intrauterine effects of ultrasound: animal studies

Intrauterine Effects of Ultrasound:Animal StudiesRONALD P. JENSH1* AND ROBERT L. BRENT2

1Jefferson Medical College, Department of Pathology, Anatomy, and Cell Biology, Thomas Jefferson University,Philadelphia, Pennsylvania 191072Alfred I. duPont Hospital for Children, Wilmington, Delaware 19899

ABSTRACT During the past several decades,the use of ultrasound technology in the clinical settinghas greatly increased. Because nearly every pregnantwoman receives at least one sonographic proceduretoday, there has been developing concern about thesafety of such procedures. Since ultrasound exposurecan result in hyperthermia and other physiologicaleffects, the determination of a threshold or no-effectexposure has become a high-priority goal. Animalresearch has been important to the study of the effectsof various exposures at all stages of pregnancy, sincethe clinical use of ultrasonography can occur during thepreimplantation, organogenic, and fetal stages. Animalexperiments using various mammalian species havebeen able to determine no-effect exposure levels forembryonic loss, congenital malformations and neurobe-havioral effects. The preponderance of evidence fromthese studies indicates that, in the absence of a thermaleffect, ultrasonography represents no measurable riskwhen used at recommended intensity levels. Teratology59:240–251, 1999. r 1999 Wiley-Liss, Inc.

There is continuing interest about possible deleteri-ous embryonic and fetal effects of exposure to diagnos-tic levels of ultrasound because of its frequent utiliza-tion. Increased attention has also been placed onmethodology for adequate characterization of ultra-sound fields produced by the imaging equipment.

Ultrasound has been used clinically to visualizeinternal structures of the human body for severaldecades. With the increasing use of this diagnostic tool,there has been renewed interest concerning possibledeleterious effects of such exposure on the developingfetus. With rapidly growing improvements in instrumen-tation technology and the lack of clear-cut adverseeffects, its use has increased significantly, especially inthe application of fetal imaging. It is estimated that oneout of every two children born in the United Statestoday has been exposed to ultrasound prenatally (NIHReport, ’84). The rate in other countries, such as theUnited Kingdom, is fast approaching 100%. In addition,increased attention has been placed on the methodol-ogy for adequate characterization of the ultrasoundfields produced by the imaging equipment.

Animal studies were designed to address questionsconcerning possible deleterious effects of exposure to

ultrasound during pregnancy. Use of animal models isof importance since various possible confounding fac-tors could be controlled in such investigations. Experi-mental designs could address such factors as the stageof pregnancy during which exposure takes place, hyper-thermia, cavitation, restraint stress, and type andintensity of the ultrasound. Postnatal psychophysiologi-cal evaluations were also possible, permitting long-term follow-up and evaluation under fully controlledconditions.

Damage to the central nervous system (CNS) indeveloping and adult mammals after prenatal exposureto high-ultrasound intensities has been reviewed re-cently (Sikov, ’86a, ’86b; Brent, ’91; Miller and Ziskin,’89; Lewin and Goldberg, ’89; Ziskin, ’90; Thomeniusand Lewin, ’91). In many cases, the damage reportedmay have been attributable to subtle biochemical alter-ations which occur during critical periods of braintissue development in the fetus (Dunn and Fry, ’71; Huand Ulrich, ’76; Vernadakis and Weiner, ’74), resultingin lesions in the CNS (Borelli et al., ’81). Often, ob-served effects were due to hyperthermia, a well knownteratogen (Warkany, ’86; Brent, ’86; Arora et al., ’79;Borrelli et al., ’86; Chernoff et al., ’83; Edwards, ’68, ’86;Edwards and Webster, ’82; Germain et al., ’85; Shiota,’88; Skreb and Franz, ’63).

Postnatal behavioral effects of prenatal exposure toultrasound also have been observed in a number ofstudies (Coyle et al., ’76; Brown et al., ’79; Murai et al.,’74, ’75a, ’75b; Sikov et al., ’77; Tarantal and Hendrickx,’89a, ’89b; Norton et al., ’91). Divergent results mayhave been due to differing methodologies or, in someinstances, monitoring techniques that may not havecompletely portrayed exposure indices.

Behavioral alterations may be the most sensitiveindicators of teratogenic activity (Vernadakis andWeiner, ’74; Spyker, ’75a, ’75b; Butcher et al., ’75; Coyleet al., ’76; Kameyama, ’77). It has been shown thatpostnatal functional evaluation can be a more sensitiveindicator of teratologic effects than physical malforma-tions (Butcher et al., ’75; Kameyama, ’77). Alteration inthe normal sequence of attainment of reflexes may

*Correspondence to: Ronald P. Jensh, Department of Pathology,Anatomy, and Cell Biology, JAH562: Thomas Jefferson University,1020 Locust Street, Philadelphia, PA 19107-6799.

TERATOLOGY 59:240–251 (1999)

r 1999 WILEY-LISS, INC.

indicate a corresponding alteration in the normal pat-terns of development and maturation of various parts ofthe brain and body. Although brain development maydiffer among mammalian species, the same basic devel-opmental stages of brain development and sequencingoccurs (Goldman, ’76; Langman et al., ’75; Davidsonand Dobbing, ’68). Bayer (’89) has stated that thedevelopment of the nervous system is highly dependentupon specific temporal sequences of neurogenic eventsrequiring precise timing, and any insult which altersthat timing may lead to permanent changes in theadult central nervous system. In relating test results inanimals to human behavior, Kimler and Norton (’88)have stated that ‘‘if those behavioral tests in rats thatmeasure aspects such as sensorimotor coordination anda response to a novel environment involve complexcerebral functioning, then extrapolation to similar cen-tral processing functions in humans may be allowed.’’Interspecies behavioral and morphologic comparisonshave been made previously (Jensh, ’83 and ’86), and ithas been shown that structural changes are oftenmanifested as behavioral alterations later in life. Post-natal behavioral tests are sensitive, noninvasive mea-sures of prenatal exposure, although their predictivevalidity for human behavioral changes has not beendefinitively determined.

REVIEWS

Smyth (’66) reviewed animal toxicity studies in whichdiagnostic levels of ultrasound were used. The hazardsof exposure of mice to diagnostic ultrasonography werereviewed by Warwick et al. (’70); Curzen (’72) discussedthe safety of exposure to diagnostic ultrasound. Leopoldand Asher (’74) discussed the use of ultrasound inobstetrics and gynecology. Thomenius and Lewin (’91)have published a review of the bioeffects of exposure toultrasound.

There are a number of reviews of the effects ofultrasound (Curzen, ’72; Brent ’84, ’86, ’94; Miller andZiskin, ’89; Brent et al., ’91, ’92). O’Brien (’86) explainsthe various factors and physical phenomena related todiagnostic ultrasound, including discussion on classifi-cation, acoustic and mechanical energy and cavitationeffects, while Miller (’91) reviews the safety of ultra-sound exposure, AIUM recommendations, and the lackof biologic effects due to exposure at diagnostic levels inwomen. The results of postnatal evaluations of childrenexposed prenatally to diagnostic ultrasound indicate nosignificant detrimental effects, although one study didreport a slight, but statistically significant, decrease inbirth weight (Falus et al., ’72; Moore et al., ’82; Stark etal., ’84). The recommendations of a National Institutesof Health Consensus Panel (’84) recommended thatdiagnostic ultrasound be used only when there werespecific indicators that it would be in the best interestsof the patient.

PHYSICAL FACTORS RELATEDTO INTRAUTERINE EXPOSURE

TO ULTRASOUND PHYSICS

Several papers have reviewed the physics related toultrasonography (O’Brien, ’86; Lewin and Goldberg,’89; MacDonald and Madsen, ’92). It is essential thatpublications concerning the biological effects of ultra-sound include a sufficient description of the physicalparameters of the ultrasound exposure to allow thereader to properly compare results from different inves-tigations. Parameters that should be included are thenature of the ultrasound (continuous or pulsed wave—ifa pulsed wave, the pulse duration and PRF), theintensity (e.g., Isptp, Istpa, Im), and the durations ofexposure and, in the case of reproductive toxicology, thetime during pregnancy during which the exposure tookplace.

RESTRAINT

Restraint stress could be a confounding variable,since it is a well-known teratogen (Rosenzweig andBlaustein, ’70; Kholkute and Udupa, ’78; Weinstock etal., ’88). There is no evidence, however, that restraint ofanesthetically induced unconscious mothers causes sig-nificant alterations in pregnancy outcome or postnatalbehavior.

Thermal Effects

Hyperthermia has been a well-known teratogen foralmost 50 years. Hsu (’48) demonstrated that elevationof body temperature during pregnancy resulted inincreased embryolethality, and Fernandez-Cano (’58)and Skreb and Franz (’63) showed that hyperthermiaalso resulted in an increase in the rate of abnormalitiesobserved at term in rats from body temperatureselevated by as little as 2° to 3°C for 1 hr.

By 1968 more studies had been completed regardingthe relationship between time of exposure during preg-nancy, level of temperature elevation, and duration ofhyperthermia. Lethality, abnormalities of various or-gan systems, and growth retardation were commonlyobserved (Edwards, ’68, ’86; Alexander and Williams,’71;Arora et al., ’79; Edwards and Webster, ’82; Lehmannand DeLateur, ’82; Chernoff et al., ’83; Germain et al.,’85; Finnell et al., ’86; Shiota, ’88).

Several reviews of the effects of hyperthermia ondevelopment among species have been published. Ed-wards (’86) discussed the clinical significance of animalstudies, while Smith et al. (’92) compared the results ofguinea pig studies with human data. In 1986 anin-depth review was published by Brent. He concludedthat a rise of at least 2.5°C could result in a significantincrease in malformations, dependent on the stage ofdevelopment during which exposure occurred and thelength of time of exposure. Warkany (’86) has statedthat ‘‘hyperthermia has been shown to be teratogenic inall species in which it has been tested systematically(chickens, rats, in vivo and in vitro, mice, rabbits,

INTRAUTERINE EFFECTS OF ULTRASOUND 241

guinea pigs, sheep, pigs, and New and Old Worldmonkeys).’’ Miller and Ziskin (’89; also Ziskin, ’91)stated that the higher the temperature and the longerthe exposure, the greater the risk of a biologic effect, buttemperature increases of less than 2°C would not resultin any adverse sequelae.

One of the best understood interactions of ultrasoundand biological tissue is the production of heat (Fry, etal., ’70; Fry et al., ’78). Several studies have beencompleted in which ultrasound was used as a method ofelevating body temperature to induce developmentalchanges. Carnes et al., ’91, demonstrated alterations intesticular cellular structure due to prenatal exposure toultrasound at levels which were known to producehyperthermia. Hartley et al. (’74) observed alterationsin central nervous system development in ewes, andLele (’75) observed abnormalities in mice. Previously, in’63, Lele had shown that peripheral nerves could bedestroyed with ultrasound and postulated that theresults were heat induced. This phenomenon is mostlikely to occur during ultrasound applications of high-intensity, uniform, continuous wave (cw) for a consider-able period of time. While useful in therapeutic ultra-sound applications, heat production is undesirable indiagnostic applications of ultrasound. This is especiallytrue during fetal imaging, since extreme temperatureelevation is known to be a teratogenic agent (Lewin andGoldberg, ’89; Thomenius and Lewin, ’91).

The Bioeffects Committee of the AIUM (’87) hasstated that fetal exposures from an in-situ temperaturerise to $41°C is considered hazardous. The committeealso recommended that diagnostic exposures shouldnot result in a temperature rise of .1°C above normalphysiological levels. In general, diagnostic pulse echoultrasound equipment used presently produces intensi-ties which are unlikely to cause a significant increase inthe temperature of exposed tissue (Nyborg, ’85). How-ever, there is growing concern about thermal bioeffectswhich may result when using pulsed wave Doppler in afetal examination. It has been demonstrated that theuse of equipment intended for nonfetal purposes, e.g.,cardiac applications, may increase the temperature ofthe fetus to surprisingly high-values in a worst-casesetting (Thomenius and Lewin, ’91). Should these tem-peratures be maintained for sufficiently long periods oftime, the possibility of bioeffects must be considered(Miller and Ziskin, ’89).

Cavitation

Another mechanism of interaction between biologicalmaterial and ultrasound is ascribed to cavitation. Forthis to occur during ultrasound exposure, gas nucleimust be present in the form of small bubbles or pocketswith dimensions of microns or smaller. Two types ofcavitation are found to exist: stable and transient.Stable cavitation is described as volume oscillation, orvibration of gas bubbles (NCRP, ’84). These vibratinggaseous bodies may lead to microstreaming in theliquid-like media adjacent to the bubbles. Microstream-

ing may then produce mechanical stresses sufficient todisrupt the cell membrane (Nyborg, ’79; Lewin andBjorno, ’82).

Until recently, it was believed that diagnostic ultra-sound pulses are too short to produce cavitation. Recentstudies have suggested that the microsecond-lengthpulses of ultrasound can cause transient cavitation(Flynn, ’82). While this type of cavitation was observedto be produced by pulsed ultrasound in vitro(Carstensen, ’82; Carstensen and Flynn, ’82), it re-mains to be seen whether it can be produced at diagnos-tic levels in vivo. There is no experimental evidencethat transient cavitation occurs in vivo at diagnosticultrasound levels.

It had been considered unlikely that stable cavitationcould occur in tissue. However, a phenomenon whichresembled stable cavitation was observed in tissueafter exposure to continuous wave (cw) ultrasound (terHar and Daniels, ’81). Although the intensity levels(approximately 150 mW/cm2 intensity (Isata) used inthis experiment were comparable to those generated bysome cw Doppler devices, the frequency used in thisexperiment (0.75 MHz) is not employed in diagnosticultrasound instrumentation. Also, the exposure timewas significantly longer than a typical diagnostic ultra-sound examination.

Transient cavitation is a more violent activity inwhich bubbles collapse during ultrasound exposure.This collapse is accompanied by localized shock wavesand generation of highly reactive chemical species; freeradicals. Production of free radicals such as the hy-droxyl (OH2) and hydrogen (H1) radicals may causesignificant cytotoxicity.

Diagnostic ultrasound pulses were believed to be tooshort to produce cavitation. However, recent evidencestrongly suggests that cavitation in mammalian tissuecannot be ruled out in clinically relevant pulsed ultra-sound conditions. Well defined, reproducible thresholdsfor lung hemorrhage in mice at pulsed ultrasoundpressures on the order of 1–2 MPa have been reported(Child et al., ’90). Since the results were observed in theabsence of excessive hyperthermia, they provide astrong suggestion that cavitation in vivo may occur atlevels encountered in clinical diagnostic instrumenta-tion. On the other hand, studies of the effect of pulsedultrasound on mouse kidney have indicated the prob-ability of a cavitation-like phenomenon in kidney tissueis extremely low, even at the highest diagnostic levels(Carstensen et al., ’90). Therefore, the currently avail-able data indicate that occurrence of cavitation intissues, with the exception of lung, is indeed veryunlikely.

Other Factors

A number of factors involved in research protocolmust be considered when assessing the possible effectsof ultrasound. Confounding factors include maternaldiseases, hereditary diseases and exposure to reproduc-tive toxicants. Maternal toxicity and nutritional defi-

242 R.P. JENSH AND R.L. BRENT

ciencies may affect the outcome of a reproductive study.Manipulation of the pregnant animal may also affectthe pregnancy. This factor is of special concern sincetechniques such as immersion of the pregnant female inwater or other specialized methodologies necessary toestablish and maintain accurate ultrasonic exposuremay also affect the fetus. As early as ’68, Nyborgemphasized that the quantitative aspects of ultrasoundare very important, particularly when considering itseffects on the developing organism.

It is essential, that these factors be considered in thedesign of the experimental protocol. A full description ofthe methodology, specifically addressing these factors,must be included in the publication of such experi-ments.

TERATOLOGIC EFFECTS

A review of the literature indicates a number ofdeficiencies in animal studies which evaluated thebiological effects of ultrasound. The primary deficiencyconcerns insufficient information relating to ultra-sound exposure conditions and the lack of precise andaccurate dosimetry techniques. A number of studies aresummarized in Table 1.

Tachibana et al. (’77) discussed continuous wave (cw)ultrasound intensity levels needed to produce specificteratogenic effects and the relationship between inten-sity level and length of time of exposure. O’Brien (’76,’83; O’Brien et al., ’82) observed the effect of geneticvariation in animals. Ultrasound exposure producedvarying results, depending on the strain of mouse beingexposed and the intensity of exposure.

Many of the animal studies relating to morphologiceffects of ultrasound used exposure levels well beyondthe diagnostic range and/or were given at a time inpregnancy when such exposure would not be clinicallyrelevant. Akamatsu (’81) exposed preimplantation ratembryos to cw ultrasound but attributed the observedexposure-related effects to hyperthermal activity.

Most of the previous studies of the effects of ultra-sound on behavior were performed using cw ultra-sound. Murai et al. (’75a) exposed pregnant rats to cwultrasound with a spatial average, temporal averageintensity of 20 mW/cm2 for a total of 300 min. Resultsshowed some possible delay in the development of theneuromotor reflex in the offspring. A similar studyperformed by the same group under the same exposureconditions resulted in altered emotional behavior in theadult rat (Murai et al., ’75b). However, the prenatal ageduring which the animals were exposed was not given,and the study did not control for possible effects ofrestraint on the offspring.

Takeuchi et al. (’70) were unable to detect anyexposure-related changes in mortality or abnormalityrates in rats due to a 20-min exposure on the third andfourth days of rat pregnancy to 2 MHz ultrasound at aspatial average intensity of 0.15 W/cm2. Shoji et al.(’71), however, observed increases in mortality andskeletal abnormalities in mice exposed to low intensity

(40 mW/cm2) 2.25 MHz ultrasound for 5 hr on theeighth day of gestation.

In a series of bioeffects experiments undertaken byBrown et al. (’81) postnatal behavioral developmentwas evaluated in mice exposed in utero on day 9 ofgestation at intensities (Isata) of 50–500 mW/cm2 for 3min. Results varied, showing both accelerated andretarded development of walking, righting, pivoting,and eye opening but, although the trends were interest-ing, they were not statistically significant.

Sikov et al. (’77) exposed rats on day 15 of postconcep-tion to a range of intensities (10 mW/cm2 to 1.0 W/cm2;0.93 MHz, cw) and observed a dose-related increase inmortality. They also observed postnatal developmentaldelays in righting and head lifting reflex acquisition atthe higher dosage levels but did not observe anyincrease in postnatal mortality or alterations in growth.They concluded that there were no permanent deficitsand the threshold for the observed alteration must bebelow 10 mW/cm2.

Sikov and colleagues exposed rats to a variety ofintensity levels from 0 to 32.4 W/cm2 on day 9 ofpregnancy for 5–15 min (Sikov and Hildebrand, ’76;Sikov et al., ’76). A few malformed fetuses resulted fromintensity levels greater than 10 W/cm2, and they deter-mined that there is an apparent threshold at 3 W/cm2

with an LD50 at about 17.6 W/cm2. Stolzenberg et al.(’80a, ’80b, ’80c) observed some effects due to ultra-sound exposure during the first 3 days of mouse preg-nancy when the duration of the exposure (2 MHz;Isata 5 1.0 W/cm2) exceeded 100 seconds. They attrib-uted the observed effects to be thermally induced and/orsecondarily related to maternal effects. Sikov and Pap-pas (’86) exposed rats on days 9, 10, 12, or 15 postconcep-tion to 0.8 MHz cw ultrasound at intensity levels of 0 to20 W/cm2, Isata. Embryolethality rates were similar forexposures on any of the first three days but less so fromexposure on day 15. They also observed a difference inthe type of malformations observed, with cardiovascu-lar and CNS malformations corresponding to earlierexposure times and skeletal and limb defects to latertimes. Kimmel et al. (’83, ’89) did not observe significantteratogenic activity in mice on day 17 due to ultrasoundexposure (1.0 MHz, cw) on day 8 of gestation for 10 minat intensities of 0–1.0 W/cm2, Isata (see below).

A number of studies have shown that fetal exposureto ultrasound can cause alterations in pregnancy out-come, while others have not observed significant expo-sure-induced changes. O’Brien and Stratmeyer (’75)observed significant postnatal weight reduction at 8weeks in mice exposed to ultrasound for 2 min on day 13of postconception. McClain et al. (’72) exposed rats to 10mW/cm2 (Isata), 2.5 MHz cw for 30 min or 2 hr on days8–13 or 11–13 and examined the fetuses on day 20 ofgestation. They did not observe any significant changesin mortality or abnormality rates.

Takabayashi et al. (’81) exposed mice prenatally topulsed ultrasound (2 MHz; Isata 5 500 mW/cm2; PRF 5150–1,000 Hz), inducing a significant increase in fetal


abnormalities, but no such increase was reported in areplicated study (Child et al., ’88). Studies of 2 MHzpulsed ultrasound (Isata 5 500 mW/cm2) in hamstersrevealed no adverse effects (Shimizu and Tanaka, ’81).

In ’89, Fry compared the effects of pulsed versus cwexposure of 8-day pregnant mice using 1 MHz ultra-sound (cw: 100., 12 W/cm22 Ispta pulsed: 450 W/cm22

Isptp, 27-µsec duration, PRF 5 1 KHz). There was nodifference between the two types of ultrasound whenthe same average intensities were compared. Maternalmortality was elevated due to hyperthermia (43%,pulsed; 19%, continuous wave).

In a series of studies, Hande (Hande and Devi, ’92,’93; Hande et al., ’93) exposed mice to 3.5-MHz ultra-sound (Isptp 5 1 W/cm2) for 10 min on days 3.5, 6.5 or11.5 of pregnancy and examined the fetuses at term andoffspring several months later for neurobehavioral defi-cits. Although they reported statistically significantalterations in some parameters at term, the changeswere minimal and were not biologically significant.

Vorhees et al. (’91) (see also Fisher et al., ’94) used aunique approach to study possible teratogenic effects ofultrasound exposure. Rats were trained to remainimmobile while exposed to ultrasound, thus obviatingthe need for anesthesia or possible complications result-ing from forced restraint. The animals were exposed to0.1, 2.0, or 30.0 W/cm2 (Ispta), 3.0 MHz cw, for about 15min per day on days 4–19 of gestation. There were nodose-dependent changes in a variety of maternal param-eters, the incidence of malformations, or fetal weights.Vorhees et al. (’94) also exposed rats to these levels ofultrasound daily for 10 min on gestational days 4–20.Although they did not observe exposure-related alter-ations in maternal parameters, offspring survival andgrowth, or neonatal psychophysiologic parameters,changes did occur in offspring adult behavior in locomo-tor activity and in two measures of multiple-T watermaze test performance at the highest dosage level. In asimilar study (Fisher et al., ’96) they concluded thatneurobehavioral development was unaltered if the ultra-sound intensity levels (Ispta) did not exceed 30 W/cm2.

Norton et al. (’90) did observe histologic changes 24hr after exposure of day 15 rat fetuses to 2.5-MHzpulsed ultrasound at an intensity level of 0.78 W/cm2

(Ispta) with a PRR of 50 KHz for 30 min. Average peaktemperature rose to 40.1°C. They observed an increasein the number of pyknotic cells and macrophages and adecrease in mitotic figures in the telencephalon. Nortonand colleagues also observed changes in several neona-tal and adult psychophysiologic parameters (Norton etal., ’91). Specifically, they observed alterations in theattainment of reflex suspension and negative geotaxis.They stated it was possible the results might have beendue entirely or in part to thermal activity.

Tarantal and Hendrickx (’89a; ’89b) exposed cynomol-gus macaques five times weekly to 7.5 MHz ultrasound(Ispta 5 12 mW/cm2) on days 21–35, three times perweek on days 36–60, and once a week on days 61–150 ofpregnancy. Most of the numerous morphologic param-

eters measured did not change significantly, but therewere alterations in birthweight, crown-rump length,and white blood cell counts. There were no exposure-induced changes in behavior, but a significant increasein muscle tone was observed among the exposed off-spring.

INVESTIGATIONS IN OURLABORATORY: PURPOSE

The overall aim of our investigations has been toexamine the effects of ultrasound exposure on thecentral nervous system (CNS) of the developing mam-mal, using highly reproducible, calibrated ultrasounddosimetry procedures; specifically to: (1) study theultrasound bioeffects studies at the 5.0-MHz (Jensh etal., ’89, ’89b, ’90, ’92, ’94, ’95), 3.5-MHz pulse echo and2.0 MHz cw Doppler frequencies (Jensh et al., a,b, inpreparation); (2) determine whether prenatal insonifica-tion of rats to ultrasound would result in alterations inpostnatal growth and neurophysiologic developmentusing well established behavioral testing procedures;(3) determine whether alterations in neonatal reflexdevelopment and adult behavior and growth resultfrom exposure to ultrasound; and (4) establish bioeffectthresholds, if any, for obstetrical Doppler applications.The selected exposure conditions were representativeof clinical practice, including pulse echo imaging and cwDoppler devices. The number of ultrasound field param-eters were carefully selected to complete the study andto comply with requirements for statistical significance.

PROCEDURES

An ultrasound chamber was constructed, and appro-priate computer programs and hardware for controllingexposure were developed. The system was capable ofdelivering highly controlled exposure dosages. Rel-evant parameters included the spatial peak temporalpeak intensity (Isptp), the spatial peak pulse averageintensity (Isppa), the spatial peak temporal averageintensity (Ispta) and the maximum intensity (Im). Inaddition, the system allowed total acoustic power emit-ted and beam profile distribution to be determined. Ananimal holding system was designed that allowed theanimal to be scanned or moved in the ultrasound field,mimicking clinical exposure conditions and avoiding astationary, prolonged insonification. The animal wasmoved in X and Y planes and software was developed tocontrol the movement of the animal within the insonifi-cation field. The acoustic output acquisition system wasdesigned to adhere to the latest measurement require-ments as defined by Food and Drug AdministrationGuidelines and AIUM/NEMA Draft Standards. Thefacility provided both carefully controlled dosages ofultrasound and a highly accurate means of measuringthe exact dosage levels and relevant ultrasound param-eters. Precise determination of the acoustic field distri-bution both immediately in front of and behind theexposed animal, using wideband PVDF polymer hydro-


TABLE 1. Reproductive effects of experimental exposures of pregnant mammals to ultrasound*

InvestigatorEffect

evaluated ExposureTime of

exposureAnimalspecies

Effectobserved

Weinland, ’63 Embryopathol-ogy

CW500 mW/cm2 SATA

2 min Hamster Malformations observed

Fry et al., ’78 Embryopathol-ogy

50 W/cm2 SPPA 20 seconds Mouse IUGR

Warwick et al., ’70 Embryopathol-ogy

27 W/cm2 P, SATA 24 seconds Mouse No IUGR; no fetalabnormalities

Kimmel et al., ’83 Teratogenesis 1 MHz0, 0.05, 0.51 W/cm2

ISATA

2 min Mouse day 8 No significant terato-genic effect

Stolzenberg et al.,’80a,b

Reproductiveeffects of acuteexposures

2 MHz1 W/cm2, CWSATA

80–400 sec-onds

Mouse exposedat 5 stages ofgestation

No effect at ,100 secexposure; lethaleffects above 100 sec

40–180 sec-onds

Mouse exposedday 8

Temperature rise to40°C and lethaleffects on embryo andgrowth retardation

Mouse on day 8of gestation

IUGR threshold at 0.5W/cm2 for 140 sec

1 W/cm2 for 60 secTachibana et al.,

’77Embryopathic

effectsCW80 mW/cm2 SATA

8 min Mouse IUGR

1000 mW/cm2 2 min Mouse CNS and abdominalabnormalities

1400 mW/cm2 CWSATA

5 min Mouse fetalabnormalities

Rugh and Mac-manaway, ’77

Embryonic andfetal effects

CW2000 mW/cm2

SATA

3 min Mouse Fetal malformations

Shimizu, ’77;Tanaka andShimzu, ’79


1400 mW/cm2 CWSATA

5 min Mouse Fetal malformations

O’Brien, ’76, ’83 Embryonic andfetal effects

CW500 mW/cm2

SATA

10 sec to 3 min Mouse IUGR

Muranaka et al.,’74


CW100 mW/cm2

SATA

4 min Mouse IUGR and elevated fetalmortality

Stratmeyer et al.,’79, ’81


CW75 mW/cm2

SATA

2 min Mouse IUGR

Kim et al., ’83 Ultrasoundexposure com-bined withproteinrestriction

1 MHz2.5 W/cm2 spatial

peak

20 sec Mouse exposedon day 8 ofgestation

IUGR in proteindeprived motherswere worsened byultrasound

Mannor et al., ’72 Embryopathol-ogy

1000 mW/cm2

CW10 min Mouse IUGR

Takabayashi etal., ’79, ’81

Embryopathol-ogy

586 mW/cm2

SATA pulsed5 min Mouse No IUGR

Embryopathol-ogy

Pulsed58.6 W/cm2; 296 m

W/cm2

59.4 W/cm2

TP intensity

5 min MouseMouse

Fetal abnormalitiesFetal abnormalities

Takabayashi etal., ’85

Embryoniceffects

Variations in peakintensity andpulse width

1 MHz 0.058–0.586W/cm2 SAI

5.8–59.4-W/cm2

peak intensity

5 min C3H/He mouse8th-daymouse expo-sure

Teratogenicity maydepend on both peakintensity and pulsewidth. Thresholdintensities on the 8thday were 56.3 W/cm2

(pulsed) SATP and 1.2W/cm2 SPTA. Bothpulse with and peakintensity were impor-tant

TABLE 1 continues on next page


TABLE 1. Reproductive effects of experimental exposures of pregnant mammals to ultrasound (continued)

InvestigatorEffect



Effectobserved

Shoji et al., ’75 Embryoniceffects

2.25 MHzcw; 4 mW/cm2

5 hr Mice combina-tion ofrestraint andexposure

A/HeMK mice had anincrease in malforma-tions, but DHS didnot

Kimmel et al., ’89 Embryonic andfetal effects,maternalweight gain,litter size mal-formation ratemean fetalweight

1 MHz pulsed, 6.5µsec 4 groups

0–1 W/cm2 SATA;90 W/cm2 SPPA

Two 10-minintervals

ICR Mice 8thgestationalday

No developmentaleffects at any expo-sure, even at expo-sures that were lethalsome of the mothers,75 litters per group

Pizzarello et al.,’78

1.5 mWcm2 pulsedSATA

5 min Rat IUGR

Child et al., ’84 Growth Pulsed12 mW/cm2

14 W/cm2, SATATemporal peak

intensity

5 min Rat No growth retardation;replicated

Pizzarello et al. studyand could not confirmresults

Akamatsu, ’81Akamatsu et al.,

’77a,b,c, ’81

Preimplantationembryo-morula andblastocyststage, tem-peraturemaintained at37°C, in vitro

2 MHz CW0.4–8.8 W/cm2

pulsed 1 mHz at#220 W/cm2

SpTp

5–720 min Rat Abnormal effects(degeneration)observed above 3W/cm2 even at 37°C(CW); no effects atany dose of pulsedultrasound, non-thermal effects

Hara et al., ’77a,b,’80


Pulsed600 W mW/cm2

SATA22 W/cm2; TP inten-

sity2,000 mW/cm2

CW, SATA

5 min Rat Reduced maternalweight; fetal abnor-malities

Sikov and Hilde-brand, ’76, ’79;Sikov et al., ’83


500 W/cm2 pulsedSATA50 w/cm2

TPI

5 min Rat Fetal abnormalities

3,000 mW/cm2

CW5 min Rat Threshold for fetal

abnormalities andprenatal death

2,350 mW/cm2

CW5 min15 min

Rat 15-min exposure wassignificantly moreembryocidal

Sikov et al., ’76,’79

Postnatal neu-robehavioraleffects

CW 5 min Rat Developmental delay ofneuromuscular devel-opment

Sikov andPappas, ’86

Malformationsat differentgestationalstages

0.8 MHz CS1–20 W/cm2 SATA

5 min Rat gestationaldays 9, 10, 12,15 transducerplaced againstembryonic siteafter exposinguterine horn

Minimal effects seen in1–5 W/cm2 range;increase fetal mor-tality in the 20 W/cm2

groups; type of mal-formation in highdose groups dependson stage of gestation

Norton et al., ’90 Embryopathiceffects

0.78 W/cm2 SPTA2.5 MHz

30 min Rat: exposed onday 15

Temperature rise to40.1°C; histologicalevidence of damage tofetal cortex

Jensh, et al.,’89a,b, ’90

PostnatalNeurophysi-

ological effects

5 mHz P0.170 µsec pulse

duration500-W/cm2 ISPTP24-mW/cm2 ISPTA

and second groupat 1,500 W/cm2

ISPTP

35 min Rat exposed on15th, 17th,19th days ofgestation

No temperature eleva-tion in tissue; no neu-rophysiologicalchanges observed

TABLE 1 continues on next page


phones, provided a direct measure of the amount ofultrasound power absorbed by the animal. Body tem-perature was monitored closely. Body ultrasound absorp-tion was also monitored to determine approximatelyhow much of the ultrasound exposure was reaching thefetus. This approach aimed at closely mimicking clini-cal conditions regarding intensity, frequency, and expo-sure time (Lewin, ’81; Lewin et al., ’87a, ’87b, ’87c;Pozcobutt et al., ’87, ’88; Schafer and Lewin, ’88).

Temperature monitoring was performed on severalreference rats to determine if any significant tempera-ture increase occurred as a result of the ultrasoundexposure. A significant temperature rise would be con-sidered if greater than 1°C elevation above the normalintrauterine temperature (AIUM, ’87). Monitoring wasnot performed during exposure of experimental off-spring and subsequent postnatal evaluation since themanipulation, itself, might contribute to postnatal ef-fects. Since a rat’s internal temperature can varybetween 34°C and 39°C, the curve showing the tempera-ture rise due to ultrasound exposure can only becompared with the stabilized temperature curve forthat particular rat. The greatest temperature increaseobserved at the 2.0 MHz, 30 mW/cm2 (Ispta) exposurewas 0.6°C. This can most likely be attributed to the factthat the 2.0-MHz exposure lasted 2 hr, while the 3.5MHz and 5.0-MHz exposures only lasted 35 min. Re-sults indicated no significant temperature increase.Therefore, if any significant alteration in postnatal

development was observed, the possibility of it beingcaused by the thermal mechanism was negligible.

The protocol consisted of exposure of pregnant rats toone of two pulse-echo frequencies (3.5 or 5.0 MHz) atone of three spatial peak-temporal peak (Isptp) inten-sity levels (500, 1,500, 5,000 W/cm2) for 35 min on days15, 17, and 19 of postconception in the rat. One cwDoppler frequency (2.0-MHz) exposure was tested at aspatial peak temporal average (Ispta) of 30 mW/cm2 for30 min or 2 hr on the same days of pregnancy.

Days 15, 17, and 19 of postconception were chosen, asthey correspond to a critical period in the developmentof portions of the CNS and, therefore, may be sensitiveto possible ultrasound disruption (Dunn and Fry, ’71;Vernadakis and Weiner, ’74; Hu and Ulrich, ’76). Thesedays also correspond to the time during human fetaldevelopment when diagnostic ultrasound examinationsare commonly performed. The ultrasound exposure wascompleted on three days as opposed to a single exposurein order to maximize the likelihood of a bioeffect.

Biological effects of ultrasound on the central ner-vous system of animals insonified in utero were moni-tored using selected behavioral characteristics evalu-ated after birth and into adult life. A primary goal wasto determine for the first time whether such subtlebiologic effects occur due to prenatal exposure to Dopp-ler ultrasound at clinically relevant levels.

Exposure conditions were carefully selected to repre-sent those found in a typical clinical environment. They

TABLE 1. Reproductive effects of experimental exposures of pregnant mammals to ultrasound (continued)

InvestigatorEffect



Effectobserved

McClain et al., ’72 Embryoniceffects

2.5 MHz CW; 9mW/cm2

30 min to 2hours

Rats No malformationincrease, some skel-etal variation

Murai et al., ’75 Postnatal effects CW20 mW/cm2

300 min Rats No IUGR; develop-mental delay of neu-romuscular develop-ment

Kellman et al., ’86 Placental func-tion, placentalblood flow

1-W/cm2 cw0.02 W/cm2 pulsed

both SATA; noeffect levels

6.3- W/cm2 cw;0.06- W/cm2

pulsed

10 min; noeffect

Guinea pig pla-centa perfu-sion

Thresholds for alteringplacental flow were100 times the diag-nostic ultrasoundlevel for imaging

Tarantal andHendrickx,’89a,b

Growth (numer-ous param-eters)behavior, neo-natal andinfant obser-vations, Apgarscores

Commercial realtime mechanicalsector scanner;7.5 MHz

12 mW/cm2 SPTA;98 W/cm2; SPPA;137 W/cm2, Im

10 min perexam or 20min perexam

Cymologousmacaqueexposed fromday 21–150: 5times perweek fromdays 21–35, 3times perweek fromdays 36–60,and onceweekly fromdays 61–150

No increase in abor-tions, stillbirth ormalformations; slightdecrease in birth-weight; better Apgarscore and ore muscletone and decreasedWBC; weight differ-ence disappeared;many behavioralchanges were tran-sient

*IUGR, intrauterine growth retardation; P, pulsed; SPPA, spatial peak pulse average intensity; SATA, spacial average,temporal average intensity; TPI, temporal peak intensity; cw, continuous wave.


included pulse echo and cw Doppler ultrasound. Inpulse echo ultrasound the frequencies, 3.5 MHz and 5.0MHz, and instantaneous peak intensity, ranging from500 to 5,000 W/cm2 (Isptp), were the principal param-eters studied. The cw Doppler studies investigated theeffects of exposure at a frequency of 2.0 MHz at anintensity level of 30 mW/cm2 (Ispta). These exposureconditions were carefully verified using calibrated ultra-sound dosimetry procedures that allowed for the studyof relevant field parameters.

All offspring were given nine physiologic parameterand reflex acquisition tests. The offspring were followedthrough young adulthood, and their postnatal weeklyweights were recorded through adulthood. Adult behav-ioral tests were also completed on all exposed, shamexposed, and control offspring.

RESULTS

Results of absorption analysis indicated that, sinceno pressure-time waveform was observed directly be-hind the subject rat, most of the power measured wasabsorbed. A small fraction of the power, however, mayhave been reflected and/or scattered as it propagatedthrough the water and/or animal. Since no reflectionswere observed, any reflected fraction of power wasconsidered negligible. Results of intrauterine tempera-ture monitoring plots confirmed that no significantincrease in temperature, greater than 1°C, occurredduring ultrasound exposure.

Several of the neonatal tests showed a statisticallysignificant difference between exposed groups and shamgroups. However, most of the statistically significanteffects were observed at the 5,000 W/cm2 (Isptp) inten-sity level which is far above that used in a clinicalenvironment. Only a few tests exhibited a difference atthe 1,500 W/cm2 (Isptp) level, which is still three timesthe typical ultrasound level for fetal monitoring. Alter-ations in the acquisition age of reflexes and physiologicmarkers may have been attributable to delays or accel-erations in development. These delays or accelerationsmay be indicators of toxicity and may suggest thatnormal cellular differentiation and growth depend on asynchronized order of cellular events.

Prenatal ultrasound did affect postnatal growth ofthe offspring, but this effect was not statistically signifi-cant. Therefore, it can be inferred from the resultsabove that clinically relevant dosages (,Isptp 5 1,500W/cm2) of prenatal ultrasound should not result insignificant changes in the appearance of selected physi-ologic markers or the acquisition of selected reflexes inWistar rats.

The threshold for observing significant changes usingthese parameters appear to be 1,500–5,000 W/cm2.Similarly, the weight of evidence indicates no signifi-cant effects due to exposure to 3.5 MHz pulse-echoultrasound at power levels less than 1,500 W/cm2,although the threshold for such effects appears to belower at the lower frequency. Postnatal growth wasaffected at the highest level (5,000 W/cm2, Isptp), with a

more pronounced effect being observed at the lower(3.5-MHz) frequency. The weight of evidence indicatesthat exposure to 2.0 MHz, cw ultrasound at 30 mW/cm2

(Ispta) for #2 hr does not result in any significantchanges in postnatal growth or psychophysiologic devel-opment or offspring adult behavior. These results sug-gest that prenatal pulse-echo ultrasound exposure atclinically relevant levels (,500 W/cm2, Isptp) does notresult in significant changes in postnatal growth, theappearance of selected physiological markers or theacquisition of selected reflexes, or adult behavior.

CONCLUSIONS

A multitude of animal studies concerning the effectsof ultrasound exposure on pregnancy outcome havebeen conducted during the past three decades. Thesestudies have included a number of different species ofanimals with exposures varying from low-intensitydiagnostic levels to intensities that cause severe hyper-thermia. The time during pregnancy when exposureswere given has also varied from the preimplantation tothe fetal period, although most of these studies havebeen conducted during the period of major organogen-esis. Often investigators have not taken into accountthe confounding effects of hyperthermia or maternalrestraint; both of which are known teratogens. A num-ber of studies did not provide sufficient data regardingthe quantitative and qualitative nature of the ultra-sound to enable proper interpretation of the results asrelated to other investigations.

Postnatal psychophysiological studies were com-pleted by several investigators. These studies are ofparticular import, since such testing permits long-termevaluation of prenatal exposure to ultrasound. Struc-tural changes are often manifested as behavioral alter-ations later in life, and testing for such changes pro-vides a sensitive, noninvasive measure of the effects ofprenatal exposure. If psychophysiological effects areobserved, then specific and appropriate anatomical andbiochemical investigations can be designed and imple-mented.

The preponderance of animal studies completed thusfar indicate that ultrasound exposure at clinicallyrelevant diagnostic levels does not result in adversematernal pregnancy outcome, significant morphologicalterations in term fetuses, or perinatal or postnataladult neurophysiologic abnormalities. In addition, thereappears to be a wide margin of safety between diagnos-tic intensities and those intensity levels which result interatogenic or neurobehavioral effects.

In the near future, almost all fetuses may be exposedto ultrasound, and for this reason even a minimal effectfrom such exposure would have implications on a largepopulation. The results of studies completed during thelast several decades continue to help formulate andmonitor guidelines for the use of prenatal ultrasonogra-phy and provide reassurance with regard to the safetyof diagnostic exposures to ultrasound.


LITERATURE CITED

AIUM. 1987. American Institute of Ultrasound in Medicine. Safetyconsiderations for diagnostic ultrasound. Newsletter of the Ameri-can Institute of Ultrasound in Medicine Bioeffects Committee.Laurel, MD: American Institute of Ultrasound in Medicine.

Akamatsu N. 1981. Ultrasound irradiation effects on pre-implantationembryos. Acta Obstet Gynaecol Jpn 33:969–978.

Alexander G, Williams D. 1971. Heat stress and development of theconceptus in domestic sheep. J Agric Sci Camb 76:53–72.

Arora KL, Cohen BJ, Beaudoin AR. 1979. Fetal and placenta re-sponses to artificially induced hyperthermia in rats. Teratology19:251–260.

Bayer SA. 1989. Cellular aspects of brain development. Neurotoxicol-ogy 10:307–320.

Borelli MJ, Bailey KI, Dunn F. 1981. Early ultrasonic effects uponmammalian CNS structures (chemical synapses). J Acoust Soc Am69:1514–1516.

Borelli MJ, Frizzell LA, Dunn F. 1986. Ultrasonically induced morpho-logical changes in the mammalian neonatal spinal cord. UltrasoundMed Bull 12:285–295.

Brent RL. 1984. The effects of ionizing radiation, microwaves, andultrasound on the developing embryo: Clinical interpretations andapplication of the data. Curr Probl Pediatr 14:4–87.

Brent RL. 1986. Is hyperthermia a direct or indirect teratogen?Teratology 3:373–374.

Brent RL. 1994. Effect of embryonic and fetal exposure to x-rays,microwaves, ultrasound, magnetic resonance, and isotopes. In:Barron WM, Lindheimer MD, editors. Medical disorders duringpregnancy. 2nd ed. St. Louis: Mosby–Yearbook. p 487–518.

Brent RL, Jensh RP, Beckman DA. 1991. Medical sonography: Repro-ductive effects and risks. Teratology 44:123–146.

Brent RL, Jensh RP Beckman DA. 1992. Medical sonography: Repro-ductive effects and risks. In: Chervenek FA, Isaacman GC, CampbellS, editors. Ultrasound in obstetrics and gynecology. Boston: Little,Brown. p 111–132.

Brown NT, Galloway WED, Henton WW. 1981. Reflex developmentfollowing in-utero exposure to ultrasound. In: Proceedings of theAIUM, San Francisco. p 119.

Brown NT, Galloway WED, Monahan JC. 1979. Postnatal behaviorand development following in utero exposure of mice to ultrasoundand microwave radiation. In: Gryer RM, Frankos VH, editors.Proceedings of the fifth FDA scientific symposium. p 161.

Butcher RE, Hauer K, Burbachi J, Scott W. 1975. Human and animalstudies. In: Ellis NR, editor. Aberrant development in infancy.Hillsdale, NJ: Erlbaum Press. p 161–167.

Carnes KI, Hess RA, Dunn F. 1991. Effects of in utero ultrasoundexposure on the development of the fetal mouse testis. Biol Reprod45:432–439.

Carstensen EL. 1982. Biological effects of low temporal averageintensity, pulsed ultrasound. Bioelectromagnetics 3:147.

Carstensen EL, Flynn HG. 1982. The potential for transient cavitationwith microsecond pulses of ultrasound. Ultrasound Med Biol 8:L720–724.

Carstensen EL, Hartman CL, Child SZ, Cox CA, Meyer R, Schenk E.1990. Test for kidney hemorrhage following exposure to intensepulsed ultrasound. Ultrasound Med Biol 16:681–685.

Chernoff GF, Golden JA, Edwards MJ. 1983. Heat induced neural tubedefects in C3H/lg-Mi mice. In: Saul RA, editor. Proceedings ofGreenwood Genetic Center. Clinton, SC: Jacobs Press. p 2.

Child SZ, Carstensen EL, Gates AH, Hall WJ. l988. Testing for theteratogenicity of pulsed ultrasound in mice. Ultrasound Med Biol14:493–498.

Child SZ, Hartman CL, Schery LA, Carstensen EL. 1990. Lungdamage from exposure to pulsed ultrasound. Ultrasound Med Biol16:817–825.

Coyle I, Wagner MJ, Singer A. 1976. Behavioral teratogenesis: Acritical evaluation. Pharmacol Biochem Behav 4:191–200.

Curzen P. 1972. The safety of diagnostic ultrasound. Practitioner209:822–824.

Davidson AN, Dobbing J. 1968. Applied neurochemistry. Philadelphia:FA Davis.

Dunn F, Fry FJ. 1971. Ultrasonic threshold dosages for the mamma-lian CNS. Transact Biomed Eng 18:253–256.

Edwards MJ. 1968. Congenital malformations in the rat followinginduced hyperthermia during gestation. Teratology 1:173–175.

Edwards MJ. 1986. Hyperthermia as a teratogen: A review of experi-mental studies and their clinical significance. Teratol CarcinogenMutagen 6:563–582.

Edwards MJ, Webster WS. 1982. Hyperthermia induction of neuraltube defects in mice. Proc Aust Soc Reprod Biol 14:19.

Falus M, Karanyi G, Sobel M, Pesti E, Trinh VB. 1972. Follow-upstudies on infants examined by ultrasound during the fetal age. OrvHetil 13:2119–2121.

FDA. 1985. Food and Drug Administration. 510K guide for measuringand reporting acoustic output of diagnostic ultrasound medicaldevices. Rockville, MD: Center for Devices and Radiological Health.

Fernandez-Cano L. 1958. Effects of increase or decrease of bodytemperature and hypoxia on pregnancy in the rat. Fertil Steril9:455–459.

Finnell RH, Moon SP, Abbott LC, Goldman JA, Chernoff GF. 1986.Strain differences in heat induced neural tube defects in mice.Teratology 33:247–252.

Fisher JE, Acuff-Smith KD, Schilling MA, Meyer RA, Smith NB,Moran MS, O’Brien WD Jr, Vorhees CV. 1996. Behavioral effects ofprenatal exposure to pulsed-wave ultrasound in unanesthetizedrats. Teratology 54:65–72.

Fisher JE, Acuff-Smith KD, Schilling MA, Vorhees CV, Meyer RA,Smith NB, Moran MS, O’Brien WD Jr. 1994. Teratologic evaluationof rats prenatally exposed to pulsed-wave ultrasound. Teratology49:150–155.

Flynn HG. 1982. Generation of transient cavities in liquids bymicrosecond pulses of ultrasound. J Acoust Soc Am 72:1926–1932.

Fry FJ. 1989. Ultrasound irradiation of pregnant mice. UltrasoundMed Biol 15:519–521.

Fry FJ, Erdman WA, Johnson LK, Baird AI. 1978. Ultrasonic toxicitystudy. Ultrasound Med Biol 3:351–366.

Fry FJ, Kosoff G, Eggleton RC, Dunn F. 1970. Threshold ultrasonicdosages for structural changes in the mammalian brain. J AcoustSoc Am 48:1413.

Germain M, Webster WS, Edwards MJ. 1985. Hyperthermia as ateratogen: Parameters determining hyperthermia-induced headdefects in the rat. Teratology 31:265–272.

Goldman PS. 1976. Maturation of the mammalian nervous system andthe ontogeny of behavior. In: Rosenblatt JS, Hind RA, Shaw E, BeerC, editors. Advances in the study of behavior. Vol 7. New York:Academic Press. p 2–91.

Hande MP, Devi PU. 1992. Effect of prenatal exposure to diagnosticultrasound on the development of mice. Radiat Res 130:125–128.

Hande MP, Devi PU. 1993. Effect of in utero exposure to diagnosticultrasound on the postnatal survival and growth of mouse. Teratol-ogy 48:405–411.

Hande MP, Devi PU, Karanth KS. 1993. Effect of prenatal ultrasoundexposure on adult behavior in mice. Teratology 15:433–438.

Hartley WJ, Alexander J, Edwards MJ. 1974. Brain cavitation andmicrocephaly in lambs exposed to prenatal hyperthermia. Teratol-ogy 9:299–303.

Hsu CY. 1948. Influence of temperature in development of rat em-bryos. Anat Rec 100:79–90.

Hu, JH, Ulrich WD. 1976. Effects of low intensity ultrasound on theCNS of primates. Aviat Space Environ Med 47:640–643.

Jensh RP. 1983. Behavioral testing procedures–a review. In: JohnsonEM, Kochhar DM, editors. Handbook of experimental pharmacol-ogy: Teratogenesis and reproductive biology. Vol 65. Berlin: Springer-Verlag. p 171–206.

Jensh RP. 1986. Effects of prenatal irradiation on postnatal psycho-physiological development. In: Riley EP, Vorhees CV, editors. Hand-book of behavioral teratology. New York: Plenum. p 471–486.

Jensh RP, Brent RL, Lewin PA, Goldberg B, Hueg CL, Oler J. 1989a.Prenatal ultrasound exposure and postnatal growth and develop-ment. Pediatr Res 25:77A.

Jensh RP, Brent RL, Lewin PA, Goldberg B, Hueg CL, Oler J. 1989b.Postnatal growth and development following prenatal 5.0 MHzultrasound exposure. Teratology 39:460–461.


Jensh RP, Lewin PA, Goldberg BB, Till MK, Eskesen MB, Oler J, BrentRL. 1990. Adult behavioral studies following prenatal 5.0 MHzultrasound exposure in the Wistar rat. Teratology 41:567.

Jensh RP, Lewin PA, Goldberg BB, Till MK, Eskesen MB, Oler J, BrentRL. 1992. Postnatal development following 5.0 MHz ultrasoundexposure during the fetal period in the Wistar rat. Teratology45:467.

Jensh RP, Lewin PA, Poczobutt MT, Goldberg BB, Oler J, Goldman M,Brent RL. 1994. Studies concerning the effects of prenatal ultra-sound exposure on postnatal growth and acquisition of reflexes.Radiat Res 140:284–293.

Jensh RP, Lewin PA, Poczobutt MT, Goldberg BB, Oler J, Goldman M,Brent RL. 1995. Effects of prenatal ultrasound exposure on adult ratoffspring in the Wistar rat. Proc Soc Exp Biol Med 210:171–179.

Kameyama E. 1977. In: Inouye E, Nishimura S. Gene interaction incommon diseases. Tokyo: University of Tokyo Press. p 167–178.

Kholkute SD, Udupa KJ. 1978. The effects of immobilization stress onpregnancy in the rat. Ind J Exp Biol 16:799–800.

Kimler BF, Norton S. 1988. Behavioral changes and structural defectsin rats irradiated in utero. Int J Radiat Oncol Biol Physiol 15:1171–1177.

Kimmel CA, Stratmeyer ME, Galloway WD, Laborde JB, Brown NT,Pinkavitch F. 1983. The embryotoxic effects of ultrasound exposurein pregnant ICR mice. Teratology 27:245–251.

Kimmel CA, Stratmeyer ME, Galloway WD, Brown NT, Laborde JB,Bates HK. 1989. Developmental exposure of mice to pulsed ultra-sound. Teratology 40:387–393.

Langman J, Webster W, Rodier P. 1975. Morphological and behavioralabnormalities caused by insults to the CNS in the perinatal period.In: Berry CL, Poswillo DE, editors. Teratology: Trends and applica-tions. Berlin: Springer-Verlag. p 182–200.

Lehmann JF, Delateur BJ. 1982. Therapeutic heat. In: Lehmann JF,editor. Therapeutic heat and cold. 3rd ed. Baltimore: Williams &Wilkins. p 404.

Lele PP. 1963. Effects of focused ultrasonic radiation peripheral nerve:With observations on local heating. Exp Neurol 8:47–83.

Lele PP. 1975. Ultrasonic teratology in mouse and man. In: Proceed-ings of the second European Congress on Ultrasound in Medicine,Munich, Germany. International Congress Series No. 363. Amster-dam: Excerpta Medica. p 22–27.

Leopold GR, Asher WM. 1974. Ultrasound in obstetrics and gynecol-ogy. Radiol Clin North Am 12:127.

Lewin PA. 1981. Calibration and performance evaluation of miniatureultrasonic probes using time delay spectrometry. In: Proceedings ofthe IEEE Ultrasonics Symposium, Chicago. p 660–664.

Lewin PA, Bjorno L. 1982. Acoustically induced shear stresses in thevicinity of microbubbles in tissue. J Acoust Soc Am 71:728–734.

Lewin PA, Goldberg BB. 1989. Ultrasound bioeffects for the perinatolo-gist. In: Sciarra JJ, editor. Gynecology and obstetrics. Vol 3. Philadel-phia: JB Lippincott. p 1–14.

Lewin PA, Poczobutt MT, Schafer ME. 1987a. Calibration of ultra-sound medical equipment: Analog versus digital approach. Ultrason-ics Int 87:686–691

Lewin PA, Poczobutt MT, Schafer ME, Goldberg BB, Jensh RP. 1987b.Improved measurement system for gravity control and ultrasoundbioeffects studies. In: 32nd annual convention of the AmericanInstitute of Ultrasound in Medicine, New Orleans. p 38–39.

Lewin PA, Schafer ME, Poczobutt MT. 1987c. Computerized acousticoutput acquisition system for imaging transducers. Proc Euroson, p324A.

Macdonald MC, Madsen EL. 1992. Method for determining the SATAand SAPA intensities for real-time ultrasonographic modes. JUltrasound 11:11–23.

McClain RM, Hoar RM, Saltzman MB. 1972. Teratologic study of ratsexposed to ultrasound. Am J Obstet Gynecol 114:39–42.

Miller DL. 1991. Update on safety of diagnostic ultrasonography. JClin Ultrasound 19:531–540.

Miller MW, Ziskin MC. 1989. Biological consequences of hyperther-mia. Ultrasound Med Biol 15:707–722.

Moore RM Jr, Barrick MK, Hamilton TM. 1982. Effects of sonicradiation on growth and development. Am J Epidemiol 116:571.

Mura N, Hoshi K, Suzuki M. 1974. Effects of prenatal exposure todiagnostic ultrasound on the behavioral development of rats. Tera-tology 10:91.

Murai N, Hoshi K, Nakamura T. 1975a. Effects of diagnostic ultra-sound irradiated during fetal stage on development of orientingbehavior and reflex ontogeny in rats. Tohoku J Exp Med 116:17–24.

Murai N, Hoshi K, Kang C-H, Suzuki M. 1975b. Effects of diagnosticultrasound irradiated during fetal stage on emotional and cognitivebehavior in rats. Tohoku J Exp Med 117:223–233.

NCRP. 1984. National Council on Radiation Protection and Measure-ments. Biological effects of ultrasound: Mechanisms and clinicalimplications, NCRP Report No. 74. Bethesda, MD: National Councilon Radiation Protection and Measurement.

NIH. 1984. National Institutes of Health. Diagnostic ultrasoundimaging in pregnancy. In: Report of NIH consensus developmentconference. Washington, DC: U.S. Government Printing Office. p84–667.

Norton S, Kimler BF, Cytacki EP, Rosenthal SJ. 1990. Acute responseof fetal rat telencephalon to ultrasound exposure in utero. ExpNeurol 107:154–163.

Norton S, Kimler BF, Cytacki EP, Rosenthal SJ. 1991. Prenatal andpostnatal consequences in the brain and behavior of rats exposed toultrasound in utero. J Ultrasound Med 10:69–75.

Nyborg WL. 1968. Mechanisms for non-thermal effects of sound. JAcoust Soc Am 44:1302–1309.

Nyborg WL. 1979. Physical mechanisms for biological effects ofultrasound. In: Repacholi MH, Benwell DA, editors. Ultrasonicshort course transactions. Ottawa, Canada: Radiation ProtectionBureau, Health Protection Branch, National Health and Welfare.p 84.

Nyborg WL. 1985. Mechanisms. In: Nyborg WL, Ziskin MC, editors.Biological effects of ultrasound. New York: Churchill Livingstone.p 24.

O’Brien WD Jr. 1976. Ultrasonically induced fetal weight reduction inmice. In: White D, Barnes R, editors. Ultrasound in medicine. Vol 2.New York: Plenum. p 531.

O’Brien WD Jr. 1983. Dose-dependent effects of ultrasound on fetalweight in mice. J Ultrasound Med 2:1–8.

O’Brien WD Jr. 1986. Biological effects of ultrasound: Rationale for themeasurement of selected ultrasound output quantities. Echocardiog-raphy 3:165–179.

O’Brien WD Jr, Stratmeyer ME. 1975. Ultrasonically induced weightreduction in mice. Congen Anomal 15:260.

O’Brien WD Jr, Janucki SJ, Dunn F. 1982. Ultrasound biologic effects:A suggestion of strain specificity. J Ultrasound Med 1:367–370.

Poczobutt MT, Lewin PA, Jensh RP. 1988. In vivo measurement ofultrasound field parameters using implanted hydrophone probes.Ultrasound in medicine and biology/second world congress of sonog-raphers, Washington, D.C.

Poczobutt MT, Lewin PA, Schafer ME. 1987. Computerized acousticoutput acquisition system. In: Proceedings of the 13th NortheastBioengineering Conference, 87-CH 2436–4. p 477–479.

Rosenzweig S, Blaustein FM. 1970. Cleft palate in A/J mice resultingfrom restraint and deprivation of food and water. Teratology 3:47–52.

Schafer ME, Lewin PA. 1988. A computerized system for measuringthe acoustic output from diagnostic ultrasound equipment. IEEETrans UFFC 35:102–109.

Shimizu T, Tanaka K. 1981. Experimental safety: Studies on sonica-tion of pulsed ultrasound. Jpn J Med Ultrasound 8:289–292.

Shiota K. 1988. Induction of neural tube defects and skeletal malforma-tions in mice following brief hyperthermia in utero. Biol Neonate53:86–97.

Shoji R, Momma E, Shimizu T, Matsuda S. 1971. An experimentalstudy on the effect of low-intensity ultrasound on developing mouseembryos. J Fac Sci Hokkaido Univ Ser VI Zool 18:51–56.

Sikov MR. 1986a. Effect of ultrasound on development. Part 1:Introduction and studies in inframammalian species. J UltrasoundMed 5:577–583.

Sikov MR. 1986b. Effect of ultrasound on development. Part 2: Studiesin mammalian species and overview. J. Ultrasound Med 5:651–661.


Sikov MR, Hildebrand BP. 1976. Effects of ultrasound on the prenataldevelopment of the rat. Part 1. 3.2 MHz continuous wave at ninedays of gestation. J Clin Ultrasound 4:357–363.

Sikov MR, Pappas RH. 1986. Stage dependence of ultrasound-inducedembryotoxicity in the rat. IEEE Trans UFFC 33:235–240.

Sikov MR, Hildebrand BP, Stearns JD. 1976. Effect of exposure ofnine-day rat embryo to ultrasound. Ultrasound Med 2:529–530.

Sikov MR, Hildebrand BP, Sterns JD. 1977. Postnatal sequelae ofultrasound exposure at fifteen days of gestation in the rat (work inprogress). Ultrasound Med. Biol. 3B:2017–2023.

Skreb N, Franz Z. 1963. Developmental abnormalities in the ratinduced by heat-shock. J Embryol Exp Morphol 5:311–323.

Smith MSR, Upfold JB, Edwards MJ, Shiota K, Cawdell-Smith J.1992. The induction of neural tube defects by maternal hyperther-mia: A comparison of the guinea pig and human. Neuropathol ApplNeurobiol 18:71–80.

Smyth MG. 1966. Animal toxicity studies with ultrasound at diagnos-tic power levels. In: Grossman CC, Holes HJ, Joyner C, et al.,editors. Diagnostic ultrasound. New York: Plenum, p 296–299.

Spyker J.M. 1975a. Assessing the impact of low level chemicals ondevelopment: Behavioral and latent effects. Fed Proc 34a:1835–1844.

Spyker JM. 1975b. Behavioral teratology and toxicology. In: Weiss B,Laties VG, editors. Behavioral toxicology. New York: Plenum. p311–349.

Stark CR, Orleans M, Haverkamp AD, Murphy J. 1984. Short andlong-term risks after exposure to diagnostic ultrasound in utero.Obstet Gynecol 63:194–200.

Stolzenberg SJ, Edmonds PD,Torbit CA, Sasmore DP. 1980a. Toxiceffects of ultrasound in mice: Damage to central and autonomicnervous systems. Toxicol Appl Pharmacol 53:432–438.

Stolzenberg SJ, Torbit CA, Edmonds PD, Taenzer JC. 1980b. Effects ofultrasound on the mouse exposed at different stages of gestation:Acute studies. Radiat Environ Biophys 17:245–270.

Stolzenberg SJ, Torbit CA, Pryer GT, Edmonds PD. 1980c. Toxicity ofultrasound in mice: Neonatal studies. Radiat Environ Biophys18:37–44.

Tachibana M, Tachibana Y, Suzuki M. 1977. The present status of thesafety of ultrasonic diagnosis in the area of obstetrics: The effect ofultrasound irradiation on pregnant mice as indicated in theirfetuses. Jpn J Med Ultrasound 4:279–283.

Takabayashi T, Abe Y, Sato S, Suzuki M. 1981. Effects of pulsed-waveultrasonic irradiation on mouse embryos. Jpn J Med Ultrasound8:286–288.

Takeuch H, Nakazawa T, Kumakiri K, Kasaud R. 1970. Experimentalstudies in ultrasonic Doppler methods in obstetrics. Acta ObstetGynaecol Jpn 17:11–16.

Tarantel AF, Hendrickx AG. 1989a. Evaluation of the bioeffects ofprenatal ultrasound exposure on cynomolgus macaque (Macacafascicularis). I. Neonatal/infant observations. Teratology 39:137–147.

Tarantel AF, Hendrickx AG. 1989b. Evaluation of the bioeffects ofprenatal ultrasound exposure on cynomolgus macaque (Macacafascicularis). II. Growth and behavior during the first year. Teratol-ogy 39:149–162.

Ter Har G, Daniels S. 1981. Evidence for ultrasonically inducedcavitation in-vivo. Phys Med Biol 26:1145–1149.

Thomenius KE, Lewin PA. 1991. Ultrasound bioeffects 1991: Anupdate. Ultrasound Q 9:111–137.

Vernadakis A, Weiner H, eds. 1974. Drugs and the developing brain.In: Advances in behavioral biology. Vol 8. New York: Plenum. pv–537.

Vorhees CV, Acuff-Smith KD, Schilling MA, Fisher JE Jr, Meyer RA,Smith NB, Ellis DS, O’Brien WD Jr. 1994. Behavioral teratologiceffects of prenatal exposure to continuous-wave ultrasound inunanesthetized rats. Teratology 50:238–249.

Vorhees CV, Acuff-Smith KD, Weisenburger WP, Meyer RA, Smith NB,O’Brien WD Jr. 1991. A teratologic evaluation of continuous-wave,daily ultrasound exposure in unanesthetized pregnant rats. Teratol-ogy 44:667–674.

Warkany J. 1986. Teratogen update: Hyperthermia. Teratology 33:365–371.

Warwick RR, Pond JB, Wood B. 1970. Hazards of diagnostic ultrasonog-raphy: A study with mice. IEEE Trans. Son Ultrasound 17:158–164.

Weinstock E, Fride E Gertzberg R. 1988. Prenatal stress effects onfunctional development of the offspring. Prog Brain Res 73:319–331.

Ziskin MC. 1990. Update on the safety of ultrasound in obstetrics.Semin Roentgenol 25:294–298.

Ziskin MC. 1991. Bioeffects related to thermal index. J UltrasoundMed 10:531.


intrauterine effects of ultrasound: animal studies

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