Tissue microarray: An effective high-throughput method to study the placenta for clinical and research purposes

Objective.?Tissue microarray (TMA) technology allows simultaneous examination of the expression of many molecular markers (protein, mRNA, DNA, etc.) with high-throughput. The application of this technology, to date, has been largely confined to the study of cancer. Placental pathology poses unique challenges because of the size of the organ, its complex anatomy, as well as its histological heterogeneity. The objective of this study was to assess the feasibility and efficiency of TMAs for immunohistochemistry and in situ hybridization of placental tissues.

Study design.?TMAs were constructed using an automated tissue arrayer. Standard 0.6-mm or 1-mm microarray needles were used. Villous parenchyma, basal plate, and chorioamniotic membranes were targeted in each block. Five μm-thick TMA sections underwent immunohistochemical analysis of both cytoplasmic and nuclear antigens using a panel of antibodies against a variety of cytoplasmic [cytokeratin-7, vascular endothelial growth factor (VEGF), and protein Z], membranous (endoglin), and nuclear (c-fos and c-jun) antigens. mRNA in situ hybridization for surfactant protein A (SP-A) and chromogenic in situ hybridization for the Y chromosome (DYZ1) were also performed.

Results.?Validation of TMA immunoreactivity demonstrated comparable results with corresponding whole sections. When a two-tiered scoring system (positive/negative) was employed, there was agreement between two and three cores and whole tissue sections (kappa>0.7). When a three-tiered scoring system (negative, weak-positive, or strong-positive) was used, the data from three cores showed the highest agreement with whole tissue sections (kappa >0.7). In situ hybridization experiments for mRNA and DNA were also successful in that the signals were readily detectable. Successful transfer from the donor block to the recipient block differed according to the anatomical compartment. The transfer efficiency of villous parenchyma, basal plate, and chorioamniotic membranes were 96.9% (875/903), 76.7% (115/150), and 75.4% (224/297), respectively.

Conclusion.?TMA is a practical and effective tool for high-throughput molecular analysis of the human placenta. Duplicate and triplicate cores offer agreement with whole tissue sections for two-category distinction immunostaining. TMA also affords relevant results from in situ hybridization experiments for mRNA and DNA. The major advantages are the conservation of tissues and reagents, simultaneous comparison of molecular markers in different anatomical compartments of the placenta, and reduction of experimental error.     

Validation of tissue microarray technology using cervical adenocarcinoma and its precursors as a model system

The tissue microarray (TMA) technology has potentiated large-scale retrospective cohort studies using archival formalin-fixed, paraffin-embedded tissues. We used a large series of cervical adenocarcinomas to investigate TMA technology in assessment of immunohistochemical staining. A TMA was constructed using 273 archival paraffin blocks from 139 patients with 119 invasive and 20 adenocarcinoma in situ and 16 normal controls. Two paired cores were obtained from specific regions of donor blocks selected at histologic review and were arrayed into a recipient blocks. The novel array blocks and some whole donor blocks were sectioned and used for immunohistochemical analysis for carcinoembryonic antigen, cytokeratin 7, and cytokeratin 20 antibodies as potential diagnostic markers. We compared staining in the microarray disks with the whole tissue sections. Two paired TM cores were found to yield good immunohistochemical staining that was concordant with that of the whole section from which it originated in about 97% of cases, and the cores accurately represented the morphology of the tumor with respect to tumor typing and differentiation in all cases. Our results suggest that TMAs can be successfully used for immunohistochemical studies of cervical adenocarcinomas. The areas sampled from donor blocks must be selected by careful review of sections from the original blocks.     

Expression of aquaporin 9 in human chorioamniotic membranes and placenta

OBJECTIVE: Aquaporin 9 (AQP9) is one of the recently identified water channels that is also permeable to neutral solutes including urea. To investigate the molecular mechanism of intramembranous pathway of amniotic fluid regulation, we sought to determine whether AQP9 is expressed, and the cellular localization of AQP9 expression in human fetal membranes. STUDY DESIGN: Fetal membranes from 5 normal term human pregnancies were studied. Northern analysis was used to determine the tissue AQP9 messenger RNA (mRNA) expression. In situ hybridization and immunohistochemical staining with specific anti-AQP9 antibody was used for cellular AQP9 localization in the human fetal membranes. RESULTS: Northern analysis detected AQP9 mRNA expression in human amnion, chorion, and placenta. In situ hybridization revealed AQP9 mRNA expression in epithelial cells of the amnion, chorion cytotrophoblasts, and syncytiotrophoblasts and cytotrophoblasts of placenta. Further immunohistochemical study confirmed the AQP9 protein expression in these cell types of fetal membranes. CONCLUSION: This study demonstrated the expression of AQP9 mRNA and protein in human chorioamniotic membranes and placenta. The AQP9 expression in fetal membranes suggests that AQP9 may be an important water channel in intramembranous amniotic fluid water regulation.     

Ontogeny of expression of insulin-like growth factor (IGF) and IGF binding protein mRNAs in the guinea-pig placenta and uterus.

To determine the temporal and spatial distribution of insulin-like growth factor (IGF) and its family of binding proteins (IGFBPs), guinea-pig yolk sac and chorioallantoic placentae were collected at 15, 20, 25, 29, 44-45, 55 and 65-66 days of gestation. Messenger RNAs for IGF I, IGF II and IGFBP 1-6 were identified in tissue sections by in situ hybridization, using 35S-cRNA probes. Epithelial and mesenchymal cell types were identified by immunohistochemistry for cytokeratin and vimentin, respectively. At 15 days of gestation, IGF-II mRNA was expressed in ectoplacental mesoderm, cytotrophoblasts and syncytiotrophoblast, and IGFBP-5 mRNA was detected in the syncytiotrophoblast. In the mid-gestation placenta, IGFBP-5 mRNA was expressed in the marginal and interlobular syncytium and IGF-II mRNA in the labyrinth. Near term, when expansion of the labyrinth was complete, IGFBP-5 mRNA was coexpressed with IGF-II mRNA in the marginal and interlobular syncytium. These observations suggest that interaction between IGF-II and IGFBP-5 plays a role in the vascularization of the placenta by fetal vessels. IGF-II mRNA was not expressed in the maternal tissues at any gestational age. IGFBP-2, -3 and -5 mRNAs were expressed in the endometrial stroma at 7-12 days of gestation but, following establishment of the placenta, IGFBP mRNAs were more abundant in the myometrium than in the decidua. IGF-II mRNA was detected in trophoblasts invading the walls of maternal vessels, and the endothelium of the preplacental vessels expressed IGFBP-4 mRNA, while IGFBP-2 and IGFBP-5 mRNAs were present in the tunica media of mesometrial arteries that had not been invaded by trophoblast. These findings suggest that IGF-II produced by the trophoblast acts in an autocrine and/or paracrine fashion to promote trophoblast invasion and that this process is modulated by interaction with IGFBPs present in maternal tissues.     

Necdin基因在妊娠高血压综合征胎盘组织中的表达及其意义

目的：研究Necdin基因在妊娠高血压综合征(妊高征)患者胎盘组织中的表达和定位，探讨Necdin基因在妊高征发病机制中的可能作用。方法：自1999年12月至2000年6月以消减杂交技术获得的Necdin基因片段为探针，采用斑点杂交检测10例正常妊娠胎盘组织和10例妊高征患者胎盘组织中Necdin基因的表达。并采用原位杂交检测Necdin mRNA在上述组织的定位。结果：正常胎盘组织和妊高征胎盘组织中均存在Necdin mRNA。妊高征胎盘组织Necdin基因的表达显著高于正常胎盘组织(P＜0．05)。在胎盘组织中，Necdin mRNA主要分布于胎盘滋养细胞。结论：胎盘组织中Necdin基因的高表达可能与妊高征的病理生理过程相关。     

人巨细胞病毒感染妊娠早期绒毛组织中即刻早期基因与晚期基因的表达及形态学变化的体外研究

目的 观察妊娠早期绒毛组织感染人巨细胞病毒（hCMV）后,即刻早期基因与晚期基因的表达及绒毛组织形态学变化。方法 采用绒毛组织体外培养技术,建立体外hCMV感染妊娠早期绒毛模型;采用间接免疫荧光法和原位杂交法,检测不同hCMV浓度、不同感染时间,即刻早期蛋白（IEP）72-IEP86和晚期基因（LG）mRNA的表达;同时应用透射电镜观察妊娠早期绒毛组织的形态学变化。结果 （1）以浓度为100半数致细胞病变滴度（TCID50）、200TCID50及300TCID50的hCMV感染绒毛组织后,细胞滋养细胞、合体滋养细胞及间质细胞均可见IEP72一IEP86表达。（2）100TCID50 hCMV感染后,绒毛组织无LGmRNA表达;200 TCID50及300 TCID50 hCMV感染第0～2天后,合体滋养细胞及间质细胞LGmRNA均呈阳性表达,细胞滋养细胞LGmRNA呈强阳性表达。（3）妊娠早期绒毛组织与经hCMV感染后的妊娠早期绒毛组织,共同于体外连续培养10d,妊娠早期绒毛组织保持了正常的形态学特征;不同浓度hCMV感染后的妊娠早期绒毛组织的形态均发生了不同程度的变化,合体滋养细胞表面微绒毛水肿、粗面内质网扩张,细胞滋养细胞多见,溶酶体增生及毛细血管腔扩张。结论 （1）hCMV感染妊娠早期绒毛组织的早期,hCMV可在绒毛组织细胞中完整复制,IEP72-IEP86可长期存在于感染后的绒毛组织中。（2）hCMV感染妊娠早期绒毛组织,可引起绒毛组织细胞的超微结构变化。     

胎盘组织中人类主要组织相容性抗原G mRNA的表达变化与特发性胎儿生长受限发病的关系

目的探讨人类主要组织相容性抗原G(HLA G) mRNA在特发性胎儿生长受限(IFGR)产妇胎盘组织中的表达及其与IFGR发病的关系。方法采用原位杂交法,检测20例IFGR产妇(IFGR组)及28例正常产妇(对照组)胎盘组织中HLA GmRNA的表达水平和表达部位,并对两组产妇的胎盘组织进行病理学观察。结果( 1 )IFGR组产妇胎盘出现病理改变的发生率为75%(15 /20), 主要为慢性绒毛膜炎、胎盘梗死及绒毛发育迟缓;明显高于对照组的18% (5 /28),两组比较,差异有统计学意义(P=0 001)。(2)IFGR组产妇胎盘HLA GmRNA的阳性表达率为45% ( 9 /20),明显低于对照组的79% ( 22 /28 )。两组比较,差异有统计学意义(P= 0 017 )。( 3 )HLA GmRNA的阳性表达部位主要位于胎盘绒毛外细胞滋养细胞及合体滋养细胞的细胞质内,呈紫蓝色的颗粒状沉淀,轮廓清晰。(4)HLA GmRNA表达阴性者,出现胎盘病理改变的例数较HLA GmRNA表达阳性者明显增多(r=-0 638,P=0 008 )。结论IFGR产妇胎盘组织中HLA GmRNA表达水平明显下降,HLA GmRNA的异常表达可能参与了IFGR的发病过程。     

Placental expression of erythropoietin mRNA, protein and receptor in sheep

In ovine placentae at 100 days gestation, we localized the expression of erythropoietin (EPO) mRNA by in situ hybridization and determined the cellular localization of EPO protein and EPO receptor protein by fluorescence immunohistochemistry. Erythropoietin mRNA was observed in maternal tissue at the apical tips of the fetal cytotrophoblastic villi at their interface with the maternal caruncle and was absent from both the centrally located fetal-maternal tissue and the more peripherally located maternal caruncle. An EPO-protein-associated fluorescent signal was observed in the same interface region as the EPO mRNA hybridization signal. An intense fluorescent signal associated with EPO receptor protein was observed in the apical fetal-maternal interface region with a lower density signal dispersed throughout the remainder of the interdigitating fetal-maternal tissue. The predominant cells in the apical fetal-maternal interface were identified as binucleate cells by immunohistochemistry. Thus the localization of the binucleate cells was the same as that for the EPO mRNA and the EPO protein, whereas the EPO receptor had a more generalized distribution. Since the binucleate cells are hormone producing cells, we speculate that the binucleate cells are the source of the EPO that is present in ovine placenta.     

Localization of mRNA for the oncotrophoblastic protein oncomodulin during implantation and early placentation in the rat

The mRNA for the oncodevelopmental calcium-binding protein oncomodulin (MW 11,700) has been detected in tissues of the rat conceptus by in situ hybridization using biotinylated RNA probes. Oncomodulin mRNA was detected in the basal zone and labyrinth of rat placenta, following a similar distribution to that shown for oncomodulin by immunohistochemistry. Oncomodulin mRNA was also detected in rat ectoplacental cone at ten days and in amnion and PYS, but not VYS from 11 days onward. Previously oncomodulin was not detected embryonically from day 14 to birth, but in the present study of oncomodulin mRNA and protein, both were detected in implantation stages from blastula through egg cylinder. Staining was also present on decidual tissue. The suggestion is made that the oncomodulin gene is initially active in all cell types, but later its activity is confined to extraembryonic tissues.     

人类白细胞抗原G、E在人胎盘组织中的表达及其意义

目的:探讨人类白细胞抗原G、E(HLA-G、E)在人胎盘组织中的表达及意义。方法:用原位杂交及免疫组化法分别检测HLA-G、E mRNA及蛋白质在人正常早孕绒毛组织、晚孕胎盘组织中的表达。结果:在人正常早孕绒毛组织中的绒毛细胞滋养细胞、合体滋养细胞及绒毛外滋养细胞(EVCT)内均有HLA-G、E mRNA及HLA-E蛋白质表达,而HLA-G蛋白质仅在EVCT内表达;晚孕胎盘组织中HLA-G、E mRNA及蛋白质表达于蜕膜板内的EVCT及羊膜上皮细胞。结论:HLA-E表达于人正常早孕绒毛组织中所有类型的滋养细胞及晚孕胎盘组织中的EVCT和羊膜上皮细胞。抗人HLA-G单克隆抗体4H84仅能检测出人胎盘组织中EVCT及羊膜上皮细胞内的HLA-G蛋白。     

Linkage of human chorionic gonadotrophin and placental lactogen biosynthesis to trophoblast differentiation and tumorigenesis

Normal trophoblast of the human placenta elaborates at least two major protein hormones, chorionic gonadotrophin (hCG) and placental lactogen (hPL). Molar and choriocarcinoma tissues characteristically synthesize large amounts of hCG and hPL. To examine the role of trophoblast differentiation in the expression of the hCG and hPL genes, we studied the cytological distribution of their mRNAs in tissue sections of human hydatidiform mole and choriocarcinoma by in situ hybridization. Histologically, these tissues are in different stages of cellular differentiation. In normal placenta, hCG alpha/beta mRNA can be localized to some cytotrophoblasts and primarily to the syncytium, whereas hPL mRNA appears only in the syncytial layer. In hydatidiform mole, which still retains placental villous morphology, the hPL gene and hCG alpha and beta genes are expressed but are poorly localized because of the admixture of cyto- and syncytiotrophoblasts. By contrast, choriocarcinoma, which is devoid of placental villous pattern but in which the cyto- and syncytiotrophoblast-like components are distinguishable, expresses hCG alpha and beta in the syncytial-like areas but little, if any, hPL. These results suggest that a certain level of trophoblast differentiation, such as villous formation, is associated with hPL expression, while the hCG alpha gene and the hCG beta gene can be expressed in more disorganized tissues which contain cytotrophoblastic elements.     

Expression of epidermal growth factor receptor-related family products in gestational trophoblastic diseases and normal placenta and its relationship with development of postmolar tumor

OBJECTIVE: The goal of this work was to study the expression of epidermal growth factor receptor (EGFR) and c-erbB-3 and c-erbB-4 oncogenes in gestational trophoblastic diseases and normal first-trimester placenta. STUDY DESIGN: Paraffin sections of 16 cases of partial mole, 25 cases of complete mole, 10 cases of gestational choriocarcinoma, and 11 cases of therapeutic abortion were studied immunohistochemically for EGFR, c-erbB-3, and c-erbB-4 proteins. The presence of EGFR mRNA was studied using in situ hybridization. RESULTS: Staining for EGFR was detected immunohistochemically in all cell types in gestational trophoblastic diseases and normal placenta. In situ hybridization for EGFR mRNA correlated with immunostaining for EGFR in all tissues studied. All 10 cases of choriocarcinoma exhibited strong immunoreactivity for EGFR. The levels of expression of EGFR in choriocarcinoma and syncytiotrophoblasts and cytotrophoblasts in complete mole were significantly greater than those in syncytiotrophoblasts and cytotrophoblasts in both normal placenta and partial mole (P < 0.01, P < 0.01). Expression of c-erbB-3 did not significantly differ among placental and gestational trophoblastic disease tissues and trophoblastic cell types except for significantly increased expression in choriocarcinoma as compared with cytotrophoblasts of partial mole (P = 0.02). The placenta, complete and partial mole, and choriocarcinoma tissues demonstrated similar immunoreactivity for c-erbB-4. Strong immunostaining for EGFR (P = 0.02) and c-erbB-3 (P < 0.01) in extravillous trophoblasts of complete mole was found to be significantly correlated with the development of persistent postmolar gestational trophoblastic tumor. CONCLUSION: The EGFR-related family of oncogenes may be important in the pathogenesis of gestational trophoblastic diseases. The increased expression of EGFR and c-erbB-3 in complete mole may also influence the development of persistent gestational trophoblastic tumor.     

人类组织相容性抗原-G mRNA表达与不明原因习惯性流产关系的初步研究

目的　探讨人类组织相容性抗原Ｇ（ＨＬＡＧ） ｍＲＮＡ 的表达与不明原因习惯性流产（ＲＳＡ） 的关系。方法　用原位杂交的方法检测正常妊娠早期、晚期孕妇胎盘和ＲＳＡ患者经免疫治疗并成功分娩的胎盘母胎界面处的ＨＬＡＧｍＲＮＡ的表达。结果　正常妊娠早期、晚期孕妇胎盘母胎界面均有ＨＬＡＧｍＲＮＡ的阳性表达,阳性表达位于绒毛内（ 或） 外细胞滋养细胞,以及合体滋养细胞的胞浆内。ＲＳＡ患者免疫治疗后的胎盘均不表达ＨＬＡＧｍＲＮＡ,免疫治疗没有调节ＨＬＡＧ ｍＲＮＡ 表达的作用。结论　ＨＬＡＧ 可能与ＲＳＡ的发病原因没有直接的关系;与胎儿存活也无直接关系。     

Interleukin-33 in the human placenta

Objective: Interleukin-33 (IL-33) is the newest member of the IL-1 cytokine family, a group of key regulators of inflammation. The purpose of this study was to determine whether IL-33 is expressed in the human placenta and to investigate its expression in the context of acute and chronic chorioamnionitis. Methods: Placental tissues were obtained from five groups of patients: 1) normal pregnancy at term without labor (n = 10); 2) normal pregnancy at term in labor (n = 10); 3) preterm labor without inflammation (n = 10); 4) preterm labor with acute chorioamnionitis and funisitis (n = 10); and 5) preterm labor with chronic chorioamnionitis (n = 10). Immunostaining was performed to determine IL-33 protein expression patterns in the placental disk, chorioamniotic membranes, and umbilical cord. mRNA expression of IL-33 and its receptor IL1RL1 (ST2) was measured in primary amnion epithelial and mesenchymal cells (AECs and AMCs, n = 4) and human umbilical vein endothelial cells (HUVECs, n = 4) treated with IL-1β (1 and 10?ng/ml) and CXCL10 (0.5 and 1 or 5?ng/ml). Results: 1) Nuclear IL-33 expression was found in endothelial and smooth muscle cells in the placenta, chorioamniotic membranes, and umbilical cord; 2) IL-33 was detected in the nucleus of CD14+ macrophages in the chorioamniotic membranes, chorionic plate, and umbilical cord, and in the cytoplasm of myofibroblasts in the Wharton’s jelly; 3) acute (but not chronic) chorioamnionitis was associated with the presence of IL-33+ macrophages in the chorioamniotic membranes and umbilical cord; 4) expression of IL-33 or IL1RL1 (ST2) mRNA in AECs was undetectable; 5) IL-33 mRNA expression increased in AMCs and HUVECs after IL-1β treatment but did not change with CXCL10 treatment; and 6) IL1RL1 (ST2) expression decreased in AMCs and increased in HUVECs after IL-1β but not CXCL10 treatment. Conclusions: IL-33 is expressed in the nucleus of placental endothelial cells, CD14+ macrophages, and myofibroblasts in the Wharton’s jelly. IL-1β can induce the expression of IL-33 and its receptor. Protein expression of IL-33 is detectable in macrophages of the chorioamniotic membranes in acute (but not chronic) chorioamnionitis.     

Cellular localization of rat placental lactogen II and rat prolactin-like proteins A and B by in situ hybridization

In situ hybridizations using 35S-labelled antisense and sense RNA probes of rPLII, rPLP-A and rPLP-B were carried out on developing rat placenta to determine which cell types synthesized each specific mRNA. Cellular localization of the sites of synthesis of these placental RNAs would help to decide whether these proteins were functioning in the mother of the fetus. The cells of the basal zone are known to have access only to the maternal blood supply, while the labyrinth region is supplied by both maternal and fetal blood vessels. The data in this paper show that at day 12 of pregnancy the rPLII mRNA is synthesized in the primary and secondary giant cells. At later days, hybridization is seen in both the giant cells of the basal zone, and cells in the labyrinth, suggesting that rPLII has a function not only in the mother, but also in the fetus. The rPLP-A mRNA is synthesized in both the giant cells and the cytotrophoblasts of the basal zone. No hybridization is seen to any cells in the labyrinth, even at the later days when it appears that all cytotrophoblasts synthesize rPLP-A mRNA. The rPLP-B mRNA is synthesized exclusively by the cytophoblasts of the fetal placenta. Like rPLP-A, all these cells synthesize this mRNA in the late term placenta. The synthesis of the rPLP-A and rPLP-B mRNAs in cells which have access only to the maternal circulation suggest that they have a role in the mother.