1. Efficient cryopreservation of testicular tissue: effect of age, sample state, and concentration of cryoprotectant 
Sreepoorna Unni, M.Sc., Sandhya Kasiviswanathan, M.Sc., Serena D’Souza, Ph.D., Sushma Khavale, B.Sc., Srabani Mukherjee, Ph.D., Sujata Patwardhan, M.D., Deepa Bhartiya, Ph.D. 
Fertility and Sterility 
Volume 97, Issue 1, Pages e1 (January 2012)
DOI: /j.fertnstert
Copyright © 2012 American Society for Reproductive Medicine Terms and Conditions

2. Figure 1
(A–F) Impact of CP and concentration on immature and adult rat testicular tissue (CS or TUB) before cryopreservation. Irrespective of age or CP, higher cell death was observed in CS than in TUB. Immature tissue (A–C) was more susceptible to cell death by increasing CP strength compared with adult, except when exposed to DMSO (F). (G–L) Cell-specific effect of CPs after cryopreservation, by ploidy analysis. Specific effects of GLY on 1N, DMSO on 2N, and EG on 4N were observed in immature rat testicular tissue (G, J). These effects were not prominent in adult rat (H, K) or human (I, L) tissue. Experiments were repeated independently three times and analyzed by two-way or one-way ANOVA. **P<.01, *P<.05, statistically significant.
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3. Figure 2
Ultrastructural observations of effects of GLY (D–F), EG (G–I), and DMSO (J–L) on 2N (A, D, G, J), 4N (B, E, H, K), and 1N (C, F, I, L) in cryopreserved immature rat testicular tubules compared with fresh controls (A–C). Spermatogonia were well preserved with DMSO and EG (J, G), Spermatocytes with EG (H); synaptonemal complex structures were also observed (arrowheads in B, H). Spermatids were few in number and were well preserved with GLY and DMSO (F, L); distinct acrosomal structures were observed (arrows in C, F, I, L). Glycerol had a deleterious effect on cell morphology: membrane integrity was highly compromised (D, E). Magnification, ×9,300 in C, F, I, and L; for all other images, ×6,800.
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4. Figure 3
Characterization of cultured human testicular tissue after thaw. Maintenance of most germ cells, including proliferating spermatogonia (PCNA positive; arrows in B, E, H) and postmeiotic spermatids (TP1 positive; arrows in C, F, I) were observed with GLY (B, C), EG (E, F), and DMSO (H, I). Tubular architecture was maintained even after culture by EG (D) and DMSO (G). Extensive cell loss, except for mature sperm (arrowheads) was observed with GLY (A). Cell viability improved when cells were cultured as CS (J) compared with TUB (K). A drop in viability of DMSO-preserved cultured TUB was noted. Resumption of normal gene expression after culture was evident in DMSO and EG explants by high expression of integrin and SCP3 transcripts. Loss of most cell types in GLY explants may account for observed poor gene expression (L). Magnification, ×40.
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5. Supplementary Figure 1
Representative images of gating and ploidy analysis for murine (A, B) and human (C, D) testicular tissue by flow cytometry. After acquisition of total viability in log scale, cells were acquired in the linear scale. The first window depicts FL2-A plotted against FL2-W (A, C). For doublet discrimination gate R1 was placed on the plot, and cells within this gate were traced and their fluorescence levels determined in the corresponding histogram (B, D). The second window is a histogram that represents the number of cells at each fluorescence level (B, D). M1 corresponds to haploid population (1N), M2 to diploid population (2N), and M3 to tetraploid population (4N). Once established the gates and voltage settings were maintained constant throughout the study; for example, settings of A and B were used for all murine samples, and settings of C and D for all human samples.
Fertility and Sterility  , e1DOI: ( /j.fertnstert )
Copyright © 2012 American Society for Reproductive Medicine Terms and Conditions