Cryopreservation of prepubertal mouse testicular tissue by vitrification  Mara Curaba, M.Sc., Magali Verleysen, M.D., Christiani Andrade Amorim, V.M.D., Ph.D., Marie-Madeleine Dolmans, M.D., Ph.D., Anne Van Langendonckt, Ph.D., Outi Hovatta, M.D., Ph.D., Christine Wyns, M.D., Ph.D., Jacques Donnez, M.D., Ph.D.  Fertility and Sterility  Volume 95, Issue 4, Pages 1229-1234.e1 (March 2011) DOI: 10.1016/j.fertnstert.2010.04.062 Copyright © 2011 American Society for Reproductive Medicine Terms and Conditions

Figure 1 (A) Number of apoptotic cells per tubule after 1 day of culture of vitrified (V D1) and slow-frozen (SF D1) tissue compared with fresh culture (FR D1) and noncultured controls (FR Ctrl). Log-transformed data are expressed as medians and percentiles (P25 and P75). N = 5 mice per group. aSignificantly different from Ctrl, P<.01; bsignificantly different from Ctrl, P<.05; and csignificantly different from FR and SF, P<.01. (B) Immunostaining of active caspase-3 apoptotic marker in prepubertal testicular tissue after 1 day of culture of vitrified (V D1) and slow-frozen (SF D1) tissue compared with fresh culture (FR D1) and noncultured controls (FR Ctrl). Human tonsils were used as positive controls (T Ctrl+). Negative control sections were processed by incubation with nonspecific IgG (T Ctrl-). Original magnification, ×200; bar = 50 μm. Fertility and Sterility 2011 95, 1229-1234.e1DOI: (10.1016/j.fertnstert.2010.04.062) Copyright © 2011 American Society for Reproductive Medicine Terms and Conditions

Figure 2 (A) Percentage of proliferative tubular cells in prepubertal testicular tissue after vitrification (V) and slow-freezing (SF); comparison with fresh cultured controls (FR) after 1 (D1) and 3 (D3) days of culture and fresh noncultured controls (FR Ctrl). N = 5 mice per group. Data are expressed as mean ± SD. aSignificantly different from Ctrl, P<.05; bsignificantly different from FR on D1, P<.01; and csignificantly different from FR on D3, P<.01. (B) Ki67 immunostaining after 3 days of in vitro culture of fresh (FR D3), slow-frozen (SF D3), and vitrified (V D3) testicular tissue. Fresh prepubertal mouse testicular tissue was used as a positive control. Negative control sections were processed by incubation with nonspecific IgG. Magnification, ×200; bar = 50 μm. Fertility and Sterility 2011 95, 1229-1234.e1DOI: (10.1016/j.fertnstert.2010.04.062) Copyright © 2011 American Society for Reproductive Medicine Terms and Conditions

Figure 3 (A) Proportion of seminiferous tubules with good (score ≥5), medium (score = 4), and poor (score ≤3) integrity after 3 (D3) days of in vitro culture of vitrified (V) and slow-frozen (SF) tissue compared with fresh culture (FR) and noncultured controls (FR Ctrl). Median values are shown for five mice per group. (B) Histology of seminiferous tubules cultured for 3 days after slow-freezing (SF) and vitrification (V) compared with fresh cultured controls (FR). Tissues were fixed in Bouin's solution and stained with hematoxylin-eosin (H & E). Magnification, ×400; bar = 25 μm. Fertility and Sterility 2011 95, 1229-1234.e1DOI: (10.1016/j.fertnstert.2010.04.062) Copyright © 2011 American Society for Reproductive Medicine Terms and Conditions

Experimental design comparing outcomes of vitrification and slow-freezing methods to cryopreserve prepubertal mouse testicular tissue. Three experimental groups were compared: fresh tissue, slow-frozen/thawed tissue, and vitrified/warmed tissue. For each group, testicular tissue was collected from 10 mice. One testis per mouse was used for viability testing. The other testis was processed for short-term organotypic culture (1 [D1] or 3 [D3] days) (n = 5 mice) or fixed for noncultured controls (4% buffered formalin or Bouin's solution) (n = 5 mice). End points assessed after each treatment are shown in Table 1. Ctrl = control; LDH = lactate dehydrogenase; IHC = immunohistochemistry; LM = light microscopy. Fertility and Sterility 2011 95, 1229-1234.e1DOI: (10.1016/j.fertnstert.2010.04.062) Copyright © 2011 American Society for Reproductive Medicine Terms and Conditions