International Journal of
Molecular Sciences
Article
Maternal Obesity Alters Placental Cell Cycle
Regulators in the First Trimester of Human Pregnancy:
New Insights for BRCA1
Denise Hoch 1 , Martina Bachbauer 1 , Caroline Pöchlauer 1 , Francisco Algaba-Chueca 2 ,
Veronika Tandl 1 , Boris Novakovic 3 , Ana Megia 2 , Martin Gauster 4 , Richard Saffery 3 ,
Andreas Glasner 5 , Gernot Desoye 1, * and Alejandro Majali-Martinez 1
1
2
3
4
5
*
Department of Obstetrics and Gynecology, Medical University of Graz, 8036 Graz, Austria;
denise.hoch@medunigraz.at (D.H.); martina.bachbauer@gmail.com (M.B.);
caroline.poechlauer@gmx.at (C.P.); veronika.tandl@stud.medunigraz.at (V.T.);
a.majali-martinez@medunigraz.at (A.M.-M.)
Department of Endocrinology and Nutrition Research Unit, University Hospital of Tarragona Joan
XXIII-Institut d´Investigació Sanitària Pere Virgili (IISPV), 43005 Tarragona, Spain;
falgabachueca@gmail.com (F.A.-C.); ana.megia@gmail.com (A.M.)
Murdoch Children’s Research Institute, Royal Children’s Hospital, 3052 Melbourne, Australia;
boris.novakovic@mcri.edu.au (B.N.); richard.saffery@mcri.edu.au (R.S.)
Division of Cell Biology, Histology and Embryology, Gottfried Schatz Research Centre for Cell Signaling,
Metabolism and Ageing, Medical University of Graz, 8036 Graz, Austria; martin.gauster@medunigraz.at
Femina Med Center, 8010 Graz, Austria; office@dr-glasner.at
Correspondence: gernot.desoye@medunigraz.at; Tel.: +43-316-385-84605
Received: 10 December 2019; Accepted: 9 January 2020; Published: 11 January 2020
Abstract: In the first trimester of pregnancy, placental development involves a wide range of cellular
processes. These include trophoblast proliferation, fusion, and differentiation, which are dependent
on tight cell cycle control. The intrauterine environment affects placental development, which also
includes the trophoblast cell cycle. In this work, we focus on maternal obesity to assess whether an
altered intrauterine milieu modulates expression and protein levels of placental cell cycle regulators in
early human pregnancy. For this purpose, we use first trimester placental tissue from lean and obese
women (gestational week 5+0 –11+6 , n = 58). Using a PCR panel, a cell cycle protein array, and STRING
database analysis, we identify a network of cell cycle regulators increased by maternal obesity in
which breast cancer 1 (BRCA1) is a central player. Immunostaining localizes BRCA1 predominantly to
the villous and the extravillous cytotrophoblast. Obesity-driven BRCA1 upregulation is not able to be
explained by DNA methylation (EPIC array) or by short-term treatment of chorionic villous explants
at 2.5% oxygen with tumor necrosis factor α (TNF-α) (50 mg/mL), leptin (100 mg/mL), interleukin
6 (IL-6) (100 mg/mL), or high glucose (25 nM). Oxygen tension rises during the first trimester, but this
change in vitro has no effect on BRCA1 (2.5% and 6.5% O2 ). We conclude that maternal obesity affects
placental cell cycle regulation and speculate this may alter placental development.
Keywords: human placenta; first trimester; early pregnancy; obesity; BRCA1; cell cycle
1. Introduction
Adequate human placental development is crucial for embryonic and fetal growth. During the
first trimester of pregnancy, placental development relies on several cellular processes, including
trophoblast proliferation, differentiation, and fusion [1,2]. Within placental villi, the structural unit
of the first trimester placenta, villous cytotrophoblasts (vCTs) proliferate and fuse to give rise to the
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syncytiotrophoblast (ST), a multinucleated cell layer with transport, endocrine, barrier, and protective
functions. vCT progenitors can lose their proliferative potential and differentiate into extravillous
cytotrophoblasts (EVTs), an invasive cell type that migrates and invades the decidua, thus anchoring
the fetoplacental unit to the uterus [3]. EVTs also reach and remodel the maternal spiral arteries to
ensure adequate blood supply to the fetus [4,5].
Villous trophoblast turnover and EVT invasion are tightly regulated both in a spatial- and
time-dependent manner [6]. Although this regulation has an autocrine and paracrine component
mediated by placenta-derived signals, including progesterone, human chorionic gonadotropin,
placental lactogen, transforming growth factor β, and kisspeptins, among others [7,8], the maternal
intrauterine environment also plays a pivotal role [9]. We have previously described how several factors
found in the maternal circulation, such as insulin, tumor necrosis factor α (TNF-α), and endothelin-1,
regulate diverse aspects of first trimester trophoblast biology [10–12]. Oxygen tension, which rises
during the first trimester of pregnancy due to spiral artery remodeling ultimately resulting in flow
onset of fully oxygenated blood, is also a major regulator of trophoblast biology [13,14]. It has become
increasingly appreciated that maternal environment derangements affecting trophoblast biology
during the first trimester of pregnancy may determine trajectories of placental growth, development,
and function throughout pregnancy, with ensuing perinatal and long-term consequences for offspring
health due to fetal programming [15].
Maternal obesity is characterized by a sustained low-grade pro-inflammatory and metabolically
altered environment and entails long term consequences for mother and offspring [16]. Obesity has
drastically increased among women of reproductive age during the last decade [17]. Pregnancy
complications such as preeclampsia and gestational diabetes mellitus are more common among obese
pregnant women, suggesting that maternal obesity affects placental function [18,19]. Additionally,
the fetus is affected by maternal obesity, as reflected by the altered fetal metabolome in maternal obesity.
This may contribute to the higher incidence of type 2 diabetes, hypertension, and obesity among obese
women’s offspring [20].
While obesity-associated adverse effects on maternal and fetal health have been studied in
depth at term of pregnancy [21], the effect of maternal obesity on the human first trimester placenta,
and specifically on the trophoblast, remains poorly understood [15]. Recent studies have suggested
that a tight control of cell cycle progression and cell cycle arrest is required during trophoblast
proliferation, fusion, and invasion [22]. Interestingly, maternal obesity has been shown to alter
trophoblast transcriptome, affecting genes involved in cell metabolism and cell function [23]. Hence,
as part of a major effort to understand effects of maternal obesity on first trimester growth and
development, we focus in this study on first trimester placental cell cycle regulation.
Although the expression of classical cell cycle regulators, e.g., cyclin A, B, and D, and Ki67, p21,
and p27, has already been described in human first trimester placenta [24,25], for the first time we
identify breast cancer 1 (BRCA1) at the core of a network of cell cycle regulators affected by maternal
obesity in early pregnancy. We assess its specific location as well as its expression levels in human
first trimester placental tissue from lean (gestational week 5+0 –11+6 ; n = 37) and obese (gestational
week 5+0 –11+2 ; n = 21) women. Finally, we determine its specific short-term regulation in vitro
by obesity-associated pro-inflammatory cytokines and oxygen tension using human chorionic first
trimester explants.
2. Results
In order to determine the effect of maternal obesity on placental cell cycle modulators, first trimester
placental tissue from non-smoking women was divided into two groups, i.e., lean (n = 37, mean body
mass index (BMI) = 22.2 kg/m2 ) and obese (n = 21, mean BMI = 32.3 kg/m2 ). Table 1 shows the sample
characteristics for each experimental approach.
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Table 1. Description of the study cohorts by experiment.
Characteristics
Sample size (n)
Gestational age (days)
Maternal age (years)
Maternal BMI (kg/m2 )
Fetal sex
PCR Panel and Protein Array
Lean
Obese
p
7
6
49.0 ± 0.0 49.8 ± 1.8
0.3
33.0 ± 5.8 36.7 ± 5.3
0.3
20.3 ± 1.6 30.3 ± 2.3
<0.0001
4 m; 3 f
3 m; 3 f
Lean
15
57.3 ± 13.5
27.8 ± 7.0
22.2 ± 1.6
6 m; 9 f
Methylation
Obese
15
56.1 ± 13.1
30.3 ± 6.0
34.3 ± 3.4
9 m; 6 f
p
0.7
0.3
<0.0001
IHC
Lean
4
53.0 ± 17.5
31.7 ± 9
24.5 ± 3.3
1 m; 3 f
Explants
Lean
11
51.9 ± 11.9
28.2 ± 7.4
21.9 ± 3.3
6 m; 5 f
Data are expressed as mean ± SD. The Mann-Whitney test was used for statistical analysis. Legend: BMI, body
mass index; IHC, immunohistochemistry; m, male; f, female.
2.1. Maternal Obesity Affects Placental Cell Cycle Regulators Already in the First Trimester of Pregnancy
A PCR panel and a protein array were used to first test the effect of maternal obesity on gene
expression and protein levels of several cell cycle regulators in placental tissue from lean and obese
women (week 7). The results were analyzed using a multivariate linear regression (MVLR) model with
BMI as the exposure variable adjusting for maternal age.
Only cell cycle regulators with a fold change (FC) > 1.3 and p < 0.05 were considered significant.
Maternal obesity increased the expression of 9 out of 187 (4.8%) cell cycle regulators (Supplementary
Table S1), with excision repair 5 (ERCC5) and BRCA1 showing the highest fold change between lean
and obese (1.5-fold, p = 0.02 and 2.0-fold, p = 0.03, Figure 1A, respectively). Among the cell cycle
regulating proteins, 25 out of 95 (26.3%) were also increased (Supplementary Table S2), with Rad52
and BRCA1 showing the highest fold change (1.5-fold, p < 0.0001 and 1.4-fold, p = 0.002, respectively,
Figure 1B). Phospho-BRCA1 (p(Ser1423) -BRCA1) was also significantly upregulated by maternal obesity
(FC = 1.3, p = 0.009, Figure 1B).
Figure 1. Induction of negative cell cycle regulators by maternal obesity in early pregnancy. Volcano
plots show fold change for genes (A) and proteins (B) differentially expressed between lean (n = 7) and
obese (n = 6) placental tissue (gestational week 7). PCR panel and protein array results were analyzed
through multivariate linear regression using BMI as exposure, adjusting for maternal age. Differences
were considered significant when p < 0.05 and the fold change threshold was set to 1.3. x axis = log2
fold change (lean versus obese), y axis = multivariate linear regression p value. Legend: BRCA1, breast
cancer 1; ERRC5, excision repair 5; p(Ser1423) -BRCA1, phospho-BRCA1.
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2.2. BRCA1 Is a Key Player in Cell Cycle Regulation in Early Pregnancy and Is Upregulated by
Maternal Obesity
Among all the cell cycle regulators analyzed in the present study, only BRCA1 mRNA and protein
were concordantly upregulated by maternal obesity (Figure 2A and Supplementary Figure S1). Using
the STRING database network analysis tool, we established a protein–protein association network
to identify possible functional interactions between these obesity-upregulated cell cycle modulators.
Within this network, BRCA1 was located at a central position (Figure 2B, arrow), interacting with
proteins involved in different cell cycle events such as Chk1, Chk2, and Myc.
Figure 2. Central role of placental BRCA1 in the first trimester of pregnancy in the context of
maternal obesity. Venn diagram depicting cell cycle genes and proteins regulated by maternal obesity
≥
(BMI ≥ 30 kg/m2 ) in the first trimester of pregnancy with BRCA1 as the only common factor (A).
Protein–protein interaction analysis of the upregulated proteins using the STRING database shows
a central role of BRCA1 in the cell cycle regulation network (B, arrow). Line shape indicates the
predicted mode of action, with nodes describing protein action effects (arrow: positive, dash: negative,
circle: unspecified) and line color describing protein action types (blue: binding, black: reaction, green:
activation, red: inhibition, pink: posttranslational modification, violet: catalysis). The minimum
required interaction score was set to medium confidence (0.4), showing up to 10 interactions in the
first shell.
BRCA1 upregulation by maternal obesity was confirmed by Nanostring analysis and Western
blotting. Nanostring data showed an increase in BRCA1 expression by 52.3% in placental tissue from
obese versus lean women (p = 0.032, Figure 3A). Similarly, placental BRCA1 and p(Ser1423) -BRCA1
protein levels were also increased within the obese cohort (by 49.1%, p = 0.044 and 57.4%, p = 0.001,
respectively) compared to the lean cohort (Figure 3B–D). The differences observed were independent
of fetal sex.
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Figure 3. Placental BRCA1 is upregulated by maternal obesity. BRCA1 expression and protein levels
were determined in first trimester placental tissue (gestational week 7) from lean (n = 6–7) and obese
(n = 6) women by Nanostring (A) and Western blotting (B–D), respectively. Gene counts of Nanostring
analysis were normalized to the mean of two different housekeeping (HK) genes (WD repeat domain 45B
(WDR45L) and TATA box binding protein (TBP, A). Immunoblots for BRCA1 and p(Ser1423) -BRCA1 (B)
were quantified by densitometric analysis (C and D). β-actin and α-tubulin were used for normalization
α using the
as loading controls. Results are presented as mean ± SD. Statistical analysisβwas performed
Mann Whitney test or t-test as appropriate.
2.3. BRCA1 DNA Methylation Is Not Altered by Maternal Obesity
Obesity influences placental gene expression through DNA methylation [26]. To test whether
obesity-associated BRCA1 upregulation is the result of epigenetic changes in early pregnancy, placental
BRCA1 methylation was assessed. We found no evidence that maternal obesity alters the DNA
methylation profile of the 86 CpGs in the BRCA1 gene in the first trimester placenta (Figure 4).
Mean beta values for the sum of all CpG sites of obese total tissue DNA samples (0.319, gestational
week 6+0 –11+6 , n = 15) were similar to those of the lean group (0.318, gestational week 5+0 –11+2 , n = 15).
The BRCA1 methylation profile was also not affected by maternal age (data not shown). A list with all
analyzed CpGs can be found in Supplementary Table S3.
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Figure 4. DNA methylation profile of BRCA1 gene is not altered by maternal obesity in the first
trimester of pregnancy (n(lean/obese) = 15/15). Genome-wide DNA methylation quantification using
Infinium MethylationEPIC Array (850 K) BeadChip included 86 CpGs annotated to the BRCA1 gene.
2.4. Placental BRCA1 Is Mainly Localized to vCTs and EVTs during the First Trimester of Pregnancy
BRCA1 location in early, mid, and late first trimester placental tissue (gestational week 5, 8,
and 12, respectively) was detected by immunostaining. Both vCTs and EVTs showed strong BRCA1
staining which was localized to the nuclei and the cytosol (Figure 5A, C, E). Stromal cells were also
stained with anti-BRCA1. BRCA1 immunostaining in the ST was located in the nuclei (Figure 5B, D, F,
arrowheads). No differences in location were observed between early, mid, and late first trimester
placental specimens. Negative (immunoglobulin G isotype) and positive (ovarian tumor section)
controls showed no signal or strong nuclear and cytosolic BRCA1 staining, respectively (Supplementary
Figure S2).
α
α
−
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Figure 5. Immunohistochemistry of placental tissue from early (gestational week 5, A and B),
mid (gestational week 8, C and D), and late (gestational week 12, E and F) first trimester localizing
BRCA1 to the nuclei and the cytoplasm of villous cytotrophoblasts (vCTs, A, C, and E) and extravillous
cytotrophoblasts (EVTs, A, C, and E). Syncytiotrophoblast (ST) BRCA1 immunostaining was located
in the nuclei (B, D, and F, arrowheads. Open arrowheads indicate vCTs). Scale bar: 100 µm (A, C
and E) or 20 µm (B, D and F). Dotted frames in A, C, and E indicate those fields shown with a higher
magnification in B, D and F. Positive (ovarian cancer specimen) and negative (IgG isotype) controls
(Supplementary Figure S2).
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2.5. Short Term Exposure to Obesity-Associated Cytokines, Hyperglycemia, and Increased Oxygen Tension Do
Not Regulate Placental BRCA1 Levels in Early Pregnancy
To identify whether potential components of the pro-inflammatory environment associated
with obesity can drive acute BRCA1 upregulation in the first trimester placenta, human chorionic
villous explants (gestational week 5+0 –10+4 , n = 6–11) were incubated with TNF-α (50 ng/mL), leptin
(100 ng/mL), and interleukin 6 (IL-6) (100 ng/mL) for 48 h. BRCA1 expression was determined by
RT-qPCR (Figure 6A) and BRCA1 and p(Ser1423) -BRCA1 protein levels were measured by Western
blotting (Figure 6D, G, J). TNF-α induced a downregulation of BRCA1 expression (−42% versus control,
p < 0.001). However, BRCA1 and p(Ser1423) -BRCA1 protein levels remained unaffected. No effects were
found upon leptin and IL-6 treatment.
Figure 6. BRCA1 is not regulated by short term exposure to obesity-associated inflammation,
hyperglycemia, or oxygen tension in early pregnancy. First trimester placental chorionic villous
explants from different placental tissues (n = 4–11, gestational week 5–11) were cultured at 2.5% O2
with tumor
α necrosis
α factor α (TNF-α) (50 ng/mL), leptin (100 ng/mL), interleukin 6 (IL-6) (100 ng/mL),
D-glucose (25 nM), and L-glucose (25 nM, osmotic control) for 48 h in triplicate. Explants (n = 10–12)
were also cultured at 6.5% O2 . BRCA1 gene expression was analyzed by RT-qPCR and normalized to the
mean of hypoxanthine phosphoribosyltransferase 1 (HPRT1) and peptidylprolyl isomerase A (PPIA),
which were used as housekeeping (HK) genes (A-C). BRCA1 and p(Ser1423) -BRCA1 protein levels were
analyzed by Western blotting (D–F). Immunoblots were quantified by densitometric analysis (G–I for
BRCA1 and J–L for pβ(Ser1423) -BRCA1), with β-actin used as a loading control.ΔData are shown as –∆Ct
β
(A–C) or ratio to β-actin (G–L) and have been presented as mean ± SD from different placental tissues
(n = 4–12). Statistical analysis included the Mann Whitney test or Friedman’s test followed by Dunn’s
post hoc analysis.
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In a similar experimental set-up, we investigated the influence of hyperglycemia on BRCA1.
Chorionic villi (gestational week 6+4 –10+4 , n = 4–8) were incubated with high glucose (D-glucose,
25 nM) and L-glucose (25 nM) used as an osmotic control. High glucose did not affect BRCA1 mRNA
(Figure 6B) or protein levels (Figure 6E, H). BRCA1 Ser1423-phosphorylation was also not altered by
glucose treatment (Figure 6E, K).
All the experiments were performed at 2.5% O2 , which was considered as a physiological O2
tension for early first trimester placenta. Since oxygen tension in the intervillous space rises during
the first trimester period covered by the explant experiments (weeks 5–10), we tested the potential
effect of oxygen on BRCA1. To this end, chorionic villi (gestational week 5+0 –9+1 , n = 10–12) were also
cultured under 6.5% O2 and the results compared to 2.5% O2 . No differences in BRCA1 expression
(Figure 6C) and BRCA1 and p(Ser1423) -BRCA1 protein levels (Figure 6F, I, L) were detected between the
two oxygen tensions.
The ratio between p(Ser1423) -BRCA1 and total BRCA1, reflecting activity of upstream ataxia
telangiectasia mutated (ATM) kinase, was also not affected by any of the treatments described above
(Supplementary Figure S3). In principle, the absence of a response to most of the treatments may have
been due to low tissue viability. This can be ruled out since human chorionic gonadotropin, a major
hormone produced by the trophoblast, was secreted into the culture medium, and its levels remained
stable during the various treatments (Supplementary Figure S4).
3. Discussion
Adequate trophoblast proliferation, differentiation, fusion, and survival are required for successful
placental development and function [22]. Although cell cycle regulation is crucial for these biological
processes [27], placental cell cycle modulators and their potential regulation by the intrauterine
environment have been scarcely investigated.
Obesity has been associated with low-grade sustained inflammation and oxidative stress,
both classical triggers of cell cycle arrest [28]. In the present study, we demonstrated that maternal
obesity increases the expression of several cell cycle regulators, i.e., 9 genes and 25 proteins, in the
first trimester of pregnancy, suggesting that obesity affects placental cell cycle control already in
early pregnancy. This might in turn compromise vCT proliferation and differentiation into EVTs,
altering invasion and spiral artery remodeling, and may, thus, set the basis for obesity-associated
pregnancy disorders such as preeclampsia [29]. Advanced maternal age is known to alter placental
cell proliferation and apoptosis [30,31] and might also modify cell cycle regulation. Thus, this was
accounted for in the present study by adjusting exposure–outcome relationships for maternal age.
This should allow for the identification of obesity-mediated effects, although residual confounding
cannot be excluded.
To subsequently characterize the effect of maternal obesity on specific cell cycle modulators,
we selected BRCA1 as a potential candidate based on its consistent upregulation at the mRNA and the
protein level. BRCA1 has been traditionally studied in the context of breast and ovarian cancer [32],
where it plays a pivotal role in establishing an adequate DNA damage response [33,34]. It also
plays an important role in cell cycle checkpoint regulation, inducing G1 cell arrest through p27
activation, blocking S phase entry through p53-dependent activation, and favoring G2/M arrest through
p53-dependent 14-3-3 zeta/delta activation [35,36]. Interestingly, we found that BRCA1 was the only
cell cycle modulator upregulated at both the gene and protein level by maternal obesity. Several
BRCA1 protein binding partners were also upregulated by maternal obesity, including 14-3-3 zeta/delta,
chk1, and chk2, and the latter is known to induce cell cycle arrest in a pathway involving cdc25 and
p-BRCA1 [37,38]. We also observed that p(Ser1423) -BRCA1 was upregulated by maternal obesity. Several
kinases are involved in BRCA1 phosphorylation, including chk2, Akt, and ATM, the latter being
directly involved in BRCA1 phosphorylation at serine 1423 [39]. Phosphorylation of BRCA1 fine-tunes
its function. The enhanced phosphorylation in obesity might reflect further BRCA1 interactions with
other cell cycle regulators [40]. Indeed, our network analysis of functional protein associations using
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the STRING database identified BRCA1 as central in the pathways controlling placental cell cycle
regulation in early pregnancy.
Only a few reports have investigated BRCA1 location in late first trimester and term human
placental tissue [41,42]. Thus, a thorough characterization of BRCA1 location in early pregnancy was
still required. We localized BRCA1 predominantly to vCTs and EVTs in early, mid, and late first
trimester placental tissue. BRCA1 immunostaining within the ST was weaker and restricted to a few
nuclei. A similar distribution pattern, i.e., absence or weak expression in ST versus high expression on
vCTs and EVTs, has been described for several other cell cycle regulators including Ki67, cyclin A, p53,
and p57 [24,43]. Considering that ST is characterized by the absence of an active cell cycle [44], BRCA1
positive nuclei within the ST might be the result of recent vCT–ST fusion.
It has been previously reported that subcellular BRCA1 location also determines its function [45].
We detected BRCA1 in the nuclei and cytosol of vCTs. Cytosolic BRCA1 location has been associated
with highly proliferating breast cancer tumors [46]. Its presence in the cytosol of vCTs reflects the high
proliferative potential, as can be expected from the trophoblast stem cell population. The essential role
of BRCA1 for vCTs is also demonstrated by BRCA1 knock-down leading to increased apoptosis in the
first trimester trophoblast cell line Swan71 [42], suggesting that BRCA1 promotes trophoblast survival.
Likewise, its presence in EVTs might reflect the role of BRCA1 in cell cycle regulation during the gradual
differentiation of vCTs into EVTs, which precedes invasion. Whether BRCA1 is directly involved in
regulating cell proliferation and invasion in primary vCTs and EVTs needs to be further studied.
We then aimed to determine which obesity-associated molecular mechanisms could explain
the differences observed in placental BRCA1 levels between lean and obese women. Among other
mechanisms obesity has been shown to alter gene expression through DNA methylation [47], which
may be a candidate mechanism to explain these differences. This hypothesis is also supported by a
recent study demonstrating BRCA1 downregulation due to promoter hypermethylation in disorders
characterized by trophoblast over-proliferation [48]. Despite the overall effects of obesity on placental
DNA methylation [26], placental BRCA1 methylation profile was not affected by maternal obesity in
early pregnancy.
To identify short-term drivers of BRCA1 changes related to obesity we chose those cytokines most
prominently associated with the pro-inflammatory environment of obesity [49] and tested them in
an in vitro villous explant culture model. TNF-α treatment altered BRCA1 gene expression without
concomitant changes of BRCA1 protein. This lack of a short-term effect on BRCA1 protein levels
does not preclude the possibility that chronic exposure to TNF-α in vivo can contribute to BRCA1
upregulation, as found in the obese cohort. Similarly, IL-6 and leptin did not regulate BRCA1 protein.
Although we carefully selected cytokine concentrations within the physiological range to mimic the
in vivo environment in first trimester placenta [50], we did not investigate a potential interplay between
these cytokines, which might also fine-tune placental BRCA1 regulation. Moreover, treatment of
placental chorionic villous explants from lean women with pro-inflammatory cytokines only allows
testing for their short-term effects (up to 48 h), which may not be long enough to induce changes seen
as the result of long-term tissue exposure to the obesity environment.
Obesity-associated hyperglycemia has been shown to alter placental development and function [51].
Interestingly, hyperglycemia is able to promote cell cycle arrest via cyclin D1 and p21 upregulation [52].
Here, high glucose treatment did not alter human placental BRCA1 gene expression or protein levels.
Intriguingly, women with BRCA1 mutations are more prone to developing diabetes, a risk that
also increases in women with a high BMI [53]. In this regard, hyperinsulinemia is also a common
feature of both obesity and diabetes, and high insulin levels have been linked to cell cycle arrest in
mouse keratinocytes [54]. Thus, further studies assessing the potential role of obesity-associated
hyperinsulinemia on BRCA1 levels are warranted.
Oxygen tension is one of the major regulators of vCT proliferation and EVT differentiation.
Hence, we determined whether oxygen might directly regulate first trimester placental BRCA1 levels.
Physiological oxygen concentrations rise from 2.5% to 6.5% O2 in early pregnancy [55]. Although we
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have shown that BRCA1 and p(Ser1423) -BRCA1 levels remained stable under both physiological oxygen
concentrations, BRCA1-associated RING domain protein 1 (BARD1) is increased in first trimester
trophoblast under physiological low oxygen tension (6.5% O2 ) [56]. The BARD1–BRCA1 dimer results
in BRCA1 stabilization [35]. Thus, BRCA1 activity might indirectly be regulated by oxygen tension.
To the best of our knowledge, this is the first study assessing the influence of maternal obesity
on placental cell cycle regulators during the first trimester of pregnancy. The results using placental
tissue, which preserves the spatial arrangement of cells and the extracellular matrix, in which they
are embedded, clearly demonstrated an effect of maternal obesity on several cell cycle modulators.
Among these we could identify BRCA1 as a central node in the network of cell cycle regulator proteins.
It localizes to vCTs and EVTs independently of maternal BMI. Key components of the obesity-associated
inflammatory and metabolic environment are unlikely to contribute to these changes. The physiological
increase in oxygen tension and DNA methylation do also not appear to drive BRCA1 upregulation
in obesity.
In the present study all the experiments were conducted on human first trimester placental tissue in
a physiological, i.e., low, oxygen environment. This is a major strength and avoids potential hyperoxic
effects of ambient oxygen (21% O2 ). The main limitation of this study was the lack of an in-depth
analysis of the cellular consequences of obesity-associated BRCA1 changes. Moreover, concentration
and time-dependent effects of pro-inflammatory cytokines and the hyperglycemic environment on
BRCA1 regulation cannot be excluded. Likewise, other maternal metabolic factors that could explain
the obesity associated long-term effects on BRCA1 levels, e.g., hyperinsulinemia and insulin resistance,
were not investigated in the present study and their contribution cannot be ruled out.
Collectively, our results suggest that BRCA1 might play a role in first trimester trophoblast biology.
Thus, its upregulation by maternal obesity might alter placental development with potentially ensuing
adverse consequences for maternal and fetal health.
4. Materials and Methods
4.1. Study Subjects
The study was approved by the institutional review board and the ethical committee of the
Medical University of Graz (29-095 ex16/17, 23 December 2016). Women with a singleton pregnancy
scheduled for legal elective pregnancy termination were recruited upon signing written informed
consent. Since smoking has major effects on metabolism and inflammation, we carefully excluded
smoking women, who were identified by serum cotinine levels ≥0.03 nmol/L [57]. Women with other
comorbidities and those under current medication were also excluded from the study.
Upon inclusion, clinical data was collected (Table 1). Maternal BMI was calculated using the
formula BMI = weight (kg)/height (m)2 . Gestational age was calculated based on the patient’s last
menstrual period and verified by measurement of the fetal crown-rump length.
4.2. Human Placental Tissue Collection
First trimester placental tissue (gestational week 5+0 –11+6 ) was obtained after surgical pregnancy
termination, washed with phosphate-buffered saline (PBS) and cryopreserved at −80 ◦ C until further
use, or was fixed in 4% paraformaldehyde (PFA) and paraffin-embedded. Fetal sex was assessed
by gene expression analysis (DDX3Y and XIST, see 4.6). Based on the maternal pre-pregnancy BMI,
gestational age matched samples were subsequently divided into two groups, i.e., lean (mean BMI =
22.2 kg/m2 ) and obese (mean BMI = 32.3 kg/m2 ).
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4.3. First Trimester Chorionic Villous Explants
Human first trimester chorionic villi were micro-dissected into small pieces (15–20 mg wet weight),
rinsed with PBS, and cultured in Dulbecco’s Modified Eagle Medium (DMEM; Gibco, Invitrogen,
Carlsbad, CA, USA) and Ham’s F-12 medium 1:1 (v/v; Gibco) supplemented with 10% fetal calf serum
(FCS, Thermo Scientific, Rockford, IL, USA) and 1% penicillin-streptomycin (Gibco). Placental explants
were cultured in a hypoxic workstation (BioSpherix; Redfeld, NY, USA). After 24 h of pre-incubation to
allow for adjustment to the in vitro conditions, samples were treated with TNF-α (50 ng/mL, Sigma
Aldrich, St. Louis, MO, USA), leptin (100 ng/mL, Sigma Aldrich), IL-6 (100 ng/mL, Sigma Aldrich),
D-Glucose (25 mM, Merck, Billerica, MA, USA), or L-Glucose (25 mM, Sigma Aldrich) at 2.5% O2 for
48 h. The effect of oxygen tension was additionally assessed at 6.5% O2 for 48 h. Thereafter, explants
were snap-frozen for subsequent RNA extraction or protein isolation. Culture supernatant was frozen
and used for human chorionic gonadotropin (hCG) analysis (see Supplementary Materials).
4.4. DNA/RNA Isolation and Reverse Transcription
First trimester placental tissue was homogenized in RLT Plus Buffer (Qiagen, Venlo, Netherlands)
with 1% β-mercaptoethanol (v/v, Merck) using a tissue lyser (MagNa Lyser, Roche, Basel, Switzerland).
DNA and total RNA was isolated with the AllPrep DNA/RNA/miRNA Universal Kit (Qiagen)
according to the manufacturer’s guidelines. After a quality check (Bioanalyzer, RNA integrity number
(RIN) > 3.0), mRNA reverse transcription was performed using the SuperScript II Reverse Transcriptase
kit (Life Technologies, Carlsbad, CA, USA) as per the manufacturer’s protocol.
4.5. PrimePCR Panel
Differential expression of 187 genes associated with DNA damage-repair and cell cycle regulation
was analyzed using a PrimePCR Collection panel (DNA damage H384 Predesigned 384-well, BioRad
Laboratories, Munich, Germany) according to the manufacturer´s guidelines. A complete list of the
genes and controls can be found on the manufacturer’s website (https://www.bio-rad.com/de-at/primepcr-assays/pathway/dna-damage-collection-panel). For each PCR reaction 10 ng of cDNA were used.
Real-time PCR was then conducted using the CFX384 PCR detection system (BioRad Laboratories)
and Ct values were generated by the associated software. Results were analyzed using the 2−∆∆Ct
method. Hypoxanthine phosphoribosyltransferase 1 (HPRT1) and TATA box binding protein (TBP)
were selected as housekeeping genes since their expression was unaffected by maternal obesity.
4.6. Real Time PCR
BRCA1 expression was determined by quantitative real-time PCR using FAM-labeled TaqMan
gene expression assays (Life Technologies, BRCA1: Hs01556193_m1). Fetal sex was determined
in a multiplex PCR setup using FAM-labelled DDX3Y and VIC-labelled XIST expression assays
(Life Technologies, DDX3Y: Hs00965254_gH, XIST: Hs01079824_m1) as described elsewhere [58].
RT-qPCR was performed using TaqMan Universal PCR Master Mix (Life Technologies) using the
CFX96 Thermocycler (BioRad Laboratories). A calibrator sample was added onto each plate to control
for inter-run variations. Ct values were generated by the BioRad CFX Manager 3.1 software and
relative gene expression was calculated by the 2−∆∆Ct method, with HPRT1 (Life Technologies, HPRT1:
Hs02800695_m1) and peptidylprolyl isomerase A (Life Technologies, PPIA: Hs04194521_s1) used as
housekeeping genes.
4.7. Nanostring
PrimePCR Panel validation was performed using the NanoString nCounter system (Nanostring
Technologies, Seattle, WA, USA), which is based on direct digital detection of mRNA molecules using
target-specific, color-coded probe pairs that hybridize directly to target molecules. Gene expression was
measured by counting the barcode for each specific molecule, which is detected by a digital analyzer.
Int. J. Mol. Sci. 2020, 21, 468
13 of 18
Positive normalization to the geo-mean of the top three positive controls and codeset normalization
on the reference genes WD repeat domain 45B (WDR45L) and TBP was performed using nSolver
software (Nanostring Technologies). Results have been expressed as gene counts of mRNA molecules
in 100 ng/µL RNA.
4.8. Protein Isolation and Quantification
Placental tissue was lysed in RIPA buffer (Sigma Aldrich) containing complete protease inhibitors
(Roche) using a tissue lyser (MagNa Lyser, Roche). Protein concentration was determined using the
bicinchoninic acid assay (BCA, Thermo Fisher Scientific) as per the manufacturer’s guidelines.
4.9. Cell Cycle Control Protein Array
Cell cycle-related protein profile was determined using the Cell Cycle Control Phospho Antibody
Array (95 site- and phospho-specific antibodies, Fullmoon Biosystems, Sunnyvale, CA, USA) according
to the manufacturer´s guidelines. Briefly, slides were treated with blocking solution (Fullmoon
Biosystems) for 30 min at room temperature and incubated with 75 µg of biotin-labelled first trimester
placental protein lysates overnight at 4 ◦ C. After washing, conjugated proteins were detected using
Cy3-conjugated streptavidin. Image analysis was performed using GenePix Pro 7.0 software (Molecular
Devices, San Jose, CA, USA). After local background subtraction, data were normalized on the median
intensity value of all antibodies on each array (excluding empty spots and negative/positive markers).
Only those signals exceeding the background intensity by two-fold were considered.
4.10. Immunoblotting
Equal amounts of total protein were mixed with Laemmli-buffer (Sigma) and denatured for
5 min at 96 ◦ C. Ten micrograms of total protein were loaded onto 4–20% SDS-PAGE gels (BioRad
Laboratories), resolved for 1 h at 110 V, and transferred to nitrocellulose membranes using the BioRad
TurboBlot (BioRad Laboratories). Blotting efficiency was determined with Ponceau staining (Ponceau
S solution, Sigma-Aldrich). Membranes were blocked for 1 h with 5 % non-fat dry milk (Bio-Rad) in
tris-buffered saline (TBS) and 0.1% Tween 20 (Sigma), and subsequently incubated with anti-BRCA1
(1:1000, Sigma-Aldrich, AB-1423), anti-p(Ser1423) -BRCA1 (1:1000, Merck, 07-635), anti-α-tubulin (1:1000,
Merck, CP06), and anti-β-actin (1:10000, ab8227, Abcam, Cambridge, UK) antibodies overnight at 4 ◦ C.
Thereafter, membranes were incubated with the appropriated horseradish peroxidase (HRP)-conjugated
secondary antibody (BioRad Laboratories, 1:2000 for BRCA1, p(Ser1423) -BRCA1, and α-tubulin, 1:25,000
for β-actin) for 1 h at room temperature. Immunodetection using SuperSignal-Pico Chemiluminescent
Substrate (Thermo Scientific) was visualized with a Fusion FX instrument (Vilber Lourmat, Collégien,
France). Band intensity was quantified using EvolutionCapt software (Vilber Lourmat). To account
for inter-membrane variation, data was normalized to an internal calibrator sample (first trimester
placental tissue) included in every gel.
4.11. Immunohistochemistry
BRCA1 immunostaining was performed on 3 µm thick sections of paraffin-embedded first trimester
placental tissue. After deparaffinization, antigen retrieval was performed using 10 mM citrate buffer
(pH 6) at 110 ◦ C for 10 min in a Decloaking Chamber (Biocare medical, Pacheco, CA, USA). BRCA1
immunohistochemistry was performed using the UltraVision LP Detection System (HRP polymer
kit, Thermo Fisher Scientific) as per the manufacturer´s instructions. Briefly, endogenous peroxidase
was blocked with UltraVision Hydrogen Peroxide Block for 10 min followed by an incubation with
UltraVision Protein Block for 5 min at room temperature. Anti-BRCA1 antibody (1:500, Sigma-Aldrich,
AB-1423) was diluted in Antibody Diluent (Dako, Glostrup, Denmark) and incubated for 1 h at room
temperature in a humidified chamber. Antibody detection was performed after incubation with
Primary Antibody Enhancer using an HRP-labeled polymer (Dako) and 3-amino-9-ethyl-carbazole
Single Solution (Thermo Scientific). Nuclei were counterstained with Mayer’s hematoxylin (Gatt Koller,
Int. J. Mol. Sci. 2020, 21, 468
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Absam, Austria) and slides were mounted with Aquatex (Merck, Darmstadt, Germany). Images were
acquired with a Zeiss Axio Z1 microscope (Zeiss, Oberkochen, Germany) equipped with a digital
camera (Olympus X, Tokyo, Japan) using AxioVision Software (Zeiss).
4.12. DNA Methylation Profiling
Genomic DNA samples were sent to HuGe-F (Erasmus MC, Rotterdam, Netherlands) for sodium
bisulfite treatment and genome-wide methylation analysis using Illumina InfiniumMethylationEPIC
BeadChips. Raw data (IDAT files) were exported from GenomeStudio (Illumina, San Diego, CA).
The Bioconductor package missMethyl was used to read the data into R and carry out quality control,
pre-processing, and normalization using the subset-quantile within array normalization (SWAN)
method [59,60]. The Bioconductor limma package was used to fit a linear model to compare lean and
obese placental tissue samples.
M values were calculated after removing poor performing probes (p value cut-off > 0.05 for all
samples) and probes on the sex chromosomes. Beta values were derived from intensities as defined by
the ratio of methylated to unmethylated probes (β = M/(U + M + 100)) and were used as a measure of
effect size.
4.13. Statistics
Statistical analysis was performed with GraphPad Prism 8, IBM SPSS Statistics 25, and R 3.6.1 [61].
Normal distribution of the data was determined using the Kolmogórov-Smirnov test. Associations
between maternal obesity and gene expression or protein levels were assessed using an MVLR
model adjusted for maternal age, since advanced maternal age might also alter placental cell cycle
regulation [30,31]. In this model, maternal age was considered a continuous variable and maternal
BMI was categorized into lean (mean BMI = 22.2 kg/m2 ) and obese (mean BMI = 32.3 kg/m2 ). Volcano
plots were generated using the linear regression p value (−log10) and a pre-calculated fold change
(log2) and visualized using the R Bioconductor packages ggplot2 [62], gridExtra [63], and plotly [64].
p < 0.05 together with a fold change exceeding 1.3 was considered statistically significant. Normally
distributed data were analyzed using a t-test and one-way ANOVA. For non-parametric analysis,
Mann Whitney or Friedman’s test followed by Dunn’s post hoc test was used. p < 0.05 was considered
statistically significant. The STRING database (https://string-db.org/) was used to visualize functional
protein interaction and protein array data is presented as a connectivity network showing action effects
and types.
Supplementary Materials: Supplementary materials can be found at http://www.mdpi.com/1422-0067/21/2/468/s1.
Author Contributions: Conceptualization, A.M.-M., R.S., and G.D.; methodology, D.H., C.P., M.B., F.A.-C., and
V.T.; formal analysis, B.N., D.H., C.P., M.B., and A.M.-M.; investigation, D.H., C.P., and M.B.; resources, A.G.; data
curation, D.H.; writing—original draft preparation, D.H. and A.M.-M.; writing—review and editing, A.M.-M.,
D.H., F.A.-C., B.N., M.G., A.M., R.S., and G.D.; visualization, A.M.-M., D.H., C.P., and M.B.; supervision, R.S. and
G.D.; funding acquisition, G.D. All authors have read and agreed to the published version of the manuscript.
Funding: D. Hoch was funded by the doctorate program MOLIN (FWF, W1241) and was supported by an Albert
Renold Travel Fellowship (EFSD, 94420). B. Novakovic was funded by an NHMRC Australia CJ Martin Fellowship.
M. Gauster was supported by the Austrian Science Fund (FWF) (P 29639, I 3304, and Doc 31-B26) and by funds of
the Oesterreichische Nationalbank (Anniversary Fund, project number 18175). A. Majali-Martinez and G. Desoye
were supported by funds of the Oesterreichische Nationalbank (Anniversary Fund, project number 17950).
Acknowledgments: The authors are grateful to the women who participated in this study. We would like to
thank Renate Michlmaier and Theresa-Maria Kaudela for their expertise and their technical assistance. We would
also like to express our gratitude to Monika Stubitsch for her administrative support.
Conflicts of Interest: The authors declare no conflict of interest.
Int. J. Mol. Sci. 2020, 21, 468
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Abbreviations
ATM
BCA
BMI
BRCA1
CRL
DMEM
EVTs
FC
hCG
HPRT1
IHC
IL-6
MVLR
PFA
PNKP
PPIA
SD
ST
T1D
TBP
TNF-α
vCTs
WDR45L
Ataxia telangiectasia mutated kinase
Bicinchoninic acid assay
Body mass index
Breast cancer 1
Crown-rump length
Dulbecco’s Modified Eagle Medium
Extravillous trophoblasts
Fold change
Human chorionic gonadotropin
Hypoxanthine phosphoribosyltransferase 1
Immunohistochemistry
Interleukin 6
Multivariate linear regression
Paraformaldehyde
Polynucleotide kinase 3′ -phosphatase
Peptidylprolyl isomerase A
Standard deviation
Syncytiotrophoblast
Type-1 diabetes
TATA box binding protein
Tumor necrosis factor α
Villous cytotrophoblasts
WD repeat domain 45B
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