3.3 GO pathway and network analysis
The functional enrichment analysis of DEGs related to the complement and coagulation signaling pathways revealed that the observed genes also participated in more than 200 statistically over-represented GO categories (Fig. 1D). All DEGs and their functions in the complement or coagulation signaling pathways are shown in Figure 2. For the complement pathway, the up-regulation of genes relating to the classical, lectin, and alternative pathways was observed. Crucial extrinsic pathway, intrinsic pathway, and fibrinolytic genes within the coagulation system were widely up-regulated, whereas important genes relating to cell adhesion, invasion, migration, and proliferation were down-regulated (Fig. 2). In addition to participating in the complement and coagulation signaling pathways, these 50 DEGs also participated in multiple other broad signaling pathways, including Staphylococcus aureus infection (n = 13, false discovery rate [FDR]: P = 2.11 × 10−23), systemic lupus erythematosus (n = 10, FDR: P = 2.02 × 10−13), prion diseases (n = 7, FDR: P = 2.03 × 10−12), pertussis (n = 7, FDR P = 2.69 × 10−10), Chagas disease (American trypanosomiasis) (n = 4, FDR: P = 6.83 × 10−5), platelet activation (n = 4, FDR: P = 1.27 × 10−4), neuroactive ligand-receptor interaction (n = 4, FDR: P = 2.37 × 10−4), phagosome (n = 3, FDR: P = .36%), hematopoietic cell lineage (n = 2, FDR: P = 1.32%), and AGE-RAGE signaling pathway in diabetic complications (n = 2, FDR: P = 1.85%) (Supplementary Table S2, http://links.lww.com/MD/D329).
The 50 DEGs were also mapped using STRING online software (Fig. 3). Their transcripts were widely distributed in the nucleus, cytoplasm, and cell membrane of placental cells. Bioinformatics analyses of the data suggest that various molecular and cellular functions were affected, and show their link to pregnancy complications.
3.4 Validation by real-time PCR
To validate the microarray results, qRT-PCR was performed. Because their biological functions were mostly related to the placenta, ten of the fifty DEGs were selected to confirm their expression (Fig. 4). qRT-PCR data confirmed the up-regulation of FGB, FGA, FGG, SERPINC1, and PROC, as well as the down-regulation of PLAU, PLAUR, SERPINE1, CD59, and CFD in placenta from IVF-ET samples. Among the 10 tested genes, we observed a significant correlation with the results obtained previously; this confirmed the observed fold changes from our microarray analysis.
To locate differentially expressed proteins related to the complement and coagulation signaling pathways in human placenta during the first trimester, 5 genes, representing different critical functions in these signaling pathways, were selected for IHC analysis: FGB, FGG, SERPINC1, PLAU, and PLAUR. These 5 proteins were found to be located in either the cytoplasm or on the cell membrane of trophoblasts in placental villous tissues (Fig. 5).
There is substantial evidence supporting the hypothesis that several adverse pregnancy outcomes observed after ART are due to suboptimal placentation caused by abnormal trophoblast function.[23,24] Indeed, in humans, after adjusting for several confounding factors, the risk of spontaneous abortion was found to be higher in ART cohorts than in spontaneous pregnancies. Notably, human studies found an increased risk of gestational hypertension, preeclampsia, placenta previa, and placental abruption in ART patients. A number of studies have previously examined and identified alterations in gene expression in placental tissues after IVF-ET treatment.[27,28] A small study investigated the global gene expression in 3 term placentas from IVF-ET pregnancies compared to that in 3 placentas from spontaneous pregnancies. They found 18 DEGs and classified them according to their role in biological process in immune response, transmembrane transport, metabolism, oxidative stress, cell differentiation, and other processes. Furthermore, several studied on the placental transcriptome after IVF-ET in animals have revealed comparable results. The aim of our study was to investigate changes in the complement and coagulation pathways due to ART, and how this would affect placental formation and function to result in placenta-related adverse pregnancy outcomes.
The complement and coagulation cascades are not only parts of the innate immune system, but also effectors of antibody-mediated immunity. The major biological functions of these systems include defense against infections, connecting the innate and adaptive immunity, and the clearance of immune complexes and apoptotic cells. The complement cascade, when activated by the classical, mannose-binding lectin, or alternative pathways, deposits several split products on the cell membrane, ultimately creating a cytotoxic cell lysis complex. The complement split products also include free circulating anaphylatoxins such as C3a and C5a, which can initiate inflammation and tissue injury. Dysregulation or over-activation of these systems are emerging as associated factors in many pregnancy complications. In our study, a total of fifty DEGs related to the complement and coagulation signaling pathways were identified in the placenta during first trimester in pregnancy after IVF-ET therapy compared to placenta samples from natural pregnancies. We mapped these DEGs to the complement and coagulation pathways, and the results showed that these systems were over-activated and uncontrolled. The classical, mannose-binding lectin, and alternative pathways in the complement cascade were all over-activated in early placental tissue after IVF-ET treatment compared to those from natural pregnancy.
In our study, the C3, C5, and CD59 genes, which are known to play a role in the complement cascade, were confirmed to be significantly differently expressed in the placenta between the 2 groups. The expression levels of C3 and C5 in IVF-ET placentae were significantly higher compared to placentae from natural pregnancies, while CD59 expression was significantly lower. The alternative pathway is triggered by the spontaneous hydrolysis of internal thioester bonds within C3 and C5 in the fluid phase, leading to the formation of C3a and C5a. C3a and C5a, which are known as anaphylatoxins, are pleiotropic inflammatory mediators. The complement cascade is controlled by several soluble membrane-bound factors, including CD59, which inhibits the complement pathway at the feto-maternal interface. In a model of spontaneous abortion, C3a and C5a were shown to be required for triggering abortion; C5a in particular was found to be critical for the induction of abortion. Our data were also consistent with those of previous studies in humans investigating C3a, C5a and CD59 levels. A recent study reported that women with unexplained fetal death displayed elevated levels of plasma C3a and C5a compared to those in healthy women. Our research further confirms that this factor at the feto-maternal interface triggered the hyperactivation of the complement cascade after IVF-ET treatment. In addition, similar studies have reported a significant association of elevated C3a and C5a levels and decreased CD59 levels with various pregnancy complications, including gestational hypertension, preterm delivery, and intrauterine growth restrictions.[39,40] Our research adds important evidence that excessive complement activation in complicated pregnancies may be associated with many pre-existing conditions, which are triggered by IVF-ET treatment.
The coagulation system is a component of the homeostatic process and a major contributor to thrombosis. Pregnancy is a physiological hypercoagulable state, and the body must prepare the mother for the hemostatic challenge of delivery. In addition, cytotrophoblast differentiation and fusion to syncytiotrophoblasts requires the initiation of apoptosis and the exposure of negatively charged phospholipids on their membrane surface. The need for the rapid inhibition of hemorrhaging in the placental intervillous spaces during gestation explains the procoagulant nature of trophoblasts. Although these conditions are heterogeneous in their pathophysiology, hereditary and acquired thrombophilia has been shown to be associated with recurrent pregnancy loss and gestational vascular complications. In the present study, KEGG pathway analysis revealed that the coagulation cascade was significantly activated through the intrinsic and extrinsic pathways in placentae after IVF-ET treatment compared to that in placentae from natural pregnancy. In early IVF-ET placentae, a state of hypercoagulability and the local formation of thrombi in the microvasculature draining the site of embryo implantation provide a competent barrier to protect the embryo from the maternal immune system. Apart from its direct role in preventing contact with maternal circulation, the coagulation system can be viewed as an intermediary that converts mechanical information from the embryo implantation site into biochemical signals that trigger cell responses, resulting in vascular biological and inflammatory responses, as well as platelet aggregation. Therefore, an over-activated coagulation system in IVF-ET early placentae may be a protective compensatory mechanism for the survival of the semi-allogeneic fetus. However, IVF-ET treatment may destroy the balance and exceed the compensatory range of the coagulation cascade, resulting in reduced nutrient supply to the embryo and increased thrombophilia-associated pregnancy complications. Moreover, the protein-protein interact network and co-expression analysis derived from STRING database also revealed that some key genes were actively interacted with each other and might be valuable biomarkers and potential novel therapeutic targets against the unfavorable effects of IVF-ET.
To our knowledge, the present study provides evidence for the first time that although the parallel over-activation of the fibrinolysis and complement systems has been observed, the expression of urokinase plasminogen activator (uPA) and urokinase plasminogen activator receptor (uPAR) was significantly reduced at the transcriptional and protein levels in placentae after IVF-ET in the first trimester compared to samples from natural pregnancies. uPA and its receptor uPAR are central molecules for uPA/uPAR/plasmin-dependent proteolysis and plasmin-dependent extracellular proteolysis. To our knowledge, this is the first study to analyze uPA and uPAR expression and localization in the early human placenta after IVF-ET treatment. These distinctive expression patterns were closely associated with their possible individual functions during the IVF-ET implantation process. Decreased expression of uPA and its receptor uPAR is thought to cause impaired trophoblast invasion and expansion, and may also affect the function of massive tissue remodeling in the interstitial endometrium during the process of uterine angiogenesis and degeneration of the epithelial plaque after IVF-ET treatment. Abnormal trophoblast invasion in IVF-ET leads to incomplete uterine vascular conversion, an inadequate fetal blood supply, and a pathological hypoxia milieu. This placental defect after IVF-ET treatment is associated with the persistence of a pro-inflammatory environment and is considered to be a failure of maternal immune tolerance, which is required for normal implantation.
This study examined the molecular mechanisms of IVF-ET-induced alterations to the complement and coagulation signaling pathways in early placenta. We found that the convergence between the complement system and the clotting system extend far beyond the chemical nature of the complement and coagulation pathways, both of which displayed an over-activated proteolytic cascade in early placentae after IVF-ET treatment compared to that in placentae from natural pregnancies. Multiple regulatory loops linking both systems were simultaneously activated to synchronize an effective response by the placenta to disrupt the IVF-ET process. Most often, this cooperative and clearly beneficial effort ensures the elimination of interference by IVF-ET technology to embryo implantation and prevents immediate abortions. However, when some regulatory mechanisms controlling complement activation or hemostasis failure, the complement and coagulation pathways become harmful, significantly contributing to various pregnancy complications, for which only complex therapies targeting multiple molecules can be effective. Indeed, in most case, pregnancies obtained through IVF-ET can be carried to term with no obvious immediate adverse outcomes. This supports the hypothesis that initial defective trophoblast functions could trigger placental adaptive response during pregnancy.
One limitation of this study was the small number of samples investigated in each group. These samples were selected from a cohort of 8 samples collected prospectively for this work. Although the sample size was small, only significantly DEGs (>2-fold) were reported and significant signal pathways were analyzed. These data allow future work to be directed toward the interference of placental formation and function, resulting in placenta-related adverse pregnancy outcomes in IVF-ET. Another limitation was that we could not formally localize the expression of more proteins through IHC, which would improve our understanding of the functional importance of these changes. Further work is needed to determine the biological plausibility of the observed variations in gene expression; however, if our results are consistent with future findings and translate to protein expression in maternal serum, they may still be of value in predicting abnormal outcomes in later pregnancy.
Our study offers a comprehensive view of dysregulated gene networks in the placental complement and coagulation signaling pathways influenced by IVF-ET treatment, although the detailed regulatory patterns were not explored. Another important result of our study was the discovery of the negative effects on trophoblast invasion, expansion, and tissue remodeling in early placentae after IVF-ET treatment through these 2 systems. Although these interconnections make it difficult to precisely define, separate, and classify biological processes, our data will enable us to focus on a small number of key genes and pathways that need to be better elucidated. Future studies with a larger sample size, focusing on these molecular and biological pathways, may lead to the development of molecular tests to predict adverse outcomes in first trimester. An improved understanding of the equilibrium between complement-mediated immune responses and thrombotic mechanisms triggered by IVF-ET may improve the safety and effectiveness of IVF-ET protocols.
Conceptualization: Liang Zhao.
Data curation: Liang Zhao, Lifang Sun, Xiuli Zheng, Rong Zheng, Rui Yang.
Formal analysis: Liang Zhao, Lifang Sun, Xiuli Zheng, Jingfang Liu, Rong Zheng, Ying Wang.
Funding acquisition: Liang Zhao.
Investigation: Liang Zhao, Lifang Sun, Rong Zheng, Rui Yang, Ying Wang.
Methodology: Liang Zhao, Lifang Sun, Xiuli Zheng, Jingfang Liu, Rui Yang.
Project administration: Liang Zhao, Rong Zheng, Ying Wang.
Resources: Liang Zhao.
Software: Liang Zhao, Lifang Sun, Xiuli Zheng, Jingfang Liu, Ying Wang.
Supervision: Liang Zhao, Lifang Sun, Jingfang Liu, Rui Yang, Ying Wang.
Validation: Liang Zhao, Lifang Sun, Xiuli Zheng, Rong Zheng, Ying Wang.
Visualization: Liang Zhao, Lifang Sun, Jingfang Liu, Rui Yang.
Writing – original draft: Liang Zhao, Lifang Sun, Jingfang Liu, Rong Zheng, Rui Yang.
Writing – review & editing: Liang Zhao.
Liang Zhao orcid: 0000-0002-0670-5242.
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complement and coagulation pathways; first trimester; in vitro fertilization and embryo transfer (IVF-ET); placenta; pregnancy complication
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