Increased placental expression and maternal serum levels of apoptosis-inducing TRAIL in recurrent miscarriage

Abstract

Introduction: Recurrent miscarriage (RM; ≥3 consecutive pregnancy losses) occurs in 1-3% of fertile couples. No biomarkers with high predictive value of threatening miscarriage have been identified. We aimed to profile whole-genome differential gene expression in RM placental tissue, and to determine the protein levels of identified loci in maternal sera in early pregnancy.

Methods: GeneChips (Affymetrix(®)) were used for discovery and Taqman RT-qPCR assays for replication of mRNA expression in placentas from RM cases (n = 13) compared to uncomplicated pregnancies matched for gestational age (n = 23). Concentrations of soluble TRAIL (sTRAIL) and calprotectin in maternal serum in normal first trimester (n = 35) and failed pregnancies (early miscarriage, n = 18, late miscarriage, n = 4; tubal pregnancy, n = 11) were determined using ELISA.

Results: In RM placentas 30 differentially expressed (with nominal P-value < 0.05) transcripts were identified. Significantly increased placental mRNA expression of TNF-related apoptosis-inducing ligand (TRAIL; P = 1.4 × 10(-3); fold-change 1.68) and S100A8 (P = 7.9 × 10(-4); fold-change 2.56) encoding for inflammatory marker calprotectin (S100A8/A9) was confirmed by RT-qPCR. When compared to normal first trimester pregnancy (sTRAIL 16.1 ± 1.6 pg/ml), significantly higher maternal serum concentration of sTRAIL was detected at the RM event (33.6 ± 4.3 pg/ml, P = 0.00027), and in pregnant women, who developed an unpredicted miscarriage 2-50 days after prospective serum sampling (28.5 ± 4.4 pg/ml, P = 0.039). Women with tubal pregnancy also exhibited elevated sTRAIL (30.5 ± 3.9 pg/ml, P = 0.035). Maternal serum levels of calprotectin were neither diagnostic nor prognostic to early pregnancy failures (P > 0.05).

Conclusions: The study indicated of sTRAIL as a potential predictive biomarker in maternal serum for early pregnancy complications.

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

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.

Fig. 3

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. 4

Concentration of sTRAIL in first trimester maternal serum in normal and failed pregnancies. Normal pregnancy (n = 35) included serum samples collected at the first trimester of gestation from individuals whose pregnancy ended with a live birth or from women's who had decided for elective termination of an uncomplicated pregnancy (ETP group). Maternal serum in early miscarriage group had been sampled either at the time of inevitable or delayed miscarriage (after clinical appearance, n = 13) or prospectively during the ongoing index pregnancy, which spontaneously terminated with a miscarriage before 13th gestational weeks (n = 5). Serum had been sampled 2–50 days before clinical appearance of pregnancy loss. Late miscarriage group (n = 4) was defined as a miscarriage after 12th gestational weeks (16 and 20 weeks) or as extremely preterm (24 and 27 weeks) delivery of a stillbirth baby; the analyzed maternal serum had been sampled prospectively in the first trimester of pregnancy. The serum from the patients with tubal pregnancy (n = 11) was taken before application of surgical or medical treatment. The boxes represent the 25 and 75th percentiles. The median is denoted as the line that bisects the boxes. The whiskers are lines extending from each end of the box covering the extent of the data on 1.5 X interquartile ranges. Circles represent the outlier values. P-values reflecting the differences between groups were estimated by logistic regression and adjusted for maternal and gestational age.