The present study was aimed to investigate the expression of tumor suppressor gene PTEN (phosphatase and tensin homolog deleted on chromosome ten) in mouse endometrium during early pregnancy and its possible role during blastocyst implantation. Real-time fluorescent quantitative PCR (FQ-PCR) and immunohistochemical techniques were applied to detect PTEN mRNA and protein expressions in endometrium in un-pregnant and pregnant mice on days 1, 3, 4, 5, 7 of pregnancy, respectively. In addition, PTEN antisense oligonucleotide was injected into the horns of uterus in pregnant mice on day 3 of pregnancy and its effects on blastocyst implantation was detected in vivo. The higher expressions of PTEN mRNA and protein were observed in pregnant mice compared with that in un-pregnant mice, with a steady increasing from day 1 to 7 and reaching the maximal level on day 5 of pregnancy. PTEN antisense oligonucleotide decreased the number of implanted blastocysts compared with saline. The results suggest that PTEN might associate with apoptosis of luminal epithelial and decidual cells, coordinating decidualization of endometrium and invasion of trophoblastic cells. Thus, PTEN may participate in the process of blastocyst implantation in mice.
Animals ; Chromosomes ; Embryo Implantation ; Endometrium ; metabolism ; Female ; Mice ; PTEN Phosphohydrolase ; metabolism ; Pregnancy ; Trophoblasts ; metabolism

Antigens, Neoplasm ; metabolism ; Antigens, Surface ; metabolism ; Colorectal Neoplasms ; immunology ; pathology ; Humans ; Prognosis ; Trophoblasts ; immunology

Protein kinase C (PKC) is a critical molecule in cellular signal transduction in mammals. It is involved in many biological processes in embryonic development, including nuclear remodeling, cell cycle adjustment and cellular polarity regulation. The present study aimed to observe the location of PKCα, an important isozyme of PKC, in fertilized, parthenogenetic and tetraploid preimplantation embryos, and compare the expression of PKCα during embryonic compaction in Kunming mice. The location of PKCα was detected by immunochemistry and laser confocal microscopy. Western blot was performed to quantify PKCα expression during embryonic compaction in the three kinds of embryos. In the experiment, fertilized embryos were flushed from oviduct or uterus at 45, 52, 69, 76 and 93 h after injection of human chorionic gonadotrophin (hCG); parthenogenetic embryos were collected by SrCl2 activation of oocytes for 6 h; and tetraploid embryos were produced by electrofusion of 2-cell embryos. Embryos were fixed at different developmental stages for immunofluorescent staining. 8-cell/4-cell embryos and morula were lysed for Western blot. The results showed that PKCα had similar location pattern in different embryos. It was distributed mainly in the nuclear aggregating around chromatin at different developmental stages. However, PKCα expressed strongly in the interphase than in mitotic blastomere. Before embryonic compaction, PKCα was localized at the blastomere boundary. At late blastocyst stage of fertilized embryos, PKCα was localized only in the polar trophoblast, but not in other trophoblast. At late stage of pathenogenetic blastocyst, there was no clear PKCα signal in the polar trophoblast. Tetraploid embryos had larger blastomere than other embryos and compacted after 4-cell stage, but not after 8-cell stage. Meanwhile, there was PKCα signal at the blastomere boundary at 4-cell stage. Our results showed that the expression of PKCα lasted through all the preimplantation stage. Although there were different expression levels among different stages, the expression increased around embryonic compaction. Quantification of expression of PKCα by Western blot demonstrated that the expression increased after compaction, indicating that the compaction was possibly dependent on the relocation of PKCα. Moreover, it was shown that the second relocation of PKCα occurred during the blastocyst formation. PKCα had different expression patterns in the three kinds of preimplantation embryos. However, the effects of PKCα on embryonic development started in early stage. There must be a necessary connection between PKCα relocation and cell adhesion starting at embryonic compaction.
Animals ; Embryonic Development ; Female ; Mice ; Parthenogenesis ; Pregnancy ; Protein Kinase C-alpha ; metabolism ; Tetraploidy ; Trophoblasts ; enzymology

OBJECTIVETo explore the possible effects of 50 Hz magnetic fields (MF) exposure on HCG and progesterone secretion of human villous trophoblasts in vitro.

METHODSThe trophoblasts were isolated from human villus by trypsin digestion and incubated in DMEM medium. Then the trophoblasts were exposed to 0.2 mT, 0.4 mT 50 Hz MF for 6 h, 12 h, 24 h, 48 h and 72 h, respectively. Each exposure group was matched to one control group which was from the same villus and cultured with the same condition except the 50 Hz MF exposure. The concentration of human chorionic gonadotropin (HCG) and progesterone in the culture medium was detected by electrochemiluminescence immunoassay. Statistical significance of differences between means was determined by one way-ANOVA with P < 0.05 considered significant.

RESULTSExposure of trophoblasts to 50 Hz MF at 0.2 mT intensity within 72 h did not affect the secretion level of HCG and progesterone (compared with blank control, P > 0.05). There was also no significant change of the secretion level of HCG and progesterone when trophoblasts were exposed to 0.4 mT 50 Hz MF within 48 h (compared with blank control, P > 0.05). However, 50 Hz MF inhibited the HCG and progesterone secretion significantly with exposure for 72 h (compared with blank control, P < 0.05).

CONCLUSIONThe exposure to 50 Hz MF for long period could inhibit trophoblasts excreting the HCG and progesterone, and the threshold intensity may be between 0.2 mT and 0.4 mT.

Cells, Cultured ; Chorionic Gonadotropin ; metabolism ; Female ; Humans ; Magnetic Fields ; adverse effects ; Pregnancy ; Progesterone ; metabolism ; Trophoblasts ; metabolism ; secretion

OBJECTIVE: To investigate the expression of Talin1 in the fallopian tube and chorionic villi in patients with tubal pregnancy and its role in regulating invasion and migration of trophoblasts. METHODS: Immunohistochemistry and Western blotting were used to detect the localization and expression level of Talin1 in the fallopian tube and chorionic villi in patients with tubal pregnancy and in women with normal pregnancy. In the cell experiment, HTR-8/SVneo cells was transfected with Talin1 siRNA and the changes in cell invasion and migration were assessed using scratch assay and Transwell assay. The expressions of MMP-2, MMP-9, N-cadherin and Snail in the transfected cells were detected by qRT-PCR and Western blotting. RESULTS: Positive expression of Talin1 was detected in both normal fallopian tube tissues and tissues from women tubal pregnancy, and its expression was localized mainly in the cytoplasm of cilia cells. The expression level of Talin1 was significantly higher in both the fallopian tube and chorionic villi in women with tubal pregnancy than in normal fallopian tube and chorionic villi samples (P < 0.01). In HTR-8/SVneo cells, transfection with Talin1 siRNA significantly inhibited cell invasion (P < 0.01) and migration (P < 0.05), down-regulated the expression of N-cadherin, MMP-2 and Snail (P < 0.05), and up-regulated the expression of MMP-9 in the cells (P < 0.05). CONCLUSION The expression of Talin1 in the fallopian tube and chorionic villi is significantly increased in women with tubal pregnancy, suggesting the association of Talin1-regulated trophoblast cell invasion with the occurrence of tubal pregnancy.
Cadherins/metabolism* ; Cell Movement ; Chorionic Villi/metabolism* ; Fallopian Tubes/metabolism* ; Female ; Humans ; Matrix Metalloproteinase 2/metabolism* ; Matrix Metalloproteinase 9/metabolism* ; Pregnancy ; Pregnancy, Tubal/metabolism* ; RNA, Small Interfering/metabolism* ; Talin/metabolism* ; Trophoblasts/metabolism*

BACKGROUNDPreeclampsia (PE) is associated with abnormal fatty acid beta-oxidation (FAO), especially metabolic disorders of long-chain fatty acid oxidation. The role of FAO dysfunction in inadequate invasion is unclear. The aim of this study was to explore the influence of various lengths fatty acids oxidation on invasiveness of trophoblasts.

METHODSPrimary human trophoblast cells and HTR8/SVneo cells were treated with fatty acids of various lengths. Morphological changes, lipid deposition and ultrastructure changes of trophoblast cells were detected. Cells invasiveness was determined by transwell insert. CPT1, CPT2 and long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD) protein expression were analyzed. The correlation between intracellular lipid droplets deposition and cells invasiveness was evaluated.

RESULTSCells treated with long-chain fatty acids showed significant increased lipid droplets deposition, severe mitochondrial damage, decreased CPT2 and LCHAD protein expression (P < 0.05) but no significant difference in CPT1 protein expression (P > 0.05). Invasiveness of the trophoblast cells of the LC-FFA group significantly decreased (P < 0.05). Intracellular lipid droplets deposition was negatively correlated with invasivenss (R = -0.745, P < 0.05).

CONCLUSIONTrophoblast cells after stimulation with long chain fatty acids exist fatty acid oxidation disorders, and reduce the ability of trophoblastic invasion.

Cell Line ; Cells, Cultured ; Fatty Acids, Nonesterified ; pharmacology ; Humans ; Lipid Metabolism ; Long-Chain-3-Hydroxyacyl-CoA Dehydrogenase ; metabolism ; Trophoblasts ; drug effects

This paper aimed to study the ability of baicalein to block human cytomegalovirus (HCMV) infection in extravillous cytotrophoblasts (EVT) and its effect on the vasoactive intestinal peptide (VIP) expression in HCMV-infected EVT in vitro. A human trophoblast cell line (HPT-8) was chosen in this study. HCMV with 100 TCID50 was added into culture medium to infect HPT-8 cells, and then HCMV pp65 antigen was assayed by immunofluorescence staining. The infection status was determined by virus titration. Real-time quantitative polymerase chain reaction (qRT-PCR) was used to detect virus DNA load in the infected cells. The expression of VIP mRNA and protein in the infected cells was measured by qRT-PCR, immunocytochemistry and Western blotting. Concentration of VIP secreted in supernatants was determined by ELISA. Red-stained HCMV pp65 antigens were found in infected HPT-8 cells 48 h after infection. HCMV replicated in large quantity in infected HPT-8 cells 4 days after infection, reaching a peak at day 6 post-infection. After treatment with baicalein, virus DNA load in infected HPT-8 cells was decreased (P<0.05), and the levels of VIP mRNA and protein, and the concentration were raised to the normal (P>0.05). Our study suggested that baicalein exerts a positive effect on the VIP expression in HCMV-infected EVT at maternal-fetal interface.
Antiviral Agents ; pharmacology ; Cell Line ; Cytomegalovirus ; drug effects ; physiology ; Flavanones ; pharmacology ; Humans ; Trophoblasts ; cytology ; drug effects ; metabolism ; virology ; Vasoactive Intestinal Peptide ; metabolism ; Virus Inactivation ; drug effects

OBJECTIVETo investigate the expression of vascular endothelial growth factor (VEGF) in human placental trophoblasts and the role of VEGF in regulating placental villous angiogenesis.

METHODSPlacental samples were obtained from 10 pregnant women receiving induced abortion in the first trimester, 10 receiving induced labor in the second trimester and 10 having cesarean section at term delivery, with gestational duration of 6-9, 18-22 and 37-38 weeks, respectively. All the samples were fixed in formalin solution and prepared for the morphological study. The expression of VEGF and vascular distribution in the placental villi were examined and evaluated by immunohistochemistry and stereomorphometry, respectively.

RESULTSIn the course of pregnancy, there was a significant decrease in the level of VEGF expression in chorionic villi (28.19+/-3.01, 18.65+/-2.43, 4.95+/-0.86, respectively, P<0.01). The radial parameters of the blood vessels showed no significant changes (26.67+/-7.74, 25.08+/-4.67, 23.36+/-5.30, respectively, P>0.05), but the length density of the blood vessels increased significantly (1.46+/-0.64, 5.58+/-1.31, 19.56+/-1.40, respectively, P<0.01).

CONCLUSIONDuring gestation, VEGF expression in chorionic villi gradually weakens but the length density of the blood vessels increases, indicating that VEGF is not the only regulatory factor of angiogenesis in the chorionic villi.

Adult ; Chorionic Villi ; blood supply ; Female ; Gestational Age ; Humans ; Neovascularization, Physiologic ; physiology ; Placenta ; metabolism ; Pregnancy ; Trophoblasts ; metabolism ; Vascular Endothelial Growth Factor A ; biosynthesis

OBJECTIVETo study the expression of matrix metalloproteinases (MMPs ) in the decidual stromal cells (DSCs) and extravillous cytotrophoblasts (EVCT) in human early pregnancy and explore the change of MMPs in endometrial stromal cell (ESC) decidualization and its impact on implantation and placentation.

METHODSThe decidua and villi from 5 women with early pregnancy and mid-secretory endometrium from 5 normal women were collected and cultured in vitro, and the supernatants of the culture media were collected after 48 hours of incubation. The expression of the MMPs in the ESCs, DSCs and EVCTs was detected using Luminex xMAP system simultaneously and the difference in MMPs expression and their correlations were analyzed with SPSS10.0 software.

RESULTSThe MMPs (MMP-1, MMP-2, MMP-3, MMP-7, MMP-8, and MMP-9) were expressed in ESCs, DSCs and EVCTs, while MMP-12 was not found in ESCs and MMP-13 not in DSCs. Expressions of MMP-8, MMP-12, and MMP-13 were lowered. Compared with the ESCs, DSCs and EVCTs showed significantly lowered expressions of MMP-1, MMP-3, and MMP-7 (P<0.05), whereas expression of MMP-2 and MMP-9 increased significantly, and the high expressions of MMP-1, MMP-3, and MMP-7 was especially obvious in the DSCs. The expressions of MMP-1, MMP-3, and MMP-7, however, were significantly decreased in the EVCTs in comparison with the DSCs. Significant correlations were noted between MMP-1, MMP-3, and MMP-7, and MMP-2 was closely correlated with MMP-9. MMP-8 was significantly lower and MMP-12 and MMP-13 showed no obvious variation in the cell culture.

CONCLUSIONMMPs are secreted by ESCs, DSCs and EVCTs. Diverse MMPs play an important role in proliferation and differentiation of the ESC to affect embryo implantation and placentation. All MMPs establish a balance to co-regulate the process of pregnancy.

Adult ; Cells, Cultured ; Decidua ; cytology ; enzymology ; Endometrium ; cytology ; enzymology ; Female ; Humans ; Isoenzymes ; metabolism ; Matrix Metalloproteinases ; metabolism ; Pregnancy ; Pregnancy Trimester, First ; Stromal Cells ; cytology ; enzymology ; Trophoblasts ; cytology ; enzymology

OBJECTIVETo investigate the role of p57 and p53 immunohistochemistry in the differential diagnosis of hydropic abortion, partial hydatidiform mole and complete hydatidiform mole.

METHODSImmunohistochemical stains (EnVision method) for p57 and p53 were performed in tissue samples of normal placenta chorionic villi (n=10), abortion chorionic villi (n=12), partial hydatidiform (n=23) and complete hydatidiform moles (n=20).

RESULTSThe expression of p57 was predominantly localized in the nuclei of villous cytotrophoblasts and stromal cells. The positive rates of p57 in normal placenta, hydropic abortion and partial hydatidiform mole were 10/10, 12/12, and 100% (23/23), respectively, with no significant difference among the groups (P>0.05). However, none of the complete hydatidiform moles analyzed exhibited p57 positivity in cytotrophoblasts and stromal cells. There was a significant difference between partial and complete hydatidiform moles (P<0.05). The expression of p53 was observed in the nuclei of cytotrophoblastic cells and intermediate trophoblasts. No p53 expression was seen in normal placenta and only 1 of 12 hydropic abortion showed p53 positivity. The positive rates of p53 expression in partial and complete hydatidiform mole were 60.9% (14/23) and 85.0% (17/20) respectively. It was significantly higher in partial hydatidiform mole than that in hydropic abortion. A significant difference was also found between partial and complete hydatidiform moles (P<0.05).

CONCLUSIONSOur findings confirm that p57 immunohistochemistry assists the differential diagnosis of complete hydatidiform mole from partial hydatidiform mole. Expression of p53 may be helpful in distinguishing partial hydatidiform mole from hydropic abortion.

Abortion, Spontaneous ; diagnosis ; metabolism ; pathology ; Cyclin-Dependent Kinase Inhibitor p57 ; metabolism ; Diagnosis, Differential ; Female ; Humans ; Hydatidiform Mole ; diagnosis ; metabolism ; pathology ; Immunohistochemistry ; Pregnancy ; Stromal Cells ; metabolism ; Trophoblasts ; metabolism ; Tumor Suppressor Protein p53 ; metabolism ; Uterine Neoplasms ; diagnosis ; metabolism ; pathology