Increased placental apoptosis in intrauterine growth restriction

OBJECTIVES: Our purpose was to investigate a possible role for apoptosis in the pathophysiologic mechanisms of intrauterine growth restriction. STUDY DESIGN: Placental samples were obtained from 43 uncomplicated third-trimester pregnancies and from 26 pregnancies complicated by intrauterine growth restriction. The definition used to identify cases of intrauterine growth restriction depended on three criteria: clinical evidence of suboptimal growth, ultrasonographic evidence of deviation from an appropriate growth percentile, and individualized birth weight ratios <10th percentile. Light microscopy was used to quantify the incidence of apoptosis. Electron microscopy and TUNEL (terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick end labeling) staining were used to confirm the occurrence of apoptosis. RESULTS: Quantification of apoptosis (medians and interquartile ranges) resulted in the following values: normal third trimester (n = 43) 0.14% of cells (0.08% to 0.20%) and intrauterine growth restriction third trimester (n  = 26) 0.24% of cells (0.16% to 0.29%). The incidence of apoptosis was significantly higher in placentas from pregnancies with intrauterine growth restriction compared with normal third-trimester placentas (p < 0.01, Mann Whitney U test). CONCLUSIONS: These results suggest that apoptosis may play a role in the pathophysiologic mechanisms of intrauterine growth restriction.(Am J Obstet Gynecol 1997;177:401)     

Apoptosis is a rare event in first-trimester placental tissue

OBJECTIVE: The aim of this study was to confirm our previous findings of an increase in the incidence of placental apoptosis with increasing gestational age by means of transmission electron microscopic assessment of first-trimester placental tissue. We also wanted to challenge the validity of terminal uracil nick end-labeling staining for the assessment of placental apoptosis. STUDY DESIGN: The incidence of apoptosis was quantified with hematoxylin and eosin staining and terminal uracil nick end-labeling staining in 6 first-trimester placental samples, 5 second-trimester placental samples, and 7 third-trimester placental samples. These results were then compared with the results that were obtained by quantifying apoptosis in 6 first-trimester placental samples with transmission electron microscopy. RESULTS: Our previous hematoxylin and eosin-staining findings of a rise in apoptosis with increasing gestational age were confirmed. Transmission electron microscopy confirmed the low incidence of apoptosis in first-trimester placental tissue. Terminal uracil nick end-labeling staining produced incongruous results. CONCLUSION: Our earlier findings of a low incidence of apoptosis in first-trimester placental tissue have been confirmed by transmission electron microscopy, which remains the criterion standard in the assessment of apoptosis. Our findings discredit terminal uracil nick end-labeling staining for the assessment of placental apoptosis.     

胎盘细胞凋亡与胎儿生长受限关系的研究

目的　探讨胎盘细胞调亡与胎儿生长受限 (FGR)发生的关系。方法　应用透射电镜和DNA缺口末端标记法检测 18例FGR患者 (FGR组 )及 14例正常妊娠妇女 (正常妊娠组 )的胎盘细胞凋亡情况。同时观察两组的胎盘重量及新生儿平均出生体重。结果　FGR组胎盘凋亡细胞核比率为12 1‰ ,电镜下凋亡细胞核呈明显致密状 ,染色质结块 ,胎盘平均重量为 (2 3 6± 2 4)g ,新生儿平均出生体重为 (2 0 71± 42 8)g;正常妊娠组胎盘凋亡细胞核比率为 7 3‰ ,胎盘平均重量为 (3 5 4± 63 )g ,新生儿平均出生体重为 (3 411± 5 88)g。FGR组胎盘凋亡细胞核比率明显高于正常妊娠组 (P <0 0 5 )。结论　胎盘细胞凋亡增加与FGR的发生有关     

Increased placental apoptosis in pregnancies complicated by preeclampsia

Placentas were obtained at delivery from 34 pregnancies complicated by preeclampsia and from 34 uncomplicated pregnancies. The incidence of apoptotic nuclei was significantly greater (P <.01) in the placentas from the pregnancies complicated by preeclampsia.     

Correlation with placental kisspeptin in postterm pregnancy and apoptosis

Postterm pregnancy represents a condition associated with trophoblast apoptosis. Kisspeptin is a peptide able to induce apoptosis by a specific receptor, GPR54, through the upregulation of proapoptotic genes. The aims of the study were to evaluate (1) the messenger RNA (mRNA) expression of kisspeptin, GPR54, Bax/Bcl2, and p21 in postterm placentas and (2) kisspeptin ability to act on apoptosis in the third trimester placental explants. Placental specimens were collected from spontaneous term and postterm delivery and kisspeptin, GPR54, Bax/Bcl2, and p21 mRNA expression levels were analyzed by real-time polymerase chain reaction. Placental explants, collected from elective term cesarean sections, treated with different doses of kisspeptin were analyzed by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL). The expression levels of all the genes studied in postterm placentas were significantly higher than in-term placentas. Kisspeptin-induced apoptosis in placental explants with a dose-dependent effect, and TUNEL assay demonstrated the kisspeptin involvement in the apoptotic placental processes. Our present findings led us to hypothesize that kisspeptin may represent a placental proapoptotic agent acting in physiological and/or pathological pregnancy conditions in which placental apoptosis mechanisms are increased.     

重度子痫前期患者胎盘合体滋养细胞凋亡的研究

目的 比较早发型与晚发型重度子痫前期患者胎盘合体滋养细胞凋亡水平变化,探讨其病因及发病机制的差异.方法 选择2008年11月至2009年5月在上海交通大学附属第六人民医院住院剖宫产分娩的早发型重度子痫前期患者15例(早发型组)、晚发型重度子痫前期患者15例(晚发型组)和健康妊娠妇女10例(对照组),采用酶联免疫吸附试验检测孕妇血浆中合体滋养细胞微粒(STBM)水平,蛋白印迹法检测胎盘组织中凋亡蛋白--半胱氨酸天冬氨酸蛋白酶3(caspase-3)蛋白表达水平.结果 (1)STBM:早发型组孕妇血浆STBM水平为(71±21)μg/L,高于晚发型组的(42±30)μg/L和对照组的(26±11)μg/L,分别比较,差异均有统计学意义(P＜0.05);晚发型组血浆STBM水平与对照组比较,差异无统计学意义(P＞0.05).(2)caspase-3蛋白:早发型组胎盘组织中caspase-3蛋白表达水平为0.85±0.61,晚发型组为0.77±0.46,对照组为0.32±0.15,早发型和晚发型组胎盘组织中caspase-3蛋白表达水平均高于对照组,差异有统计学意义(P＜0.05);但早发型组和晚发型组比较,差异无统计学意义(P＞0.05).结论 早发型与晚发型重度子痫前期可能存在不同的发病机制,早发型重度子痫前期可能是一种胎盘疾病,而晚发型则可能与母体因素有关.     

妊娠不同时期铅暴露对大鼠胎盘细胞凋亡的影响

目的 观察妊娠不同时期铅暴露对大鼠胎盘细胞凋亡的影响。方法 研究共分为4组,每组27只Wistar大鼠,雌性18只,雄性9只。雌雄2:1合笼,以发现雌鼠阴栓次日为妊娠第0天。妊娠期全程铅暴露组大鼠妊娠期全程（妊娠1～20 d）饮服o.025％醋酸铅溶液染毒,妊娠甲.期铅暴露组大鼠妊娠早期（妊娠1～10 d）饮服0.025％醋酸铅溶液染毒,妊娠晚期铅暴露组大鼠妊娠晚期（妊娠11～20 d）饮服0.025％醋酸铅溶液染毒,对照组大鼠妊娠期全程饮服蒸馏水。原子吸收光谱法测定妊娠末期（妊娠19～21 d）血铅水平;Hoechst染色法和脱氧核糖核苷酸末端转移酶介导的缺口末端标记技术检测胎盘细胞凋亡并计算凋亡指数。各组间比较采用单因素方差分析,两两比较采用q检验。 结果妊娠末期血铅水平在妊娠期全程铅暴露组[(1.74±0.19) μmol/L]、妊娠早期铅暴露组[（1.27±0.26） μmol/L.]、妊娠晚期铅暴露组[（0.60±0.11）μmol/L]和对照组[(0.04±0.01) μmol/L]依次降低,差异均有统计学意义（F=12.10,P＜0.01）。Hoechst染色法显示,铅暴露组胎盘滋养层凋亡细胞核浓染,部分核内可见致密块状荧光颗粒,对照组凋亡细胞呈散在分布。脱氧核糖核苷酸末端转移酶介导的缺口末端标记技术检测显示,铅暴露组大鼠胎盘滋养层凋亡细胞核呈棕黄色,部分核中有棕黄色颗粒。凋亡细胞核较正常增大,呈椭圆形或不规则形,核内染色质聚集于边缘处。2种凋亡检测方法均显示各铅暴露组胎舷细胞凋亡指数均高于对照组,且妊娠期全程铅暴露组高于妊娠早期和妊娠晚期铅暴露组（P均＜0.05）。 结论 妊娠期铅暴露大鼠血铅水平升高,促进胎盘细胞凋亡的发生。     

妊娠高血压综合征患者胎盘滋养细胞凋亡基因表达谱的研究

目的　采用基因芯片技术筛查妊娠高血压综合征 (妊高征 )患者胎盘滋养细胞凋亡基因的差异表达 ,探讨细胞凋亡与妊高征发病的关系。方法　妊高征患者 2 0例为妊高征组 ,正常孕妇 10例为对照组。采用脱氧核糖核苷酸末端转移酶介导的缺口末端标记 (TUNEL)方法检测两组孕妇胎盘组织滋养细胞凋亡情况。同时采用上海博星公司生产的基因芯片进行凋亡基因表达谱的筛查。判断基因差异表达的标准 :(1)cy5 (妊高征组 ) /cy3(对照组 )的自然对数绝对值 >0 6 9,cy3与cy5的信号相差 2倍以上。 (2 )cy3或cy5、cy3和cy5的信号值其中之一必须 >80 0。 结果　 (1)TUNEL检测显示 ,每 10 0 0 0 μm2 绒毛面积中滋养细胞凋亡数 ,妊高征组为 1 5 84个 ,对照组为 0 0 32个 ,两组比较 ,差异有极显著性 (P <0 0 1)。 (2 )基因芯片筛查有差异表达的凋亡基因 10个 ,占基因芯片筛查凋亡基因总数的 5 %。妊高征组胎盘组织中的凋亡基因表达均明显下调 ,即cy5 /cy3比值 <1。其中包括具有明显抗凋亡作用的SFRP2 、IAP2 、DHCY2 4及ATPIA1基因。结论　妊高征患者胎盘滋养细胞存在显著的凋亡现象 ,具有抗凋亡作用的基因表达明显下调 ,将可能导致胎盘滋养细胞凋亡 ,从而导致妊高征发病。     

Apoptosis: (2) characteristics of apoptosis.

The importance of apoptosis has been increasingly appreciated during the last decade. Apoptosis and cell proliferation are two opposing and tightly-controlled events in physiology and pathology. In the 21st century, therapy through either enhancement or suppression of the apoptotic process, depending on the type of disease, will be one important task in medicine. Learning methods to document and quantify apoptotic events is the first step in embracing the approaching era in which modulation of apoptosis may become a goal of clinical treatment. In this article, the second part of two related reviews (first review, J Formos Med. Assoc. 1999; 98:381-93), characteristics of apoptosis, including morphology, biochemistry, and the efficient elimination of apoptotic cells by tissues are discussed with a special emphasis on the quantitative analyses of apoptosis in biomedical research.     

Physiologic concentrations of magnesium and placental apoptosis: prevention by antioxidants

OBJECTIVE:To identify the role of physiologic magnesium concentrations on the induction of placental apoptosis in vitro and test the anti-apoptotic action of antioxidants. METHODS: Placental tissue was obtained from normal pregnancies after cesarean delivery. Placental explants were incubated with increasing concentrations of extracellular magnesium (range 0-2.0 mM). Placental apoptosis was evaluated by tissue morphology, DNA fragmentation, cytokeratin-18 neoepitope formation, and cleavage of plasminogen activator inhibitor type 2. RESULTS: Physiologic concentrations of extracellular magnesium stimulated placental apoptosis. Magnesium stimulated apoptosis within the physiologic range (0.8-1.2 mM) (n = 6, P <.001) and was associated with cleavage of plasminogen activator inhibitor type 2 and cytokeratin-18 neoepitope formation. These data implicate caspase activation in the transduction of the magnesium-induced apoptotic signal. Therapeutic concentrations of vitamin C, vitamin E, and acetylcysteine (all at 25 microg/mL) inhibited DNA fragmentation and attenuated cleavage of plasminogen activator inhibitor type 2 and cytokeratin-18 neoepitope formation. CONCLUSION: Magnesium-induced placental apoptosis is a potent mechanism of placental degeneration in vitro and may represent an important regulator of placental tissue dynamics in vivo. The ability of antioxidants to prevent magnesium-induced placental apoptosis implicates oxidation-reduction-dependent signaling events in this process. Furthermore, these findings provide a basis for further studies of antioxidants in mitigating the adverse effects of preeclampsia.     

Prenatal vitamin C status is associated with placental apoptosis in normal-term human pregnancies

Pregnancy is associated with increased susceptibility to oxidative stress. Deficiencies in antioxidants during pregnancy and placental oxidant-antioxidant imbalance may impair the development of the fetoplacental unit or the eventual offspring. In order to elucidate the association of prenatal status of vitamin C with the oxidative stress and apoptotic activity in normal full-term placentas, we evaluated the content of placental lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) and the trophoblast apoptotic index in normal-term human pregnancies. Tissue samples of placentas obtained from 80 normal-term pregnancies were categorized into 40 cases with a lower level of prenatal vitamin C (< 8.997 microg/ml) and 40 cases with a higher level of prenatal vitamin C (> or =11.734 microg/ml). We evaluated the placental LOX-1 content and the trophoblast apoptotic index with Western blot analysis and immunohistochemistry, and then determined their correlation with the prenatal status of vitamin C. We confirmed that the trophoblast expression for the endothelial scavenger receptor LOX-1 and the apoptotic activity were significantly lower in the group with a higher prenatal level of vitamin C, indicating that placental oxidative stress and the apoptotic index were associated with the maternal status of vitamin C. We therefore postulate that the maternal status of antioxidant vitamins during pregnancy can affect fetal development.     

Heparin and aspirin attenuate placental apoptosis in vitro: implications for early pregnancy failure

OBJECTIVE: Live birth rates are increased by treatment with heparin and aspirin in cases of poor pregnancy outcome such as antiphospholipid syndrome. Both drugs may attenuate miscarriage by inhibiting aberrant coagulation or by modulating trophoblast apoptosis. Here we assessed their roles in trophoblast apoptosis in vitro. STUDY DESIGN: BeWo cells and placental villi were cultured in sera from women with successful or failing in vitro fertilization, with and without heparin or aspirin. Apoptosis was assessed by using DNA laddering, cytokeratin 18 neoepitope formation, Bcl-2, and caspase 7 expression. RESULTS: In BeWo cells, sera from in vitro fertilization failure increased trophoblast apoptosis, whereas heparin and aspirin reversed these effects. In villous trophoblast, heparin increased Bcl-2 and cytokeratin 18 protein expression. Heparin and aspirin inhibited DNA laddering. CONCLUSION: Heparin and aspirin modulate trophoblast apoptosis suggesting a direct impact on trophoblast biology, thus providing an additional mechanism to explain the clinical benefits of heparin and aspirin on recurrent pregnancy loss.     

Effects of oxygen on cell turnover and expression of regulators of apoptosis in human placental trophoblast

Pre-eclampsia (PE) and intrauterine growth restriction (IUGR) are associated with aberrant cell turnover, including increased apoptosis, in placental villous trophoblast. The increased apoptosis is associated with exaggerated expression of p53, which promotes cell cycle arrest or apoptosis via downstream proteins such as p21 or Bax. These changes in apoptosis and p53 expression are purported to result from exposure to altered oxygen tension. Using a model of villous trophoblast turnover, we examined the effect of 20%, 6% and 1% ambient oxygen (O(2)) on apoptosis, necrosis, proliferation and expression of p53 and related regulators of cell turnover, compared to both fresh tissue. Altered O(2) tension exerted an effect on cell turnover in cultured term villous tissue: cytotrophoblast proliferation was increased by culture in 20% O(2) and reduced in 1% O(2) (median proliferative index: fresh tissue=0.32%, 20% O(2)=0.9%, 6% O(2)=0.28%, 1% O(2)=0.07%). Apoptosis was increased in all culture environments, but was significantly enhanced by culture in 1% O(2) (median apoptotic index: fresh tissue=0.64%, 20% O(2)=2.96%, 6% O(2)=3.81%, 1% O(2)=9.2%). Necrotic cell death was also increased by culture in 1% O(2) compared to 6% and 20% O(2). The expression of p53, p21 and Mdm2 in both cytotrophoblast and stromal cells was increased following culture in 1% O(2). There was no alteration in the expression of Bax or Bcl-2. This study provides evidence that p53 is elevated in trophoblast following exposure to hypoxia. The potential role of the p53-pathway in the control of cell turnover in villous trophoblast and the regulation of p53 by altered O(2) tension merits further investigation.     

特发性胎儿生长受限与胎盘细胞凋亡及bcl-2基因表达的关系

目的 探讨特发性胎儿生长受限(FGR)与胎盘细胞凋亡及bcl-2基因表达的关系.方法 应用透射电镜、脱氧核苷酸末端转移酶介导的脱氧尿苷三磷酸标记法(TUNEL)、流式细胞技术(FCM)检测特发性FGR孕妇(FGR组)及正常妊娠妇女(对照组)各15例的胎盘细胞凋亡情况,并采用RT-PCR技术观察两组胎盘细胞中bcl-2基因相对表达量.结果 电镜下观察到FGR组胎盘合体滋养细胞核膜皱缩,核仁消失,染色质致密、凝聚;TUNEL 检测 FGR 组胎盘细胞凋亡率为13.68%,高于对照组的4.05%,两组比较,差异有统计学意义(P<0.05);FCM检测胎盘S期细胞比率FGR组为3.4%,对照组为2.2%,两组比较,差异有统计学意义(P<0.05),FCM检测的胎盘细胞凋亡率变化趋势与TUNEL检测结果一致;FGR组胎盘细胞bcl-2基因相对表达量为0.19±0.13,对照组为0.55±0.17,两组比较,差异也有统计学意义(P<0.05).结论 特发性FGR的发生与胎盘细胞凋亡增加有关,胎盘细胞凋亡增加与胎盘细胞中bcl-2基因表达下调有一定关系.     

妊娠肝内胆汁淤积症患者血清甘胆酸水平变化与胎盘细胞凋亡调节基因表达的关系

目的　探讨妊娠肝内胆汁淤积症 (ICP)患者血清甘胆酸水平变化与胎盘细胞凋亡的关系 ,进一步分析ICP患者胎盘功能减退的可能机制。方法　应用放射免疫方法 ,测定 30例ICP患者(ICP组 )及 2 7例正常孕妇 (对照组 )分娩前血清甘胆酸水平 ,应用免疫组化方法测定两组孕妇胎盘细胞凋亡调节基因bax和bcl 2的表达。结果　 (1)ICP组甘胆酸水平为 (5 1 8± 5 9) μmol/L ,对照组为(9 4± 5 6 ) μmol/L ,两组比较 ,差异有显著性 (P <0 0 5 )。 (2 )ICP组胎盘合体滋养细胞bax阴性 2例 ,弱阳性 9例 ,中等阳性 11例 ,强阳性 8例 ;bcl 2弱阳性 12例 ,中等阳性 12例 ,强阳性 6例。对照组bax阴性 9例 ,弱阳性 13例 ,中等阳性 4例 ,强阳性 1例 ;bcl 2弱阳性 3例 ,中等阳性 4例 ,强阳性 2 0例。两组比较 ,ICP组bax表达阳性率显著高于对照组 (P <0 0 0 0 5 ) ,而bcl 2的表达阳性率又显著低于对照组 (P <0 0 0 0 5 )。 (3)孕妇血甘胆酸水平与胎盘bax表达阳性率呈线性正相关 (P <0 0 0 5 ) ,与胎盘bcl 2表达呈线性负相关 (P <0 0 0 5 )。结论　ICP患者血中高水平的甘胆酸使胎盘合体滋养细胞bax基因过度表达 ,这可能是ICP患者胎盘功能减退的机制之一。