Hum. Reprod. Advance Access published online on January 23, 2008
Human Reproduction, doi:10.1093/humrep/dem414
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Ovarian tissue viability following whole ovine ovary cryopreservation: assessing the effects of sphingosine-1-phosphate inclusion
1 Division of Agricultural and Environmental Sciences, School of Biosciences, University of Nottingham, Sutton Bonington, Loughborough, Leicestershire, LE12 5RD, UK 2 Division of Obstetrics and Gynaecology, School of Human Development, University of Nottingham, Queens Medical Centre, Nottingham, Nottinghamshire, NG7 2UH, UK
3 Correspondence address. Tel: +44-115-951-6061; E-mail: bob.webb{at}nottingham.ac.uk
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Abstract |
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BACKGROUND: Cryopreservation is hypothesized to result in apoptosis, contributing to stromal damage and follicle loss in ovarian tissue. This study investigated tissue viability following whole ovine ovary cryopreservation and examined the effects of the anti-apoptotic agent sphingosine-1-phosphate (S-1-P) on ovarian cryopreservation efficiency.
METHODS: Whole ovine ovaries were cryoperfused and subjected to slow-freeze, rapid-thaw cryopreservation before a range of functional viability tests were performed. The effects of 20 µmol–1 S-1-P, in the cryopreservation media, were then assessed against a control cryopreservation media and non-frozen tissue.
RESULTS: Granulosa cell viability (assessed by trypan blue) was not significantly affected, however, Ki67 expression, indicative of cellular proliferation, was reduced following cryopreservation (P< 0.05). Following S-1-P supplementation, granulosa cell viability was not affected by either cryopreservation or S-1-P inclusion. Bromodeoxyuridine uptake, demonstrating DNA synthesis, was seen in both cryopreserved and fresh cortical tissue and the viability stain, 5(6)carboxyfluorescein diacetate succinimidyl ester, showed many viable small follicles. Cryopreservation increased arterial endothelial disruption (P< 0.01), but not internal elastic lamina rupture or venous damage. However, S-1-P supplementation did not improve ovarian or vascular tissue survival.
CONCLUSION: These results are encouraging for whole ovary cryopreservation, demonstrating maintained cell viability, however, they do not support S-1-P inclusion at this concentration to improve tissue viability following cryopreservation.
Key words: cryopreservation/whole ovary/viability/sphingosine-1-phosphate/ovarian graft
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Introduction |
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Ovarian cortical strip and whole ovary cryopreservation to allow the long-term storage of ovarian tissue has many applications including restoring ovarian function following iatrogenic failure, preserving endangered species and in advancing knowledge of early folliculogenesis (Nugent et al., 1997
; Newton, 1998; Shaw et al., 2000; Kim et al., 2001; Demirci et al., 2003; Falcone et al., 2004). Historically, the successful cryopreservation and thawing of ovarian tissue was first carried out in rodents during the 1950's (Parkes and Smith, 1953; Deansely, 1954; Green et al., 1956). However it was not until the early 1990's that the first live birth was reported in a large animal species following the grafting of previously cryopreserved cortical strips of ovarian tissue in sheep (Gosden et al., 1994). A subsequent study confirmed these results with a further live birth (Baird et al., 1999), but also demonstrated abnormal endocrine levels and severe depletion of follicle reserves within 22 months of transplantation.
The limited functional lifespan experienced with autografted ovarian tissue is not unexpected as the use of cortical strips reduces the follicle population present in the graft compared with the whole ovary. In addition, at least 70% of primordial follicles in the graft tissue are lost as a result of cryopreservation damage and ischaemia during graft revascularization (Baird et al., 1999). The potential to extend the period of ovarian function may therefore lie in whole ovary cryopreservation with vascular reanastomosis as this allows the preservation and transfer of the maximum follicle reserve and also provides the opportunity to immediately revascularize the graft, thus reducing the follicle loss associated with post-grafting ischaemia.
Whole ovary cryopreservation itself poses several challenges. While there have been numerous successful reports of whole ovary cryopreservation in rats and mice (Gunasena et al., 1997; Candy et al., 2000; Wang et al., 2002; Yin et al., 2003), in these species effective cryoprotectant penetration can occur through simple diffusion and grafts can be non-vascular. This is not the case for larger species whose ovaries are larger and more fibrous. Therefore a technique of cryoperfusion has to be adopted where the cryoprotectant is also perfused though the ovary via the ovarian artery and this has proved an effective method of cryopreservation (Bedaiwy et al., 2003; Arav et al., 2005; Imhof et al., 2006). In addition, freezing the whole ovary and pedicle requires the cryopreservation protocol to be optimized for many different tissue and cell types, i.e. ovarian stroma, follicular cells, oocytes and vascular tissue, some of which may be contradictory to each other.
Few studies involving freeze–thawing of whole human ovaries have been reported (Martinez-Madrid et al., 2004; Bedaiwy et al., 2006), however, the sheep is considered a suitable model for the human due to the similar size and fibrous nature of their ovaries (Gosden et al., 1994; Oktay et al., 2000). Although studies have been published on whole ovine ovary cryopreservation, it remains a relatively novel technique and while good follicle survival rates and some restoration of ovarian hormone production have been documented (Bedaiwy et al., 2003; Revel et al., 2004; Arav et al., 2005; Courbiere et al., 2005; Imhof et al., 2006), some loss of tissue viability is almost always evident and consistent, long-term ovarian function has yet to be achieved. Most of these studies have reported good follicle viability following cryopreservation, comparable with fresh tissue; however this has mostly been assessed by trypan blue exclusion and histological staining; neither of which measures cell viability in terms of its functional capabilities. Therefore there is a need for more in-depth functional assessments of ovarian tissue viability following perfusion, cryopreservation and thawing. In this study, Ki67 expression and bromodeoxyuridine (BrdU) uptake in cultured ovarian cortical tissue were used as cell proliferation markers to give an indirect assessment of cell viability. In addition, the viability stain 5(6) carboxyfluorescein diacetate succinimidyl ester (CFDA SE) was used to indicate cellular metabolic activity in ovarian cortical tissue. Trypan blue exclusion and 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) metabolism assays were also performed on isolated granulosa cells to assess viability in terms of cell membrane integrity and cell metabolism, respectively.
The loss of cell and follicle viability following cryopreservation has been attributed to necrosis as a result of intracellular ice formation and osmotic stress (Mazur, 1984; Gao and Critser, 2000), however, it is also thought that a proportion of cells are lost due to apoptosis (Stroh et al., 2002). A review of ovarian tissue cryopreservation studies shows that there is no general agreement on the effects of cryopreservation with respect to follicular morphology and apoptotic signalling (Oktay et al., 1997; Gook et al., 1999; Demirci et al., 2002; Fabbri et al., 2003; Rimon et al., 2005; Hussein et al., 2006). In this current study, it was hypothesized that apoptosis may be induced by cryopreservation and the inclusion of anti-apoptotic substances in the cryopreservation media may act to prevent some of this apoptosis and hence improve tissue survival. Sphingosine-1-phosphate (S-1-P) is one such anti-apoptotic agent. It is able to block the ceramide-induced apoptotic pathway (Cuvillier et al., 1996) and has been shown to suppress both radiation- and chemotherapy-induced follicular apoptosis (Perez et al., 1997; Morita et al., 2000; Paris et al., 2002) and increase follicle survival following the cryopreservation of ovarian tissue in sheep (H.A. Almislimani, H.M. Picton and B.K. Campbell, unpublished data). It is therefore possible that S-1-P could be utilized in whole ovary cryopreservation by its inclusion in the cryoprotective media to reduce the depletion of the follicle reserve by apoptosis, in order to lengthen the functional lifespan of the graft.
In the present study, we initially used a range of functional assessments to examine the effects of a slow-freezing protocol on tissue viability and then used the same functional tests to determine if exposure of the ovary and its vascular pedicle to the anti-apoptotic agent S-1-P, prior to cryopreservation, had any beneficial effect.
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All materials purchased from Sigma-Aldrich, Dorset, UK unless otherwise specified.
Cryoprotectant and S-1-P solution
The cryopreservation media was based on that previously shown to be effective for the cryopreservation of ovarian tissue autografts (Gosden et al., 1994); Leibovitz L-15 media supplemented with 1.5 mol l–1 dimethyl sulphoxide (DMSO; Fisher Scientific, Loughborough, UK), 0.1 mol l–1 sucrose (Fisher Scientific) and 10% heat inactivated fetal calf serum (FCS).
S-1-P (1 mg ml–1) was prepared according to the supplier's instructions by dissolving in 100% methanol at 50–60°C. The methanol was evaporated off to leave a thin film of S-1-P which was stored prior to use at –20°C. When required, the S-1-P was dissolved in the cryopreservation media to give a 20 µmol l–1 (7.59 µg ml–1) solution of S-1-P.
Tissue cryopreservation
For the initial study, whole ovine reproductive tracts (n = 9), complete with ovarian vasculature extending to the origin of the ovarian artery at the dorsal aorta, were collected from a local abattoir and transported to the laboratory in cold (0–4°C) Ringer's solution. The origin of the ovarian artery of one ovary from each tract was cannulated using a 2.5F (0.75 mm outer diameter) i.v. cannula (Sims Portex Limited, Kent, UK) (Fig. 1) and tied securely in place using 2/0 mersilk suture (Ethicon, Edinburgh, UK). The ovary and cannulated pedicle section were dissected free of the tract, with subsidiary vessels ligated using 2/0 mersilk suture, before being placed in a perfusion tray and immersed in the cryoprotective solution. The cryoprotective solution was perfused through the ovary via the cannula for 60 min at a rate of 0.5 ml min–1 using a syringe-driven perfusion pump (Precidor Infors Ag Basel; ChemLab Scientific Products Ltd, Hornchurch, UK) fitted with a 50 ml syringe. All solutions were refrigerated before use and all perfusions were carried out on ice.
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Each ovary, with its cannulated pedicle, was transferred to a 15 ml cryogenic vial (Scientific Laboratory Supplies, Nottingham, UK) and covered with cryoprotective solution. The tissue was then frozen with a planer freezer (Kryo 10 Series, Model 10–20, Planer Products Ltd, Sunbury-on-Thames, UK) using a programme based on that developed for cortical patches of ovarian tissue (Gosden et al., 1994
; Newton et al., 1996). The tissue was cooled to –9°C at –1°C min–1, when manual seeding was performed by holding the cryogenic vial over liquid nitrogen until the first signs of ice formation were observed. The tissue was then cooled to –40°C at a rate of –0.2°C min–1 and then to –140°C at a rate of –10°C min–1. The cryogenic vials were stored in liquid nitrogen. The second study, which examined the effects of S-1-P inclusion in the cryopreservation media, used a similar protocol to the first with minor changes. Reproductive tracts were collected from yearling fat lambs (n = 8) slaughtered at the on-site abattoir with cannulation and dissection of the ovary and pedicle completed within 60 min of slaughter. Each ovary was immersed in and perfused with cold Leibovitz L-15 media until all ovaries were cannulated. One ovary from each tract was then immersed in and perfused with a cold (0–4°C) cryoprotective solution as described above. Prior to cryopreservation the other ovary from each tract was immersed in and perfused with a cold, cryoprotective solution containing S-1-P, prepared as described above.
Tissue thawing
In both studies, the cryogenic vials were removed from liquid nitrogen and held at room temperature for 2 min before being plunged into a 37°C water bath and gently agitated for 30–40 min. Each ovary was then transferred to a perfusion tray and immersed in and, via the pre-existing cannula, perfused with warm (37°C) thawing media. In the first study three thawing media were used; the first consisted of Leibovitz L-15 media supplemented with 1 mol l–1 DMSO, 0.1 mol l–1 sucrose and 10% FCS, the second was supplemented with 0.5 mol l–1 DMSO and 10% FCS and the third with just 10% FCS. Each thawing media was perfused for 10 min at 1 ml min–1. In the second study BrdU labelling reagent [3 mg ml–1 BrdU in phosphate-buffered saline (PBS); Zymed® Laboratories; Cambridge Bioscience, Cambridge, UK] was also included in all the thawing media at 1 in 100 dilution and the perfusion times were increased to 30 min for thawing media 1 and 2 and to 60 min for the final thawing media to enable BrdU uptake studies. All perfusions were carried out at 37°C and the ovaries were kept at 37°C in the final thawing media until analysis.
Positive control tissue
In the first study, a fresh ovary was collected from a local abattoir on each thawing day and transported to the laboratory in 37°C PBS (n = 5). For the second study a whole reproductive tract, complete with vasculature, was collected from a local abattoir and transported in 37°C PBS (n = 6). On arrival, the ovarian artery of one ovary was cannulated and the ovary and pedicle dissected as described above, before being immersed in and perfused with the final thawing media for 2 h at 1 ml min–1. This tissue was subjected to the same viability tests as the frozen-thawed ovaries (for details, see next section).
Tissue dissection and viability tests
The ovarian pedicle was first separated from the ovary using a single-edged razor blade and samples were taken from around the area of cannulation, the mid-section and the hilus region. These samples were fixed in Bouin's fixative for later analysis of vascular tissue damage (this was done for the second study only; see details later).
Under aseptic conditions, the ovary was bisected and one-half further divided to give two quarters. The cortex of one quarter was fixed in Bouin's fixative and used for the detection of proliferating cell nuclear antigen (PCNA) expression using an immunohistochemistry method based on that described by Oktay et al. (1995). PCNA is a nuclear antigen only expressed in actively proliferating cells and because of its relatively long half life (Bravo and Macdonald-Bravo, 1987), its expression was used to give an indication of pre-freezing viability.
From the other quarter, thin strips of cortex (2 mm x 2 mm x <1 mm) were taken and divided between use for CFDA SE and propidium iodide (PI) viability staining and a longer-term viability study measuring Ki67 expression and BrdU uptake in cultured cortical tissue. Cell proliferation markers were studied as indirect indicators of cell viability; the assumption being that if a cell is involved in the cell cycle then it is viable. Ki67 is a nuclear antigen specifically expressed during the late G1, S, M and G2 phases of the cell cycle (but not G0) and its expression is positively correlated with cell proliferation (Gerdes et al., 1984). Ki67 expression has been used, more specifically, as an indicator of follicular cell proliferation (Fabbri et al., 2003) and therefore, expression was used here as an indicator of ovarian cell viability. Similarly, BrdU is incorporated into DNA in place of uridine during DNA replication and therefore its uptake indicates cellular activity in terms of DNA synthesis (Muskhelishvili et al., 2003). Its use in determining follicular cell proliferation has also been reported following both in vivo and in vitro tissue labelling (Jablonka-Shariff et al., 1994; Biron-Shental et al., 2004). The metabolic activity of cells following cryopreservation was also studied by incubation with the viability stain CFDA SE, which fluoresces green following its cleavage by intracellular esterases (Wang et al., 2005). Typically this stain, in conjunction with PI (which stains non-viable tissue in a similar way to trypan blue), is used with isolated cells or follicles (Oktay et al., 1997), however, in this study it was used with thin slices of cortex which also allowed the identification of viable follicles.
Collection of cortical strips was achieved using a clinical skin graft knife and was performed under warm culture media to maintain viability. The culture media was based on that developed by Campbell et al. (1996) for serum-free granulosa cell culture; 
-modified essential media (with sodium bicarbonate, but without L-glutamine) supplemented with 0.1% (w/v) bovine serum albumin, 100 kIU l–1 penicillin, 0.1 ug l–1 streptomycin, 3 mmol l–1 L-glutamine, 2.5 mg l–1 bovine holo-transferrin, 4 µg l–1 selenium, 10 ng ml–1 human recombinant Long R3 insulin-like growth factor-I (Novozymes GroPep Ltd., Adelaide, Australia), 10 ng ml–1 bovine insulin and 10 ng ml–1 ovine FSH [NIDDK-oFSH-S20; kind donation from the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)].
The remaining hemi-ovary was used to recover granulosa cells from antral follicles. The granulosa cells were isolated in Dulbecco's PBS (DPBS), washed in culture media and a final cell suspension was made in culture media (1 ml) in preparation for use both in a trypan blue exclusion test (Freshney, 2000) and an MTS metabolism assay using the CellTitre96® AQueous One Solution Cell Proliferation Assay kit (Promega UK, Southampton, UK), as used in previous studies (Brankin et al., 2005). The trypan blue exclusion test was used to give an assessment of cell membrane integrity (only entering those cells with damaged or non-intact cell membranes), while the CellTitre assay demonstrates cellular metabolic activity in terms of the metabolism of MTS to a coloured formazan product which can be detected and measured, giving a functional assessment of cell viability.
Longer-term viability study
An explant culture of cortical strips was set-up similar to that described previously for uterine tissue (Sheldrick and Flicksmith, 1993). Briefly, cortical strips were cultured on squares of sterile lens paper (Whatman #105, Fisher Scientific) placed on small stainless steel grids in 6-well plates and culture media was added to a level that just made contact with the tissue. The tissue was cultured at 37°C and 5% CO2 for 48 h, with a media change after 24 h when BrdU labelling reagent was included in the culture media (1 in 100 dilution; as recommended by the manufacturer). Following culture, the tissue strips were fixed in 4% paraformaldehyde solution before being processed and embedded in paraffin wax in preparation for Ki67 expression and BrdU uptake detection using immunohistochemistry.
Immunohistochemistry methods
Tissue sections (5 µm) were dewaxed and rehydrated through an ascending series of alcohols before undergoing heat and citrate buffer (10 mM; pH 6.0) antigen retrieval. To detect PCNA expression, anti-PCNA mouse monoclonal antibody (38 mg ml–l; PCNA Clone PC10; Sigma-Aldrich) was diluted 1:2000 in PBS and incubated on sections overnight at 4°C. For Ki67 expression, anti-Ki67 mouse monoclonal antibody (85 µg ml–l; Vector Laboratories; Peterborough, UK), diluted 1:100 in PBS, was added to the tissue sections for 1 h at 37°C. For detection of BrdU uptake, anti-BrdU mouse monoclonal antibody (24 µg ml–l; Novocastra Laboratories; Newcastle, UK) was diluted 1:200 in PBS and incubated on sections overnight at 4°C. Negative controls were also included where the primary antibody was substituted for mouse immunoglobulin (Ig)G at the same concentration. A standard avidin–biotin complex technique was then carried out on all slides using a Vectastain Elite ABC Mouse IgG kit (Vector Laboratories). Visualization of positively stained cells was achieved using 3, 3'-diaminobenzidine substrate (Vector Laboratories) and counterstaining with Mayer's haematoxylin.
Image analysis
Analysis was carried out on several random images taken from Ki67 expression and BrdU uptake slides using an "OpenLab" image package (Improvision®). All positively stained and counterstained cells in each image were counted and the percentage of cells expressing Ki67 or demonstrating uptake of BrdU was then calculated.
Follicle counts
Sections of ovarian cortex (5 µm) fixed either immediately after thawing (or collection; in terms of fresh tissue) or following culture for 48 h were stained with haematoxylin and eosin (H&E). The numbers of each type of follicle (defined below) were counted in 20 consecutive magnification fields (x400) covering the ovarian cortex. The average count for each type of follicle was then calculated and compared between pre-culture and post-culture cortical tissue. The follicles were categorized by the following classification based on that by Lundy et al. (1999):
- Primordial—single layer of flattened pre-granulosa cells.
- Transitional/Primary—single layer of granulosa cells; one or more being cuboidal.
- Secondary and Preantral—two or more layers of cuboidal granulosa cells; no antrum present.
- Antral—multiple layers of cuboidal granulosa cells; antrum present.
CFDA SE/PI viability staining
Slices of ovarian cortical tissue were incubated with CFDA SE solution (1 mg ml–1 in DMSO; 100 µg ml–1 in DPBS) for 20 min at 37°C followed by PI solution (1 mg ml–1 in DMSO; 100 µg ml–l in DPBS) for 5 min at 37°C. The tissue was washed in Leibovitz L-15 medium with 10% (v/v) FCS and, working in restricted light, a whole mount of the tissue was prepared and viewed under a fluorescent microscope (x100). Viable tissue fluoresced green, whereas non-viable tissue appeared red.
Images of the tissue from the S-1-P study were captured using "Simple PCI" image software (Compix Inc., Imaging systems, PA, USA) and follicle counts were carried out on these images using Adobe® Photoshop® CS2 (Version 9.0; Adobe Systems Incorporated, USA). The area of highest follicle density within each image was identified and the number of fluorescing viable follicles within five consecutive fields of view were counted across the tissue. The average number of follicles per field of view was then calculated for each ovary.
Pedicle tissue analysis
For each pedicle region, 5 µm sections, spaced 100 µm apart, were stained using H&E. On each section the gross tissue morphology was studied, similar to that described previously (Courbiere et al., 2005). For every artery in each section, the following were recorded: percentage endothelial cell disruption, evidence of internal elastic lamina (IEL) rupture (Fig. 2) and damage to the smooth muscle tissue. Percentage endothelial cell disruption was also recorded for every vein seen in each section.
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Statistical analyses
Statistical analyses of both the granulosa cell viability data and endothelial layer detachment data were performed using a general analysis of variance on the GenStat statistical computer package (VSN International Ltd, Hemel Hempstead, UK) having confirmed that the data was normally distributed. Statistical analyses for the Ki67 expression and BrdU uptake cell count data in addition to the IEL rupture and smooth muscle damage data for the pedicle tissue were performed on GenStat using a generalized linear regression with binomial distribution following a logit transformation. Follicle count data generated following both CFDA SE and H&E staining were analysed using a generalized linear mixed model with a Poisson distribution using residual maximum likelihood on the GenStat program. A value of P < 0.05 was considered significant.
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Results |
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Study 1: assessing current efficiency
Pre-freezing viability—PCNA expression
PCNA expression was analysed in cryopreserved cortical tissue fixed immediately after thawing as an indicator of the pre-freezing viability of the ovaries used. Abundant PCNA expression was observed in both follicular and stromal cells in tissue taken from all the ovaries used (Fig. 3), which suggested that the ovarian tissue and follicles were viable at the start of the study.
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Longer-term viability study—Ki67 expression
Cultured cortical strips demonstrated positive staining for Ki67 expression in granulosa cells of preantral follicles and stromal cells in both cryopreserved and fresh ovaries. Where possible, six sections of tissue from each ovary were studied for Ki67-positive cells. In total, eight primordial, seven primary and three preantral follicles were seen with Ki67-positive granulosa cells in the frozen-thawed ovarian tissue compared with two primary, three secondary and one preantral follicle in the fresh tissue examined. The percentage of stromal cells expressing Ki67 in each image was also calculated. In the cryopreserved tissue, on average 1.6% of cells expressed the Ki67 antigen, and were therefore assumed to be proliferating. This was significantly less than for fresh tissue sections, in which 3.4% of cells expressed Ki67 (P < 0.05).
Granulosa cell viability
Granulosa cell viability was not significantly affected by cryopreservation, as determined by trypan blue exclusion. On average, frozen-thawed tissue yielded 39.1% (SEM 6.0) viable granulosa cells compared with 22.7% (SEM 3.7) in fresh, control tissue. In addition, neither trypan blue exclusion nor CellTitre® viability assay results showed any significant effect of cryopreservation on the total number of viable granulosa cells recovered. Following trypan blue exclusion, cryopreserved tissue yielded on average 419 000 viable granulosa cells ml–1 (SEM 171 000); which although less than that recovered from the fresh tissue (652 000 viable cells ml–1; SEM 145 000), was not found to be significantly different. The CellTitre® viability assay also showed no significant reduction in the average number of metabolically active granulosa cells recovered from frozen-thawed tissue (509 000 viable cells ml–1; SEM 98 000) compared with fresh control ovarian tissue (715 000 viable cells ml–1; SEM 102 000).
Study 2: effects of S-1-P supplementation
Pre-freezing viability—PCNA expression
As in the first study, abundant PCNA expression was observed in both follicular and stromal cells in ovarian tissue fixed immediately after thawing (Fig. 3), suggesting that all the ovaries used in this study contained viable tissue and follicles prior to cryopreservation.
Longer-term viability study
Ki67 expression
Most of the positive staining for Ki67 expression was observed in the ovarian stroma, however, evidence of granulosa cell Ki67 expression was also seen in all classifications of early follicles (Fig. 4). From the sections studied, three out of eight S-1-P supplemented ovaries contained follicles with granulosa cells positive for Ki67 expression; in total, three primary follicles, one secondary follicle and one preantral follicle. Follicles with Ki67-positive granulosa cells were observed in six out of eight control frozen ovaries, comprising four primordial, seven primary and five preantral follicles. Fifty per cent of the tissue studied from fresh, positive control ovaries contained follicles with Ki67-positive granulosa cells; two primary follicles, two secondary and three preantral follicles were observed.
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Looking at the ovarian stroma, neither cryopreservation nor S-1-P supplementation had a significant effect on Ki67 expression. On average, 2.0% of cells from S-1-P supplemented ovaries expressed the Ki67 antigen compared with 2.9% in control cryopreserved ovaries. The fresh, control ovaries contained the lowest amount of positive staining, 1.7% of cells, though this difference was not statistically significant (P > 0.3).
BrdU incorporation
Initially, no positive staining was detected for BrdU uptake in either frozen-thawed or fresh ovaries following the culture of cortical strips with BrdU label. Following a period of method development, culminating in the inclusion of BrdU label in the thawing media and perfusion through the ovary for a total of 2 h in addition to the culture period (see Tissue thawing section of Materials and Methods), positive evidence of BrdU uptake was seen in fresh and cryopreserved whole ovaries. Therefore this method was adopted in the second study.
Uptake of BrdU, indicative of DNA synthesis (Muskhelishvili et al., 2003) and therefore suggestive of cell viability, was observed in cultured cortical tissue from ovaries subjected to all treatments (Fig. 5). Evidence of BrdU uptake by granulosa cells was seen in tissue from five out of eight S-1-P supplemented ovaries, seven out of eight control frozen ovaries and four out of six fresh positive control ovaries. In S-1-P supplemented tissue, 1 primordial, 10 primary, 4 secondary and 4 preantral follicles were seen to contain positively stained granulosa cells. In the control frozen tissue, granulosa cells in 7 primordial, 26 primary, 4 secondary and 5 preantral follicles were seen to have taken up the BrdU label. Fewer follicles were seen with proliferating granulosa cells in the fresh positive control tissue; eight in total comprising two primordial, two primary, two secondary and two preantral follicles.
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The majority of BrdU uptake was seen in stromal cells. Following image analysis, in fresh, non-cryopreserved ovarian tissue on average 4.4% of stromal cells had taken up the BrdU label compared with 3.5% of cells in the S-1-P supplemented cryopreserved ovarian tissue. These results were not found to be significantly different. However in control frozen ovaries, on average 7.9% of cells had taken up the BrdU label, which was significantly greater than that in either the S-1-P cryopreserved or fresh groups (P < 0.05).
Follicle count
Table I shows the average number of follicles of each classification contained within 20 magnification fields of ovarian cortex both immediately after thawing and following 48-h culture. Within all follicle classifications and across the treatment groups, follicle density was significantly lower following 48-h culture (P < 0.001). However between the treatments, control cryopreserved and S-1-P cryopreserved, there was no significant difference in follicle density either immediately after thawing or following 48-h culture (P > 0.2). While follicle density decreased following culture, the extent of this reduction was not significantly affected by cryopreservation or S-1-P inclusion.
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CFDA SE/PI viability staining
In the first study, green fluorescence was observed in both fresh and cryopreserved cortical tissue, indicating viable tissue. However, an improvement in the use of CFDA SE and PI viability staining in the second study also allowed the visualization of a substantial number of primordial and primary follicles in tissue from all treatment groups (Fig. 6). Follicle counting was performed on several images from each ovary; between three and eight images per ovary. Only images with sufficient tissue in focus were included. S-1-P tissue yielded on average 2.2 viable follicles per 0.3 mm2 area, whereas in control cryopreserved tissue, on average 1.6 viable follicles were counted over the same area. These results show a trend of increased viable follicle density following S-1-P supplementation of the cryopreservation media, however, this was not found to be statistically significant. Encouragingly, there was also no statistically significant detrimental effect of cryopreservation on viable follicle density as fresh control tissue contained 2.1 viable follicles per 0.3 mm2 area.
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Granulosa cell viability
The results of the trypan blue exclusion assay showed S-1-P inclusion in the cryopreservation media had no significant effect on average granulosa cell viability (25.3%; SEM 1.7) compared with ovaries frozen with the control cryoprotectant (24.7%; SEM 2.9). The fresh, positive control ovaries yielded on average 24.7% (SEM 4.5) viable granulosa cells, which again was not found to be significantly different.
In terms of the average total number of viable granulosa cells recovered per follicle (Table II), results from S-1-P cryoperfused ovaries were not significantly different from either control cryopreserved ovaries or the fresh, positive control ovaries when using either trypan blue exclusion or CellTitre® assays.
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Vascular tissue
Cryopreservation had a significant effect on the extent of endothelial cell detachment suffered by the arterial tissue (P< 0.01). The arterial endothelial layer disruption experienced by cryopreserved pedicles taken from ovaries perfused with S-1-P supplemented cryoprotectant was not found to be significantly different from that seen in the ovarian pedicles frozen following perfusion with a control cryoprotectant. However, both groups of frozen-thawed pedicles showed a significantly greater amount of arterial endothelial damage than the non-frozen pedicles (P < 0.01; Table III). This was seen across all the pedicle regions and suggested that freeze-thawing had a detrimental effect on the integrity of the endothelial layer which was not benefited by the inclusion of S-1-P. In both cryopreserved groups the cannulated region sustained significantly more endothelial damage than the hilus region (P < 0.05), with the mid-section values intermediate to the other two regions. In the fresh tissue however, damage to the endothelial layer was not significantly different between pedicle regions.
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Disruption of the endothelial layer in the venous tissue was less extensive than in the arterial tissue for both of the frozen-thawed treatment groups. There was also no significant difference in venous endothelial layer disruption between frozen and fresh pedicles or between any of the pedicle regions studied (Table III).
In terms of IEL rupture there was no statistically significant effect of cryopreservation or S-1-P inclusion observed at any pedicle region. Also within treatment groups, pedicle region did not significantly affect the extent of IEL damage sustained.
When using the combined results from the cryopreserved tissue compared with the fresh tissue, cryopreservation was seen to result in greater smooth muscle damage across all the pedicle regions studied (P < 0.02). However between the two cryopreserved groups, supplementation of the cryopreservation media with S-1-P had no significant effect. Damage to the smooth muscle was also not found to be significantly more prevalent in any one pedicle region (Table III).
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Discussion |
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Overall, these results are extremely encouraging for whole ovary cryopreservation with good evidence of survival and restored cell proliferation in frozen-thawed ovarian tissue. No significant detrimental effect of cryopreservation was found on granulosa cell viability in terms of either cell membrane integrity or metabolic activity. Following the culture of ovarian cortical strips, evidence of cell proliferation was demonstrated by Ki67 expression and BrdU uptake in both fresh and cryopreserved tissue to a similar degree. The presence of metabolically active primordial and primary follicles in frozen-thawed tissue was confirmed using CFDA SE viability staining and viable follicle density also appeared unaffected by the cryopreservation process. While cryopreservation was shown to significantly increase arterial endothelial cell disruption and smooth muscle damage, no significant damaging effect was seen on the venous endothelial cell layers or arterial IEL. However, S-1-P inclusion was not shown to have a beneficial effect on any of these aspects of ovarian or vascular tissue viability.
PCNA expression was examined in freshly thawed cortical tissue to assess pre-freezing cell proliferation. Staining for PCNA expression was evident in both follicular and stromal cells in ovarian tissue taken from all the ovaries used in these studies (Fig. 3), implying that they all contained viable, proliferating cells at the commencement of the study.
Ki67 expression and BrdU uptake were also used as measures of cell proliferation (Gerdes et al., 1984; Muskhelshvili et al., 2003). Ki67 expression and BrdU uptake were seen in both follicular and stromal cells in tissue from cryopreserved whole ovaries, suggesting not only that these follicles would be capable of further growth and development, but also that the stroma would be able to provide structure and support for developing follicles following grafting. The majority of proliferative cells were stromal cells and in contrast to the first study, where Ki67 expression was significantly lower in cryopreserved stromal tissue, the second study found no significant effect of cryopreservation on Ki67 expression or BrdU uptake in stromal cells. In fact, a significantly greater percentage of stromal cells in the control cryopreserved ovaries took up BrdU label than in either the S-1-P cryopreserved or fresh control groups which suggests some sort of stimulus, as a result of cryopreservation, for cells to undergo DNA synthesis or possibly repair. This follows a similar, though more significant pattern to that described for Ki67 expression both in the second study of this paper and also in a study on cryopreserved human ovarian tissue (Fabbri et al., 2003).
Further evidence of sustained cell viability following whole ovary cryopreservation was demonstrated by CFDA SE/PI staining. CFDA SE fluorescence indicates that a cell is metabolically active (Wang et al., 2005) and the images obtained clearly showed many viable primordial and primary follicles following cryopreservation (Fig. 6). This is an important result as it is the small follicle population whose survival is paramount to long-term ovarian function (Gosden et al., 1994; Baird et al., 1999).
The viability of granulosa cells isolated from antral follicles was unaffected by cryopreservation, implying that these follicles were able to withstand freeze-thawing. This is in contrast to the common finding that only primordial follicles are able to survive cryopreservation (Gosden et al., 1994; Newton et al., 1996; Aubard et al., 1999; Baird et al., 1999). However, this present study also found evidence of primary, secondary and preantral follicles capable of and actively undergoing proliferation, as determined by Ki67 expression and BrdU uptake, following only 48-h culture. Other studies support these findings, detailing the survival of primordial through to antral stage follicles in cryopreserved ovarian tissue in several species (Candy et al., 1995; Salle et al., 1998; Fabbri et al., 2003; Almodin et al., 2004a, b). However, where follicle survival has been measured after a period of either culture or grafting into a host, results show that the vast majority of growing follicles past the primary stage are in fact irrecoverably damaged with very few observed within one week of grafting (Gosden et al., 1994; Aubard et al., 1999; Baird et al., 1999).
During the first trial, the collection and transport of reproductive tracts from a local abattoir could take 2–3 h. Therefore in the subsequent trial, ewes were slaughtered at the on-site abattoir, ensuring that cannulation and perfusion occurred within 60 min of slaughter. Comparison of granulosa cell viability results between the two trials (38.7 and 24.6%, respectively) showed that the longer ischaemia time had no significant effect on cell viability and confirmed that tissue viability had not been compromised in the first study.
It is important to consider vascular tissue survival following whole ovary cryopreservation, particularly as one of its main advantages over the use of cortical strips is the ability to reanastomose the ovarian artery and vein during regrafting thereby providing an immediate and sustained revascularization of the tissue. Data collected here has shown a significant detrimental effect of cryopreservation on the extent of arterial endothelial cell layer detachment (P < 0.01) and arterial smooth muscle damage (P < 0.02), with no beneficial effect of S-1-P inclusion. These results, in particular the effect of cryopreservation on the endothelial cell layer, may give some insight as to why previous autograft studies have suffered vascular occlusions and thromboses (Bedaiwy et al., 2003; Arav et al., 2005) and suggest that protection of the endothelial layer during cryopreservation represents an important area for further research.
While ovarian tissue is more commonly thawed rapidly, studies involving the cryopreservation of both mini-pig and rabbit arterial tissue have shown that vascular tissue is best preserved using a slower, more controlled thawing technique and demonstrate the damaging effect of rapid thawing on both the endothelial layer and general vascular structure (Pegg et al., 1997; Bujan et al., 2001). A further study also attributed the high rate of fracture formation in ovarian arteries following vitrification of whole ovine ovaries to the rapid rate of thaw used (Courbiere et al., 2005). The development of an optimal thawing rate for both ovarian and vascular tissues may therefore represent an important area to consider for future research.
The majority of whole ovine ovary cryopreservation studies have utilized slow-freeze, rapid-thaw protocols (Bedaiwy et al., 2003; Arav et al., 2005; Imhof et al., 2006) as in this current study. Difficulties arise when comparing the results of these studies with the current study due to the variety of viability assessments used and the depth of analysis. However, Arav et al. (2005) documented no significant effect of whole ovary cryopreservation on follicle viability following fluorescent live/dead staining, echoing the results obtained here. Despite no comparisons of follicle viability made between fresh and frozen tissue, Bedaiwy et al. (2003) found no significant difference in follicle viability, using the trypan blue method, between cryopreserved whole ovaries and cortical strips. Possible detrimental effects of the freeze-thaw process on the ovarian vasculature are suggested by the occurrence of vascular occlusions reported by both Bedaiwy et al. (2003) and Arav et al. (2005) following autotransplantation of cryopreserved whole ovine ovaries. Imhof et al. (2006) made no in vitro measurements of ovarian tissue survival prior to ovarian transplantation, however, the encouraging results following the autotransplantation of cryopreserved whole ovaries suggest some tissue survival. Despite this, evidence of tissue damage and follicle losses were evident following grafting. A further study, (Courbiere et al., 2005) employed vitrification of whole ovine ovaries followed by rapid thawing and, as with this current study, overall found no significant effect on follicle viability. Again similar to this study, evidence of vascular tissue damage was reported in terms of smooth muscle and endothelial cell disruption which was attributed to the rapid rate of thawing. Therefore, in conclusion, it can be seen from this, and other studies, that further development and optimization of cryopreservation protocols for whole ovaries in sheep, and other large animal species, is still required.
Supplementation of the cryopreservation media with the anti-apoptotic agent S-1-P at a 20 µmol l–1 dose had no significant beneficial effects on cell survival and proliferation. Ki67 expression levels were not significantly different between control and S-1-P supplemented cryopreserved tissue, nor were the viable follicle counts following CFDA SE viability staining significantly improved. In addition, S-1-P supplementation did not provide any improvement in isolated granulosa cell viability or the total number of viable granulosa cells recovered. This observation supports the hypothesis that the majority of cell loss sustained following cryopreservation is due to necrosis as a result of either intracellular ice formation or osmotic stress during freezing and thawing (Mazur, 1984; Gao and Critser, 2000), rather than apoptosis (Bedaiwy et al., 2006; Hussein et al., 2006). However, other reports have suggested that cryopreservation does induce the expression of apoptosis-related genes (mouse; Liu et al., 2003) and increases the percentage of apoptotic follicles (human; Rimon et al., 2005) in frozen-thawed ovarian tissue. In addition, Rimon et al. (2005) also commented that the percentage of apoptotic follicles observed in cryopreserved human ovarian tissue, following terminal deoxynucleotidyltransferase-mediated dUTP nick-end labelling and 4',6-Diamidino-2-phenylindole staining, was significantly greater than the percentage of follicles deemed "morphologically normal" following H&E staining. This, therefore, suggests that the morphological assessments commonly used in cryopreservation studies may underestimate the extent of cryoinjury sustained by ovarian tissue. However, it must also be considered that Rimon et al. (2005) cryopreserved ovarian tissue taken from ovarian cancer patients and therefore this may have affected or altered the apoptotic mechanisms away from the norm.
An alternative explanation for the lack of response to S-1-P supplementation is that this factor may be targeting the wrong apoptotic pathway. Previous published examples of S-1-P's ability to reduce follicular apoptosis have been in the counteraction of either chemo- or radiotherapy-induced apoptosis in murine ovarian tissue (Perez et al., 1997; Morita et al., 2000). Chemo- and radiotherapies are known to induce ceramide-activated apoptotic pathways (Haimovitz-Friedman et al., 1994; Perez et al., 1997; Morita et al., 2000) and therefore if cryopreservation does not activate the same apoptotic pathway, this would explain the lack of effect of S-1-P. Caspase-3 activation has been implicated in the apoptosis seen following sperm cryopreservation (Stroh et al., 2002; Martin et al., 2004; Paasch et al., 2004) and is also associated with follicular atresia (Nicholas et al., 2005). The inhibition of caspase-3 may therefore represent an alternative route to improve cryopreservation efficiency in whole ovaries. Finally, it is possible that the methanol used to dissolve the S-1-P could have had a detrimental effect on tissue viability due to its toxicity, thereby masking any positive effects of S-1-P. However, the dissolution was carried out as per the supplier's instructions and was evaporated to dryness and, therefore no methanol should have been present in the perfusion media.
One of the main aims of this study was to utilize a wide range of functional viability assessments following whole ovary cryopreservation to obtain a clearer picture of its effects on ovarian tissue viability and in doing so, determine the causes behind the lack of success in terms of long-term ovarian function currently seen following autografting. In conclusion, this study has extended previous research findings to show that the results of these functional assessments of ovarian cell viability are comparable between fresh and cryopreserved whole ovine ovaries. However, longer-term cultures or grafting studies are still required to confirm fully long-term follicular viability. In contrast, cryopreservation was seen to have a significantly detrimental effect on the endothelial layer of arterial tissue contained within the ovarian vascular pedicle, suggesting that more research needs to be focused on the development of optimal cryopreservation protocols for both ovarian and, in particular, endothelial vascular tissue. However, this study cannot support the inclusion of 20 µmol l–1 S-1-P as an anti-apoptotic agent in a cryoprotective solution to improve either ovarian or vascular cell viability following whole ovary cryopreservation.
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V.J.O was supported by a post-graduate scholarship from the University of Nottingham.
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The authors would like to thank Mrs Catherine Pincott-Allen, Mrs Anne Skinner and Miss Sylwia Adamiak for technical assistance, Mr John Corbett for tissue collection and Dr Jim Craigon for advice on statistical analyses.
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Submitted on March 2, 2007; resubmitted on November 20, 2007; accepted on December 9, 2007.
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