Optimization of protocols for human ovarian tissue cryopreservation with sucrose, 1,2- propanediol and human serum  Raffaella Fabbri, Gianandrea Pasquinelli, Declan Keane, Valentina Magnani, Roberto Paradisi, Stefano Venturoli  Reproductive BioMedicine Online  Volume 21, Issue 6, Pages 819-828 (December 2010) DOI: 10.1016/j.rbmo.2010.07.008 Copyright © 2010 Reproductive Healthcare Ltd. Terms and Conditions

Figure 1 Flow-chart of study design. Reproductive BioMedicine Online 2010 21, 819-828DOI: (10.1016/j.rbmo.2010.07.008) Copyright © 2010 Reproductive Healthcare Ltd. Terms and Conditions

Figure 2 Statistical analysis, showing scores for (A) follicle number per high power field, (B) follicle preservation, (C) stroma preservation, and (D) follicle and stroma preservation in fresh and frozen–thawed ovarian cortical tissue. Values are expressed as mean±SD. Score interpretation for A: 0=0 follicles, 1=1–5 follicles, 2=6–10 follicles, 3=more than 10 follicles; follicle number was not significantly different among the samples analysed. Score interpretation for B–D: 0=good preservation, 1=poor preservation, 2=bad preservation, 3=worst preservation. Follicle preservation seemed to be better with B, L1 and M2 freezing–thawing solutions; procedure O1 was statistically significantly worse (P<0.01) when compared with fresh samples (B). Stromal preservation seemed to be better with procedure B; procedures O1, O2, P2, Q1 and Q2 were statistically significantly worse (P<0.05) as compared with fresh samples (C). Together follicle and stroma preservation seemed to be better in samples cryopreserved in solutions B, L1, M2 and P1. The other categories were statistically significantly worse (P<0.05) as compared with fresh control samples (D). For definitions of the freezing and dilution solution categories, see Table 1. F=fresh control samples. * indicates a statistically significant difference compared with fresh control. Reproductive BioMedicine Online 2010 21, 819-828DOI: (10.1016/j.rbmo.2010.07.008) Copyright © 2010 Reproductive Healthcare Ltd. Terms and Conditions

Figure 3 Light microscopy of ovarian cortex showing (A) fresh and (B–F) frozen–thawed follicles. (A) Fresh follicles showed a round nucleus, dispersed chromatin, mitochondria at the nuclear periphery and follicular cells of healthy appearance. (B) Frozen–thawed follicles with normal morphological appearance from procedure B, comparable to that seen in fresh samples (groups B, L1, M2). (C) Frozen–thawed follicles with cytoplasmic vacuoles (v) from group L2. (D) Follicular cell vacuolization (arrows) from group O2. (E) Follicular cell shrinkage (arrowhead) (groups L2, M1, N, O, P, Q) from P1. (F) Example of a ballooned follicle (asterisk) from group Q2. For definitions of the freezing and dilution solution categories, see Table 1. Bar=50μm. Reproductive BioMedicine Online 2010 21, 819-828DOI: (10.1016/j.rbmo.2010.07.008) Copyright © 2010 Reproductive Healthcare Ltd. Terms and Conditions

Figure 4 Light microscopy of ovarian cortex showing (A) fresh and (B–F) frozen–thawed stromal cells. Stromal cells treated with freezing–thawing solution B were moderately damaged (B) when compared with fresh samples (A) and greatly damaged by all the other procedures. Interstitial oedema and nuclear pyknosis were moderate in groups M, N (C) (group M1 is shown), patchy in groups L, P1 (D) (group L2 is shown) and widespread and marked in groups O, P2, Q (E and F) (group P2 is shown in E and group Q1 is shown in F). Follicle damage reflects the degree of stromal injury (asterisk). For definitions of the freezing and dilution solution categories, see Table 1. Bar=100μm. Reproductive BioMedicine Online 2010 21, 819-828DOI: (10.1016/j.rbmo.2010.07.008) Copyright © 2010 Reproductive Healthcare Ltd. Terms and Conditions

Figure 5 Transmission electron microscopy of ovarian cortex showing follicles and stromal cells from fresh non-cryopreserved control group (A and B), group cryopreserved in freezing-dilution solution B (C and D), and two other freezing/dilution solutions (P2 and Q, respectively) (E and F). (A) Fresh ovarian tissue showing a well-preserved oocyte, with nucleus (N) centrally placed in the cell, regularly shaped and with the membrane intact. Mitochondria (m) were located around the nucleus and had a matrix of intermediate density. The follicles were surrounded by stromal cells (S). (B) Fresh ovarian cortex with stromal cells with moderately dispersed chromatin and delicate long and slender cytoplasmic projection; with occasional perinuclear vacuoles (arrows). (C) Tissue cryopreserved with protocol B primordial follicles showing ultrastructural features comparable to those observed in fresh non-cryopreserved control group. (D) Stromal cells with peripheral chromatin condensation (arrows); some cells retained their native ultrastructural morphology. (E) Freezing/dilution solutions other than group B yielded primordial follicles (asterisk) with fairly well-preserved oocytes (O) and follicular cells (F) with minimal areas of injury (clear vacuoles) embedded in markedly damaged stroma (S). (F) Stroma damage was characterized by marked interstitial oedema (asterisk), and stromal cells with pycnotic nuclei and disrupted cytoplasm. For definitions of the freezing and dilution solution categories, see Table 1. Bar=10μm. Reproductive BioMedicine Online 2010 21, 819-828DOI: (10.1016/j.rbmo.2010.07.008) Copyright © 2010 Reproductive Healthcare Ltd. Terms and Conditions