All the steps in this protocol follow the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health and have been approved by the Institutional Animal Care and Use Committee of New York University Grossman School of Medicine.
1. Mating of mice for timed pregnancies
<ol>
	<li>Pair the appropriate female mouse (usually a heterozygote) in a cage with the appropriate male mouse (usually a heterozygote) for overnight mating.</li>
	<li>Separate the mice the next morning. Alternatively, continuously mate the female and male mice, thus increasing the chances of pregnancy. 
	NOTE: However, an accurately timed pregnancy cannot be assured with the alternative, and staging of mouse embryos by ultrasound is not clear, especially when the disease process results in intrauterine growth retardation. If the embryos carrying the gene variant are assumed to be small, look for the larger wildtype littermates to gauge the gestational age.
	<ol>
		<li>Optional: Look for the vaginal plug. If there is no vaginal plug, the female mouse has not been mated. If there is a vaginal plug, it is still likely that the female mouse does not become pregnant.</li>
	</ol>
	</li>
	<li>Time the day after the overnight mating as E0.5.</li>
	<li>Perform imaging at E6.5&#8211;E.8.5 to determine pregnancy, and re-mate the mice if the mouse is not pregnant (see step 1.1). 
	NOTE: At this stage, the female dam is not obviously pregnant to the eye; hence, the ultrasound imaging allows early determination and re-mating.</li>
</ol>
2. Anesthesia and preparation of mouse
<ol>
	<li>Place the pregnant mouse in the anesthetic induction chamber.</li>
	<li>Mix isoflurane with either room air or medical oxygen, at a concentration of 2%&#8211;3% at 1 L/min flow rate to induce sedation of the pregnant mouse in the induction chamber. 
	NOTE: Sedation typically occurs within 1&#8211;2 min. The mouse will be lying still, and her breathing will have slowed.</li>
	<li>Quickly transfer the mouse to the imaging platform. The imaging platform typically has heating elements as well, which can help keep the mouse warm.</li>
	<li>Place the nose of the mouse into the anesthetic nosecone.</li>
	<li>Quickly re-route the isoflurane/oxygen mixture to the imaging platform nosecone. Maintain isoflurane at 2%&#8211;3% at 1 L/min flow.</li>
	<li>Determine the level of sedation by paw pinch, corneal reflex, level of respiration, and any movement. 
	NOTE: The corneal reflex can be initially determined by applying moisturizing ointment to the eyes to keep them from drying out while the mouse is anesthetized (the mouse does not close her eyes).</li>
	<li>With the mouse lying supine (on its back), tape the paws to the imaging platform&#8217;s electrocardiogram (EKG) pads. 
	NOTE: However, an EKG is not necessary for this kind of imaging.</li>
	<li>Remove the fur from the pregnant dam&#8217;s abdomen as follows:
	<ol>
		<li>Wet the abdominal fur thoroughly with 70% ethanol, including up to the lateral edges. Do not apply so much that there is run-off onto the platform. 
		NOTE: Ethanol works better as a shaving lubricant than water.</li>
		<li>Use the razor blade to carefully shave the abdomen. Be careful not to cut the nipples.</li>
		<li>Wipe the shaved fur off the abdomen with gauze or some wipes.</li>
		<li>Alternatively, use a depilatory cream after using the fur clippers to remove most of the fur.</li>
	</ol>
	</li>
</ol>
3. Transabdominal imaging of the (presumed) pregnant mouse
<ol>
	<li>After the abdomen is shaved, reduce the isoflurane to 1%&#8211;1.5%, still maintaining a flow rate of 1 L/min. Monitor the level of sedation by the level of respiration and any movements as well as paw pinch and/or corneal reflex. 
	NOTE: The heart rate of the anesthetized mouse will typically be 400&#8211;500/min, depending on the core (rectal) temperature. For the purpose of a rapid pregnancy check, do not warm the mouse to a physiological core temperature; the heart rate will be closer to 400&#8211;450/min, but may drop further with an irregular rhythm should the mouse become cold with prolonged imaging.
	<ol>
		<li>Importantly, make sure that the respirations are moderate in depth and regularity, not erratic or agonal (gasping: deep, slow, and erratic, with deep intercostal and subcostal retractions). 
		NOTE: The respiratory rate of the non-ventilated, anesthetized mouse will typically be 60&#8211;100/min. See https://ahcs.ninds.nih.gov/ACUC_pages/pg_003_anesth_animals.html and https://az.research.umich.edu/animalcare/guidelines/guidelines-anesthesia-and-analgesia-mice.</li>
	</ol>
	</li>
	<li>Apply ultrasound (acoustic coupling) gel to the abdomen generously.</li>
	<li>Place the imaging transducer on the abdomen to orient it in a horizontal plane: orient the probe to obtain a left-right orientation on the imaging system; the &#8220;dot&#8221; or ridge indicated on the side of the imaging probe should be facing right. 
	NOTE: Sliding the imaging probe to the mouse&#8217;s right should shift the corresponding image on the ultrasound system to the mouse&#8217;s right (imagine looking &#8220;up&#8221; from the mouse&#8217;s tail&#8212;the mouse&#8217;s right will be left on the monitor).
	<ol>
		<li>Typically, use the imaging system&#8217;s transducer mount and rail-manipulator system. Here, &#8220;free hand&#8221; imaging has been used in which the imaging transducer is hand-held (two hands are steadier than one) and which allows for more rapid movements around the abdomen. These movements, as will be outlined below, include both rotational and translational movements. However, this requires more practice.</li>
	</ol>
	</li>
	<li>Identify the bladder on the screen (Video 1).</li>
	<li>Scanning caudally from the bladder, identify the vagina. Then, scanning slowly and smoothly in a cranial direction, identify the bifurcation of the vagina into the left and right uterine horns (Figure 2; Video 1).</li>
	<li>Begin the survey of the uterus (left and right uterine horns)13 (Figure 3; Video 2). 
	NOTE: Up to mid-gestation (E10.5 or E11.5), the mouse embryos will be positioned along the right and left peripheries. As they grow, the more distal portions of the uterus and their corresponding embryos will turn outwards and posteriorly. As the embryos grow further (E15.5 and later, generally), the mouse fetuses will be positioned almost randomly in various directions, and it becomes difficult to &#8220;track&#8221; a uterus from proximal to distal.
	<ol>
		<li>Scan quickly simply to check if a mouse is pregnant or not. This rapid method requires only recognition of a gravid uterus and mouse embryos.</li>
		<li>Alternatively, take extra time to enumerate the embryos (live, dead, resorbed) in each uterine horn. 
		NOTE: In general, a live embryo will exhibit distinct organs such as a heart, limbs, head with ventricles, and eyes. A dead embryo takes on a homogeneous, &#8220;mushy&#8221; appearance unless just dead. Resorbed embryos have a pinpoint echogenic spot in the middle of a gravid-looking uterus (Figure 4, Figure 5, Figure 6, Figure 7, Figure 8).</li>
		<li>To determine if an embryo is truly alive, look for the heartbeat and/or blood flow. 
		NOTE: Color Doppler flow mapping can aid in determining the presence of blood flow, both in the embryo and in the umbilical cord. In general, this may be applied to embryos older than E8.5.</li>
		<li>Recognize potential artifacts that may mimic a gravid uterus (Figure 9, Figure 10). In addition, as bowel gas and other ultrasound &#8220;shadow&#8221; artifacts may obscure segments of the uterine horn, perform the imaging from multiple vantage points to ensure adequate visualization of the uterus.</li>
	</ol>
	</li>
	<li>After the survey is complete, wipe the gel from the abdomen with gauze or wipes. Try to remove as much as possible as the gel tends to cool the mouse down.</li>
	<li>Untape the paws, and remove the mouse from the anesthetic nosecone.</li>
	<li>Gently move the mouse back into her cage. She should wake up and start moving around within a minute or so. 
	NOTE: Genetically modified mice may take a little longer to recover from anesthesia and need to be watched more closely.</li>
</ol>

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(128), e56207 (2017).</li><li>Greco, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+frequency+ultrasound+for+in+vivo+pregnancy+diagnosis+and+staging+of+placental+and+fetal+development+in+mice.">High frequency ultrasound for in vivo pregnancy diagnosis and staging of placental and fetal development in mice.</a> PLoS One. 8 (10), 77205 (2013).</li><li>Flores, L. E., Hildebrandt, T. B., K&#252;hl, A. A., Drews, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+detection+and+staging+of+spontaneous+embryo+resorption+by+ultrasound+biomicroscopy+in+murine+pregnancy.">Early detection and staging of spontaneous embryo resorption by ultrasound biomicroscopy in murine pregnancy.</a> Reproductive Biology and Endocrinology. 12, 38 (2014).</li><li>Norton, W. 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The authors have nothing to disclose.

The most important first step in the imaging is to identify the vagina and then to determine the bifurcation of the uterine horn to the left and right. By following each uterine horn, the imager is less likely to mis-identify loops of the bowel as the uterus. Moreover, understanding the variations in the appearance of the bowel (with/without fecal matter) is important to distinguish these from the uterus; occasionally, fecal &#8220;balls&#8221; in bowel loops may mimic a gravid (pregnant) uterus. Although other authors h...

The mouse is the preferred mammalian model for many human diseases and biological processes1,2,3,4. Research in developmental biology often requires staged-pregnant mice to determine evolving processes at various timepoints5,6,7,8. Moreover, optimal and effective breeding of model mice requires an assessment of timed pregnancies, particularly when investigators are studying the effects of a gene mutation on development. Typically, investigators mate heterozygous mice overnight, look for a vaginal plug early the next morning, and hope that a pregnancy ensues9. Determining intrauterine loss typically starts with checking a newborn litter for Mendelian ratios of genotypes, then working backwards by sacrificing pregnant mice at various gestational stages, and recovering the embryos. Investigators may determine weight gain as a metric of a positive pregnancy10,11; however, especially with genetically-engineered mice, the litters may be very small and subsequently resorbed when there is intrauterine loss due to which the weight gain may not be obvious (particularly early in pregnancy, ~E6.5&#8211;8.5). A mouse may appear falsely pregnant due, for example, to a benign abdominal tumor. In essence, one works &#8220;blind&#8221;.
High-resolution ultrasound biomicroscopy allows for direct visualization of the gravid uterus and developing mouse embryos12,13,14,15,16. Although we had initially developed methods to assess embryonic mouse cardiovascular physiology16,17, we recognized the utility of this imaging modality to streamline our mouse breeding. Specifically, we no longer had to wait to &#8220;see&#8221; if a mouse were pregnant, based on either the obvious weight gain or delivery of a litter; we could determine the gravid state and re-mate mice quickly if the dam was not pregnant. Moreover, intrauterine losses could also be easily imaged, and a timeline of loss could be determined without sacrificing the mouse (see Figure 1 for a schematic). Time, valuable model mice, and funds can thus be saved.

<table>
	<tbody>
		<tr>
			<td>Depilatory cream</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol, 70%</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fur clippers</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Gauze or KimWipes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Medical oxygen (optional)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Medical tape</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse imaging system (including anesthesia set-up and imaging platform)</td>
			<td>Fujifilm Visual Sonics</td>
			<td>Various</td>
			<td>Any system with 40 MHz center-frequency ultrasound transducer probe</td>
		</tr>
		<tr>
			<td>Razor blade (not a safety razor)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Scale (to weigh mouse)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrasound gel</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

is my mouse pregnant? high-frequency ultrasound assessment

The mouse is the mammalian animal model of choice for many human diseases and biological processes. Developmental biology often requires staged-pregnant mice to determine evolving processes at various timepoints. Moreover, optimal and efficient breeding of model mice requires an assessment of timed pregnancies. Most commonly, mice are mated overnight, and the presence of a vaginal plug is determined; however, the positive predictive value of this technique is suboptimal, and one needs to wait to know if the mouse is truly pregnant. High-resolution ultrasound biomicroscopy is an effective and efficient tool for imaging: 1) Whether a mouse is pregnant; 2) What gestational stage the mouse has reached; and 3) Whether there are intrauterine losses. In addition to the embryos and fetuses, the investigator must also recognize common artifacts in the abdominal cavity so as not to mistake these for a gravid uterus. This article provides a protocol for imaging along with illustrative examples.

This protocol will allow an investigator to determine confidently whether a mouse is pregnant, including during the early stages and to determine whether there are obvious prenatal embryonic or fetal losses without needing to sacrifice the pregnant dam. This protocol is especially useful when breeding genetically engineered mice; typically, heterozygous x heterozygous crosses to yield homozygous offspring leads to failure of proper development, which causes prenatal lethality. Figure 1 depic...

Watch this Scientific Journal Video about Is My Mouse Pregnant? High-Frequency Ultrasound Assessment at JoVE.com

Is My Mouse Pregnant? High-Frequency Ultrasound Assessment

High-resolution ultrasound can help streamline experiments requiring timed-pregnant mice by determining the state of pregnancy, gestational age, and pregnancy losses. Presented here is a protocol to illustrate methods to assess mouse pregnancies as well as potential pitfalls (image artifacts) that may mimic pregnancy.

The mouse is the mammalian animal model of choice for many human diseases and biological processes. Developmental biology often requires staged-pregnant ...

is-my-mouse-pregnant-high-frequency-ultrasound-assessment

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Developmental Biology

6.9K Views.  New York University Grossman School of Medicine. High-resolution ultrasound can help streamline experiments requiring timed-pregnant mice by determining the state of pregnancy, gestational age, and pregnancy losses. Presented here is a protocol to illustrate methods to assess mouse pregnancies as well as potential pitfalls (image artifacts) that may mimic pregnancy.

Video: Is My Mouse Pregnant? High-Frequency Ultrasound Assessment

Contrast Imaging in Mouse Embryos Using High-frequency Ultrasound

Ultrasound contrast-enhanced imaging can convey essential quantitative information regarding tissue vascularity and perfusion and, in targeted applications, facilitate the detection and measure of vascular biomarkers at the molecular level. Within the mouse embryo, this noninvasive technique may be used to uncover basic mechanisms underlying vascular development in the early mouse circulatory system and in genetic models of cardiovascular disease. The mouse embryo also presents as an excellent model for studying the adhesion of microbubbles to angiogenic targets (including vascular endothelial growth factor receptor 2 (VEGFR2) or &#945;v&#946;3) and for assessing the quantitative nature of molecular ultrasound. We therefore developed a method to introduce ultrasound contrast agents into the vasculature of living, isolated embryos. This allows freedom in terms of injection control and positioning, reproducibility of the imaging plane without obstruction and motion, and simplified image analysis and quantification. Late gestational stage (embryonic day (E)16.6 and E17.5) murine embryos were isolated from the uterus, gently exteriorized from the yolk sac and microbubble contrast agents were injected into veins accessible on the chorionic surface of the placental disc. Nonlinear contrast ultrasound imaging was then employed to collect a number of basic perfusion parameters (peak enhancement, wash-in rate and time to peak) and quantify targeted microbubble binding in an endoglin mouse model. We show the successful circulation of microbubbles within living embryos and the utility of this approach in characterizing embryonic vasculature and microbubble behavior.

Here, we present a protocol to inject ultrasound microbubble contrast agents into living, isolated late-gestation stage murine embryos. This method enables the study of perfusion parameters and of vascular molecular markers within the embryo using contrast-enhanced high-frequency ultrasound imaging.

Ultrasound contrast-enhanced imaging can convey essential quantitative information regarding tissue vascularity and perfusion and, in targeted ...

Assessment of Endothelial Cell Migration After Exposure to Toxic Chemicals

Exposure to chemical substances (including alkylating chemical warfare agents like sulfur and nitrogen mustards) cause a plethora of clinical symptoms including wound healing disorder. The physiological process of wound healing is highly complex. The formation of granulation tissue is a key step in this process resulting in a preliminary wound closure and providing a network of new capillary blood vessels &#8211; either through vasculogenesis (novel formation) or angiogenesis (sprouting of existing vessels). Both vasculo- and angiogenesis require functional, directed migration of endothelial cells. Thus, investigation of early endothelial cell (EEC) migration is important to understand the pathophysiology of chemical induced wound healing disorders and to potentially identify novel strategies for therapeutic intervention.
We assessed impaired wound healing after alkylating agent exposure and tested potential candidate compounds for treatment. We used a set of techniques outlined in this protocol. A modified Boyden chamber to quantitatively investigate chemokinesis of EEC is described. Moreover, the use of the wound healing assay in combination with track analysis to qualitatively assess migration is illustrated. Finally, we demonstrate the use of the fluorescent dye TMRM for the investigation of mitochondrial membrane potential to identify underlying mechanisms of disturbed cell migration. The following protocol describes basic techniques that have been adapted for the investigation of EEC.

Investigation of early endothelial cell (EEC) migration is important to understand the pathophysiology of certain illnesses and to potentially identify novel strategies for therapeutic intervention. The following protocol describes techniques to assess cell migration that have been adapted for the investigation of EEC.

Exposure to chemical substances (including alkylating chemical warfare agents like sulfur and nitrogen mustards) cause a plethora of clinical symptoms ...

Assessment of Maternal Vascular Remodeling During Pregnancy in the Mouse Uterus

The placenta mediates the exchange of factors such as gases and nutrients between mother and fetus and has specific demands for supply of blood from the maternal circulation. The maternal uterine vasculature needs to adapt to this temporary demand and the success of this arterial remodeling process has implications for fetal growth. Cells of the maternal immune system, especially natural killer (NK) cells, play a critical role in this process. Here we describe a method to assess the degree of remodeling of maternal spiral arteries during mouse pregnancy. Hematoxylin and eosin-stained tissue sections are scanned and the size of the vessels analysed. As a complementary validation method, we also present a qualitative assessment for the success of the remodeling process by immunohistochemical detection of smooth muscle actin (SMA), which normally disappears from within the arterial vascular media at mid-gestation. Together, these methods enable determination of an important parameter of the pregnancy phenotype. These results can be combined with other endpoints of mouse pregnancy to provide insight into the mechanisms underlying pregnancy-related complications.

This protocol describes a technique to assess changes in the maternal vasculature during pregnancy in mice. Using stereological methods, remodeling of the decidual spiral arteries is assessed quantitatively and the results confirmed qualitatively using immunohistochemistry.

The placenta mediates the exchange of factors such as gases and nutrients between mother and fetus and has specific demands for supply of blood from the ...

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.

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 ...

Fetal Mouse Cardiovascular Imaging Using a High-frequency Ultrasound (30/45MHZ) System

Congenital heart defects (CHDs) are the most common cause of childhood morbidity and early mortality. Prenatal detection&#160;of the underlying molecular mechanisms of CHDs is&#160;crucial for inventing new preventive and therapeutic strategies. Mutant mouse models are powerful tools to discover new&#160;mechanisms&#160;and environmental stress modifiers that drive cardiac development and their potential&#160;alteration in CHDs. However, efforts to establish the causality of these putative contributors have been limited to histological&#160;and molecular studies&#160;in non-survival animal experiments, in which&#160;monitoring the key physiological and hemodynamic parameters is&#160;often absent. Live imaging technology has become an essential tool to establish the&#160;etiology of CHDs. In particular,&#160;ultrasound imaging can be used prenatally without surgically exposing the fetuses,&#160;allowing maintaining&#160;their baseline physiology while monitoring the impact of environmental stress&#160;on the hemodynamic and structural aspects of cardiac chamber development. Herein, we use the High-Frequency Ultrasound (30/45)&#160;system to examine the cardiovascular system in fetal mice at E18.5 in utero at the baseline and in response to prenatal hypoxia exposure. We demonstrate the feasibility of the system to measure cardiac chamber size, morphology, ventricular function, fetal heart rate, and umbilical artery flow indices, and their alterations in fetal mice exposed to systemic chronic hypoxia in utero in real time.

Fetal Mouse Cardiovascular Imaging Using a High-frequency Ultrasound 30/45MHZ System

High-frequency ultrasound imaging of the fetal mouse has improved imaging resolution and can provide precise non-invasive characterization of cardiac development and structural defects. The protocol outlined herein is designed to perform real-time fetal mice echocardiography in vivo.

Congenital heart defects (CHDs) are the most common cause of childhood morbidity and early mortality. Prenatal detection&#160;of the underlying molecular ...

High Frequency Ultrasound for the Analysis of Fetal and Placental Development In Vivo

Ultrasound imaging is a widespread method used to detect organ anomalies and tumors in human and animal tissues. The method is non-invasive, harmless, and painless, and the application is easy, fast, and can be done anywhere, even with mobile devices. During pregnancy, ultrasound imaging is standardly used to closely monitor fetal development. The technique is important to assess intrauterine growth restriction (IUGR), a pregnancy complication with short- and long-term health consequences for both the mother and fetus. Understanding the process of IUGR is indispensable for developing effective therapeutic strategies. 
The ultrasound system used in this manuscript is an ultrasound device produced for the analysis of small animals and can be used in various research fields, including pregnancy research. Here we describe the usage of the system for in vivo analysis of fetuses from natural killer (NK) cell/mast cell (MC)-deficient mothers that give birth to growth-restricted pups. The protocol includes preparation of the system, handling of the mice before and during measurements, and the usage of the B-mode, color doppler mode, and pulse-wave doppler mode. Fetal size, placental size, and blood supply to the fetus were analyzed. We found reduced implantation sizes and smaller placentas in NK/MC-deficient mice from mid-gestation onwards. In addition, MC/NK-deficiency was associated with absent and reversed end diastolic flow in the fetal Arteria umbilicalis(UmA) and an elevated resistance index. The methods described in the protocol can easily be used for related and non-related research topics.

High Frequency Ultrasound for the Analysis of Fetal and Placental Development In Vivo

Here we describe the technique of high frequency ultrasound for in vivo analysis of fetuses in mice. This method allows the follow-up of fetuses and the analysis of placental parameters as well as maternal and fetal blood flow throughout pregnancy.

Ultrasound imaging is a widespread method used to detect organ anomalies and tumors in human and animal tissues. The method is non-invasive, harmless, and ...

Fluorimetric Techniques for the Assessment of Sperm Membranes

Standard spermiograms describing sperm quality are mostly based on the physiological and visual parameters, such as ejaculate volume and concentration, motility and progressive motility, and sperm morphology and viability. However, none of these assessments is good enough to predict the semen quality. Given that maintenance of sperm viability and fertilization potential depends on membrane integrity and intracellular functionality, evaluation of these parameters might enable a better prediction of sperm fertilization competence. Here, we describe three feasible methods to evaluate sperm quality using specific fluorescent probes combined with fluorescence microscopy or flow cytometry analyses. Analyses assessed plasma membrane integrity using 4&#39;,6-diamidino-2-phenylindole (DAPI) and propidium iodide (PI), acrosomal membrane integrity using fluorescein isothiocyanate-conjugated Pisum sativum agglutinin (FITC-PSA) and mitochondrial membrane integrity using 5,5&#39;,6,6&#39;-tetra-chloro-1,1&#39;,3,3&#39;-tetraethylbenzimidazolyl carbocyanine iodide (JC-1). Combinations of these methods are also presented. For instance, use of annexin V combined with PI fluorochromes enables assessing apoptosis and calculating the proportion of apoptotic sperm (apoptotic index). We believe that these methodologies, which are based on examining spermatozoon membranes, are very useful for the evaluation of sperm quality.

Here, we present methodologies to evaluate spermatozoan membrane integrity, a cellular feature associated with sperm fertilization competence. We describe three techniques for the fluorimetric assessment of sperm membranes: simultaneous staining with specific fluorescent probes, fluorescence microscopy, and advanced sperm-dedicated flow cytometry. Examples of combining the methodologies are also presented.

Standard spermiograms describing sperm quality are mostly based on the physiological and visual parameters, such as ejaculate volume and concentration, ...

Assessment of Ultrastructural Neuroplasticity Parameters After In Utero Transduction of the Developing Mouse Brain and Spinal Cord

The present study combines in utero transduction with transmission electron microscopy (TEM) aiming at a precise morphometrical analysis of ultrastructural parameters in unambiguously identified topographical structures, affected by a protein of interest that is introduced into the organism via viral transfer. This combined approach allows for a smooth transition from macrostructural to ultrastructural identification by following topographical navigation maps in a tissue atlas. High-resolution electron microscopy of the in-utero-transduced tissue reveals the fine ultrastructure of the neuropil and its plasticity parameters, such as cross-sectioned synaptic bouton areas, the number of synaptic vesicles and mitochondria within a bouton profile, the length of synaptic contacts, cross-sectioned axonal areas, the thickness of myelin sheaths, the number of myelin lamellae, and cross-sectioned areas of mitochondria profiles. The analysis of these parameters reveals essential insights into changes of ultrastructural plasticity in the areas of the nervous system that are affected by the viral transfer of the genetic construct. This combined method can not only be used for studying the direct effect of genetically engineered biomolecules and/or drugs on neuronal plasticity but also opens the possibility to study the in utero rescue of neuronal plasticity (e.g., in the context of neurodegenerative diseases).

The combination of transmission electron microscopy and in utero transduction is a powerful approach for studying morphological changes in the fine ultrastructure of the nervous system during development. This combined method allows deep insights into changes in structural details underlying neuroplasticity with respect to their topographical representation.

The present study combines in utero transduction with transmission electron microscopy (TEM) aiming at a precise morphometrical analysis of ...

Assessment of Oxidative Damage in the Primary Mouse Ocular Surface Cells/Stem Cells in Response to Ultraviolet-C (UV-C) Damage

The ocular surface is subjected to regular wear and tear due to various environmental factors. Exposure to UV-C radiation constitutes an occupational health hazard. Here, we demonstrate the exposure of primary stem cells from the mouse ocular surface to UV-C radiation. Reactive oxygen species (ROS) formation is the readout of the extent of oxidative stress/damage. In an experimental in vitro setting, it is also essential to assess the percentage of dead cells generated due to oxidative stress. In this article, we will demonstrate the 2&#39;,7&#39;-Dichlorofluoresceindiacetate (DCFDA) staining of UV-C exposed mouse primary ocular surface stem cells and their quantification based on the fluorescent images of DCFDA staining. DCFDA staining directly corresponds to ROS generation. We also demonstrate the quantification of dead and live cells by simultaneous staining with propidium iodide (PI) and Hoechst 3332 respectively and the percentage of DCFDA (ROS positive) and PI positive cells.

Assessment of Oxidative Damage in the Primary Mouse Ocular Surface Cells/Stem Cells in Response to Ultraviolet-C UV-C Damage

This protocol demonstrates the simultaneous detection of reactive oxygen species (ROS), live cells, and dead cells in live primary cultures from mouse ocular surface cells. 2&#39;,7&#39;-Dichlorofluoresceindiacetate, propidium iodide, and Hoechst staining are used to assess the ROS, dead cells, and live cells, respectively, followed by imaging and analysis.

The ocular surface is subjected to regular wear and tear due to various environmental factors. Exposure to UV-C radiation constitutes an occupational ...

Human Egg Maturity Assessment and Its Clinical Application

The optimal timing of intracytoplasmic sperm injection (ICSI) is of a serious concern for fertility programs because untimely sperm entry diminishes the egg&#39;s developmental competence. Presence of the first polar body (PB) together with the meiotic spindle indicates completion of the oocyte maturation and the egg&#39;s readiness for fertilization. In clinical practice, it is customary to assume that all oocytes displaying a PB are mature metaphase (MII) oocytes. However, PB extrusion precedes the formation of the bipolar MII spindle. This asynchrony makes the mere presence of PB an unreliable marker of oocyte maturity. Noninvasive spindle imaging using polarized light microscopy (PLM) allows quick and easy inspection of whether the PB-displaying oocyte actually reassembled a meiotic spindle prior to ICSI. Here, we present a standard protocol to perform human egg maturity assessment in the clinical laboratory. We also show how to optimize the time of ICSI with respect to the oocyte&#39;s developmental stage in order to prevent premature sperm injection of late-maturing oocytes. Using this approach, even immature oocytes extruding PB in vitro can be clinically utilized. Affirmation that MII spindle is present prior to sperm injection and individual adjustment of the time of ICSI is particularly important in poor prognosis in-vitro fertilization (IVF) cycles with a low number of oocytes available for fertilization.

We provide an outline of the clinical protocol for non-invasive assessment of human egg maturity using polarized light microscopy.

The optimal timing of intracytoplasmic sperm injection (ICSI) is of a serious concern for fertility programs because untimely sperm entry diminishes the ...