Advanced mass spectrometry analysis showed that not only SCM but also unconditioned IVF medium contains a large number of undeclared proteins known to play roles in cellular regulatory pathways. To eliminate the impact of protein background in embryo-free media, SCM samples were normalized to corresponding controls. We observed no significant difference in the proteome fingerprint of individual embryos that developed to blastocyst and their cleavage stage-arrested siblings. Instead, we observed lot-to-lot variability in specific protein abundance. To test whether the protein composition variation can be attributed to the addition of a plasma-derived protein, we supplemented a protein-free medium with HSA of different origins. This experiment revealed that adding HSA purified from plasma introduces batch-related divergence in undeclared protein content, contributing to variability in the media's protein composition. In contrast, the analyzed contaminating proteins of human origin were undetectable after the addition of recombinant HSA, highlighting its potential as a alternative for embryo culture. The findings of this study indicate that the protein background in commercial IVF media is an overlooked variable in human embryo cultures, which compromises the reproducibility and interpretation of embryonic secretome analyses.

A cross-sectional proteomic study involving a total of 85 samples of (un)used monophasic IVF culture media purchased from 2 manufacturers as 13 different production lots. The protein composition of 53 SCM samples from 22 IVF cycles collected between February 2022 and June 2023 was analyzed together with corresponding controls and available blank media samples.

Untargeted (nanoLC-TIMS-TOF-MS/MS) and targeted (UHPLC-QqQ-MS/MS) proteomic analyses were used to identify the protein content of media from extended embryo cultures with known embryo quality. The SCM sampling from individual embryo culture droplets was performed along with their controls. Each new batch of culture media was sampled before use (blank samples). In addition, the protein composition of HSA-free IVF media supplemented with either serum-derived HSA or recombinant HSA (rec-HSA) was examined.