MSACL 2022 Abstract

Novel First Trimester Biomarkers of Fetal Growth Restriction Identified by Next Generation Proteomics of Maternal Plasma

Nandor Gabor Than (1,2), Gergo Orosz (3) Daniel Gyorffy (1), Laszlo Orosz (3), Mate Posta (1), Andras Szilagyi (1), Magdalena Bober (4), Claudia Escher (4), Oliver Rinner (4), Petronella Hupuczi (2), Sandor Nagy (5), Zoltan Papp (2), Olga Torok (3)
(1) Systems Biology of Reproduction Research Group, Institute of Enzymology, Research Centre for Natural Sciences, Budapest, Hungary; (2) Maternity Private Clinic of Obstetrics and Gynecology, Budapest, Hungary; (3) Department of Obstetrics and Gynecology, University of Debrecen, Debrecen, Hungary; (4) Biognosys AG, Schlieren, Switzerland; (5) Faculty of Health and Sport Sciences, Szechenyi Istvan University, Gyor, Hungary

Nandor Gabor Than, MD, PhD (Presenter) Research Centre for Natural Sciences, Budapest, Hungary

Presenter Bio: Nandor Gabor Than, MD, PhD is senior research fellow at the Research Centre for Natural Sciences (RCNS, Budapest, Hungary). He is/was also affiliated with Semmelweis University (Hungary), University of Southern California (USA) and Wayne State University (USA) as associate professor. He is an obstetrician with PhD in molecular biology and 20+ years of experience with human placenta and placental protein research. He also gained experience in reproductive immunology, evolutionary and systems biology. He is past director of the Placental Laboratory at the Perinatology Research Branch (NIH, USA) and director of the Laboratory of Systems Biology in Reproduction and the Perinatal Biobank at RCNS. Dr. Than has been Editor or on Editorial Board of 8 journals. He is author of >160 research papers/books, many published in high impact journals. He has an H index of 53 and received >7,700 citations. He is co-inventor on >10 patents for novel diagnostic/therapeutic tools for pregnancy complications. He is on the review panel of >50 scientific journals and 9 grant agencies.
Dr. Than’s team is focusing on the systems biology study of placental pathologic pathways in pregnancy complications, the identification of biomarkers for the early detection of women at risk, and the development of in vitro / in vivo models to test drug candidates for the personalized treatment of these diseases. His team plans to develop specific and reliable clinical proteomics biomarkers for the early detection and prevention of pregnancy complications. Their major achievements involve the discovery and sequencing of genes encoding 91 placental proteins in 15 species. They discovered a placenta-specific galectin family that evolved in primates, is important in regulating maternal-fetal immune interactions, and is dysregulated in preeclampsia. Their integrated systems biology study explored early maternal/placental molecular pathways of preeclampsia. Recently, they have discovered plasma protein biomarkers for preeclampsia and miscarriages using mass spectrometry.

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INTRODUCTION: Fetal growth restriction (FGR) is one of the obstetrical syndromes, caused by the failure of the fetus to achieve its genetically determined growth potential. It is clinically defined as a birthweight less than 10th centile and affects 7–10% of pregnancies. FGR is a major cause of perinatal morbidity and mortality by its association with spontaneous and medically indicated preterm birth and neonatal death. Since impaired fetal growth leads to metabolic, neurodevelopmental or cardiovascular changes in the fetus that persist until adulthood, FGR leads to life-long predisposition to illness. Indeed, FGR increases the risk for cardiovascular and metabolic diseases including diabetes, hypertension, ischemic heart disease and stroke in adulthood; therefore, it imposes a major burden on health and healthcare. FGR has two clinical subgroups that are distinct by the presence or absence of Doppler ultrasound abnormalities in the placental/fetal circulation. Placental dysfunction is a major cause of FGR and maternal adaptations to placental insufficiency may also play a role in the pathophysiology. However, the exact molecular pathways of these FGR subgroups and their biomarkers which may assist in the early screening and in driving tailored treatments are not yet known. Here, we aimed to discover novel early biomarkers of these two clinical subgroups of FGR by investigating maternal blood using next generation proteomics technology to help in the development of early and effective screening (diagnostic) and preventive (therapeutic) tools.
METHODS: First trimester maternal blood samples were collected from women whose pregnancies were subsequently affected by FGR with (n=24) or without (n=24) Doppler ultrasound abnormalities as well as gestational age-matched healthy pregnant control women (n=24). Plasma samples were prepared and stored at -80oC. After immunodepletion of 14 highly abundant proteins, plasma samples were reduced, alkylated and trypsin digested. Then, samples were analyzed using hyper reaction monitoring (HRM). For the LC-MS/MS HRM measurements, nano-flow reversed-phase chromatography was applied to separate peptides, and then mass spectrometric analysis was performed in data-independent acquisition (DIA) mode. HRM mass spectrometric data were analyzed using Spectronaut Pulsar software (Biognosys). The false discovery rate (FDR) on peptide and protein level was set to 1%, and the data was filtered using row-based extraction. The HRM measurements analyzed with Spectronaut were normalized using local regression normalization. Initial analyses with Spectronaut used peptide level data and included multivariate analysis and statistical testing to identify differentially expressed (DE) proteins, which we expanded with protein level data assessment. Downstream analyses included Gene Ontology (GO) enrichment and protein network analyses using String-db.
RESULTS: On average, 636 proteins were quantified per sample, and a total of 827 proteins represented by 18,164 peptide ion variants were quantified across all samples. Protein level data analysis identified 49 DE proteins in FGR with Doppler abnormalities, while 41 DE proteins in FGR without Doppler changes compared to healthy controls. Fourteen DE proteins commonly changed in both FGR subgroups, while 35 or 27 proteins were uniquely changing in the two FGR subgroups, respectively. We also identified 40 DE proteins when comparing the two FGR subgroups. Partial Least Squares Discriminant Analysis allowed considerable separation of the two FGR subgroups from controls. Downstream analysis found common and separate biological functions changing in the two FGR subgroups. This indicates partially distinct molecular pathways in the two FGR subgroups, and more disturbance in placental development in cases with ultrasound abnormalities.
CONCLUSIONS: We identified novel biomarkers of the two FGR subgroups in early pregnancy. Our results may facilitate the better understanding of the molecular pathways of these FGR subgroups and promote the identification of potential drug target molecules. Larger clinical studies need to investigate the true predictive power of the identified biomarkers, and to test drugs for the modulation of the identified molecular disease pathways and the prevention of FGR.