Light element distribution in fresh and frozen-thawed human ovarian tissues: a preliminary study

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 electron 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 of light elements, could be useful in establishing new cryopreservation protocols.

Keywords: Cryopreservation; Ovarian tissue; Slow freezing; Vacuolisation; X-ray fluorescence microscopy.