Anti-CTLA-and Anti-PD-1 Immune Checkpoint Inhibitor Antibodies Impair Human Sperm Motility

Ilaria Cosci ¹ Luca De Toni ¹ Paolo Del Fiore ² Andrea Di Nisio ³ Samuela Carraro ¹ Caludia Maria Radu ¹ Loris Bertazza ¹ Simone Mocellin ^1,2 Jacopo Pigozzo ² Giovanna Crivellaro ² Marina Coppola ² Alberto Ferlin ^1*

• ¹ University of Padua, Padua, Italy
• ² Veneto Institute of Oncology (IRCCS), Padua, Veneto, Italy
• ³ Pegaso University, Naples, Campania, Italy

Background: Immune checkpoint inhibitors (ICIs), namely anti-cytotoxic T lymphocyte-associated protein 4 (CTLA-4) monoclonal antibody Ipilimumab and anti-and programmed cell death 1 (PD-1) monoclonal antibodies Nivolumab, and Pembrolizumab, have improved the treatment outcomes for many other cancer types. However, their impact on fertility remains under-explored.The possible direct effects of ICIs on human sperm was investigated. Spermatozoa from ten normozoospermic donors were exposed to Ipilimumab, Nivolumab, or Pembrolizumab at concentrations ranging from 1 to 100 ng/mL. Sperm motility was assessed through standard laboratory process. Cell viability and apoptosis markers were evaluated by flow-cytometry using fluorescent Annexin-V probe and Terminal Uridine Nick-End Label (TUNEL) assays. Protein-A-purified therapeutic antibodies (IgG) were also evaluated.Results: Spermatozoa had high PD-1 (>99%) and negligible CTLA-4 expression. Exposure to ICIs, was associated with a concentration-dependent impairment of sperm motility, noticeable for Pembrolizumab and Ipilimumab since 10 ng/mL, and for Nivolumab since 100 ng/mL. However, no significant effect on cell apoptosis or viability was shown. Purified IgG from ICIs maintained the adverse effect on cell motility without affecting viability, Conclusion: ICIs, specifically Pembrolizumab, Nivolumab, and Ipilimumab, adversely affect human sperm motility in vitro. Further research is required to understand the underlying mechanisms and clinical implications.