Ultrasound Molecular Imaging of Vascular Endothelial Growth Factor Receptor 2 Expression for Endometrial Receptivity Evaluation

Purpose: Ultrasound (US) molecular imaging by examining the expression\nof vascular endothelial growth factor receptor 2 (VEGFR2) on uterus\nvascular endothelium was applied to evaluate the endometrial\nreceptivity.\nMethods: VEGFR2-targeted ultrasound contrast agents (UCA) and the\ncontrol UCA (without VEGFR2) were prepared and characterized. Adhesion\nexperiment in vitro was performed with mouse microvascular endothelial\ncells (bEnd.3) and the ratio of the number of UCA to that of cells at\nthe same field was compared. In vivo study, randomized boluses of\ntargeted or control UCA were injected into the animals of non-pregnancy\n(D0), pregnancy on day 2 (D2) and day 4 (D4), respectively. Sonograms\nwere acquired by an ultrasound equipment with a 40-MHz high-frequency\ntransducer (Vevo 2100; VisualSonics, Toronto, Canada). The ultrasonic\nimaging signals were quantified as the video intensity amplitudes\ngenerated by the attachment of VEGFR2-targeted UCA. Immunoblotting and\nimmunofluorescence assays were used for confirmation of VEGFR2\nexpression.\nResults: Our results showed that VEGFR2-targeted UCA could bind to\nbEnd.3 cells with significantly higher affinity than the control UCA\n(9.8 +/- 1.0 bubbles/cell versus 0.7 +/- 0.3 bubbles/cell, P < 0.01) in\nvitro. The mean video intensity from the US backscattering of the\nretained VEGFR2-targeted UCA was significantly higher than that of the\ncontrol UCA in D2 and D4 mice (D2, 10.5 +/- 2.5 dB versus 1.5 +/- 1.1\ndB, P < 0.01; D4, 15.7 +/- 4.0 dB versus 1.5 +/- 1.2 dB, P < 0.01), but\nnot significantly different in D0 mice (1.0 +/- 0.8 dB versus 0.9 +/-\n0.6 dB, P > 0.05). Moreover, D4 mice showed the highest video intensity\namplitude, indicating the highest VEGFR2 expression when compared with\nD2 and D0 mice (P < 0.01). This was further confirmed by our\nimmunoblotting and immunofluorescence experiments.\nConclusion: Ultrasound molecular imaging with VEGFR2-targeted UCA may be\nused for non-invasive evaluation of endometrial receptivity in murine\nmodels.