Placental Bed Biopsies Research Articles

Fetal–maternal interface impedance parallels local NADPH oxidase related superoxide production

Blood flow assessment employing Doppler techniques is a useful procedure in pregnancy evaluation, as it may predict pregnancy disorders coursing with increased uterine vascular impedance, as pre-eclampsia. While the local causes are unknown, emphasis has been put on reactive oxygen species (ROS) excessive production. As NADPH oxidase (NOX) is a ROS generator, it is hypothesized that combining Doppler assessment with NOX activity might provide useful knowledge on placental bed disorders underlying mechanisms.A prospective longitudinal study was performed in 19 normal course, singleton pregnancies. Fetal aortic isthmus (AoI) and maternal uterine arteries (UtA) pulsatility index (PI) were recorded at two time points: 20–22 and 40–41 weeks, just before elective Cesarean section. In addition, placenta and placental bed biopsies were performed immediately after fetal extraction.NOX activity was evaluated using a dihydroethidium-based fluorescence method and associations to PI values were studied with Spearman correlations. A clustering of pregnancies coursing with higher and lower PI values was shown, which correlated strongly with placental bed NOX activity, but less consistently with placental tissue.The study provides evidence favoring that placental bed NOX activity parallels UtA PI enhancement and suggests that an excess in oxidation underlies the development of pregnancy disorders coursing with enhanced UtA impedance.

Mesenchymal stem cells reside in a vascular niche in the decidua basalis and are absent in remodelled spiral arterioles

IntroductionMaternal decidua basalis tissue attached to the placenta following delivery is a source of decidual mesenchymal stem cells (DMSCs). The in vitro characteristics of DMSCs have been partly defined but their in vivo function(s) are poorly understood. The anatomic location, or niche, provides clues regarding potential in vivo function(s) of DMSCs, but the niche has not been described. MethodsCells were isolated from the decidua basalis and flow cytometric analyses showed the expected phenotypic profile for MSC cell surface markers. In vitro, the cells differentiated into adipocytes, osteocytes, and chondrocytes. DMSCs were then stained with antibodies by immunofluorescence detection. ResultsImmunocytochemistry revealed that DMSCs were positive for FZD-9, STRO-1, 3G5, and α-SMA as expected and lacked expression of vWF and Ck7. Fluorescence in situ hybridization analysis showed the cultured cells were of maternal origin. Immunofluorescence was carried out on placental bed biopsies using the FZD-9, STRO-1, 3G5, and α-SMA antibodies. DMSCs were located in the vascular niche in decidua basalis. Immunofluorescence with antibodies to FZD-9, Ck7 and vWF revealed DMSCs in the vascular niche surrounding intact non-transformed spiral arterioles but DMSCs were absent in fully transformed spiral arterioles. DiscussionSpiral arteriole remodelling is a critical feature of human pregnancy. The DMSC niche was investigated in fully transformed and non-transformed spiral arterioles. DMSCs have not been previously implicated in spiral arteriole remodelling. The absence of DMSCs around fully transformed spiral arterioles suggests they are a target for replacement or destruction by invading placental extravillous trophoblast cells, which carry out spiral arteriole remodelling.

The frequency of acute atherosis in normal pregnancy and preterm labor, preeclampsia, small-for-gestational age, fetal death and midtrimester spontaneous abortion*

Objective: Acute atherosis is characterized by subendothelial lipid-filled foam cells, fibrinoid necrosis and perivascular lymphocytic infiltration. This lesion is generally confined to non-transformed spiral arteries and is frequently observed in patients with preeclampsia. However, the frequency of acute atherosis in the great obstetrical syndromes is unknown. The purpose of this study was to determine the frequency and topographic distribution of acute atherosis in placentas and placental bed biopsy samples obtained from women with normal pregnancy and those affected by the “great obstetrical syndromes”. We also examined the relationship between acute atherosis and pregnancy outcome in patients with preeclampsia. Material and methods: A retrospective cohort study of pregnant women who delivered between July 1998 and July 2014 at Hutzel Women’s Hospital/Detroit Medical Center was conducted to examine 16 345 placentas. Patients were classified into the following groups: (1) uncomplicated pregnancy; (2) spontaneous preterm labor (sPTL) and preterm prelabor rupture of membranes (PPROM); (3) preeclampsia; (4) gestational hypertension; (5) small-for-gestational age (SGA); (6) chronic hypertension; (5) fetal death; (6) spontaneous abortion and (7) others. A subset of patients had placental bed biopsy. The incidence of acute atherosis was compared among the different groups. Results: (1) The prevalence of acute atherosis in uncomplicated pregnancies was 0.4% (29/6961) based upon examination of nearly 7000 placentas; (2) the frequency of acute atherosis was 10.2% (181/1779) in preeclampsia, 9% (26/292) in fetal death, 2.5% (3/120) in midtrimester spontaneous abortion, 1.7% (22/1,298) in SGA neonates and 1.2% (23/1,841) in sPTL and PPROM; (3) among patients with preeclampsia, those with acute atherosis than in those without the lesion had significantly more severe disease, earlier onset, and a greater frequency of SGA neonates (p < 0.05 all) and (4) the lesion was more frequently observed in the decidua (parietalis or basalis) than in the decidual segment of the spiral arteries in patients with placental bed biopsies. Conclusions: Acute atherosis is rare in normal pregnancy, and occurs more frequently in patients with pregnancy complications, including preeclampsia, sPTL, preterm PROM, midtrimester spontaneous abortion, fetal death and SGA.

Dysregulated expression of matrix metalloproteinases and their inhibitors may participate in the pathogenesis of pre-eclampsia and fetal growth restriction

BackgroundTrophoblast invasion into the maternal endometrium serves an important function in human pregnancy. Dysregulation of the finely controlled process of trophoblast invasion can result in a wide spectrum of pregnancy abnormalities. AimsWe aimed to elucidate the relationship between the expression of matrix metalloproteinases and pregnancy complication. Study designThe study group consisted of placental bed biopsy tissues obtained from normal vaginal deliveries (N=15), normal cesarean deliveries (N=15), pre-eclampsia (N=24) and fetal growth restriction (FGR) (N=10). We evaluated the expressions of MMP-2, -8, -9, -11, -19, -15 (MT2-MMP), -16 (MT3-MMP), and -24 (MT5-MMP), as well as TIMP-1 and -3, by applying Western blot and immunohistochemistry methods. SubjectsHuman placental tissues were used for this study. Outcome measuresThe expressions of MMP-2, -8, -9, -11, -19, -15 (MT2-MMP), -16 (MT3-MMP), and -24 (MT5-MMP), as well as TIMP-1 and -3 in human placenta tissues. ResultsCompared with those in normal pregnancies, the expression of MMP-2, -8, -9 and -11 was downregulated in villous tissues of pre-eclampsia and FGR cases (p<0.05). TIMP-1 and -3 were increased in pre-eclampsia and FGR (p<0.05). No significant difference was found between normal vaginal deliveries and cesarean deliveries. ConclusionsWe speculate that the change in invasion-associated proteinase expression will affect placental development and may thus contribute to the development of complicated pregnancies.

Correlation between First and Second Trimester Uterine Artery Doppler Velocimetry and Placental Bed Histopathology.

Aim. To evaluate the relationship between uterine artery Doppler indices and placental bed histopathology independent of clinical outcome. Materials and Methods. Uterine artery measurements were performed to 510 pregnant women who had come for routine antenatal care in 11–14th and 20–24th weeks. Placental bed biopsies from 141 cases were taken during cesarean section. Physiological changes and abnormal placental histology findings were investigated and compared with Doppler findings. Results. 116 biopsies were accepted as adequate biopsy and included in the study. Physiological changes were seen in 100 biopsies. Statistically significant higher PI and RI values in second trimester and higher notch rate in both trimesters were detected in the abnormal placental histology group (P < 0,001). Conclusion. Strong relationship between uterine artery Doppler indices and preeclampsia or intrauterine growth retardation has been shown in previous studies. In our study, we concluded that there is significant relationship between Doppler findings and placental bed histopathology independent of clinical course.

Spiral Artery Remodeling and Trophoblast Invasion in Preeclampsia and Fetal Growth Restriction

Failure to transform uteroplacental spiral arteries is thought to underpin disorders of pregnancy, including preeclampsia and fetal growth restriction (FGR). In this study, spiral artery remodeling and extravillous-cytotrophoblast were examined in placental bed biopsies from normal pregnancy (n=25), preeclampsia (n=22), and severe FGR (n=10) and then compared with clinical parameters. Biopsies were immunostained to determine vessel wall integrity, extravillous-cytotrophoblast location/density, periarterial fibrinoid, and endothelium. Muscle disruption was reduced in myometrial spiral arteries in preeclampsia ( P =0.0001) and FGR ( P =0.0001) compared with controls. Myometrial vessels from cases with birth weight &lt;5th percentile ( P &lt;0.001), abnormal uterine Doppler ( P &lt;0.01), abnormal umbilical artery Doppler ( P &lt;0.001), and preterm delivery ( P &lt;0.001) had less muscle destruction compared with &gt;5th percentile. Fewer extravillous-cytotrophoblast surrounded both decidual and myometrial vessels in the normal group and preeclampsia group compared with the FGR group ( P =0.001). For myometrial vessels, the normal group contained more intramural extravillous-cytotrophoblast than in preeclampsia ( P =0.015). Decidual vessels in the FGR group had less fibrinoid deposition compared with controls ( P =0.013). For myometrial vessels, less fibrinoid was deposited in both the preeclampsia group ( P =0.0001) and the FGR group ( P =0.01) when compared with controls, and less fibrinoid was deposited in the preeclampsia group when compared with FGR group ( P &lt;0.001). Myometrial vessels obtained from birth weights &lt;5th percentile had less periarterial fibrinoid than those with &gt;5th percentile ( P &lt;0.02). A major defect in myometrial spiral artery remodeling occurs in preeclampsia and FGR that is linked to clinical parameters. Interstitial extravillous-cytotrophoblast is not reduced in preeclampsia but is increased in FGR.

Spiral Artery Remodeling in Preeclampsia Revisited

See related article, pp 1046–1054 The first observations that placental bed defects were potentially linked to clinical outcomes such as preeclampsia (PE) were made nearly 50 years ago.1 This study followed prior findings that the placental clearance of radiolabeled saline was significantly decreased in women with hypertensive gestations.2 Histological examination of placentas and placental bed biopsies obtained from complicated gestations and compared with normotensive controls were performed in hopes to discern histopathologic causes for clinical outcomes. Decades of research into placental histopathology have held fast to certain dogma; defects in spiral artery remodeling and trophoblast invasion are characteristic of hypertensive disorders of pregnancy and fetal growth restriction (FGR).3,4 These 2 processes, deemed essential for normal pregnancy, are mechanistically not understood. Our current understanding of spiral artery remodeling and trophoblast invasion is that they are highly inter-related, but separate processes. Regulation is likely multifactorial, including genetic, immune, maternal, fetal, endocrine, and cardiovascular influences. Temporal regulation is known, making timing of study and sampling a large issue, particularly in women. Typically, samples are only available as terminations in first trimester, before presentation of maternal disease, or at term, well after disease presentation. There is no opportunity for serial sampling, which is an understandable limitation, to our current knowledge, in humans. Data from animal models have yielded important findings, although the potential for translation to humans is questionable. Even in studies of animals with hemochorial placentation, several differences are apparent. Perhaps the best studied are mice, with short (19–20) …

The Role of Apoptosis on Trophoblast Cell Invasion in the Placental Bed of Normotensive and Preeclamptic Pregnancies

Background. Placental development depends on careful coordination of trophoblast proliferation and apoptosis; however, the synchrony of its effect on trophoblast invasion is unknown. Objective. To examine the relationship between trophoblast apoptosis and proliferation in placental bed tissue of preeclamptic and normotensive pregnancies. Methods. Serial sections from archived placental bed biopsies of 12 normotensive (group 1) and 12 preeclamptic (group 2) were immunolabeled with a rabbit anti-Ki67 antibody, a mouse anti-cytokeratin 18 and its neo-epitope, and a monoclonal cytodeath M30 antibody. Results. The immunoexpression of Ki67 for all trophoblast cell subpopulations within the myometrium was non-reactive in both study groups. Smooth muscle cells of the microvasculature reflected a moderate degree of proliferation in both groups. Morphometric image analysis of the wall of the spiral artery revealed a mean area of 31,1729 ± 51,180 µm2 compared to 35,795 ± 8045 µm2 in groups 1 and 2, respectively. An elevation of intramural trophoblast was evident within the spiral artery of group 1 (13%). Comparative analyses of M30 distribution on corresponding serial sections were 0.06% versus 0% in groups 1 and 2, respectively. The mean field area percentage of interstitial trophoblast invasion was 10.79% versus 2.87% with corresponding areas of apoptosis been 0.8 % versus 1.9 % in groups 1 and 2, respectively. Conclusions. This study demonstrates an increased trophoblast apoptosis in placental bed of preeclamptic compared to normotensive pregnancies with concurrent absence of proliferation at term.

Altered expression of interleukin-6, interleukin-8 and their receptors in decidua of women with sporadic miscarriage

Are alterations in decidual expression of interleukin (IL)-6 and IL-8 associated with sporadic miscarriage? IL-6 and IL-8 secretion from decidual uterine natural killer (uNK) cells and macrophages isolated from women with spontaneous miscarriage was reduced compared with normal controls. Miscarriage is a common gynaecological problem with huge financial and personal implications. Eleven to twenty per cent of all clinically recognized pregnancies are lost before the 20th week of gestation, with miscarriages often being divided into early (≤ 12 completed weeks from last menstrual period) and late (≥ 13 weeks). Spiral artery remodelling is a key feature of early pregnancy; failure of this process has been implicated in sporadic miscarriage. The molecular triggers that initiate spiral artery remodelling are not clear, although cytokines such as IL-6 and IL-8 may play a role. This was a laboratory-based study using decidual and placental bed biopsy samples from women with sporadic miscarriage (n = 30) and termination of pregnancy controls (n = 30). Total adherent decidual cells, CD10(+) stromal cells, CD14(+) macrophages and CD56(+) uNK cells were isolated from decidua from apparently normal pregnancies that were terminated at either 8-10 or 12-14 weeks' gestation. In addition, CD14(+) macrophages and CD56(+) uNK cells were isolated from decidua from sporadic miscarriage at 8-10 weeks' gestation. Secreted IL-8 was measured in all isolated cell populations, while IL-6 was measured in CD14(+) macrophages and CD56(+) uNK cells from both sporadic miscarriage and normal controls. Placental bed biopsies were taken from women after sporadic miscarriage or termination of pregnancy at ≤ 12 completed weeks' or >13 weeks' gestational age, formalin-fixed, paraffin-embedded and immunostained for IL-6, IL-6Rα, GP130, IL-8, CXCR1, CXCR2 and CD13 (aminopeptidase N). Staining intensity for each factor was assessed in extravillous trophoblast cell populations, myometrial and decidual stroma, myometrial and decidual spiral arteries and decidual glandular epithelium. A CPA model was used to assess the potential role of IL-6 and IL-8 in spiral artery remodelling. IL-8 was secreted by total adherent decidual cells, CD10(+) stromal cells and CD14(+) macrophages at both 8-10 and 12-14 weeks' gestation, with CD14(+) cells secreting the highest levels. Both CD14(+) and CD56(+) cells isolated from decidua of early sporadic miscarriage produced lower IL-6 (P = 0.04, P = 0.01, respectively) and IL-8 levels (P = 0.0007, P = 0.002, respectively) compared with normal cases. In addition, altered expression of IL-6, IL-8 and their receptors was observed in various cell types in placental bed (myometrial stroma, glandular epithelium, interstitial extravillous trophoblast cells, vascular smooth muscle cells and endothelial cells) in sporadic miscarriage, particularly from later gestational ages. IL-6 and IL-8 disrupted vascular smooth muscle morphology and organization in an in vitro model of spiral artery remodelling. By the nature of sampling at the time of miscarriage, it was not possible to ascertain the cause or effect in the observed alterations of levels of IL-6 and IL-8 in sporadic miscarriage. Alterations in the expression of IL-6, IL-8 and their receptors may be associated with the aetiology of sporadic miscarriage, especially given the potential role of these cytokines in the regulation of trophoblast invasion and spiral artery remodelling. This project was supported by funding from Wellbeing of Women (RG1000). The authors have no competing interests to declare. Not applicable.

PP016. Relation of apoptosis, proliferation and angiogenesis in early and late onset of preeclampsia

The placental bed plays a key role in placentation during gestation. Most studies investigated the expression of angiogenic factors in the placenta, but their expression and potential role in the placental bed have not been investigated adequately. The aim of the study was to examine the expression of the fact is that Apo-Cas is apoptotic marker and VEGF in placental bed of pregnancy with early, late-onset PE and without PE. Placental bed biopsy tissues obtained during Cesarean Section from patients with early-onset (n=15), late-onset (n=15) and without (n=15) PE. The normotensive controls without PE were matched for gestational age at delivery with patients with PE. The expression of Apo-Cas and VEGF in placental bed tissues were evaluated using reverse transcriptase PCR, real-time PCR, immunohistochemistry and Western blot. There was no statistical difference between the PE group and normotensive control group in age and body mass index. The level of apoptotic marker Apo-Cas was higher in early-compared to late-onset of PE (5%±1.4, and 15%±2.7). The expression of VEGF was significantly decreased in both PE groups compare to control (p<0.05), but not statistically significant between groups with PE. We also revealed reduction of VEGF receptors in endometrial stroma and its absence in endothelial cells. This study showed the prevalence of apoptosis and decreased expression of VEGF in the placental bed of pregnancies complicated by PE compared with control. Further research will help to create the pathogenetic basis for early prediction, recognition and management of PE.

Corin, an enzyme with a putative role in spiral artery remodeling, is up-regulated in late secretory endometrium and first trimester decidua

What is the nature of cellular Corin expression in human gestational tissues? CORIN is expressed in non-pregnant late secretory phase endometrium, first trimester human implantation sites and is up-regulated with decidualization ex vivo. Adequate trophoblast invasion and spiral artery remodeling/transformation is critical for successful implantation. CORIN, best known for its role in activating atrial natruietic peptide (ANP) to regulate blood pressure, has recently been proposed to be centrally involved in trophoblast invasion and spiral artery remodeling. It is postulated that ANP, activated by CORIN, promotes trophoblast invasion and that a deficiency causes pre-eclampsia. Mice deficient in either Corin or ANP displayed poor trophoblast invasion, impaired spiral artery remodeling and phenocopied human pre-eclampsia. However, the precise cellular localization of CORIN within human gestational tissues has not been well characterized. We measured CORIN protein localization in a number of human gestational tissues relevant to early embryo/placental implantation: non-pregnant (NP) endometrial biopsies (n = 5 per phase of the menstrual cycle), first trimester placental bed biopsies (n = 12) and pre-term control (n = 10) and severe early onset preeclamptic placentas (n = 15). Endometrial stromal cells were isolated from human endometrial biopsies (n = 5) and induced to decidualize ex vivo. Finally, CORIN concentrations were measured in serum obtained from pregnant women during the first trimester of whom, 56 subsequently ended up with a healthy term delivery (controls), 18 developed fetal growth restriction (FGR) and 21 had a miscarriage. We performed immunohistochemistry to assess CORIN localization. Changes in Corin mRNA expression in human endometrial stromal cells decidualized ex vivo were measured by quantitative RT-PCR, and levels of CORIN within human sera were measured by ELISA. CORIN was expressed in both NP late secretory phase endometrium and first trimester decidua within placental bed biopsies. Importantly, decidualization of primary human endometrial cells ex vivo significantly increased Corin expression (P < 0.05). CORIN was also detected within the villous cytotrophoblast, but there was no change in mRNA levels in placentas complicated by severe preterm pre-eclampsia when compared with pre-term controls. Although CORIN was detected in first trimester serum, levels did not change across gestation, nor could they predict miscarriage or FGR (other disorders of impaired placental invasion). Owing to the fact that we utilized early pregnancy human specimens, this is mainly a descriptive study with a limited amount of functional experiments. This is the first study to thoroughly characterize Corin mRNA and protein expression in human gestational tissue. Our findings support recent data from murine studies collectively suggesting that CORIN plays a critical role in trophoblast migration and spiral artery remodeling during early pregnancy in humans. Therefore, further studies of CORIN biology in early pregnancy may identify new therapeutic targets to improve implantation quality in early pregnancy and potentially reduce the rates of pregnancy complications caused by inadequate implantation (pre-eclampsia, FGR and miscarriage). This study was supported by The National Health and Medical Research Council of Australia (Salary support #490970, #490995). The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript. The authors declare that no competing interests exist.

Aberrant expression of corticotropin-releasing hormone in pre-eclampsia induces expression of FasL in maternal macrophages and extravillous trophoblast apoptosis

Corticotropin-releasing hormone (CRH) and its receptors are expressed in human placenta. Recently, the impaired function of this system has been associated with a number of complications of pregnancy, including pre-eclampsia. The aim of the study was to test the hypothesis that CRH participates in the pathophysiology of pre-eclampsia through the induction of macrophage-mediated apoptosis of extravillous trophoblasts (EVTs). We found that the expression of CRH was increased in the EVT of the placental bed biopsy specimens from pre-eclamptic pregnancies (1.8-fold increase; P < 0.05). In addition, significantly larger numbers of apoptotic EVT were detected in pre-eclamptic placentas compared with normal ones (P < 0.05), and only in pre-eclamptic placentas, decidual macrophages were found to be Fas ligand (FasL)-positive. In vitro studies on the effect of CRH on human macrophages suggested that CRH induced the expression of the FasL protein in human macrophages and potentiated their ability to induce the apoptosis of a Fas-expressing EVT-based hybridoma cell line in co-cultures. These findings demonstrate a possible mechanism by which the aberrant expression of CRH in pre-eclampsia may activate the FasL-positive decidual macrophages, impair the physiological turnover of EVT and eventually disturb placentation.

The role of vascular smooth muscle cell apoptosis and migration during uterine spiral artery remodeling in normal human pregnancy

During human uterine spiral artery (SpA) remodeling, vascular smooth muscle cells (VSMCs) are lost and replaced by fibrinoid, incorporating extravillous trophoblast (EVT) cells. The aim of the current study was to determine the relative contributions of apoptosis and migration to VSMC loss during SpA remodeling. Immunohistochemistry (Apoptag, active caspase 3, lamin) of placental bed biopsies (8-20 wk gestation) demonstrated apoptotic cells in all samples; double immunolabeling identified these as trophoblasts, leukocytes, and endothelial cells. In total, 294 SpAs were studied, and only one apoptotic VSMC was identified. H-caldesmon-immunopositive VSMCs were observed surrounding and separate from SpA walls in partially remodeled vessels; the highest level of VSMC migration was observed in vessels with associated EVT cells (number of migrated cells 6.4 ± 1.2; distance migrated 3.5 ± 0.3 pixels) compared with those without (number of migrated cells 3.6 ± 0.5, P<0.001; distance migrated 2.8 ± 0.1 pixels, P<0.0001). VEGF-A, VEGF-C, TGF-β1, and Ang-2 all stimulated human aorta VSMC invasion in vitro, although EVT cell culture supernatants did not. In summary, apoptosis is unlikely to play a major role in loss of VSMCs from SpAs during remodeling in normal pregnancy, but VSMCs appear to migrate away from the wall of the SpA, an effect enhanced by the presence of EVT cells.

Decreased expressions of vascular endothelial growth factor and visfatin in the placental bed of pregnancies complicated by preeclampsia

The aim of the present study was to examine the expression of vascular endothelial growth factor (VEGF) and visfatin in the third trimester placental bed of pregnancies with and without preeclampsia (PE). The study group consisted of placental bed biopsy tissues obtained from pregnancies with (n = 20) and without (n = 20) PE. The normotensive controls without PE were matched for gestational age at delivery with patients with PE. The expression of VEGF and visfatin in the placental bed tissues were evaluated using reverse transcriptase-polymerase chain reaction (RT-PCR), real-time polymerase chain reaction (PCR), immunohistochemistry and Western blot. There was no statistical difference between the PE group and the normotensive control group in age and body mass index (BMI). The expression of VEGF and visfatin was significantly decreased in the PE group compared with the normotensive control group (P < 0.05). This study showed decreased expressions of VEGF and visfatin in the third trimester placental bed of pregnancies with PE compared with the normotensive controls. This result suggests that decreased expression of these angiogenic factors in placental bed may be associated with the pathogenesis of PE.

Effects of interleukin-6 on extravillous trophoblast invasion in early human pregnancy

Invasion of uterine tissues by extravillous trophoblast cells (EVT) is essential for successful human pregnancy. EVT invasion is tightly regulated by a number of factors, including growth factors and cytokines, but the mechanisms that underlie their regulatory effect remain poorly understood. Interleukin (IL)-6 has been suggested to play a role in controlling EVT invasion. We hypothesized that IL-6 produced by cells in uterine decidua would regulate EVT invasiveness via IL-6Rα and gp130 receptors expressed by trophoblast cells. The effect of IL-6 on EVT signalling and cytokine production was also studied. Supernatants from disaggregated 'total' decidual cells, CD8(+) T cells, CD10(+) decidual stromal cells, CD14 macrophages, CD56(+) uterine natural killer cells, cytotrophoblast and EVT cells contained large quantities of IL-6 protein at both 8-10 and 12-14 weeks gestational age. IL-6Rα and gp130 were immunolocalized to EVT in placental bed biopsies from 8 to 20 weeks gestation and IL-6Rα expression was confirmed by western blotting. IL-6 had no effect on the invasive potential of EVT from chorionic villi or the immortalized EVT cell line HTR-8/SVneo in a Matrigel(®) invasion assay. IL-6 stimulated phosphorylation of several cell signalling proteins in EVT (8-14 weeks' gestation), although significance was lost after correction for multiple comparisons. Incubation with IL-6 decreased secretion of regulated upon activation, normal T-cell expressed and secreted (RANTES) by EVT cells. In conclusion, although IL-6 did not affect trophoblast cell invasion, it stimulated EVT cellular cascades and inhibited secretion of RANTES involved in a number of cellular processes.

Increased Nerve Fibers in Placental Bed Myometrium in Women With Preeclampsia

Narrowing of the uterine spiral arterioles below the deciduomyometrial junction is 1 of the key pathophysiological changes in women with preeclampsia. The contribution of pelvic autonomic nerves to decidualization and impaired placentation in preeclampsia is not clear. Placental bed biopsies were obtained from 10 women with preeclampsia and 23 nornotensive women at caesarean section. We stained them with anti-S100 and CD34 antibodies to detect the presence of nerve fibers and blood vessels, respectively. We detected S100-immunoactive nerve fibers in the myometrium but not in the decidua in both groups of women. S100-immunoactive nerve fiber density in the placental bed myometrium was significantly increased in women with preeclampsia compared to normotensive women. There was no clear relationship between the densities of nerve fibers and CD34-positive blood vessels in these biopsies. These results suggest increased nerve fibers in the placental bed myometrium may play a role in the pathogenesis of the preeclampsia.

Lymphatics in the human endometrium disappear during decidualization

The mammalian placenta plays a central role in maternal tolerance of the semi-allogeneic fetus and fluid balance between the maternal and fetal compartments. The lymphatics play a role in both these function. The aim of this study was to describe the distribution of lymphatic vessels in human decidua, with particular focus on the lymphatics that surround remodelling spiral arteries during decidualization and trophoblast invasion. Placental bed and non-placental bed (decidua parietalis) biopsies were obtained from 41 women undergoing elective termination of pregnancy at 6-18 weeks gestational age as well as placental bed biopsies from 5 women undergoing elective Caesarean section at term. In addition to routine haematoxylin and eosin staining, double immunohistochemical labelling was performed on serial 3-µm sections to identify lymphatic vessels in conjunction with one of the following: blood vessels, smooth muscle, epithelial and trophoblast cells or proliferating cells. Representative photomicrographs of all sections were obtained from a total of 273 areas (46 samples, average 6 range 3-15 areas per sample). Descriptive findings of the organization of lymphatics in human placental bed and decidua parietalis were made from a total of 1638 images. Lymphatic vessels positive for podoplanin were abundant in non-decidualized hypersecretory endometrium at all stages of gestation. By contrast, the decidua was nearly always devoid of lymphatics. In some samples, structures that appeared to be regressing lymphatics could be observed at the boundary between non-decidualized hypersecretory and decidualized endometrium. Lymphatic vessels were notably absent from the vicinity of spiral arteries that were surrounded by decidualized stromal cells. Lymphatic vessels in non-decidualized hypersecretory endometrium appeared larger and more elongated as gestation progressed. Proliferating lymphatic vascular endothelial cells were identified in both large vessels, and in streaks of D2-40 positive cells that could have been newly forming lymphatic vessels. Placental bed lymphatics exhibited limited and variable staining with LYVE-1 at all stages of pregnancy apart from term. We have made novel observations on lymphatics in the placental bed and their relationship with other structures throughout pregnancy. Endometrial stromal cell decidualization results in a loss of lymphatics, with this phenomenon being particularly apparent around the spiral arteries.

Role of Interleukin 8 in Uterine Natural Killer Cell Regulation of Extravillous Trophoblast Cell Invasion

Background Extravillous trophoblast cell (EVT) invasion of maternal tissues is critical for successful pregnancy. Decidual factors, including uterine natural killer (uNK) and T cell derived cytokines play a role in regulating this process. Interleukin (IL) 8 has been implicated as a regulator of EVT invasion. Hypothesis uNK cell stimulation of EVT invasion is associated with IL-8 levels. Methods CD8+, total decidual and CD56 + uNK cells (8–10 and 12–14 weeks gestational age) were cultured. IL-8 mRNA and protein levels were determined. IL-8 receptors (IL-8RA and IL-8RB) were localised in first trimester placental bed biopsies. The effect of IL-8 ± IL-8 neutralising antibodies and CD8+ T cell or uNK cell supernatants ± IL-8 neutralising antibodies on EVT invasion was assessed. EVT secreted levels of MMP-2, MMP-9, uPA, PAI-1 and PAI-2 were assessed by substrate zymography or Western Blot. Results High levels of IL-8 protein and mRNA were detected in all samples. IL-8RA and IL-8RB were expressed by EVT. Exogenous IL-8 stimulated EVT invasion in a paracrine manner. uNK cell supernatants, but not CD8+ cell supernatants, stimulated EVT invasion. IL-8 neutralising antibody partially abrogated this uNK cell stimulated invasion. IL-8 increased levels of secreted MMP-2, but did not alter any of the other proteases or protease inhibitors tested. Conclusion uNK cell stimulation of EVT invasion is partially mediated by IL-8. Unstimulated CD8+ T cells do not alter EVT invasion despite secreting similar levels of IL-8 as uNK cells.

157. LYMPHATICS IN THE HUMAN PLACENTAL BED AND SURROUNDING THE SPIRAL ARTERIOLES DISAPPEAR DURING ENDOMETRIAL DECIDUALISATION

The mammalian placenta plays central roles in maternal tolerance of the semi-allogeneic fetus and fluid balance between maternal and fetal compartments. The lymphatics play a role in both immune function and fluid balance. The aim of this study was to describe the distribution of lymphatic vessels in human placental bed and decidua, with particular focus on the lymphatics that surround the remodelling spiral arteries during decidualisation and trophoblast invasion. Placental bed, non-placental bed and decidual biopsies were obtained from women undergoing elective pregnancy termination (6–18 weeks gestational age) and from women undergoing elective caesarean section at term. Double immunohistochemical labeling was performed on serial sections to identify lymphatic vessels in conjunction with blood vessels, smooth muscle, epithelial and trophoblast cells, or proliferating cells. Using representative photomicrographs, descriptive findings of the organisation of the human placental bed lymphatics were made. Lymphatic vessels positive for podoplanin (D2-40) were abundant in hypersecretory endometrium (lacking stromal decidualisation) at all stages of gestation. By contrast, the decidua was nearly always devoid of lymphatics. In some samples, structures that appeared to be regressing lymphatics were observed at the boundary between hypersecretory and decidual tissues. Lymphatic vessels were notably absent from the vicinity of spiral arteries that were surrounded by decidualised stromal cells. Lymphatic vessels in hypersecretory endometrium appeared larger and more elongated as gestation progressed. Proliferating lymphatic vascular endothelial cells were identified in both large vessels, and in streaks of D2-40 positive cells that could have been newly forming lymphatic vessels. In conclusion endometrial stromal cell decidualisation results in a loss of lymphatics, with this phenomenon being particularly apparent around the spiral arterioles; the functional consequences of this loss have yet to be elucidated.

Decidual leucocyte populations in early to late gestation normal human pregnancy

Most research on human decidual leucocytes to date has focused on the predominant CD56+ uterine natural killer (uNK) cell population in early pregnancy. Few reports have documented decidual leucocyte populations after 13 weeks gestation and in late pregnancy. Placental bed (decidua basalis) and non-placental bed (decidua parietalis) biopsies from normal pregnancies were taken from women undergoing termination of pregnancy in the 1st and 2nd trimesters and following Caesarean section in the 3rd trimester. Immunohistochemistry was used to quantify the numbers of decidual cells expressing CD56, CD3, CD8, CD94, NKG2A and CD14 and double labelled CD161+CD3+ NKT-like cells. Although a significant reduction in CD56+ uNK cells was found in 3rd trimester samples compared with 1st and 2nd trimester decidua, a substantial residual CD56+ leucocyte population was identified in 3rd trimester decidua. Expression of the KIR CD94/NKG2A mirrored that of CD56 at all gestational ages, providing an explanation for the absence of cytotoxic responses at the fetal-maternal interface. There was no difference in leucocyte populations between decidua basalis and decidua parietalis. Double immunohistochemical labelling revealed small numbers of decidual CD3+CD56+ and CD8+CD56+ cells, which decreased in number at term, and CD161+CD3+ cells, which increased in number at term. No differences in leucocyte populations were detected between decidua parietalis and decidua basalis. In contrast to previous reports, a substantial residual CD56+ cell population was demonstrated in 3rd trimester decidua. Decidual cytotoxic T-lymphocytes did not alter in number during gestation, while in contrast CD14+ macrophages decreased at term, representing the smallest decidual population assessed.