Miscarriages affect 50–70% of all conceptions and 15–20% of clinically recognized pregnancies. Recurrent pregnancy loss (RPL, ≥2 miscarriages) affects 1–5% of recognized pregnancies. Nevertheless, our knowledge about the etiologies and pathophysiology of RPL is incomplete, and thus, reliable diagnostic/preventive tools are not yet available. Here, we aimed to define the diagnostic value of three placental proteins for RPL: human chorionic gonadotropin free beta-subunit (free-β-hCG), pregnancy-associated plasma protein-A (PAPP-A), and placental growth factor (PlGF). Blood samples were collected from women with RPL (n = 14) and controls undergoing elective termination of pregnancy (n = 30) at the time of surgery. Maternal serum protein concentrations were measured by BRAHMS KRYPTOR Analyzer. Daily multiple of median (dMoM) values were calculated for gestational age-specific normalization. To obtain classifiers, logistic regression analysis was performed, and ROC curves were calculated. There were differences in changes of maternal serum protein concentrations with advancing healthy gestation. Between 6 and 13 weeks, women with RPL had lower concentrations and dMoMs of free β-hCG, PAPP-A, and PlGF than controls. PAPP-A dMoM had the best discriminative properties (AUC = 0.880). Between 9 and 13 weeks, discriminative properties of all protein dMoMs were excellent (free β-hCG: AUC = 0.975; PAPP-A: AUC = 0.998; PlGF: AUC = 0.924). In conclusion, free-β-hCG and PAPP-A are valuable biomarkers for RPL, especially between 9 and 13 weeks. Their decreased concentrations indicate the deterioration of placental functions, while lower PlGF levels indicate problems with placental angiogenesis after 9 weeks.

The prevention of pre-eclampsia is difficult due to the syndromic nature and multiple underlying mechanisms of this severe complication of pregnancy. The current clinical distinction between early- and late-onset disease, although clinically useful, does not reflect the true nature and complexity of the pathologic processes leading to pre-eclampsia. The current gaps in knowledge on the heterogeneous molecular pathways of this syndrome and the lack of adequate, specific diagnostic methods are major obstacles to early screening and tailored preventive strategies. The development of novel diagnostic tools for detecting the activation of the identified disease pathways would enable early, accurate screening and personalized preventive therapies. We implemented a holistic approach that includes the utilization of different proteomic profiling methods of maternal plasma samples collected from various ethnic populations and the application of systems biology analysis to plasma proteomic, maternal demographic, clinical characteristic, and placental histopathologic data. This approach enabled the identification of four molecular subclasses of pre-eclampsia in which distinct and shared disease mechanisms are activated. The current review summarizes the results and conclusions from these studies and the research and clinical implications of our findings.

Introduction: Preeclampsia (PE) is a severe obstetrical syndrome characterized by new-onset hypertension and proteinuria and it is often associated with fetal intrauterine growth restriction (IUGR). PE leads to long-term health complications, so early diagnosis would be crucial for timely prevention. There are multiple etiologies and subtypes of PE, and this heterogeneity has hindered accurate identification in the presymptomatic phase. Recent investigations have pointed to the potential role of small regulatory RNAs in PE, and these species, which travel in extracellular vesicles (EVs) in the circulation, have raised the possibility of non-invasive diagnostics. The aim of this study was to investigate the behavior of exosomal regulatory small RNAs in the most severe subtype of PE with IUGR. Methods: We isolated exosomal EVs from first-trimester peripheral blood plasma samples of women who later developed preterm PE with IUGR (n=6) and gestational age-matched healthy controls (n=14). The small RNA content of EVs and their differential expression were determined by next-generation sequencing and further validated by quantitative real-time PCR. We also applied the rigorous exceRpt bioinformatics pipeline for small RNA identification, followed by target verification and Gene Ontology analysis. Results: Overall, >2700 small RNAs were identified in all samples and, of interest, the majority belonged to the RNA interference (RNAi) pathways. Among the RNAi species, 16 differentially expressed microRNAs were up-regulated in PE, whereas up-regulated and down-regulated members were equally found among the six identified Piwi-associated RNAs. Gene ontology analysis of the predicted small RNA targets showed enrichment of genes in pathways related to immune processes involved in decidualization, placentation and embryonic development, indicating that dysregulation of the induced small RNAs is connected to the impairment of immune pathways in preeclampsia development. Finally, the subsequent validation experiments revealed that the hsa_piR_016658 piRNA is a promising biomarker candidate for preterm PE associated with IUGR. Discussion: Our rigorously designed study in a homogeneous group of patients unraveled small RNAs in circulating maternal exosomes that act on physiological pathways dysregulated in preterm PE with IUGR. Therefore, our small RNA hits are not only suitable biomarker candidates, but the revealed biological pathways may further inform us about the complex pathology of this severe PE subtype.

Preeclampsia is a syndromic disease of the mother, fetus, and placenta. The main limitation in early and accurate diagnosis of preeclampsia is rooted in the heterogeneity of this syndrome as reflected by diverse molecular pathways, symptoms, and clinical outcomes. Gaps in our knowledge preclude successful early diagnosis, personalized treatment, and prevention. The advent of “omics” technologies and systems biology approaches addresses this problem by identifying the molecular pathways associated with the underlying mechanisms and clinical phenotypes of preeclampsia. Here, we provide a brief overview on how the field has progressed, focusing on studies utilizing state-of-the-art transcriptomics and proteomics methods. Moreover, we summarize our systems biology studies involving maternal blood proteomics and placental transcriptomics, which identified early maternal and placental disease pathways and showed that their interaction influences the clinical presentation of preeclampsia. We also present an analysis of maternal blood proteomics data which revealed distinct molecular subclasses of preeclampsia and their molecular mechanisms. Maternal and placental disease pathways behind these subclasses are similar to those recently reported in studies on the placental transcriptome. These findings may promote the development of novel diagnostic tools for the distinct subtypes of preeclampsia syndrome, enabling early detection and personalized follow-up and tailored care of patients.

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Introduction: Miscarriages affect 50-70% of all gestations and 15-20% of clinically recognized pregnancies. Recurrent pregnancy loss (RPL) occurs in 1-5% of clinical pregnancies and has an enormous demographic impact. However, the etiologies and molecular pathways of RPL are scarcely understood, and therefore, reliable diagnostic and preventive methods are not yet available. Here, we aimed to discover novel biomarkers for RPL using next-generation proteomics technology to help develop early and effective diagnostic tools. Methods: First-trimester blood samples were collected from women with RPL (n=11) and controls with elective termination of pregnancy (n=11) between 6–13 weeks of gestation. After immunodepleting 14 highly abundant proteins, plasma samples were reduced, alkylated, and trypsin digested. For the separation of peptides, nano-flow reversed-phase chromatography was applied, and then mass spectrometric analysis was performed. Differentially abundant (DA) proteins were identified using strict criteria and analyzed by protein network and Gene Ontology (GO) enrichment analyses, and two biomarker candidates (CGB and PAPPA) were validated by immunoassay. Biomarker predictive properties were demonstrated using Receiver Operating Characteristic (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: Altogether, 651 proteins were identified and quantified across all samples. When comparing “early control” and “late control” samples, 60 proteins [11 predominantly placenta-expressed (PPE)] were DA. When analyzing all cases, 50 DA proteins were found in RPL (top 3 down: PZP, PSG9, CGB; top 3 up: C4BPA, HBA, HBB), among which 11 PPE proteins were found, all downregulated. Enriched GO terms included ‘placental function’, ‘oxidative processes’, ‘immune function’, and ‘blood coagulation’ related biological processes. When cases were split into early and late RPL groups, 40 DA proteins were identified in early RPL (top 3 down: SHBG, CGB, CGA; top 3 up: C4BPA, SAMP, C4BPB) and 90 in late RPL (top 3 down: PZP, PAPPA, PSG9; top 3 up: THBS1, ECM1, HBB), among which only 15 were shared by both RPL groups. In early RPL, only ‘placental function’ and ‘immune function’ related biological processes were enriched, while in late RPL the top enriched GO terms included ‘placental function’, ‘oxidative processes’, ‘immune function’, ‘blood coagulation’, ‘angiogenesis’, ‘cell migration’, and ‘blood circulation’ related biological processes. Among GO terms, only ‘placental function’ related biological processes were enriched when early- and late RPL DA proteins were analyzed together. Furthermore, the areas under the ROC curves were >0.9 for two protein candidates in all RPL, for five proteins in early RPL, and for ten proteins in late RPL. Among these candidates, CGB and PAPPA were validated by immunoassay which showed a good correlation with MS data (RCGB=0.795 and RPAPPA=0.965). Conclusion: We discovered distinct as well as shared molecular pathways associated with RPL pathogenesis before and after the start of placental circulation and identified novel biomarkers for these pathways which have outstanding discriminative properties. Our results may facilitate a better understanding of the molecular pathways of RPL. However, larger clinical studies are needed to investigate whether the identified biomarkers also have predictive power for RPL before pregnancies fail and to test drugs for the modulation of the identified disease pathways and the prevention of RPL. Our findings highlight the importance of the maternal immune system in maintaining successful pregnancy and suggest that targeting immune pathways may offer novel therapeutic approaches for RPL.