Anti-CTLA-and anti-PD-1 immune checkpoint inhibitor antibodies impair human sperm motility in-vitro

Abstract

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.

Methods: 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.

Keywords: Annexin-V; Nivolumab; Pembrolizumab; TUNEL; ipilimumab.

FIGURE 1

Representative images of flow-cytometry analysis for the phenotypic characterization, in peripheral blood mononucelar cells (PBMC) (A) and in human spermatozoa (B), of anti-cytotoxic T lymphocyte-associated protein 4 (CTLA-4) monoclonal and anti- and programmed cell death 1 (PD-1) expression. Expression of CTLA-4 and PD-1 in PBMC was evaluated in both naïve (PHA-) and upon stimulation with Phytohemagglutinin (PHA+) for 3 days. (C) Representative images of CTLA-4 and PD-1 localization in human sperm cells evaluated by immunofluorescence through imaging flow cytometer. Cell nucleus was counterstained with propidium iodide (PI).

FIGURE 2

Effect of the in vitro exposure to Nivolumab, Ipilimubab or Pembrolizumab, of human spermatozoa from freshly ejaculated semen (A) or motile sperm fraction isolated by swim up selection (B). Cells were exposed to drugs for 4 h at 37°C at the concentrations indicated. Singular results and mean values ± standard deviations are reported. Significance: P values for the comparison of single conditions are reported.

FIGURE 3

(A) Flow-cytometry evaluation of sperm cell apoptosis by Annexin-V staining/propidium iodide (PI) counterstaining exposed to immune checkpoint inhibitors Pembrolizumab, Nivolumab or Ipilimumab for 4 h at 37°Cs. In control condition (CTRL), drugs were omitted. Single positive Annexin V⁺/PI⁻ events were considered as early apoptotic cells. Double positive Annexin V⁺/PI⁺ events were considered as dead cells. (B) Flow-cytometry evaluation of DNA fragmentation by Terminal Uridine Nick-End Label (TUNEL). Cells were counterstained with PI and double positive TUNEL⁺/PI⁺ events were considered as cells with fragmented DNA. Representative images of the morphological gating strategies (Morpho) are reported. Scatterplots of experimental replicates show the quantitative comparisons among conditions. Significance: n.s. = not significant among the indicated conditions.

FIGURE 4

(A) Effect of the in vitro exposure to terapeutic antibodies isolated from Nivolumab, Ipilimubab or Pembrolizumab, isolated by protein-A approach from medicinal specialties, on human spermatozoa from freshly ejaculated semen. Class G Immunoglobulins (IgG) from peripheral blood were used as reference. Cells were exposed to drugs for 4 h at 37°C at the concentrations indicated. Singular results and mean values ± standard deviations are reported. Significance: P values for the comparison of single conditions are reported; n.s. = not significant. (B) Flow-cytometry evaluation of sperm cell apoptosis by Annexin-V staining/propidium iodide (PI) counterstaining exposed to purified immunoglobulins for 4 h at 37°Cs. In control condition (CTRL), drugs were omitted. Single positive Annexin V⁺/PI⁻ events were considered as early apoptotic cells. Double positive Annexin V⁺/PI⁺ events were considered as dead cells. (C) Flow-cytometry evaluation of DNA fragmentation by Terminal Uridine Nick-End Label (TUNEL). Cells were counterstained with PI and double positive TUNEL⁺/PI⁺ events were considered as cells with fragmented DNA. Representative images of the morphological gating strategies (Morpho) are reported. Scatterplots of experimental replicates show the quantitative comparisons among conditions. Significance: n.s. = not significant among the indicated conditions.