Department of Reproductive Medicine, Westmead Hospital, Westmead, Sydney, NSW 2145, Australia
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Abstract |
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Key words: cryopreservation/culture/follicle/ovary
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Introduction |
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The first studies of ovarian tissue cryopreservation were carried out in rodents during the 1950s (Parkes and Smith, 1953; Green et al., 1956
; Parkes, 1956
; Parrott, 1960
). Ovarian follicles were found to survive freezethawing, albeit in low numbers, and if tissue was transplanted into ovariectomized recipients cyclical function and fertility were restored. Over recent years significant improvements in the field of cryobiology have been made, most importantly the introduction of controlled rate freezing apparatus and more efficient cryoprotective agents. A corresponding improvement in the success of murine ovarian tissue freezing has been demonstrated (Harp et al., 1994
; Cox et al., 1996
; Gunasena et al., 1997
). The technique has also proved successful with primate and ovine tissue (Gosden et al., 1994
; Candy et al., 1995
). A high proportion of viable follicles survive in human tissue after freezethawing (Hovatta et al., 1996
; Newton et al., 1996
; Hovatta et al., 1997
; Oktay et al., 1997
; Gook et al., 1999
) and this has lead to interest in the procedure as a potential strategy for preserving the fecundity of patients at risk of premature ovarian failure (Wood et al., 1997
; Newton, 1998
; Oktay et al., 1998
).
One method of harvesting mature oocytes from the frozenthawed tissue may be to isolate small follicles from the surrounding stroma and grow them to maturity in vitro (Gosden et al., 1993; Hartshorne, 1997
; Smitz and Cortvrindt, 1999
). The dissection of a small piece of ovarian tissue harvests large numbers of immature follicles, many of which are destined never to undergo ovulation. In the murine model, gently teasing apart the stroma with a needle yields a number of pre-antral follicles which can be grown to antral sizes in vitro (Qvist et al., 1990
; Nayudu and Osborn, 1992
). In response to human chorionic gonadotrophin (HCG), oocytes are released from the antral follicles and can be fertilized, producing blastocysts (Cortvrindt et al., 1996a
; Rose et al., 1999
) which, after transfer to pseudopregnant females, have resulted in the birth of viable pups (Spears et al., 1994
). If follicles are frozenthawed after isolation from the ovarian tissue antral stages still develop and hatched blastocysts have been produced after fertilization of the mature oocytes (Cortvrindt et al., 1996b
). Less progress has been made in other species, although techniques have been developed to grow follicles isolated from human, pig, sheep, rat and hamster tissue to antral stages in vitro (Roy and Greenwald, 1989
; Roy and Treacy, 1993
; Hirao et al., 1994
; Cain et al., 1995
; Abir et al., 1997
; Newton et al., 1999
). In the ovine model, freezethawing the tissue prior to follicle isolation did not reduce the ability of follicles to develop to antral stages (Newton et al., 1999
). Nevertheless, to date, live births have only been recorded from in-vitro grown murine pre-antral follicles.
Optimization of the freezing protocol for ovarian tissue should reduce the number of follicles irreversibly damaged during cooling and thus increase the size of the population available for isolation and in-vitro growth. In this study murine follicles were isolated from frozenthawed tissue and their survival and growth was studied during 8 days of in-vitro culture. Different cryoprotective agents and seeding/thawing temperatures were tested to investigate their effect on the rate of follicle survival. In order to study further the viability of frozenthawed follicles the size of the granulosa cell population was studied with a cell proliferation assay and endocrine function after 8 days was assessed by measurement of dimeric inhibin.
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Materials and methods |
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Slow freeze cryopreservation
Dimethyl sulphoxide (DMSO), propylene glycol (PROH) and glycerol (GLY) were prepared at a concentration of 1.5 mol/l in Leibovitz medium supplemented with 10% FCS and 0.1 mol/l sucrose. The cryoprotectant solutions (1.5 ml) were aliquotted into 1.8 ml cryovials (Nalgene; New York, NY, USA). Ovarian pieces were transferred into cryovials (two per tube) and gently rolled on a Coulter mixer (Coulter Electronics Ltd., Herts, UK) for 25 min at 4°C to allow equilibration of the cryoprotective agent. The vials were cooled in a programmable freezer (Cryomed 1010) as follows: (i) cooled from 4°C at 2°C/min to 9°C; (ii) soaked for 6 min at 9°C; (iii) seeded manually; (iv) held for 4 min at 9°C; (v) cooled at 0.3°C/min to 40°C; (vi) cooled to 100°C at 10°C/min; (vii) plunged into liquid nitrogen and stored for up to 2 weeks. Three freezes were carried out on separate days. To investigate the effect of seeding temperature ovarian tissue was equilibrated with DMSO, as described above, and the freezing programme was altered to allow manual seeding at either 5°C or 7°C. For each seeding temperature three freezes were carried out on separate days.
Thawing
To compare different cryoprotective agents the vials containing tissue frozenthawed in DMSO, PROH or GLY were thawed by plunging into a water bath at room temperature (~22°C), as described in previous protocols (Newton et al., 1996). To compare seeding temperatures, vials containing tissue frozen in DMSO and seeded at either 5°, 7° or 9°C were thawed at 37°C. The higher thawing temperature was chosen to investigate whether overall follicular survival rates were affected. The results suggested that the thawing temperature did have an effect, therefore tissue frozen in DMSO and seeded at 7°C was thawed at either 27°, 37° or 47°C. In all cases vials were removed from the water bath as soon as the sample was fully thawed, thus avoiding overheating of tissue thawed at 47°C. Dilution of the cryoprotectant was carried out in a stepwise manner by transferring tissue into Petri dishes containing 3 ml of antibiotic medium supplemented with: (i) 1 mol/l cryoprotectant/0.1 mol/l sucrose for 5 min; (ii) 0.5 mol/l cryoprotectant/0.1 mol/l sucrose for 5 min; (iii) 0.1 mol/l sucrose for 3 min. Tissue was finally transferred into fresh antibiotic medium for 5 min before follicle isolation. Each step was carried out at 37°C on a heated stage.
Vitrification (rapid freeze)
The vitrification method was based on the protocol of Vajta et al. for the vitrification of bovine embryos (Vajta et al., 1996). Ovarian pieces were transferred to cryovials (1.8 ml) containing 1.5 ml of Leibovitz medium supplemented with 0.03 mol/l sucrose, 20% FCS, 10% (v/v) DMSO and 10% (v/v) PROH for 10 min. The tissue was subsequently moved into cryovials containing 1.5 ml of Leibovitz medium supplemented with 0.03 mol/l sucrose, 20% FCS, 20% DMSO/20% PROH and equilibrated for 5 min. Throughout the procedure the samples were gently rolled on a Spiramix at 4°C to allow equilibration of the cryoprotective agent. Vials were then transferred directly into liquid nitrogen. The freezing procedure was repeated three times on separate days. Thawing was carried out by plunging vials into a water bath at room temperature. The tissue was retrieved and transferred to Petri dishes containing 3 ml of Leibovitz medium supplemented with 0.03 mol/l sucrose, 20% FCS, 10% DMSO and 10% PROH for 5 min, tissue was then moved to antibiotic medium containing 0.03 mol/l sucrose for 5 min and finally to antibiotic medium alone for 5 min before follicle isolation. Each step was carried out at 37°C on a heated stage.
Follicle isolation
Murine pre-antral follicles measuring 100135 µm in diameter were isolated under sterile conditions using 27 gauge insulin needles (Terumo, Leuven, Belgium) and a dissecting microscope with a heated stage (37°C). Isolated follicles were transferred into antibiotic medium at 37°C and washed twice before culture. Morphology was assessed under the dissecting microscope and only follicles with an intact round structure and a spherical centrally located oocyte were used in the study.
For comparative purposes follicles from fresh murine ovarian tissue were isolated and cultured in an identical manner to those which had been isolated from frozenthawed tissue.
Follicle culture
All follicles were cultured in minimal essential medium (Life Technologies, Melbourne, Victoria, Australia), supplemented with 50 IU/ml penicillin, 50 µg/ml streptomycin, 5.5 µg/ml sodium pyruvate, 3 mmol/l L-glutamine, 10 µg/ml transferrin, 5 µg/ml insulin, 5 ng/ml sodium selenite and 90 IU/ml recombinant FSH (Gonal-F®; Serono, Frenchs Forest, Sydney, Australia) (Cortvrindt et al., 1996a
).
Follicles were cultured individually in 96-well sterile flat bottomed plates (Falcon, Becton Dickinson Labware, Franklin, NJ, USA) containing 200 µl of culture medium supplemented with 10% FCS at 37°C under an atmosphere of 5% CO2 and 95% air. The period of in-vitro growth was 8 days. On day 4 of culture half the medium was removed and replenished with fresh prewarmed medium. Late on day 7, 180 µl of medium was carefully removed, taking care to leave a small volume over the follicle and replaced with 100 µl of fresh medium. After 24 h of culture 100 µl of spent medium was removed and stored at 20°C for measurements of day 8 inhibin A and B production. Follicle diameter was measured throughout the culture period on a dissection microscope (x100) with an ocular micrometer.
Ovulation
On day 8 of culture follicles were stimulated by the addition of 100 µl of medium containing 2.5 IU/ml Chorulon® (chorionic gonadotrophin; Intervet Australia, Sydney, NSW, Australia) and 5 ng/ml human epidermal growth factor (Smitz and Cortvrindt, 1998). Cumulusoocyte complexes were removed from the culture wells, stripped and assessed for maturation 17 h later.
Assays
After careful removal of the cumulusoocyte complex on day 8 the number of granulosa cells remaining in the culture well was measured using the CellTiter 96® non-radioactive cell proliferation assay (Promega, Madison, WI, USA). The assay is based on the cellular conversion of tetrazolium salt into a formazan product which is detected at an absorbance 550 nm with a 630 nm reference filter on an enzyme-linked immunosorbent assay plate reader (Bio-Rad®, Regents Park, NSW, Australia). To estimate cell number a standard curve was prepared from murine granulosa cells. Antral follicles, isolated from murine ovarian tissue, were ruptured with 27 gauge insulin needles and the granulosa cells were released into a small volume of minimal essential medium. Cell concentration was estimated using haemocytometer counts of Trypan Blue treated cells. Cells were seeded into 96-well culture plates at concentrations of 100010 000 and incubated immediately with the tetrazolium viability stain.
Inhibin A and B were measured according to previously described protocols (Groome, 1991; Groome et al., 1996
) using plates supplied by Professor Nigel Groome (Oxford Brookes University, Oxford, UK). We found, in contrast to previous publications (Kananen et al., 1996
; Smitz and Cortvrindt, 1998
), non-parallelism between serial dilutions of human inhibin standards and serial dilutions of a series of murine inhibin preparations (Wang et al., unpublished). Specific mouse standards for both inhibin A and inhibin B were therefore prepared using media from fresh murine follicles cultured individually as described above and used to generate standard curves for murine inhibin A and B. These standards exhibited parallelism with serial dilutions of murine testicular extract and serum from superovulated mice. The mouse standard preparations were calibrated with human inhibin A and B standard samples. The concentration of mouse standard that gave equivalent immunofluorescence to 1000 pg/ml was defined as 1000 arbitrary mouse units per millilitre (amu/ml).
The mouse inhibin B standard curve consisted of spent media at 100% and then dilutions to 50, 37.5, 25, 18.75, 12.5, 6.25 and 4.5% in minimal essential medium. This gave a concentration curve with values ranging from 1500 to 35 amu/ml. The limit of detection of the assay was 35 amu/ml. All day 8 test samples were diluted to 40% in
minimal essential medium and were measured in one assay. The intra-assay coefficient of variation was 6.3%.
The inhibin A standard curve consisted of spent media at dilutions of 75, 37.5, 25, 18.75, 12.5, 4.5, 2.34 and 1.17% in minimal essential medium. This gave a concentration curve with values ranging from 3000 to 47 amu/ml. The limit of detection of the assay was 47 amu/ml. All day 8 test samples were diluted to 40% in
minimal essential medium and were measured in one assay. The intra-assay coefficient of variation was 8.3%.
Statistical analysis
The data were analysed with the Student's t-test. All values presented are mean ± SEM.
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Results |
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Growing follicles retained their spherical shape for the first 5 days of culture (Figure 1a), and thereafter became flattened in appearance as the granulosa cell population expanded and spread out on the culture well. Patches of granulosa cells became less densely attached and formed antral cavity-like structures (Figure 1b
). After 8 days in vitro, follicle survival was defined as a healthy expanded granulosa cell mass, with antral cavities and a visible oocyte.
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To investigate the efficiency of different cryoprotective agents murine tissue was frozenthawed in DMSO, PROH or GLY. The percentage of follicles which survived 8 days of in-vitro culture after isolation from fresh ovarian tissue was 79 ± 3% (n = 112). Following isolation from tissue frozenthawed in DMSO, PROH or GLY respectively, 43 ± 5% (n = 95), 24 ± 2% (n = 92) and 0 ± 0% (n = 63) of follicles survived and developed to the antral stage. All the treatment groups differed significantly (Figure 3a) (P < 0.001). Follicles isolated from tissue frozen using the vitrification protocol survived in low numbers (9 ± 7%, n = 76). Of the six follicles which survived the 8 day in-vitro growth period only one released a mature oocyte after HCG stimulation.
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Discussion |
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Pre-antral follicles measuring 100135 µm rapidly increased in diameter as a result of granulosa cell proliferation during in-vitro culture and antral-like cavities formed. In contrast, follicles <100 µm did not increase in diameter and became dark and necrotic in appearance. One explanation for this observation may be that early stages of follicle growth cannot survive as isolated structures and require physical and/or nutritional support from the surrounding stroma cells. The culture of these small follicles to maturity will probably require a multi-step strategy, such as the approach used by Eppig and O'Brien to grow murine primordial follicles to maturity (Eppig and O'Brien, 1996).
A comparison of follicles isolated from fresh and frozenthawed tissue showed that after 8 days of culture fresh follicles had a larger diameter than their frozenthawed counterparts. This observation was supported by a cell proliferation assay which indicated that fresh follicles had a larger granulosa cell population at the end of culture. This may be due to delayed proliferation or initial cell death of the granulosa cells as a result of the freezethawing process. Frozenthawed follicles also produced lower concentrations of inhibin A and B, which may be a reflection of the lower numbers of granulosa cells. A study which investigated the growth and function of murine pre-antral follicles frozenthawed after isolation from ovarian tissue reported similar findings (Cortvrindt et al., 1996b).
Seventeen hours after the addition of HCG to the culture medium, oocytes were assessed for maturation. All follicles released a morphologically normal oocyte and the numbers which progressed to metaphase II were similar in fresh and frozenthawed groups. The mature oocytes were not fertilized, nevertheless, a previous study has shown that oocytes from frozenthawed follicles grown to maturity in vitro are capable of fertilization and development to the hatched blastocyst stage (Cortvrindt et al., 1996b).
Follicle survival after isolation from tissue frozenthawed in different cryoprotective agents was assessed. DMSO was found to be the most efficient solute, whilst no follicles survived after isolation from tissue frozen in glycerol. These results are supported by the work of Candy et al. who exposed murine ovarian tissue to different cryoprotectants for various time periods prior to freezethawing and grafting under the kidney capsules of ovariectomized immunodeficient recipients (Candy et al., 1995, 1997
). Histological analysis of grafts recovered 15 days after transplantation showed many morphologically normal follicles survived in tissue frozenthawed in DMSO or PROH. In contrast fewer follicles were recorded in tissue frozenthawed in glycerol (Candy et al., 1997
). Similar results were obtained in human studies. Ovarian tissue was cryopreserved in different cryoprotectants and grafted under the kidney capsules of immunodeficient mice for 18 days. The highest follicle survival rates were observed in tissue frozenthawed in DMSO or PROH (4484%), whilst few follicles survived freezethawing in glycerol and grafting (Newton et al., 1996
). One explanation for the poor survival rates recorded after freezing tissue in glycerol may be that the solute has a slow permeation rate, thus follicles at the centre of the tissue are not adequately protected from freezing damage. This theory is supported by nuclear magnetic resonance studies of human ovarian tissue in which DMSO was found to permeate tissue faster than glycerol (Newton et al., 1998
). Furthermore, in murine studies, extending the equilibration period with glycerol from 5 to 30 min prior to freezing increased the number of morphologically normal follicles surviving cryopreservation and grafting, suggesting that the extent of cryoprotectant penetration is proportional to follicle survival (Candy et al., 1997
).
Vitrification is an alternative method of freezing in which cells are frozen in high molar concentrations of cryoprotectant which, when cooled rapidly, become increasingly viscous, eventually forming a glass. The complete absence of ice formation eliminates the risk of intracellular freezing and the build up of salts in the extracellular medium. The technique has commonly been applied to oocytes and embryos. After vitrification murine embryos have exhibited good rates of survival (Rall and Fahy, 1985; Rall and Wood, 1994
) and in human studies one live birth has been reported from a vitrified oocyte (Kuleshova et al., 1999
). The high concentrations of cryoprotectant required necessitate short equilibration times to minimize toxicity. This presents a greater problem in tissue than single cells because of the need to permeate fully the sample with cryoprotectant. Nevertheless, neonatal rat ovarian tissue has been frozen using vitrification and viable follicles were still present after 4 days of culture following thawing (Sugimoto et al., 1996
). In the present study tissue was frozen using a protocol based on the method used to freeze bovine embryos (Vajta et al., 1996
). Follicles isolated from the tissue after thawing appeared morphologically normal but only six of the 76 follicles survived and grew to antral stages in culture. After HCG stimulation one of the vitrified follicles produced a morphologically normal mature oocyte, which suggests that the technique is feasible but improvements in the protocol may be required to improve efficiency.
The induction of seeding at 5°C, rather than at 7° and 9°C, significantly improved follicle survival rates. The survival of murine follicles after freezethawing depends on preserving the viability of both the oocyte and the granulosa cells. It is likely that, because of differences in size and membrane characteristics, optimal freezing conditions will vary for each cell type. In murine ovarian tissue, 5°C may be the most optimal seeding temperature for the follicular unit as a whole. Seeding temperature also influences the survival of human oocytes after cryopreservation. In a recent study the formation of intracellular ice, within oocytes, was visualized under a cryomicroscope after seeding was carried out at different temperatures. Simply increasing the seeding temperature from 8° to 4.5°C improved the 24 h post-thaw survival rate from 32 to 93% (Trad et al., 1998).
The rate at which frozen samples are thawed is also important in maintaining cell viability. In this study, the highest follicle survival rates were recorded when samples were thawed at 27°C. Increasing the thawing temperature reduced the number of follicles surviving the 8 day in-vitro growth period. It may be that at temperatures of 37° and 47°C, greater follicle damage was induced because of stress caused by the rapid increase in temperature.
The significance of these findings for the future of in-vitro human follicle culture is unclear. The majority of follicles within human tissue are at the primordial or primary stages and pre-antral follicles are more scarce (Lass et al., 1997). To date, complete in-vitro growth from the primordial follicle up to ovulation has only been achieved in the mouse (Eppig and O'Brien, 1996
) and even then the success rate was very low. Furthermore, the factors which initiate follicle growth are unknown and once development has begun it may take more than 200 days for a human primary follicle to reach ovulatory size (Gougeon, 1986
). In-vitro growth protocols currently in use are inadequate to sustain this length of culture. Nevertheless since the number of patients having ovarian tissue stored is increasing it may be prudent at this early stage to optimize freezing protocols. This would ensure that if in-vitro growth of human follicles becomes a possibility, the success rate will not be jeopardized by poor initial freezing techniques.
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Notes |
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References |
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Submitted on September 19, 2000; accepted on November 24, 2000.