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ARTICLE IN PRESSGModelRTX-6428; No.of Pages8Reproductive Toxicology xxx (2010) xxxxxx

Contents lists available at ScienceDirect

Reproductive Toxicology

journa l homepage: www.e lsev ier .com

Semen quality and sperm DNA damage in relatiomen from an infertility clinic,

John D. Meekera,, Shelley Ehrlichb, Thomas L. Tothc, Diane L.Ana T. Trisinib, Xiaoyun Yed, Russ Hauserb,c

a Department ob Department oc Vincent Mem nitedd Centers for Di

a r t i c l

Article history:Received 28 JaReceived in reAccepted 12 JuAvailable online xxx

Keywords:BiomarkersEndocrine disruptionEnvironmentEpidemiologyEstrogenicExposureFertilityMaleReproduction

genesalitydete

[IQR]) concentration of 1.3 (0.82.5) ng/mL. Urinary BPA concentration was associated with slightly ele-vated, though not statistically signicant, odds for below reference sperm concentration, motility, andmorphology. When modeled as continuous dependent variables, an IQR increase in urinary BPA concen-tration was associated with declines in sperm concentration, motility, and morphology of 23% (95%CI40%, 0.3%), 7.5% (17%, +1.5%), and 13% (26%, 0.1%), respectively, along with a 10% (0.03%, 19%)increase in sperm DNA damage measured as the percentage of DNA in comet tail. In conclusion, urinary

1. Introdu

Bisphenin the manin baby antainer lininglead to humduced annu[2]. Dietarypopulation

Work suppof Environmenauthors gratefGA) for measu Disclaimeand do not neControl and Pr

Corresponversity of MichAnn Arbor, MI

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0890-6238/$ doi:10.1016/j.this article in press as: Meeker JD, et al. Semen quality and sperm DNA damage in relation to urinary bisphenol A among men fromty clinic. Reprod Toxicol (2010), doi:10.1016/j.reprotox.2010.07.005

BPAmay be associatedwith declined semen quality and increased spermDNA damage, but conrmatorystudies are needed.

2010 Elsevier Inc. All rights reserved.

ction

ol A (BPA) is a high production volume chemical usedufacture of polycarbonate plastics, which can be usedd water bottles, and epoxy resins used in food con-s and other applications; the use of these products canan exposure [1]. Over 6 billion pounds of BPA are pro-ally, with a 610% growth in demand expected per yearingestion is considered the primary source of generalexposure to BPA. Other exposure sources may include

orted by grants ES009718 and ES00002 from the National Institutetal Health Sciences (NIEHS), National Institutes of Health (NIH). Theully acknowledge Amber Bishop, Tao Jia, and Jack Reidy (CDC, Atlanta,ring the urinary concentrations of BPA.r: The ndings and conclusions in this report are those of the authorcessarily represent the ofcial position of the Centers for Diseaseevention.ding author at: Department of Environmental Health Sciences, Uni-igan School of Public Health, 6635 SPH Tower, 109 S. Observatory St.,48109, United States. Tel.: +1 734 764 7184; fax: +1 734 936 7283.ress: [email protected] (J.D. Meeker).

water, air, and dust [1,3]. As a result, there is widespread generalpopulation exposure to BPA [1,4]. BPA has been shown to alterendocrine function throughmultiple pathways [5], and anumber ofanimal studies have reported adverse reproductive effects inmalesexposed to low levels of BPA in early life or in adulthood [6]. To ourknowledge no human studies of BPA exposure and semen qualityor sperm DNA damage have been conducted to date. In the presentstudy we assessed the relationship between urinary BPA concen-trations, which have been used as a biomarker of exposure to BPA[4], and semen quality and sperm DNA damage in men recruitedthrough a United States (US) infertility clinic.

2. Methods

Subjects were recruited during 20002004 from an ongoing study on the rela-tionship between environmental agents and reproductive health. Participatingmenwere partners in subfertile couples seeking treatment from the Vincent Androl-ogy Lab at Massachusetts General Hospital. The study was approved by the HumanStudies Institutional Review Boards of the Massachusetts General Hospital, Har-vard School of Public Health, the Centers for Disease Control and Prevention (CDC),and the University of Michigan. After the study procedures were explained and allquestions answered, subjects signed an informed consent. Men between the ages of1855yearswithoutpost-vasectomystatuswhopresented to theAndrologyLabora-torywereeligible toparticipate.Of thoseapproached, approximately65%consented.

see front matter 2010 Elsevier Inc. All rights reserved.reprotox.2010.07.005f Environmental Health Sciences, University of Michigan, Ann Arbor, MI, United Statesf Environmental Health, Harvard School of Public Health, Boston, MA, United Statesorial Obstetrics and Gynecology Service, Massachusetts General Hospital, Boston, MA, Usease and Control and Prevention, Atlanta, GA, United States

e i n f o

nuary 2010vised form 16 April 2010ly 2010

a b s t r a c t

Bisphenol A (BPA) impairs spermatourinary BPA concentrations, semen quthrough an infertility clinic. BPA was/ locate / reprotox

n to urinary bisphenol A among

Wrightc, Antonia M. Calafatd,

States

is in animals, but human studies are lacking. We measured, and sperm DNA damage (comet assay) in 190 men recruitedcted in 89% of samples, with a median (interquartile range

Please cite A daman infertili

ARTICLE IN PRESSGModelRTX-6428; No.of Pages82 J.D. Meeker et al. / Reproductive Toxicology xxx (2010) xxxxxx

Most men that declined to participate in the study cited lack of time on the day oftheir clinic visit as the reason for not participating.

2.1. Urinary BPA

A single spclinic visit in afrom the bodyexposure in ththird urine samally collectedbclinic visit. Af(National Instrdivided in aliqto the CDC, Atlplus conjugatecoupled to isotandem massAgilent 1100 mquadrupole APFirst, 100L oH1; Sigma ChBPA was thenSPE column (Mnents using anegative ion-ation (LOD) for(4ng/mL) anwith pooled huand unknowncerning subjecgravity (SG) usSG-adjusted ution, and SG ishandheld refra

2.2. Semen sa

Semenwaspecimen cuption at 37 C fomeasured at tcryogenic strathe straws dirshowed that tcorrelated witanalyzed in babath for 10 s a

2.3. Semen qu

In theMGHfor spermconcaided semen aSetting paramCASA were esconcentrationwarmed (37 CAminimum ofeach specimensperm concendened as Wowith a velocitywith a velocityteristics has bmeasured, onl(VCL) and lineto a high degre

At least twsmear was alling kit (Dade Bperformed witCompany, Tokfor each specimnormal or belo

2.4. Neutral co

The cometage has beenp

(0.7%3:1high resolutionagarose;Amresco, Solon,OH,USA)wasembeddedbetweentwo additional layers of agarose onmicrogel electrophoresis glass slides (Erie Scien-tic, Portsmouth, NH, USA). Slides were then immersed in a cold lysing solution todissolve the cellmembraneandmake chromatin accessible for the enzymedigestionsteps. After 1h cold lysis, slides were transferred to a solution for enzyme treat-

th 10s wer(Am

n a hoent eleand dxtent,eremCompsure ohe taiil. TDMof co

(I

I isthe x-

tistica

analyescripributiomeaspotenconc

s. DiffswherBPAdaANESd amoassociand mwererm corm ms usedat werationn) priore assspeciution.day o0a.m.es, anonsidelogicnged todel

tabilitere ereasetiple lconc

ers, ansforotioncentrogisticas ainterd as ameas.sets o

ingle uetricthreeetric

3) usithatsinglethis article in press as: Meeker JD, et al. Semen quality and sperm DNty clinic. Reprod Toxicol (2010), doi:10.1016/j.reprotox.2010.07.005

ot urine sample was collected from each subject on the day of theirsterile polypropylene cup. Because BPA is metabolized and excretedrapidly, and a single urinary measure likely reects an individualse hours to days leading up to urine sample collection [7], a second andple were collected from a subset of men. These samples were gener-etween1week and2months following therst sample at a follow-upter measuring specic gravity (SG) using a handheld refractometerument Company, Inc., Baltimore, MD, USA), each urine sample wasuots and frozen at 80 C. Samples were shipped on dry ice overnightanta, Georgia, USA, where the total urinary concentration of BPA (freed species) was measured using online solid-phase extraction (SPE)tope dilution high performance liquid chromatography (HPLC) spectrometry (MS/MS) on a system constructed from several HPLCodules (Agilent Technologies, Wilmington, DE) coupled to a tripleI 4000 mass spectrometer (Applied Biosystems, Foster City, CA) [8].f urine was treated with -glucuronidase/sulfatase (Helix pomatia,emical Co., St. Louis, MO) to hydrolyze the BPA-conjugated species.retained and concentrated on a C18 reversed-phase size-exclusionerck KGaA, Germany), separated from other urine matrix compo-pair of monolithic HPLC columns (Merck KGaA), and detected bytmospheric pressure chemical ionization-MS/MS. The limit of detec-BPA in a 0.1-mL urine sample was 0.4ng/mL. Low-concentration

dhigh-concentration (20ng/mL) quality controlmaterials, preparedman urine, were analyzed with analytical standards, reagent blanks,samples [8]. Analysts at the CDC were blind to all information con-ts. BPA concentrations were corrected for urine dilution by specicing the following formula: Pc =P[(1.0241)/SG1)], where Pc is therinary BPA concentration (ng/mL), P is the observed BPA concentra-the specic gravity of the urine sample. SG was measured using actometer (National Instrument Company, Inc., Baltimore, MD, USA).

mple collection

s collectedon site atMassachusettsGeneralHospital in a sterile plasticafter a recommended period of abstinence of 48h. After liquefac-r 30min, semen quality parameters and motion characteristics werehe clinic. The remaining unprocessed semen was frozen in 0.25mLws (CryoBiosystem, I.M.V. Division, San Diego, CA) by immersingectly into liquid nitrogen (196 C). Previous work in our laboratoryhis freezing method produced comet assay results that were highlyh results from fresh, unfrozen samples [9]. Semen samples were latertches, where straws were thawed by gently shaking in a 37 C waternd the semen was immediately processed for the comet assay.

ality

Andrology Laboratory, trained clinical staff analyzed semen samplesentration, total spermcount, andmotionparametersusingcomputer-nalyzer (CASA, HTM-IVOS Version 10HTM-IVOS, Beverly, MA, USA).eters and the denition of measured sperm motion parameters fortablished by the Hamilton-Thorn Company. To measure both spermand motility, 5L of semen from each sample was placed into a pre-)Makler counting chamber (Se Medical instruments, Haifa, Israel).200 sperm cells from at least four different elds was analyzed from. Total sperm count (106/ejaculate) was calculated by multiplyingtration (106/mL) by semen sample volume (mL). Motile sperm wasrld Health Organization (WHO) grade a sperm (rapidly progressive25m/s at 37 C) plus b grade sperm (slow/sluggish progressive5m/s but 10%) in any of the statistical mod-s were adjusted for the same covariates for consistency. To improvey, all models were adjusted for the same covariates and effect esti-xpressed as odds ratios (OR) associated with an interquartile rangein urinary BPA concentrations.inear regression was also used to assess associations between uri-entrations and continuous measures of semen quality, spermmotionnd sperm DNA damage. Total sperm count and sperm concentrationmed using the natural logarithm, whereas all other semen quality,, and DNA damage measures were modeled untransformed. Urinaryations were also ln-transformed. The same covariates listed aboveregression models were considered, and specic gravity was againcontinuous variable in all models to adjust for urinary dilution. Topretability, the regression coefcients were back transformed andchange in the dependent variable (i.e., semen quality or sperm DNA

ures) for an interquartile range (IQR) increase in urinary BPA concen-

fmodelswere constructed: (1) using only urinary BPA concentrationsrine sample collected on the same day as the semen sample; (2) usingmean urinary BPA concentration for each participant, where betweenvalues were used to calculate each individuals geometric mean (i.e.mean for men with only one urine sample was equal to that singleng the geometric mean urinary BPA concentration among only par-contributed BPA data from at least two urine samples; and (4) usingurinary BPAmeasure collected on the same day as the semen sample


ARTICLE IN PRESSGModelRTX-6428; No.of Pages8J.D. Meeker et al. / Reproductive Toxicology xxx (2010) xxxxxx 3

Table 1Demographic categories by semen parameters (N=190 men).

Comparisonsubjects (N=76)a,b

Semen parameters

Total sperm count



Table 4Adjusted odds ratios (95% condence intervals) for below reference semen quality parameter associated with an interquartile range increase in urinary BPA concentration.Adjusted for specic gravity, age, BMI, abstinence period, current smoking, and time of urine sample.

Semen quality parameter (1) BPA measure from (2) Geometric mean BPAs

(3) Geometric mean BPA (4) BPA from same day as

Total spermSperm conceSperm motilSperm morp

a Among meb Geometric monc Geometricd Ln-transfo

from whomin the twop-value =0.within thelection withstrengthen

When cand DNA dinversely atively assoc(p-values 0.4).

on

d that urinary BPA concentrations measured in spotles collected on the same day as a semen sample were

20g/tion ofSwissspermwere scontroin adufor 6 dthis article in press as: Meeker JD, et al. Semen quality and sperm DNA damty clinic. Reprod Toxicol (2010), doi:10.1016/j.reprotox.2010.07.005

with suggestive declines in semen quality parameterscreased spermDNAdamage (measured as Tail%). For theity parameters, these associations were only observedling the parameters as continuous variables in linearmodels but not when dichotomizing them accordingccepted WHO reference values in logistic regressiono, in linear regression models stratied by semen qual-he relationships were only found among men with atmen parameter below WHO reference values. Whensubsequent urine samples collected in the weeks andowing the semen samplewere taken into considerationions were inconsistent and weakened, perhaps due totistical power since repeated urineswere only availablebset of the men, or because the repeated samples weretside the most relevant exposure window of interesttcome measures (i.e. weeks and months prior to the

ple).ings of suggestive relationships between urinary BPAons and semen quality parameters are consistent withies reporting adverse effects on Sertoli cell function anduction in relation to BPA exposure [1929]. Thoughese studies were conducted in vitro or report effectsrom in utero or early postnatal BPA exposure, and thusbility to directly compare with our ndings, severale assessed effects following in vivo exposure in adult-3]. Sakaue et al. [20] showed that oral administrationdult SpragueDawley rats at concentrations as low as

didymal spingestion odays [23].

In the pbetween urassociationor TDM. Inmeasures osame indepies, and it hparametersSpecicallyreported, alikely to beTail% maystudy, the pand Tail% msingle-strannumber ofbeen fully cremain uncreproductiodepletion oepididymal

Urinarythan those rgeneral popge measure associated with increasing quartiles of urinary BPA con-tility (p-value for trend=0.04); (c) sperm morphology (p-value forod, current smoking status, and time of day of urine sample.

w/day resulted in a reduction in daily sperm produc-to 40%. This was conrmed in a later study on adultexposed to 25100ng/kg bw/day for 1 month. Dailyuction, epididymal sperm concentration, and fertilitycantly decreased in the exposed groups compared to]. Toyamaet al. [22] alsonoted spermatid abnormalitiesstar rats and CD-1 mice injected with 20g/kg bw/dayFinally, there was a dose-dependent reduction in epi-age in relation to urinary bisphenol A among men from

erm motility and sperm count in male rats followingf 0.2, 2, and 20g/kg bw/day of BPA for a period of 45

resent study, we found evidence for an associationinary BPA concentrations and increased Tail%, but nos betweenurinaryBPA concentrations and comet extentconsistent results between the various DNA damagebtained by the neutral comet assay in relation to theendent variable have been observed in previous stud-as been hypothesized that the different comet assaymay reect different types of DNA strand breaks [30]., a lack of correlation between TDM and Tail% has beennd it was hypothesized that a high TDM may be moreassociated with double-strand breaks, whereas a highreect single-strand breaks [30]. Thus, in the presentositive association between urinary BPA concentrationay reect a relationship between BPA exposure andd breaks. While BPA has expressed genotoxicity in ain vitro and in vivo models [31,32], ndings have notonsistent and details of the directmechanisms involvedlear. However, the adverse effects of BPA on adult malen may be through the induction of oxidative stress andf antioxidant defense mechanisms, as was reported insperm of rats orally dosed with BPA [23].BPA concentrations in the present study were lowereported for adultmen in the sameage range fromtheUSulation in NHANES 20052006 (Table 3). In our study


ARTICLE IN PRESSGModelRTX-6428; No.of Pages8J.D. Meeker et al. / Reproductive Toxicology xxx (2010) xxxxxx 7

the median and 95th percentile BPA concentrations (uncorrectedfor specic gravity)were 1.3 and 9.3ng/mL, respectively, comparedto 2.3 and 12.2ng/mL among the 540male participants aged 1855years in NHANES 20052006. One potential reason for the discrep-ancy may ievening insamples colIn additionincome betto the diffin NHANESamong nonhigher amoferences inin the presewith2005BPA levels r20032004

Since ththrough anresults to thof couples srelated to apopulationfertility proour resultswould haveto BPA expodiagnosis/tring that meexposure, wconcentratiwith at leaour data sugsensitive tomore studieis importanthe results.

We founman weeksrelated (r=the likelihowithin-indiavailabilityticipants. Hnon-differeassociationusing broadmeasuremaover a longsemen samsample to rnot well chdence that oseveral wee

Anotherdue to theparticipant,ple from ovcausation inunderlyingto altered B

In conclureduced semever, due toand exposu

be assessed in other large and appropriately designed human epi-demiologic studies that measure BPA in multiple urine samplesacross the exposure window of interest.

t of

auth

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denbispheridgensT,RA, Trgium.afat Aon tospecttherill. In7;24(hter CIn vivo7;24(halingal varwomX, Kutchinge envity SMty of2;17(rld Htion obridg

eker Jshipan se

ty SM,weenrm anger TFvalue8;49(gh NPrm. Mty SMshiphuma3;111ional]. Atlap://wwinbauationic Grss AWthodsOccupghes Pic alky2+) puaue Mphenolth 20Hiyasause feama Ybisph4;67(tra Kss b3;185uchiresseitro muchi Yertoli3;305uchithe cthis article in press as: Meeker JD, et al. Semen quality and sperm DNty clinic. Reprod Toxicol (2010), doi:10.1016/j.reprotox.2010.07.005

nvolve the inclusion of urine samples collected in theNHANES, which had higher BPA concentrations thanlected in the afternoon in earlier NHANES datasets [4]., differences in the distribution of race/ethnicity andween the two study populations may also contributeerence in urinary BPA concentrations. For example,20032004 urinary BPA concentrations were higher

-hispanic blacks compared to non-hispanic whites, andng participants with lower household incomes [4]. Dif-study years should also be considered. Urine samplesnt study were collected in years 20002004 compared2006 in themost recentNHANESdata.However, urinaryeported in thepreviousNHANES investigation (NHANES) were even higher than in NHANES 20052006 [4].e present study was conducted among men recruitedinfertility clinic itmay limit our ability to generalize thee general population.However, themenwerememberseeking infertility diagnosis and treatment potentiallymale factor, a female factor, or both, resulting in a studythat included both fertile men and men with a range ofblems. In addition, in order for the generalizability ofto be limited, men recruited through an infertility clinicto respond differently (i.e. be more or less susceptible)sure compared tomen not in couples seeking infertilityeatment. Althoughwe are not aware of evidence show-n from an infertility clinic are more sensitive to BPAe did nd stronger relationships between urinary BPAons and reduced semen quality parameters amongmenst one parameter below WHO reference values. Thus,gest the possibility thatmenwith subfertility aremoreBPA-related effects than men with normal fertility. Ass are published on BPA exposure and semen quality, itt to consider the subject populationswhen interpreting

d that repeated urine samples collected from the sameto months apart from one another were weakly cor-0.18). Thus, another limitation in the present study isod of exposure measurement error due to the highvidual temporal variability in BPA exposure and theof multiple BPA measures from only a subset of par-owever, measurement error would be expected to bential, which would tend to reduce the ability to detects between exposure and outcome. In addition, whenexposure categories (e.g. quartiles in Fig. 1), a singley adequately predict an individuals exposure categoryer period of time [7]. We also collected only a singleple from each man, and the reliability of a single semenepresent semen quality over a longer period of time isaracterized. However, two recent reports provide evi-ne samplemay be representative of semenquality overks in large epidemiologic studies [33,34].limitation of our study was its cross-sectional designavailability of only a single semen sample from eachas well as the availability of only a single urine sam-er half of the men. Thus, we cannot rule out reversethe explanation of our ndings in the event that an

condition that causes poor semen quality may also leadPA metabolism.sion, human exposure to BPA may be associated withen quality and increased sperm DNA damage. How-some inconsistencies in our results between statisticalre assessment approaches, these relationships need to

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interest statement

ors declare that there are no conicts of interest.

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[33] Francavilla F, Barbonetti A, Necozione S, Santucci R, Cordeschi G, Macerola B,et al. Within-subject variation of seminal parameters in men with infertilemarriages. Int J Androl 2007;30(3):17481.

[34] Stokes-Riner A, Thurston SW, Brazil C, Guzick D, Liu F, Overstreet JW, etal. One semen sample or 2? Insights from a study of fertile men. J Androl2007;28(5):63843.this article in press as: Meeker JD, et al. Semen quality and sperm DNty clinic. Reprod Toxicol (2010), doi:10.1016/j.reprotox.2010.07.005age in relation to urinary bisphenol A among men from

Semen quality and sperm DNA damage in relation to urinary bisphenol A among men from an infertility clinicIntroductionMethodsUrinary BPASemen sample collectionSemen qualityNeutral comet assayStatistical analysis

ResultsDiscussionConflict of interest statementReferences

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