High-frequency Ultrasonography Based Early Pregnancy Characterization: A Technique to Study Embryo Implantation and Pregnancy Progression in Pregnant Murine Model

Overview

In this video, we demonstrate high-frequency ultrasonography to image embryo implantation sites in a pregnant murine model. This imaging technique successfully detects viable pregnancy and characterizes embryo-implantation sites during early murine pregnancy.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Imaging the Pregnant Mouse

1. Place mouse supine on the monitoring platform. The mouse continuously receives isoflurane anesthetic (between 1.5–2.5%) and oxygen (1 L/min) via the nose cone. Gently tape all paws to heart rate monitoring pads on the platform.
   1. Apply 1–2 mL of ultrasound transmission gel on abdomen.
2. Using the manual motor stage, position the ultrasound probe on the lower abdomen.
   1. Locate the bladder, which should appear as a fluid-filled dark circle just cephalad to the vaginal opening.
   2. Once the bladder is located, move the probe very slowly cephalad to visualize the pregnant uterus, which should appear as a cylindrical shape with round areas at pregnancy sites. This can be described as looking like pearls on a chain.
   3. Once the pregnant uterus has been identified, begin 2D imaging.

2. The 2D Ultrasound Imaging (Figure 1)

1. Once the pregnant uterus has been identified, begin at the pregnancy site closest to the bladder and slowly and sequentially move cephalad to determine number and location of pregnancy sites.
2. If the kidney, spleen, or liver are visualized, reposition the probe closer caudal (closer to the bladder) as the user has gone too far cephalad.
3. Image the contralateral uterine horn in the same manner.
4. Freeze the image when the ultrasound frame is in the center of the implantation/pregnancy site to save for later analysis and measurements.
   NOTE: It takes less than one second for the image to be frozen and saved for later analysis.
5. Measure implantation distance using the hyperechoic decidualization reaction as a marker by manually clicking on the "measure" tool first, and then clicking on the location of one implantation site. Next, drag the cursor to next implantation site and click to draw a line that the computer will automatically report its distance. The software will then report what the manual measurement is. This step is not computerized but relies on the user to mark the distance between decidualized sites by drawing a line between the decidualized sites, which the program will provide a measurement for.
6. Measure implantation size, gestational sac size, and fetal pole size.

Representative Results

Figure 1: High-frequency ultrasound detection of implantation site development and monitoring of fetal growth throughout gestation. As shown in Figure 1A, on 5.5 d.p.c. the lighter hyperechoic decidualized endometrium (D) allows the number of implantation sites and spacing to be quantified. As shown in Figure 1B, the decidualization sites (D) in another uterine horn are joined by the uterine horn (U). By 7.5 d.p.c., the darker hypoechoic sac (GS) and fetal pole (F) are easily identifiable Figure 1C.

Materials

Name	Company	Catalog Number	Comments
VisualSonics Vevo 2100 Ultrasound Imaging Platform/ Machine	VisualSonics, inc.	VS-11945
Vevo Imaging Station	VisualSonics, inc.	SA-11982
Aquasonic 100 Ultrasound Transmission Gel	Parker	#SKU PLI 01-08
Isoflurane (IsoThesia) 100mL bottle	Henry Shein	#29404
PuraLubenAnimal Ophthalmic Ointment	Dechra	#12920060