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Assessment of vitrification outcome by xenotransplantation of ovarian cortex pieces in γ-irradiated mice: morphological and molecular analyses of apoptosis

  • Fertility Preservation
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Abstract

Purpose

The aim of this study was the investigation of caspase-3/7 activity and apoptosis related gene expression after vitrification and xenotransplantation of human ovarian fragments.

Methods

Ovarian specimens were obtained from normal female-to-male transsexual women during laparoscopic surgery and cut into small pieces and were considered as vitrified and non-vitrified groups. The morphological study, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, caspase-3/7 activity and apoptosis related gene expression analysis were done in both non-vitrified and vitrified groups in two steps (before transplantation of ovarian tissues and 30 days after transplantation).

Result(s)

In spite of high rate of normal follicles in both non-transplanted tissues these rates were significantly decreased in vitrified and non-vitrified grafted tissues, moreover grafted-vitrified tissue showed significantly less normal follicles than grafted-non-vitrified group (P < 0.05). The expression of some pro and anti-apoptotic genes in vitrified-warmed tissues were not changed compared to non-vitrified ones but the expression of Fas and caspase8 was increased and the expression of BRIC5 was decreased in this group (P < 0.05). In transplanted vitrified group the Bcl2, FasL and BRIC5 gene expression was high and caspase8 was low (P < 0.05). The expression of all genes in both grafted groups was more than non-grafted tissues except for caspase8 (P < 0.05). The TUNEL positive signals and caspase-3/7 activity were increased in both grafted groups compared to non-grafted groups and this enzyme activity in grafted-vitrified group was more than grafted-non-vitrified group (P < 0.05).

Conclusion(s)

This study provides the first evidence on the significant effect of vitrification on follicular apoptosis of grafted human ovarian tissue at mRNA level. The signs of follicular survival or degeneration detected by morphological assessment and caspase-3/7 activity were closely correlated to the changes in expression of apoptosis-related genes.

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References

  1. Jeong K, Aslan E, Ozkaya E, Sonmezer M, Oktay K. Ovarian cryopreservation. Minerva Med. 2012;103(1):37–42.

    CAS  PubMed  Google Scholar 

  2. Rodriguez-Wallberg KA, Oktay K. Recent advances in oocyte and ovarian tissue cryopreservation and transplantation. Best Pract Res Clin Obstet Gynaecol. 2012;26(3):391–405.

    Article  PubMed Central  PubMed  Google Scholar 

  3. Donnez J, Dolmans M-M. Fertility preservation in women. Nat Rev Endocrinol. 2013;9(12):735–49.

    Article  CAS  PubMed  Google Scholar 

  4. Hovatta O, Silye R, Krausz T, Abir R, Margara R, Trew G, et al. Cryopreservation of human ovarian tissue using dimethylsulphoxide and propanediol-sucrose as cryoprotectants. Hum Reprod. 1996;11(6):1268–72.

    Article  CAS  PubMed  Google Scholar 

  5. Hovatta O. Methods for cryopreservation of human ovarian tissue. Reprod Biomed Online. 2005;10(6):729–34.

    Article  PubMed  Google Scholar 

  6. Herraiz S, Novella-Maestre E, Rodríguez B, Díaz C, Sánchez-Serrano M, Mirabet V, et al. Improving ovarian tissue cryopreservation for oncologic patients: slow freezing versus vitrification, effect of different procedures and devices. Fertil Steril. 2014;101(3):775–84.

    Article  CAS  PubMed  Google Scholar 

  7. Isachenko V, Lapidus I, Isachenko E, Krivokharchenko A, Kreienberg R, Woriedh M, et al. Human ovarian tissue vitrification versus conventional freezing: morphological, endocrinological, and molecular biological evaluation. Reproduction. 2009;138(2):319–27.

    Article  CAS  PubMed  Google Scholar 

  8. Keros V, Xella S, Hultenby K, Pettersson K, Sheikhi M, Volpe A, et al. Vitrification versus controlled-rate freezing in cryopreservation of human ovarian tissue. Hum Reprod. 2009;24(7):1670–83.

    Article  CAS  PubMed  Google Scholar 

  9. Rahimi G, Isachenko E, Sauer H, Isachenko V, Wartenberg M, Hescheler J, et al. Effect of different vitrification protocols for human ovarian tissue on reactive oxygen species and apoptosis. Reprod Fertil Dev. 2003;15(6):343–9.

    Article  PubMed  Google Scholar 

  10. Fabbri R, Vicenti R, Macciocca M, Pasquinelli G, Paradisi R, Battaglia C, et al. Good preservation of stromal cells and no apoptosis in human ovarian tissue after vitrification. Biomed Res Int. 2014; 673537.

  11. Xiao Z, Wang Y, Li L, Luo S, Li S-W. Needle immersed vitrification can lower the concentration of cryoprotectant in human ovarian tissue cryopreservation. Fertil Steril. 2010;94(6):2323–8.

    Article  CAS  PubMed  Google Scholar 

  12. Zhou X-H, Wu Y-J, Shi J, Zheng S-S. Cryopreservation of human ovarian tissue: comparison of novel direct cover vitrification and conventional vitrification. Cryobiology. 2010;60(2):101–5.

    Article  CAS  PubMed  Google Scholar 

  13. Amorim CA, Curaba M, Van Langendonckt A, Dolmans M-M, Donnez J. Vitrification as an alternative means of cryopreserving ovarian tissue. Reprod Biomed Online. 2011;23(2):160–86.

    Article  PubMed  Google Scholar 

  14. Amorim CA, David A, Van Langendonckt A, Dolmans M-M, Donnez J. Vitrification of human ovarian tissue: effect of different solutions and procedures. Fertil Steril. 2011;95(3):1094–7.

    Article  CAS  PubMed  Google Scholar 

  15. 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.

    Article  PubMed  Google Scholar 

  16. Sheikhi M, Hultenby K, Niklasson B, Lundqvist M, Hovatta O. Preservation of human ovarian follicles within tissue frozen by vitrification in a xeno-free closed system using only ethylene glycol as a permeating cryoprotectant. Fertil Steril. 2013;100(1):170–7.

    Article  CAS  PubMed  Google Scholar 

  17. 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.

    Article  CAS  PubMed  Google Scholar 

  18. 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.

    PubMed Central  CAS  PubMed  Google Scholar 

  19. Andreu-Vieyra C, Habibi H. Factors controlling ovarian apoptosis. Can J Physiol Pharmacol. 2000;78(12):1003–12.

    Article  CAS  PubMed  Google Scholar 

  20. Gook DA, Edgar D, Borg J, Archer J, McBain J. Diagnostic assessment of the developmental potential of human cryopreserved ovarian tissue from multiple patients using xenografting. Hum Reprod. 2005;20(1):72–8.

    Article  PubMed  Google Scholar 

  21. Depalo R, Lorusso F, Bettocchi S, Selvaggi L, Cavallini A, Valentini AM, et al. Assessment of estrogen receptors and apoptotic factors in cryopreserved human ovarian cortex. Syst Biol Reprod Med. 2009;55:236–43.

    Article  CAS  PubMed  Google Scholar 

  22. Rahimi G, Isachenko V, Kreienberg R, Sauer H, Todorov P, Tawadros S, et al. Re-vascularisation in human ovarian tissue after conventional freezing or vitrification and xenotransplantation. Eur J Obstet Gynecol Reprod Biol. 2010;149(1):63–7.

    Article  PubMed  Google Scholar 

  23. Amorim CA, Dolmans M-M, David A, Jaeger J, Vanacker J, Camboni A, et al. Vitrification and xenografting of human ovarian tissue. Fertil Steril. 2012;98(5):1291–8.e1-2.

    Article  PubMed  Google Scholar 

  24. Silber SJ. Ovary cryopreservation and transplantation for fertility preservation. Mol Hum Reprod. 2012;18(2):59–67.

    Article  CAS  PubMed  Google Scholar 

  25. Rahimi G, Isachenko E, Isachenko V, Sauer H, Wartenberg M, Tawadros S, et al. Comparison of necrosis in human ovarian tissue after conventional slow freezing or vitrification and transplantation in ovariectomized SCID mice. Reprod Biomed Online. 2004;9(2):187–93.

    Article  CAS  PubMed  Google Scholar 

  26. Donnez J, Dolmans M-M, Pellicer A, Diaz-Garcia C, Sanchez Serrano M, Schmidt KT, et al. Restoration of ovarian activity and pregnancy after transplantation of cryopreserved ovarian tissue: a review of 60 cases of reimplantation. Fertil Steril. 2013;99(6):1503–13.

    Article  PubMed  Google Scholar 

  27. Rahimi G, Isachenko V, Todorov P, Tawadros S, Mallmann P, Nawroth F, et al. Apoptosis in human ovarian tissue after conventional freezing or vitrification and xenotransplantation. Cryoletters. 2009;30(4):300–9.

    CAS  PubMed  Google Scholar 

  28. Salehnia M, Moghadam EA, Velojerdi MR. Ultrastructure of follicles after vitrification of mouse ovarian tissue. Fertil Steril. 2002;78(3):644–5.

    Article  PubMed  Google Scholar 

  29. Klug CA, Jordan C. Hematopoietic stem cells protocols. Totowa: Hummana Press Inc; 2002. p. 155–60.

    Google Scholar 

  30. Gougeon A. Dynamics of follicular growth in the human: a model from preliminary results. Hum Reprod. 1986;1(2):81–7.

    CAS  PubMed  Google Scholar 

  31. Boldizsár F, Pálinkás L, Czömpöly T, Bartis D, Németh P, Berki T. Low glucocorticoid receptor (GR), high Dig2 and low Bcl-2 expression in double positive thymocytes of BALB/c mice indicates their endogenous glucocorticoid hormone exposure. Immunobiology. 2006;211(10):785–96.

    Article  PubMed  Google Scholar 

  32. 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.

    Article  CAS  PubMed  Google Scholar 

  33. Dath C, Van Eyck AS, Dolmans MM, Romeu L, Delle Vigne L, Donnez J, et al. Xenotransplantation of human ovarian tissue to nude mice: comparison between four grafting sites. Hum Reprod. 2010;25(7):1734–43.

    Article  CAS  PubMed  Google Scholar 

  34. Luyckx V, Scalercio S, Jadoul P, Amorim CA, Soares M, Donnez J, et al. Evaluation of cryopreserved ovarian tissue from prepubertal patients after long-term xenografting and exogenous stimulation. Fertil Steril. 2013;100(5):1350–7.

    Article  CAS  PubMed  Google Scholar 

  35. Kim SS, Kang HG, Kim NH, Lee HC, Lee HH. Assessment of the integrity of human oocytes retrieved from cryopreserved ovarian tissue after xenotransplantation. Hum Reprod. 2005;20(9):2502–8.

    Article  PubMed  Google Scholar 

  36. Soleimani R, Heytens E, Van den Broecke R, Rottiers I, Dhont M, Cuvelier CA, et al. Xenotransplantation of cryopreserved human ovarian tissue into murine back muscle. Hum Reprod. 2010;25(6):1458–70.

    Article  CAS  PubMed  Google Scholar 

  37. Liu J, Van der Elst J, Van den Broecke R, Dhont M. Early massive follicle loss and apoptosis in heterotopically grafted newborn mouse ovaries. Hum Reprod. 2002;17(3):605–11.

    Article  PubMed  Google Scholar 

  38. Laschke MW, Menger MD, Vollmar B. Cryopreservation does not affect neovascularization of freely transplanted ovarian follicles. Fertil Steril. 2003;79(6):1458–60.

    Article  PubMed  Google Scholar 

  39. Donnez J, Martinez-Madrid B, Jadoul P, Van Langendonckt A, Demylle D, Dolmans M-M. Ovarian tissue cryopreservation and transplantation: a review. Hum Reprod Update. 2006;12(5):519–35.

    Article  PubMed  Google Scholar 

  40. Inoue N, Maeda A, Matsuda-Minehata F, Fukuta K, Manabe N. Expression and localization of Fas ligand and Fas during atresia in porcine ovarian follicles. J Reprod Dev. 2006;52(6):723–30.

    Article  CAS  PubMed  Google Scholar 

  41. Miyashita T, Reed JC. Tumor suppressor p53 is a direct transcriptional activator of the human bax gene. Cell. 1995;80(2):293–9.

    Article  CAS  PubMed  Google Scholar 

  42. Miyashita T, Harigai M, Hanada M, Reed JC. Identification of a p53-dependent negative response element in the bcl-2 gene. Cancer Res. 1994;54(12):3131–5.

    CAS  PubMed  Google Scholar 

  43. Knudson CM, Tung KS, Tourtellotte WG, Brown GA, Korsmeyer SJ. Bax-deficient mice with lymphoid hyperplasia and male germ cell death. Science. 1995;270(5233):96–9.

    Article  CAS  PubMed  Google Scholar 

  44. Ambrosini G, Adida C, Altieri DC. A novel anti-apoptosis gene, survivin, expressed in cancer and lymphoma. Nat Med. 1997;3(8):917–21.

    Article  CAS  PubMed  Google Scholar 

  45. Shin S, Sung BJ, Cho YS, Kim HJ, Ha NC, Hwang JI, et al. An anti-apoptotic protein human survivin is a direct inhibitor of caspase-3 and -7. Biochemistry. 2001;40(4):1117–23.

    Article  CAS  PubMed  Google Scholar 

  46. Johnson AL, Bridgham JT. Caspase-mediated apoptosis in the vertebrate ovary. Reproduction. 2002;124(1):19–27.

    Article  CAS  PubMed  Google Scholar 

  47. Eguchi Y, Ewert DL, Tsujimoto Y. Isolation and characterization of the chicken bcl-2 gene: expression in a variety of tissues including lymphoid and neuronal organs in adult and embryo. Nucleic Acids Res. 1992;20(16):4187–92.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  48. Tilly J, Tilly K, Kenton M, Johnson A. Expression of members of the bcl-2 gene family in the immature rat ovary: equine chorionic gonadotropin-mediated inhibition of granulosa cell apoptosis is associated with decreased bax and constitutive bcl-2 and bcl-xlong messenger ribonucleic acid levels. Endocrinology. 1995;136(1):232–41.

    CAS  PubMed  Google Scholar 

  49. Hsu SY, Hsueh AJ. Tissue-specific Bcl-2 protein partners in apoptosis: an ovarian paradigm. Physiol Rev. 2000;80(2):593–614.

    CAS  PubMed  Google Scholar 

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Acknowledgments

This work was supported by grants from Tarbiat Modares University and Tehran University of Medial Sciences.

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Authors and Affiliations

  1. Reproductive Health Research Center, Tehran University of Medical Sciences, Tehran, Iran

    Mina Jafarabadi

  2. Department of Anatomical Sciences, Faculty of Medicine, Qom University of Medical Science, Qom, Iran

    Maasoume Abdollahi

  3. Department of Anatomical Sciences, Medical Sciences Faculty, Tarbiat Modares University, P. O. BOX: 14115-111, Tehran, Iran

    Mojdeh Salehnia

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  1. Mina Jafarabadi
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  2. Maasoume Abdollahi
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  3. Mojdeh Salehnia
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Correspondence to Mojdeh Salehnia.

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Capsule Our study demostrated for the first time the signs of follicular survival or degeneration detected by morphological assessment and caspase-3/7 activity were closely correlated to the changes in expression of apoptosis-related genes.

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Jafarabadi, M., Abdollahi, M. & Salehnia, M. Assessment of vitrification outcome by xenotransplantation of ovarian cortex pieces in γ-irradiated mice: morphological and molecular analyses of apoptosis. J Assist Reprod Genet 32, 195–205 (2015). https://doi.org/10.1007/s10815-014-0382-1

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  • DOI: https://doi.org/10.1007/s10815-014-0382-1

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