Immunohistological characterization of endometrial gland epithelium and extravillous fetal trophoblast in third trimester human placental bed tissues

Summary. The distribution, morphology and antigen expression of endometrial glands, uterine vessels and fetal trophoblast have been studied in third trimester placental bed tissues with a panel of monoclonal antibodies in immunohistochemical techniques. Residual endometrial glands were numerous but often were attenuated or compressed and could only be identified clearly with epithelial cell markers. These glands must be clearly distinguished from vessels and trophoblast in immunological studies of cells in the placental bed. The changing pattern of antigen expression of both maternal glands and fetal trophoblast in placental bed tissues may indicate a form of local regulation of gene expression.     

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.     

Villous culture of first trimester human placenta--model to study extravillous trophoblast (EVT) differentiation.

During implantation and subsequent placentation the human extravillous trophoblast (EVT) cells invade the endometrium and maternal vasculature within the uterus. The origin of the EVT and signals triggering its differentiation, migration and invasion are poorly understood. First and second trimester human chorionic villi explants were used as a source of EVT and a variety of substrates which resemble extracellular matrix (ECM) in vivo have been tested to induce EVT differentiation and migration. The obtained results demonstrate that villous explants from both 5-7 and 8-10 weeks of gestation give rise to EVT cells in vitro if maintained on the surface of Matrigel or decidual extract supplemented collagen gel. Fetal calf serum (FCS) supplemented media was essential for EVT differentiation and villous trophoblast viability. Immunostaining of both EVT cells and cells from the cytotrophoblastic column with monoclonal antibody Ki67 (cell proliferation marker) indicate that EVT cells differentiate in vitro by proliferation from the tip of anchoring villi. These mononucleated, round-shaped, migrating cells are HLA-A,B,C class I antigen (W6/32) antibody and low molecular weight cytokeratin positive, and do not immunostain with PAI-1 (plasminogen activator inhibitor) and HPL antibodies. Differentiation of EVT was restricted to first trimester villous tissue; explants from second trimester placentae did not give rise to EVT. Tissue viability as monitored by glucose utilization, lactate, progesterone and hCG production rates correlated with EVT differentiation. The production rates for hCG demonstrated significant variation among individual placentae and was maintained constant for 10 days consistently only in explants cultured on decidual extract supplemented collagen matrix. The described villous tissue culture system may be, therefore, a unique in vitro model to study proliferation and differentiation of EVT from cytotrophoblastic columns, the regulation of EVT proliferation and differentiation, the role of ECM in the induction of the migration and the interaction of extravillous and villous trophoblast at the level of the cytotrophoblastic column.     

Bone-marrow origin of endometrial granulocytes in the early human placental bed

A large number of cells in the first trimester human placenta have been shown to express leucocyte-common antigen by the use of a monoclonal antibody in an immunoperoxidase technique. The distribution of endometrial granulocytes in the decidua of early pregnancy indicates that a large proportion of these cells bear the leucocyte-common antigen and are thus bone-marrow derived.     

Apoptosis of extravillous trophoblast cells limits the trophoblast invasion in uterine but not in tubal pregnancy during first trimester

During the first trimester of pregnancy extravillous trophoblast cells (EVT) invade the maternal decidua. Invasion normally is reduced from the second trimester onwards and stops in the inner third of the myometrium. By contrast, in extrauterine tubal pregnancy, trophoblast invasion may even penetrate the tubal wall, which ultimately leads to the rupture of the fallopian tube. Induction of apoptosis of EVT cells, by maternal immune competent cells, may be an important mechanism to limit EVT invasion in uterine pregnancy. Tissue specimens from first and second trimester uterine pregnancy and first trimester tubal pregnancy were analyzed for apoptosis by TUNEL- and M30-staining. By immunohistochemical double labelling, maternal leukocyte subtypes were co-localized to apoptotic cells and in this context, the number of CD56(+)NK cells was analyzed. Our data show that apoptosis is confined to the decidua basalis. Most apoptotic cells are single cytokeratin-positive epithelial cells residing in the stromal compartment. Consequently these cells can only be EVT cells. Maternal leukocytes are not apoptotic. They are located in close contact to apoptotic cells. The number of apoptotic cells in the second trimester (1.8+/-0.7 per cent) is reduced compared to first trimester (5.6+/-0.7 per cent) of uterine pregnancy. In parallel, the number of NK cells declines from first (24.4+/-2.9) to second (12.4+/-1.8) trimester. Furthermore, apoptosis is significantly reduced in ectopic (0.9+/-0.3 per cent) compared to eutopic first trimester pregnancies. Consequently, we suggest that in first trimester uterine pregnancy, induction of EVT cell apoptosis by the maternal immune system is one mechanism to limit EVT invasion. During the second trimester, in parallel to declining numbers of NK cells, the mechanism changes. However, in tubal pregnancy due to differing immunological microenvironments at the ectopic implantation site, apoptosis induction fails, which deleteriously may result in uncontrolled invasion and penetration of the tubal wall.     

Confined placental mosaicism for trisomy 14 and maternal uniparental disomy in association with elevated second trimester maternal serum human chorionic gonadotrophin and third trimester fetal growth restriction

A case of confined placental mosaicism (CPM) and maternal uniparental isodisomy 14 identified after placental karyotype revealed trisomy 14 in a newborn with intrauterine growth restriction (IUGR) and minor dysmorphic features is reported. During the second trimester of the pregnancy, multiple marker screening revealed an increased risk for Down syndrome of > 1 in 10. The maternal serum human chorionic gonadotrophin (MShCG) was markedly elevated at 4.19 MoM. Amniocentesis revealed a normal 46,XX karyotype. Fetal growth restriction has been associated with elevated MShCG and placental aneuploidy with CPM for chromosomes 2, 7, 9 and 16. The present case of CPM for chromosome 14 was also associated with fetal growth restriction and elevated second trimester MShCG, suggesting a common link. Further studies need to be done to determine if indeed elevation of second trimester MShCG is associated with increased risk of CPM. The present case again demonstrates the need to perform placental karyotype in unexplained fetal growth restriction.     

Immunohistochemical localization of HLA antigens and placental proteins (alpha hCG, beta hCG CTP, hPL and SP1 in villous and extravillous trophoblast in normal human pregnancy: a distinctive pathway of differentiation of extravillous trophoblast

Immunohistochemical localization of HLA antigens and placental proteins (alpha hCG, beta hCG CTP, hPL and SP1) in villous and extravillous trophoblast at various stages of normal human gestation were studied, using hysterectomy specimens. In the chorionic villi, the capacity for synthesizing placental proteins seemed to develop in parallel with the morphological change from mononuclear cells to multinucleated syncytiotrophoblast and no villous trophoblast expressed HLA antigens. In contrast, extravillous trophoblast, including the multinucleated trophoblastic cells at the deciduomuscular junction, expressed HLA-A, -B, and -C, and their capacity for synthesizing placental proteins did not seem to correspond with the degree of morphological change: the location of alpha hCG, beta hCG CTP and SP1 was restricted to mononuclear trophoblast in the superficial decidua, while hPL was present extensively in extravillous trophoblast. These findings strongly suggest that extravillous trophoblast possesses many distinctive biological features and differentiates in an independent manner. Mononuclear trophoblast forming the cell columns was also positive for HLA-A, -B, and -C, and no placental protein was demonstrated in these cells; this, together with previous morphological observations, may indicate the germinative nature of these cells.     

Myoglobin in third trimester amniotic fluid of human pregnancy--a potential indicator of fetal hypoxia

195 amniotic fluid samples from the third trimester were examined for their content of myoglobin by means of radio-immunoassay. 151 of the samples were obtained intrapartum, the rest (44) was taken antepartum by transabdominal amniocentesis within one week prior to delivery. Depending on the myoglobin levels measured, different amniotic fluid groups were defined: (A) amniotic fluids containing no myoglobin or traces of it (less than 3 ng/ml), (B) amniotic fluids with moderately elevated myoglobin levels (3-10 ng/ml), (C) amniotic fluids with high myoglobin levels (greater than 10 ng/ml). Myoglobin levels above 3 ng/ml could be measured in 98 of the 151 samples taken intrapartum. In the amniotic fluids drawn antepartum the proportion of "myoglobin-positive" samples (greater than 3 ng/ml) amounted to only 22.7% (10 out of 44 samples). In pregnancies with amniotic fluids showing high myoglobin levels intrapartum, the prevalence of meconium staining of the samples, pathological cardiotocograms intrapartum and metabolic acidoses in umbilical artery blood samples was significantly higher than in pregnancies with myoglobin-negative amniotic fluids. The frequency of cesarean sections for fetal distress rose with increasing myoglobin levels in amniotic fluid, being 5.7, 13, and 18.2% in Group A, B and C respectively. In the pregnancies in which the amniotic fluid samples were taken antepartum, the prevalence of meconium-stained amniotic fluid increased with elevating amniotic fluid myoglobin (p less than 0.05, Group A vs. Group C). The frequency of cesarean sections for fetal distress and of neonatal depression immediately after delivery was considerably heightened in the cases with myoglobin-positive amniotic fluids antepartum compared to those with myoglobin-negative liquor.(ABSTRACT TRUNCATED AT 250 WORDS)     

Transabdominal placental biopsy in the second and third trimester of pregnancy.

Transabdominal placental biopsy under ultrasound guidance was carried out in 260 cases in the second trimester and 50 cases in the third trimester of pregnancy. Placental tissue was aspirated using an 18 or 20 gauge needle. In a total of 310 placental biopsies in the second and third trimester, 100 were performed because of suspicious ultrasonographic findings. Placental biopsy is simple in the presence of severe oligohydramnios where fetal blood sampling is usually more difficult. Oligohydramnios and polyhydramnios were the ultrasonographic findings in 50% of cases and were found to be associated with 30% of abnormal chromosomal findings. There was one (0.3%) abortion within two weeks following placental biopsy. Placental biopsy did not affect the outcome of the pregnancy.     

Expression of urocortin in the extravillous human trophoblast at the implantation site

Urocortin (UCN) is a 40 amino acid peptide which is closely related to corticotropin-releasing hormone and binds with high affinity to both CRH type 1 and type 2 receptors. UCN is expressed in human reproductive tissues including endometrium, ovary, and placenta. This study was designed to investigate the cellular localization of UCN at the implantation site of the human blastocyst, as well as the regulation of the UCN promoter by two major intracellular signaling pathways, the cAMP/PKA and diacylglycerol/PKC pathways, in cells of placental origin. For this reason, immunohistochemistry was performed on tissue sections from paraffin-embedded human first trimester placentas and freshly isolated human invasive extravillous trophoblast cells (EVT) were analyzed for UCN expression using RT-PCR and immunofluorescence. Finally, UCN promoter activity was analyzed in the JEG3 human choriocarcinoma cell line. Immunohistochemistry revealed expression of UCN in the cytotrophoblast, the EVT and decidual cells. Both UCN mRNA and peptide were detectable in freshly isolated EVT. Finally, a human UCN promoter luciferase reporter construct transfected into JEG3 cells was significantly inducible by phorbol ester plus ionomycin, but not by phorbol ester alone or by forskolin. Collectively, the present study reports the expression of UCN in EVT and the activation of the UCN gene promoter by the diacylglycerol/PKC pathway. The functional significance of urocortin for the physiology of EVT requires further investigation.     

Evidence for the expression of granulocyte-macrophage colony-stimulating factor receptors by human first trimester extravillous trophoblast and its response to this cytokine.

The present study has demonstrated that human extravillous trophoblast cells isolated from first trimester placentae can bind granulocyte-macrophage colony-stimulating factor (GM-CSF). We have used a technique which incorporates a phycoerythrin (PE)-conjugated GM-CSF in association with a monoclonal antibody against trophoblast (BC-1) in single and double flow cytometric analysis. Trophoblast cells are also observed to show increased DNA synthesis in response to GM-CSF as assayed by [3H]thymidine incorporation and proliferative activity as demonstrated by double immunocytochemical staining of cells with a trophoblast cytokeratin marker (PKK1) and for the nuclear proliferation antigen (Ki-67). These findings provide evidence that human extravillous trophoblast expresses receptors for GM-CSF and responds to this cytokine.     

Galectin-8 is expressed by villous and extravillous trophoblast of the human placenta

Galectins (gals) as multifunctional animal lectins are of great potential significance for establishment and function of the placenta, due to their capacity to modulate cellular functions including proliferation, adhesion, and invasion. Human trophoblast is known to express gal-1, gal-3 and gal-13 proteins, as well as mRNAs for gal-14, -16 and -17. This study was undertaken to establish cellular distribution of gal-8, not previously associated with trophoblast, since we have recently detected gal-8 RNA and protein in cytotrophoblast cell material. Results of immunolocalization showed that gal-8 was expressed by villous and extravillous cytotrophoblast.     

孕晚期营养素摄入与B超下胎儿各生长指标的关系

目的:探讨妊娠晚期孕妇不同营养素摄入量与B超下胎儿各部位测量值的关系。方法:对799例无妊娠合并症及并发症的健康孕妇进行前瞻性营养调查,在妊娠32、34、36周调查3天。用食物日志形式记录3天摄入食物的种类及重量。将食物摄入量转换成营养素摄入量。于妊娠28周及38周左右行两次B超检查,测量胎儿双顶径、头围、腹围、股骨及肱骨长度,并用两次测量之差值反映胎儿孕晚期的生长情况。采用多元线性回归分析孕晚期营养与胎儿生长的关系。结果:孕晚期脂肪供能比与胎儿腹围、头围和股骨增长有关,脂肪供能比每增加1%,胎儿腹围增加2.27mm(P=0.023),胎儿头围增加3.86mm(P=0.01),胎儿股骨长度增加1.35mm(P=0.04)。钙的摄入量与胎儿股骨增长有关;锌、维生素B1、、B2及维生素E与胎儿双顶径关系密切。结论:脂肪供能比与胎儿多个生长指标密切相关,是影响胎儿出生体重的重要因素。不同的营养素对B超测量的胎儿生长指标的影响不完全相同的。     

Tests of fetal wellbeing in the third trimester of pregnancy

Five different products secreted by the fetoplacental unit into the maternal circulation were measured in 272 patients when they were 34 weeks pregnant. The most useful indicator of present pathology or future complications of pregnancy was a placental protein, pregnancy associated plasma protein. A which was raised in pre-eclamptic toxaemia, antepartum haemorrhage and premature labour. The highest values were recorded in pre-eclampsia before any signs of the disease were evident. Schwangerschafts protein 1 was also raised in pre-eclampsia and antepartum haemorrhage but only after the disease had presented. Placental lactogen was also raised in pre-eclampsia and its measurement may have some predictive value. Total oestriol was lowered in fetal growth retardation and the unconjugated steroid raised in pre-eclampsia and lowered in retarded fetal growth.