Evaluation of the Gonadotoxicity of Cancer Therapies to Improve Counseling of Patients About Fertility and Fertility Preservation Measures: Protocol for a Retrospective Systematic Data Analysis and a Prospective Cohort Study.

BACKGROUND: Cytotoxic treatments such as chemo- and radiotherapy and immune therapies are required in cancer diseases. These therapies have the potential to cure patients but may also have an impact on gonadal function and, therefore, on fertility. Consequently, fertility preservation treatments such as freezing of gametes and gonadal tissue might be required. However, as detailed data about the necessity to perform fertility preservation treatment are very limited, this study was designed to fill this data gap. OBJECTIVE: Primary objective of this study is to analyze the impact of cancer therapies and chemotherapies on the ovarian reserve and sperm quality. Secondary objectives are to analyze the (1) impact of cancer therapies and chemotherapies on other fertility parameters and (2) probability of undergoing fertility preservation treatments in relation to specific cancer diseases and treatment protocols and the probability to use the frozen gametes and gonadal tissue to achieve pregnancies. METHODS: First, previously published studies on the gonadotoxicity of chemo- and radiotherapies among patients with cancer will be systematically analyzed. Second, a prospective cohort study set up by approximately 70 centers in Germany, Switzerland, and Austria will collect the following data: ovarian function by analyzing anti-Müllerian hormone (AMH) concentrations and testicular function by analyzing sperm parameters and total testosterone immediately before and around 1 year after gonadotoxic therapies (short-term fertility). A follow-up of these fertility parameters, including history of conceptions, will be performed 5 and 10 years after gonadotoxic therapies (long-term fertility). Additionally, the proportion of patients undergoing fertility-preserving procedures, their satisfaction with these procedures, and the amount of gametes and gonadal tissue and the children achieved by using the frozen material will be analyzed. Third, the data will be merged to create the internet-based data platform FertiTOX. The platform will be structured in accordance with the ICD (International Classification of Diseases) classification of cancer diseases and will be easily be accessible using a specific App. RESULTS: Several funding bodies have funded this study. Ten systematic reviews are in progress and the first one has been accepted for publication. All Swiss and many German and Austrian ethics committees have provided their approval for the prospective cohort study. The study registry has been set up, and a study website has been created. In total, 50 infertility centers have already been prepared for data collection, which started on December 1, 2023. CONCLUSIONS: The study can be expected to bridge the data gap regarding the gonadotoxicity of cancer therapies to better counsel patients about their infertility risk and their need to undergo fertility preservation procedures. Initial data are expected to be uploaded on the FertiTOX platform in 2026. TRIAL REGISTRATION: ClinicalTrials.gov NCT05885048; https://clinicaltrials.gov/study/NCT05885048. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): DERR1-10.2196/51145.

Isolation of Human Small Extracellular Vesicles and Tracking of their Uptake by Retinal Pigment Epithelial Cells In Vitro.

Small extracellular vesicles (EVs) are among the most frequently investigated EVs and play major roles in intercellular communication by delivering various cargo molecules to target cells. They could potentially represent an alternative delivery strategy to treat ocular toxoplasmosis, a parasitosis affecting the retinal pigment epithelium (RPE). To date, the uptake of human small EVs by RPE cells has never been reported. In this study, we report on the intracellular uptake of fluorescently labelled human urine and fibroblast-derived small EVs by human RPE cells. In summary, both dye-labelled urinary small EVs and small EVs obtained from fibroblasts stably expressing membrane-bound green fluorescent protein were successfully internalized by RPE cells as revealed by immunohistochemistry. In recipient ARPE19 cells, BODIPY-labelled small EVs were found in close vicinity to the parasite Toxoplasma gondii. Additionally, an ultrastructural method was enabled to distinguish between labelled exogenous and endogenous small EVs within target cells.

Slow conduction in mixed cultured strands of primary ventricular cells and stem cell-derived cardiomyocytes.

Modern concepts for the treatment of myocardial diseases focus on novel cell therapeutic strategies involving stem cell-derived cardiomyocytes (SCMs). However, functional integration of SCMs requires similar electrophysiological properties as primary cardiomyocytes (PCMs) and the ability to establish intercellular connections with host myocytes in order to contribute to the electrical and mechanical activity of the heart. The aim of this project was to investigate the properties of cardiac conduction in a co-culture approach using SCMs and PCMs in cultured cell strands. Murine embryonic SCMs were pooled with fetal ventricular cells and seeded in predefined proportions on microelectrode arrays to form patterned strands of mixed cells. Conduction velocity (CV) was measured during steady state pacing. SCM excitability was estimated from action potentials measured in single cells using the patch clamp technique. Experiments were complemented with computer simulations of conduction using a detailed model of cellular architecture in mixed cell strands. CV was significantly lower in strands composed purely of SCMs (5.5 ± 1.5 cm/s, n = 11) as compared to PCMs (34.9 ± 2.9 cm/s, n = 21) at similar refractoriness (100% SCMs: 122 ± 25 ms, n = 9; 100% PCMs: 139 ± 67 ms, n = 14). In mixed strands combining both cell types, CV was higher than in pure SCMs strands, but always lower than in 100% PCM strands. Computer simulations demonstrated that both intercellular coupling and electrical excitability limit CV. These data provide evidence that in cultures of murine ventricular cardiomyocytes, SCMs cannot restore CV to control levels resulting in slow conduction, which may lead to reentry circuits and arrhythmias.

Functional Characterization and Comparison of Intercellular Communication in Stem Cell-Derived Cardiomyocytes.

One novel treatment strategy for the diseased heart focuses on the use of pluripotent stem cell-derived cardiomyocytes (SC-CMs) to overcome the heart's innate deficiency for self-repair. However, targeted application of SC-CMs requires in-depth characterization of their true cardiogenic potential in terms of excitability and intercellular coupling at cellular level and in multicellular preparations. In this study, we elucidated the electrical characteristics of single SC-CMs and intercellular coupling quality of cell pairs, and concomitantly compared them with well-characterized murine native neonatal and immortalized HL-1 cardiomyocytes. Firstly, we investigated the electrical properties and Ca(2+) signaling mechanisms specific to cardiac contraction in single SC-CMs. Despite heterogeneity of the new cardiac cell population, their electrophysiological activity and Ca(2+) handling were similar to native cells. Secondly, we investigated the capability of paired SC-CMs to form an adequate subunit of a functional syncytium and analyzed gap junctions and signal transmission by dye transfer in cell pairs. We discovered significantly diminished coupling in SC-CMs compared with native cells, which could not be enhanced by a coculture approach combining SC-CMs and primary CMs. Moreover, quantitative and structural analysis of gap junctions presented significantly reduced connexin expression levels compared with native CMs. Strong dependence of intercellular coupling on gap junction density was further confirmed by computational simulations. These novel findings demonstrate that despite the cardiogenic electrophysiological profile, SC-CMs present significant limitations in intercellular communication. Inadequate coupling may severely impair functional integration and signal transmission, which needs to be carefully considered for the prospective use of SC-CMs in cardiac repair. Stem Cells 2015;33:2208-2218.

Simultaneous impedimetric and amperometric interrogation of renal cells exposed to a calculus-forming salt.

The complexity of the cellular response, induced even by the simplest experimental stimulus, requires an increased number of cellular parameters to be simultaneously monitored. An all electrochemical system allowing the simultaneous and real-time monitoring of both cell adherence and superoxide release into the extracellular space was developed to address this challenge. Cell adherence (to neighboring cells and to substrate) was monitored using non-faradaic impedance spectroscopy while the superoxide release was monitored using a cytochrome c-based amperometric biosensor. The system was used to observe for the first time how these two cellular parameters are changing in real-time for renal cells exposed to calcium oxalate, a calculus-forming salt. It was discovered that calcium oxalate crystals decrease cell adherence and in the same time induce oxidative stress by an overproduction of superoxide. Subconfluent cells, without fully developed tight junctions, appear to be more vulnerable than confluent cells with tight junctions indicating the important protective role of these junctions.

Effect of calcium oxalate on renal cells as revealed by real-time measurement of extracellular oxidative burst.

Calcium oxalate is one of the main constituents of kidney stones and has a proved deleterious effect on renal cells that is mediated by oxidative stress. However, the subcellular source of this oxidative stress, and whether it is extending to the extracellular space or not, is still disputed. Therefore, an electrochemical superoxide biosensor was constructed, positioned above A6 renal cells, and used to measure in real-time the extracellular oxidative burst following addition of calcium oxalate crystals. It was observed that A6 cells do secrete superoxide into their extracellular space in few minutes after encountering calcium oxalate crystals. The amount of released superoxide peaks at about 20 min. Superoxide is cleared away from the extracellular space after approximately 3h. Superoxide secretion depends on the presence of superoxide-scavenging enzyme superoxide dismutase, the age of the cells, the amount of calcium oxalate crystals, and the temperature. Moreover, the effect of calcium oxalate crystals was mimicked by phorbol 12-myristate 13-acetate. The developed sensing system can be a useful tool for biologists investigating nephrolithiasis at cellular level.