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Gene Expression Profiling of Rat Fetuses
Exposed to 2-Dimensional Ultrasound
ltrasound (US) is a crucial tool in clinical obstetrics and in
vitro fertilization (IVF) procedures. It allows assessment of
fetal anatomy, behavior, and function. Accordingly, diag-
nostic US use in obstetrics has been growing rapidly to become an
integral part of prenatal care today.1,2 Most epidemiologic studies
tend to support the safety of diagnostic US use during pregnancy.1,3
In a study by Stark et al,4a total of 425 children exposed to diag-
nostic US and 381 matched control children were studied for
adverse effects at birth and again at examination between 7 and 12
years of age. No biologically significant differences between exposed
and unexposed children were found. However, advances in US tech-
nology enabled development of high-power devices that are used
to scan the fetus, and increasingly, this procedure is performed early
in gestation, a time when the fetus is known to be particularly sen-
sitive to external influences.3,5,6 Current US technology has become
more powerful, and its safety profile is largely unknown.1,2 It has
been estimated that fetal exposure using modern equipment could be
up to 8 times greater than that with older US systems.3,7 Unfortu-
nately, most studies about US safety were conducted in the past, using
older equipment. Thus, there is a basis for concern, especially because
few studies have researched the bioeffects of modern US devices on
the molecular level.3Studies regarding US-tissue interactions have led
Zvonko Hocevar, MD, Janez Rozman, PhD, Alja Videtic Paska, PhD, Robert Frangez, PhD, DVM,
Tomaz Vaupotic, PhD, Petra Hudler, PhD
Received July 21, 2011, from the University Med-
ical Center Ljubljana, Ljubljana, Slovenia (Z.H.);
Center for Implantable Technology and Sensors,
Ljubljana, Slovenia (J.R.); Faculty of Medicine,
University of Ljubljana, Institute of Biochemistry,
Medical Center for Molecular Genetics, Ljubljana,
Slovenia (A.V.P., T.V., P.H.); and Veterinary Fac-
ulty, University of Ljubljana, Ljubljana, Slovenia
(R.F.). Revision requested August 29, 2011.
Revised manuscript accepted for publication
December 19, 2011.
This study was supported by the Ministry of
Higher Education and Science, Republic of Slovenia
(research project J3-0259).
Address correspondence to Petra Hudler,
PhD, Faculty of Medicine, University of Ljubljana,
Institute of Biochemistry, Medical Center for
Molecular Genetics, Vrazov Trg 2, 1000 Ljubljana,
Slovenia.
E-mail: petra.hudler@mf.uni-lj.si
Abbreviations
CNS, central nervous system; PCR, polymerase chain reaction;
RT, reverse transcription; US, ultrasound
U
©2012 by the American Institute of Ultrasound in Medicine |J Ultrasound Med 2012; 31:923–932 |0278-4297 |www.aium.org
ORIGINAL RESEARCH
Objectives—This study evaluated the possible effects of ultrasound (US) on gene
expression in brain tissue of rat embryos.
Methods—Four groups (n = 5 each) of pregnant Wistar Han rats were exposed to US
for different durations (55, 100, 145, and 195 seconds) via a multifrequency transducer
in the 2-dimensional imaging mode with a pulse duration of 1.29 microseconds, a pulse
repetition frequency of 1 kHz, and a derated spatial-peak pulse-average intensity of
222.4 W/cm2on day 5, 9, 7, or 13 of gestation. Gene expression profiling was performed
in fetal brain tissue (n = 5 per group) by quantitative reverse transcription–polymerase
chain reaction arrays.
Results—The results indicated substantial alterations in gene expression. The most
differentially expressed genes were Adamts5, Gadd45a, Npy2r, and Chrna1, which are
implicated in important developmental signaling pathways.
Conclusions—On the basis of our findings, routine short US examinations for moni-
toring fetal development are not contraindicated, but prolonged exposures should be
used only when needed to obtain important diagnostic information.
Key Words—central nervous system; fetal development; gene expression profiling;
safety; ultrasound
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to a better understanding of its mechanisms and side effects.
As a result, these studies revealed the nature of the undesired
side effects of US therapy and imaging, but only a handful of
them searched for aberrant gene expression patterns in fetal
tissues exposed to US.
In some older studies, biological effects of US were
reported in animals,6but very few harmful effects have
been shown in humans.4,8 For example, in a study using
monkeys as a model and testing older equipment, Taran-
tal and Hendrickx6found significant differences in birth
weight, crown-rump length, and white blood cell counts.
Although it is possible that these findings were chance
effects, it is also plausible that frequent exposure to US
could influence fetal growth.1,9–14
The main goal of our study was to examine whether
the exposure of pregnant rats to US influences the gene
expression in the central nervous system (CNS) of the
embryos. We identified differences in gene expression
related to different levels of US exposure. We wanted to
gather the differences in gene expression with a focus on
genes that encode the crucial molecular components of the
CNS, such as extracellular matrix and adhesion molecules,
components of signal transduction pathways, neuropep-
tides and their receptors, and neurotransmitters and their
receptors, using novel and pathway-focused gene expres-
sion profiling technology.
Materials and Methods
Preparation of Animals
All procedures and protocols were approved by the
Veterinary Administration of the Republic of Slovenia,
Ministry of Agriculture, Forestry, and Food (number
34401-44/2008/2). Forty-eight 11-week-old outbred
Wistar Han rats (HsdRccHan:WIST) were included in the
study. Rats were housed individually and provided with
food and water ad libitum. The mean weight of the animals
± SD at 11 weeks was 190 ± 20.87 g. The female animals
were mated with 6 male animals, and forty female ani-
mals became pregnant. An acrylic US exposure chamber
was specifically designed for experimental bioeffects stud-
ies. The rats were trained to use this chamber. Unanes-
thetized pregnant rats were held in position within the
exposure chamber, and the lateral surfaces of the chamber
were cut to allow exposure of the abdominal wall to the US
transducer (Figure 1). The animals were divided into 8
groups with 5 animals in each group. Four groups (U1–
U4) were exposed to US in the 2-dimensional imaging
mode at gestational days 5, 7, 9, and 13 (described in detail
below and in Table 1), and 4 groups (C1–C4) were not
exposed to US. The animals in the control groups were
handled the same way as the animals exposed to US regard-
ing other procedures, such as depilation of the abdomen
and restraint in the US exposure chamber.
Before US exposure, the pregnant rats’ abdomens
were shorn. Afterward, a chemical depilatory cream (Veet;
Reckitt Benckiser, Hull, Yorkshire, England) was used.
The cream was tested on a small patch of skin before appli-
cation to avoid allergic reactions to the ingredients in the
product. The cream and dissolved hair were removed with
a spatula and sponge.
Ultrasound Exposure
For US exposure, we selected a portable all-digital soft-
ware-controlled broadband multifrequency US imaging
system, which has been routinely used in human medical
clinics (M-Turbo; SonoSite, Inc, Bothell, WA) and an
HFL38x transducer (13–6 MHz, linear array, 6-cm scan
depth). The intensity of the acoustic signal was calibrated
as recommended by the manufacturer on the US machine
without the transducer; therefore, we note that the values
given in the text are those reported by the manufacturer
for this device, transducer, and imaging mode. The system
configurations were 2-dimensional/tissue imaging, small
parts, and auto gain automatic image optimization with a
scan depth of 6 cm. The mechanical index in all of our
scans was 0.71. The transducer operates in the multifre-
quency mode. We used a 2.5-cm tissue standoff pad
(spacer) between the transducer and animals, filled with
an acoustic gel (Figure 1) to enable secure contact with the
animal skin and eliminate air pockets between the trans-
ducer and the skin. Because the fetal rat brain is small, a
small spread of the US beam perpendicular to the scan
plane is sufficient to include the entire brain. A water bag
was acoustically coupled to the side of the rat opposite the
transducer to minimize the possibility of standing waves
or reflections that might have affected the exposure. Both
the left and right sides of the animals were exposed to equal
durations of US to ensure exposure in both uterine horns.
An actual range of acoustic intensities to which fetuses are
exposed was provided by the producer of the US machine.
Instead of measurements performed in a water bath (free-
field measurements), the engineers of the US machine
defined the acoustic intensities experimentally using their
own reference MicroMaxx system and HFL US trans-
ducer, replicating our experimental setup. The testing of
the US system showed that the derated spatial-peak pulse-
average intensity was 222.4 W/cm2, and the derated spa-
tial-peak temporal-average intensity was 13.64 mW/cm2.
The acoustic output power was 16.21 mW.
Hocevar et al—Gene Expression in Rat Fetuses Exposed to Ultrasound
J Ultrasound Med 2012; 31:923–932924
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The exposure schemes were as follows: the first group
of pregnant rats was exposed to diagnostic US in one ses-
sion for a total of 55 seconds (group U1) per side on ges-
tational day 5; group U2 was exposed to US for 100
seconds per side on gestational days 5 and 13; group 3 was
exposed to US for a total of 145 seconds per side on gesta-
tional days 5, 9, and 13; and group 4 was exposed to US for
a total of 195 seconds per side on gestational days 5, 7, 9,
and 13. Detailed data on US exposures per session are pre-
sented in Table 1. The sessions were performed on differ-
ent days to simulate human scanning conditions. Day 5
was chosen because in humans and rats, the blastula
attaches to the uterus lining (5–8 days after fertilization
in humans and 5 days after fertilization in rats). Days 7, 9,
and 13 were chosen because the rat embryo brain is most
sensitive to teratogenic influences during this period. The
embryos were monitored on the screen, and the sono-
grams were stored. Four groups of pregnant animals, des-
ignated C1–C4, were used as controls, one control group
for the US-exposed group of the corresponding number;
control animals were not exposed to US.
Euthanasia
Euthanasia of all animals was performed strictly by following
the “Recommendations for Euthanasia of Experimental Ani-
mals” (part 2, directive 86/609/EEC, number L358, ISSN
0378-6978). The animals from the treated and control
groups were euthanized with embutramide (T61; Merck
Animal Health, Summit, NJ) administered intraperitoneally
at the same day of pregnancy as shown in Table 1 to match
the day of gestation between these groups of animals.
Because embryos were to be removed, an increased amount
of embutramide (1 mL) was administered to the dam and
maintained for a longer period to ensure that the euthanasia
J Ultrasound Med 2012; 31:923–932 925
Hocevar et al—Gene Expression in Rat Fetuses Exposed to Ultrasound
Figure 1. a, Ultrasound wave transducer (HFL38x, 13–6 MHz). b, Ultrasound transducer with tissue spacer. c, Ultrasound transducer with tissue
spacer filled with acoustic gel. d, Chamber used for exposure of pregnant rats to ultrasound.
3106jumonline.qxp:Layout 1 5/22/12 3:32 PM Page 925
solution had crossed the placenta. Afterward, the animals
were placed in a box that was completely filled with inhala-
tional carbon dioxide from a commercially available gas
cylinder.15 Death was confirmed by extraction of the heart.
Extraction of Brain Tissue and Preparation of Samples
Immediately after euthanasia, a full-length median laparo-
tomy was performed in all groups of animals. The exposed
uterus with embryos was carefully extracted and opened.
The brains of the embryos were extracted using a micro-
surgical technique, stored in TRIzol reagent (Invitrogen,
Carlsbad, CA), and snap frozen in liquid nitrogen. The
frozen tissues were stored at –70°C. To preserve RNA in
brain tissue samples, which could be degraded by harmful
heat generated during microsurgical extraction by infrared
waves produced by the tungsten lamps used in ordinary
optic microscopes, white light-emitting diodes were used
for illumination. All carcasses were temporarily collected
and kept at –70°C until they were destroyed according to
valid internal regulations at the Veterinary Faculty of the
University of Ljubljana (V89, V155, and V171).
Biochemical Procedures for Quantitative Gene
Expression Profiling
RNA was isolated with TRIzol reagent according to the
manufacturer’s protocol. Briefly, 5 embryo brains (from 1
selected maternal animal) per group were homogenized
in 1 mL of TRIzol reagent. After the addition of 200 µL of
chloroform and centrifugation, the RNA was precipitated
with isopropanol and washed twice with ethanol. Air-dried
RNA was dissolved in 10 µL of RNase-free water (Invitro-
gen). RNA was then purified using an RNeasy Mini kit
(Qiagen, Valencia, CA). The 260:280 ratio of RNA was in
the range from 1.8 to 2.0. In each tested and control group,
RNA was isolated in duplicate (5 brains from 2 selected
maternal animals per group) to obtain biological duplicates.
Synthesis of complementary DNA was performed from 1 µg
of total RNA using an RT2first-strand kit (SABiosciences,
Valencia, CA) according to the manufacturer’s instructions.
Pathway-focused RT2Profiler quantitative reverse
transcription–polymerase chain reaction (RT-PCR)
arrays (SABiosciences) were used to determine gene
expression profiles. Each array represents 84 genes
involved in specific pathways, 5 internal controls/house-
keeping genes, and 3 controls for detecting DNA con-
tamination, RNA quality, and general PCR performance.
The following RT Profiler quantitative PCR arrays were
used: (1) rat extracellular matrix and adhesion molecule
array (PARN-013A-12), (2) rat signal transduction Path-
wayFinder array (PARN-014A-12), (3) rat neurotrophin
and receptor array (PARN-031A-12), and (4) rat neuro-
transmitter receptors and regulator array (PARN-060A-12).
Quantitative PCR was performed using the RT Profiler
arrays and RT SYBR Green/ROX Fast PCR MasterMix
(SABiosciences) in a real-time PCR 7500 instrument
(Applied Biosystems, Foster City, CA). Real-time PCR
signals were evaluated using SDS version 1.4 software
(Applied Biosystems). Melting curve analysis was performed
(1°C/s increases from 60°C to 95°C, with continuous fluo-
rescence readings) at the end of the run to ensure that single
PCR products were obtained. Gene expression was nor-
malized to 5 internal controls/housekeeping genes
prespotted on the RT Profiler arrays: (1) ribosomal pro-
tein P1, large subunit (MGC72935), (2) hypoxanthine
guanine phosphoribosyl transferase (Hprt1), (3) riboso-
mal protein L13A (Rpl13a), (4) lactate dehydrogenase A
(Ldh), and (5) β-actin (Actx), using the RT2Profiler array
data analysis software (SABiosciences). The relative
expression of each RNA was calculated by the compara-
tive Ct (Ct) method where Ct = Ct (tested group
gene) – Ct (control group gene). The fold change in
expression of the gene of interest between the two samples
is then equal to 2^(–Ct). The relative expression ratios
of the target genes normalized to internal controls and rel-
Hocevar et al—Gene Expression in Rat Fetuses Exposed to Ultrasound
J Ultrasound Med 2012; 31:923–932926
Table 1. Ultrasound Exposure Scheme for the Pregnant Wistar Han Rats
Time of Exposure per Session Day of
(Total Time of Day of Exposure Pregnancy at
Animal Group Animals, n Exposure Per Side), s (Day of Gestation) Euthanasia
U1 5 55 (55) 9 16
U2 5 55, 45 (100) 5, 13 17
U3 5 55, 45, 45 (145) 5, 9, 13 15
U4 5 55, 45, 45, 50 (195) 5, 7, 9, 13 14
C1 5 NA NA 16
C2 5 NA NA 17
C3 5 NA NA 15
C4 5 NA NA 14
C indicates control animals; NA, not applicable, and U, ultrasound-exposed animals.
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ative to expression of genes in the control groups are pre-
sented in Figures 2–5. This relative expression is reported
as fold regulation, which is the inverse of the fold change in
the case of upregulated genes and the negative inverse of
the fold change in the case of downregulated genes.
Results
We gathered qualitative and quantitative information on
differential gene expression in brains of rat embryos
exposed to US compared to control groups. We found sub-
stantial differences in expression of genes belonging to 4
functional gene groups, namely, (1) extracellular matrix and
adhesion molecules, (2) signal transduction pathways, (3)
neuropeptides and their receptors, and (4) neurotransmit-
ters and their receptors.
Altogether, 336 different genes belonging to 4 func-
tional groups of 84 genes were tested. A total of 64 genes
were assigned as differentially expressed, showing a greater
than 2-fold difference in their transcript levels in any of ani-
mal groups U1–U4. The 5 most differentially expressed
genes from each of the functional groups and their levels of
expression based on CNS-isolated RNA from embryos of
different US-treated animal groups (U1–U4) relative to
nontreated animal groups (C1–C4) are graphically pre-
sented in Figures 2–5. Genes were assigned their protein
products and physiological functions together with fold
regulation as detected by the quantitative PCR arrays.
Because of the fold regulation axis adjustment, the most
differentially expressed gene from each of the functional
groups is shown in a separate chart.
Among genes coding for extracellular matrix and
adhesion molecules (Figure 2) 45-fold upregulation was
observed for Adamts5 in the CNS of embryos extracted
from animal group U4, with the longest exposure to US.
In the CNS of embryos from groups U1, U2, and U3, it
was almost undetectable. Four other differentially expressed
genes, namely, Postn, Tgfbi, Thbs2, and Mmp1a, were all
found to be substantially downregulated on exposure to US,
with Postn and Thbs2 showing the strongest downregula-
tion in the CNS of embryos from group U3, Tgfbi showing
the strongest downregulation in the CNS of embryos from
groups U1 and U3, and Mmp1a showing the strongest
downregulation in the CNS of embryos from group U1.
J Ultrasound Med 2012; 31:923–932 927
Hocevar et al—Gene Expression in Rat Fetuses Exposed to Ultrasound
Figure 2. Differentially expressed genes coding for extracellular matrix and adhesion molecules (RT² Profiler quantitative polymerase chain reaction
array PARN-013A-12). The left chart represents the most differentially expressed gene, and the right chart represents other highly differentially
expressed genes in the group. U1a–U4a and U1b–U4b represent biological replicates.
3106jumonline.qxp:Layout 1 5/22/12 3:32 PM Page 927
Among genes coding for signal transduction path-
way molecules (Figure 3), the strongest upregulation, at
several hundred-fold, was observed for Gadd45a in the
CNS of embryos from group U3, whereas it was unde-
tectable in the CNS of embryos from groups U1, U2,
and U4. Approximately 7-fold upregulation was
observed for Birc1b in the CNS of embryos from groups
U3 and U4. Nos2, Tnf, and Cxcl1 were substantially
downregulated in groups U1, U2, and U4, respectively.
Among genes coding neuropeptides and their recep-
tors (Figure 4), the strongest upregulation was observed
for Npy2r in the CNS of embryos extracted from group
U2 and Maged1 in the CNS of embryos from group U4.
The strongest downregulation was observed for Gfra3 in
the CNS of embryos extracted from group U3. Bdnf was
most evidently downregulated in group U4. Ppyr1
showed a promiscuous profile of upregulation and down-
regulation between groups U1 and U2–U4.
Among genes coding neurotransmitters and their
receptors (Figure 5), most of the differentially expressed
genes were downregulated, with Chrna1, Chrnd, and Glra1
showing greater than 10-fold downregulation in the CNS
of embryos extracted from groups U3 and U4. Chat was
also substantially downregulated in group U4, whereas
Brs3 was evidently downregulated only in group U1.
Discussion
This study was intended to provide new insight for the
medical/scientific community regarding the effects of US
on the gene expression patterns in the CNS of rat fetuses.
More precisely, it was aimed at making experimental con-
tributions to the research on US effects at the molecular level.
Although it is generally assumed that prenatal US use
is safe, very few studies have focused on possible side effects
at the molecular level, and much data have been obtained
using older US machines with far less output potential.1,2,7,9
Hocevar et al—Gene Expression in Rat Fetuses Exposed to Ultrasound
J Ultrasound Med 2012; 31:923–932928
Figure 3. Differentially expressed genes coding for signal transduction molecules (RT² Profiler quantitative polymerase chain reaction array
PARN-014A-12). The left chart represents the most differentially expressed gene, and the right chart represents other highly differentially expressed
genes in the group. U1a–U4a and U1b–U4b represent biological replicates.
3106jumonline.qxp:Layout 1 5/22/12 3:32 PM Page 928
The introduction of Doppler US, the use of more power-
ful equipment in obstetrics, and the trend of increased
use of US for nonmedical purposes requires continued
scrutiny of the safety issues of US.1,7 Recently, Ang et al10
showed that exposure of embryonic mice to US could
affect neuronal migration in the cerebral cortex. They
determined that a small, but statistically significant,
number of neurons failed to reach their proper positions
and remained scattered within inappropriate cortical layers
or in the subjacent white matter. They also emphasized the
fact that the cellular and molecular mechanisms of this
effect are unknown.
In our study, we found different gene expression pro-
files in the brains of fetuses exposed to US. In the group of
extracellular genes and adhesion molecules, we observed
45-fold upregulation of Adamts5, which was found to be
implicated in the destruction of the cartilage proteoglycan
aggrecan in arthritis (Figure 2). Its expression profile during
embryogenesis was recently studied in mice and was found
to have a role in the development of nervous system, head,
and neck structures and limbs, although its exact physio-
logic functions remain unclear.11 Nevertheless, the extra-
cellular matrix is a network that provides a substrate for cell
anchorage, serves as a tissue scaffold, guides cell migration
during embryonic development and wound repair, and has
a key role in tissue morphogenesis. The extracellular matrix
is also responsible for modulating signal transduction path-
ways, which ultimately affects cell proliferation, differentia-
tion, and death. Other differentially expressed genes from
this group, Postn and Thbs2, belong to cell adhesion mole-
cules, which are involved in cell-cell and cell-extracellular
matrix binding. Thbs2, for example, was shown to be impli-
cated in the inhibition of angiogenesis and promotion of
CNS synaptogenesis.12,13 Christopherson et al12 suggested
that human homologs thrombospondin 1 and 2 (rat Thbs2)
could act as permissive switches that time CNS synapto-
genesis by enabling neuronal molecules to assemble into
synapses within a specific window of CNS development.
J Ultrasound Med 2012; 31:923–932 929
Hocevar et al—Gene Expression in Rat Fetuses Exposed to Ultrasound
Figure 4. Differentially expressed genes coding neuropeptides and their receptors (RT² Profiler quantitative polymerase chain reaction array
PARN-031A-12). The left chart represents the most differentially expressed gene, and the right chart represents other highly differentially expressed
genes in the group. U1a–U4a and U1b–U4b represent biological replicates.
3106jumonline.qxp:Layout 1 5/22/12 3:32 PM Page 929
Clearly, the disruption of these molecular mechanisms
could have prominent effects on developing fetuses.
Gadd45a probably induces DNA repair, and the
upregulation of the corresponding gene in our study could
indicate that US exposure caused DNA damage in rat
fetuses (Figure 3). Interestingly, Birc1b, which is impli-
cated in inhibition of apoptosis, was also upregulated in
our study. This finding contradicts the action of Gadd45a,
thereby indicating that US exposure could disrupt normal
signaling in cells. Cxcl1 plays a fundamental role in cell traf-
ficking of various leukocytes and development of immune
system and has also effects on cells of the CNS as well as
endothelial cells involved in angiogenesis or angiostasis.
Tsai et al14 showed a role for rodent Cxcl1 in patterning the
developing spinal cord, namely, signaling through Cxcl1
and Cxcr2 inhibited oligodendrocyte precursor migration.
Neuropeptides directly or indirectly modulate synap-
tic activity and may also function as primary neurotrans-
mitters. In our study, we detected abnormally upregulated
Npy2r, a neuropeptide receptor (Figure 4). Its role in
embryogenesis is unknown, although it was reported that
it could exert inhibition of cyclic adenosine monophosphate
production, an important second messenger of several sig-
naling pathways (Gene; National Center for Biotechnology
Information; http://www.ncbi.nlm.nih.gov/gene). We also
found downregulation of Gfra3, which promotes the sur-
vival and maintenance of different neuronal cell types and
could be responsible for cell survival in the inner ear.16 In
addition, we detected reduced levels of Bdnf, a member of
the nerve growth factor family. Among several other func-
tions, Bdnf is necessary for survival of striatal neurons, and
it was also shown that Bdnf could be effective in preserving
photoreceptors from the cell death that normally accom-
panies retinal degeneration.17,18 Because the bioeffects of
more powerful modern US devices have not been exten-
sively studied, it would be interesting to assess exposure
to modern US systems and vision in infants and children.
We also found upregulation of Maged1, which is involved in
Hocevar et al—Gene Expression in Rat Fetuses Exposed to Ultrasound
J Ultrasound Med 2012; 31:923–932930
Figure 5. Differentially expressed genes coding neurotransmitters and their receptors (RT² Profiler quantitative polymerase chain reaction array
PARN-060A-12). The left chart represents the most differentially expressed gene, and the right chart represents other highly differentially expressed
genes in the group. U1a–U4a and U1b–U4b represent biological replicates.
3106jumonline.qxp:Layout 1 5/22/12 3:32 PM Page 930
mediating nerve growth factor–dependent apoptosis.
Whole-mount in situ hybridization analysis showed low-
level expression of this gene throughout embryonic day 11.5
in rat embryos.19 In our study, its expression was 8-fold
higher in group U4, which was exposed to US 4 times
longer than in embryos that were not exposed to US.
These results could suggest impairment of neuronal devel-
opment in US-exposed embryos.
Among neurotransmitters, we found aberrant expres-
sion of Chrna1, which encodes the α subunit of the acetyl-
choline receptor that plays a role in acetylcholine binding
and channel gating (Figure 5). In a study by Scheffer et al,20
this gene was active in vestibular and cochlear hair cells
during early development of the inner ear innervations.
Although several studies have been performed in the past,
none associated US with impaired hearing.1,21 Interest-
ingly, Chrnd and Chat, the first gene coding another
subunit of the acetylcholine receptor and the second impli-
cated in biosynthesis of acetylcholine, were both underex-
pressed in fetal rat tissue exposed to US. Mutations in
Chrnd and Chrna1 and, consequently, impaired function
of their protein products are associated with congenital
myasthenic and multiple pterygium syndromes.22,23
Glra1 encodes the α1 subunit of the glycine receptor, a
ligand-gated chloride channel. This inhibitory glycine
receptor mediates postsynaptic inhibition in the spinal
cord and other regions of the CNS.24 Hirzel et al25 showed
that mutant mice with a homozygous deletion in this
gene had a phenotype that was similar to human hyper-
ekplexia, with increased neuromuscular tone, inducible
tremors, and an abnormal gait. In our study, this gene was
downregulated; thus, it could be implicated in abnormal
development of the CNS.
Our results show that US could probably affect expres-
sion of highly important genes implicated in embryonic
development. The exact mechanisms and the consequences
of this aberrant expression remain to be elucidated.
Our study also showed on the molecular level that US should
be used with caution, particularly in early pregnancy and in
vitro fertilization procedures. Repeated prenatal US imag-
ing and Doppler flow examinations should be restricted to
those women for whom the information is likely to be of
clinical benefit. Because there are no set rules for determin-
ing the correct exposure for every situation, it is reasonable
for the US user to use the ALARA (as low as reasonably
achievable) principle and follow the information concern-
ing the US procedures prudently.26 The qualified US user
should determine the most appropriate way to keep the
exposure low and bioeffects to a minimum while obtaining
a diagnostic examination. The nonmedical use of powerful
2-dimensional, Doppler, 3-dimensional, and 4-dimensional
US for keepsake imaging should be discouraged. Our results
show that assessment of the safety of diagnostic US should
be approached in two directions: by conducting long-term
epidemiologic studies of exposed infants and by studying the
interactions of US in tissues of animal models and attempt-
ing to extrapolate the results to clinical practice.
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