Three dimensional microvascular measurements in human endometrium using optical slices from laser scanning confocal microscopy (LSCM)

Abstract

There is increasing interest in the structure of the microvascular environment in human endometrium because of the recognition of the complexity and functional importance of this tissue. Endometrial microcirculatory networks and their relationships have rarely been studied in three-dimensions. Longitudinal uterine slices containing endometrial tissue were carefully selected from women undergoing a hysterectomy. Formalin-fixed endometrial sections (≤ 50 μm) representing the fundal and isthmic regions were immunofluorescently labeled with monoclonal antibody (CD34) to target the endothelium of microvessel and fluorescein isothiocyanate (FITC) labeled goat anti-mouse. Digital images were acquired using a Nikon Eclipse E800 microscope equipped with a Radiance 2000 confocal scanning laser attachment. ImarisBasic 4.1 visualization suite was utilized for qualitative interpretation. NeuronTracer 1.0 software was utilized to derive the length and numerical densities. There were significant changes across the phases of the menstrual cycle in functional and basal endometrial layers in vessel length density (LD(v)) and branch point density (ND(v)) within both fundal and isthmic regions of the uterus (P<0.001). There was also a significant effect of menstrual cycle phase on mean vessel segment length (SL(v)) within each region and within each of the layers (P<0.001). The capillary radial diffusion distance r(diff) was negatively correlated with LD(v). In general, within each of the menstrual cycle phases, LD(v), ND(v) were greater in the fundal than the isthmic regions while, in contrast, SL(v) was found to be greatest in the isthmic region. Utilization of immunofluorescence and laser scanning confocal microscopy has enabled us to demonstrate significant vascular changes in human endometrial layers illustrating that in general, within each of the menstrual cycle phases, vessel length and branch point densities were greater in the fundal than the isthmic regions, while vessel segment lengths were found to be greatest in the isthmic region.