Ultrastructural deformity of ovarian follicles induced by different cryopreservation protocols  Gil Ah Kim, M.S., Hwa Young Kim, B.Sc., Jee Woong Kim, M.S., Gene Lee, D.D.S., Ph.D., Eunsong Lee, D.V.M., Ph.D., Jeong Mook Lim, D.V.M., Ph.D.  Fertility and Sterility  Volume 94, Issue 4, Pages 1548-1550.e1 (September 2010) DOI: 10.1016/j.fertnstert.2009.12.029 Copyright © 2010 American Society for Reproductive Medicine Terms and Conditions

Figure 1 Ultrastructure of preantral follicles derived from intact ovaries (A, E), and thawed ovaries that had been frozen slowly (B–D) or vitrified (F–H). A comparison of injuries seen in cryopreserved and fresh tissues (I–L). In fresh ovaries (A, E), an oocyte (O), flattened or cuboidal granulosa cells (GC), theca cells (TC), the zona pellucida (ZP), and the basement membrane (BM) of both preantral follicles and follicular cells can be seen. Mutivesicular bodies (MB) and the nuclear envelop (NE) are visible, and organelles, such as smooth endoplasmic reticulum (SER), were also observed in ooplasm. There are clusters of undamaged mitochondria (white arrow). In the thawed ovaries that had been frozen slowly (B–D) or vitrified (F–H), numerous intracytoplasmic vacuoles (V) derived from ice crystals can be seen. The basement membranes of granulosa and theca cells were damaged (white arrowhead), and both undamaged and collapsed mitochondria (black arrow) were observed. Numerous vacuoles and debris (D) were observed inside the zona pellucida. L = lacunae; LB = lamellar body; Lip = lipid; MV = microvillus; N = nucleus; PVS = perivitelline space. Magnification: ×4,000 (A), ×4,000 (B), ×25,000 (C), ×50,000 (D), ×12,000 (E), ×4,000 (F), ×18,000 (G), ×50,000 (H). For numerical analysis of the cryodamage, the mean number of degenerated mitochondria (MT) per 100 μm2 of a follicular cell (FC) (I), mean number of degenerated MT per 100 μm2 of ooplasm (J), mean size of vacuoles per 100 μm2 of ooplasm (K), and mean number of vacuoles per 1 μm2 of ZP (I) were counted and compared among the vitrified–warmed, slow frozen–thawed, and fresh follicles. Data are shown as mean ± SD. жP<.0001, ΨP<.05 between two comparison groups. Fertility and Sterility 2010 94, 1548-1550.e1DOI: (10.1016/j.fertnstert.2009.12.029) Copyright © 2010 American Society for Reproductive Medicine Terms and Conditions

Morphology of the ovaries cryopreserved with a slow freezing or a vitrification protocol. Section of the paraffin embedded intact ovaries (A, D and G), or ovaries cryopreserved with a slow freezing (B, E, H, J) and a vitrification (C, F, I, K) method were stained with haematoxylin and eosin. Cryodamage such as shrunk (E and F) or deformed (H and I) cytoplasm of intrafollicular oocytes, vacuoles in ovarian stromal tissue (K) and dissociation of follicular cells from basement membrane of preantral follicles (J and K) were prominent in cryopreserved follicles, regardless of freezing methods. Scale bar = 50 μm. Fertility and Sterility 2010 94, 1548-1550.e1DOI: (10.1016/j.fertnstert.2009.12.029) Copyright © 2010 American Society for Reproductive Medicine Terms and Conditions