Fetal medicine
DOI: 10.1111/j.1471-0528.2007.01653.x
www.blackwellpublishing.com/bjog
Fetal exposure to herpesviruses may be
associated with pregnancy-induced hypertensive
disorders and preterm birth in a Caucasian
population*
CS Gibson,a PN Goldwater,b AH MacLennan,a EA Haan,c K Priest,d GA Dekkera
for the South Australian Cerebral Palsy Research Group
a Discipline of Obstetrics and Gynaecology, The University of Adelaide, Adelaide, South Australia, Australia b Discipline of Microbiology
and Infectious Diseases and c Discipline of Genetic Medicine, Children, Youth and Women’s Health Service, Adelaide, South Australia,
Australia d Epidemiology Branch, Department of Health, Adelaide, South Australia, Australia
Correspondence: Dr CS Gibson, Department of Obstetrics and Gynaecology, The University of Adelaide, Women’s and Children’s Hospital,
1st Floor Queen Victoria Building, 72 King William Road, North Adelaide 5006, Adelaide, South Australia. Email catherine.s.gibson@adelaide.edu.au
Accepted 6 December 2007.
Objective To investigate the role of fetal viral infection in the
development of a range of adverse pregnancy outcomes (APOs),
including pregnancy-induced hypertensive disorders (PIHD),
antepartum haemorrhage (APH), birthweight <10th percentile
(small for gestational age, SGA) and preterm birth (PTB).
Design Population-based case–control study.
Setting Laboratory-based study.
Population The newborn screening cards of 717 adverse pregnancy
cases and 609 controls.
Methods Newborn screening cards were tested for RNA from
enteroviruses and DNA from herpesviruses using polymerase chain
reaction (PCR). The herpesviruses were detected using two PCRs,
one detecting nucleic acids from herpes simplex virus (HSV)-1,
HSV-2, Epstein–Barr virus (EBV), cytomegalovirus (CMV) and
human herpesvirus (HHV)-8, hereafter designated Herpes PCR
group A viruses, and the other detecting nucleic acids from
varicella-zoster virus (VZV), HHV-6 and HHV-7, hereafter
designated Herpes PCR group B viruses.
Main outcome measure Odds ratios and 95% CIs for specific APOs.
Results For both term and PTBs, the risk of developing PIHD was
increased in the presence of DNA from Herpes PCR group B
viruses (OR 3.57, 95% CI 1.10–11.70), CMV (OR 3.89, 95% CI
1.67–9.06), any herpesvirus (OR 5.70, 95% CI 1.85–17.57) and any
virus (OR 5.17, 95% CI 1.68–15.94). The presence of CMV was
associated with PTB (OR 1.61, 95% CI 1.14–2.27). No significant
association was observed between SGA or APH and exposure to
viral infection.
Conclusions Fetal exposure to herpesvirus infection was associated
with PIHD for both term and PTBs in this exploratory study.
Exposure to CMV may also be associated with PTB. These findings
need confirmation in future studies.
Keywords Adverse pregnancy outcomes, fetal viral infection,
pregnancy-induced hypertension, preterm birth.
Please cite this paper as: Gibson C, Goldwater P, MacLennan A, Haan E, Priest K, Dekker G for the South Australian Cerebral Palsy Research Group. Fetal
exposure to herpesviruses may be associated with pregnancy-induced hypertensive disorders and preterm birth in a Caucasian population. BJOG 2008;
115:492–500.
Introduction
Clinical maternal viral infection is not uncommon in pregnancy and when it occurs, either as a primary or reactivational
* This research was conducted in Adelaide, South Australia, Australia.
492
infection with viraemia, the fetus is placed at risk of infection
through transplacental transmission.1 Subclinical maternal
viral infection must be common as there is nucleic acid evidence of viral exposure in 40–44% of healthy newborns.2,3 It
has been postulated that fetal viral infection in utero may
increase the risk of adverse pregnancy outcomes (APOs),
such as pregnancy-induced hypertensive disorders (PIHD),
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Viruses and adverse pregnancy outcomes
birthweight <10th percentile (small for gestational age, SGA)
and preterm birth (PTB).4–6
The placenta acts as a potential barrier to the transfer of
viruses from mother to fetus during the viraemic phase of
maternal infection.7 The placental barrier may be less effective
in early pregnancy and when the placenta is damaged, for
example by infarction caused by vascular disease. Placental
dysfunction has also been associated with acquired and genetic
thrombophilia, systemic lupus erythematosus and pre-existing
diabetes. Clinical syndromes associated with placental insufficiency and/or placental vasculopathy include pre-eclampsia,
SGA, PTB and fetal demise. Very little is known about the
passage of viruses across the placenta or the role of placental
viral infection in adverse pregnancy and fetal outcomes. However, it is postulated that viral infection of extravillous trophoblast cells may alter the process of placental invasion and
predispose the mother and fetus to adverse reproductive outcomes that result from placental dysfunction.8
Herpesviruses (including cytomegalovirus (CMV), herpes
simplex viruses (HSV) 1 and 2, varicella-zoster virus (VZV),
Epstein–Barr virus (EBV) and human herpesviruses (HHV) 6,
7 and 8) and enteroviruses are capable of crossing the placenta
and causing in utero infection9–16 and could potentially contribute directly or indirectly to APOs. It has been shown that
CMV infection impairs critical aspects of cytotrophoblast function, which may explain some of the deleterious effects of this
virus on pregnancy outcome.17 The likelihood of maternal
infection resulting in fetal infection varies according to the
specific virus, whether the infection is primary or recurrent,
and the gestational age of the fetus at the time of infection.
Once the infection has crossed the placenta into the fetal circulation, there is the potential for adverse fetal outcomes. These
can be caused by the infectious agent directly or indirectly
through the fetal and/or placental inflammatory response to
infection, where proinflammatory cytokines may adversely
affect the developing brain and perhaps also placental function.
This study investigated the role of fetal exposure to viral
infection (detected through the presence of viral nucleic acids
in newborn screening cards) in APOs, including SGA, PIHD,
antepartum haemorrhage (APH) and PTB. This is the largest
case–control study to date investigating the role of maternal
and fetal infection in APOs.
Methods
Patient selection
The cases and controls in this cohort were selected as part of
a study investigating the role of genetic polymorphisms and
viruses in the development of cerebral palsy (CP). The selection process for these cases and controls has been detailed previously.18 For this analysis, we disregarded CP as an outcome
and combined our cohort of 443 CP cases and 883 controls
(total 1326) before separating them on the basis of APOs.19
A total of 717 of the 1326 babies (54.1%) met the following
selection criteria for cases. Some cases had more than one
condition:
1 PTB <37 weeks of gestation (451/717, 62.9%).
2 SGA <10th percentile calculated from Roberts and Lancaster20 (241/717, 33.6%).
3 APH (any recorded bleeding at or after 20 weeks of gestation) (340/717, 47.4%). The classification of APH within
the South Australian Perinatal Data Collection of births
includes diagnosis of placenta praevia and placental abruption, as well as other and unknown causes of APH.
4 PIHD (blood pressure ‡140/90 mmHg or higher on two
occasions at least 4 hours apart, or ‡170/110 mmHg or
higher on one occasion, first noted after 20 weeks of
gestation). The South Australian Pregnancy Outcome
Database does not contain data on proteinuria; therefore,
in this study, cases with PIHD include both gestational
hypertension and pre-eclampsia (23/717, 3.2%).
The remaining 609 babies (45.9%) had none of the above
selection criteria and were used as the comparison group for
analysis.
Subanalysis was also performed, using the following selection criteria. These subgroups were identified a priori, before
the data were analysed.
1 All PIHD plus SGA (9/717, 1.3%).
2 All APH plus SGA (108/717, 15.1%).
3 All PTB <37 weeks of gestation plus SGA (82/717, 11.4%).
Ethical considerations
This research was approved by the Research Ethics Committee of the Children, Youth and Women’s Health Service in
Adelaide, Australia, and followed the National Health and
Medical Research Council of Australia Guidelines. The ethics
committee deemed that cases and controls must be deidentified, and collection of clinical data was limited to that contained within the South Australian Supplementary Birth
Record. No linkage was allowed with case notes or other
neonatal outcomes. The supplementary birth record unfortunately does not collect data on perinatal infectious morbidity.
The collection uses notifications of births in South Australia
made by hospital and homebirth midwives and hospital neonatal nurses and includes comprehensive data on medical
conditions present in pregnancy and obstetric complications.
Validation of this perinatal data collection form has been
undertaken, which showed that the data, such as major pregnancy events, are accurate and reliable in comparison with
hospital medical records.21
Virus detection
The viruses of interest were categorised into DNA and RNA
viruses. The DNA viruses included: HSV-1, HSV-2, VZV,
EBV, CMV, HHV-6, HHV-7 and HHV-8. The RNA viruses
included members of the enterovirus family. DNA viruses
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493
Gibson et al.
were detected using two polymerase chain reactions (PCRs)
using previously published primers,22 and results were
assigned to the respective PCR test groups: the first detected
nucleic acids of HSV-1, HSV-2, EBV, CMV and HHV-8,
hereafter designated Herpes PCR group A, and the second
detected nucleic acids of VZV, HHV-6 and HHV-7, hereafter
designated Herpes PCR group B. The separation of the herpesviruses into these groups by primer pairs corresponded to
the G+C content of their DNA and not to the phylogenetic
grouping based on the complete genome.22 Within the Herpes
PCR group A, differentiation between CMV and the remaining viruses (HSV-1, HSV-2, EBV and HHV-8) was possible
because of differences in PCR product band size visually
determined by agarose gel electrophoresis.
Punches of dried blood (1.2 mm) on newborn screening
cards (collected by heel-prick at approximately 3–5 days of
life) were extracted for DNA viruses using the NucleoSpinÒ
Tissue Extraction Kit (Macherey-Nagel, Düren, Germany).
The newborn screening cards were extracted for RNA using
a phenolic wash method.23
All amplification conditions were optimised using reference
RNA and DNA samples extracted from viral stocks. These
reference samples were included as positive controls for all
subsequent amplifications in addition to no template controls.
Sensitivity of virus detection
The minimum number of detectable viral nucleic acid copies
was determined for each PCR and extrapolated back to a minimum number of detectable viral nucleic acid copies per millilitre of blood. The minimum number of detectable viral
nucleic acid copies was 2.8/bloodspot (5.6 · 103/ml blood)
for enterovirus, 1.6/bloodspot (3.2 · 103/ml blood) for herpes
PCR group A viruses and 15/ml (3.2 · 103/ml blood) for
herpes PCR group B viruses. Viral nucleic acids were detected
from newborn screening samples that had been stored for up
to 18 years. Not all samples had a valid test result for all
viruses, and therefore the numbers in the tables may add up
to less than the total number of cases and controls available
for testing. The storage time of the newborn samples did not
affect the ability to amplify genomic DNA, which was amplified from all samples tested.
Statistical analysis
As controls were not matched for important covariates, such as
gestational age, analysis was undertaken using all controls without taking account of matching. Data analysis (GraphPad Instat
version 3.06) then considered cases by gestational age range
(<32 weeks, 32–36 weeks, <37 weeks, ‡37 weeks and all gestational ages). Results were expressed as odds ratios with 95% CIs
comparing positive with negative virus detection. Tables S1–S8
detail all calculated odds ratios and confidence intervals; only
significant results are presented in the main text. P values of
<0.05 are highlighted in the tables. No adjustments were made
494
for multiple testing in this largely exploratory study into the
associations of in utero exposure to viral infection and APOs.
Results
All APOs versus non-CP APO
Significantly, more babies with a subsequent diagnosis of CP
were in the case group of this study (OR 2.00, 95% CI 1.56–
2.54). We therefore investigated whether the CP babies were
overrepresented in the APOs studied and found that CP
babies were overrepresented in the PTB group, irrespective
of whether the PTB was spontaneous or iatrogenic (Table 1).
There were no significant differences for APH, SGA, or PIHD.
As a result of these findings, PTB was analysed only using
non-CP babies in the case (n = 251) and control (n = 455)
groups.
We also investigated whether the prevalence of viral infection
differed between the APOs comparison group and the non-CP
APOs comparison group. Table 2 illustrates the prevalences of
viral exposure for each of these groups. No significant differences were observed between the two control populations.
SGA <10th percentile
Two hundred and forty-one babies in the case cohort (33.6%)
were classified as SGA. There were no associations between
any of the viruses tested and SGA.
Pregnancy-induced hypertensive disorders
The mothers of 42 babies in the case cohort (5.9%) suffered
from hypertension, either pregnancy-induced (23) or preexisting (20). One mother suffered from both. The low frequency of PIHD in this study is most likely explained by the
high incidence of PTB (62.9% of the overall study population).
Detection of Herpes PCR group B viruses was associated
with PIHD (OR 3.57, 95% CI 1.10–11.57) (Table 3). This
significant association was also observed in preterm babies.
The detection of CMV was also significantly associated with
PIHD (OR 3.89, 95% CI 1.67–9.06) (Table 3). The detection
of any herpesvirus or any virus was also associated with
PIHD, with odds ratios of 5.70 (95% CI 1.85–17.57) and
5.17 (95% CI 1.68–15.94), respectively (Table 3).
PIHD and SGA <10th percentile
Of the 23 mothers of babies in the case cohort who developed
PIHD, 9 (39.1%) also gave birth to an SGA baby. No significant associations were observed between exposure to viral
infection and PIHD plus SGA.
Antepartum haemorrhage
The mothers of 340 of 717 babies in the case cohort (47.4% of
cases) suffered from APH. No significant associations were
observed between exposure to viral infection and APH.
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Table 1. Odds ratios (95% CI) for CP versus non-CP babies in the APO case group for the APOs of interest
Adverse pregnancy
outcome
APO cases (n 5 717)
CP babies (n 5 289)
Non-CP babies (n 5 428)
132
157
91
198
200
89
118
82
8
281
208
220
150
278
251
177
148
103
15
413
APH (n 5 340)
No APH (n 5 377)
SGA (n 5 241)
No SGA (n 5 476)
PTB (n 5 451)
No PTB (n 5 266)
Spontaneous PTB (n 5 266)
Iatrogenic PTB (n 5 185)
PIHD (n 5 23)
No PIHD (n 5 694)
APH and SGA <10th percentile
Of the 340 mothers of babies in the case cohort diagnosed
with APH, 108 (31.8%) gave birth to an SGA baby. The
majority of associations investigated were nonsignificant.
Babies born at 32–36 weeks of gestation who tested positive
for Herpes PCR group B viral DNA were at greater risk of
APH and SGA (OR 2.79, 95% CI 1.08–7.25) (Table 3).
PTB <37 weeks—non-CP
Four hundred and fifty one (62.9%) of the case cohort were
born prematurely at a gestational age of less than 37 weeks. Of
these premature babies, 251 (55.7%) were not diagnosed with
CP. Because of the significant association observed between
PTB and CP diagnosis within the PTB cohort (Table 1), all
results for prematurity were calculated for only the non-CP
population to avoid skewing of the results. Detection of CMV
DNA was significantly associated with PTB (OR 1.61, 95% CI
1.14–2.27). The detection of Herpes PCR group A viruses and
OR (95% CI)
P value
0.89 (0.65–1.21)
0.49
0.85 (0.61–1.19)
0.36
1.58 (1.14–2.20)
0.005
1.00 (0.67–1.49)
0.93
0.78 (0.30–2.00)
0.74
any herpesvirus was also associated with PTB (OR 1.51, 95%
CI 1.08–2.10 and OR 1.43, 95% CI 1.02–2.01, respectively).
PTB and SGA <10th percentile
Eighty two (18.2%) of the 451 preterm babies were classified
as SGA. Of these, 43 (52.4%) were not diagnosed with CP.
Detection of any herpesvirus was associated with combined
PTB at 32–36 weeks of gestation and SGA (OR 2.21, 95% CI
1.03–4.73) (Table 3). No other significant associations were
observed between PTB with SGA and exposure to viral
infection.
Discussion
This is the largest study to investigate the associations
between perinatal exposure to viral infection, detected by
the presence of viral nucleic acids in blood collected within
the first 3–5 days of neonatal life, and APOs—SGA, PIHD,
Table 2. Prevalence of viral infections in the total APO control population and the non-CP APO control population, expressed as percentage
positive of the total tested*
Virus
Herpes PCR group B
Herpes PCR group A
CMV
HSV
Enterovirus
Any herpesvirus
Any virus
All APO comparison group
Non-CP APO comparison group
Positive/total
Prevalence % (95% CI)
Positive/total
Prevalence % (95% CI)
41/480
167/587
147/587
25/587
19/585
191/502
203/503
8.5 (6.2–11.4)
28.5 (24.8–32.3)
25.0 (21.6–28.8)
4.3 (2.8–6.2)
3.4 (2.0–5.2)
38.0 (33.8–42.5)
40.4 (36.0–44.8)
26/362
120/441
104/441
20/441
14/442
136/380
145/382
7.2 (4.7–10.3)
27.2 (23.1–31.6)
23.6 (19.7–27.8)
4.5 (2.8–6.9)
3.2 (1.7–5.3)
35.8 (31.0–40.8)
38.0 (33.1–43.0)
*No significant differences were observed between the two control populations. Not all samples had a valid test result for all viruses, therefore
the total number of samples included in analyses may be less than the total number of cases and controls available for study.
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Gibson et al.
Table 3. Significant odds ratios (95% CI) for all APOs and virus exposures*
APO
Virus
PIHDs
Herpes PCR group B
CMV
Herpes PCR group A
Any herpesvirus
Any virus
APH 1 SGA
PTB (non-CP APO)
Herpes PCR group B
CMV
Herpes PCR group A
Any herpesvirus
PTB 1 SGA (non-CP APO)
Any herpesvirus
Gestation (weeks)
Cases (positive/total)
OR (95% CI)
P value
All
,37
,32
All
37
,37
All
37
All
37
,37
All
37
,37
32–36
,37
32–36
,32
,37
32–36
,37
32–36
32–36
4/16
4/12
4/8
13/23
5/6
8/17
13/23
5/6
14/18
5/5
9/13
14/18
5/5
9/13
6/29
82/247
48/145
34/102
89/247
54/145
98/221
62/134
16/29
3.57 (1.10–11.57)
5.35 (1.55–18.55)
10.71 (2.58–44.42)
3.89 (1.67–9.06)
14.97 (1.73–129.21)
2.66 (1.00–7.02)
3.27 (1.41–7.60)
12.58 (1.46–108.50)
5.70 (1.85–17.57)
17.89 (0.98–325.63)
3.66 (1.11–12.06)
5.17 (1.68–15.94)
16.24 (0.89–295.57)
3.33 (1.01–10.95)
2.79 (1.08–7.25)
1.61 (1.14–2.27)
1.60 (1.06–2.42)
1.62 (1.02–2.58)
1.51 (1.08–2.10)
1.59 (1.07–2.36)
1.43 (1.02–2.01)
1.55 (1.04–2.30)
2.21 (1.03–4.73)
,0.05
,0.05
,0.01
,0.01
,0.01
0.05
,0.01
,0.01
,0.01
,0.01
,0.05
,0.01
,0.05
,0.05
,0.05
,0.01
,0.05
,0.05
,0.05
,0.05
,0.05
,0.05
,0.05
Positive, total number of samples testing positive for the viruses; total, total number of samples with a valid test result. Herpes PCR group B: VZV,
HHV-6, HHV-7; Herpes PCR group A: HSV-1, HSV-2, EBV, CMV, HHV-8; Any Herpesvirus: HSV-1, HSV-2, EBV, CMV, VZV, HHV-6, HHV-7, HHV-8;
Any Virus: any herpesvirus or enterovirus.
*Not all samples had a valid test result for all viruses, therefore the total number of samples included in analyses may be less than the total
number of cases and controls available for study.
APH and PTB. The link between prematurity and infection is
well established.24–26 The role of infection in other APOs is
not so well studied, although over the past decade, the potential involvement of infection and inflammatory responses in
the placenta and mother in the pathogenesis of pre-eclampsia
has received attention.27–31
Using this large cohort of pathology-enriched cases and
term controls, specific viral DNA and RNA nucleic acid
sequences have been identified, and an association has been
shown between exposure to viral infection and APOs, particularly PIHD.
Caveats
While this is the largest study of its kind, there are a number
of caveats. Our cases and controls were derived from another
study hypothesis and were not matched to each other. Furthermore, 217 separate analyses were performed on the
individual viruses, with 10.6% (23) yielding significant
associations. Such multiple analyses increase the likelihood
of identifying chance statistical associations (type 1 error)
and because of small numbers in some of the subanalyses,
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associations cannot be confidently excluded (type 2 error).
Limitations imposed by our ethics committee meant that
we were unable to access case notes or other relevant clinical
information about our cases and controls. Information such
as Doppler studies on umbilical and uterine arteries, if available, would have enhanced this study. Our chosen outcomes
may causally interact to result in an APO, and there are inherent difficulties in determining the roles played by the individual
outcomes. Finally, although the viruses detected in the neonatal
blood spots probably reflected exposure in utero, neonatal
exposure is also possible. Findings must be reported but interpreted with caution, and further large-scale prospective studies
are necessary to confirm these associations.
Despite these caveats, this study has demonstrated that the
presence of viral nucleic acids, in particular Herpes PCR
group B and CMV, in newborn screening blood samples
may be associated with PIHD over a wide range of gestational
ages. One mechanism by which such associations could be
explained is the inflammatory response caused by these
viruses. Vessel inflammation and artery wall thickening, as
a result of viral infection, can contribute to the increased
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Viruses and adverse pregnancy outcomes
resistance of blood vessels,4 thus promoting a hypertensive
state. HSV-1, HSV-2 and CMV, members of the herpesvirus
family, are capable of causing thrombogenic changes to host
cells and initiating the clotting cascade through the generation
of thrombin,32 thus promoting vascular disease. A large crosssectional study by Sun et al.4 found that HSV-2 infection was
associated with essential hypertension.
Our results demonstrated associations between CMV exposure and PIHD in all infants and term-born infants born to
mothers who had PIHD. A marginally significant trend was
observed (P = 0.05) between CMV exposure and PIHD in
preterm infants born to mothers who had PIHD. We did
not observe significant findings for the small subgroup PIHD
with SGA. This may be because of the very small numbers in
this subgroup, and large prospective studies would be needed
to rule out such associations. von Dadelszen et al.33 showed
that women with early-onset pre-eclampsia had higher levels
of anti-CMV antibodies than those with late-onset preeclampsia, those with normotensive intrauterine growth
restriction (IUGR) and those with normal pregnancies. Exposure to CMV and subsequent production of anti-CMV antibodies may generate pathogenic antiphospholipid antibodies,
which are capable of binding and activating endothelial
cells.34 This in turn may enhance thrombus formation and
increase inflammation, resulting in hypertension. A similar
connection has been identified for enteroviral infection and
atherosclerosis,35 suggesting that several viral infections are
capable of increasing blood pressure and causing hypertension. A study reported by Belfort et al.36 demonstrated a significant association between evidence of CMV infection and
pre-eclampsia (P < 0.01), thus supporting an association
between pre-eclampsia and viral infection during pregnancy.
An alternative hypothesis to explain the link between CMV
and APOs is that CMV may affect the biology of cytotrophoblasts and thus critical cytotrophoblast function.17
Our study demonstrated associations between PTB and
exposure to viral infection, particularly CMV. This provides
further evidence that the presence of infectious agents in utero
is associated with subsequent PTB.25,37–39
Our study did not show any associations between exposure
to viral nucleic acids and SGA, despite other research suggesting possible links.5,6 Fetal viral infection, in particular CMV,
can cause SGA, however, no association was found in the
present study. The results do, however, agree with van
Dongen et al.,40 who found no associations between IUGR
and adenoviruses or enteroviruses. Discrepancies may be
explained by study design differences. Our study used nonquantitative PCR methodology on archived newborn screening cards, and there remains the possibility of the results
reflecting differing viral loads. Low viral loads may not invoke
significant inflammatory/cytokine responses and therefore
may not create susceptibility to APOs, such as PTB or SGA;
alternatively, high viral loads, possibly associated with major
inflammation and/or damaging cytokine production, could
be necessary before such adverse outcomes are observed. Furthermore, these results may reflect different gestational ages at
which exposure to the virus first occurred. Infections that
occur earlier in intrauterine life tend to be associated with
more severe clinical sequelae compared with those occurring
later.41 Positive amniotic fluid viral DNA PCR results have
been associated with an increased rate of fetal structural malformations, IUGR, hydrops and other fetal abnormalities.6
These tests were performed between 19–20 weeks of gestation
compared with 3–5 days after birth in the current study,
which may explain the differing results. Prospective studies
designed to quantify viral loads at various gestational ages are
planned to investigate this hypothesis further. Such prospective studies will also determine whether these infections are
primary infections or reactivation of latent viral infection.
This study was unable to test every potential virus worthy
of investigation (e.g. adenoviruses, rotaviruses, human coronaviruses, parvovirus, paramyxoviruses and lymphocytic choriomeningitis virus), and these should be investigated in
future studies.
No associations with APH were identified, an outcome not
previously investigated for associations with viral infection.
Within our pathology-enriched case cohort of 717 babies, the
mothers of 340 (47.4%) were diagnosed with some form of
APH. The classification of APH within the South Australian
Perinatal Data Collection of births includes diagnosis of
placenta praevia and placental abruption, as well as other
and unknown causes of APH. However, no associations were
evident in this large group.
A high prevalence of viral DNA was observed in the control
group, with 203/503 (40%) controls with a valid PCR result
for all viruses testing positive for at least one virus. A similar
control group prevalence of 44% was reported in 2005 by
other authors,42 suggesting that antenatal viral exposure is
common but not clinically relevant unless other factors are
present to initiate infection and/or an inflammatory response.
It is important to recognise that the presence of viral DNA
does not necessarily indicate active congenital or neonatal
infection. Newborn screening cards were tested for the presence of viral DNA and RNA. While detection of viral nucleic
acids in the blood of neonates indicates exposure to and
replication of the respective virus or viruses, this study was
not designed to detect evidence of an accompanying inflammatory response. Given the small sample volume (1.2 mm
diameter) of dried blood and the limit of detection of 1–10
viral nucleic acid copies, it could not be confirmed that true
viraemic infection was occurring. It would seem unlikely that
the detection of viral nucleic acid in such small samples
merely reflected maternal and fetal cell trafficking or maternal
blood contamination. The possibility of nosocomial infection
needs to be considered, although it is thought exceedingly
unlikely that this infection would have had time to incubate
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Gibson et al.
and cause infection in the newborn infant within the first days
of life without clinical symptoms. Clinically, a nosocomial
infection rate of 40% in our control population would have
been identified as a major epidemic, which was not evident.
Prospective investigations are required to follow women
through pregnancy, quantitatively testing antenatal blood
samples for viral nucleic acids and determining if there is
active infection in the fetus/neonate by examining leukocytes
and sera for the presence of viral antigens associated with
active viral replication. PCR contamination was excluded by
working in two designated separate laboratories for preparation of PCR samples (to eliminate the possibility of PCR
product contamination) and by using appropriate controls
in all assays. Furthermore, no other viral PCR work was being
conducted by other users of the PCR laboratories.
In summary, we have demonstrated that exposure to viral
infection (as demonstrated by the presence of viral nucleic
acids in blood on newborn screening cards) may be associated
with PIHD and PTB. These exploratory findings require confirmation in other studies. These findings support the
previously published indirect findings (such as antibody
responses) of others that pointed to a relationship between
CMV and hypertension of pregnancy. Our findings suggest
that some APOs could potentially be prevented by immunisation or passive immunity through virus-specific immunoglobulin. It is likely that APOs are multifactorial and that
other factors, such as genetic susceptibility to infection, genetically regulated proinflammatory cytokine responses and
inherited thrombophilia19 are needed for the adverse phenotypes to be expressed.
Funding
This research was supported by the Australian National
Health and Medical Research Council, The Channel 7 Children’s Research Foundation, The University of Adelaide and
The South Australian Government Captive Insurance Corporation. The supporting sources had no influence on the
analysis, writing or submission of the manuscript.
Details of ethics approval
The procedures of the study received ethics approval from the
Women’s and Children’s Hospital Human Research Ethics
Committee, South Australia. The date of approval was 6 June
2002, reference number REC1323/5/2008.
Contribution to authorship
All authors listed on this paper fulfil the uniform requirements for authorship. No one who fulfils these criteria has
498
been omitted from authorship. C.S.G. contributed to the
design of the study, collection, analysis and interpretation
of data, writing of the manuscript and gave final approval
of the version to be published. P.N.G. contributed to the
study design and interpretation of the data, writing of the
manuscript and gave final approval of the version to be published. A.H.M. contributed to the study design and interpretation of the data, writing of the manuscript and gave final
approval of the version to be published. E.A.H. contributed to
the study design, collection and interpretation of the data,
writing of the manuscript and gave final approval of the version to be published. K.P. contributed to the collection and
analysis of data, writing of the manuscript and gave final
approval of the version to be published. G.A.D. contributed
to the study design, analysis and interpretation of data, writing of the manuscript and gave final approval of the version to
be published. Other members of the South Australian Cerebral Palsy Research Group were involved in the design of the
study and are listed as follows: A/Prof Annabelle Chan, Dr
William Hague, Dr Zbigniew Rudzki, Ms Phillipa van Essen,
A/Prof T Yee Khong, Dr Mark R Morton, Mr Enzo Ranieri,
Ms Heather Scott, Dr Heather Tapp, Mr Graeme Casey.
Acknowledgements
We thank the staff of the Neonatal Screening Laboratory
(Women’s and Children’s Hospital) for their technical assistance. We also thank Barry Slobedman (Westmead Millenium
Institute, NSW, Australia) for providing us with positive control viral material.
Supplementary material
The following supplementary materials are available for this
article:
Table S1. Odds Ratios (95% CI) for Herpes PCR Group B
Results for specified adverse pregnancy outcomes.
Table S2. Odds Ratios (95% CI) for Herpes PCR Group A
Results for specified adverse pregnancy outcomes.
Table S3. Odds Ratios (95% CI) for CMV results for specified
adverse pregnancy outcomes.
Table S4. Odds Ratios (95% CI) for HSV results for specified
adverse pregnancy outcomes.
Table S5. Odds Ratios (95% CI) for Enterovirus results
for specified adverse pregnancy outcomes.
Table S6. Odds Ratios (95% CI) for any herpesvirus result
for specified adverse pregnancy outcomes.
Table S7. Odds Ratios (95% CI) for any virus result for
specified adverse pregnancy outcomes.
Table S8. Odds Ratios (95% CI) for all preterm birth
and non-CP preterm birth for the specified virus groups.
ª 2008 The Authors Journal compilation ª RCOG 2008 BJOG An International Journal of Obstetrics and Gynaecology
Viruses and adverse pregnancy outcomes
These materials are available as part of the online article
from: http://www.blackwell-synergy.com/doi/abs/10.1111/j.
1471-0528.2007.01653.x.
(This link will take you to the article abstract).
Please note: Blackwell Publishing is not responsible for the
content or functionality of any supplementary materials
supplied by the authors. Any queries (other than missing
material) should be directed to the corresponding author
for the article. j
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