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• W2079056593 abstract "Placental apoptosis is increased in vivo in preeclampsia (PE) and intrauterine growth restriction (IUGR). The cause and pathological implications of this phenomenon are unknown. This study considers the apoptotic susceptibility of villous trophoblasts from normal, PE, and IUGR pregnancies. Cultured cytotrophoblasts (CTs) and an in vitro model of syncytialization were used. CTs were isolated from term placentas of 12 normal, 12 PE, and 12 IUGR pregnancies. Apoptosis was determined by terminal dUTP nick-end labeling (TUNEL), Annexin V binding, and ADP:ATP ratios. Cells were stimulated with tumor necrosis factor-α/interferon-γ or reduced oxygen (<5 KPa). For CTs, ADP:ATP <1 correlates with Annexin V binding. For normal pregnancy, tumor necrosis factor-α and depleted oxygen significantly increased TUNEL, Annexin V binding and ADP:ATP in CTs and syncytiotrophoblasts (STs). Spontaneous apoptosis was similar between groups for both cell types. After stimulation, TUNEL and Annexin V binding of CTs were significantly raised in PE and IUGR as compared with normal pregnancy. After oxygen reduction, ADP:ATP in CTs and STs were significantly elevated in IUGR. TUNEL was also increased in STs in PE after oxygen depletion and was significantly raised in STs from IUGR pregnancies after stimulation with both agonists. This is the first description of enhanced apoptosis in isolated villous trophoblasts in PE and IUGR. These intrinsic differences may represent an important factor in the pathophysiology of these conditions. Placental apoptosis is increased in vivo in preeclampsia (PE) and intrauterine growth restriction (IUGR). The cause and pathological implications of this phenomenon are unknown. This study considers the apoptotic susceptibility of villous trophoblasts from normal, PE, and IUGR pregnancies. Cultured cytotrophoblasts (CTs) and an in vitro model of syncytialization were used. CTs were isolated from term placentas of 12 normal, 12 PE, and 12 IUGR pregnancies. Apoptosis was determined by terminal dUTP nick-end labeling (TUNEL), Annexin V binding, and ADP:ATP ratios. Cells were stimulated with tumor necrosis factor-α/interferon-γ or reduced oxygen (<5 KPa). For CTs, ADP:ATP <1 correlates with Annexin V binding. For normal pregnancy, tumor necrosis factor-α and depleted oxygen significantly increased TUNEL, Annexin V binding and ADP:ATP in CTs and syncytiotrophoblasts (STs). Spontaneous apoptosis was similar between groups for both cell types. After stimulation, TUNEL and Annexin V binding of CTs were significantly raised in PE and IUGR as compared with normal pregnancy. After oxygen reduction, ADP:ATP in CTs and STs were significantly elevated in IUGR. TUNEL was also increased in STs in PE after oxygen depletion and was significantly raised in STs from IUGR pregnancies after stimulation with both agonists. This is the first description of enhanced apoptosis in isolated villous trophoblasts in PE and IUGR. These intrinsic differences may represent an important factor in the pathophysiology of these conditions. Apoptosis, or programmed cell death, is crucial to the development and homeostasis of human tissues, including the human placenta. In combination with mitosis, apoptosis regulates the number of cells in any given tissue and therefore its size and function. In normal pregnancy, cytotrophoblasts (CTs) and syncytiotrophoblasts (STs) can be considered to be in a steady state, however it is likely that placental insults can alter this relationship, possibly by modulating trophoblast cell turnover.1Mayhew TM Leach L McGee R Ismail WW Myklebust R Lammiman MJ Proliferation, differentiation and apoptosis in villous trophoblast at 13–41 weeks of gestation (including observations on annulate lamellae and nuclear pore complexes).Placenta. 1999; 20: 407-422Abstract Full Text PDF PubMed Scopus (141) Google Scholar Our previous investigations have demonstrated that trophoblast apoptosis is a normal event in placental aging,2Smith SC Baker PN Symonds EM Placental apoptosis in normal human pregnancy.Am J Obstet Gynecol. 1997; 177: 57-65Abstract Full Text Full Text PDF PubMed Scopus (362) Google Scholar but we have also shown elevated levels of placental apoptosis in intrauterine growth restriction (IUGR) and preeclampsia (PE).3Leung DN Smith SC To KF Sahota DS Baker PN Increased placental apoptosis in pregnancies complicated by preeclampsia.Am J Obstet Gynecol. 2001; 184: 1249-1250Abstract Full Text Full Text PDF PubMed Scopus (249) Google Scholar, 4Smith SC Baker PN Symonds EM Increased placental apoptosis in intrauterine growth restriction.Am J Obstet Gynecol. 1997; 177: 1395-1401Abstract Full Text Full Text PDF PubMed Scopus (329) Google Scholar As there is no fall in the incidence of proliferating cells,5Smith SC Price E Hewitt MJ Symonds EM Baker PN Cellular proliferation in the placenta in normal human pregnancy and pregnancy complicated by intrauterine growth restriction.J Soc Gynecol Invest. 1998; 5: 317-323Crossref PubMed Scopus (25) Google Scholar it could be postulated that accelerated apoptosis may represent either a primary pathological event, or a secondary adaptation to maintain and optimize the syncytial barrier. Although extensively studied in other systems, the mechanism and control of apoptosis in the human trophoblast is unclear.6Levy R Nelson DM To be, or not to be, that is the question. Apoptosis in human trophoblast.Placenta. 2000; 21: 1-13Abstract Full Text PDF PubMed Scopus (124) Google Scholar Differences in the morphological features of cell death between CTs and STs, and their susceptibility to apoptosis in vitro, have suggested disparate apoptotic pathways in differentiated and undifferentiated cells.7Crocker IP Barratt S Kaur M Baker PN The in-vitro characterization of induced apoptosis in placental cytotrophoblasts and syncytiotrophoblasts.Placenta. 2001; 22: 822-830Abstract Full Text PDF PubMed Scopus (61) Google Scholar For most experiments, we have used isolated cultured CTs and an in vitro model of syncytialization and we have demonstrated that trophoblast apoptosis can be induced by a number of physiological and nonphysiological agonists.7Crocker IP Barratt S Kaur M Baker PN The in-vitro characterization of induced apoptosis in placental cytotrophoblasts and syncytiotrophoblasts.Placenta. 2001; 22: 822-830Abstract Full Text PDF PubMed Scopus (61) Google Scholar Of these the most pertinent in healthy pregnancy, PE, and IUGR are tumor necrosis factor (TNF)-α and conditions of depleted oxygen.8Heyborne KD Witkin SS McGregor JA Tumor necrosis factor-alpha in midtrimester amniotic fluid is associated with impaired intrauterine fetal growth.Am J Obstet Gynecol. 1992; 167: 920-925Abstract Full Text PDF PubMed Scopus (85) Google Scholar, 9Conrad KP Miles TM Benyo DF Circulating levels of immunoreactive cytokines in women with preeclampsia.Am J Reprod Immunol. 1998; 40: 102-111Crossref PubMed Scopus (365) Google Scholar, 10Lyall F Bulmer JN Duffie E Cousins F Theriault A Robson SC Human trophoblast invasion and spiral artery transformation: the role of PECAM-1 in normal pregnancy, preeclampsia, and fetal growth restriction.Am J Pathol. 2001; 158: 1713-1721Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar It is believed that these conditions can arise in the placenta as a result of placental underperfusion, oxidative stress, and inflammatory assault. In previous experiments, we and others, have shown that TNF-α stimulates apoptosis in villous CTs and STs via the TNF-α receptor, TNF-RI.7Crocker IP Barratt S Kaur M Baker PN The in-vitro characterization of induced apoptosis in placental cytotrophoblasts and syncytiotrophoblasts.Placenta. 2001; 22: 822-830Abstract Full Text PDF PubMed Scopus (61) Google Scholar, 11Yui J Hemmings D Garcia-Lloret M Guilbert LJ Expression of the human p55 and p75 tumor necrosis factor receptors in primary villous trophoblasts and their role in cytotoxic signal transduction.Biol Reprod. 1996; 55: 400-409Crossref PubMed Scopus (77) Google Scholar We have also shown, along with others, that TNF-α-induced apoptosis can be inhibited by epidermal growth factor (EGF) and to a lesser extent by platelet-derived growth factor, insulin-like growth factor-1, and basic fibroblast growth factor.12Garcia-Lloret MI Yui J Winkler-Lowen B Guilbert LJ Epidermal growth factor inhibits cytokine-induced apoptosis of primary human trophoblasts.J Cell Physiol. 1996; 167: 324-332Crossref PubMed Scopus (133) Google Scholar, 13Smith SC Francis R Guilbert L Baker PN Growth factor rescue of cytokine mediated trophoblast apoptosis.Placenta. 2002; 23: 322-330Abstract Full Text PDF PubMed Scopus (72) Google Scholar Therefore, overall placental apoptosis occurs in the physiological context of excessive stimulation by death factors and inadequate protection by survival factors. Whether these survival factors can influence the apoptotic machinery directly or whether they act indirectly by regulating cell differentiation is not yet fully established. By identifying and inducing apoptosis in CTs and STs in vitro, we have studied the apoptotic susceptibility of placental villous trophoblasts in healthy pregnancy and pregnancies complicated by PE and IUGR. In these experiments, we have compared spontaneous apoptosis and induced apoptosis in response to TNF-α and depleted oxygen. Measurements have been made using DNA fragmentation, phosphatidylserine expression and the ratios of ADP:ATP (a form of apoptotic recognition reliant on differences in mitochondrial output). The overall aim was to investigate whether variations in trophoblast responsiveness could explain the elevated levels of placental apoptosis in IUGR and PE. The Local Ethics Committee gave approval for this work and participants gave informed consent. Twelve women with PE, 12 women with pregnancies complicated with IUGR, and 12 normal pregnant women were studied. PE was defined as a blood pressure of >140/90 mmHg on two or more occasions after the 20th week of pregnancy, in a previously normotensive woman in the presence of significant proteinuria (either >300 mg/L in a 24-hour collection or >2+ on a voided random urine sample in the absence of urinary tract infection). Women with essential hypertension and with medical complications such as diabetes and renal disease were excluded. IUGR was identified by antenatal ultrasound scans and confirmed after delivery by an individualized birth weight ratio below the 10th centile. The individualized birth weight ratio is relative to predicted birth weight and is calculated using independent coefficients for gestation at delivery, fetal sex, parity, ethnicity, maternal height, and booking weight. The individualized birth weight ratio enables a more accurate prediction of pregnancies ending in poor outcome than birth weight for gestational age alone.14Wilcox MA Johnson IR Maynard PV Smith SJ Chilvers CE The individualised birthweight ratio: a more logical outcome measure of pregnancy than birthweight alone.Br J Obstet Gynaecol. 1993; 100: 342-347Crossref PubMed Scopus (159) Google Scholar Unless otherwise stated all reagents were purchased from Sigma Chemical Co., Poole, UK. Cells were isolated from whole term placentas taken from women at elective cesarean section. A CT-enriched population was isolated from placentas using a modified method of Kliman and colleagues.15Kliman HJ Nestler JE Sermasi E Sanger JM Strauss III, JF Purification, characterization, and in vitro differentiation of cytotrophoblasts from human term placentae.Endocrinology. 1986; 118: 1567-1582Crossref PubMed Scopus (1408) Google Scholar In brief, membranes, connective tissues, and surfaces adjacent to decidua were removed and the remaining tissue digested with trypsin (0.25%) and DNase (0.2 mg/ml) in prewarmed Hanks' balanced salt solution, containing 25 mmol/L of HEPES in a shaking water bath at 37°C for 30 minutes. The tissue fragments were allowed to settle and 25-ml aliquots of the supernatant were layered over 5 ml of newborn calf serum and centrifuged at 400 × g for 10 minutes at room temperature. The pelleted cells were then resuspended in Dulbecco's modified Eagle's medium. The remaining tissue was subjected to the digestion procedure twice more, always with the addition of fresh trypsin-DNase solution each time. All resultant cell suspensions were pooled, centrifuged at 400 × g and resuspended in 6 ml of Dulbecco's modified Eagle's medium. The cells were then divided in half and layered over discontinuous Percoll density gradients (3 ml 70%, 2 ml 60%, 2 ml 55%, 2 ml 50%, 2 ml 45%, 2 ml 40%, 2 ml 35%, 4 ml 30%, 2 ml 20%, and 2 ml 10% Percoll in Hanks' balanced salt solution). The Percoll gradients were centrifuged at 1200 × g at room temperature for 30 minutes. The band of cells between 55% and 35% Percoll contained an enriched population of CTs. These cells were collected, washed once, and preserved in liquid nitrogen in 10% dimethyl sulfoxide in fetal bovine serum. For each experiment, cells were removed from frozen and cultured in Dulbecco's modified Eagle's medium:Ham's F12 (1:1) with 10% fetal bovine serum, 25 mmol/L HEPES (pH 7.4), 2 mmol/L glutamine, and antibiotics in a humidified 5% CO2/95% air incubator at 37°C. Adherent CTs were assessed for purity after 24 hours in culture. Cells were washed twice with warmed phosphate-buffered saline (PBS) to remove nonadherent cells and fragments, then fixed with methanol for 10 minutes, washed twice again with PBS and then stained. The biological markers used were cytokeratin-7, which is expressed on trophoblasts but not other villous components,16Haigh T Chen C Jones CJ Aplin JD Studies of mesenchymal cells from 1st trimester human placenta: expression of cytokeratin outside the trophoblast lineage.Placenta. 1999; 20: 615-625Abstract Full Text PDF PubMed Scopus (94) Google Scholar vimentin that is expressed by mesenchymal cells, leukocyte common antigen (LCA/CD45) that identifies macrophages, and von Willebrand Factor that reacts with human endothelial cells and platelets. CTs were preincubated for 30 minutes in 20% goat serum in PBS, then incubated for 1 hour with either anti-human cytokeratin-7 (OV/TL1230; DAKO Ltd., High Wycombe, UK) diluted 1:50 (v/v) in 20% goat serum, anti-human vimentin (1:20, V9; DAKO Ltd.), anti-human LCA (1:10, T29/33; DAKO Ltd.), or anti-human von Willebrand Factor (1:25, F8/86; DAKO Ltd.). After incubations, cells were washed and incubated for 30 minutes with fluorescein-conjugated goat anti-mouse IgG diluted 1:50 (v/v) in 10% goat serum. Cells were viewed with an inverted fluorescent microscope. The purity of the CTs within these adherent preparations was estimated from the proportion of cytokeratin-7-positive cells. Syncytialization was induced by treating CTs with 5 ng/ml of recombinant human EGF (PeproTech EC Ltd., London, UK). This procedure is known to enhance trophoblast differentiation in vitro.17Levy R Smith SD Chandler K Sadovsky Y Nelson DM Apoptosis in human cultured trophoblasts is enhanced by hypoxia and diminished by epidermal growth factor.Am J Physiol. 2000; 278: C982-C988Google Scholar After a period of 5 days, spent medium was aspirated and any remaining EGF was removed by washing. Syncytialization was confirmed by immunostaining of fixed cells with monoclonal anti-desmosomal protein and counterstaining with hematoxylin and by human chorionic gonadotrophin (hCG) production. All stimulation was conducted in the absence of EGF. Agents and concentrations were chosen because of their apoptotic effects in previous studies12Garcia-Lloret MI Yui J Winkler-Lowen B Guilbert LJ Epidermal growth factor inhibits cytokine-induced apoptosis of primary human trophoblasts.J Cell Physiol. 1996; 167: 324-332Crossref PubMed Scopus (133) Google Scholar, 17Levy R Smith SD Chandler K Sadovsky Y Nelson DM Apoptosis in human cultured trophoblasts is enhanced by hypoxia and diminished by epidermal growth factor.Am J Physiol. 2000; 278: C982-C988Google Scholar and their suggested relevance to PE and IUGR. Adherent populations of CTs and STs were prepared as outlined. After the initial culture of CTs for 24 hours, and 5 days in the case of STs, cells were further maintained in either media alone or in the presence of TNF-α with interferon gamma (IFN-γ) (10 ng/ml and 100 U/ml, respectively; PeproTech EC Ltd., London, UK) for 24 hours; or under reduced oxygen conditions, generated using Anaerocult bags (Merck, Poole, UK) for 48 hours at 37°C. After this incubation, oxygen concentrations (kPa) were determined using a Chiron/Diagnostics 840 blood gas analyzer. The concentration of hCG in culture medium was assessed by quantitative immunoradiometric determination using a commercially available kit (hCG solid-phase component system; ICN Pharmaceuticals, Basingstoke, UK). The hCG assay uses the sandwich technique in which the solid phase binds the α subunit of hCG and a radiolabeled antibody in the liquid phase binds the β subunit. Apoptosis was assessed using Annexin V binding with propidium iodide (PI) staining, terminal dUTP nick-end labeling (TUNEL), and the measurement of ATP and ADP levels. For Annexin V, PI, and ATP/ADP, cells were seeded at 100,000/well in 96-well cell-culture plates. For TUNEL measurements cells were seeded at 200,000/well. All TUNEL measurements were made on the FACScan flow cytometer (Becton Dickinson, Oxford, UK) using the FlowTACS Apoptosis Detection Kit (R&D Systems, Oxon, UK). For cultured CTs, cells were first dissociated with ethylenediaminetetraacetic acid solution (2 mmol/L) with mild agitation, washed in fresh media, and resuspended in 100 μl of freshly prepared 3.7% paraformaldehyde solution. After a 10-minute incubation at room temperature, the cells were centrifuged at 500 × g for 10 minutes and the fixative removed. The cells were washed once with 200 μl of PBS and then resuspended in 100 μl of permeabilization solution for 30 minutes at room temperature. The cells were then centrifuged again, resuspended in 25 μl of the labeling reaction mixture, and incubated for 60 minutes at 37°C. The reaction was stopped by the addition of 100 μl of stop buffer and the cells were centrifuged and resuspended in 25 μl of diluted streptavidin-fluorescein isothiocyanate. After incubation for 10 minutes in the dark, the cells were centrifuged, resuspended in 300 μl of PBS, and analyzed by flow cytometry. Negative controls were set by resuspending fixed and permeabilized cells in 50 μl of label solution in the absence of terminal deoxynucleotidyl transferase. Positive controls were included; washed and permeabilized cells were resuspended in 25 μl of nuclease solution for 30 minutes at 37°C before being labeled. For STs, TUNEL analysis was performed on isolated nuclei using the method of Garcia-Lloret and colleagues.12Garcia-Lloret MI Yui J Winkler-Lowen B Guilbert LJ Epidermal growth factor inhibits cytokine-induced apoptosis of primary human trophoblasts.J Cell Physiol. 1996; 167: 324-332Crossref PubMed Scopus (133) Google Scholar STs were washed twice in PBS before the addition of 200 μl of 2× Krishan buffer (0.2% Na citrate, 0.2% Igepal CA-630, 40 μg/ml RNase, pH 7.4). Culture plates were incubated for 10 minutes at 37°C and the lysates were aspirated and transferred to Eppendorf tubes. An equal volume of 70% ethanol was then added and the tubes were allowed to stand on ice for 5 minutes. Isolated nuclei were pelleted by centrifugation at 800 × g, washed once in 500 μl of 70% ethanol, once in 500 μl of PBS, and finally resuspended in 25 μl of the labeling reaction mixture. Further TUNEL labeling was conducted as outlined above. During early apoptosis, a loss of membrane asymmetry occurs when phosphatidylserine is exposed on the outer leaflet of the plasma membrane. Annexin V will preferentially bind to phosphatidylserine and can therefore be used as an early indicator of apoptosis. In addition, PI can be used to assess plasma membrane integrity and cell viability. PI fluoresces red when bound to DNA or double-stranded RNA, but is excluded from cells with intact plasma membranes. An in situ Apoptosis Detection Kit was used for Annexin V binding and PI staining (R&D Systems). Cultured CTs in each well of a 96-well microtiter plate, (Corning Costar, High Wycombe, UK) were washed twice in fresh media, dissociated as described above and resuspended in 80 μl of binding buffer (10 mmol/L Hepes, 140 mmol/L sodium chloride, 2.5 mmol/L calcium chloride, pH 7.4). To each cell suspension was added 10 μl of fluorescein-conjugated Annexin V (10 μg/ml) and 10 μl of PI reagent (50 μg/ml). The cells were mixed and incubated in the dark for 15 minutes at room temperature. At the end of the incubation, a further 400 μl of binding buffer was added and the cells were analyzed immediately by flow cytometry. Control tubes of unstained cells, cells stained with PI alone, and cells stained with Annexin V, were included to set flow cytometric compensation. Direct and indirect measurements of ATP and ADP were made using the bioluminescence Apoglow Kit (LumiTech Ltd., Nottingham, UK). For these experiments, cells were cultured in clear-bottomed white-walled 96-well culture plates (Corning Costar) and the bases covered before readings were taken. Nucleotides were first released from the cultured cells by addition of an equal volume (in this case 100 μl) of somatic cell nucleotide-releasing reagent. This releasing reagent also contains the luciferin-luciferase nucleotide-monitoring reagent. The ATP levels were then measured using the luminometer (1450 Microbeta jet; EG & G Wallac Ltd., Milton Keynes, UK) and expressed as the number of relative light units (RLU). The ATP signal was allowed to decay for 10 minutes to a steady state. After 10 minutes the ADP in the wells was converted to ATP by the addition of 20 μl of ADP-converting reagent. An immediate reading was taken to determine the baseline ADP RLU (ADP 0). A third reading was taken after 5 minutes incubation to allow for conversion of ADP to ATP (ADP 5). Finally, the ratio of ADP:ATP was calculated from these three readings as follows: (ADP 5 − ADP 0) ÷ ATP. Unless otherwise stated, statistical significance of difference for normally distributed data were determined using a Student's t-test (with or without a Bonferroni correction for multiple comparisons). Normal distribution was assessed by the Shapiro-Wilk significance level normality test and results are presented as means and standard errors of the means (±SEM), with the data considered significant at P < 0.05. The median values and ranges for participant demographics are given in Table 1. No differences were established between pregnant groups for maternal age, gravidity, or parity. Both systolic and diastolic blood pressures were significantly higher at sampling in women with PE compared with normal controls. Gestational age at delivery and individualized birth weight ratio values were lower in PE and IUGR—statistical significance was only reached in the case of IUGR.Table 1Patient DemographicsPregnancyPEIUGRMaternal Age30 (40–26)29 (40–20)31 (40–19)Gravidity2 (8–1)3 (6–1)3 (4–1)Parity1 (4–0)0 (3–0)1 (2–0)Sample BP Systolic (mmHg)120 (140–110)158 (195–150)*(P < 0.01 Mann-Whitney U-test).140 (150–107) Diastolic (mmHg)72 (80–70)100 (110–90)*(P < 0.01 Mann-Whitney U-test).80 (105–64)Gestation (weeks)38 (35–40)35 (31–38)33 (27–35)*(P < 0.01 Mann-Whitney U-test).IBR62 (94–11)18 (99–3)4 (9–0)*(P < 0.01 Mann-Whitney U-test).Results represented as medians (ranges)* (P < 0.01 Mann-Whitney U-test). Open table in a new tab Results represented as medians (ranges) The purity of CTs in culture was 90%, with a range of 85 to 100%. All contaminating cells were vimentin-positive only. Between 30% and 65% of the cells adhered to tissue culture surfaces after overnight incubation before initiation of the experiments (45 ± 9%, normals; 54 ± 12%, PE; 47 ± 8%, IUGR). Contamination by leukocytes and endothelial cells was negligible. Before culturing, enriched CT viabilities were 76% ± 4%. After 5 days in the presence of EGF, CTs were seen to form multinucleated syncytia. These differentiated cells were observed by light microscopy, showed a reduction in staining for desmosomal protein, gave a significant increase in hCG production (from 569 ± 194 to 3639 ± 1739 mIU/5 × 105cells/hour) and could also be identified from cytokeratin-7 staining as previously described.18Crocker IP Strachan BK Lash GE Cooper S Warren AY Baker PN Vascular endothelial growth factor but not placental growth factor promotes trophoblast syncytialization in vitro.J Soc Gynecol Invest. 2001; 8: 341-346Crossref PubMed Scopus (41) Google Scholar A significant decrease in pO2 was recorded in the media of cells cultured in Anaerocult bags (normoxic, 23.6 (22.1 to 25.2) kPa; reduced oxygen, 4.9 (3.9 to 5.8) kPa (P < 0.05). All TUNEL results are presented in Figure 1. For normal pregnancy, unstimulated values of apoptosis in CTs and STs were 12.0 ± 1% and 6.1 ± 1.1%, respectively. Cytotoxic agents increased these measurements to 22.4% and 20.1% in the case of TNF-α and to 47.9% and 20.9% in the case of oxygen depletion. After TNF-α induction and oxygen depletion, TUNEL positivity was significantly elevated in CTs from PE and IUGR above those of normal pregnancy. Apoptosis was increased in PE to 31.7% (TNF-α) and 64.5% (reduced oxygen), and in IUGR to 29.4% (TNF-α) and 57.1% (reduced oxygen). Although increases in response to diminished oxygen were less marked in STs than CTs, apoptosis was still significantly enhanced over normal pregnant controls. For IUGR, TNF-α and oxygen depletion raised these levels to 28.4% and 37.3%. For PE, apoptotic levels were increased to 27.3% after oxygen reductions. Basal apoptosis for CTs and STs were unchanged between all study groups. Baseline apoptosis for normal CTs, as measured by Annexin V binding, was increased in response to TNF-α and oxygen depletion from 4.0 ± 2.8% to 17.1 ± 2.4% and 35.1 ± 1.6%, respectively (Figure 2). Again, baseline values between PE (7.3 ± 1.1%), IUGR (6.3 ± 0.4%), and normal pregnant groups were not significantly affected. For PE and IUGR, Annexin V binding of CTs was significantly increased above normals in response to both TNF-α and oxygen depletion. This is illustrated in Figure 2. For PE, apoptosis was elevated above normal to 27.2% in response to TNF-α and to 47.9% in response to oxygen reduction. For IUGR, these levels were significantly higher at 46.6% after oxygen depletion but were comparable to the normal pregnant group in response to TNF-α. Syncytialization was confirmed in CT cultures after 5 days by measuring the production of hCG. Figure 3 shows that there were no significant differences in hCG release between control cultures and those of PE and IUGR before stimulation. After treatment with TNF-α/IFN-γ and 3% O2, hCG production fell in the control group by 32.5% and 55.6%, respectively. Although reduced, these levels were again statistically similar to those of PE and IUGR. As previously reported (and reproduced by permission),7Crocker IP Barratt S Kaur M Baker PN The in-vitro characterization of induced apoptosis in placental cytotrophoblasts and syncytiotrophoblasts.Placenta. 2001; 22: 822-830Abstract Full Text PDF PubMed Scopus (61) Google Scholar ADP:ATP ratios of less than 1 correlate significantly with Annexin V binding of CTs on the flow cytometer (Figure 4). In response to TNF-α, ADP:ATPs were raised in normal CTs from 0.19 ± 0.03 to 0.25 ± 0.04 and in normal STs from 0.19 ± 0.04 to 0.39 ± 0.04 (Figure 5). By reducing oxygen in culture, ADP:ATP values were raised in CTs and STs to 0.47 ± 0.06 and 0.30 ± 0.06, respectively. After oxygen reductions, the elevations in the ratios of both CTs and STs were significantly higher in the IUGR preparations (0.69 ± 0.09 and 0.48 ± 0.09 for CTs and STs, respectively) as compared to the normal pregnant preparations. No significant differences between PE and normal pregnant cells were found.Figure 5The effect of TNF-α/IFN-γ and oxygen depletion on the ADP:ATP ratios of CTs and STs from the placentas of normal pregnancies and pregnancies complicated by PE and IUGR. Light gray columns, normal pregnancy; dark gray columns, PE; white columns, IUGR. *, P < 0.05; **, P < 0.01 (Student's t-test).View Large Image Figure ViewerDownload Hi-res image Download (PPT) The etiology of IUGR and PE is unclear, however placental dysfunction is considered a common underlying cause in both conditions. It has been suggested that these placentas display altered cell kinetics19Paine CG Observations on placental histology in normal and abnormal pregnancy.J Obstet Gynaecol. 1957; 64 (672): 688Google Scholar and also, in the case of PE, an inappropriate release of harmful material into the maternal circulation.20Redman CW Sargent IL Placental debris, oxidative stress and pre-eclampsia.Placenta. 2000; 21: 597-602Abstract Full Text PDF PubMed Scopus (433) Google Scholar As already described, we and others have reported an increase in placental apoptosis in both PE and IUGR.2Smith SC Baker PN Symonds EM Placental apoptosis in normal human pregnancy.Am J Obstet Gynecol. 1997; 177: 57-65Abstract Full Text Full Text PDF PubMed Scopus (362) Google Scholar, 3Leung DN Smith SC To KF Sahota DS Baker PN Increased placental apoptosis in pregnancies complicated by preeclampsia.Am J Obstet Gynecol. 2001; 184: 1249-1250Abstract Full Text Full Text PDF PubMed Scopus (249) Google Scholar, 21Erel CT Dane B Calay Z Kaleli S Aydinli K Apoptosis in the placenta of pregnancies complicated with IUGR.Int J Gynaecol Obstet. 2001; 73: 229-235Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar Throu" @default.
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• W2079056593 date "2003-02-01" @default.
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• W2079056593 title "Differences in Apoptotic Susceptibility of Cytotrophoblasts and Syncytiotrophoblasts in Normal Pregnancy to Those Complicated with Preeclampsia and Intrauterine Growth Restriction" @default.
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