Glucose/lactate metabolism of cryopreserved intact bovine ovaries as a novel quantitative marker to assess tissue cryodamage R. Gerritse, C.C.M. Beerendonk,

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Glucose/lactate metabolism of cryopreserved intact bovine ovaries as a novel quantitative marker to assess tissue cryodamage R. Gerritse, C.C.M. Beerendonk, J.R. Westphal, L. Bastings, D.D.M. Braat, R. Peek Reproductive BioMedicine Online Volume 23, Issue 6, Pages 755-764 (December 2011) DOI: 10.1016/j.rbmo.2011.08.008 Copyright © 2011 Reproductive Healthcare Ltd. Terms and Conditions

Figure 1 Preparation of ovarian fragments from different tissue layers. Tissue rods (shown at the bottom) were obtained by taking end-to-end punch biopsies from the ovary, with a diameter of 6mm. Next, tissue rods were divided into two cortical, two subcortical and two medullar fragments of comparable length (2–3mm). Reproductive BioMedicine Online 2011 23, 755-764DOI: (10.1016/j.rbmo.2011.08.008) Copyright © 2011 Reproductive Healthcare Ltd. Terms and Conditions

Figure 2 (a) Schematic representation of the different temperature monitoring points of an ovary frozen in a passive cooling device. (b) Temperature kinetics at the monitoring points indicated in (a), shown at 4-min intervals. DMSO = dimethylsulphoxide. Reproductive BioMedicine Online 2011 23, 755-764DOI: (10.1016/j.rbmo.2011.08.008) Copyright © 2011 Reproductive Healthcare Ltd. Terms and Conditions

Figure 3 Glucose uptake (a) and lactate production (b) of cultured ovarian fragments derived from cortex, subcortex and medulla. Glucose/lactate metabolism was determined at two different time points of culture; days 1–4 and days 5–7. Condition A: fresh (non-cryopreserved) tissue; condition B: tissue frozen–thawed without cryoprotective measures; condition C: tissue frozen–thawed after submersion in DMSO for 15min; condition D: tissue frozen–thawed after submersion in DMSO for 180min. Values are mean and SD. Numbers above bars represent the number of ovaries analysed per condition. +=treatment applied; −=treatment not applied; NA=not applicable. Reproductive BioMedicine Online 2011 23, 755-764DOI: (10.1016/j.rbmo.2011.08.008) Copyright © 2011 Reproductive Healthcare Ltd. Terms and Conditions

Figure 4 Glucose uptake (a) and lactate production (b) of cultured ovarian fragments derived from cortex, subcortex and medulla. Glucose/lactate metabolism was determined at two different time points of culture; days 1–4 and days 5–7. Condition A: fresh (non-cryopreserved) tissue; condition E: tissue frozen–thawed after 30min perfusion, without submersion; condition F: tissue frozen–thawed after submersion in, and perfusion with, DMSO for 5min; condition G: tissue frozen–thawed after submersion in and perfusion with DMSO for 30min. Values are mean and SD. Numbers above bars represent the number of ovaries analysed per condition. +=treatment applied; −=treatment not applied; NA=not applicable. Reproductive BioMedicine Online 2011 23, 755-764DOI: (10.1016/j.rbmo.2011.08.008) Copyright © 2011 Reproductive Healthcare Ltd. Terms and Conditions

Figure 5 Haematoxylin–eosin-stained ovarian medullar tissue fragments derived from intact ovaries. (A) Tissue derived from unfrozen non-cultured ovary. (B–D) Fragments were cultured for 7 days. (B) Tissue derived from fresh (unfrozen) ovary; (C) tissue derived from a frozen–thawed ovary in the absence of DMSO; (D) tissue derived from a frozen–thawed ovary in the presence of DMSO. Some pycnotic cells are indicated by arrowheads. Bar=100μm. Reproductive BioMedicine Online 2011 23, 755-764DOI: (10.1016/j.rbmo.2011.08.008) Copyright © 2011 Reproductive Healthcare Ltd. Terms and Conditions