Effect of cooling on the quality of follicles (expressed as quantity and normality of follicles).

<p>Group 1: freezing of tissue without pre-cooling, then thawing and evaluation of their quality. Group 2: freezing of tissue after 24h pre-cooling to 5°C, then thawing and evaluation of their quality. Group 3: freezing of tissue without pre-cooling, then thawing, xenografting and evaluation of their quality. Group 4: freezing of tissue without pre-cooling, then thawing, xenografting and evaluation of their quality. No statistical differences between respective groups (Group 1 <i>vs</i> Group 2 and Group 3 <i>vs</i> Group 4 (P>0.1).</p

Xenografting of cryopreserved human ovarian tissue.

<p>(A) Ovarian pieces after thawing, (B, C, D) Transplantation of these pieces, (E, F) Ovarian pieces after 45 d xenografting. Scale bar: 2 mm.</p

Translocation of phosphatidylserine in ovarian tissue pre-cooled to 5°C for 24 h before the freezing and then xenografted in SCID mice: representative example of one experiment.

<p>(a, b, c, d) Group 1: pieces were frozen immediately after operation, thawed and just after thawing their quality was FACS analyzed, (e, f, g, h) Group 2: pieces after operation were cooled to 5°C for 24 h, then frozen, thawed and just after thawing their quality was FACS analyzed, (i, j, k, l), pieces were frozen immediately after operation, without pre-cooling, thawed, transplanted to SCID mice and then, after 45 d of culture their quality was FACS analyzed. (m, n, o, p) pieces were frozen after 24 h pre-cooling to 5°C, thawed, transplanted to SCID mice and then, after 45 d their quality was FACS analyzed, (a, e, i, m) forward and scatter dot plot used to select the interest population, (c, g, k, o) histograms displaying fluorescence of PE channel, used to measure fluorescence intensity of Propidium Iodide (PI), (d, h, i, p) dot plot analysis of FITC-Annexin V and PE channels, (Q1) cells negative to Annexin V (FITC A) and positive to PI (could indicate necrotic cells), (Q2) cells positive to both Annexin V and PI (could indicate late apoptotic stage), (Q3) cells negative for both Annexin V and PI (could indicate viable cells), (Q4) cells positive to FITC-Annexin V and negative to PI (could indicate early apoptotic state).</p

Cryopreserved ovarian medulla-free and medulla-containing pieces before and after 5 days culture with chorioallantoic membrane (CAM) system.

<p>(a, b, c, d, e) medulla-free piece, (a, b) just after thawing and seeding on CAM marked by silicone ring, (c, d, e) the same piece after culture, (c) piece on CAM, (d, e) piece in Petri dish; (f, g, h, i, j) medulla-containing piece, (f, g) just after thawing and seeding on CAM marked by silicone ring, (h, i, j) the same piece after culture, (c) piece on CAM, (d, e) piece in Petri dish; (e) outer CAM-layer with medulla-free piece, (j) inner CAM-layer with medulla-containing piece. Different intensiveness of the avian vascularisation in the place of the seeding of pieces was noted: (e) versus (j). Bar = 1 mm.</p

Vascularisation (von Willebrand factor expression) in cryopreserved medulla-containing ovarian tissue after in vitro and CAM-culture.

<p>Immonostained after in vitro (a, A) and CAM (b, B) culture. Bar = 0.25 mm.</p

Cryopreserved ovarian medulla-free and medulla-containing ovarian pieces after thawing and 8 days in vitro culture.

<p>(a, b) medulla-free piece, (a) just after thawing, (b) the same piece after culture, (c, d) medulla-containing piece, (c) just after thawing, (d) the same piece after culture. Bar = 1 mm.</p

Quality of follicles (expressed as quantity and percentage of normal follicles) after in vitro and CAM-culture of cryopreserved ovarian pieces.

<p>Respective rates are not statistical different (P>0.1).</p