Soluble CD163 and TWEAK in early pregnancy gestational diabetes and later glucose intolerance
Soluble CD163 and TWEAK in early pregnancy gestational diabetes and later glucose intolerance
Jonatan DerekeID 0 2
Jacob Nilsson 0 2
Charlotta Nilsson 0 2
Helena Strevens 1 2
Mona Landin-Olsson 0 2
Magnus Hillman 0 2
0 Lund University, Faculty of Medicine, Department of Clinical Sciences Lund, Diabetes Research Laboratory, Lund, Sweden, 2 Department of Paediatrics, Helsingborg Hospital , Helsingborg , Sweden
1 Department of Obstetrics, Ska?ne University Hospital Lund, Lund, Sweden, 4 Department of Endocrinology, Ska?ne University Hospital Lund , Lund , Sweden
2 Editor: David J. Garry, Stony Brook University Health Sciences Center School of Medicine , UNITED STATES
Gestational diabetes mellitus (GDM) is today universally diagnosed during late pregnancy. Treating hyperglycaemia during pregnancy reduces the risk of complications, the effect of interventions is however limited due to the late diagnosis. It is thus important to identify biomarkers reaching a high precision for GDM development in early pregnancy. Here we aim to investigate soluble CD163 (sCD163) and soluble tumour necrosis factor-like weak inducer of apoptosis (sTWEAK) in early pregnancy GDM and their association to the development of later glucose intolerance. In this case-control study, women diagnosed with GDM in early pregnancy (n = 70) at Lund University Hospital, Lund, Sweden in 2011-2015 were age- and BMI matched to pregnant volunteers without diabetes (n = 70) recruited in early pregnancy from maternal health care centres in 2014-2015. Plasma levels of sCD163 and sTWEAK were analysed using commercial ELISA. Plasma levels of sCD163 did not differ between patients with and without GDM in early pregnancy (p = 0.86), plasma levels of sTWEAK however was decreased in women with GDM (0.71 [0.4-1.75] ng/ml) compared to controls (1.38 [0.63-4.86] ng/ml; p = 0.003). Women with sTWEAK levels in the lowest tertile had an increased risk of GDM in early pregnancy (p = 0.014). Neither sCD163 nor sTWEAK were associated with later glucose intolerance in women with GDM. This study reports decreased levels of sTWEAK in women with early pregnancy GDM, independent of age and BMI. Neither sCD163 nor sTWEAK were found to be associated to later glucose intolerance.
Data Availability Statement: All relevant data are
within the manuscript and its Supporting
Funding: MH received funding for this study by the
Swedish Diabetes Foundation, MLO received
funding from Sk?ne University Hospital Funding
and Donations, and CN received funding from the
Gorthon Foundation. The funders had no role in
study design, data collection and analysis, decision
to publish, or preparation of the manuscript.
Gestational diabetes mellitus (GDM) affects approximately 2% of all pregnant women in
southern Sweden [
]. Pregnancies complicated by GDM reveal an increased risk of
hypertensive disorders and future manifest diabetes for the mother. GDM may also have adverse effects
on foetal development, increasing the risk of macrosomia, perinatal complications and the risk
for future obesity in the offspring [
Increased levels of free fatty acids and advanced glycation end products, which lead to the
production of pro-inflammatory cytokines from adipose tissue and subsequent chronic
inflammation, are typical for GDM [
]. Inflamed adipose tissue causes monocyte infiltration
and activation of tissue resident macrophages. The endocytic scavenger receptor CD163 is
expressed on macrophages active in adipose tissue [
]. A large prospective study showed that
increased serum levels of soluble CD163 (sCD163) could predict future development of type 2
diabetes independent of age and body mass index (BMI) [
]. Studies on sCD163 in GDM are
few and inconclusive with one study reporting increased levels in GDM [
], while another
found no difference compared to in pregnant women with normal glucose tolerance (NGT)
Soluble tumour necrosis factor-like weak inducer of apoptosis (sTWEAK) exert multiple
different cellular activities on a wide array of cells, which are important in regulating the
antiinflammatory/inflammatory balance, and has also been suggested as a ligand for CD163 [
Studies made on circulating sTWEAK in diabetes report significantly decreased serum levels
in both type 1 diabetes and type 2 diabetes [
]. One study reported lower levels of
sTWEAK in women with GDM compared to women with NGT [
GDM is today universally diagnosed during late pregnancy. Treating hyperglycaemia
during pregnancy is known to reduce the risk of complications [
], but the effect of intervention
is often limited due to the late diagnosis. Finding early pregnancy biomarkers capable of
identifying women at increased risk for GDM could motivate earlier and more efficient
interventions, minimising the risk for adverse outcomes [
]. Identifying GDM patients at increased
risk of later glucose intolerance development is of importance and could act as an incentive for
necessary lifestyle changes and pharmacological interventions in these women [
The aims of this study was to investigate plasma levels of sCD163 and sTWEAK in pregnant
women with and without GDM in early pregnancy and their association to later glucose
Participants and methods
In the catchment area of Lund University Hospital, Lund, Sweden, pregnant women are
offered an OGTT as part of a general GDM screening. The glucose load for the OGTT is 75-g
following overnight fast and a 2-hour capillary plasma glucose value above 10mmol/l was used
as the diagnostic cut-off for GDM at the time of patient recruitment as recommended by the
European Association for the Study of Diabetes (EASD) [
]. Between 2011 and 2015 a total of
519 pregnant women were diagnosed with GDM with 140 receiving the diagnosis in early
pregnancy. The patients included in this study were diagnosed in early pregnancy (gestational
age 14 ? 4 weeks) (n = 70). Information regarding family history of diabetes, previous GDM
and later development of glucose intolerance was retrieved when available from the patients?
primary and/or secondary records both by studying journals and ICD-10 codes in 2018.
Thirteen (19%) of the patients developed glucose intolerance up to four years after delivery. The
mean time to development was 1.7 ? 1.1 years. Age and BMI matched pregnant controls
(n = 70) were recruited at their first visit to the maternal health care centres (around week 12)
from the same geographical region in 2014?2015. Controls with previously diagnosed diabetes
or GDM were not included in this study. Blood samples were drawn into
ethylenediaminetetraacetic acid (EDTA) plasma tubes and centrifuged at 2000 x g for 10 minutes. The soluble
component was separated from the blood cells and stored at -70?C until time of analysis.
This study was approved by the regional ethical committee in Lund (Regionala
etikpro?vningsna?mnden Lund) (849/2005, 2014/78 and 2014/744) and performed in accordance with
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the principles of the 1964 Declaration of Helsinki. All participants received oral and written
information about this study before providing written informed consent.
Plasma levels of sCD163 and sTWEAK were measured using commercially available DuoSet
enzyme linked immuno-sorbent assay (ELISA) kits (R&D Systems, Minneapolis, Minnesota,
USA) and optimised for human plasma. The analyses were run according to the
manufacturer?s instructions. Samples were diluted 1:200 and 1:5 respectively for the sCD163 and sTWEAK
analysis. The intra-assay coefficient of variation was 8.7% for sCD163 and 7.8% for sTWEAK
respectively. All samples were run as duplicates, and patient and control samples were
alternated to minimise bias caused by intra-assay variation.
Mean ? standard deviation (SD) were used to present data for continuous variables where
normality was accepted and median followed by interquartile range in brackets where normality
was rejected. Student?s t-test or the Mann-Whitney U-test were used to compare differences
between groups. Spearman?s rho was used in order to assess the degree of correlation between
variables. Odds-ratios (OR) were calculated for the association of GDM with regard to plasma
sTWEAK tertiles. P-values less than 0.05 were considered statistically significant. All statistical
analyses were performed using MedCalc Statistical Software version 18.9 (MedCalc Software
bvba, Ostend, Belgium; http://www.medcalc.org; 2018).
The data set containing raw data of sCD163, sTWEAK and postpartum diabetes
development is provided as Supporting Information (S1 Dataset).
No difference in plasma levels of sCD163 could be observed between women with or without
GDM diagnosed in early pregnancy (318 [246?395] ng/ml and 321 [243?432] ng/ml respectively;
p = 0.86). Plasma levels of sTWEAK were however decreased in women with early pregnancy
GDM (0.71 [0.4?1.75] ng/ml) compared to controls (1.38 [0.63?4.86] ng/ml; p = 0.003). Plasma
sTWEAK levels were not associated to participant age (p = 0.61) or BMI (p = 0.55), and no
correlation between sCD163 and sTWEAK could be observed (p = 0.86). There was no association
between sCD163 (p = 0.16) or sTWEAK (p = 0.27) and glucose intolerance development
following GDM in this study. Women with GDM had more often a family history of diabetes (57% (36/
63)) compared to controls (31% (22/70; p = 0.003). Neither sCD163 nor sTWEAK levels were
found to differ in women with or without a family history of diabetes (p = 0.48 and p = 0.27
respectively). Some of the patients have had previous GDM (10% (6/59)). No association could be
observed between previous GDM and sCD163 (p = 0.76) or sTWEAK (p = 0.74) levels.
In order to increase reproducibility through different modes of analysis, sTWEAK levels
were arranged into tertiles (T1-T3). Plasma sTWEAK levels in the lowest tertile (T1 (<0.63ng/
ml)) were associated with an increased risk of GDM (OR: 2.48, CI95 (1.20?5.10); p = 0.014),
while sTWEAK levels in the highest tertile T3 ( 1.75ng/ml) showed a tendency for a decreased
risk of GDM (OR: 0.49, CI95 (0.24?1.00; p = 0.05) (Table 1).
This study has investigated sCD163 and sTWEAK levels in women diagnosed with GDM in
early pregnancy. While no difference in sCD163 levels could be observed, decreased levels of
sTWEAK were associated with the development of GDM. A previous study has found
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increased levels of sCD163 at delivery in women diagnosed with GDM in late pregnancy [
while another study where sCD163 was measured in late pregnancy did not observe a
difference when compared to women with NGT [
] which is in accordance with our findings in
early pregnancy. The study by Bari et al. [
] investigated sCD163 levels at parturition in
women with elective caesarean section in a small group of GDM patients (n = 18) and BMI
matched controls (n = 20). They suggested sCD163 to be a significant predictor of GDM,
although the precision was not presented. The late sampling and small sample size could be a
reason for the discrepancy among the studies. Adipose tissue explants and placenta showed
increased sCD163 release in GDM and the placenta was suggested to be a significant source of
sCD163 in GDM. Simon-Muela et al. measured sCD163 levels in late pregnancy GDM
(n = 66) and NGT (n = 71) [
]. They found sCD163 levels to be associated to a poor metabolic
profile, but could not find any significant difference in sCD163 levels between the groups. In
contrast to the study by Bari et al, sCD163 release from adipose tissue and placenta was not
studied by Simon-Muela et al. nor by this study.
GDM is a condition characterised by chronic inflammation and decreased sTWEAK levels
have been observed in diseases associated with chronic inflammation and insulin resistance
]. Circulating sTWEAK was in this study decreased in early pregnancy GDM
independent of participant age and BMI and family history of diabetes. Our results are in agreement
with a Spanish study made in later pregnancy . Membrane bound CD163 has been reported
to bind sTWEAK in circulation [
]. We could however, in contrast to the Spanish study [
not observe any correlation between circulating sCD163 and sTWEAK. By arranging the
sTWEAK levels into tertiles, we facilitate the possibility to reproduce the findings in this study.
Women with sTWEAK levels in the lowest tertile had significantly increased risk of GDM,
while there was a tendency for a decreased risk of GDM with sTWEAK levels in the highest
tertile. In this study we also sought to determine any associations between sCD163, sTWEAK
and the development of glucose intolerance following a pregnancy complicated by GDM.
Neither sCD163 nor sTWEAK were however found to be associated to such development, possibly
explained by the low number of patients who developed later glucose intolerance.
A strength with this study is that the GDM patients included are part of a unique screening
program that enables all pregnant women in Lund to undergo an OGTT, resulting in a
heterogeneous group of participants. The women included as controls are matched in age and BMI,
and they are recruited from the same geographical region as the patients, further minimising
confounding effects. Limitations include a limited number of participants and lacking the
possibility for a follow-up during late pregnancy. A power calculation suggests 145 participants
per group for sTWEAK and 759 for sCD163 with an alpha value of 0.05 and beta value of 0.8
for later glucose intolerance. It is important to notice however that the distribution for these
molecules in non-parametric which may skew the power calculation.
This study reports decreased levels of sTWEAK in women with early pregnancy GDM,
independent of age and BMI. No difference could be observed with regard to sCD163 levels, and
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neither sTWEAK nor sCD163 was associated with later glucose intolerance. A future
prospective cohort study should focus on elucidating the potential for sTWEAK to be used as an early
pregnancy pre-screening biomarker for GDM development as this could possibly alleviate the
need for onerous screening procedures in women with low risk for developing
hyperglycaemia. More focus should also lie in earlier identification of women at risk of developing GDM
as this could motivate more efficient treatment interventions at an earlier time point,
minimizing adverse outcomes.
S1 Dataset. Minimal data set underlying the results of this study.
The authors would like to thank Ms. Birgitte Ekholm, the Diabetes Research Laboratory,
Lund, for her excellent technical assistance. The authors also want to thank the midwives at
the maternal healthcare centres in Linda?ngen in Malmo?, Dalby and Paletten in Staffanstorp.
Data curation: Magnus Hillman.
Formal analysis: Jonatan Dereke.
Conceptualization: Jonatan Dereke, Charlotta Nilsson, Mona Landin-Olsson, Magnus
Funding acquisition: Charlotta Nilsson, Mona Landin-Olsson, Magnus Hillman.
Investigation: Jonatan Dereke, Jacob Nilsson, Magnus Hillman.
Methodology: Jonatan Dereke, Helena Strevens, Magnus Hillman.
Project administration: Jonatan Dereke, Mona Landin-Olsson, Magnus Hillman.
Resources: Magnus Hillman.
Software: Magnus Hillman.
Supervision: Jonatan Dereke, Charlotta Nilsson.
Writing ? original draft: Jonatan Dereke.
vens, Mona Landin-Olsson, Magnus Hillman.
Writing ? review & editing: Jonatan Dereke, Jacob Nilsson, Charlotta Nilsson, Helena
Stre5 / 6
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