A Novel Use of Three-dimensional High-frequency Ultrasonography for Early Pregnancy Characterization in the Mouse

The overall goal of this ultrasound imaging procedure is to detect, monitor and characterize early pregnancy in the mouse. This method can help answer key questions in the field of murine reproductive biology and establishing the growth, development and viability of early mouse pregnancies. The main advantage of this technique is that these pregnancies can be characterized and phenotyped in vivo, without interruption of the mouse pregnancy, allowing for longitudinal collection of data.

Generally, individuals new to this method will struggle with getting all the images in one plane. This is due to the small incremental movements of the ultrasound probe that is necessary. Begin with preparing the ultrasound machine.

Turn on the transducer and then attach the 3-D motor stage to the transducer. Next, attach the probe to the transducer clamp. Now, position the mouse, supine, on the monitoring platform.

Provide it anesthetic via nose cone and gently tape all of its paws to the heart rate monitoring pads on the platform. Next, rub about one or two milliliters of ultrasound transmission gel onto the abdomen. Then position the ultrasound probe on the lower abdomen using the manual motor stage.

Using the ultrasound, locate the bladder, which should appear as a food-filled dark circle just cephalad to the vaginal opening. 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.

Now, proceed with 2-D imaging. Once the pregnant uterus has been identified, begin at the pregnancy site closest to the bladder and slowly and sequentially move cephalad to determine location of all the pregnancy sites. Save the image.

If the kidney, spleen or liver can be seen, then the scan is too far cephalad. Reposition the probe closer to the bladder and save the image. Next, image the contralateral uterine horn in the same manner.

Freeze the image when the ultrasound frame is at the center of the pregnancy or implantation site and save the image for analysis. Now or later, use the saved images to measure implantation positions using the hyperechoic decidualization reaction as a marker. Manually click on the measure tool and click on the location of an implantation site.

Then, drag the cursor to the next implantation site and click again to draw a line. The software then reports the length of the line. After all the implantation sites are measured, use the same technique and proceed to measure the gestational sac size and the fetal pole size.

After the 2-D imaging, position the probe in an area that is at the approximate mid-point of the desired 3-D image. For example, if one were imaging an apple in 3-D, position the probe at the core. Once the probe is properly positioned, obtain a 3-D acquisition.

The 3-D motor stage will travel this distance across the uterus in a series of steps or frames. As the scan is obtained, ensure that the intended structures are completely captured in the scan. After collecting the scan, load it up for image processing.

Choose the parallel and rotational methods for processing, which will load all the 3-D image frames into one 3-D box. Next, choose the step size of 0.08 millimeters, then begin at one end of the image block and scroll through all the frames to become familiar with the data. Next, start at one end of the stack and manually trace the outline of an object of interest.

Then, proceed to the next frame and manually trace the outline of the object again. Continue this process until all frames have been manually annotated. Once complete, click finish to obtain a 3-D image and a calculation of the object's volume.

For the image, choose between a mesh or a solid overlay. Then, choose to keep or remove the surrounding image information for clarity. The high frequency ultrasound can detect the development of an implantation site as early as the 5.5 days post-conception.

Using the lighter hyperechoic decidualized endometrium as a marker of implantation sites, the number of implantation sites and spacing of these sites, to be quantified at 6.5 days post-conception. As the pregnancy progresses to 7.5 days post-conception, a darker, hypoechoic gestational sac and fetal pole are both easily identifiable. Three-dimensional reconstructions of the mouse uterus at 6.5 days post-conception, were made in both mesh and solid overlay formats.

At 7.5 days post-conception, the 3-D scan was just as revealing. After watching this video, you should have a good understanding of how to obtain accurate ultrasound images in both 2-D and 3-D, to characterize a pregnancy without interruption. The total time needed for an ultrasound acquisition, in both 2-D and 3-D, is approximately 10 to 20 minutes in the hands of an experienced user.

While attempting this procedure, it's important to remember to keep your movements small and incremental. This technique paved the way for researchers to explore normal and abnormal pregnancy phenotypes in the mouse.