Human Trophoblast Invasion and Spiral Artery Transformation
2001; Elsevier BV; Volume: 158; Issue: 5 Linguagem: Inglês
10.1016/s0002-9440(10)64127-2
ISSN1525-2191
AutoresFiona Lyall, Judith N. Bulmer, Elizabeth Duffie, Frances Cousins, A Theriault, Stephen C. Robson,
Tópico(s)Gestational Diabetes Research and Management
ResumoDuring early human pregnancy extravillous cytotrophoblasts invade the uterus and spiral arteries transforming them into large vessels of low resistance. Failure of trophoblast invasion and spiral artery transformation occurs in preeclampsia and fetal growth restriction (FGR); these processes are not well understood. Recent studies have suggested that cytotrophoblasts that invade spiral arteries mimic the endothelial cells they replace and express PECAM-1. It was also reported that in preeclampsia, cytotrophoblasts fail to express PECAM-1 and that failure to express endothelial cell adhesion molecules may account for failed trophoblast invasion. Despite the possible importance of adhesion molecules in trophoblast invasion, no study has systematically investigated the expression of PECAM-1 in the placental bed throughout the period of invasion, particularly in the myometrial segments where the key failure occurs. There are no studies on PECAM-1 expression in the placental bed in FGR. We have examined the expression of PECAM-1 in placental bed biopsies and placentas from 8 to 19 weeks of gestation and in the placenta and placental bed in the third trimester in cases of preeclampsia, FGR, and control pregnancies. PECAM-1 was expressed on endothelium of vessels in the placenta and placental bed but not by villous or extravillous trophoblasts in normal or pathological samples. These findings do not support a role for PECAM-1 in normal invasion or in the pathophysiology of preeclampsia or FGR. During early human pregnancy extravillous cytotrophoblasts invade the uterus and spiral arteries transforming them into large vessels of low resistance. Failure of trophoblast invasion and spiral artery transformation occurs in preeclampsia and fetal growth restriction (FGR); these processes are not well understood. Recent studies have suggested that cytotrophoblasts that invade spiral arteries mimic the endothelial cells they replace and express PECAM-1. It was also reported that in preeclampsia, cytotrophoblasts fail to express PECAM-1 and that failure to express endothelial cell adhesion molecules may account for failed trophoblast invasion. Despite the possible importance of adhesion molecules in trophoblast invasion, no study has systematically investigated the expression of PECAM-1 in the placental bed throughout the period of invasion, particularly in the myometrial segments where the key failure occurs. There are no studies on PECAM-1 expression in the placental bed in FGR. We have examined the expression of PECAM-1 in placental bed biopsies and placentas from 8 to 19 weeks of gestation and in the placenta and placental bed in the third trimester in cases of preeclampsia, FGR, and control pregnancies. PECAM-1 was expressed on endothelium of vessels in the placenta and placental bed but not by villous or extravillous trophoblasts in normal or pathological samples. These findings do not support a role for PECAM-1 in normal invasion or in the pathophysiology of preeclampsia or FGR. During early human pregnancy, extravillous cytotrophoblasts (CTBs) from anchoring villi invade the decidualized endometrium and myometrium (interstitial trophoblasts) and also migrate in a retrograde direction along the spiral arteries (endovascular trophoblasts) transforming them into large diameter conduit vessels of low resistance.1Pijnenborg R Bland JM Robertson WB Brosens I Uteroplacental arterial changes related to interstitial trophoblast migration in early human pregnancy.Placenta. 1983; 4: 397-414Abstract Full Text PDF PubMed Scopus (602) Google Scholar Endovascular trophoblast invasion has been reported to occur in two waves; the first into the decidual segments of spiral arteries at 8 to 10 weeks of gestation and the second into myometrial segments at 16 to 18 weeks of gestation.1Pijnenborg R Bland JM Robertson WB Brosens I Uteroplacental arterial changes related to interstitial trophoblast migration in early human pregnancy.Placenta. 1983; 4: 397-414Abstract Full Text PDF PubMed Scopus (602) Google Scholar This physiological transformation is characterized by a gradual loss of the normal musculoelastic structure of the arterial wall and replacement by amorphous fibrinoid material in which trophoblast cells are embedded.2Brosens I Robertson WB Dixon HG The physiological response of the vessels of the placental bed to normal pregnancy.J Pathol Bacteriol. 1967; 93: 569-579Crossref PubMed Scopus (596) Google Scholar, 3Pijnenborg R Dixon G Robertson WB Brosens I Trophoblastic invasion of human decidua from 8 to 18 weeks of pregnancy.Placenta. 1980; 1: 3-19Abstract Full Text PDF PubMed Scopus (696) Google Scholar, 4Sheppard BL Bonnar J The ultrastructure of the arterial supply of the human placenta in pregnancy complicated by fetal growth retardation.J Obstet Gynaecol Br Cwlth. 1976; 83: 948-959Crossref Scopus (186) Google Scholar, 5De Wolf F De Wolf-Peeters C Brosens I Ultrastructure of the spiral arteries in human placental bed at the end of normal pregnancy.Am J Obstet Gynecol. 1973; 117: 833-848Abstract Full Text PDF PubMed Scopus (98) Google Scholar, 6Khong TY De Wolf F Robertson WB Brosens I Inadequate maternal vascular response to placentation in pregnancies complicated by pre-eclampsia and by small-for-gestational age infants.Br J Obstet Gynaecol. 1986; 93: 1049-1059Crossref PubMed Scopus (1434) Google Scholar, 7Blankenship TN Enders AC King BF Trophoblastic invasion and modification of uterine veins during placental development macaques.Cell Tissue Res. 1993; 274: 135-144Crossref PubMed Scopus (71) Google Scholar These physiological changes are required for a successful pregnancy. Failure of trophoblast invasion and spiral artery transformation has been documented in preeclampsia (PE), one of the leading causes of maternal death. In this syndrome reduced uteroplacental perfusion is associated with widespread endothelial dysfunction and fetal growth restriction (FGR) leading to significant maternal and perinatal morbidity.8Roberts JM Redman CW Pre-eclampsia: more than pregnancy-induced hypertension.Lancet. 1993; 341: 1447-1451Abstract PubMed Scopus (1160) Google Scholar Similar spiral artery abnormalities have been reported in the placental bed of women with FGR and spontaneous abortion in the absence of maternal hypertension.4Sheppard BL Bonnar J The ultrastructure of the arterial supply of the human placenta in pregnancy complicated by fetal growth retardation.J Obstet Gynaecol Br Cwlth. 1976; 83: 948-959Crossref Scopus (186) Google Scholar, 9Michel MZ Khong TY Clark DA Beard RW A morphological and immunological study of human placental bed biopsies in miscarriage.Br J Obstet Gynaecol. 1990; 97: 984-988Crossref PubMed Scopus (62) Google Scholar, 10Jauniaux E Zaidi J Jurkovic D Campbell S Hustin J Comparison of colour Doppler features and pathological findings in complicated early pregnancy.Hum Reprod. 1994; 12: 2432-2437Google Scholar, 11Hustin J Jauniaux E Schaaps JP Histological study of the materno-embryonic interface in spontaneous abortion.Placenta. 1990; 11: 477-486Abstract Full Text PDF PubMed Scopus (268) Google Scholar, 12Khong TY Liddell HS Robertson W Defective haemochorial placentation as a cause of miscarriage: a preliminary study.Br J Obstet Gynaecol. 1987; 94: 649-655Crossref PubMed Scopus (194) Google Scholar, 13Khong TY Placental changes in fetal growth retardation. Fetus and Neonate. Physiology and Clinical Applications, vol 3.in: Hanson MA Spencer JAD Rodeck CH Cambridge University Press, Cambridge1995Google Scholar, 14McFadyen IR Price AB Geirsson RT The relation of birth-weight to histological appearances in vessels of the placental bed.Br J Obstet Gynaecol. 1986; 93: 476-481Crossref PubMed Scopus (81) Google Scholar, 15Pijnenborg R Anthony J Davey DA Rees A Tiltman AL Van Assche FA Placental bed spiral arteries in the hypertensive disorders of pregnancy.Br J Obstet Gynaecol. 1991; 98: 648-655Crossref PubMed Scopus (591) Google Scholar, 16Sheppard BL Bonnar J An ultrastructural study of utero placental arteries in hypertensive and normotensive pregnancy and fetal growth retardation.Br J Obstet Gynaecol. 1981; 88: 695-705Crossref PubMed Scopus (278) Google Scholar Thus failure of the spiral arteries to undergo physiological transformation may lead to a spectrum of pregnancy failures. Despite the importance of trophoblast invasion and vascular remodeling, these processes are still not well understood. However, they are thought to include changes in expression of cell adhesion molecules, matrix metalloproteinases and their tissue inhibitors, and growth factors and their receptors.17Lyall F Robson SC Defective extravillous trophoblast function and pre-eclampsia.in: Kingdom JCP Jauniaux ERM O'Brien SPM The Placenta: Basic Science and Clinical Practice. RCOG Press, London2000: 79-96Google Scholar, 18Lyall F Kaufmann P The uteroplacental circulation: extravillous trophoblast.in: Baker PN Kingdom JCP Intrauterine Growth Restriction. Springer-Verlag, London2000: 85-119Crossref Google Scholar Platelet endothelial cell adhesion molecule (PECAM-1) is a member of the immunoglobulin family and is a transmembrane glycoprotein of ∼130 kd.19De Lisser HM Newman PJ Albelda SM Molecular and functional aspects of PECAM-1/CD31.Immunol Today. 1994; 15: 490-495Abstract Full Text PDF PubMed Scopus (289) Google Scholar PECAM-1 is expressed by a wide variety of cells including endothelial cells, platelets, neutrophils, monocytes, and lymphocytes19De Lisser HM Newman PJ Albelda SM Molecular and functional aspects of PECAM-1/CD31.Immunol Today. 1994; 15: 490-495Abstract Full Text PDF PubMed Scopus (289) Google Scholar and appears early in the development of the vascular system.20Baldwin HS Shen HM Yan H-C DeLisser HM Chung HM Mickanin A Trask T Kirschbaum NE Newman PJ Albelda SM Buck CA Platelet-endothelial adhesion molecule (PECAM-1/CD31) and its alternatively spliced isoforms are expressed during early mammalian cardiovascular development.Development. 1994; 120: 2539-2553PubMed Google Scholar PECAM-1 is localized to cell-cell borders of adjacent endothelial cells suggesting a role in angiogenesis.21Muller WA Ratti CM McDonnell SL Cohn ZA A human endothelial cell-restricted externally disposed plasmalemmal protein enriched in intercellular junctions.J Exp Med. 1989; 170: 399-414Crossref PubMed Scopus (307) Google Scholar Several studies also support a role for PECAM-1 in leukocyte-endothelial interactions during leukocyte margination at times of inflammation.22Muller WA SAW Deng X Phillips DM PECAM-1 is required for transendothelial migration of leukocytes.J Exp Med. 1993; 178: 439-447Crossref PubMed Scopus (999) Google Scholar Recent studies have suggested that PECAM-1 and other endothelial cell adhesion molecules (CAMs) may also play a role in spiral artery transformation.23Zhou Y Fisher SJ Janatpour M Genbacev O Dejana E Wheelock M Damsky CH Human cytotrophoblasts adopt a vascular phenotype as they differentiate. A strategy for successful endovascular invasion?.J Clin Invest. 1997; 99: 2139-2151Crossref PubMed Scopus (841) Google Scholar It was suggested that CTBs that invade spiral arteries mimic the adhesion phenotype of the endothelial cells they replace and that extravillous CTBs in cell columns, interstitial and endovascular CTBs express PECAM-1. The authors also reported that in PE, the CTBs fail to express PECAM-1.23Zhou Y Fisher SJ Janatpour M Genbacev O Dejana E Wheelock M Damsky CH Human cytotrophoblasts adopt a vascular phenotype as they differentiate. A strategy for successful endovascular invasion?.J Clin Invest. 1997; 99: 2139-2151Crossref PubMed Scopus (841) Google Scholar, 24Zhou Y Damsky CH Fisher SJ Preeclampsia is associated with failure of human cytotrophoblasts to mimic a vascular adhesion phenotype.J Clin Invest. 1997; 99: 2152-2164Crossref PubMed Scopus (818) Google Scholar It was suggested that failure to express endothelial CAMs in PE may account for the failure of trophoblast invasion. Despite the possible importance of cell adhesion molecules such as PECAM-1 in trophoblast invasion, no study has systematically investigated the expression of PECAM-1 in the placental bed throughout the period of trophoblast invasion and spiral artery transformation, particularly in the myometrial segments where the key failure in invasion in PE occurs. There are also no studies on PECAM-1 expression in the placental bed in FGR. Thus in this study we have used immunohistochemistry to examine the expression of PECAM-1 in placental bed biopsies and placentas from 7 to 19 weeks of gestation and in placenta and placental bed in the third trimester in cases of PE, FGR, and matched control pregnancies. Samples were obtained from pregnant women at the Royal Victoria Infirmary, Newcastle-on-Tyneside. The study was approved by the Joint Ethics Committee of Newcastle and North Ty Authority and the University of Newcastle. The procedure for collection of placentas and placental bed biopsies from first, second, and term pregnancies has been described previously.25Lyall F Robson SC Bulmer JN Kelly H Duffie E Human trophoblast invasion and spiral artery transformation: the role of nitric oxide.Am J Pathol. 1999; 154: 1105-1114Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 26Lyall F Barber A Myatt L Bulmer JN Robson SC Hemeoxygenase expression in human placenta and placental bed n implies a role in regulation of trophoblast invasion and placental function.FASEB J. 2000; 14: 208-219PubMed Google Scholar First and second trimester samples were obtained from women undergoing termination of an apparently normal pregnancy. An initial ultrasound scan was performed to confirm fetal viability and to determine gestational age and placental position. After evacuation of the uterine contents, three placental bed biopsies were taken under ultrasound guidance using biopsy forceps (Wolf, Wimbleton, UK) introduced through the cervix. Forty-three placental bed biopsies spread evenly between 8 to 18 weeks of gestation were studied. Placental samples were collected from all cases. For the third trimester study three groups of women were studied; control pregnancies with no hypertension or FGR (n = 18), women with pregnancies complicated by PE (n = 17), and women with pregnancies complicated by FGR in the absence of maternal hypertension (n = 8). Briefly, after delivery of the infant, the position of the placenta was determined by manual palpation. Six placental bed biopsies were then taken under direct vision using biopsy forceps. Placental samples were collected from all cases. PE was defined as pregnancy-induced hypertension (blood pressure, 140/90) and proteinuria (300 mg/24 hours) in women who were normotensive before pregnancy and had no other underlying clinical problems such as renal disease. FGR was defined ultrasonically as fetal abdominal circumference (AC) 1.5 SDS27Robson SC Chang TC Measurement of human fetal growth. Fetus and Neonate, vol 3.in: Hanson MA Spencer JAD Rodeck CH Cambridge University Press, Cambridge1995Google Scholar and umbilical artery pulsatility index equaling the 95th centile.28Arduini D Rizzo G Normal values of pulsatility index from fetal vessels; a cross-sectional study of 1556 healthy fetuses.J Perinat Med. 1990; 18: 165-172Crossref PubMed Scopus (446) Google Scholar We have previously shown that a fall in AC SDS of >1.5 SDS is the optimal cut-off to define a group of fetuses with evidence of wasting at birth and morbidity associated with FGR.27Robson SC Chang TC Measurement of human fetal growth. Fetus and Neonate, vol 3.in: Hanson MA Spencer JAD Rodeck CH Cambridge University Press, Cambridge1995Google Scholar Birth weight centiles were obtained from charts of the Northern Region population of England.29Tin W Wariyar UK Hey EN Selection biases invalidate current low birthweight-for-gestation standards.Br J Obstet Gynaecol. 1997; 104: 180-185Crossref PubMed Scopus (54) Google Scholar Clinical details were compared using analysis of variance and post hoc testing was performed using the Fisher's PLSD test. All samples were frozen in liquid nitrogen-cooled isopentane and stored sealed at −70°C until required. Cryostat sections (7 μm) from each specimen were stained with hematoxylin and eosin for histological analysis. Placental bed biopsies were included in this study if they contained decidual and/or myometrial spiral arteries with interstitial trophoblasts. Desmin (NCL-DES-DERII) and cytokeratin (NCL-LP34) monoclonal antibodies were obtained from Novocastra, Newcastle-upon-Tyne, UK. The PECAM-1 monoclonal antibody was obtained from R&D Systems, Abingdon, UK. The fluorescein isothiocyanate-conjugated anti-cytokeratin monoclonal antibody was obtained from Sigma Chemical Company (Poole, UK) and the Texas red anti-mouse IgG antibody was obtained from Vector Laboratories (Peterborough, UK). Aqueous mounting medium (Citifluor) was purchased from UKC Chemical Laboratory (Canterbury, UK) and diamidino-2-phenylindole from the Sigma Chemical Company. All other reagents were purchased from Sigma unless stated otherwise. Western blotting was used to determine that the PECAM-1 antibody detected the correct molecular weight species. Placental samples comprising full thickness blocks from chorionic plate through to basal plate were snap-frozen in liquid nitrogen. Tissue samples were ground to a fine powder in liquid nitrogen with a mortar and pestle and added to 4 volumes of cold lysis buffer (25 mmol/L Tris/0.25 mol/L sucrose/1 mmol/L ethylenediaminetetraacetic acid, pH 7.6 and 50 μl/g tissue protease inhibitor cocktail) (Sigma). Using a Polytron homogenizer at setting 10, the sample containers were surrounded by ice and homogenized for 3 × 10 second intervals. The homogenate was spun at 5000 × g for 10 minutes at 4°C to remove debris and the resultant supernatant was aliquoted and stored at −70°C. Protein concentrations were determined by the method of Bradford30Bradford MM A refined and sensitive method for the quantitation of proteins utilizing the principle of protein-dye binding.Anal Biochem. 1976; 72: 248-254Crossref PubMed Scopus (222621) Google Scholar using bovine serum albumin as a standard, and diluted to the required concentration. Samples were mixed 1:1 with loading buffer (1.2 ml of 1 mol/L Tris, pH 6.8, 2 ml of glycerol, 4 ml of 10% sodium-dodecyl-sulfate, 2 ml of 1 mol/L dithiothreitol, and 0.8 ml of distilled water with bromophenol blue added to give a deep blue color) and boiled for 5 minutes before loading. Samples were separated on 10% sodium-dodecyl-sulfate polyacrylamide resolving gels with a 4% stacking gel using a minigel kit (BioRad, Hemelhempstead, UK)31Laemmli UK Cleavage of structural proteins during the assembly of the head of bacteriophage T4.Nature. 1970; 227: 680-685Crossref PubMed Scopus (213238) Google Scholar at a constant current of 15 mA. Each well was loaded with 50 μg of protein. Molecular weight markers (SDS-7B prestained 33- to 205-kd range; Sigma) were loaded beside the samples. Protein was transferred overnight in buffer containing 25 mmol/L Tris, 190 mmol/L glycine, 20% methanol at a constant 30 V to Hybond ECL nitrocellulose membranes (Amersham, UK). Filters were blocked for 1 hour at room temperature in PBST buffer (1 × PBS, 0.1% Tween 20) (containing 5% Marvel). The PECAM-1 antibody (1:1000) was prepared in PBST containing 1% Marvel and left to stand for 1 hour at room temperature before use to reduce nonspecific binding. The antibody was added for 1 hour at room temperature. The filters were rinsed twice for 5 minutes in PBST and were then incubated with horseradish peroxidase-conjugated sheep anti-mouse IgG (Diagnostics Scotland, Carluke, UK) diluted 1:1000 in PBST containing 1% Marvel for 1 hour at room temperature. Blots were then rinsed again and washed twice for 10 minutes in PBST followed by one 5-minute wash in distilled water. Proteins were detected using the Amersham ECL detection system and filters were exposed to Hyperfilm ECL (Amersham, Buckinghamshire, UK). Sections were all stained on the same day (for each antibody) to eliminate day to day variations in immunostaining. Immunohistochemistry was performed using the Vectastain Universal kit (Vector Laboratories). Cryostat sections (7 μm) were mounted on 3-aminopropyl-triethoxysilane-coated glass slides. In addition, to assist in identification of spiral arteries and trophoblasts, sections from placental bed biopsies were immunostained for cytokeratin (1:200) to detect trophoblast and desmin (1:100) to detect muscle. Two separate immunohistochemical methods were used. In the first method, sections were fixed in acetone for 5 minutes, ethanol for 5 minutes, and then rehydrated in water for 5 minutes. Nonspecific binding sites were blocked with the universal kit horse serum at 37°C and after washing in phosphate-buffered saline (PBS) for 5 minutes, the sections were incubated with the PECAM-1 antibody (1:10,000) for 90 minutes in PBS at 37°C. After 2 × 5 minute PBS washes the biotinylated secondary antibody was added for 30 minutes at 37°C. Two more PBS washes were performed and then endogenous peroxidase activity was quenched by incubating the sections in 1% (v/v) hydrogen peroxide in methanol for 15 minutes. The remaining steps were performed according to the instructions supplied with the kit and were performed at room temperature. Immunoreactive proteins were detected with Fast diaminobenzidine tablets (Sigma). Sections were counterstained in Harris's hematoxylin (BDH, Poole, UK) and mounted in synthetic resin. Omission of primary antibody or substitution of nonimmune serum for the primary antibody were both included as controls and resulted in no immunostaining. In the second method a double-immunofluorescence method was used. Samples were fixed as above and then the PECAM-1 antibody, diluted 1:10,000 in the blocking buffer supplied with the kit used in the first method, was added for 1 hour at 37°C. After 3 × 5-minute washes in PBS the second antibody (Texas Red anti-mouse IgG) was added at 1:100 in PBS for 60 minutes at 37°C. Next the cytokeratin-fluorescein isothiocyanate antibody (diluted 1:50 in blocking buffer) was added for 60 minutes at 37°C. After three further 5-minute washes in PBS the sections were mounted in aqueous mounting medium containing diamidino-2-phenylindole. Mounting medium was prepared by mixing 3 volumes of diamidino-2-phenylindole with 100 volumes of Citiflour. Coverslips were added and sealed with clear nail varnish. Sections were viewed using a Quips LS PathVysion Workstation,/Zeiss Axioplan epifluorescence microscope equipped with cooled charged coupled device camera equipped and filters that allow viewing of Texas red or fluorescein isothiocyanate labeling without cross-contamination (Applied Imaging, Newcastle, UK). The clinical details for patients used for the third trimester immunohistochemistry studies are shown in Table 1. Umbilical artery PI was abnormally elevated in all of the FGR fetuses; five had absent and one had reversed end-diastolic frequencies. Birth weight was significantly reduced in the FGR and the PE group when compared with the control group. All infants in the FGR group had a birth weight less than the 10th centile with five less than the third centile. Two of the infants in the PE group had birth weights <10th centile.Table 1Clinical Details for Placental Immunohistochemistry StudiesControl (n = 18)PE (n = 17)FGR (n = 8)Age (years)30.77 ± 6.0027.62 ± 7.3830.14 ± 8.45Gestational age at delivery (weeks)37 ± 2.9333.94 ± 4.11*P < 0.05 compared with control pregnant group.34.12 ± 2.47Birth weight (kg)3.13 ± 0.842.18 ± 1.04*P < 0.05 compared with control pregnant group.1.34 ± 0.38†P < 0.005 compared with control pregnant group.Systolic BP (mm Hg)118.07 ± 8.8155.94 ± 13.93†P < 0.005 compared with control pregnant group.120.63 ± 7.76Diastolic BP (mm Hg)70 ± 5.67105.56 ± 7.46†P < 0.005 compared with control pregnant group.70.63 ± 7.76Plasma urate (mmol/L)—422 ± 68—Values are shown as mean ± SD.* P < 0.05 compared with control pregnant group.† P < 0.005 compared with control pregnant group. Open table in a new tab Values are shown as mean ± SD. A representative Western blot of placental villous tissue is shown in Figure 1. A band of ∼130 kd was identified in the samples that is consistent with the reported molecular weight for PECAM-1.32Newman PJ The biology of PECAM-1.J Clin Invest. 1997; 99: 3-8Crossref PubMed Scopus (441) Google Scholar As shown in later immunohistochemistry experiments this band reflects PECAM-1 expressed on the villous endothelium. The overall findings showed that CTB did not express PECAM-1 across gestation. The findings were consistent with both staining methods. Figure 2 summarizes the findings. Figures 2A is a positive control for the antibodies and shows a double-immunofluorescence result for a placenta at 38 weeks of gestation. The villous endothelial cells are positive for PECAM (red) but villous CTBs and syncytiotrophoblasts (green) did not express PECAM. These results were confirmed by the second single staining ABC method (Figure 2D). Similar results were obtained for all placentas examined across gestation. Next we examined the expression of PECAM-1 in cell columns and superficial decidua. A representative case at 16 weeks of gestation stained using the double-immunofluorescence method is shown in Figure 2B. The start of the cell column is indicated by the arrow. None of the cells within the column expressed PECAM-1, however endothelial cells of blood vessels in the adjacent villi are clearly PECAM-1-positive. At the distal end of the columns and within the decidua CTBs were also PECAM-1-negative. Few occasional smaller cells that were cytokeratin-negative (presumably lymphocytes) were PECAM-1-positive and endothelium of blood vessels within the decidua were also PECAM-1-positive. These results were confirmed by the ABC method (Figure 2E). A cell island adjacent to villous tissue is shown in Figure 2C. This case was at 8 weeks of gestation. Extravillous CTBs (EVT) in the cell island were also PECAM-1-negative and this was confirmed by the ABC method (Figure 2F). Note the PECAM-1-positive blood vessels in the villous tissue in Figure 2C. Similar results were obtained for cell columns and cell islands at all gestations. CTB in the basal plate (38-week gestation sample shown) were cytokeratin-positive (Figure 2G, bottom) and PECAM-1-negative (Figure 2G, top). Within the placental bed CTBs were consistently PECAM-1-negative using both methods of immunostaining. We first used the ABC method to examine the expression of PECAM-1. Figure 2H (left) shows EVT surrounding a myometrial blood vessel. This example is at 9 weeks of gestation. The adjacent section shows that whereas the endothelium of the blood vessel is PECAM-1-positive, the CTBs are PECAM-1-negative. A section showing deep decidua at 9 weeks of gestation is shown in Figure 2I. A cytokeratin-positive gland and many EVT including giant cells can be seen (left panel) but all are PECAM-1-negative (right panel). Similar findings for all gestations were found using both staining methods. A representative double-immunofluorescence example of a myometrial spiral artery at 18 weeks of gestation that has undergone extensive invasion by CTBs is shown in Figure 2; J, K, and L. The endothelium is intact in places but in others has disappeared completely (Figure 2K). Some of the CTBs have replaced the endothelium, some are lying on top and to the outside of the endothelium, and some are within the lumen itself (Figure 2L). All of the CTBs shown are PECAM-1-negative. Because there was no double staining these findings show that invasive CTBs do not express PECAM-1. By the third trimester the majority of spiral arteries from both normal and abnormal pregnancies had an intact endothelium that was PECAM-1-positive. In normal cases where CTBs were embedded in the vessel wall the majority were separated from the lumen by endothelium and few intraluminal CTBs were evident. None of the CK-positive CTB expressed PECAM-1. Figure 3 shows immunofluorescence results for a representative case from a placental bed from a placental bed biopsy obtained from a control pregnancy at 36 weeks of gestation (Figure 3; A, B, and C), a case complicated by PE at 27 weeks of gestation (Figure 3; D, E, and F) and a case complicated by FGR at 37 weeks of gestation (Figure 3; G, H, and I). The normal case selected shows CTBs surrounding a myometrial vessel that has undergone complete physiological change; this vessel had almost no muscle remaining as assessed by desmin immunostaining (not shown). The inset shows another area of the same biopsy where CTBs are in contact with the endothelium. The biopsy shown from the case complicated by PE contains a myometrial vessel that has not undergone physiological change; most of the muscle surrounding this vessel was still intact. Despite the retention of the muscle, this and the other cases of PE still contained abundant interstitial CTBs. These CTBs were also PECAM-1-negative. Finally a case of FGR is shown. Note that the endothelium is complete and almost all of the CTBs are separated from the lumen by the endothelium. As for the control and PE groups, the CTBs in FGR placental bed biopsies did not express PECAM-1 as demonstrated by no double staining. We believe that the present study is the most comprehensive investigation of PECAM-1 expression in the placenta and placental bed. Because no population of extravillous CTBs expressed PECAM-1 our findings do not support a role for PECAM-1 in the process of normal trophoblast invasion. We also found no differences in PECAM-1 expression on trophoblast from carefully select
Referência(s)