Kang Youn-Jung, Forbes Karen, Carver Janet, Aplin John D
Maternal and Fetal Health Research Centre, Institute of Human Development, University of Manchester, St. Mary's Hospital, Central Manchester University Hospitals NHS Foundation Trust, Manchester Academic Health Science Centre, Level 5-Research, Manchester, UK.
Hum Reprod. 2014 Apr;29(4):739-49. doi: 10.1093/humrep/det433. Epub 2014 Jan 17.
Does the interaction between integrin and its ligand osteopontin (OPN) mediate embryonic attachment to endometrial epithelium at implantation?
OPN of epithelial origin binds the receptor integrin αvβ3 at the maternal surface to support adhesion during the early stages of implantation.
Integrin αvβ3 and OPN are both present in the endometrial luminal epithelium in the mid-secretory phase.
STUDY DESIGN, SIZE, DURATION: Microscopy of attachment sites of blastocysts (mouse, n = 151, human, n = 8) and OPN- or BSA-coated beads (n = 488) interacting with Ishikawa cell monolayers at 24 and 48 h. Levels of epithelial OPN or integrin αvβ3 were altered by siRNA-mediated targeting and the results compared with non-targeting siRNA or mock-transfected controls.
PARTICIPANTS/MATERIALS, SETTING, METHODS: In vitro modelling of early implantation with human endometrial cells (Ishikawa) and mouse or human embryos or ligand-coated beads. Immunolocalization of antigen around attached embryos was measured by image analysis with multiple repeats (n > 3), allowing a gradient of relative intensity to be detected. Attachment was quantified using a stability scale and protein expression documented by indirect immunofluorescence. Protein associations were probed by pulldown assays.
Integrin and OPN levels were increased in epithelial cells near to attached embryos. The pulldown assay confirmed OPN-integrin αvβ3 binding (n > 3). Decreased attachment stability of mouse embryos observed after siRNA knock-down of integrin αvβ3 or OPN itself, or OPN-coated beads after knock-down of integrin αvβ3, was tested for significance using Kruskal-Wallis with Dunn's post hoc tests.
LIMITATIONS, REASONS FOR CAUTION: In vitro model. Attachment data using human embryos is limited by embryo availability. Mouse embryo attachment to human cells involves a species crossover so must be interpreted with caution. Ligand-coated beads allow specific molecular interactions mediating attachment to be probed, but obviously lack the adhesion and signaling repertoire of a live embryo.
Some of the literature identifies reduced integrin αvβ3 expression in infertile endometrium; these findings predict that embryo attachment stability will be reduced in vivo if integrin levels are low. We suggest that the robustness of the initial attachment of the embryo affects its ability to progress to the post-epithelial phase of implantation; some poorly attached embryos will be lost.
STUDY FUNDING/COMPETING INTEREST(S): No external funds were used for this study, which was supported by funds from the Universities of Manchester and Oxford. None of the authors has any conflict of interest to declare.
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整合素及其配体骨桥蛋白（OPN）之间的相互作用是否介导胚胎在着床时与子宫内膜上皮的附着？
上皮来源的OPN在母体表面结合受体整合素αvβ3，以支持着床早期的黏附。
整合素αvβ3和OPN在分泌中期的子宫内膜腔上皮中均有表达。
研究设计、规模、持续时间：在24小时和48小时时，对囊胚（小鼠，n = 151；人类，n = 8）以及与石川细胞单层相互作用的OPN或牛血清白蛋白（BSA）包被的珠子（n = 488）的附着位点进行显微镜观察。通过小干扰RNA（siRNA）介导的靶向作用改变上皮OPN或整合素αvβ3的水平，并将结果与非靶向siRNA或模拟转染对照进行比较。
参与者/材料、环境、方法：用人子宫内膜细胞（石川细胞）以及小鼠或人类胚胎或配体包被的珠子进行早期着床的体外建模。通过多次重复（n > 3）的图像分析测量附着胚胎周围抗原的免疫定位，从而检测相对强度梯度。使用稳定性量表对附着进行定量，并通过间接免疫荧光记录蛋白质表达。通过下拉试验探究蛋白质相互作用。
附着胚胎附近的上皮细胞中整合素和OPN水平升高。下拉试验证实了OPN与整合素αvβ3的结合（n > 3）。使用Kruskal-Wallis检验和Dunn事后检验对整合素αvβ3或OPN本身的siRNA敲低后观察到的小鼠胚胎附着稳定性降低，以及整合素αvβ3敲低后OPN包被珠子的附着稳定性降低进行显著性检验。
局限性、谨慎的原因：体外模型。使用人类胚胎的附着数据受胚胎可用性限制。小鼠胚胎与人细胞的附着涉及物种交叉，因此必须谨慎解释。配体包被的珠子能够探究介导附着的特定分子相互作用，但显然缺乏活胚胎的黏附及信号传导机制。
一些文献指出不育子宫内膜中整合素αvβ3表达降低；这些发现预测，如果整合素水平较低，胚胎在体内的附着稳定性将会降低。我们认为胚胎初始附着的稳健性会影响其进入着床上皮后阶段的能力；一些附着不佳的胚胎将会丢失。
研究资金/利益冲突：本研究未使用外部资金，由曼彻斯特大学和牛津大学的资金支持。所有作者均无利益冲突需要声明。
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