Vitrification does not Increase the Incidence of Apoptosis and Caspase 3/7 Activity in Human Ovarian Tissues

Authors
M. Abdollahi 1
M. Salehnia 1
S. Salehpour 2
1 Department of Anatomical Sciences, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran
2 Infertility and Reproductive Health Research Center (IRHRC), Ayatollah Taleghani Hospital, Shahid Beheshti University of Medical Sciences, Tehran, Iran
Abstract
Objective: This study aimed to evaluate the incidence of apoptosis in vitrified and non-vitrified human ovarian tissue by the use of morphological analysis and apoptosis assay techniques. Methods: We obtained human ovarian tissue biopsies from 30 women who underwent elective caesarean sections. Tissues were transported to the laboratory in pre-warmed, equilibrated Leibovitz L-15 medium within 2 hours. The tissues were cut into small pieces and divided into two groups, vitrified and non-vitrified (control). Apoptosis incidence was assessed by light microscope and the TUNEL assay and DNA laddering. Evaluation of caspase 3/7 protein levels was performed by the luminescent assay. Results: We observed no morphological signs of apoptosis in the vitrified samples. There were no apoptosis signals as evidenced by TUNEL staining and no DNA laddering pattern observed in the vitrified group. Caspase 3/7 activity was 2294±169.19 RLU/µg protein in the non-vitrified control group and 2231±89.271 RLU/µg protein in the vitrified group, which was not significantly different. Conclusion: The structure of human vitrified ovarian tissue was well preserved. Vitrification could not increase apoptosis and caspase 3/7 activity in human ovarian tissue.

Keywords

[1]     Deanesly R. Immature rat ovaries grafted after freezing and thawing. J Endocrinol 1954; 11(2): 197-200.
[2]     Zhang J, Liu J, Xu KP, Liu B, DiMattina M. Extracorporeal development and ultrarapid freezing of human fetal ova. J Assist Reprod Genet 1995; 12(6): 361-8.
[3]     Kelly SM, Buckett WM, Abdul-Jalil AK, Tan SL. The cryobiology of assisted reproduction. Minerva Ginecol 2003; 55(5): 389-98.
[4]     Newton H, Aubard Y, Rutherford A, Sharma V, Gosden R. Low temperature storage and grafting of human ovarian tissue. Hum Reprod 1996; 11(7): 1487-91.
[5]     Hovatta O, Silye R, Krausz T, Abir R, Margara R, Trew G, Lass A, Winston RM. Cryopreservation of human ovarian tissue using dimethylsulphoxide and propanediol-sucrose as cryoprotectants. Hum Reprod 1996; 11(6): 1268-72.
[6]     Amorim CA, Curaba M, Van Langendonckt A, Dolmans MM, Donnez J. Vitrification as an alternative means of cryopreserving ovarian tissue. Reprod Biomed Online 2011; 23(2): 160-86.
[7]     Amorim CA, Dolmans MM, David A, Jaeger J, Vanacker J, Camboni A, Donnez J, Van Langendonckt A. Vitrification and xenografting of human ovarian tissue. Fertil Steril 2012; 98(5): 1291-8.e1-2.
[8]     Isachenko E, Isachenko V, Nawroth F, Rahimi G, Kreienberg R, Reinsberg J, Weiss J. Human ovarian tissue preservation: is vitrification acceptable method for assisted reproduction? Cryo Letters 2008; 29(4): 301-14.
[9]     Nawroth F, Isachenko V, Isachenko E, Rahimi G. Vitrification Of Ovarian Tissue. In: Tucker Mj, Liebermann J (Editors). Vitrification In Assisted Reproduction: A User’s Manual And Trouble-Shooting Guide. London: Informa Uk Ltd, 2007: 261-72.
[10]  Morita Y, Tilly JL. Oocyte apoptosis: like sand through an hourglass. Dev Biol 1999; 213(1): 1-17.
[11]  Zhou XH, Wu YJ, Shi J, Xia YX, Zheng SS. Cryopreservation of human ovarian tissue: comparison of novel direct cover vitrification and conventional vitrification. Cryobiology 2010; 60(2): 101-5.
[12]  Chang HJ, Moon JH, Lee JR, Jee BC, Suh CS, Kim SH. Optimal condition of vitrification method for cryopreservation of human ovarian cortical tissues. J Obstet Gynaecol Res 2011; 37(8): 1092-101.
[13]  Xiao Z, Wang Y, Li L, Luo S, Li SW. Needle immersed vitrification can lower the concentration of cryoprotectant in human ovarian tissue cryopreservation. Fertil Steril 2010; 94(6): 2323-8.
[14]  Salehnia M, Sheikhi M, Pourbeiranvand S, Lundqvist M. Apoptosis of human ovarian tissue is not increased by either vitrification or rapid cooling. Reprod Biomed Online 2012; 25(5): 492-9.
[15]  Abdollahi M, Salehnia M, Salehpour S, Ghorbanmehr N. Human ovarian tissue vitrification/warming has minor effect on the expression of apoptosis-related genes. Iran Biomed J 2013; 17(4): 179-86.
[16]  Salehnia M, Abbasian Moghadam E, Rezazadeh Velojerdi M. Ultrastructure of follicles after vitrification of mouse ovarian tissue. Fertil Steril 2002; 78(3): 644-5.
[17]  Carrasco RA, Stamm NB, Patel BK. One-step cellular caspase-3/7 assay. Biotechniques 2003; 34(5): 1064-7.
[18]  Fathi R, Valojerdi MR, Salehnia M. Effects of different cryoprotectant combinations on primordial follicle survivability and apoptosis incidence after vitrification of whole rat ovary. Cryo Letters 2013; 34(3): 228-38.
[19]  Mazoochi T, Salehnia M, Valojerdi MR, Mowla SJ. Morphologic, ultrastructural, and biochemical identification of apoptosis in vitrified-warmed mouse ovarian tissue. Fertil Steril 2008; 90(4 Suppl): 1480-6.
[20]  Mazoochi T, Salehnia M, Pourbeiranvand S, Forouzandeh M, Mowla SJ, Hajizadeh E. Analysis of apoptosis and expression of genes related to apoptosis in cultures of follicles derived from vitrified and non-vitrified ovaries. Mol Hum Reprod 2009; 15(3): 155-64.
[21]  Nyachieo A, Spiessens C, Chai DC, Kiulia NM, Willemen D, Mwenda JM, Bourgain C, D'Hooghe TM. Ovarian tissue cryopreservation by vitrification in olive baboons (Papio anubis): a pilot study. Gynecol Obstet Invest 2013; 75(3): 157-62.
[22]  Liu J, Cheng KM, Silversides FG. Novel needle-in-straw vitrification can effectively preserve the follicle morphology, viability, and vascularization of ovarian tissue in Japanese quail (Coturnix japonica). Anim Reprod Sci 2012; 134(3-4): 197-202.
[23]  Depalo R, Lorusso F, Bettocchi S, Selvaggi L, Cavallini A, Valentini AM, Caruso ML, Lippolis C. Assessment of estrogen receptors and apoptotic factors in cryopreserved human ovarian cortex. Syst Biol Reprod Med 2009; 55(5-6): 236-43.
[24]  Huang L, Mo Y, Wang W, Li Y, Zhang Q, Yang D. Cryopreservation of human ovarian tissue by solid-surface vitrification. Eur J Obstet Gynecol Reprod Biol 2008; 139(2): 193-8.
[25]  Li YB, Zhou CQ, Yang GF, Wang Q, Dong Y. Modified vitrification method for cryo-preservation of human ovarian tissues. Chin Med J (Engl)  2007; 120(2): 110-4.
[26]  Keros V, Xella S, Hultenby K, Pettersson K, Sheikhi M, Volpe A, Hreinsson J, Hovatta O. Vitrification versus controlled-rate freezing in cryopreservation of human ovarian tissue. Hum Reprod 2009; 24(7): 1670-83.
[27]  Sheikhi M, Hultenby K, Niklasson B, Lundqvist M, Hovatta O. Clinical grade vitrification of human ovarian tissue: an ultrastructural analysis of follicles and stroma in vitrified tissue. Hum Reprod 2011; 26(3): 594-603.
[28]  Gandolfi F, Paffoni A, Papasso Brambilla E, Bonetti S, Brevini TA, Ragni G. Efficiency of equilibrium cooling and vitrification procedures for the cryopreservation of ovarian tissue: comparative analysis between human and animal models. Fertil Steril 2006; 85 Suppl 1: 1150-6.
[29]  Isachenko V, Lapidus I, Isachenko E, Krivokharchenko A, Kreienberg R, Woriedh M, Bader M, Weiss JM. Human ovarian tissue vitrification versus conventional freezing: morphological, endocrinological, and molecular biological evaluation. Reproduction 2009; 138(2): 319-27.
[30]  Brodowska A, Laszczyńska M, Starczewski A. Apoptosis in ovarian cells in postmenopausal women. Folia Histochem Cytobiol 2007; 45(2): 99-105.
[31]  Rahimi G, Isachenko V, Todorov P, Tawadros S, Mallmann P, Nawaroth F, Isachenko E. Apoptosis in human ovarian tissue after conventional freezing or vitrification and xenotransplantation. Cryo Letters 2009; 30(4): 300-9.
Pathobiology Reserach
Volume 16, Issue 4
February 2014
Pages 47-58