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Dimethyl sulfoxide perfusion in caprine ovarian tissue and its relationship with follicular viability after cryopreservation
Dimethyl sulfoxide perfusion in caprine ovarian tissue and its relationship with follicular viability after cryopreservation 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. (Fertil Steril 2009;91:1513–5. 2009 by American Society for Reproductive Medicine.) Key Words: DMSO, cryopreservation, HPLC
Despite the encouraging results obtained after cryopreservation of human ovarian tissue (1), 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 (2) and proton nuclear magnetic resonance analysis (3). 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 (4), 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 (5). 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, Received July 1, 2008; revised July 22, 2008; accepted July 29, 2008; published online October 18, 2008. V.B.L. is the recipient of a grant from CNPq, Brazil. R.R.S. has nothing to disclose. L.C.P. has nothing to disclose. A.A.X.S. has nothing to disclose. J.J.H.C. has nothing to disclose. J.M. has nothing to disclose. C.C.C. has nothing to disclose. J.R.F. has nothing to disclose. A.P.R.R. has nothing to disclose. Supported by FUNCAP, Brazil (No. 257/06). Reprint requests: Regiane R. Santos, Ph.D., Yalelaan 104, 3584CM, Utrecht, The Netherlands (FAX: þ 31 30 253 4811; E-mail: [email protected]).
0015-0282/09/$36.00 doi:10.1016/j.fertnstert.2008.07.1778
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 (6). 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) (HMþ). For cryoprotectant removal, fragments were washed three times (5 minutes each) in HMþ 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 (4) 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 (7). 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 (8); 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, earlystage follicles were isolated from the tissue, followed by trypan blue staining as quality control in ovarian cryopreservation procedures (9).
Fertility and Sterility Vol. 91, No. 4, Supplement, April 2009 Copyright ª2009 American Society for Reproductive Medicine, Published by Elsevier Inc.
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FIGURE 1 Percentage (mean SD) of (A) morphologically normal early-stage follicles after exposure to dimethyl sulfoxide (DMSO), (B) DMSO perfusion (mg) into the ovarian tissue after exposure and (C) viable follicles after cryopreservation. *Statistically significant difference from control. a,bStatistically significant difference among incubation times (10, 20, 30, and 40 minutes). A,BStatistically significant difference among DMSO concentrations (1.0, 1.5, and 2.0 M).
sis 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 ¼ –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 earlystage 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.
Luz. Correspondence. Fertil Steril 2009.
Mean percentages of morphologically normal (experiment 1) and viable (experiment 2) early-stage follicles in the different treatments were compared using Dunnet test. Analy-
1514
Luz et al.
Correspondence
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 (10, 11) and 1.5 M (3, 8). In the studies using goat ovarian tissue, exposure to DMSO has been carried out at 20 C (8, 10), as in our present study, but for a longer period (20 minutes). Exposure in previous human studies (3, 11) has been performed for 30 minutes but at a lower temperature (4 C). Only the study from Newton et al. (3) evaluated the permeation rate of the cryoprotectant. Our study obtained viability rates similar to those observed in fresh tissue (control). To assess the permeation Vol. 91, No. 4, Supplement, April 2009
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.
Acknowledgments: The authors thank Fortaleza University (UNIFOR) for the logistical support.
Valesca B. Luz, D.V.M.a Regiane R. Santos, Ph.D.b Leonardo C. Pinto, D.V.M.a Alison A. X. Soares, D.V.M.a Juliana J. H. Celestino, M.S.a Jair Mafezoli, Ph.D.c Cla´udio C. Campello, Ph.D.a Jose R. Figueiredo, Ph.D.a Ana P. R. Rodrigues, Ph.D.a a Laboratory of Manipulation of Oocytes Enclosed in Preantral Follicles, Faculty of Veterinary Medicine, University of Ceara´, Fortaleza, Brazil; b Department of Farm Animal Health, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The
Fertility and Sterility
Netherlands; and c Department of Life Sciences, Fortaleza University, Fortaleza, Brazil REFERENCES 1. Donnez J, Dolmans MM, Demylle D, Jadoul P, Pirard C, Squifflet J, et al. Livebirth after orthotopic transplantation of cryopreserved ovarian tissue. Lancet 2004;364:1405–10. 2. Clark P, Fahy GM, Karow AM. Factors influencing renal cryopreservation. II. Toxic effects of three cryoprotectants in combination with three vehicle solutions in non frozen rabbit cortical slices. Cryobiology 1984;21:274–84. 3. Newton H, Fisher J, Arnold JR, Pegg DE, Faddy MJ, Gosden RG. Permeation of human ovarian tissue with cryoprotective agents in preparation for cryopreservation. Hum Reprod 1998;13:376–80. 4. Carpenter JF, Dawson PE. Quantitation of dimethylsulfoxide in solutions and tissues by high performance liquid chromatography. Cryobiology 1991;28:210–5. 5. Santos 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. 6. Silva JR, Tharasanit T, Taverne MA, Van der Weijden GC, Santos RR, Figueiredo JR, et al. The activin-follistatin system and in vitro early follicle development in goats. J Endocrinol 2006;1889:113–25. 7. Santos RR, Van Haeften T, Roelen BAJ, Knijn HM, Colenbrander B, Gadella BM, et al. Osmotic tolerance and freezability of isolated caprine early-staged follicles. Cell Tissue Res 2008;333:323–31. 8. Rodrigues APR, Amorim CA, Costa SHF, Matos MHT, Santos RR, Lucci CM, et al. Cryopreservation of caprine ovarian tissue using dimethylsulphoxide and propanediol. Anim Reprod Sci 2004;84:211–27. 9. Fauque P, Ben Amor A, Joanne C, Agnani G, Bresson JL, Roux C. Use of trypan blue staining to assess the quality of ovarian cryopreservation. Fertil Steril 2007;87:1200–7. 10. Santos RR, Tharasanit T, Figueiredo JR, 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: 523–31. 11. Qu J, Godin PA, 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;15:302–10.
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Despite the encouraging results obtained after cryopreservation of human ovarian tissue (1), 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 (2) and proton nuclear magnetic resonance analysis (3). 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 (4), 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 (5). 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, Received July 1, 2008; revised July 22, 2008; accepted July 29, 2008; published online October 18, 2008. V.B.L. is the recipient of a grant from CNPq, Brazil. R.R.S. has nothing to disclose. L.C.P. has nothing to disclose. A.A.X.S. has nothing to disclose. J.J.H.C. has nothing to disclose. J.M. has nothing to disclose. C.C.C. has nothing to disclose. J.R.F. has nothing to disclose. A.P.R.R. has nothing to disclose. Supported by FUNCAP, Brazil (No. 257/06). Reprint requests: Regiane R. Santos, Ph.D., Yalelaan 104, 3584CM, Utrecht, The Netherlands (FAX: þ 31 30 253 4811; E-mail: [email protected]).
0015-0282/09/$36.00 doi:10.1016/j.fertnstert.2008.07.1778
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 (6). 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) (HMþ). For cryoprotectant removal, fragments were washed three times (5 minutes each) in HMþ 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 (4) 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 (7). 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 (8); 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, earlystage follicles were isolated from the tissue, followed by trypan blue staining as quality control in ovarian cryopreservation procedures (9).
Fertility and Sterility Vol. 91, No. 4, Supplement, April 2009 Copyright ª2009 American Society for Reproductive Medicine, Published by Elsevier Inc.
1513
FIGURE 1 Percentage (mean SD) of (A) morphologically normal early-stage follicles after exposure to dimethyl sulfoxide (DMSO), (B) DMSO perfusion (mg) into the ovarian tissue after exposure and (C) viable follicles after cryopreservation. *Statistically significant difference from control. a,bStatistically significant difference among incubation times (10, 20, 30, and 40 minutes). A,BStatistically significant difference among DMSO concentrations (1.0, 1.5, and 2.0 M).
sis 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 ¼ –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 earlystage 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.
Luz. Correspondence. Fertil Steril 2009.
Mean percentages of morphologically normal (experiment 1) and viable (experiment 2) early-stage follicles in the different treatments were compared using Dunnet test. Analy-
1514
Luz et al.
Correspondence
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 (10, 11) and 1.5 M (3, 8). In the studies using goat ovarian tissue, exposure to DMSO has been carried out at 20 C (8, 10), as in our present study, but for a longer period (20 minutes). Exposure in previous human studies (3, 11) has been performed for 30 minutes but at a lower temperature (4 C). Only the study from Newton et al. (3) evaluated the permeation rate of the cryoprotectant. Our study obtained viability rates similar to those observed in fresh tissue (control). To assess the permeation Vol. 91, No. 4, Supplement, April 2009
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.
Acknowledgments: The authors thank Fortaleza University (UNIFOR) for the logistical support.
Valesca B. Luz, D.V.M.a Regiane R. Santos, Ph.D.b Leonardo C. Pinto, D.V.M.a Alison A. X. Soares, D.V.M.a Juliana J. H. Celestino, M.S.a Jair Mafezoli, Ph.D.c Cla´udio C. Campello, Ph.D.a Jose R. Figueiredo, Ph.D.a Ana P. R. Rodrigues, Ph.D.a a Laboratory of Manipulation of Oocytes Enclosed in Preantral Follicles, Faculty of Veterinary Medicine, University of Ceara´, Fortaleza, Brazil; b Department of Farm Animal Health, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The
Fertility and Sterility
Netherlands; and c Department of Life Sciences, Fortaleza University, Fortaleza, Brazil REFERENCES 1. Donnez J, Dolmans MM, Demylle D, Jadoul P, Pirard C, Squifflet J, et al. Livebirth after orthotopic transplantation of cryopreserved ovarian tissue. Lancet 2004;364:1405–10. 2. Clark P, Fahy GM, Karow AM. Factors influencing renal cryopreservation. II. Toxic effects of three cryoprotectants in combination with three vehicle solutions in non frozen rabbit cortical slices. Cryobiology 1984;21:274–84. 3. Newton H, Fisher J, Arnold JR, Pegg DE, Faddy MJ, Gosden RG. Permeation of human ovarian tissue with cryoprotective agents in preparation for cryopreservation. Hum Reprod 1998;13:376–80. 4. Carpenter JF, Dawson PE. Quantitation of dimethylsulfoxide in solutions and tissues by high performance liquid chromatography. Cryobiology 1991;28:210–5. 5. Santos 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. 6. Silva JR, Tharasanit T, Taverne MA, Van der Weijden GC, Santos RR, Figueiredo JR, et al. The activin-follistatin system and in vitro early follicle development in goats. J Endocrinol 2006;1889:113–25. 7. Santos RR, Van Haeften T, Roelen BAJ, Knijn HM, Colenbrander B, Gadella BM, et al. Osmotic tolerance and freezability of isolated caprine early-staged follicles. Cell Tissue Res 2008;333:323–31. 8. Rodrigues APR, Amorim CA, Costa SHF, Matos MHT, Santos RR, Lucci CM, et al. Cryopreservation of caprine ovarian tissue using dimethylsulphoxide and propanediol. Anim Reprod Sci 2004;84:211–27. 9. Fauque P, Ben Amor A, Joanne C, Agnani G, Bresson JL, Roux C. Use of trypan blue staining to assess the quality of ovarian cryopreservation. Fertil Steril 2007;87:1200–7. 10. Santos RR, Tharasanit T, Figueiredo JR, 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: 523–31. 11. Qu J, Godin PA, 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;15:302–10.
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