Hypoxia inhibits invasion of extravillous trophoblast cells through reduction of matrix metalloproteinase (MMP)-2 activation in the early first trimester of human pregnancy

During early pregnancy, extravillous trophoblast (EVT) cells are exposed to very low pO₂ values. In this study, we investigated the proteolytic functions and invasiveness of human primary EVT cells under hypoxic conditions to show the early placental pathophysiology.Placental samples (from 5 to 10 weeks gestation) were obtained at termination of pregnancy. Cytotrophoblast cells were separated by Percoll^® gradient method and cultured on Matrigel^® to obtain an invasive phenotype (similar to EVT). The invasion capacity (Matrigel-coated invasion assay), migration of the cells (wound healing assay), activity and expression of matrix metalloproteinase (MMP)-2 and tissue inhibitor for MMP (TIMP)-2 (gelatin gel zymography, ELISA, and quantitative RT-PCR), and expression of membrane-type (MT)1-MMP (western blot) were investigated. All cultures (except for quantitative RT-PCR) were performed under 20% oxygen, 5% oxygen, and 5% oxygen with 3 repetitions of 0.1% oxygen hypoxic stimulation for 1 h.Invasion and MMP2 activity of the cells were significantly increased in 20% and decreased in 0.1% oxygen. There was no significant difference in cell migration among the oxygen environments. Concentrations of MMP2 in the supernatant and expression of MT1-MMP were increased in both the 0.1% and 20% oxygen environments. The MMP2 mRNA level was increased after 1-h stimulation with 0.1% oxygen. The TIMP2 concentration was increased only in 20% oxygen, but the mRNA level was decreased in 0.1% oxygen.These results suggested that hypoxia might inhibit the invasive capacity and MMP2 activation of EVT cells in the early first trimester of pregnancy. Decrease in TIMP2 production may reduce the MMP2/TIMP2/MT1-MMP complex and lead to this unique behavior of EVT cells under hypoxic conditions.     

Recurrent first trimester pregnancy loss is associated with uterine septum but not with bicornuate uterus

OBJECTIVE: Determine the relative frequency of having a uterine septum or bicornuate uterus in women presenting with first trimester recurrent pregnancy loss and a divided uterine cavity by hysterosalpingography (HSG) or hysteroscopy. DESIGN: Retrospective analysis of the frequency of a uterine septum or a bicornuate uterus in all patients undergoing surgery (laparoscopy and hysteroscopy) with the clinical presentation of recurrent pregnancy loss and a divided uterine cavity between January 1995 and October 2002. SETTING: An academic medical center. PATIENT(S): Thirty-five consecutive, premenopausal women with a divided uterine cavity on preoperative HSG or hysteroscopy. INTERVENTION(S): Diagnostic laparoscopy and diagnostic hysteroscopy. MAIN OUTCOME MEASURE (S): A fused uterine fundus at laparoscopy indicating a uterine septum. RESULT(S): All 35 patients had a uterine septum. CONCLUSION(S): When presenting with recurrent first trimester pregnancy loss and a divided uterine cavity by HSG or hysteroscopy, virtually all the women have a uterine septum.     

Suppression of extravillous trophoblast invasion of uterine spiral arteries by estrogen during early baboon pregnancy

The present study determined whether estrogen plays a role in regulating invasion and remodeling of the uterine spiral arteries by extravillous trophoblasts during early baboon pregnancy. The level of trophoblast invasion of spiral arteries was assessed on day 60 of gestation (term is 184 days) in baboons untreated or treated on days 25-59 with estradiol or aromatizable androstenedione. The administration of estradiol or androstenedione increased (P<0.01) maternal serum estradiol levels approximately 3-fold above normal. The mean+/-SE percentage of spiral arteries/arterioles invaded by extravillous cytotrophoblasts in estradiol-treated baboons for vessels with diameters of 26-50 microm (0.0+/-0.0), 51-100 microm (1.2+/-0.7) and >100 microm (13.2+/-5.5) was 100%, 90%, and 75% lower (P<0.001), respectively, than in untreated baboons (2.4+/-1.2%; 11.0+/-5.5%, and 54.5+/-8.5%, respectively). Similar results were obtained with androstenedione treatment. However, the distribution of uterine spiral arteries grouped by diameter or number of arteries per basal plate area, i.e. microvessel density, were similar in untreated and estrogen-treated baboons. We suggest, therefore, that the low levels of estrogen exhibited during early primate pregnancy are required to permit normal progression of trophoblast vascular invasion and that the surge in estrogen which occurs during the second-third of normal pregnancy has a physiological role in suppressing further arterial trophoblast invasion. Consequently, we propose that the estrogen-dependent restraint of spiral artery invasion/remodeling ensures optimal blood flow dynamics across the uteroplacental vascular bed to promote normal fetal growth and development.     

Glycodelin A and differentiation of first trimester trophoblast cells in vitro

Aim The glycoprotein, glycodelin A (GdA) is a main product of the maternal decidua in the first trimester of pregnancy and is secreted into the amniotic fluid. The purpose of this study was to investigate the effect of GdA on secretion and surface markers of isolated first trimester trophoblasts in vitro.Methods Cytotrophoblasts were prepared from human first trimester placentae and incubated with varying concentrations of GdA or transfected separately with the expression plasmid of GdA. Supernatants were assayed for human chorionic gonadotropin (hCG) protein concentrations. Expression of human placental lactogen (hPL), mucin 1 (MUC1) and the Thomsen–Friedenreich (TF) epitope was analysed in stimulated trophoblast cells and in unstimulated controls by immunocytochemistry.Results Glycodelin A induced a reduced expression of hPL compared with unstimulated controls. Expression of MUC1 was not affected by GdA. Freshly isolated trophoblast cells showed no TF expression but became positive for this antigen after 96 h of cultivation. GdA-stimulated trophoblast cells inhibited TF expression after 96 h of cultivation. GdA plasmids induced a significantly higher hCG production in transfected cells than in cells transfected with the empty plasmid.Conclusions The results obtained in this study suggest that GdA is involved in the differentiation of trophoblast cells. The treatment of GdA plasmid transfected trophoblast cells stimulated hCG production in isolated trophoblast cells and inhibited hPL and TF expression, suggesting a functional link between hCG and GdA.     

输卵管妊娠患者巨细胞病毒感染分析

目的 探讨输卵管妊娠患者生殖道巨细胞病毒感染情况。方法 应用酶联免疫吸附技术(ELLSA)检测输卵管妊娠患者直清中巨细胞病毒(cytomegalovius, CMV)IgG抗体和特异性IgM抗体,同时应用多聚酶链反应(PCR)检测输卵管妊娠患者宫颈分泌物、宫腔冲洗物以及手术时输卵管妊娠组织标本中CMV DNA。结果 49例输卵管妊娠患者血清IgG抗体阳性21例,阳性率42.86％;宫颈分泌物CMV DNA阳性10例,阳性率20.40％;宫腔冲洗物CMV DNA阳性9例,阳性率18.36％;输卵管妊娠组织CMV DNA阳性4例,阳性率8.16％;其中1例血清IGg抗体阳性并伴有宫颈、宫腔分泌物及输卵管妊娠组织CMV DNA阳性;4例宫颈组织与宫腔组织同时阳性。结论 既往曾有过巨细胞病毒感染者发生输卵管妊娠的风险增加,巨细胞病毒感染,尤其是生殖道巨细胞病毒感染可以导致输卵管妊娠。     

Secretion of cytokines by villous cytotrophoblast and extravillous trophoblast in the first trimester of human pregnancy

Cytokines are proposed to play roles in regulation of trophoblast invasion, spiral artery remodeling and immunoregulation during early pregnancy. Secretion of 12 cytokines (interleukin (IL)-1β, IL-2, IL-4, IL-5, IL-8, IL-10, IL-12p70, IL-13, IFNγ, GM-CSF, MCP-1 and RANTES) by first trimester extravillous trophoblast and villous cytotrophoblast cells was examined using multiplex cytokine array technology. Seven (IL-1β, IL-8, IL-12p70, IL-13, GM-CSF, MCP-1 and RANTES) of the 12 cytokines examined were detectable in the samples studied (n=10 each group). Villous cytotrophoblast production of IL-1β and IL-8 increased with gestational age. Extravillous trophoblast production of IL-8, IL-13 and RANTES increased with gestational age. At 12-14 weeks gestation extravillous trophoblast cells secreted higher levels of IL-8, IL-13 and RANTES than villous cytotrophoblast cells.     

Cytokine microenvironments in human first trimester decidua are dependent on trophoblast cells

OBJECTIVE: To compare cytokine expression profiles of decidua basalis (containing trophoblast cells) and decidua parietalis (without trophoblast cells) for determination of microenvironments in human first trimester decidua. DESIGN: Retrospective study. SETTING: School of Medicine, RWTH University of Aachen, Aachen Germany, and Bourgognekliniek Maastricht, Maastricht, The Netherlands. PATIENT(S): Forty-six women who had undergone elective first-trimester termination of viable pregnancy at 5 to 12 weeks. MAIN OUTCOME MEASURE(S): Quantitative cytokine protein analysis in decidual tissues by enzyme-linked immunosorbent assay, qualitative cytokine messenger (m)RNA analysis in isolated decidual cell samples, and comparative mRNA and protein analysis in tissues of decidua basalis compared with decidua parietalis. RESULT(S): Interleukin-2, interferon-gamma (Th-1), interleukin-4 (Th-2), and interleukin-1beta proteins are expressed in the human first-trimester decidua. Interleukin-2, interferon-gamma, and interleukin-4 mRNA mainly derive from the decidual tissue leukocytes. Interleukin-1beta mRNA is expressed by all decidual cell types. Interferon-gamma mRNA and protein is detected predominantly in the decidua basalis, which contains trophoblast cells. CONCLUSION(S): Microenvironments are established topographically by different expression of cytokines in decidua basalis and decidua parietalis. These locally specific patterns are indicative of fetomaternal cross-talk. Higher interferon-gamma concentrations in decidua basalis may influence leukocyte differentiation (e.g., macrophage activation) and trophoblast invasion (e.g., by induction of expression of major histocompatibility complex).     

甲氨蝶呤和米非司酮联合治疗非破裂型输卵管妊娠

目的探讨甲氨蝶呤(MTX)和米非司酮联合治疗非破裂型输卵管妊娠的效果。方法米非司酮300mg一次顿服,MTX20mg静注×5d。单用MTX的病人设为对照组。结果MTX和米非司酮联合治疗的成功率为87.5％,明显高于对照组。观察治疗期间病情变化,发现疗效与血β-hCG高低及有无心管搏动有关。结论MTX和米非司酮联合治疗非破裂型输卵管妊娠安全有效,适用于生命体征平稳、无剧烈腹痛、无心管搏动及血β-hCG＜30μg/L的非破裂型输卵管妊娠。     

NK and trophoblast cells interaction: cytotoxic activity on recurrent pregnancy loss

Abstract

The trial objective was to determine the peripheral blood NK cells cytotoxic activity effect on trophoblast cells at recurrent pregnancy loss (RPL). The investigation involved non-pregnant women with PRL in proliferating and secretory menstrual cycle phases (PMCPh and SMCPh, respectively); women of 6–7 weeks pregnancy with RPL in past medical history; healthy fertile non-pregnant women in PMCPh and SMCPh, women of 6–7 weeks physiological pregnancy, nulliparity healthy women with regular menstrual function in PMCPh and SMCPh. NK cells cytotoxic activity was determined using peripheral blood mononuclear cells. The target cells were JEG-3?line trophoblasts. It has been established that NK cells cytotoxic activity effect on trophoblasts is lower in SMCPh than in PMCPh in non-pregnant fertile women. The NK cells cytotoxic activity was higher in SMCPh than in PMCPh in non-pregnant women with PRL and also higher than the same value in SMCPh in non-pregnant fertile women. The increased NK cells cytotoxic activity values in SMCPh in women with RPL may be the reason for miscarriage.     

A case of tubal endometriosis mimicking the appearances of tubal pregnancy at laparoscopy

Tubal pregnancy is a common gynaecological emergency. Due to the accuracy of transvaginal ultrasonography and serum human chorionic gonadotrophin (hCG) analysis is being managed more frequently by laparoscopy in asymptomatic patients. A case of presumed tubal pregnancy is presented in which the evidence generated by ultrasonography, hCG levels and laparoscopy all suggested tubal pregnancy. However, histological examination showed a tubal endometrioma. This case highlights the importance of histological analysis of all surgical specimens and provides a rare differential diagnosis of tubal mass.     

Rupture of tubal pregnancy in the Vilnius population

OBJECTIVES: To evaluate the determinants of tubal rupture in women who suffered from ectopic pregnancy in relation to their demographic profile and medical history. STUDY DESIGN: This retrospective observational clinical study was conducted in five general hospitals in Vilnius, Lithuania. The population was composed of 879 women with surgically proven ectopic pregnancy. Tubal rupture was diagnosed at the time of surgery. Univariate and multivariate logistic regression analyses were performed to identify the risk factors for tubal rupture. RESULTS: The occurrence of tubal rupture was 29.5% (259/879). It was encountered significantly more often in women with age of > or =35 years (odds ratio 1.9 [1.3-2.8]). Patients whose EP was located in the isthmus were at higher risk of having tubal rupture (odds ratio 3.2 [2.2-4.5]) while known risk factors for EP were not associated with an elevated risk. CONCLUSIONS: Our data suggest that age of > or =35 years and implantation in the straightest segment of the tube could be associated with increased rate of tubal rupture. Of particular interest is the overall tubal rupture prevalence (29.5%) observed since these women were managed in an environment where transvaginal ultrasound equipment and quantitative assessment of beta-human chorionic gonadotrophin were not routinely available.     

Uterine NK cells and macrophages in pregnancy

The presence of immune cells in the placental bed is important for both mother and child. Although various immune cells can be found in the placental bed, such as regulatory T cells and dendritic cells, uterine NK cells and macrophages are the most prominent immune cells in the placental bed in early pregnancy. uNK cell and macrophage numbers in the placental bed decrease in the third trimester. These cells seem to be specifically adapted for their function and environment. uNK cells do not show cytotoxic activity, but are producers of cytokines, growth factors and many other factors. uNK cell function is regulated by inhibitory and activating receptors binding to HLA class I on trophoblast cells. uNK cells are also involved in regulating trophoblast invasion. Macrophages mainly show an M2-like phenotype and also produce cytokines and various other factors. They are important in phagocytosis of various cells and cell debris in the placental bed. Both cell types are also involved in angiogenesis and spiral artery remodeling in the placental bed. In this review we will elaborate on the most important functions of uNK cells and macrophages in the placental bed in humans. We will also discuss animal models, since they may provide clues for function of uNK cells and macrophages in humans.     

Docosahexaenoic acid stimulates tube formation in first trimester trophoblast cells, HTR8/SVneo

Angiogenesis is a key factor in the placentation process and vascular remodeling that involves several growth factors such as vascular endothelial growth factor (VEGF) and angiopoietin-like protein 4 (ANGPTL4). PPARs are involved in the placentation process but not much information is available on whether their ligands such as fatty acids have any effects on these processes. We therefore investigated the effect of fatty acids (arachidonic acid, 20:4 n-6(ARA), eicosapentaenoic acid, 20:5 n-3(EPA), docosahexaenoic acid, 22:6 n-3 (DHA) and oleic acid, 18:1 n-9 (OA)) on tube formation (as a measure of angiogenesis) on matrigel in the first trimester trophoblast cells, HTR8/SVneo. In addition we also investigated the effects of fatty acids on expression of genes involved in angiogenesis (VEGF and ANGPTL4) and lipid metabolism in these cells. Gene expression was determined after incubating these cells with different fatty acids for 24 h using real-time qRT-PCR, whereas VEGF and ANGPTL4 proteins were measured by respective ELISA kits. Of all the fatty acids tested, DHA increased tube formation to the greatest extent. DHA-induced increase in tube length was 583%, 247% and 70% over control, OA and EPA, respectively (p < 0.05). In addition, DHA stimulated cell proliferation by 150% of these cells. Of all fatty acids investigated, only DHA stimulated VEGF mRNA expression and protein secretion compared with control. Unlike DHA, other fatty acids (OA, EPA, ARA) stimulated ANGPTL4 mRNA expression and protein secretion in these cells. An inhibitor of VEGF decreased DHA stimulated tube formation in these cells. Altogether these data indicate that DHA may potently influence the placentation process by stimulating tube formation and this effect may be mediated in part via VEGF in first trimester trophoblast cells.     

Interleukin-2 receptor positive cells in human decidua during the first trimester of pregnancy and their association with macrophages

Summary Maternal immune rejection of the fetus could sometimes be the cause of unexplained recurrent spontaneous abortion. Components of the T cell-mediated immunity were investigated in the first trimester of pregnancy in women having spontaneous abortion and compared to those with normal pregnancy having a termination on social grounds. Interleukin 2 (IL-2) had no proliferative effect on the trophoblasts of chorionic villi and there were also no IL-2 receptors in these cells. However IL-2 receptor positive cells were found in the decidua in 7 out of 24 women with normal pregnancy and in 12 out of 18 with spontaneous abortion. A high density of macrophages showed an association with IL-2 receptors in both groups. There were no differences with respect to T-cytotoxis cells and T-helper cells in cases of normal pregnancy (with and without IL-2 receptor positive cells), whereas in women having spontaneous abortion we found lower densities of T-helper and T-cytotoxic cells in those without IL-2 receptors than in those with IL-2 receptors.     

环孢素A对正常早孕期细胞滋养细胞体外生长的调节作用

目的　探讨环孢素A(cyclosporinA ,CsA)对正常早孕期细胞滋养细胞生长的调节作用。方法　选择孕 6～ 8周行人工流产术妇女绒毛 ,应用硅化聚乙酰胺吡咯烷酮 (percoll)密度梯度离心法 ,分离细胞滋养细胞 ;采用氚标记胸腺嘧啶摄入法 ,评价细胞滋养细胞的增殖能力 ;流式细胞仪分析其细胞周期 ;并应用扫描电子显微镜观察其细胞形态。结果　 (1)CsA在有效剂量范围内 (1× 10 -4 ～ 1μmol/L)可刺激细胞滋养细胞增殖 ,当细胞培养液中CsA浓度为 10 μmol/L时 ,则显著抑制细胞滋养细胞增殖。 (2 )细胞滋养细胞在浓度为 1μmol/LCsA培养液中培养 2 4h时 ,G2 M期细胞从 (2 8± 0 5 ) %增至 (6 7± 1 3) % ,当培养 48h时 ,则S期细胞从 (14± 5 ) %增至 (2 8± 8) %、凋亡细胞从 (8 7± 2 2 ) %减至 (3 6± 1 0 ) %。 (3)在浓度为 1μmol/LCsA培养液中培养 48h可使细胞滋养细胞伪足增多、变长 ,提示细胞侵袭力增加 ;细胞培养液中CsA浓度为 10 μmol/L时伪足减少。 结论　CsA在有效剂量范围内可刺激早孕期细胞滋养细胞生长 ,细胞形态改变 ,侵袭力增加。     

Cardiotonic steroids induce anti-angiogenic and anti-proliferative profiles in first trimester extravillous cytotrophoblast cells

ObjectivePreeclampsia (preE), is characterized by abnormal placental invasion and function. Marinobufagenin (MBG), a cardiotonic steroid (CTS), inhibits cytotrophoblast (CTB) cell functions that are critical for normal placental development. This study tests the hypothesis that CTSs induce anti-angiogenic and anti-proliferative effects in CTB cells.MethodsHuman extravillous CTB cells of the line Sw-71, derived from first trimester chorionic villus tissue, were incubated with 0, 0.1, 1, 10, and 100 nM of each of three CTSs (MBG, cinobufatalin (CINO) and ouabain (OUB)) for 48 h. Thereafter, levels of pro-angiogenic (vascular endothelial growth factor (VEGF165), placental growth factor (PlGF)) and anti-angiogenic (soluble fms-like tyrosine kinase-1 (sFlt-1), soluble endoglin (sEng)) factors were measured in culture media using ELISA kits. Expression of three receptors (VEGF receptor 1 (VEGFR1), angiogenic angiotensin type 1 receptor (AT₁) and anti-angiogenic angiotensin type 2 receptor (AT₂)) were assayed using immunoblotting (western blots) in cell lysates.ResultssFlt-1 and sEng secretion were increased while VEGF165 and PIGF were decreased in the culture media of CTB cells treated with 1 nM or more of each CTSs (p < 0.01 for each). The AT₂ receptor expression was up-regulated (p < 0.05) in CTB cells treated with 1 nM or more of MBG and CINO and with 100 nM OUB, while AT₁ and VEGFR1 expressions decreased (p < 0.05) with 1 nM or more of MBG and 10 nM or more of CINO and OUB.ConclusionsCTSs influence extravillous CTB cells to induce an anti-angiogenic and anti-proliferative profile.     

Systemic interleukin-10 concentrations do not alter between the first and second trimester in normal pregnancy

Background: Interleukin 10 (IL-10) is involved in normal fecundity and systemic IL-10 changes during gestation might reflect an immunologic shift at the maternal–fetal interface. Methods: Serum IL-10 levels were measured in the first and second trimester of uncomplicated pregnancy in 32 women. The low interassay coefficient of variation of the low adjustor of the IL-10 assay (5.2%) enabled us to detect IL-10 concentrations between 0.50 pg/ml and 4.0 pg/ml. Results: There was no statistically significant difference between serum IL-10 levels in the first trimester (median 1.10; range 0.53–4.60 pg/ml) and second trimester (median 1.05; range 0.64–3.30 pg/ml). Conclusion: IL-10 is not systemically activated to a detectable degree between the first and second trimester of normal pregnancy.