Confocal laser scanning microscopy analysis of bioenergetic potential and oxidative stress in fresh and frozen-thawed human ovarian tissue from oncologic.

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Confocal laser scanning microscopy analysis of bioenergetic potential and oxidative stress in fresh and frozen-thawed human ovarian tissue from oncologic patients Raffaella Fabbri, B.Sc., Rossella Vicenti, B.Sc., Nicola Antonio Martino, Ph.D., Maria Elena Dell'Aquila, Ph.D., Gianandrea Pasquinelli, M.D., Maria Macciocca, Ph.D., Valentina Magnani, Ph.D., Roberto Paradisi, M.D., Stefano Venturoli, M.D. Fertility and Sterility Volume 101, Issue 3, Pages 795-804.e1 (March 2014) DOI: 10.1016/j.fertnstert.2013.11.032 Copyright © 2014 American Society for Reproductive Medicine Terms and Conditions

Figure 1 Light microscopy of ovarian cortex showing fresh (A, G, O) and frozen-thawed (B, H, P) follicles and stroma. In group A, the oocytes of fresh (A) and frozen-thawed (B) samples showed increased chromatin density, cytoplasmic vacuolization, and detachment from surrounding follicular cells. The stroma displayed interstitial edema, marked chromatin clumping, and widespread vacuolization both in fresh (A) and frozen-thawed (B) tissue. In groups B and C, the follicular cells showed normal morphologic characteristics and the oocytes displayed round nuclei with lightly stained chromatin and perinuclear mitochondrial aggregates in fresh (G, H) and frozen-thawed (O, P) samples. The stromal cells showed regular nuclei without chromatin clumping and interstitial edema in all fresh (G, H) and frozen-thawed (O, P) samples. Transmission electron microscopy of ovarian tissue showing fresh (C, E, I, M, Q, S) and frozen-thawed (D, F, L, N, R, T) follicles and stroma. In group A, the follicles appeared dysmorphic with increased chromatin clumping in the oocyte and granulosa cell nucleus, cytoplasmic vacuolization, loss of cytoplasm organelles, and oocyte detachment from surrounding follicular cells in fresh (C) and frozen-thawed tissue (D). The stromal cells had pyknotic nuclei, abundant cytoplasm fragmentation, marked interstitial edema, cytoplasmic vacuolization with loss of intracellular organelles in fresh (E) and frozen-thawed (F) tissue. In group B and C, the oocytes and granulosa cells showed regularly shaped nuclei with nucleoli and finely dispersed chromatin; the cytoplasm contained perinuclear aggregates of mitochondria. The oocytes were tightly adherent to the surrounding flat or rounded granulosa cells with the usual cytoplasmic organelles in fresh (I, Q) and in frozen-thawed (L, R) samples. Stromal cells had oval shaped nuclei without chromatin clumping, presence of small mitochondria and moderate fragmentation in the cytoplasm and slight interstitial edema in fresh (M, S) and frozen-thawed (N, T) samples. Fertility and Sterility 2014 101, 795-804.e1DOI: (10.1016/j.fertnstert.2013.11.032) Copyright © 2014 American Society for Reproductive Medicine Terms and Conditions

Figure 2 Tissue light microscopy (LM)-scored as severely damaged showed heavy functional damage after freezing/thawing with significant fluorescence intensity reduction in MitoTracker Orange CMTMRos (A, B) and 2′,7′-dichlorodihydrofluorescein diacetate (DCF) (C, D) both in fresh (A, C) and frozen-thawed (B, D) tissue. Slightly damaged LM-scored samples showed significant fluorescence intensity increase in MitoTracker Orange CMTMRos (E, F) and DCF (G, H) both in fresh (E, G) and frozen-thawed (F, H) tissue. Tissue LM-scored as unaffected by cryopreservation showed no significant fluorescent variation in MitoTracker Orange CMTMRos (I, L) and DCF (M, N) both in fresh (I, M) and frozen-thawed (L, N) tissue. Fertility and Sterility 2014 101, 795-804.e1DOI: (10.1016/j.fertnstert.2013.11.032) Copyright © 2014 American Society for Reproductive Medicine Terms and Conditions

Supplemental Figure 1 Confocal laser scanning microscopy of ovarian cortex showing fresh (A, C, E) and frozen-thawed (B, D, F) follicles. The oocytes showed small mitochondria aggregates localized in the pericortical region both in fresh (A) and cryopreserved (B) samples. The reactive oxygen species signal indicated a low level of hydrogen peroxide in fresh (C) and cryopreserved samples (D). Merge images revealed that reactive oxygen species colocalize with mitochondria and the damage level was very low both in fresh (E) and frozen-thawed (F) samples. Fertility and Sterility 2014 101, 795-804.e1DOI: (10.1016/j.fertnstert.2013.11.032) Copyright © 2014 American Society for Reproductive Medicine Terms and Conditions