Novel proteomics biomarkers of recurrent pregnancy loss reflect the dysregulation of immune interactions at the maternal-fetal interface

Eszter Tóth¹Máté Posta^1,2Dániel Györffy^1,3Orsolya Oravecz^1,4Emese Farkas^1,2Andrea Balogh¹Claudia Escher⁵Magdalena Bober⁵András Szilágyi¹Petronella Hupuczi⁶Lajos Veress⁷Olga Torok⁸Sandor Nagy⁹Oliver Rinner⁵Offer Erez¹⁰Zoltán Papp^11,6Nandor Acs¹¹Nandor Gabor Than MD, PhD^1,11,6*

• ¹Systems Biology of Reproduction Research Group, Institute of Molecular Life Sciences, Research Centre for Natural Sciences, Hungarian Academy of Sciences (MTA), Budapest, Hungary
• ²Károly Rácz School of Ph.D. Studies, Semmelweis University, Budapest, Hungary
• ³Faculty of Information Technology and Bionics, Pázmány Péter Catholic University, Budapest, Hungary
• ⁴Institute of Biology, Doctoral School of Biology, Eötvös Loránd University, Budapest, Hungary
• ⁵Biognosys (Switzerland), Schlieren, Switzerland
• ⁶Maternity Obstetrics and Gynecology Private Clinic, Budapest, Hungary
• ⁷Department of Pharmacology and Pharmacotherapy, Faculty of Medicine, University of Debrecen, Debrecen, Hungary
• ⁸Department of Obstetrics and Gynecology, Faculty of Medicine, University of Debrecen, Debrecen, Hungary
• ⁹Széchenyi István University, Gyor, Gyor-Moson-Sopron, Hungary
• ¹⁰Bun Gurion University, Department of Obstetrics and Gynecology, Beer Sheba, Israel
• ¹¹Department of Obstetrics and Gynaecology, Faculty of Medicine, Semmelweis University, Budapest, Hungary

INTRODUCTION Miscarriages affect up to 70% of all gestations, with recurrent pregnancy loss (RPL) occurring in 1–5% of clinical pregnancies. Despite its prevalence and demographic impact, the molecular mechanisms underlying RPL remain poorly understood, and reliable diagnostic tools are not yet available. This study aimed to identify novel biomarkers for RPL using next-generation proteomics to support the development of early diagnostic and preventive strategies.METHODS: First-trimester blood samples were collected from women with RPL and controls undergoing elective termination between 6–13 weeks of gestation. After immunodepleting 14 abundant plasma proteins, samples were digested and analyzed by nano-flow liquid chromatography coupled with mass spectrometry. Differentially abundant (DA) proteins were identified and analyzed using protein network and Gene Ontology (GO) enrichment. Two candidate biomarkers (CGB, PAPPA) were validated by immunoassay, and predictive performance was assessed using ROC curves. Assessments were performed for all cases and then for two gestational age groups, before and after the start of placental circulation [“early RPL”: gestational weeks (GW) 6–9, “late RPL”: GW 9–13].RESULTS: A total of 651 proteins were quantified. Comparing early and late control samples revealed 60 DA proteins, including 11 placenta-specific (PPE). In RPL cases, 50 DA proteins were identified (top: PZP, PSG9, CGB), with 11 PPE proteins all downregulated. Enriched GO terms included ‘placental function’, ‘oxidative stress’, ‘immune response’, and ‘coagulation’. Subgroup analysis revealed 40 DA proteins in early RPL and 90 in late RPL, among which only 15 were shared by both groups. Early RPL was associated with placental and immunopathological processes, while late RPL showed broader enrichment, also including abnormalities with angiogenesis and circulation. ROC curves showed excellent performance (AUC>0.9) for several biomarker candidates. CGB and PAPPA were successfully validated (RCGB=0.795, RPAPPA=0.965).CONCLUSION: This study identifies distinct and shared molecular pathways underlying RPL before and after placental circulation begins, and highlights biomarkers with strong discriminative power. These findings enhance our understanding of RPL pathogenesis and may guide future therapeutic approaches, though larger studies are required to confirm predictive power and explore drug targets.