Therapeutic ultrasound as a potential male contraceptive: power, frequency and temperature required to deplete rat testes of meiotic cells and epididymides of sperm determined using a commercially available system.

1.4 MB·English

by  Tsuruta, James K

Tsurutaetal.ReproductiveBiologyandEndocrinology2012,10:7
http://www.rbej.com/content/10/1/7
RESEARCH Open Access
Therapeutic ultrasound as a potential male
contraceptive: power, frequency and temperature
required to deplete rat testes of meiotic cells and
epididymides of sperm determined using a
commercially available system
James K Tsuruta1*, Paul A Dayton3, Caterina M Gallippi3, Michael G O’Rand1,2, Michael A Streicker4,
Ryan C Gessner3, Thomas S Gregory3,6, Erick JR Silva1,2, Katherine G Hamil1,2, Glenda J Moser4 and David C Sokal5
Abstract
Background: Studies published in the 1970s by Mostafa S. Fahim and colleagues showed that a short treatment
with ultrasound caused the depletion of germ cells and infertility. The goal of the current study was to determine
if a commercially available therapeutic ultrasound generator and transducer could be used as the basis for a male
contraceptive.
Methods: Sprague-Dawley rats were anesthetized and their testes were treated with 1 MHz or 3 MHz ultrasound
while varying power, duration and temperature of treatment.
Results: We found that 3 MHz ultrasound delivered with 2.2 Watt per square cm power for fifteen minutes was
necessary to deplete spermatocytes and spermatids from the testis and that this treatment significantly reduced
epididymal sperm reserves. 3 MHz ultrasound treatment reduced total epididymal sperm count 10-fold lower than
the wet-heat control and decreased motile sperm counts 1,000-fold lower than wet-heat alone. The current
treatment regimen provided nominally more energy to the treatment chamber than Fahim’s originally reported
conditions of 1 MHz ultrasound delivered at 1 Watt per square cm for ten minutes. However, the true spatial
average intensity, effective radiating area and power output of the transducers used by Fahim were not reported,
making a direct comparison impossible. We found that germ cell depletion was most uniform and effective when
we rotated the therapeutic transducer to mitigate non-uniformity of the beam field. The lowest sperm count was
achieved when the coupling medium (3% saline) was held at 37 degrees C and two consecutive 15-minute
treatments of 3 MHz ultrasound at 2.2 Watt per square cm were separated by 2 days.
Conclusions: The non-invasive nature of ultrasound and its efficacy in reducing sperm count make therapeutic
ultrasound a promising candidate for a male contraceptive. However, further studies must be conducted to
confirm its efficacy in providing a contraceptive effect, to test the result of repeated use, to verify that the
contraceptive effect is reversible and to demonstrate that there are no detrimental, long-term effects from using
ultrasound as a method of male contraception.
Keywords: Male contraception, therapeutic ultrasound, testis, epididymis, wet-heat
*Correspondence:[email protected]
1TheLaboratoriesforReproductiveBiology,DepartmentofPediatrics,220
TaylorHall,CB7500,TheUniversityofNorthCarolinaatChapelHill,Chapel
Hill,NorthCarolina27599,USA
Fulllistofauthorinformationisavailableattheendofthearticle
©2012Tsurutaetal;licenseeBioMedCentralLtd.ThisisanOpenAccessarticledistributedunderthetermsoftheCreativeCommons
AttributionLicense(http://creativecommons.org/licenses/by/2.0),whichpermitsunrestricteduse,distribution,andreproductionin
anymedium,providedtheoriginalworkisproperlycited.
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Background Maleratswereanesthetizedwithisoflurane/oxygen(4%
An ideal male contraceptive would be inexpensive, reli- for induction, 2 - 2.5% to maintain anesthesia) prior to
able and reversible. Other desirable qualities include a andduringultrasoundtreatment.Aligaturewasusedto
low incidence of side effects, prolonged duration of the preventretraction ofthe testesintothe abdomen by the
contraceptive effect and no need for invasive surgical cremastermuscleduringtreatment.
procedures or hormonal treatments. Men have not had
many options for non-invasive, side-effect-free, reliable Ultrasound
contraception without resorting to the use of condoms. Atherapeuticultrasoundgenerator(ME740,MettlerElec-
While the barrier method has proven to be a reliable tronics, Anaheim, CA) and two different transducers
method to prevent the spread of sexually transmitted (ME7413:5cm2surfacearea,250mmdiameter;ME7410:
diseases [1], it is not always accepted as a family plan- 10 cm2 surface area, 360 mm diameter; Mettler Electro-
ning method for committed, monogamous couples nics, Anaheim, CA) were used to treat rat testes. This
[1,2]. instrument was capable of producing ultrasound of
Ultrasound’spotentialasamalecontraceptivewasfirst 1 or 3 MHz frequency with power up to a maximum of
reportedbyFahimetal.[3].Inaseriesofpublications,it 2.2 W/cm2 at a duty cycle of 100%. While the ME7413
was shown that a single application of ultrasound could transduceroperatedatboth1MHzand3MHz,thelarger
result in a dramatic loss of germ cells from testes and ME7410transduceronlyproduced1MHzultrasound.
that this loss of germ cells was reversible. No notable
side effects other than infertility were reported during Treatment apparatus
studies with rats, dogs and monkeys [4]. This method APlexiglas cylinder wasused as the ultrasound chamber
was tested on several human subjects who were already (70mmdiameter,25mmtall).Thebottomofthischam-
scheduled for orchiectomy to treat prostate cancer. ber was a singlelayerofacoustically transparent latex.A
These men reported that the procedure was pain-free, single layer of acoustically transparent polypropylene
onlycreatingagentlefeelingofwarmth[4,5]. meshwasheldinplaceapproximately1cmabovethebot-
Fahimusedfrequencies,powersandadutycycleasso- tom of the chamber to provide a reproducible distance
ciatedwiththetherapeuticuseofultrasoundratherthan betweenthetransducerandthescrotum.[Figure1].
parameters used for imaging tissue. In addition, Fahim The ultrasound chamber was plumbed to allow input
had an ultrasound generator and transducer built by of coupling medium across the bottom of the chamber
Whitewater Electronics (Helenville, WI) specifically for to dissipate any heat built up in the transducer. The
use as a contraceptive device [4,5]. Unfortunately, this transducer was affixed to an offset cam to allow it to
manufacturer is no longer in business and efforts to rotate in a horizontal plane against the bottom of the
locate Fahim’s original instrumentation have proved ultrasound chamber during treatment. Ultrasound gel
fruitless [personal communication, David Sokal, Family was used to coat the transducer face and the underside
HealthInternational]. of the latex sheet used as the bottom of the ultrasound
Thus, the objective of this study was to determine if chamber to achieve acoustic coupling.
commerciallyavailabletherapeuticultrasoundgenerators
and transducers could replicate the loss of germ cells Beam field mapping
demonstrated by Fahim. We report that a present-day The spatial distribution of acoustic pressures delivered
therapeuticultrasoundinstrumentwascapableofinducing by the ME7413 transducer to the testis was mapped as
a nearly complete loss of germ cells from rat testes only follows: a needle hydrophone (Onda, Sunnyvale, CA)
whenFahim’soriginaltreatmentconditionsweremodified. was held vertically over the operating transducer and
raster scanned 1.5 cm from the transducer’s face
Methods (approximating the distance to the center of the testis)
Animals in 0.5 mm increments using a computer controlled
All animal work was approved by the Institutional Ani- motion stage (Newport, Irvine, CA). The beam field was
mal Care and Use Committee (IACUC) of Integrated mapped at 1 MHz and at 3 MHz with the transducer
Laboratory Systems(ILS,ResearchTriangle Park, North centered against the acoustically transparent latex sheet
Carolina, USA) or by the IACUC of the University of used as the bottom of the treatment chamber. Distilled
North Carolina (UNC, Chapel Hill, North Carolina, water (DW), degassed distilled water and degassed 3%
USA). Pilot Studies and Study 1 were performed at ILS (w/v) sodium chloride were tested as coupling media.
while Study 2 was performed at UNC. Sprague Dawley Both the ME7410 and ME7413 transducers were also
rats (retired male breeders and adult females) were mapped at 1 MHz frequency at distances of 0.5 cm to
obtainedfromCharlesRiversLaboratories. 3.5 cm from the transducer face.
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(cid:0) (cid:4) (cid:2)
(cid:6) (cid:7) (cid:8) (cid:12)
(cid:13)
(cid:3)
(cid:10) (cid:7) (cid:11)
(cid:5)
(cid:9)
Figure1Apparatususedtopositionratsforultrasoundtreatment.PartswerecutfromPlexiglasunlessotherwisenoted.Aslantedsection
supportedmostoftherat’sbodyabovethelevelreachedbyre-circulatingcouplingmedium.Therat’sscrotumwasplacedwithinthe
ultrasoundtreatmentchamberafterusingaligaturetoretainthetesteswithinthescrotum(notshown).Thebottomofthetreatmentchamber
wasformedofasinglelayeroflatex,whichwasheldinplaceagainstarubberO-ringbyanaluminumringsecuredbymachinescrews.This
formedaliquid-tightseal,allowingcouplingmediumtobere-circulatedthroughthetreatmentchamberandaholdingvesselcontainedwithin
atemperature-regulatedwaterbath(tubing,waterbath,plumbinginputandoutputhavebeenomittedforclarity).Aringofultrasound
absorbingmaterialwassuspended1cmfromthebottomofthetreatmentchambertoaidpositioningofthetestesandtoreducereflectionof
ultrasoundenergy.Anultrasound-transparent,nylonmeshwasattachedtothebottomoftheringtomaintainaminimumdistanceof1cm
betweenthebottomoftheultrasoundchamberandtheproximalportionofthescrotum.
Determining the true effective radiating area (ERA) of our Sonicator 740 to be 1 W/cm2 and 2 W/cm2. The trans-
transducers ducer face was centered 2 cm directly above the pres-
Beam plots acquired with the transducer - hydrophone sure-sensing cone and the radiant force method was
separation set at 5 mm were used to determine the used to determine the total output in Watts.
actual effective radiating area of both transducers used
in our studies. Both transducers were driven at 1 MHz Temperature data
frequency and 1 W/cm2 intensity with the Mettler Soni- An implantable copper-constantan thermocouple (IT-21,
cator 740 used in our studies. The beam area was Physitemp Instruments, Clifton, NJ) was inserted down
defined as the contiguous region with intensity greater the long axis of the testis at an oblique angle to avoid
than 5% of the peak value. piercing the epididymis to record testis temperature.
The bimetal probe was connected to an analog-to-digital
Determining the true power output of our transducers converter (Thermes USB, Physitemp Instruments, Clif-
An Ultrasound Power Meter (model UPM-DT-1AV, ton, NJ) and data was collected using Labview software
Ohmic Instrument Co., Easton, MD) was used to mea- (National Instruments, Austin, TX). Additional thermo-
sure the power output of our transducers at 1 or 3 MHz couples were used to record the temperature of the cou-
frequency, at intensities indicated by the Mettler pling medium and the surface of the scrotum.
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Ultrasound treatment Study 2: Sperm were collected from both cauda epidi-
The treatment frequency (1 MHz or 3 MHz), intensity dymides for determining sperm count, as described
setting (1 W/cm2 to 2.2 W/cm2), duty cycle (100%) and above. The total sperm count was determined using a
duration were selected on the ultrasound generator hemocytometer by counting all sperm heads; the intact
[Tables 1, 2, and 3]. Rats were anesthetized and main- sperm count was calculated after tallying the number of
tained on 2 - 2.5% isoflurane/oxygen. A ligature to sperm heads without an attached tail. Computer-aided
retain the testes was tied tightly enough only to prevent sperm analysis performed with a CEROS sperm analysis
the retraction of the testes from the scrotum during system (software version 12.3; Hamilton Thorne Bios-
treatment. If testis temperature was recorded, the ther- ciences, Beverly, MA) was used to determine sperm
mocouple was inserted at this time. The rat was posi- motility.
tioned so that his scrotum was centered on the mesh
layer of the ultrasound chamber. The appropriate cou- Sperm count index
pling medium was circulated through the ultrasound Sperm counts (106 per cauda epididymis) were assigned
chamber [Tables 1, 2, and 3]. The temperature of the tooneoffivearbitrary countranges(<11,11-20,21-40,
coupling medium was controlled by re-circulating it 41-80, > 80). The count ranges were assigned values
through a holding vessel contained within a tempera- (fromlowtohigh)of:0,1,2,4and10.TheSpermCount
ture-controlled bath. Temperature recording was Index was calculated as a weighted average using the
initiated one minute prior to the start of ultrasound arbitraryvaluesassignedtothecountrangesandtheper-
treatment and continued for one minute after the con- centage of counts that fell within each range. For exam-
clusion of ultrasound treatment to record pre- and post- ple, if 75% of a group’s sperm counts fell in the second
treatment baseline temperatures. range of 11-20 × 106 and the remaining 25% of the
counts fell in the fourth range of 41-80 × 106 the count
Sperm count and motility were assessed two weeks after indexwouldbe(0.75×1)+(0.25×4)=1.75.
treatment
PreliminaryStudies andStudy 1: A testisandepididymis Fertility testing
wereremovedpriortowhole-bodycardiacperfusionwith Foreachmatingtrial,asinglemalewashousedwithapair
Bouin’s fixative. The cauda epididymis was carefully offemalesforoneweek.InPilotStudy2,thefirstmating
removed andseveralcuts weremadetoallow the release trialwasinitiated the day of the ultrasound treatment.A
of sperm. The incised cauda epididymis was placed in second mating trial with a new pair of females occurred
10mlofM16medium(Sigma,St.Louis,MO)foratleast duringthesecondweekafterultrasoundtreatment.Sperm
one half hour to allow motile sperm to be released. For parameterswereassessedattheconclusionofthesecond
determiningspermcount,adilutionwasmadeindistilled mating trial. Females were held for at least four weeks
waterandcountedonahemocytometer.Spermcountwas aftertheconclusionoftheirmatingtrialtocompletepreg-
expressedasmillionsofspermpercaudaepididymis.For nanciestoterm.
estimating sperm motility, a dilution was made in M16
medium. Motile and non-motile sperm were scored Untreated, sham-treated or wet-heat controls
visuallyusingahemocytometer. Three different controls were used for comparison of
spermcountsandmotilities.Untreated,retiredbreeders
served as untreated controls. Sham-treated animals
underwent all preparations for ultrasound treatment as
Table 1Treatment parameters forpreliminary studies treated animals: anesthesia was administered and main-
Parameter Preliminary Preliminary tained at 2 - 2.5% isoflurane/oxygen, scrotal fur was
Study#1 Study#2 shaved, a ligature was used to retain the testes in the
Couplingmedium(°C) N.R. N.R. scrotum,roomtemperaturecouplingmediumwasplaced
Treatments 1 1 in the treatment chamber, animal was placed on the
Duration(minutes) 10 10 treatmentapparatusandthescrotumwascenteredinthe
Couplingmedium DWorPBS dg-DW treatment chamber. The temperature of the coupling
Intensity(W/cm2) 1 2.2 medium was not regulated, the coupling medium was
Frequency(MHz) 1 1 not re-circulated and the ultrasound generator was not
Transducer(cm2) 5 5 turned on for the sham-treated animals. The wet-heat
FertilityTrial No Yes control animals were treated like the sham-treated con-
Thetemperatureofthecouplingmediumwasnotregulated(N.R.)inthese trolsexceptthatthetemperatureofthecouplingmedium
studies.Couplingmediumwasdistilledwater(DW),phosphatebufferedsaline was held constant at 45°C while it was re-circulated
(PBS),ordegassed,distilledwater(dg-DW).Fertilitytrialwasconductedas
describedintheMethods. throughthetreatmentchamber.
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Table 2Treatment parameters forStudy 1
Groupname Sham Wetheat 1MHz, 3MHz, 3MHz, 1MHz, 1MHz,
highpower highpower highpower, lowpower lowpower,
Na+ Na+
Groupnumber 1 2 3 4 5 6 7
Couplingmedium(°C) NR 45 37 37 37 NR NR
Treatments 2 2 2 2 2 2 2
Duration(minutes) 15 15 15 15 15 15 15
Couplingmedium dg-DW dg-DW dg-DW dg-DW dg-Na+ dg-DW dg-Na+
Intensity(W/cm2) - - 2.2 2.2 2.2 1 1
Frequency(MHz) - - 1 3 3 1 1
Transducer(cm2) n/a n/a 5 5 5 10 10
Rotation n/a n/a + + + - -
Animals 2 3 3 4 4 3 3
Degassed,3%sodiumchloridewasusedasthecouplingmedium(dg-Na+),otherwisedegasseddistilledwaterwasused(dg-DW).Temperatureofthecoupling
mediumwasnoted;otherwisetherewasnoregulation(NR).TransducerME7413hadasurfaceareaof~5cm2(5)whilemodelME7410hadasurfaceareaof~
10cm2(10).Transducerswerestationary(-)orwererotatedinaplaneparalleltothebottomoftheultrasoundchamber(+).Allgroupsreceivedtwoconsecutive
treatmentsseparatedbytwodays.
Histology cut into 0.5 cm thick cross-sections to facilitate penetra-
PilotStudiesandStudy1:Ratswereanesthetizedwithiso- tion of Bouin’s fixative. Fixed tissues were processed for
flurane prior to cardiac perfusion with Bouin’s fixative. histology as described above for Study 1. Digital micro-
One testis and one epididymis per animal were fixed for graphs were assembled into larger montages using an
histological examination. An additional 24 hours of Olympus BX51 microscope and motorized 2-dimen-
immersion fixation in Bouin’s solution was performed sional stage controlled by MetaMorph software (Mole-
prior to 2 days of washing in 70% ethanol. Tissues were cular Devices, Sunnyvale, CA).
processed into paraffin and 8 μm sections were stained
withhematoxylinandeosinusingstandardmethods.Digi- Statistical analyses
tal micrographs were assembled into larger montages One-way ANOVA analyses with post-tests were per-
usingthephotomerge functioninPhotoshopCS(Adobe, formed using GraphPad Prism version 5.0 d, GraphPad
SanJose,CA). Software, San Diego California USA [6]. If data failed
Study 2: Testes and epididymides were drop-fixed in Bartlett’s test for equal variances, significance was evalu-
Bouin’s fixative for 24 hours to prepare them for histol- ated using the Kruskal-Wallis test and Dunn’s multiple
ogy. After an initial fixation of three hours, testes were comparison post-test. In Study 1, sham-treated animals
Table 3Treatment parameters forStudy 2
Groupname Untreated 37C, 37C, 37C, 35C, 35C, 35C,
2×15, 2×10, 1×10 2×15, 2×15, 2×10,
saline saline saline water saline,
2W/cm2
Groupnumber 8 9 10 11 12 13 14
Couplingmedium(°C) NR 37 37 37 35 35 35
Treatments - 2 2 1 2 2 2
Duration(minutes) - 15 10 10 15 15 10
Couplingmedium - dg-Na+ dg-Na+ dg-Na+ dg-Na+ dg-DW dg-Na+
orDW
Intensity(W/cm2) - 2.2 2.2 2.2 2.2 2.2 2.0
Frequency(MHz) - 3 3 3 3 3 3
Transducer(cm2) - 5 5 5 5 5 5
Rotation - + + + + + +
Animals 2 7 4 5 8 4 4
Degassed,3%sodiumchloridewasusedasthecouplingmedium(dg-Na+),otherwisedistilledwater(DW)ordegassed,distilledwaterwasused(dg-DW).
Temperatureofthecouplingmediumwasnoted,otherwisetherewasnoregulation(NR).TransducerME7413hadasurfaceareaof~5cm2(5).Transducers
werestationary(-)orwererotatedinaplaneparalleltothebottomoftheultrasoundchamber(+).Allgroupsreceivedtwoconsecutivetreatmentsseparatedby
twodays,exceptasnotedforGroups8and11.
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(n = 2) were excluded from analysis but the remaining 1W/cm2andanoutputof20.0Wattsatanominalinten-
treatment groups (n = 3 or 4) were analyzed for statisti- sitysettingof2W/cm2.
cal differences.
True spatially averaged intensities were determined for
Results our transducers
Field mapping and measuring the true ERA and power The5-cm2transducer(ME7413)hadaneffectiveradiating
output of our transducers areaof4.4cm2.Atboth1and3MHzfrequencytheactual
Fieldmappingoftheoutputfromthetherapeutictransdu- intensityforthistransduceratanindicated1W/cm2was
cershowedthattherewasadonutshaped“hotspot”inthe 1.05W/cm2.Theactualintensityforthistransduceratan
5-cm2transducer’soutput(ME7413)at3MHz[Figure2]. indicated2W/cm2variedfrom2.02W/cm2at1MHzto
The field map was the same regardless of the coupling 2.11W/cm2at3MHz.The spatially averagedintensities
mediumused(DW,degassedDWor3%(w/v)saline).The determined for this transducer were all within 6% of the
beamfieldofthe 5-cm2 transducer changedwhenit was valuesindicatedbytheMettlerSonicator740.
mapped at 1 MHz: instead of a donut shaped hotspot, The 10-cm2 transducer (ME7410) was only capable of
therewasadiscretepeakofenergynearthecenterofthe operatingat1MHzfrequencyandhadaneffectiveradiat-
transducerface[Additionalfile1FigureS1]. ing area of 9.3 cm2. The actual intensity determined for
Beam plots from both transducers [Additional file 1 this transducer at an indicated 1W/cm2 was 1.1 W/cm2
Figure S1] were used to determine the area of the beam andatanindicated2W/cm2theactualvaluewas2.15W/
withenergyequaltoatleast5%ofthepeakbeamenergy cm2.Thespatiallyaveragedintensitiesdeterminedforthis
whenthedistancebetweenthehydrophoneandtransdu- transducerwerewithin10%ofthevaluesindicatedbythe
cer was set to 0.5 cm. The ME7413 transducer with a MettlerSonicator740.
nominalareaof5cm2had atrueeffectiveradiating area
of 4.4 cm2; the ME7410 transducer with a nominal area Mitigating thermal bio-effects
of10cm2hadatrueeffectiveradiatingareaof9.3cm2. In order to create a more even field of ultrasound at
Thepoweroutputofourtransducerswasdeterminedat both frequencies, we devised a method to rotate the
intensities indicated by the Mettler Sonicator 740 to be transducer in a horizontal plane coincident with the
1W/cm2and2W/cm2.The5cm2transducer(ME7413) bottom surface of the ultrasound chamber with the cen-
at a nominal intensity settingof 1 W/cm2 had an output ter of rotation offset 8 mm from the center of the trans-
of 4.6 W at either 1 or 3 MHz; with a nominal intensity ducer face. The movement of the transducer mimics its
setting of 2 W/cm2 the output varied from 8.9 Watts at use as a therapeutic device and results in an averaging
1 MHz to 9.3 Watts at 3 MHz. The 10-cm2 transducer of the field output over time.
(ME7410) was only measured at 1 MHz and had an The distance betweenthe transducer andthe scrotum
output of 10.2 Watts at a nominal intensity setting of was initially set to 3 cm. In an attempt to increase the
energy delivered to the testes, the distance between the
scrotum and the transducer was successively decreased.
Some rats’ testes actually rested on the bottom of the
ultrasound chamber,separatedfromthetransducer only
by a layer of latex. This may have been responsible for
(cid:22) some localized heat damage to the scrotum; these rats
(cid:26)(cid:16) (cid:27) (cid:28)(cid:31)
(cid:25) wouldoccasionallydevelopasmallcirculardiscoloration
(cid:25)
(cid:22)
(cid:16) ontheirscrotum.
(cid:24) (cid:31) (cid:28)(cid:29)
(cid:23) Constructing a mesh support provided a reproducible
(cid:22)
(cid:19)(cid:20)(cid:18)(cid:21) (cid:31) offset of 1 cm between the bottom of the treatment
(cid:17)
(cid:16) chamber and the scrotum; recirculating the coupling
(cid:14)(cid:15) (cid:31)(cid:27)(cid:28)(cid:28)(cid:29)(cid:29) (cid:27) (cid:28)(cid:29) medium eliminated any thermal bio-effects localized to
(cid:27) (cid:28)(cid:31) (cid:27) (cid:28)(cid:31)
(cid:31) (cid:28)(cid:29) (cid:31) (cid:28)(cid:29) the scrotum.
(cid:31) (cid:31)
(cid:30)(cid:31) (cid:28)(cid:29) (cid:30)(cid:31) (cid:28)(cid:29)
(cid:0) (cid:2) (cid:3)(cid:4) (cid:5) (cid:6) (cid:7) (cid:8) (cid:9) (cid:10)(cid:8) (cid:11) (cid:12) (cid:30)(cid:27) (cid:28)(cid:31) (cid:30)(cid:27) (cid:28)(cid:31) (cid:13) Pilot study 1: published treatment parameters did not
(cid:30)(cid:27) (cid:28)(cid:29) (cid:30)(cid:27) (cid:28)(cid:29) (cid:2) (cid:3)(cid:4) (cid:5) (cid:6) (cid:7) (cid:8) (cid:9) (cid:10)(cid:8) (cid:11) (cid:12) alter testis histology
Attempts to cause germ cell loss using a single ten min-
Figure 2 Beam field map of the Model ME7413 therapeutic
ultrasoundtransduceracquiredat3MHz.Normalizedacoustic ute dose of ultrasound at 100% duty cycle, 1 MHz and
pressureisplottedontheY-axis.TheXandY-axesrepresentthe 1 W/cm2 (Pilot Study 1) did not alter testis histology.
coordinatesusedtomeasureacousticpressuredeliveredbythe These were the original parameters that were reported
ultrasoundtransducer.
by Fahim to cause the loss of almost all germ cells from
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the testis [4]. Pilot study 1 used phosphate buffered Callowedustoachieveinternaltestistemperaturescom-
saline or distilled water as the coupling medium filling parable to theultrasoundtreated testes [Figure 3].Inter-
the ultrasound chamber. The coupling medium sur- estingly,heatalone[Table4Group2]wasmoreeffective
rounded the scrotum and allowed ultrasound to be effi- at reducing epididymal sperm count than the use of
ciently transmitted from the transducer to the scrotum; 1 MHz ultrasound either when the temperature of the
ultrasound passed through the scrotum and was coupling medium was held constant at 37°C [Table 4
absorbed by the testes. Group 3, Tukey’s post-test, p < 0.001] or when the tem-
peratureofthecouplingmediumwasnotregulated[Table
Pilot study 2: increased power and degassed coupling 4Group6,Tukey’spost-test,p<0.001],howeverwhen1
medium MHz ultrasound was applied through 3% saline at low
An experiment using a single treatment of 1 MHz at power,spermcountwasreducedsufficientlysothatthere
2.2 W/cm2 and 100% duty cycle through degassed water wasnosignificantdifferencefromwetheat.
was performed (Pilot Study 2). Treating with 2.2 W/cm2 Incontrast,the use of3MHzultrasoundresulted ina
was more successful than treating with 1 W/cm2. Two total epididymal sperm count ~10-fold lower than wet
weeks after ultrasound treatment, the testis was depleted heat alone but with almost 1,000 times fewer motile
of developing germ cells and sperm count was reduced sperm recovered from the epididymis: 3 MHz treated
to 200 × 103 sperm per cauda epididymis. These sperm animals [Table 4 Group 4] had ~ 6 × 103 motile sperm
were not motile when analyzed in M16 medium. per cauda epididymis while wet heat treated animals
Fahim reported that his ultrasound conditions caused [Table 4Group2]had~5×106 motile spermpercauda
ratstoimmediatelylosetheirfertility[4].Whenwetreated epididymis (derived from data presented in Table 4;
withlow frequencyand high power(PilotStudy 2), pups motilesperm=totalsperm×%motile).
were sired during the first and second weeks after treat-
ment.However,therewerenomotilespermattheendof Study 1: combining heat and ultrasound more effective
this pair of one-week mating trials. Hypothetically, if than heat alone
anothermatingtrialhadbeenperformedduringthethird The normal testis[Figure4,A-D] hada complex epithe-
week after treatment, the rat would have been infertile. lium consisting of many spermatogenic cells in various
This demonstrated that even though motile sperm were stagesofspermatogenesis.Twoweeksafterusingwetheat
not detected at the end of the second mating trial, there toelevatetestistemperaturetherewasasignificantlossof
were sufficient motile sperm during the initial two-week spermatogenic cells although most seminiferous tubules
periodaftertreatmentforfertility. stillretainedsomespermatogeniccells[Figure4,E-H].
Incontrast,combiningelevatedtemperatureand3MHz
Study 1: two consecutive treatments ultrasound [Table 4 Group 4 or 5] caused testis-wide
Inanattempttobringpost-treatmentspermcountscloser depletionofgermcells[Figure5].The lossofdeveloping
tozero,theeffectoftwoconsecutivetreatmentsseparated spermatocytes and spermatids from the seminiferous
by two days were tested [Study 1, Table 2]. Two weeks epithelium was extensive; almost all tubules examined
aftertreatment,totalspermcountinthecaudaepididymis were effectively depleted by this treatment [Additional
dropped below 2 × 106 total sperm with essentially no file 2 Figure S2]. The loss of spermatogenic output was
motility when 3 MHz ultrasound was applied at 2.2 W/ reflectedbyspermcountsintheseanimalsbelow2×106
cm2 through 37°C distilled water at 100% duty cycle sperm per cauda epididymis, two weeks after ultrasound
[Table 4Group4].Usingcouplingmediumheatedto45° treatment[Table4Groups4and5].
Table 4Testistemperatures andsperm parameters fromStudy 1
Group Treatment n Testistemperature(°C) Spermcount Motility
(106) (%)
1 Sham 2 30.1±0.8 380±33 45±3
2 Wetheat 3 42.6±0.1 23±4 22±5.8
3 Lowfreq.,highpower 3 40.5±1.2 84±3§ 54±2
4 Highfreq.,highpower 3 41.8±0.6 1.9±0.9† 0.3±0.3
5 Highfreq.,highpower,Na+ 4 nd 1.5±0.8† nd
6 Lowfreq.,lowpower 3 42.1±2.8 96±17§ 39±2
7 Lowfreq.,lowpower,Na+ 3 35.4±1.9 51±5 40±2
Spermanalyseswereperformedtwoweeksafterultrasoundtreatment.Averagespermcountrepresentsmillionsofspermpercaudaepididymis±SEM.%
Motilityrepresentsthepercentageofrecoveredspermwithforwardmotility±SEM.Theaveragemaximumtestistemperatureduringtreatmentislistedin
degreesCelsius±SEM.Notdetermined(nd).§,statisticallygreaterthanthewet-heatcontrol(Group2)byTukey’spost-test(p<0.001).†,statisticallylowerthan
1MHz,highpower(Group3)and1MHz,lowpower(Group6)byTukey’spost-test(p<0.001).
Tsurutaetal.ReproductiveBiologyandEndocrinology2012,10:7 Page8of15
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p-values].Group12hadaSpermCountIndexequalto3.9
42
andapproximatelyonethirdofthisgroup’sspermcounts
fellintotherangeof41-80millionspermpercaudaepidi-
dymis. Group 10 had a Sperm Count Index of 6.0 with a
mean sperm count of 67 ± 7 million motile sperm per
40
caudaepididymis.AsthehigherSpermCountIndexindi-
cated,amuchlargerproportion(7/8)ofthisgroup’ssperm
counts fell into the range of 41 - 80 million sperm per
)
C 38 caudaepididymis.
(

e
r
u Study 2: saline was a more effective coupling medium
t
a
r than distilled water at 35°C
e
p 36 Whenanimalsweretreatedonceat37°Cfor10minutesat
m
e 2.2W/cm2therewasnotasignificantdifferenceinsperm
t
countasafunctionofcouplingmedium(degasseddistilled
wet heat water versusdegassed3% saline) so this data was pooled
34 1 MHz (Group11).However,whenanimalsweretreatedtwiceat
35°Cfor15minutesat2.2W/cm2thecompositionofthe
coupling medium did make a significant difference in
spermcount(Tukey’spost-test,p<0.01):degassed3%sal-
32 ine(Group12)resultedinaspermcount50%lowerthan
0 5 10 15 degassed distilled water (Group 13). The use of saline
resultedinabouthalfofthespermcountsforGroup12to
minutes
be lower than 41 × 106 per cauda epididymis (Sperm
Figure3Representativetemperaturecurvesduringultrasound CountIndex=3.9)whiletheuseofdistilledwater(Group
orwetheat.Athermalcouplewasinserteddownthelongaxisofthe
13) resulted in only about 12% of counts below that
testisandanotherwasplacedinthecouplingmedium.Coupling
threshold [Figure 6, Sperm Count Index = 8.1]. In addi-
mediumwasre-circulatedat37°Cduringultrasoundtreatmentsandat
45°Cforthewetheatcontrol.Therotationfrequencyofthetransducer tion, the number of intact sperm was significantly lower
correlatedwithtemperaturefluctuationsatthesiteofthethermal (Tukey’spost-test,p<0.05)whentreatingat35°Cthrough
couple.Thewetheatcontrolyieldedatestistemperatureprofile 3%saline[Figure7,Group12]thanthroughdegasseddis-
similartoanultrasoundtreatedtestis.
tilledwater[Figure7,Group13].
Study 2: varying 3 MHz ultrasound treatments Most effective treatment
All animals in Study 2 were treated with 3 MHz ultra- Whenthefourtreatmentsgroups(Groups9-12)withthe
sound. We varied the temperature of the couplingmed- lowest mean spermcountsinStudy 2 were compared by
ium (35 or 37°C), its composition (DW or saline), the one-way ANOVA, Group 9 was found to have a signifi-
number(1or2)ordurationoftreatments(10or15min- cantly lower mean motile sperm count than the other
utes) to determine the effect of these changes in treat- threegroups(Kruskal-WalliswithDunn’spost-test, refer
mentonmeanmotilespermcountpercaudaepididymis to Figure 6 for p-values). In addition, the percentage of
[Figure6].Exceptforthegrouptreatedthroughdegassed intactsperminGroup9[Figure7]wassignificantlylower
distilledwaterat35°C(Group13),alltreatmentsresulted (Tukey’s post-test, p < 0.01) than the untreated control
in a significantly lower mean motile sperm count than [Figure 7, Group 8]. Thus, the treatment that reduced
the untreated group (Group 8) according to Dunnett’s caudaepididymisspermcounttwoweeksaftertreatment
multiplecomparisontest(p<0.001). tothelowestlevelswasthesameinStudy1(Group5)and
ThemosteffectivetreatmentinStudy2(Group9:treat- in Study 2 (Group 9): two 15- minute treatments with 3
ingtwicefor15minutesat3MHzand2.2W/cm2inten- MHzultrasoundat2.2W/cm2throughdegassed3%saline
sitythroughdegassed3%salineheldat37°C)resultedin3 maintainedat37°C.
± 1 million motile sperm per cauda epididymis and a
Sperm Count Index equal to 0. The next three lowest Discussion
sperm counts were in Groups 10 - 12; all of these treat- Rat as a model system
mentsresultedinmeanmotilespermcountsgreaterthan Rats are reported to retain fertility even with extremely
50millionspermpercaudaepididymiswhichwassignifi- low sperm counts [7]. In contrast to rats, the World
cantly higherthanobservedfor Group9[Figure 6,Krus- Health Organization has defined oligospermia in men as
kal-Wallis with Dunn’s post-test, refer to figure for less than 20 million sperm/ml in the ejaculate and men
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Figure4Representativehistologyofnormalorwet-heat-treatedtestesandseminiferoustubules.A-D:hematoxylinandeosinstained
cross-sectionsofuntreatedtestis.Thetallseminiferousepitheliumcontainsmanyspermatocytes(sp),roundspermatids(rs)andcondensing
spermatids(cs).Tails(t)ofcondensingspermatidsandnewlyreleasedtesticularspermareseeninthelumen(Lu)ofsometubules.E-H:testis
cross-sectionstainedtwoweeksafterwetheattreatment.Almostalltubuleshaveenlargedluminaldiametersaftertreatmentwithheatalone.
Theseminiferousepithelium(e)isreducedinheightduetothelossofmanyspermatocytesandspermatids.Sometubuleshavedisorganized
epithelium(*).
are generally considered sub-fertile when their sperm that combining elevated temperature, high power and
concentration drops below 10 million sperm/ml [8]. highfrequencywasthemosteffectivemethodforreducing
Thus, we anticipate that decreasing sperm count suffi- spermcount.
ciently to cause infertility in rats would also cause infer-
tility in men. However, sperm counts or concentrations Variation between ultrasound transducers
that would represent infertility in men could allow rats A direct comparison between our treatments and those
to retain their fertility. Our second pilot study showed of Fahim are not possible without measuring the true
that the absence of motile sperm at the end of a mating effective radiating area (ERA, cm2) and power output
trial did not rule out the ability to sire pups. With the (Watts)forallofthetransducersusedinthesestudiesin
mating scheme used in our study, it appeared that ordertocalculatethetruespatialaverageintensity (SAI,
sperm count was changing rapidly and that the count W/cm2)deliveredduringtreatment.TheSAIreportedby
on the day of conception could be higher than the clinical therapeutic ultrasound systems is not directly
count determined at necropsy. Consequently, in lieu of regulated in the United States by the Food and Drug
testing fertility we decided to assay epididymal sperm Administration(FDA)even thoughthisistheparameter
reserves to monitor the efficacy of our treatment most often used clinically to determine dosing during
conditions. treatment.The FDAdoesrequire the true poweroutput
Our results clearly show that therapeutic ultrasound tobewithin±20%ofthevaluereportedbythemanufac-
treatment depleted developing germ cellsfromthe testis turerhowevernospecificguidelinewaspresentedforthe
andsubsequentlydecreasedthesize of spermreservesin accuracy in reporting ERA [9]; most manufacturers
theepididymiswhenratsweretreatedwithtwoconsecu- report ERA with an error of ± 20 - 25%. Therefore, the
tiveultrasoundtreatmentsseparatedbytwodays[Table4 trueSAIforatransducercouldvarybyupto150%from
Figure6].Thisdiffersfromreportsinthe1970sbyFahim the displayed value while still satisfying FDA guidelines
et al. [3,4], which reported that a single treatment of forERAandpoweroutput.Astudyofsixty-sixtherapeu-
1MHzultrasoundwassufficienttoinduceacontraceptive tic ultrasound transducers showed that their true SAI
effectofapproximately sixmonthsduration.Nomention varied from -43% to +63% of the displayed value [10].
of controlling the temperature of the coupling medium The effects of ultrasound are dose-dependent, thus
appeared inthose original reports. Incontrast,we found reproducible clinical dosing of therapeutic ultrasound
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Figure5Testishistologytwoweeksafter3MHzultrasound(Group4).(A)Thelossofspermatogeniccellsafterthistreatmentwasmore
completethanafterthewetheattreatment.Thisresultedinashorterepitheliumandalargerdiameterlumen.(B)Anisolatedclusteroftubules
inthisparticularanimalshowedevidenceofthermaldamage(td)inadditiontothelossofspermatogeniccells.(C)Mosttubuleshadavery
shortepitheliallayerandincreasedlumendiameterduetothelossofallspermatocytesandspermatids.(D)Tubulesthatappeartohavealarger
epitheliallayerandsmallerdiameterlumenwerestillmissingspermatocytesandspermatids.
requires determining the actual ERA, power output and comparisonofourstudyresultswithfuturestudiesandto
SAI of the generator and transducers being used for begin to standardize the clinical dosing of therapeutic
treatment. ultrasoundwhenusedasamalecontraceptive.
In some cases, more advanced monitoring techniques Since Fahim’s custom-built generator and transducer
such as quantitative Schlieren assessment may be werenotavailablefortesting,wecannotruleoutthepos-
required to discern differences in output of transducers sibility that hissystemdelivered more ultrasoundenergy
operated under identical nominal parameters [11]. This tothetestesthanourtherapeuticultrasoundinstrument.
method can measure the power distribution in discrete Accordingly,wemodifiedourcouplingmediumandtreat-
portionsoftheultrasoundbeamthatarenotcapturedby ment parameters to increase the delivery of ultrasound
measurementsmandatedbytheFDAsuchasbeamnon- energytothetestes.Whileattemptingtomaximizeenergy
uniformity ratio (BNR) and the aforementioned total delivery, we also took steps to mitigate any thermal bio-
powerandERA.Differencesinthedistributionofpower effectsobservedonthescrotalepithelium.Thetransducer
within an ultrasoundfieldmay account forthe ability of face became quite hot to the touch by the end of each
nominally identical transducers to heat tissue at signifi- treatmentsowereasonedthatconductivetransferofheat
cantlydifferentrates[11]. caused occasional circular discolorations when the scro-
We determined the actual effective radiatingareasand tum was pressed against the bottom of the treatment
poweroutputofthetransducers usedinourstudies.The chamber.Wemodifiedtheinteriorofourchambertopro-
trueSAIofourtransducersweredeterminedtobewithin vide a reproducible offset between the scrotum and the
10%ofthevaluesreportedbyourtherapeuticultrasound chamberbottom/transducer.Thisalsoprovidedaspaceto
generator.InadditiontodeterminingthetrueERA,power re-circulate coupling medium between the scrotum and
outputandSAIforourtransducers,wehavealsoprovided chamber bottom/transducer to dissipate any localized
beam plots [Additional file 1 Figure S1] to facilitate buildupofheat.Irregularitiesinthebeamfieldprompted