PURPOSE: To explore the protein compositional variability of IVF media and identify sources of undeclared contaminants that interfere with the detection of embryo-derived signals. METHODS: Untargeted and targeted mass spectrometry techniques were used to analyze protein composition in 85 samples of used and unused monophasic IVF media across 13 production lots from two manufacturers. Samples included spent culture media (SCM) from individual embryo cultures, matched controls, and unused (blank) media. Protein-free base media was supplemented with either serum-derived or recombinant human serum albumin (HSA) to evaluate their impact on protein contamination. RESULTS: Proteomic analysis revealed that not only SCM but also unconditioned media contained over 700 undeclared human proteins, many of which are known to be implicated in key cellular pathways. No significant differences were observed between the protein profiles of embryos that reached the blastocyst stage (n = 29) and those arrested at cleavage (n = 24). Instead, protein level variation strongly correlated with media production lot, as shown by targeted analysis of 14 candidate proteins and principal component clustering of 53 SCM samples. Analysis of blank media confirmed substantial lot-to-lot heterogeneity. Supplementation experiments demonstrated that serum-derived HSA introduces undeclared, batch-variable proteins into IVF media, contributing to a non-standardized culture environment and confounding the detection of embryo-derived signals. CONCLUSION: Serum-derived HSA was identified as the primary source of protein contamination in IVF media. This overlooked protein background contributes to variability in clinical culture conditions, undermines the reproducibility of secretome analyses, and complicates the discovery of reliable biomarkers in SCM.

Department of Histology and Embryology Faculty of Medicine Masaryk University Brno Czech Republic

Proteomics Core Facility Central European Institute for Technology Masaryk University Brno Czech Republic

RECETOX Faculty of Science Masaryk University Brno Czech Republic

Reprofit International Clinic of Reproductive Medicine and Gynecology Brno Czech Republic

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