Clinical Biochemistry 40 (2007) 793 – 797

Association between oxidative stress in pregnancy and preterm

premature rupture of membranes
Mariangela Longini, Serafina Perrone, Piero Vezzosi, Barbara Marzocchi, Antonio Kenanidis,
Giovanni Centini, Lucia Rosignoli, Giuseppe Buonocore ⁎
Department of Pediatrics, Obstetrics and Reproductive Medicine, University of Siena, Policlinico “Le Scotte”, V.le Bracci 36, 53100 Siena, Italy
Received 4 January 2007; received in revised form 27 February 2007; accepted 12 March 2007
Available online 20 March 2007

Abstract

Background: Premature rupture of membranes (PROM) is caused by collagen damage in the chorioamniotic sac leading to tearing. Reactive
oxygen species (ROS) may be the cause of collagen damage. Isoprostanes (F2-IP) are produced by ROS attack on polyunsaturated fatty acids and
are sensitive and specific biomarkers of lipid-peroxidation in vivo.
Aim: To verify whether oxidative stress occurs in pregnancies associated with preterm PROM.
Methods: F2-IPs were measured in amniotic fluid of 16 pregnancies with preterm PROM (Group II) and 97 without PROM (Group I).
Results: F2-IP concentrations (pg/mL) were significantly higher in group II than group I (p < 0.0001). The ROC curve showed a sensitivity of
100% and a specificity of 84.5% at a cut-off of 124.4 pg/mL.
Conclusions: An association exists between oxidative stress in pregnancy and preterm PROM. The detection of amniotic fluid F2-IP
concentrations seems to be a reliable predictive index of risk of preterm PROM.
© 2007 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

Keywords: Oxidative stress; Lipid peroxidation; Reactive oxygen species; Amniotic fluid; Chorioamniotic sac; High risk pregnancies

Introduction cigarette smoking, urinary tract infection, sexually transmitted

Premature rupture of the membrane (PROM) prior to onset
of labour complicates approximately 10% of all pregnancies;
about a third of which before term. Preterm premature rupture of
the membranes (P-PROM) is defined as a spontaneously rupture
in the chorioamnion, before the 37 weeks' gestation. Preterm
premature rupture of the membranes occurs in approximately
1% of all pregnancies and is associated with 30% to 40% of
preterm deliveries [1,2].
Membrane rupture is related to biochemical processes in-
volving collagen synthesized by fibroblasts in the foetal mem-
branes. Since the strength and the elasticity of the foetal
membranes is primarily due to collagen, reduced concentrations
and/or an altered collagen cross-link profile can induced PROM
[3]. Known clinical risk factors for PROM include low socio-
economic status, low body mass index, prior preterm birth,
⁎ Corresponding author. Fax: +39 577 586182.
E-mail address: buonocore@unisi.it (G. Buonocore).
0009-9120/$ - see front matter © 2007 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.
doi:10.1016/j.clinbiochem.2007.03.004
diseases, cervical conization or cerclage, overdistended uterus,
amniocentesis in the current pregnancy, prior preterm labour and
symptomatic contractions in the current pregnancy [4–7].
Neonatal complications are related to the gestational age at
which membrane rupture and delivery occur. Respiratory distress
syndrome is the most common serious complication after PROM
at any gestational age. Other serious morbidities, including
necrotizing enterocolitis, intraventricular haemorrhage, sepsis,
bronchopulmonary dysplasia and retinopathy of prematurity,
decrease with advancing gestational age at delivery [8–11].
Maintenance of the chorioamniotic sac throughout normal
pregnancy requires a balance between collagen synthesis by
fibroblasts and collagenolytic activity by controlled responses
of enzymes expressed in the foetal membrane. The membranes
are reported to be stronger before term than at term, which
suggests that preterm rupture may be related to loss of strength
and elasticity [3].
Reactive oxygen species (ROS), unstable molecules gener-
ated continuously in the body, could be responsible for collagen
794 M. Longini et al. / Clinical Biochemistry 40 (2007) 793–797

damage in the chorioamniotic sac leading to tearing. Normally a
balance exists between production and elimination of ROS.
Oxidative stress (OS) occurs when prooxidants exceed anti-
oxidants [12–14]. OS caused by increased ROS formation may
modify the strength and the elasticity of collagen and cause
PROM [3,15].
In vitro exposure of cultured amniocytes and amniotic
membranes to ROS provides evidence of altered intracellular
biology that could predispose to PROM [3,16,17].
Isoprostanes (F2-IP) can be used as direct markers of OS
[18], quantifying oxidative damage to lipids [19,20]. F2-IP are
newly discovered markers of free radical-catalyzed lipid
peroxidation [21]. They are prostaglandin-like products formed
in vivo by free radical catalyzed nonenzymic peroxidation of
arachidonic acid [22]. Unlike prostaglandins, they do not
require cyclooxygenase for their formation and are generated by
removal of a bis-allylic hydrogen atom and addition of an
oxygen molecule to arachidonic acid to form a peroxyl radical
[23,24].
In this paper we test “in vivo” the hypothesis that OS occurs in
pregnancies associated with preterm PROM predisposing to
preterm delivery.
Materials and methods
Between 1st January 2001 and 31st December 2004 we
studied 260 women consecutively visiting the Prenatal
Diagnosis Centre, University of Siena, Italy, for amniocent-
esis. Indications for amniocentesis were advanced maternal
age or maternal anxiety. The women were at 15–18 weeks of
pregnancy. All gave their written informed consent to
enrolment in the present study. The study was approved by
the Human Ethics Committee of Siena University Medical
Faculty.
On presentation for amniocentesis all women underwent
ultrasound examination to assess foetal growth, viability and
gestational age. Foetal biometry was obtained by a single
specially trained examiner using an ATL HDI 3000 (Philips
Medical Systems, The Netherlands) and was assessed in relation
to local reference values for biparietal diameter, head circum-
ference, abdominal circumference, femur length and humerus
length. The data was recorded on a form designed for women
undergoing amniocentesis in our centre.
The neonatal characteristics recorded were sex, birth weight,
gestational age at birth, Apgar score at 1 and 5 min, neonatal
mortality and NICU stay if more than 3 days (Table 1).
The following exclusion criteria were chosen to eliminate the
effects of potential confounding factors on associations between
oxidative stress and P-PROM: parity more than 3 (n = 19),
repeated amniocentesis or cordocentesis (n = 14), chromosomal
abnormalities (n = 7), positivity for genetic diseases (n = 4),
family history of karyotype anomalies or congenital malforma-
tions (n = 9), routine clinical and laboratory evidence of infection
(n = 19), bleeding (n = 16), cigarette smoking (n = 39), drug
addict (n = 1), prior preterm birth (n = 14), and insufficient
follow-up (n = 5).
Of the 113 women enrolled, 97 delivered after normal labour
(Group I) and 16 delivered after P-PROM (Group II).
Amniotic fluid collected at amniocentesis (15–18 weeks of
gestation) for determination of F2-IP was spiked with butylhy-
droxytoluene (BHT) to prevent oxidation during processing.
Samples were centrifuged at 1000 rpm and the supernatant
stored at − 80 °C until analysis. Free F2-IP concentration was
determined in a single matrix. Ethanol was added to remove
precipitated protein and pH was adjusted to 4.0 with acetate
buffer prior to F2-IsoP determination. Lipids were separated
from other metabolites with C18 and silica Sep-Paks cartridges
(Waters Co., Milford, MA, USA) before colorimetric enzyme
immunoassay (Cayman Chemical, Ann Arbor, MI, USA). The
EIA used a monoclonal antibody highly specific (100%) for 8-
F2-IsoP. The range of the standard curve was 3.9 to 500 pg/mL
and the detection limit 5 pg/mL. Serial sample dilutions were
parallel to the standard curve. The intra- and inter-assay
coefficients of variation of free F2-IP were ≤4% and = 6%,
respectively.
Results were expressed as mean ± S.D., median and CI. The
Kolmogorov–Smirnov test, Mann Whitney U-test and receiver
operating characteristic (ROC) curves were used for statistical
analysis of the data with STATA SE 8.2 software (Stata Corpo-
ration, College Station, Texas 77845, USA).

Table 1
Characteristics of patients
Normal pregnancy (Group I) Pregnancies with P-PROM (Group II) p-value
Newborns (no.) 97 16
Maternal age (years) 30.8 ± 4.8 (18–44) 31.4 ± 5.6 (18–44) NS
Amniocentesis (weeks) 15.4 ± 0.6 15.6 ± 0.3 NS
Gestational age at birth (weeks) 37.8 ± 2.9 (34–40) 32.1 ± 2.8 (29–35) 0.002
Mode of delivery
Vaginal delivery 78 (80.4%) 14 (87.5%) NS
Cesarean section 19 (19.6%) 2 (12.5%) NS
Apgar 1′ > 7 63 (64.9%) 4 (25%) 0.006
Apgar 5′ > 7 95 (97.9%) 15 (94.7%) NS
Males 59 (60.8%) 10 (62.5%) NS
Females 38 (39.1%) 6 (37.5%) NS
Mortality 0 0
Stay in NICU > 3 days 2 (2%) 6 (37.5%) 0.0001
Neonatal birth weight (g) 3000.5 ± 757.2 (910–4100)* 1857 ± 686.5 (910–2800) < 0.0001
 M. Longini et al. / Clinical Biochemistry 40 (2007) 793–797 795

Results

The two groups did not differ significantly in maternal age or

foetal gestational age at amniocentesis (Table 1). The mean age
of the women at amniocentesis was 31.6 ± 3.8 (30.8 ± 4.8 in
group I and 31.4 ± 5.6 in group II).
Six of the 16 babies in the P-PROM group stayed in the NICU
for more than 3 days, whereas only two from the other group
were admitted to the NICU. There were no cases of perinatal
mortality in either group.
The mean concentration of F2-IP collected from amniotic
fluid at 15–18 weeks of gestational age (pg/mL) was
significantly higher in group II (172.1 ± 38.9 mean ± S.D.;
157.5 median; 148.5–188.5 25th–75th percentiles; normal
distribution of the sample by Kolmogorov–Smirnov Test) than
in group I (83.2 ± 48.7 mean ± S.D.; 62 median; 52–95 25th–
75th percentiles; non-normal distribution of sample by Kolmo-
gorov–Smirnov Test) (p < 0.0001) (Fig. 1).
The ROC curve, drawn to evaluate the reliability of F2-IP
as a marker of P-PROM, allowed us to differentiate no-P- Fig. 2. Receiver operating characteristic curve analysis for P-PROM. The area
PROM from P-PROM deliveries (Fig. 2). The area under the under curve indicates that a randomly selected P-PROM pregnancy has a higher
curve was 0.905 (95% CI: 0.835–0.952) with p < 0.0001. test value than a randomly selected normal pregnancy in 90.5% of cases.
The best balanced threshold for test sensitivity and specificity The confidence interval was 0.835–0.952. The best balanced threshold is
was 124.4 pg/mL with 100% sensitivity (95% CI: 79.2–100) 124.4 pg/mL with 100% sensitivity and 84.5% specificity.
and 84.5% specificity (95% CI: 75.8–91.1). This threshold
gave a positive predictive value of 51.6% and a negative
predictive value of 100% for the 14% prevalence of P- contractions or vaginal bleeding before the 37 weeks' gestation
PROM at the Obstetrics and Gynaecology Department of Siena [25]. P-PROM is characterized by reduced collagen concentra-
University hospital. The Odds ratio was 71.75 with CI 8.8 and tions, altered collagen cross-link profiles and increased con-
584.9. centrations of biomarkers of oxidative damage [3]. It is
associated with extensive changes in collagen metabolism,
Discussion collagenolytic activity and collagen solubility suggesting that
weakening of the amnion in preparation for rupture may be
P-PROM is diagnosed when clinically apparent leakage of determined partly by factors controlling the synthesis and
amniotic fluid is confirmed in pregnant women without uterine degradation of collagen [26].

Fig. 1. Amniotic fluid F2-Isoprostanes concentrations in pregnancies with (Group II) and without P-PROM (Group I).
796 M. Longini et al. / Clinical Biochemistry 40 (2007) 793–797
The amniotic membrane is a complex tissue in which
collagen is presumably essential for mechanical integrity and
stress tolerance. Some studies suggested that foetal membranes
grow actively until mid-pregnancy, laying down new collagen.
In the last half of pregnancy, research suggests that the mem-
brane no longer grows but stretches to accommodate foetal
growth [27]. Other investigators have reported a progressive
decrease in amniotic concentrations of collagen towards term.
This would weaken the amnion in preparation for membrane
rupture [28].
P-PROM may be an effect of collagen damage caused
by increased ROS formation and/or antioxidant depletion.
Increased concentrations of markers of OS in tissue from de-
liveries with P-PROM have been reported [11,15,28].
In the present study, 97/113 women had physiological
pregnancies with normal delivery and the other 16/113
experienced P-PROM with a significantly higher F2-IP levels
in amniotic fluid.
These findings show that F2-IP levels in amniotic fluid at 15–
18 weeks of gestation are an early, reliable, predictive indicator
of PROM in premature deliveries.
ROS are generated spontaneously under various biological
conditions in all aerobic organisms and they damage all classes
of macromolecules. The extent of the damage depends on the
type and concentration of ROS as well as on host antioxidant
defences. ROS may disrupt amino acid binding in proteins and
destabilize DNA, causing cell dysfunction. Polyunsaturated
fatty acids of the membrane lipid bilayers are particularly sus-
ceptible targets. The body uses various endogenous and diet-
derived antioxidants to limit free radical-induced tissue damage
[29,30,31].
In vitro exposure of cultured amniocytes to ROS has
provided additional evidence of modification of intracellular
biology that could predispose to PROM. Other models of
amniotic membrane exposure provide further evidence that
ROS alter chorioamniotic biology [15,28,32].
The role of antioxidants in protecting the chorioamniotic sac
from damage by ROS is suggested by several studies [15,32–
36]. Researchers have also confirmed that ascorbic acid
deficiency leads to poor collagen production in foetal mem-
branes [33]. A recent study found that supplementation with
antioxidant vitamins, including vitamin E, decreases markers of
OS at delivery [36].
Previously we found that plasma F2-IP was significantly
higher in neonates than adults. Because no increase in F2-IP
levels was found in plasma of mothers at delivery or during
pregnancy, and no correlation was found between plasma F2-IP
in mothers and newborns, the results suggested that some form
of lipid peroxidation is active in the foetus [37]. The process of
lipid peroxidation is well known. It mainly concerns polyun-
saturated fatty acids present in cell membranes. Peroxidation of
membrane lipids decreases membrane fluidity and impairs
membrane barrier function. We demonstrated that an oxidative
stress occurring in a phase of developmental growth may injure
many foetal organs and tissues, impairing the normal growth
potential [25]. To what extent oxidative stress can induce meta-
bolic and developmental foetal growth deregulation is unknown.
The present data clarifies yet another aspect of the
relationship between ROS-induced damage to foetal mem-
branes and P-PROM. The oxidative stress in pregnancy may
enhance the risks of P-PROM that is mediated by excessive or
undamped peroxidation of amniotic epithelium and chorio-
amniotic collagen.
We hypothesize that when an oxidative stress develops early
in pregnancy, intrauterine growth restriction or P-PROM or both
may occur depending on its entity and length.
A topic for future prospective clinical trials is whether dietary
supplementation with antioxidants may protect the foetal
membranes and decrease the risk of P-PROM and preterm
delivery.
Acknowledgments
Grants from the Italian Ministry for the University
and Scientific-Technological Research (MIUR 2005) are
acknowledged.
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