BIOPRESERVATION AND BIOBANKING 6:269–276 (2008) © Mary Ann Liebert, Inc. DOI: 10.1089/bio.2008.0014
Quantification of Dimethyl Sulfoxide Perfusion in Sheep Ovarian Tissue: A Predictive Parameter for Follicular Survival to Cryopreservation Leonardo C. Pinto,1 Regiane R. Santos,2 Luciana R. Faustino,1 Cleidson M.G. da Silva,1 Valesca B. Luz,1 José E. Maia Júnior,1 Alison A.X. Soares,1 Juliana J.H. Celestino,1 Jair Mafezoli,3 Cláudio C. Campello,1 José R. Figueiredo,1 and Ana P.R. Rodrigues1
The aim of the present study was to determine the amount of dimethyl sulfoxide (DMSO) present in sheep ovarian tissue after exposure to cryoprotectant at different times (5, 10, 20, or 30 min) and at different concentrations (1.0, 1.5, or 2.0 M). To quantify the levels of DMSO in the ovarian tissue, the high-performance liquid chromatography (HPLC) method was applied. In addition, viability of preantral follicles after toxicity test and cryopreservation of ovarian tissue using the above mentioned concentrations of DMSO and exposure times was evaluated. We have observed that the presence of ~0.6 mg of DMSO into the ovarian tissue may be deleterious to the sheep preantral follicles. In addition, the application of a short exposure time (5 min at 1.5 or 2.0 M DMSO) or low concentration (1.0 M for 10 min) of DMSO successfully preserves sheep preantral follicles following cryopreservation.
Introduction
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ince the discovery of the use of glycerol as a cryoprotectant in 1949,1 the preservation of cells and tissues at extremely low temperatures has become a goal for many researchers. Nowadays, cryopreservation is routinely applied for the preservation of gametes, embryos, and, in the near future, also ovarian tissue. Cryopreservation of ovarian tissue has been employed to preserve early-stage gametes from mice,2 livestock animals such as cattle,3 sheep,4,5 and goats,6–8 as well as from nonhuman primates9 and humans.10 Although normal live birth has been obtained after cryopreservation and autotransplantation of human ovarian tissue, Donnez et al.11 may indicate routine clinical application; there are still many questions related to the cryopreservation procedure itself that must be elucidated. Therefore, studies to properly understand the mechanisms involved in the cryopreservation process are performed in animal models such as mice2 and small ruminants, which present more similarities with humans, that is, ovarian/cells structures and folliculogenesis.8,12
Ovarian tissue from sheep, the most commonly used animal model for human, has been successfully cryopreserved in the presence of dimethyl sulfoxide (DMSO), an intracellular cryoprotectant, generally used at the concentrations of 1.0,13 1.5,6,14 or 2.0 M.15 Several authors have demonstrated a satisfactory rate of morphologically normal6 and viable14 follicles, restoration of the ovarian function,16 as well as the offspring of normal lambs after autotransplantation of cryopreserved ovine ovarian tissue.15 Although it is postulated that 1.5 M DMSO is efficient for the preservation of ovarian tissue, the real amount is unknown of cryoprotectant that penetrates the tissue. Recently, our group has adapted a high-performance liquid chromatography (HPLC) protocol to evaluate the concentrations of DMSO in the goat ovarian tissue.17 We have shown that indeed only a small amount of the cryoprotectant permeate the tissue (around 1%) and can be responsible for the preservation of the preantral follicles during cooling and freezing process. On the other hand, it is known that cryoprotectant may impair cell viability if used in higher
1 Laboratory of Manipulation of Oocytes Enclosed in Preantral Follicles–LAMOFOPA, Faculty of Veterinary, State University of Ceará, Fortaleza, Ceará, Brazil. 2 Department of Equine Sciences, Veterinary Pharmaceuticals, Pharmacology, and Toxicology Division, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands. 3 Laboratory of Physical Chemical analysis, Fortaleza University, Fortaleza, Ceará, Brazil.
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concentrations than those needed, or inefficient in too low concentrations. Thus, based on the lack of information over the real permeation of DMSO in sheep ovarian tissue, as well as its relationship with follicle survival after freezingthawing procedure, we aimed to investigate the permeation rate of DMSO in ovine ovarian tissue submitted to different exposure times (5, 10, 20, or 30 min) and concentrations (1.0, 1.5, or 2.0 M) on the morphology and viability of frozenthawed preantral follicles enclosed in the ovarian tissue.
in Fig. 1). For cryoprotectant removal, ovarian fragments were washed three times (5 min each) in HM+ and fixed for routine histological analysis. The other 12 fragments were exposed to DMSO as described previously, and then they immediately underwent HPLC analysis to evaluate the DMSO perfusion into the ovarian fragments. We used the same procedure previously adapted by our group.17
Experiment 2: Toxicity test and cryopreservation of sheep ovarian tissue
Materials and Methods
In a second experiment, 25 ovarian cortical fragments (3 × 3 × 1 mm) were removed from each ovarian pair and placed in the HM. For viability analysis, one fragment immediately underwent follicular isolation (control), and preantral follicles were classified as viable (not stained) or nonviable (stained) by using Trypan blue dye.18 The remaining fragments were exposed to DMSO as described in the first experiment. After exposure, 12 fragments were washed from the cryoprotectants and subjected to a viability test, while the other 12 fragments were conventionally cryopreserved using a protocol previously tested by our group5; afterward, the samples were thawed and the DMSO washed out from the ovarian tissue according to the procedure described in the first experiment. To assess viability, preantral follicles were isolated from the tissue, followed by Trypan blue staining for quality control in ovarian cryopreservation procedures (Fig. 2).
Source and preparation of ovarian tissue This study comprised two experiments. For each experiment, ovarian pairs (n = 5 experiment 1; n = 6 experiment 2) from cycling adult mixed-breed sheep (Ovis aries) were obtained at a local slaughterhouse in Brazil. Ovarian pairs were washed in 70% alcohol, washed twice in HEPESbuffered modified Eagle medium (MEM; Sigma, St. Louis, MO), the holding medium (HM), supplemented with 0.1% (v/v) penicillin/streptomycin (GIBCO BRL, Paisley, United Kingdom). The pairs then were transported to the laboratory in thermo flasks at 20°C within 1 h.
Experiment 1: Relationship between perfusion of ovarian tissue with DMSO and follicular morphology In a first experiment, 25 ovarian cortical fragments (3 × 3 × 1 mm) were removed from each ovarian pair and placed in the HM. One fragment was immediately fixed (control) in Carnoy for routine histological analysis. The preantral follicles were classified as normal or atretic.5 The remaining 24 fragments were divided as follows. We exposed 12 fragments at 20°C, for 5, 10, 20, or 30 min to 1.0, 1.5, or 2.0 M (140.4, 210.6, or 280.8 mg, respectively) of DMSO (Vetec, Rio de Janeiro, Brazil) in macrotubes containing 1.8 mL of HM plus 10% fetal bovine serum (FBS) (HM+) (see protocol
Cryopreservation For freezing, ovarian fragments were individually placed in 2.0 mL macrotubes containing 1.8 mL MEM+ with DMSO at the same concentrations and equilibration period as used in the toxicity test (see experiment 1). After equilibration, the macrotubes with the ovarian tissue were transferred to a computerized programmable freezer (Freeze Control, CryoLogic Pty Ltd., Waverley, Australia) at 20°C.
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FIG. 1. Protocol design for experiment 1: relationship between perfusion of ovarian tissue with DMSO and follicular morphology.
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experiment 2: toxicity test and cryopreservation of sheep ovarian tissue.
Follicular isolation and viability analysis
The vials were cooled at –2°C/min to −7°C and ice crystal formation (seeding) was induced manually by touching the vials with forceps prechilled in liquid nitrogen. The specimens were held at this temperature for 15 min, and then cooled at –0.3°C/min to −30°C and thereafter at –0.15°C/ min to −33°C. The samples were then plunged directly into liquid nitrogen (−196°C) and stored for up to 5 days before thawing. For thawing, the samples were taken from the liquid nitrogen, warmed at room temperature for ~1 min, and immersed in a water bath at 37°C until the cryopreservation solution melted. The cryoprotectant was then removed as described earlier for the toxicity test; thereafter, the ovarian fragments were submitted to follicular isolation for the viability test.
Histology After fixation in Carnoy’s fluid, ovarian fragments were dehydrated in ethanol, clarified with xylene, and embedded in paraffin wax. Serial sections (7 μm) of ovarian tissue were cut and every fifth section was mounted on glass slides and stained with periodic acid Schiff (PAS)-hematoxylin. All sections were examined using a light microscope (Leica) at magnifications 20× and 40×. Preantral follicles were defined as follicles with an oocyte surrounded either by one flattened and/or cuboidal layer or several layers of only cuboidal granulosa cells. To avoid counting a follicle more than once, preantral follicles were counted only in the sections where their oocyte nucleus was observed. Follicular quality was evaluated based on the morphological integrity of the oocyte, the granulosa cells, and the basement membrane. Preantral follicles were classified as (i) histologically/morphologically normal when they contain an intact oocyte and intact granulosa cells, (ii) degenerated when their oocyte nucleus has become pyknotic, the oocyte was shrunken, and granulosa cells have detached from the basement membrane and have enlarged in volume (presence of at least one of these mentioned features was indicative of degeneration).
FIG. 2. Protocol design for
Viability test Preantral follicles were mechanically isolated from ovarian tissue by applying a mechanical procedure for the isolation of ovine follicles as described by Amorim et al.19 Briefly, the ovarian cortex was cut into small fragments using a tissue-chopper (The Mickle Laboratory Engineering Co., Gomshal, Surrey, UK). The ovarian fragments were then placed in PBS supplemented with 0.1% (v/v) penicillin/streptomycin (GIBCO, Paisley, UK) at room temperature (25°C) and then pipetted 40 times using a Pasteur pipette. The suspension was successively filtered through 500 and 100 μm nylon mesh filters. Viability of follicles was evaluated by adding 5 μL of 0.4% Trypan blue (Sigma, St. Louis, MO) to each 100 μL of solution and incubating for 1 min (RT). Thereafter, follicles were examined using an inverted microscope and classified as nonviable when stained with Trypan blue and viable when not stained with Trypan blue (Fig. 3).
High-performance liquid chromatography (HPLC) The analytical system used in the experiments was a highperformance liquid chromatograph (Gilson 321) consisting of a chromatographic interface, binary pump, UV/vis 152 detector, vacuum degasser, Rheodyne injection valve, and a 3.0 Unipoint program. A pre-column Hichrom-5 C18-10C was connected to a Hichrom-5 C18 column (250 × 4.6 mm, 5 μm, end capped). The analyses were performed at room temperature at a flow rate of 1 mL/min, and the elution profiles were monitored at λ = 214 ηm. The separation column was equilibrated with the mobile phase until baseline stabilization, at which point sample injections (20 μL) were made. The mobile phase consisted of water:methanol (9:1 v:v). To obtain the calibration curve, standard DMSO solutions (0; 0.00305; 0.01218; 0.04875; 0.19500; and 0.78000 mg/mL) were prepared from a DMSO stock solution (1.56 mg/mL) in the mobile phase. Based on the calibration curve, the detection (DL) and quantification (QL) limits were 0.012 and 0.03647 mg/mL,
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Statistical analysis A
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The percentages of morphologically normal preantral follicles (experiment 1) were compared among the treatments and with the control by applying the Dunnett test. The percentages of viable preantral follicles (experiment 2) were compared by applying frequency dispersion and chisquare. Comparisons within concentrations and exposure time were performed by using ANOVA and t-test. Values were considered significant when p < 0.05.
Results Relationship between perfusion of ovarian tissue with DMSO and follicular morphology
FIG. 3. Trypan blue staining of sheep preantral follicles. Viable follicles (A and B) and nonviable follicles (C and D).
Figure 5 shows the levels of DMSO in the sheep ovarian tissue after exposure at different times and concentrations used in the present experiment. We observed that time had no consequential effect on the tissue levels containing DMSO within any of the tested concentrations. On the other hand, a significant concentration effect was observed for all exposure times, except for 30 min, where there was no difference between 1.5 and 2.0 M DMSO on the ovarian tissue perfusion. To compare the rates of perfusion with follicular morphology, a total of 1,950 preantral follicles (150 follicles per treatment) were histologically evaluated (Fig. 6). Normal and degenerated preantral follicles were observed in all fragments subjected to perfusion, as well as those from the control (see Fig. 7). However, only ovarian cortex perfusion with 1.0 M DMSO for 5 min presented similar percentages (66%) of morphologically normal follicles when compared to the control (80%). There was no time effect on the tissue levels of DMSO within any of the tested concentrations, although a significant reduction on the percentage of morphologically normal follicles was observed after perfusion for 20 and 30 min when compared with 5 and 10 min perfusion (concentrations of 1.5 and 2.0 M).
respectively. The time retention was standardized for DMSO (3.4 min) (Fig. 4). All the solutions used were prepared in triplicates, and the correlation coefficient was 0.99985. Each ovarian cortical tissue previously exposed to DMSO (see experiment 1) was individually and quickly dried out with a paper to remove the remaining solution on the surface. After the weight of each ovarian fragment was recorded, the cortical pieces were immersed into tubes containing methanol 10%. Based on a pilot (data not published), after 6 h the fragments were removed carefully from each tube. The tubes containing methanol 10% were submitted to HPLC, while the cortical tissues were incubated for 6 h in methanol 100%, followed by 3 h in ether until complete evaporation, to recover the dry weight of the ovarian fragment. To obtain the DMSO tissue levels, the calculations were performed as follows: Concentration = FC1 + FC2 × peak area DMSO tissue levels = Concentration × ∆fragment weight × methanol 10% volume
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% Morphologically Normal Early-stage Follicles
The viability of 3,774 preantral follicles was evaluated by using Trypan blue dye (at least 100 follicles per treatment), and according to Santos et al.14 follicles were classified as viable (nonstained) or nonviable (stained). We have observed a toxic effect of the cryoprotectants immediately after exposure, which leads to a significant decrease in the percentages of viable follicles and, consequently after freeze-thawing, when compared with control values (83.89%) (see Fig. 8). Regarding the toxicity test, it was observed that no significant differences among the DMSO concentrations for each condition tested for exposure time. However, a significant reduction of follicular viability was observed when the exposure time was extended to 30 min. After cryopreservation of ovarian tissue previously exposed to DMSO for 5, 10, and 20 min, it was observed that there was no significant
FIG. 5. DMSO levels in sheep ovarian tissue. Values with different lower-case letters (a– c) differ significantly among the DMSO concentrations (1.0, 1.5, and 2.0 M) within the same exposure time (p < 0.05).
difference in the viability percentages for each concentration tested. Comparing the DMSO concentration within each exposure time, it was observed that follicles exposed to DMSO for 5 min were better cryopreserved when the cryoprotectant concentration employed was 1.5 M instead of 1.0 M. On the other hand, follicles exposed to cryoprotectant for 30 min were cryopreserved more efficiently when the DMSO concentration was 1.0 M instead of 2.0 M (p < 0.05). Although all the treatments presented percentages of viable follicles significantly inferior to those of the control, the follicular viability was efficiently cryopreserved when ovarian tissue was previously exposed to 1.5 and 2.0 M DMSO for 5 min or 1.0 M for 10 min.
Discussion In this study we evaluated, for the first time, the perfusion of DMSO on sheep ovarian tissue and its relationship with follicular morphology and viability after exposure of
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FIG. 6. Percentage of morphologically normal nonexposed (control) and DMSO exposed preantral follicles (experiment 1; n = minimum of 150 follicles per repetition). *Differs significantly from control (p < 0.05). Values with different lower-case letters (a, b) differ significantly among the DMSO concentrations (1.0, 1.5, and 2.0 M) within the same exposure time (p < 0.05). Values with different upper-case letters (A, B) differ significantly among the exposure times within the same DMSO concentration tested (p < 0.05).
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FIG. 7. Histological sections of preantral follicles in which morphologically normal (A) primordial, (B) primary, and (C) secondary follicles are shown. (D, E) Degenerating preantral follicles after exposure to 1.5 M DMSO for 30 min, and degenerating preantral follicle from control (F). (G) Morphologically normal preantral follicles after exposure to 1.0 M DMSO (Note: normal follicular structure as well as from ovarian cortical cells). (H, I) Degenerating preantral follicles after exposure to 2.0 M DMSO for 30 min. Arrows indicate oocyte retraction and pyknotic nucleus.
% Viable Preantral Follicles
ovarian cortical fragments to DMSO (1.0, 1.5, or 2.0 M) for 5, 10, 20, or 30 min, and subsequent cryopreservation. The exposure of cells or tissues to a cryoprotectant solution, in a denominated equilibrium period, consists of an important step in the cryopreservation process since this may lead the cells to reach a balance between the concentrations of intracellular water and cryoprotectant. Consequently, this acts in minimizing the cryodamage caused by intracellular ice crystal formation during freezing (Candy et al., 1994). When one considers ovarian tissue permeation, however, most of the studies evaluate the effects of cryoprotectant 100 90 80 70 60 50 40 30 20 10 0
exposure without quantifying the real amount of cryoprotectant in the tissue. Recently, we have adapted a HPLC protocol to calculate the permeation of DMSO into goat ovarian tissue after exposure for 10, 20, 30, and 40 min to DMSO at concentrations of 1.0, 1.5, and 2.0 M.17 Based on our last study, and considering the toxic effects of DMSO on the cells during exposure,18 we have evaluated a reduced equilibration time (5 min) and quantified the amount of DMSO in the sheep ovarian tissue. We have observed that not the exposure time but the cryoprotectant concentration affects the DMSO perfusion into the ovarian tissue. The similar perfusion observed when exposure was performed in presence of 1.5 and 2.0 M DMSO for 30 min may be due to a saturation of DMSO into the ovarian tissue. When histological features of preantral follicles from ovaries submitted to the mentioned exposure were evaluated, we have found a significant decrease in the percentages of morphologically normal follicles after exposure to 2.0 M DMSO when compared to 1.0 M DMSO. It is well known that apart from its cryoprotective effect, DMSO can be toxic to the cells if used in high concentrations.5,6 However, as we have shown, not only the concentration of the DMSO may be deleterious to the cells, but also the time of exposure. In our previous study using caprine ovarian tissue,17 we have observed that only after 40 min of exposure the preantral follicles presented a significant impairment of follicular morphology, while in sheep such a effect is observed after 30 min. Thus, we suggest that a slight balance among exposure time, cryoprotectant concentration, and quantification of ovarian tissue perfusion must be applied to develop efficient cryopreservation protocols, always considering species-specific differences. Apart from the follicular morphology, viability of cells must be preserved from the first step of cryopreservation (exposure or equilibration period). Therefore, we have performed the toxicity test and cryopreservation of ovarian tissue followed by follicular viability analysis using Trypan blue dye.20 Although in all the treatments the percentages of viable follicles was significantly inferior to those observed in control, such data were expected given that the ovarian tissue is subjected to many stressful steps and hypoxia during collection, laboratorial preparation, and freezing procedures. Hence, we also have evaluated the rates of viable follicles among the treatments, and the higher percentages of viable follicles were
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FIG. 8. Viability based on Trypan blue staining of isolated sheep preantral follicles exposed to DMSO for 5, 10, 20, or 30 min at different concentrations and subsequently cryopreserved (experiment 2; n = minimum of 100 follicles per repetition). *Differs significantly from control (p < 0.05). Values with different lower-case letters (a, b) differ significantly among the DMSO concentrations within the same exposure time (p < 0.05). Values with different upper-case letters (A–B) differ significantly among the exposure times (toxicity test) within the same DMSO concentration tested (p < 0.05).
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DIFFERENT DMSO LEVELS AFFECTING FOLLICULAR QUALITY obtained after exposure and freezing of ovarian tissue in presence of 1.0 M DMSO for 10, 20, or 30 min, or 1.5 M DMSO for 5, 10, or 20 min. When sheep ovarian tissue was cryopreserved in the presence of 1.5 M DMSO for a maximum time of 20 min, Santos et al.5 suggested the use of short periods of exposure to avoid toxic damage to the follicular cells. By our present study, we have proved it possible to maintain follicular viability after cryopreservation of ovarian tissue exposed to low concentrations of DMSO (1.0 M, for 10 min), as well as a short period of equilibrium (5 min at 1.5 or 2.0 M DMSO). In conclusion, the presence of low concentrations of DMSO (~0.6 mg) into the sheep ovarian tissue are responsible for morphological damages in the preantral follicles, and the reduction of exposure time or concentration of the cryoprotectant may avoid follicular impairment during cryopreservation procedure.
Acknowledgments Leonardo C. Pinto and Ana P.R. Rodrigues are recipients of the Brazilian grants from FUNCAP and CNpQ, respectively. The authors thank Fortaleza University (UNIFOR) for the logistical support, and two anonymous reviewers for their helpful suggestions.
References 1. Polge C, Smith AU, Parkes AS. Revival of spermatozoa after vitrification and dehydration at low temperatures. Nature 1949;164:666–676. 2. Sauvat F, Capito C, Sarnacki S, et al. Immature cryopreserved ovary restores puberty and fertility in mice without alteration of epigenetic marks. PLoS ONE 2008;16:3–4. 3. Celestino JJ, Santos RR, Lopes CA, et al. Preservation of bovine preantral follicle viability and ultra-structure after cooling and freezing of ovarian tissue. Anim Reprod Sci 2008;108: 309–318. 4. Amorim CA, Rondina D, Lucci CM, et al. Cryopreservation of sheep primordial follicles. Reprod Domest Anim 2007;42: 53–57. 5. Santos RR, Rodrigues APR, Costa SH, et al. Histological and ultrastructural analysis of cryopreserved sheep preantral follicles. Anim Reprod Sci 2006;91:249–263. 6. Rodrigues APR, Amorim CA, Costa SH, et al. Cryopreservation of caprine ovarian tissue using dimethyl sulphoxide and propanediol. Anim Reprod Sci 2004a;84:211–227. 7. Rodrigues APR, Amorim CA, Costa SH, et al. Cryopreservation of caprine ovarian tissue using glycerol and ethylene glycol. Theriogenology 2004b;15:1009–1024. 8. Santos RR, Knijn HM, Vos PL, et al. Complete follicular development and recovery of ovarian function of frozen-thawed, autotransplanted caprine ovarian cortex. Fertil Steril 2008. [Epub ahead of print]
9. Li G, Thirumala S, Leibo SP, et al. Subzero water transport characteristics and optimal rates of freezing rhesus monkey (Macaca mulatta) ovarian tissue. Mol Reprod Dev 2006;73:1600–1611. 10. Amorim CA, Van Langendonckt A, David A, et al. Survival of human preantral follicles after cryopreservation of ovarian tissue, follicular isolation and in vitro culture in a calcium alginate matrix. Hum Reprod 2008. [Epub ahead of print] 11. Donnez J, Dolmans MM, Demylle D, et al. Livebirth after orthotopic transplantation of cryopreserved ovarian tissue. Lancet 2004;16:1405–1410. 12. Imhof M, Bergmeister H, Lipovac M, et al. Orthotopic microvascular reanastomosis of whole cryopreserved ovine ovaries resulting in pregnancy and live birth. Fertil Steril 2006;85:1208–1215. 13. Demirci B, Lornage J, Salle B, et al. Follicular viability and morphology of sheep ovaries after exposure to cryoprotectant and cryopreservation with different freezing protocols. Fertil Steril 2001;75:754–762. 14. Santos RR, Van Den Hurk R, Rodrigues APR, et al. Effect of cryopreservation on viability, activation and growth of in situ and isolated ovine early-stage follicles. Anim Reprod Sci 2007;99:53–64. 15. Salle B, Demirci B, Franck M, et al. Normal pregnancies and live births after autograft of frozen-thawed hemi-ovaries into ewes. Fértil Steril 2002;77:403–408. 16. Salle B, Lornage J, Demirci B, et al. Restoration of ovarian steroid secretion and histologic assessment after freezing, thawing, and autograft of a hemi-ovary in sheep. Fertil Steril 1999;72:366–370. 17. Luz VB, Santos RR, Pinto LC, et al. DMSO perfusion in caprine ovarian tissue and its relationship with follicular viability after cryopreservation. Fertil Steril 2008. [Epub ahead of print] 18. Rodrigues APR, Costa SH, Santos RR, et al. In vitro culture of cryopreserved caprine ovarian tissue pieces and isolated follicles. Cell Preserv Technol 2006;4:290–298. 19. Amorim CA, Rodrigues APR, Lucci CM, et al. Effect of sectioning on the number of isolated ovine preantral follicles. Small Rumin Res 2000;37:269–277. 20. Fauque P, Ben Amor A, Joanne C, et al. Use of trypan blue staining to assess the quality of ovarian cryopreservation. Fertil Steril 2007;87:1200–1207.
Address reprint requests to: Dr. Regiane R. Santos Department of Equine Sciences Veterinary Pharmaceuticals, Pharmacology, and Toxicology Division Faculty of Veterinary Medicine Utrecht University Yalelaan 114 Utrecht, 3584 CM The Netherlands
E-mail: [email protected] Received 5 November, 2008/Accepted 5 January, 2009
Quantification of Dimethyl Sulfoxide Perfusion in Sheep Ovarian Tissue: A Predictive Parameter for Follicular Survival to Cryopreservation Leonardo C. Pinto,1 Regiane R. Santos,2 Luciana R. Faustino,1 Cleidson M.G. da Silva,1 Valesca B. Luz,1 José E. Maia Júnior,1 Alison A.X. Soares,1 Juliana J.H. Celestino,1 Jair Mafezoli,3 Cláudio C. Campello,1 José R. Figueiredo,1 and Ana P.R. Rodrigues1
The aim of the present study was to determine the amount of dimethyl sulfoxide (DMSO) present in sheep ovarian tissue after exposure to cryoprotectant at different times (5, 10, 20, or 30 min) and at different concentrations (1.0, 1.5, or 2.0 M). To quantify the levels of DMSO in the ovarian tissue, the high-performance liquid chromatography (HPLC) method was applied. In addition, viability of preantral follicles after toxicity test and cryopreservation of ovarian tissue using the above mentioned concentrations of DMSO and exposure times was evaluated. We have observed that the presence of ~0.6 mg of DMSO into the ovarian tissue may be deleterious to the sheep preantral follicles. In addition, the application of a short exposure time (5 min at 1.5 or 2.0 M DMSO) or low concentration (1.0 M for 10 min) of DMSO successfully preserves sheep preantral follicles following cryopreservation.
Introduction
S
ince the discovery of the use of glycerol as a cryoprotectant in 1949,1 the preservation of cells and tissues at extremely low temperatures has become a goal for many researchers. Nowadays, cryopreservation is routinely applied for the preservation of gametes, embryos, and, in the near future, also ovarian tissue. Cryopreservation of ovarian tissue has been employed to preserve early-stage gametes from mice,2 livestock animals such as cattle,3 sheep,4,5 and goats,6–8 as well as from nonhuman primates9 and humans.10 Although normal live birth has been obtained after cryopreservation and autotransplantation of human ovarian tissue, Donnez et al.11 may indicate routine clinical application; there are still many questions related to the cryopreservation procedure itself that must be elucidated. Therefore, studies to properly understand the mechanisms involved in the cryopreservation process are performed in animal models such as mice2 and small ruminants, which present more similarities with humans, that is, ovarian/cells structures and folliculogenesis.8,12
Ovarian tissue from sheep, the most commonly used animal model for human, has been successfully cryopreserved in the presence of dimethyl sulfoxide (DMSO), an intracellular cryoprotectant, generally used at the concentrations of 1.0,13 1.5,6,14 or 2.0 M.15 Several authors have demonstrated a satisfactory rate of morphologically normal6 and viable14 follicles, restoration of the ovarian function,16 as well as the offspring of normal lambs after autotransplantation of cryopreserved ovine ovarian tissue.15 Although it is postulated that 1.5 M DMSO is efficient for the preservation of ovarian tissue, the real amount is unknown of cryoprotectant that penetrates the tissue. Recently, our group has adapted a high-performance liquid chromatography (HPLC) protocol to evaluate the concentrations of DMSO in the goat ovarian tissue.17 We have shown that indeed only a small amount of the cryoprotectant permeate the tissue (around 1%) and can be responsible for the preservation of the preantral follicles during cooling and freezing process. On the other hand, it is known that cryoprotectant may impair cell viability if used in higher
1 Laboratory of Manipulation of Oocytes Enclosed in Preantral Follicles–LAMOFOPA, Faculty of Veterinary, State University of Ceará, Fortaleza, Ceará, Brazil. 2 Department of Equine Sciences, Veterinary Pharmaceuticals, Pharmacology, and Toxicology Division, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands. 3 Laboratory of Physical Chemical analysis, Fortaleza University, Fortaleza, Ceará, Brazil.
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concentrations than those needed, or inefficient in too low concentrations. Thus, based on the lack of information over the real permeation of DMSO in sheep ovarian tissue, as well as its relationship with follicle survival after freezingthawing procedure, we aimed to investigate the permeation rate of DMSO in ovine ovarian tissue submitted to different exposure times (5, 10, 20, or 30 min) and concentrations (1.0, 1.5, or 2.0 M) on the morphology and viability of frozenthawed preantral follicles enclosed in the ovarian tissue.
in Fig. 1). For cryoprotectant removal, ovarian fragments were washed three times (5 min each) in HM+ and fixed for routine histological analysis. The other 12 fragments were exposed to DMSO as described previously, and then they immediately underwent HPLC analysis to evaluate the DMSO perfusion into the ovarian fragments. We used the same procedure previously adapted by our group.17
Experiment 2: Toxicity test and cryopreservation of sheep ovarian tissue
Materials and Methods
In a second experiment, 25 ovarian cortical fragments (3 × 3 × 1 mm) were removed from each ovarian pair and placed in the HM. For viability analysis, one fragment immediately underwent follicular isolation (control), and preantral follicles were classified as viable (not stained) or nonviable (stained) by using Trypan blue dye.18 The remaining fragments were exposed to DMSO as described in the first experiment. After exposure, 12 fragments were washed from the cryoprotectants and subjected to a viability test, while the other 12 fragments were conventionally cryopreserved using a protocol previously tested by our group5; afterward, the samples were thawed and the DMSO washed out from the ovarian tissue according to the procedure described in the first experiment. To assess viability, preantral follicles were isolated from the tissue, followed by Trypan blue staining for quality control in ovarian cryopreservation procedures (Fig. 2).
Source and preparation of ovarian tissue This study comprised two experiments. For each experiment, ovarian pairs (n = 5 experiment 1; n = 6 experiment 2) from cycling adult mixed-breed sheep (Ovis aries) were obtained at a local slaughterhouse in Brazil. Ovarian pairs were washed in 70% alcohol, washed twice in HEPESbuffered modified Eagle medium (MEM; Sigma, St. Louis, MO), the holding medium (HM), supplemented with 0.1% (v/v) penicillin/streptomycin (GIBCO BRL, Paisley, United Kingdom). The pairs then were transported to the laboratory in thermo flasks at 20°C within 1 h.
Experiment 1: Relationship between perfusion of ovarian tissue with DMSO and follicular morphology In a first experiment, 25 ovarian cortical fragments (3 × 3 × 1 mm) were removed from each ovarian pair and placed in the HM. One fragment was immediately fixed (control) in Carnoy for routine histological analysis. The preantral follicles were classified as normal or atretic.5 The remaining 24 fragments were divided as follows. We exposed 12 fragments at 20°C, for 5, 10, 20, or 30 min to 1.0, 1.5, or 2.0 M (140.4, 210.6, or 280.8 mg, respectively) of DMSO (Vetec, Rio de Janeiro, Brazil) in macrotubes containing 1.8 mL of HM plus 10% fetal bovine serum (FBS) (HM+) (see protocol
Cryopreservation For freezing, ovarian fragments were individually placed in 2.0 mL macrotubes containing 1.8 mL MEM+ with DMSO at the same concentrations and equilibration period as used in the toxicity test (see experiment 1). After equilibration, the macrotubes with the ovarian tissue were transferred to a computerized programmable freezer (Freeze Control, CryoLogic Pty Ltd., Waverley, Australia) at 20°C.
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FIG. 1. Protocol design for experiment 1: relationship between perfusion of ovarian tissue with DMSO and follicular morphology.
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experiment 2: toxicity test and cryopreservation of sheep ovarian tissue.
Follicular isolation and viability analysis
The vials were cooled at –2°C/min to −7°C and ice crystal formation (seeding) was induced manually by touching the vials with forceps prechilled in liquid nitrogen. The specimens were held at this temperature for 15 min, and then cooled at –0.3°C/min to −30°C and thereafter at –0.15°C/ min to −33°C. The samples were then plunged directly into liquid nitrogen (−196°C) and stored for up to 5 days before thawing. For thawing, the samples were taken from the liquid nitrogen, warmed at room temperature for ~1 min, and immersed in a water bath at 37°C until the cryopreservation solution melted. The cryoprotectant was then removed as described earlier for the toxicity test; thereafter, the ovarian fragments were submitted to follicular isolation for the viability test.
Histology After fixation in Carnoy’s fluid, ovarian fragments were dehydrated in ethanol, clarified with xylene, and embedded in paraffin wax. Serial sections (7 μm) of ovarian tissue were cut and every fifth section was mounted on glass slides and stained with periodic acid Schiff (PAS)-hematoxylin. All sections were examined using a light microscope (Leica) at magnifications 20× and 40×. Preantral follicles were defined as follicles with an oocyte surrounded either by one flattened and/or cuboidal layer or several layers of only cuboidal granulosa cells. To avoid counting a follicle more than once, preantral follicles were counted only in the sections where their oocyte nucleus was observed. Follicular quality was evaluated based on the morphological integrity of the oocyte, the granulosa cells, and the basement membrane. Preantral follicles were classified as (i) histologically/morphologically normal when they contain an intact oocyte and intact granulosa cells, (ii) degenerated when their oocyte nucleus has become pyknotic, the oocyte was shrunken, and granulosa cells have detached from the basement membrane and have enlarged in volume (presence of at least one of these mentioned features was indicative of degeneration).
FIG. 2. Protocol design for
Viability test Preantral follicles were mechanically isolated from ovarian tissue by applying a mechanical procedure for the isolation of ovine follicles as described by Amorim et al.19 Briefly, the ovarian cortex was cut into small fragments using a tissue-chopper (The Mickle Laboratory Engineering Co., Gomshal, Surrey, UK). The ovarian fragments were then placed in PBS supplemented with 0.1% (v/v) penicillin/streptomycin (GIBCO, Paisley, UK) at room temperature (25°C) and then pipetted 40 times using a Pasteur pipette. The suspension was successively filtered through 500 and 100 μm nylon mesh filters. Viability of follicles was evaluated by adding 5 μL of 0.4% Trypan blue (Sigma, St. Louis, MO) to each 100 μL of solution and incubating for 1 min (RT). Thereafter, follicles were examined using an inverted microscope and classified as nonviable when stained with Trypan blue and viable when not stained with Trypan blue (Fig. 3).
High-performance liquid chromatography (HPLC) The analytical system used in the experiments was a highperformance liquid chromatograph (Gilson 321) consisting of a chromatographic interface, binary pump, UV/vis 152 detector, vacuum degasser, Rheodyne injection valve, and a 3.0 Unipoint program. A pre-column Hichrom-5 C18-10C was connected to a Hichrom-5 C18 column (250 × 4.6 mm, 5 μm, end capped). The analyses were performed at room temperature at a flow rate of 1 mL/min, and the elution profiles were monitored at λ = 214 ηm. The separation column was equilibrated with the mobile phase until baseline stabilization, at which point sample injections (20 μL) were made. The mobile phase consisted of water:methanol (9:1 v:v). To obtain the calibration curve, standard DMSO solutions (0; 0.00305; 0.01218; 0.04875; 0.19500; and 0.78000 mg/mL) were prepared from a DMSO stock solution (1.56 mg/mL) in the mobile phase. Based on the calibration curve, the detection (DL) and quantification (QL) limits were 0.012 and 0.03647 mg/mL,
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Statistical analysis A
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The percentages of morphologically normal preantral follicles (experiment 1) were compared among the treatments and with the control by applying the Dunnett test. The percentages of viable preantral follicles (experiment 2) were compared by applying frequency dispersion and chisquare. Comparisons within concentrations and exposure time were performed by using ANOVA and t-test. Values were considered significant when p < 0.05.
Results Relationship between perfusion of ovarian tissue with DMSO and follicular morphology
FIG. 3. Trypan blue staining of sheep preantral follicles. Viable follicles (A and B) and nonviable follicles (C and D).
Figure 5 shows the levels of DMSO in the sheep ovarian tissue after exposure at different times and concentrations used in the present experiment. We observed that time had no consequential effect on the tissue levels containing DMSO within any of the tested concentrations. On the other hand, a significant concentration effect was observed for all exposure times, except for 30 min, where there was no difference between 1.5 and 2.0 M DMSO on the ovarian tissue perfusion. To compare the rates of perfusion with follicular morphology, a total of 1,950 preantral follicles (150 follicles per treatment) were histologically evaluated (Fig. 6). Normal and degenerated preantral follicles were observed in all fragments subjected to perfusion, as well as those from the control (see Fig. 7). However, only ovarian cortex perfusion with 1.0 M DMSO for 5 min presented similar percentages (66%) of morphologically normal follicles when compared to the control (80%). There was no time effect on the tissue levels of DMSO within any of the tested concentrations, although a significant reduction on the percentage of morphologically normal follicles was observed after perfusion for 20 and 30 min when compared with 5 and 10 min perfusion (concentrations of 1.5 and 2.0 M).
respectively. The time retention was standardized for DMSO (3.4 min) (Fig. 4). All the solutions used were prepared in triplicates, and the correlation coefficient was 0.99985. Each ovarian cortical tissue previously exposed to DMSO (see experiment 1) was individually and quickly dried out with a paper to remove the remaining solution on the surface. After the weight of each ovarian fragment was recorded, the cortical pieces were immersed into tubes containing methanol 10%. Based on a pilot (data not published), after 6 h the fragments were removed carefully from each tube. The tubes containing methanol 10% were submitted to HPLC, while the cortical tissues were incubated for 6 h in methanol 100%, followed by 3 h in ether until complete evaporation, to recover the dry weight of the ovarian fragment. To obtain the DMSO tissue levels, the calculations were performed as follows: Concentration = FC1 + FC2 × peak area DMSO tissue levels = Concentration × ∆fragment weight × methanol 10% volume
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Toxicity test and cryopreservation of sheep ovarian tissue
% Morphologically Normal Early-stage Follicles
The viability of 3,774 preantral follicles was evaluated by using Trypan blue dye (at least 100 follicles per treatment), and according to Santos et al.14 follicles were classified as viable (nonstained) or nonviable (stained). We have observed a toxic effect of the cryoprotectants immediately after exposure, which leads to a significant decrease in the percentages of viable follicles and, consequently after freeze-thawing, when compared with control values (83.89%) (see Fig. 8). Regarding the toxicity test, it was observed that no significant differences among the DMSO concentrations for each condition tested for exposure time. However, a significant reduction of follicular viability was observed when the exposure time was extended to 30 min. After cryopreservation of ovarian tissue previously exposed to DMSO for 5, 10, and 20 min, it was observed that there was no significant
FIG. 5. DMSO levels in sheep ovarian tissue. Values with different lower-case letters (a– c) differ significantly among the DMSO concentrations (1.0, 1.5, and 2.0 M) within the same exposure time (p < 0.05).
difference in the viability percentages for each concentration tested. Comparing the DMSO concentration within each exposure time, it was observed that follicles exposed to DMSO for 5 min were better cryopreserved when the cryoprotectant concentration employed was 1.5 M instead of 1.0 M. On the other hand, follicles exposed to cryoprotectant for 30 min were cryopreserved more efficiently when the DMSO concentration was 1.0 M instead of 2.0 M (p < 0.05). Although all the treatments presented percentages of viable follicles significantly inferior to those of the control, the follicular viability was efficiently cryopreserved when ovarian tissue was previously exposed to 1.5 and 2.0 M DMSO for 5 min or 1.0 M for 10 min.
Discussion In this study we evaluated, for the first time, the perfusion of DMSO on sheep ovarian tissue and its relationship with follicular morphology and viability after exposure of
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FIG. 6. Percentage of morphologically normal nonexposed (control) and DMSO exposed preantral follicles (experiment 1; n = minimum of 150 follicles per repetition). *Differs significantly from control (p < 0.05). Values with different lower-case letters (a, b) differ significantly among the DMSO concentrations (1.0, 1.5, and 2.0 M) within the same exposure time (p < 0.05). Values with different upper-case letters (A, B) differ significantly among the exposure times within the same DMSO concentration tested (p < 0.05).
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FIG. 7. Histological sections of preantral follicles in which morphologically normal (A) primordial, (B) primary, and (C) secondary follicles are shown. (D, E) Degenerating preantral follicles after exposure to 1.5 M DMSO for 30 min, and degenerating preantral follicle from control (F). (G) Morphologically normal preantral follicles after exposure to 1.0 M DMSO (Note: normal follicular structure as well as from ovarian cortical cells). (H, I) Degenerating preantral follicles after exposure to 2.0 M DMSO for 30 min. Arrows indicate oocyte retraction and pyknotic nucleus.
% Viable Preantral Follicles
ovarian cortical fragments to DMSO (1.0, 1.5, or 2.0 M) for 5, 10, 20, or 30 min, and subsequent cryopreservation. The exposure of cells or tissues to a cryoprotectant solution, in a denominated equilibrium period, consists of an important step in the cryopreservation process since this may lead the cells to reach a balance between the concentrations of intracellular water and cryoprotectant. Consequently, this acts in minimizing the cryodamage caused by intracellular ice crystal formation during freezing (Candy et al., 1994). When one considers ovarian tissue permeation, however, most of the studies evaluate the effects of cryoprotectant 100 90 80 70 60 50 40 30 20 10 0
exposure without quantifying the real amount of cryoprotectant in the tissue. Recently, we have adapted a HPLC protocol to calculate the permeation of DMSO into goat ovarian tissue after exposure for 10, 20, 30, and 40 min to DMSO at concentrations of 1.0, 1.5, and 2.0 M.17 Based on our last study, and considering the toxic effects of DMSO on the cells during exposure,18 we have evaluated a reduced equilibration time (5 min) and quantified the amount of DMSO in the sheep ovarian tissue. We have observed that not the exposure time but the cryoprotectant concentration affects the DMSO perfusion into the ovarian tissue. The similar perfusion observed when exposure was performed in presence of 1.5 and 2.0 M DMSO for 30 min may be due to a saturation of DMSO into the ovarian tissue. When histological features of preantral follicles from ovaries submitted to the mentioned exposure were evaluated, we have found a significant decrease in the percentages of morphologically normal follicles after exposure to 2.0 M DMSO when compared to 1.0 M DMSO. It is well known that apart from its cryoprotective effect, DMSO can be toxic to the cells if used in high concentrations.5,6 However, as we have shown, not only the concentration of the DMSO may be deleterious to the cells, but also the time of exposure. In our previous study using caprine ovarian tissue,17 we have observed that only after 40 min of exposure the preantral follicles presented a significant impairment of follicular morphology, while in sheep such a effect is observed after 30 min. Thus, we suggest that a slight balance among exposure time, cryoprotectant concentration, and quantification of ovarian tissue perfusion must be applied to develop efficient cryopreservation protocols, always considering species-specific differences. Apart from the follicular morphology, viability of cells must be preserved from the first step of cryopreservation (exposure or equilibration period). Therefore, we have performed the toxicity test and cryopreservation of ovarian tissue followed by follicular viability analysis using Trypan blue dye.20 Although in all the treatments the percentages of viable follicles was significantly inferior to those observed in control, such data were expected given that the ovarian tissue is subjected to many stressful steps and hypoxia during collection, laboratorial preparation, and freezing procedures. Hence, we also have evaluated the rates of viable follicles among the treatments, and the higher percentages of viable follicles were
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FIG. 8. Viability based on Trypan blue staining of isolated sheep preantral follicles exposed to DMSO for 5, 10, 20, or 30 min at different concentrations and subsequently cryopreserved (experiment 2; n = minimum of 100 follicles per repetition). *Differs significantly from control (p < 0.05). Values with different lower-case letters (a, b) differ significantly among the DMSO concentrations within the same exposure time (p < 0.05). Values with different upper-case letters (A–B) differ significantly among the exposure times (toxicity test) within the same DMSO concentration tested (p < 0.05).
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DIFFERENT DMSO LEVELS AFFECTING FOLLICULAR QUALITY obtained after exposure and freezing of ovarian tissue in presence of 1.0 M DMSO for 10, 20, or 30 min, or 1.5 M DMSO for 5, 10, or 20 min. When sheep ovarian tissue was cryopreserved in the presence of 1.5 M DMSO for a maximum time of 20 min, Santos et al.5 suggested the use of short periods of exposure to avoid toxic damage to the follicular cells. By our present study, we have proved it possible to maintain follicular viability after cryopreservation of ovarian tissue exposed to low concentrations of DMSO (1.0 M, for 10 min), as well as a short period of equilibrium (5 min at 1.5 or 2.0 M DMSO). In conclusion, the presence of low concentrations of DMSO (~0.6 mg) into the sheep ovarian tissue are responsible for morphological damages in the preantral follicles, and the reduction of exposure time or concentration of the cryoprotectant may avoid follicular impairment during cryopreservation procedure.
Acknowledgments Leonardo C. Pinto and Ana P.R. Rodrigues are recipients of the Brazilian grants from FUNCAP and CNpQ, respectively. The authors thank Fortaleza University (UNIFOR) for the logistical support, and two anonymous reviewers for their helpful suggestions.
References 1. Polge C, Smith AU, Parkes AS. Revival of spermatozoa after vitrification and dehydration at low temperatures. Nature 1949;164:666–676. 2. Sauvat F, Capito C, Sarnacki S, et al. Immature cryopreserved ovary restores puberty and fertility in mice without alteration of epigenetic marks. PLoS ONE 2008;16:3–4. 3. Celestino JJ, Santos RR, Lopes CA, et al. Preservation of bovine preantral follicle viability and ultra-structure after cooling and freezing of ovarian tissue. Anim Reprod Sci 2008;108: 309–318. 4. Amorim CA, Rondina D, Lucci CM, et al. Cryopreservation of sheep primordial follicles. Reprod Domest Anim 2007;42: 53–57. 5. Santos RR, Rodrigues APR, Costa SH, et al. Histological and ultrastructural analysis of cryopreserved sheep preantral follicles. Anim Reprod Sci 2006;91:249–263. 6. Rodrigues APR, Amorim CA, Costa SH, et al. Cryopreservation of caprine ovarian tissue using dimethyl sulphoxide and propanediol. Anim Reprod Sci 2004a;84:211–227. 7. Rodrigues APR, Amorim CA, Costa SH, et al. Cryopreservation of caprine ovarian tissue using glycerol and ethylene glycol. Theriogenology 2004b;15:1009–1024. 8. Santos RR, Knijn HM, Vos PL, et al. Complete follicular development and recovery of ovarian function of frozen-thawed, autotransplanted caprine ovarian cortex. Fertil Steril 2008. [Epub ahead of print]
9. Li G, Thirumala S, Leibo SP, et al. Subzero water transport characteristics and optimal rates of freezing rhesus monkey (Macaca mulatta) ovarian tissue. Mol Reprod Dev 2006;73:1600–1611. 10. Amorim CA, Van Langendonckt A, David A, et al. Survival of human preantral follicles after cryopreservation of ovarian tissue, follicular isolation and in vitro culture in a calcium alginate matrix. Hum Reprod 2008. [Epub ahead of print] 11. Donnez J, Dolmans MM, Demylle D, et al. Livebirth after orthotopic transplantation of cryopreserved ovarian tissue. Lancet 2004;16:1405–1410. 12. Imhof M, Bergmeister H, Lipovac M, et al. Orthotopic microvascular reanastomosis of whole cryopreserved ovine ovaries resulting in pregnancy and live birth. Fertil Steril 2006;85:1208–1215. 13. Demirci B, Lornage J, Salle B, et al. Follicular viability and morphology of sheep ovaries after exposure to cryoprotectant and cryopreservation with different freezing protocols. Fertil Steril 2001;75:754–762. 14. Santos RR, Van Den Hurk R, Rodrigues APR, et al. Effect of cryopreservation on viability, activation and growth of in situ and isolated ovine early-stage follicles. Anim Reprod Sci 2007;99:53–64. 15. Salle B, Demirci B, Franck M, et al. Normal pregnancies and live births after autograft of frozen-thawed hemi-ovaries into ewes. Fértil Steril 2002;77:403–408. 16. Salle B, Lornage J, Demirci B, et al. Restoration of ovarian steroid secretion and histologic assessment after freezing, thawing, and autograft of a hemi-ovary in sheep. Fertil Steril 1999;72:366–370. 17. Luz VB, Santos RR, Pinto LC, et al. DMSO perfusion in caprine ovarian tissue and its relationship with follicular viability after cryopreservation. Fertil Steril 2008. [Epub ahead of print] 18. Rodrigues APR, Costa SH, Santos RR, et al. In vitro culture of cryopreserved caprine ovarian tissue pieces and isolated follicles. Cell Preserv Technol 2006;4:290–298. 19. Amorim CA, Rodrigues APR, Lucci CM, et al. Effect of sectioning on the number of isolated ovine preantral follicles. Small Rumin Res 2000;37:269–277. 20. Fauque P, Ben Amor A, Joanne C, et al. Use of trypan blue staining to assess the quality of ovarian cryopreservation. Fertil Steril 2007;87:1200–1207.
Address reprint requests to: Dr. Regiane R. Santos Department of Equine Sciences Veterinary Pharmaceuticals, Pharmacology, and Toxicology Division Faculty of Veterinary Medicine Utrecht University Yalelaan 114 Utrecht, 3584 CM The Netherlands
E-mail: [email protected] Received 5 November, 2008/Accepted 5 January, 2009
