Fig 1: Study design. A. Patient groups analyzed in the study included patients diagnosed with recurrent miscarriage (RM), women with no miscarriages in their reproductive history as well as or patients with tubal pregnancy. RM was defined as three and more consecutive pregnancy losses. Index pregnancies at the recruitment were classified as ongoing or terminated. B. Biological material analyzed in the study included placental tissue (boxes in gray background, solid line) and maternal serum samples (unfilled boxes, dashed line). Trophoblastic tissue for genome-wide expression profiling was obtained during the surgical evacuation of uterus in patients with recurrent (=3rd case) miscarriage or elective termination of normal uncomplicated pregnancy (ETP) before 12th gestational weeks. Serum samples were taken during an office visit at the first trimester of pregnancy. The course of the index pregnancy was followed until successful delivery or pregnancy loss. Treatment with methotrexate or laparoscopic surgery due to tubal pregnancy was applied after blood sampling. C. Experimental design included mRNA expression analysis and functional assays. Differentially expressed genes identified from genome-wide expression profile in trophoblastic tissue were confirmed and replicated by real-time RT-qPCR using locus-specific Taqman assays. Concentration of proteins sTRAIL and S100A8/A9 (calprotectin) coded by the loci exhibiting significant overexpression in RM placentas, was measured in maternal blood serum samples.
Fig 2: Dynamics of sTRAIL (A,B) and heterocomplex S100A8/S100A9 (C,D) level in first trimester maternal serum in normal and miscarried pregnancies. Normal pregnancy (a, c; n = 35) group included serum samples collected at the first trimester of gestation (gestational age shown in days) from individuals whose pregnancy ended with a live birth of a singleton baby or from women's who had decided for elective termination of an uncomplicated pregnancy (ETP group). Early miscarriage group (b, d; n = 18) included maternal sera sampled either at the time of inevitable or delayed miscarriage (RM group) or prospectively during the ongoing index pregnancy, which spontaneously terminated with a miscarriage before 13th gestational weeks. A logarithmic trendline describes the dynamics of sTRAIL and S100A8/S100A9 (calprotectin) concentration in serum during the pregnancy and R2 estimate equals the square of the correlation coefficient between the observed and modeled (predicted) data values. Serial serum measurements during the first trimester from four individuals, numbered (1) to (4) are shown with dashed line.
Fig 3: Significantly increased expression of TRAIL and S100A8 in placental tissue from recurrent miscarriage cases. TaqMan primer/probe sets were applied for quantification of gene expression of studied using total RNA isolated from placental tissue of recurrent miscarriage (RM) and electively terminated uncomplicated pregnancies (ETP) in discovery (RM cases, n = 4; ETP controls, n = 6) and in replication (RM cases, n = 9; ETP controls, n = 17) sample-sets. Samples of total RNA isolated from placentas of recurrent miscarriage (RM cases; n = 13) and electively terminated uncomplicated pregnancies (ETP controls, n = 23) were analyzed using TaqMan primer/probe sets. Presented boxplots summarize the distribution of relative mRNA expression in the joint sample set of the discovery and the replication specimen. The median expression level of the ETP group was selected as calibrator and relative mRNA expression levels are shown on logarithmic scale. P-values for the comparison of gene expression between RM and ETP groups were estimated by logistic regression model with gestational age and maternal age as cofactors.
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