Identification of novel prognostic protein biomarkers from maternal serum of women with recurrent pregnancy loss

Background: The underlying cause of recurrent pregnancy loss (RPL) is often not understood. Abnormal receptivity of the endometrium or chromosomal aberrations of the embryo are the usual determining factors in the cascade leading to early miscarriages. The risk of miscarriage declines with ongoing pregnancy, however the anxious wait of couples until the end of the first trimester is an important cause of stress which can be minimized if reliable prediction models exist for fetal viability.

Methods: Serum samples were obtained at gestational age of 5-7 post-menstrual weeks from 8 women that suffered subsequently a miscarriage within the trimester of pregnancy, and 10 healthy control subjects (HC) with continuing viable pregnancy. Using bottom-up untargeted proteomics with LC-MS/MS in data-independent acquisition (DIA) mode, maternal serum samples were analyzed in order to investigate their proteome profiles. We utilized Spectronaut 18 for protein quantification and a combination of analytical parameters, including statistical significance, fold change, number of unique peptides detected, number of missing values and the coefficient of variation of the candidate biomarkers among the QC samples to detect reliable prognostic biomarkers which were further evaluated using receiver operating characteristic curve (ROC) curve analysis.

Results: In total, 411 proteins from 3698 peptides were quantified in the studied serum samples. Among them, 13 proteins were significantly downregulated in RPL cases and 2 proteins were significantly upregulated. Two low-abundance proteins involved in extracellular matrix remodeling and in antioxidant defense mechanisms were selected after filtering for further analysis. The combination of the two proteins showed excellent diagnostic accuracy (AUC=0.95), while individual AUC values were 0.9 and 0.763, respectively.

Conclusion: Shotgun proteomics revealed that two low-abundance proteins have the potential to serve as prognostic biomarkers for the early screening of RPL. These promising findings will be further validated in an independent cohort by enzyme-linked immunosorbent assay (ELISA), providing strong evidence for the reliability of the new biomarkers discovered.