1 Department of Obstetrics and Gynaecology, University of Wales College of Medicine, Heath Park, Cardiff CF14 4XN, UK
2 Present address: Academic Division of Obstetrics and Gynaecology, Derby City General Hospital, Uttoxeter Road, Derby DE22 3NE, UK
3 To whom correspondence should be addressed. e-mail: Ruppert-LinghamCJ{at}cardiff.ac.uk
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Abstract |
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Key words: cryopreservation/cumulusoocyte complex/dimethylsulphoxide/GV-stage oocytes/maturation
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Introduction |
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Regimes for the collection of fully-grown GV-stage human oocytes have been devised that involve modification of mature oocyte collection techniques (Trounson et al., 1994) and reduction in hormonal stimulation. However, at present only a limited number of successful pregnancies have arisen from immature human oocytes (Cha et al., 1991
; Tucker et al., 1998
; Wu et al., 2001
). The clinical use of GV-stage oocytes to be matured for IVF may be preferable to traditional IVF treatment as the risk of ovarian hyperstimulation syndrome is reduced. The cost and duration of hormonal treatment could also be reduced. The development of a reliable method for long-term storage would allow these immature oocytes to be banked for future use. One major application would be the storage of oocytes from cancer patients prior to their receiving high-dose chemotherapy and total body irradiation, which can impair ovarian function. As well as allowing storage of oocytes for a patients own use, oocyte banking would simplify the process of oocyte donation by avoiding the need to synchronize donor and recipient cycles.
Spindle abnormalities have been reported following cryopreservation of GV-stage human oocytes (Park et al., 1997). However, other cryopreservation studies have emphasized the high levels of spindle and chromosome normality (
80%) observed following cryopreservation and in-vitro maturation (IVM) of immature human oocytes (Baka et al., 1995
). Cryopreserved GV-stage human oocytes have been shown to be capable of completing nuclear maturation and becoming fertilized (Toth et al., 1994
; Wu et al., 2001
). However, the development of embryos resulting from cryopreserved immature human oocytes was impaired (Toth et al., 1994
; Son et al., 1996
; Wu et al., 2001
). The proportion of immature human oocytes that reach maturity in vitro remains low, even among non-cryopreserved GV-stage human oocytes.
The refinement of IVM protocols for fully-grown murine GV-stage oocytes has resulted in oocytes with a similar level of developmental competence to that observed among in-vivo-matured oocytes when the donors were pre-treated with gonadotrophins (Schroeder and Eppig, 1984, 1989). High rates of nuclear maturation have been reported following cryopreservation and IVM of murine GV-stage oocytes using a variety of cryopreservation techniques (Schroeder et al., 1990
; Van der Elst et al., 1993
; Candy et al., 1994
; Van Blerkom and Davis, 1994
). In some cases, the rate of fertilization was similar for thawed and fresh control oocytes (Candy et al., 1994
). However, there have also been reports of decreased rates of fertilization following cryopreservation (Schroeder et al., 1990
; Van der Elst et al., 1993
; Van Blerkom and Davis, 1994
). In general, the developmental capacity of cryopreserved GV-stage murine oocytes is poor (Schroeder et al., 1990
; Van der Elst et al., 1992
, 1993; Candy et al., 1994
; Van Blerkom and Davis, 1994
).
It has been established that coupling of somatic cumulus granulosa cells with the GV-stage oocyte is vital to the progression of oocyte maturation and subsequent embryo development (Fagbohun and Downs, 1991). Studies have shown that GV-stage oocytes which are stripped of cumulus cells have a reduced developmental capacity compared with that of cumulus-enclosed GV-stage oocytes (Schroeder and Eppig, 1984
). Cryopreservation has been reported to cause cumulus cell loss (Van der Elst et al., 1993
; Cooper et al., 1998
; Goud et al., 2000
). The three-dimensional COC is likely to be particularly prone to physical disruption caused by ice crystal formation. Even when ice crystal formation is avoided in the process of vitrification, the vast difference in size between the oocyte and its associated cumulus cells means that they are likely to react very differently to the stresses applied during cryopreservation. The high levels of survival, morphological normality and maturation combined with the apparently low levels of developmental competence observed in freezethawed GV-stage oocytes could be an indication of damage and/or disruption to the somatic cumulus cells and their association with the oocyte.
The present study aimed to investigate the extent of damage inflicted on the cumulus cells of the COC by cryoprotectant loading/unloading either alone or with the additional stress of ice formation during slow-cooling and transfer to 196°C using dimethylsulphoxide (DMSO) as a cryoprotectant. The effect of this damage on the survival and subsequent development of the oocytes was examined. The effect of co-culture of fresh cumulus cells with fresh denuded GV-stage oocytes and frozenthawed COCs was also investigated.
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Materials and methods |
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Source of oocytes
Immature/GV-stage oocytes
Female CBAxC57 (6- to 8-week-old) mice bred from stock (obtained from Harlan, Bicester, UK) were kept under controlled conditions (14 h light, 10 h dark) and fed water and pellets ad libitum. The ovaries of the animals were stimulated by i.p. injection of 0.1 ml pregnant mares serum (PMS), 50 IU/ml (Folligon; Intervet UK Ltd, Milton Keynes, UK). After 46 h, the animals were killed by cervical dislocation and the ovaries removed immediately into modified phosphate-buffered saline (PBS) (Invitrogen, Paisley, UK) supplemented with 4 mg/ml bovine serum albumin (BSA) (Albumin fraction V powder; Sigma, Poole, Dorset, UK). The contents of the ovarian follicles were released by repeated puncturing with a 28 G micro-inject needle into 1 ml of standard maturation medium (SMM) which consisted of MEM Earles (Invitrogen, Paisley, UK) containing 10% fetal bovine serum (FBS) heat-inactivated (Invitrogen), 27.5 mg/l sodium pyruvate (Invitrogen), 50 mg/l streptomycin (streptomycin-sulphate BP; Evans, UK), 60 mg/l penicillin (crystapen benzyl penicillin sodium BP; Britannia, Redhill, Surrey, UK), 1 µg/l epidermal growth factor (Sigma), 1 mol/l L-glutamine (Invitrogen) and 0.1 mg/ml dcAMP (Sigma).
GV-stage oocytes, surrounded by at least two layers of cumulus cells, were selected and placed into 30 µl droplets of SMM (as detailed previously) containing 0.1 mg/ml dcAMP, using a pulled Pasteur pipette. COCs were held in this medium, under mineral oil (Sigma) at 37°C in an atmosphere of 5% CO2 in air until all COCs had been isolated from the ovaries (a maximum of 120 min).
In-vivo matured oocytes
Female CBAxC57 (6- to 8-week-old) mice were stimulated by i.p. injection of 0.1 ml PMS as detailed above, followed by 0.1 ml hCG (100 IU/ml) (Chorulon; Intervet UK Ltd) administered 53 h later. After a further 13 h, these animals were killed by cervical dislocation and the oviducts removed immediately into modified PBS supplemented with 4 mg/ml BSA.
Removal of cumulus cells
Freshly collected GV-stage COCs, held in 30 µl droplets of SMM + 10% FBS + 0.1 mg/ml dcAMP, were denuded by being drawn up and down a pulled Pasteur pipette with an internal diameter of 8090 µm. Oocytes and cumulus cells were transferred into separate 30 µl droplets of SMM + 10% FBS + 0.1 mg/ml dcAMP. dcAMP was included in all media and cryopreservation solutions with the exception of the hormone-supplemented maturation media. This was to ensure that the COCs remained at the GV stage throughout the manipulations. Morphologically normal oocytes were selected for IVM either in isolation or in the presence of pooled cumulus cells; that is, cumulus cells derived from twice the number of COCs to be included in the maturation group (to allow for cell loss during isolation). Freshly removed cumulus cells were also added, in similar proportions, to thawed COCs prior to entry into the IVM protocol.
In-vitro maturation (IVM)
Intact COCs, denuded oocytes, denuded oocytes with cumulus cells, thawed oocytes or thawed oocytes with cumulus cells were held in 30 µl droplets of SMM + 10% FBS + 0.1 mg/ml dcAMP at 37°C in an atmosphere of 5% CO2 in air. Each group was then placed into a 30 µl droplet of SMM + 10% FBS containing 0.75 IU/ml Gonal-F® (rFSH, Serono, London, UK), under mineral oil for 4 h at 37°C in an atmosphere of 5% CO2 in air. After this time, the medium was replaced, using a Gilson pipette, with 30 µl of SMM + 10% FBS containing 7.5 IU/ml Humegon® (FSH/LH at a 1:1 ratio; Organon, Cambridge, UK) at 37°C in an atmosphere of 5% CO2 in air for 18 h.
Cryopreservation of immature oocytes
Slow-freezing
Freshly collected COCs were transferred, by pulled Pasteur pipette, from 30 µl droplets of SMM supplemented with dcAMP into 1 ml of cryoprotectant solution containing 1.5 mol/l DMSO (Sigma) diluted in modified PBS +10% FBS + 0.1 mg/ml dcAMP. After 5 min of exposure to the solution at 0°C, the oocytes were pipetted into a small column (1520 µl) of the same cryoprotectant solution within freezing straws, that had been partially filled with the diluent, 0.1 mol/l sucrose (Aristar; BDH, Poole, UK; sucrose was added to minimize the toxic effects of DMSO) made up in modified PBS +10% FBS + 0.1 mg/ml dcAMP. Straws had been prepared previously, and stored on ice. Approximately 20 COCs were placed into each straw.
The cryopreservation protocol used slow-cooling and warming rates. The straws were sealed with wet plastic plugs and placed on ice. When the COCs had been in the presence of DMSO at 0°C for a total of 15 min, the straws were cooled at 2°C/min to 6°C in a controlled rate freezer (Planer Kryo10, Series III) which had been precooled to 4°C. The straws were held at this temperature (6°C) for 10 min, after which ice nucleation was instigated by touching each straw with cooled forceps. The straws were held at 6°C for a further 10 min. Cooling was then continued at a rate of 0.3°C/min to 60°C. When the samples reached a temperature of 60°C the straws were removed from the controlled-rate freezing machine and plunged into liquid nitrogen. The straws were stored at 196°C for between 1 and 12 weeks.
Thawing
The straws were removed from liquid nitrogen storage and placed in a controlled-rate freezing machine precooled to 70°C. They were then warmed to 4°C at a rate of 8°C/min. The contents of the straws were flushed with 1 ml of 0.1 mol/l sucrose made up in modified PBS +10% FBS + 0.1 mg/ml dcAMP into a culture dish. After being held in 0.1 mol/l sucrose solution at room temperature for 5 min, the COCs were placed into modified PBS + 10% FBS + 0.1 mg/ml dcAMP at room temperature for 5 min. They were finally placed into a fresh droplet of the same solution on a hot plate at 37°C for 5 min. After thawing, the oocytes were either stained for membrane integrity or were placed into the IVM protocol.
Exposure of COCs to cryoprotectant without freezing
COCs were transferred, by pulled Pasteur pipette, from SMM supplemented with dcAMP into 1 ml of cryoprotectant solution (1.5 mol/l DMSO +10% FBS + 0.1 mg/ml dcAMP) and held on ice for 15 min. After this time, the COCs were transferred into a dish containing 1 ml of 0.1 mol/l sucrose at room temperature. After 5 min the COCs were treated in a manner identical to thawed COCs. Following exposure to and dilution of the cryoprotectant, the COCs were either stained for membrane integrity or were placed into the IVM protocol.
Assessment of COCs and oocytes
Membrane integrity staining
Freshly collected GV-stage COCs, COCs which had been exposed to the cryoprotective agent without freezing, and thawed COCs were incubated in the dark (immediately after the 5 min incubation in modified PBS) at 37°C in modified PBS +10% FBS + 0.1 mg/ml dcAMP, containing 0.1 mg/ml carboxy fluorescein diacetate (Sigma) and 0.1 mg/ml propidium iodide (Sigma) for 10 min. The COCs were then washed twice in modified PBS +10% FBS + 0.1 mg/ml dcAMP. The COCs were then placed into a droplet of the same solution on a cavity slide and viewed using an Optiphot-2 microscope (Nikon, Tokyo, Japan) fitted with a filter capable of detecting fluorescence in the range 450490 nm. Cells with an intact cell membrane fluoresce green, whereas those with a damaged cell membrane fluoresce red. Each COC was double-blind scored for membrane integrity of the cumulus cells using the following scoring system. COCs were scored according to the proportion of the oocyte surface covered by cumulus cells with intact membranes: score 1 = 76100% coverage; score 2 = 5175% coverage; score 3 = 2650% coverage; and score 4 = 025% coverage.
IVF and assessment of development
In-vivo- or in-vitro-matured oocytes were released or placed (respectively) into 0.9 ml of Tyrodes medium (Invitrogen) supplemented with 16 mg/ml BSA, and then incubated for 10 min at 37°C in an atmosphere of 5% CO2 in air. After this time, 0.1 ml of capacitated sperm (sperm incubated for 11.5 h in Tyrodes medium supplemented with 16 mg/ml BSA at 37°C) was added to the oocytes, and the mixture was further incubated at 37°C in an atmosphere of 5% CO2 in air for 5 h. Oocytes were then transferred through three droplets of Tyrodes medium supplemented with 4 mg/ml BSA under mineral oil and incubated in the final droplet for a further 15 h at 37°C, in an atmosphere of 5% CO2 in air. At this point, the cells were assessed for normality and progression to the 2-cell stage. The number of blastocysts and hatching blastocysts was counted 4 days later.
Statistical analysis
For membrane integrity data, comparisons were made between freshly collected COCs, COCs which had been exposed to cryoprotectant without freezing and cryopreserved COCs using the 2-test. With data obtained following IVF, three data sets were compared using the KruskalWallis test: either two treatment groups and in-vitro-matured controls for maturation experiments; or one treatment group (either exposure to cryoprotectant or cryopreservation) and in-vivo- and in-vitro-matured control groups. Where a significant difference was found using the KruskalWallis test, pair-wise comparisons were made for this data set using the MannWhitney U-test.
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Results |
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Discussion |
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The current study aimed to determine at what stage of the cryopreservation process damage occurred, and whether damage was inflicted on the oocyte or the cumulus cells, or both. Following membrane integrity staining, the scores assigned to fresh control COCs and to COCs exposed to DMSO were similar. Thus, no substantial disassociation of the oocyte and cumulus cells occurred as a result of exposure to 1.5 mol/l DMSO for 15 min at 0°C. Exposure to DMSO also had no effect on the morphological normality of the oocytes or on the developmental capacity of the oocytes compared with fresh control COCs. One group (Schroeder et al., 1990) found a decrease in morphological normality of GV-stage oocytes following exposure to DMSO at 0°C for 30 min. The lower rates of normality can be attributed to the longer duration of exposure that, due to the kinetics of chilling injury, could lead to more extensive damage. This has been demonstrated in GV-stage bovine oocytes (Zeron et al., 1999
), while others (Van der Elst et al., 1992
) reported significantly fewer oocytes with normal spindle morphology following exposure to DMSO at 0°C at the GV stage than in control groups. However, no chromosomal abnormalities were reported, and in all cases the chromosomes were located at the equatorial plane of the spindle (Van der Elst et al., 1992
).
Following cryopreservation, normality of oocytes was found to be highly variable. This parameter was assessed at 47 h post-treatment and, therefore reflects the ability of the oocyte to survive the freezethaw process, to achieve maturation to metaphase II and to survive in culture for this period. In contrast, high survival at 15 h post-thaw has been reported (Schroeder et al., 1990; Van der Elst et al., 1992
). However, fertilization was significantly reduced compared with non-frozen controls. In another study, 93% survival at 16 h post-thaw with 83% maturation in vitro and 70% fertilization were reported (Candy et al., 1994
). In the current study, oocytes that were morphologically normal following cryopreservation were fertilized in similar proportions to controls.
Membrane integrity staining revealed extensive loss of plasma membrane integrity of the cumulus cells of thawed COCs. Thawed COCs therefore had significantly less direct contact with intact cumulus cells compared with fresh COCs and COCs exposed to DMSO without freezing. Following cryopreservation, embryo development was poor. Developmental impairment as a result of cryopreservation has been demonstrated following post-fertilization culture both in vitro (Van der Elst et al., 1993) and in vivo (Candy et al., 1994
). In the current study, it was observed that the blastocysts derived from fresh COCs were larger than those derived following slow cooling. A similar delay in development has been reported following vitrification of GV-stage COCs (Van Blerkom and Davis, 1994
).
Some studies have reported a loss of cumulus cells from the COC following cryopreservation and thawing (Cooper et al., 1998; Goud et al., 2000
). In the present study, careful pipetting allowed retention of the majority of cumulus cells and assessment of the membrane integrity of COCs immediately after thawing. However, most of the cumulus cells of the thawed COCs became disassociated from the oocyte following a short period of culture. The reduced developmental capacity of the thawed oocytes could therefore be due to a loss of integrity and/or functionality of cumulus cells.
The addition of fresh cumulus cells to thawed COCs did not improve maturation or development of the oocytes. Therefore, as with denuded oocytes, no benefit of co-culture with fresh cumulus cells during IVM was established. This is evidence against the significance of a secretory maturational factor that is lacking in thawed COCs, but supports the assertion that the metabolic coupling which exists between the cumulus cells and the oocyte has a maturational role that is disrupted by the process of slow-cooling and thawing. This evidence also supports the assertion that direct physical contact between the oocyte and cumulus cells, possibly via intercellular junctions, is a requirement for the completion of cytoplasmic maturation and subsequent embryo development.
In conclusion, at some stage during the process of cryopreservation a loss of association between the oocyte and cumulus cells occurred, and the integrity of cumulus cells was compromised. Disassociation of these two cell types prior to the completion of maturation led to a decrease in the developmental capacity of the oocyte. This developmental deficit was not overcome by co-culture with fresh disassociated cumulus cells. The stages in the cryopreservation profile at which this damage occurs need to be identified so that cryopreservation protocols can be designed which avoid or minimize such disruption. This could be achieved for example, by changing the rates of cooling/warming and employing alternative methods of cryopreservation such as vitrification (Hong et al., 1999).
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Acknowledgement |
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References |
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Submitted on August 1, 2002; accepted on October 22, 2002.