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Dimethyl sulfoxide perfusion in caprine ovarian tissue and its relationship with follicular viability after cryopreservation

2008; Elsevier BV; Volume: 91; Issue: 4 Linguagem: Inglês

10.1016/j.fertnstert.2008.07.1778

1556-5653

Valesca Barreto Luz, Regiane R. Santos, Leonardo C. Pinto, Alison A.X. Soares, J.J.H. Celestino, Jair Mafezoli, C.C. Campello, J.R. Figueiredo, Ana Paula Ribeiro Rodrigues,

Pluripotent Stem Cells Research

Ovarian cortical fragments (3 × 3 × 1 mm) were exposed to dimethyl sulfoxide (DMSO) in different concentrations for further analysis of cryoprotectant perfusion by applying high-performance liquid chromatography (HPLC) and conventional cryopreservation. This simple perfusion test can predict the efficiency of the cryopreservation procedure. Ovarian cortical fragments (3 × 3 × 1 mm) were exposed to dimethyl sulfoxide (DMSO) in different concentrations for further analysis of cryoprotectant perfusion by applying high-performance liquid chromatography (HPLC) and conventional cryopreservation. This simple perfusion test can predict the efficiency of the cryopreservation procedure. Despite the encouraging results obtained after cryopreservation of human ovarian tissue (1Donnez J. Dolmans M.M. Demylle D. Jadoul P. Pirard C. Squifflet J. et al.Livebirth after orthotopic transplantation of cryopreserved ovarian tissue.Lancet. 2004; 364: 1405-1410Abstract Full Text Full Text PDF PubMed Scopus (1341) Google Scholar), several events related to the procedure itself must be properly controlled. The first step before freezing consists of permeating the ovarian tissue with a cryoprotectant agent. Because the ovary is a complex tissue, optimal perfusion is indicative of a successful freezing procedure. To verify the diffusion of a cryoprotectant into the tissue, several sophisticated procedures have been used such as radioactive tracers (2Clark P. Fahy G.M. Karow A.M. Factors influencing renal cryopreservation. II. Toxic effects of three cryoprotectants in combination with three vehicle solutions in non frozen rabbit cortical slices.Cryobiology. 1984; 21: 274-284Crossref PubMed Scopus (32) Google Scholar) and proton nuclear magnetic resonance analysis (3Newton H. Fisher J. Arnold J.R. Pegg D.E. Faddy M.J. Gosden R.G. Permeation of human ovarian tissue with cryoprotective agents in preparation for cryopreservation.Hum Reprod. 1998; 13: 376-380Crossref PubMed Scopus (171) Google Scholar). However, high-performance liquid chromatography (HPLC), a simpler and more inexpensive method, has been used successfully to evaluate perfusion of dimethyl sulfoxide (DMSO) into liver fragments (4Carpenter J.F. Dawson P.E. Quantitation of dimethylsulfoxide in solutions and tissues by high performance liquid chromatography.Cryobiology. 1991; 28: 210-215Crossref PubMed Scopus (34) Google Scholar), and HPLC can be performed rapidly and routinely in any laboratory.To evaluate the HPLC method in ovarian tissue, we used the goat, an excellent animal model for humans (5Santos RR, Knijn HM, Vos PLAM, Oei CHY, Van Loon T, Colenbrander B, et al. Complete follicular development and recovery of ovarian function of frozen-thawed, autotransplanted caprine ovarian cortex. Fertil Steril. Published online August 22, 2008.Google Scholar). Ovarian tissue from goats was exposed to DMSO and then underwent HPLC analysis and cryopreservation. Our aim was to determine the ovarian tissue levels of DMSO and examine the follicular viability after exposure and cryopreservation.Two experiments were performed using ovaries from eight adult mixed-breed goats. For each experiment, four ovarian pairs were used, which means that each of the experimental conditions was repeated four times. Ovarian pairs obtained at a local slaughterhouse were washed in 70% alcohol, washed twice in HEPES-buffered 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 hour.In a first experiment, 25 cortical fragments (3 × 3 × 1 mm) were removed from each ovarian pair and placed in the HM. One fragment was immediately fixed (control) in 4% paraformaldehyde for routine histologic and apoptotic (terminal deoxynucleotidyl transferase dUTP nick-end labeling, TUNEL) studies. The early-stage follicles were classified as normal or atretic (6Silva J.R. Tharasanit T. Taverne M.A. Van der Weijden G.C. Santos R.R. Figueiredo J.R. et al.The activin-follistatin system and in vitro early follicle development in goats.J Endocrinol. 2006; 1889: 113-125Crossref Scopus (45) Google Scholar).The remaining 24 fragments were divided as follows. [1] We exposed 12 fragments at 20°C, for 10, 20, 30, or 40 minutes 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 1.8 mL of HM plus 10% fetal bovine serum (FBS) (HM+). For cryoprotectant removal, fragments were washed three times (5 minutes each) in HM+ and fixed for routine histologic and TUNEL analysis. [2] 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 adapted a procedure previously described elsewhere for liver tissue (4Carpenter J.F. Dawson P.E. Quantitation of dimethylsulfoxide in solutions and tissues by high performance liquid chromatography.Cryobiology. 1991; 28: 210-215Crossref PubMed Scopus (34) Google Scholar) to create a method for ovarian tissue.In a second experiment, seven cortical fragments (3 × 3 × 1 mm) were randomly removed from each pair of ovaries and placed in HM. For viability analysis, one fragment immediately underwent follicular isolation (control), and early-stage follicles were classified as viable (not stained) or nonviable (stained) by using trypan blue dye (7Santos R.R. Van Haeften T. Roelen B.A.J. Knijn H.M. Colenbrander B. Gadella B.M. et al.Osmotic tolerance and freezability of isolated caprine early-staged follicles.Cell Tissue Res. 2008; 333: 323-331Crossref PubMed Scopus (11) Google Scholar). The remaining six fragments were exposed to DMSO as described in the first experiment, excluding the exposure times of 30 and 40 minutes, and we performed the conventional cryopreservation procedure previously tested by our group (8Rodrigues A.P.R. Amorim C.A. Costa S.H.F. Matos M.H.T. Santos R.R. Lucci C.M. et al.Cryopreservation of caprine ovarian tissue using dimethylsulphoxide and propanediol.Anim Reprod Sci. 2004; 84: 211-227Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar); 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, early-stage follicles were isolated from the tissue, followed by trypan blue staining as quality control in ovarian cryopreservation procedures (9Fauque P. Ben Amor A. Joanne C. Agnani G. Bresson J.L. Roux C. Use of trypan blue staining to assess the quality of ovarian cryopreservation.Fertil Steril. 2007; 87: 1200-1207Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar).Mean percentages of morphologically normal (experiment 1) and viable (experiment 2) early-stage follicles in the different treatments were compared using Dunnet test. Analysis of variance (ANOVA) and comparison using a t test and Pearson correlation were performed for all data. P<.05 was considered statistically significant.In the first experiment, a total of 1560 early-stage follicles were examined for their morphology, and the mean percentages of normal follicles were calculated (Fig. 1A ). Follicular exposure to DMSO did not statistically significantly reduce (P>.05) the percentage of normal early-stage follicles when compared with untreated (control) follicles or among the treatments. However, exposure to 2.0 M DMSO for 30 and 40 minutes statistically significantly reduced the percentage of normal follicles when compared with control.To determine the DMSO perfusion into the ovarian tissue, HPLC analysis has been performed (see Fig. 1B). Except for 1.5 M DMSO exposure for 40 minutes and 2.0 M DMSO exposure for 30 minutes, tissue levels were not statistically significantly affected by increase in time and concentration. This was confirmed by the low correlation among follicular morphology, cryoprotectant concentration, and time of exposure (r2 = –0.36). The mean minimum (0.63 mg) and maximum (1.63 mg) DMSO tissue levels were observed after ovarian tissue exposure to 1.0 M DMSO for 10 minutes and 2.0 M DMSO for 30 minutes, respectively.The second experiment followed the results obtained in the experiment 1, and a total of 840 early-stage follicles were examined for viability using trypan blue stain. The mean percentage of viable follicles was calculated (see Fig. 1C). Cryopreservation of ovarian tissue after exposure to 1.0 and 1.5 M DMSO for 10 minutes did not statistically significantly reduce (P>.05) the percentage of viable early-stage follicles when compared with control. After TUNEL, no high percentages of apoptotic follicles were observed for any treatment.Follicular morphology was not negatively affected when ovarian tissue was exposed to 1.0 and 1.5 M DMSO until 40 minutes, or to 2.0 M DMSO for 10 and 20 minutes. However, follicular survival after cryopreservation was similar to control values with exposure to 1.0 or 1.5 M DMSO for a maximum period of 10 minutes.Previous experiments had demonstrated that the ovarian tissue of large mammals (e.g., caprine and human) can be efficiently cryopreserved in low concentrations of DMSO, such as approximately 1.0 M (10Santos R.R. Tharasanit T. Figueiredo J.R. Van Haeften T. Van den Hurk R. Preservation of caprine preantral follicles viability after cryopreservation in sucrose and ethylene glycol.Cell Tissue Res. 2006; 325: 523-531Crossref PubMed Scopus (41) Google Scholar, 11Qu J. Godin P.A. Nisolle M. Donnez J. Distribution and epidermal growth factor receptor expression of primordial follicles in human ovarian tissue before and after cryopreservation.Hum Reprod. 2000; 15: 302-310Crossref PubMed Scopus (88) Google Scholar) and 1.5 M (3Newton H. Fisher J. Arnold J.R. Pegg D.E. Faddy M.J. Gosden R.G. Permeation of human ovarian tissue with cryoprotective agents in preparation for cryopreservation.Hum Reprod. 1998; 13: 376-380Crossref PubMed Scopus (171) Google Scholar, 8Rodrigues A.P.R. Amorim C.A. Costa S.H.F. Matos M.H.T. Santos R.R. Lucci C.M. et al.Cryopreservation of caprine ovarian tissue using dimethylsulphoxide and propanediol.Anim Reprod Sci. 2004; 84: 211-227Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). In the studies using goat ovarian tissue, exposure to DMSO has been carried out at 20°C (8Rodrigues A.P.R. Amorim C.A. Costa S.H.F. Matos M.H.T. Santos R.R. Lucci C.M. et al.Cryopreservation of caprine ovarian tissue using dimethylsulphoxide and propanediol.Anim Reprod Sci. 2004; 84: 211-227Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 10Santos R.R. Tharasanit T. Figueiredo J.R. Van Haeften T. Van den Hurk R. Preservation of caprine preantral follicles viability after cryopreservation in sucrose and ethylene glycol.Cell Tissue Res. 2006; 325: 523-531Crossref PubMed Scopus (41) Google Scholar), as in our present study, but for a longer period (20 minutes). Exposure in previous human studies (3Newton H. Fisher J. Arnold J.R. Pegg D.E. Faddy M.J. Gosden R.G. Permeation of human ovarian tissue with cryoprotective agents in preparation for cryopreservation.Hum Reprod. 1998; 13: 376-380Crossref PubMed Scopus (171) Google Scholar, 11Qu J. Godin P.A. Nisolle M. Donnez J. Distribution and epidermal growth factor receptor expression of primordial follicles in human ovarian tissue before and after cryopreservation.Hum Reprod. 2000; 15: 302-310Crossref PubMed Scopus (88) Google Scholar) has been performed for 30 minutes but at a lower temperature (4°C). Only the study from Newton et al. (3Newton H. Fisher J. Arnold J.R. Pegg D.E. Faddy M.J. Gosden R.G. Permeation of human ovarian tissue with cryoprotective agents in preparation for cryopreservation.Hum Reprod. 1998; 13: 376-380Crossref PubMed Scopus (171) Google Scholar) evaluated the permeation rate of the cryoprotectant.Our study obtained viability rates similar to those observed in fresh tissue (control). To assess the permeation of cryoprotectant into ovarian tissue using a simple, inexpensive, and rapid procedure, we performed HPLC analysis. We found that a minimum amount of DMSO in ovarian tissue (0.63 mg) associated with a short and optimal exposure time (10 minutes) was sufficient to protect the early-stage follicles against chilling injuries. Thus, the toxic effect of the cryoprotectant can be reduced without compromising the success of freezing as confirmed by viability analysis and the absence of apoptotic cells, which was confirmed by TUNEL. Our study demonstrates the possibility of successful cryopreservation of ovarian tissue after exposure to 1.0 or 1.5 M DMSO for 10 minutes. Based on this, further studies evaluating other cryoprotectants as well as reducing the cryoprotectant concentration of the freezing medium will help to develop efficient procedures for isolated early-stage follicles before clinical application. Despite the encouraging results obtained after cryopreservation of human ovarian tissue (1Donnez J. Dolmans M.M. Demylle D. Jadoul P. Pirard C. Squifflet J. et al.Livebirth after orthotopic transplantation of cryopreserved ovarian tissue.Lancet. 2004; 364: 1405-1410Abstract Full Text Full Text PDF PubMed Scopus (1341) Google Scholar), several events related to the procedure itself must be properly controlled. The first step before freezing consists of permeating the ovarian tissue with a cryoprotectant agent. Because the ovary is a complex tissue, optimal perfusion is indicative of a successful freezing procedure. To verify the diffusion of a cryoprotectant into the tissue, several sophisticated procedures have been used such as radioactive tracers (2Clark P. Fahy G.M. Karow A.M. Factors influencing renal cryopreservation. II. Toxic effects of three cryoprotectants in combination with three vehicle solutions in non frozen rabbit cortical slices.Cryobiology. 1984; 21: 274-284Crossref PubMed Scopus (32) Google Scholar) and proton nuclear magnetic resonance analysis (3Newton H. Fisher J. Arnold J.R. Pegg D.E. Faddy M.J. Gosden R.G. Permeation of human ovarian tissue with cryoprotective agents in preparation for cryopreservation.Hum Reprod. 1998; 13: 376-380Crossref PubMed Scopus (171) Google Scholar). However, high-performance liquid chromatography (HPLC), a simpler and more inexpensive method, has been used successfully to evaluate perfusion of dimethyl sulfoxide (DMSO) into liver fragments (4Carpenter J.F. Dawson P.E. Quantitation of dimethylsulfoxide in solutions and tissues by high performance liquid chromatography.Cryobiology. 1991; 28: 210-215Crossref PubMed Scopus (34) Google Scholar), and HPLC can be performed rapidly and routinely in any laboratory. To evaluate the HPLC method in ovarian tissue, we used the goat, an excellent animal model for humans (5Santos RR, Knijn HM, Vos PLAM, Oei CHY, Van Loon T, Colenbrander B, et al. Complete follicular development and recovery of ovarian function of frozen-thawed, autotransplanted caprine ovarian cortex. Fertil Steril. Published online August 22, 2008.Google Scholar). Ovarian tissue from goats was exposed to DMSO and then underwent HPLC analysis and cryopreservation. Our aim was to determine the ovarian tissue levels of DMSO and examine the follicular viability after exposure and cryopreservation. Two experiments were performed using ovaries from eight adult mixed-breed goats. For each experiment, four ovarian pairs were used, which means that each of the experimental conditions was repeated four times. Ovarian pairs obtained at a local slaughterhouse were washed in 70% alcohol, washed twice in HEPES-buffered 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 hour. In a first experiment, 25 cortical fragments (3 × 3 × 1 mm) were removed from each ovarian pair and placed in the HM. One fragment was immediately fixed (control) in 4% paraformaldehyde for routine histologic and apoptotic (terminal deoxynucleotidyl transferase dUTP nick-end labeling, TUNEL) studies. The early-stage follicles were classified as normal or atretic (6Silva J.R. Tharasanit T. Taverne M.A. Van der Weijden G.C. Santos R.R. Figueiredo J.R. et al.The activin-follistatin system and in vitro early follicle development in goats.J Endocrinol. 2006; 1889: 113-125Crossref Scopus (45) Google Scholar). The remaining 24 fragments were divided as follows. [1] We exposed 12 fragments at 20°C, for 10, 20, 30, or 40 minutes 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 1.8 mL of HM plus 10% fetal bovine serum (FBS) (HM+). For cryoprotectant removal, fragments were washed three times (5 minutes each) in HM+ and fixed for routine histologic and TUNEL analysis. [2] 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 adapted a procedure previously described elsewhere for liver tissue (4Carpenter J.F. Dawson P.E. Quantitation of dimethylsulfoxide in solutions and tissues by high performance liquid chromatography.Cryobiology. 1991; 28: 210-215Crossref PubMed Scopus (34) Google Scholar) to create a method for ovarian tissue. In a second experiment, seven cortical fragments (3 × 3 × 1 mm) were randomly removed from each pair of ovaries and placed in HM. For viability analysis, one fragment immediately underwent follicular isolation (control), and early-stage follicles were classified as viable (not stained) or nonviable (stained) by using trypan blue dye (7Santos R.R. Van Haeften T. Roelen B.A.J. Knijn H.M. Colenbrander B. Gadella B.M. et al.Osmotic tolerance and freezability of isolated caprine early-staged follicles.Cell Tissue Res. 2008; 333: 323-331Crossref PubMed Scopus (11) Google Scholar). The remaining six fragments were exposed to DMSO as described in the first experiment, excluding the exposure times of 30 and 40 minutes, and we performed the conventional cryopreservation procedure previously tested by our group (8Rodrigues A.P.R. Amorim C.A. Costa S.H.F. Matos M.H.T. Santos R.R. Lucci C.M. et al.Cryopreservation of caprine ovarian tissue using dimethylsulphoxide and propanediol.Anim Reprod Sci. 2004; 84: 211-227Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar); 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, early-stage follicles were isolated from the tissue, followed by trypan blue staining as quality control in ovarian cryopreservation procedures (9Fauque P. Ben Amor A. Joanne C. Agnani G. Bresson J.L. Roux C. Use of trypan blue staining to assess the quality of ovarian cryopreservation.Fertil Steril. 2007; 87: 1200-1207Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). Mean percentages of morphologically normal (experiment 1) and viable (experiment 2) early-stage follicles in the different treatments were compared using Dunnet test. Analysis of variance (ANOVA) and comparison using a t test and Pearson correlation were performed for all data. P<.05 was considered statistically significant. In the first experiment, a total of 1560 early-stage follicles were examined for their morphology, and the mean percentages of normal follicles were calculated (Fig. 1A ). Follicular exposure to DMSO did not statistically significantly reduce (P>.05) the percentage of normal early-stage follicles when compared with untreated (control) follicles or among the treatments. However, exposure to 2.0 M DMSO for 30 and 40 minutes statistically significantly reduced the percentage of normal follicles when compared with control. To determine the DMSO perfusion into the ovarian tissue, HPLC analysis has been performed (see Fig. 1B). Except for 1.5 M DMSO exposure for 40 minutes and 2.0 M DMSO exposure for 30 minutes, tissue levels were not statistically significantly affected by increase in time and concentration. This was confirmed by the low correlation among follicular morphology, cryoprotectant concentration, and time of exposure (r2 = –0.36). The mean minimum (0.63 mg) and maximum (1.63 mg) DMSO tissue levels were observed after ovarian tissue exposure to 1.0 M DMSO for 10 minutes and 2.0 M DMSO for 30 minutes, respectively. The second experiment followed the results obtained in the experiment 1, and a total of 840 early-stage follicles were examined for viability using trypan blue stain. The mean percentage of viable follicles was calculated (see Fig. 1C). Cryopreservation of ovarian tissue after exposure to 1.0 and 1.5 M DMSO for 10 minutes did not statistically significantly reduce (P>.05) the percentage of viable early-stage follicles when compared with control. After TUNEL, no high percentages of apoptotic follicles were observed for any treatment. Follicular morphology was not negatively affected when ovarian tissue was exposed to 1.0 and 1.5 M DMSO until 40 minutes, or to 2.0 M DMSO for 10 and 20 minutes. However, follicular survival after cryopreservation was similar to control values with exposure to 1.0 or 1.5 M DMSO for a maximum period of 10 minutes. Previous experiments had demonstrated that the ovarian tissue of large mammals (e.g., caprine and human) can be efficiently cryopreserved in low concentrations of DMSO, such as approximately 1.0 M (10Santos R.R. Tharasanit T. Figueiredo J.R. Van Haeften T. Van den Hurk R. Preservation of caprine preantral follicles viability after cryopreservation in sucrose and ethylene glycol.Cell Tissue Res. 2006; 325: 523-531Crossref PubMed Scopus (41) Google Scholar, 11Qu J. Godin P.A. Nisolle M. Donnez J. Distribution and epidermal growth factor receptor expression of primordial follicles in human ovarian tissue before and after cryopreservation.Hum Reprod. 2000; 15: 302-310Crossref PubMed Scopus (88) Google Scholar) and 1.5 M (3Newton H. Fisher J. Arnold J.R. Pegg D.E. Faddy M.J. Gosden R.G. Permeation of human ovarian tissue with cryoprotective agents in preparation for cryopreservation.Hum Reprod. 1998; 13: 376-380Crossref PubMed Scopus (171) Google Scholar, 8Rodrigues A.P.R. Amorim C.A. Costa S.H.F. Matos M.H.T. Santos R.R. Lucci C.M. et al.Cryopreservation of caprine ovarian tissue using dimethylsulphoxide and propanediol.Anim Reprod Sci. 2004; 84: 211-227Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). In the studies using goat ovarian tissue, exposure to DMSO has been carried out at 20°C (8Rodrigues A.P.R. Amorim C.A. Costa S.H.F. Matos M.H.T. Santos R.R. Lucci C.M. et al.Cryopreservation of caprine ovarian tissue using dimethylsulphoxide and propanediol.Anim Reprod Sci. 2004; 84: 211-227Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 10Santos R.R. Tharasanit T. Figueiredo J.R. Van Haeften T. Van den Hurk R. Preservation of caprine preantral follicles viability after cryopreservation in sucrose and ethylene glycol.Cell Tissue Res. 2006; 325: 523-531Crossref PubMed Scopus (41) Google Scholar), as in our present study, but for a longer period (20 minutes). Exposure in previous human studies (3Newton H. Fisher J. Arnold J.R. Pegg D.E. Faddy M.J. Gosden R.G. Permeation of human ovarian tissue with cryoprotective agents in preparation for cryopreservation.Hum Reprod. 1998; 13: 376-380Crossref PubMed Scopus (171) Google Scholar, 11Qu J. Godin P.A. Nisolle M. Donnez J. Distribution and epidermal growth factor receptor expression of primordial follicles in human ovarian tissue before and after cryopreservation.Hum Reprod. 2000; 15: 302-310Crossref PubMed Scopus (88) Google Scholar) has been performed for 30 minutes but at a lower temperature (4°C). Only the study from Newton et al. (3Newton H. Fisher J. Arnold J.R. Pegg D.E. Faddy M.J. Gosden R.G. Permeation of human ovarian tissue with cryoprotective agents in preparation for cryopreservation.Hum Reprod. 1998; 13: 376-380Crossref PubMed Scopus (171) Google Scholar) evaluated the permeation rate of the cryoprotectant. Our study obtained viability rates similar to those observed in fresh tissue (control). To assess the permeation of cryoprotectant into ovarian tissue using a simple, inexpensive, and rapid procedure, we performed HPLC analysis. We found that a minimum amount of DMSO in ovarian tissue (0.63 mg) associated with a short and optimal exposure time (10 minutes) was sufficient to protect the early-stage follicles against chilling injuries. Thus, the toxic effect of the cryoprotectant can be reduced without compromising the success of freezing as confirmed by viability analysis and the absence of apoptotic cells, which was confirmed by TUNEL. Our study demonstrates the possibility of successful cryopreservation of ovarian tissue after exposure to 1.0 or 1.5 M DMSO for 10 minutes. Based on this, further studies evaluating other cryoprotectants as well as reducing the cryoprotectant concentration of the freezing medium will help to develop efficient procedures for isolated early-stage follicles before clinical application. The authors thank Fortaleza University (UNIFOR) for the logistical support.