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Abnormal trophoblast function is associated with fetal growth restriction (FGR). The JAK-STAT pathway is one of the principal signalling mechanisms by which cytokines and growth factors modulate cell proliferation, differentiation, cell migration and apoptosis. The expression of placental JAK-STAT genes in human idiopathic FGR is unknown. In this study, we propose the hypothesis that JAK-STAT pathway genes are differentially expressed in idiopathic FGR-affected pregnancies and contribute to abnormal feto-placental growth by modulating the expression of the amino acid transporter SNAT2, differentiation marker CGB/human chorionic gonadotrophin beta-subunit (β-hCG) and apoptosis markers caspases 3 and 8, and TP53. Expression profiling of FGR-affected placentae revealed that mRNA levels of STAT3, STAT2 and STAT5B decreased by 69, 52 and 50%, respectively, compared with gestational-age-matched controls. Further validation by real-time PCR and immunoblotting confirmed significantly lower STAT3 mRNA and STAT3 protein (total and phosphorylated) levels in FGR placentae. STAT3 protein was localised to the syncytiotrophoblast (ST) in both FGR and control placentae. ST differentiation was modelled by in vitro differentiation of primary villous trophoblast cells from first-trimester and term placentae, and by treating choriocarcinoma-derived BeWo cells with forskolin in cell culture. Differentiation in these models was associated with increased STAT3 mRNA and protein levels. In BeWo cells treated with siRNA targeting STAT3, the mRNA and protein levels of CGB/β-hCG, caspases 3 and 8, and TP53 were significantly increased, while that of SNAT2 was significantly decreased compared with the negative control siRNA. In conclusion, we report that decreased STAT3 expression in placentae may contribute to abnormal trophoblast function in idiopathic FGR-affected pregnancies., (© 2015 Society for Reproduction and Fertility.)

Introduction: Solithromycin is a 4th generation macrolide/fluoroketolide antibiotic that has potential applications in the treatment and prevention of intrauterine and fetal infections in pregnancy; it has also been reported to exert anti-inflammatory effects. The objective of the present study was to determine its ability to cross the human placenta and inhibit cytokine production by placental and decidual cells in culture., Methods: Maternal-to-fetal passage of solithromycin was determined using the dual recycling ex vivo placental perfusion model; normal healthy term placentas delivered by Caesarean section were employed for the study. Creatinine transfer was also assessed as a diffusion-limited perfusion control. Purified primary decidual and trophoblast cells were treated in vitro for 20 h with solithromycin (0-100 μg/mL) and cytokine production and cell viability were assessed., Results: The mean ± SD maternal-to-fetal transfer ratio (TRf: concentration in maternal ÷ fetal circuit) of solithromycin after 3 h perfusion was 40.3 ± 23.6% (n = 4 placentas), with values from individual experiments ranging from 18 to 65%. The peak TRf of creatinine was 54%, and the clearance index for solithromycin (TRfsoli/TRfcreat) was 87% at 3 h. Solithromycin did not inhibit production of IL-6 and TNF-α by trophoblasts and decidual cells at non-toxic pharmacological concentrations (≤ 11 μg/mL)., Discussion: Solithromycin is the first antibiotic of its class to exhibit efficient maternal-to-fetal transfer across the human placenta and is thus an ideal candidate for evaluation for the treatment of intrauterine and fetal infections in pregnancy. At pharmacological concentrations it does not appear to inhibit pro-inflammatory cytokine production by placental cells., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Dlx3, a member of the large homeobox gene family of transcription factors, is important for murine placental development. Targeted deletion of Dlx3 in the mouse results in embryonic death due to placental failure. This study investigated the role of human DLX3 in villous cytotrophoblast (VCT) differentiation in the placenta. Primary VCT from human term placentae, which spontaneously differentiate when maintained in culture over 72 h, showed a significant increase in mRNA and protein expression of DLX3 and 3βHSD. The functional role of DLX3 was determined using trophoblast derived-cell line, BeWo. Forskolin treated BeWo cells showed significantly increased DLX3 mRNA and protein expression. Forskolin stimulation also showed a significant increase in syncytin and 3βHSD mRNA expression, and increased release of βhCG into the cell culture supernatant. To determine whether DLX3 had a direct or indirect effect on VCT differentiation, mRNA and protein expression of DLX3 was increased using a plasmid DLX3 over-expression construct. Over-expression of DLX3 resulted in increased mRNA expression of 3βHSD and syncytin, as well as increased secretion of β-hCG protein in the cell culture medium. In conclusion, we provide evidence that DLX3 acts upstream of syncytin, 3βHSD and βhCG and that DLX3 has a regulatory role in VCT differentiation., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Oxygenated cholesterol metabolites known as oxysterols display potent biological activities ranging from regulation of lipid homeostasis to cytotoxicity. Oxysterols have previously been shown to inhibit the invasion of first trimester trophoblasts, an effect which involves activation of the nuclear liver X receptors (LXRs). In the present study, we investigated the effects of several oxysterols on syncytialisation (differentiation and fusion) in term placental trophoblasts. Treatment of cultured term primary trophoblast cells with oxysterols [25-hydroxycholesterol, 7-ketocholesterol, 22(R)-hydroxycholesterol] and the synthetic LXR agonist T0901317 at non-toxic doses decreased expression of GCM-1 and HERV-W mRNA and reduced hCG secretion and placental alkaline phosphatase activity, indicative of diminished trophoblast differentiation. Furthermore, treatment with these compounds also decreased cell fusion measured by E-cadherin immunostaining and quantification of syncytialised nuclei. Treatment with an LXR antagonist (geranylgeranyl diphosphate) abrogated the inhibitory effects of oxysterols and T0901317 on trophoblast syncytialisation indicating that these effects are mediated by LXR. These findings suggest that oxysterols impair differentiation and fusion of term trophoblast cells via an LXR-dependent mechanism., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Trophoblast cells undergo loss of plasma membrane lipid asymmetry during cell fusion without further progression to terminal phases of apoptosis. The nature of the anti-apoptotic mechanisms providing cell survival during this process is unknown. Using a BeWo cell model, we explored the role of the xenobiotic/lipid transporter ABCG2 in promoting cell survival during forskolin-induced differentiation. Suppression of ABCG2 expression by siRNA led to a marked increase in phosphatidylserine externalisation followed by accumulation of ceramides and increased apoptosis. Expression of markers of syncytial formation (beta-hCG and HERV-W) was decreased by ABCG2 silencing, although fusion was unaffected. These findings suggest that ABCG2 protects cells during the period of transient membrane instability associated with cell differentiation and fusion, highlighting a novel, previously unrecognised role of ABCG2 as a survival factor during the formation of the placental syncytium.

Elevated activin A and inhibin A levels have been associated with pre-eclampsia, a pregnancy-related disorder associated with placental hypoxaemia. We investigated the effect of in vitro hypoxia on the production of inhibin A, activin A and its binding protein follistatin in term villous placental explants (n=4-7) and trophoblast monolayer cultures (n=4). Explants and trophoblasts were incubated for 24-72 h under either normoxic (21 per cent O(2)) or hypoxic (2 per cent O(2)) conditions. Production of activin A, inhibin A, and follistatin was determined by specific ELISA. After 48 h of hypoxia, villous explants exhibited a significant reduction in activin A production rates to 53.2 +/- 8.9 per cent (mean +/- SEM, P<0.05) of normoxic controls which was sustained after 72 h in culture (46.8 +/- 5.9 per cent), whereas production by trophoblast monolayers was not affected by hypoxia. Follistatin production was decreased to 53.7 +/- 9.2 per cent of control (P<0.05) after 48 h of hypoxia. Inhibin A production remained unaltered in both culture systems. Our data demonstrate for the first time that hypoxia lowers term placental activin A and follistatin production in vitro. These findings do not support the notion that elevated circulating activin A levels in pre-eclampsia originate from the placenta as a result of placental hypoxia. Other as yet unknown maternal/placental factors may contribute to elevated activin A production in women with severe pre-eclampsia., (Copyright 2002 Elsevier Science Ltd.)

Prostanoids have been proposed to play a major role in the regulation of uteroplacental blood flow. We examined the effect of hypoxia on the production of prostaglandin E(2)(PGE(2)) thromboxane B(2)(TXB(2)), and prostacyclin (measured as 6-keto-PGF(1alpha)) by human term trophoblast cells and villous placental explants. Explants (n=8) and purified trophoblast cells (n=5) were incubated for 24-72 h under either normoxic (21 per cent O(2)) or hypoxic (2 per cent O(2)) conditions. In trophoblast monolayer cultures, hypoxia attentuated PGE(2)production rates to 52+/-9.4 per cent (mean+/-sem, P< 0.05) but recovered to control rates within 48 h. In villous explants, PGE(2)production was significantly decreased after 48 and 72 h of hypoxia versus the normoxic control, accompanied by increased production of 6-keto-PGF(1alpha)to 173.9+/-26.7 per cent after 48 h. TXB(2)production was increased to 172.3+/-25.9 per cent and 653.2+/-135.7 per cent (P< 0.05) control after 48 and 72 h of hypoxia, respectively. These results were confirmed in villous explants (n=3) cultured in the presence of exogenous 10 microm arachidonic acid. Hypoxia had no significant effect on TXB(2)and 6-keto-PGF(1alpha)in trophoblast cells. In summary, our findings suggest that hypoxia could be responsible for abnormal profiles of prostanoid production commonly observed in women with pre-eclampsia. These results indicate a putative link between hypoxia and compromised placental perfusion., (Copyright 2001 Harcourt Publishers Ltd.)

Transforming growth factor-beta (TGF-beta) and activin-A, two members of a ubiquitous family of regulators of growth, differentiation and hormonogenesis, are produced by the human placenta. Their effects on placental hCG, inhibin, and oestrogen production in vitro, either alone or in combination, were investigated using cultured Percoll-purified placental trophoblasts. Inhibin and hCG were measured by immunoassay, while aromatase activity (i.e. oestrogen production) was measured using the tritiated water method. Aromatase activity and production of hCG, but not inhibin, were inhibited (up to approximately 30 per cent) in a dose-dependent fashion by 48 h treatment with TGF-beta. The effects were significant at all doses tested, from 0.1-10 ng/ml. In contrast, activin stimulated hCG production and aromatase activity over the doses tested (0.25-25 ng/ml). The maximum effect (approximately 50 per cent stimulation above control) was seen at the 2.5 ng/ml dose, with lesser effects seen at the lower and higher doses. This characteristic bell-shaped dose-response curve was maintained in the presence of TGF-beta (10 ng/ml) or a maximally-effective dose of forskolin (6.7 microM). This suggests that the actions of activin were independent of those of TGF-beta, and were not mediated by the protein kinase-A pathway. Activin had a weak stimulatory effect on inhibin production. The results indicate that in the placenta activin and TGF-beta have opposing actions on hormonogenesis. Both factors may play a role in regulating placental function and the timing and progression of labour.

In the present study, we investigated the roles of cyclic adenosine monophosphate (cAMP), intracellular calcium, glucocorticoids, protein kinase-C and gonadotrophin-releasing hormone (GnRH) in regulating human chorionic gonadotrophin (hCG), inhibin and activin production in cultured human term placental trophoblast cells. Inhibin and hCG were measured in conditioned media by radioimmunoassay, while putative forms of inhibin and activin were characterized by western blotting using affinity-purified antisera directed against the inhibin alpha- and beta A-subunits. Inhibin and hCG secretion were stimulated by dexamethasone (0.2 microM), GnRH (5-25 microM), calcium ionophore A23187 (0.2-1 microM), phorbol-12-myristate-13-acetate (22 nM) and epinephrine (1 microM), with increasing response over successive 24-h treatment periods. Two molecules Mr approximately 30 and 32 kDa appeared to be the predominant dimeric forms of inhibin secreted by the cells, while 26 kDa activin was present in excess over inhibin. Large amounts of 40-44 kDa protein were detected by the alpha-directed antisera only, which may be a form of the inhibin alpha-subunit precursor protein. Secretion of activin was responsive to phorbol ester-mediated stimulation but not to the presence of GnRH or elevated cAMP concentrations. The divergence in maternal serum inhibin and hCG concentrations during late pregnancy remains unexplained by these findings.