As a kind of endocrine-disrupting chemicals, BPA may affect the human placenta. Due to consumer unease about BPA, many manufacturers are using alternatives to BPA, such as BPS. However, some reports suggest that BPS may produce similar results to BPA. To understand how BPA/BPS leads to reduced synthesis of placental estradiol (E2), we conducted studies using a human choriocarcinoma cell (JEG-3) model for research. In this study. Elisa assay revealed that both BPA/BPS exposures decreased E2 synthesis in JEG-3 cells. The results of RT-PCR showed that both BPA and BPS could reduce the mRNA expression of CYP19A1, a key enzyme for E2 synthesis in JEG-3 cells. In addition, Western blot assay showed that BPA/BPS-induced ER-stress PERK/eIF2α/ATF4 signaling protein expression was increased. The expression of ROS in cells after exposure to BPA/BPS was detected using the 2,7-dichlorodihydrofluorescein diacetate (DCF-DA) method. The results of this experiment showed that BPA/BPS significantly induced an inhibition of ROS in JEG-3 cells. The present study concluded that, firstly, BPS exposure induced almost the same effect as BPA in reducing E2 synthesis in JEG-3 cells. Second, BPA/BPS exposure may reduce E2 synthesis in JEG-3 cells by increasing ROS levels and thus activating endoplasmic reticulum stress.

Hepatitis E virus (HEV) has become one of the important pathogens that threaten the global public health. Type 3 and 4 HEV are zoonotic, which can spread vertically and cause placental damage. At the same time, autophagy plays an important role in the process of embryo development and pregnancy maintenance. However, the relationship between HEV and autophagy, especially in the placenta tissue, has not been clarified. We found lower litter rates in HEV-infected female mice, with significant intrauterine abortion of the embryo (24.19%). To explore the effects of HEV infection on placenta autophagy, chorionic cells (JEG-3) and mice placenta have been employed as research objects, while the expression of autophagy-related proteins (ATGs) has been detected in JEG-3 cells with different times of HEV inoculation. The results demonstrated that the expression of protein LC3 decreased and p62 accumulated, meanwhile ATGs such as ATG4B, ATG5, and ATG9A in JEG-3 cells have decreased significantly. In addition, the maturation of autophagosomes, which referred to the process of the combination of autophagosomes and lysosomes was prevented by HEV infection as well. All processes of autophagic flux, which include the initiation, development, and maturation three stages, were suppressed in JEG-3 cells after HEV infection. Similarly, the protein and gene expression of LC3 were significantly decreased in the placenta of pregnant mice with HEV infection. In summary, our results suggested that HEV inhibited autophagy in JEG-3 cells and placenta of pregnant mice, which might be the important pathogenic mechanisms of HEV infection leading to embryo abortion.

Oxygen tension varies during placental and fetal development. Although hypoxia drives early trophoblast invasion, low placental oxygen levels during pregnancy show association with pregnancy complications including fetal growth restriction and preeclampsia. JEG-3 cells are often used as a trophoblast model. We studied transcriptional changes of JEG-3 cells on a uterine leiomyoma derived matrix Myogel. This might be the closest condition to the real uterine environment that we can get for an in vitro model. We observed that culturing JEG-3 cells on the leiomyoma matrix leads to strong stimulation of ribosomal pathways, energy metabolism, and ATP production. Furthermore, Myogel improved JEG-3 cell adherence in comparison to tissue culture treated plastic. We also included PDMS microchip hypoxia creation, and observed changes in oxidative phosphorylation, oxygen related genes and several hypoxia genes. Our study highlights the effects of Myogel matrix on growing JEG-3 cells, especially on mitochondria, energy metabolism, and protein synthesis.

The placenta is a vital fetal organ that plays an important role in maintaining fetal sex hormone homeostasis. Xenobiotics can alter placental sex-steroidogenic enzymes and transporters, including enzymes such as aromatase (CYP19A1) and the hydroxysteroid dehydrogenases (HSDs) but studying how compounds disrupt in vivo placental metabolism is complex. Utilizing high-throughput in vitro models is critical to predict the disruption of placental sex-steroidogenic enzymes and transporters, particularly by drug candidates in the early stages of drug discovery. JAR and JEG-3 cells are the most common, simple, and cost-effective placental cell models that are capable of high-throughput screening, but how well they express the sex-steroidogenic enzymes and transporters is not well known. Here, we compared the proteomes of JAR and JEG-3 cells in the presence and absence of physiologically relevant concentrations of dehydroepiandrosterone (DHEA, 8 µM) and testosterone (15 nM) to aid the characterization of sex-steroidogenic enzymes and transporters in these cell models. Global proteomics analysis detected 2931 and 3449 proteins in JAR cells and JEG-3 cells, respectively. However, dramatic differences in sex-steroidogenic enzymes and transporters were observed between these cells. In particular, the basal expression of steroid sulfatase (STS), HSD17B1, and HSD17B7 were unique to JEG-3 cells. JEG-3 cells also showed significantly higher protein levels of aldo-keto reductase (AKR) 1A1 and AKR1B1, while JAR cells showed significantly higher levels of HSD17B4 and HSDB12. Aldehyde dehydrogenase (ALDH) 3A2 and HSD17B11 enzymes as well as the transporters sterol O-acyltransferase (SOAT) 1 and ATP binding cassette subfamily G2 (ABCG2) were comparable between the cell lines, whereas sulfotransferases (SULTs) were uniquely present within JAR cells. Androgen treatments significantly lowered HSD17B11, HSD17B4, HSD17B12, and ALDH3A2 levels in JAR cells. DHEA treatment significantly raised the level of HSD17B1 by 51 % in JEG-3 cells, whereas CYP19A1 was increased to significant levels in both JAR and JEG-3 cells after androgen treatments. The proteomics data were supported by a complementary targeted metabolomics analysis of culture media in the DHEA (8 µM) and testosterone (15 nM) treated groups. This study has indicated that untreated JEG-3 cells express more sex-steroidogenic enzymes and transporters. Nevertheless, JEG-3 and JAR cells are unique and their respective proteomics data can be used to select the best model depending on the hypothesis.

During pregnancy, uterine NK cells interact with trophoblast cells. In addition to contact interactions, uterine NK cells are influenced by cytokines, which are secreted by the cells of the decidua microenvironment. Cytokines can affect the phenotypic characteristics of NK cells and change their functional activity. An imbalance of pro- and anti-inflammatory signals can lead to the development of reproductive pathology. The aim of this study was to assess the effects of cytokines on NK cells in the presence of trophoblast cells in an in vitro model. We used TNFα, IFNγ, TGFβ and IL-10; the NK-92 cell line; and peripheral blood NK cells (pNKs) from healthy, non-pregnant women. For trophoblast cells, the JEG-3 cell line was used. In the monoculture of NK-92 cells, TNFα caused a decrease in CD56 expression. In the coculture of NK cells with JEG-3 cells, TNFα increased the expression of NKG2C and NKG2A by NK-92 cells. Under the influence of TGFβ, the expression of CD56 increased and the expression of NKp30 decreased in the monoculture. After the preliminary cultivation of NK-92 cells in the presence of TGFβ, their cytotoxicity increased. In the case of adding TGFβ to the PBMC culture, as well as coculturing PBMCs and JEG-3 cells, the expression of CD56 and NKp44 by pNK cells was reduced. The differences in the effects of TGFβ in the model using NK-92 cells and pNK cells may be associated with the possible influence of monocytes or other lymphoid cells from the mononuclear fraction.

Pregnancy success depends greatly on a balanced immune homeostasis. The detection of bacterial components in the upper reproductive tract in non-pregnant and pregnant women raised questions on its possible beneficial role in reproductive health. The local conditions that allow the presence of bacteria to harmonize with the establishment of pregnancy are still unknown. Among the described bacterial species in endometrial and placental samples, Fusobacterium nucleatum was found. It has been observed that F. nucleatum can induce tumorigenesis in colon carcinoma, a process that shares several features with embryo implantation. We propose that low concentrations of F. nucleatum may improve trophoblast function without exerting destructive responses. Inactivated F. nucleatum and E. coli were incubated with the trophoblastic cell lines HTR8/SVneo, BeWo, and JEG-3. Viability, proliferation, migratory capacity, invasiveness and the secretion of chemokines, other cytokines and matrix metalloproteinases were assessed. The presence of F. nucleatum significantly induced HTR8/SVneo invasion, accompanied by the secretion of soluble mediators (CXCL1, IL-6 and IL-8) and metalloproteinases (MMP-2 and MMP-9). However, as concentrations of F. nucleatum increased, these did not improve invasiveness, hindered migration, reduced cell viability and induced alterations in the cell cycle. Part of the F. nucleatum effects on cytokine release were reverted with the addition of a TLR4 blocking antibody. Other effects correlated with the level of expression of E-cadherin on the different cell lines tested. Low amounts of F. nucleatum promote invasion of HTR8/SVneo cells and induce the secretion of important mediators for pregnancy establishment. Some effects were independent of LPS and correlated with the expression of E-cadherin on trophoblasts.

NK cell development is affected by their cellular microenvironment and cytokines, including IL-15 and IL-18. NK cells can differentiate in secondary lymphoid organs, liver and within the uterus in close contact with trophoblast cells. The aim was to evaluate changes in the NK cell phenotype and function in the presence of IL-15, IL-18 and JEG-3, a trophoblast cell line. When cocultured with JEG-3 cells, IL-15 caused an increase in the number of NKG2D+ NK-92 cells and the intensity of CD127 expression. IL-18 stimulates an increase in the amount of NKp44+ NK-92 cells and in the intensity of NKp44 expression by pNK in the presence of trophoblast cells. NK-92 cell cytotoxic activity against JEG-3 cells increased only in presence of IL-18. Data on changes in the cytotoxic activity of NK-92 cells against JEG-3 cells in the presence of IL-15 and IL-18 indicate the modulation of NK cell function both by the cytokine microenvironment and directly by target cells. IL-15 and IL-18 were present in conditioned media (CM) from 1st and 3rd trimester placentas. In the presence of 1st trimester CM and JEG-3 cells, NK-92 cells showed an increase in the intensity of NKG2D expression. In the presence of 3rd trimester CM and JEG-3 cells, a decrease in the expression of NKG2D by NK-92 cells was observed. Thus, culturing of NK-92 cells with JEG-3 trophoblast cells stimulated a pronounced change in the NK cell phenotype, bringing it closer to the decidual NK cell-like phenotype.

OBJECTIVE: The present study was to estimate the role of cytokines for trophoblast death in NK cells presence.
METHODS: This study involves assessment of NK-92 line NK cell cytotoxic activity against JEG-3 line cells, in presence of cytokines. We also assessed the effect of secretory placenta products on NK cell cytotoxic activity toward JEG-3 line cells.
RESULTS: Uteroplacental contact zone cytokines are able to enhance trophoblast mortality both by themselves in case of IL-1β, IL-6, IFNγ, IL-4, TGFβ, bFGF, and also through increasing the cytotoxic potential of NK cells in case of IL-1β, IFNγ, IL-8, TGFβ, and GM-CSF. PLGF decreases NK cell cytotoxicity for trophoblasts. Secretory products of first trimester placenta enhance NK cell cytotoxic potential for trophoblasts.
CONCLUSIONS: Cytokines of the uteroplacental contact zone can appear a mechanism ensuring trophoblast mortality dynamics throughout pregnancy.

Lamotrigine (LTG) is an important antiepileptic drug for the treatment of seizures in pregnant women with epilepsy. However, it is not known if the transport of LTG into placental cells occurs via a carrier-mediated pathway. The aim of this study was to investigate the uptake properties of LTG into placental cell lines (BeWo and JEG-3), and to determine the involvement of organic cation transporters (OCTs, SLC22A1-3) and organic cation/carnitine transporter (OCTNs, SLC22A4-5) in the uptake process. The uptake of LTG at 37 °C was higher than that at 4 °C. OCT1 and OCTNs were detected in both cell lines. The uptake of LTG was not greatly affected by the extracellular pH, Na+-free conditions, or the presence of l-carnitine, suggesting that OCTNs were not involved. Although several potent inhibitors of OCTs (chloroquine, imipramine, quinidine, and verapamil) inhibited LTG uptake, other typical inhibitors had no effect. In addition, siRNA targeted to OCT1 had no significant effect on LTG uptake. The mRNA expression in human term placenta followed the order OCTN2 > OCT3 > OCTN1 > OCT1 ≈ OCT2. These observations suggested that LTG uptake into placental cells was carrier-mediated, but that OCTs and OCTNs were not responsible for the placental transport process.

Steroid sulfatase (STS) has recently emerged as a drug target for management of hormone-dependent malignancies. In the present study, a new series of twenty-one aryl amido-linked sulfamate derivatives 1a-u was designed and synthesized, based upon a cyclohexyl lead compound. All members were evaluated as STS inhibitors in a cell-free assay. Adamantyl derivatives 1h and 1p-r were the most active with more than 90% inhibition at 10 µM concentration and, for those with the greatest inhibitory activity, IC50 values were determined. These compounds exhibited STS inhibition within the range of ca 25-110 nM. Amongst them, compound 1q possessing a o-chlorobenzene sulfamate moiety exhibited the most potent STS inhibitory activity with an IC50 of 26 nM. Furthermore, to assure capability to pass through the cell lipid bilayer, compounds with low IC50 values were tested against STS activity in JEG-3 whole-cell assays. Consequently, 1h and 1q demonstrated IC50 values of ca 14 and 150 nM, respectively. Thus, compound 1h is 31 times more potent than the corresponding cyclohexyl lead (IC50 value = 421 nM in a JEG-3 whole-cell assay). Furthermore, the most potent STS inhibitors (1h and 1p-r) were evaluated for their antiproliferative activity against the estrogen-dependent breast cancer cell line T-47D. They showed promising activity with single digit micromolar IC50 values (ca 1-6 µM) and their potency against T-47D cells was comparable to that against STS enzyme. In conclusion, this new class of adamantyl-containing aryl sulfamate inhibitor has potential for further development against hormone-dependent tumours.