Abstract

In testicular germ cell tumour (TGCT) patients, sperm cryopreservation prior to anti-cancer treatment represents the main fertility preservation approach. However, it is associated with low sperm recovery rate after thawing. Since sperm is a high-energy demanding cell, which is supplied by glycolysis and oxidative phosphorylation (OXPHOS), mitochondrial dysfunctionality can directly result in sperm anomalies. In this study, we investigated the bioenergetic pattern of cryopreserved sperm of TGCT patients in comparison with normozoospermic samples using two state-of-the-art methods; the Extracellular Flux Analyzer (XF Analyzer) and Two-Photon Fluorescence Lifetime imaging (2P-FLIM), in order to assess the contributions of OXPHOS and glycolysis to energy provision. A novel protocol for combined measurement of OXPHOS (Oxygen Consumption Rate - OCR) and glycolysis (Extracellular Acidification Rate - ECAR) using the XF Analyzer was developed together with a unique customized AI-based approach for semiautomated processing of 2P-FLIM images. Our study delivers optimized Low-HEPES modified Human Tubal Fluid media (mHTF) for sperm handling during pre-analytical and analytical phases to maintain sperm physiological parameters and optimal OCR, equivalent of OXPHOS. The negative effect of cryopreservation was signified by deterioration of both bioenergetic pathways represented by modified OCR and ECAR curves and the derived parameters. This was true for normozoospermic as well as TGCT samples, which showed even a stronger damage within the respiratory chain compared to the level of glycolytic activity impairment. The impact of cryopreservation and pathology are supported by 2P-FLIM analysis showing a significant decrease in bound NADH in contrast to unbound NAD(P)H which reflects decreased metabolic activity in samples from TGCT patients. Our study provides novel insight into the impact of TGCT on sperm bioenergetics and delivers a verified protocol to be used for assessment of human sperm metabolic activity, which can be a valuable tool for further research and clinical andrology. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=182 SRC="FIGDIR/small/595824v2_ufig1.gif" ALT="Figure 1"> View larger version (47K): org.highwire.dtl.DTLVardef@130b9eeorg.highwire.dtl.DTLVardef@1fec841org.highwire.dtl.DTLVardef@d401forg.highwire.dtl.DTLVardef@1eccff8_HPS_FORMAT_FIGEXP M_FIG C_FIG