1. 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 e1 (March 2011)
DOI: /j.fertnstert
Copyright © 2011 American Society for Reproductive Medicine Terms and Conditions

2. 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  , e1DOI: ( /j.fertnstert )
Copyright © 2011 American Society for Reproductive Medicine Terms and Conditions

3. 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  , e1DOI: ( /j.fertnstert )
Copyright © 2011 American Society for Reproductive Medicine Terms and Conditions

4. 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  , e1DOI: ( /j.fertnstert )
Copyright © 2011 American Society for Reproductive Medicine Terms and Conditions

5. 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  , e1DOI: ( /j.fertnstert )
Copyright © 2011 American Society for Reproductive Medicine Terms and Conditions