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Perinatal outcomes, mitochondrial toxicity andapoptosis in HIV-treated pregnant women and

in-utero-exposed newborn

Sandra Herna`ndeza,b,c,d, Constanza Morenb,c,d, Marta Lopeza,

Oriol Colla, Francesc Cardellachb,c,d, Eduard Gratacosa,

O`scar Mirob,c,d and Glo`ria Garraboub,c,d

Objectiv

mitochonewborn

Design:tion.

Methodfrommatreatedclinicalcontentdrial fun

Results:gestation7.33, 5.drial parmtDNAglycerolrespectilated, exwomen,correlate

Conclusdamageapoptosdemons

ity, pregnancy

aMaternoLaboratodCentro

CorrespoDepartm

Tel: +34Received

DOI:10.-Fetal Medicine Department, Clinical Institute of Gynaecology, Obstetrics and Neonatology, bMitochondrial Researchry, IDIBAPS-University of Barcelona, cInternal Medicine Department, Hospital Clinic of Barcelona, Barcelona, andde Investigacion Biomedica en Red de Enfermedades Raras, CIBERER, Valencia, Spain.

ndence to Glo`ria Garrabou, Mitochondrial Research Laboratory, IDIBAPS, University of Barcelona, Internal Medicineent, Hospital Clinic of Barcelona, CIBERER, Villarroel 170 08036, Barcelona, Spain.

932275400x2907; fax: +34 932279365; e-mail: garrabou@clinic.ub.es: 17 August 2011; revised: 13 October 2011; accepted: 14 November 2011.

1097/QAD.0b013e32834f3232

ISSN 0269-9370 Q 2012 Wolters Kluwer Health | Lippincott Williams & Wilkins 419Keywords: antiretroviral treatment, apoptoDNA, mitochondrial toxicight Lippinconpregnant adults, andHIV, additionally, to apoptosis.We determinedwhetherndrial toxicity and apoptosis are present in HIV-pregnant women and theirs and could be the basis of adverse pregnancy outcome.

Single-site, cross-sectional, controlled observational study without interven-

s: We studied mitochondrial and apoptotic parameters in mononuclear cellsternal peripheral blood and infant cord blood at delivery in 27HIV-infected andpregnant women, and 35 uninfected controls and their infants, to correlateoutcome with experimental findings: mitochondrial number (CS), mtDNA(ND2/18SrRNA), mitochondrial protein synthesis (COX-II/V-DAC), mitochon-ction (enzymatic activities) and apoptotic rate (caspase-3/b-actin).

Global adverse perinatal outcome, preterm births and small newborn foral age were significantly increased in HIV pregnancies [odds ratio (OR)77 and 9.71]. Mitochondrial number was unaltered. The remaining mitochon-ameters were reduced in HIV mothers and their newborn; especially newbornlevels, maternal and fetal mitochondrial protein synthesis and maternal-3-phosphate complex III function (38.6, 25.8, 13.6 and 31.2% reduced,vely, P

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Introduction

Antiretroviral therapy (ART) during pregnancy is criticalto prevhumanto delayantiretroMTCT,pregnan

The poexposurbeen restrengthAntiretrpregnanpretermtroversia

Severalposed tpregnaneffects(NRTIs(HAARnonpregdependg-polym(and toDNA (mmtDNAfinally c

Such mthe effeccause diin vitro,mechanAdditiodemonstissue fr

The kntoxicitythat somrisk of dexposurpregnanuninfectmodelNRTIaltered mand mit

Blanchemitochoperinatamental

mothers. Although few abnormal clinical findings areusually found in NRTI-exposed infants, the incidence ofmitochondrial dysfunction is increased in these children

26%e belysinlears (PBosedbeenNRTwn mreas,30]tentasseienct int

sideletion in newborn from HIV-infected mothers andlack of assessment of alternative markers of

ochondrial lesion we measured the real impact ofochoosedwledctionrelat

hyptoserlyinatastionmeth reslts w.

ien

signperervat

dy py-twsecutineternoic o

ocho-1-t coelive

420 AIDS 2012, Vol 26 No 4t Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.ent mother-to-child transmission (MTCT) ofimmunodeficiency virus (HIV) infection anddisease progression [1]. Widespread use of

virals has been accepted for the prevention ofdespite the lack of safety data related to humancies [24].

tential clinical risks associated with antiretrovirale in HIV-pregnant women, fetus and infants haveported by observational studies with varyings of evidence and conflicting results [59].oviral drugs have been associated with adversecy outcomes such as preeclampsia, fetal death,birth and low birth weight, although con-l results have been published [1015].

physiopathological mechanisms have been pro-o explain the adverse clinical effects in HIVcies. The negative mitochondrial and clinicalof nucleoside reverse transcriptase inhibitors) used in highly active antiretroviral therapyT) have been firmly established in HIV-infectednant adults [1618]. These negative effectson the capacity of the NRTI to inhibit DNAerase, the only enzyme devoted to the replicationa lesser extent, the repair) of the mitochondrialtDNA) genome, thus leading to a decrease incopy number and quality, which, in turn, mayause mitochondrial dysfunction [16].

itochondrial abnormalities may be magnified byts of HIV itself, since it has been demonstrated toffuse mitochondrial alterations, either in vivo orprobably through the activation of apoptoticisms triggered by HIV proteins [1921].nally, increased apoptosis rates have also beentrated after in-vitro exposure to NRTI and inom patients using antiretroviral drugs [22].

own patterns of mitochondrial and apoptoticin HIV patients receiving NRTI therapy suggeste children, though HIV-uninfected, may be ateveloping sequels from in-utero HIV and NRTIe [515,2332]. Mitochondrial toxicity alongcy has been studied in animal models includinged pregnant monkeys exposed to ART. Thisdemonstrates the association between in-uteroexposure and fetal tissue mtDNA depletion,itochondrial respiratory chain (MRC) function

ochondrial dysmorphology [23].

et al. [24] associated, for the first time,ndrial dysfunction with the manifestation ofl hyperlactaemia and neurological and develop-sequels in NRTI-exposed children from HIV

byhavananuccellexphasofshowhe[25connotefficligh

Condepthemitmitexpknofuncor

Weapoundperqueparawitresudata

Pat

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MitHIVnanat dndrial dysfunction on CBMCs of antiretroviral-HIV-uninfected children. Additionally, to ourge, no study has evaluated mitochondrialand apoptosis in HIV-pregnant women or has

ed these data with pregnancy outcome.

pothesized that mitochondrial toxicity andis caused by HIV and ART could be theing pathophysiological mechanism of adversel outcome in HIV pregnancy. To address thiswe compared mitochondrial and apoptotic

ers of HIV-infected women and their childrenpect to healthy controls to correlate experimentalith immunovirological, therapeutic and obstetric

ts and methods

formed a single-site, cross-sectional, controlledional study without intervention.

opulationo pregnant women were prospectively andtively included in the present study during theirprenatal care at first trimester of gestation, in the-Fetal Medicine Department of the Hospitalf Barcelona (Barcelona, Spain).

ndrial and apoptotic studies of 27 asymptomaticinfected pregnant women, 35 uninfected preg-ntrols and their newborns were performedry.[25]. However, conflicting experimental resultsen reported in the few studies currently availableg mitochondrial toxicity in cord blood mono-cells (CBMCs), peripheral blood mononuclearMCs) or placenta of asymptomatic antiretroviral-newborn [2432]. Consequently, no concernraised about the obstetric and perinatal safetyIs in human pregnancies. Some studies havetDNA depletion compared to controls [2629],others have reported no significant changes

and the remaining have even described increased[31,32]. Additionally, most of these studies didss mitochondrial number, function, translationy or apoptosis development, which would bringo the cell consequences of mtDNA depletion.

ring the controversial results regarding mtDNA

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Controls and cases were matched by age and parity. Theinclusion criteria for pregnant women were: age at least18 years, single pregnancy and delivery after at least22 weepatients

All indivwas obtby the E

In orderpatientschondriwith fam

An eleclogical,data, pe

Maternaincludedsubstanc

Immunowomenflow cytcopy quDiagnosalong pr

Antiretrinfectedlines. Hof theradrugs ddouble-or one n

Informainfectedtional dhypertendiastolicfetal deadelivery,birth wpercentiadmissioperinata

Finally,materna

SampleImmedicollected20ml oEDTA t

designed to prevent invasive sample collection and increasestudy participation. In both samples mononuclear cellswere isolated by Ficoll density gradient centrifugation,

ded

eletnocythe

teintein-

ochod mod mochoevalustandmennuclicatchetentochorRN

ochoasse

formteinterioodedecttedC;ndan

ochosphcomevalymaetryl. [3plexffectodedhineC ciallyndriressestraterote

ochoassetrop

Mitotoxicity and apoptosis in HIV pregnancy Herna`ndez et al. 421ks of gestation and, in case of HIV-infected, previous diagnosis of HIV-1 infection.

iduals were informed and signed written consentained for inclusion in this protocol and approvedthical Committee of our hospital.

to avoid confounders of mitochondrial toxicity,taking other potentially toxic drugs for mito-a were excluded from the study as were patientsilial history of mitochondrial disease.

tronic database was created to collect epidemio-obstetric, immunovirological and therapeuticrinatal outcome and experimental results.

l epidemiological and obstetric parametersinformation on maternal age, race, parity, illicite abuse and mode of delivery.

virological parameters for HIV-1-infectedconsisted in measuring CD4 T-cell count (byometry) and plasmatic viral load by HIV-1 RNAantification (Amplicor HIV Monitor; Rochetic Systems, Branchburg, New Jersey, USA)egnancy.

oviral therapy was administered to all HIV-pregnant women following international guide-IV women were stratified into different categoriespeutic care according to the use of antiretroviraluring pregnancy. HAART always consisted of aNRTI schedule and either one protease inhibitoron-NRTI drug (NNRTI).

tion regarding perinatal outcome for both HIV-pregnant women and controls included: gesta-iabetes mellitus, preeclampsia (new onset ofsion of >140mmHg systolic or >90mmHgpressure and >300mg proteins/24 h of urine),th (>22 weeks of pregnancy), gestational age atpreterm birth (

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activity [citrate synthase (CS): EC 4.1.3.7], a mitochon-drial enzyme of the Krebs cycle widely considered as areliable marker of mitochondrial content [34].

ApoptoTo evalublot anaPAGE eactive (csion (17proteinChemio3/b-actin relative content and were interpreted as amarker of advanced apoptotic events.

StatistiClinicalas meanmeans apercentacontrols

AdverseresultscomparechildrenproblemHIV inlations wmitochoproperome dapoptoton newbdata (toobstetric

Differenamongnonparafor indecalculateintervalcoefficiecance).statistica

Result

ClinicaThe clistudy padata of

All pregwith an

differences were observed in maternal epidemiologicaldata between HIV-positive and HIV-negative women.

HIVARTnths,

st HnanARTationvery

either ofor perc

delivble-nt w

inatobsdeta

infptomtationeoease, toelive

itioificacies0.04ifica7 vh 5735

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rease

-prerease.9%Tab

.5%inutle 1

422 AIDS 2012, Vol 26 No 4cal analysisand epidemiological parameters were expresseds and range interval and experimental results asnd standard error of the mean (SEM) or as age of increase/decrease with respect to healthy.

perinatal outcome, mitochondrial and apoptoticof HIV-infected women and newborn wered to those of uninfected women and theirto assess the presence of obstetric/perinatal

s and mitochondrial or apoptotic damage due tofection and ART. Additionally, different corre-ere sought between: molecular and functionalndrial parameters (to ascertain dependence ofmitochondrial function on mitochondrial gen-epletion); mother-to-child mitochondrial oric parameters (to determine maternal influenceorn cellular status); and clinical and experimentalassess mitochondrial or apoptotic basis ofproblems and perinatal outcome).

ces between cases and controls and correlationsquantitative parameters were assessed usingmetric tests: MannWhitney analysis to searchpendent sample differences, chi-squared test toodds ratio (OR) values [OR; 95% confidence(CI); significance] and the Spearmans ranknt for parameter correlation (R2 and signifi-The level of significance was set at 0.05 for all thel tests.

s

l datanical and epidemiological characteristics of therticipants and immunovirological and therapeuticHIV mothers are summarized in Table 1.

nant women were mainly of Caucasian origin,age ranging from 25 to 42 years. Nonsignificant

Atdoucou

PerTheare

AllsymGesandincrciesat d

AddsignnanPsign(7/2witthan32forHIV86.5

MitbloComwomdec1a).

HIVdec(25and(18dimTabtic studiesate apoptotic cell death we performed Westernlysis of 20mg of total cell protein by 7/13% SDS-lectrophoresis and posterior immunoanalysis ofleaved) caspase-3 pro-apoptotic protein expres-19 KDa) normalized by the content on b-actinsignal (47 KDa) as a cell loading control.luminescence results were expressed as caspase-

ForHAmo

Mopregforgestdelit Lippincott Williams & Wilkins. Unauthorized rparticipants, the mean time of HIV infection andtreatment prior to delivery was 84 and 48

respectively.

IV women were under HAART beforecy (85%) and only four cases (15%) were naiveand started HAART at the second trimester of. HAARTwas given to all patients at the time ofto avoid MTCTand consisted of two NRTI andne protease inhibitor or one NNRTI (see Table 1entages of treatment assignment).

ery, all women had received at least 6 months ofNRTI treatment and the mean CD4 T-cellas 560 cells/ml with an undetectable viral load.

al outcomestetric and neonatal outcomes of the study cohortiled in Table 2.

ants were HIV-uninfected with no clinicals compatible with mitochondrial toxicity.nal diabetes mellitus, gestational age at deliverynatal intensive care unit admission tended to bed, albeit not significantly, among HIV pregnan-gether with the reduction of birth weightry.

nally, global adverse perinatal outcomes werently more frequent among HIV-positive preg-(11/27 vs. 3/35, OR 7.33; 95% CI 1.830.1;8). The overall prematurity rate was alsontly increased for newborns to HIV motherss. 2/35, OR 5.77; 95% CI 1.130.6; P 0.034)% (4/7) of the premature deliveries being at lessweeks, 50% of which (2/4) were at less thans. Finally, the number of infants who were smalltional age was also significantly increased amonggnancies (6/27 vs. 1/35, OR 9.71; 95% CI 1.1 0.036).

ondrial and apoptotic maternal peripheralononuclear cell analysis

ed with uninfected controls, HIV-pregnantshowed a marked but nonsignificant trend to ad mtDNA content of 26.3% (PNS, and Table

gnant women also exhibited a significantin the mitochondrial protein synthesis rate

COX-II/V-DAC content reduction, P 0.042;le 1a and b) and all MRC enzyme activitiesfor CIV and 31.2% for G3PdhCIII functionion; PNS and P 0.049, respectively; anda). The only mitochondrial enzymatic activityeproduction of this article is prohibited.

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which wII (increas a comtDNA

No diffactivity)mtDNAMRC ewhen nshown).

HIV-infalbeit ncompareand b).

Materna(G3Pdhcorrelat

No signmaternaparamet

com), exre pnthshosetent

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tent0.0

ilaribitethesi0.04reasetrolsvityordi

Mitotoxicity and apoptosis in HIV pregnancy Herna`ndez et al. 423

Table 1. Clinic, epidemiologic, immunovirologic and therapeutic characteristics of HIV-infected and uninfected pregnant women.

HIV-positive (n27) HIV-negative (n35) P

Maternal age at deliverya 34.7 (2742) 33.7 (2541) NSHCV infection [N (%)] 3 (11.1) 1 (2.6) NSIllicit drug use [N (%)] 0 (0) 0 (0) Alcohol use [N (%)] 0 (0) 0 (0) HIV RNA copies per ml at deliverya 62.3 (49250) CD4 T-cell count per ml at deliverya 560.2 (971242) NRTI prior pregnancy (months)a 48 (0106) NNRTI prior pregnancy (months) a 3 (086) PI prior pregnancy (months)a 12 (097) Time from diagnosis of HIV infection to delivery (months)a 84 (4228) HAART second and third trimesters2NRTI1PI [No. (%)] 4 (15)

HAART all trimesters2 NRTI1 PI [No. (%)] 15 (55.5) 2 NRTI1 NNRTI [N (%)] 8 (29.5)

HCV, hepatitis C virus; HIV, human immunodeficiency virus; NRTI, nucleoside-analogue reverse transcriptase inhibitor; NNRTI, non-NRTI reversetranscriptase inhibitor; NS, not significant; PI, protease inhibitors; RNA, ribonucleic acid.aData are presented as means and range interval.

Table 2.

GestationPreeclamFetal deaGestationPreterm bBirth weiSmall for5-min ApNeonatalGlobal ad

95% CI, 9aData areas preserved in HIV women was MRC complexased 13.7% compared to controls, PNS), usedntrol parameter because it is independent todepletion.

erences were found in mitochondrial mass (CSbetween cases and controls and, consequently,content, mitochondrial protein synthesis or

nzymatic activities maintained the same trendsormalized to mitochondrial mass (data not

ected pregnant women presented a marked,onsignificantly increased apoptotic rate of 100%d with healthy controls (PNS; and Table 1a

l mtDNA levels and mitochondrial functionCIII activity) were positively and significantlyed (Fig. 2).

ificant correlation was, however, found betweenl experimental (mitochondrial or apoptotic)

outagebefomoto tcon

MitbloInfaandcon(P

SimexhsynPdecconactiAccight Lippincott Williams & Wilkins. Unauthorize

ers and clinical results (including perinatal enzyma

Obstetric and neonatal outcomes of the study cohorts.

HIV-positive (n27)

al diabetes mellitus [N (%)] 2 (7.4)psia [N (%)] 0 (0)th [N (%)] 0 (0)al age at delivery (weeks)a 37.5 (32.241.2)irth (

Copyright Lippincott Williams & Wilkins. Unauthorized r

cases andand HA

Again, n(CS actsequentsynthesiwhen nshown).

Contrarlevelsactivity)infants bapoptot(PNS

424 AIDS 2012, Vol 26 No 4

-50

0

50

100

150

MtDNA contentMt Protein synthesis

Mt activity (CIV)Mt activity (G3Pdh+CIII)

**% re

spec

t to

cont

rols Maternal(a)

(b)

% respect to c

Newborn

50

100

150

**

Pregnant women

Control HIV-infected

MtDNA

(ND2mtDNA/18SrRNAnDNA)4.450.59 3.280.35

26.3%, p=NS

Mt Protein Synthesis

(COX-II/V-DAC relative units)1.430.23 1.060.14

25.9%, p=0.042

Mt activity CIV

(nmol/min.mg prot)47.725.91 38.893.90

18.5%, p=NS

Mt activity G3Pdh+CIII

(nmol/min.mg prot)27.893.58 19.192.41

31.2%, p=0.049

Mt control activity CII

(nmol/min.mg prot)22.242.33 25.283.22

+13.7%, p=NS

Apoptosis

(Caspase-3/-Actine relative units)0.180.05 0.360.09

+100%, p=NS

V- DAC (31 KDa) COX -II (25.6 KDa)

HIV+HIV-M N M N

Act.Casp -3 (17-19 KDa) -Actin (47 KDa)

Fig. 1. Mitochondrial and apoptotic parameters. (a) Percentage of incrparticipants with respect to mean values of uninfected controls. MtDNA, mitoprotein synthesis (COX-II/V-DAC); CIV, G3PdhCIII or CII enzymatic acglycerol-3-phosphate dehydrogenase and complex III or complex II enzymatand activation (caspase-3/b-Actin). () P 0.042; () P0.049; (y) P0.006synthesis and apoptosis quantification. HIV: human immunodeficiencymothers/newborn.

100

75

50

G3P

dh+C

III(nm

ol/mi

n.mg p

rot)

25

00.0 2.5 5.0

mtDNA(ND2 mtDNA/18 SrRNA nDNA)

mtDNA(ND2 mtDNA/18 SrRNA nDNA)

7.5 10.0

100

75

50

G3P

dh+C

III(nm

ol/mi

n.mg p

rot)

25

00.0 2.5 5.0 7.5 10.0

NewbornMaternal

P = 0.474

P = 0.590

P = 0.108

R2 = 0.130

R2 = 0.001

R2 = 0.107

P = 0.216

P = 0.016

P = 0.077

R2 = 0.113

R2 = 0.166

R2 = 0.158

Fig. 2. Correlation between genetic and functional mito-chondrial parameters.Association between genetic and func-tional parameters is shown in the graph for HIV-pregnantwomen or newborn from HIV pregnancies (black line andsignificance), for control women and newborn (grey line andsignificance) and for all included women or children (dis-continued line and significance in bold).Mt control activity (CII)

ontrols-500

Apoptosis

Newborn

Control HIV-mothers

3.060.26 1.880.32

38.6%, p=0.006

1.800.34 1.280.16

28.9, p=0.045

43.695.54 37.244.10

14.8%, p=NS

21.844.75 15.522.26

28.9%, p=NSeproduction of this article is prohibited.

controls was complex II (0.1% decreased in HIVART-exposed infants, PNS).

o differences were found in mitochondrial massivity) between cases and controls and, con-ly, mtDNA content, mitochondrial proteins or MRC function preserved identical trendsormalized to mitochondrial mass (data not

ily to their mothers, however, newborn mtDNAand mitochondrial function (G3PdhCIIIwere not correlated (Fig. 2) and seronegativeorn from HIV women presented an unalteredic rate (18.9% decreased) with respect to controls; and Table 1a and b).

28.383.41 28.363.36

0.1%, p=NS

0.560.13 0.460.13

18.9%, p=NS

ease/decrease of mitochondrial parameters in HIVchondrial DNA;Mt Protein synthesis, mitochondrialtivity, mitochondrial respiratory chain complex IV,ic function, respectively; apoptosis, caspase cleavage; (z) 0.045. (b) Western blot for mitochondrial proteinvirus infected/not-infected pregnant-women; M/N:

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Additiocorrelatand apoeither co(Fig. 3).

No signnewborimmunoperinata

Discus

Animaltoxicity

Mitotoxicity and apoptosis in HIV pregnancy Herna`ndez et al. 425

P = NSR2 = 0.04

P = 0.00012

9

NA

(NA

nDNA

)

0

5.0

7.5

2.5

0.0

V-DA

C)

0.0

75

00

50

25

Fig. 3. Cfetal parsignificaprotein schondriaCasp3/bP = 0.0004

P = 0.0001

P = 0.0091

R2 = 0.352

R2 = 0.362

R2 = 0.374

0Maternal mtDNA

(ND2 mtDNA/18SrRNA nDNA)

3 6 9

1

New

born

G3P

dh+

CIII

(nmol/

min.m

g prot

)

100

150

50

New

born

mtD

NA

ND2 m

tDNA

/18Sr

RNA

nDNA

)6P = NS

P = NSR2 = 0.04

R2 = 0.003

New

born

mtD

D2 m

tDNA

/18Sr

RN

New

born

COX

-II/

(relat

ive u

nitsight Lippincott Williams & Wilkins. Unauthorize

nally, there was a positive and significantion between all maternal and fetal mitochondrialptotic parameters except in mtDNA content,nsidering exclusively HIV cases, controls or both

ificant correlation was, however, found betweenn mitochondrial parameters and maternallogical status, ART regimen, age or adversel outcome.

sion

models have shown evidence of mitochondrialfrom in-utero NRTI exposure [23]. However,

clinicalbeen lacconflictiHIVandto mitoreducedand MRwomentheir inmitochodifferenreducedsuggestiOur resmtDNAtreated m

P = 0.0462

P = 0.0002

P = 0.0007

R2 = 0.193

R2 = 0.324

R2 = 0.572

00

1.5

2.0

2.5

1.0

0.5

0.0

New

born

Cas

p3/-

Actin

(relat

ive u

nits

)

0.0

50

75

25

0

New

born

MR

C CI

I(nm

ol/mi

n.mg p

rot)

0Maternal MRC CII(nmol/min.mg prot)

25 50 75

0

(

0Maternal MRC CIV

(nmol/min.mg prot)

50 100 150

orrelation between materno-fetal mitochondrial or apoptotic parameters are shown in the graphs for HIV patients (grey line andnce) or for both patients and controls (discontinued line and signynthesis, mitochondrial protein synthesis (COX-II/V-DAC); MRCl respiratory chain complex IV, glycerol-3-phosphate dehydrogena-actin, apoptotic development (caspase-3/b-actin).P = 0.0084

P = 0.0001

P = 0.0009

R2 = 0.312

R2 = 0.360

R2 = 0.56

P = 0.0001

P = 0.0001

R = 0.860

R2 = 0.848

R2 = 0.821

Maternal COX-II/V-DAC(relative units)

2.5 5.0 7.5d reproduction of this article is prohibited.

evidence of mitochondrial toxicity has largelyking in HIVmothers and their infants and there isng evidence regarding the association of in-uteroARTexposure with mortality and morbidity duechondrial dysfunction [2432]. We observed amtDNA content and derived protein synthesisC function in both PBMC of HIV-pregnantat delivery receiving ARTas well as in CBMC of-utero-exposed newborns. Although not allndrial parameters showed statistically significantces between cases and controls, all measures werein HIV-pregnant women and their newborn,

ng NRTI or HIV-mediated mitochondrial lesion.ults are in concordance with the studies reportingdepletion in newborn from HIV-infected andothers [2629]. However, none of these studies

P = 0.0484

P = 0.0049

P = 0.0073

R2 = 0.155

R2 = 0.205

R2 = 0.353

Maternal G3Pdh+CIII(nmol/min.mg prot)

25 50 75 100

Maternal Casp3/-Actin(relative units)

0.5 1.0 1.5 2.0 2.5

ameters. Association between quantitative materno-significance), uninfected controls (black line and

ificance in bold). MtDNA, mitochondrial DNA; MtCIV, G3PdhCIII or CII enzymatic activity, mito-se and complex III or complex II enzymatic function;

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parallely evaluated mitochondrial number, mtDNA-encoded MRC protein expression and mitochondrialfunction in HIV and HAART-exposed newborn.The ptoxicitydepletiomitochomitochogenetics

Althoug18], thireportedMitochotreated wetiopathand couobstetric

Whetheand theiof doubreceivedguidelindepletioistrationmediatechondriapoptosderivedindirect[1921]previouinfants.increasein theirsuggestsphenomwomen,becausepregnanstudies apresentnewborpopulaticould btherebyHIV innewborof HAA

Despiteand apo(PBMCand signWe attriof mitooocyte

In our opinion, different theories may explain materno-fetal correlation of mitochondrial parameters andstrengthen the hypothesis of NRTI-mediated mitochon-

l toxage.ld limth

exclentludi], be

restiitiveochowevemetciaticies.NAeen

epenparapacort,freqificadrennotainicomassocectonsed aoutborabolclini

studnonuptotictlyll sizotheinataof pctedlicatborveryur inreceioupletlternard

426 AIDS 2012, Vol 26 No 4resent work strengthens the mitochondrialhypothesis by confirming that fetal mtDNAn is not an isolated finding and subclinicalndrial lesion is present in several markers ofndrial function downstream from mitochondrial.

h previously reported in nonpregnant adults [16s is the first time that similar results have beenin HIV-infected and treated pregnant women.ndrial lesion in oocytes of HIV-infected andomen has previously been suggested to play anological role in the infertility of these women [17]ld, potentially, be the cause of their associatedproblems.

r mitochondrial lesion in both infected mothersr newborn is due to HIVor ART is still a mattert because, in our study, all HIV-pregnant womenART prior delivery following international

es. Interestingly, we found increased mtDNAn in HIV-pregnant women with NRTI admin-extended over 100 months, suggesting NRTI-d injury. However, HIV implication in mito-al lesion can not be discarded, especially becauseis was increased in HIV-infected women. HIV-mitochondrial toxicity has been reported to bely caused by the activation of apoptotic pathways. To our knowledge, apoptotic studies have neversly been performed in HIV mothers and theirWe observed that apoptotic events tend to bed in HIV-pregnant women but remain unalterednewborn. This finding, although not significant,that HIV itself could trigger the apoptoticenon exclusively in HIV-infected pregnantwhereas their infants, which are uninfectedof the therapeutic activity of HAART alongcy, show an unaltered apoptotic rate. Furtherre required to elucidate if apoptotic lesion is alsoin naive pregnant women and in HIV-infectedns. If apoptotic lesions are increased in theseons, obstetric problems of HIV pregnanciese attributed, at least in part, to apoptotic means,confirming the prevention of not only MTCToffection but also HIV-mediated apoptosis inn from HIV-infected women with the useRT.

studying maternal and newborn mitochondrialptotic parameters in different kinds of tissuess and CBMCs, respectively), all were positivelyificantly correlated, except for mtDNA content.bute this concordance to the maternal heritancechondria organelles, exclusively provided by thealong the fecundation process [17].

driadamcouis innonpres(inc[36

InteposmitHoparaassonanmtDbetwindcomor ccohandsignchilwasremoutberespdemplayrulenewmetsub

Themoapodiresmahypperlackinfeimpnewdeliin ohadprevcomin adisct Lippincott Williams & Wilkins. Unauthorized ricity, in detriment of HIV-induced mitochondrialFirst, mitochondrial injury inmaternal blood cellsit its transplacental function once maternal blood

e fetus. Second, mitochondrial toxicity may beusively restricted tomaternal cells and could also bein tissues more related to fetal developmentng placenta) [29]. Finally, NRTIs cross the placentacoming direct fetal-damaging agents.

ngly, maternal mtDNA levels and functionly correlated, demonstrating the dependence ofndrial functionalism on mitochondrial genetics.r, mtDNA levels and function were independenters in their newborn, with no trend towardson, especially in infant born from HIV preg-This finding may explain why maternal and fetallevels were the only parameters not associatedmother and child; mtDNA levels seem to be

dently regulated in maternal and newborntments, perhaps due to distinct biological needsities of response to toxic exposure. In our HIVglobal adverse perinatal outcome, preterm birthuency of small for gestational age newborns werently increased. However, in these women and, mitochondrial and/or apoptotic compromiseincreased compared to uninfected controls or theng HIV cohort. Although all these adversees are unspecific of HIV and HAART and couldiated with other pathological situations, withto HIV pregnancies, our findings failed totrate that mitochondria or apoptosis disturbancesrole in infant outcome. This assertion does notthe possibility that mitochondrial dysfunction inns could contribute to the future development ofic problems in adulthood, independently of itscal perinatal consequences [37].

y may have some limitations. First, the analysis ofclear cells could show different mitochondrial andc results compared to those present in other tissuesrelated to pregnancy development. Second, thee of our sample could hinder the testing of oursis (mitochondrial or apoptotic basis of adversel outcome), independently of its veracity. Third,regnant HIV women naive for HAARTor HIV-children could limit the assessment of HIV-

ion in adverse perinatal outcome. Fourth, lack ofn symptomatic of mitochondrial toxicity atcould reduce the presence of molecular lesionfant population. Fifth, although all treatedwomenived at least 6 months of double-NRTI schedules,s and current treatment options were notely uniform and the results could therefore differative treatment interventions. Finally, we cannotthe possibility that adverse effects of HIV oreproduction of this article is prohibited.

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HAART, alternative to mitochondrial dysfunction orapoptosis, may play a role.

DespiteART adtransplacin bothnewborcorrelated mitochondrial protein synthesis and enzymaticfunction. On the contrary, antiretroviral drugs maypreventbenefici

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S.H., Mfollow-uinformaand G.Gimentalparticipanalyses

We alsohelp of Ester Tobias and Mireia Nicola`s as laboratorytechnicians and Donna Pringle for language assistance.

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1. VolmAntimissCD0

2. Watts DH. Management of human immunodeficiency virusinfection in pregnancy. N Engl J Med 2002; 346:18791891.

3. Public Health Service Task Force. Recommendations for the useof antiretroviral drugs in pregnant HIV-1 infected women formattranSturingDataBrocHIVthe105

6. ThooutcantiTuoHugthe187Tuoet alareactimunLamBethandinfeClinSuyet ainfetherWimThoapyRudPopmatHaeBogimmacti201TowAntHIV200

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Mitotoxicity and apoptosis in HIV pregnancy Herna`ndez et al. 427dy has been funded by Fundacion para laacion y la Prevencion del SIDA en Espana (FIPSE09 and 360982/10), Fondo de Investigaciona (FIS 0229/08 and 00462/11), Red de Sida (RD, Suports a Grups de Recerca de la Generalitat deya (SGR 09/1158 and 09/1385) and CIBER deedades Raras (CIBERER, an initiative of ISCIII).

ts of interestf the authors has any financial, consultant,onal and other relationship that might lead to biasflict of interest for the present manuscript.

nces

ink J, Siegfried NL, van der Merwe L, Brocklehurst P.retrovirals for reducing the risk of mother-to-child trans-ion of HIV infection. Cochrane Database Syst Rev 2007;03510.

16.

17.

18.

19.

20.

21.

22.newborn apoptotic lesions, thereby leading toal effects independent of MTCT prevention.

clusion, no relationship was found betweenndrial and apoptotic abnormalities and adversel outcome. However, mitochondrial toxicityd with NRTI administration and newbornty should be further investigated in tissuesrelated to pregnancy in larger cohorts of patientsg naive pregnant HIV women or women underparing regimens, as well as HIV-infected new-d newborn with symptomatic manifestation ofndrial or apoptotic lesion.

wledgments

.L. and O.C. performed patient inclusion andp and collected samples and all the clinicaltion of the study participants. C.M., F.C., O.M.. processed samples and performed the exper-analysis of experimental parameters. All authorsated in the study design, statistical and resultand manuscript production.

wish to thank the collaboration and the valuable

7.

8.

9.

10.

11.

12.

13.

14.these limitations, we can, however, conclude thatministration along pregnancy may cause theental subclinical mitochondrial lesions observedpregnant women and their in-utero-exposed

n. This finding is confirmed by materno-fetal

4.

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428 AIDS 2012, Vol 26 No 4t Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.

Perinatal outcomes, mitochondrial toxicity and apoptosis in HIV-treated pregnant women and in-utero-exposednewbornIntroductionPatients and methodsDesignStudy populationSample collection and processingMitochondrial studies in maternal peripheral blood mononuclear cells and newborn cord blood mononuclear cellsMitochondrial DNA quantificationMitochondrial protein synthesisMitochondrial respiratory chain complex II, glycerol-3-phosphate dehydrogenase+complex III (G3Pdh+III) and complex IV (COX) enzyme activityMitochondrial mass

Apoptotic studiesStatistical analysis

ResultsClinical dataPerinatal outcomesMitochondrial and apoptotic maternal peripheral blood mononuclear cell analysisMitochondrial and apoptotic newborn cord blood mononuclear cell analysis

DiscussionAcknowledgmentsConflicts of interest