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Human idiopathic fetal growth restriction (FGR) is associated with placental insufficiency. Previously, we reported that the expression of homeobox gene Distal-less 3 (DLX3) is increased in idiopathic FGR placentae and is a regulator of villous trophoblast differentiation. Here, we identify the downstream targets of DLX3 in trophoblast-derived cell lines. We modelled the high levels of DLX3 in FGR using an over-expression plasmid construct and complemented this using short-interference RNA (siRNA) for inactivation in cultured cells. Using a real-time PCR-based gene profiling, candidate target genes of DLX3 over-expression and inactivation were identified as regulators of trophoblast differentiation; GATA2 and PPARγ. The expression of GATA2 and PPARγ were further assessed in placental tissues and showed increased mRNA and protein levels in FGR-affected tissues compared with gestation-matched controls. We conclude that DLX3 orchestrates the expression of multiple regulators of trophoblast differentiation and that expression of these regulatory genes is abnormal in FGR.

Abnormal trophoblast function is associated with human fetal growth restriction (FGR). Targeted disruption of homeobox gene transforming growth β-induced factor (TGIF-1) results in placental dysfunction in the mouse. The role of human TGIF-1 in placental cell function is unknown. The aims of this study were to determine the expression of TGIF-1 in human idiopathic FGR-affected placentae compared with gestation-matched controls (GMC), to elucidate the functional role of TGIF-1 in trophoblasts and to identify its downstream targets. Real-time PCR and immunoblotting revealed that TGIF-1 mRNA and protein expression was significantly increased in FGR-affected placentae compared with GMC (n = 25 in each group P < 0.05). Immunoreactive TGIF-1 was localized to the villous cytotrophoblasts, syncytiotrophoblast, microvascular endothelial cells and in scattered stromal cells in both FGR and GMC. TGIF-1 inactivation in BeWo cells using two independent siRNA resulted in significantly decreased mRNA and protein of trophoblast differentiation markers, human chorionic gonadotrophin (CGB/hCG), syncytin and 3β-hydroxysteroid dehydrogenase/3β-honest significant difference expression. Our data demonstrate that homeobox gene TGIF-1 is a potential up-stream regulator of trophoblast differentiation and the altered TGIF-1 expression may contribute to aberrant villous trophoblast differentiation in FGR.

International audience; Background: The sequence of the beta-subunit of human chorionic gonadotropin (hCG beta varies depending on whether hCG beta is encoded by type I or type II genes. Type II genes are upregulated in trophoblast and cancer but hCG beta can be detected in the serum of nonpregnant women and healthy individuals. We aimed to determine whether monoclonal antibody (mAb) FBT11-II specifically detects hCG beta encoded by type II genes (type II hCG beta). Methods: Competitive inhibition assays with synthetic peptides, immunocytochemical and immunohistochemical studies, type II hCG beta dosing immunoassays and sequencing of CGB genes were performed. Results: Competitive inhibition assays determined that mAb FBT11-II recognizes the type II hCG beta derived peptide. CGB mRNA sequencing of JEG-3 (trophoblastic) and 124 (bladder) cell lines confirmed that JEG-3 expresses type II genes while T24 expresses exclusively type I. FBT11-II only recognizes JEG-expressed hCG beta. Placenta immunohistochemical studies confirmed that type II hCG beta expression is restricted to the syncytiotrophoblast. Immunoassays detected type II hCG beta in serum of patients with either nontrophoblastic cancers or fetal Down syndrome. Conclusion: Type II gene expression can be detected using FBT11-II. This specific recognition could improve the clinical usefulness of assays aimed at either managing aggressive tumors or screening for Down syndrome.

Human chorionic gonadotropin (hCG) displays a major role in pregnancy initiation and progression and is involved in trophoblast differentiation and fusion. However, the site and the type of dimeric hCG production during the first trimester of pregnancy is poorly known. At that time, trophoblastic plugs present in the uterine arteries disappear, allowing unrestricted flow of maternal blood to the intervillous space. The consequence is an important modification of the trophoblast environment, including a rise of oxygen levels from about 2.5% before 10 wk of amenorrhea (WA) to ∼8% after 12 WA. Two specific β-hCG proteins that differ from three amino acids have been described: type 1 (CGB7) and type 2 (CGB3, -5, and -8). Here, we demonstrated in situ and ex vivo on placental villi and in vitro in primary cultures of human cytotrophoblasts that type 1 and 2 β-hCG RNAs and proteins were expressed by trophoblasts and that these expressions were higher before blood enters in the intervillous space (8–9 vs. 12–14 WA). hCG was immunodetected in villous mononucleated cytotrophoblasts (VCT) and syncytiotrophoblast (ST) at 8–9 WA but only in ST at 12–14 WA. Furthermore, hCG secretion was fourfold higher in VCT cultures from 8–9 WA compared with 12–14 WA. Interestingly, VCT from 8–9 WA placentas were found to exhibit more fusion features. Taken together, we showed that type 1 and type 2 β-hCG are highly expressed by VCT in the early first trimester, contributing to the high levels of hCG found in maternal serum at this term.

The placenta provides critical transport functions between the maternal and fetal circulations during intrauterine development. Formation of this interface is controlled by nuclear transcription factors including homeobox genes. Here we summarize current knowledge regarding the expression and function of homeobox genes in the placenta. We also describe the identification of target transcription factors including PPARγ, biological pathways regulated by homeobox genes and their role in placental development. The role of the nuclear receptor PPARγ, ligands and target genes in human placental development is also discussed. A better understanding of these pathways will improve our knowledge of placental cell biology and has the potential to reveal new molecular targets for the early detection and diagnosis of pregnancy complications including human fetal growth restriction.

Human idiopathic foetal growth restriction (FGR) is frequently associated with placental insufficiency. In our previous studies, we have reported the isolation and characterisation of the homeobox gene Distal-less 3 (DLX3) in the human placenta. In this study, we have investigated the level of DLX3 expression in idiopathic FGR-affected placentae and determined its functional role in villous trophoblast differentiation. FGR-affected placentae (n = 25) were collected based on well-defined clinical criteria and matched for gestation with control uncomplicated pregnancies (n = 25). Real-time polymerase chain reaction and immunoblotting showed increased DLX3 mRNA and protein expression in FGR-affected placentae compared with gestation-matched controls. Qualitative immunohistochemistry revealed DLX3 localisation in the syncytiotrophoblast, cytotrophoblasts and endothelial cells surrounding the foetal capillaries in both FGR-affected and control placentae. Down-regulation of DLX3 in primary villous trophoblast cells and a trophoblast-derived cell line showed decreased expression of differentiation markers, 3βHSD, βhCG and syncytin. Therefore, we conclude that increased DLX3 expression in FGR may contribute to trophoblast dysfunction observed in FGR.