Transcriptional profiling of the response to the trichloroethylene metabolite S-(1,2-dichlorovinyl)-L-cysteine revealed activation of the eIF2α/ATF4 integrated stress response in two in vitro placental models.

Trichloroethylene (TCE) is an industrial solvent and widespread environmental contaminant. Although TCE exposure is prevalent, epidemiological studies of TCE exposure associations with adverse birth outcomes are inconclusive. Prior studies show that the TCE metabolite S-(1,2-dichlorovinyl)-L-cysteine (DCVC) exhibits toxicity in a placental cell line. In the current study, genome-wide gene expression and gene set enrichment analyses were used to identify novel genes and pathway alterations in the HTR-8/SVneo human trophoblast cell line and human placental villous explants treated with DCVC at concentrations relevant to human exposures. In the cells, concentration- and time-dependent effects were observed, as evidenced by the magnitude of altered gene expression after treatment with 20 µM DCVC versus 10 µM, and 12-h versus 6-h of treatment. Comparing the two models for the transcriptional response to 12-h 20 µM DCVC treatment, no differentially expressed genes reached significance in villous explants, whereas 301 differentially expressed genes were detected in HTR-8/SVneo cells compared with non-treated controls (FDR < 0.05 + LogFC > 0.35 [FC > 1.3]). GSEA revealed five upregulated enriched pathways in common between explants and cells (FDR < 0.05). Moreover, all 12-h DCVC treatment groups from both models contained upregulated pathways enriched for genes regulated by the ATF4 transcription factor. The overrepresentation of ATF4 regulation of differentially expressed genes indicated activation of the integrated stress response (ISR), a condition triggered by multiple stress stimuli, including the unfolded protein response. DCVC-induced ISR activation was confirmed by elevated eIF2α phosphorylation, ATF4 protein concentrations, and decreased global protein synthesis in HTR-8/SVneo cells. This study identifies a mechanism of DCVC-induced cytotoxicity by revealing the involvement of a specific stress signaling pathway.

Baboon placental heparin-binding epidermal growth factor-like growth factor.

Placental extravillous trophoblast remodeling of the uterine spiral arteries is important for promoting blood flow to the placenta and fetal development. Heparin-binding EGF-like growth factor (HB-EGF), an EGF family member, stimulates differentiation and invasive capacity of extravillous trophoblasts in vitro. Trophoblast expression and maternal levels of HB-EGF are reduced at term in women with preeclampsia, but it is uncertain whether HB-EGF is downregulated earlier when it may contribute to placental insufficiency. A nonhuman primate model has been established in which trophoblast remodeling of the uterine spiral arteries is suppressed by shifting the rise in estrogen from the second to the first trimester of baboon pregnancy. In the present study, we used this model to determine if placental HB-EGF is altered by prematurely elevating estrogen early in baboon gestation. Uterine spiral artery remodeling and placental expression of HB-EGF and other EGF family members were assessed on day 60 of gestation in baboons treated with estradiol (E2) daily between days 25 and 59 of gestation (term = 184 days). The percentages of spiral artery remodeling were 90, 84 and 70% lower (P < 0.01), respectively, for vessels of 26-50, 51-100 and >100 µm diameter in E2-treated compared with untreated baboons. HB-EGF protein quantified by immunocytochemical staining/image analysis was decreased three-fold (P < 0.01) in the placenta of E2-treated versus untreated baboons, while amphiregulin (AREG) and EGF expression was unaltered. Therefore, we propose that HB-EGF modulates the estrogen-sensitive remodeling of the uterine spiral arteries by the extravillous trophoblast in early baboon pregnancy.

Extraembryonic heparin-binding epidermal growth factor-like growth factor deficiency compromises placentation in mice.

Heparin-binding epidermal growth factor (EGF)-like growth factor (HBEGF) is expressed in the embryo and uterus at the implantation site, stimulating trophoblast invasive activity essential for placentation. The effect of extraembryonic HBEGF deficiency on placental development was investigated by breeding mice heterozygous for the Hbegf null mutation. On gestation day 13.5, the average placental weights of the wild-type (Hbegf+/+) and heterozygous (Hbegf+/-) mice were approximately 76 and 77 mg, respectively, as opposed to reduced average placental weights of approximately 61 mg in homozygous null (Hbgef-/-) females. In contrast, fetal weights were not significantly affected by genotype. HBEGF immunostaining in placental sections was Hbegf gene dosage-dependent, while expression of other EGF family members was comparable in Hbegf+/+ and Hbegf-/- placentas. Histological analysis revealed no apparent differences in trophoblast giant cells, but the spongiotrophoblast region was reduced compared to labyrinth (P < 0.05) in Hbegf null placentas. While no differences in cell apoptosis were noted, proliferation as assessed by nuclear Ki67 staining was elevated in the labyrinth and decreased in the spongiotrophoblast region of Hbegf-/- placentas. Labyrinth morphology appeared disrupted in Hbegf -/- placentas stained with laminin, a marker for capillary basement membrane, and the capillary density was reduced. Immunohistochemical staining revealed reduced vascular endothelial growth factor (VEGF) levels in both spongiotrophoblast and labyrinth (P < 0.01) regions of Hbegf-/- placentas. In vitro, HBEGF supplementation increases the expression of VEGF in a human trophoblast cell line. These findings suggest that trophoblast HBEGF promotes placental capillary formation by inducing VEGF in the developing placenta of mice.

Rosiglitazone augments antioxidant response in the human trophoblast and prevents apoptosis†.

Insufficient perfusion of the trophoblast by maternal blood is associated with an increased generation of reactive oxygen species and complications of the placenta. In this study, we first examined whether rosiglitazone, an agonist of the peroxisome proliferator-activated receptor-γ (PPARγ), protects the human trophoblast from oxidative injury by regulating key antioxidant proteins, catalase (CAT) and the superoxide dismutases (SOD1 and SOD2). In first trimester placental explants, localization of CAT was limited to cytotrophoblasts, whereas SOD1 was expressed in both the cyto- and syncytiotrophoblasts. In first trimester placental explants, hypoxia decreased the expression of both SOD1 and SOD2, and increased apoptosis. Treatment with rosiglitazone dose-dependently upregulated anti-oxidative CAT and SOD2, and rescued hypoxic injury in first trimester villous explants and JEG-3 cells, strongly suggesting the involvement of the PPARγ in regulating their expressions. Rosiglitazone facilitated transcription activity of PPARγ, and enhanced promotor binding, increased transcriptional activity at the CAT promoter, and elevated protein expression/activity. Treatment of hypoxic JEG-3 cells with rosiglitazone resulted in mitochondrial membrane potential increase and a reduction of caspase 9 and caspase 3 activity which is consistent with improved cell survival. To complement PPARγ activation data, we also utilized the antagonist (SR-202) and siRNA to suppress PPARγ expression and demonstrate the specific role of PPARγ in reducing ROS and oxidative stress. Ex vivo examination of term human placenta revealed lower expression of antioxidant proteins in pathologic compared to healthy placental tissues, which could be rescued by rosiglitazone, indicating that rosiglitazone can improve survival of the trophoblast under pathological conditions. These findings provide evidence that the PPARγ pathway directly influences cellular antioxidants production and the pathophysiology of placental oxidative stress.

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

BACKGROUND: 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. METHODS: Human first-trimester chorionic villous explants and the human trophoblast cell line HTR-8/SVneo (HTR) were pretreated with 12.5 to 50 nM of the Ca2+ channel blocker nifedipine for 1 hour before exposure to 50 mM 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. RESULTS: 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. CONCLUSIONS: 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.

Enhancement of trophoblast differentiation and survival by low molecular weight heparin requires heparin-binding EGF-like growth factor.

STUDY QUESTION: Does low molecular weight heparin (LMWH) require heparin-binding epidermal growth factor (EGF)-like growth factor (HBEGF) signaling to induce extravillous trophoblast differentiation and decrease apoptosis during oxidative stress? SUMMARY ANSWER: LMWH increased HBEGF expression and secretion, and HBEGF signaling was required to stimulate trophoblast extravillous differentiation, increase invasion in vitro and reduce trophoblast apoptosis during oxidative stress. WHAT IS KNOWN ALREADY: Abnormal trophoblast differentiation and survival contribute to placental insufficiency syndromes, including preeclampsia and intrauterine growth restriction. Preeclampsia often manifests as a pro-thrombotic state, with unsuccessful transformation of the spiral arteries that reduces oxygen supply and can produce placental infarction. LMWH improves placental function by increasing blood flow. Recent data suggest that the actions of LMWH transcend its anti-coagulative properties, but the molecular mechanism is unknown. There is evidence that LMWH alters the expression of human HBEGF in trophoblast cells, which regulates human trophoblast pathophysiology. HBEGF, itself, is capable of increasing trophoblast survival and invasiveness. STUDY DESIGN, SIZE, DURATION: First-trimester placental explants and the HTR-8/SVneo cell line, established using extravillous trophoblast outgrowths from first-trimester villous explants, were treated in vitro with LMWH to examine the effects on HBEGF signaling and trophoblast function under normal physiological and pathological conditions. A highly specific antagonist of HBEGF and other inhibitors of HBEGF downstream signaling were used to determine the relationship between LMWH treatment and HBEGF. PARTICIPANTS/MATERIALS, SETTING, METHODS: Placental tissues (n = 5) were obtained with IRB approval and patient consent from first-trimester terminations. Placental explants and HTR-8/SVneo cells were cultured on plastic or Matrigel™ and treated with a therapeutic dose of LMWH (Enoxaparin; 10 IU/ml), with or without CRM197, pan Erb-B2 Receptor Tyrosine Kinase (ERBB) inhibitor, anti-ERBB1 or ERBB4 blocking antibodies, or pretreatment of cells with heparitinase I. Extravillous differentiation was assessed by immunocytochemistry to determine the relative levels of integrins α6ß4 and α1ß1. Trophoblast invasiveness was assessed in villous explants by measuring outgrowth from villous tips cultured on Matrigel, and by invasion assays with HTR-8/SVneo cells cultured on Matrigel-coated transwell insert. Placental explants and HTR-8/SVneo cells were exposed to oxidative stress in a hypoxia-reoxygenation (H-R) model, measuring cell death by TUNEL assay, caspase 3 cleavage, and BCL-2α expression. MAIN RESULTS AND THE ROLE OF CHANCE: LMWH induced extravillous differentiation, according to trophoblast invasion assays and integrin (α6ß4-α1ß1) switching. Treatment with LMWH rescued cytotrophoblasts and HTR-8/SVneo cells from apoptosis during exposure to reoxygenation injury, based on TUNEL, caspase 3 cleavage and BCL-2α expression. Experiments using CRM197, ERBB1 and ERBB4 blocking antibodies, pan-ERBB inhibitor and removal of cell surface heparin demonstrated that the effects of LMWH on trophoblast invasion and survival were dependent upon HBEGF signaling. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: The primary limitation of this study was the use of only in vitro experiments. Patient demographics from elective terminations were not available. WIDER IMPLICATIONS OF THE FINDINGS: These data provide new insights into the non-coagulation-related aspects of perinatal LMWH treatment in the management of placental insufficiency disorders. STUDY FUNDING/COMPETING INTEREST(S): This research was supported by grants from the National Institutes of Health (HD071408 and HL128628), the March of Dimes, and the W. K. Kellogg Foundation. There were no conflicts or competing interests.

Reduced homeobox protein MSX1 in human endometrial tissue is linked to infertility.

STUDY QUESTION: Is protein expression of the muscle segment homeobox gene family member MSX1 altered in the human secretory endometrium by cell type, developmental stage or fertility? SUMMARY ANSWER: MSX1 protein levels, normally elevated in the secretory phase endometrium, were significantly reduced in endometrial biopsies obtained from women of infertile couples. WHAT IS KNOWN ALREADY: Molecular changes in the endometrium are important for fertility in both animals and humans. Msx1 is expressed in the preimplantation mouse uterus and regulates uterine receptivity for implantation. The MSX protein persists a short time, after its message has been down-regulated. Microarray analysis of the human endometrium reveals a similar pattern of MSX1 mRNA expression that peaks before the receptive period, with depressed expression at implantation. Targeted deletion of uterine Msx1 and Msx2 in mice prevents the loss of epithelial cell polarity during implantation and causes infertility. STUDY DESIGN, SIZE DURATION: MSX1 mRNA and cell type-specific levels of MSX1 protein were quantified from two retrospective cohorts during the human endometrial cycle. MSX1 protein expression patterns were compared between fertile and infertile couples. Selected samples were dual-labeled by immunofluorescence microscopy to localize E-cadherin and ß-catenin in epithelial cells. PARTICIPANTS/MATERIALS, SETTING METHODS: MSX1 mRNA was quantified by PCR in endometrium from hysterectomies (n = 14) determined by endometrial dating to be in the late-proliferative (cycle days 10-13), early-secretory (cycle days 14-19) or mid-secretory (cycle days 20-24) phase. MSX1 protein was localized using high-throughput, semi-quantitative immunohistochemistry with sectioned endometrial biopsy tissues from fertile (n = 89) and infertile (n = 89) couples. Image analysis measured stain intensity specifically within the luminal epithelium, glands and stroma during the early-, mid- and late- (cycle days 25-28) secretory phases. MAIN RESULTS AND THE ROLE OF CHANCE: MSX1 transcript increased 5-fold (P < 0.05) between the late-proliferative and early secretory phase and was then down-regulated (P < 0.05) prior to receptivity for implantation. In fertile patients, MSX1 protein displayed strong nuclear localization in the luminal epithelium and glands, while it was weakly expressed in nuclei of the stroma. MSX1 protein levels accumulated throughout the secretory phase in all endometrial cellular compartments. MSX1 protein decreased (P < 0.05) in the glands between mid- and late-secretory phases. However, infertile patients demonstrated a broad reduction (P < 0.001) of MSX1 accumulation in all cell types throughout the secretory phase that was most pronounced (∼3-fold) in stroma and glands. Infertility was associated with persistent co-localization of E-cadherin and ß-catenin in epithelial cell junctions in the mid- and late-secretory phases. LIMITATIONS, REASONS FOR CAUTION: Details of the infertility diagnoses and other patient demographic data were not available. Therefore, patients with uterine abnormalities (Mullerian) could not be distinguished from other sources of infertility. Antibody against human MSX2 is not available, limiting the study to MSX1. However, both RNAs in the human endometrium are similarly regulated. In mice, Msx1 and Msx2 are imperative for murine embryo implantation, with Msx2 compensating for genetic ablation of Msx1 through its up-regulation in a knockout model. WIDER IMPLICATIONS OF THE FINDINGS: This investigation establishes that the MSX1 homeobox protein accumulation is associated with the secretory phase in endometrium of fertile couples, and is widely disrupted in infertile patients. It is the first study to examine MSX1 protein localization in the human endometrium, and supported by genetic findings in mice, suggests that genes regulated by MSX1 are linked to the loss of epithelial cell polarity required for uterine receptivity during implantation. STUDY FUNDING/COMPETING INTERESTS: This research was supported by the NICHD National Cooperative Reproductive Medicine Network grant HD039005 (M.P.D.), NIH grants HD068524 (S.K.D.), HD071408 (D.R.A., M.P.D.), and HL128628 (S.D.), the Intramural Research Program of the NICHD, March of Dimes (S.K.D., S.D.) and JSPS KAKENHI grant 26112506 (Y.H.). There were no conflicts or competing interests.

Estradiol Elicits Proapoptotic and Antiproliferative Effects in Human Trophoblast Cells.

During the first trimester of pregnancy, appropriate regulation of estradiol (E2) is essential for normal placental development. Previous studies demonstrate that premature elevation in E2 concentrations can lead to abnormal placentation, but have not fully elaborated the mechanism of this effect in the first-trimester trophoblast. Our aim was to determine whether E2 elicits trophoblast cell death or inhibits proliferation. The first-trimester human cytotrophoblast cell line HTR-8/SVneo was cultured in phenol red-free medium containing charcoal-stripped serum and treated with 17beta-E2 at concentrations between 0 and 100 nM. TUNEL and invasion assays indicated that E2 significantly increased cell death and reduced cell invasion at 10 nM, and nuclear Ki67 expression revealed that it decreased cell proliferation at 1 nM. A similar effect on cell death was observed in first-trimester placental explants. The E2 antagonist fulvestrant blocked all effects of E2. Immunohistochemistry showed that protein expression of proapoptotic caspases 3, 8, and 9 increased at E2 concentrations of 25 nM and greater, whereas expression of antiapoptotic BCL2-alpha decreased at E2 concentrations of 10 nM and greater. Additionally, treatments with estrogen receptor (ER) alpha-specific and ERbeta-specific agonists at concentrations between 0 and 1000 nM indicated that only ERalpha mediates E2's effects, although immunohistochemistry and Western immunoblotting showed that HTR-8/SVneo cells and placental explants express both ERalpha and ERbeta. Taken together, these findings reveal the interplay between elevated serum E2 and apoptosis in the first trimester of pregnancy. These factors could be associated with pregnancy complications including infertility and uteroplacental insufficiency.

Trophoblast retrieval and isolation from the cervix (TRIC) is unaffected by early gestational age or maternal obesity.

OBJECTIVE: The objective of this study is to evaluate whether trophoblast yield obtained by trophoblast retrieval and isolation from the cervix (TRIC) is affected by pregnancy outcome, gestational age (GA) at retrieval, maternal body mass index (BMI), parity, or maternal age. METHODS: TRIC was performed on 224 ongoing pregnancies between 5 and 20 weeks of GA. Trophoblast cells were isolated from cervical cells using anti-human leukocyte antigen-G antibody coupled to magnetic nanoparticles. Purity was assessed by the percentage of isolated cells that express ß-hCG. Patient records were monitored until delivery, and pregnancy outcomes were determined. Trophoblast yield was compared with GA at time of collection, maternal BMI, parity, maternal age, and outcome of pregnancy, using linear regression. RESULTS: There was no effect of GA, maternal BMI, parity, and maternal age on trophoblast yield. Trophoblast yield decreased significantly with early pregnancy loss compared with uncomplicated pregnancies that delivered at term. Trophoblast yield with preeclampsia or intrauterine growth restriction was decreased compared with healthy term outcomes; however, they did not reach statistical significance. CONCLUSIONS: If TRIC becomes available as a method for non-invasive prenatal testing, our data demonstrate that it is unaffected by BMI and is useful as early as 5 weeks of GA.

Intracellular Ca2+ signaling and preimplantation development.

The key, versatile role of intracellular Ca2+ signaling during egg activation after fertilization has been appreciated for several decades. More recently, evidence has accumulated supporting the concept that cytoplasmic Ca2+ is also a major signaling nexus during subsequent development of the fertilized ovum. This chapter will review the molecular reactions that regulate intracellular Ca2+ levels and cell function, the role of Ca2+ signaling during egg activation and specific examples of repetitive Ca2+ signaling found throughout pre- and peri-implantation development. Many of the upstream and downstream pathways utilized during egg activation are also critical for specific processes that take place during embryonic development. Much remains to be done to elucidate the full complexity of Ca2+ signaling mechanisms in preimplantation embryos to the level of detail accomplished for egg activation. However, an emerging concept is that because this second messenger can be modulated downstream of numerous receptors and is able to bind and activate multiple cytoplasmic signaling proteins, it can help the coordination of development through up- and downstream pathways that change with each embryonic stage.

Apoptosis of alcohol-exposed human placental cytotrophoblast cells is downstream of intracellular calcium signaling.

BACKGROUND: Apoptosis is induced by ethanol (EtOH) in human placental trophoblast cells, possibly disrupting placentation and contributing to intrauterine growth restriction in fetal alcohol spectrum disorder (FASD). EtOH induces programmed cell death in several embryonic tissues by raising intracellular Ca(2+) . Therefore, the role of Ca(2+) signaling in EtOH-induced apoptosis was examined using human first trimester cytotrophoblast cell lines, examining the hypothesis that apoptosis is dependent on intracellular Ca(2+) signaling. METHODS: Using HTR-8/SVneo and SW.71 cytotrophoblast cell lines, real-time intracellular Ca(2+) concentration was monitored by fluo-4 epifluorescence microscopy and apoptosis was assessed by flow cytometry of cells fluorescently labeled for DNA fragmentation (TUNEL) and annexin V binding. RESULTS: Intracellular Ca(2+) concentrations increased synchronously in all cells within 10 seconds of exposure to 50 mM EtOH, but not at lower EtOH concentrations (10 to 25 mM) incapable of inducing apoptosis. Trophoblast cells treated with inhibitors of Ca(2+) signaling (BAPTA-AM, U73122, xestospongin D, BAPTA, SKF-96365) produced no intracellular Ca(2+) transients after exposure to 50 mM EtOH and were protected from cell death induced by EtOH. CONCLUSIONS: EtOH-induced apoptosis in human cytotrophoblast cells, identified by DNA fragmentation and externalized phosphatidylserine, was dependent upon Ca(2+) signaling. Both intracellular Ca(2+) mobilization and extracellular Ca(2+) influx were required, as well as phosphatidylinositol signaling. Inhibition by SKF-96365 suggests that the capacitative Ca(2+) entry mechanism that utilizes TRPC channels was activated by EtOH. Apoptosis occurs downstream of Ca(2+) signaling in trophoblasts and may contribute to placental insufficiency and poor fetal growth associated with FASD.

High throughput, cell type-specific analysis of key proteins in human endometrial biopsies of women from fertile and infertile couples.

BACKGROUND: Although histological dating of endometrial biopsies provides little help for prediction or diagnosis of infertility, analysis of individual endometrial proteins, proteomic profiling and transcriptome analysis have suggested several biomarkers with altered expression arising from intrinsic abnormalities, inadequate stimulation by or in response to gonadal steroids or altered function due to systemic disorders. The objective of this study was to delineate the developmental dynamics of potentially important proteins in the secretory phase of the menstrual cycle, utilizing a collection of endometrial biopsies from women of fertile (n = 89) and infertile (n = 89) couples. METHODS AND RESULTS: Progesterone receptor-B (PGR-B), leukemia inhibitory factor, glycodelin/progestagen-associated endometrial protein (PAEP), homeobox A10, heparin-binding EGF-like growth factor, calcitonin and chemokine ligand 14 (CXCL14) were measured using a high-throughput, quantitative immunohistochemical method. Significant cyclic and tissue-specific regulation was documented for each protein, as well as their dysregulation in women of infertile couples. Infertile patients demonstrated a delay early in the secretory phase in the decline of PGR-B (P < 0.05) and premature mid-secretory increases in PAEP (P < 0.05) and CXCL14 (P < 0.05), suggesting that the implantation interval could be closing early. Correlation analysis identified potential interactions among certain proteins that were disrupted by infertility. CONCLUSIONS: This approach overcomes the limitations of a small sample number. Protein expression and localization provided important insights into the potential roles of these proteins in normal and pathological development of the endometrium that is not attainable from transcriptome analysis, establishing a basis for biomarker, diagnostic and targeted drug development for women with infertility.

Toxicity assessments of selected trichloroethylene and perchloroethylene metabolites in three in vitro human placental models.

Residential and occupational exposures to the industrial solvents perchloroethylene (PERC) and trichloroethylene (TCE) present public health concerns. In humans, maternal PERC and TCE exposures can be associated with adverse birth outcomes. Because PERC and TCE are biotransformed to toxic metabolites and placental dysfunction can contribute to adverse birth outcomes, the present study compared the toxicity of key PERC and TCE metabolites in three in vitro human placenta models. We measured cell viability and caspase 3 + 7 activity in the HTR-8/SVneo and BeWo cell lines, and caspase 3 + 7 activity in first trimester villous explant cultures. Cultures were exposed for 24 h to 5-100 µM S-(1,2-dichlorovinyl)-L-cysteine (DCVC) and S-(1,2,2-trichlorovinyl)-L-cysteine (TCVC), or 5-200 µM trichloroacetate (TCA) and dichloroacetate (DCA). DCVC significantly reduced cell viability and increased caspase 3 + 7 activity in HTR-8/SVneo cells at a lower concentration (20 µM) compared with concentrations toxic to BeWo cells and villous explants. Similarly, TCVC reduced cell viability and increased caspase 3 + 7 activity in HTR-8/SVneo cells but not in BeWo cells. TCA and DCA had only negligible effects on HTR-8/SVneo or BeWo cells. This study advances understanding of potential risks of PERC and TCE exposure during pregnancy by identifying metabolites toxic in placental cells and tissues.

Regulation of proteinases during mouse peri-implantation development: urokinase-type plasminogen activator expression and cross talk with matrix metalloproteinase 9.

Trophoblast cells express urokinase-type plasminogen activator (PLAU) and may depend on its activity for endometrial invasion and tissue remodeling during peri-implantation development. However, the developmental regulation, tissue distribution, and function of PLAU are not completely understood. In this study, the expression of PLAU and its regulation by extracellular matrix proteins was examined by RT-PCR, immunocytochemistry, and plasminogen-casein zymography in cultured mouse embryos. There was a progressive increase in Plau mRNA expression in blastocysts cultured on gestation days 4-8. Tissue-type plasminogen activator (55 kDa) and PLAU (a triplet of 40, 37, and 31 kDa) were present in conditioned medium and embryo lysates, and were adsorbed to the culture plate surface. The temporal expression pattern of PLAU, according to semi-quantitative gel zymography, was similar in non-adhering embryos and embryos cultured on fibronectin, laminin, or type IV collagen, although type IV collagen and laminin upregulated Plau mRNA expression. Immunofluorescence revealed PLAU on the surface of the mural trophectoderm and in non-spreading giant trophoblast cells. Exogenous human plasminogen was transformed to plasmin by cultured embryos and activated endogenous matrix metalloproteinase 9 (MMP9). Indeed, the developmental expression profile of MMP9 was similar to that of PLAU. Our data suggest that the intrinsic developmental program predominantly regulates PLAU expression during implantation, and that PLAU could be responsible for activation of MMP9, leading to localized matrix proteolysis as trophoblast invasion commences.

Function-specific intracellular signaling pathways downstream of heparin-binding EGF-like growth factor utilized by human trophoblasts.

Heparin-binding EGF-like growth factor (HBEGF) is expressed by trophoblast cells throughout gestation. First-trimester cytotrophoblast cells are protected from hypoxia-induced apoptosis because of the accumulation of HBEGF through a posttranscriptional autocrine mechanism. Exogenous application of HBEGF is cytoprotective in a hypoxia/reoxygenation (H/R) injury model and initiates trophoblast extravillous differentiation to an invasive phenotype. The downstream signaling pathways induced by HBEGF that mediate these various cellular activities were identified using two human first-trimester cytotrophoblast cell lines, HTR-8/SVneo and SW.71, with similar results. Recombinant HBEGF (1 nM) induced transient phosphorylation of MAPK3/1 (ERK), MAPK14 (p38), and AKT within 15 min and JNK after 1-2 h. To determine which downstream pathways regulate the various functions of HBEGF, cells were treated with specific inhibitors of the ERK upstream regulator MEK (U0126), the AKT upstream regulator phosphoinositide-3 (PI3)-kinase (LY294002), MAPK14 (SB203580), and JNK (SP600125), as well as with inactive structural analogues. Only SB203580 specifically prevented HBEGF-mediated rescue during H/R, while each inhibitor attenuated HBEGF-stimulated cell migration. Accumulation of HBEGF at reduced oxygen was blocked only by a combination of U0126, SB203580, and SP600125. We conclude that HBEGF advances trophoblast extravillous differentiation through coordinate activation of PI3 kinase, ERK, MAPK14, and JNK, while only MAPK14 is required for its antiapoptotic activity. Additionally, hypoxia induces an autocrine increase in HBEGF protein levels through MAPK14, JNK or ERK. These experiments reveal a complexity of the intracellular signaling circuitry that regulates trophoblast functions critical for implantation and placentation.

Rhesus macaque embryos derived from MI oocytes maturing after retrieval display high rates of chromosomal anomalies.

BACKGROUND: Rhesus macaque and human preimplantation embryos display similar rates of chromosomal abnormalities. The aim of this study was to determine whether embryos developing from MI oocytes that mature post-retrieval display more chromosomal anomalies than those embryos that are generated from oocytes that are at MII at the time of retrieval. METHODS: Rhesus macaque oocytes were obtained after hormonal ovarian stimulation. Immediately after retrieval, the oocytes were classified according to their maturational status. Following in vitro fertilization, Day 3 embryos with good morphology and development derived from oocytes maturing post-retrieval and those from oocytes that were mature at the time of retrieval were cytogenetically assessed using a five-color fluorescent in situ fluorescent hybridization assay developed for rhesus macaque chromosomes homologous to human chromosomes 13, 16, 18, X and Y. RESULTS: Blastomeres from 53 embryos were analyzed. Of the 27 embryos that developed from oocytes that were mature at collection, 18 embryos were chromosomally normal (66.7%), while from the 26 embryos that developed from oocytes that matured post-retrieval, only 9 embryos were chromosomally normal (34.6%). CONCLUSIONS: These results indicate that embryos developing from oocytes maturing post-retrieval display high rates of chromosomal abnormalities and have therefore a reduced developmental competence. As a result, the clinical relevance of using immature oocytes that are retrieved after stimulated cycles in human IVF warrants further investigation.

Retrieval of trophoblast cells from the cervical canal for prediction of abnormal pregnancy: a pilot study.

BACKGROUND: Fetal cells are shed from the regressing chorionic villi and it is possible to retrieve extravillous cytotrophoblast cells by transcervical sampling. The abundance of trophoblast cells in transcervical samples suggests that this non-invasive approach could distinguish between normal and abnormal pregnancies, such as an ectopic pregnancy (EP) and blighted ovum (BO). We aim to identify and quantify fetal trophoblast cells in the cervical canal during the first trimester to assess their usefulness to predict an abnormal pregnancy. METHODS: Patients, age 18-45, presenting with a normal intrauterine pregnancy (IUP; n = 37), diagnosis of EP (n = 10) or BO (n = 5) were enrolled for collection of transcervical specimens using a cytobrush and fixative rinse. Non-pregnant, nulliparous women (n = 7) were included as negative controls. Cells were cleared of mucus by acidification, prepared on microscope slides and labeled with a monoclonal antibody recognizing the trophoblast marker, human leukocyte antigen (HLA)-G. HLA-G positive and negative cells were counted to calculate the ratio of trophoblast cells to total cervical cells. RESULTS: Trophoblast cells were observed in 35/37 normal IUP, 6/10 EP and 4/5 BO specimens. The average frequency of HLA-G positive cells in the normal IUP cervical samples was approximately 1 in 2000, which was 4-fold higher than samples from patients with EP or BO (P < 0.001). Receiver operating characteristic analysis showed that EP and BO pregnancies were distinguishable from normal pregnancies with 93% sensitivity, 95% specificity, 97% positive predictive value and 87% negative predictive value. CONCLUSIONS: This pilot study presents evidence that trophoblast cells can be reliably obtained and identified among cervical cells in the first trimester by immunohistochemical staining for HLA-G, and suggests for the first time that abnormal pregnancies may be predictable based on the abundance of trophoblast cells in the cervical canal.

Diverse functions of HBEGF during pregnancy.

The establishment of pregnancy requires an intimate physical interaction and a molecular dialogue between the conceptus and the maternal reproductive tract that commences at implantation and continues until the placenta is formed and fully functional. Failure of the regulatory processes that ensure the fidelity of this relationship can precipitate a catastrophic pregnancy loss. One of the earliest identified molecular mediators of blastocyst implantation is heparin-binding epidermal growth factor (EGF)-like growth factor (HBEGF), which signals between the endometrium and implanting trophoblast cells to synchronize their corresponding developmental programs. HBEGF expression by trophoblast cells of the developing placenta appears to regulate extravillous differentiation and provide cytoprotection in a sometimes-hostile environment. This versatile member of the EGF signaling system will be examined in light of its associations with key events during early pregnancy.

Endocervical trophoblast for interrogating the fetal genome and assessing pregnancy health at five weeks.

Prenatal testing for fetal genetic traits and risk of obstetrical complications is essential for maternal-fetal healthcare. The migration of extravillous trophoblast (EVT) cells from the placenta into the reproductive tract and accumulation in the cervix offers an exciting avenue for prenatal testing and monitoring placental function. These cells are obtained with a cervical cytobrush, a routine relatively safe clinical procedure during pregnancy, according to published studies and our own observations. Trophoblast retrieval and isolation from the cervix (TRIC) obtains hundreds of fetal cells with >90% purity as early as five weeks of gestation. TRIC can provide DNA for fetal genotyping by targeted next-generation sequencing with single-nucleotide resolution. Previously, we found that known protein biomarkers are dysregulated in EVT cells obtained by TRIC in the first trimester from women who miscarry or later develop intrauterine growth restriction or preeclampsia. We have now optimized methods to stabilize RNA during TRIC for subsequent isolation and analysis of trophoblast gene expression. Here, we report transcriptomics analysis demonstrating that the expression profile of TRIC-isolated trophoblast cells was distinct from that of maternal cervical cells and included genes associated with the EVT phenotype and invasion. Because EVT cells are responsible for remodeling the maternal arteries and their failure is associated with pregnancy disorders, their molecular profiles could reflect maternal risk, as well as mechanisms underlying these disorders. The use of TRIC to analyze EVT genomes, transcriptomes and proteomes during ongoing pregnancies could provide new tools for anticipating and managing both fetal genetic and maternal obstetric disorders.

Diminished survival of human cytotrophoblast cells exposed to hypoxia/reoxygenation injury and associated reduction of heparin-binding epidermal growth factor-like growth factor.

OBJECTIVE: The antiapoptotic action of heparin-binding epidermal growth factor (HBEGF)-like growth factor and its regulation by O(2) constitutes a key factor for trophoblast survival. The hypothesis that cytotrophoblast survival is compromised by exposure to hypoxia-reoxygenation (H/R) injury, which may contribute to preeclampsia and some missed abortions, prompted us to investigate HBEGF regulation and its role as a survival factor during H/R in cytotrophoblast cells. STUDY DESIGN: A transformed human first-trimester cytotrophoblast cell line HTR-8/SVneo was exposed to H/R (2% O(2) followed by 20% O(2)) and assessed for HBEGF expression and cell death. RESULTS: Cellular HBEGF declined significantly within 30 minutes of reoxygenation after culture at 2% O(2). H/R significantly reduced proliferation and increased cell death when compared with trophoblast cells cultured continuously at 2% or 20% O(2). Restoration of cell survival also was achieved by adding recombinant HBEGF during reoxygenation. HBEGF inhibited apoptosis through its binding to either human epidermal receptor (HER)-1 or HER4, its cognate receptors. CONCLUSION: These results provide evidence that cytotrophoblast exposure to H/R induces apoptosis and decreased cell proliferation. HBEGF accumulation is diminished under these conditions, whereas restoration of HBEGF signaling improves trophoblast survival.