Contrast-enhanced ultrasound reveals real-time spatial changes in vascular perfusion during early implantation in the macaque uterus  Christopher S. Keator, Ph.D., Jonathan R. Lindner, M.D., J. Todd Belcik, B.S., Cecily V. Bishop, Ph.D., Ov D. Slayden, Ph.D.  Fertility and Sterility  Volume 95, Issue 4, Pages 1316-1321.e3 (March 2011) DOI: 10.1016/j.fertnstert.2011.01.040 Copyright © 2011 American Society for Reproductive Medicine Terms and Conditions

Figure 1 Sonograms showing changes in the macaque uterus during early pregnancy. CEU images represent frames captured after microbubble destruction (0 seconds) and then microbubble replenishment over time (1.5 to 30 seconds). Yellow arrowheads indicate uterine changes detected by DUS: an endometrial perturbation on day 27; endometrial thickening on day 30; gestational sacs on day 33; and the detection and confirmation of the fetal heartbeat on day 36. Blue arrows indicate the primary disc, and pink arrows indicate the secondary disc. Fertility and Sterility 2011 95, 1316-1321.e3DOI: (10.1016/j.fertnstert.2011.01.040) Copyright © 2011 American Society for Reproductive Medicine Terms and Conditions

Figure 2 Replenishment kinetic curves derived from the perfusion of the corresponding uterine regions of interest (ROIs) during early implantation in a single macaque. ROIs were drawn over the uterine images shown after 30 seconds of infusion as in Figure 1. Note that peak video intensities for the ROIs are lowest on day 30, which corresponded with the nadir in P for this animal. Fertility and Sterility 2011 95, 1316-1321.e3DOI: (10.1016/j.fertnstert.2011.01.040) Copyright © 2011 American Society for Reproductive Medicine Terms and Conditions

Figure 3 Effects of P on intrauterine vascular perfusion. Values for each animal were aligned relative to the nadir (day 0) in serum P. Data are presented as the mean ± SE. Different letters denote daily differences (P<0.05) in blood flow within each region. Fertility and Sterility 2011 95, 1316-1321.e3DOI: (10.1016/j.fertnstert.2011.01.040) Copyright © 2011 American Society for Reproductive Medicine Terms and Conditions

Diagram illustrating the experimental timeline Diagram illustrating the experimental timeline. See text for detailed explanations. P = serum progesterone; HB = heartbeat. Fertility and Sterility 2011 95, 1316-1321.e3DOI: (10.1016/j.fertnstert.2011.01.040) Copyright © 2011 American Society for Reproductive Medicine Terms and Conditions

Mathematical formula used to calculate blood flow in the manually identified ROIs in the macaque uterus. AROI is the plateau in VIROI (representing microvascular blood volume); βROI is the microvascular flux rate constant (representing blood velocity); 6 is a scaling factor for the different infusion rates for brachial artery and vascular perfusion (used to avoid saturation of the dynamic range) (1); 1.03 is the density of the uterus (31); and 60 is a conversion from seconds to minutes. Fertility and Sterility 2011 95, 1316-1321.e3DOI: (10.1016/j.fertnstert.2011.01.040) Copyright © 2011 American Society for Reproductive Medicine Terms and Conditions

Sonograms of a macaque uterus on day 30 of the fertile cycle, illustrating hypertrophied vessels supplying the primary placental disc. Grayscale images represent frames captured after microbubble destruction (0 seconds) and replenishment over time (1.5–6 seconds). The myometrium (myo) and hypertrophied artery (yellow arrow) exhibit strong video intensity after 1.5 seconds of replenishment. After 3 seconds, a second hypertrophied vessel (upper arrow) can be detected, and by 6 seconds both vessels and the primary placental disc (yellow arrowhead) exhibit strong video intensity. The lower images have been pseudocolored to illustrate the relative intensity of the microbubble replenishment. Fertility and Sterility 2011 95, 1316-1321.e3DOI: (10.1016/j.fertnstert.2011.01.040) Copyright © 2011 American Society for Reproductive Medicine Terms and Conditions