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Non-standardized protein background in IVF media linked to serum-derived albumin supplementation

Nezvedová, Markéta, Porokh, Volodymyr, Bočková, Tami, Pustka, Václav, Kyjovská, Drahomíra, Maierová, Barbora, Kloudová, Soňa, Otevřel, Pavel +1 more·2025·Journal of Assisted Reproduction and Genetics
DOI 10.1007/s10815-025-03616-0PMC PMC12602788
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Abstract

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.

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  • The undefined or incompletely characterized composition and batch-to-batch variability of animal-derived reagents and human blood-derived products can compromise reproducibility and limit translational relevance.

    “These animal-derived reagents, and also human blood-derived products, are frequently associated with undefined or incompletely characterized composition and batch-to-batch variability, which can compromise reproducibility and limit translational relevance (Aisenbrey and Murphy, 2020; Cassotta et al., 2022; Nezvedova et al., 2025; Wolff and Hendrix, 2025; Akuta et al., 2026).”

    From Challenges and solutions in transitioning to animal-free standards: a comprehensive analysis of components in human cell-based developmental neurotoxicity assays (2026) · cites this paper as Nezvedova et al., 2025

    The Results section (‘Blood-derived albumin as the source of undeclared proteins in commercial IVF media’; page unavailable) identifies undeclared proteins and batch-specific differences in human blood-derived albumin. The Discussion reports inconsistent culture conditions and concerns about the validity of protein studies, supporting the reproducibility point. The paper does not establish the same claim for animal-derived reagents or address translational relevance. (LLM, conf=0.87)

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  • Animal-derived reagents and human blood-derived products are frequently associated with undefined or incompletely characterized composition and batch-to-batch variability.

    “These animal-derived reagents, and also human blood-derived products, are frequently associated with undefined or incompletely characterized composition and batch-to-batch variability, which can compromise reproducibility and limit translational relevance (Aisenbrey and Murphy, 2020; Cassotta et al., 2022; Nezvedova et al., 2025; Wolff and Hendrix, 2025; Akuta et al., 2026).”

    From Challenges and solutions in transitioning to animal-free standards: a comprehensive analysis of components in human cell-based developmental neurotoxicity assays (2026) · cites this paper as Nezvedova et al., 2025

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Cited by

Sorted by citations

Authors
Nezvedová, M., Porokh, V., Bočková, T., Pustka, V., Kyjovská, D., Maierová, B., Kloudová, S., Otevřel, P., & Holubcová, Z.
ORCID
Holubcová, Zuzana
Venue
Journal of Assisted Reproduction and Genetics
Publisher
Springer Science and Business Media LLC
Published
2025-09-13
Volume
42
Issue
10
Pages
3535-3549
ISSN
  • 1058-0468
  • 1573-7330
DOI
10.1007/s10815-025-03616-0
PMC
PMC12602788
Cited by
6
References
0
Language
en
Licence
Funders
  • Ministerstvo Zdravotnictví Ceské Republiky (NU22-08-00543)
  • Lékařská fakulta, Masarykova univerzita (MUNI/A/1738/2024)
  • Masaryk University

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