involvement of inflammation in normal pregnancy
TRANSCRIPT
AOGS SHORT RESEARCH REPORT
Involvement of inflammation in normal pregnancyMARIA PALM1, OVE AXELSSON1,2, LISA WERNROTH3, ANDERS LARSSON4 & SAMAR BASU5,6
1Department of Women’s and Children’s Health, Obstetrics and Gynecology, 2Centre for Clinical Research S€ormland,3Uppsala Clinical Research Center, 4Department of Medical Sciences, Clinical Chemistry,5Oxidative Stress and Inflammation, Department of Public Health and Caring Sciences, Uppsala University, Uppsala,
Sweden, and 6Laboratory of Biochemistry, Molecular Biology and Nutrition, Faculty of Pharmacy, University of Auvergne,
Clermont-Ferrand, France
Key words
Interleukin-6, inflammation, pregnancy, tumor
necrosis factor-a, prostaglandin F2a
Correspondence
Maria Palm, Department of Women’s and
Children’s Health, Obstetrics and Gynecology,
Uppsala University, SE-751 85 Uppsala,
Sweden. E-mail: [email protected]
Conflict of interest
The authors report no conflicts of interest.
The authors alone are responsible for the
content and writing of the paper.
Please cite this article as: Palm M, Axelsson
O, Wernroth L, Larsson A, Basu S. Involvement
of inflammation in normal pregnancy.
Acta Obstet Gynecol Scand 2013; 92:
601–605.
Received: 23 February 2012
Accepted: 7 January 2013
DOI: 10.1111/aogs.12093
Abstract
To study the role of inflammation throughout normal pregnancy and post-
partum, 37 women with normal pregnancies, including normal neonatal
outcome, participated. Blood and urine samples were collected from each
woman at least six times during pregnancy and postpartum. Plasma levels of
interleukin-6 (IL-6) and tumor necrosis factor-a (TNF-a) and urinary levels of
a prostaglandin-F2a (PGF2a) metabolite were measured. Median, 25th to 75th
centile and average change per gestational week of IL-6, TNF-a and the PGF2ametabolite were measured. Levels of IL-6 increased significantly throughout
pregnancy and remained high postpartum. No change in TNF-a could be seen.
The PGF2a metabolite levels increased significantly throughout pregnancy and
decreased postpartum. These results suggest that mild but significant inflam-
matory activity is involved in the development of normal pregnancy, which
might have important physiological roles.
Abbreviations: COX, cyclooxygenase; IL-6, interleukin-6; PGF2a, prostaglandin-
F2a; TNF-a, tumor necrosis factor-a.
Introduction
Inflammation is a process associated with various diseases
but is also involved in normal physiological states, such
as pregnancy. The maternal inflammatory response is
supposed to be modulated to allow establishment and
maintenance of a viable pregnancy (1). Existing human
pregnancy data on inflammation are mostly limited to
individuals with a history of reproductive failure or are
hampered by cross-sectional study designs (2,3). Longitu-
dinal data on inflammatory factors in normal pregnancy
and the normal postpartum period are scarce and the role
of inflammation in human pregnancy is not fully under-
stood. Data on inflammatory factors in normal pregnancy
are a prerequisite to understand their role in complicated
pregnancy. In recent years, studies on pre-eclampsia,
intrauterine growth restriction and prematurity have
mainly been focused on the role of inflammation and the
maternal immune response.
In the inflammatory process, circulating leucocytes
interact locally, adhere and migrate through the endothe-
lium into the tissue with the help of adhesion molecules
and cytokines, such as interleukins and tumor necrosis
factor-a (TNF-a). This process influences vascular tone,
increases vasopermeability and leads to tissue edema. The
cytokines stimulate the formation of cyclooxygenase
(COX) -mediated products mainly by inducing COX-2
expression in damaged tissue. Interleukin 6 (IL-6), a pro-
inflammatory and anti-inflammatory cytokine, is pro-
duced by white blood cells, fibroblasts and endothelial
cells and it initiates a systemic response to a local inflam-
matory stimulus. Conflicting results are found regarding
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the role of circulating IL-6 during normal pregnancy
(4,5), but many authors report that increased IL-6 levels
are found in pre-eclampsia compared with normal
pregnancy (2,6). Interleukin-6 increases in hypoxic condi-
tions (7) and is thought to contribute to the endothelial
cell activation/damage associated with pre-eclampsia. The
proinflammatory cytokine TNF-a belongs to the TNF
superfamily and is produced by monocytes, neutrophils
and macrophages in placental tissues (8). Several studies
have reported higher concentrations of TNF-a in pre-
eclampsia (6,9). The TNF-a increases in hypoxic condi-
tions (7) and it has been postulated to be a mediator of
endothelial cell activation/damage in pre-eclampsia. It
alters the balance between endothelium-derived vasocon-
strictors and vasodilators and impairs endothelium-
dependent relaxation.
Prostaglandin-F2a (PGF2a) is formed in vivo in both
physiological and pathophysiological situations by activa-
tion of COX and has mainly potent vasoconstrictive and
proinflammatory properties (10). COX is generally upreg-
ulated at sites of inflammation and the prostaglandins
produced are presumed to be regulators of such inflam-
matory reaction. However, the role of COX-catalyzed
PGF2a in normal human pregnancy is relatively unknown.
It is a well-known luteolytic compound in many animal
species, where it controls ovarian function and is involved
in the parturition process but their exact role in human
pregnancy is less studied (11). In an earlier cross-sectional
study, PGF2a levels were higher in pregnant than in non-
pregnant Japanese women (12), and in a recent longitudi-
nal study in pregnant British women, it was shown that
PGF2a metabolite levels increased until week 34 of preg-
nancy (13).
Normal pregnancy is known to evoke a systemic
inflammatory response, which is associated with evidence
of increasing oxidative stress as pregnancy advances in
terms of several circulating markers, particularly oxidized
lipids and F2-isoprostanes (14). Inflammation can be esti-
mated by various indicators reflecting different parts of
the inflammatory reaction in vivo.
The primary aim of this study was to elucidate the
inflammatory process during normal pregnancy and the
postpartum period, by measuring plasma levels of IL-6
and TNF-a and urinary levels of a PGF2a metabolite from
longitudinally collected biological samples. The secondary
aim was to calculate the change per gestational week for
these inflammatory biomarkers.
Material and methods
Details on the participants and the blood sample collection
regimen were published previously (14). Plasma IL-6 and
TNF-a were analyzed by commercial sandwich enzyme-
linked immunosorbent assays (human IL-6 Quantikine HS
ELISA Kit HS600B, human TNF-a Quantikine HS ELISA
HSTA00D; R&D Systems, Minneapolis, MN, USA),
according to the recommendations of the manufacturer.
The total coefficients of variation of the methods were 5–7%. The urine samples were analyzed for 15-keto-dihydro-
PGF2a, a major metabolite of PGF2a, by a radioimmunoas-
say described in detail elsewhere (15).
Statistical calculations
Because the data were not normally distributed, non-
parametric methods were used for analysis. The median,
the 25th to 75th centiles and the average change per ges-
tational week of IL-6, TNF-a and 15-keto-dihydro-PGF2awere calculated. Statistical methods were as previously
described (14).
Results
All pregnancies were singleton and all deliveries took
place at Uppsala University Hospital 2004–05. The moth-
ers had normal body mass index at first visit to the ante-
natal clinic and half of them were primiparas. The
majority of the deliveries were vaginal at term and birth-
weights of the newborns were normal. Blood and urine
samples were collected six to eight times from all partici-
pating women.
Median levels of plasma IL-6 throughout pregnancy
and during the postpartum period are presented in
Table 1 and Figure 1a. An increase in IL-6 with advanc-
ing gestational age was revealed. The trend analysis
showed a significant increase of 0.026 pg/mL per week
with advancing gestational age (p < 0.001). The median
levels observed 9–10 weeks postpartum corresponded to
levels in gestational weeks 39–40 (p = 0.903) and were
higher, though not significantly, than levels observed in
gestational weeks 9–10 (p = 0.084) (Figure 1a).
Median levels of plasma TNF-a throughout pregnancy
and during the postpartum period are presented in
Table 1 and Figure 1b. No significant change of TNF-awas recorded with advancing gestational age. There
seemed to be an increase of TNF-a during the second
half of pregnancy. The median levels observed 9–10 weeks
postpartum were in the same range as levels in gestational
weeks 39–40 (p = 0.634) and gestational weeks 9–10(p = 0.128) (Figure 1b).
Median levels of urinary 15-keto-dihydro-PGF2athroughout pregnancy and during the postpartum period
are presented in Table 1 and Figure 1c. An increase in
15-keto-dihydro-PGF2a with advancing gestational age
was revealed. The trend analysis showed a significant
increase of 0.001 nmol/mmol 15-keto-dihydro-PGF2a/
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Acta Obstetricia et Gynecologica Scandinavica ª 2013 Nordic Federation of Societies of Obstetrics and Gynecology 92 (2013) 601–605602
Inflammation in normal pregnancy M. Palm et al.
creatinine per week with advancing gestational age
(p < 0.001). The median levels observed in the post-
partum period were lower than in gestational weeks 39–40 (p = 0.037) and corresponded to levels in gestational
weeks 9–10 (p = 0.306) (Figure 1c).
Discussion
In the current study, normal pregnancy was characterized
by a mild, but significant systemic inflammatory
response, shown by increased levels of the cytokine IL-6
and the COX-mediated marker PGF2a metabolite with
advancing gestational age. In addition, IL-6 remained
high while PGF2a metabolite diminished in the postpar-
tum period.
The physical activity during labor implies both mechan-
ical and metabolic stress that can increase inflammatory
activity, which in turn could be involved in the process of
parturition (16). To be able to accurately diagnose inflam-
matory disorders during pregnancy it is important to have
basal reference values from normal pregnancies. Moreover,
such reference values are also important and essential
when studying inflammatory factors in complicated preg-
nancies. Most published studies from normal pregnancies
have been limited by relatively small sample sizes, cross-
sectional designs or restricted longitudinal sampling peri-
ods. This longitudinal study describes the inflammatory
biomarkers of the cytokine and COX routes, the two
important pathways of inflammation, throughout normal
pregnancy and during the postpartum period.
Previous results from longitudinal studies regarding IL-6
throughout pregnancy are conflicting. Both increases
(4,6) and decreases (5) with advancing gestational age
have been reported. Current results showed an increase of
IL-6 levels in line with earlier reports (4–6). Increased
levels of IL-6 indicate a proinflammatory cytokine
response of the immune system throughout normal preg-
nancy and the high levels seen before delivery might be of
importance concerning labor onset (16). The sustained
high levels 9–10 weeks postpartum are in the same range
as those found 12–14 weeks after delivery in a cross-
sectional study (9) and support the presence of a remaining
systemic inflammatory response weeks after delivery (9).
Earlier longitudinal studies regarding TNF-a (5,6,17)
throughout pregnancy are also conflicting. No statistically
significant changes in TNF-a could be seen with advanc-
ing gestational age or in the postpartum period in our
study. This lack of change is in agreement with the results
from one longitudinal study (6), whereas another study
reported decreasing levels of TNF-a in pregnancy (5).
Moreover, increasing TNF-a receptor levels have been
reported with advancing gestational age (17). We found
that TNF-a levels remained unchanged 9–10 weeks post-
partum, a result consistent with one earlier study (9).
Such results suggest that TNF-a is not produced by the
placenta but by white blood cells (9).
The increase in PGF2a metabolite levels is in agreement
with a cross-sectional study in Japanese women (12) and
a recent longitudinal study in British women (13). During
pregnancy PGF2a, which acts both as luteolysin and
inflammatory response indicator in many species, is pro-
duced in several tissues, specifically in endometrium and
ovaries, and has essential roles concerning initiation of
delivery, cervical dilation and uterine contractions (18).
PGF2a analogs have also been used for induction of labor
(19) as well as for treatment of postpartum hemorrhage.
The PGF2a metabolite levels 9–10 weeks postpartum were
significantly lower compared with gestational weeks 39–40 and lower, although not significant, compared with
levels in gestational weeks 9–10. One cross-sectional study
has reported apparently higher levels of 13,14-dihydro-
Table 1. Median plasma interleukin-6 and tumor necrosis factor-a,
and urinary 15-keto-dihydro-prostaglandin-F2a levels at 2-week
intervals throughout pregnancy and during the postpartum period
(n = 37).
Pregnancy
week
Pregnancy
day n
IL-6
(pg/mL)
TNF-a
(pg/mL)
15-keto-dihydro-
PGF2a (nmol/mmol
creatinine)
7–8 49–62 4 0.92 1.20 0.18
9–10 63–76 10 0.99 1.17 0.21
11–12 77–90 19 1.04 1.26 0.20
13–14 91–104 1 0.52 1.80 0.40
15–16 105–118 3 0.76 1.35 0.24
17–18 119–132 1 0.82 1.94 0.19
19–20 133–146 23 0.91 1.05 0.24
21–22 147–160 12 0.67 1.12 0.20
23–24 161–174 8 1.17 0.87 0.25
25–26 175–188 28 0.97 1.14 0.23
27–28 189–202 15 1.45 1.01 0.24
29–30 203–216 19 1.02 1.20 0.26
31–32 217–230 26 1.09 1.26 0.23
33–34 231–244 12 1.01 1.26 0.23
35–36 245–258 28 1.57 1.44 0.23
37–38 259–272 10 1.16 1.43 0.25
39–40 273–286 20 1.75 1.68 0.22
Postpartum
week
Postpartum
day
5–6 35–48 1 1.05 1.37 0.16
7–8 49–62 7 1.16 2.22 0.12
9–10 63–76 14 1.77 1.78 0.16
11–12 77–90 4 0.73 1.96 0.21
13–14 91–104 5 0.81 1.34 0.14
15–16 105–118 3 1.19 1.27 0.20
17–18 119–132 2 1.13 1.70 0.17
19–20 133–146 1 1.27 4.39 0.48
IL-6, interleukin-6; PGF2a, prostaglandin-F2a; TNF-a, tumor necrosis
factor-a.
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M. Palm et al. Inflammation in normal pregnancy
15-keto PGF2a in saliva collected postpartum (20). The
measurements were, however, performed as early as
1–4 days after delivery.
The increased levels of IL-6 and PGF2a metabolite are
important because they indicate both a proinflammatory
cytokine response of the immune system as well as an
increase of the vasoconstrictive, inflammatory and luteo-
lytic PGF2a throughout normal pregnancy. The specific
properties of these compounds certainly have an as yet
undefined vital role in pregnancy progression and perhaps
successful parturition. The diverging data published on
cytokine levels are most probably a result of technical
differences. It may also reflect the diversity that lies
within the physiology of normal pregnancy.
One of the strengths of our study is that all women
were healthy and all pregnancies were uncomplicated.
Moreover, all infants were of normal birthweight, born at
term and had an uneventful early neonatal course.
Another strength is the high compliance with the sam-
pling schedule and the number of collection periods. A
minor limitation is that although a majority of samples
was taken during the scheduled time-frames, a substantial
number was collected at other times. Most often,
however, samples were collected within 1 week (Table 1)
15-k
eto-
dihy
dro-
PG
F 2α (n
mol
/mm
ol c
reat
inin
e)
9-10
11-1
213
-14
15-1
617
-18
19-2
021
-22
23-2
425
-26
27-2
829
-30
31-3
233
-34
35-3
637
-38
39-4
0
9-10
0.00
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0.40TN
F-α
(pg/
mL)
9-10
11-1
213
-14
15-1
617
-18
19-2
021
-22
23-2
425
-26
27-2
829
-30
31-3
233
-34
35-3
637
-38
39-4
0
9-10
0.0
0.5
1.0
1.5
2.0
2.5
IL-6
(pg/
mL)
9-10
11-1
213
-14
15-1
617
-18
19-2
021
-22
23-2
425
-26
27-2
829
-30
31-3
233
-34
35-3
637
-38
39-4
0
9-10
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
Gestational age (weeks)
P = 0.037P = 0.306
Trend P < 0.001
Gestational age (weeks) Postpartum(Weeks)
P = 0.634P = 0.128
Trend P = 0.260
Gestational age (weeks) Postpartum (weeks)
P = 0.903
P = 0.084
Trend P < 0.001
Postpartum (weeks)
(a)
(c)
(b)
Figure 1. (a) Plasma levels of interleukin-6 (IL-6), (b) plasma levels of tumor necrosis factor-a (TNF-a) and (c) urinary levels of 15-keto-dihydro-
prostaglandin-F2a (15-keto-dihydro PGF2a) in uncomplicated pregnancy and 9–10 weeks postpartum (n = 10 to n = 28). Only values from
intervals including samples from at least 10 women are given. Data are presented as median and the 25th centile to the 75th centile.
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Acta Obstetricia et Gynecologica Scandinavica ª 2013 Nordic Federation of Societies of Obstetrics and Gynecology 92 (2013) 601–605604
Inflammation in normal pregnancy M. Palm et al.
of the scheduled time, which is reasonable for this type of
study. Concerning the comparisons between early and late
pregnancy with postpartum levels, data were analyzed
using a cross-sectional approach because only a limited
number of women were sampled on all occasions.
In conclusion, this study presents IL-6, TNF-a and
PGF2a metabolite levels throughout normal pregnancy
and during the postpartum period and implicates mild
involvement of inflammation in normal pregnancy. The
physiological function of this inflammatory process dur-
ing pregnancy has yet to be elucidated.
Acknowledgments
We thank Ulla Geifalk and the late Eva Seiby for technical
assistance.
Funding
This study was supported by financial grants from the
foundation of Gillbergska, Uppsala, the Perinatal Research
Foundation, Uppsala and the General Maternity Hospital
Foundation, Stockholm.
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M. Palm et al. Inflammation in normal pregnancy