Name: a novel use of three-dimensional high-frequency ultrasonography for early pregnancy characterization in the mouse
Uploaded: 2017-10-24T13:00:00
Description: Watch this Scientific Journal Video about A Novel Use of Three-dimensional High-frequency Ultrasonography for Early Pregnancy Characterization in the Mouse at JoVE.com

We very much appreciate the assistance of Rong Zhao, Jie Li, and Yan Ying.

These studies were conducted in accordance with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health and animal protocols approved by the Institutional Animal Care and Use Committee (IACUC) of Baylor College of Medicine under protocol number AN-4203.
1. Preparation of the Pregnant Mouse for Ultrasound
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	<li>Timed mating
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		<li>Place the dam with a proven fertile male mouse overnight beginning after 1700. Separate dam an...

<ol>
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This novel use of 3-D HFUS imaging demonstrates the ability to successfully detect, visualize, and characterize embryo-implantation sites during early murine pregnancy in a non-invasive manner. The technology offers a significant improvement over current methods, which rely on the interruption of pregnancies for gross tissue and histopathologic characterization. However it should be noted that histological methods would still be considered more optimal when characterization at a more magnified and more cellular level is ...

Recurrent early pregnancy loss is one of the most common complications after conception and affects approximately 1% of couples trying to conceive1,2. The underlying mechanisms of early pregnancy loss are varied: from intrinsic embryonic abnormalities and maternal comorbidities to defects in endometrial receptivity1,3,4. Because of their genetic tractability, mouse models have been widely utilized for investigations of early embryo implantation and pregnancy. Furthermore, the short gestational time of the mouse and the ability to perform large-scale studies have ensured the growing utility of the mouse in addressing key clinical questions in human reproductive medicine5. That said, the vast majority of murine experimental designs still require numerous dams to be euthanized on sequential gestational days to quantify and analyze implantation site location, number, size, and spacing patterns during pregnancy6,7,8, thereby precluding longitudinal studies on the same animal.
In the clinic, ultrasound is a reliable and invaluable tool to monitor human fetal viability and development in a non-invasive manner9,10,11. More recently, high-frequency ultrasound (HFUS) has begun to find limited applications in the mouse as a method for monitoring fetal viability and growth during pregnancy12,13,14. The recent technological advances in ultrasound imaging have permitted the application of three-dimensional (3-D) data for visual reconstruction of animal organs and subsequent monitoring of pathologies15,16,17. Use of this advanced imaging technology has markedly improved the power to detect smaller volume fluctuations, to reduce inter-animal variability, and to monitor the progression of a pathology or the efficacy of a therapeutic intervention17. While the primary utility of this technology has been to monitor malignancy progression in oncomouse models15,16, 3-D HFUS imaging has only recently been used to quantitate and monitor the active growth of embryo implantation and fetal development in the mouse uterus18.
Here, we demonstrate how to perform HFUS imaging to produce 2-D and 3-D data to generate reconstructions of the early pregnant mouse uterus. We demonstrate the utility of this novel method to detect these early embryonic implantation events without the need for pregnancy termination, allowing researchers to collect data in a non-invasive manner.

<table>
	<tbody>
		<tr>
			<td>VisualSonics Vevo 2100 Ultrasound Imaging Platform/Machine</td>
			<td>VisualSonics, inc.</td>
			<td>VS-11945</td>
			<td></td>
		</tr>
		<tr>
			<td>Vevo Imaging Station</td>
			<td>VisualSonics, inc.</td>
			<td>SA-11982</td>
			<td></td>
		</tr>
		<tr>
			<td>Aquasonic 100 Ultrasound Transmission Gel</td>
			<td>Parker</td>
			<td>#SKU PLI 01-08</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane (IsoThesia) 100mL bottle</td>
			<td>Henry Shein</td>
			<td>#29404</td>
			<td></td>
		</tr>
		<tr>
			<td>PuraLubenAnimal Ophthalmic Ointment</td>
			<td>Dechra</td>
			<td>#12920060</td>
			<td></td>
		</tr>
	</tbody>
</table>

a novel use of three-dimensional high-frequency ultrasonography for early pregnancy characterization in the mouse

High-frequency ultrasonography (HFUS) is a common method to non-invasively monitor the real-time development of the human fetus in utero. The mouse is routinely used as an in vivo model to study embryo implantation and pregnancy progression. Unfortunately, such murine studies require pregnancy interruption to enable follow-up phenotypic analysis. To address this issue, we used three-dimensional (3-D) reconstruction of HFUS imaging data for early detection and characterization of murine embryo implantation sites and their individual developmental progression in utero. Combining HFUS imaging with 3-D reconstruction and modeling, we were able to accurately quantify embryo implantation site number as well as monitor developmental progression in pregnant C57BL6J/129S mice from 5.5 days post coitus (d.p.c.) through to 9.5 d.p.c. with the use of a transducer. Measurements included: number, location, and volume of implantation sites as well as inter-implantation site spacing; embryo viability was assessed by cardiac activity monitoring. In the immediate post-implantation period (5.5 to 8.5 d.p.c.), 3-D reconstruction of the gravid uterus in both mesh and solid overlay format enabled visual representation of the developing pregnancies within each uterine horn. As genetically engineered mice continue to be used to characterize female reproductive phenotypes derived from uterine dysfunction, this method offers a new approach to detect, quantify, and characterize early implantation events in vivo. This novel use of 3-D HFUS imaging demonstrates the ability to successfully detect, visualize, and characterize embryo-implantation sites during early murine pregnancy in a non-invasive manner. The technology offers a significant improvement over current methods, which rely on the interruption of pregnancies for gross tissue and histopathologic characterization. Here we use a video and text format to describe how to successfully perform ultrasounds of early murine pregnancy to generate reliable and reproducible data with reconstruction of the uterine form in mesh and solid 3-D images.

As demonstrated in Figure 1, the high frequency ultrasound can detect implantation site development beginning as early as the 5.5 d.p.c time point. Using the lighter hyperechoic decidualized endometrium as a marker of implantation sites at 6.5 d.p.c allows the number of implantation sites and spacing of these sites to be quantified. As the pregnancy progresses to 7.5 d.p.c., a darker hypoechoic gestational sac and fetal pole are also easily identifiable.
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Watch this Scientific Journal Video about A Novel Use of Three-dimensional High-frequency Ultrasonography for Early Pregnancy Characterization in the Mouse at JoVE.com

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

Mice are widely used to study gestational biology. However, pregnancy termination is required for such studies which precludes longitudinal investigations and necessitates the use of large numbers of animals. Therefore, we describe a non-invasive technique of high-frequency ultrasonography for early detection and monitoring of post-implantation events in the pregnant mouse.

High-frequency ultrasonography (HFUS) is a common method to non-invasively monitor the real-time development of the human fetus in utero. The mouse is ...

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.

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