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
	<ol>
		<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 ...

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