Expression In Villi Research Articles

Impaired decidualization and relative increase of PROK1 expression in the decidua of patients with unexplained recurrent pregnancy loss showing insulin resistance

A recent meta-analysis revealed that patients with unexplained recurrent pregnancy loss (RPL) show higher insulin resistance compared to healthy controls. However, the etiology of RPL remains unknown. Prokineticin (PROK1), a pleiotropic uterine endometrial protein, is important for implantation and decidualization and is regulated by hypoxia and insulin. In this study, we investigated the decidualization status and the role of PROK1 in the decidua of patients with unexplained RPL showing insulin resistance. Thirty-two patients with unexplained RPL were included in this study. Following the diagnosis of a miscarriage, the decidua and villi of the patient were surgically collected. Fasting blood glucose and insulin levels were measured, and HOMA-β was calculated. Using IHC and ELISA, the expression of IGFBP-1, PRL and PROK1 in the decidua and IGF-2 in the villi were analyzed in patients with euploid miscarriage with a high HOMA-β index (n = 8) and compared to controls (euploid miscarriage with normal HOMA-β: n = 12, aneuploid miscarriage with normal HOMA-β: n = 12). The co-localization of PROK1 and IGFBP-1 was observed in the decidua by IHC. In the decidua of RPL patients with high HOMA-β, the expression levels of IGFBP-1 and PRL were significantly lower, whereas the PROK1/IGFBP-1 ratio was significantly higher compared to that of the controls. IGF-2 expression in villi was significantly lower in RPL patients with high HOMA-β. Impaired decidualization and excessive PROK1 production may have pathological implications in patients with unexplained RPL with insulin resistance, especially under the state of hyper insulin production.

Aquaporin 9 causes recurrent spontaneous abortion by inhibiting trophoblast cell epithelial-mesenchymal transformation and invasion through the PI3K/AKT pathway†.

Invasion of the endometrium by trophoblast cells is a key event during pregnancy, although the underlying mechanism remains unclear. Aquaporin 9 (AQP 9) is expressed in many eukaryotes and is associated with cell invasion. The objective of this study was to evaluate the significance of AQP9 in recurrent spontaneous abortion. We screened the GSE22490 dataset and further differentiated aquaporin 9 expression in villi. AQP9 was evaluated as one of the key factors in abortion by injecting AQP9 overexpressed plasmid into the uterus of CD1 mice. Trophoblast cells were transfected with AQP9-overexpressing plasmid or siAQP9 to measure cell proliferation, migration, invasion, and apoptosis. Western blot was used to measure changes in the expression of invasion, epithelial-mesenchymal transformation process, and PI3K/AKT pathway. Finally, the role of AQP9 in PI3K/AKT signaling pathway was determined using the PI3K/AKT inhibitor, LY294002, and activator, 740Y-P. AQP9 is highly expressed in recurrent spontaneous abortion villus. Intrauterine injections of AQP9-overexpressing plasmid into CD1 mice resulted in atrophy and blackness of the gestational sac and increased the absorption rate, it is the causative factor of abortion. AQP9 upregulation inhibited the proliferation, invasion, migration, and epithelial-mesenchymal transformation process in vitro of trophoblast cells and increased cell apoptosis. The opposite result was observed after silencing AQP9. AQP9 overexpression also inhibited the PI3K/AKT pathway. LY294002 and 740Y-P partially recovered AQP9-induced trophoblast invasion and migration via the PI3K/AKT pathway. AQP9 reduces the invasive ability of trophoblast cells by regulating PI3K/AKT signaling pathway, participating in recurrent spontaneous abortion.

Alterted SLIT2/ROBO1 signalling is linked to impaired placentation of missed and threatened miscarriage in early pregnancy.

Two-thirds of early pregnancy failures present with reduced trophoblast invasion, and SLIT2/ROBO1 signalling is considered to play an important role in trophoblast function during pregnancy. We investigated SLIT2/ROBO1 signalling associated with missed and threatened miscarriage during early gestation. Human placenta samples were collected from women with missed miscarriage (n = 25), threatened miscarriage (n = 22) and termination of pregnancy controls (n = 32). Corresponding decreases in beta human chorionic gonadotrophin (β-hCG) levels and shallow trophoblast invasion were observed in patients with missed and threatened miscarriage, immunohistological staining revealed abnormal Slit2 and Robo1, as well as E-cadherin and activating protein-2 alpha (AP-2α) expression in villi and extravillous trophoblasts, and the expression of these proteins were confirmed in villi and decidua of miscarriage material by Western blotting. Using HTR8/SVneo cells, blocking SLIT2/ROBO1 signalling promoted cell migration, proliferation and suppressed differentiation. Moreover, blocking SLIT2/ROBO1 signalling in HTR8/SVneo cells altered trophoblast differentiation-related and angiogenesis-related gene mRNA expression, which also occurred in the tissues of missed and threatened miscarriage. SLIT2/ROBO1 signalling may regulate trophoblast differentiation and invasion causing restricting β-hCG production, shallow trophoblast invasion and inhibiting placental angiogenesis in missed and threatened miscarriage during the first trimester.

Decreased expression of the vitamin D receptor in women with recurrent pregnancy loss

The multiple functions of vitamin D3 have stimulated interest in the role that this vitamin may play during pregnancy. The present study investigated the expression of the vitamin D receptor (VDR) in women during the first trimester of pregnancy in order to determine whether VDR is associated with recurrent pregnancy loss (RPL). Forty women at 7–10 weeks gestation with RPL and 40 women of similar gestational age with a healthy pregnancy were recruited. VDR mRNA and protein in chorionic villi and decidua were evaluated by immunohistochemistry, confocal laser scanning microscopy (CLSM), western blot, and quantitative real-time polymerase chain reaction. The serum levels of VDR were measured by an enzyme-linked immunosorbent assay. Women with RPL had a significantly weaker expression of VDR mRNA in villi and decidual tissues compared with the control women (both p < 0.0001). Western blot analysis showed an approximately 46% decrease in VDR expression in villi and a 52% decrease in decidua in the RPL vs. the controls. Serum VDR levels were also significantly lower in the RPL group than in the control group (p = 0.003). Compared with the controls, immunohistochemical and CLSM analysis revealed significantly lower VDR expression in villous cytotrophoblasts and stromal cells, as well as in decidual glandular epithelial and stromal cells (all p < 0.05). In conclusion, these observations show that women with RPL have lower levels of VDR expression in chorionic villi, decidua and serum compared with normal pregnant women, suggesting that decreased VDR expression in the first trimester pregnancy may be associated with RPL.

Adrenomedullin 2 (ADM2) Regulates Mucin 1 at the Maternal-Fetal Interface in Human Pregnancy.

Association of an altered expression of placental mucin 1 (MUC1) with first-trimester spontaneous abortion and its regulation in placenta by an invasion-promoting peptide, adrenomedullin 2 (ADM2), is not known. The objective of this study was to assess 1) the association of MUC1 mRNA expression in the placental villi and decidua with first-trimester spontaneous abortion, 2) the effects of ADM2 on the expression of MUC1 in trophoblast cells in the presence or absence of hypoxia, 3) the effects of ADM2 on expression of MUC1 in decidual stromal cells (DSCs), and 4) if ADM2 regulates the expression of MUC1 and MMP2 protein in trophoblastic spheroids. Data demonstrate that 1) expression of MUC1 mRNA in villous tissue is higher in spontaneous abortion compared to age-matched electively terminated pregnancies (P > 0.05), 2) ADM2 decreases the expression of MUC1 mRNA and protein in trophoblast cells and spheroids with concomitant increases in MMP2 immunoreactivity in the spheroids, 3) ADM2 decreases hypoxia-induced increases in MUC1 immunoreactivity in trophoblast cells, 4) decidual MUC1 mRNA expression is lower in spontaneous compared to elective abortions (P < 0.05), and 5) DSCs express MUC1 mRNA and protein and ADM2 decreases the expression of MUC1 mRNA and protein in DSCs. Taken together, this study demonstrates that first-trimester spontaneous abortion is associated with increases in MUC1 expression in villi and decreases in the decidual tissues, and suggests that ADM2 may contribute to the physiology of embryo implantation and placental growth via increasing MMP2 and decreasing MUC1 expression to facilitate trophoblast invasion.

Prospective study evaluating the effect of mifepristone on E-cadherin expression in villi in early pregnancy

BackgroundE-cadherin plays an important regulatory role in implantation, embryo development and placentation. This study aimed to determine the effect of mifepristone on E-cadherin expression in human villi in early pregnancy. Study designForty healthy women seeking elective pregnancy termination at 5–7 weeks of gestation were recruited. Of these, 22 women chose medical termination (mifepristone-treated group) and took 25mg mifepristone every 12h for 3 days and 600μg buccal misoprostol on the morning of the fourth day. The other 18 women underwent vacuum aspiration (control group). Following collection of villi, E-cadherin protein expression was assessed by immunohistochemical analysis, and E-cadherin mRNA expression was assessed by reverse transcription-polymerase chain reaction. ResultsE-cadherin protein expression was significantly higher (p<0.05) in villous cytotrophoblast cells in the mifepristone-treated group compared with the control group. E-cadherin mRNA expression was also significantly higher (p<0.01) in the mifepristone-treated group compared with the control group. ConclusionE-cadherin expression in villi may be involved in mifepristone-induced pregnancy termination.

NOD1 and NOD2 control the invasiveness of trophoblast cells via the MAPK/p38 signaling pathway in human first-trimester pregnancy

IntroductionNucleotide binding and oligomerization leucine-rich repeats (NLR) are recognized as members of pattern-recognition receptors that play important roles in host innate immune defenses. In this study, we have investigated the expression and regulation of nucleotide-binding oligomerization domains 1 and 2 (NOD1 and NOD2) on the invasiveness of primary human trophoblasts during the first trimester of pregnancy. MethodsThe expression of NOD1 and NOD2 by human first trimester villi from recurrent spontaneous abortion (RSA) patients and normal pregnancy was analyzed by immunochemistry, western blot and real-time RT-PCR, respectively. The effects of ligands on the regulation of their receptors and invasiveness of trophoblasts were examined. The ELISA was used to measure the secretion of matrix matallopeptidase-9 (MMP9) and matrix matallopeptidase-2 (MMP2) by trophoblasts. We also investigated the signaling pathways involved in invasion of trophoblasts. ResultsThe higher NOD1 and NOD2 were observed in villi from patients who experienced RSA compared with those who experienced a normal pregnancy. The ligands can up-regulate the expression of their receptors. After activation, NOD1 and NOD2 inhibited the invasion of trophoblast cells by decreasing MMP9 but not MMP2. After the activation of NOD1 and NOD2, the mitogen-activated protein kinase (MAPK)/p38 pathway was activated time dependently. Blocking this pathway with a specific inhibitor (SB203580) attenuated NOD1-and NOD2-regulated trophoblast invasions. DiscussionThese results suggest that abnormal elevated NOD1 and NOD2 expression in villi relate to pregnancy outcomes. Further, activation of NOD1 and NOD2 could have a bearing on our understanding of the invasive ability of trophoblasts.

Functional consequences of copy number variants in miscarriage.

BackgroundThe presence of unique copy number variations (CNVs) in miscarriages suggests that their integral genes have a role in maintaining early pregnancy. In our previous work, we identified 19 unique CNVs in ~40% of studied euploid miscarriages, which were predominantly familial in origin. In our current work, we assessed their relevance to miscarriage by expression analysis of 14 genes integral to CNVs in available miscarriage chorionic villi. As familial CNVs could cause miscarriage due to imprinting effect, we investigated the allelic expression of one of the genes (TIMP2) previously suggested to be maternally expressed in placenta and involved in placental remodelling and embryo development.ResultsSix out of fourteen genes had detectable expression in villi and for three genes the RNA and protein expression was altered due to maternal CNVs. These genes were integral to duplication on Xp22.2 (TRAPPC2 and OFD1) or disrupted by a duplication mapping to 17q25.3 (TIMP2). RNA and protein expression was increased for TRAPPC2 and OFD1 and reduced for TIMP2 in carrier miscarriages. The three genes have roles in processes important for pregnancy development such as extracellular matrix homeostasis (TIMP2 and TRAPPC2) and cilia function (OFD1). TIMP2 allelic expression was not affected by the CNV in miscarriages in comparison to control elective terminations.ConclusionWe propose that functional studies of CNVs could help determine if and how the miscarriage CNVs affect the expression of integral genes. In case of parental CNVs, assessment of the function of their integral genes in parental reproductive tissues should be also considered in the future, especially if they affect processes relevant for pregnancy development and support.Electronic supplementary materialThe online version of this article (doi:10.1186/s13039-015-0109-8) contains supplementary material, which is available to authorized users.

Abstract 2293: KLF5 is essential for oncogenesis of intestinal tumors and control of intestinal stem cells.

Abstract Genetic changes in intestinal epithelial stem cells are more important for oncogenesis of intestinal tumors than such changes in non-stem cells. A large-scale sequencing analysis of human colorectal carcinoma (CRC) revealed that Wnt/β-catenin signal activation is the most frequent genomic changes (92-97%) in human colorectal carcinoma (CRC). However, fundamental significance of its signal activation of intestinal stem cells is still remains unsolved. Kruppel-like factor 5 (KLF5) is one of KLF zinc finger transcription factor with diverse biological functions. KLF5 has stronger expression in the bottom of intestinal crypts, where the stem cells reside, and weaker expression in villi as the epithelial cells differentiate into more mature cells, which is complementary pattern of KLF4. KLF5 is implicated in the renewal and maintenance of ES cells, and has the potential to induce iPS cells, although weaker than KLF4. In this study, we found that inducible deletion of KLF5, specifically in Lgr5+ intestinal epithelial stem cells, suppressed the proliferation and nuclear localization of β-catenin, and generated some apoptotic cells in the intestinal crypts of mice. Furthermore, we demonstrated that inducible deletion of KLF5 in all intestinal epithelia caused mislocalization of Paneth cells, which constitute the stem cell niche. These data indicate that KLF5, rather than KLF4, is required for the maintenance and differentiation of Lgr5+ intestinal stem cells. Moreover, we detected frequent genome amplification at the KLF5 locus in human colorectal cancers (CRCs), and enhanced expression of KLF5 in CRC samples. In mice, Lgr5+ stem cell-specific induction of an active β-catenin mutant generated severe lethal adenomas and carcinomas. By contrast, when the induction of active β-catenin was performed simultaneously with deletion of KLF5 in stem cells, oncogenesis was almost completely suppressed. The simultaneous deletion of KLF5 under the induction of the active mutant of β-catenin in the stem cells suppressed the transcription of HDAC1and Sphk1, which promotes CRCs, and upregulated the transcription of Aim2 and Foxo3, tumor suppressors in CRCs, in both the FACS sorted stem cells and the transit amplifying cells. These results indicate that oncogenesis of intestinal tumors induced by Wnt/β-catenin signal activation (92-97% in CRCs) in stem cells requires KLF5, as in the case of Myc, and that KLF5 is also an essential controlling factor for homeostatic turnover and maintenance, niche formation, and differentiation of the intestinal epithelial stem cells. These suggest that KLF5 is a promising target oncogene for CRC therapy directed at colon cancer stem cells. Citation Format: Takeo Nakaya, Masahiko Kuroda, Ryozo Nagai. KLF5 is essential for oncogenesis of intestinal tumors and control of intestinal stem cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2293. doi:10.1158/1538-7445.AM2013-2293

Significance of IGFBP-4 in the Development of Fetal Growth Restriction

Fetal growth restriction (FGR) is a leading cause of perinatal mortality and morbidity. Animal studies suggest dysregulation of IGF-binding protein (IGFBP)-4 is significant in the development of FGR, although human data are lacking. We postulated that IGFBP-4 is expressed at the maternal fetal interface and plays a role in regulating IGF bioavailability. Thus, maternal serum levels of IGFBP-4 may be associated with complications of abnormal placental growth and development including FGR. Circulating levels of IGFBP-4 and its protease, pregnancy-associated plasma protein-A (PAPP-A), were examined in healthy pregnancies. Their expression in villi and bed as possible sources of the circulating products were examined by immunohistochemistry. From the large Ottawa and Kingston (OaK) Birth Cohort, a nested case-control study was conducted to examine circulating levels of IGBP-4, PAPP-A, IGF-I, and IGF-II by Western blot in early gestation in 36 women who went on to develop FGR and 36 controls having normal-weight babies. IGFBP-4 was elevated in early pregnancy compared with nonpregnant women and women in later pregnancy, consistent with the presence of abundant extravillous trophoblasts and decidual cells that highly expressed IGFBP-4. High expression of PAPP-A was observed in extravillous trophoblasts and decidual cells in early pregnancy but hardly detectable in the circulation at this time, suggesting maternal circulating PAPP-A originates more likely from syncytiotrophoblasts. Increased IGFBP-4 in the maternal circulation in early pregnancy was associated with the development of FGR [0.48 (0.28-0.74) in control vs. 1.22 (0.66-1.65) in FGR; odds ratio = 22 (95% confidence interval = 2.7-181)]. No difference was observed in circulating PAPP-A, IGF-I and IGF-II in the FGR vs. control group. Our findings support the role of IGFBP-4 in regulating IGF bioavailability and provide new clues for the prevention and treatment of FGR, raising the possibility of clinical use of IGFBP-4 as an early biomarker for this condition.

The expression and activity of prostaglandin H synthase-2 is enhanced in trophoblast from women with preeclampsia.

Preeclampsia is associated with altered biosynthesis of vasoactive prostanoids in placental villi. The two isozymes of prostaglandin H synthase (PGHS) are essential for prostanoid synthesis. We tested the hypothesis that PGHS-2 expression is elevated in trophoblast from preeclamptic women, compared with trophoblast from healthy women. Using immunofluorescent staining, we demonstrated a higher PGHS-2 expression in villi from preeclampsia, compared with normal pregnancy. Cytotrophoblasts cultured from placentas of preeclamptic women expressed higher levels of PGHS-2 compared with cytotrophoblasts from normal placentas. This enhanced expression of PGHS-2 correlated with increased media levels of both thromboxane and prostaglandin E2, two products of PGHS activity. The increased prostanoid production by trophoblast from preeclamptic women was markedly reduced by NS-398, a specific inhibitor of PGHS-2. We conclude that both expression and activity of PGHS-2 are enhanced in trophoblasts from preeclamptic women compared with trophoblast from normal pregnancies. The increased production of prostanoids may contribute to the clinical syndrome of preeclampsia. Our data suggest that a selective inhibitor of PGHS-2 might provide a therapeutic alternative to prophylactic low-dose aspirin in modifying the prostanoid profile in preeclampsia.