Needle immersed vitrification can lower the concentration of cryoprotectant in human ovarian tissue cryopreservation  Zhun Xiao, M.D., Yan Wang, Ph.D., Lei Li, Ph.D., Shan Luo, M.D., Shang-Wei Li, M.D.  Fertility and Sterility  Volume 94, Issue 6, Pages 2323-2328 (November 2010) DOI: 10.1016/j.fertnstert.2010.01.011 Copyright © 2010 American Society for Reproductive Medicine Terms and Conditions

Figure 1 Histologic analyses by hematoxylin-eosin and transmission electron microscopy before and after cryopreservation. (A) The morphologically normal primordial follicles consisting of intact oocytes and compact granulosa cells before cryopreservation (arrows). (B) Well preserved primordial follicles in group B (arrows). (C) The morphologically abnormal primordial follicles in the slow freezing group (arrows). Granulosa cells were strongly adhered to the shrunken and damaged oocyte. (A, B, and C: original magnification ×400; bar = 5 μm.) (D) The oocyte in primordial follicle from fresh tissue showing an abundance of mitochondria (mt) with normal arching cristae in the oocyte cytoplasm near the germinal vesicle (GV) membrane (×8,000; bar = 1 μm). (E) The oocyte in primordial follicle from group B, showing morphologically normal mitochondria in the oocyte cytoplasm (×12,000; bar = 1 μm). (F) The flat pregranulosa cells (PGC) in primordial follicle from fresh tissue, showing distinct cell membranes and nuclei with nuclear content normally distributed (×6,000; bar = 2 μm). (G) Poor preservation of the PGC in primordial follicle from the slow freezing group. The cytoplasm in PGC consisted of large areas devoid of organelles, and the mitochondria and ribosome were absent (×5,000; bar = 2 μm). (H) The stromal cells (SC) and the collagen bundles (CB) in group B, showing moderately dispersed nuclear chromatin and few small vacuoles (SV) in the cytoplasm. The intercellular space was slightly expanded (×10,000; bar =1 μm). (I) The presence of large vacuoles (LV) was more easily seen in the slow freezing group. The intercellular space expanded obviously. The SC showed markedly condensed nuclear chromatin, and the CB were disappeared (×8,000; bar = 1 μm). Fertility and Sterility 2010 94, 2323-2328DOI: (10.1016/j.fertnstert.2010.01.011) Copyright © 2010 American Society for Reproductive Medicine Terms and Conditions

Figure 2 Identification of apoptosis by TUNEL and in vitro culture of ovarian tissues. (A) In fresh tissue, the apoptosis was only observed in morphologically abnormal primordial follicle, which was characterized by pyknotic nuclei in follicular cells or irregular deformed shape of the oocyte (arrow). (B) In fresh tissue, apoptosis was not observed in morphologically normal primordial follicle (arrow). (C) In cryopreserved tissue, apoptosis was observed in morphologically normal primordial follicle (arrow). (D and E) Decreased apoptosis of stromal cells (TUNEL-positive areas of ovarian cortex) was observed in group B (D) compared with the slow freezing group (E). (F) The ovarian tissues in group B grew in in vitro culture system in 6 days. The ovarian tissue slices became round in shape. The stromal tissue began to outgrow from the verge of tissue. (G) The ovarian tissues in group B grew in in vitro culture system in 14 days. The stromal tissue clearly outgrew from the verge of tissue. (H) The ovarian tissues in the slow freezing group grew in in vitro culture system in 6 days. The ovarian tissue slices became round in shape. (I) The ovarian tissues in the slow freezing group grew in in vitro culture system in 14 days. The stromal tissue slowly outgrew from the verge of tissue. A, B, and C: magnification ×400; bar=20 μm; D and E: magnification ×40; bar=100 μm; F, G, H, and I: magnification ×50; bar =400 μm). Fertility and Sterility 2010 94, 2323-2328DOI: (10.1016/j.fertnstert.2010.01.011) Copyright © 2010 American Society for Reproductive Medicine Terms and Conditions

Figure 3 Evaluation of ovarian tissue damage by measuring the level of lactate dehydrogenase (LDH) in culture medium. Fertility and Sterility 2010 94, 2323-2328DOI: (10.1016/j.fertnstert.2010.01.011) Copyright © 2010 American Society for Reproductive Medicine Terms and Conditions