Mitochondrial toxicity in HIV type-1-exposed pregnancies in the era of highly active antiretroviral therapy.
ABSTRACT The aim of this study was to determine the effects of HIV type-1 (HIV-1) infection and antiretroviral therapy (ART) on placental mitochondria.
HIV-1-infected pregnant women and HIV-1-uninfected controls were enrolled prospectively. Placental mitochondrial DNA (mtDNA) copy numbers were determined by quantitative PCR, subunits II and IV of cytochrome c oxidase (COX) were quantified by western blot and mitochondrial ultrastructure was evaluated by electron microscopy. Venous blood lactate was measured in newborns.
In total, 45 HIV-1-infected pregnant women on ART and 32 controls were included. Mean +/-sd mtDNA copy numbers were significantly reduced in ART and HIV-1-exposed placentas (240 +/-118 copies/cell) in comparison with controls (686 +/-842 copies/cell; P<0.001). The mean COX II/IV ratio was 48% lower in the investigational group compared with controls (P<0.001). There was no evidence of severe ultrastructural damage within mitochondria of HIV-1-infected ART-exposed placentas. Although lactate levels between newborns did not differ, they were negatively correlated with placental mtDNA levels. There was no clear association between mitochondrial parameters and a particular nucleoside reverse transcriptase inhibitor (NRTI), the number of NRTIs or time of NRTI exposure.
Placental tissue of HIV-1-infected ART-exposed pregnancies shows evidence of mtDNA depletion with secondary respiratory chain compromise. The clinical effects of this finding warrant further investigation.
- [Show abstract] [Hide abstract]
ABSTRACT: Oxidative stress has been suggested to play a key role in the neuropathogenesis of HIV infection. HIV proteins (gp120, Tat) and proinflammatory cytokines can trigger the production of reactive oxygen species (ROS), resulting in DNA and RNA lesions. Among all the lesions induced by ROS, one of the most abundant lesions in DNA and RNA is 8-hydroxydeoxyguanosine (8-oxoG). Here, we studied accumulated DNA oxidative damage induced by ROS in the central nervous system (CNS) in tissue from neuro-AIDS patients. The frontal cortex of autopsy tissue from HIV-1 infected patients was adopted for analysis for HIV-1 subtype, nuclear and mitochondrial DNA lesions by immunofluorescence staining, qPCR and sequencing of PCR cloning. This study provides evidence that HIV infection in the CNS leads to nuclear and mitochondrial genomic DNA damage in the brain. High level of nuclear and mtDNA 8-oxoG damage were identified in the cortex autopsy tissue of HAND patients. Increased accumulation of mtDNA mutations and depletion occurs in brain tissue in a subset of HAND cases, and is significantly different from that observed in control cases. These findings suggest that higher level of ROS in the CNS of HAND patients would contribute to the HIV induced neuro-inflammation and apoptosis of neuronal and glial cells.Brain research 04/2012; 1458:1-11. · 2.83 Impact Factor
Article: HIV-Infektion in der Schwangerschaft[Show abstract] [Hide abstract]
ABSTRACT: Zusammenfassung Mit Hilfe der modernen antiretroviralen Therapie kann die vertikale Transmission von HIV-1 inzwischen auf unter 1% reduziert werden. Dafür ist eine interdisziplinäre Betreuung der HIV-positiven Schwangeren in einem HIV-erfahrenem Zentrum durch einen HIV-Behandler, Gynäkologen und Pädiater erforderlich. Bei einer effektiven Viruslastsenkung der Mutter erfolgt auch bei einer HIV-Infektion die Wahl des Geburtsmodus nur nach geburtshilflichen Kriterien. Der Stillverzicht gehört in Ländern mit ausreichenden Ressourcen weiterhin zum Standard.Der Gynäkologe 08/2011; 44(8).
- [Show abstract] [Hide abstract]
ABSTRACT: Despite a wide body of literature supporting the use of antenatal antiretrovirals (ARV) for the prevention of mother to child transmission, there remains a need for continued monitoring as the intrauterine interval is a critical period during which fetal programming influences the future health and development of the child. We conducted a systematic review of the current literature addressing potential metabolic complications of in utero HIV and ARV exposure. We describe studies evaluating metabolic outcomes such as intrauterine and early postnatal growth, bone health, and mitochondrial toxicity. Overall, infants exposed to HIV/ARV do not appear to exhibit vastly compromised intrauterine or early postnatal growth. However, some studies on the effect of combination antiretroviral therapy (cART) on small for gestational age (SGA) and low birth weight (LBW) outcomes in low-middle income countries show a risk for SGA/LBW while those in the U.S. do not. Postnatal growth to 1 year does not appear to be affected by intrauterine tenofovir exposure in African studies, but a U.S. study found statistically significant differences in length for age z scores (LAZ) at 1 year. Little data exists on long term bone health. Mitochondrial toxicity including abnormal mitochondrial morphology and DNA content, as well as neurologic deficits and death have been demonstrated in HIV/ARV-exposed infants. Though gross measures of metabolic well-being appear to be reassuring, careful vigilance of even small risks for potential serious adverse effects to infants exposed to intrauterine HIV/ARVs is warranted as intrauterine fetal metabolic programming may substantially impact the future health of the child.The Pediatric Infectious Disease Journal 12/2013; · 3.14 Impact Factor
©2009 International Medical Press 1359-6535 (print) 2040-2058 (online)331
Antiviral Therapy 14:331–338
Background: The aim of this study was to determine the
effects of HIV type-1 (HIV-1) infection and antiretroviral
therapy (ART) on placental mitochondria.
Methods: HIV-1-infected pregnant women and HIV-1
-uninfected controls were enrolled prospectively. Pla-
cental mitochondrial DNA (mtDNA) copy numbers were
determined by quantitative PCR, subunits II and IV of
cytochrome c oxidase (COX) were quantified by western
blot and mitochondrial ultrastructure was evaluated by
electron microscopy. Venous blood lactate was measured
Results: In total, 45 HIV-1-infected pregnant women on
ART and 32 controls were included. Mean ±sd mtDNA copy
numbers were significantly reduced in ART and HIV-1-
exposed placentas (240 ±118 copies/ cell) in comparison
with controls (686 ±842 copies/cell; P<0.001). The mean
COX II/IV ratio was 48% lower in the investigational
group compared with controls (P<0.001). There was no
evidence of severe ultrastructural damage within mito-
chondria of HIV-1-infected ART-exposed placentas.
Although lactate levels between newborns did not differ,
they were negatively correlated with placental mtDNA
levels. There was no clear association between mito-
chondrial parameters and a particular nucleoside reverse
transcriptase inhibitor (NRTI), the number of NRTIs or
time of NRTI exposure.
Conclusions: Placental tissue of HIV-1-infected ART-
exposed pregnancies shows evidence of mtDNA depletion
with secondary respiratory chain compromise. The clinical
effects of this finding warrant further investigation.
Highly active antiretroviral therapy has effectively
reduced mother-to-child HIV type-1 (HIV-1) trans-
mission in developed countries . Current guidelines
recommend the use of nucleoside reverse transcriptase
inhibitors (NRTIs) during pregnancy and emphasize
zidovudine as a routine part of the antiretroviral regi-
men . Apart from the undisputed benefits of NRTIs,
concerns arise from their potential risk for the fetus,
which is highly exposed because NRTIs readily cross
the placenta . Blanche et al.  were the first to report
on severe encephalomyopathies and mitochondrial
dysfunction in HIV-1-negative newborns exposed pre-
natally to antiretrovirals. Although such mitochondrial
syndromes might be the exception rather than the rule,
as suggested by another big perinatal cohort , ani-
mal models and human studies raise concern about a
possible compromise of mitochondrial function in the
fetal compartment and a high incidence of hyperlac-
tataemia during the first months of life . A quanti-
tative reduction of mitochondrial DNA (mtDNA), for
example, was seen in peripheral blood mononuclear
cells (PBMCs) of HIV-1-uninfected children exposed
to zidovudine in utero, and was shown to persist even
at 2 years of age . It is, however, still unclear to what
extent such mitochondrial damage influences the life
of children born to HIV-1-infected mothers. In clinical
Mitochondrial toxicity in HIV type-1-exposed
pregnancies in the era of highly active antiretroviral
Andrea Gingelmaier1*, Thomas A Grubert2, Bernd P Kost1, Bernhard Setzer3, Dirk Lebrecht3, Ioannis Mylonas1,
Josef Mueller-Hoecker4, Udo Jeschke1, Stephan Hiedl5, Klaus Friese1 and Ulrich A Walker3,6
1Department of Obstetrics and Gynaecology, Ludwig Maximilians University of Munich, Munich, Germany
2Medical Practice of Obstetrics and Gynaecology, Ravensburg, Germany
3Department of Rheumatology and Clinical Immunology, Albert Ludwigs University of Freiburg, Freiburg, Germany
4Department of Pathology, Ludwig Maximilians University of Munich, Munich, Germany
5Department of Paediatrics, Ludwig Maximilians University of Munich, Munich, Germany
6Department of Rheumatology, Basel University, Basel, Switzerland
*Corresponding author: E-mail: firstname.lastname@example.org
© 2009 International Medical Press
A Gingelmaier et al.
practice, zidovudine is not the only drug used to pre-
vent vertical HIV-1 transmission; rather, a wide variety
of different NRTI combination therapies are frequently
administered, even in the first trimester. Concern also
arises from the in vitro evidence that antiretrovirals
that per se do not have any mitochondrial effect on
their own can confer unpredictable mitochondrial
toxicity when given in combination . In order to
elucidate the fetal risk, we examined biomarkers of
mitochondrial toxicity in placentas of HIV-1-infected
antiretroviral therapy (ART)- exposed individuals, and
compared them to placental tissue of healthy women.
After approval from the local ethics committee and
informed written consent from participants, pregnant
HIV-1-infected women were prospectively enrolled
from March 2003 until November 2006 in the outpa-
tient clinic of the Department of Obstetrics and Gynae-
cology, University of Munich, Munich, Germany. The
HIV-1-infected ART-exposed mothers had a planned
caesarean section 2 weeks prior to the estimated date of
delivery according to the German-Austrian Guidelines
for the Care of HIV-infected Pregnant Women . Their
newborns received zidovudine for 4 weeks post-partum
and were considered to be HIV-1-uninfected if HIV-1
RNA was undetectable in three repeat blood samples
by the age of 3 months. All newborns remained in regu-
lar follow-up until 18 months of age. The control group
consisted of HIV-1-uninfected healthy pregnant women
who delivered during the study period at the same insti-
tution. The human experimentation guidelines of the
US Department of Health and Human Services and of
the authors’ institutions were followed in the conduct
of clinical research.
Peripheral blood lactate levels of the newborns were
measured between days 3 and 5 post-partum. Venous
blood was drawn in a fluorinated tube and care was
taken to draw the sample at rest, without the use of a
tourniquet if possible, to transport the blood to the lab-
oratory on ice and to insure its immediate automated
analysis (Cobas Integra 800; F Hoffmann–La Roche,
Ltd., Basel, Switzerland).
Mitochondrial DNA copy number
Immediately after delivery, a full-thickness placental sec-
tion was collected and, after removal of the decidua, was
frozen at -80°C until batched processing for further anal-
ysis. Total DNA was extracted from the placenta with the
QIAamp DNA isolation kit (Qiagen, Hilden, Germany).
mtDNA and nuclear DNA (nDNA) copy numbers were
determined by quantitative PCR as previously described
. Amplifications of mitochondrial and nuclear prod-
ucts were performed in triplicates. Absolute mtDNA and
nDNA copy numbers were calculated using serial dilu-
tions of plasmids with known copy numbers.
Mitochondiral DNA-encoded respiratory chain
The subunit II of cytochrome c oxidase (COX) is encoded
by mtDNA, whereas the subunit IV of COX is encoded
by nDNA. COX II and COX IV western blot analyses
were conducted as follows: placental extracts were pre-
pared with RIPA buffer (50 mM Tris, pH 8.0, 150 mM
NaCl, 1% NP40, 0.5% doxycholine and 0.1% SDS)
and 20 µg of protein (BioRad Bradford Assay; BioRad,
Munich, Germany), and were subjected to SDS-PAGE.
Proteins were transferred to polyvinylidenefluoride
membranes in a BioRad Mini Protean II Cell (BioRad) at
1 mA/cm2 membrane in 10% methanol, 50 mM borate
and pH 8.0. Membranes were blocked with 4% non-
fat milk powder for 1 h. Primary antibodies (murine
monoclonal anti-COX II antibody [Sigma Aldrich Co.,
St Louis, MO, USA] or murine monoclonal anti-COX IV
antibody [Molecular Probes; Invitrogen, Carlsbad, CA,
USA]) were applied in blocking buffer (COX II dilution
1:1,000 and COX IV dilution 1:200) and incubated at
room temperature overnight. After washing in Tris buffer
saline (TBS)/Tween (20 mM Tris/HCl, pH 7.2, 1 M NaCl
and 0.05% Tween 20), membranes were incubated with
a secondary antibody (rabbit anti-mouse immunoglob-
ulin G; Vectastain Elite ABC Kit; Vector Laboratories,
Burlingame, CA, USA; dilution 1:200). After incuba-
tion with ABC reagent (Vectastain Elite ABC kit; Vector
Laboratories) for 1 h and repeated washings with TBS/
Tween, visualization was performed by applying the
colorimetric alkaline phosphatase reaction (BCIP/NBT;
Promega, Mannheim, Germany). The intensities of the
signals were quantified by scanning densitometry using
the image analyser Quantity One (BioRad).
We randomly selected placental tissue from four HIV-1-
infected women exposed to the combination of zidovu-
dine plus lamivudine, four HIV-1-infected mothers coex-
posed to emtricitabine and tenofovir disoproxil fumarate,
and six HIV-1-uninfected controls for ultrastructural
studies. Immediately after delivery, placental tissues
were separated into amnion, chorion and decidua, and
fixed for 2 h at room temperature in 6.25% Soerensen
phosphate-buffered glutaraldehyde. After washing in
saccharose (0.2 M in Soerensen phosphate buffer), the
tissue was post-fixed for 1.5 h in osmium (2% in distilled
water), dehydrated in graded acetone and embedded in
Epon. Semi-thin sections were stained with methylene
blue. Ultra-thin sections were counterstained with uranyl
Mitochondrial toxicity in HIV-1-exposed pregnancies
Antiviral Therapy 14.3
acetate and lead citrate and viewed with a Philips EM
420 microscope (Phillips, Eindhoven, the Netherlands).
The normal distribution of the group variables was
tested with the Kolmogorov–Smirnov test. Subse-
quently, group means were compared using unpaired
t-tests or Wilcoxon tests as appropriate. Similarly,
Pearson or Spearman regressions were computed. The
threshold of significance was set at P=0.05. All calcu-
lations were performed using SPSS version 16.0 (SPSS
Inc., Chicago, IL, USA).
A total of 77 pregnant women were enrolled in the
study. Overall, 45 pregnancies were from HIV-1-infected
mothers and 32 were from healthy controls. The demo-
graphic and HIV-1-related characteristics of both study
groups are detailed in Table 1. There were no HIV-1-in-
fected mothers who did not receive perinatal ART. The
composition of the ART is detailed in Table 2. The most
prevalent antiretroviral combination therapy (33% of
mothers) consisted of two NRTIs plus nevirapine, a non-
NRTI. Among the NRTIs, zidovudine plus lamivudine
were the drugs most commonly used (54%). Overall,
20 (44%) HIV-1-infected mothers had commenced an
antiretroviral regimen prior to the onset of pregnancy.
All newborns were confirmed to be HIV-1-uninfected.
In the group of babies born to HIV-1-infected mothers
and the group of their HIV-1-uninfected counterparts,
nearly all newborns were of good health and had no obvi-
ous pathological findings. Only one HIV-1-exposed girl
had a stenosis of the ureteropelvic junction. Her mother
was treated with the triple combination of zidovudine,
Characteristic HIV-1-infected mothers (n=45) HIV-1-uninfected mothers (n=32)
Mean maternal age, years (±sd)
Maternal CDC classification
A 1-3, n
B 1-3, n
C 1-3, n
Mean maternal CD4+ T-cell count, cells/µl (±sd)
Mean maternal plasma HIV-1 RNA, log10 copies/ml (±sd)
Mothers with HIV-1 RNA<50 copies/ml, n
Mean gestational age at delivery, weeks (±sd)
Mean birth weight, g (±sd)
HIV-1-infected newborns, n
Table 1. Maternal and fetal characteristics at delivery
CDC, Centers of Disease Control and Prevention; HIV-1, HIV type-1; NA, not applicable.
AZT+3TC D-drug +ABC
AZT+3TC 3TC or
NRTIs NRTIs NRTIs with NVP with PI
only FTC+TDF AZT NRTI NVP+PI
First trimester, n
Second trimester, n –
Third trimester, n
Any trimester, n
Mean COX II/IV
Table 2. Onset and composition of antiretroviral treatment in 45 pregnancies of HIV type-1-infected women
ABC, abacavir; AZT, zidovudine; COX, cytochrome c oxidase; D-drugs, the dideoxynucleosides, didanosine and stavudine; FTC, emtricitabine; mtDNA, mitochondrial DNA;
NA, not applicable; NRTI, nucleoside reverse transcriptase inhibitor; NVP, nevirapine; PI, protease inhibitor; TDF, tenofovir disoproxil fumarate; 3TC, lamivudine.
A Gingelmaier et al.
© 2009 International Medical Press
lamivudine and abacavir during the entire pregnancy. No
other birth defects were observed.
Venous lactate levels were available only from 35 HIV-1-
infected ART-exposed children and 30 newborns from
the control group because this parameter was amended
in 2004. The mean ±sd lactate of HIV-1-exposed new-
borns was 2.6 ±1.1 mM/l (range 0.2–5.9) and no sig-
nificant difference was demonstrated when compared
with the 30 babies born to the HIV-1-uninfected moth-
ers (mean ±sd lactate 2.5 ±0.8 mM/l, range 1.2–3.8).
Interestingly, all the HIV-1-exposed children (28.6%)
whose lactate was >3.0 mM/l were prenatally exposed
to the combination of zidovudine and lamivudine.
Only two out of eight newborns prenatally exposed
to didanosine or stavudine showed an increased lac-
tate ≥2.5 mM/l (2.5 and 2.7 mM/l, respectively). The
mean ±sd lactate levels of children exposed to zidovu-
dine without a second NRTI were significantly higher
(2.5 ±0.6 mM/l) than those of children exposed to any
regimen with didanosine or stavudine (three and five
patients respectively, 1.7 ±0.8 mM/l; P=0.006). Fur-
thermore, the mean ±sd lactate of newborns exposed
to the combination of zidovudine plus lamivudine (3.2
±1.3 mM/l) was significantly higher than those of new-
borns exposed to zidovudine, lamivudine and abacavir
(2.5 ±0.4 mM/l; P=0.038; Table 2). Mean ±sd lactate
levels of the newborns were significantly higher if ART
was commenced in the second (3.4 ±0.8 mM/l) or third
(3.1 ±1.4 mM/l) trimester in comparison with ART
from the first trimester (2.2 ±0.8 mM/l; P=0.014 and
Mitochondrial DNA copy number
There was a significant reduction (P<0.001) of mean
±sd mtDNA copies/cell in the placentas of 45 ART-
exposed pregnancies (240 ±118 copies/cell) compared
with placentas of 32 ART-unexposed controls (686
±842 copies/cell; Figure 1). The comparison of mean
±sd mtDNA copies in placentas with ART exposure in
the first trimester (241 ±131 copies/cell, n=26) showed
no significant difference versus second (271 ±76 copies/
cell, n=7) or third (221 ±112 copies/cell, n=12) trimes-
ter. Among the placentas exposed to different numbers
of NRTI, there was also no significant difference of
mtDNA copy numbers (Table 2); however, a significant
depletion of mtDNA could be seen in placentas exposed
to zidovudine, lamivudine and abacavir (168 ±40 cop-
ies/cell) versus those exposed to lamivudine or emtricit-
abine and tenofovir disoproxil fumarate (208 ±76 cop-
ies/cell; P=0.025; Table 2). Two placentas which were
HIV-1-infected and ART-exposed, but had no exposure
to NRTIs, had 187 and 356 mtDNA copies.
There were no significant correlations between
mtDNA copy numbers, HIV-1 RNA, CD4+ T-cell
count or gestational age (data not shown); however,
the mtDNA levels in placental tissue of HIV-1-infected
Figure 1. mtDNA copies/cell in placental tissue
Data represent group means ±se. HIV-1, HIV type-1; mtDNA, mitochondrial DNA.
Figure 2. Inverse correlation of mtDNA levels in placental
tissue of HIV-1-infected mothers with the lactate levels of
HIV-1, HIV type-1; mtDNA, mitochondrial DNA.
Mitochondrial toxicity in HIV-1-exposed pregnancies
Antiviral Therapy 14.3
mothers correlated negatively with the lactate levels of
the infants (r=-0.40, P=0.016; Figure 2).
Mitochondrial DNA-encoded respiratory chain subunits
The mean ±sd COX II/IV ratio of the investigational
group was 0.46 ±0.26 and was significantly lower than
that of the control group (0.89 ±0.41; P<0.001). The
comparison of the COX II/IV ratio between different
NRTI regimen showed no significant difference, except
for placentas exposed to zidovudine only (0.59 ±0.6)
versus zidovudine or lamivudine plus didanosine or sta-
vudine (0.32 ±0.2; P=0.038; Table 2).
Mitochondrial morphology by electron microscopy
The ultrastructural investigation focussed on the mito-
chondrial network. In the placental tissue of both the
controls (n=6) and of the HIV-1-infected mothers (n=8),
the mitochondrial network was best developed in the
cytotrophoblast and in the decidua (Figure 3). The
organelles had normal cristae and were predominantly
of the lamellar type in both regions and there were
no differences in the appearance of the mitochondria
between HIV-1 patients and controls. Neither in the
trophoblast, nor in the decidua, were abnormal mito-
chondria with loss or severely abnormal cristae profiles
seen; there were also no circular profiles or paracristal-
line inclusions. By contrast, the mitochondria in the
syncytiotrophoblast of the HIV-1-infected patients
appeared slightly increased in number and size. Occa-
sionally rather large mitochondria were seen and their
cristae were irregularly orientated and slightly swol-
len. In the capillaries of the chorionic villi, most mito-
chondria appeared normal. Only incidentally enlarged
mitochondria were found, but the organelles had a
regular substructure. There were no clear differences
between the mitochondria exposed to the combination
of zidovudine plus lamivudine and those coexposed to
emtricitabine and tenofovir disoproxil fumarate.
The data presented in our study demonstrate mtDNA
depletion in HIV-1-infected ART-exposed placentas,
whereas severe mitochondrial morphological damage
was not observed. By contrast to previous publica-
tions, the HIV-1-infected women in this investigation
were not only exposed to zidovudine or the combina-
tion of zidovudine plus lamivudine, but were treated
with a wide variety of antiretroviral regimens, which
also included newer NRTIs, such as emtricitabine and
tenofovir disoproxil fumarate. Our study also extends
the findings of these studies by demonstrating loss of
mtDNA-encoded respiratory chain subunits.
In other studies, only a minority of HIV-1 and
NRTI exposed children were reported to have clinical
symptoms compatible with mitochondrial disease
[4,11]. Nevertheless, transient increases of postnatal
lactate levels have been reported by several authors
in association with NRTI use in pregnancy [12–14].
This hyperlactataemia was usually reversible with
Figure 3. Mitochondrial ultrastructure by electron microscopy
Mitochondrial ultrastructure in cytotrophoblasts (CT) and syncytiotrophoblasts
(ST) from placentas of HIV type-1 (HIV-1)-infected antiretroviral-exposed
patients (left column) and HIV-1-uninfected controls (right column). (A) In
the CT, the mitochondria (indicated by arrows) appear unchanged. In the ST of
HIV-1-infected individuals, the mitochondria are increased in number, but have
a regular substructure. (B) Higher magnification of mitochondria in the ST reveal
enlarged mitochondria with distortion and swelling of the cristae (indicated by
stars) in antiretroviral-exposed placentas. (C) The mitochondria of the decidua
appear normal. The size bar for the top row is 0.5 µm and for the middle and
bottom rows is 0.16 µm. ER, endoplasmic reticulum; Mv, microvilli; N, nucleus.
A Gingelmaier et al.
© 2009 International Medical Press
treatment cessation and is of unknown relevance for
the future life of these children. In this study a relevant
hyperlactataemia in the first days of life could not be
demonstrated in HIV-1-infected NRTI-exposed babies
in comparison with those born to HIV- 1- uninfected
mothers. There was, however, an inverse correlation
between placental mtDNA copy numbers and perina-
tal lactate levels in the newborns.
Our results with regard to NRTI-induced mtDNA
depletion in human placental tissue are consistent with
other studies that examined the placenta or umbilical
cord [15–17] and showed decreased mtDNA copy num-
bers with zidovudine and lamivudine exposure. Divi
et al.  found a ranking of treatment-induced mtDNA
depletion in umbilical cords from infant monkeys such
that it was greatest with stavudine/lamivudine, followed
by zidovudine/didanosine, zidovudine/lamivudine then
lamivudine. The interactions between antiretrovirals are
very complex and, at times, unpredictable. For exam-
ple, in vitro, tenofovir and lamivudine, two compounds
previously believed to be inert with respect to mito-
chondrial toxicity, both attenuated didanosine-related
cytotoxicity but worsened the effects of abacavir and
zidovudine . These effects might result from antago-
nistic or synergistic effects on kinases, transporters or
mitochondrial nucleoside pools. The differential expres-
sion of these nuclear factors in different tissues can also
explain the tissue specificity of mitochondrial toxicity.
Even the newer NRTIs can induce major mitochondrial
toxicity, especially when combined with other NRTIs
as recently demonstrated in vitro . We therefore also
focused on the issue, but there was no significant dif-
ference among placentas exposed to different NRTI
numbers, nor a specific ranking of individual NRTIs or
their combinations. However, care must be applied in
the interpretation of these data, as the statistical power
of most comparisons is limited.
The addition of protease inhibitor might also enhance
the mitochondrial toxicity of some NRTIs. Lopinavir/
ritonavir, for example, increases tenofovir serum levels
and also might inhibit MDR4, with both mechanisms
then contributing to intracellular tenofovir accumula-
tion as demonstrated for proximal tubular cells [18,19].
We have not identified such interactions.
One study  compared the mitochondria from
placentas of seven smoking and seven non-smoking
mothers. Smoking was associated with a decrease in the
mtDNA content and a reduction of complex III enzy-
matic activity. Whereas the data from this small study
are interesting, we were not able to assess the influence of
smoking because we did not measure smoking status.
The synthesis of proteins, which are needed for
oxidative phosphorylation, is impaired by mtDNA
depletion. Few studies examined the effect of NRTIs
on oxidative phosphorylation in ART-exposed fetal
tissues. Gerschenson et al.  measured the oxida-
tive phosphorylation enzymatically in heart muscle,
skeletal muscle and placental tissue of zidovudine- and
lamivudine- exposed monkey fetuses and found a sub-
stantial decrease of complex I and IV activity in cardiac
and skeletal muscle. Another research group  meas-
ured complex IV activity in cardiac muscle of 18-month-
old mice exposed transplacentally to zidovudine and
found it to be even higher than in unexposed mice. Our
study is the first to report on a significant decrease of
mitochondrial-encoded COX II expression in ART- and
HIV-1-exposed placental tissue in comparison with
HIV-1-uninfected and ART-unexposed controls.
In a recent study , mtDNA copy numbers were
analysed in PBMCs of infants born to HIV-1-infected
women and found to be increased compared with those
from infants born to healthy HIV-1-negative controls.
There was, however, no difference in mtDNA-encoded
respiratory chain protein. It is not unlikely that the
disparate regulation of mtDNA levels between the pla-
centa and PBMCs reflects the tissue specificity of mito-
chondrial toxicity, possibly because of disparate NRTI
concentrations in the maternal and fetal circulation, dif-
ferent nucleoside pools in mitochondria or other causes
. Such upregulated mitochondrial genome in some
fetal compartments might also account for or contrib-
ute to the lack of significant differences in lactate levels
between the newborns in our study. The finding that
newborns from mothers treated for three trimesters had
lower lactate level than those from mothers treated for
only one trimester, however, remains puzzling.
Other investigators have examined human umbilical
cords after transplacental NRTI exposure and found
evidence of ultrastructural damage in the mitochon-
dria [16,17]. In animal models, similar morphological
alterations were detected in the organelles of myocardial
and skeletal muscle [21,22,25]. We have now analysed
the different parts of ART-exposed placentas (amnion,
chorion and decidua) in detail, but did not detect severe
ultrastructural damage of the organelles. In the syn-
cytiotrophoblast of HIV-1 patients, however, a minor
increase in the number and size of mitochondria was
shown. This lack of significant alterations in mitochon-
drial morphology in light of the relatively pronounced
mtDNA depletion might be explained by the threshold
effect , according to which it takes an even higher
percentage of defective mtDNA molecules for a signifi-
cant phenotype to develop. Divi et al.  postulated a
correlation between a longer duration of NRTI exposure
and a higher degree of mitochondrial damage observed
by electron microscopy, but six of eight HIV-1 -infected
women in this investigation had already taken their
ART in the first trimenon.
It is unethical to withhold ART from HIV-1- infected
pregnant women. We were, therefore, not able to compare
Mitochondrial toxicity in HIV-1-exposed pregnancies
Antiviral Therapy 14.3
our findings with placentas from HIV-1- infected women
not receiving ART. Thus, this study cannot distinguish
the effect of HIV-1 infection itself versus the effect of
ART. There were, however, two placentas exposed to an
NRTI-free regimen that showed no significant difference
in mtDNA copy numbers or COX II expression in com-
parison with NRTI-exposed placentas. In Erythrocebus
patas monkeys, neonates that were NRTI-exposed trans-
placentally showed mitochondrial compromise, as deter-
mined by mtDNA depletion and ultrastructural altera-
tions . These findings must be induced by NRTIs,
because the monkeys had no retroviral infection. Pre-
vious clinical investigations, however, also suggest that
HIV-1 infection might cause mtDNA depletion in infants
in the absence of NRTI therapy, but that the additional
exposure to zidovudine could significantly contribute to
mtDNA depletion .
In summary, we found a significant reduction in
mtDNA copy numbers, as well as a significant decrease
of mtDNA-encoded COX II expression in HIV-1-
infected ART-exposed placentas compared with pla-
centas of healthy controls. The mitochondrial compro-
mise was not dependent on either the number of NRTIs
administered, on the length of NRTI application or on
a certain NRTI. Although there was an inverse cor-
relation between lactate levels and placental mtDNA
copy numbers, the clinical significance of our findings
NRTIs have substantially lowered the risk of
mother- to-child transmission of HIV-1 but their risk
of mitochondrial toxicity for the future life of new-
borns warrants further vigilance.
We thank Irene Krienke, Susanne Kunze, Sandra Schulze
and Christina Kuhn for their research and assistance.
There was no particular financial support for this study.
Part of the results of this investigation were presented
at the German-Austrian AIDS Conference, 27–30 June
2007, Frankfurt, Germany.
The authors declare no competing interests.
1. European Collaborative Study. Mother-to-child
transmission of HIV infection in the era of highly active
antiretroviral therapy. Clin Infect Dis 2005; 40:458–465.
Perinatal HIV Guidelines Working Group. Public
Health Service Task Force recommendations for use of
antiretroviral drugs in pregnant HIV-infected women for
maternal health and interventions to reduce perinatal HIV
transmission in the United States. (Updated 8 July 2008.
Accessed 10 August 2008.) Available from http://aidsinfo.
3. Chappuy H, Tréluyer JM, Jullien V, et al. Maternal-fetal
transfer and amniotic fluid accumulation of nucleoside
analogue reverse transcriptase inhibitors in human
immunodeficiency virus-infected pregnant women.
Antimicrob Agents Chemother 2004; 48:4332–4336.
Blanche S, Tardieu M, Rustin P, et al. Persistent mitochondrial
dysfunction and perinatal exposure to antiretroviral
nucleoside analogues. Lancet 1999; 354:1084–1099.
Perinatal Safety Working Group. Nucleoside exposure in
the children of HIV-infected women receiving antiretroviral
drugs: absence of clear evidence for mitochondrial disease in
children who died before 5 years of age in five United States
cohorts. J Acquir Immune Defic Syndr 2000; 25:261–268.
Venhoff N, Walker UA. Mitochondrial disease in the
offspring as a result of antiretroviral therapy. Expert Opin
Drug Saf 2006; 5:373–381.
Poirier MC, Divi RL, Al-Harthi L, et al. Long-term
mitochondrial toxicity in HIV-uninfected infants born to
HIV-infected mothers. J Acquir Immune Defic Syndr 2003;
Venhoff N, Setzer B, Melkaoui K, Walker UA.
Mitochondrial toxicity of tenofovir, emtricitabine and
abacavir alone and in combination with additional
nucleoside reverse transcriptase inhibitors. Antivir Ther
Buchholz B, Beichert M, Marcus U, et al. German-Austrian
recommendations for HIV-therapy in pregnancy and in
HIV-exposed newborn - update 2005. Eur J Med Res 2006;
10. Lebrecht D, Vargas-Infante YA, Setzer B, Kirschner J,
Walker UA. Uridine supplementation antagonizes
zalcitabine-induced microvesicular steatohepatitis in mice.
Hepatology 2007; 45:72–79.
11. Benhammou V, Tardieu M, Warszawski J, Rustin P,
Blanche S. Clinical mitochondrial dysfunction in uninfected
children born to HIV-infected mothers following perinatal
exposure to nucleoside analogues. Environ Mol Mutagen
12. Giaquinto C, De Romeo A, Giacomet V, et al. Lactic acid
levels in children perinatally treated with antiretroviral agents
to prevent HIV transmission. AIDS 2001; 15:1074–1075.
13. Alimenti A, Burdge DR, Ogilvie GS, Money DM,
Forbes JC. Lactic acidemia in human immunodeficiency
virus-uninfected infants exposed to perinatal antiretroviral
therapy. Pediatr Infect Dis J 2003; 22:782–789.
14. Noguera A, Fortuny C, Muñoz-Almagro C, et al.
Hyperlactatemia in human immunodeficiency virus-
uninfected infants who are exposed to antiretrovirals.
Pediatrics 2004; 114:e598–e603.
15. Shiramizu B, Shikuma KM, Kamemoto L, et al. Placenta
and cord blood mitochondrial DNA toxicity in HIV-
infected women receiving nucleoside reverse transcriptase
inhibitors during pregnancy. J Acquir Immune Defic Syndr
16. Divi RL, Leonard SL, Kuo MM, et al. Transplacentally
exposed human and monkey newborn infants show similar
evidence of nucleoside reverse transcriptase inhibitor-
induced mitochondrial toxicity. Environ Mol Mutagen
17. Divi RL, Walker VE, Wade NA, et al. Mitochondrial
damage and DNA depletion in cord blood and umbilical
cord from infants exposed in utero to Combivir. AIDS
18. Cihlar T, Ray AS, Laflamme G. Molecular assessment of the
potential for renal drug interactions between tenofovir and
HIV protease inhibitors. Antivir Ther 2007; 12:267–272.
19. Kearney BP, Mathias A, Mittan A, Sayre J, Ebrahimi R,
Cheng AK. Pharmacokinetics and safety of tenofovir
disoproxil fumarate on coadministration with lopinavir/
ritonavir. J Acquir Immune Defic Syndr 2006; 43:278–283.
20. Bouhours-Nouet N, May-Panloup P, Coutant R, et al.
Maternal smoking is associated with mitochondrial DNA
depletion and respiratory chain complex III deficiency
in placenta. Am J Physiol Endocrinol Metab 2005;
A Gingelmaier et al.
© 2009 International Medical Press
21. Gerschenson M, Nguyen V, Ewings EL, et al. Mitochondrial
toxicity in fetal Erythrocebus patas monkeys exposed
transplacentally to zidovudine plus lamivudine. AIDS Res
Hum Retroviruses 2004; 20:91–100.
22. Walker DM, Poirier MC, Campen MJ, et al. Persistence of
mitochondrial toxicity in hearts of female B6C3F1 mice
exposed in utero to 3′-azido-3′-deoxythymidine. Cardiovasc
Toxicol 2004; 4:133–153.
23. McComsey GA, Kang M, Ross AC, et al. Increased
mtDNA levels without change in mitochondrial enzymes in
peripheral blood mononuclear cells of infants born to HIV-
infected mothers on antiretroviral therapy. HIV Clin Trials
24. Walker UA, Venhoff N, Koch EC, Olschewski M,
Schneider J, Setzer B. Uridine abrogates mitochondrial
toxicity related to nucleoside analogue reverse transcriptase
inhibitors in HepG2 cells. Antivir Ther 2003; 8:463–470.
25. Bishop JB, Tani Y, Witt K, et al. Mitochondrial damage
revealed by morphometric and semiquantitative analysis of
mouse pup cardiomyocytes following in utero and postnatal
exposure to zidovudine and lamivudine. Toxicol Sci 2004;
26. Clark KM, Taylor RW, Johnson MA, et al. An mtDNA
mutation in the initiation codon of the cytochrome c
oxidase subunit II gene results in lower levels of the protein
and a mitochondrial encephalomyopathy. Am J Hum Genet
Accepted for publication 8 December 2008