WHOLE TESTES CRYOPRESERVATION FOR FUTURE AUTO-TRANSPLANTATION: COMPARISON OF DIFFERENT FREEZING METHODS

Chenchu Nagarakanti, MD1; Robert R.A. Wilson, Bsc1; Elizabeth D Greene, LATG.2; Ibrahim Hacibey, MD1; Megan Escott, MD1,4; Yalcin Kulahci1; Vijay Gorantla1; Anthony Atala, MD1,3; Kelvin G.M. Brockbank, PhD2,3; Hooman Sadri-Ardekani, MD, PhD1,3

1Wake Forest Institute for Regenerative Medicine (WFIRM), Wake Forest School of Medicine, Winston-Salem, NC.

2Tissue Testing Technologies LLC, North Charleston, SC

3Department of Bioengineering, Clemson University, Clemson, SC

4Department of Urology, Wake Forest School of Medicine, Winston-Salem, NC.

Background:

In the past decades, survival rates of cancer patients have increased significantly, though infertility remains a long-term complication of cancer therapy. Whole testes (WT) cryopreservation for successive transplantation is one of the potential fertility-sparing strategies in childhood cancer patients with solid tumors.

Methods:

The study involved eight rats, each were sacrificed and submitted to orchiectomy. WT organs from each rat were randomly assigned to four groups: Group A, fresh tissue (control); Group B, slow freezing with 10% DMSO and rewarmed at 37°C; Group C, slow freezing with 10% DMSO and rewarmed on ice, and Group D, vitrified using VS55, a complex mixture containing DMSO, formamide, and 1,2-propanedio. Diluted VS55 (4°C) was added sequentially to achieve full-strength VS55. The samples were rapidly cooled to -100°C in 2-methylbutane and then transferred to vapor phase nitrogen for slower cooling and storage below -135°C for >24 h before testing (vitrification). Vitrified testes were slowly warmed to -100°C followed by rapid warming to melting. Each group had two replicates from different rats. Each testicle was measured metrically and with an orchidometer. Histological examination was conducted to assess morphological changes in the testicular tissues. Immunohistochemical staining techniques, specifically targeting PGP, Calretinin, Vimentin and Smooth Muscle Actine (SMA) were employed to identify undifferentiated spermatogonia, Leydig cells, Sertoli cells and peritubular cells. Additionally, micro-CT scanning was used to evaluate the distribution and effectiveness of DMSO within the testicular tissue across different loading times (from 6 hours to 4 days).

Results:

Slight morphological alterations were observed in the experimental groups compared to the control, including testicle tubule tissue shrinkage in vitrified samples. Frozen tissue demonstrated more significant tubule tissue shrinkage, minor disruption of the epididymal tissues, and more significant disruption of the endothelial layers of the blood vessels in the vascular pedicle. Arterial vasospasm was present in all four groups. PGP 9.5 IHC identified undifferentiated spermatogonial cells. 1-way ANOVA test showed a statistically significantly lower number of cells in the three experimental groups than in the control. It found that 10% DMSO gave 10% concentration in the testes after 12 hours of loading time, which is close to optimum.

Conclusion:

Slow freezing with loading 10% DMSO for 12 hours could preserve WT structure cell-specific sensitivity to the freeze-thaw process has been detected. All methods revealed significantly decreased PGP9.5 and calretinin expression, and further studies are necessary to optimize PGP 9.5 and calretinin expression and confirm testicular tissue function for possible auto-transplantation