Gene expression analysis of ovine prepubertal testicular tissue vitrified with a novel cryodevice (E.Vit)

  • Daniela BebbereEmail author
  • Sara Pinna
  • Stefano Nieddu
  • Dity Natan
  • Amir Arav
  • Sergio Ledda
Fertility Preservation



Testicular tissue cryopreservation prior to gonadotoxic therapies is a method to preserve fertility in children. However, the technique still requires development, especially when the tissue is immature and rather susceptible to stress derived from in vitro manipulation. This study aimed to investigate the effects of vitrification with a new cryodevice (E.Vit) on cell membrane integrity and gene expression of prepubertal testicular tissue in the ovine model.


Pieces of immature testicular tissue (1 mm3) were inserted into “E.Vit” devices and vitrified with a two-step protocol. After warming, tissues were cultured in vitro and cell membrane integrity was assessed after 0, 2, and 24 h by trypan blue exclusion test. Controls consisted of non-vitrified tissue analyzed after 0, 2, and 24 h in vitro culture (IVC). Expression of genes involved in transcriptional stress response (BAX, SOD1, CIRBP, HSP90AB1), cell proliferation (KIF11), and germ- (ZBDB16, TERT, POU5F1, KIT) and somatic- (AR, FSHR, STAR) cell specific markers was evaluated 2 and 24 h after warming.


Post-warming trypan blue staining showed the survival of most cells, although membrane integrity immediately after warming (66.00% ± 4.73) or after 2 h IVC (59.67% ± 4.18) was significantly lower than controls (C0h 89.67% ± 1.45). Extended post-warming IVC (24 h) caused an additional decrease to 31% ± 3.46 (P < 0.05). Germ- and somatic-cell specific markers showed the survival of both cell types after cryopreservation and IVC. All genes were affected by cryopreservation and/or IVC, and moderate stress conditions were indicated by transcriptional stress response.


Vitrification with the cryodevice E.Vit is a promising strategy to cryopreserve prepubertal testicular tissue.


Cryopreservation Testicular tissue Gene expression Prepubertal In vitro culture 



Research was supported by Regione Autonoma della Sardegna.- L.R. 7-MIGLIOVINGENSAR Project and by Bando Competitivo Fondazione di Sardegna – 2016, CUP J86C18000800005.

DB is the recipient of an RTDa contract at the University of Sassari, Italy, granted by “P.O.R. SARDEGNA F.S.E. 2014–2020” – CUP J86C18000270002.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

All applicable international, national, and/or institutional guidelines for the care and use of animals were followed.


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© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of Veterinary MedicineUniversity of SassariSassariItaly
  2. 2.FertileSAFE LtdNess ZionaIsrael

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