteristics [4,21,22]. Pooled data from the literature showed no advantage of immature oocyte freezing in terms of survival rates, fertilisation rates of in vitro matured oocytes and developmental ability of the resulting embryos, especially in unstimulated cycles. Moreover, concerns have been raised that GV oocyte freezing can increase chromosomal abnormalities even at this early immature stage with the DNA in a decondensed state. Park et al. [23] have shown that 128 frozen immature oocytes from unstimulated ovaries had an increased frequency of chromosomal and spindle abnormalities (77.8 and 70%, respectively) compared with 91 control oocytes (31.8 and 22.2%). Boiso et al. [24] also observed a deleterious effect of cryopreservation at the GV or MII (after in vitro maturation) stages on the organisation of the meiotic spindle. In 67 control oocytes from stimulated ovaries, 22% of the spindles were abnormal or absent, compared to more than 70% in cryopreserved oocytes, despite high rates of survival and in vitro maturation. This may result from a defective spindle assembly checkpoint induced by cryopreservation and reducing the accuracy of chromosome segregation in meiosis I during the in vitro maturation of the thawed GVoocytes [25]. Other authors, however, have not found any increase in abnormalities of spindle formation and chromosome configuration after progression in vitro to the MII stage of GV frozen–thawed oocytes [18,26]. We applied the classical protocol used for early cleaved human embryos and comprising propanediol (1.5 M) and sucrose (01 M) to 217 immature oocytes either at the GV stage or at the MII stage after a 24–48 h period of in vitro maturation. Survival rates reached 78% and maturation rates 55%, without any significant difference according to the stage of maturation at freezing (GV or MII). Following cryopreservation and in vitro maturation, immunostaining of tubulin (with an anti a-tubulin monoclonal antibody) and of chromatin (with propidium iodide) was performed.
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Localisation of tubulin and chromatin, revealed by FITC and propidium fluorescence, was done on a laser-scanning confocal microscope (Leica, TCP-SP), with an argon-krypton laser. Following fixation and staining procedures, only 32% of the oocytes could be analysed. Oocytes cryopreserved at the GV stage exhibited normal spindle-chromosome configuration in 87% of cases (13/15) compared with 29% only when oocytes were cryopreserved after in vitro maturation (2/7). At present, only one child has been born after the use of cryopreserved immature oocytes at the GV stage, matured in vitro and fertilised by ICSI [27], demonstrating at least the feasibility of this technique.
[6]
[7] [8]
[9]
[10]
[11]
6. Conclusion [12]
The human meiotic oocyte is highly sensitive to cooling. Paradoxically, the most dangerous situation seems to be inappropriate handling of human MII oocytes at room temperature. Under adequate conditions and with improved freezing protocols, the preservation or recovery of the structure and function of the meiotic spindle seem rather good. It appears essential, however, to assess the effects of every new freeze–thaw procedure not only by evaluating survival rates but also by checking the integrity of the human meiotic spindle. Immature oocyte cryopreservation has not proved to be safer. More studies are needed, so as to improve mature and immature oocyte survival and make oocyte freezing an established and safe technique for every type of ART including fertility preservation in cancer patients.
[13]
[14] [15]
[16]
[17]
[18]
7. Condensation [19]
Oocyte metaphase spindles, as tubulin structures, are highly sensitive to changes in temperature. In humans, under adequate conditions, meiotic spindle structure and function may be well preserved after freezing and thawing.
[20]
[21]
References [22] [1] Chen C. Pregnancy after human oocyte cryopreservation. Lancet 1986;II:884–6. [2] Van Uem JF, Siebzehnrubl ER, Schuh B, Koch R, Trotnow S, Lang N. Birth after cryopreservation of unfertilized oocytes. Lancet 1987;8535:752–3. [3] Porcu E, Fabbri R, Seracchioli R, Ciotti PM, Magrini O, Flamigni C. Birth of a healthy female after intracytoplasmic sperm injection of cryopreserved human oocytes. Fertil Steril 1997;68:724–6. [4] Mandelbaum J, Belaisch-Allart J, Junca AM, Antoine JM, Plachot M, Alvarez S, et al. Cryopreservation in human assisted reproduction is now routine for embryos but remains a research procedure for oocytes. Hum Reprod 1998;13(Suppl 3):161–74. [5] Wang WH, Meng L, Hackett RJ, Odenbourg R, Keefe DL. Limited recovery of meiotic spindles in living human oocytes after cooling-
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rewarming observed using polarized light microscopy. Hum Reprod 2001;16:2374–8. Bernard A, Fuller BJ. Cryopreservation of human oocytes: a review of current problems and perspectives. Hum Reprod Update 1996;2:193–207. Sathananthan AH, Trounson A, Freeman L, Brady T. The effects of cooling human oocytes. Hum Reprod 1988;3:968–77. Pickering SJ, Braude PR, Johnson MH, Cant A, Currie J. Transient cooling to room temperature can cause irreversible disruption to the meiotic spindle in human oocytes. Fertil Steril 1990;54:102–8. Almeida PA, Bolton VN. The effect of temperature fluctuations on the cytoskeletal organization and chromosomal constitution of the human oocyte. Zygote 1995;3:357–65. George MA, Pickering SJ, Braude PR, Johnson MH. The distribution of a- and g-tubulin in fresh and aged mouse and human oocytes exposed to cryoprotectant. Mol Hum Reprod 1996;2:445–56. Zenzes MT, Bielecki R, Casper RF, Leibo SP. Effects of chilling to 0 8C on the morphology of meiotic spindles in human metaphase II oocytes. Fertil Steril 2001;75:769–77. Aigner S, Van der Elst J, Siebzehnrubl E, Wildt L, Lang N, Van Steirteghem AC. The influence of slow and ultra-rapid freezing on the organization of the meiotic spindle in the mouse oocyte. Hum Reprod 1992;7:857–64. Joly C, Bchini O, Boulekbache H, Testart J, Maro B. Effects of 1,2propanediolon the cytoskeletal organisation of the mouse oocyte. Hum Reprod 1992;3:374–8. Sandalinas M, Marquez C, Munne S. Spectral karyotyping of fresh, non-inseminated oocytes. Mol Hum Reprod 2002;8:580–5. Battaglia DE, Goodwin P, Klein NA, Soules MR. Influence of maternal age on meiotic spindle assembly in oocytes from naturally cycling women. Hum Reprod 1996;13:2217–22. Volarcik K, Sheean L, Goldfarb J, Woods L, Abdul-Karim FW, Hunt P. The meiotic competence of in-vitro matured human oocytes is influenced by donor age: evidence that folliculogenesis is compromised in the reproductively aged ovary. Hum Reprod 1998;13:154– 60. Gook DA, Osborn SM, Johnston WI. Cryopreservation of mouse and human oocytes using 1,2-propanediol and the configuration of the meiotic spindle. Hum Reprod 1993;8:1101–9. Van Blerkom J, David PW. Cytogenetic, cellular and developmental consequences of cryopreservation of immature and mature mouse and human oocytes. Microsc Res Technol 1994;27:165–93. Gook DA, Osborn SM, Bourne H, Johnston WIH. Fertilization of human oocytes following cryopreservation normal karyotypes and absence of stray chromosomes. Hum Reprod 1994;9:684–91. Cobo A, Rubio C, Gerli S, Ruiz A, Pellicer A, Remohi J. Use of fluorescence in situ hybridization to assess the chromosomal status of embryos obtained from cryopreserved oocytes. Fertil Steril 2001;75:354–60. Toth TL, Lanzendorf SE, Sandow BA, Veeck LL, Hassen WA, Hansen K, et al. Cryopreservation of human prophase I oocytes collected from unstimulated follicles. Fertil Steril 1994;61:1077–82. Son WY, Park SE, Lee KA, Lee WS, Ko JJ, Yoon TK, et al. Effects of 1,2-propanediol and freezing-thawing on the in vitro developmental capacity of human immature oocytes. Fertil Steril 1996;66:995–9. Park SE, Son WY, Lee SH, Lee KA, Ko JJ, Cha KY. Chromosome and spindle configurations of human oocytes matured in vitro after cryopreservation at the germinal vesicle stage. Fertil Steril 1997;68:920–6. Boiso I, Mati M, Santalo J, Ponsa M, Barri P, Veiga A. A confocal microscopy analysis of the spindle and chromosome configurations of human oocytes cryopreserved at the germinal vesicle and metaphase II stage. Hum Reprod 2002;17:1885–91. Steuerwald N, Cohen J, Herrera RJ, Sandalinas M, Brenner CA. Association between spindle assembly checkpoint expression and maternal age in human oocytes. Mol Hum Reprod 2001;7:49–55.
J. Mandelbaum et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 113S (2004) S17–S23 [26] Baka SG, Toth TL, Veeck LL, Jones Jr. HW, Muasher SJ, Lanzendorf SE. Evaluation of the spindle apparatus of in-vitro matured human oocytes following cryopreservation. Hum Reprod 1995;10:1816– 20. [27] Tucker MJ, Morton PC, Wright G, Sweitzer CL, Massey JB. Clinical application of human egg cryopreservation. Hum Reprod 1998;13: 3156–9.
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[28] Kim NH, Chung HM, Cha KY, Chung KS. Microtubule and microfilament organizaion in maturing human oocytes. Human Reproduction, 1998. [29] George MA, Johnson MH. Use of fetal bovine serum substitutes for the protection of mouse zone pellucida against hardening, during, cryoprotectant addition. Human Reproduction, 1993, Nov, 8(11), 1998–9000.
Effects of cryopreservation on the meiotic spindle of human oocytes J. Mandelbauma,b,*, O. Anastasioua,b, R. Le´vyd, J.F. Gue´rind, V. de Larouzie`rea,b, J.M. Antoinec a
Service d’Histologie, Biologie de la Reproduction et Cytoge´ne´tique (Pr. Vendrely), Hoˆpital Tenon, 4 rue de la Chine, 75020 Paris, France b EA1533, Faculte´ de Me´decine Saint-Antoine, Paris, France c Service de Gyne´cologie–Obste´trique (Pr. S. Uzan), Hoˆpital Tenon, Paris, France d De´partement de Me´decine de la Reproduction, Hoˆpital Edouard Herriot, Lyon, France
Abstract The microtubular meiotic spindle of most mammals, including humans, is very sensitive to cooling [Hum. Reprod. 16 (2001) 2374; Fertil. Steril. 54 (1990) 102; Fertil. Steril. 75 (2001) 769; Zygote 3 (1995) 357] and is rapidly depolymerised even after a slight reduction in temperature to 33 8C. Spindle disassembly is dependent on the extent of temperature decrease and its duration. After rewarming, the recovery is far from complete. Cryoprotectants themselves may alter the spindle structure, depending on the duration and temperature of exposure, the duration of recovery at 37 8C and the species [Hum. Reprod. Update 2 (1996) 193]. Damage to the meiotic spindle is considered to be the cause of aneuploid embryos, by inducing chromatid non-disjunction and chromosome scattering and by disturbing the sequence of events leading to the completion of meiosis and fertilisation. Nevertheless, a consensus arose from all the studies: appropriate exposure to cryoprotectants and appropriate rates of cooling and thawing allow the cryopreservation of mature oocytes without any significant changes in their second meiotic spindle organisation and without any increase in the rate of aneuploid embryos [Mol. Hum. Reprod. 2 (1996) 445; Hum. Reprod. 8 (1993) 1101; Hum. Reprod. 9 (1994) 684; Microsc. Res. Technol. 27 (1994) 165; Fertil. Steril. 75 (2001) 354]. These fundamental studies in humans, showing good preservation of cell structures after freeze–thaw procedures opened the way to new successful clinical trials with embryos derived from cryopreserved mature oocytes [Fertil. Steril. 68 (1997) 724]. Considering immature oocyte freezing at prophase I (germinal vesicle (GV) stage), a stage which was thought to be less sensitive to cryoinjury, pooled data from the literature showed no advantage in terms of survival rates, fertilisation rates of in vitro matured oocytes and developmental ability of the resulting embryos, especially in unstimulated cycles. Moreover, conflicting results are reported on the effects of freezing on the spindle-chromosome configuration of immature oocytes or in vitro matured oocytes, highlighting the need for large scale studies [Hum. Reprod. 10 (1995) 1816; Hum. Reprod. 13 (Suppl. 3) (1998) 161; Hum. Reprod. 17 (2002) 1885; Microsc. Res. Technol. 27 (1994) 165; Fertil. Steril. 68 (1997) 920]. One child has been born after the use of cryopreserved immature oocytes at GV stage, matured in vitro and fertilised by ICSI [Hum. Reprod. 13 (1998) 3156], demonstrating at least the feasibility of this technique. Improvements are required so as to make mature and immature oocyte cryopreservation an established and safe technique for ART. # 2003 Elsevier Ireland Ltd. All rights reserved. Keywords: Oocyte; Cryopreservation; Meiotic spindle; Human
1. Introduction Whereas frozen storage of human semen and embryos has become routine, consistent cryopreservation of oocytes has remains an elusive goal. Routine oocyte cryopreservation would greatly benefit assisted reproductive technologies in general, by offering an alternative to embryo freezing, allowing donated eggs to be used in a more flexible manner and providing a means of preserving women’s fertility after *
Corresponding author. Tel.: þ33-1-56016832; fax: þ33-1-56017803. E-mail address: [email protected] (J. Mandelbaum).
pelvic diseases, surgery or radio-/chemotherapy and consequent ovarian damage. The initial reports by Chen [1] and Van Uem et al. [2] of the first births following human mature oocyte cryopreservation were highly encouraging. However, their results could not be reproduced until 1997 [3], since all the teams who subsequently attempted oocyte cryopreservation failed or obtained only short-lived pregnancies. The combined data indicate that 383 mature oocytes had been thawed, resulting in the birth of four babies, i.e. 1% of living babies per thawed oocyte (see [4], for review). Various factors were responsible for this lack of success: low survival rates (25–40%), low fertilisation rates after classical insemination, a high inci-
0301-2115/$ – see front matter # 2003 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.ejogrb.2003.11.005
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dence of polyploidy and poor developmental ability of embryos. This led several teams to reinvestigate the effects of cooling, freezing and cryoprotectants on oocyte structures, especially on the meiotic spindle.
2. Structure of the meiotic spindle (1) The mature mammalian oocyte is arrested at the metaphase II (MII) stage of the second meiotic division with the chromosomes equatorially located on a microtubular spindle which is associated with the oocyte cortex and its subcortical microfilamentous net. The meiotic spindle is a temporary and dynamic structure of microtubules (MT). Two types of microtubule are observed: those running from pole to pole, passing between the chromosomes, through the metaphase plate and those attached to the kinetochores of chromosomes. (2) At the pachytene stage of oocyte prophase I, centrioles disappear while there remains a filamentous pericentriolar material (PCM) containing microtubule organising centres (MTOC). When oocyte meiosis is reinitiated in vivo (after the ovulatory gonadotropic discharge) or in vitro (after removal from the inhibitory follicular environment), tubulin polymerisation begins as the first breaks appear in the envelope of the oocyte nucleus, the germinal vesicle (GV). After germinal vesicle breakdown GVBD), microtubules emanate from the MTOCs and join the kinetochores of the condensing chromosomes. As the microtubules polymerise, the MTOCs migrate to form the poles of this acentriolar spindle and this determines the architecture of the first meiotic spindle The detailed behaviour of MTOCs during anaphase I, telophase I and the very brief prophase II is poorly known but they are also present at both poles of the second meiotic spindle. Other foci of PCM material can be seen in the ooplasm but they lack any MTOC activity, except in the mouse. (3) Human meiotic spindles are symmetrical, barrelshaped with anastral poles, slightly pointed at each pole, with the pole adjacent to the oolemma being smaller than that directed towards the centre of the ooplasm. The second meiotic spindle is narrower and more pointed as the components of the MTOCs are more closely assembled. Both spindles are peripheral, radially oriented towards the oocyte plasma membrane, unlike rodents in which the spindle is parallel to the surface and must rotate by 908 to extrude the polar body. The size of the human spindle from pole to pole, has been evaluated around 18–21 mm by Kim et al. [28] and 11 mm by Wang et al. [5]. (4) The microtubules, forming the meiotic spindle, are constituted by the assembly of heterodimeric units of a- and b-tubulin composing 13 protofilaments arranged
side by side to form a cylindrical wall. These polymers are labile and polarised. Associated proteins Microtubule Associated Proteins (MAPs) regulate the assembly of microtubules and participate in interactions with other cell components. The polymerised tubulin is in equilibrium with the free tubulin pool in the ooplasm. Microtubules are therefore highly sensitive to any physical (cooling, exposure to cryoprotectants) or physiological (ageing) changes which may trigger tubulin depolymerisation and microtubular disassembly. Chemical and physical stresses have been shown, in several species, to affect the microtubular structure of the oocyte meiotic spindle with deleterious consequences on chromosomal organisation (see [6], for review). Similar studies have been performed in humans.
3. Assessment of the integrity of the meiotic spindle Most of our knowledge about spindle structure is obtained from analysis of fixed samples imaged by classical staining on serial sections (Fig. 1), by immunocytochemistry or confocal microscopy after immunostaining of tubulin and chromatin (Figs. 2 and 3) and by electron microscopy. These techniques provide static images and do not allow study of the dynamic behaviour of spindles in individual oocytes. The Polscope, an orientation independent polarised light microscope, gives a non-invasive opportunity to analyse spindle architecture kinetics in the same living oocytes, based on the birefringence of the microtubular spindle (Fig. 4). Intensity of light is similar to differential interference contrast, used safely in IVF for two decades. Moreover, it is possible to measure microtubule density [5]. It is also possible to indirectly assess spindle integrity by analysing the chromosomal consequences of damage to the meiotic spindle. It is considered to be the cause of aneuploid embryos by inducing non-disjunction, unbalanced disjunction or premature disjunction of chromatids and chromo-
Fig. 1. Serial semi-thin sections of a human oocyte cryopreserved at the GV stage, matured in vitro to the MII stage, and stained with hematoxylin–eosin. The spindle and chromosome configuration appears totally preserved.
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Fig. 2. Confocal microscopy analysis of an oocyte stained immunocytochemically with anti a-tubulin monoclonal antibody and FITC to visualise the spindle (green) and counterstained with propidium iodide to visualise the chromosomes (red). After freezing at the GV stage and in vitro maturation, the spindle configuration appears normal with chromosomes equatorially arranged on a regular plate. On the left, the first polar body can be seen.
some scattering and by disturbing the sequence of events leading to the completion of meiosis and fertilisation. Cytogenetic analysis of resulting embryos or children gives the ultimate proof of the normality of the meiotic spindle.
4. Effects of cooling on the human meiotic spindle Human oocytes (n ¼ 576) have been cooled to various temperatures from room temperature (RT) to 0 8C in order to
Fig. 3. Confocal microscopy analysis of a GV oocyte, cryopreserved at the MII stage, after in vitro maturation, thawed and cultured for 4 h before fixation. Complete destabilisation of the spindle is associated with a still compact metaphase plate.
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oocytes were kept for 10 min at 33, 28 or 25 8C and then warmed, spindle recovery occurred in 5/5, 2/5, 0/5 oocytes, respectively. Spindle disassembly is dependent on the extent of temperature decrease and its duration. 4.2. Cooling to 0 8C
Fig. 4. Spindle image in a human oocyte at 37 8C, using the Polscope (picture by K. Rink).
assess spindle changes [5,7–11]. They were either donated to research at recovery or obtained after in vitro maturation or assessed after fertilisation failure (unfertilised aged oocytes). 4.1. Cooling to room temperature In the study by Pickering et al. [8], 52 freshly recovered oocytes were analysed by immunocytochemistry: 50% of spindles were abnormal after 10 min at room temperature as compared to 8% in control oocytes. Thirty minutes at RT caused disruption in all oocytes (5/5) and some chromosomal dispersal. Return to 37 8C for 1 or 4 h restored normal morphology in 28% of oocytes only while 11% displayed chromosomal dispersal. Human oocytes are rapidly affected by brief exposure to room temperature. Almeida and Bolton [9] showed that abnormalities were identical when oocytes were exposed to 32 8C (2 min at RT) or 27–25 8C (10–30 min at RT). In cooled oocytes, 77–89% of spindles displayed anomalies and 50% had chromosomal dispersal versus 69 and 13%, respectively, in control oocytes. These high rates of abnormalities resulted from the use of unfertilised aged oocytes but, nevertheless, control results were significantly different in this large series (n ¼ 340). The effects were reversed when oocytes returned to 37 8C, but only after exposure to RT for 2 min. Microtubule depolymerisation begins even with a fall in temperature of only 5 8C in humans and seems irreversible when oocytes are exposed to 27 8C. The study by Wang et al. [5], using the Polscope in living human oocytes, confirmed the results of Pickering and Bolton: depolymerisation begins at 32 8C and complete disassembly is observed after 5 min at 27 8C or 10 min at 33 8C. Rewarming to 37 8C restores the spindle integrity by 20 min if started soon after RT has been reached. When
Not surprisingly, exposure to 0 8C is highly detrimental to the meiotic spindle. The study by Zenzes et al. [11], on 55 in vitro matured oocytes, again confirmed that damage to the spindle in metaphase II stage (MII) oocytes is time dependent: negligible after 1 min, complete disappearance after 10 min. In control unchilled oocytes, 19% of spindles only, were abnormal after in vitro maturation. Chromosomes remained close together, a usual feature suggesting that chromosome separation triggered by spindle dynamics does not occur after the rapid disassembly of the spindle. Achievement of meiosis may, however, be impaired in the absence of spindle restoration. Another possible explanation might be the fact that microtubules attached to the kinetochores appeared less sensitive to chilling than pole to pole MT which were damaged within 3 min. In the report by Sathananthan et al. [7], cooling to 0 8C was performed in the presence of a cryoprotectant (DMSO, 1.5 mol) which did not provide substantial stabilisation of the spindle. Although no rewarming had been performed, the studies of both Zenzes et al. and Sathananthan et al. are highly disturbing. These conclusions may be reappraised after a study by George et al. [10]. They used a protocol that had been developed to improve the survival and developmental potential of cryopreserved mouse oocytes. Donated fresh human oocytes were cooled to 4 8C with or without DMSO (1.5 mol). Exposure of oocytes to 4 8C in the absence of DMSO caused dismantling of the spindle. In the presence of DMSO, only a slight reduction in size was observed, indicating depolymerisation of some microtubules, especially when oocytes were stripped enzymatically of their cumulus cells prior to DMSO exposure. Restoration to control medium at 37 8C for 3 h restored normal spindle structure. The consensus from all these studies including studies in mice [6,12,13,29] appears to be that inappropriate exposure to cryoprotectants and/or cooling may induce deleterious anomalies in the spindle microtubules. It seems possible, however, to use protocols which minimise these potentially dangerous alterations. One must also be aware of the numerous biases that may lead to overestimation of the true rate of damage caused by cooling and/ or cryoprotectants to the oocyte spindle. Among these biases, one can find interspecies variation suggesting that the human oocyte should be its own model; inappropriate freezing protocols or lack of adequate rewarming, leading to spindle alterations. Varying criteria of what is an abnormal spindle-chromosome configuration and varying techniques of analysis make comparisons between studies difficult. These difficulties are increased by the methodology of
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existing reports: small numbers of oocytes, inadequate material (aged unfertilised oocytes, in vitro matured oocytes) and low efficiency of fixation and staining procedures, rarely clearly evaluated by authors. The age of the patients included in the studies is an important parameter, as it has been shown that spindle and chromosomal anomalies are significantly and positively correlated to increasing age. More non-disjunction and predivision of chromatids are found in oocytes of patients older than 35 years [14]. Battaglia et al. [15] reported that the meiotic spindle in older women (40–45 years) is frequently abnormal: 79% of the oocytes exhibit abnormal tubulin placement and one or more chromosomes are displaced from the metaphase II plate. In contrast, 83% of the oocytes of younger patients (20–25 years) possess well ordered meiotic spindles with fully aligned chromosomes. Similar results were also reported by Volarcik et al. [16].
5. Oocyte cryopreservation and effects on the meiotic spindle 5.1. The mature human oocyte In the 1990s, reappraisal of the effects of freezing on human oocyte structures gave a more optimistic outlook [17,18]. Cryopreservation by a slow freeze–rapid thaw method, using propanediol as cryoprotectant allowed 64% of MII oocytes to survive after thawing and 60% of surviving oocytes had normal spindle and chromosome configurations. Moreover, there was no evidence of an increased incidence of freezing-associated aneuploidy, as assessed by fluorescence and cytogenetics [19]. After fertilised cryopreserved oocytes were analysed for lack of incorporation of chromosomes into the pronuclei or the second polar body; no stray chromosomes or micronuclei were detected using DNA staining by Hoechst dye. Van Blerkom and Davis [18] performed karyotyping on 268 control mature human oocytes and 182 oocytes cryopreserved at the metaphase II stage, with the propanediol– sucrose, slow cooling protocol. Survival rates reached 65%. Seven percent of control oocytes were aneuploid and there was no increase in abnormal karyotypes in cryopreserved mature oocytes, despite a severe impairment in early embryonic development after in vitro fertilisation of these thawed oocytes. Cobo et al. [20] reported the first study in which the rate of chromosomal abnormalities in embryos resulting from frozen oocytes was determined by blastomere biopsy and analysis by FISH of chromosomes X, Y, 13, 18 and 21, and compared to a control group of patients undergoing preimplantation genetic diagnosis for sex chromosomelinked disease. The overall survival rate was 59%. There was no significant difference between the control group (n ¼ 18) and the cryopreserved group (43 donated oocytes) in fertilisation rates (90% versus 76%) or blastocyst devel-
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opment (35% versus 30%). Aneuploid embryos were observed at the same rate in both groups (26% and 29%). Fundamental studies in humans showing good preservation of oocyte structures after freeze–thaw procedures opened the way to new clinical trials and to several live births of normal children suggesting that these studies were reliable. Taking together the normal births after mature oocyte cryopreservation [1–3] and the euploid embryos derived from frozen oocytes [19,20], almost 40 conceptuses displayed no chromosomal abnormalities and no aneuploid embryos had arisen from oocyte freezing, although large scale studies are needed to confirm these reassuring data. 5.2. The immature human oocyte cryopreservation Nevertheless, the concerns that had been raised over mature oocyte cryopreservation prompted some teams to turn to immature oocyte freezing at prophase I (GV stage), which was thought to be less sensitive to cryoinjury, lacking a mitotic spindle and possessing different membrane permeability characteristics [4,21,22]. Pooled data from the literature showed no advantage of immature oocyte freezing in terms of survival rates, fertilisation rates of in vitro matured oocytes and developmental ability of the resulting embryos, especially in unstimulated cycles. Moreover, concerns have been raised that GV oocyte freezing can increase chromosomal abnormalities even at this early immature stage with the DNA in a decondensed state. Park et al. [23] have shown that 128 frozen immature oocytes from unstimulated ovaries had an increased frequency of chromosomal and spindle abnormalities (77.8 and 70%, respectively) compared with 91 control oocytes (31.8 and 22.2%). Boiso et al. [24] also observed a deleterious effect of cryopreservation at the GV or MII (after in vitro maturation) stages on the organisation of the meiotic spindle. In 67 control oocytes from stimulated ovaries, 22% of the spindles were abnormal or absent, compared to more than 70% in cryopreserved oocytes, despite high rates of survival and in vitro maturation. This may result from a defective spindle assembly checkpoint induced by cryopreservation and reducing the accuracy of chromosome segregation in meiosis I during the in vitro maturation of the thawed GVoocytes [25]. Other authors, however, have not found any increase in abnormalities of spindle formation and chromosome configuration after progression in vitro to the MII stage of GV frozen–thawed oocytes [18,26]. We applied the classical protocol used for early cleaved human embryos and comprising propanediol (1.5 M) and sucrose (01 M) to 217 immature oocytes either at the GV stage or at the MII stage after a 24–48 h period of in vitro maturation. Survival rates reached 78% and maturation rates 55%, without any significant difference according to the stage of maturation at freezing (GV or MII). Following cryopreservation and in vitro maturation, immunostaining of tubulin (with an anti a-tubulin monoclonal antibody) and of chromatin (with propidium iodide) was performed.
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Localisation of tubulin and chromatin, revealed by FITC and propidium fluorescence, was done on a laser-scanning confocal microscope (Leica, TCP-SP), with an argon-krypton laser. Following fixation and staining procedures, only 32% of the oocytes could be analysed. Oocytes cryopreserved at the GV stage exhibited normal spindle-chromosome configuration in 87% of cases (13/15) compared with 29% only when oocytes were cryopreserved after in vitro maturation (2/7). At present, only one child has been born after the use of cryopreserved immature oocytes at the GV stage, matured in vitro and fertilised by ICSI [27], demonstrating at least the feasibility of this technique.
[6]
[7] [8]
[9]
[10]
[11]
6. Conclusion [12]
The human meiotic oocyte is highly sensitive to cooling. Paradoxically, the most dangerous situation seems to be inappropriate handling of human MII oocytes at room temperature. Under adequate conditions and with improved freezing protocols, the preservation or recovery of the structure and function of the meiotic spindle seem rather good. It appears essential, however, to assess the effects of every new freeze–thaw procedure not only by evaluating survival rates but also by checking the integrity of the human meiotic spindle. Immature oocyte cryopreservation has not proved to be safer. More studies are needed, so as to improve mature and immature oocyte survival and make oocyte freezing an established and safe technique for every type of ART including fertility preservation in cancer patients.
[13]
[14] [15]
[16]
[17]
[18]
7. Condensation [19]
Oocyte metaphase spindles, as tubulin structures, are highly sensitive to changes in temperature. In humans, under adequate conditions, meiotic spindle structure and function may be well preserved after freezing and thawing.
[20]
[21]
References [22] [1] Chen C. Pregnancy after human oocyte cryopreservation. Lancet 1986;II:884–6. [2] Van Uem JF, Siebzehnrubl ER, Schuh B, Koch R, Trotnow S, Lang N. Birth after cryopreservation of unfertilized oocytes. Lancet 1987;8535:752–3. [3] Porcu E, Fabbri R, Seracchioli R, Ciotti PM, Magrini O, Flamigni C. Birth of a healthy female after intracytoplasmic sperm injection of cryopreserved human oocytes. Fertil Steril 1997;68:724–6. [4] Mandelbaum J, Belaisch-Allart J, Junca AM, Antoine JM, Plachot M, Alvarez S, et al. Cryopreservation in human assisted reproduction is now routine for embryos but remains a research procedure for oocytes. Hum Reprod 1998;13(Suppl 3):161–74. [5] Wang WH, Meng L, Hackett RJ, Odenbourg R, Keefe DL. Limited recovery of meiotic spindles in living human oocytes after cooling-
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[28] Kim NH, Chung HM, Cha KY, Chung KS. Microtubule and microfilament organizaion in maturing human oocytes. Human Reproduction, 1998. [29] George MA, Johnson MH. Use of fetal bovine serum substitutes for the protection of mouse zone pellucida against hardening, during, cryoprotectant addition. Human Reproduction, 1993, Nov, 8(11), 1998–9000.