maternal obesity and its effect on placental cell turnover
TRANSCRIPT
2013
http://informahealthcare.com/jmfISSN: 1476-7058 (print), 1476-4954 (electronic)
J Matern Fetal Neonatal Med, 2013; 26(8): 783–788! 2013 Informa UK Ltd. DOI: 10.3109/14767058.2012.760539
Maternal obesity and its effect on placental cell turnover
Lucy Higgins1, Tracey A. Mills1,2, Susan L. Greenwood1, Elizabeth J. Cowley1, Colin P. Sibley1, and Rebecca L. Jones1
1Maternal and Fetal Health Research Centre, Institute of Human Development, Manchester Academic Health Science Centre, St. Mary’s Hospital,
Central Manchester University Hospitals NHS Foundation Trust, University of Manchester, Manchester, UK, and 2School of Nursing, Midwifery and
Social Work, University of Manchester, Manchester, UK
Abstract
Background: Maternal obesity is a frequent obstetric risk factor, linked with short- and long-termconsequences for mother and child, including foetal overgrowth, growth restriction andstillbirth. The mechanisms underlying these pathologies remain unknown but likely involve theplacenta.Aims: To study placental cell turnover in relation to maternal body mass index (BMI).Methods: Term placental villous tissue was randomly sampled from 24 pregnancies, with arange of maternal BMI of 19.5–49.6. Immunohistochemistry was performed for human chorionicgonadotropin, Ki67 and M30 and image analysis used to calculate syncytiotrophoblast area andproliferative and apoptotic indices. Results were compared categorically between women ofBMI 18.5–24.9 (normal), BMI 30.0–39.9 (obese classes 1and 2) and BMI 40þ (obese class 3) andcontinuously against BMI; p50.05 by the Kruskal–Wallis test or linear regression was consideredstatistically significant.Results: Increased maternal BMI was associated with categorical (normal versus obese class 3and obese classes 1 and 2 versus obese class 3, both p50.05) and continuous (r2¼ 0.24,p¼ 0.016) reductions in the proliferative index and a continuous reduction (r2¼ 0.17, p¼ 0.047)in the apoptotic index.Discussion: Maternal obesity is associated with a dose-dependent reduction in placental villousproliferation and apoptosis which may increase susceptibility to adverse pregnancy outcomes.
Keywords
Adiopokine, apoptosis, body mass index,immunohistochemistry, pregnancy,proliferation, syncytiotrophoblast, villous
History
Received 28 August 2012Revised 13 December 2012Accepted 17 December 2012Published online 31 January 2013
Introduction
Obesity is now the most frequent risk factor in obstetric care
[1]. Rates of maternal obesity (defined as a body mass index
(BMI) of �30, calculated as weight in kilograms divided by
height in metres squared [2]) at first antenatal contact have
been rising in line with worldwide adult obesity trends. In the
North West of England, the prevalence of maternal obesity
almost doubled between 1990 and 2004 rising from 10% to
16–19% [3].
Maternal obesity is linked to short- and long-term health
problems in both mother and offspring, including preeclamp-
sia, foetal growth restriction (FGR), foetal overgrowth and
stillbirth (summarised by Higgins et al. [4]) with risks
escalating as the maternal BMI increases further (e.g. the risk
of preeclampsia is tripled at BMI 30–39.9 but quadrupled
when the BMI is 40 or more [5]). Following delivery,
intrauterine programming of the offspring’s metabolic state
leads to an increased risk of childhood obesity and
cardiovascular disease, thus propagating a vicious cycle of
obesity-related adverse health outcomes [6].
Assessing the appropriateness of foetal growth in the
context of maternal obesity is troublesome, as current birth
weight normograms were predominantly created before the
onset of the obesity epidemic [7,8]; indeed in some
populations, obesity was so uncommon that obese women
were excluded from centile calculations. Thus, the commonly
observed range of birth weights in obese women is unclear,
and it is likely that many seemingly ‘‘appropriate for
gestational age’’ (AGA) foetuses have failed to achieve their
own individual growth potential, and are in fact FGR,
contributing towards the increased incidence of stillbirth
amongst lower-centile-‘‘AGA’’ infants of obese women [9].
As the placenta is the principle determinant of foetal
growth, differences in placental structure and function may
contribute to aberrant foetal growth states (both FGR and
foetal overgrowth). Differences in placental cell turnover have
been documented in pregnancies complicated by preeclamp-
sia (increased trophoblast proliferation and apoptosis [10,11])
and FGR (decreased trophoblast proliferation [12] and
increased apoptosis [13]). Such aberrations in trophoblast
cell turnover are likely to influence the size or integrity of the
placental exchange barrier, potentially impacting on nutrient
and oxygen transfer to the foetus. Placental cell turnover has
Address for correspondence: Lucy Higgins, Maternal and FetalHealth Research Centre, Institute of Human Development, ManchesterAcademic Health Science Centre, St. Mary’s Hospital, CentralManchester University Hospitals NHS Foundation Trust, University ofManchester, Level 5 (Research), Oxford Road, Manchester M13 9WL,UK. Tel: þ161 7016951. Fax: þ161 7016971. E-mail: [email protected]
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not previously been investigated in the context of maternal
obesity. We tested the hypothesis that syncytiotrophoblast
area and villous turnover are altered in obese compared with
normal-BMI women.
Methods
Patient recruitment
The study was approved by Central Manchester Research
Ethics Committee (08/H1008/80). Healthy women with
known first trimester BMI, delivering between 37þ0 and
42þ0 weeks’ gestation were eligible for inclusion. Those with
known maternal or foetal health problems (including
antenatally detected FGR or foetal overgrowth) were excluded
to ensure that any observed differences in placental structure
and function were not accounted for by co-pathology or
differences in management of the pregnancy (such as early
delivery, administration of corticosteroids, non-routine glu-
cose tolerance testing and subsequent treatment of gestational
diabetes). Basic demographic (maternal age, ethnicity, parity
and smoking status) and outcome data (gestational age at
delivery, birth weight, gender and mode of delivery) were
collected to ensure groups were comparable in other respects.
Birth weight was expressed as an individualised birth weight
centile (IBC) after adjustment for maternal ethnicity, parity,
height and weight, gestational age and foetal gender using
Gestation-Related Optimal Weight software (Customised
Weight Centile Calculator v5.12/6.2 2009 downloaded from
www.gestation.net). IBCs above the 90th centile were
classified as large for gestational age (LGA) and those
below the 5th centile were classified as FGR.
Three study groups were planned; normal-BMI (18.5–
24.9), obese classes 1 and 2 (BMI 30.0–39.9) and obese class
3 (BMI� 40.0). Based on similar studies of syncytiotropho-
blast area and villous turnover in FGR and preeclampsia [14],
we calculated that eight women per group would be required
to detect a similar magnitude of difference with a power of
90% at a type one error probability of 0.05.
Tissue collection and processing
The placenta was collected as soon as possible following
delivery, trimmed and weighed and three villous biopsies
from randomly selected sites of the placenta were fixed in
10% neutral buffered formalin and paraffin embedded.
Researchers were blinded to maternal BMI until the data
analysis stage, by random identification number allocation.
Sections of villous tissue were immunostained with colori-
metric detection using an antihuman chorionic gonadotrophin
(hCG) antibody (rabbit polyclonal 5 mg/ml; Dako, Ely, UK) to
identify the syncytiotrophoblast, and antibodies against Ki67
(mouse monoclonal 0.16 mg/ml; Dako) and cytokeratin M30
(mouse monoclonal 1 mg/ml; Roche Diagnostics, Burgess
Hill, UK) as markers of proliferation and apoptosis,
respectively, as previously described [15]. Substitution of
the primary antibody with non-immune species-specific
immunoglobulin at equal concentration was performed for
negative controls. Positive localization was performed with
biotinylated species-specific secondary antibodies
(swine antirabbit 0.88mg/ml at 1:200 dilution; Dako and
goat antimouse 0.79 mg/ml at 1:200 dilutions; Dako for hCG
and Ki67/M30, respectively) and chromogen diaminobenzi-
dine. Tissue sections were counter-stained with haematoxylin.
Representative images are shown in Figure 1.
Image analysis
Ten randomly selected fields of view were captured from each
tissue section using a Leitz Dialux 22 Microscope (Ernst Leitz
Wetzlar GMBH, Germany) and Qicam Fast 1394 camera
(Qimaging, Surrey, Canada) using a �25 objective. Images
were then analysed using Image Pro Plus 6.0 software (Media
Cybernetics UK, Marlow, UK). Villous and syncytiotropho-
blast areas were calculated (mm2) and the total number of
nuclei per field of view was calculated using pseudo-
colouring. In this technique, the software’s histogram function
was used to highlight the total area of villous tissue by
adjustment of a three-colour sliding scale to superimpose a
pseudo-colour overlay in each field of view. Following this,
areas staining positive for hCG were highlighted using the
‘‘colour-dropper’’ function in which individual pixels of
colour were selected to create a new pseudo-colour overlay
restricted to the syncytiotrophoblast area. The total nuclei
count was calculated using the histogram function and sliding
scale to highlight haematoxylin-stained nuclei; the number of
Ki67þ- and M30þ-stained nuclei per field of view were each
manually counted.
Syncytiotrophoblast area was expressed as raw hCGþ area
and as a percentage of total villous area per field of view. The
proliferative and apoptotic indices were expressed as Ki67þ
and M30þ percentages of the total nuclei count, respectively.
A mean value was calculated across 10 fields of view per
villous biopsy, and the median value of all three villous
biopsies from each placenta was taken for comparison
between placentas.
Data analysis
Following image analysis, the researcher was un-blinded to
maternal BMI. Statistical analysis was performed in two ways.
Categorical differences between normal-BMI, obese classes 1
and 2 and obese class 3 groups were analysed by the Kruskal–
Wallis test with Dunn’s post hoc test. Continuous relation-
ships between BMI and syncytiotrophoblast area, proliferative
and apoptotic indices were assessed using linear regression
and were conducted on the whole cohort and were repeated
after exclusion of FGR and LGA pregnancies. Linear
regression was also performed between IBC and syncytio-
trophoblast area, proliferative and apoptotic indices.
Statistical significance was set at the level of p50.05.
Results
Other than for maternal BMI (p50.0001), the experimental
groups were well matched for baseline and pregnancy
outcome characteristics with statistically significant differ-
ences only in the percentage of Caucasian women (with fewer
Caucasian obesity classes 1 and 2 women than in other
groups) and a two-day increase in gestational age in the
normal-BMI group in comparison to obese groups (Table 1).
There were no statistically significant differences between
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BMI categories in birth weight, IBC, placental weight or feto–
placental weight ratio (p40.05). Whilst antenatally detected
foetal overgrowth and FGR cases were excluded, postnatally
identified FGR (one) and LGA (two) infants were born to
mothers in the obesity class 3 cohort and were included in
analyses. Data from these pregnancies are shown as squares
(FGR) and triangles (LGA) to aid identification.
Syncytiotrophoblast and villous area
No categorical differences were observed between groups in
raw syncytiotrophoblast (Kruskal–Wallis p¼ 0.072; data not
shown) or villous areas (Kruskal–Wallis p¼ 0.65; data
not shown). When expressed as a percentage of villous area,
no significant relationship was observed between maternal
BMI and syncytiotrophoblast area (r2¼ 0.035, p¼ 0.85;
Figure 2a and b) or between IBC and syncytiotrophoblast
area (r2¼ 0.0083, p¼ 0.68; data not shown).
Villous cell turnover
No categorical differences were observed between groups in
the total number of nuclei per field of view in sections stained
for Ki67 (Kruskal–Wallis p¼ 0.18; data not shown) and M30
(p¼ 0.27; data not shown). A negative relationship was
detected between villous proliferation and maternal BMI
(Kruskal–Wallis p¼ 0.026; Figure 2c), with placentas from
women with class 3 obesity demonstrating significantly fewer
Figure 1. Representative images of immunohistochemical staining in term placental villous tissue. In images (a–c) brown staining demonstratespositivity for human chorionic gonadotropin in (a) normal-BMI and (b) obese women; and (c) a negative control. In images (d–f) the black arrowsindicate villous nuclei staining positive for the proliferation marker Ki67 in (d) normal-BMI and (e) obese women and (f) a negative control. In images(g–i) the black arrows indicate trophoblast staining positive for the apoptosis marker cytokeratin M30 in (g) normal-BMI and (h) obese women and (i) anegative control.
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proliferating nuclei per field of view compared with either
normal-BMI (Dunn’s post hoc test p50.001) or obesity
classes 1 and 2 (Dunn’s post hoc test p50.05) women’s
placentas. An inverse linear relationship was detected
between BMI and the proliferative index (r2¼ 0.24,
p¼ 0.016; Figure 2d). Whilst categorical differences in the
apoptotic index between maternal BMI groups did not reach
statistical significance (Kruskal–Wallis p¼ 0.16; Figure 2e),
an inverse relationship was found between BMI and the
apoptotic index (r2¼ 0.17, p¼ 0.047; Figure 2f).
After exclusion of FGR and LGA infants a significant
negative relationship persisted between BMI and proliferative
index (r2¼ 0.20, p¼ 0.040; data not shown), but no relation-
ship remained between BMI and apoptotic index (r2¼ 0.11,
p¼ 0.14; data not shown). No significant relationship was
detected between IBC and proliferative (r2¼ 0.036, p¼ 0.38;
data not shown) and apoptotic indices (r2¼ 0.0026, p¼ 0.82;
data not shown).
Discussion
This is the first study to examine syncytiotrophoblast area and
villous turnover in the context of maternal obesity, to explore
whether aberrant placental cell turnover may contribute to the
susceptibility of obese women to pregnancy complications.
This research area is fundamentally complicated by the
increased prevalence of two contrasting foetal growth
phenotypes, FGR and LGA, in obese women. Whilst
syncytiotrophoblast area was unaffected in maternal obesity,
we detected a significant negative relationship between
maternal BMI and both villous proliferative and apoptotic
indexes, in a dose-dependent manner. These findings cannot
be explained by differences in villous area and the total nuclei
number per field of view, as these parameters remained
constant between groups, and thus the findings of this study
are not a result of the ‘‘reference trap’’ [16].
The findings of this study are directly opposite to those of
similar studies in preeclampsia which demonstrates an
increase in both trophoblast proliferation and apoptosis.
This is surprising as preeclampsia is a pregnancy complica-
tion that is both more frequent in obesity and is associated
with similar circulating inflammatory endocrine [17] and
placental gene transcription [18] profiles. In particular,
maternal circulating leptin concentrations and placental
leptin gene transcription are several-fold higher in preeclamp-
tic compared with normotensive pregnancies. The endocrine
imbalance in preeclampsia is, however, more transient than
that observed in prepregnancy maternal obesity [19] and it
may be that a difference in duration of exposure to deranged
adipokine levels may explain the contrasting placental
turnover phenotypes in these two conditions.
A total of 75% of infants from class 3 obese mothers had
an IBC below the 50th centile. This skewing of birth weights
amongst the offspring of the most obese women may
represent a relative failure of these foetuses to achieve their
own birth weight potential despite remaining above a birth
weight apparently ‘‘appropriate’’ for their gestational age. If
these pregnancies were in fact sub-clinically growth
restricted, one would expect to see a similar pattern of
placental proliferation and apoptosis to that previously
reported in FGR pregnancies. This is the case for placental
proliferation, which was significantly decreased with increas-
ing BMI, consistent with a decrease in placental proliferation
in FGR [12]. These differences persisted even after the
exclusion of FGR and LGA pregnancies, supporting a causal
relationship with maternal BMI. However, the finding of
decreased apoptosis with increasing BMI contrasts with those
of comparable studies in FGR which report an increase in
apoptosis [13]. Interestingly, placentas from LGA infants
displayed apoptotic indices within the lowest quartile of the
entire cohort and when these were excluded from the analysis
of the apoptotic index, the significant negative relationship
with BMI was lost. These findings suggest that lower rates of
apoptosis in the placentas of LGA infants may positively
influence placental and foetal growth, and indeed in this study
their placental weights were within the highest quartile.
Further studies are required to delineate the frequently
overlapping influences of maternal BMI and LGA infants.
The observed alteration in placental proliferation and
apoptosis could affect placental development and function,
resulting in altered placental size and integrity of the placental
exchange barrier. This may have consequent effects on
placental efficiency and may increase susceptibility of the
foetus to poor pregnancy outcomes. Indeed, Oliva et al. [20]
have demonstrated a reduction in the expression of cellular
integrity proteins in homogenised placental tissue as maternal
Table 1. Demographic and pregnancy outcome data for the study cohort.
BMI 18.5–24.9, (n¼ 8) BMI 30.0–39.9, (n¼ 8) BMI 40þ , (n¼ 8) p
BMI (kg/m2) 22.3 (19.5–23.9) 32.5 (30.1–37.8) 42.4 (40.6–49.6) 50.0001Maternal age (years) 33 (29–38) 31 (24–35) 33 (27–36) 0.42Parity 0 (0–5) 2 (0–3) 2.5 (0–4) 0.35Caucasian (%) 100.0 50.0 87.5 0.040Smoker (%) 37.5 12.5 12.5 0.21Gestation (weeksþdays) 40þ0 (38þ0–41þ0) 38þ5 (37þ0–41þ0) 38þ5 (37þ3–39þ5) 0.042Male (%) 25.0 50.0 50.0 0.50Laboured (%) 50.0 37.5 0.0 0.073Caesarean (%) 75.0 75.0 100.0 0.30Birth weight (g) 3280 (2960–3640) 3270 (2880–3745) 3380 (2655–4845) 0.53IBC 34.6 (10.8–83.1) 36.5 (24.9–80.7) 38.7 (1.1–100.0) 0.87Trimmed placental weight (g) 538 (439–703) 480 (410–550) 583 (446–911) 0.13Feto–placental weight ratio 5.8 (4.9–7.7) 6.9 (6.0–7.7) 6.0 (4.9–7.5) 0.08
Data are expressed as median (range in parentheses) unless otherwise specified. Continuous data are compared between BMI categories using theKruskal–Wallis test and categorical data are compared using the chi-squared test. Statistical significance was set at the level of p50.05.
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BMI increases. Despite this, we did not see any significant
differences in the feto–placental weight ratio between BMI
categories, although the current study was underpowered to
detect subtle differences in this parameter.
Adipose tissue is now widely recognised as an endocrino-
logically active organ, and differences in the volume of
adipose tissue in the body (accurately reflected by BMI in
early pregnancy [21]) are responsible for disturbances in
maternal and foetal circulating levels of adipokines including
leptin and adiponectin (reviewed by Higgins et al. [4]). It is
proposed that poor pregnancy outcomes related to maternal
obesity may result, at least in part, from the effects of the
deranged hormonal milieu of obesity [4]. The effects of two
key adipokines, leptin and adiponectin, have been investigated
on placental cell lines (JEG and BeWo cells). Circulating
maternal leptin levels are increased in maternal obesity
[22,23], and leptin treatment of placental cell lines results in
an increase in proliferation [24] and decrease in apoptosis
BMI 18.5
-24.9
BMI 30.0
- 39
.9
BMI 40+
0
10
20
30
40
50
% o
f Vill
ous
area
20 30 40 50 600
10
20
30
40
50
r2 = 0.0016, p = 0.85
BMI
% o
f Vill
ous
are
a
BMI 1
8.5-
24.9
BMI 3
0.0
- 39.
9
BMI 4
0+0
2
4
6
****
% o
f to
tal n
ucle
i Ki6
7 po
sitiv
e
20 30 40 50 600
2
4
6
r2 = 0.24, p = 0.016
BMI
% o
f tot
al n
ucle
i Ki6
7 po
sitiv
e
BMI 18.
5-24
.9
BMI 30.0
- 39.9
BMI 4
0+0.0
0.2
0.4
0.6
0.8
1.0
% o
f tot
al n
ucle
i M30
pos
itive
20 30 40 50 600.0
0.2
0.4
0.6
0.8
1.0
r2 = 0.17, p = 0.047
BMI
% o
f tot
al n
ucle
i M30
pos
itive
(a) (b)
(c) (d)
(e) (f)
Figure 2. Relationship between maternal BMI and syncytiotrophoblast area, and villous proliferative and apoptotic indices. Whilst syncytiotrophoblastarea is unchanged by increasing BMI (a–b), a significant dose-dependent reduction in proliferative (c–d) and apoptotic indices (e–f) is demonstrated(proportion of all nuclei stained positive for each marker). Circles represent infants with IBC between the 5th and 90th centiles, squares representinfants with IBC55th centile, and triangles represent infants with IBC490th centile. Data are compared between BMI categories using Kruskal–Wallis test with Dunn’s post hoc test and are compared to BMI using linear regression. Statistical significance was set at the level of p50.05, *p50.05and ***p50.001.
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[25,26]. Maternal adiponectin levels are suppressed in
maternal obesity. Adiponectin treatment of placental tropho-
blast cell lines results in decreased proliferation [27], thus
lower circulating adiponectin concentrations might reason-
ably be expected to promote placental proliferation. Possible
reasons for the discrepancy between these findings and those
of the current study may include the development of placental
leptin resistance with prolonged in vivo exposure [28,29],
opposing the effects of co-deranged adipokines and growth
factors and the inherent differences between fresh tissue and
immortalised cell lines, especially with respect to prolifera-
tion. The effects of other adipokines (e.g. interleukin-6,
TNFa, free IGF-1 and resistin) on placental cell turnover have
not been examined.
This study highlights the potential effects of the maternal
obesogenic environment on placental cell turnover, and thus
the potential role of the placenta in the pathogenesis of
aberrant foetal growth in these pregnancies. Its findings are
strengthened by the exclusion of maternal obesity-related
co-pathologies such as preeclampsia. Future work in this area
should aim to confirm these findings, to examine whether
such differences in nuclear turnover are present earlier in
pregnancy (prior to the onset of foetal growth abnormalities)
and to determine whether (acute or more prolonged) in vitro
exposure of placental villous tissue from normal-BMI women
to deranged adipokine concentrations (singularly via use of
commercially available products or collectively via use of
obese maternal serum) replicates the obese phenotype
described in this study.
Acknowledgements
We would like to thank Andrea Ditchfield, Helen Miller and
Linda Peacock for their assistance in recruitment and tissue
sampling, and the staff and patients of St. Mary’s Hospital,
Manchester, for their participation in the study.
Declaration of interest
The authors report no declarations of interest. This work was
funded by Tommy’s the Baby Charity, with infrastructure
support provided by the NIHR Manchester Biomedical
Research Centre. Dr Mills is supported by an Action
Medical Research Training Fellowship.
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