Problem This study aims to address sperm reproductive parameters and testis germ cell pathologies related to testicular germ cell tumour (TGCT) from both the clinical and experimental angle. The goal is to deliver novel diagnostic strategies for the evaluation of sperm samples collected from TGCT men prior to surgery and after the therapeutic intervention, in order to monitor and compare the quality of fertility parameters and recommend the first choice of cryopreserved semen sample for assisted reproduction.

Methods of study Combination of Ki67, a marker of cell proliferation was newly used for TGCT severity assessment accompanied by Hematoxylin-eosin-histopathological assessment. Epigenetic profiling and analysis of sperm and testicular germ cell epigenome was addressed through monitoring of H3K9ac, H3K36me3 and n H3K27me3 pattern. Assessment of tumour progression and sperm quality parameters such as acrosome integrity (PNA lectin), DNA integrity (TUNEL), motility (CASA) and apoptosis(ApoFlowEx) were addressed. Assessment of testicular and sperm mitochondrial activity was evaluated by Oxygen consumption by complex I and complex II. The 2-photon FLIM was performed to assess NAD(P)H lifetime in mitochondria.

Results Sperm analyses of acrosome integrity and sperm motility provided detailed analysis of impaired sperm function which corelates with elevated DNA fragmentation. Sperm parameters corelated with severity of the TGCT tumour assessed by Ki67 marker. The epigenetic profile of sperm is severely altered and Multi-parametric correlation of TGCT sperm pathology assessment is in needed. Oxygen consumption by mitochondrial complex I and complex II, (CI, CII) revealed that CI and CII dependent respiration is higher in tumour than normal tissue. A high heterogeneity in the TGCT tissue for NAD(P)H was discovered, and patients specific unique lifetimes appeared, ranging between 0-5 ns, indicating differences in CI-dependent respiration.

Conclusions Using a Ki67 proliferation marker is a promising method for assessment of severity of TGCT.Histone modification assessment in testicular tissue and sperm shows differences between tumour and nontumour tissue. Total and progressive sperm motility by CASA points to the best sperm quality in the sample prior to 1st and 2nd chemotherapy. Assessment of acrosome damage is crucial for prediction of optimal IVF method, and corelates with DNA fragmentation. The altered mitochondrial activity in TGCT patients’sperm and testicular tissue compared to sperm of healthy donors and non-tumour tissue points to potential use of mitochondrial markers as a diagnostic tool of reproductive parameters of TGCT affected men.