It is now well established that maternal serum markers are often abnormal in fetal trisomy 21. Their determination is recommended for prenatal screening and pregnancy follow-up. However, mechanisms leading to abnormal maternal serum levels of such markers are still debated. Our objective was to help clinicians and scientists unravel the pathophysiology of these markers via a review of the main studies published in this field, both in vivo and in vitro, focusing on the six most widely used markers (hCG, its free subunit hCGβ, PAPP-A, AFP, uE3, and inhibin A) as well as cell-free feto-placental DNA. Analysis of the literature shows that mechanisms underlying each marker's regulation are multiple and not necessarily directly linked with the supernumerary chromosome 21. The crucial involvement of the placenta is also highlighted, which could be defective in one or several of its functions (turnover and apoptosis, endocrine production, and feto-maternal exchanges and transfer). These defects were neither constant nor specific for trisomy 21, and might be more or less pronounced, reflecting a high variability in placental immaturity and alteration. This explains why maternal serum markers can lack both specificity and sensitivity, and are thus restricted to screening.
The effects of fluid shear stress (FSS) on the human syncytiotrophoblast and its biological functions have never been studied. During pregnancy, the syncytiotrophoblast is the main source of placental growth factor (PlGF), a proangiogenic factor involved in the placental angiogenesis and the vascular adaptation to pregnancy. The role of FSS in regulating PlGF expression in syncytiotrophoblasts is unknown. We investigated the impact of FSS on the production and secretion of the PlGF by the human syncytiotrophoblasts in primary cell culture. Laminar and continuous FSS (1 dyn cm −2 ) was applied to human syncytiotrophoblasts cultured in a parallel-plate flow chambers. Secreted levels of PlGF, sFlt-1 (soluble fms-like tyrosin kinase-1), and prostaglandin E2 were tested by immunologic assay. PlGF levels of mRNA and intracellular protein were examined by RT-PCR and Western blot, respectively. Intracellular cAMP levels were examined by time-resolved fluorescence resonance energy transfer cAMP accumulation assay. Production of cAMP and PlGF secretion was significantly increased in FSS conditions compared with static conditions. Western blot analysis of cell extracts exposed to FSS showed an increased phosphorylation of protein kinase A substrates and cAMP response element-binding protein on serine 133. FSS-induced phosphorylation of cAMP response element-binding protein and upregulation of PlGF were prevented by inhibition of protein kinase A with H89 (3 μmol/L). FSS also triggers intracellular calcium flux, which increases the synthesis and release of prostaglandin E2. The enhanced intracellular cAMP in FSS conditions was blocked by COX1/COX2 (cyclooxygenase) inhibitors, suggesting that the increase in prostaglandin E2 production could activate the cAMP/protein kinase A pathway in an autocrine/paracrine fashion. FSS activates the cAMP/protein kinase A pathway leading to upregulation of PlGF in human syncytiotrophoblast.
Protease Inhibitors (PI e.g., ritonavir (RTV) and lopinavir (LPV)) used to treat pregnant mothers infected by HIV induce prematurity and endocrine dysfunctions. The maintenance of pregnancy relies on placental hormone production (human Chorionic Gonadotrophin (hCG) and progesterone (P4)). Those functions are ensured by the villous trophoblast and are mainly regulated by the Unfolded Protein Response (UPR) pathway and mitochondria. We investigated, in vitro, if PI impair hCG and P4 production and the potential intracellular mechanisms involved. Term villous cytotrophoblast (VCT) were cultured with or without RTV or LPV from 6 to 48 h. VCT differentiation into syncytiotrophoblast (ST) was followed measuring hCG and P4 secretion. We evaluated the expression of P4 synthesis partners (Metastatic Lymph Node 64 (MLN64), cholesterol side-chain cleavage (P450SCC), Hydroxy-delta-5-Steroid Dehydrogenase and 3 Beta-and steroid delta-isomerase 1 (HSD3B1)), of mitochondrial pro-fusion factors (Mitofusin 2 (Mfn2), Optic Atrophy 1 (OPA1)) and of UPR factors (Glucose-Regulated Protein 78 (GRP78), Activating Transcription Factor 4 (ATF4), Activating Transcription Factor 6 (ATF6), spliced X-box Binding Protein 1 (sXBP1)). RTV had no significant effect on hCG and P4 secretion, whereas lopinavir significantly decreased both secretions. LPV also decreased P450SCC and HSD3B1 expression, whereas it increased Mfn2, GRP78 and sXBP1 expression in ST. RTV has no effect on the endocrine placenta. LPV impairs both villous trophoblast differentiation and P4 production. It is likely to act via mitochondrial fusion and UPR pathway activation. These trophoblastic alterations may end in decreased P4 levels in maternal circulation, inducing prematurity.
The glucose-regulated protein 78 (GRP78) is a molecular chaperone that is responsible for protein folding, which belongs to the heat shock protein 70 kDa (HSPA/HSP70). Because of the conjunction of GRP78 transcription with endoplasmic reticulum stress, the chaperone plays an important role in the unfolded protein response (UPR), which is induced after the accumulation of misfolded proteins. In the last years, a significant body of research concentrated on interplay between GRP78 and sexual steroid hormones. Throughout this review, we describe the mechanisms by which GRP78 regulates steroidogenesis at multiple levels and how steroids modulate GRP78 expression in different mammalian reproductive organs. Finally, we discuss the cooperation between GRP78 and steroids for cell survival and proliferation in the context of reproduction and tumorigenesis. This new paradigm offers significant opportunities for future exploration.
Preeclampsia is a hypertensive pregnancy disease associated with a massive increase in sFlt-1 (soluble form of the vascular endothelial growth factor 1) in the maternal circulation, responsible for angiogenic imbalance and endothelial dysfunction. Pilot studies suggest that extracorporeal apheresis may reduce circulating sFlt-1 and prolong pregnancy. Nonspecific apheresis systems have potential adverse effects because of the capture of many other molecules. Our concept is based on a specific and competitive apheresis approach using VEGF (vascular endothelial growth factor) functionalized magnetic beads to capture sFlt-1 while releasing endogenous PlGF (placental growth factor) to restore a physiological angiogenic balance. Magnetic beads were functionalized with VEGF to capture sFlt-1. Experiments were performed using PBS, conditioned media from human trophoblastic cells, and human plasma. The proof of concept was validated in dynamic conditions in a microfluidic device as an approach mimicking real apheresis. Magnetic beads were functionalized with VEGF and characterized to evaluate their surface ligand density and recognition capabilities. VEGF-coated magnetic beads proved to be an efficient support in capturing sFlt-1 and releasing PlGF. In static conditions, sFlt-1 concentration decreased by 33±13%, whereas PlGF concentration increased by 27±10%. In dynamic conditions, the performances were improved, with 40% reduction of sFlt-1 and up to 2-fold increase of free PlGF. The sFlt-1/PlGF ratio was reduced by 63% in the plasma of preeclamptic patients. Apheresis was also associated with VEGF release. A ligand-based approach using VEGF-coated beads is an effective approach to the capture of sFlt-1 and the release of endogenous PlGF. It offers new perspectives for the treatment of preeclampsia.
