Article Light element distribution in fresh and frozen–thawed human ovarian tissues: a preliminary study Lorella Pascolo a, *, Irene Venturin b , Alessandra Gianoncelli c , Roberta Bortul b , Gabriella Zito a , Elena Giolo a , Murielle Salomé d , Diana E Bedolla c , Matteo Altissimo c , Marina Zweyer b , Giuseppe Ricci a,b a Institute for Maternal and Child Health, IRCCS Burlo Garofolo, 34137 Trieste, Italy b Department of Medical, Surgical, and Health Sciences, University of Trieste, 34149 Trieste, Italy c Elettra-Sincrotrone Trieste S.C.p.A., Strada Statale SS14, km 163.5, Basovizza 34149, Italy d European Synchrotron Radiation Facility, 38000 Cedex 9 Grenoble, France Lorella Pascolo obtained her PhD in biochemistry from the University of Trieste. She has published more than 50 international scientific articles and is currently working as researcher at the Institute IRCCS Burlo Garofolo (Trieste). Her research interests are related to the application of advanced synchrotron techniques in clinical contests, particularly toxicology and reproductive medicine. KEY MESSAGE This study describes novel synchrotron-based X-ray fluorescence analyses in human ovarian tissues and reports on the utility of monitoring light element distribution in these samples to reveal the tissue integrity and oocyte quality. For the first time, the light element composition of ooplasmic vacuoles is identified. ABSTRACT Research question: Does synchrotron X-ray fluorescence (XRF) provide novel chemical information for the evaluation of human ovarian tissue cryopreservation protocols? Design: Tissues from five patients undergoing laparoscopic surgery for benign gynaecological conditions were fixed for microscopic analysis either immediately or after cryopreservation. After fixation, fresh and slowly frozen samples were selected by light microscopy and transmission electron microscopy, and subsequently analysed with synchrotron XRF microscopy at different incident energies. Results: The distributions of elements detected at 7.3 keV (S, P, K, Cl, Fe, and Os) and 1.5 keV (Na and Mg) were related to the changes revealed by light microscopy and transmission electron microscopy analyses. The light elements showed highly informative findings. The S distribution was found to be an indicator of extracellular component changes in the stromal tissues of the freeze–stored samples, further revealed by the transmission elec- tron microscopy analyses. Low-quality follicles, frequent in the freeze–thawed tissues, showed a high Na level in the ooplasm. On the contrary, good- quality follicles were detected by a homogeneous Cl distribution. The occurrence of vacuolated follicles increased after cryopreservation, and the XRF analyses showed that the vacuolar structures contained mainly Cl and Na. Conclusions: The study demonstrates that elemental imaging techniques, particularly revealing the distribution light elements, could be useful in es- tablishing new cryopreservation protocols. © 2018 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved. * Corresponding author. E-mail address: lorella.pascolo@gmail.com (L Pascolo). https://doi.org/10.1016/j.rbmo.2018.04.051 1472-6483/© 2018 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved. ARTICLE IN PRESS Please cite this article in press as: Lorella Pascolo, Irene Venturin, Alessandra Gianoncelli, Roberta Bortul, Gabriella Zito, Elena Giolo, Murielle Salomé, Diana E. Bedolla, MatteoAltissimo, Marina Zweyer, Giuseppe Ricci, Light element distribution in fresh and frozen–thawed human ovarian tissues: a preliminary study, Reproductive BioMedicine Online (2018), doi: 10.1016/j.rbmo.2018.04.051 Q2 Q1 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46