Trophoblast Cell Apoptosis Research Articles

Leptin protects placental cells from apoptosis induced by acidic stress.

Development of the human placenta is critical for a successful pregnancy. The placenta allows the exchange of oxygen and carbon dioxide and is crucial to manage acid-base balance within a narrow pH. It is known that low pH levels are a risk of apoptosis in several tissues. However, there has been little discussion about the effect of acidic stress in the placenta. Leptin is produced by the placenta with a trophic autocrine effect. Previous results of our group have demonstrated that leptin prevents apoptosis of trophoblast cells under different stress conditions such as serum deprivation and hyperthermia. The purpose of the present work is to evaluate acidic stress consequences in trophoblast explant survival and to determine leptin action in these conditions. For this objective, term human trophoblast explants were cultured at physiological pH (pH 7.4) and at acidic pH (pH 6.8) in the presence or absence of leptin. Western blot assays were performed to study the abundance of active caspase-3 and the p89 fragment of PARP-1. Pro-apoptotic and pro-survival members of Bcl-2 family, as Bax, t-Bid, and Bcl-2, were studied. Moreover, p53 pathway was also evaluated including Mdm-2, the main p53 regulator. Active caspase-3 and cleaved PARP-1 abundances were increased at low extracellular pH. Moreover, t-Bid levels were also augmented as well as p53 expression and phosphorylation on S46. Leptin treatment prevents the consequences of acidosis, decreasing p53 expression and increasing Mdm-2 expression. In summary, this work demonstrated for first time that low pH induces apoptosis of human trophoblast explants involving apoptotic intrinsic pathway, and leptin impairs this effect.

Curcumin induces apoptosis in trophoblast model cell line

Background: Several studies have reported that curcumin exerts chemopreventive effects in various type of cancers, through several mechanisms, however, the effect of curcumin on carcinogenesis in patients with hydatidiform mole has not yet been investigated. This study was conducted to evaluate the effect of curcumin on apoptosis, proliferation, and nuclear translocation of endothelial nitricoxide synthase in trophoblast cells induced by estradiol in complete hydatidiform mole (CHM).Methods: In this in vitro study, trophoblast cells were divided into six groups, the control group (trophoblast cells were exposed to 100 pg/mL of 17-β estradiol) and the treatment group (trophoblast cells were exposed to 100 pg/mL of 17-β estradiol in the presence of curcumin with doses: 50, 100, 200, 400, and 800 µM). At the end of study, the cell proliferation was analyzed using MTT assay and apoptosis with TUNEL test in each group thropoblast cell. eNOS translocation was assayed using confocal laser scanning microscopy at the various dose of curcumin.Results: Curcumin at the doses of 200, 400, and 800 µM significantly decreased the proliferation and increased the apoptotic index in curcumin-treated group compared to those in the control group (p<0.05). All doses of curcumin treatment significantly decreased the nuclear eNOS expression compared to that in the control group. The three highest doses of curcumin increased cytoplasmic eNOS expression compared to that in control group.Conclusion: Curcumin inhibits the proliferation and modulates the apoptosis of trophoblast cells induced by estradiol in CHM involvement.

MicroRNA-221/222 participates in the pathogenesis of intrahepatic cholestasis of pregnancy via promoting the apoptosis of human placental trophoblast HTR-8 cells

Objective: MicroRNA-221/222 is involved in the pathogenesis of intrahepatic cholestasis of pregnancy (ICP) to promote the apoptosis of placental bile acids through human trophoblastic cells. This study investigates the effects of miR-221/222 on proliferation, apoptosis and apoptosis-related proteins of human trophoblast HTR-8/SVneo (HTR-8 cells) to understand its role in promoting trophoblastic apoptosis. Methods: The experiment was divided into transfection group and negative control group. Transient transfection method was used in both groups. The transfection efficiency was detected by RT-QPCR after 48 h transfection. CCK-8 was used to detect the proliferation of HTR-8 cells and the apoptosis of HTR-8 cells were analyzed by flow cytometry. Western blot was used to detect the expression of B-cell Lymphoma 2 (Bcl-2) in HTR-8 cells. Data were compared with t-test. Results: The expression of miR-221/222 transfected group (25.43±0.80, 22.70±0.95) was increased significantly in the HTR-8 cells than that to negative control group (1.14±0.14, 1.58±0.14), and P value was < 0.01, the difference was statistically significant. The expression of Bcl-2 protein in mir-221/222 transfection group was (0.56 ± 0.03, 0.53 ± 0.03), and the protein expression was decreased compared with negative control group (0.72 ± 0.003, 0.76 ± 0.04). P value was < 0.05, the difference was statistically significant, and compared with the mir-221/222 negative control group (8.827 ± 0.48, 11.80 ± 0.45), cell apoptosis of mir-221/222 transfection group (42.53 ± 4.47, 24.09 ± 2.53) increased significantly, P value was < 0.01, and the difference was statistically significant. Proliferation rate in mir-221/222 transfection group was (0.82 ± 0.02, 0.74±0.01), and proliferation was inhibited, when compared with control group (1.15 ± 0.08, 1.06 ± 0.08), P value was < 0.05, and the difference was statistically significant. Conclusion: miR-221/222 may promote the apoptosis of human trophoblastic cells by down regulating the expression of apoptosis inhibitory protein bcl-2, leading to placental dysfunction and impairing the normal bile acid transport function of placenta. This mechanism may be involved in the occurrence and development of ICP.

MicroRNA-454 is involved in regulating trophoblast cell proliferation, apoptosis, and invasion in preeclampsia by modulating the expression of ephrin receptor B4

Preeclampsia (PE) is a pregnancy-specific disorder representing a major cause for maternal and perinatal morbidity and mortality. The dysfunction of trophoblast cells plays an important role in the pathogenesis of PE. In recent years, microRNAs (miRNAs) have been suggested to play an important role in regulating trophoblast cell biological functions involved in the pathogenesis of PE. Accumulating evidence has showed that miR-454 plays an important role in regulating cell functions. However, whether miR-454 is involved in regulating cell functions of trophoblast cells during PE remains unclear. In this study, we found that miR-454 expression was significantly downregulated in placental tissues from PE patients. in vitro experiments showed that miR-454 overexpression significantly increased proliferation, inhibited apoptosis, and promoted invasion of trophoblast cells, whereas miR-454 inhibition markedly suppressed proliferation, increased apoptosis, and inhibited invasion of trophoblast cells. Interestingly, bioinformatics analysis predicted that ephrin receptor B4 (EPHB4), an important gene for regulating trophoblast cell function in PE, was a potential target gene of miR-454. Dual-luciferase reporter assay showed that miR-454 directly targeted the 3’-untranslated region of EPHB4. Real-time quantitative polymerase chain reaction and Western blot analysis demonstrated that miR-454 negatively regulated EPHB4 expression in trophoblast cells. Moreover, miR-454 expression was found inversely correlated with EPHB4 expression in placental tissues from PE patients. Importantly, EPHB4 overexpression partially reversed the promotion effect of miR-454 overexpression on trophoblast cell proliferation and invasion. Taken together, these findings demonstrate that miR-454 promotes the proliferation and invasion of trophoblast cells by inhibiting EPHB4 expression, and the decreased miR-454 expression may contribute to PE by promoting EPHB4 expression. Our study provides novel insights into understanding the molecular pathogenesis of PE.

Hypermethylation of endoplasmic reticulum disulfide oxidase 1α leads to trophoblast cell apoptosis through endoplasmic reticulum stress in preeclampsia.

Abnormal trophoblast cell apoptosis is implicated in the pathogenesis of pregnancy-related disorders including preeclampsia (PE), and endoplasmic reticulum (ER) stress has been considered as a novel pathway in the regulation of cell apoptosis. In this study, we observed that both apoptosis and ER stress are triggered in trophoblast cells under hypoxia as well as in the placenta of PE rats. Quantitative polymerase chain reaction and Western blot analysis showed that the expression of endoplasmic reticulum disulfide oxidase 1α (ERO1α) is suppressed in trophoblast cells under hypoxia due to the hypermethylation of the ERO1α promoter region, and the inhibition of ERO1α expression plays an important role in ER stress and trophoblast cell apoptosis. Furthermore, we found that DNA methyltransferase 1 (DNMT1) is a key methyltransferase for DNA methylation in the regulation of ERO1α expression, and the binding level of DNMT1 to the ERO1α promoter is markedly elevated under hypoxia although DNMT1 expression is inhibited by hypoxia, suggesting that the binding level of DNMT1 to the ERO1α promoter region rather than the DNMT1 expression level contributes to the hypermethylation of ERO1α. Taken together, these results demonstrate that the hypermethylation of ERO1α mediated by increased binding of DNMT1 to the ERO1α promoter leads to trophoblast cell apoptosis through ER stress in the placenta of PE rats, which shed insight into the etiology of PE and might present a validated therapeutic target for the treatment of PE.

Synergistic effects of tumor necrosis factor-α and insulin-like growth factor-I on survival of human trophoblast-derived BeWo cell line

ObjectiveTrophoblast survival is regulated by cytokines and growth factors. While the pharmacological levels (10–100 ng/mL) of tumor necrosis factor (TNF)- α affect trophoblasts survival in vitro, the effects of the physiological levels (1–10 pg/mL) of TNF-α remain unknown. We investigated the effects of the physiological levels of TNF-α on proliferation and apoptosis of human trophoblast cells by using BeWo cells. Insulin-like growth factor (IGF)-I is also a potent regulator of trophoblast survival and has been known to exert synergistic effects with other hormones. The interaction of IGF-I and TNF-α on BeWo cells survival was also examined. MethodsAfter incubating BeWo under the presence of TNF-α (10–105 pg/mL) and IGF-I (102 ng/mL), we assessed cell number by WST-1 assay and cell proliferation by BrdU uptake assay and immunocytochemistry with anti-Ki67 antibody. Apoptosis was evaluated by TUNEL assay and caspase-3, 8 activity assays. ResultsUnder the presence of IGF–I, cell number, BrdU uptake, and Ki-67 expression of BeWo were dose-dependently enhanced by low TNF-α (10–102 pg/mL), while no such effects were detected without IGF–I. Higher levels of TNF-α (104–105 pg/mL) showed inhibiting effects on cell number and cell proliferation. The number of TUNEL positive cells were decreased and caspase activities were suppressed by lower levels (10–102 pg/mL) of TNF-α and IGF-I independently. Higher levels of TNF-α (104–105 pg/mL) showed promoting effects on apoptosis irrespective of IGF–I. ConclusionThe physiological levels of TNF-α and IGF-I had synergetic effects on enhancing cell proliferation and also independently inhibited apoptosis of Bewo cells.

P38: MicroRNA‐184 promotes apoptosis of trophoblast cells by up‐regulating Fas expression and inhibits differentiation of decidual regulatory T cells at the maternal‐fetal interface

P38: MicroRNA‐184 promotes apoptosis of trophoblast cells by up‐regulating Fas expression and inhibits differentiation of decidual regulatory T cells at the maternal‐fetal interface

P-239 - Underpinning the endocannabinoid 2-AG induced-apoptosis in trophoblast cells: the role of oxidative stress

The endocannabinoid system (ECS) regulates several reproductive events such as decidualization, implantation and placentation. Placental development involves a balance between proliferation, differentiation and cell death of trophoblasts. We have reported that 2-AG induces apoptosis in trophoblast cells [1], on which endoplasmic reticulum (ER)-stress might play a role. Prolonged ER-stress may activate pancreatic ER kinase (PERK) protein and trigger apoptotic pathways. Therefore, in this work it was evaluated the role of PERK branch on 2-AG induced-apoptosis in BeWo cell line, a model of cytotrophoblasts. Through q-PCR it was observed that 2-AG increased mRNA levels of crucial ER-stress markers: CHOP and ATF4. Through Western-blot we found that 2-AG also increased CHOP protein levels and activation/phospohorylation of eIF2α. The involvement of cannabinoid receptors was also investigated. Herein we found that 2-AG induced apoptosis is mediated by activation of PERK branch of ER-stress response which may impair placental development, and lead to miscarriage, fetal growth-restriction and pregnancy-related diseases.

Toxoplasma gondii dense granule protein 15 induces apoptosis in choriocarcinoma JEG-3 cells through endoplasmic reticulum stress

BackgroundToxoplasma gondii, a single-celled parasite commonly found in mammals, has been shown to induce trophoblast cell apoptosis and subsequently cause fetal damage and abortion. Although dense granule protein 15 (GRA15) has been identified as a key component in innate immunity to T. gondii infection and its pathogenesis, its role in host cell apoptosis remains unclarified.MethodsType II GRA15 (GRA15II) cDNA was inserted into a plasmid encoding enhanced green fluorescent protein (pEGFP). Choriocarcinoma JEG-3 cells were transfected with either pEGFP or pEGFP-GRA15II and cultured for 24 h. Cell apoptosis and endoplasmic reticulum stress (ERS) responses were assessed. Inhibitors targeting inositol-requiring kinase 1α (IRE1α; 4μ8C, 100 nM) or c-Jun N-terminal kinase (JNK; SP6000125, 20 μM) were added 12 h after plasmid transfection, followed by testing the effect of GRA15II on ERS.ResultsWhen compared to pEGFP, pEGFP-GRA15II transfection facilitated cell apoptosis (P < 0.05), increased mRNA expression of caspase-3, caspase-4, 78-kDa glucose-regulated protein (GRP78), C/EBP homologous protein (CHOP) and X-box binding protein-1 (XBP1) (all P < 0.05), and promoted protein expression of cleaved caspase-3, cleaved poly(ADP-ribose) polymerase, Bax, CHOP, GRP78, phospho-JNK, and phospho-IRE1α (all P < 0.05). The 4μ8C and SP6000125 decreased apoptosis and protein expression of XBP1s, CHOP, TNF receptor-associated factor 2 (TRAF2), phosphorylated apoptosis signal-regulating kinase 1 (ASK1), cleaved caspase-3, phospho-JNK, and Bax (all P < 0.05) in pEGFP-GRA15II transfected cells.ConclusionsToxoplasma GRA15II induced ERS and subsequently caused apoptosis of choriocarcinoma JEG-3 cells.

Role of X-linked inhibitor of apoptosis (XIAP) in frozen and thawed dormant and normal-hatched murine blastocysts

Cryo-injury of mammalian blastocysts occurs during cryopreservation and induces apoptosis in trophoblast cells. This damage affects subsequent embryo development or may even cause death before implantation. X-linked inhibitor of apoptosis (XIAP) is an anti-apoptosis gene that has been widely studied in cancer research. However, only a few studies have investigated the activity of XIAP in cryopreservation. In this study, we investigate the role of XIAP in frozen and thawed murine blastocysts. A total of 1630 blastocysts were divided into fresh and freeze-thaw groups, and XIAP expression was investigated using qPCR, Western blot and confocal analyses. In addition, the effect of the embelin (a XIAP inhibitor) was also evaluated by co-culturing 390 dormant blastocysts. XIAP protein is primarily localized to the mitochondria of trophoblastic cells. Gene and protein expression is significantly down-regulated in blastocysts after cryopreservation, whereas embelin has negative effect on their survivals. These findings further broaden the understanding of mammalian embryonic cryopreservation.

Cell Cycle Arrest and Apoptosis Induced by Porphyromonas gingivalis Require Jun N-Terminal Protein Kinase- and p53-Mediated p38 Activation in Human Trophoblasts.

Porphyromonas gingivalis, a periodontal pathogen, has been implicated as a causative agent of preterm delivery of low-birth-weight infants. We previously reported that P. gingivalis activated cellular DNA damage signaling pathways and ERK1/2 that lead to G1 arrest and apoptosis in extravillous trophoblast cells (HTR-8 cells) derived from the human placenta. In the present study, we further examined alternative signaling pathways mediating cellular damage caused by P. gingivalis. P. gingivalis infection of HTR-8 cells induced phosphorylation of p38 and Jun N-terminal protein kinase (JNK), while their inhibitors diminished both G1 arrest and apoptosis. In addition, heat shock protein 27 (HSP27) was phosphorylated through both p38 and JNK, and knockdown of HSP27 with small interfering RNA (siRNA) prevented both G1 arrest and apoptosis. Furthermore, regulation of G1 arrest and apoptosis was associated with p21 expression. HTR-8 cells infected with P. gingivalis exhibited upregulation of p21, which was regulated by p53 and HSP27. These results suggest that P. gingivalis induces G1 arrest and apoptosis via novel molecular pathways that involve p38 and JNK with its downstream effectors in human trophoblasts.

MiR-195 modulates oxidative stress-induced apoptosis and mitochondrial energy production in human trophoblasts via flavin adenine dinucleotide-dependent oxidoreductase domain-containing protein 1 and pyruvate dehydrogenase phosphatase regulatory subunit.

Preeclampsia is a severe pregnancy-specific syndrome defined as newly onset hypertension and proteinuria. Abnormal placental development has been generally accepted as the initial cause of the disorder. Recently, miR-195 was identified as one of the downregulated small RNAs in preeclamptic placentas. The potential targets of miR-195 in human trophoblast cells were screened by isobaric tags for relative and absolute quantification-based mass spectrum analysis. Localization of miR-195 and its targets was examined by in-situ hybridization and immunohistochemistry in human placenta. Real-time PCR, western blotting and luciferase assay were used for target validation. Apoptosis was accessed by Annexin V/PI costaining, whereas mitochondrial function by ATP measurement and tetramethylrhodamine ethyl ester fluorescence. Two mitochondria-associated proteins, flavin adenine dinucleotide-dependent oxidoreductase domain-containing protein 1 (FOXRED1) and pyruvate dehydrogenase phosphatase regulatory subunit (PDPR), were identified as targets of miR-195. Overexpression of miR-195 in HTR8/SVneo cells resulted in enhanced apoptosis, decreased mitochondrial membrane potential and cellular ATP content upon hydrogen peroxide stimulation. The effects could be partially rescued by FOXRED1 or PDPR. In preeclamptic patients, lowered circulating level of miR-195 were found at early-to-mid gestation and term pregnancy, and marked increase in FOXRED1 and PDPR expression were observed in the placenta when compared with gestational week-matched controls. In addition, chronic hydrogen peroxide stimuli suppressed miR-195 expression in trophoblast cells. MiR-195 could suppress mitochondrial energy production via targeting FOXRED1 and PDPR, and lead to trophoblast cell apoptosis under oxidative stress. In preeclamptic placenta, lowered level of miR-195 might be induced by chorionic oxidative stress and subsequently form a compensation mechanism to defend the disturbed energy production and cell apoptosis upon oxidative stress.

Pengaruh Insulin, Jahe dan Kombinasi Keduanya Terhadap Jumlah Sel Trofoblas Rattus Norvegicus Model Diabetes Pragestasional

Diabetes mellitus (DM) is a metabolic disease with characteristics of hyperglycemia that has an effect on increasing morbidity and mortality in the mother. One of the complications of pregestational DM (pGDM) is preeclampsia (PE). The perinatal mortality rate increases 20x in pGDM women with PE complication. Exposure to hyperglycemia causes an increase in oxidative stress that increases apoptosis of trophoblast cells which in turn causes the decrease of proliferation and the number of trophoblastic cells and leads to endothelial dysfunction and hypoxia. The purpose of this study was to distinguish the effect of insulin administration and ginger extract on the number of trophoblast cells of R. norvegicus. This research was an experimental research using post-test only control group design. The population used were pregnant R. norvegicus. The sampling technique was simple random sampling with independent variables are insulin and Z. officinale extract and the dependent variable is the number of trophoblast cells. Data analysis of the number of trophoblast cells used wereKruskal-Wallis and Mann-Whitney test. The result showed that there was a significant difference in the trophoblast cell number with p-value <0,05 (p = 0,001) on positive control with insulin treatment group. The conclusion of this study was the presence of ginger extract can be used as a single therapy or combination with insulin for pregestational diabetes.

Butyl paraben promotes apoptosis in human trophoblast cells through increased oxidative stress-induced endoplasmic reticulum stress.

Butyl paraben (BP) has antimicrobial effects and is widely used as a preservative in cosmetics, foods, and pharmaceuticals. It is also absorbed into various tissues of the human body. It is known that BP is measurable in maternal and fetal tissues during pregnancy, but the effects of BP on placental development, essential for maintaining normal pregnancy, are unclear. Therefore, we investigated the effect of BP on the proliferation, apoptosis, and invasiveness of human trophoblast cells, using an HTR8/SVneo cell line. BP inhibited cell proliferation and induced both apoptosis and endoplasmic reticulum stress. In addition, BP promoted the production of intracellular reactive oxygen species, increased Ca2+ concentration in HTR8/SVneo cells, and induced mitochondrial membrane depolarization. BP also inhibited the activation of PI3K/AKT pathways including AKT, ribosomal protein S6, P70 S6 kinase, and glycogen synthase kinase 3β. Furthermore, pretreatment of cells with LY294002 (an AKT inhibitor) and U0126 (ERK1/2 inhibitor) revealed that ERK1/2 activity is also involved in BP-mediated signal transduction in HTR8/SVneo cells. We therefore suggest that exposing human trophoblast cells to BP diminishes normal physiological activity, leading to apoptosis and problems with early placental development.

Benzo(a)pyren-7,8-dihydrodiol-9,10-epoxide induces human trophoblast Swan 71 cell dysfunctions due to cell apoptosis through disorder of mitochondrial fission/fusion.

Benzo(a)pyren-7,8-dihydrodiol-9,10-epoxide (BPDE) is an endocrine disrupter and ultimate carcinogenic product of benzo(a)pyrene (BaP). Numerous studies have shown that BPDE causes trophoblast-related diseases, such as preeclampsia, growth restriction or miscarriages. However, the underlying mechanism, especially the mitochondria-related BPDE-induced trophoblast dysfunction remains unknown. In this study, we examined mitochondrial functions in BPDE-induced human trophoblast cell line Swan 71. BPDE decreased cell ability, attenuated cell invasion and HCG secretion, induced cell apoptosis, decreased mitochondrial membrane potential, increased reactive oxygen species (ROS) and MDA, and decreased SOD activity in a dose-dependent manner. In the mechanism, BPDE significantly increased pro-apoptosis protein (P53 and Bak1) and decreased anti-apoptosis protein (Bcl-2). Furthermore, the protein expression levels of mitochondrial fusion genes (Mfn1, Mfn2, and OPA1) were decreased and those of fission genes (Fis1 and Drp1) were increased with increasing concentrations of BPDE and incubation time, resulting in the release of Cyt c and activation of Caspase 3, which irreversibly induced trophoblast cell apoptosis. This study reveals the mechanism of dysfunction of trophoblast cells through cell apoptosis due to the disorder of mitochondrial fission/fusion after exposure to BPDE, providing a further experimental understanding the adverse effects of BaP on trophoblast cells in early pregnancy.

English

Preeclampsia is one of the main causes of maternal and perinatal mortality and morbidity. The pathogenesis of preeclampsia remains unclear until now. It is believed that regulation of apoptosis in trophoblast cells plays an important role in the pathophysiology of preeclampsia. Failure of spiral arteries remodeling will eventually lead to placental hypoxia lead to excessive trophoblast apoptosis. The molecular mechanism of apoptosis is very complicated involving many signaling molecules included Bcl-2 proteins. The Bcl- 2 protein group consists of proapoptosis proteins (Bax) and apoptosis inhibitor proteins (Bcl-2 and Bcl-xL). The aimed of this stuty was to compare the expression of Bcl-xL protein in placental trophoblast cells of pregnancy complicated by severe preeclampsia with that normotensive pregnancy. This study was an observational study with cross sectional design involving 43 pregnancy patients with severe preeclampsia and 38 normotensive pregnancy who treated in Dr. Sardjito General Hospital, Yogyakarta from October 2011 until March 2012. Placenta samples were obtained from all subjects for Bcl-xL protein expression analysis using immunohistochemistry technique. Data were analyzed using independent t-test, chi-square test, and logistic regression. A p value <0.05 was considered significant. Significant difference in Bcl-xL protein expression in trophoblast cells of pregnancy complicated by severe preeclampsia (1.29 ± 0.12) compared to that normotensive pregnancy (1.71 ± 0.14) was reported (p = 0.00). In addition, logistic regression test showed that diagnosis of severe preeclampsia had a statistically significant role in Bcl-xL protein expression (p= 0.000). In conclusion, the expression of Bcl-xL protein is lower in pregnancy complicated by severe preeclampsia compared to normotensive pregnancy.

Proliferation and apoptosis of human placental cells exposed to aromatic hydrocarbons.

The objective of this study is to assess the effect of elevated urinary levels of aromatic hydrocarbons (AH) on the proliferation and apoptosis of human placental trophoblast cells obtained in the course of normal pregnancy in an AH-polluted region. Tissue material was obtained for study purposes from 50 afterbirths from Płock as the study group and 50 afterbirths from Kutno as the control group. The extent and intensity of reactions were analyzed. The levels of phenol and 1-hydroxypyrene in the excreted urine of pregnant (in labor) patients were determined by gas chromatography and colorimetry. The proliferative activity of trophoblast cells was assessed using MPM-2 antibodies against phosphoprotein synthesized upon mitotic induction and Ki-67 antigen while the intensity of apoptosis in trophoblast cells was assessed using p53 and bcl-2 oncoproteins involved in apoptosis-regulating mechanisms. The immunohistochemical reactions were assessed for their extent and intensity. The levels of phenol and 1-hydroxypyrene excreted in the urine were statistically significantly higher in patients from Płock region. The proliferative activity of trophoblast cells was statistically significantly higher in the study group (p < 0.05). The activity of oncoprotein bcl-2 was significantly higher in the study group while the activity of p53 was sig¬nificantly higher in the control group. Pregnancy in an aromatic hydrocarbon-polluted environment has a significantly negative impact on placental tissue. Ad¬aptation mechanisms are induced as manifested by increased proliferative activity within the trophoblast and extensive inhibition of apoptosis in the study group.

Nifedipine Prevents Apoptosis of Alcohol-Exposed First-Trimester Trophoblast Cells.

Maternal alcohol abuse leading to fetal alcohol spectrum disorder (FASD) includes fetal growth restriction (FGR). Ethanol (EtOH) induces apoptosis of human placental trophoblast cells, possibly disrupting placentation and contributing to FGR in FASD. EtOH facilitates apoptosis in several embryonic tissues, including human trophoblasts, by raising intracellular Ca2+ . We previously found that acute EtOH exposure increases trophoblast apoptosis due to signaling from both intracellular and extracellular Ca2+ . Therefore, nifedipine, a Ca2+ channel blocker that is commonly administered to treat preeclampsia and preterm labor, was evaluated for cytoprotective properties in trophoblast cells exposed to alcohol. Human first-trimester chorionic villous explants and the human trophoblast cell line HTR-8/SVneo (HTR) were pretreated with 12.5 to 50nM of the Ca2+ channel blocker nifedipine for 1hour before exposure to 50mM EtOH for an additional hour. Intracellular Ca2+ concentrations were monitored in real time by epifluorescence microscopy, using fluo-4-AM. Apoptosis was assessed by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), accumulation of cytoplasmic cytochrome c, and cleavage rates of caspase 3 and caspase 9. The increase in intracellular Ca2+ upon exposure to EtOH in both villous explants and HTR cells was completely blocked (p<0.05) when pretreated with nifedipine, accompanied by inhibition of EtOH-induced release of cytochrome c, caspase activities, and TUNEL. This study indicates that nifedipine can interrupt the apoptotic pathway downstream of EtOH exposure and could provide a novel strategy for future interventions in women with fetuses at risk for FASD.

MicroRNA-30a-3p is overexpressed in the placentas of patients with preeclampsia and affects trophoblast invasion and apoptosis by its effects on IGF-1

Preeclampsia (PE) affects many women globally and remains a primary cause of neonatal and maternal morbidity and mortality. Aberrant placental microRNA (miRNA) expression might be associated with PE. Previously, 33 PE-related miRNAs, 11 up-regulated and 23 down-regulated, were detected in placentas of women with severe PE when compared with those of normal patients. One of the most up-regulated miRNAs in PE is miR-30a-3p. The predicted target of it is insulin-like growth factor 1 (IGF-1), which has been reported to have a relatively low expression level in PE patients. This study was conducted to determine the aberrant increased of miR-30a-3p in the placentas of women with preeclampsia and to elucidate the target and function of it in trophoblast cells. miR-30a-3p expression in placenta tissues was compared between women with preeclampsia (n= 25) and normal pregnant women (n= 20). The miRNA target was studied by in silico and functional assay. The effects of the miRNA were verified by apoptosis assay and invasion assay in the trophoblast cell line. miR-30a-3p was increased significantly in the placenta of women with preeclampsia when compared to those with normal pregnancies. Luciferase assay confirmed direct regulation of miR-30a-3p on the expression of IGF-1. Forced expression of miR-30a-3p suppressed IGF-1 protein expression in the HTR-8/SVneo cells. The functional assay suggests that the over-expression of miR-30a-3p alter the invasive capacity of JEG-3 cells and induce the apoptosis of HTR-8/SVneo cells (Figure). Expression of miR-30a-3p was significantly increased in the placentas of patients with preeclampsia. miR-30a-3p might be involved in the pathogenesis of preeclampsia by targeting IGF-1 and regulating the invasion and apoptosis of trophoblast cells.

Elevated microRNA-34a contributes to trophoblast cell apoptosis in preeclampsia by targeting BCL-2

Preeclampsia (PE) is one of the most common pregnancy-specific pathologic complications, and is characterised by onset of hypertension and proteinuria. Placental trophoblast cell apoptosis is generally accepted as a major cause of PE. However, the details of the mechanism underlying the condition remain unclear. Here, we aimed to investigate a possible association between microRNA (miR)-34a and human trophoblast cell apoptosis during PE. We evaluated miR-34a expression in placentas from patients with PE compared with those from healthy pregnant individuals. Furthermore, we measured apoptosis rate after miR-34a mimic and/or inhibitor transfection in vitro, and identified B-cell CLL/lymphoma 2 (BCL-2) as a target of miR-34a. We found that miR-34a levels were significantly higher in placental tissues from patients with PE than in normal placentas. Upregulation of miR-34a induced trophoblast cell apoptosis in PE by inhibiting expression of BCL-2 protein. miR-34a inhibition reversed miR-34a-induced apoptosis in the HTR-8/SVneo human trophoblast cell line. Our findings indicate that miR-34a may be linked to the occurrence of PE via effects on BCL-2 in the human placenta, and may therefore provide a potential therapeutic target for PE.