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Abstract:
In patients with testicular germ cell tumours (TGCT), sperm cryopreservation prior to anti-cancer treatment represents the main fertility preservation approach. However, it is associated with a 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 microscopy (2P-FLIM), in order to assess the contributions of OXPHOS and glycolysis to energy provision. A novel protocol for the 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 to OXPHOS. The negative effect of cryopreservation was signified by the deterioration of both bioenergetic pathways represented by modified OCR and ECAR curves and the derived parameters. This was true for normozoospermic as well as samples from TGCT patients, which showed even 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 insights into the impact of TGCT on sperm bioenergetics and delivers a verified protocol to be used for the assessment of human sperm metabolic activity, which can be a valuable tool for further research and clinical andrology.

Keywords:
TGCT, spermatozoa, sperm biochemistry, sperm function, energetic metabolism, oxidative phosphorylation, XF Analyzer, 2P-FLIM, infertility, cancer

Abstract:
The long-term storage of boar sperm presents an ongoing challenge, and the modification of the cryoprotective compounds in semen extenders is crucial for improving cryopreservation’s success rate. The aim of our study was to reduce the percentage of glycerol in the extender by elimination or substitution with biocompatible, non-toxic polysaccharides. For boar semen extender improvement, we tested a novel modification with the polysaccharides dextran and pentaisomaltose in combination with unique in silico predictive modeling. We targeted the analysis of in vitro qualitative sperm parameters such as motility, viability, mitochondrial activity, acrosome integrity, and DNA integrity. Non-penetrating polysaccharide-based cryoprotective agents interact with sperm surface proteins such as spermadhesins, which are recognized as fertility markers of boar sperm quality. The in silico docking study showed a moderate binding affinity of dextran and pentaisomaltose toward one specific spermadhesin known as AWN, which is located in the sperm plasma membrane. Pentaisomaltose formed a hydrophobic pocket for the AWN protein, and the higher energy of this protein–ligand complex compared with dextran was calculated. In addition, the root mean square deviation (RMSD) analysis for the molecular dynamics (MD) of both polysaccharides and AWN simulation suggests their interaction was highly stable. The in silico results were supported by in vitro experiments. In the experimental groups where glycerol was partially or entirely substituted, the use of pentaisomaltose resulted in improved sperm mitochondrial activity and DNA integrity after thawing when compared with dextran. In this paper, we demonstrate that pentaisomaltose, previously used for cryopreservation in hematopoietic stem cells, represents a promising compound for the elimination or reduction of glycerol in extenders for boar semen cryopreservation. This novel approach, using in silico computer prediction and in vitro testing, represents a promising technique to help identify new cryoprotectants for use in animal breeding or genetic resource programs.

Keywords:
cryopreservation, boar sperm, dextran, pentaisomaltose, polysaccharide, reproduction, glycerol, AWN spermadhesin